Patrick W Mantyh

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• Chartier, S. R., Mitchell, S. A., Majuta, L. A., & Mantyh, P. W. (2018). The Changing Sensory and Sympathetic Innervation of the Young, Adult and Aging Mouse Femur. Neuroscience, 387, 178-190.
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  Although bone is continually being remodeled and ultimately declines with aging, little is known whether similar changes occur in the sensory and sympathetic nerve fibers that innervate bone. Here, immunohistochemistry and confocal microscopy were used to examine changes in the sensory and sympathetic nerve fibers that innervate the young (10 days post-partum), adult (3 months) and aging (24 months) C57Bl/6 mouse femur. In all three ages examined, the periosteum was the most densely innervated bone compartment. With aging, the total number of sensory and sympathetic nerve fibers clearly declines as the cambium layer of the periosteum dramatically thins. Yet even in the aging femur, there remains a dense sensory and sympathetic innervation of the periosteum. In cortical bone, sensory and sympathetic nerve fibers are largely confined to vascularized Haversian canals and while there is no significant decline in the density of sensory fibers, there was a 75% reduction in sympathetic nerve fibers in the aging vs. adult cortical bone. In contrast, in the bone marrow the overall density/unit area of both sensory and sympathetic nerve fibers appeared to remain largely unchanged across the lifespan. The preferential preservation of sensory nerve fibers suggests that even as bone itself undergoes a marked decline with age, the nociceptors that detect injury and signal skeletal pain remain relatively intact.
• Enomoto, M., Mantyh, P. W., Murrell, J., Innes, J. F., & Lascelles, B. D. (2019). Anti-nerve growth factor monoclonal antibodies for the control of pain in dogs and cats. The Veterinary record, 184(1), 23.
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  Nerve growth factor (NGF) is essential for the survival of sensory and sympathetic neurons during development. However, in the adult, NGF and its interaction with tropomyosin receptor kinase A receptor (TrkA) has been found to play a critical role in nociception and nervous system plasticity in pain conditions. Thus, various monoclonal antibody (mAb) therapies targeting this pathway have been investigated in the development of new pharmacotherapies for chronic pain. Although none of the mAbs against NGF are yet approved for use in humans, they look very promising for the effective control of pain. Recently, species-specific anti-NGF mAbs for the management of osteoarthritis (OA)-associated pain in dogs and cats has been developed, and early clinical trials have been conducted. Anti-NGF therapy looks to be both very effective and very promising as a novel therapy against chronic pain in dogs and cats. This review outlines the mechanism of action of NGF, the role of NGF in osteoarthritis, research in rodent OA models and the current status of the development of anti-NGF mAbs in humans. Furthermore, we describe and discuss the recent development of species-specific anti-NGF mAbs for the treatment of OA-associated pain in veterinary medicine.
• Majuta, L. A., Mitchell, S. A., Kuskowski, M. A., & Mantyh, P. W. (2018). Anti-nerve growth factor does not change physical activity in normal young or aging mice but does increase activity in mice with skeletal pain. Pain, 159(11), 2285-2295.
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  Anti-nerve growth factor (anti-NGF) therapy has shown significant promise in attenuating several types of skeletal pain. However, whether anti-NGF therapy changes the level of physical activity in individuals with or without skeletal pain is largely unknown. Here, automated day/night activity boxes monitored the effects of anti-NGF treatment on physical activity in normal young (3 months old) and aging (18-23 months old) mice and mice with bone fracture pain. Although aging mice were clearly less active and showed loss of bone mass compared with young mice, anti-NGF treatment had no effect on any measure of day/night activity in either the young or aging mice. By contrast, in mice with femoral fracture pain, anti-NGF treatment produced a clear increase (10%-27%) in horizontal activity, vertical rearing, and velocity of travel compared with the Fracture + Vehicle group. These results suggest, just as in humans, mice titrate their level of physical activity to their level of skeletal pain. The level of skeletal pain may in part be determined by the level of free NGF that seems to rise after injury but not normal aging of the skeleton. In terms of bone healing, animals that received anti-NGF showed an increase in the size of calcified callus but no increase in the number of displaced fractures or time to cortical union. As physical activity is the best nondrug treatment for many patients with skeletal pain, anti-NGF may be useful in reducing pain and promoting activity in these patients.
• Mantyh, P. W. (2018). Mechanisms that drive bone pain across the lifespan. British journal of clinical pharmacology.
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  Disorders of the skeleton are frequently accompanied by bone pain and a decline in the functional status of the patient. Bone pain occurs following a variety of injuries and diseases including; bone fracture, osteoarthritis, low back pain, orthopedic surgery, fibrous dysplasia, rare bone diseases, sickle cell disease, and bone cancer. In the past two decades, significant progress has been made in understanding the unique population of sensory and sympathetic nerves that innervate bone and the mechanisms that drive bone pain. Following physical injury of bone, mechanotranducers expressed by sensory nerve fibers that innervate bone are activated and sensitized so that even normally non-noxious loading or movement of bone is now being perceived as noxious. Injury of the bone also causes release of factors that; directly excite and sensitize sensory nerve fibers, up-regulate pro-algesic neurotransmitters, receptors and ion channels expressed by sensory neurons, induce ectopic sprouting of sensory and sympathetic nerve fibers resulting in a hyper-innervation of bone, and central sensitization in the brain that amplifies pain. Many of these mechanisms appear to be involved in driving both non-malignant and malignant bone pain. Results from human clinical trials suggest that mechanism-based therapies that attenuate one type of bone pain are often effective in attenuating pain in other seemingly unrelated bone diseases. Understanding the specific mechanisms that drive bone pain in different diseases and developing mechanism-based therapies to control this pain has the potential to fundamentally change the quality of life and functional status of patients suffering from bone pain.
• Mitchell, S. A., Majuta, L. A., & Mantyh, P. W. (2018). New Insights in Understanding and Treating Bone Fracture Pain. Current osteoporosis reports, 16(4), 325-332.
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  This paper describes recent advances in understanding the mechanisms that drive fracture pain and how these findings are helping develop new therapies to treat fracture pain.
• Chartier, S. R., Mitchell, S. A., Majuta, L. A., & Mantyh, P. W. (2017). Immunohistochemical localization of nerve growth factor, tropomyosin receptor kinase A, and p75 in the bone and articular cartilage of the mouse femur. Molecular pain, 13, 1744806917745465.
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  Sequestration of nerve growth factor (NGF) significantly attenuates skeletal pain in both animals and humans. However, relatively little is known about the specific cell types that express NGF or its cognate receptors tropomyosin receptor kinase A (TrkA) and p75 in the intact bone and articular cartilage. In the present study, antibodies raised against NGF, TrkA, and p75 (also known as CD271) were used to explore the expression of these antigens in the non-decalcified young mouse femur. In general, all three antigens displayed a remarkably restricted expression in bone and cartilage with less than 2% of all DAPI+ cells in the femur displaying expression of any one of the three antigens. Robust NGF immunoreactivity was found in mostly CD-31- blood vessel-associated cells, a small subset of CD-31+ endothelial cells, an unidentified group of cells located at the subchondral bone/articular cartilage interface, and a few isolated, single cells in the bone marrow. In contrast, p75 and TrkA were almost exclusively expressed by nerve fibers located nearby NGF+ blood vessels. The only non-neuronal expression of either p75 or TrkA in the femur was the expression of p75 by a subset of cells located in the deep and middle zone of the articular cartilage. Understanding the factors that tightly regulate the basal level of expression in normal bone and how the expression of NGF, TrkA, and p75 change in injury, disease, and aging may provide insights into novel therapies that can reduce skeletal pain and improve skeletal health.
• Majuta, L. A., Guedon, J. G., Mitchell, S. A., Ossipov, M. H., & Mantyh, P. W. (2017). Anti-nerve growth factor therapy increases spontaneous day/night activity in mice with orthopedic surgery-induced pain. Pain, 158(4), 605-617.
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  Total knee arthroplasty (TKA) and total hip arthroplasty (THA) are 2 of the most common and successful surgical interventions to relieve osteoarthritis pain. Control of postoperative pain is critical for patients to fully participate in the required physical therapy which is the most influential factor in effective postoperative knee rehabilitation. Currently, opiates are a mainstay for managing postoperative orthopedic surgery pain including TKA or THA pain. Recently, issues including efficacy, dependence, overdose, and death from opiates have made clinicians and researchers more critical of use of opioids for treating nonmalignant skeletal pain. In the present report, a nonopiate therapy using a monoclonal antibody raised against nerve growth factor (anti-NGF) was assessed for its ability to increase the spontaneous activity of the operated knee joint in a mouse model of orthopedic surgery pain-induced by drilling and coring the trochlear groove of the mouse femur. Horizontal activity and velocity and vertical rearing were continually assessed over a 20 hours day/night period using automated activity boxes in an effort to reduce observer bias and capture night activity when the mice are most active. At days 1 and 3, after orthopedic surgery, there was a marked reduction in spontaneous activity and vertical rearing; anti-NGF significantly attenuated this decline. The present data suggest that anti-NGF improves limb use in a rodent model of joint/orthopedic surgery and as such anti-NGF may be useful in controlling pain after orthopedic surgeries such as TKA or THA.
• Majuta, L. A., Mitchell, S. A., Kuskowski, M. H., & Mantyh, P. W. (2017). Mice with cancer-induced bone pain show a marked decline in day/night activity. PAIN Reports, 2(5), e614. doi:10.1097/PR9.0000000000000614
• Guedon, J. M., Longo, G., Majuta, L. A., Thomspon, M. L., Fealk, M. N., & Mantyh, P. W. (2016). Dissociation between the relief of skeletal pain behaviors and skin hypersensitivity in a model of bone cancer pain. Pain, 157(6), 1239-47.
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  Recent studies have suggested that in humans and animals with significant skeletal pain, changes in the mechanical hypersensitivity of the skin can be detected. However, whether measuring changes in skin hypersensitivity can be a reliable surrogate for measuring skeletal pain itself remains unclear. To explore this question, we generated skeletal pain by injecting and confining GFP-transfected NCTC 2472 osteosarcoma cells unilaterally to the femur of C3H male mice. Beginning at day 7 post-tumor injection, animals were administered vehicle, an antibody to the P2X3 receptor (anti-P2X3) or anti-NGF antibody. Pain and analgesic efficacy were then measured on days 21, 28, and 35 post-tumor injection using a battery of skeletal pain-related behaviors and von Frey assessment of mechanical hypersensitivity on the plantar surface of the hind paw. Animals with bone cancer pain treated with anti-P2X3 showed a reduction in skin hypersensitivity but no attenuation of skeletal pain behaviors, whereas animals with bone cancer pain treated with anti-NGF showed a reduction in both skin hypersensitivity and skeletal pain behaviors. These results suggest that although bone cancer can induce significant skeletal pain-related behaviors and hypersensitivity of the skin, relief of hypersensitivity of the skin is not always accompanied by attenuation of skeletal pain. Understanding the relationship between skeletal and skin pain may provide insight into how pain is processed and integrated and help define the preclinical measures of skeletal pain that are predictive end points for clinical trials.
• Hanlon, K. E., Lozano-Ondoua, A. N., Umaretiya, P. J., Symons-Liguori, A. M., Chandramouli, A., Moy, J. K., Kwass, W. K., Mantyh, P. W., Nelson, M. A., & Vanderah, T. W. (2016). Modulation of breast cancer cell viability by a cannabinoid receptor 2 agonist, JWH-015, is calcium dependent. Breast cancer (Dove Medical Press), 8, 59-71.
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  Cannabinoid compounds, both nonspecific as well as agonists selective for either cannabinoid receptor 1 (CB1) or cannabinoid receptor 2 (CB2), have been shown to modulate the tumor microenvironment by inducing apoptosis in tumor cells in several model systems. The mechanism of this modulation remains only partially delineated, and activity induced via the CB1 and CB2 receptors may be distinct despite significant sequence homology and structural similarity of ligands.
• Majuta, L. A., Guedon, J. G., Mitchell, S. A., Ossipov, M. H., & Mantyh, P. W. (2016). Anti-nerve growth factor therapy increases spontaneous day/night activity in mice with orthopedic surgery induced pain. Pain.
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  Total knee arthroplasty (TKA) and total hip arthroplasty (THA) are two of the most common and successful surgical interventions to relieve osteoarthritis pain. Control of postoperative pain is critical for patients to fully participate in the required physical therapy which is the most influential factor in effective postoperative knee rehabilitation. Currently opiates are a mainstay for managing postoperative orthopedic surgery pain including TKA/THA pain. Recently, issues including efficacy, dependence, overdose and death from opiates have made clinicians and researchers more critical of use of opioids for treating non-malignant skeletal pain. In the present report, a non-opiate therapy using a monoclonal antibody raised against nerve growth factor (anti-NGF) was assessed for its ability to increase the spontaneous activity of the operated knee joint in a mouse model of orthopedic surgery pain induced by drilling and coring the trochlear groove of the mouse femur. Horizontal activity & velocity and vertical rearing were continually assessed over a 20 hour day/night period using automated activity boxes in an effort to reduce observer bias and capture night activity when the mice are most active. At days 1 and 3 post-orthopedic surgery there was a marked reduction in spontaneous activity and vertical rearing; anti-NGF significantly attenuated this decline. The present data suggests that anti-NGF improves limb use in a rodent model of joint/orthopedic surgery and as such anti-NGF may be useful in controlling pain following orthopedic surgeries such as TKA/THA.
• Slosky, L. M., BassiriRad, N. M., Symons, A. M., Thompson, M., Doyle, T., Forte, B. L., Staatz, W. D., Bui, L., Neumann, W. L., Mantyh, P. W., Salvemini, D., Largent-Milnes, T. M., & Vanderah, T. W. (2016). The cystine/glutamate antiporter system xc- drives breast tumor cell glutamate release and cancer-induced bone pain. Pain, 157(11), 2605-2616.
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  Bone is one of the leading sites of metastasis for frequently diagnosed malignancies, including those arising in the breast, prostate and lung. Although these cancers develop unnoticed and are painless in their primary sites, bone metastases result in debilitating pain. Deeper investigation of this pain may reveal etiology and lead to early cancer detection. Cancer-induced bone pain (CIBP) is inadequately managed with current standard-of-care analgesics and dramatically diminishes patient quality of life. While CIBP etiology is multifaceted, elevated levels of glutamate, an excitatory neurotransmitter, in the bone-tumor microenvironment may drive maladaptive nociceptive signaling. Here, we establish a relationship between the reactive nitrogen species peroxynitrite, tumor-derived glutamate, and CIBP. In vitro and in a syngeneic in vivo model of breast CIBP, murine mammary adenocarcinoma cells significantly elevated glutamate via the cystine/glutamate antiporter system xc. The well-known system xc inhibitor sulfasalazine significantly reduced levels of glutamate and attenuated CIBP-associated flinching and guarding behaviors. Peroxynitrite, a highly reactive species produced in tumors, significantly increased system xc functional expression and tumor cell glutamate release. Scavenging peroxynitrite with the iron and mangano-based porphyrins, FeTMPyP and SRI10, significantly diminished tumor cell system xc functional expression, reduced femur glutamate levels and mitigated CIBP. In sum, we demonstrate how breast cancer bone metastases upregulate a cystine/glutamate co-transporter to elevate extracellular glutamate. Pharmacological manipulation of peroxynitrite or system xc attenuates CIBP, supporting a role for tumor-derived glutamate in CIBP and validating the targeting of system xc as a novel therapeutic strategy for the management of metastatic bone pain.
• Thompson, M. L., Chartier, S. R., Mitchell, S. A., & Mantyh, P. W. (2016). Preventing painful age-related bone fractures: Anti-sclerostin therapy builds cortical bone and increases the proliferation of osteogenic cells in the periosteum of the geriatric mouse femur. Molecular pain, 12.
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  Age-related bone fractures are usually painful and have highly negative effects on a geriatric patient's functional status, quality of life, and survival. Currently, there are few analgesic therapies that fully control bone fracture pain in the elderly without significant unwanted side effects. However, another way of controlling age-related fracture pain would be to preemptively administer an osteo-anabolic agent to geriatric patients with high risk of fracture, so as to build new cortical bone and prevent the fracture from occurring. A major question, however, is whether an osteo-anabolic agent can stimulate the proliferation of osteogenic cells and build significant amounts of new cortical bone in light of the decreased number and responsiveness of osteogenic cells in aging bone. To explore this question, geriatric and young mice, 20 and 4 months old, respectively, received either vehicle or a monoclonal antibody that sequesters sclerostin (anti-sclerostin) for 28 days. From days 21 to 28, animals also received sustained administration of the thymidine analog, bromodeoxyuridine (BrdU), which labels the DNA of dividing cells. Animals were then euthanized at day 28 and the femurs were examined for cortical bone formation, bone mineral density, and newly borne BrdU+ cells in the periosteum which is a tissue that is pivotally involved in the formation of new cortical bone. In both the geriatric and young mice, anti-sclerostin induced a significant increase in the thickness of the cortical bone, bone mineral density, and the proliferation of newly borne BrdU+ cells in the periosteum. These results suggest that even in geriatric animals, anti-sclerostin therapy can build new cortical bone and increase the proliferation of osteogenic cells and thus reduce the likelihood of painful age-related bone fractures.
• Kim, J. V., Tillu, D. V., Quinn, T. L., Mejia, G. L., Shy, A., Asiedu, M. N., Murad, E., Schumann, A. P., Totsch, S. K., Sorge, R. E., Mantyh, P. W., Dussor, G., & Price, T. J. (2015). Spinal dopaminergic projections control the transition to pathological pain plasticity via a D1/D5-mediated mechanism. The Journal of neuroscience : the official journal of the Society for Neuroscience, 35(16), 6307-17.
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  The mechanisms that lead to the maintenance of chronic pain states are poorly understood, but their elucidation could lead to new insights into how pain becomes chronic and how it can potentially be reversed. We investigated the role of spinal dorsal horn neurons and descending circuitry in plasticity mediating a transition to pathological pain plasticity suggesting the presence of a chronic pain state using hyperalgesic priming. We found that when dorsal horn neurokinin 1 receptor-positive neurons or descending serotonergic neurons were ablated before hyperalgesic priming, IL-6- and carrageenan-induced mechanical hypersensitivity was impaired, and subsequent prostaglandin E2 (PGE2) response was blunted. However, when these neurons were lesioned after the induction of priming, they had no effect on the PGE2 response, reflecting differential mechanisms driving plasticity in a primed state. In stark contrast, animals with a spinally applied dopaminergic lesion showed intact IL-6- and carrageenan-induced mechanical hypersensitivity, but the subsequent PGE2 injection failed to cause mechanical hypersensitivity. Moreover, ablating spinally projecting dopaminergic neurons after the resolution of the IL-6- or carrageenan-induced response also reversed the maintenance of priming as assessed through mechanical hypersensitivity and the mouse grimace scale. Pharmacological antagonism of spinal dopamine D1/D5 receptors reversed priming, whereas D1/D5 agonists induced mechanical hypersensitivity exclusively in primed mice. Strikingly, engagement of D1/D5 coupled with anisomycin in primed animals reversed a chronic pain state, consistent with reconsolidation-like effects in the spinal dorsal horn. These findings demonstrate a novel role for descending dopaminergic neurons in the maintenance of pathological pain plasticity.
• Majuta, L. A., Longo, G., Fealk, M. N., McCaffrey, G., & Mantyh, P. W. (2015). Orthopedic surgery and bone fracture pain are both significantly attenuated by sustained blockade of nerve growth factor. Pain, 156(1), 157-65.
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  The number of patients suffering from postoperative pain due to orthopedic surgery and bone fracture is projected to dramatically increase because the human life span, weight, and involvement in high-activity sports continue to rise worldwide. Joint replacement or bone fracture frequently results in skeletal pain that needs to be adequately controlled for the patient to fully participate in needed physical rehabilitation. Currently, the 2 major therapies used to control skeletal pain are nonsteroidal anti-inflammatory drugs and opiates, both of which have significant unwanted side effects. To assess the efficacy of novel therapies, mouse models of orthopedic and fracture pain were developed and evaluated here. These models, orthopedic surgery pain and bone fracture pain, resulted in skeletal pain-related behaviors that lasted 3 weeks and 8 to 10 weeks, respectively. These skeletal pain behaviors included spontaneous and palpation-induced nocifensive behaviors, dynamic weight bearing, limb use, and voluntary mechanical loading of the injured hind limb. Administration of anti-nerve growth factor before orthopedic surgery or after bone fracture attenuated skeletal pain behaviors by 40% to 70% depending on the end point being assessed. These data suggest that nerve growth factor is involved in driving pain due to orthopedic surgery or bone fracture. These animal models may be useful in developing an understanding of the mechanisms that drive postoperative orthopedic and bone fracture pain and the development of novel therapies to treat these skeletal pains.
• Thompson, M. L., Jimenez-Andrade, J. M., & Mantyh, P. W. (2015). Sclerostin Immunoreactivity Increases in Cortical Bone Osteocytes and Decreases in Articular Cartilage Chondrocytes in Aging Mice. The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.
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  Sclerostin is a 24-kDa secreted glycoprotein that has been identified as a negative modulator of new bone formation and may play a major role in age-related decline in skeletal function. Although serum levels of sclerostin markedly increase with age, relatively little is known about whether cells in the skeleton change their expression of sclerostin with aging. Using immunohistochemistry and confocal microscopy, we explored sclerostin immunoreactivity (sclerostin-IR) in the femurs of 4-, 9-, and 24-month-old adult C3H/HeJ male mice. In the femur, the only two cell types that expressed detectable levels of sclerostin-IR were bone osteocytes and articular cartilage chondrocytes. At three different sites along the diaphysis of the femur, only a subset of osteocytes expressed sclerostin-IR and the percentage of osteocytes that expressed sclerostin-IR increased from approximately 36% to 48% in 4- vs. 24-month-old mice. In marked contrast, in the same femurs, there were ~40% fewer hypertrophic chondrocytes of articular cartilage that expressed sclerostin-IR when comparing 24- vs. 4-month-old mice. Understanding the mechanism(s) that drive these divergent changes in sclerostin-IR may provide insight into understanding and treating the age-related decline of the skeleton.
• Thompson, M. L., Jimenez-Andrade, J. M., Chartier, S., Tsai, J., Burton, E. A., Habets, G., Lin, P. S., West, B. L., & Mantyh, P. W. (2015). Targeting cells of the myeloid lineage attenuates pain and disease progression in a prostate model of bone cancer. Pain, 156(9), 1692-702.
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  Tumor cells frequently metastasize to bone where they can generate cancer-induced bone pain (CIBP) that can be difficult to fully control using available therapies. Here, we explored whether PLX3397, a high-affinity small molecular antagonist that binds to and inhibits phosphorylation of colony-stimulating factor-1 receptor, the tyrosine-protein kinase c-Kit, and the FMS-like tyrosine kinase 3, can reduce CIBP. These 3 targets all regulate the proliferation and function of a subset of the myeloid cells including macrophages, osteoclasts, and mast cells. Preliminary experiments show that PLX3397 attenuated inflammatory pain after formalin injection into the hind paw of the rat. As there is an inflammatory component in CIBP, involving macrophages and osteoclasts, the effect of PLX3397 was explored in a prostate model of CIBP where skeletal pain, cancer cell proliferation, tumor metastasis, and bone remodeling could be monitored in the same animal. Administration of PLX3397 was initiated on day 14 after prostate cancer cell injection when the tumor was well established, and tumor-induced bone remodeling was first evident. Over the next 6 weeks, sustained administration of PLX3397 attenuated CIBP behaviors by approximately 50% and was equally efficacious in reducing tumor cell growth, formation of new tumor colonies in bone, and pathological tumor-induced bone remodeling. Developing a better understanding of potential effects that analgesic therapies have on the tumor itself may allow the development of therapies that not only better control the pain but also positively impact disease progression and overall survival in patients with bone cancer.
• Chartier, S. R., Thompson, M. L., Longo, G., Fealk, M. N., Majuta, L. A., & Mantyh, P. W. (2014). Exuberant sprouting of sensory and sympathetic nerve fibers in nonhealed bone fractures and the generation and maintenance of chronic skeletal pain. Pain, 155(11), 2323-36.
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  Skeletal injury is a leading cause of chronic pain and long-term disability worldwide. While most acute skeletal pain can be effectively managed with nonsteroidal anti-inflammatory drugs and opiates, chronic skeletal pain is more difficult to control using these same therapy regimens. One possibility as to why chronic skeletal pain is more difficult to manage over time is that there may be nerve sprouting in nonhealed areas of the skeleton that normally receive little (mineralized bone) to no (articular cartilage) innervation. If such ectopic sprouting did occur, it could result in normally nonnoxious loading of the skeleton being perceived as noxious and/or the generation of a neuropathic pain state. To explore this possibility, a mouse model of skeletal pain was generated by inducing a closed fracture of the femur. Examined animals had comminuted fractures and did not fully heal even at 90+days post fracture. In all mice with nonhealed fractures, exuberant sensory and sympathetic nerve sprouting, an increase in the density of nerve fibers, and the formation of neuroma-like structures near the fracture site were observed. Additionally, all of these animals exhibited significant pain behaviors upon palpation of the nonhealed fracture site. In contrast, sprouting of sensory and sympathetic nerve fibers or significant palpation-induced pain behaviors was never observed in naïve animals. Understanding what drives this ectopic nerve sprouting and the role it plays in skeletal pain may allow a better understanding and treatment of this currently difficult-to-control pain state.
• Fillingim, R. B., Bruehl, S., Dworkin, R. H., Dworkin, S. F., Loeser, J. D., Turk, D. C., Widerstrom-Noga, E., Arnold, L., Bennett, R., Edwards, R. R., Freeman, R., Gewandter, J., Hertz, S., Hochberg, M., Krane, E., Mantyh, P. W., Markman, J., Neogi, T., Ohrbach, R., , Paice, J. A., et al. (2014). The ACTTION-American Pain Society Pain Taxonomy (AAPT): an evidence-based and multidimensional approach to classifying chronic pain conditions. The journal of pain : official journal of the American Pain Society, 15(3), 241-9.
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  Current approaches to classification of chronic pain conditions suffer from the absence of a systematically implemented and evidence-based taxonomy. Moreover, existing diagnostic approaches typically fail to incorporate available knowledge regarding the biopsychosocial mechanisms contributing to pain conditions. To address these gaps, the Analgesic, Anesthetic, and Addiction Clinical Trial Translations Innovations Opportunities and Networks (ACTTION) public-private partnership with the U.S. Food and Drug Administration and the American Pain Society (APS) have joined together to develop an evidence-based chronic pain classification system called the ACTTION-APS Pain Taxonomy. This paper describes the outcome of an ACTTION-APS consensus meeting, at which experts agreed on a structure for this new taxonomy of chronic pain conditions. Several major issues around which discussion revolved are presented and summarized, and the structure of the taxonomy is presented. ACTTION-APS Pain Taxonomy will include the following dimensions: 1) core diagnostic criteria; 2) common features; 3) common medical comorbidities; 4) neurobiological, psychosocial, and functional consequences; and 5) putative neurobiological and psychosocial mechanisms, risk factors, and protective factors. In coming months, expert working groups will apply this taxonomy to clusters of chronic pain conditions, thereby developing a set of diagnostic criteria that have been consistently and systematically implemented across nearly all common chronic pain conditions. It is anticipated that the availability of this evidence-based and mechanistic approach to pain classification will be of substantial benefit to chronic pain research and treatment.
• Mantyh, P. W. (2014). Bone cancer pain: from mechanism to therapy. Current opinion in supportive and palliative care, 8(2), 83-90.
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  To review how common cancers such as breast, lung, and prostate cancers drive significant and frequently life-altering pain when the cells metastasize to bones.
• Mantyh, P. W. (2014). The neurobiology of skeletal pain. The European journal of neuroscience, 39(3), 508-19.
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  Disorders of the skeleton are one of the most common causes of chronic pain and long-term physical disability in the world. Chronic skeletal pain is caused by a remarkably diverse group of conditions including trauma-induced fracture, osteoarthritis, osteoporosis, low back pain, orthopedic procedures, celiac disease, sickle cell disease and bone cancer. While these disorders are diverse, what they share in common is that when chronic skeletal pain occurs in these disorders, there are currently few therapies that can fully control the pain without significant unwanted side effects. In this review we focus on recent advances in our knowledge concerning the unique population of primary afferent sensory nerve fibers that innervate the skeleton, the nociceptive and neuropathic mechanisms that are involved in driving skeletal pain, and the neurochemical and structural changes that can occur in sensory and sympathetic nerve fibers and the CNS in chronic skeletal pain. We also discuss therapies targeting nerve growth factor or sclerostin for treating skeletal pain. These therapies have provided unique insight into the factors that drive skeletal pain and the structural decline that occurs in the aging skeleton. We conclude by discussing how these advances have changed our understanding and potentially the therapeutic options for treating and/or preventing chronic pain in the injured, diseased and aged skeleton.
• McCaffrey, G., Thompson, M. L., Majuta, L., Fealk, M. N., Chartier, S., Longo, G., & Mantyh, P. W. (2014). NGF blockade at early times during bone cancer development attenuates bone destruction and increases limb use. Cancer research, 74(23), 7014-23.
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  Studies in animals and humans show that blockade of nerve growth factor (NGF) attenuates both malignant and nonmalignant skeletal pain. While reduction of pain is important, a largely unanswered question is what other benefits NGF blockade might confer in patients with bone cancer. Using a mouse graft model of bone sarcoma, we demonstrate that early treatment with an NGF antibody reduced tumor-induced bone destruction, delayed time to bone fracture, and increased the use of the tumor-bearing limb. Consistent with animal studies in osteoarthritis and head and neck cancer, early blockade of NGF reduced weight loss in mice with bone sarcoma. In terms of the extent and time course of pain relief, NGF blockade also reduced pain 40% to 70%, depending on the metric assessed. Importantly, this analgesic effect was maintained even in animals with late-stage disease. Our results suggest that NGF blockade immediately upon detection of tumor metastasis to bone may help preserve the integrity and use, delay the time to tumor-induced bone fracture, and maintain body weight.
• Lozano-Ondoua, A. N., Hanlon, K. E., Symons-Liguori, A. M., Largent-Milnes, T. M., Havelin, J. J., Ferland, H. L., Chandramouli, A., Owusu-Ankomah, M., Nikolich-Zugich, T., Bloom, A. P., Jimenez-Andrade, J. M., King, T., Porreca, F., Nelson, M. A., Mantyh, P. W., & Vanderah, T. W. (2013). Disease modification of breast cancer-induced bone remodeling by cannabinoid 2 receptor agonists. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 28(1), 92-107.
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  Most commonly originating from breast malignancies, metastatic bone cancer causes bone destruction and severe pain. Although novel chemotherapeutic agents have increased life expectancy, patients are experiencing higher incidences of fracture, pain, and drug-induced side effects; furthermore, recent findings suggest that patients are severely undertreated for their cancer pain. Strong analgesics, namely opiates, are first-line therapy in alleviating cancer-related pain despite the severe side effects, including enhanced bone destruction with sustained administration. Bone resorption is primarily treated with bisphosphonates, which are associated with highly undesirable side effects, including nephrotoxicity and osteonecrosis of the jaw. In contrast, cannabinoid receptor 2 (CB(2) ) receptor-specific agonists have been shown to reduce bone loss and stimulate bone formation in a model of osteoporosis. CB(2) agonists produce analgesia in both inflammatory and neuropathic pain models. Notably, mixed CB(1) /CB(2) agonists also demonstrate a reduction in ErbB2-driven breast cancer progression. Here we demonstrate for the first time that CB(2) agonists reduce breast cancer-induced bone pain, bone loss, and breast cancer proliferation via cytokine/chemokine suppression. Studies used the spontaneously-occurring murine mammary cell line (66.1) implanted into the femur intramedullary space; measurements of spontaneous pain, bone loss, and cancer proliferation were made. The systemic administration of a CB(2) agonist, JWH015, for 7 days significantly attenuated bone remodeling, assuaged spontaneous pain, and decreased primary tumor burden. CB(2) -mediated effects in vivo were reversed by concurrent treatment with a CB(2) antagonist/inverse agonist but not with a CB(1) antagonist/inverse agonist. In vitro, JWH015 reduced cancer cell proliferation and inflammatory mediators that have been shown to promote pain, bone loss, and proliferation. Taken together, these results suggest CB(2) agonists as a novel treatment for breast cancer-induced bone pain, in which disease modifications include a reduction in bone loss, suppression of cancer growth, attenuation of severe bone pain, and increased survival without the major side effects of current therapeutic options.
• Mantyh, P., & Mantyh, P. W. (2013). Bone cancer pain: Causes, consequences, and therapeutic opportunities. Pain.
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  Common cancers, including cancers of the breast, lung, and prostate, frequently metastasize to multiple bones where they can cause significant and life-altering pain. Similar to cancer itself, the factors that drive bone cancer pain evolve and change with disease progression. Once cancer cells have metastasized to bone, both the cancer cells and their associated stromal cells generate pain by releasing algogenic substances including protons, bradykinin, endothelins, prostaglandins, proteases, and tyrosine kinase activators. The release of these factors by cancer/stromal cells can induce sensitization and activation of nerve fibers that innervate the bone. Additionally, these factors can drive a remarkable increase in the number, size, and activity of bone-destroying osteoclasts, which can ultimately result in fracture of the tumor-bearing bone. Tumor growth in bone can also generate a neuropathic pain by directly injuring nerve fibers as well as inducing an active and highly pathological sprouting of both sensory and sympathetic nerve fibers that normally innervate the bone. This structural reorganization of sensory and sympathetic nerve fibers in the bone, combined with the cellular and neurochemical reorganization that occurs in the spinal cord and brain, appears to contribute to the peripheral and central sensitization that is common in advanced bone cancer pain. These mechanistic insights have begun to lead to advances in both how we understand and treat bone cancer pain.
• Ghilardi, J. R., Freeman, K. T., Jimenez-Andrade, J. M., Coughlin, K. A., Kaczmarska, M. J., Castaneda-Corral, G., Bloom, A. P., Kuskowski, M. A., & Mantyh, P. W. (2012). Neuroplasticity of sensory and sympathetic nerve fibers in a mouse model of a painful arthritic joint. Arthritis and rheumatism, 64(7), 2223-32.
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  Many forms of arthritis are accompanied by significant chronic joint pain. This study was undertaken to investigate whether there is significant sprouting of sensory and sympathetic nerve fibers in the painful arthritic knee joint and whether nerve growth factor (NGF) drives this pathologic reorganization.
• Mantyh, P., Jimenez-Andrade, J. M., & Mantyh, P. W. (2012). Sensory and sympathetic nerve fibers undergo sprouting and neuroma formation in the painful arthritic joint of geriatric mice. Arthritis research & therapy, 14(3).
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  Although the prevalence of arthritis dramatically increases with age, the great majority of preclinical studies concerning the mechanisms that drive arthritic joint pain have been performed in young animals. One mechanism hypothesized to contribute to arthritic pain is ectopic nerve sprouting; however, neuroplasticity is generally thought to be greater in young versus old nerves. Here we explore whether sensory and sympathetic nerve fibers can undergo a significant ectopic nerve remodeling in the painful arthritic knee joint of geriatric mice.
• Mantyh, P., Jimenez-Andrade, J. M., Mantyh, W. G., Bloom, A. P., Freeman, K. T., Ghilardi, J. R., Kuskowski, M. A., & Mantyh, P. W. (2012). The effect of aging on the density of the sensory nerve fiber innervation of bone and acute skeletal pain. Neurobiology of aging, 33(5).
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  As humans age there is a decline in most sensory systems including vision, hearing, taste, smell, and tactile acuity. In contrast, the frequency and severity of musculoskeletal pain generally increases with age. To determine whether the density of sensory nerve fibers that transduce skeletal pain changes with age, calcitonin gene related peptide (CGRP) and neurofilament 200 kDa (NF200) sensory nerve fibers that innervate the femur were examined in the femurs of young (4-month-old), middle-aged (13-month-old) and old (36-month-old) male F344/BNF1 rats. Whereas the bone quality showed a significant age-related decline, the density of CGRP(+) and NF200(+) nerve fibers that innervate the bone remained remarkably unchanged as did the severity of acute skeletal fracture pain. Thus, while bone mass, quality, and strength undergo a significant decline with age, the density of sensory nerve fibers that transduce noxious stimuli remain largely intact. These data may in part explain why musculoskeletal pain increases with age.
• Reilly, R. M., McDonald, H. A., Puttfarcken, P. S., Joshi, S. K., Lewis, L., Pai, M., Franklin, P. H., Segreti, J. A., Neelands, T. R., Han, P., Chen, J., Mantyh, P. W., Ghilardi, J. R., Turner, T. M., Voight, E. A., Daanen, J. F., Schmidt, R. G., Gomtsyan, A., Kort, M. E., , Faltynek, C. R., et al. (2012). Pharmacology of modality-specific transient receptor potential vanilloid-1 antagonists that do not alter body temperature. The Journal of pharmacology and experimental therapeutics, 342(2), 416-28.
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  The transient receptor potential vanilloid-1 (TRPV1) channel is involved in the development and maintenance of pain and participates in the regulation of temperature. The channel is activated by diverse agents, including capsaicin, noxious heat (≥ 43°C), acidic pH (< 6), and endogenous lipids including N-arachidonoyl dopamine (NADA). Antagonists that block all modes of TRPV1 activation elicit hyperthermia. To identify efficacious TRPV1 antagonists that do not affect temperature antagonists representing multiple TRPV1 pharmacophores were evaluated at recombinant rat and human TRPV1 channels with Ca(2+) flux assays, and two classes of antagonists were identified based on their differential ability to inhibit acid activation. Although both classes of antagonists completely blocked capsaicin- and NADA-induced activation of TRPV1, select compounds only partially inhibited activation of the channel by protons. Electrophysiology and calcitonin gene-related peptide release studies confirmed the differential pharmacology of these antagonists at native TRPV1 channels in the rat. Comparison of the in vitro pharmacological properties of these TRPV1 antagonists with their in vivo effects on core body temperature confirms and expands earlier observations that acid-sparing TRPV1 antagonists do not significantly increase core body temperature. Although both classes of compounds elicit equivalent analgesia in a rat model of knee joint pain, the acid-sparing antagonist tested is not effective in a mouse model of bone cancer pain.
• Castañeda-Corral, G., Jimenez-Andrade, J. M., Bloom, A. P., Taylor, R. N., Mantyh, W. G., Kaczmarska, M. J., Ghilardi, J. R., & Mantyh, P. W. (2011). The majority of myelinated and unmyelinated sensory nerve fibers that innervate bone express the tropomyosin receptor kinase A. Neuroscience, 178, 196-207.
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  Although skeletal pain is a leading cause of chronic pain and disability, relatively little is known about the specific populations of nerve fibers that innervate the skeleton. Recent studies have reported that therapies blocking nerve growth factor (NGF) or its cognate receptor, tropomyosin receptor kinase A (TrkA) are efficacious in attenuating skeletal pain. A potential factor to consider when assessing the analgesic efficacy of targeting NGF-TrkA signaling in a pain state is the fraction of NGF-responsive TrkA+ nociceptors that innervate the tissue from which the pain is arising, as this innervation and the analgesic efficacy of targeting NGF-TrkA signaling may vary considerably from tissue to tissue. To explore this in the skeleton, tissue slices and whole mount preparations of the normal, adult mouse femur were analyzed using immunohistochemistry and confocal microscopy. Analysis of these preparations revealed that 80% of the unmyelinated/thinly myelinated sensory nerve fibers that express calcitonin gene-related peptide (CGRP) and innervate the periosteum, mineralized bone and bone marrow also express TrkA. Similarly, the majority of myelinated sensory nerve fibers that express neurofilament 200 kDa (NF200) which innervate the periosteum, mineralized bone and bone marrow also co-express TrkA. In the normal femur, the relative density of CGRP+, NF200+ and TrkA+ sensory nerve fibers per unit volume is: periosteum>bone marrow>mineralized bone>cartilage with the respective relative densities being 100:2:0.1:0. The observation that the majority of sensory nerve fibers innervating the skeleton express TrkA+, may in part explain why therapies that block NGF/TrkA pathway are highly efficacious in attenuating skeletal pain.
• Ghilardi, J. R., Freeman, K. T., Jimenez-Andrade, J. M., Mantyh, W. G., Bloom, A. P., Bouhana, K. S., Trollinger, D., Winkler, J., Lee, P., Andrews, S. W., Kuskowski, M. A., & Mantyh, P. W. (2011). Sustained blockade of neurotrophin receptors TrkA, TrkB and TrkC reduces non-malignant skeletal pain but not the maintenance of sensory and sympathetic nerve fibers. Bone, 48(2), 389-98.
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  Current therapies for treating skeletal pain have significant limitations as available drugs (non-steroidal anti-inflammatory drugs and opiates) have significant unwanted side effects. Targeting nerve growth factor (NGF) or its cognate receptor tropomysin receptor kinase A (TrkA) has recently become an attractive target for inhibition of adult skeletal pain. Here we explore whether sustained administration of a selective small molecule Trk inhibitor that blocks TrkA, TrkB and TrkC kinase activity with nanomolar affinity reduces skeletal pain while allowing the maintenance of sensory and sympathetic neurons in the adult mouse. Twice-daily administration of a Trk inhibitor was begun 1 day post fracture and within 8 h of acute administration fracture pain-related behaviors were reduced by 50% without significant sedation, weight gain or inhibition of fracture healing. Following administration of the Trk inhibitor for 7 weeks, there was no significant decline in the density of unmyelinated or myelinated sensory nerve fibers, sympathetic nerve fibers, measures of acute thermal pain, acute mechanical pain, or general neuromuscular function. The present results suggest that sustained administration of a peripherally selective TrkA, B and C inhibitor significantly reduces skeletal pain without having any obvious detrimental effects on adult sensory and sympathetic nerve fibers or early fracture healing. As with any potential therapeutic advance, understanding whether the benefits of Trk blockade are associated with any risks or unexpected effects will be required to fully appreciate the patient populations that may benefit from this therapeutic approach.
• Mantyh, P. W., Koltzenburg, M., Mendell, L. M., Tive, L., & Shelton, D. L. (2011). Antagonism of nerve growth factor-TrkA signaling and the relief of pain. Anesthesiology, 115(1), 189-204.
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  Nerve growth factor (NGF) was originally discovered as a neurotrophic factor essential for the survival of sensory and sympathetic neurons during development. However, in the adult NGF has been found to play an important role in nociceptor sensitization after tissue injury. The authors outline mechanisms by which NGF activation of its cognate receptor, tropomyosin-related kinase A receptor, regulates a host of ion channels, receptors, and signaling molecules to enhance acute and chronic pain. The authors also document that peripherally restricted antagonism of NGF-tropomyosin-related kinase A receptor signaling is effective for controlling human pain while appearing to maintain normal nociceptor function. Understanding whether there are any unexpected adverse events and how humans may change their behavior and use of the injured/degenerating tissue after significant pain relief without sedation will be required to fully appreciate the patient populations that may benefit from these therapies targeting NGF.
• Mantyh, P., Bloom, A. P., Jimenez-Andrade, J. M., Taylor, R. N., Castañeda-Corral, G., Kaczmarska, M. J., Freeman, K. T., Coughlin, K. A., Ghilardi, J. R., Kuskowski, M. A., & Mantyh, P. W. (2011). Breast cancer-induced bone remodeling, skeletal pain, and sprouting of sensory nerve fibers. The journal of pain : official journal of the American Pain Society, 12(6).
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  Breast cancer metastasis to bone is frequently accompanied by pain. What remains unclear is why this pain tends to become more severe and difficult to control with disease progression. Here we test the hypothesis that with disease progression, sensory nerve fibers that innervate the breast cancer bearing bone undergo a pathological sprouting and reorganization, which in other nonmalignant pathologies has been shown to generate and maintain chronic pain. Injection of human breast cancer cells (MDA-MB-231-BO) into the femoral intramedullary space of female athymic nude mice induces sprouting of calcitonin gene-related peptide (CGRP(+)) sensory nerve fibers. Nearly all CGRP(+) nerve fibers that undergo sprouting also coexpress tropomyosin receptor kinase A (TrkA(+)) and growth-associated protein-43 (GAP43(+)). This ectopic sprouting occurs in periosteal sensory nerve fibers that are in close proximity to breast cancer cells, tumor-associated stromal cells, and remodeled cortical bone. Therapeutic treatment with an antibody that sequesters nerve growth factor (NGF), administered when the pain and bone remodeling were first observed, blocks this ectopic sprouting and attenuates cancer pain. The present data suggest that the breast cancer cells and tumor-associated stromal cells express and release NGF, which drives bone pain and the pathological reorganization of nearby CGRP(+)/TrkA(+)/GAP43(+) sensory nerve fibers.
• Mantyh, P., Jimenez-Andrade, J. M., Ghilardi, J. R., Castañeda-Corral, G., Kuskowski, M. A., & Mantyh, P. W. (2011). Preventive or late administration of anti-NGF therapy attenuates tumor-induced nerve sprouting, neuroma formation, and cancer pain. Pain, 152(11).
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  Early, preemptive blockade of nerve growth factor (NGF)/tropomyosin receptor kinase A (TrkA) attenuates tumor-induced nerve sprouting and bone cancer pain. A critical unanswered question is whether late blockade of NGF/TrkA can attenuate cancer pain once NGF-induced nerve sprouting and neuroma formation has occurred. By means of a mouse model of prostate cancer-induced bone pain, anti-NGF was either administered preemptively at day 14 after tumor injection when nerve sprouting had yet to occur, or late at day 35, when extensive nerve sprouting had occurred. Animals were humanely killed at day 70 when, in vehicle-treated animals, significant nerve sprouting and neuroma formation was present in the tumor-bearing bone. Although preemptive and sustained administration (days 14-70) of anti-NGF more rapidly attenuated bone cancer nociceptive behaviors than late and sustained administration (days 35-70), by day 70 after tumor injection, both preemptive and late administration of anti-NGF significantly reduced nociceptive behaviors, sensory and sympathetic nerve sprouting, and neuroma formation. In this model, as in most cancers, the individual cancer cell colonies have a limited half-life because they are constantly proliferating, metastasizing, and undergoing necrosis as the parent cancer cell colony outgrows its blood supply. Similarly, the sensory and sympathetic nerve fibers that innervate the tumor undergo sprouting at the viable/leading edge of the parent tumor, degenerate as the parent cancer cell colony becomes necrotic, and resprout in the viable, newly formed daughter cell colonies. These results suggest that preemptive or late-stage blockade of NGF/TrkA can attenuate nerve sprouting and cancer pain.
• Ghilardi, J. R., Freeman, K. T., Jimenez-Andrade, J. M., Mantyh, W. G., Bloom, A. P., Kuskowski, M. A., & Mantyh, P. W. (2010). Administration of a tropomyosin receptor kinase inhibitor attenuates sarcoma-induced nerve sprouting, neuroma formation and bone cancer pain. Molecular pain, 6, 87.
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  Pain often accompanies cancer and most current therapies for treating cancer pain have significant unwanted side effects. Targeting nerve growth factor (NGF) or its cognate receptor tropomyosin receptor kinase A (TrkA) has become an attractive target for attenuating chronic pain. In the present report, we use a mouse model of bone cancer pain and examine whether oral administration of a selective small molecule Trk inhibitor (ARRY-470, which blocks TrkA, TrkB and TrkC kinase activity at low nm concentrations) has a significant effect on cancer-induced pain behaviors, tumor-induced remodeling of sensory nerve fibers, tumor growth and tumor-induced bone remodeling. Early/sustained (initiated day 6 post cancer cell injection), but not late/acute (initiated day 18 post cancer cell injection) administration of ARRY-470 markedly attenuated bone cancer pain and significantly blocked the ectopic sprouting of sensory nerve fibers and the formation of neuroma-like structures in the tumor bearing bone, but did not have a significant effect on tumor growth or bone remodeling. These data suggest that, like therapies that target the cancer itself, the earlier that the blockade of TrkA occurs, the more effective the control of cancer pain and the tumor-induced remodeling of sensory nerve fibers. Developing targeted therapies that relieve cancer pain without the side effects of current analgesics has the potential to significantly improve the quality of life and functional status of cancer patients.
• Lozano-Ondoua, A. N., Wright, C., Vardanyan, A., King, T., Largent-Milnes, T. M., Nelson, M., Jimenez-Andrade, J. M., Mantyh, P. W., & Vanderah, T. W. (2010). A cannabinoid 2 receptor agonist attenuates bone cancer-induced pain and bone loss. Life sciences, 86(17-18), 646-53.
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  Cannabinoid CB(2) agonists have been shown to alleviate behavioral signs of inflammatory and neuropathic pain in animal models. AM1241, a CB(2) agonist, does not demonstrate central nervous system side effects seen with CB(1) agonists such as hypothermia and catalepsy. Metastatic bone cancer causes severe pain in patients and is treated with analgesics such as opiates. Recent reports suggest that sustained opiates can produce paradoxical hyperalgesic actions and enhance bone destruction in a murine model of bone cancer. In contrast, CB(2) selective agonists have been shown to reduce bone loss associated with a model of osteoporosis. Here we tested whether a CB(2) agonist administered over a 7day period inhibits bone cancer-induced pain as well as attenuates cancer-induced bone degradation.
• Mantyh, P., Jimenez-Andrade, J. M., Bloom, A. P., Stake, J. I., Mantyh, W. G., Taylor, R. N., Freeman, K. T., Ghilardi, J. R., Kuskowski, M. A., & Mantyh, P. W. (2010). Pathological sprouting of adult nociceptors in chronic prostate cancer-induced bone pain. The Journal of neuroscience : the official journal of the Society for Neuroscience, 30(44).
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  Pain frequently accompanies cancer. What remains unclear is why this pain frequently becomes more severe and difficult to control with disease progression. Here we test the hypothesis that with disease progression, sensory nerve fibers that innervate the tumor-bearing tissue undergo a pathological sprouting and reorganization, which in other nonmalignant pathologies has been shown to generate and maintain chronic pain. Injection of canine prostate cancer cells into mouse bone induces a remarkable sprouting of calcitonin gene-related peptide (CGRP(+)) and neurofilament 200 kDa (NF200(+)) sensory nerve fibers. Nearly all sensory nerve fibers that undergo sprouting also coexpress tropomyosin receptor kinase A (TrkA(+)). This ectopic sprouting occurs in sensory nerve fibers that are in close proximity to colonies of prostate cancer cells, tumor-associated stromal cells and newly formed woven bone, which together form sclerotic lesions that closely mirror the osteoblastic bone lesions induced by metastatic prostate tumors in humans. Preventive treatment with an antibody that sequesters nerve growth factor (NGF), administered when the pain and bone remodeling were first observed, blocks this ectopic sprouting and attenuates cancer pain. Interestingly, reverse transcription PCR analysis indicated that the prostate cancer cells themselves do not express detectable levels of mRNA coding for NGF. This suggests that the tumor-associated stromal cells express and release NGF, which drives the pathological reorganization of nearby TrkA(+) sensory nerve fibers. Therapies that prevent this reorganization of sensory nerve fibers may provide insight into the evolving mechanisms that drive cancer pain and lead to more effective control of this chronic pain state.
• Mantyh, P., Jimenez-Andrade, J. M., Mantyh, W. G., Bloom, A. P., Ferng, A. S., Geffre, C. P., & Mantyh, P. W. (2010). Bone cancer pain. Annals of the New York Academy of Sciences, 1198.
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  In the United States, cancer is the second most common cause of death and it is expected that about 562,340 Americans will have died of cancer in 2009. Bone cancer pain is common in patients with advanced breast, prostate, and lung cancer as these tumors have a remarkable affinity to metastasize to bone. Once tumors metastasize to bone, they are a major cause of morbidity and mortality as the tumor induces significant skeletal remodeling, fractures, pain, and anemia. Currently, the factors that drive cancer pain are poorly understood. However, several recently introduced models of bone cancer pain, which closely mirror the human condition, are providing insight into the mechanisms that drive bone cancer pain and guide the development of mechanism-based therapies to treat the cancer pain. Several of these mechanism-based therapies have now entered human clinical trials. If successful, these therapies have the potential to significantly enlarge the repertoire of modalities that can be used to treat bone cancer pain and improve the quality of life, functional status, and survival of patients with bone cancer.
• Mantyh, P., Jimenez-Andrade, J. M., Mantyh, W. G., Bloom, A. P., Xu, H., Ferng, A. S., Dussor, G., Vanderah, T. W., & Mantyh, P. W. (2010). A phenotypically restricted set of primary afferent nerve fibers innervate the bone versus skin: therapeutic opportunity for treating skeletal pain. Bone, 46(2).
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  Although musculoskeletal pain is one of the most common causes of chronic pain and physical disability in both developing and developed countries, relatively little is known about the nerve fibers and mechanisms that drive skeletal pain. Small diameter sensory nerve fibers, most of which are C-fiber nociceptors, can be separated into two broad populations: the peptide-rich and peptide-poor nerve fibers. Peptide-rich nerve fibers express substance P (SP) and calcitonin gene-related peptide (CGRP). In contrast, the peptide-poor nerve fibers bind to isolectin B4 (IB(4)) and express the purinergic receptor P(2)X(3) and Mas-related G protein-coupled receptor member d (Mrgprd). In the present report, we used mice in which the Mrgprd(+) nerve fibers express genetically encoded axonal tracers to determine the peptide-rich and peptide-poor sensory nerve fibers that innervate the glabrous skin of the hindpaw as compared to the bone marrow, mineralized bone and periosteum of the femur. Whereas the skin is richly innervated by CGRP(+), SP(+), P(2)X(3)(+) and Mrgprd(+) sensory nerve fibers, the bone marrow, mineralized bone and periosteum receive a significant innervation by SP(+) and CGRP(+), but not Mrgprd(+) and P(2)X(3)(+) nerve fibers. This lack of redundancy in the populations of C-fibers that innervate the bone may present a unique therapeutic opportunity for targeting skeletal pain as the peptide-rich and peptide-poor sensory nerve fibers generally express a different repertoire of receptors and channels to detect noxious stimuli. Thus, therapies that target the specific types of C-nerve fibers that innervate the bone may be uniquely effective in attenuating skeletal pain as compared to skin pain.
• Mantyh, W. G., Jimenez-Andrade, J. M., Stake, J. I., Bloom, A. P., Kaczmarska, M. J., Taylor, R. N., Freeman, K. T., Ghilardi, J. R., Kuskowski, M. A., & Mantyh, P. W. (2010). Blockade of nerve sprouting and neuroma formation markedly attenuates the development of late stage cancer pain. Neuroscience, 171(2), 588-98.
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  For many patients, pain is the first sign of cancer and, while pain can be present at any time, the frequency and intensity of pain tend to increase with advancing stages of the disease. Thus, between 75 and 90% of patients with metastatic or advanced-stage cancer will experience significant cancer-induced pain. One major unanswered question is why cancer pain increases and frequently becomes more difficult to fully control with disease progression. To gain insight into this question we used a mouse model of bone cancer pain to demonstrate that as tumor growth progresses within bone, tropomyosin receptor kinase A (TrkA)-expressing sensory and sympathetic nerve fibers undergo profuse sprouting and form neuroma-like structures. To address what is driving the pathological nerve reorganization we administered an antibody to nerve growth factor (anti-NGF). Early sustained administration of anti-NGF, whose cognate receptor is TrkA, blocks the pathological sprouting of sensory and sympathetic nerve fibers, the formation of neuroma-like structures, and inhibits the development of cancer pain. These results suggest that cancer cells and their associated stromal cells release nerve growth factor (NGF), which induces a pathological remodeling of sensory and sympathetic nerve fibers. This pathological remodeling of the peripheral nervous system then participates in driving cancer pain. Similar to therapies that target the cancer itself, the data presented here suggest that, the earlier therapies blocking this pathological nerve remodeling are initiated, the more effective the control of cancer pain.
• Bove, S. E., Flatters, S. J., Inglis, J. J., & Mantyh, P. W. (2009). New advances in musculoskeletal pain. Brain research reviews, 60(1), 187-201.
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  Non-malignant musculoskeletal pain is the most common clinical symptom that causes patients to seek medical attention and is a major cause of disability in the world. Musculoskeletal pain can arise from a variety of common conditions including osteoarthritis, rheumatoid arthritis, osteoporosis, surgery, low back pain and bone fracture. A major problem in designing new therapies to treat musculoskeletal pain is that the underlying mechanisms driving musculoskeletal pain are not well understood. This lack of knowledge is largely due to the scarcity of animal models that closely mirror the human condition which would allow the development of a mechanistic understanding and novel therapies to treat this pain. To begin to develop a mechanism-based understanding of the factors involved in generating musculoskeletal pain, in this review we present recent advances in preclinical models of osteoarthritis, post-surgical pain and bone fracture pain. The models discussed appear to offer an attractive platform for understanding the factors that drive this pain and the preclinical screening of novel therapies to treat musculoskeletal pain. Developing both an understanding of the mechanisms that drive persistent musculoskeletal pain and novel mechanism-based therapies to treat these unique pain states would address a major unmet clinical need and have significant clinical, economic and societal benefits.
• Honore, P., Chandran, P., Hernandez, G., Gauvin, D. M., Mikusa, J. P., Zhong, C., Joshi, S. K., Ghilardi, J. R., Sevcik, M. A., Fryer, R. M., Segreti, J. A., Banfor, P. N., Marsh, K., Neelands, T., Bayburt, E., Daanen, J. F., Gomtsyan, A., Lee, C., Kort, M. E., , Reilly, R. M., et al. (2009). Repeated dosing of ABT-102, a potent and selective TRPV1 antagonist, enhances TRPV1-mediated analgesic activity in rodents, but attenuates antagonist-induced hyperthermia. Pain, 142(1-2), 27-35.
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  Transient receptor potential vanilloid type 1 (TRPV1) is a ligand-gated ion channel that functions as an integrator of multiple pain stimuli including heat, acid, capsaicin and a variety of putative endogenous lipid ligands. TRPV1 antagonists have been shown to decrease inflammatory pain in animal models and to produce limited hyperthermia at analgesic doses. Here, we report that ABT-102, which is a potent and selective TRPV1 antagonist, is effective in blocking nociception in rodent models of inflammatory, post-operative, osteoarthritic, and bone cancer pain. ABT-102 decreased both spontaneous pain behaviors and those evoked by thermal and mechanical stimuli in these models. Moreover, we have found that repeated administration of ABT-102 for 5-12 days increased its analgesic activity in models of post-operative, osteoarthritic, and bone cancer pain without an associated accumulation of ABT-102 concentration in plasma or brain. Similar effects were also observed with a structurally distinct TRPV1 antagonist, A-993610. Although a single dose of ABT-102 produced a self-limiting increase in core body temperature that remained in the normal range, the hyperthermic effects of ABT-102 effectively tolerated following twice-daily dosing for 2 days. Therefore, the present data demonstrate that, following repeated administration, the analgesic activity of TRPV1 receptor antagonists is enhanced, while the associated hyperthermic effects are attenuated. The analgesic efficacy of ABT-102 supports its advancement into clinical studies.
• Jimenez-Andrade, J. M., Bloom, A. P., Mantyh, W. G., Koewler, N. J., Freeman, K. T., Delong, D., Ghilardi, J. R., Kuskowski, M. A., & Mantyh, P. W. (2009). Capsaicin-sensitive sensory nerve fibers contribute to the generation and maintenance of skeletal fracture pain. Neuroscience, 162(4), 1244-54.
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  Although skeletal pain can have a marked impact on a patient's functional status and quality of life, relatively little is known about the specific populations of peripheral nerve fibers that drive non-malignant bone pain. In the present report, neonatal male Sprague-Dawley rats were treated with capsaicin or vehicle and femoral fracture was produced when the animals were young adults (15-16 weeks old). Capsaicin treatment, but not vehicle, resulted in a significant (>70%) depletion in the density of calcitonin-gene related peptide positive (CGRP(+)) sensory nerve fibers, but not 200 kDa neurofilament H positive (NF200(+)) sensory nerve fibers in the periosteum. The periosteum is a thin, cellular and fibrous tissue that tightly adheres to the outer surface of all but the articulated surface of bone and appears to play a pivotal role in driving fracture pain. In animals treated with capsaicin, but not vehicle, there was a 50% reduction in the severity, but no change in the time course, of fracture-induced skeletal pain-related behaviors as measured by spontaneous flinching, guarding and weight bearing. These results suggest that both capsaicin-sensitive (primarily CGRP(+) C-fibers) and capsaicin-insensitive (primarily NF200(+) A-delta fibers) sensory nerve fibers participate in driving skeletal fracture pain. Skeletal pain can be a significant impediment to functional recovery following trauma-induced fracture, osteoporosis-induced fracture and orthopedic surgery procedures such as knee and hip replacement. Understanding the specific populations of sensory nerve fibers that need to be targeted to inhibit the generation and maintenance of skeletal pain may allow the development of more specific mechanism-based therapies that can effectively attenuate acute and chronic skeletal pain.
• Freeman, K. T., Koewler, N. J., Jimenez-Andrade, J. M., Buus, R. J., Herrera, M. B., Martin, C. D., Ghilardi, J. R., Kuskowski, M. A., & Mantyh, P. W. (2008). A fracture pain model in the rat: adaptation of a closed femur fracture model to study skeletal pain. Anesthesiology, 108(3), 473-83.
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  Because of the relative lack of understanding of the mechanisms that drive skeletal pain, the purpose of this study was to adapt a previously validated closed femur fracture model to quantitatively evaluate skeletal pain in female and male rats.
• Halvorson, K. G., Sevcik, M. A., Ghilardi, J. R., Sullivan, L. J., Koewler, N. J., Bauss, F., & Mantyh, P. W. (2008). Intravenous ibandronate rapidly reduces pain, neurochemical indices of central sensitization, tumor burden, and skeletal destruction in a mouse model of bone cancer. Journal of pain and symptom management, 36(3), 289-303.
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  Over half of all chronic cancer pain arises from metastases to bone and bone cancer pain is one of the most difficult of all persistent pain states to fully control. Currently, bone pain is treated primarily by opioid-based therapies, which are frequently accompanied by significant unwanted side effects. In an effort to develop nonopioid-based therapies that could rapidly attenuate tumor-induced bone pain, we examined the effect of intravenous administration of the bisphosphonate, ibandronate, in a mouse model of bone cancer pain. Following injection and confinement of green fluorescent protein-transfected murine osteolytic 2472 sarcoma cells into the marrow space of the femur of male C3H/HeJ mice, ibandronate was administered either as a single dose (300 microg/kg), at Day 7 post-tumor injection, when tumor-induced bone destruction and pain were first evident, or in three consecutive doses (100 microg/kg/day) at Days 7, 8, and 9 post-tumor injection. Intravenous ibandronate administered once or in three consecutive doses reduced ongoing and movement-evoked bone cancer pain-related behaviors, neurochemical markers of central sensitization, tumor burden, and tumor-induced bone destruction. These results support limited clinical trials that suggest the potential of ibandronate to rapidly attenuate bone pain and illuminate the mechanisms that may be responsible for limiting pain and disease progression.
• Mantyh, P., Jimenez-Andrade, J. M., Herrera, M. B., Ghilardi, J. R., Vardanyan, M., Melemedjian, O. K., & Mantyh, P. W. (2008). Vascularization of the dorsal root ganglia and peripheral nerve of the mouse: implications for chemical-induced peripheral sensory neuropathies. Molecular pain, 4.
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  Although a variety of industrial chemicals, as well as several chemotherapeutic agents used to treat cancer or HIV, preferentially induce a peripheral sensory neuropathy what remains unclear is why these agents induce a sensory vs. a motor or mixed neuropathy. Previous studies have shown that the endothelial cells that vascularize the dorsal root ganglion (DRG), which houses the primary afferent sensory neurons, are unique in that they have large fenestrations and are permeable to a variety of low and high molecular weight agents. In the present report we used whole-mount preparations, immunohistochemistry, and confocal laser scanning microscopy to show that the cell body-rich area of the L4 mouse DRG has a 7 fold higher density of CD31+ capillaries than cell fiber rich area of the DRG or the distal or proximal aspect of the sciatic nerve. This dense vascularization, coupled with the high permeability of these capillaries, may synergistically contribute, and in part explain, why many potentially neurotoxic agents preferentially accumulate and injure cells within the DRG. Currently, cancer survivors and HIV patients constitute the largest and most rapidly expanding groups that have chemically induced peripheral sensory neuropathy. Understanding the unique aspects of the vascularization of the DRG and closing the endothelial fenestrations of the rich vascular bed of capillaries that vascularize the DRG before intravenous administration of anti-neoplastic or anti-HIV therapies, may offer a mechanism based approach to attenuate these chemically induced peripheral neuropathies in these patients.
• Budai, D., Khasabov, S. G., Mantyh, P. W., & Simone, D. A. (2007). NK-1 receptors modulate the excitability of ON cells in the rostral ventromedial medulla. Journal of neurophysiology, 97(2), 1388-95.
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  The role of neurokinin-1 (NK-1) receptors in the rostral ventromedial medulla (RVM) was studied using extracellular single-unit recording combined with microiontophoresis. In rats, on- and off-type neurons were identified using noxious heat or mechanical stimuli applied to the tail. Responses evoked by iontophoretic application of N-methyl-d-aspartate (NMDA) were determined before and after intraplantar injection of capsaicin or iontophoretic application of substance P. In off cells, capsaicin produced an extended pause in ongoing activity but did not alter the subsequent spontaneous discharge rate or NMDA-evoked responses. In contrast, spontaneous discharge rates of on cells increased after capsaicin, and their responses to NMDA increased >100% above control values. The increased responses to NMDA after capsaicin were attenuated by iontophoretic application of the selective NK-1 receptor antagonist L-733,060. Similarly to capsaicin, iontophoretic application of the selective NK-1 receptor agonist, [Sar(9),Met(O(2))(11)]-substance P (SM-SP), increased the spontaneous discharge rate and NMDA-evoked responses of on cells by >100% of control values. These effects were antagonized by L-733,060. Immunohistochemical studies showed that a subset of neurons in the RVM labeled NK-1 receptors and that nearly all of these neurons were immunoreactive for the NMDAR1 subunit of the NMDA receptor. These results demonstrate that activation of NK-1 receptors in the RVM enhances responses of on cells evoked by NMDA. It is suggested that activation of NK-1 receptors in the RVM and the ensuing sensitization of on cells may contribute to the development of central sensitization and hyperalgesia after tissue injury and inflammation.
• Jimenez-Andrade, J. M., Martin, C. D., Koewler, N. J., Freeman, K. T., Sullivan, L. J., Halvorson, K. G., Barthold, C. M., Peters, C. M., Buus, R. J., Ghilardi, J. R., Lewis, J. L., Kuskowski, M. A., & Mantyh, P. W. (2007). Nerve growth factor sequestering therapy attenuates non-malignant skeletal pain following fracture. Pain, 133(1-3), 183-96.
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  Current therapies to treat skeletal fracture pain are extremely limited. Some non-steroidal anti-inflammatory drugs have been shown to inhibit bone healing and opiates induce cognitive dysfunction and respiratory depression which are especially problematic in the elderly suffering from osteoporotic fractures. In the present report, we developed a closed femur fracture pain model in the mouse where skeletal pain behaviors such as flinching and guarding of the fractured limb are reversed by 10mg/kg morphine. Using this model we showed that the administration of a monoclonal antibody against nerve growth factor (anti-NGF) reduced fracture-induced pain-related behaviors by over 50%. Treatment with anti-NGF reduced c-Fos and dynorphin up-regulation in the spinal cord at day 2 post-fracture. However, anti-NGF treatment did not reduce p-ERK and c-Fos expression at 20 and 90 min, respectively, following fracture. This suggests NGF is involved in maintenance but not the acute generation of fracture pain. Anti-NGF therapy did not inhibit bone healing as measured by callus formation, bridging of the fracture site or mechanical strength of the bone. As the anti-NGF antibody does not appreciably cross the blood-brain barrier, the present data suggest that the anti-hyperalgesic action of anti-NGF therapy results from blockade of activation and/or sensitization of the CGRP/trkA positive fibers that normally constitute the majority of sensory fibers that innervate the bone. These results demonstrate that NGF plays a significant role in driving fracture pain and that NGF sequestering therapies may be efficacious in attenuating this pain.
• Koewler, N. J., Freeman, K. T., Buus, R. J., Herrera, M. B., Jimenez-Andrade, J. M., Ghilardi, J. R., Peters, C. M., Sullivan, L. J., Kuskowski, M. A., Lewis, J. L., & Mantyh, P. W. (2007). Effects of a monoclonal antibody raised against nerve growth factor on skeletal pain and bone healing after fracture of the C57BL/6J mouse femur. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 22(11), 1732-42.
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  A closed femur fracture pain model was developed in the C57BL/6J mouse. One day after fracture, a monoclonal antibody raised against nerve growth factor (anti-NGF) was delivered intraperitoneally and resulted in a reduction in fracture pain-related behaviors of approximately 50%. Anti-NGF therapy did not interfere with bone healing as assessed by mechanical testing and histomorphometric analysis.
• Kristich, C. J., Chandler, J. R., & Dunny, G. M. (2007). Development of a host-genotype-independent counterselectable marker and a high-frequency conjugative delivery system and their use in genetic analysis of Enterococcus faecalis. Plasmid, 57(2), 131-44.
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  Enterococcus faecalis is a gram-positive commensal bacterium of the gastrointestinal tract. E. faecalis is also an opportunistic pathogen that frequently exhibits resistance to available antibiotics. Despite the clinical significance of the enterococci, genetic analysis has been restricted by limitations inherent in the available genetic tools. To facilitate genetic manipulation of E. faecalis, we developed a conjugative delivery system for high-frequency introduction of cloned DNA into target strains of E. faecalis and a host-genotype-independent counterselectable marker for use in markerless genetic exchange. We used these tools to construct a collection of E. faecalis mutant strains carrying defined mutations in several genes, including ccfA, eep, gelE, sprE, and an alternative sigma factor (sigH). Furthermore, we combined these mutations in various permutations to create double mutants, triple mutants, and a quadruple mutant of E. faecalis that enabled tests of epistasis to be conducted on the pheromone biosynthesis pathway. Analysis of cCF10 pheromone production by the mutants revealed that both the ccfA2 and delta eep10 mutations are epistatic to mutations in gelE/sprE. To our knowledge, this represents the first example of epistasis analysis applied to a chromosomally encoded biosynthetic pathway in enterococci. Thus, the advanced tools for genetic manipulation of E. faecalis reported here enable efficient and sophisticated genetic analysis of these important pathogens.
• Martin, C. D., Jimenez-Andrade, J. M., Ghilardi, J. R., & Mantyh, P. W. (2007). Organization of a unique net-like meshwork of CGRP+ sensory fibers in the mouse periosteum: implications for the generation and maintenance of bone fracture pain. Neuroscience letters, 427(3), 148-52.
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  Although bone fracture frequently results in significant pain and can lead to increased morbidity and mortality, it is still not clearly understood how sensory neurons are organized to detect fracture pain. In the present report we focused on the periosteum, as this thin tissue is highly innervated and tightly adherent to the outer surface of bone. To define the organization and distribution of the sensory and sympathetic fibers in the mouse femoral periosteum, we used whole-mount preparations, transverse sections, immunofluoresence and laser scanning confocal microscopy. While both the outer fibrous layer and the inner more cellular cambium layer of the periosteum receive an extensive innervation by calcitonin gene-related peptide (CGRP) and 200-kDa neurofilament (NF200) positive sensory fibers as well as tyrosine hydroxylase (TH) positive sympathetic fibers, there is a differential organization of sensory vs. sympathetic fibers within the periosteum. In both layers, the great majority of TH+ fibers are closely associated with CD31+ blood vessels and wind around the larger vessels in a corkscrew pattern. In contrast, the majority of CGRP+ and NF200+ sensory fibers in both layers lack a clear association with CD31+ blood vessels and appear to be organized in a dense net-like meshwork to detect mechanical distortion of periosteum and bone. This organization would explain why stabilization/fixation causes a marked attenuation of movement-evoked fracture pain. Understanding the organization, plasticity and molecular characteristics of sensory and sympathetic nerve fibers innervating the skeleton may permit the development of novel mechanism-based therapies for treating non-malignant skeletal pain.
• Peters, C. M., Jimenez-Andrade, J. M., Jonas, B. M., Sevcik, M. A., Koewler, N. J., Ghilardi, J. R., Wong, G. Y., & Mantyh, P. W. (2007). Intravenous paclitaxel administration in the rat induces a peripheral sensory neuropathy characterized by macrophage infiltration and injury to sensory neurons and their supporting cells. Experimental neurology, 203(1), 42-54.
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  Paclitaxel-induced peripheral neuropathy (PN) can be a significant problem for patients receiving chemotherapeutic regimens for the treatment of breast, ovarian, and lung cancer as PN can influence the quality of life and survivorship in these patients. To begin to understand the cellular changes that occur within the peripheral and central nervous system as PN develops, we intravenously infused rats with clinically relevant doses of paclitaxel. Ten days later, behavioral changes indicative of PN became evident that included mechanical allodynia, cold hyperalgesia, and deficits in ambulation/coordination. These behaviors were accompanied by increased expression of activating transcription factor 3 (ATF3; a marker of cellular injury) in a population of large>medium>small diameter sensory neurons, a population of satellite cells in the lumbar dorsal root ganglia (DRG) and in myelinating Schwann cells in the sciatic nerve. In addition, there was an increase in the expression of glial fibrillary acidic protein (GFAP) in DRG satellite cells and an increase in the number of CD68 positive activated macrophages within the DRG and peripheral nerve. Within lamina III-IV of the lumbar spinal cord, there was an increase in OX42 positive microglia. These data suggest that intravenous infusion of paclitaxel induces a peripheral neuropathy characterized by injury of neuronal and non-neuronal cells in the peripheral nervous system, macrophage activation in both the DRG and peripheral nerve, and microglial activation within the spinal cord. An understanding of the factors involved in the development and maintenance of PN may lead to mechanism based therapies that prevent/treat PN and thus improve the survival and quality of life of patients receiving chemotherapy.
• Peters, C. M., Jimenez-Andrade, J. M., Kuskowski, M. A., Ghilardi, J. R., & Mantyh, P. W. (2007). An evolving cellular pathology occurs in dorsal root ganglia, peripheral nerve and spinal cord following intravenous administration of paclitaxel in the rat. Brain research, 1168, 46-59.
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  Paclitaxel (Taxol) is a frontline antineoplastic agent used to treat a variety of solid tumors including breast, ovarian, or lung cancer. The major dose limiting side effect of paclitaxel is a peripheral sensory neuropathy that can last days to a lifetime. To begin to understand the cellular events that contribute to this neuropathy, we examined a marker of cell injury/regeneration (activating transcription factor 3; ATF3), macrophage hyperplasia/hypertrophy; satellite cell hypertrophy in the dorsal root ganglia (DRG) and sciatic nerve as well as astrocyte and microglial activation within the spinal cord at 1, 4, 6 and 10 days following intravenous infusion of therapeutically relevant doses of paclitaxel. At day 1 post-infusion, there was an up-regulation of ATF3 in a subpopulation of large and small DRG neurons and this up-regulation was present through day 10. In contrast, hypertrophy of DRG satellite cells, hypertrophy and hyperplasia of CD68(+) macrophages in the DRG and sciatic nerve, ATF3 expression in S100beta(+) Schwann cells and increased expression of the microglial marker (CD11b) and the astrocyte marker glial fibrillary acidic protein (GFAP) in the spinal cord were not observed until day 6 post-infusion. The present results demonstrate that using the time points and markers examined, DRG neurons show the first sign of injury which is followed days later by other neuropathological changes in the DRG, peripheral nerve and dorsal horn of the spinal cord. Understanding the cellular changes that generate and maintain this neuropathy may allow the development of mechanism-based therapies to attenuate or block this frequently painful and debilitating condition.
• Tracey, I., & Mantyh, P. W. (2007). The cerebral signature for pain perception and its modulation. Neuron, 55(3), 377-91.
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  Our understanding of the neural correlates of pain perception in humans has increased significantly since the advent of neuroimaging. Relating neural activity changes to the varied pain experiences has led to an increased awareness of how factors (e.g., cognition, emotion, context, injury) can separately influence pain perception. Tying this body of knowledge in humans to work in animal models of pain provides an opportunity to determine common features that reliably contribute to pain perception and its modulation. One key system that underpins the ability to change pain intensity is the brainstem's descending modulatory network with its pro- and antinociceptive components. We discuss not only the latest data describing the cerebral signature of pain and its modulation in humans, but also suggest that the brainstem plays a pivotal role in gating the degree of nociceptive transmission so that the resultant pain experienced is appropriate for the particular situation of the individual.
• Allen, J. W., Mantyh, P. W., Horais, K., Tozier, N., Rogers, S. D., Ghilardi, J. R., Cizkova, D., Grafe, M. R., Richter, P., Lappi, D. A., & Yaksh, T. L. (2006). Safety evaluation of intrathecal substance P-saporin, a targeted neurotoxin, in dogs. Toxicological sciences : an official journal of the Society of Toxicology, 91(1), 286-98.
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  Intrathecal (IT) substance P-Saporin (SP-SAP), a 33-kDa-targeted neurotoxin, produces selective destruction of superficial neurokinin 1 receptor (NK1r)-bearing cells in the spinal dorsal horn. In rats, SP-SAP prevents the formation of hyperalgesia and can reverse established neuropathic pain behavior in rodents. To determine the safety of this therapeutic modality in a large animal model, beagles received bolus IT lumbar injections of vehicle, SP-SAP (1.5, 15, 45, or 150 microg), or a nontargeted preparation of saporin (SAP, 150 microg) for immunohistological analysis of spinal cords. Doses of 15 microg SP-SAP and above produced a significant and equivalent loss of NK1r-bearing cells and dendrites in lumbar laminae II and I compared to vehicle- or SAP-treated animals. Cervical regions in all animals displayed no loss of NK1r immunoreactivity as compared to controls. Total numbers of neurons in the lumbar dorsal horn or alpha-motor neurons in the ventral horn demonstrated no significant changes. No increases in the astrocytic marker glial fibrillary acidic protein were noted following treatment with SP-SAP, suggesting a lack of generalized neurotoxicity. Additional dogs received doses of 1.5-150 microg SP-SAP or SAP and were sacrificed after 28 or 90 days to assess behavioral and physiological parameters. Although some acute motor signs were observed with both SP-SAP and SAP, no long-lasting significant events were noted in any of these animals. These data indicate no adverse toxicity at doses up to 10 times those necessary for producing loss of superficial NK1r-bearing neurons in a large animal model.
• Halvorson, K. G., Sevcik, M. A., Ghilardi, J. R., Rosol, T. J., & Mantyh, P. W. (2006). Similarities and differences in tumor growth, skeletal remodeling and pain in an osteolytic and osteoblastic model of bone cancer. The Clinical journal of pain, 22(7), 587-600.
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  More than 1.3 million cases of cancer will be diagnosed in 2006 in the United States alone, and 90% of patients with advanced cancer will experience significant, life-altering cancer-induced pain. Bone cancer pain is the most common pain in patients with advanced cancer as most common tumors including breast, prostate, and lung have a remarkable affinity to metastasize to bone. Once tumors metastasize to bone they are a major cause of morbidity and mortality as the tumor induces significant skeletal remodeling, pain and anemia, which reduce the survival and quality of life of the patient. Currently, the factors that drive cancer pain are poorly understood; however, several recently introduced models of cancer pain are not only providing insight into the mechanisms that drive bone cancer pain but are guiding the development of novel mechanism-based therapies to treat the pain and skeletal remodeling that accompanies metatstatic bone cancer. As analgesics can also influence disease progression, findings from these studies may lead to therapies that have the potential to improve the quality of life and survival of patients with skeletal malignancies.
• Jimenez-Andrade, J. M., Peters, C. M., Mejia, N. A., Ghilardi, J. R., Kuskowski, M. A., & Mantyh, P. W. (2006). Sensory neurons and their supporting cells located in the trigeminal, thoracic and lumbar ganglia differentially express markers of injury following intravenous administration of paclitaxel in the rat. Neuroscience letters, 405(1-2), 62-7.
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  Paclitaxel-induced peripheral neuropathy is a sensory neuropathy that affects thousands of cancer patients each year as paclitaxel is commonly used to treat breast, non-small cell lung and ovarian cancer. To begin to define the type and location of sensory neurons most impacted by paclitaxel, we examined rat trigeminal ganglion, thoracic and lumbar dorsal root ganglion (DRG) 10 days following intravenous infusion of clinically relevant doses of paclitaxel. To define the population of cells injured by paclitaxel, we examined the expression of activating transcription factor-3 (ATF3), a marker of cell injury; to define the hypertrophy of satellite cells, we quantified the expression of the intermediate filament protein glial fibrillary acidic protein (GFAP); and to define the activation of macrophages, we examined the expression of the lysosomal protein CD68. Intravenous infusion of paclitaxel induced a significant increase of ATF3 in mainly but not exclusively large and medium sensory neurons in all sensory ganglia. An increase in both GFAP immunofluorescence in satellite cells and the number of activated macrophages occurred in lumbar>thoracic>trigeminal ganglia of paclitaxel-treated rats. This differential expression of cellular markers suggests that the largest sensory cell bodies with the longest axons are the most at risk of being injured by paclitaxel (size and length dependent pathology). These results provide a pathological basis for the anatomical distribution of paclitaxel-induced symptoms in patients receiving therapeutic regimens of paclitaxel.
• Lindsay, T. H., Halvorson, K. G., Peters, C. M., Ghilardi, J. R., Kuskowski, M. A., Wong, G. Y., & Mantyh, P. W. (2006). A quantitative analysis of the sensory and sympathetic innervation of the mouse pancreas. Neuroscience, 137(4), 1417-26.
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  Pain from pancreatitis or pancreatic cancer can be both chronic and severe although little is known about the mechanisms that generate and maintain this pain. To define the peripheral sensory and sympathetic fibers involved in transmitting and modulating pancreatic pain, immunohistochemistry and confocal microscopy were used to examine the sensory and sympathetic innervation of the head, body and tail of the normal mouse pancreas. Myelinated sensory fibers were labeled with an antibody raised against 200 kD neurofilament H (clone RT97), thinly myelinated and unmyelinated peptidergic sensory fibers were labeled with antibodies raised against calcitonin gene-related peptide (CGRP) and post-ganglionic sympathetic fibers were labeled with an antibody raised against tyrosine hydroxylase (TH). RT97, CGRP, and TH immunoreactive fibers were present in parenchyma of the head, body and tail of the pancreas with the relative density of both RT97 and CGRP expressing fibers being head>body>tail, whereas for TH, a relatively even distribution was observed. In all three regions of the pancreas, RT97 fibers were associated mainly with large blood vessels, the CGRP fibers were associated with the large- and medium-sized blood vessels and the TH were associated with the large- and medium-sized blood vessels as well as capillaries. In addition to this extensive set of sensory and sympathetic nerve fibers that terminate in the pancreas, there were large bundles of en passant nerve fibers in the dorsal region of the pancreas that expressed RT97 or CGRP and were associated with the superior mesenteric plexus. These data suggest the pancreas receives a significant sensory and sympathetic innervation. Understanding the factors and disease states that sensitize and/or directly excite the nerve fibers that terminate in the pancreas as well as those that are en passant may aid in the development of therapies that more effectively modulate the pain that frequently accompanies diseases of the pancreas, such as pancreatitis and pancreatic cancer.
• Mantyh, P. W. (2006). Cancer pain and its impact on diagnosis, survival and quality of life. Nature reviews. Neuroscience, 7(10), 797-809.
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  Cancer pain significantly affects the diagnosis, quality of life and survival of patients with cancer. During the past decade, preclinical and clinical data has begun to provide insight into the mechanisms that drive and mask cancer pain and the mechanisms by which anti-neoplastic agents induce peripheral neuropathy. Developing a mechanism-based understanding and mechanism-based therapies to treat cancer-associated pain and sensory neuropathy, and incorporating these into mainstream cancer research and therapy, will be crucial to improving the quality of life and survival of patients with cancer.
• Sevcik, M. A., Jonas, B. M., Lindsay, T. H., Halvorson, K. G., Ghilardi, J. R., Kuskowski, M. A., Mukherjee, P., Maggio, J. E., & Mantyh, P. W. (2006). Endogenous opioids inhibit early-stage pancreatic pain in a mouse model of pancreatic cancer. Gastroenterology, 131(3), 900-10.
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  The endogenous opioid system is involved in modulating the experience of pain, the response to stress, and the action of analgesic therapies. Recent human imaging studies have shown a significant tonic modulation of visceral pain, raising the question of whether endogenous opioids tonically modulate the pain of visceral cancer.
• Ghilardi, J. R., Röhrich, H., Lindsay, T. H., Sevcik, M. A., Schwei, M. J., Kubota, K., Halvorson, K. G., Poblete, J., Chaplan, S. R., Dubin, A. E., Carruthers, N. I., Swanson, D., Kuskowski, M., Flores, C. M., Julius, D., & Mantyh, P. W. (2005). Selective blockade of the capsaicin receptor TRPV1 attenuates bone cancer pain. The Journal of neuroscience : the official journal of the Society for Neuroscience, 25(12), 3126-31.
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  Cancer colonization of bone leads to the activation of osteoclasts, thereby producing local tissue acidosis and bone resorption. This process may contribute to the generation of both ongoing and movement-evoked pain, resulting from the activation of sensory neurons that detect noxious stimuli (nociceptors). The capsaicin receptor TRPV1 (transient receptor potential vanilloid subtype 1) is a cation channel expressed by nociceptors that detects multiple pain-producing stimuli, including noxious heat and extracellular protons, raising the possibility that it is an important mediator of bone cancer pain via its capacity to detect osteoclast- and tumor-mediated tissue acidosis. Here, we show that TRPV1 is present on sensory neuron fibers that innervate the mouse femur and that, in an in vivo model of bone cancer pain, acute or chronic administration of a TRPV1 antagonist or disruption of the TRPV1 gene results in a significant attenuation of both ongoing and movement-evoked nocifensive behaviors. Administration of the antagonist had similar efficacy in reducing early, moderate, and severe pain-related responses, suggesting that TRPV1 may be a novel target for pharmacological treatment of chronic pain states associated with bone cancer metastasis.
• Goblirsch, M., Lynch, C., Mathews, W., Manivel, J. C., Mantyh, P. W., & Clohisy, D. R. (2005). Radiation treatment decreases bone cancer pain through direct effect on tumor cells. Radiation research, 164(4 Pt 1), 400-8.
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  The most used treatment for bone cancer pain is radiation; however, the mechanism responsible for analgesia after irradiation is unknown. The mechanistic influence of a single, localized 10-, 20- or 30-Gy dose of radiation on painful behaviors, osteolysis, histopathology and osteoclast number was evaluated in mice with painful femoral sarcomas. Dramatic reductions in pain behaviors (P < 0.05) and osteolysis (P < 0.0001) were seen in mice irradiated with 20 and 30 Gy. Irradiation reduced the tumor area by more than 75% (P < 0.05) but did not affect osteoclast frequency per mm2 tumor. Treatment with 20 Gy prior to tumor injection had no effect on tumor growth or pain behaviors, suggesting that radiation reduces osteolysis and pain through direct tumor effects. To demonstrate that tumor elimination was responsible for reduction in osteolysis and pain, sarcoma cells containing the suicide gene cytosine deaminase (CD) were inoculated into femora. After onset of bone cancer pain, mice were treated with the prodrug 5-fluorocytosine (5-FC). 5-FC treatment significantly reduced both osteolysis (P < 0.0005) and bone cancer pain (P < 0.05). The findings in this study demonstrate that one mechanism through which radiation decreases bone cancer pain is by direct effects on tumor cells.
• Halvorson, K. G., Kubota, K., Sevcik, M. A., Lindsay, T. H., Sotillo, J. E., Ghilardi, J. R., Rosol, T. J., Boustany, L., Shelton, D. L., & Mantyh, P. W. (2005). A blocking antibody to nerve growth factor attenuates skeletal pain induced by prostate tumor cells growing in bone. Cancer research, 65(20), 9426-35.
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  Prostate cancer is unique in that bone is often the only clinically detectable site of metastasis. Prostate tumors that have metastasized to bone frequently induce bone pain which can be difficult to fully control as it seems to be driven simultaneously by inflammatory, neuropathic, and tumorigenic mechanisms. As nerve growth factor (NGF) has been shown to modulate inflammatory and some neuropathic pain states in animal models, an NGF-sequestering antibody was administered in a prostate model of bone cancer where significant bone formation and bone destruction occur simultaneously in the mouse femur. Administration of a blocking antibody to NGF produced a significant reduction in both early and late stage bone cancer pain-related behaviors that was greater than or equivalent to that achieved with acute administration of 10 or 30 mg/kg of morphine sulfate. In contrast, this therapy did not influence tumor-induced bone remodeling, osteoblast proliferation, osteoclastogenesis, tumor growth, or markers of sensory or sympathetic innervation in the skin or bone. One rather unique aspect of the sensory innervation of bone, that may partially explain the analgesic efficacy of anti-NGF therapy in relieving prostate cancer-induced bone pain, is that nearly all nerve fibers that innervate the bone express trkA and p75, and these are the receptors through which NGF sensitizes and/or activates nociceptors. The present results suggest that anti-NGF therapy may be effective in reducing pain and enhancing the quality of life in patients with prostate tumor-induced bone cancer pain.
• Khasabov, S. G., Ghilardi, J. R., Mantyh, P. W., & Simone, D. A. (2005). Spinal neurons that express NK-1 receptors modulate descending controls that project through the dorsolateral funiculus. Journal of neurophysiology, 93(2), 998-1006.
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  Selective ablation of spinal neurons possessing substance P receptors (NK-1 receptors) using the selective cytotoxin conjugate substance P-saporin (SP-SAP) decreases hyperalgesia and central sensitization. The mechanisms by which NK-1 expressing neurons modulate the excitability of other dorsal horn neurons are unclear. Because the majority of NK-1 expressing spinal neurons project rostrally, it is possible that they are part of a spinal-supraspinal circuitry that contributes to descending modulation of excitability of spinal nociceptive neurons. We therefore determined whether ablation of spinal neurons that possess the NK-1 receptor altered descending systems that travel via the dorsolateral funiculus (DLF). Spontaneous activity and responses of dorsal horn neurons evoked by mechanical (von Frey monofilaments) and heat (35-51 degrees C) stimuli were determined before and after transection of the DLF and were compared in rats pretreated with intrathecal application of vehicle or SP-SAP. In vehicle-treated rats, transection of the DLF caused a 233% increase in mean spontaneous activity of neurons and enhanced their responses to mechanical and heat stimuli, whereas these increases in excitation were blocked in rats pretreated with SP-SAP. Importantly, SP-SAP alone had no effect on spontaneous or evoked activity in the absence of DLF transection. These results demonstrate that spinal neurons expressing the NK-1 receptor appear to play a pivotal role in regulating descending systems that modulate activity of nociceptive dorsal horn neurons.
• Lindsay, T. H., Jonas, B. M., Sevcik, M. A., Kubota, K., Halvorson, K. G., Ghilardi, J. R., Kuskowski, M. A., Stelow, E. B., Mukherjee, P., Gendler, S. J., Wong, G. Y., & Mantyh, P. W. (2005). Pancreatic cancer pain and its correlation with changes in tumor vasculature, macrophage infiltration, neuronal innervation, body weight and disease progression. Pain, 119(1-3), 233-46.
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  To begin to understand the relationship between disease progression and pain in pancreatic cancer, transgenic mice that develop pancreatic cancer due to the expression of the simian virus 40 large T antigen under control of the rat elastase-1 promoter were examined. In these mice precancerous cellular changes were evident at 6 weeks and these included an increase in: microvascular density, macrophages that express nerve growth factor and the density of sensory and sympathetic fibers that innervate the pancreas, with all of these changes increasing with tumor growth. In somatic tissue such as skin, the above changes would be accompanied by significant pain; however, in mice with pancreatic cancer, changes in pain-related behaviors, such as morphine-reversible severe hunching and vocalization only became evident at 16 weeks of age, by which time the pancreatic cancer was highly advanced. These data suggest that in mice as well as humans, there is a stereotypic set of pathological changes that occur as pancreatic cancer develops, and while weight loss generally tracks disease progression, there is a significant lag between disease progression and behaviors indicative of pancreatic cancer pain. Defining the mechanisms that mask this pain in early and mid-stage disease and drive the pain in late-stage disease may aid in earlier diagnosis, survival, and increased quality of life of patients with pancreatic cancer.
• Luger, N. M., Mach, D. B., Sevcik, M. A., & Mantyh, P. W. (2005). Bone cancer pain: from model to mechanism to therapy. Journal of pain and symptom management, 29(5 Suppl), S32-46.
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  Pain is the cancer-related event that is most disruptive to the cancer patient's quality of life. Although bone cancer pain is one of the most severe and common of the chronic pains that accompany breast, prostate, and lung cancers, relatively little is known about the mechanisms that generate and maintain this pain. Recently, we developed a mouse model of bone cancer pain. Ten days following tumor implantation into the intramedullary space of the femur, significant bone destruction and bone cancer pain-related behaviors were observed and progressed in severity over time. A critical question is how closely this model mirrors human bone cancer pain. In a recent publication, we show that, as in humans, pain-related behaviors are diminished by systemic morphine administration in a dose-dependent fashion that is naloxone-reversible. Humans suffering from bone cancer pain generally require significantly higher doses of morphine as compared to individuals with inflammatory pain and in the mouse model the doses of morphine required to block bone cancer pain-related behaviors were 10 times that required to block peak inflammatory pain behaviors of comparable magnitude induced by hindpaw injection of complete Freund's adjuvant (CFA; 1-3 mg/kg). As these animals were treated acutely, there was not time for morphine tolerance to develop and the rightward shift in analgesic efficacy observed in bone cancer pain versus inflammatory pain suggests a fundamental difference in the underlying mechanisms that generate bone cancer versus inflammatory pain. These results indicate that this model will be useful in defining drug therapies that are targeted for complex bone cancer pain syndromes.
• Peters, C. M., Ghilardi, J. R., Keyser, C. P., Kubota, K., Lindsay, T. H., Luger, N. M., Mach, D. B., Schwei, M. J., Sevcik, M. A., & Mantyh, P. W. (2005). Tumor-induced injury of primary afferent sensory nerve fibers in bone cancer pain. Experimental neurology, 193(1), 85-100.
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  Bone is the most common site of chronic pain in patients with metastatic cancer. What remains unclear are the mechanisms that generate this pain and why bone cancer pain can be so severe and refractory to treatment with opioids. Here we show that following injection and confinement of NCTC 2472 osteolytic tumor cells within the mouse femur, tumor cells sensitize and injure the unmyelinated and myelinated sensory fibers that innervate the marrow and mineralized bone. This tumor-induced injury of sensory nerve fibers is accompanied by an increase in ongoing and movement-evoked pain behaviors, an upregulation of activating transcription factor 3 (ATF3) and galanin by sensory neurons that innervate the tumor-bearing femur, upregulation of glial fibrillary acidic protein (GFAP) and hypertrophy of satellite cells surrounding sensory neuron cell bodies within the ipsilateral dorsal root ganglia (DRG), and macrophage infiltration of the DRG ipsilateral to the tumor-bearing femur. Similar neurochemical changes have been described following peripheral nerve injury and in other non-cancerous neuropathic pain states. Chronic treatment with gabapentin did not influence tumor growth, tumor-induced bone destruction or the tumor-induced neurochemical reorganization that occurs in sensory neurons or the spinal cord, but it did attenuate both ongoing and movement-evoked bone cancer-related pain behaviors. These results suggest that even when the tumor is confined within the bone, a component of bone cancer pain is due to tumor-induced injury to primary afferent nerve fibers that innervate the tumor-bearing bone. Tumor-derived, inflammatory, and neuropathic mechanisms may therefore be simultaneously driving this chronic pain state.
• Sabino, M. A., & Mantyh, P. W. (2005). Pathophysiology of bone cancer pain. The journal of supportive oncology, 3(1), 15-24.
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  The most common cancers, such as those affecting the breast, prostate, and lung have a strong predilection to metastasize to bone. Bone metastasis frequently results in pain, pathologic fractures, hypercalcemia, and spinal cord compression. Pain can have a devastating effect on the quality of life in advanced cancer patients and is a serious complication of cancer. Although significant advances are being made in cancer treatment and diagnosis, the basic neurobiology of bone cancer pain is poorly understood. New insights into the mechanisms that induce cancer pain now are coming from animal models. Chemicals derived from tumor cells, inflammatory cells, and cells derived from bone appear to be involved simultaneously in driving this frequently difficult-to-control pain state. Understanding the mechanisms involved in the pathophysiology of bone cancer pain will improve both our ability to provide mechanism-based therapies and the quality of life of cancer patients.
• Sevcik, M. A., Ghilardi, J. R., Halvorson, K. G., Lindsay, T. H., Kubota, K., & Mantyh, P. W. (2005). Analgesic efficacy of bradykinin B1 antagonists in a murine bone cancer pain model. The journal of pain : official journal of the American Pain Society, 6(11), 771-5.
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  Cancer pain is a significant clinical problem because it is the first symptom of disease in 20% to 50% of all cancer patients, and 75% to 90% of patients with advanced or terminal cancer must cope with chronic pain syndromes related to failed treatment and/or tumor progression. One of the most difficult to treat cancer pains is metastatic invasion of the skeleton that can generate ongoing and bone breakthrough pain, which represents one of the most debilitating cancer-related events. Because bradykinin has been shown to be released in response to tissue injury and plays a significant role in driving acute and chronic inflammatory pain, we focused on bradykinin antagonists in a model of bone cancer pain. In our model of bone cancer, which involves the injection and confinement of 2472 sarcoma cells to the mouse femur, pharmacologic blockade of the bradykinin B1 receptor is effective in reducing pain-related behaviors at both early and advanced stages of bone cancer.
• Sevcik, M. A., Ghilardi, J. R., Peters, C. M., Lindsay, T. H., Halvorson, K. G., Jonas, B. M., Kubota, K., Kuskowski, M. A., Boustany, L., Shelton, D. L., & Mantyh, P. W. (2005). Anti-NGF therapy profoundly reduces bone cancer pain and the accompanying increase in markers of peripheral and central sensitization. Pain, 115(1-2), 128-41.
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  Bone cancer pain can be difficult to control, as it appears to be driven simultaneously by inflammatory, neuropathic and tumorigenic mechanisms. As nerve growth factor (NGF) has been shown to modulate inflammatory and neuropathic pain states, we focused on a novel NGF sequestering antibody and demonstrated that two administrations of this therapy in a mouse model of bone cancer pain produces a profound reduction in both ongoing and movement-evoked bone cancer pain-related behaviors that was greater than that achieved with acute administration of 10 or 30 mg/kg of morphine. This therapy also reduced several neurochemical changes associated with peripheral and central sensitization in the dorsal root ganglion and spinal cord, whereas the therapy did not influence disease progression or markers of sensory or sympathetic innervation in the skin or bone. Mechanistically, the great majority of sensory fibers that innervate the bone are CGRP/TrkA expressing fibers, and if the sensitization and activation of these fibers is blocked by anti-NGF therapy there would not be another population of nociceptors, such as the non-peptidergic IB4/RET-IR nerve fibers, to take their place in signaling nociceptive events.
• Clohisy, D. R., & Mantyh, P. W. (2004). Bone cancer pain and the role of RANKL/OPG. Journal of musculoskeletal & neuronal interactions, 4(3), 293-300.
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  Cancer-induced bone diseases are common and can have a devastating impact at the end of life. One of the most difficult sequelae of cancer is metastases to the skeleton, an event that results in bone destruction and bone cancer pain. Bone cancer pain is usually progressive as the disease advances, and is particularly difficult to treat. Recently, experimental models of bone cancer pain have been developed and have provided seminal insight in understanding the pathophysiology of bone cancer pain. Animal models of bone cancer provided the finding that bone destruction (osteolysis) is associated with pain, and it has been determined that cancer-induced osteolysis is mediated by osteoclasts. Having established that RANK ligand contributed to cancer-induced osteoclastogenesis, it was determined that disruption of the RANKL-RANK axis with OPG inhibited tumor-induced osteoclastogenesis and decreased bone cancer pain.
• Ghilardi, J. R., Svensson, C. I., Rogers, S. D., Yaksh, T. L., & Mantyh, P. W. (2004). Constitutive spinal cyclooxygenase-2 participates in the initiation of tissue injury-induced hyperalgesia. The Journal of neuroscience : the official journal of the Society for Neuroscience, 24(11), 2727-32.
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  Inhibitors of the isozyme cyclooxygenase-2 (COX-2) represent an important advance in pain management, although where and when these inhibitors can exert their antihyperalgesic actions are not completely understood. Here we show that unlike many peripheral tissues in which COX-2 is only expressed in physiologically significant levels after tissue injury, in the normal rat lumbar spinal cord, the majority of neurons and radial glia constitutively express high levels of COX-2 protein. Immediately after peripheral tissue injury and before any measurable upregulation of COX-2 protein in peripheral tissue or spinal cord, inhibition of constitutively expressed spinal COX-2 reduced injury-induced activation of primary afferent neurons, activation of spinal neurons, and the mechanical and thermal hyperalgesia that normally occurs after peripheral tissue injury. The present data demonstrate that constitutively expressed spinal COX-2 plays an important role in the initial hyperalgesia that follows peripheral tissue injury. These results suggest that blocking constitutive spinal COX-2 before tissue injury may reduce the initial peripheral and central sensitization that occurs after tissue injury.
• Goblirsch, M., Mathews, W., Lynch, C., Alaei, P., Gerbi, B. J., Mantyh, P. W., & Clohisy, D. R. (2004). Radiation treatment decreases bone cancer pain, osteolysis and tumor size. Radiation research, 161(2), 228-34.
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  Radiotherapy is the cornerstone of palliative treatment for primary bone cancer in animals and metastatic bone cancer in humans. However, the mechanism(s) responsible for pain relief after irradiation is unknown. To identify the mechanism through which radiation treatment decreases bone cancer pain, the effect of radiation on mice with painful bone cancer was studied. Analysis of the effects of a 20-Gy treatment on localized sites of painful bone cancers was performed through assessments of animal behavior, radiographs and histological analysis. The findings indicated that radiation treatment reduced bone pain and supported reduced cancer burden and reduced osteolysis as mechanisms through which radiation reduces bone cancer pain.
• Mantyh, P. W. (2004). A mechanism-based understanding of bone cancer pain. Novartis Foundation symposium, 261, 194-214; discussion 214-9, 256-61.
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  Although bone cancer pain can be severe and is relatively common, as it frequently arises from metastases from breast, prostate and lung tumours, relatively little is known about the basic mechanisms that generate and maintain this chronic pain. To begin to define the mechanisms that give rise to bone cancer pain, we developed a mouse model using the intramedullary injection and containment of osteolytic sarcoma cells into the mouse femur. These tumour cells induced bone destruction as well as ongoing and movement evoked pain behaviours similar to that found in patients with bone cancer pain. In addition, there was a significant neurochemical reorganization of sensory neurons that innervate the tumour bearing bone as well as in the spinal cord segments that received sensory input from the cancerous bone. This reorganization generated a neurochemical signature of bone cancer pain that was different from that observed in mouse models of chronic neuropathic or inflammatory pain. These data suggest that there is an inflammatory, neuropathic and tumorigenic component to bone cancer pain. Therefore defining when and how these different components drive bone cancer pain may allow the development of more selective analgesic agents to treat this chronic pain state.
• Mantyh, P. W., & Hunt, S. P. (2004). Mechanisms that generate and maintain bone cancer pain. Novartis Foundation symposium, 260, 221-38; discussion 238-40, 277-9.
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  Although bone cancer pain can be severe and is relatively common, as it frequently arises from metastases from breast, prostate, and lung tumours, very little is known about the basic mechanisms that generate and maintain this chronic pain. To begin to define the mechanisms that give rise to bone cancer pain, we have developed mouse and rat models using the intramedullary injection and containment of tumour cells into the femur. These tumour cells induced bone remodelling as well as ongoing and movement evoked pain behaviours similar to that found in patients with bone cancer pain. In addition there is a significant reorganization of the spinal cord that received sensory input from the cancerous bone and this reorganization generated a neurochemical signature of bone cancer pain that is both dramatic and significantly different from that observed in mouse and rat models of chronic neuropathic or inflammatory pain. These models have provided insight into the mechanisms that drive cancer pain and have begun to allow the development of mechanism-based therapies. Together these advances should reduce tumour-induced pain and suffering and significantly improve the quality of life of cancer patients.
• Mantyh, P. W., & Hunt, S. P. (2004). Setting the tone: superficial dorsal horn projection neurons regulate pain sensitivity. Trends in neurosciences, 27(10), 582-4.
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  The neurokinin-1 receptor is expressed by lamina I projection neurons of the spinal cord that are crucial for regulating pain behavior. These neurons are nocispecific, support long-term potentiation and appear to downregulate the K(+)-Cl(-) exporter channel KCC2 following peripheral nerve damage, leading to increased excitability. These lamina I neurons project to the brainstem and thalamus and modulate descending inhibitory and excitatory pathways to the dorsal horn that regulate nociceptive traffic.
• Peters, C. M., Lindsay, T. H., Pomonis, J. D., Luger, N. M., Ghilardi, J. R., Sevcik, M. A., & Mantyh, P. W. (2004). Endothelin and the tumorigenic component of bone cancer pain. Neuroscience, 126(4), 1043-52.
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  Tumors including sarcomas and breast, prostate, and lung carcinomas frequently grow in or metastasize to the skeleton where they can induce significant bone remodeling and cancer pain. To define products that are released from tumors that are involved in the generation and maintenance of bone cancer pain, we focus here on endothelin-1 (ET-1) and endothelin receptors as several tumors including human prostate and breast have been shown to express high levels of ETs and the application of ETs to peripheral nerves can induce pain. Here we show that in a murine osteolytic 2472 sarcoma model of bone cancer pain, the 2472 sarcoma cells express high levels of ET-1, but express low or undetectable levels of endothelin A (ETAR) or B (ETBR) receptors whereas a subpopulation of sensory neurons express the ETAR and non-myelinating Schwann cells express the ETBR. Acute (10 mg/kg, i.p.) or chronic (10 mg/kg/day, p.o.) administration of the ETAR selective antagonist ABT-627 significantly attenuated ongoing and movement-evoked bone cancer pain and chronic administration of ABT-627 reduced several neurochemical indices of peripheral and central sensitization without influencing tumor growth or bone destruction. In contrast, acute treatment (30 mg/kg, i.p.) with the ETBR selective antagonist, A-192621 increased several measures of ongoing and movement evoked pain. As tumor expression and release of ET-1 has been shown to be regulated by the local environment, location specific expression and release of ET-1 by tumor cells may provide insight into the mechanisms that underlie the heterogeneity of bone cancer pain that is frequently observed in humans with multiple skeletal metastases.
• Sevcik, M. A., Luger, N. M., Mach, D. B., Sabino, M. A., Peters, C. M., Ghilardi, J. R., Schwei, M. J., Röhrich, H., De Felipe, C., Kuskowski, M. A., & Mantyh, P. W. (2004). Bone cancer pain: the effects of the bisphosphonate alendronate on pain, skeletal remodeling, tumor growth and tumor necrosis. Pain, 111(1-2), 169-80.
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  Patients with metastatic breast, lung or prostate cancer frequently have significant bone cancer pain. In the present report we address, in a single in vivo mouse model, the effects the bisphosphonate alendronate has on bone cancer pain, bone remodeling and tumor growth and necrosis. Following injection and confinement of green fluorescent protein-transfected murine osteolytic tumor cells into the marrow space of the femur of male C3H/HeJ mice, alendronate was administered chronically from the time the tumor was established until the bone cancer pain became severe. Alendronate therapy reduced ongoing and movement-evoked bone cancer pain, bone destruction and the destruction of sensory nerve fibers that innervate the bone. Whereas, alendronate treatment did not change viable tumor burden, both tumor growth and tumor necrosis increased. These data emphasize that it is essential to utilize a model where pain, skeletal remodeling and tumor growth can be simultaneously assessed, as each of these can significantly impact patient quality of life and survival.
• Yezierski, R. P., Yu, C., Mantyh, P. W., Vierck, C. J., & Lappi, D. A. (2004). Spinal neurons involved in the generation of at-level pain following spinal injury in the rat. Neuroscience letters, 361(1-3), 232-6.
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  Using a conjugate of substance P and the ribosome-inactivating protein saporin, neurons expressing the neurokinin-1 receptor in lamina I of the spinal cord were targeted to determine their role in the expression of a spontaneous pain behavior following intraspinal injections of quisqualic acid in the rat. Treatment was carried out at the time of injury in order to prevent the onset of the behavior, and following onset in order to evaluate the potential clinical utility of this intervention. Treatment at the time of injury resulted in significant decreases in onset-time and severity of pain behavior, while treatment at the time of onset led to a significant reduction of the spontaneous self-directed behavior. The results suggest that the substrate for at-level pain following spinal cord injury includes a population of spinal neurons expressing the neurokinin-1 receptor in the superficial laminae of the spinal cord.
• Clohisy, D. R., & Mantyh, P. W. (2003). Bone cancer pain. Cancer, 97(3 Suppl), 866-73.
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  Bone cancer pain is very common, and patients with this type of pain may be difficult to treat. Development of an experimental model for studying this condition is critical to advancing an understanding of the mechanisms that cause pain in patients with malignant disease.
• Clohisy, D. R., & Mantyh, P. W. (2003). Bone cancer pain. Clinical orthopaedics and related research, S279-88.
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  Bone cancer pain is very common, and patients with this type of pain may be difficult to treat. Development of an experimental model for studying this condition is critical to advancing an understanding of the mechanisms that cause pain in patients with malignant disease.
• Egnaczyk, G. F., Pomonis, J. D., Schmidt, J. A., Rogers, S. D., Peters, C., Ghilardi, J. R., Mantyh, P. W., & Maggio, J. E. (2003). Proteomic analysis of the reactive phenotype of astrocytes following endothelin-1 exposure. Proteomics, 3(5), 689-98.
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  Reactive gliosis is an invariant feature of the pathology of central nervous system (CNS) injury and a major determinant of neuronal survival and regeneration. To begin to understand the alterations in astrocyte protein expression that drive glial changes that occur following injury, we used an established model system (endothelin-1 stimulation of hypertrophy) and proteomic analysis to define a discrete set of differentially expressed proteins and post-translational modifications that occur as the astrocytes change from a quiescent to a reactive state. This orchestrated set of changes included proteins involved in cytoskeletal reorganization (caldesmon, calponin, alpha B-crystallin, stathmin, collapsing response mediator protein-2), cell adhesion (vinculin, galectin-1), signal transduction (RACK-1) and astrocyte differentiation (glutamine synthetase). Using proteomic analysis to understand what drives astrocyte expression of these functionally divergent molecules may offer insight into the mechanisms by which astrocytes can exhibit both pro-regenerative and anti-regenerative activities following CNS injury.
• Morcuende, S., Gadd, C. A., Peters, M., Moss, A., Harris, E. A., Sheasby, A., Fisher, A. S., De Felipe, C., Mantyh, P. W., Rupniak, N. M., Giese, K. P., & Hunt, S. P. (2003). Increased neurogenesis and brain-derived neurotrophic factor in neurokinin-1 receptor gene knockout mice. The European journal of neuroscience, 18(7), 1828-36.
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  It has previously been shown that chronic treatment with antidepressant drugs increases neurogenesis and levels of brain-derived neurotrophic factor in the hippocampus. These changes have been correlated with changes in learning and long-term potentiation and may contribute to the therapeutic efficacy of antidepressant drug treatment. Recently, antagonists at the neurokinin-1 receptor, the preferred receptor for the neuropeptide substance P, have been shown to have antidepressant activity. Mice with disruption of the neurokinin-1 receptor gene are remarkably similar both behaviourally and neurochemically to mice maintained chronically on antidepressant drugs. We demonstrate here that there is a significant elevation of neurogenesis but not cell survival in the hippocampus of neurokinin-1 receptor knockout mice. Neurogenesis can be increased in wild-type but not neurokinin-1 receptor knockout mice by chronic treatment with antidepressant drugs which preferentially target noradrenergic and serotonergic pathways. Hippocampal levels of brain-derived neurotrophic factor are also two-fold higher in neurokinin-1 receptor knockout mice, whereas cortical levels are similar. Finally, we examined hippocampus-dependent learning and memory but found no clear enhancement in neurokinin-1 receptor knockout mice. These data argue against a simple correlation between increased levels of neurogenesis or brain-derived neurotrophic factor and mnemonic processes in the absence of increased cell survival. They support the hypothesis that increased neurogenesis, perhaps accompanied by higher levels of brain-derived neurotrophic factor, may contribute to the efficacy of antidepressant drug therapy.
• Peters, C. M., Rogers, S. D., Pomonis, J. D., Egnaczyk, G. F., Keyser, C. P., Schmidt, J. A., Ghilardi, J. R., Maggio, J. E., Mantyh, P. W., & Egnazyck, G. F. (2003). Endothelin receptor expression in the normal and injured spinal cord: potential involvement in injury-induced ischemia and gliosis. Experimental neurology, 180(1), 1-13.
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  The endothelins (ETs) are a family of peptides that exert their biological effects via two distinct receptors, the endothelin A receptor (ET(A)R) and the endothelin B receptor (ET(B)R). To more clearly define the potential actions of ETs following spinal cord injury, we used immunohistochemistry and confocal microscopy to examine the protein expression of ET(A)R and ET(B)R in the normal and injured rat spinal cord. In the normal spinal cord, ET(A)R immunoreactivity (IR) is expressed by vascular smooth muscle cells and a subpopulation of primary afferent nerve fibers. ET(B)R-IR is expressed primarily by radial glia, a small population of gray and white matter astrocytes, ependymal cells, vascular endothelial cells, and to a lesser extent in smooth muscle cells. Fourteen days following compression injury to the spinal cord, there was a significant upregulation in both the immunoexpression and number of astrocytes expressing the ET(B)R in both gray and white matter and a near disappearance of ET(B)R-IR in ependymal cells and ET(A)R-IR in primary afferent fibers. Conversely, the vascular expression of ET(A)R and ET(B)R did not appear to change. As spinal cord injury has been shown to induce an immediate increase in plasma ET levels and a sustained increase in tissue ET levels, ETs would be expected to induce an initial marked vasoconstriction via activation of vascular ET(A)R/ET(B)R and then days later a glial hypertrophy via activation of the ET(B)R expressed by astrocytes. Strategies aimed at blocking vascular ET(A)R/ET(B)R and astrocyte ET(B)Rs following spinal cord injury may reduce the resulting ischemia and astrogliosis and in doing so increase neuronal survival, regeneration, and function.
• Rogers, S. D., Peters, C. M., Pomonis, J. D., Hagiwara, H., Ghilardi, J. R., & Mantyh, P. W. (2003). Endothelin B receptors are expressed by astrocytes and regulate astrocyte hypertrophy in the normal and injured CNS. Glia, 41(2), 180-90.
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  The ability of mammalian central nervous system (CNS) neurons to survive and/or regenerate following injury is influenced by surrounding glial cells. To identify the factors that control glial cell function following CNS injury, we have focused on the endothelin B receptor (ET(B)R), which we show is expressed by the majority of astrocytes that are immunoreactive for glial acid fibrillary protein (GFAP) in both the normal and crushed rabbit optic nerve. Optic nerve crush induces a marked increase in ET(B)R and GFAP immunoreactivity (IR) without inducing a significant increase in the number of GFAP-IR astrocytes, suggesting that the crush-induced astrogliosis is due primarily to astrocyte hypertrophy. To define the role that endothelins play in driving this astrogliosis, artificial cerebrospinal fluid (CSF), ET-1 (an ET(A)R and ET(B)R agonist), or Bosentan (a mixed ET(A)R and ET(B)R antagonist) were infused via osmotic minipumps into noninjured and crushed optic nerves for 14 days. Infusion of ET-1 induced a hypertrophy of ET(B)R/GFAP-IR astrocytes in the normal optic nerve, with no additional hypertrophy in the crushed nerve, whereas infusion of Bosentan induced a significant decrease in the hypertrophy of ET(B)R/GFAP-IR astrocytes in the crushed but not in the normal optic nerve. These data suggest that pharmacological blockade of astrocyte ET(B)R receptors following CNS injury modulates glial scar formation and may provide a more permissive substrate for neuronal survival and regeneration.
• Sabino, M. A., Luger, N. M., Mach, D. B., Rogers, S. D., Schwei, M. J., & Mantyh, P. W. (2003). Different tumors in bone each give rise to a distinct pattern of skeletal destruction, bone cancer-related pain behaviors and neurochemical changes in the central nervous system. International journal of cancer, 104(5), 550-8.
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  Pain is the most common presenting symptom in patients with bone cancer and bone cancer pain can be both debilitating and difficult to control fully. To begin to understand the mechanisms involved in the generation and maintenance of bone cancer pain, we implanted 3 well-described murine tumor cell lines, 2472 sarcoma, B16 melanoma and C26 colon adenocarcinoma into the femur of immunocompromised C3H-SCID mice. Although each of the tumor cell lines proliferated and completely filled the intramedullary space of the femur within 3 weeks, the location and extent of bone destruction, the type and severity of the pain behaviors and the neurochemical reorganization of the spinal cord was unique to each tumor cell line injected. These data suggest that bone cancer pain is not caused by a single factor such as increased pressure induced by intramedullary tumor growth, but rather that multiple factors are involved in generating and maintaining bone cancer pain.
• Esler, W. P., Marshall, J. R., Stimson, E. R., Ghilardi, J. R., Vinters, H. V., Mantyh, P. W., & Maggio, J. E. (2002). Apolipoprotein E affects amyloid formation but not amyloid growth in vitro: mechanistic implications for apoE4 enhanced amyloid burden and risk for Alzheimer's disease. Amyloid : the international journal of experimental and clinical investigation : the official journal of the International Society of Amyloidosis, 9(1), 1-12.
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  The transition from the partially folded soluble Abeta monomer to insoluble Abeta amyloidfibrils is seminal to the formation and growth of amyloid plaques in Alzheimer's disease (AD). A detailed understanding of the role of AD risk factors in these processes is essential to understanding the physiochemical nature of this conformational rearrangement. The apolipoprotein E epsilon4 allele, a risk factor for AD, affects AD pathology by increasing amyloid burden relative to the much more common epsilon3 allele. In the present study, in vitro models were employed to probe the effect of these proteins on kinetically distinct steps in Abeta fibrillogenesis. Formation of Abeta amyloid was faster in the presence of apoE4 than apoE3, while growth of existing plaques was unaffected by either isoform. Further, experiments with Abeta stereoisomers establish that this effect of apoE3 is mediated through interaction with oligomeric fibrillogenic intermediates rather than through specific contacts with monomeric Abeta. Consistent with the altered pathology and enhanced risk for AD associated with inheritance of the epsilon4 allele, we conclude that APOE epsilon4 is a risk factor for AD not due to a pathological gain of function of apoE4 but to a loss of protective function of apoE3.
• Honoré, P., Kamp, E. H., Rogers, S. D., Gebhart, G. F., & Mantyh, P. W. (2002). Activation of lamina I spinal cord neurons that express the substance P receptor in visceral nociception and hyperalgesia. The journal of pain : official journal of the American Pain Society, 3(1), 3-11.
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  Spinal lamina I neurons expressing the substance P receptor (SPR) have been shown to play a role in the transmission of somatic inflammatory and neuropathic pain. To evaluate their involvement in visceral nociception in both the noninflamed and inflamed colon, we examined the expression and ligand-induced internalization of the SPR in the rat spinal cord after distention of the noninflamed colon and in rats with inflammation induced by intracolonic instillation of zymosan (3 hours). In the noninflamed animal, acute noxious but not non-noxious colorectal distention induced SPR internalization in lamina I neurons at the thoracolumbar (T13) and lumbosacral (S1) spinal levels, whereas SPR internalization was not detected in lamina I neurons at spinal lumbar segment L4. Although zymosan-induced colorectal inflammation alone did not induce SPR internalization in lamina I neurons, there was an increased number of SPR-expressing lamina I neurons showing SPR internalization in segments T12 through S2 of the spinal cord after colorectal distention. These results show that acute noxious visceral stimuli induce activation of spinal lamina I neurons expressing the SPR and, that after visceral inflammation, there is a marked increase in both the number and rostrocaudal extent of lamina I SPR neurons activated in response to both normally non-noxious and noxious distention of the colon.
• Khasabov, S. G., Rogers, S. D., Ghilardi, J. R., Peters, C. M., Mantyh, P. W., & Simone, D. A. (2002). Spinal neurons that possess the substance P receptor are required for the development of central sensitization. The Journal of neuroscience : the official journal of the Society for Neuroscience, 22(20), 9086-98.
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  In previous studies, we have shown that loss of spinal neurons that possess the substance P receptor (SPR) attenuated pain and hyperalgesia produced by capsaicin, inflammation, and nerve injury. To determine the role of SPR-expressing neurons in modulating pain and hyperalgesia, responses of superficial and deep lumbar spinal dorsal horn neurons evoked by mechanical and heat stimuli and by capsaicin were made after ablation of SPR-expressing neurons using the selective cytotoxin conjugate substance P-saporin (SP-SAP). Morphological analysis and electrophysiological recordings were made after intrathecal infusion of vehicle, saporin alone, or SP-SAP. SP-SAP, but not vehicle or SAP alone, produced an approximately 62% decrease in SPR-expressing neurons in the dorsal horn. Loss of SPR-expressing neurons diminished the responses of remaining neurons to intraplantar injection of capsaicin. Peak responses to 10 microg of capsaicin were approximately 65% lower in animals pretreated with SP-SAP compared with controls. Additionally, sensitization to mechanical and heat stimuli that normally follows capsaicin was rarely observed. Importantly, responses to mechanical and heat stimuli in the absence of capsaicin were not altered after SP-SAP treatment. In addition, nociceptive neurons did not exhibit windup in the SP-SAP-treated group. These results demonstrate that SPR-expressing neurons located in the dorsal horn are a pivotal component of the spinal circuits involved in triggering central sensitization and hyperalgesia. It appears that this relatively small population of neurons can regulate the physiological properties of other nociceptive neurons and drive central sensitization.
• Luger, N. M., Sabino, M. A., Schwei, M. J., Mach, D. B., Pomonis, J. D., Keyser, C. P., Rathbun, M., Clohisy, D. R., Honore, P., Yaksh, T. L., & Mantyh, P. W. (2002). Efficacy of systemic morphine suggests a fundamental difference in the mechanisms that generate bone cancer vs inflammatory pain. Pain, 99(3), 397-406.
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  Pain is the cancer related event that is most disruptive to the cancer patient's quality of life. Although bone cancer pain is one of the most severe and common of the chronic pains that accompany breast, prostate and lung cancers, relatively little is known about the mechanisms that generate and maintain this pain. Recently, we developed a mouse model of bone cancer pain and 16 days following tumor implantation into the intramedullary space of the femur, significant bone destruction and bone cancer pain-related behaviors were observed. A critical question is how closely this model mirrors human bone cancer pain. In the present study we show that, as in humans, pain-related behaviors are diminished by systemic morphine administration in a dose dependent fashion that is naloxone-reversible. Humans suffering from bone cancer pain generally require significantly higher doses of morphine as compared to individuals with inflammatory pain and in the mouse model, the doses of morphine required to block bone cancer pain-related behaviors were ten times that required to block peak inflammatory pain behaviors of comparable magnitude induced by hindpaw injection of complete Freund's adjuvant (CFA) (1-3mg/kg). As these animals were treated acutely, there was not time for morphine tolerance to develop and the rightward shift in analgesic efficacy observed in bone cancer pain vs. inflammatory pain suggests a fundamental difference in the underlying mechanisms that generate bone cancer vs. inflammatory pain. These results indicate that this model may be useful in defining drug therapies that are targeted for complex bone cancer pain syndromes.
• Mach, D. B., Rogers, S. D., Sabino, M. C., Luger, N. M., Schwei, M. J., Pomonis, J. D., Keyser, C. P., Clohisy, D. R., Adams, D. J., O'Leary, P., & Mantyh, P. W. (2002). Origins of skeletal pain: sensory and sympathetic innervation of the mouse femur. Neuroscience, 113(1), 155-66.
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  Although skeletal pain plays a major role in reducing the quality of life in patients suffering from osteoarthritis, Paget's disease, sickle cell anemia and bone cancer, little is known about the mechanisms that generate and maintain this pain. To define the peripheral fibers involved in transmitting and modulating skeletal pain, we used immunohistochemistry with antigen retrieval, confocal microscopy and three-dimensional image reconstruction of the bone to examine the sensory and sympathetic innervation of mineralized bone, bone marrow and periosteum of the normal mouse femur. Thinly myelinated and unmyelinated peptidergic sensory fibers were labeled with antibodies raised against calcitonin gene-related peptide (CGRP) and the unmyelinated, non-peptidergic sensory fibers were labeled with the isolectin B4 (Bandeira simplicifolia). Myelinated sensory fibers were labeled with an antibody raised against 200-kDa neurofilament H (clone RT-97). Sympathetic fibers were labeled with an antibody raised against tyrosine hydroxylase. CGRP, RT-97, and tyrosine hydroxylase immunoreactive fibers, but not isolectin B4 positive fibers, were present throughout the bone marrow, mineralized bone and the periosteum. While the periosteum is the most densely innervated tissue, when the total volume of each tissue is considered, the bone marrow receives the greatest total number of sensory and sympathetic fibers followed by mineralized bone and then periosteum. Understanding the sensory and sympathetic innervation of bone should provide a better understanding of the mechanisms that drive bone pain and aid in developing therapeutic strategies for treating skeletal pain.
• Mantyh, P. W. (2002). A mechanism based understanding of cancer pain. Pain, 96(1-2), 1-2.
• Mantyh, P. W. (2002). Neurobiology of substance P and the NK1 receptor. The Journal of clinical psychiatry, 63 Suppl 11, 6-10.
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  Substance P belongs to a group of neurokinins (NKs), small peptides that are broadly distributed in the central nervous system (CNS) and peripheral nervous system (PNS). The biological effects of substance P in the CNS, namely regulation of affective behavior and emesis in the brain and nociception in the spinal cord, are mediated by its binding to the NK1 receptor. The substance P-NK1 (SP-NK1) receptor system is the most extensively studied NK pathway, and in contrast to receptors for other neurotransmitters, such as glutamate, which have high expression throughout the CNS, only a minority of neurons (5% to 7%) in certain CNS areas express the NK1 receptor. The NK1 receptor is distributed in the plasma membrane of cell bodies and dendrites of unstimulated neurons, but upon substance P binding, the NK1 receptor undergoes rapid internalization, followed by rapid recycling to the plasma membrane. Release of substance P is induced by stressful stimuli, and the magnitude of its release is proportional to the intensity and frequency of stimulation. More potent and more frequent stimuli allow diffusion of substance P farther from the site of release, allowing activation of an approximately 3- to 5-times greater number of NK1 receptor-expressing neurons. Recent studies employing pharmacologic or genetic inactivation of NK1 receptors demonstrate the important role of the SP-NK1 receptor system in the regulation of affective behavior and suggest that inhibition of this pathway may be a useful approach to treatment of depression and associated anxiety.
• Mantyh, P. W., Clohisy, D. R., Koltzenburg, M., & Hunt, S. P. (2002). Molecular mechanisms of cancer pain. Nature reviews. Cancer, 2(3), 201-9.
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  Pain is the most disruptive influence on the quality of life of cancer patients. Although significant advances are being made in cancer treatment and diagnosis, the basic neurobiology of cancer pain is poorly understood. New insights into these mechanisms are now arising from animal models, and have the potential to fundamentally change the way that cancer pain is controlled.
• Marshall, J. R., Stimson, E. R., Ghilardi, J. R., Vinters, H. V., Mantyh, P. W., & Maggio, J. E. (2002). Noninvasive imaging of peripherally injected Alzheimer's disease type synthetic A beta amyloid in vivo. Bioconjugate chemistry, 13(2), 276-84.
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  The pathological hallmark of Alzheimer's disease (AD) is accumulation in the brain of amyloid composed of the 40-mer peptide A beta. Many fundamental questions about the biology of (AD) remain unanswered because there is currently no method of quantifying A beta amyloid in vivo. A noninvasive method of detecting and quantifying A beta amyloid in vivo would have wide application for the premortem diagnosis of AD and the efficient evaluation of candidate therapeutics aimed at inhibiting the formation and growth of A beta amyloid. Taking advantage of the extraordinarily high affinity of A beta for itself, we have synthesized an N'-terminal diethylenetriaminepentaacetic acid (DTPA) derivative of A beta possessing the kinetic activity and specificity for A beta amyloid desired of a probe to be used for noninvasive imaging. DTPA-A beta(3-40) is readily labeled with (111)InOAc(3) to yield a stable probe with exquisite specificity for naturally occurring and synthetic A beta amyloid in vitro. Moreover, (111)In-DTPA-A beta(3-40), administered intravascularly can specifically deposit onto and label previously injected synthetic A beta amyloid and be imaged in vivo with a gamma camera. The present results demonstrate the design, synthesis, and use of an A beta amyloid-specific probe and methods for its use as a noninvasive imaging agent. In vivo imaging of A beta amyloid represents an important step toward the development of biochemically based objective tools for the assessment of progression of AD and efficacy of potential therapeutics.
• Sabino, M. A., Ghilardi, J. R., Jongen, J. L., Keyser, C. P., Luger, N. M., Mach, D. B., Peters, C. M., Rogers, S. D., Schwei, M. J., de Felipe, C., & Mantyh, P. W. (2002). Simultaneous reduction in cancer pain, bone destruction, and tumor growth by selective inhibition of cyclooxygenase-2. Cancer research, 62(24), 7343-9.
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  More than half of all chronic cancer pain arises from metastases to bone, and bone cancer pain is one of the most difficult of all persistent pain states to fully control. Several tumor types including sarcomas and breast, prostate, and lung carcinomas grow in or preferentially metastasize to the skeleton where they proliferate, and induce significant bone remodeling, bone destruction, and cancer pain. Many of these tumors express the isoenzyme cycloxygenase-2 (COX-2), which is involved in the synthesis of prostaglandins. To begin to define the role COX-2 plays in driving bone cancer pain, we used an in vivo model where murine osteolytic 2472 sarcoma cells were injected and confined to the intramedullary space of the femur in male C3HHeJ mice. After tumor implantation, mice develop ongoing and movement-evoked bone cancer pain-related behaviors, extensive tumor-induced bone resorption, infiltration of the marrow space by tumor cells, and stereotypic neurochemical alterations in the spinal cord reflective of a persistent pain state. Thus, after injection of tumor cells, bone destruction is first evident at day 6, and pain-related behaviors are maximal at day 14. A selective COX-2 inhibitor was administered either acutely [NS398; 100 mg/kg, i.p.] on day 14 or chronically in chow [MF. tricyclic; 0.015%, p.o.] from day 6 to day 14 after tumor implantation. Acute administration of a selective COX-2 inhibitor attenuated both ongoing and movement-evoked bone cancer pain, whereas chronic inhibition of COX-2 significantly reduced ongoing and movement-evoked pain behaviors, and reduced tumor burden, osteoclastogenesis, and bone destruction by >50%. The present results suggest that chronic administration of a COX-2 inhibitor blocks prostaglandin synthesis at multiple sites, and may have significant clinical utility in the management of bone cancer and bone cancer pain.
• Sabino, M. A., Honore, P., Rogers, S. D., Mach, D. B., Luger, N. M., & Mantyh, P. W. (2002). Tooth extraction-induced internalization of the substance P receptor in trigeminal nucleus and spinal cord neurons: imaging the neurochemistry of dental pain. Pain, 95(1-2), 175-86.
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  Although pains arising from the craniofacial complex can be severe and debilitating, relatively little is known about the peripheral and central mechanisms that generate and maintain orofacial pain. To better understand the neurons in the trigeminal complex and spinal cord that are activated following nociceptive stimuli to the orofacial complex, we examined substance P (SP) induced internalization of substance P receptors (SPR) in neurons following dental extraction in the rat. Unilateral gingival reflection or surgical extraction of a rat maxillary incisor or molar was performed and tissues harvested at various time points post-extraction. Immunohistochemical analysis of brainstem and cervical spinal cord sections was performed using an anti-SPR antibody and confocal imaging. Both the number and location of neurons showing SPR internalization was dependent on the location and extent of tissue injury. Whereas extraction of the incisor induced internalization of SPR in neurons bilaterally in nucleus caudalis and the spinal cord, extraction of the molar induced strictly unilateral internalization of SPR-expressing neurons in the same brain structures. Minor tissue injury (retraction of the gingiva) activated SPR neurons located in lamina I whereas more extensive and severe tissue injury (incisor or molar extraction) induced extensive SPR internalization in neurons located in both laminae I and III-V. The rostrocaudal extent of the SPR internalization was also correlated with the extent of tissue injury. Thus, following relatively minor tissue injury (gingival reflection) neurons showing SPR internalization were confined to the nucleus caudalis while procedures which cause greater tissue injury (incisor or molar extraction), neurons showing SPR internalization extended from the interpolaris/caudalis transition zone through the C7 spinal level. Defining the population of neurons activated in orofacial pain and whether analgesics modify the activation of these neurons should provide insight into the mechanisms that generate and maintain acute and chronic orofacial pain.
• Sabino, M. C., Ghilardi, J. R., Feia, K. J., Jongen, J. L., Keyser, C. P., Luger, N. M., Mach, D. B., Peters, C. M., Rogers, S. D., Schwei, M. J., De Filipe, C., & Mantyh, P. W. (2002). The involvement of prostaglandins in tumorigenesis, tumor-induced osteolysis and bone cancer pain. Journal of musculoskeletal & neuronal interactions, 2(6), 561-2.
• Hunt, S. P., & Mantyh, P. W. (2001). The molecular dynamics of pain control. Nature reviews. Neuroscience, 2(2), 83-91.
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  Pain is necessary for survival, but persistent pain can result in anxiety, depression and a reduction in the quality of life. The discriminative and affective qualities of pain are both thought to be regulated in an activity-dependent fashion. Recent studies have identified cells and molecules that regulate pain sensitivity and the parallel pathways that distribute nociceptive information to limbic or sensory areas of the forebrain. Here, we emphasize the cellular and neurobiological consequences of pain, especially those that are involved in the generation and maintenance of chronic pain. These new insights into pain processing will significantly alter our approach to pain control and the development of new analgesics.
• Khasabov, S. G., Cain, D. M., Thong, D., Mantyh, P. W., & Simone, D. A. (2001). Enhanced responses of spinal dorsal horn neurons to heat and cold stimuli following mild freeze injury to the skin. Journal of neurophysiology, 86(2), 986-96.
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  The effects of a mild freeze injury to the skin on responses of nociceptive dorsal horn neurons to cold and heat stimuli were examined in anesthetized rats. Electrophysiological recordings were obtained from 72 nociceptive spinal neurons located in the superficial and deep dorsal horn. All neurons had receptive fields (RFs) on the glabrous skin of the hindpaw, and neurons were functionally divided into wide dynamic range (WDR) and high-threshold (HT) neurons. Forty-four neurons (61%) were classified as WDR and responded to both innocuous and noxious mechanical stimuli (mean mechanical threshold of 12.8 +/- 1.6 mN). Twenty-eight neurons (39%) were classified as HT and were excited only by noxious mechanical stimuli (mean mechanical threshold of 154.2 +/- 18.3 mN). Neurons were characterized for their sensitivity heat (35 to 51 degrees C) and cold (28 to -12 degrees C) stimuli applied to their RF. Among WDR neurons, 86% were excited by both noxious heat and cold stimuli, while 14% responded only to heat. For HT neurons, 61% responded to heat and cold stimuli, 32% responded only to noxious heat, and 7% responded only to noxious cold. Effects of a mild freeze injury (-15 degrees C applied to the RF for 20 s) on responses to heat and cold stimuli were examined in 30 WDR and 22 HT neurons. Skin freezing was verified as an abrupt increase in skin temperature at the site of injury due to the exothermic reaction associated with crystallization. Freezing produced a decrease in response thresholds to heat and cold stimuli in most WDR and HT neurons. WDR and HT neurons exhibited a mean decrease in response threshold for cold of 9.0 +/- 1.3 degrees C and 10.0 +/- 1.6 degrees C, respectively. Mean response thresholds for heat decreased 4.0 +/- 0.4 degrees C and 4.3 +/- 1.3 degrees C in WDR and HT neurons, respectively. In addition, responses to suprathreshold cold and heat stimuli increased. WDR and HT neurons exhibited an 89% and a 192% increase in response across all cold stimuli, and a 93 and 92% increase in responses evoked across all heat stimuli, respectively. Our results demonstrate that many spinal neurons encode intensity of noxious cold as well as noxious heat over a broad range of stimulus temperatures. Enhanced responses of WDR and HT neurons to cold and heat stimuli after a mild freeze injury is likely to contribute to thermal hyperalgesia following a similar freeze injury in humans.
• Luger, N. M., Honore, P., Sabino, M. A., Schwei, M. J., Rogers, S. D., Mach, D. B., Clohisy, D. R., & Mantyh, P. W. (2001). Osteoprotegerin diminishes advanced bone cancer pain. Cancer research, 61(10), 4038-47.
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  Bone cancer pain most commonly occurs when tumors originating in breast, prostate, or lung metastasize to long bones, spinal vertebrae, and/or pelvis. Primary and metastatic cancers involving bone account for approximately 400,000 new cancer cases per year in the United States alone, and >70% of patients with advanced breast or prostate cancer have skeletal metastases. Whereas pain resulting from bone cancer can dramatically impact an individual's quality of life, very little is known about the mechanisms that generate and maintain this pain. To begin to define the mechanisms that give rise to advanced bone cancer pain, osteolytic 2472 sarcoma cells or media were injected into the intramedullary space of the femur of C3H/HeJ mice, and the injection hole was sealed using dental amalgam, confining the tumor cells to the bone. Twelve days after injection of 2472 tumor cells, animals showed advanced tumor-induced bone destruction of the injected femur, bone cancer pain, and a stereotypic set of neurochemical changes in the spinal cord dorsal horn that receives sensory inputs from the affected femur. Administration of osteoprotegerin, a naturally secreted decoy receptor that inhibits osteoclast maturation and activity and induces osteoclast apoptosis, or vehicle was begun at 12 days, when significant bone destruction had already occurred, and administration was continued daily until day 21. Ongoing pain behaviors, movement-evoked pain behaviors, and bone destruction were assessed on days 10, 12, 14, 17, and 21. The neurochemistry of the spinal cord was evaluated at days 12 and 21. Results indicated that osteoprotegerin treatment halted further bone destruction, reduced ongoing and movement-evoked pain, and reversed several aspects of the neurochemical reorganization of the spinal cord. Thus, even in advanced stages of bone cancer, ongoing osteoclast activity appears to be involved in the generation and maintenance of ongoing and movement-evoked pain. Blockade of ongoing osteoclast activity appears to have the potential to reduce bone cancer pain in patients with advanced tumor-induced bone destruction.
• Mantyh, P. W., & Yaksh, T. L. (2001). Sensory neurons are PARtial to pain. Nature medicine, 7(7), 772-3.
• Pomonis, J. D., Rogers, S. D., Peters, C. M., Ghilardi, J. R., & Mantyh, P. W. (2001). Expression and localization of endothelin receptors: implications for the involvement of peripheral glia in nociception. The Journal of neuroscience : the official journal of the Society for Neuroscience, 21(3), 999-1006.
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  The endothelins (ETs) are peptides that have a diverse array of functions mediated by two receptor subtypes, the endothelin A receptor (ET(A)R) and the endothelin B receptor (ET(B)R). Pharmacological studies have suggested that in peripheral tissues, ET(A)R expression may play a role in signaling acute or neuropathic pain, whereas ET(B)R expression may be involved in the transmission of chronic inflammatory pain. To begin to define the mechanisms by which ET can drive nociceptive signaling, autoradiography and immunohistochemistry were used to examine the distribution of ET(A)R and ET(B)R in dorsal root ganglia (DRG) and peripheral nerve of the rat, rabbit, and monkey. In DRG and peripheral nerve, ET(A)R-immunoreactivity was present in a subset of small-sized peptidergic and nonpeptidergic sensory neurons and their axons and to a lesser extent in a subset of medium-sized sensory neurons. However, ET(B)R-immunoreactivity was not seen in DRG neurons or axons but rather in DRG satellite cells and nonmyelinating ensheathing Schwann cells. Thus, when ETs are released in peripheral tissues, they could act directly on ET(A)R-expressing sensory neurons and on ET(B)R-expressing DRG satellite cells or nonmyelinating Schwann cells. These data indicate that ETs can have direct, nociceptive effects on the peripheral sensory nervous system and that peripheral glia may be directly involved in signaling nociceptive events in peripheral tissues.
• Esler, W. P., Felix, A. M., Stimson, E. R., Lachenmann, M. J., Ghilardi, J. R., Lu, Y. A., Vinters, H. V., Mantyh, P. W., Lee, J. P., & Maggio, J. E. (2000). Activation barriers to structural transition determine deposition rates of Alzheimer's disease a beta amyloid. Journal of structural biology, 130(2-3), 174-83.
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  Brain amyloid composed of the approximately 40-amino-acid human beta-amyloid peptide A beta is integral to Alzheimer's disease pathology. To probe the importance of a conformational transition in Abeta during amyloid growth, we synthesized and examined the solution conformation and amyloid deposition activity of A beta congeners designed to have similar solution structures but to vary substantially in their barriers to conformational transition. Although all these peptides adopt similar solution conformations, a covalently restricted Abeta congener designed to have a very high barrier to conformational rearrangement was inactive, while a peptide designed to have a reduced barrier to conformational transition displayed an enhanced deposition rate relative to wild-type A beta. The hyperactive peptide, which is linked to a heritable A beta amyloidosis characterized by massive amyloid deposition at an early age, displayed a reduced activation barrier to deposition consistent with a larger difference in activation entropy than in activation enthalpy relative to wild-type A beta. These results suggest that in Alzheimer's disease, as in the prion diseases, a conformational transition in the depositing peptide is essential for the conversion of soluble monomer to insoluble amyloid, and alterations in the activation barrier to this transition affect amyloidogenicity and directly contribute to human disease.
• Esler, W. P., Stimson, E. R., Jennings, J. M., Vinters, H. V., Ghilardi, J. R., Lee, J. P., Mantyh, P. W., & Maggio, J. E. (2000). Alzheimer's disease amyloid propagation by a template-dependent dock-lock mechanism. Biochemistry, 39(21), 6288-95.
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  Amyloid plaques composed of the peptide Abeta are an integral part of Alzheimer's disease (AD) pathogenesis. We have modeled the process of amyloid plaque growth by monitoring the deposition of soluble Abeta onto amyloid in AD brain tissue or synthetic amyloid fibrils and show that it is mediated by two distinct kinetic processes. In the first phase, "dock", Abeta addition to the amyloid template is fully reversible (dissociation t(1/2) approximately 10 min), while in the second phase, "lock", the deposited peptide becomes irreversibly associated (dissociation t(1/2) > 1000 min) with the template in a time-dependent manner. The most recently deposited peptide dissociates first while Abeta previously deposited becomes irreversibly "locked" onto the template. Thus, the transition from monomer to neurotoxic amyloid is mediated by interaction with the template, a mechanism that has also been proposed for the prion diseases. Interestingly, two Abeta peptides bearing primary sequence alterations implicated in heritable Abeta amyloidoses displayed faster lock-phase kinetics than wild-type Abeta. Inhibiting the initial weak docking interaction between depositing Abeta and the template is a viable therapeutic target to prevent the critical conformational transition in the conversion of Abeta((solution)) to Abeta((amyloid)) and thus prevent stable amyloid accumulation. While thermodynamics suggest that inhibiting amyloid assembly would be difficult, the present study illustrates that the protein misfolding diseases are kinetically vulnerable to intervention.
• Honore, P., & Mantyh, P. W. (2000). Bone cancer pain: from mechanism to model to therapy. Pain medicine (Malden, Mass.), 1(4), 303-9.
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  Although bone cancer pain can be severe and is relatively common, very little is known about the basic mechanisms that generate and maintain this debilitating pain. To begin to define the mechanisms that give rise to bone cancer pain, a mouse model was developed using the intramedullary injection and containment of osteolytic sarcoma cells in the mouse femur. These tumor cells induced bone destruction as well as ongoing and movement-evoked pain behaviors similar to that found in patients with bone cancer pain. In addition, there was a significant reorganization of the spinal cord that received sensory input from the cancerous bone, and this reorganization was significantly different from that observed in mouse models of chronic neuropathic or inflammatory pain. To determine whether this mouse model of bone cancer could be used to define the basic mechanisms giving rise to bone cancer pain, we targeted excessive osteoclast activity using osteoprotegerin, a secreted decoy receptor that inhibits osteoclast activity. Osteoprotegerin blocked excessive tumor-induced, osteoclast-mediated bone destruction, and significantly reduced ongoing and movement-evoked pain, and the neurochemical reorganization of the spinal cord. These data suggest that this model can provide insight into the mechanisms that generate bone cancer pain and provide a platform for developing and testing novel analgesics to block bone cancer pain.
• Honore, P., Luger, N. M., Sabino, M. A., Schwei, M. J., Rogers, S. D., Mach, D. B., O'keefe, P. F., Ramnaraine, M. L., Clohisy, D. R., & Mantyh, P. W. (2000). Osteoprotegerin blocks bone cancer-induced skeletal destruction, skeletal pain and pain-related neurochemical reorganization of the spinal cord. Nature medicine, 6(5), 521-8.
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  Bone cancer pain is common among cancer patients and can have a devastating effect on their quality of life. A chief problem in designing new therapies for bone cancer pain is that it is unclear what mechanisms drive this distinct pain condition. Here we show that osteoprotegerin, a secreted 'decoy' receptor that inhibits osteoclast activity, also blocks behaviors indicative of pain in mice with bone cancer. A substantial part of the actions of osteoprotegerin seems to result from inhibition of tumor-induced bone destruction that in turn inhibits the neurochemical changes in the spinal cord that are thought to be involved in the generation and maintenance of cancer pain. These results demonstrate that excessive tumor-induced bone destruction is involved in the generation of bone cancer pain and that osteoprotegerin may provide an effective treatment for this common human condition.
• Honore, P., Rogers, S. D., Schwei, M. J., Salak-Johnson, J. L., Luger, N. M., Sabino, M. C., Clohisy, D. R., & Mantyh, P. W. (2000). Murine models of inflammatory, neuropathic and cancer pain each generates a unique set of neurochemical changes in the spinal cord and sensory neurons. Neuroscience, 98(3), 585-98.
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  The aim of this investigation was to determine whether murine models of inflammatory, neuropathic and cancer pain are each characterized by a unique set of neurochemical changes in the spinal cord and sensory neurons. All models were generated in C3H/HeJ mice and hyperalgesia and allodynia behaviorally characterized. A variety of neurochemical markers that have been implicated in the generation and maintenance of chronic pain were then examined in spinal cord and primary afferent neurons.Three days after injection of complete Freund's adjuvant into the hindpaw (a model of persistent inflammatory pain) increases in substance P, calcitonin gene-related peptide, protein kinase C gamma, and substance P receptor were observed in the spinal cord. Following sciatic nerve transection or L5 spinal nerve ligation (a model of persistent neuropathic pain) significant decreases in substance P and calcitonin gene-related peptide and increases in galanin and neuropeptide Y were observed in both primary afferent neurons and the spinal cord. In contrast, in a model of cancer pain induced by injection of osteolytic sarcoma cells into the femur, there were no detectable changes in any of these markers in either primary afferent neurons or the spinal cord. However, in this cancer-pain model, changes including massive astrocyte hypertrophy without neuronal loss, increase in the neuronal expression of c-Fos, and increase in the number of dynorphin-immunoreactive neurons were observed in the spinal cord, ipsilateral to the limb with cancer. These results indicate that a unique set of neurochemical changes occur with inflammatory, neuropathic and cancer pain in C3H/HeJ mice and further suggest that cancer induces a unique persistent pain state. Determining whether these neurochemical changes are involved in the generation and maintenance of each type of persistent pain may provide insight into the mechanisms that underlie each of these pain states.
• Honoré, P., Menning, P. M., Rogers, S. D., Nichols, M. L., & Mantyh, P. W. (2000). Neurochemical plasticity in persistent inflammatory pain. Progress in brain research, 129, 357-63.
• Honoré, P., Schwei, J., Rogers, S. D., Salak-Johnson, J. L., Finke, M. P., Ramnaraine, M. L., Clohisy, D. R., & Mantyh, P. W. (2000). Cellular and neurochemical remodeling of the spinal cord in bone cancer pain. Progress in brain research, 129, 389-97.
• Allen, B. J., Li, J., Menning, P. M., Rogers, S. D., Ghilardi, J., Mantyh, P. W., & Simone, D. A. (1999). Primary afferent fibers that contribute to increased substance P receptor internalization in the spinal cord after injury. Journal of neurophysiology, 81(3), 1379-90.
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  Upon noxious stimulation, substance P (SP) is released from primary afferent fibers into the spinal cord where it interacts with the SP receptor (SPR). The SPR is located throughout the dorsal horn and undergoes endocytosis after agonist binding, which provides a spatial image of SPR-containing neurons that undergo agonist interaction. Under normal conditions, SPR internalization occurs only in SPR+ cell bodies and dendrites in the superficial dorsal horn after noxious stimulation. After nerve transection and inflammation, SPR immunoreactivity increases, and both noxious as well as nonnoxious stimulation produces SPR internalization in the superficial and deep dorsal horn. We investigated the primary afferent fibers that contribute to enhanced SPR internalization in the spinal cord after nerve transection and inflammation. Internalization evoked by electrical stimulation of the sciatic nerve was examined in untreated animals, at 14 days after sciatic nerve transection or sham surgery and at 3 days after hindpaw inflammation. Electrical stimulation was delivered at intensities to excite Abeta fibers only, Abeta and Adelta fibers or A and C fibers as determined by the compound action potential recorded from the tibial nerve. Electrical stimuli were delivered at a constant rate of 10 Hz for a duration of 5 min. Transection of the sciatic nerve and inflammation produced a 33.7 and 32.5% increase in SPR and immunoreactivity in lamina I, respectively. Under normal conditions, stimulation of Adelta or C fibers evoked internalization that was confined to the superficial dorsal horn. After transection or inflammation, there was a 20-24% increase in the proportion of SPR+ lamina I neurons that exhibited internalization evoked by stimulation of Adelta fibers. The proportion of lamina I SPR+ neurons that exhibited internalization after stimulation of C-fibers was not altered by transection or inflammation because this was nearly maximal under normal conditions. Moreover, electrical stimulation sufficient to excite C fibers evoked SPR internalization in 22% of SPR+ lamina III neurons after nerve transection and in 32-36% of SPR+ neurons in lamina III and IV after inflammation. Stimulation of Abeta fibers alone never evoked internalization in the superficial or deep dorsal horn. These results indicate that activation of small-caliber afferent fibers contributes to the enhanced SPR internalization in the spinal cord after nerve transection and inflammation and suggest that recruitment of neurons that possess the SPR contributes to hyperalgesia.
• Esler, W. P., Stimson, E. R., Fishman, J. B., Ghilardi, J. R., Vinters, H. V., Mantyh, P. W., & Maggio, J. E. (1999). Stereochemical specificity of Alzheimer's disease beta-peptide assembly. Biopolymers, 49(6), 505-14.
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  The formation and growth of insoluble amyloid deposits composed primarily of the human beta-amyloid peptide (A beta) in brain is an essentially invariant feature of Alzheimer's disease (AD) and is widely believed to contribute to the progressive neurodegeneration of the disorder. To probe the specificity of amyloid formation and growth, we synthesized and examined the self-assembly of D- and L-stereoisomers of A beta in vitro. While both enantiomers formed insoluble aggregates at similar rates with amyloid-like fibrillar morphology, deposition of soluble A beta peptide onto preexisting A beta aggregates was stereospecific. Although the L-peptide deposited readily onto immobilized L-A beta aggregates with first-order kinetic dependence on soluble peptide concentration, essentially no association between the D-peptide and L-template was observed. Similarly, the D-peptide deposited with first-order kinetics onto a D-A beta aggregate template but did not deposit onto a similar template composed of aggregates of the L-enantiomer. Furthermore, although the L-A beta isomer deposited onto authentic AD amyloid in preparations of unfixed AD brain, no focal association between the D-peptide and brain amyloid was detected. These results establish that deposition of soluble A beta onto preexisting amyloid template is stereospecific, likely involving direct docking interactions between peptide backbone and/or side chains rather than simple hydrophobic association.
• Esler, W. P., Stimson, E. R., Mantyh, P. W., & Maggio, J. E. (1999). Deposition of soluble amyloid-beta onto amyloid templates: with application for the identification of amyloid fibril extension inhibitors. Methods in enzymology, 309, 350-74.
• Honor, P., Menning, P. M., Rogers, S. D., Nichols, M. L., Basbaum, A. I., Besson, J. M., & Mantyh, P. W. (1999). Spinal substance P receptor expression and internalization in acute, short-term, and long-term inflammatory pain states. The Journal of neuroscience : the official journal of the Society for Neuroscience, 19(17), 7670-8.
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  Inflammatory pain involves the sensitization of both primary afferent and spinal cord neurons. To explore the neurochemical changes that contribute to inflammatory pain, we have examined the expression and ligand-induced internalization of the substance P receptor (SPR) in the spinal cord in acute, short-term, and long-term inflammatory pain states. These inflammatory models included unilateral injection of formalin (8-60 min), carrageenan (3 hr), and complete Freund's adjuvant (CFA; 3 d) into the rat hindpaw as well as adjuvant-induced polyarthritis (21 d). In acute inflammatory pain there is ongoing release of substance P (SP) as measured by SPR internalization in lamina I neurons at both 8 and 60 min after formalin injection. Although there is no tonic release of SP in short-term inflammatory pain, at 3 hr after carrageenan injection, SP is released in response to both noxious and non-noxious somatosensory stimulation with SPR internalization being observed in neurons located in both laminae I and III-IV. In long-term inflammatory pain models (CFA and polyarthritis) the same pattern of SP release and SPR activation occurs as is observed in short-term inflammation with the addition that there is a significant upregulation of the SPR in lamina I neurons. These results suggest that SPR internalization might serve as a marker of the contribution of ongoing primary afferent input in acute and persistent pain states. These stereotypical neurochemical changes suggest that there are unique neurochemical signatures for acute, short-term, and long-term inflammatory pain.
• Nichols, M. L., Allen, B. J., Rogers, S. D., Ghilardi, J. R., Honore, P., Luger, N. M., Finke, M. P., Li, J., Lappi, D. A., Simone, D. A., & Mantyh, P. W. (1999). Transmission of chronic nociception by spinal neurons expressing the substance P receptor. Science (New York, N.Y.), 286(5444), 1558-61.
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  Substance P receptor (SPR)-expressing spinal neurons were ablated with the selective cytotoxin substance P-saporin. Loss of these neurons resulted in a reduction of thermal hyperalgesia and mechanical allodynia associated with persistent neuropathic and inflammatory pain states. This loss appeared to be permanent. Responses to mildly painful stimuli and morphine analgesia were unaffected by this treatment. These results identify a target for treating persistent pain and suggest that the small population of SPR-expressing neurons in the dorsal horn of the spinal cord plays a pivotal role in the generation and maintenance of chronic neuropathic and inflammatory pain.
• O'Hare, E., Weldon, D. T., Mantyh, P. W., Ghilardi, J. R., Finke, M. P., Kuskowski, M. A., Maggio, J. E., Shephard, R. A., & Cleary, J. (1999). Delayed behavioral effects following intrahippocampal injection of aggregated A beta (1-42). Brain research, 815(1), 1-10.
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  Beta amyloid protein (A beta) is the major extracellular component of Alzheimer's disease (AD) plaques. In the current study, A beta (1-42) was aggregated in vitro using a method which produces A beta aggregates similar to those found in the AD brain. Twelve male Sprague-Dawley rats were trained in two-lever operant chambers under an alternating lever cyclic-ratio (ALCR) schedule. When performance was stable on the ALCR schedule, six subjects were injected (bilaterally into the CA3 area of the dorsal hippocampus) with 5.0 microliters aggregated A beta in suspension, and the remaining six subjects were injected with 5.0 microliters sterile water. Behavioral testing resumed 5 days after surgery and continued for 90 days post-injection. Aggregated A beta injection did not affect the number of lever switching errors made in a daily session but did affect the number of incorrect lever response perseverations. After approximately 30 days post-injection, aggregated A beta injection detrimentally affected ability to track the changing parameters of the schedule, and decreased the efficiency by which subjects obtained reinforcers. From approximately day 50 post-injection onward, A beta-injected subjects demonstrated significantly higher numbers of incorrect lever response perseverations than did sterile water-injected subjects. These effects appeared to be central rather than peripheral, as A beta injection did not decrease running response rates under the ALCR schedule. The delayed onset of behavioral effects seen in this and other behavioral studies may be a result of a cascade of potentially harmful responses induced through glial activation following aggregated A beta injection.
• Oyamada, H., Takatsuji, K., Senba, E., Mantyh, P. W., & Tohyama, M. (1999). Postnatal development of NK1, NK2, and NK3 neurokinin receptors expression in the rat retina. Brain research. Developmental brain research, 117(1), 59-70.
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  The biological effects of tachykinins are mediated by three distinct receptors, the neurokinin 1 receptor (NK1-R), NK2-R, and NK3-R. There is no information available concerning the development of these receptors in the retina. In the present study, we investigated the localization of tachykinin receptors, using antisera directed against NK1-R, NK2-R, and NK3-R in the adult and developing rat retinas. Numerous NK1-R immunoreactive (NK1-R IR) cells were already observed in the proximal part of the neuroblastic layer in the retina at postnatal day 5 (P5). The distribution and intensity of NK1-R IR cells and processes in the inner nuclear layer (INL) and inner plexiform layer (IPL) at P10 were similar to those of adult retina. Most NK1-R IR cells located in the proximal part of INL, which were morphologically amacrine cells. In the contrast to the early expression of NK1-R IR cells, no NK3-R IR structures existed in the neuronal elements of the retina until P10. NK3-R IR processes were first detected in the outer plexiform layer (OPL) at P10. At P15, NK3-R IR somata were slightly stained in the distal and middle parts of the INL, and NK3-R IR processes were present in the OPL and the upper part of the IPL. During P15-P30, the number of NK3-R IR somata located in the INL remarkably increased. These NK3-R IR cells were morphologically bipolar and amacrine cells. This study provides differential cellular distribution of NK1-R IR cells and NK3-R IR cells in the INL of the rat retina. Our findings suggest that NK1-R and NK3-R are involved in different visual circuits and retinal maturation, and NK3-R may play previously unknown important roles in the visual processes of the rat.
• Schwei, M. J., Honore, P., Rogers, S. D., Salak-Johnson, J. L., Finke, M. P., Ramnaraine, M. L., Clohisy, D. R., & Mantyh, P. W. (1999). Neurochemical and cellular reorganization of the spinal cord in a murine model of bone cancer pain. The Journal of neuroscience : the official journal of the Society for Neuroscience, 19(24), 10886-97.
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  The cancer-related event that is most disruptive to the cancer patient's quality of life is pain. To begin to define the mechanisms that give rise to cancer pain, we examined the neurochemical changes that occur in the spinal cord and associated dorsal root ganglia in a murine model of bone cancer. Twenty-one days after intramedullary injection of osteolytic sarcoma cells into the femur, there was extensive bone destruction and invasion of the tumor into the periosteum, similar to that found in patients with osteolytic bone cancer. In the spinal cord, ipsilateral to the cancerous bone, there was a massive astrocyte hypertrophy without neuronal loss, an expression of dynorphin and c-Fos protein in neurons in the deep laminae of the dorsal horn. Additionally, normally non-noxious palpation of the bone with cancer induced behaviors indicative of pain, the internalization of the substance P receptor, and c-Fos expression in lamina I neurons. The alterations in the neurochemistry of the spinal cord and the sensitization of primary afferents were positively correlated with the extent of bone destruction and the growth of the tumor. This "neurochemical signature" of bone cancer pain appears unique when compared to changes that occur in persistent inflammatory or neuropathic pain states. Understanding the mechanisms by which the cancer cells induce this neurochemical reorganization may provide insight into peripheral factors that drive spinal cord plasticity and in the development of more effective treatments for cancer pain.
• Trafton, J. A., Abbadie, C., Marchand, S., Mantyh, P. W., & Basbaum, A. I. (1999). Spinal opioid analgesia: how critical is the regulation of substance P signaling?. The Journal of neuroscience : the official journal of the Society for Neuroscience, 19(21), 9642-53.
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  Although opioids can reduce stimulus-evoked efflux of Substance P (SP) from nociceptive primary afferents, the consequences of this reduction on spinal cord nociceptive processing has not been studied. Rather than assaying SP release, in the present study we examined the effect of opioids on two postsynaptic measures of SP release, Fos expression and neurokinin-1 (NK-1) receptor internalization, in the rat. The functional significance of the latter was first established in in vitro studies that showed that SP-induced Ca(2+) mobilization is highly correlated with the magnitude of SP-induced NK-1 receptor internalization in dorsal horn neurons. Using an in vivo analysis, we found that morphine had little effect on noxious stimulus-evoked internalization of the NK-1 receptor in lamina I neurons. However, internalization was reduced when we coadministered morphine with a dose of an NK-1 receptor antagonist that by itself was without effect. Thus, although opioids may modulate SP release, the residual release is sufficient to exert maximal effects on the target NK-1 receptors. Morphine significantly reduced noxious stimulus-induced Fos expression in lamina I, but the Fos inhibition was less pronounced in neurons that expressed the NK-1 receptor. Taken together, these results suggest that opioid analgesia predominantly involves postsynaptic inhibitory mechanisms and/or presynaptic control of non-SP-containing primary afferent nociceptors.
• Tseng, B. P., Esler, W. P., Clish, C. B., Stimson, E. R., Ghilardi, J. R., Vinters, H. V., Mantyh, P. W., Lee, J. P., & Maggio, J. E. (1999). Deposition of monomeric, not oligomeric, Abeta mediates growth of Alzheimer's disease amyloid plaques in human brain preparations. Biochemistry, 38(32), 10424-31.
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  Senile plaques composed of the peptide Abeta contribute to the pathogenesis of Alzheimer's disease (AD), and mechanisms underlying their formation and growth may be exploitable as therapeutic targets. To examine the process of amyloid plaque growth in human brain, we have utilized size exclusion chromatography (SEC), translational diffusion measured by NMR, and in vitro models of Abeta amyloid growth to identify the oligomerization state of Abeta that is competent to add onto an existing amyloid deposit. SEC of radiolabeled and unlabeled Abeta over a concentration range of 10(-)(10)-10(-)(4) M demonstrated that the freshly dissolved peptide eluted as a single low molecular weight species, consistent with monomer or dimer. This low molecular weight Abeta species isolated by SEC was competent to deposit onto preexisting amyloid in preparations of AD cortex, with first-order kinetic dependence on soluble Abeta concentration, establishing that solution-phase oligomerization is not rate limiting. Translational diffusion measurements of the low molecular weight Abeta fraction demonstrate that the form of the peptide active in plaque deposition is a monomer. In deliberately aged (>6 weeks) Abeta solutions, a high molecular weight (>100 000 M(r)) species was detectable in the SEC column void. In contrast to the active monomer, assembled Abeta isolated from the column showed little or no focal association with AD tissue. These studies establish that, at least in vitro, Abeta exists as a monomer at physiological concentrations and that deposition of monomers, rather than of oligomeric Abeta assemblies, mediates the growth of existing amyloid in human brain preparations.
• Cao, Y. Q., Mantyh, P. W., Carlson, E. J., Gillespie, A. M., Epstein, C. J., & Basbaum, A. I. (1998). Primary afferent tachykinins are required to experience moderate to intense pain. Nature, 392(6674), 390-4.
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  The excitatory neurotransmitter glutamate coexists with the peptide known as substance P in primary afferents that respond to painful stimulation. Because blockers of glutamate receptors reliably reduce pain behaviour, it is assumed that 'pain' messages are mediated by glutamate action on dorsal horn neurons. The contribution of substance P, however, is still unclear. We have now disrupted the mouse preprotachykinin A gene (PPT-A), which encodes substance P and a related tachykinin, neurokinin A. We find that although the behavioural response to mildly painful stimuli is intact in these mice, the response to moderate to intense pain is significantly reduced. Neurogenic inflammation, which results from peripheral release of substance P and neurokinin A, is almost absent in the mutant mice. We conclude that the release of tachykinins from primary afferent pain-sensing receptors (nociceptors) is required to produce moderate to intense pain.
• Mantyh, P. W., & Hunt, S. P. (1998). Hot peppers and pain. Neuron, 21(4), 644-5.
• Weldon, D. T., Rogers, S. D., Ghilardi, J. R., Finke, M. P., Cleary, J. P., O'Hare, E., Esler, W. P., Maggio, J. E., & Mantyh, P. W. (1998). Fibrillar beta-amyloid induces microglial phagocytosis, expression of inducible nitric oxide synthase, and loss of a select population of neurons in the rat CNS in vivo. The Journal of neuroscience : the official journal of the Society for Neuroscience, 18(6), 2161-73.
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  To determine the stability of beta-amyloid peptide (Abeta) and the glial and neuronal changes induced by Abeta in the CNS in vivo, we made single injections of fibrillar Abeta (fAbeta), soluble Abeta (sAbeta), or vehicle into the rat striatum. Injected fAbeta is stable in vivo for at least 30 d after injection, whereas sAbeta is primarily cleared within 1 d. After injection of fAbeta, microglia phagocytize fAbeta aggregates, whereas nearby astrocytes form a virtual wall between fAbeta-containing microglia and the surrounding neuropil. Similar glial changes are not observed after sAbeta injection. Microglia and astrocytes near the injected fAbeta show a significant increase in inducible nitric oxide synthase (iNOS) expression compared with that seen with sAbeta or vehicle injection. Injection of fAbeta but not sAbeta or vehicle induces a significant loss of parvalbumin- and neuronal nitric oxide synthase-immunoreactive neurons, whereas the number of calbindin-immunoreactive neurons remains unchanged. These data demonstrate that fAbeta is remarkably stable in the CNS in vivo and suggest that fAbeta neurotoxicity is mediated in large part by factors released from activated microglia and astrocytes, as opposed to direct interaction between Abeta fibrils and neurons.
• Abbadie, C., Trafton, J., Liu, H., Mantyh, P. W., & Basbaum, A. I. (1997). Inflammation increases the distribution of dorsal horn neurons that internalize the neurokinin-1 receptor in response to noxious and non-noxious stimulation. The Journal of neuroscience : the official journal of the Society for Neuroscience, 17(20), 8049-60.
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  Although the neurokinin-1 (NK-1)/substance P (SP) receptor is expressed by neurons throughout the spinal dorsal horn, noxious chemical stimulation in the normal rat only induces internalization of the receptor in cell bodies and dendrites of lamina I. Here we compared the effects of mechanical and thermal stimulation in normal rats and in rats with persistent hindpaw inflammation. Electron microscopic analysis confirmed the upregulation of receptor that occurs with inflammation and demonstrated that in the absence of superimposed stimulation, the increased receptor was, as in normal rats, concentrated on the plasma membrane. In general, noxious mechanical was more effective than noxious thermal stimulation in inducing NK-1 receptor internalization, and this was increased in the setting of inflammation. Although a 5 sec noxious mechanical stimulus only induced internalization in 22% of lamina I neurons in normal rats, after inflammation, it evoked near-maximal (98%) internalization in lamina I, produced significant changes in laminae III-VI, and expanded the rostrocaudal distribution of neurons with internalized receptor. Even non-noxious (brush) stimulation of the inflamed hindpaw induced internalization in large numbers of superficial and deep neurons. For thermal stimulation, the percentage of cells with internalized receptor increased linearly at >45 degrees C, but in normal rats, these were restricted to lamina I. After inflammation, however, the 52 degrees C stimulus also induced internalization in 25% of laminae III-IV cells. These studies provide a new perspective on the reorganization of dorsal horn circuits in the setting of persistent injury and demonstrate a critical contribution of SP.
• Allen, B. J., Rogers, S. D., Ghilardi, J. R., Menning, P. M., Kuskowski, M. A., Basbaum, A. I., Simone, D. A., & Mantyh, P. W. (1997). Noxious cutaneous thermal stimuli induce a graded release of endogenous substance P in the spinal cord: imaging peptide action in vivo. The Journal of neuroscience : the official journal of the Society for Neuroscience, 17(15), 5921-7.
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  Dorsal root ganglia (DRG) neurons synthesize and transport substance P (SP) to the spinal cord where it is released in response to intense noxious somatosensory stimuli. We have shown previously that SP release in vivo causes a rapid and reversible internalization of SP receptors (SPRs) in dorsal horn neurons, which may provide a pharmacologically specific image of neurons activated by SP. Here, we report that noxious heat (43 degrees, 48 degrees, and 55 degrees C) and cold (10 degrees, 0 degrees, -10 degrees, and -20 degrees C) stimuli, but not innocuous warm (38 degrees C) and cold (20 degrees C) stimuli, applied to the hindpaw of anesthetized rats induce SPR internalization in spinal cord neurons that is graded with respect to the intensity of the thermal stimulus. Thus, with increasing stimulus intensities, both the total number of SPR+ lamina I neurons showing SPR internalization and the number of internalized SPR+ endosomes within each SPR immunoreactive neuron showed a significant increase. These data suggest that thermal stimuli induce a graded release of SP from primary afferent terminals and that agonist dependent receptor endocytosis provides evidence of a spatially and pharmacologically unique "neurochemical signature" after specific somatosensory stimuli.
• Esler, W. P., Stimson, E. R., Ghilardi, J. R., Felix, A. M., Lu, Y. A., Vinters, H. V., Mantyh, P. W., & Maggio, J. E. (1997). A beta deposition inhibitor screen using synthetic amyloid. Nature biotechnology, 15(3), 258-63.
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  The formation, growth, and maturation of brain amyloid "senile" plaques are essential pathological processes in Alzheimer's disease (AD) and key targets for therapeutic intervention. The process of in vitro deposition of A beta at physiological concentrations onto plaques in AD brain preparations has been well characterized, but is cumbersome for drug discovery. We describe here a high-through put screen for inhibitors of A beta deposition onto a synthetic template (synthaloid) of fibrillar A beta immobilized in a polymer matrix. Synthaloid is indistinguishable from plaques in AD brain (the natural template) in deposition kinetics, pH profile, and structure-activity relationships for both A beta analogs and inhibitors. Synthaloid, in contrast to current A beta aggregation screens, accurately predicted inhibitor potency for A beta deposition onto AD cortex preparations, validating its use in searching for agents that can slow the progression of AD and exposing a previously inaccessible target for drug discovery.
• Liu, H., Mantyh, P. W., & Basbaum, A. I. (1997). NMDA-receptor regulation of substance P release from primary afferent nociceptors. Nature, 386(6626), 721-4.
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  Severe or prolonged tissue or nerve injury can induce hyperexcitability of dorsal horn neurons of the spinal cord, resulting in persistent pain, an exacerbated response to noxious stimuli (hyperalgesia), and a lowered pain threshold (allodynia). These changes are mediated by NMDA (N-methyl-D-aspartate)-type glutamate receptors in the spinal cord. Here we report that activation of the NMDA receptor causes release of substance P, a peptide neurotransmitter made by small-diameter, primary, sensory 'pain' fibres. Injection of NMDA in the cerebrospinal fluid of the rat spinal cord mimicked the changes that occur with persistent injury, and produced not only pain, but also a large-scale internalization of the substance P receptor into dorsal horn neurons, as well as structural changes in their dendrites. Both the pain and the morphological changes produced by NMDA were significantly reduced by substance P-receptor antagonists or by elimination of substance P-containing primary afferent fibres with the neurotoxin capsaicin. We suggest that presynaptic NMDA receptors located on the terminals of small-diameter pain fibres facilitate and prolong the transmission of nociceptive messages, through the release of substance P and glutamate. Therapies directed at the presynaptic NMDA receptor could therefore ameliorate injury-evoked persistent pain states.
• Mantyh, P. W., Rogers, S. D., Honore, P., Allen, B. J., Ghilardi, J. R., Li, J., Daughters, R. S., Lappi, D. A., Wiley, R. G., & Simone, D. A. (1997). Inhibition of hyperalgesia by ablation of lamina I spinal neurons expressing the substance P receptor. Science (New York, N.Y.), 278(5336), 275-9.
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  Substance P is released in the spinal cord in response to painful stimuli, but its role in nociceptive signaling remains unclear. When a conjugate of substance P and the ribosome-inactivating protein saporin was infused into the spinal cord, it was internalized and cytotoxic to lamina I spinal cord neurons that express the substance P receptor. This treatment left responses to mild noxious stimuli unchanged, but markedly attenuated responses to highly noxious stimuli and mechanical and thermal hyperalgesia. Thus, lamina I spinal cord neurons that express the substance P receptor play a pivotal role in the transmission of highly noxious stimuli and the maintenance of hyperalgesia.
• Rogers, S. D., Demaster, E., Catton, M., Ghilardi, J. R., Levin, L. A., Maggio, J. E., & Mantyh, P. W. (1997). Expression of endothelin-B receptors by glia in vivo is increased after CNS injury in rats, rabbits, and humans. Experimental neurology, 145(1), 180-95.
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  Previous studies have demonstrated that neonatal cultures of astrocytes express functional endothelin (ET) receptors. To determine if similar ET receptors are expressed by adult glia we used 125I-ET-1 to examine the expression of ET receptors both in vivo in the normal and transected optic nerves of the rabbit and rat and in vitro in cultures of astrocytes, microglia, or oligodendrocytes. Additionally, we examined the expression of ET receptors in the human optic nerve. Moderate levels of ET(B) receptors were identified in the rabbit and rat forebrain, whereas in the normal rabbit, rat, and human optic nerves a low density of ET(B) receptors was observed, mainly in association with glial fibrillary acidic protein + (GFAP+) astrocytes. After unilateral optic nerve transection, or damage to the retina, the density of glial ET(B) receptors in the optic nerve is significantly increased in all species examined. Thus, at 7 days posttransection there is a significant increase in ET(B) receptors, and by 90 days posttransection the density of ET(B) receptors in the rabbit or rat optic nerve was among the highest of any area in the central nervous system (CNS). Primary cultures of astrocytes or microglia, but not oligodendrocytes, express 125I-ET-1 binding sites. These data demonstrate that in the normal CNS, astrocytes express low but detectable levels of ET(B) receptors, and, after CNS injury, both astrocytes and microglia express high levels of ET(B) receptors. ET(B) receptors provide a therapeutic target for regulating glial proliferation and the release of neurotrophic factors from glia that occur in response to neuronal injury.
• Weldon, D. T., Maggio, J. E., & Mantyh, P. W. (1997). New insights into the neuropathology and cell biology of Alzheimer's disease. Geriatrics, 52 Suppl 2, S13-6.
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  Several lines of evidence, including newly discovered genetic mutations, suggest that beta-amyloid (A beta) is directly involved in the neuropathology observed in familial and sporadic forms of Alzheimer's disease (AD). Rather than exerting its neurotoxicity directly, results from our laboratory suggest that fibrillar A beta (fA beta) activates microglia and astrocytes upon injection into the rat brain. The microglia and astrocytes, in turn, form a functional barrier between A beta and surrounding neurons. An increase in inducible nitric oxide synthase (iNOS) immunoreactivity is observed in activated microglia and astrocytes, and specific subpopulations of neurons are lost in fA beta injection areas versus controls. These data, coupled with recent discoveries of the A beta association with the receptor for advanced glycation end products (RAGE) and the class A scavenger receptors (SR), support the hypothesized role of inflammatory mechanisms in AD neurotoxicity.
• Abbadie, C., Brown, J. L., Mantyh, P. W., & Basbaum, A. I. (1996). Spinal cord substance P receptor immunoreactivity increases in both inflammatory and nerve injury models of persistent pain. Neuroscience, 70(1), 201-9.
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  Numerous studies have implicated the primary afferent derived neuropeptide, substance P, which exerts its effects via the neurokinin-1/substance P receptor, in the transmission of nociceptive messages at the level of the spinal cord. Immunocytochemical studies demonstrate that the substance P receptor is concentrated in neurons of lamina I of the superficial dorsal horn. Since alterations in the number and distribution of the receptor may underlie persistent pain conditions, we have used immunocytochemistry to study the distribution of the receptor in two very different rat models of persistent pain: chronic inflammation, which is associated with increased levels of substance P, and sciatic nerve section, which is associated with decreased levels of substance P in the dorsal horn. Inflammation was produced by unilateral hindpaw injection of complete Freund's adjuvant. We report that there is an up-regulation of substance P receptor immunoreactivity in the superficial laminae of the dorsal horn in both injury models. The increase was found at all time points studied (up to one week after induction of inflammation and up to two weeks after sciatic nerve section). The increase in substance P receptor immunoreactivity was not only present in the medial part of the dorsal horn at segment L4, which is the region of input of the afferents from the hindpaw, but also in the lateral parts of the dorsal horn, and at segments rostral (L1) and caudal (S1) to the afferent input from the hindpaw. These results indicate that the up-regulation of the receptor is not predictable merely by the change in the concentration of substance P in the dorsal horn. Furthermore, the non-topographic up-regulation of substance P receptor in these different conditions may contribute to the central sensitization of dorsal horn nociceptors under conditions of persistent pain.
• Esler, W. P., Stimson, E. R., Ghilardi, J. R., Lu, Y. A., Felix, A. M., Vinters, H. V., Mantyh, P. W., Lee, J. P., & Maggio, J. E. (1996). Point substitution in the central hydrophobic cluster of a human beta-amyloid congener disrupts peptide folding and abolishes plaque competence. Biochemistry, 35(44), 13914-21.
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  Alzheimer's disease (AD) is pathologically characterized by the presence of numerous insoluble amyloid plaques in the brain composed primarily of a 40-43 amino acid peptide, the human beta-amyloid peptide (A beta). The process of A beta deposition can be modeled in vitro by deposition of physiological concentrations of radiolabeled A beta onto preexisting amyloid in preparations of unfixed AD cerebral cortex. Using this model system, it has been shown that A beta deposition is biochemically distinct from A beta aggregation and occurs readily at physiological A beta concentrations, but which regions and conformations of A beta are essential to A beta deposition is poorly understood. We report here that an active congener, A beta (10-35)-NH2, displays time dependence, pH-activity profile, and kinetic order of deposition similar to A beta (1-40), and is sufficiently soluble for NMR spectroscopy in water under conditions where it actively deposits. To examine the importance of the central hydrophobic cluster of A beta (LVFFA, residues 17-21) for in vitro A beta deposition, an A beta (10-35)-NH2 analog with a single point substitution (F19T) in this region was synthesized and examined. Unlike A beta (10-35)-NH2, the F19T analog was plaque growth incompetent, and NMR analysis indicated that the mutant peptide was significantly less folded than wild-type A beta. These results support previous studies suggesting that the plaque competence of A beta correlates with peptide folding. Since compounds that alter A beta folding may reduce amyloid deposition, the central hydrophobic cluster of A beta will be a tempting target for structure-based drug design when high-resolution structural information becomes available.
• Esler, W. P., Stimson, E. R., Ghilardi, J. R., Vinters, H. V., Lee, J. P., Mantyh, P. W., & Maggio, J. E. (1996). In vitro growth of Alzheimer's disease beta-amyloid plaques displays first-order kinetics. Biochemistry, 35(3), 749-57.
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  A salient pathological feature of Alzheimer's disease (AD) is the presence of amyloid plaques in the brains of affected patients. The plaques are predominantly composed of human beta-amyloid peptide (A beta). Although the aggregation of synthetic A beta has been extensively studied, the mechanism of AD plaque growth is poorly understood. In order to address this question, we used an in vitro model of plaque growth to determine if assembly or aggregation of A beta is required for deposition. Labeled A beta at physiological concentrations readily deposited onto both neuritic and diffuse plaques and cerebrovascular amyloid in unfixed AD brain tissue, whereas essentially no deposition was detected in tissue without performed amyloid. Using this in vitro model of plaque growth, the kinetics of A beta deposition onto plaques was examined in two independent but complementary systems. Intact sections of unfixed AD brain cortex (analyzed by autoradiographic densitometry) allowed definitive morphological analysis of the site of deposition, while homogenates of the same tissue (analyzed by radioisotope counting) allowed precise quantitation of deposition over a wide range of conditions. Essentially identical results were obtained for both systems. Growth of preexisting tissue plaques by deposition of A beta was found to follow first-order dependence on A beta concentration and exhibited a pH optimum of 7. In sharp contrast, A beta aggregation in the absence of template follows higher order kinetics and shows a pH optimum of 5. On the basis of criteria of kinetic order, pH dependence, and structure-activity relationships, we conclude that aggregation of A beta (template-independent initial nidus formation) and deposition of A beta (template-dependent subsequent plaque growth) are fundamentally distinct biochemical processes. The process of plaque growth and maturation by A beta deposition may be an important target for therapeutic intervention to block the progression of AD.
• Esler, W. P., Stimson, E. R., Jennings, J. M., Ghilardi, J. R., Mantyh, P. W., & Maggio, J. E. (1996). Zinc-induced aggregation of human and rat beta-amyloid peptides in vitro. Journal of neurochemistry, 66(2), 723-32.
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  The major pathological feature of Alzheimer's disease is the presence of a high density of amyloid plaques in the brain tissue of patients. The plaques are predominantly composed of human beta-amyloid peptide (A beta), a 39-43-mer peptide the neurotoxicity of which is related to its aggregation state. Previous work has demonstrated that certain metals that have been implicated as risk factors for Alzheimer's disease (Al, Fe, and Zn) also cause substantial aggregation of A beta. In particular, we reported that zinc cations at concentrations of > 10(-4) M dramatically accelerate the rate of A beta aggregation at physiological peptide concentrations at 37 degrees C in vitro. In the present study, we investigate the effect of Zn2+ on aggregation of radiolabeled and unlabeled human and rat A beta over a wide range of peptide concentrations in the presence and absence of salt and blocking protein. Aggregation was assayed by centrifugation and filtration using amino acid analysis, immunoassay, and gamma-counting for quantification over a wide range of concentrations of Zn2+ and A beta above and below physiological values. The results of this study demonstrate the following: (a) Radioiodinated A beta accurately tracked unlabeled A beta, (b) zinc concentrations of at least 10(-4) M were required to induce significant aggregation of A beta, and (c) rat and human A beta species were cleared from aqueous solutions by similar concentrations of zinc. These results stand in significant quantitative disagreement (approximately 100-fold in zinc concentration) with one previous study that reported significant aggregation of A beta by < 1 microM Zn2+. Differences between the present study and the latter study from another laboratory appear to result from inappropriate reliance on optical density to measure A beta concentrations and nonspecific loss of A beta to plastic in the absence of blocking protein.
• Ghilardi, J. R., Catton, M., Stimson, E. R., Rogers, S., Walker, L. C., Maggio, J. E., & Mantyh, P. W. (1996). Intra-arterial infusion of [125I]A beta 1-40 labels amyloid deposits in the aged primate brain in vivo. Neuroreport, 7(15-17), 2607-11.
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  Alzheimer's disease is characterized by extracellular amyloid deposits in the brain at both vascular sites (cerebrovascular amyloid, CVA) and within the neuropil (plaques). In the present study we demonstrated that brain amyloid of aged non-human primates is efficiently detected by [125I]A beta in vitro, and assessed the detection of that amyloid in vivo by intravascular infusion of [125I]A beta. Aged squirrel monkeys (Saimiri sciureus) were anesthetized and infused intra-arterially with [125I]A beta, and sacrificed 2 h later. Analysis of the anterior frontal and temporal cortices by autoradiography demonstrated that [125I]A beta was deposited on CVA and that essentially every amyloid deposit which could be detected with thioflavin S or anti-A beta antibodies was also labeled by [125I]A beta. These experiments suggest that intravascular infusion of radiolabeled A beta can be used to detect and image amyloid deposits in the human AD brain.
• Hodges-Savola, C., Rogers, S. D., Ghilardi, J. R., Timm, D. R., & Mantyh, P. W. (1996). Beta-adrenergic receptors regulate astrogliosis and cell proliferation in the central nervous system in vivo. Glia, 17(1), 52-62.
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  Astrocytes express several cell surface receptors including the beta 2 -adrenergic receptor. To explore whether beta-adrenergic receptors (beta-ARs) directly regulate astrogliosis and glial scar formation, we evaluated the effects of beta-AR activation and blockade on astrocyte hypertrophy and cell proliferation in rabbit optic nerves in vivo. Artificial cerebrospinal fluid (CSF), isoproterenol (ISO; a beta-agonist), or propranolol (PROP; a beta-antagonist) were infused via osmotic minipumps into non-injured and crushed optic nerves for 14 days. Changes in nerve cell numbers and astroglial hypertrophy were monitored by ethidium bromide nuclear staining and glial fibrillary acidic protein (GFAP) immunohistochemistry, respectively. In non-injured nerves infused with CSF or PROP, there were no alterations in GFAP-immunoreactivity or cell numbers compared to normal optic nerves; however, in non-injured nerves infused with ISO, there was a significant increase in both GFAP-immunoreactivity and cell number. In crushed optic nerves, there was a significant increase in both GFAP-immunoreactivity and cell number compared to normal nerves, and this increase was not altered by infusion of either CSF or ISO. In contrast, PROP infusion significantly reduced the crush-induced increase in GFAP-immunofluorescence and cell number. These findings suggest that a) beta-AR activation, in the absence of injury, can promote astroglial hypertrophy and cell proliferation; b) after injury, beta-AR activation drives injury-induced astrogliosis and cell proliferation; c) astrocyte beta-ARs are maximally stimulated after neuronal injury; and d) neuronal regeneration may be influenced, both positively and negatively, through the pharmacological manipulation of glial receptors.
• Maggio, J. E., & Mantyh, P. W. (1996). Brain amyloid--a physicochemical perspective. Brain pathology (Zurich, Switzerland), 6(2), 147-62.
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  The ability to form stable cross-beta fibrils is an intrinsic physicochemical characteristic of the human beta-amyloid peptide (A beta), which forms the brain amyloid of Alzheimer's disease (AD). The high amyloidogenicity and low solubility of this hydrophobic approximately 40-mer have been barriers to its study in the past, but the availability of synthetic peptide and new physical methods has enabled many novel approaches in recent years. Model systems for A beta aggregation (relevant to initial nidus formation) and A beta deposition (relevant to plaque growth and maturation) in vitro have allowed structure/activity relationships and kinetics to be explored quantitatively, and established that these processes are biochemically distinct. Different forms of the peptide, with different physiochemical characteristics, are found in vascular and parenchymal amyloid. Various spectroscopic methods have been used to explore the three-dimensional conformation of A beta both in solution and in solid phase, and demonstrated that the peptide adopts a different configuration in each state. A significant conformational transition is essential to the transformation of A beta from solution to fibril. These observations suggest new therapeutic targets for the treatment of AD.
• Mantyh, C. R., Maggio, J. E., Mantyh, P. W., Vigna, S. R., & Pappas, T. N. (1996). Increased substance P receptor expression by blood vessels and lymphoid aggregates in Clostridium difficile-induced pseudomembranous colitis. Digestive diseases and sciences, 41(3), 614-20.
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  Pseudomembranous colitis is most often caused by toxins secreted by Clostridium difficile following bowel flora overgrowth after antibiotic use. The secretory and inflammatory effects observed in C. difficile toxin A-induced enterocolitis in the rat ileum are inhibited by CP-96,345, a substance P (SP) receptor antagonist. To determine if SP plays a role in the pathogenesis of human pseudomembranous colitis, SP receptor distribution was examined in a toxin A-positive specimen of bowel. Quantitative receptor autoradiography was used to examine SP receptors in tissue from a patient who tested positive for C. Difficile toxin. SP receptors were massively increased in small blood vessels and lymphoid aggregates in the pseudomembranous colitis bowel in comparison to control specimens. The SP binding was saturable and exhibited similar affinities for SP and CP-96,345. SP may contribute to the inflammatory response in pseudomembranous colitis via a massive increase in SP receptor antagonists may offer a novel therapeutic intervention for pseudomembranous colitis.
• Mantyh, C. R., Pappas, T. N., Lapp, J. A., Washington, M. K., Neville, L. M., Ghilardi, J. R., Rogers, S. D., Mantyh, P. W., & Vigna, S. R. (1996). Substance P activation of enteric neurons in response to intraluminal Clostridium difficile toxin A in the rat ileum. Gastroenterology, 111(5), 1272-80.
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  Nerves have been suggested to mediate the effects of bacterial toxins in intestinal diseases. However, the mechanisms involved are unknown. This study examined endogenous substance P (SP) activation of the substance P receptor (SPR) on enteric neurons in the rat ileum after exposure to intraluminal Clostridium difficile toxin A.
• Mantyh, P. W., Rogers, S. D., Ghilardi, J. R., Maggio, J. E., Mantyh, C. R., & Vigna, S. R. (1996). Differential expression of two isoforms of the neurokinin-1 (substance P) receptor in vivo. Brain research, 719(1-2), 8-13.
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  Recent pharmacological and biochemical studies have suggested that there may be more than one molecular form of the neurokinin-1 receptor (NK-1), a long and short isoform differing in the length of their cytoplasmic carboxyl-terminal tails, but no definitive evidence of the existence of such NK-1 receptor isoforms in tissue has been presented. To examine whether these different isoforms are expressed in vivo we have compared the distribution of high affinity substance P (SP) binding sites (visualized by autoradiography with [125I]SP), with the distribution of the C-terminal epitope of the full length receptor (visualized with a specific antibody against the extreme C-terminal sequence). The former method labels both long and short forms of the NK-1 receptor, while the latter labels only the long form of the protein. In the rat there is a close correspondence of [125I]SP binding and NK-1 immunoreactivity in the striatum, suggesting that the long isoform predominates in this tissue. In the parotid and submaxillary gland, there are very high levels of [125I]SP binding but only low levels of NK-1 immunoreactivity, suggesting that expression of the short form predominates in these tissues. These results imply that different tissues express different ratios of the two isoforms of the NK-1 receptor. This differential expression provides the theoretical basis for tissue specific pharmacological targeting of NK-1 receptors.
• Brown, J. L., Liu, H., Maggio, J. E., Vigna, S. R., Mantyh, P. W., & Basbaum, A. I. (1995). Morphological characterization of substance P receptor-immunoreactive neurons in the rat spinal cord and trigeminal nucleus caudalis. The Journal of comparative neurology, 356(3), 327-44.
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  Although there is considerable evidence that primary afferent-derived substance P contributes to the transmission of nociceptive messages at the spinal cord level, the population of neurons that expresses the substance P receptor, and thus are likely to respond to substance P, has not been completely characterized. To address this question, we used an antibody directed against the C-terminal portion of the rat substance P receptor to examine the cellular distribution of the receptor in spinal cord neurons. In a previous study, we reported that the substance P receptor decorates almost the entire dendritic and somatic surface of a subpopulation of spinal cord neurons. In the present study we have taken advantage of this labeling pattern to identify morphologically distinct subpopulations of substance P receptor-immunoreactive neurons throughout the rostral-caudal extent of the spinal cord. We observed a dense population of fusiform substance P receptor-immunoreactive neurons in lamina I at all segmental levels. Despite having the highest concentration of substance P terminals, the substantia gelatinosa (lamina II) contained almost no substance P receptor-immunoreactive neurons. Several distinct populations of substance P receptor-immunoreactive neurons were located in laminae III-V; many of these had a large, dorsally directed dendritic arbor that traversed the substantia gelatinosa to reach the marginal layer. Extensive labeling was also found in neurons of the intermediolateral cell column. In the ventral horn, we found that labeling was associated with clusters of motoneurons, notably those in Onuf's nucleus in the sacral spinal cord. Finally, we found no evidence that primary afferent fibers express the substance P receptor. These results indicate that relatively few, but morphologically distinct, subclasses of spinal cord neurons express the substance P receptor. The majority, but not all, of these neurons are located in regions that contain neurons that respond to noxious stimulation.
• Cleary, J., Hittner, J. M., Semotuk, M., Mantyh, P., & O'Hare, E. (1995). Beta-amyloid(1-40) effects on behavior and memory. Brain research, 682(1-2), 69-74.
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  Beta amyloid 1-40 is a primary protein in plaques found in the brains of patients with Alzheimer's disease. There is evidence that unaggregated soluble beta-amyloid may be neurotoxic and may have behavioral effects on some types of memory. In the current study, the 1-40 fragment of beta-amyloid protein (beta A4), or vehicle, was bilaterally injected into the rostral hippocampus of rats performing under stable food-maintained schedules of reinforcement or under a delayed conditional discrimination procedure. Under the first procedure, rats were trained to stability under a multiple fixed interval 15 s, fixed ratio 30 reinforcement schedule. This reinforcement schedule has proven sensitive to low-dose drug effects. Acute bilateral hippocampal beta A4 (1.0, 2.0 and 3.0 microliters of 10(-3) M) administration did not significantly alter responding, compared to vehicle, under either reinforcement condition. Following the acute single-injection regimen, rats were administered chronic daily beta A4 (1 microliter of 10(-3) M), bilaterally, for 15 days. No significant changes in lever-pressing performance were observed during the chronic injection regimen, but performance declined significantly 30 days after termination of the chronic daily regimen. Histological examination revealed three of six rats showed positive reactions under Thioflavin S staining in and around the area of cannulae termination. The second assessment employed a delayed conditional discrimination procedure to evaluate the effects of intrahippocampal injections of beta A4 on short-term working memory. This conditional discrimination procedure assesses appropriate responding, dependent on a previously presented stimulus, after delays of various lengths have been imposed between the stimulus and the response opportunity.(ABSTRACT TRUNCATED AT 250 WORDS)
• Lee, J. P., Stimson, E. R., Ghilardi, J. R., Mantyh, P. W., Lu, Y. A., Felix, A. M., Llanos, W., Behbin, A., Cummings, M., & Van Criekinge, M. (1995). 1H NMR of A beta amyloid peptide congeners in water solution. Conformational changes correlate with plaque competence. Biochemistry, 34(15), 5191-200.
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  To begin to examine the structural basis for the deposition of soluble A beta amyloid peptide onto senile plaques in Alzheimer's disease, we have prepared A beta congeners and measured their activity in an in vitro plaque growth assay. The N-terminal fragment, A beta (1-28)-OH, was inactive at all pH values tested. While the central fragment, A beta (10-35)-NH2, and the full length peptide, A beta (1-40)-OH, were inactive below pH 4, both were active (plaque competent) between pH 5 and 9. The active and inactive fragments were studied by nuclear magnetic resonance spectroscopy in water at submillimolar concentrations at pH 2.1 and 5.6. Changes in chemical shifts, coupling constants, and nuclear Overhauser enhancements indicate a pH dependent folding transition in A beta (10-35)-NH2 as it becomes active. The conformation of the active fragment is not helical, and preliminary data indicate the presence of several turns and at least two short strands. In contrast, the inactive fragment A beta (1-28)-OH did not undergo a similar folding transition. Earlier nuclear magnetic resonance studies of amyloid peptides in fluorinated alcohols or detergent micelles at low pH described a helical conformation and proposed a helix to sheet transition in plaque formation; the present study demonstrates that no such conformations are present in water under conditions where the peptides can adhere to authentic amyloid plaques.
• Maggio, J. E., Esler, W. P., Stimson, E. R., Jennings, J. M., Ghilardi, J. R., & Mantyh, P. W. (1995). Zinc and Alzheimer's disease. Science (New York, N.Y.), 268(5219), 1920-1; author reply 1921-3.
• Mantyh, C. R., Vigna, S. R., Bollinger, R. R., Mantyh, P. W., Maggio, J. E., & Pappas, T. N. (1995). Differential expression of substance P receptors in patients with Crohn's disease and ulcerative colitis. Gastroenterology, 109(3), 850-60.
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  Although clinical and pathological differences exist between Crohn's disease (CD) and ulcerative colitis (UC), distinguishing features are often absent, making diagnosis and treatment problematic. This study evaluated the differences in the expression of substance P (SP) receptors in patients with CD or UC.
• Mantyh, P. W., Allen, C. J., Ghilardi, J. R., Rogers, S. D., Mantyh, C. R., Liu, H., Basbaum, A. I., Vigna, S. R., & Maggio, J. E. (1995). Rapid endocytosis of a G protein-coupled receptor: substance P evoked internalization of its receptor in the rat striatum in vivo. Proceedings of the National Academy of Sciences of the United States of America, 92(7), 2622-6.
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  Studies on cultured cells have shown that agonists induce several types of G protein-coupled receptors to undergo internalization. We have investigated this phenomenon in rat striatum, using substance P (SP)-induced internalization of the SP receptor (SPR) as our model system. Within 1 min of a unilateral striatal injection of SP in the anesthetized rat, nearly 60% of the SPR-immunoreactive neurons within the injection zone display massive internalization of the SPR--i.e., 20-200 SPR+ endosomes per cell body. Within the dendrites the SPR undergoes a striking translocation from the plasma membrane to endosomes, and these dendrites also undergo a morphological reorganization, changing from a structure of rather uniform diameter to one characterized by large, swollen varicosities connected by thin fibers. In both cell bodies and dendrites the number of SPR+ endosomes returns to baseline within 60 min of SP injection. The number of neurons displaying substantial endosomal SPR internalization is dependent on the concentration of injected SP, and the SP-induced SPR internalization is inhibited by the nonpeptide neurokinin 1 receptor antagonist RP-67,580. These data demonstrate that in the central nervous system in vivo, SP induces a rapid and widespread SPR internalization in the cell bodies and dendrites and a structural reorganization of the dendrites. These results suggest that many of the observations that have been made on the internalization and recycling of G protein-coupled receptors in in vitro transfected cell systems are applicable to similar events that occur in the mammalian central nervous system in vivo.
• Mantyh, P. W., DeMaster, E., Malhotra, A., Ghilardi, J. R., Rogers, S. D., Mantyh, C. R., Liu, H., Basbaum, A. I., Vigna, S. R., & Maggio, J. E. (1995). Receptor endocytosis and dendrite reshaping in spinal neurons after somatosensory stimulation. Science (New York, N.Y.), 268(5217), 1629-32.
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  In vivo somatosensory stimuli evoked the release of substance P from primary afferent neurons that terminate in the spinal cord and stimulated endocytosis of substance P receptors in rat spinal cord neurons. The distal dendrites that showed substance P receptor internalization underwent morphological reorganization, changing from a tubular structure to one characterized by swollen varicosities connected by thin segments. This internalization and dendritic structural reorganization provided a specific image of neurons activated by substance P. Thus receptor internalization can drive reversible structural changes in central nervous system neurons in vivo. Both of these processes may be involved in neuronal plasticity.
• Mantyh, P. W., Rogers, S. D., Allen, C. J., Catton, M. D., Ghilardi, J. R., Levin, L. A., Maggio, J. E., & Vigna, S. R. (1995). Beta 2-adrenergic receptors are expressed by glia in vivo in the normal and injured central nervous system in the rat, rabbit, and human. The Journal of neuroscience : the official journal of the Society for Neuroscience, 15(1 Pt 1), 152-64.
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  Previous studies have demonstrated that glial cells in culture express several subtypes of functional adrenergic receptors. To determine if similar receptors are expressed by glia in vivo, we examined the expression of adrenergic receptors in the normal, crushed, and transected optic nerves of the rabbit and rat using quantitative receptor autoradiography. Additionally, we examined the expression of adrenergic receptors in the normal and damaged human optic nerve. High levels of alpha 1-, alpha 2-, beta 1-, and beta 2-adrenergic receptors were identified in the rabbit and rat forebrain. In the normal rabbit, rat, and human optic nerves, only alpha 1 and beta 2 receptors were observed, and these were present in low to moderate densities. Combined immunohistochemistry and autoradiography suggests that the majority of beta 2-adrenergic receptors in the rabbit, rat, and human optic nerve are expressed by astrocytes. After unilateral optic nerve crush or transection, only beta 2- adrenergic receptors were significantly increased. This increase in beta 2 receptors was first detectable at days 7 and 28 post-transection in the rabbit and rat, respectively. The expression of beta 2 receptors in the transected optic nerve continued to increase with time, so that by 90 d post-transection the density of beta 2 receptors in both the rabbit and rat optic nerve was among the highest of any area in the forebrain. Taken together with previous studies, these results suggest that in vivo, beta 2-adrenergic receptors may provide a therapeutic target for regulation of astrocyte functions including glycogen metabolism, cytokine release, and the hypertrophy and proliferation that occurs in response to neuronal injury.
• Takatsuji, K., Senba, E., Mantyh, P. W., & Tohyama, M. (1995). A relationship between substance P receptor and retinal fibers in the rat suprachiasmatic nucleus. Brain research, 698(1-2), 53-61.
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  The suprachiasmatic nucleus (SCN) in the hypothalamus controls many of the circadian rhythms in mammalian species. In the present study, we investigated the location of substance P receptor (SPR)-containing neurons in the rat SCN, using a specific antibody against SPR, which corresponds to the NK-1 subtype of tachykinin receptors, and also examined the synaptic relationship between SPR-containing neurons and retinal fibers at the ultrastructural level. An SPR-immunoreactive meshwork of labeled somata and dendrites was identified in the SCN. The strongest SPR-immunoreactivity was observed in the dorsal and lateral parts of the SCN. Many labeled somata were identified there and their dendrites protruded ventrally from their somata. A few SPR-immunoreactive somata were observed also in the ventral part of the SCN and within the optic tract. In the SCN of eye-enucleated animals, degenerating retinal fibers were shown to terminate on SPR-immunoreactive dendrites forming asymmetrical axo-dendritic contacts.
• Ghilardi, J. R., Allen, C. J., Vigna, S. R., McVey, D. C., & Mantyh, P. W. (1994). Cholecystokinin and neuropeptide Y receptors on single rabbit vagal afferent ganglion neurons: site of prejunctional modulation of visceral sensory neurons. Brain research, 633(1-2), 33-40.
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  A [125I]cholecystokinin (CCK) analog and [125I]peptide YY (PYY) were used to localize and characterize CCK and neuropeptide Y (NPY) receptor binding sites in the rabbit vagal afferent (nodose) ganglion. High concentrations of CCK and NPY binding sites were observed in 10.6% and 9.2% of the nodose ganglion neurons, respectively. Pharmacological experiments using CCK or NPY analogs suggest that both subtypes of CCK (CCK-A and CCK-B) and NPY (Y1 and Y2) receptor binding sites are expressed by discrete populations of neurons in the nodose ganglion. These results suggest sites at which CCK or NPY, released in either the nucleus of the solitary tract or a peripheral tissue, may modulate the release of neurotransmitters from a select population of visceral primary afferent neurons. Possible functions mediated by these receptors include modulation of satiety, opiate analgesia, and the development of morphine tolerance.
• Liu, H., Brown, J. L., Jasmin, L., Maggio, J. E., Vigna, S. R., Mantyh, P. W., & Basbaum, A. I. (1994). Synaptic relationship between substance P and the substance P receptor: light and electron microscopic characterization of the mismatch between neuropeptides and their receptors. Proceedings of the National Academy of Sciences of the United States of America, 91(3), 1009-13.
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  Light microscopic studies have demonstrated significant mismatches in the location of neuropeptides and their respective binding sites in the central nervous system. In the present study we used an antiserum raised against a synthetic peptide corresponding to the carboxyl-terminal tail of the substance P (SP) receptor (SPR) to further explore the relationship between a neuropeptide and its receptor. Light microscopy revealed an excellent correlation between the patterns of SPR immunoreactivity and of 125I-labeled SPR-binding sites in the central nervous system. The SPR appeared to be exclusively expressed by neurons; in fact, the SPR decorates the somatic and dendritic surface of neurons, producing Golgi-like images. Electron microscopic analysis in cortex, striatum, and spinal cord revealed that approximately 70% of the surface membrane of immunoreactive neurons is SPR laden. Simultaneous electron microscopic labeling of SP and SPR demonstrated significant mismatch at the synaptic level. Although some SP terminals contacted SPR-immunoreactive membrane, no more than 15% of the SPR-laden membrane apposed synaptic terminals. These results suggest that in contrast to more "classical" central and peripheral nervous system synapses, wherein the receptor immediately apposes the site of neurotransmitter storage and release, much of the surface of SPR-expressing neurons can be targeted by SP that diffuses a considerable distance from its site of release.
• Mantyh, C. R., Vigna, S. R., Maggio, J. E., Mantyh, P. W., Bollinger, R. R., & Pappas, T. N. (1994). Substance P binding sites on intestinal lymphoid aggregates and blood vessels in inflammatory bowel disease correspond to authentic NK-1 receptors. Neuroscience letters, 178(2), 255-9.
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  Previous reports have described the ectopic expression of substance P binding sites on lymphoid aggregates and small blood vessels in inflammatory bowel disease. In this report, three non-peptide NK-1 receptor antagonists, CP-96,345, RP-67,580, and L-703,606 abolished saturable 125I-Bolton-Hunter substance P binding to the ectopically expressed receptors in frozen sections of surgically resected bowel from five patients with either Crohn's disease or ulcerative colitis. The rank order of affinity was approximately substance P approximately CP-96,345 approximately L-703,606 > RP-67,580. These results suggest that: (i) the ectopically expressed substance P binding sites in inflammatory bowel disease are authentic NK-1 receptors, (ii) all ectopically expressed receptors on small blood vessels, and lymphoid aggregates as well as normally expressed receptors on the bowel circular muscle have similar receptor affinities and specificities for substance P and the non-peptide antagonists, and (iii) non-peptide antagonists may be therapeutically beneficial in inflammatory bowel disease by inhibiting the pro-inflammatory effects of substance P acting via the NK-1 receptor.
• Mantyh, P. W., Allen, C. J., Rogers, S., DeMaster, E., Ghilardi, J. R., Mosconi, T., Kruger, L., Mannon, P. J., Taylor, I. L., & Vigna, S. R. (1994). Some sensory neurons express neuropeptide Y receptors: potential paracrine inhibition of primary afferent nociceptors following peripheral nerve injury. The Journal of neuroscience : the official journal of the Society for Neuroscience, 14(6), 3958-68.
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  Neuropeptide Y (NPY) has been suggested to exert antinociceptive actions by inhibiting the release of neurotransmitters from trigeminal and dorsal root ganglia (DRG) neurons, but the site of direct NPY action in vivo and the NPY receptor subtype mediating these effects are unknown. 125I-peptide YY (PYY) was used to localize and characterize NPY receptor binding sites in trigeminal ganglia, DRG, and spinal cord of the rat, rabbit, and monkey. In the rat, rabbit, and monkey, 5-20% of trigeminal ganglia and DRG neurons express NPY binding sites. Unilateral cuff-induced neuropathy or transection of the rat sciatic nerve did not significantly alter the density or number of DRG neurons expressing NPY receptors. A unimodal size distribution for L4 and L5 DRG neurons expressing NPY binding sites in the rat was determined, with a mean cross-sectional area of 947 microns 2. In the spinal cord the highest concentration of NPY receptors is found in laminae I, II, V, X, and Onuf's nucleus. Pharmacological experiments using selective Y1 and Y2 receptor antagonists suggest that Y2 is the prominent NPY receptor subtype expressed in trigeminal ganglia neurons, DRG neurons, and spinal cord. Previous studies have demonstrated that a population of large-diameter, presumably myelinated primary afferents express NPY after peripheral nerve injury. NPY released from these injured large-diameter DRG neurons may act in a paracrine fashion to block the transmission of nociceptive information from the small- and medium-diameter DRG neurons that constitutively express NPY receptors. NPY receptors are therefore uniquely positioned to inhibit primary afferent nociceptors directly, especially after peripheral nerve injury.
• Allen, C. J., Ghilardi, J. R., Vigna, S. R., Mannon, P. J., Taylor, I. L., McVey, D. C., Maggio, J. E., & Mantyh, P. W. (1993). Neuropeptide Y/peptide YY receptor binding sites in the heart: localization and pharmacological characterization. Neuroscience, 53(3), 889-98.
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  [125I]Peptide YY was used to localize and characterize peptide YY and neuropeptide Y receptor binding sites in the heart. In the rat and rabbit heart, nearly every artery and arteriole that could be histologically identified also expressed saturable binding sites for [125I]peptide YY. In the arteries, these [125I]peptide YY binding sites were primarily associated with the smooth muscle layer. Pharmacological experiments demonstrated that peptide YY and neuropeptide Y were equipotent in competing for [125I]peptide YY binding in the heart. In another competition series, [Leu31,Pro34]-neuropeptide Y (a Y1 receptor-specific agonist when used with [125I]peptide YY) was significantly more potent than neuropeptide Y (a Y2 receptor-specific agonist when used with [125I]peptide YY) in competing for [125I]peptide YY binding from coronary arteries, suggesting that the receptor binding sites on cardiac arteries and arterioles are of the Y1 subtype. These results demonstrate that smooth muscle cells of the atrial and ventricular arteries and arterioles in rat and rabbit heart express Y1 receptors and suggest a possible direct effect of neuropeptide Y on coronary blood vessels to induce vasoconstriction.
• Greeno, E. W., Mantyh, P., Vercellotti, G. M., & Moldow, C. F. (1993). Functional neurokinin 1 receptors for substance P are expressed by human vascular endothelium. The Journal of experimental medicine, 177(5), 1269-76.
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  Substance P (SP), a neurotachykinin, is important in a number of inflammatory processes in which the endothelial cell also plays a critical role. SP receptors have previously been identified only on arterial endothelium, and the scant in vitro evidence for direct effects of SP on human endothelium is based on studies using nonarterial cells. To better understand SP's role in inflammation, we sought to identify functional SP receptors on human endothelium in situ and in culture. Autoradiographic ligand binding to human umbilical cord sections demonstrates the presence of SP binding sites with characteristics of the neurokinin 1 (NK-1) receptor (displacement by GTP analogues and the NK-1 specific antagonist CP-96,345) on human umbilical arterial, but not venous, endothelium. In culture, human umbilical venous endothelial cells (HUVECs) and human aortic endothelial cells express low levels of available SP binding sites. However, HUVECs, which are serum starved and refed, undergo a dramatic increase in SP binding. SP binding to starved/refed HUVECs induces a transient increase in intracellular calcium. This calcium flux is dose dependent over appropriate SP concentrations and can be blocked by NK-1 specific antagonists. The proinflammatory effects of SP may be mediated in part through the NK-1 receptor on endothelium.
• Kilgore, W. R., Mantyh, P. W., Mantyh, C. R., McVey, D. C., & Vigna, S. R. (1993). Bombesin/GRP-preferring and neuromedin B-preferring receptors in the rat urogenital system. Neuropeptides, 24(1), 43-52.
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  Bombesin binding sites were localized in the rat urogenital system by autoradiography of 125I-Tyr4-bombesin binding to frozen tissue sections. Saturable binding was observed in the bladder, seminal vesicle, uterus, and oviduct. In all organs, the binding sites corresponded to layers of smooth muscle. Radioligand binding studies were performed on homogenized membrane preparations from bladder, uterus, and seminal vesicle. Membrane binding was saturable, reversible, time- and temperature-dependent, and specific for bombesin and related peptides. Analysis of saturable equilibrium binding from all three organs yielded a best fit to a one-site model of high affinity binding with apparent KdS of 720 pM for bladder, 470 pM for uterus, and 700 pM for seminal vesicle. Neuromedin B was potent in displacing saturable 125I-Tyr4-bombesin binding from bladder and seminal vesicle but not uterus membranes. In order to characterize these binding sites further, the ability of these membranes to interact with a specific bombesin receptor antagonist, [Leu13-psi-CH2NH-Leu14]-bombesin, and with GTP-gamma-S was determined. [Leu13-psi-CH2NH-Leu14]-bombesin was much more potent in displacing saturable 125I-Tyr4-bombesin binding from uterus than from bladder and seminal vesicle membranes, further supporting the distinction between the uterus and the bladder/seminal vesicle binding sites as bombesin receptor subtypes. GTP-gamma-S inhibited saturable 125I-Tyr4-bombesin binding to membranes from all three organs, indicating that both receptor subtypes are linked to GTP-binding proteins. We conclude that smooth muscle in the rat urogenital system expresses bombesin receptors and that endogenous GRP and neuromedin B may regulate some reproductive and excretory functions. The bladder and seminal vesicle express the neuromedin B-preferring subtype and the uterus expresses the bombesin/GRP-preferring subtype of bombesin receptor.
• Mantyh, P. W., Ghilardi, J. R., Rogers, S., DeMaster, E., Allen, C. J., Stimson, E. R., & Maggio, J. E. (1993). Aluminum, iron, and zinc ions promote aggregation of physiological concentrations of beta-amyloid peptide. Journal of neurochemistry, 61(3), 1171-4.
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  A major pathological feature of Alzheimer's disease (AD) is the presence of a high density of amyloid plaques in the brain tissue of patients. The plaques are predominantly composed of human beta-amyloid peptide beta A4, a 40-mer whose neurotoxicity is related to its aggregation. Certain metals have been proposed as risk factors for AD, but the mechanism by which the metals may exert their effects is unclear. Radioiodinated human beta A4 has been used to assess the effects of various metals on the aggregation of the peptide in dilute solution (10(-10) M). In physiological buffers, 10(-3) M calcium, cobalt, copper, manganese, magnesium, sodium, or potassium had no effect on the rate of beta A4 aggregation. In sharp contrast, aluminum, iron, and zinc under the same conditions strongly promoted aggregation (rate enhancement of 100-1,000-fold). The aggregation of beta A4 induced by aluminum and iron is distinguishable from that induced by zinc in terms of rate, extent, pH and temperature dependence. These results suggest that high concentrations of certain metals may play a role in the pathogenesis of AD by promoting aggregation of beta A4.
• Ghilardi, J. R., Allen, C. J., Vigna, S. R., McVey, D. C., & Mantyh, P. W. (1992). Trigeminal and dorsal root ganglion neurons express CCK receptor binding sites in the rat, rabbit, and monkey: possible site of opiate-CCK analgesic interactions. The Journal of neuroscience : the official journal of the Society for Neuroscience, 12(12), 4854-66.
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  125I-Bolton-Hunter sulfated cholecystokinin-8 was used to localize and characterize cholecystokinin (CCK) receptor binding sites in trigeminal and dorsal root ganglia, and in the spinal cord of the rat, rabbit, and monkey. In the rabbit and monkey, a substantial number, 90 +/- 21% and 24 +/- 8%, respectively, of trigeminal and dorsal root ganglion neurons express CCK binding sites. In the spinal cord, the highest concentration of CCK receptors is found in laminae I and II, which is the major termination site of dorsal root ganglia neurons expressing CCK receptor binding sites. Neonatal capsaicin treatment of the rat results in a 70% decline in CCK receptor binding sites in laminae I and II of the spinal cord, indicating that dorsal root ganglia neurons are a major source of CCK receptors in the spinal cord. Pharmacological experiments using selective CCK-A and CCK-B receptor antagonists demonstrate that CCK-B is the prominent CCK receptor subtype in trigeminal and dorsal root ganglia neurons in the rat, rabbit, and monkey. In the rat and rabbit spinal cord, CCK-B binding sites are the prominent subtype, whereas in the monkey cord, CCK-A is the prominent receptor subtype. These results demonstrate that CCK-B receptors are expressed by a substantial percentage of dorsal root ganglion neurons at all spinal levels, and that CCK may antagonize opiate analgesia at the level of the primary afferent neuron itself.
• Maggio, J. E., Stimson, E. R., Ghilardi, J. R., Allen, C. J., Dahl, C. E., Whitcomb, D. C., Vigna, S. R., Vinters, H. V., Labenski, M. E., & Mantyh, P. W. (1992). Reversible in vitro growth of Alzheimer disease beta-amyloid plaques by deposition of labeled amyloid peptide. Proceedings of the National Academy of Sciences of the United States of America, 89(12), 5462-6.
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  The salient pathological feature of Alzheimer disease (AD) is the presence of a high density of amyloid plaques in the brain tissue of victims. The plaques are predominantly composed of human beta-amyloid peptide (beta A4), a 40-mer whose neurotoxicity is related to its aggregation. Radioiodinated human beta A4 is rapidly deposited in vitro from a dilute (less than 10 pM) solution onto neuritic and diffuse plaques and cerebrovascular amyloid in AD brain tissue, whereas no deposition is detectable in tissue without performed plaques. This growth of plaques by deposition of radiolabeled beta A4 to plaques is reversible, with a dissociation half-time of approximately 1 h. The fraction of grey matter occupied by plaques that bind radiolabeled beta A4 in vitro is dramatically larger in AD cortex (23 +/- 11%) than in age-matched normal controls (less than 2%). In contrast to the human peptide, rat/mouse beta A4 (differing at three positions from human beta A4) does not affect the deposition of radiolabeled human beta A4. beta A4 has no detectable interaction with tachykinin receptors in rat or human brain. The use of radioiodinated beta A4 provides an in vitro system for the quantitative evaluation of agents or conditions that may inhibit or enhance the growth or dissolution of AD plaques. This reagent also provides an extremely sensitive method for visualizing various types of amyloid deposits and a means for characterizing and locating sites of amyloid peptide binding to cells and tissues.
• Mantyh, P. W., Catton, M. D., Allen, C. J., Labenski, M. E., Maggio, J. E., & Vigna, S. R. (1992). Receptor binding sites for cholecystokinin, galanin, somatostatin, substance P and vasoactive intestinal polypeptide in sympathetic ganglia. Neuroscience, 46(3), 739-54.
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  Sympathetic ganglia are innervated by neuropeptide-containing fibers originating from pre- and postganglionic sympathetic neurons, dorsal root ganglion neurons, and in some cases, myenteric neurons. In the present report receptor autoradiography was used to determine whether sympathetic ganglia express receptor binding sites for several of these neuropeptides including bombesin, calcitonin gene-related peptide-alpha, cholecystokinin, galanin, neurokinin A, somatostatin, substance P, and vasoactive intestinal polypeptide. The sympathetic ganglia examined included the rat and rabbit superior cervical ganglia and the rabbit superior mesenteric ganglion. High levels of receptor binding sites for cholecystokinin, galanin, somatostatin, substance P, and vasoactive intestinal polypeptide were observed in all sympathetic ganglia examined, although only discrete neuronal populations within each ganglion appeared to express receptor binding sites for any particular neuropeptide. These data suggest that discrete populations of postganglionic sympathetic neurons may be regulated by neuropeptides released from pre- and postganglionic sympathetic neurons, dorsal root ganglion neurons, and myenteric neurons.
• Mantyh, P. W. (1991). Substance P and the inflammatory and immune response. Annals of the New York Academy of Sciences, 632, 263-71.
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  These findings suggest that SP may have proinflammatory actions in both the peripheral tissue and the central nervous system after tissue injury. Although the possibility that the same neuropeptide could have actions in both the brain and the peripheral tissues is certainly not without precedent, there is a key difference in the source of the ligand in these tissues. Unlike peripheral tissues such as the gastrointestinal tract or skin, where there is a dense innervation by SP-containing dorsal root ganglion neurons, the brain lacks such a sensory innervation. This important difference raises the question as to the possible origin of the SP that could occupy the SP receptors expressed by the CNS glia after neuronal injury. Whereas the answer to this question is currently unknown, an important clue may be the findings that circulating leukocytes have been reported to synthesize neuropeptides such as ACTH, opiates, and SP. To begin to fully understand the role that SP may play in coordinating the inflammatory and immune response to tissue injury, we must first understand where SP fits into the cascade of events that occur after tissue injury, what events lead to nociceptor sensitization (which may lead to an increase in SP release), and what regulates SP receptor expression (which may be involved in the direction of leukocytes to the site of injury, plasma extravasation, or the proliferation/hypertrophy of reactive astrocytes). Although this may seem like a daunting task, several recent advances including the cloning of the three mammalian tachykinin receptors and the introduction of highly potent and specific SP receptor antagonists should make this a highly fruitful field of investigation.
• Mantyh, P. W., Catton, M., Maggio, J. E., & Vigna, S. R. (1991). Alterations in receptors for sensory neuropeptides in human inflammatory bowel disease. Advances in experimental medicine and biology, 298, 253-83.
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  Glutamate and several neuropeptides are synthesized and released by subpopulations of primary afferent neurons. These sensory neurons play a role in regulating the inflammatory and immune responses in peripheral tissues. We have explored what changes occur in the location and concentration of receptor binding sites for sensory neurotransmitters in two human inflammatory diseases, ulcerative colitis and Crohn's disease, using quantitative receptor autoradiography. The sensory neurotransmitter receptors included bombesin, calcitonin gene-related peptide-alpha, cholecystokinin, galanin, glutamate, somatostatin, neurokinin A (substance K), substance P, and vasoactive intestinal polypeptide. Of the nine receptor binding sites examined only binding sites for substance P and vasoactive intestinal peptide were significantly altered in the inflamed tissue. These data suggest that substance P is involved in regulating the inflammatory and immune responses in human inflammatory diseases and indicate a specificity of efferent action for each sensory neurotransmitter in peripheral tissues.
• Helke, C. J., Krause, J. E., Mantyh, P. W., Couture, R., & Bannon, M. J. (1990). Diversity in mammalian tachykinin peptidergic neurons: multiple peptides, receptors, and regulatory mechanisms. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 4(6), 1606-15.
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  The tachykinins comprise a family of closely related peptides that participate in the regulation of diverse biological processes. The tachykinin peptides substance P, neurokinin A, neurokinin A(3-10), neuropeptide K, and neuropeptide gamma are produced from a single preprotachykinin gene as a result of differential RNA splicing and differential posttranslational processing. Another tachykinin, neurokinin B, is produced from a separate preprotachykinin gene. These preprotachykinin mRNAs and peptide products are differentially distributed throughout the nervous system. Three distinct G protein-coupled tachykinin receptors exist for these tachykinin peptides. The three receptors interact differentially with the tachykinin peptides and are uniquely distributed throughout the nervous system. The NK-1 receptor preferentially interacts with substance P, the NK-2 receptor prefers neurokinin A, neuropeptide K, and neuropeptide gamma, and the NK-3 receptor interacts best with neurokinin B. Examples of the roles of tachykinin peptidergic neuronal systems are taken from the spinal cord sensory system and the nigrostriatal extrapyramidal motor system. Analysis of the functional significance of multiple tachykinin peptide systems, receptor-second messenger coupling mechanisms, and developmental and regulatory mechanisms underlying peptide mRNA and receptor expression represent areas of current and future investigation.
• Vigna, S. R., Giraud, A. S., Mantyh, P. W., Soll, A. H., & Walsh, J. H. (1990). Characterization of bombesin receptors on canine antral gastrin cells. Peptides, 11(2), 259-64.
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  Dispersed canine antral mucosal cells were prepared by sequential steps of collagenase digestion and EDTA treatment. Cell preparations enriched in gastrin cells were made by centrifugal elutriation followed by step density gradient centrifugation. Specific, saturable, and reversible binding of 125I-[Tyr4]-bombesin was found in all preparations. This saturable binding was time, temperature, and cell number dependent. In both velocity (elutriator) and density cell separation experiments, saturable binding of bombesin correlated with the distribution of cells containing gastrin- but not somatostatin-like immunoreactivity. Maximal specific binding to gastrin (G) cell-enriched fractions was reached in 45 min at 37 degrees C and constituted 90% of total binding. Addition of 100 nM nonradioactive bombesin to cells incubated with 50 pM 125I-[Tyr4]-bombesin for 45 min resulted in time-dependent dissociation of specifically bound tracer to about 40% of the maximal equilibrium binding. Analysis of saturable equilibrium binding yielded a best fit to a one-site model of high affinity binding sites with an apparent Kd of 85 +/- 14 pM and a Bmax of 231,000 +/- 71,000 receptors/gastrin cell. Nonradioactive [Tyr4]-bombesin and related analogs inhibited the specific binding of the tracer in a dose-related manner. The rank order of potency, determined at the IC50, of [Tyr4]-bombesin and related analogs for inhibition of specific binding was bombesin greater than [Tyr4]-bombesin = hGRP-27 greater than GRP-10 greater than ranatensin much greater than neuromedin B. Cholecystokinin, somatostatin, substance K, and kassinin each tested at a concentration of 1 microM did not inhibit bombesin binding.(ABSTRACT TRUNCATED AT 250 WORDS)
• Bacay, A. C., Mantyh, C. R., Cohen, A. H., Mantyh, P. W., & Fine, L. G. (1989). Glomerular atrial natriuretic factor receptors in primary glomerulopathies: studies on human renal biopsies. American journal of kidney diseases : the official journal of the National Kidney Foundation, 14(5), 386-95.
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  Human renal biopsies are currently used to provide information about morphologic changes, chronicity of disease, patterns of inflammation, and immunoglobulin deposition. This practice has provided only limited insight into functional aberrations and has failed to provided information necessary for disease classification based on pathophysiology. To expand the potential of the renal biopsy in this regard and to determine whether differences in glomerular atrial natriuretic factor (ANF) binding exist in different forms of primary renal disease, quantitative autoradiography and 125I-human ANF (1-28) were used to determine the location and pharmacological characteristics of ANF binding sites in the normal human kidney. Specific ANF binding was highest in the glomeruli, but lower levels of specific binding were localized to the inner medulla and the interlobular arteries. ANF binding sites in the human kidney were found to be highly stable and similar in both location and pharmacology to those observed in experimental animals. As determined by saturation experiments, the equilibrium dissociation constants for glomeruli, inner medulla, and interlobular arteries were almost identical at 4.0 x 10(-11) mol/L. Competitive binding inhibition studies with unlabeled human ANF (1-28) demonstrated highly specific binding shared by the glomerulus, inner medulla, and interlobular artery, with apparent half-maximal inhibition concentrations of 9.2 x 10(-10) mol/L, 8.0 x -10 mol/L, and 8.2 x 10(-10) mol/L, respectively. Quantitation of specific binding of ANF to glomeruli in needle biopsy specimens of three primary glomerulopathies, ie, minimal-change disease, membranous nephropathy, and focal glomerulosclerosis, showed no differences among the groups. This study demonstrates the feasibility of studying receptor physiology on biopsy specimens of the human kidney and should allow renal diseases, particularly of glomerular origin, to be characterized according to differences in hormone binding and hormone responsiveness. The absence of significant differences in glomerular ANF binding in the primary glomerulopathies studied is consistent with other studies that have failed to delineate important pathophysiological differences in renal function and volume homeostasis in these disease states.
• Boehmer, C. G., Norman, J., Catton, M., Fine, L. G., & Mantyh, P. W. (1989). High levels of mRNA coding for substance P, somatostatin and alpha-tubulin are expressed by rat and rabbit dorsal root ganglia neurons. Peptides, 10(6), 1179-94.
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  Oligonucleotide probes complementary to alpha-tubulin, preprotachykinin A (PPT A), preprosomatostatin (PPSOM), and preproarginine-vasopressin (PPAVP) mRNA were hybridized to sections of rat and rabbit brain and dorsal root ganglia (DRG) at all spinal levels. Approximately 100% of the DRG neurons in the rat and rabbit express alpha-tubulin mRNA, 20-30% express PPT A mRNA and 5-17% express PPSOM mRNA. Whereas neurons which express PPSOM mRNA are of relative uniform size, the neurons which express PPT A mRNA segregate into two broad groups. One group is composed of smaller neurons (200-2,000 microns 2) which contain an extremely dense concentration of PPT A mRNA. The second group is composed of larger neurons (2,000-3,500 microns 2) which contain a moderate concentration of PPT A mRNA. PPAVP mRNA is present in very high concentrations in the paraventricular and supraoptic nucleus of the rat hypothalamus but is not detected in any DRG neurons. In both the rat and the rabbit the density of PPT A and PPSOM mRNA is high in individual DRG neurons in comparison to PPT A and PPSOM mRNA levels contained in most forebrain neurons. These results suggest that although the level of neuropeptide present in DRG neurons is relatively low in comparison to other brain areas, the rate of sensory neuropeptide synthesis and turnover, as reflected by mRNA content, is extremely high.
• Gates, T. S., Zimmerman, R. P., Mantyh, C. R., Vigna, S. R., & Mantyh, P. W. (1989). Calcitonin gene-related peptide-alpha receptor binding sites in the gastrointestinal tract. Neuroscience, 31(3), 757-70.
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  Calcitonin gene-related peptide-alpha (CGRP alpha) is a putative neurotransmitter in the brain and in peripheral tissues. Quantitative receptor autoradiography was used to localize and quantify the distribution of specific binding sites for radiolabeled human CGRP alpha in the canine gastrointestinal tract. The canine gastrointestinal tract was chosen as a model since it is similar in both size and structure to the human gastrointestinal tract. In the stomach CGRP alpha binding sites were localized to smooth muscle cells in the muscularis mucosa and muscularis externa, the smooth muscle and endothelium of medium and small arteries, neurons in the myenteric plexus, mucosal epithelial cells and the germinal centers of lymph nodules. In the intestines, the prominent cells types expressing CGRP alpha receptors were myenteric neurons and the germinal centers of lymph nodules. Since previous studies have demonstrated that CGRP-containing sensory neurons innervate the muscularis externa in the stomach and since CGRP alpha receptors are expressed by smooth muscle cells in the muscularis externa, these results suggest that sensory neurons may directly regulate gastric motility by releasing CGRP. In correlation with previous physiological data, the present study suggests that CGRP is involved in the regulation of a variety of gastrointestinal functions including gastric motility, mucosal ion transport, hemodynamics, digestive enzyme secretion, neuronal excitability, and the inflammatory and immune response.
• Kruger, L., Silverman, J. D., Mantyh, P. W., Sternini, C., & Brecha, N. C. (1989). Peripheral patterns of calcitonin-gene-related peptide general somatic sensory innervation: cutaneous and deep terminations. The Journal of comparative neurology, 280(2), 291-302.
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  The distribution of calcitonin-gene-related peptide (CGRP) immunoreactivity (IR) was studied in peripheral tissues of rats. The ganglionic origin, somatosensory nature, and anatomic relations of this thin-axon population were evaluated with particular emphasis on possible nociceptive roles. In animals untreated with colchicine, CGRP-IR is found in a vast proportion of small- and medium-diameter sensory ganglion cells that give rise to numerous thinly myelinated and unmyelinated axons that display CGRP-IR throughout the body. The integumentary innervation consists, in part, of an extensive subpapillary network largely traced to dermal blood vessels, sweat glands, and "free" nerve endings, some of which are found within regions containing only mast cells, fibroblasts, and collagen. Dermal papillae contain CGRP-IR axons surrounding each vascular loop; other papillary axons end freely or occasionally surround Meissner corpuscles. Intraepithelial axons enter glabrous epidermal pegs, branching and exhibiting terminals throughout the stratum spinosum. A similar pattern is found in hairy skin with additional innervation entering the base and surrounding the lower third of each hair follicle, but apparently not supplying sebaceous glands and arrector pili muscle. Axons innervating nonkeratinized oral epithelium are similar or greater in number and distribution compared to epidermis, often with more extensive branching. The high density of intraepithelial CGRP-IR innervation does not appear to correlate with the sensitive mechanoreceptor-based increase in spatial sensory discriminative capacities in the distal portions of the limb. In deep somatic tissues, CGRP-IR is principally related to vasculature and motor end plates of striated muscle, but there is an extensive network of thin axons within bone, principally in the periosteum, and focally in joint capsules, but not in relation to muscle spindles or tendon organs. These findings, together with the distribution in cranial tissues described in an accompanying paper (Silverman and Kruger: J. Comp. Neurol. 280:303-330, '89), are considered in the context of a "noceffector" concept incorporating the efferent role of these sensory axons in various tissues. It is suggested that involvement in tissue maintenance and renewal during normal function, as well as following injury, may predominate over the relatively infrequent nociceptive role of this peptidergic sensory system.
• Mantyh, P. W., Catton, M. D., Boehmer, C. G., Welton, M. L., Passaro, E. P., Maggio, J. E., & Vigna, S. R. (1989). Receptors for sensory neuropeptides in human inflammatory diseases: implications for the effector role of sensory neurons. Peptides, 10(3), 627-45.
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  Glutamate and several neuropeptides are synthesized and released by subpopulations of primary afferent neurons. These sensory neurons play a role in regulating the inflammatory and immune responses in peripheral tissues. Using quantitative receptor autoradiography we have explored what changes occur in the location and concentration of receptor binding sites for sensory neurotransmitters in the colon in two human inflammatory diseases, ulcerative colitis and Crohn's disease. The sensory neurotransmitter receptors examined included bombesin, calcitonin gene related peptide-alpha, cholecystokinin, galanin, glutamate, somatostatin, neurokinin A (substance K), substance P, and vasoactive intestinal polypeptide. Of the nine receptor binding sites examined only substance P binding sites associated with arterioles, venules and lymph nodules were dramatically up-regulated in the inflamed tissue. These data suggest that substance P is involved in regulating the inflammatory and immune responses in human inflammatory diseases and indicate a specificity of efferent action for each sensory neurotransmitter in peripheral tissues.
• Mantyh, P. W., Gates, T., Mantyh, C. R., & Maggio, J. E. (1989). Autoradiographic localization and characterization of tachykinin receptor binding sites in the rat brain and peripheral tissues. The Journal of neuroscience : the official journal of the Society for Neuroscience, 9(1), 258-79.
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  Quantitative receptor autoradiography using several radiolabeled tachykinins was used to localize and characterize tachykinin peptide receptor binding sites in rat CNS and peripheral tissues. Autoradiographic localization and displacement experiments using several radiolabeled tachykinins indicate that in the rat there are at least 3 distinct tachykinin receptor binding sites. One of these is present in both the CNS and peripheral tissues, one is present only in the CNS, and one is present only in peripheral tissues. The first tachykinin receptor binding site, which is detectable in both the CNS and peripheral tissues, appears to prefer substance P (SP) as an endogenous ligand. Areas expressing high concentrations of this binding site include the medial septum, superior colliculus, inferior olive, inner plexiform layer of the retina, external muscle of the bladder, and the muscularis externa of the esophagus. The second type of tachykinin receptor binding site, which is detectable only in the CNS appears to prefer either neuromedin K (NK) and/or substance K (SK) as the endogenous ligand. This receptor binding site is labeled by Bolton-Hunter conjugates of NK, SK, eledoisin, or kassinin and is found in high concentrations in laminae 4 and 5 of the cerebral cortex, the ventral tegmental area, laminae 1 and 2 of the spinal cord, and the inner plexiform layer of the retina. The third type of tachykinin receptor binding site is detectable only in peripheral tissues and appears to prefer SK as the endogenous ligand. This receptor binding site is labeled by SK, eledoisin, or kassinin radioligands and tissues that express high concentrations include the muscularis mucosae of the esophagus, the circular muscle of the colon, and the external muscle of the bladder. These data suggest that SP receptors are expressed in the brain and peripheral tissues, NK receptors are expressed in the CNS, and SK receptors are expressed in peripheral tissue. These data fit well with radioimmunoassay data that suggest that, whereas in the CNS SP, SK and NK are present in high concentrations, in peripheral tissues only SP and SK are present in detectable concentrations. The present classification of tachykinin receptors places a lower limit on the number of mammalian tachykinin receptor types and provides a functional/morphological framework for exploring the diverse actions of tachykinin peptides in both the CNS and peripheral tissues.
• Mantyh, P. W., Johnson, D. J., Boehmer, C. G., Catton, M. D., Vinters, H. V., Maggio, J. E., Too, H. P., & Vigna, S. R. (1989). Substance P receptor binding sites are expressed by glia in vivo after neuronal injury. Proceedings of the National Academy of Sciences of the United States of America, 86(13), 5193-7.
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  In vitro studies have demonstrated that glia can express functional receptors for a variety of neurotransmitters. To determine whether similar neurotransmitter receptors are also expressed by glia in vivo, we examined the glial scar in the transected optic nerve of the albino rabbit by quantitative receptor autoradiography. Receptor binding sites for radiolabeled calcitonin gene-related peptide, cholecystokinin, galanin, glutamate, somatostatin, substance P, and vasoactive intestinal peptide were examined. Specific receptor binding sites for each of these neurotransmitters were identified in the rabbit forebrain but were not detected in the normal optic nerve or tract. In the transected optic nerve and tract, only receptor binding sites for substance P were expressed at detectable levels. The density of substance P receptor binding sites observed in this glial scar is among the highest observed in the rabbit forebrain. Ligand displacement and saturation experiments indicate that the substance P receptor binding site expressed by the glial scar has pharmacological characteristics similar to those of substance P receptors in the rabbit striatum, rat brain, and rat and canine gut. The present study demonstrates that glial cells in vivo express high concentrations of substance P receptor binding sites after transection of retinal ganglion cell axons. Because substance P has been shown to regulate inflammatory and immune responses in peripheral tissues, substance P may also, by analogy, be involved in regulating the glial response to injury in the central nervous system.
• Vigna, S. R., Mantyh, C. R., Soll, A. H., Maggio, J. E., & Mantyh, P. W. (1989). Substance P receptors on canine chief cells: localization, characterization, and function. The Journal of neuroscience : the official journal of the Society for Neuroscience, 9(8), 2878-86.
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  Saturable binding sites for 125I-Bolton-Hunter substance P were observed in frozen sections of the oxyntic mucosa of the canine stomach using quantitative autoradiography. The cell type possessing substance P binding sites in this region was identified as the chief cell in 2 ways. First, the saturable binding of radioiodinated substance P correlated with chief cell content (and not with parietal cell content, for example) in dispersed oxyntic mucosal cells fractionated by centrifugal elutriation. Second, saturable binding of radioiodinated substance P was localized to dispersed chief cells by autoradiography using emulsion-coated preparations of isolated cells affixed to glass slides. Parietal and mucous cells did not bind substance P. In studies of enriched chief cell preparations, the binding of radiolabeled substance P was found to be time- and cell number-dependent, specific, saturable, reversible, and of high affinity. Equilibrium binding analysis revealed a single class of binding sites with an apparent Kd of 105 pM and a Bmax of 3000 receptors per cell. In competitive displacement studies, the order of potency of analogs for inhibition of the saturable binding of radiolabeled substance P to chief cells was substance P = physalaemin greater than substance K greater than neuromedin K; thus, the chief cell has a substance P-preferring tachykinin binding site. Bombesin, cholecystokinin, and somatostatin had no effect on substance P binding. Substance P stimulated pepsinogen secretion from isolated canine oxyntic glands in dose-dependent fashion with a half-maximal response occurring at a substance P dose of about 1 mM.(ABSTRACT TRUNCATED AT 250 WORDS)
• Zimmerman, R. P., Gates, T. S., Mantyh, C. R., Vigna, S. R., Welton, M. L., Passaro, E. P., & Mantyh, P. W. (1989). Vasoactive intestinal polypeptide receptor binding sites in the human gastrointestinal tract: localization by autoradiography. Neuroscience, 31(3), 771-83.
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  Vasoactive intestinal polypeptide (VIP) is a putative neurotransmitter in both the brain and peripheral tissues. To define possible target tissues of VIP we have used quantitative receptor autoradiography to localize and quantify the distribution of [125I]VIP receptor binding sites in histologically normal human surgical specimens. While the distribution of VIP binding sites was different for each gastrointestinal segment examined, specific vasoactive intestinal polypeptide binding sites were localized to the mucosa, the muscularis mucosa, the smooth muscle of submucosal arterioles, the circular and longitudinal smooth muscle of the muscularis externa, the myenteric plexus, and lymph nodules. In most segments, the mucosal layer expressed the highest concentration of VIP binding sites, with the duodenal and jejunal mucosa showing the highest density of receptors. These results identify putative VIP target tissues in the human gastrointestinal tract. In correlation with physiological data, VIP binding sites appear to be involved in the regulation of a variety of gastrointestinal functions including mucosal ion transport, gastric secretion, hemodynamic regulation, gastric and intestinal motility, neuronal excitability, and modulation of the immune system.
• Bacay, A. C., Mantyh, C. R., Vigna, S. R., & Mantyh, P. W. (1988). Receptor binding sites for atrial natriuretic factor are expressed by brown adipose tissue. Peptides, 9(5), 1021-6.
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  To explore the possibility that atrial natriuretic factor (ANF) is involved in thermoregulation we used quantitative receptor autoradiography and homogenate receptor binding assays to identify ANF bindings sites in neonatal rat and sheep brown adipose tissue, respectively. Using quantitative receptor autoradiography were were able to localize high levels of specific binding sites for 125I-rat ANF in neonatal rat brown adipose tissue. Homogenate binding assays on sheep brown fat demonstrated that the radioligand was binding to the membrane fraction and that the specific binding was not due to a lipophilic interaction between 125I-rat ANF and brown fat. Specific binding of 125I-rat ANF to the membranes of brown fat cells was inhibited by unlabeled rat ANF with a Ki of 8.0 x 10(-9) M, but not by unrelated peptides. These studies demonstrate that brown fat cells express high levels of ANF receptor binding sites in neonatal rat and sheep and suggest that ANF may play a role in thermoregulation.
• Gates, T. S., Zimmerman, R. P., Mantyh, C. R., Vigna, S. R., Maggio, J. E., Welton, M. L., Passaro, E. P., & Mantyh, P. W. (1988). Substance P and substance K receptor binding sites in the human gastrointestinal tract: localization by autoradiography. Peptides, 9(6), 1207-19.
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  Quantitative receptor autoradiography was used to localize and quantify the distribution of binding sites for 125I-radiolabeled substance P (SP), substance K (SK) and neuromedin K (NK) in the human GI tract using histologically normal tissue obtained from uninvolved margins of resections for carcinoma. The distribution of SP and SK binding sites is different for each gastrointestinal (GI) segment examined. Specific SP binding sites are expressed by arterioles and venules, myenteric plexus, external circular muscle, external longitudinal muscle, muscularis mucosa, epithelial cells of the mucosa, and the germinal centers of lymph nodules. SK binding sites are distributed in a pattern distinct from SP binding sites and are localized to the external circular muscle, external longitudinal muscle, and the muscularis mucosa. Binding sites for NK were not detected in any part of the human GI tract. These results demonstrate that: 1) surgical specimens from the human GI tract can be effectively processed for quantitative receptor autoradiography; 2) of the three mammalian tachykinins tested, SP and SK, but not NK binding sites are expressed in detectable levels in the human GI tract; 3) whereas SK receptor binding sites are expressed almost exclusively by smooth muscle, SP binding sites are expressed by smooth muscle cells, arterioles, venules, epithelial cells of the mucosa and cells associated with lymph nodules; and 4) both SP and SK binding sites expressed by smooth muscle are more stable than SP binding sites expressed by blood vessels, lymph nodules, and mucosal cells.
• Kruger, L., Mantyh, P. W., Sternini, C., Brecha, N. C., & Mantyh, C. R. (1988). Calcitonin gene-related peptide (CGRP) in the rat central nervous system: patterns of immunoreactivity and receptor binding sites. Brain research, 463(2), 223-44.
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  The distribution of immunoreactive (IR) axons and neurons in the rat central nervous system (CNS) has been studied with an antiserum directed against the C-terminal sequence of rat a-calcitonin gene-related peptide (CGRP) and a durable peroxidase reaction product for detailed analysis in relation to normal cytoarchitecture. These materials were studied and illustrated in the three principal axes in relation to cell-stained adjacent sections in normal as well as colchicine- and capsaicin-treated animals, although no fundamental differences in pattern were evident in neurotoxin-treated rats. The patterns of CGRP-IR were then compared with autoradiograms of specific, high affinity receptor binding sites for 125I-human a-CGRP. CGRP-IR labeling in motor systems includes the vast majority of motoneurons, enabling facile identification of isolated 'accessory' populations. Preganglionic parasympathetic nuclei revealed only labeling of a small proportion of neurons. By contrast, the sensory systems revealed a diversity of labeling patterns precluding simple generalizations. Peripheral input ranges from extensive labeling of thin somatic afferents, feeble to moderate gustatory and olfactory afferents to a total absence of auditory afferents, yet IR axons and neurons can be found in selective distribution within each of these sensory systems. Patterns of IR in various integrative centers, e.g. cerebellum, basal ganglia and hypothalamus, reveal selectivity that fails to conform to conventional descriptions of functional systems. Some regions display unexpected patterns, e.g. vertical stripes in cerebellar cortex. CGRP receptor binding sites (RB) are found in many of the sites where IR axons terminate and in some cases, e.g. motor neurons, which express intraneuronal IR. The main sensory systems reveal a variety of RB patterns, only a few of which can be related to sites of IR axon terminals. Many apparent 'mismatches' between IR and RB are illustrated and discussed in the context of functional peptide expression or in quasi-hormonal terms. It is suggested that the principle of CGRP-IR axon distribution in peripheral tissues, where synapses are lacking, might also apply to the CNS and that neither the locus of IR-axon terminals nor RB sites need indicate transmitter action for impulse information transfer. CGRP is a widely distributed neuromodulator probably subserving a role in both synaptic and metabolic regulation, depending on the specific requirements of the diverse distribution of its receptors.
• Kruger, L., Sternini, C., Brecha, N. C., & Mantyh, P. W. (1988). Distribution of calcitonin gene-related peptide immunoreactivity in relation to the rat central somatosensory projection. The Journal of comparative neurology, 273(2), 149-62.
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  The distribution of the neuropeptide calcitonin gene-related peptide (CGRP) was studied in relation to the known subcortical somatosensory pathways and contiguous systems in the central nervous system (CNS) of rats by using peroxidase histochemical methods in order to relate zones of immunoreactivity (IR) to cytoarchitecture. CGRP is the most ubiquitous peptide found to date in sensory ganglion cells: principally small and medium-size neurons emitting thin axons inferred to be largely nociceptive in function on the basis of the peripheral distribution of their terminals. Its apparent absence in sympathetic axons provides an especially useful sensory marker. The distribution of CGRP-IR axons displays remarkable selectivity at each level of the CNS. The trigeminal root distributes axons primarily to the pericornual layers (laminae I and II) of spinal V nucleus caudalis and to subnucleus oralis, evading the subnucleus interpolaris and contributing only few axons to principal V. Although there are only a few CGRP-IR somata at each level, heavily labeled axon trajectories can be traced to the nuclei of the solitary tract, the parabrachial nuclei, several sectors of the caudal medial thalamus, and the central nucleus of the amygdala. A sector of labeled neuron somata lies contiguous to each of these axon terminal zones, the largest of which is a thalamic nucleus containing cells of distinctive dendritic architecture extending from the periaqueductal gray across the posterior group nuclei to the peripeduncular nucleus, forming a linear array at the mesodiencephalic junction. The relation of CGRP-IR axonal distribution to spinothalamic, visceral, and gustatory systems is discussed in the context of a specialized "chemosensory" component of the thin-fiber somatosensory system.
• Mantyh, C. R., Gates, T. S., Zimmerman, R. P., Welton, M. L., Passaro, E. P., Vigna, S. R., Maggio, J. E., Kruger, L., & Mantyh, P. W. (1988). Receptor binding sites for substance P, but not substance K or neuromedin K, are expressed in high concentrations by arterioles, venules, and lymph nodules in surgical specimens obtained from patients with ulcerative colitis and Crohn disease. Proceedings of the National Academy of Sciences of the United States of America, 85(9), 3235-9.
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  Several lines of evidence indicate that tachykinin neuropeptides [substance P (SP), substance K (SK), and neuromedin K (NK)] play a role in regulating the inflammatory and immune responses. To test this hypothesis in a human inflammatory disease, quantitative receptor autoradiography was used to examine possible abnormalities in tachykinin binding sites in surgical specimens from patients with inflammatory bowel disease. Surgical specimens of colon were obtained from patients with ulcerative colitis (n = 4) and Crohn disease (n = 4). Normal tissue was obtained from uninvolved areas of extensive resections for carcinoma (n = 6). In all cases, specimens were obtained less than 5 min after removal to minimize influences associated with degradation artifacts and were processed for quantitative receptor autoradiography by using 125I-labeled Bolton-Hunter conjugates of NK, SK, and SP. In the normal colon a low concentration of SP receptor binding sites is expressed by submucosal arterioles and venules and a moderate concentration is expressed by the external circular muscle, whereas SK receptor binding sites are expressed in low concentrations by the external circular and longitudinal muscle. In contrast, specific NK binding sites were not observed in any area of the human colon. In colon tissue obtained from ulcerative colitis and Crohn disease patients, however, very high concentrations of SP receptor binding sites are expressed by arterioles and venules located in the submucosa, muscularis mucosa, external circular muscle, external longitudinal muscle, and serosa. In addition, very high concentrations of SP receptor binding sites are expressed within the germinal center of lymph nodules, whereas the concentrations of SP and SK binding sites expressed by the external muscle layers are not altered significantly. These results demonstrate that receptor binding sites for SP, but not SK or NK, are ectopically expressed in high concentrations (1000-2000 times normal) by cells involved in mediating inflammatory and immune responses. These data suggest that SP may be involved in the pathophysiology of inflammatory bowel disease and might provide some insight into the interaction between the nervous system and the regulation of inflammation and the immune response in human inflammatory disease.
• Mantyh, P. W., Mantyh, C. R., Gates, T., Vigna, S. R., & Maggio, J. E. (1988). Receptor binding sites for substance P and substance K in the canine gastrointestinal tract and their possible role in inflammatory bowel disease. Neuroscience, 25(3), 817-37.
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  The mammalian tachykinins, substance P, substance K (neurokinin A) and neuromedin K (neurokinin B), are putative peptide neurotransmitters in both the brain and peripheral tissues. We used quantitative receptor autoradiography to localize and quantify the distribution of binding sites for radiolabeled substance P, substance K and neuromedin K in the canine gastrointestinal tract. Substance P binding sites were localized to smooth muscle cells in the muscularis mucosa and muscularis externa, the smooth muscle and endothelium of arterioles and venules, neurons in the myenteric plexus, mucosal epithelial cells, exocrine cells and lymph nodules. Substance K binding sites were distributed in a pattern distinct from substance P binding sites and were localized to smooth muscle cells in the muscularis mucosa and muscularis externa, the smooth muscle and endothelium of arterioles and venules, and neurons of the myenteric plexus. Neuromedin K binding sites were not observed in any area of the canine gastrointestinal tract although they were localized with high specific/non-specific binding ratios in the canine spinal cord. These results indicate that there are at least two distinct types of tachykinin receptor binding sites in the canine gastrointestinal tract, one of which probably recognizes substance P and the other substance K as endogenous ligands. In correlation with previous physiological data, these substance P and substance K receptor binding sites appear to be involved in the regulation of a variety of gastrointestinal functions including gastric motility, mucosal ion transport, hemodynamics, digestive enzyme secretion and neuronal excitability. In addition these results demonstrate that receptor binding sites for substance P and substance K are expressed by cells involved in mediating inflammatory and immune responses. These data, together with our studies on surgical specimens from patients with inflammatory bowel disease, suggest that in a pathophysiological state tachykinins and their receptors may play a role in inflammatory bowel disease and should permit a rational approach to designing neuropeptide antagonists which may prove effective in treating inflammatory diseases.
• Popper, P., Mantyh, C. R., Vigna, S. R., Maggio, J. E., & Mantyh, P. W. (1988). The localization of sensory nerve fibers and receptor binding sites for sensory neuropeptides in canine mesenteric lymph nodes. Peptides, 9(2), 257-67.
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  Previous work has established that the central nervous system can modulate the immune response. Direct routes through which this regulation may occur are the sympathetic and sensory innervation of lymphoid organs. We investigated the innervation of canine mesenteric lymph nodes using immunohistochemistry and the expression of binding sites for sensory neuropeptides using quantitative receptor autoradiography. The sympathetic innervation of lymph nodes was examined by immunohistochemical methods using an antiserum directed against tyrosine hydroxylase (TOH), the rate limiting enzyme in catecholamine synthesis. TOH-containing fibers were associated with 90% of the blood vessels (arteries, veins, arterioles and venules) in the hilus, medullary and internodular regions of lymph nodes and in trabeculae with no obvious relationship to blood vessels. The sensory innervation of lymph nodes was investigated using antisera directed against the putative sensory neurotransmitters calcitonin gene-related peptide (CGRP) and substance P (SP). CGRP- and SP-containing fibers were detected in the hilus, the medullary region, and the internodular region of lymph nodes usually in association with arterioles and venules. About 50% of the arterioles and venules exhibited a CGRP innervation and a smaller fraction (5-10%) were innervated by SP-containing fibers. Few if any TOH, CGRP, and SP nerve fibers were detected in the germinal centers of lymph nodes. Using quantitative receptor autoradiography we studied the distribution of receptor binding sites for the sensory neuropeptides CGRP, SP, substance K (SK), vasoactive intestinal peptide (VIP), somatostatin (SOM), and bombesin. Specific CGRP binding sites were expressed throughout lymph nodes by trabeculae, arterioles, venules and 25% of the germinal centers. SP receptor binding sites were localized to arterioles and venules in the T cell regions and 25-30% of the germinal centers. VIP binding sites were localized to the internodular and T cell regions, to medullary cords, and to 10-20% of germinal centers. SK, SOM, and bombesin binding sites were not detected in the lymph nodes, although receptor binding sites for these peptides were detected with high specific/nonspecific binding ratios in other canine peripheral tissues. Taken together with previous results these findings suggest that the sympathetic and sensory innervation of mesenteric lymph nodes appears to be involved with the regulation of their blood and lymph flow. The neuropeptide receptor binding sites in lymph node germinal centers may be expressed by lymphocytes upon activation by antigens.(ABSTRACT TRUNCATED AT 400 WORDS)
• Vigna, S. R., Giraud, A. S., Soll, A. H., Walsh, J. H., & Mantyh, P. W. (1988). Bombesin receptors on gastrin cells. Annals of the New York Academy of Sciences, 547, 131-7.
• Zimmerman, R. P., Gates, T. S., Boehmer, C. G., & Mantyh, P. W. (1988). Epidermal growth factor receptors in the canine antrum. Peptides, 9(6), 1411-4.
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  In this study we localized receptor binding sites for 125I-human epidermal growth factor (hEGF) in the antrum of the adult canine stomach. High levels of specific 125I-hEGF binding sites were observed over the mucosa and muscularis mucosa, whereas specific binding sites were not detectable over the submucosa, external circular and longitudinal muscle or myenteric neurons. These results are in agreement with previous studies which indicated that EGF stimulates the proliferation of cultured epithelial cells and inhibits gastric acid secretion. This suggests that EGF may be a useful therapeutic agent in the healing of gastric ulcers.
• Zimmerman, R. P., Gates, T. S., Boehmer, C. G., & Mantyh, P. W. (1988). Epidermal growth factor receptors in the human colon. European journal of pharmacology, 150(1-2), 201-2.
• Zimmerman, R. P., Gates, T. S., Mantyh, C. R., Vigna, S. R., Boehmer, C. G., & Mantyh, P. W. (1988). Vasoactive intestinal peptide (VIP) receptors in the canine gastrointestinal tract. Peptides, 9(6), 1241-53.
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  Vasoactive intestinal peptide (VIP) is a putative neurotransmitter in both the brain and peripheral tissues. To define possible target tissues of VIP we have used quantitative receptor autoradiography to localize and quantify the distribution of 125I-VIP receptor binding sites in the canine gastrointestinal tract. While the distribution of VIP binding sites was different for each segment examined, specific VIP binding sites were localized to the mucosa, the muscularis mucosa, the smooth muscle of submucosal arterioles, lymph nodules, and the circular and longitudinal smooth muscle of the muscularis externa. These results identify putative target tissues of VIP action in the canine gastrointestinal tract. In correlation with physiological data, VIP sites appear to be involved in the regulation of a variety of gastrointestinal functions including epithelial ion transport, gastric secretion, hemodynamic regulation, immune response, esophageal, gastric and intestinal motility.
• Akesson, T. R., Mantyh, P. W., Mantyh, C. R., Matt, D. W., & Micevych, P. E. (1987). Estrous cyclicity of 125I-cholecystokinin octapeptide binding in the ventromedial hypothalamic nucleus. Evidence for downmodulation by estrogen. Neuroendocrinology, 45(4), 257-62.
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  Although there is a good correlation between the levels of gonadal steroids and sex differences in reproductive behavior and food intake, the neurochemical mechanisms by which a gonadal steroid may regulate these behaviors remain unknown. An important central nervous site for steroid modulation of food intake and reproduction is the ventromedial nucleus of the hypothalamus (VMH). Recently it has been suggested that some of the activities of the VMH are dependent on the neuroactive peptide cholecystokinin octapeptide (CCK). High levels of CCK in the VMH have been measured by radioimmunoassay, and immunohistochemical analysis has revealed a dense plexus of CCK fibers and terminals which appose and make synaptic contact with VMH neurons. There is, however, a paucity of information concerning the presence of CCK-binding sites in the VMH. This study demonstrates high levels of 125I-CCK8 binding in the VMH of male and female rats. Tissue sampled on the morning of estrus revealed depressed levels of CCK binding, and, while ovariectomy itself did not affect binding, ovariectomy followed by estrogen replacement depressed CCK binding. These results provide evidence for an estrogenic effect on CCK-binding sites that may help elucidate a role of CCK in the VMH.
• Mantyh, C. R., Kruger, L., Brecha, N. C., & Mantyh, P. W. (1987). Localization of specific binding sites for atrial natriuretic factor in the central nervous system of rat, guinea pig, cat and human. Brain research, 412(2), 329-42.
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  Specific, high-affinity binding sites for atrial natriuretic factor (ANF) were identified and localized in the rat and guinea pig central nervous system (CNS), the cat brainstem, and the rat, guinea pig, cat and human spinal cord using quantitative autoradiographic techniques. The radioligands tested were rat 125I-ANF(1-28) in guinea pig, rat, cat and human tissues, human 125I-ANF in rat and human, and rat [3H]atriopeptin III in rat. All 3 radioligands labeled essentially the same structures in the brain and spinal cord of all species in which they were tested. In guinea pig very high concentrations of ANF binding sites were observed in the olfactory bulb, lateral olfactory tract and the granule cell layer of the cerebellum, high concentrations were observed in the fasciculus retroflexus, interpeduncular nucleus and subfornical organ. Moderate concentrations were observed in the nucleus accumbens, dorsomedial and suprachiasmatic hypothalamic nuclei, paraventricular thalamic nuclei, primary olfactory cortex and the subcommissural organ. High concentrations of ANF binding sites were also observed in the choroid plexus and the leptomeninges. Low concentrations were observed in the pineal gland. In the rat the same structures were labeled as in the guinea pig except that suprachiasmatic and dorsomedial hypothalamic nuclei, paraventricular thalamus and cerebellum were unlabeled. In the lower brainstem of the cat and all levels of the rat, guinea pig, cat and human spinal cord, the only site where specific binding was observed was in the pia/arachnoid. These findings suggest that ANF binding sites constitute several functional classes in the CNS as well as in a variety of other tissues. Outside the blood-brain barrier binding sites are prominent in glandular tissues implicated in the production of hormones involved in fluid and electrolyte balance, e.g. adrenal glomerulosa, neurohypophysis and subfornical organ, unstratified epithelia involved in ion gradient exchange, e.g. renal glomerulus, ciliary body, choroid plexus and pia mater; crossing the blood-brain barrier are sites in the anterior hypothalamus, e.g. organum vasculosum, regions of the brain parenchyma associated with angiotensin II binding sites, e.g. dorsomedial nucleus of hypothalamus, some of which may be occupied by brain rather than cardiac synthesized ANF, regions of brain lacking an obvious role in fluid and ion exchange or regulation, e.g. cerebellum, although association with K+,Na+-ATPase in guinea pig cerebellum may be a relevant clue and brain regions possibly implicated in an integrative and/or indirect regulatory role in fluid and electrolyte balance.(ABSTRACT TRUNCATED AT 400 WORDS)
• Vigna, S. R., Mantyh, C. R., Giraud, A. S., Soll, A. H., Walsh, J. H., & Mantyh, P. W. (1987). Localization of specific binding sites for bombesin in the canine gastrointestinal tract. Gastroenterology, 93(6), 1287-95.
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  The goal of these studies was to determine the tissue and cell types possessing specific binding sites for bombesin/gastrin-releasing peptide in the canine gastrointestinal tract. Monoiodinated, biologically active (Tyr-4)-bombesin 14 (100 pM) was applied to sections of canine gut and localized using quantitative autoradiography. The highest density of bombesin/gastrin-releasing peptide specific binding sites occurred over endocrine cells in the antral mucosa. Specific binding sites were also found on the circular muscle layer of the gastric fundus, gastric antrum, and ileum, on longitudinal muscle of the gastric fundus and antrum, and on neuronal elements in the myenteric plexus in the gastric fundus, antrum, and small intestine. No evidence for specific binding of 125I-(Tyr-4)-bombesin 14 was found in sections of canine esophagus, gastric cardia, gallbladder, pancreas, or colon. These results suggest sites of direct action of bombesin and endogenous gastrin-releasing peptide for gastrin release and gastrointestinal motility.
• Mantyh, C. R., Kruger, L., Brecha, N. C., & Mantyh, P. W. (1986). Localization of specific binding sites for atrial natriuretic factor in peripheral tissues of the guinea pig, rat, and human. Hypertension, 8(8), 712-21.
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  Specific, high affinity atrial natriuretic factor (ANF) binding sites were identified and localized by autoradiographic techniques in peripheral tissues of the guinea pig, rat, and human. In the guinea pig kidney, high concentrations of ANF binding sites were located in the glomerular apparatus, outer medulla, and small renal arteries. Other peripheral tissues containing ANF binding sites included the zona glomerulosa of the adrenal cortex, the smooth muscle layer of the aorta and gallbladder, the lung parenchyma, the posterior lobe of the pituitary, the ciliary body of the eye, and the leptomeninges and choroid plexus of the brain. The distribution of ANF binding sites in the rat and human kidney was nearly identical to those seen in the guinea pig kidney; high concentrations were present in the glomerular apparatus, outer medulla, and small renal arteries. These results are consistent with earlier physiological and pharmacological studies that suggested that ANF plays a functional role in the regulation of extracellular fluid volume and blood pressure. There appears to be little species variation in the location and concentration of renal ANF binding sites, suggesting that, at least in the kidney, the results in experimental animals are relevant to the actions of ANF in humans. The finding that ANF binding sites were stable and present in high concentrations in human postmortem kidneys further suggests that these tissues may be amenable to testing for the involvement of ANF receptor dysfunction in diseases such as hypertension and congestive heart failure.
• Mantyh, P. W., & Hunt, S. P. (1986). Changes in 3H-substance P receptor binding in the rat brain after kainic acid lesion of the corpus striatum. The Journal of neuroscience : the official journal of the Society for Neuroscience, 6(6), 1537-44.
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  Previous studies have indicated that the substantia nigra contains the highest concentration of substance P-like immunoreactivity (SPLI) in the brain. Paradoxically, it also appears to contain one of the lowest concentrations of substance P receptors in the brain. One possibility is that the massive amount of SPLI blocks the binding of the radioligand to the substance P receptor and/or "down-regulates" the number of substance P receptors present in this structure. Since greater than 95% of the SPLI within the substantia nigra originates from the corpus striatum, we have lesioned this area and measured the changes in substance P receptor concentration in the substantia nigra and other corpus striatal projection areas. A semiquantitative autoradiographic technique for measuring the binding of 3H-substance P to substance P receptors was used in conjunction with tritium-sensitive film. 3H-substance P binding was measured in both the corpus striatum and its projection areas after kainic acid lesion of the corpus striatum. At either 4 or 21 d after the lesion there was approximately a 90% loss of substance P receptors in the rostral striatum, a 74% loss in the globus pallidus, a 57% increase in receptor number in lamina I and II of the ipsilateral somatosensory cortex, and no apparent change in the number of receptors in the substantia nigra pars reticulata, superior colliculus, and central gray. These findings suggest that the low concentration of substance P receptors found within the substantia nigra is not due the massive SPLI innervation, since removal of greater than 95% of the SPLI had no measurable effect on the concentration of substance P receptors.(ABSTRACT TRUNCATED AT 250 WORDS)
• Goedert, M., Hunt, S. P., Mantyh, P. W., & Emson, P. C. (1985). The ontogenetic development of neurotensin-like immunoreactivity and neurotensin receptors in the cat striatum. Brain research, 352(1), 127-31.
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  At birth, striatal neurotensin-like immunoreactivity amounted to 10% of the adult values which were reached at the age of 5 weeks. In the caudate nucleus neurotensin-like immunoreactivity presented a patchy distribution throughout development that was in register with Met-enkephalin staining, whereas [3H]neurotensin binding sites were most heavily concentrated in the background matrix. Thus, the adult distribution pattern of neurotensin-like immunoreactivity and [3H]neurotensin binding sites is already established at birth.
• Goedert, M., Lightman, S. L., Mantyh, P. W., Hunt, S. P., & Emson, P. C. (1985). Neurotensin-like immunoreactivity and neurotensin receptors in the rat hypothalamus and in the neurointermediate lobe of the pituitary gland. Brain research, 358(1-2), 59-69.
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  In the rat hypothalamus, cell bodies containing neurotensin-like immunoreactivity were mainly found in the medial preoptic area, the periventricular nucleus, the paraventricular nucleus, the supraoptic nucleus and the arcuate nucleus. [3H]neurotensin binding sites were observed throughout the hypothalamus with a dense accumulation of silver grains over the paraventricular nucleus, the arcuate nucleus and the median eminence region. By radioimmunoassay neurotensin-like immunoreactivity was also found in the neurointermediate lobe of the pituitary gland of various mammalian species and in human postmortem posterior pituitary glands. In the rat studies involving pituitary stalk transections and the neurotoxin monosodium glutamate indicated the presence of a neurotensinergic pathway from the arcuate nucleus to the neurointermediate lobe of the pituitary gland. [3H]neurotensin binding sites were found to be concentrated over the intermediate lobe of the pituitary gland and their presence was not affected by pituitary stalk transection, indicating their localization on endocrine cells of the intermediate lobe of the pituitary gland.
• Kalivas, P. W., Deutch, A. Y., Maggio, J. E., Mantyh, P. W., & Roth, R. H. (1985). Substance K and substance P in the ventral tegmental area. Neuroscience letters, 57(3), 241-6.
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  A comparison was made between substance P (SP) and substance K (SK) in the ventral tegmental area (VTA) (A10 dopamine cell group) of the rat. Approximately equal densities of SP and SK-immunoreactive neuronal fibers were observed. However, while previous reports demonstrate negligible density of autoradiographically defined SP receptors in the VTA, we observed a high density of SK receptors. SK or SP was microinjected into the VTA, and changes in spontaneous motor activity were measured using a photocell apparatus. SK was found to be at least 10 times more potent than SP in producing an increase in motor activity. These data suggest that while both SK and SP are present in the VTA, SK may have a more significant physiological role in modulating dopamine neurons in the ventromedial mesencephalon.
• Mantyh, C. R., & Mantyh, P. W. (1985). Differential localization of cholecystokinin-8 binding sites in the rat vs. the guinea pig brain. European journal of pharmacology, 113(1), 137-9.
• Mantyh, C. R., Brecha, N. C., Soon-Shiong, P., & Mantyh, P. W. (1985). Specific binding sites for atrial natriuretic factor in the human kidney. The New England journal of medicine, 312(26), 1710.
• Mantyh, P. W., & Hunt, S. P. (1985). The autoradiographic localization of substance P receptors in the rat and bovine spinal cord and the rat and cat spinal trigeminal nucleus pars caudalis and the effects of neonatal capsaicin. Brain research, 332(2), 315-24.
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  Substance P (SP) is a putative neurotransmitter in the central nervous system. In the present report we have used autoradiographic receptor binding techniques to investigate the distribution of SP receptor binding sites in the rat and bovine spinal cord and in the rat and cat spinal trigeminal nucleus pars caudalis. Although some quantitative differences were evident, all species appeared to have a similar distribution of SP receptor binding sites in both the spinal cord and in the spinal trigeminal nucleus pars caudalis. In the spinal cord the heaviest concentration of SP receptors is located in lamina X, while moderate to heavy concentrations were found in laminae I, II and V-IX. Very low concentrations of SP receptors were present in laminae III and IV. Examination of the cat and rat spinal trigeminal nucleus pars caudalis revealed a moderate density of SP receptor binding sites in laminae I and II, very low concentrations in laminae III and IV, and low to moderate concentrations in lamina V. Rats treated neonatally with capsaicin showed a small (11%) but significant (P less than 0.02) increase in the levels of SP receptor binding sites in laminae I and II of the cervical and lumbar spinal cord while in all other laminae the levels remained unchanged.
• Mantyh, P. W., & Hunt, S. P. (1985). Thyrotropin-releasing hormone (TRH) receptors. Localization by light microscopic autoradiography in rat brain using [3H][3-Me-His2]TRH as the radioligand. The Journal of neuroscience : the official journal of the Society for Neuroscience, 5(2), 551-61.
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  Thyrotropin releasing hormone (TRH) is a putative neurotransmitter in both the central and peripheral nervous system. In the present report, we have used autoradiography coupled with densitometric analysis of tritium-sensitive film to investigate the distribution of [3H][3-Me-His2]TRH [( 3H]MeTRH)-binding sizes in the rat brain. Previous pharmacological reports have established that many of these [3H]MeTRH-binding sites have a structure-activity profile consistent with being a physiological TRH receptor. A high level of TRH receptors were observed in the accessory olfactory bulb, lateral nucleus of the amygdala, dentate gyrus, and entorhinal cortex. Moderate levels of TRH receptors were observed in the rhinal cortex, hypothalamus, superior colliculus, several brainstem motor nuclei, and lamina I of the spinal trigeminal nucleus pars candalis, while low concentrations of receptors are present in the cerebral cortex, striatum and ventral horn of the spinal cord. Very low levels of receptors were observed in the globus pallidus and in most nuclei of the dorsal thalamus. Comparisons of the distribution of TRH receptors to TRH-immunoreactive content indicates that, while in some areas of the brain there is a rough correlation between levels of TRH peptide and its receptor, in most brain areas there is little obvious correlation between the two. While such a discrepancy has been observed for other peptides and their receptors, the extensive distribution of TRH receptors in the central nervous system does provide an explanation for the variety of behavioral effects observed when TRH is infused into the central nervous system.
• Goedert, M., Mantyh, P. W., Emson, P. C., & Hunt, S. P. (1984). Inverse relationship between neurotensin receptors and neurotensin-like immunoreactivity in cat striatum. Nature, 307(5951), 543-6.
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  The adult corpus striatum in mammals is divided into distinct histochemical compartments. If the cat caudate nucleus is stained for acetylcholinesterase a number of macroscopically visible zones appear that have lower acetylcholinesterase activity than the surrounding tissue. These patches, called 'striosomes', correspond to regions of high [Met]-enkephalin-like immunoreactivity and dense opiate receptor binding and are related to the uneven distribution of striatal efferent neurones and cortical afferent terminations. One of the highest concentrations of neurotensin-like immunoreactivity is in the striatum and the immunoreactive material co-elutes with synthetic neurotensin on gel chromatography. Recently, we have found that neurotensin-like immunoreactivity in the cat caudate nucleus coincides with the striosomes. We have now localized neurotensin receptors in the cat caudate nucleus by autoradiography and found low density in the neurotensin-rich striosomes and a high density in the neurotensin-poor surrounding tissue.
• Goedert, M., Mantyh, P. W., Hunt, S. P., & Emson, P. C. (1984). Localization of specific neurotensin binding sites in the rat adrenal gland. Brain research, 299(2), 389-92.
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  Specific tritiated neurotensin binding sites were localized in the rat adrenal gland by receptor autoradiography and characterized using a tissue homogenate receptor binding assay. High levels of specific neurotensin binding sites were found in the inner layer of the adrenal cortex and lower amounts in the adrenal medulla with only background labeling in the outer cortical layers. The structure-activity profile of the specific neurotensin binding was consistent with binding to a physiological neurotensin receptor.
• Goedert, M., Pinnock, R. D., Downes, C. P., Mantyh, P. W., & Emson, P. C. (1984). Neurotensin stimulates inositol phospholipid hydrolysis in rat brain slices. Brain research, 323(1), 193-7.
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  Neurotensin stimulated inositol phospholipid hydrolysis in matched coronal vibratome sections through the rat brain. This effect was tetrodotoxin-resistant and a good correlation was noticed between the magnitude of neurotensin-stimulated inositol phospholipid hydrolysis and the number of specific [3H]neurotensin binding sites in various brain regions. Neurotensin produced no significant effect on either basal or stimulated cAMP levels.
• Hunt, S. P., & Mantyh, P. W. (1984). Radioimmunocytochemistry with [3H]biotin. Brain research, 291(2), 203-17.
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  The methods presented in this paper grew out of the current need for a more quantitative approach to immunocytochemistry. The problem was approached by exploiting the high affinity of biotin for avidin in the design of radioimmunocytochemical methods using [3H]biotin. [3H]Biotin and avidin D form a radioactive complex which can be linked onto a primary antibody by means of a biotinylated anti-rabbit IgG or biotinylated protein A link. With both approaches it was possible to localize a number of antigens such as somatostatin, substance P, avian pancreatic polypeptide, tyrosine hydroxylase, and enkephalin-like immunoreactivity in various regions of the rat and human brain. By using tritium-sensitive film, large regions of the brain could be studied and analyzed semiquantitatively using computerized microdensitometry. The technique was also taken to the electron microscopic level, and in the case of substance P immunoreactivity within the rat substantia nigra silver grains were found to be highly localized over axons and axon terminals. It was also possible to demonstrate co-existence or lack of co-existence of a number of different antigens within neurones. The first primary antibody was localized with biotinylated protein A followed by avidin-peroxidase, while the second primary antibody was linked to the [3H]biotin again with biotinylated protein A. As an example of the potential of these methods for semiquantification, the distribution of substance P within postmortem human spinal cord was examined 24 months after amputation. A 49% loss of peptide was found in the corresponding dorsal horn. In summary these methods using [3H]biotin have proved successful in quantification, electron microscopy and double labelling studies.
• Hunter, J. C., Maggio, J. E., & Mantyh, P. W. (1984). Evidence for vasoactive intestinal polypeptide as a neurotransmitter in smooth muscle of the urogenital tract. Brain research, 305(2), 221-9.
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  The localization of vasoactive intestinal polypeptide (VIP) in the anococcygeus muscle of the rat, rabbit and cat was investigated by radioimmunoassay and immunohistochemistry. High concentrations of VIP-like immunoreactivity were found in the anococcygeus muscle of all 3 species. VIP-like immunoreactivity was observed in a network of nerve fibres branching throughout the muscle which were unaffected by treatment with 6-hydroxydopamine. VIP, when applied exogenously to isolated preparations of the anococcygeus, produced dose-related relaxations of tone in all 3 species with a time course and maximum response similar to non-adrenergic, non-cholinergic inhibitory nerve stimulation. These observations provide further evidence in support of VIP as a suitable candidate for the non-adrenergic, non-cholinergic inhibitory neurotransmitter in the anococcygeus muscle.
• Mantyh, P. W., & Hunt, S. P. (1984). Evidence for cholecystokinin-like immunoreactive neurons in the rat medulla oblongata which project to the spinal cord. Brain research, 291(1), 49-54.
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  The distribution of cholecystokinin-like immunoreactive (CCK-LI) neurons has been mapped in the rat medulla after local and intracerebroventricular colchicine injections. CCK-positive neurons were found in the nucleus raphe magnus, nucleus raphe pallidus, nucleus raphe obscurus, nucleus paragigantocellularis pars alpha, and a population of ventral medullary neurons. Combined retrograde tracing with the fluorescent dye True Blue and indirect immunofluorescence for visualizing CCK neurons suggested that there was a CCK-LI system originating in the medulla and projecting to the spinal cord. Additional double labelling experiments established that some of these CCK-LI containing neurons also contain 5-HT.
• Mantyh, P. W., & Hunt, S. P. (1984). Neuropeptides are present in projection neurones at all levels in visceral and taste pathways: from periphery to sensory cortex. Brain research, 299(2), 297-312.
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  Using combined immuno-staining and retrograde tracing techniques many of the ascending visceral and taste pathways within the rat central nervous system have been shown to be composed of a variety of neuropeptide and catecholamine synthesizing enzyme containing neurones. The pathway we examined extended from the periphery to sensory cortex and included: the nodose ganglion (periphery)----solitary nucleus (medulla)----parabrachial nucleus (pons)----ventral posterior medial nucleus (thalamus)----visceral and taste sensory areas (cortex). In the solitary nucleus of the medulla many neuronal cell bodies could be shown to be both immuno-positive for one of 6 neuropeptides including avian pancreatic peptide (APP), cholecystokinin (CCK), enkephalin (ENK), neurotensin (NT), somatostatin (SOM) and substance P (SP) or the catecholamine synthesizing enzyme tyrosine hydroxylase (TOH) and to have a projection to the parabrachial nucleus of the pons. In the parabrachial nucleus of the pons many neuronal cell bodies could be shown to be immuno-positive for one of 5 neuropeptides (CCK, ENK, NT, SOM, SP) and have a projection to the ventral posterior medial nucleus of the thalamus. In the ventral posterior medial nucleus of the thalamus several neuronal cell bodies were shown to be immuno-positive for one of 3 neuropeptides (CCK, ENK, SOM) and project to the visceral and taste sensory cortex. This is the first report of neuropeptides being present in the projection neurones of any sensory system in the central nervous system and for the first time describes an entire set of putative neurotransmitters which extends from the periphery to the sensory cortex. From previous studies it also appears that in all cases examined the relevant receptors are present in these visceral and taste relay nuclei in order for the neuropeptide or catecholamine to produce an effect upon release. Comparisons between rat and other animals suggest that a similar organization of these visceral and taste pathways may also be present in other mammals including man. Functionally these neuropeptides containing projection neurones appear to be primarily involved in relaying visceral information rather than taste information. In this capacity activation of these neurones may produce such visceral sensations as malaise, well being, hunger, satiety or thirst.
• Mantyh, P. W., Goedert, M., & Hunt, S. P. (1984). Autoradiographic visualization of receptor binding sites for substance P in the gastrointestinal tract of the guinea pig. European journal of pharmacology, 100(1), 133-4.
• Mantyh, P. W., Hunt, S. P., & Maggio, J. E. (1984). Substance P receptors: localization by light microscopic autoradiography in rat brain using [3H]SP as the radioligand. Brain research, 307(1-2), 147-65.
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  Substance P (SP) is a putative neurotransmitter in both the peripheral and central nervous systems. In the present report we have used a modification of the Young and Kuhar technique to investigate some of the SP receptors binding properties and the distribution of SP receptors in rat brain. Tritiated SP [( 3H]SP) absorbed extensively to glass but this adsorbtion was greatly reduced by preincubating the slide-mounted tissue sections in a solution containing the cationic polymer polyethylenimine. [3H]SP was found to bind to rat tissue in a saturable fashion with a Bmax of 14.7 fmol/mg tissue wet weight and a Kd of 1.1 nM. The rank order of potencies for displacing [3H]SP binding from rat tissue sections was SP greater than SP sulphoxide greater than DiMeC7 greater than Eledoisin greater than SP(5-11) greater than SP(COOH) greater than SP(1-9) amide. Using autoradiography coupled with LKB tritium-sensitive Ultrofilm or the dry emulsion-coated coverslip technique the distribution of [3H]SP binding sites was found to be very dense within olfactory bulb, amygdalo-hippocampal area and the nucleus of the solitary tract. Heavy concentrations of receptors were observed in the septum, diagonal band of Broca, striatum subiculum, hypothalamus, locus coeruleus, parabrachial nucleus and lobule 9 and 10 of the cerebellum. Moderate to low concentrations of receptors were observed in the cerebral cortex, globus pallidus, raphe nuclei and the trigeminal nucleus. Very low densities were observed in most aspects of the dorsal thalamus, substantia nigra and cerebellum (other than lobule 9 and 10). Comparisons of the present data with SP peptide levels indicate that in some areas of the brain there is a rough correlation between peptide and receptor levels. However, in other brain areas (olfactory bulb, globus pallidus and substantia nigra) there is little obvious correlation between the two.
• Mantyh, P. W., Maggio, J. E., & Hunt, S. P. (1984). The autoradiographic distribution of kassinin and substance K binding sites is different from the distribution of substance P binding sites in rat brain. European journal of pharmacology, 102(2), 361-4.
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  In the present report we have used autoradiographic receptor binding techniques to compare the distribution of substance K (SK), kassinin (K) and substance P (SP) binding sites in the rat brain. Whereas the distribution of K and SK binding sites appeared to be identical, notable differences are apparent when comparing these binding sites to the distribution of SP binding sites. These results demonstrate that in many areas of the rat brain K and SK binding sites have a different distribution than SP binding sites. These results further suggest that different classes of mammalian tachykinins may each have their own set of receptors.
• Mantyh, P. W., Pinnock, R. D., Downes, C. P., Goedert, M., & Hunt, S. P. (1984). Correlation between inositol phospholipid hydrolysis and substance P receptors in rat CNS. Nature, 309(5971), 795-7.
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  The undecapeptide substance P is a neurotransmitter candidate in the mammalian central and peripheral nervous system. Although the distribution of substance P-like immunoreactivity within the central nervous system (CNS) is well established, the recent identification and autoradiographic localization of specific substance P-binding sites has revealed numerous areas of mismatch between peptide levels and numbers of such sites. Previous studies have shown that substance P stimulates the hydrolysis of inositol phospholipids in peripheral tissues and in the hypothalamus, probably through stimulation of a polyphosphoinositide-specific phospholipase C (refs 9-11). Inositol phospholipid hydrolysis has been implicated in the mobilization of cytosolic calcium following receptor activation in several neurotransmitter and hormonal systems. We have therefore investigated the distribution of 3H-labelled substance P binding sites within various rat brain regions and correlated this with the rate of substance P-induced hydrolysis of inositol phospholipids in the same areas of the CNS. We found that the rate of inositol phospholipid hydrolysis was proportional to the number of binding sites specific for 3H-substance P, suggesting that binding sites revealed by 3H-substance P autoradiography correspond to functional substance P receptors.
• Goedert, M., Mantyh, P. W., Hunt, S. P., & Emson, P. C. (1983). Mosaic distribution of neurotensin-like immunoreactivity in the cat striatum. Brain research, 274(1), 176-9.
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  Neurotensin-like immunoreactivity was found in nerve fibers and terminals throughout the corpus striatum of the adult cat. The densest staining was observed in the globus pallidus followed by the caudate nucleus and the putamen. In the caudate nucleus neurotensin-like immunoreactivity had a patchy distribution which was in register with a similar pattern of enkephalin-like immunoreactivity.
• Mantyh, P. W. (1983). Connections of midbrain periaqueductal gray in the monkey. I. Ascending efferent projections. Journal of neurophysiology, 49(3), 567-81.
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  1. To obtain a comprehensive map of the ascending projection of the midbrain periaqueductal gray, small amounts of [3H]leucine were injected into discrete regions of the periaqueductal gray (PAG) of the monkey. 2. Despite the fact that different regions of the PAG were injected in separate animals, the majority of the PAG's efferent projections to the forebrain remained constant. 3. The diencephalic projection areas include the nucleus reticularis thalami, the nucleus medialis dorsalis, the midline thalamic nuclei, the intralaminar thalamic nuclei, the preoptic area, and the anterior, dorsal, periventricular, ventromedial, periarcurate, lateral, and posterior hypothalamic nuclei. 4. More caudal areas receiving a projection from the PAG include the zona incerta and the mesencephalic reticular formation. 5. The main route for PAG-diencephalic projections is through the periventricular bundle. This implies that if a stimulating electrode was placed in this region, both the diencephalic leads to PAG and PAG leads to diencephalic pathways would be stimulated. This observation may explain the wide variety of side effects often encountered in stimulation-produced analgesia in humans. 6. These results support our hypothesis that the PAG functions as a visceral, nociceptive, and cognitive integrator.
• Mantyh, P. W. (1983). Connections of midbrain periaqueductal gray in the monkey. II. Descending efferent projections. Journal of neurophysiology, 49(3), 582-94.
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  1. We have defined the descending efferent projections of the midbrain periaqueductal gray (PAG) by injecting small amounts of [3H]leucine into the various regions of the squirrel monkey PAG. 2. Despite the fact that different regions of the PAG were injected in separate animals, the majority of the brain stem areas labeled remained constant. 3. The PAG exhibited a dense projection to the superior colliculus, the nucleus cuneiformis, and the locus ceruleus. Parts of the reticular formation (nucleus reticularis: pontis oralis, pontis caudalis, gigantocellularis, magnocellularis, and ventralis) received a projection from the PAG, as did the nucleus parabrachial pars lateralis, ambiguous, the nucleus raphe magnus, and raphe pallidus. 4. In contrast to the brain stem, the deep laminae of the nucleus caudalis and the deep laminae of the cervical spinal cord were labeled only after injections of the lateral aspect of the PAG. 5. The main route for the PAG leads to brain stem projections is through the lateral edge of the paramedian reticular formation. The great majority of the anterograde labeling was ipsilateral to the injection although a small contralateral projection was present. 6. These results indicate that the PAG projects to the brain stem and spinal cord in the monkey. Many of the brain stem areas that the PAG projects to are known to project to the spinal cord. These secondary spinal projections coupled with the direct PAG leads to spinal projection provide a wide variety of routes through which the PAG may influence spinal cord activity.
• Mantyh, P. W. (1983). The spinothalamic tract in the primate: a re-examination using wheatgerm agglutinin conjugated to horseradish peroxidase. Neuroscience, 9(4), 847-62.
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  The sites of termination of the primate spinothalamic tract have been reinvestigated using the anterograde transport of wheatgerm agglutinin conjugated to horseradish peroxidase. Monkeys which received an injection of the conjugate at the spinal cervical level (C7-C8) displayed a "patchy" pattern of labelling in the coronal plane in the ventral posterior lateral and caudal ventrolateral nucleus. In three dimensional reconstructions this labelling appeared to be rod-like in shape. A more homogeneous pattern of labelling was present in parts of the central lateral, posterior, suprageniculate, limitans, submedius, medial dorsal, paracentral, central medial, reuniens and periventricular nucleus. Lumbar injections (L2-L3) produced a similar although less intense pattern of labelling with only the ventral posterior lateral and ventrolateral nuclei displaying an obvious topological organization. Comparison of these results with previous physiological and pharmacological reports suggests several morphological-functional correlations: first, that both the discriminative and motivational/arousal aspects of spinothalamic tract function, associated with the lateral and medial thalamic nuclei, respectively, may be conveyed by direct spinothalamic tract projections. In support of this hypothesis medial spinothalamic tract termination sites receive a homogeneous input which does not have an obvious topographical organization, whereas lateral spinothalamic tract termination sites receive a "patchy" pattern of terminals which are topographically organized; second, that the patchy pattern of labelling observed in the coronal plane in the lateral thalamus corresponds to a "rodlike" pattern of labelling in three dimensions. This "rodlike" pattern of labelling has previously been observed for medial lemniscal projections to the thalamus and has been postulated to be the thalamic equivalent of cortical "columns"; third, that there appears to be a tight overlap between spinothalamic tract terminals and opiate receptor binding in some medial but not lateral thalamic nuclei. Such an overlap may be indicative of a pharmacological difference in the types of spinothalamic tract inputs which could be modulated by opiates at the thalamic level.
• Mantyh, P. W. (1983). The terminations of the spinothalamic tract in the cat. Neuroscience letters, 38(2), 119-24.
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  The termination sites of the spinothalamic tract (STT) in the cat have been examined using the anterograde transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). Cats which received an injection of WGA-HRP at the spinal cervical level (C7-C8) displayed anterograde labelling in the ventral posterior lateral (VPL), ventrolateral, lateral posterior, limitans, suprageniculate, medial geniculate pars magnocellularis, central lateral, paracentral, centrum medianum, submedius, central medial, reuniens, rhomboid and periventricular thalamic nuclei. Lumbar injection (L2-L3) produced a similar although less intense pattern of labelling, with only the VPL displaying an obvious topological organization. Although the STT terminations in the cat appear to be less dense than those observed in the rat or monkey the present results demonstrate that in the cat a wide variety of thalamic nuclei receive a direct STT projection and that the cat VPL, like rat, monkey and man, receives a direct STT input.
• Mantyh, P. W., & Kemp, J. A. (1983). The distribution of putative neurotransmitters in the lateral geniculate nucleus of the rat. Brain research, 288(1-2), 344-8.
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  Immunocytochemical techniques were used to examine the distribution of several putative peptidergic and aminergic neurotransmitters within the various subdivisions of the rat lateral geniculate nucleus (LG). Neuronal cell bodies, immunoreactive for enkephalin and neuropeptide Y and neuronal fibers immunoreactive for enkephalin, neuropeptide Y, substance P, vasoactive intestinal polypeptide and 5-HT were each localized within distinct subdivisions of the LG. These results suggest that the anatomical and functional differences of LG neurons are also reflected by differences in the transmitters which they utilize.
• Mantyh, P. W., & Peschanski, M. (1983). The use of wheat germ agglutinin--horseradish peroxidase conjugates for studies of anterograde axonal transport. Journal of neuroscience methods, 7(2), 117-28.
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  Injection of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP), into the hemisected spinal cord of the rat, cat and monkey consistently resulted in the intense anterograde labeling of ascending spinal projections such as the spinothalamic tract and spinocerebellar tracts and their terminal fields. Injections of WGA-HRP in the dorsal column nuclei resulted in the anterograde labeling of the medial lemniscus and its terminal fields in the thalamus. Injection of similar amounts of horseradish peroxidase alone (HRP) in hemisected animals or the dorsal column nuclei resulted in little anterograde labeling. The rate of the anterograde transport of WGA-HRP in cut axons appears to be greater than 200 mm/day. Small amounts of transneuronal labeling appeared to occur after injection of WGA-HRP in both cut axons and undamaged cell bodies. These results suggest that the amount of anterograde labeling observed after injection of WGA-HRP into both cut axons and undamaged cell bodies is significantly greater than the anterograde labeling observed after injections of HRP alone. Therefore, in the central nervous system WGA-HRP appears to be a far more effective anterograde tracer than HRP alone.
• Peschanski, M., & Mantyh, P. W. (1983). Efferent connections of the subfascicular area of the mesodiencephalic junction and its possible involvement in stimulation-produced analgesia. Brain research, 263(2), 181-90.
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  Stimulation-produced analgesia (SPA) can be induced in animals and humans from an ill-defined area of the mesodiencephalic junction lying beneath the parafascicular complex of the medial thalamus. Neurons projecting to the spinal cord, the subnucleus caudalis of the trigeminal complex, the nuclei raphé magnus and dorsalis, the inferior olivary complex and the amygdala could be observed in this area, using the retrograde transport of wheat germ agglutinin conjugated to horseradish peroxidase. On the basis of the locations of the neurons projecting to these different areas, 3 subnuclei were delineated: the rostral interstitial nucleus of the MLF lying laterally along the medial tip of the medial lemniscus, containing a few neurons projecting to the raphé nuclei and the inferior olivary complex; the subparafascicular nucleus (spf) lying medially in the rostralmost part of the area and containing neurons projecting to the amygdala and basal ganglia; the subfascicular area of the mesodiencephalic junction lying medially and caudal to the spf and containing neurons projecting to the raphé nuclei, the inferior olive, the caudalis subnucleus of the trigeminal complex and the spinal cord. The possibility that the subfascicular area of the mesodiencephalic junction is the effective site for SPA is discussed.
• Peschanski, M., Mantyh, P. W., & Besson, J. M. (1983). Spinal afferents to the ventrobasal thalamic complex in the rat: an anatomical study using wheat-germ agglutinin conjugated to horseradish peroxidase. Brain research, 278(1-2), 240-4.
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  The anterograde axonal transport of wheat-germ agglutinin conjugated to horseradish peroxidase was used to reassess the spinal afferents to the ventrobasal complex in the rat. Somatotopically-organized labeled fibers and fiber terminals could be observed in the lateral portion of the complex. The distribution of the labeling corresponded to that previously described for afferents originating from the dorsal column nuclei. Terminal fibers were drawn and their morphology analyzed in comparison to that observed for dorsal column nuclei terminals. These results are discussed with reference to the involvement of the ventrobasal complex in the transmission of nociceptive information in the rat.
• Mantyh, P. W. (1982). Forebrain projections to the periaqueductal gray in the monkey, with observations in the cat and rat. The Journal of comparative neurology, 206(2), 146-58.
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  There is considerable evidence that the midbrain periaqueductal gray (PAG) is involved in visceral, emotive, and sexual responses and in endogenous analgesic effects. To see which of the forebrain areas directly influence the PAG, small injections of horseradish peroxidase were made into the various regions of monkey, cat, and rat PAG. Despite the fact that regions of the PAG were injected in separate animals the majority of the forebrain areas labeled remained constant. Retrogradely filled pyramidal neurons in layer V were found in the frontal lobe in areas 6, 8, 9, 10, 13, and 24. Labeled neurons also appeared in the amygdala, preoptic area, and the anterior, dorsal, periventricular, ventromedial, periarcuate lateral, and posterior hypothalamic nuclei. The main route for the hypothalamic leads to PAG projection appeared to be via the periaqueductal bundle which immediately borders on the cerebral aqueduct. Labeled neurons were also observed in the zona incerta, mesencephalic reticular formation, deep layers of the superior colliculus, and the nucleus cuneiformis. Most labeling was ipsilateral to the injection site although a small but consistent contralateral labeling was present. Therefore a strict subdivision of the PAG based on each subnucleus having its own unique set of connections seems inappropriate. There were few striking differences found in the forebrain areas that project to the PAG in the three species examined. These results are discussed in terms of the possible contribution these forebrain areas have in regulating the PAG with regard to its functions as a visceral, nociceptive, and cognitive integrator.
• Mantyh, P. W. (1982). The ascending input to the midbrain periaqueductal gray of the primate. The Journal of comparative neurology, 211(1), 50-64.
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  To obtain a comprehensive map of the brainstem and spinal cord areas that project to the mesencephalic central gray small injections of horseradish peroxidase were made into various regions of the periaqueductal gray in a series of monkeys. Despite the fact that different regions of the central gray were injected in separate animals, the majority of the brainstem areas containing retrogradely filled neurons remained the same. Labeled neurons were observed in the superior colliculus, periaqueductal gray, lateral parabrachial, locus coeruleus, nucleus raphe magnus and pallidus, and a variety of brainstem reticular nuclei. In contrast to labeled brainstem areas, where labeled neurons were present predominantly ipsilateral to the injection site, the spinal trigeminal nucleus pars caudalis and the spinal cord displayed labeled cells chiefly on the side contralateral to the injection. Also in contrast to the labeled brainstem sites, where medial and lateral injection sites produced a similar pattern of labeling, medial injections in the PAG labeled almost exclusively neurons in the deep laminae (V-X) in the spinal trigeminal nucleus pars caudalis and spinal cord while more lateral injections labeled neurons in both the deep (V-X) and superficial (I) laminae. No consistent differences were noted in the location of labeled neurons in either brainstem or spinal sites after dorsal vs. ventral injections or caudal vs. rostral injection sites. The present study has demonstrated that the central gray receives afferent projections from a number of brainstem and spinal areas which are known to be involved in the modulation and/or conduction of nociception, while other inputs are probably involved in the regulation of visceral functions. These data support the hypothesis that the mesencephalic periaqueductal gray functions as a visceral, nociceptive, and cognitive integrator.
• Mantyh, P. W. (1982). The midbrain periaqueductal gray in the rat, cat, and monkey: a Nissl, Weil, and Golgi analysis. The Journal of comparative neurology, 204(4), 349-63.
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  Anatomical staining methods including Nissl, Weil, Golgi, and horseradish peroxidase stain have been used to elucidate the cyto- and myeloarchitectural organization of the periaqueductal gray in monkey, cat, and rat. From these various staining methods it appears that the periaqueductal gray is composed of a tightly packed group of cells, which show a slight increase in soma size, dendritic diameter, and degree of myelinization from central to peripheral borders. This central gray region contains a wide variety of cell types including multipolar, fusiform, stellate, and pyramidal neurons. Clearly delineated subnuclei, distinguished on the basis of soma size, dendritic arborizations, pigmentation, or evidence of cytological individuality could not be discerned in this study. Together with the immunohistochemical and connectivity studies the present data suggest that the neuronal organization of the PAG could be described as a mosaic of clusters of functional related neurons rather than as three distinct subnuclei, each with its own unique cytoarchitecture and connectivity.
• Mantyh, P. W., & Peschanski, M. (1982). Spinal projections from the periaqueductal grey and dorsal raphe in the rat, cat and monkey. Neuroscience, 7(11), 2769-76.
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  There is considerable evidence that the periaqueductal grey and the dorsal raphe contribute to an endogenous analgesia system and to the regulation of a wide variety of other responses, many of which involve spinal sites of action. To map the areas of the periaqueductal grey and dorsal raphe which contain neurons that project to the spinal cord, wheat germ agglutinin conjugated to horseradish peroxidase was injected into hemisected spinal cords in rat, cat, and monkey. After cervical or lumbar injections labelled neurons were found in the periaqueductal grey and dorsal raphe in all species examined. In the rat, labelling of the dorsal raphe is sparse but numerous labelled neurons are present in the mid and rostral periaqueductal grey. In the cat, the number of retrogradely-labelled neurons in both the dorsal raphe and the periaqueductal gray are considerable. In the monkey, like the rat, the labelling in the dorsal raphe was light but numerous labelled neurons were present in the periaqueductal grey and the adjacent nucleus cuneiformis. Injections into the lumbar spinal cord produced the same pattern of labelling as seen after cervical level injections with approximately 40% fewer labelled cells in all areas. Thus, while each species had a similar pattern of spinal projections from the periaqueductal grey and dorsal raphe, quantitative differences were evident among the species examined. These results suggest that the number of periaqueductal grey and dorsal raphe neurons projecting to the spinal cord in the rat, cat and monkey are considerably more numerous than previously reported and that the effects described during the stimulation of these regions could be, at least partly, due to the involvement of these direct pathways.

Reviews

• Paice, J. A., Mulvey, M., Bennett, M., Dougherty, P. M., Farrar, J. T., Mantyh, P. W., Miaskowski, C., Schmidt, B., & Smith, T. J. (2016. AAPT Diagnostic Criteria for Chronic Cancer Pain Conditions.
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  Chronic cancer pain is a serious complication of malignancy or its treatment. Currently, no comprehensive, universally accepted cancer pain classification system exists. Clarity in classification of common cancer pain syndromes would improve clinical assessment and management. Moreover, an evidence-based taxonomy would enhance cancer pain research efforts by providing consistent diagnostic criteria, ensuring comparability across clinical trials. As part of a collaborative effort between the Analgesic, Anesthetic, and Addiction Clinical Trial Translations Innovations Opportunities and Networks (ACTTION) and the American Pain Society (APS), the ACTTION-APS Pain Taxonomy initiative worked to develop the characteristics of an optimal diagnostic system. After the establishment of these characteristics, a working group consisting of clinicians and clinical and basic scientists with expertise in cancer and cancer-related pain was convened to generate core diagnostic criteria for an illustrative sample of 3 chronic pain syndromes associated with cancer (ie, bone pain and pancreatic cancer pain as models of pain related to a tumor) or its treatment (ie, chemotherapy-induced peripheral neuropathy). A systematic review and synthesis was conducted to provide evidence for the dimensions that comprise this cancer pain taxonomy. Future efforts will subject these diagnostic categories and criteria to systematic empirical evaluation of their feasibility, reliability, and validity and extension to other cancer-related pain syndromes.