Thomas P Davis

Interests

Research

Barriers of the CNS in Health and Disease. Central P-glycoprotein trafficking from the nucleus to cerebral vascular lumen in pain states. Pharmacology of the blood brain barrier: Targeting CNS Disorders.

Teaching

Pharmacology, Toxicology and Drug Delivery.

Courses

Scholarly Contributions

Chapters

• Davis, T. P. (2016). Development and Maintenance of the Blood Brain Barrier. In Caplan's Primer on Cerebrovascular Diseases. 2nd Edition. Academic Press.(pp 50-70). Philadelphia, PA: Elsevier Inc.
• Davis, T. P. (2016). “Glial Support of Blood-Brain Barrier Integrity: Molecular Targets for Novel Therapeutic Strategies in Stroke”. Jun Chen Ph.D. John Zhang Ph.D., and Xiaoming Hu, Ph.D., Editors. pp. 45-80. Springer International Press. Published August 16, 2016.. In Non-Neuronal Mechanisms of Brain Damage and Repair after Stroke.(pp Chapter 3.). Springer International Publishing.
• Ronaldson, P. T., & Davis, T. P. (2016). Mechanisms of Endothelial Injury and Blood-Brain Barrier Dysfunction in Stroke. In Caplan Primer on Cerebrovascular Diseases, 2nd Edition(pp TBD). Philadelphia, PA: Elsevier Inc.

Journals/Publications

• Davis, T. P. (2019). A multi-center, Phase 2 study using a continual reassessment method to determine the safety and tolerability of 3K3A-APC, a Recombinant Variant of Human Activated Protein C, in combination with tissue plasminogen activator, mechanical thrombectomy or both in moderate to severe acute ischemic stroke.. Annals of Neurology, 85, 125-136..
• Davis, T. P. (2019). Distribution of insulin in trigeminal nerve and brain after intranasal administration.. Scientific Reports, 9(1), 2621-2622.
• Davis, T. P. (2019). Functional NHE1 expression is critical to blood brain barrier integrity and sumatriptan blood to brain uptake.. PLOS ONE.
• Davis, T. P. (2019). Perivascular and perineural pathways involved in brain delivery and distribution of drugs after intranasal administration.. Pharmaceutics, 12.
• Davis, T. P. (2019). Vascular Dysfunction -The disregarded partner of Alzheimers Disease.. Alzheimers and Dementia, 15(1), 158-167.
• Davis, T. P., & Ronaldson, P. (2020). Regulation of blood brain barrier integrity by microglia in health and disease. Journal of Cerebral Blood Flow and Metabolism.
• Davis, T. P., Lyden, P., Zlokovic, B., & Griffin, J. (2020). Stroke treatment with PAR1 agents that minimize recanalization related hemorrhage.. Frontiers in Neurology.
• Davis, T. P., Ranaldson, P. T., Williams, E. I., & Batterten, R. D. (2019). Transporter-Mediated Delivery of Small Molecule Drugs to the Brain: A Critical Mechanism that can advance Therapeutic Development for Ischemic Stroke.. Pharmaceutics, 12(11), 675-695.
• Davis, T. P., Ronaldson, P., Reilly, B., & Abdullah, W. (2020). Transport properties of statins by OATP1A2 and regulation by transforming growth factor Beta signaling in human endothelial cells.. J. Pharmacology Experimental Therapeutics.
• Davis, T. P., & Lochhead, J. (2020). Structure, function and regulation of the blood brain barrier tight junction in CNS disorders.. Frontiers in Physiology, 11(914), 1-17.
• Davis, T. P., & Mukherjee, P. (2020). 3K3A - activated protein C variant does not interfere with the plasma clot lysis activity of tenecteplace.. Stroke, 51(1), 2236 - 2239..
• Davis, T. P., Williams, E., & Ronaldson, P. (2020). Transporter - mediated delivery of small molecule drugs to the brain: A critical mechanism that can advance therapeutic development for stroke. Pharmaceutics, 12(2), 154-172.
• Davis, T. P., Abdullahi, W., Lochhead, J. J., & Ronaldson, P. T. (2019). Organic anion transporting polypeptide (OATP)-mediated transport at the blood-brain barrier is required for atorvastatin-induced neuroprotection in experimental ischemic stroke. JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM, 39, 59-59.
• Cottier, K. E., Galloway, E. A., Calabrese, E. C., Tome, M. E., Liktor-Busa, E., Kim, J., Davis, T. P., Vanderah, T. W., & Largent-Milnes, T. M. (2018). Loss of Blood-Brain Barrier Integrity in a KCl-Induced Model of Episodic Headache Enhances CNS Drug Delivery. eNeuro, 5(4).
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  Cortical spreading depression (CSD) in the CNS is suggested as a common mechanism contributing to headache. Despite strong evidence for CNS involvement in headache disorders, drug development for headache disorders remains focused on peripheral targets. Difficulty in delivering drugs across the blood-brain barrier (BBB) may partially account for this disparity. It is known, however, that BBB permeability is increased during several CNS pathologies. In this study, we investigated BBB changes in response to KCl-induced CSD events and subsequent allodynia in rats. Cortical KCl injection in awake, freely moving rats produced facial allodynia with peak intensity between 1.5 and 3 h and CSD induction within 0.5-2 h postinjection. Brain perfusion of C-sucrose as a marker of BBB paracellular permeability revealed increased leak in the cortex, but not brainstem, beginning 0.5 h post-KCl injection and resolving within 6 h; no changes in tight junction (TJ) proteins occludin or claudin-5 expression were observed. Acute pretreatment with topiramate to inhibit CSD did not prevent the increased BBB paracellular permeability. CNS delivery of the abortive anti-migraine agent sumatriptan was increased in the cortex 1.5 h post-KCl injection. Surprisingly, sumatriptan uptake was also increased in the brainstem following CSD induction, suggesting regulation of active transport mechanisms at the BBB. Together, these results demonstrate the ability of CSD events to produce transient, time-dependent changes in BBB permeability when allodynia is present and to mediate access of clinically relevant therapeutics (i.e., sumatriptan) to the CNS.
• Davis, T. P. (2018). Loss of Blood-Brain Barrier Integrity in a KCl induced model of Episodic Headache enhances CNS drug delivery.. eNEuro, 0116-18.
• Davis, T. P., Time, M., & Schaefer, C. (2018). Acute pain alters P-glycoprotein-containing protein complexes in rat cerebral complexes: Implications for P-glycoprotein trafficking.. J. Cerebral Blood Flow and Metabolism, 38, 2209-2222.
• Lyden, P., Pryor, K. E., Coffey, C. S., Cudkowicz, M., Conwit, R., Jadhav, A., Sawyer, R. N., Claassen, J., Adeoye, O., Song, S., Hannon, P., Rost, N. S., Hinduja, A., Torbey, M., Lee, J. M., Benesch, C., Rippee, M., Rymer, M., Froehler, M. T., , Haley, E. C., et al. (2018). Final Results of the RHAPSODY trial: A multi-center, Phase 2 trial using a continual reassessment method to determine the safety and tolerability of 3K3A-APC, a Recombinant Variant of Human Activated Protein C, in combination with tissue plasminogen activator, mechanical thrombectomy or both in moderate to severe acute ischemic stroke. Annals of neurology.
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  Agonism of the protease activated receptor (PAR) 1 by activated protein C (APC) provides neuroprotection and vasculoprotection in experimental neuro-injury models. The pleiotropic PAR1 agonist, 3K3A-APC, reduces neurologic injury and promotes vascular integrity; 3K3A-APC proved safe in human volunteers. We performed a randomized, controlled, blinded, trial to determine the maximally tolerated dose (MTD) of 3K3A-APC in ischemic stroke patients.
• Schaefer, C. P., Arkwright, N. B., Jacobs, L. M., Jarvis, C. K., Hunn, K. C., Largent-Milnes, T. M., Tome, M. E., & Davis, T. P. (2018). Chronic morphine exposure potentiates p-glycoprotein trafficking from nuclear reservoirs in cortical rat brain microvessels. PloS one, 13(2), e0192340.
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  The rates of opioid prescription and use have continued to increase over the last few decades resulting in a greater number of opioid tolerant patients. Treatment of acute pain from surgery and injury is a clinical challenge for these patients. Several pain management strategies including prescribing increased opioids are used clinically with limited success; all currently available strategies have significant limitations. Many opioids are a substrate for p-glycoprotein (p-gp), an efflux transporter at the blood-brain barrier (BBB). Increased p-gp is associated with a decreased central nervous system uptake and analgesic efficacy of morphine. Our laboratory previously found that acute peripheral inflammatory pain (PIP) induces p-gp trafficking from the nucleus to the luminal surface of endothelial cells making up the BBB concomitant with increased p-gp activity and decreased morphine analgesic efficacy. In the current study, we tested whether PIP-induced p-gp trafficking could contribute to decreased opioid efficacy in morphine tolerant rats. A 6-day continuous dosing of morphine from osmotic minipumps was used to establish morphine tolerance in female rats. PIP induced p-gp trafficking away from nuclear stores showed a 2-fold increase in morphine tolerant rats. This observation suggests that p-gp trafficking contributes to the decreased morphine analgesic effects in morphine tolerant rats experiencing an acute pain stimulus. Attenuating p-gp trafficking during an acute pain stimulus could improve pain management by increasing the amount of opioid that could reach CNS analgesic targets and decrease the need for the dose escalation that is a serious challenge in pain management.
• Yang, J., Reilly, B. G., Davis, T. P., & Ronaldson, P. T. (2018). Modulation of Opioid Transport at the Blood-Brain Barrier by Altered ATP-Binding Cassette (ABC) Transporter Expression and Activity. Pharmaceutics, 10(4).
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  Opioids are highly effective analgesics that have a serious potential for adverse drug reactions and for development of addiction and tolerance. Since the use of opioids has escalated in recent years, it is increasingly important to understand biological mechanisms that can increase the probability of opioid-associated adverse events occurring in patient populations. This is emphasized by the current opioid epidemic in the United States where opioid analgesics are frequently abused and misused. It has been established that the effectiveness of opioids is maximized when these drugs readily access opioid receptors in the central nervous system (CNS). Indeed, opioid delivery to the brain is significantly influenced by the blood-brain barrier (BBB). In particular, ATP-binding cassette (ABC) transporters that are endogenously expressed at the BBB are critical determinants of CNS opioid penetration. In this review, we will discuss current knowledge on the transport of opioid analgesic drugs by ABC transporters at the BBB. We will also examine how expression and trafficking of ABC transporters can be modified by pain and/or opioid pharmacotherapy, a novel mechanism that can promote opioid-associated adverse drug events and development of addiction and tolerance.
• Abdullahi, W., Brzica, H., Ibbotson, K., Davis, T. P., & Ronaldson, P. T. (2017). Bone morphogenetic protein-9 increases the functional expression of organic anion transporting polypeptide 1a4 at the blood-brain barrier via the activin receptor-like kinase-1 receptor. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism, 37(7), 2340-2345.
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  Targeting uptake transporters such as organic anion transporting polypeptide 1a4 (Oatp1a4) at the blood-brain barrier (BBB) can facilitate central nervous system (CNS) drug delivery. Effective blood-to-brain drug transport via this strategy requires characterization of mechanisms that modulate BBB transporter expression and/or activity. Here, we show that activation of activin receptor-like kinase (ALK)-1 using bone morphogenetic protein (BMP)-9 increases Oatp1a4 protein expression in rat brain microvessels in vivo. These data indicate that targeting ALK1 signaling with BMP-9 modulates BBB Oatp1a4 expression, presenting a unique opportunity to optimize drug delivery and improve pharmacotherapy for CNS diseases.
• Abdullahi, W., Davis, T. P., & Ronaldson, P. T. (2017). Functional Expression of P-glycoprotein and Organic Anion Transporting Polypeptides at the Blood-Brain Barrier: Understanding Transport Mechanisms for Improved CNS Drug Delivery?. The AAPS journal, 19(4), 931-939.
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  Drug delivery to the central nervous system (CNS) is greatly limited by the blood-brain barrier (BBB). Physical and biochemical properties of the BBB have rendered treatment of CNS diseases, including those with a hypoxia/reoxygenation (H/R) component, extremely difficult. Targeting endogenous BBB transporters from the ATP-binding cassette (ABC) superfamily (i.e., P-glycoprotein (P-gp)) or from the solute carrier (SLC) family (i.e., organic anion transporting polypeptides (OATPs in humans; Oatps in rodents)) has been suggested as a strategy that can improve delivery of drugs to the brain. With respect to P-gp, direct pharmacological inhibition using small molecules or selective regulation by targeting intracellular signaling pathways has been explored. These approaches have been largely unsuccessful due to toxicity issues and unpredictable pharmacokinetics. Therefore, our laboratory has proposed that optimization of CNS drug delivery, particularly for treatment of diseases with an H/R component, can be achieved by targeting Oatp isoforms at the BBB. As the major drug transporting Oatp isoform, Oatp1a4 has demonstrated blood-to-brain transport of substrate drugs with neuroprotective properties. Furthermore, our laboratory has shown that targeting Oatp1a4 regulation (i.e., TGF-β signaling mediated via the ALK-1 and ALK-5 transmembrane receptors) represents an opportunity to control Oatp1a4 functional expression for the purpose of delivering therapeutics to the CNS. In this review, we will discuss limitations of targeting P-gp-mediated transport activity and the advantages of targeting Oatp-mediated transport. Through this discussion, we will also provide critical information on novel approaches to improve CNS drug delivery by targeting endogenous uptake transporters expressed at the BBB.
• Davis, T. P., Schaefer, C., & Tome, M. (2017). The opioid epidemic: A central role for the Blood Brain Barrier in opioid analgesia and abuse.. Fluids and Barriers of the CNS, 14(32), 1-11. doi:10.1186/s12987-017-0080-3
• Lochhead, J. J., Ronaldson, P. T., & Davis, T. P. (2017). Hypoxic Stress and Inflammatory Pain Disrupt Blood-Brain Barrier Tight Junctions: Implications for Drug Delivery to the Central Nervous System. The AAPS journal, 19(4), 910-920.
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  A functional blood-brain barrier (BBB) is necessary to maintain central nervous system (CNS) homeostasis. Many diseases affecting the CNS, however, alter the functional integrity of the BBB. It has been shown that various diseases and physiological stressors can impact the BBB's ability to selectively restrict passage of substances from the blood to the brain. Modifications of the BBB's permeability properties can potentially contribute to the pathophysiology of CNS diseases and result in altered brain delivery of therapeutic agents. Hypoxia and/or inflammation are central components of a number of diseases affecting the CNS. A number of studies indicate hypoxia or inflammatory pain increase BBB paracellular permeability, induce changes in the expression and/or localization of tight junction proteins, and affect CNS drug uptake. In this review, we look at what is currently known with regard to BBB disruption following a hypoxic or inflammatory insult in vivo. Potential mechanisms involved in altering tight junction components at the BBB are also discussed. A more detailed understanding of the mediators involved in changing BBB functional integrity in response to hypoxia or inflammatory pain could potentially lead to new treatments for CNS diseases with hypoxic or inflammatory components. Additionally, greater insight into the mechanisms involved in TJ rearrangement at the BBB may lead to novel strategies to pharmacologically increase delivery of drugs to the CNS.
• Sandweiss, A. J., Cottier, K. E., McIntosh, M. I., Dussor, G., Davis, T. P., Vanderah, T. W., & Largent-Milnes, T. M. (2017). 17-β-Estradiol induces spreading depression and pain behavior in alert female rats. Oncotarget, 8(69), 114109-114122.
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  Test the putative contribution of 17-β-estradiol in the development of spreading depression (SD) events and head pain in awake, non-restrained rats.
• Bosetti, F., Galis, Z. S., Bynoe, M. S., Charette, M., Cipolla, M. J., Del Zoppo, G. J., Gould, D., Hatsukami, T. S., Jones, T. L., Koenig, J. I., Lutty, G. A., Maric-Bilkan, C., Stevens, T., Tolunay, H. E., Koroshetz, W., & , “. B. (2016). "Small Blood Vessels: Big Health Problems?": Scientific Recommendations of the National Institutes of Health Workshop. Journal of the American Heart Association, 5(11).
• Tome, M. E., Herndon, J. M., Schaefer, C. P., Jacobs, L. M., Zhang, Y., Jarvis, C. K., & Davis, T. P. (2016). P-glycoprotein traffics from the nucleus to the plasma membrane in rat brain endothelium during inflammatory pain. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism, 36(11), 1913-1928.
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  P-glycoprotein (PgP), a drug efflux pump in blood-brain barrier endothelial cells, is a major clinical obstacle for effective central nervous system drug delivery. Identifying PgP regulatory pathways that can be exploited clinically is critical for improving central nervous system drug delivery. We previously found that PgP activity increases in rat brain microvessels concomitant with decreased central nervous system drug delivery in response to acute peripheral inflammatory pain. In the current study, we tested the hypothesis that PgP traffics to the luminal plasma membrane of the microvessel endothelial cells from intracellular stores during peripheral inflammatory pain. Using immunofluorescence microscopy, we detected PgP in endothelial cell nuclei and in the luminal plasma membrane in control animals. Following peripheral inflammatory pain, luminal PgP staining increased while staining in the nucleus decreased. Biochemical analysis of nuclear PgP content confirmed our visual observations. Peripheral inflammatory pain also increased endothelial cell luminal staining of polymerase 1 and transcript release factor/cavin1 and serum deprivation response protein/cavin2, two caveolar scaffold proteins, without changing caveolin1 or protein kinase C delta binding protein/cavin3 location. Our data (a) indicate that PgP traffics from stores in the nucleus to the endothelial cell luminal membrane in response to peripheral inflammatory pain; (b) provide an explanation for our previous observation that peripheral inflammatory pain inhibits central nervous system drug uptake; and (c) suggest a novel regulatory mechanism for PgP activity in rat brain.
• Davis, T. P., Abbruscato, T. J., & Egleton, R. D. (2015). Peptides at the blood brain barrier: Knowing me knowing you. Peptides, 72, 50-6.
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  When the Davis Lab was first asked to contribute to this special edition of Peptides to celebrate the career and influence of Abba Kastin on peptide research, it felt like a daunting task. It is difficult to really understand and appreciate the influence that Abba has had, not only on a generation of peptide researchers, but also on the field of blood brain barrier (BBB) research, unless you lived it as we did. When we look back at our careers and those of our former students, one can truly see that several of Abba's papers played an influential role in the development of our personal research programs.
• Ronaldson, P. T., & Davis, T. P. (2015). Targeting transporters: promoting blood-brain barrier repair in response to oxidative stress injury. Brain research, 1623, 39-52.
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  The blood-brain barrier (BBB) is a physical and biochemical barrier that precisely regulates the ability of endogenous and exogenous substances to accumulate within brain tissue. It possesses structural and biochemical features (i.e., tight junction and adherens junction protein complexes, influx and efflux transporters) that work in concert to control solute permeation. Oxidative stress, a critical component of several diseases including cerebral hypoxia/ischemia and peripheral inflammatory pain, can cause considerable injury to the BBB and lead to significant CNS pathology. This suggests a critical need for novel therapeutic approaches that can protect the BBB in diseases with an oxidative stress component. Recent studies have identified molecular targets (i.e., putative membrane transporters, intracellular signaling systems) that can be exploited for optimization of endothelial drug delivery or for control of transport of endogenous substrates such as the antioxidant glutathione (GSH). In particular, targeting transporters offers a unique approach to protect BBB integrity by promoting repair of cell-cell interactions at the level of the brain microvascular endothelium. This review summarizes current knowledge in this area and emphasizes those targets that present considerable opportunity for providing BBB protection and/or promoting BBB repair in the setting of oxidative stress. This article is part of a Special Issue entitled SI: Cell Interactions In Stroke.
• Tome, M. E., Schaefer, C. P., Jacobs, L. M., Zhang, Y., Herndon, J. M., Matty, F. O., & Davis, T. P. (2015). Identification of P-glycoprotein co-fractionating proteins and specific binding partners in rat brain microvessels. Journal of neurochemistry, 134(2), 200-10.
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  Drug delivery to the brain for the treatment of pathologies with a CNS component is a significant clinical challenge. P-glycoprotein (PgP), a drug efflux pump in the endothelial cell membrane, is a major factor in preventing therapeutics from crossing the blood-brain barrier (BBB). Identifying PgP regulatory mechanisms is key to developing agents to modulate PgP activity. Previously, we found that PgP trafficking was altered concomitant with increased PgP activity and disassembly of high molecular weight PgP-containing complexes during acute peripheral inflammatory pain. These data suggest that PgP activity is post-translationally regulated at the BBB. The goal of the current study was to identify proteins that co-localize with PgP in rat brain microvessel endothelial cell membrane microdomains and use the data to suggest potential regulatory mechanisms. Using new density gradients of microvessel homogenates, we identified two unique pools (1,2) of PgP in membrane fractions. Caveolar constituents, caveolin1, cavin1, and cavin2, co-localized with PgP in these fractions indicating the two pools contained caveolae. A chaperone (Hsc71), protein disulfide isomerase and endosomal/lysosomal sorting proteins (Rab5, Rab11a) also co-fractionated with PgP in the gradients. These data suggest signaling pathways with a potential role in post-translational regulation of PgP activity at the BBB.
• Davis, T., Ronaldson, P. T., & Davis, T. P. (2013). Targeted drug delivery to treat pain and cerebral hypoxia. Pharmacological reviews, 65(1).
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  Limited drug penetration is an obstacle that is often encountered in treatment of central nervous system (CNS) diseases including pain and cerebral hypoxia. Over the past several years, biochemical characteristics of the brain (i.e., tight junction protein complexes at brain barrier sites, expression of influx and efflux transporters) have been shown to be directly involved in determining CNS permeation of therapeutic agents; however, the vast majority of these studies have focused on understanding those mechanisms that prevent drugs from entering the CNS. Recently, this paradigm has shifted toward identifying and characterizing brain targets that facilitate CNS drug delivery. Such targets include the organic anion-transporting polypeptides (OATPs in humans; Oatps in rodents), a family of sodium-independent transporters that are endogenously expressed in the brain and are involved in drug uptake. OATP/Oatp substrates include drugs that are efficacious in treatment of pain and/or cerebral hypoxia (i.e., opioid analgesic peptides, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors). This clearly suggests that OATP/Oatp isoforms are viable transporter targets that can be exploited for optimization of drug delivery to the brain and, therefore, improved treatment of CNS diseases. This review summarizes recent knowledge in this area and emphasizes the potential that therapeutic targeting of OATP/Oatp isoforms may have in facilitating CNS drug delivery and distribution. Additionally, information presented in this review will point to novel strategies that can be used for treatment of pain and cerebral hypoxia.
• Davis, T., Slosky, L. M., Thompson, B. J., Sanchez-Covarrubias, L., Zhang, Y., Laracuente, M., Vanderah, T. W., Ronaldson, P. T., & Davis, T. P. (2013). Acetaminophen modulates p-glycoprotein functional expression at the blood-brain barrier by a constitutive androstane receptor-dependent mechanism. Molecular pharmacology, 84(5).
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  Effective pharmacologic treatment of pain with opioids requires that these drugs attain efficacious concentrations in the central nervous system (CNS). A primary determinant of CNS drug permeation is P-glycoprotein (P-gp), an endogenous blood-brain barrier (BBB) efflux transporter that is involved in brain-to-blood transport of opioid analgesics (i.e., morphine). Recently, the nuclear receptor constitutive androstane receptor (CAR) has been identified as a regulator of P-gp functional expression at the BBB. This is critical to pharmacotherapy of pain/inflammation, as patients are often administered acetaminophen (APAP), a CAR-activating ligand, in conjunction with an opioid. Our objective was to investigate, in vivo, the role of CAR in regulation of P-gp at the BBB. Following APAP treatment, P-gp protein expression was increased up to 1.4-1.6-fold in a concentration-dependent manner. Additionally, APAP increased P-gp transport of BODIPY-verapamil in freshly isolated rat brain capillaries. This APAP-induced increase in P-gp expression and activity was attenuated in the presence of CAR pathway inhibitor okadaic acid or transcriptional inhibitor actinomycin D, suggesting P-gp regulation is CAR-dependent. Furthermore, morphine brain accumulation was enhanced by P-gp inhibitors in APAP-treated animals, suggesting P-gp-mediated transport. A warm-water (50°C) tail-flick assay revealed a significant decrease in morphine analgesia in animals treated with morphine 3 or 6 hours after APAP treatment, as compared with animals treated concurrently. Taken together, our data imply that inclusion of APAP in a pain treatment regimen activates CAR at the BBB and increases P-gp functional expression, a clinically significant drug-drug interaction that modulates opioid analgesic efficacy.
• Fernandez, J. A., Mosnier, L. O., Davis, T. P., Zlokovic, B. V., & Griffin, J. H. (2013). Influence of the 3K3A-activated protein C variant on the in vitro fibrinolytic activity of tPA. JOURNAL OF THROMBOSIS AND HAEMOSTASIS, 11, 713-714.
• Mosnier, L. O., Fernandez, J. A., Davis, T. P., Zlokovic, B. V., & Griffin, J. H. (2013). Influence of the 3K3A-activated protein C variant on the plasma clot lysis activity of t-PA and of t-PA on the variant's anticoagulant activity. JOURNAL OF THROMBOSIS AND HAEMOSTASIS, 11(11), 2059-2062.
• Davis, T., McCaffrey, G., & Davis, T. P. (2012). Physiology and pathophysiology of the blood-brain barrier: P-glycoprotein and occludin trafficking as therapeutic targets to optimize central nervous system drug delivery. Journal of investigative medicine : the official publication of the American Federation for Clinical Research, 60(8).
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  The blood-brain barrier (BBB) is a physical and metabolic barrier that separates the central nervous system from the peripheral circulation. Central nervous system drug delivery across the BBB is challenging, primarily because of the physical restriction of paracellular diffusion between the endothelial cells that comprise the microvessels of the BBB and the activity of efflux transporters that quickly expel back into the capillary lumen a wide variety of xenobiotics. Therapeutic manipulation of protein trafficking is emerging as a novel means of modulating protein function, and in this minireview, the targeting of the trafficking of 2 key BBB proteins, P-glycoprotein and occludin, is presented as a novel, reversible means of optimizing central nervous system drug delivery.
• Davis, T., McCaffrey, G., Staatz, W. D., Sanchez-Covarrubias, L., Finch, J. D., Demarco, K., Laracuente, M., Ronaldson, P. T., & Davis, T. P. (2012). P-glycoprotein trafficking at the blood-brain barrier altered by peripheral inflammatory hyperalgesia. Journal of neurochemistry, 122(5).
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  P-glycoprotein (ABCB1/MDR1, EC 3.6.3.44), the major efflux transporter at the blood-brain barrier (BBB), is a formidable obstacle to CNS pharmacotherapy. Understanding the mechanism(s) for increased P-glycoprotein activity at the BBB during peripheral inflammatory pain is critical in the development of novel strategies to overcome the significant decreases in CNS analgesic drug delivery. In this study, we employed the λ-carrageenan pain model (using female Sprague-Dawley rats), combined with confocal microscopy and subcellular fractionation of cerebral microvessels, to determine if increased P-glycoprotein function, following the onset of peripheral inflammatory pain, is associated with a change in P-glycoprotein trafficking which leads to pain-induced effects on analgesic drug delivery. Injection of λ-carrageenan into the rat hind paw induced a localized, inflammatory pain (hyperalgesia) and simultaneously, at the BBB, a rapid change in colocalization of P-glycoprotein with caveolin-1, a key scaffolding/trafficking protein. Subcellular fractionation of isolated cerebral microvessels revealed that the bulk of P-glycoprotein constitutively traffics to membrane domains containing high molecular weight, disulfide-bonded P-glycoprotein-containing structures that cofractionate with membrane domains enriched with monomeric and high molecular weight, disulfide-bonded, caveolin-1-containing structures. Peripheral inflammatory pain promoted a dynamic redistribution between membrane domains of P-glycoprotein and caveolin-1. Disassembly of high molecular weight P-glycoprotein-containing structures within microvascular endothelial luminal membrane domains was accompanied by an increase in ATPase activity, suggesting a potential for functionally active P-glycoprotein. These results are the first observation that peripheral inflammatory pain leads to specific structural changes in P-glycoprotein responsible for controlling analgesic drug delivery to the CNS.
• Davis, T., Ronaldson, P. T., & Davis, T. P. (2012). Blood-brain barrier integrity and glial support: mechanisms that can be targeted for novel therapeutic approaches in stroke. Current pharmaceutical design, 18(25).
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  The blood-brain barrier (BBB) is a critical regulator of brain homeostasis. Additionally, the BBB is the most significant obstacle to effective CNS drug delivery. It possesses specific charcteristics (i.e., tight junction protein complexes, influx and efflux transporters) that control permeation of circulating solutes including therapeutic agents. In order to form this "barrier," brain microvascular endothelial cells require support of adjacent astrocytes and microglia. This intricate relationship also occurs between endothelial cells and other cell types and structures of the CNS (i.e., pericytes, neurons, extracellular matrix), which implies existence of a "neurovascular unit." Ischemic stroke can disrupt the neurovascular unit at both the structural and functional level, which leads to an increase in leak across the BBB. Recent studies have identified several pathophysiological mechanisms (i.e., oxidative stress, activation of cytokine-mediated intracellular signaling systems) that mediate changes in the neurovascular unit during ischemic stroke. This review summarizes current knowledge in this area and emphasizes pathways (i.e., oxidative stress, cytokine-mediated intracellular signaling, glial-expressed receptors/targets) that can be manipulated pharmacologically for i) preservation of BBB and glial integrity during ischemic stroke and ii) control of drug permeation and/or transport across the BBB. Targeting these pathways present a novel opportunity for optimization of CNS delivery of therapeutics in the setting of ischemic stroke.
• Davis, T., Ronaldson, P. T., & Davis, T. P. (2011). Targeting blood-brain barrier changes during inflammatory pain: an opportunity for optimizing CNS drug delivery. Therapeutic delivery, 2(8).
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  The blood-brain barrier (BBB) is the most significant obstacle to effective CNS drug delivery. It possesses structural and biochemical features (i.e., tight-junction protein complexes and, influx and efflux transporters) that restrict xenobiotic permeation. Pathophysiological stressors (i.e., peripheral inflammatory pain) can alter BBB tight junctions and transporters, which leads to drug-permeation changes. This is especially critical for opioids, which require precise CNS concentrations to be safe and effective analgesics. Recent studies have identified molecular targets (i.e., endogenous transporters and intracellular signaling systems) that can be exploited for optimization of CNS drug delivery. This article summarizes current knowledge in this area and emphasizes those targets that present the greatest opportunity for controlling drug permeation and/or drug transport across the BBB in an effort to achieve optimal CNS opioid delivery.
• Davis, T., Ronaldson, P. T., Finch, J. D., Demarco, K. M., Quigley, C. E., & Davis, T. P. (2011). Inflammatory pain signals an increase in functional expression of organic anion transporting polypeptide 1a4 at the blood-brain barrier. The Journal of pharmacology and experimental therapeutics, 336(3).
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  Pain is a dominant symptom associated with inflammatory conditions. Pharmacotherapy with opioids may be limited by poor blood-brain barrier (BBB) permeability. One approach that may improve central nervous system (CNS) delivery is to target endogenous BBB transporters such as organic anion-transporting polypeptide 1a4 (Oatp1a4). It is critical to identify and characterize biological mechanisms that enable peripheral pain/inflammation to "transmit" upstream signals and alter CNS drug transport processes. Our goal was to investigate, in vivo, BBB functional expression of Oatp1a4 in animals subjected to peripheral inflammatory pain. Inflammatory pain was induced in female Sprague-Dawley rats (200-250 g) by subcutaneous injection of 3% λ-carrageenan into the right hind paw; control animals were injected with 0.9% saline. In rat brain microvessels, Oatp1a4 expression was increased during acute pain/inflammation. Uptake of taurocholate and [d-penicillamine(2,5)]-enkephalin, two established Oatp substrates, was increased in animals subjected to peripheral pain, suggesting increased Oatp1a4-mediated transport. Inhibition of inflammatory pain with the anti-inflammatory drug diclofenac attenuated these changes in Oatp1a4 functional expression, suggesting that inflammation in the periphery can modulate BBB transporters. In addition, diclofenac prevented changes in the peripheral signaling cytokine transforming growth factor-β1 (TGF-β1) levels and brain microvascular TGF-β receptor expression induced by inflammatory pain. Pretreatment with the pharmacological TGF-β receptor inhibitor 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide (SB431542) increased Oatp1a4 functional expression in λ-carrageenan-treated animals and saline controls, suggesting that TGF-β signaling is involved in Oatp1a4 regulation at the BBB. Our findings indicate that BBB transporters (i.e., Oatp1a4) can be targeted during drug development to improve CNS delivery of highly promising therapeutics.
• Willis, C. L., Brooks, T. A., & Davis, T. P. (2011). Chronic Inflammation Pain and the Neurovascular Unit: A Central Role for Glia in Maintaining BBB Integrity (vol 14, pg 1625, 2008). CURRENT PHARMACEUTICAL DESIGN, 17(22), 2367-2367.
• Davis, T., Lochhead, J. J., McCaffrey, G., Quigley, C. E., Finch, J., DeMarco, K. M., Nametz, N., & Davis, T. P. (2010). Oxidative stress increases blood-brain barrier permeability and induces alterations in occludin during hypoxia-reoxygenation. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism, 30(9).
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  The blood-brain barrier (BBB) has a critical role in central nervous system homeostasis. Intercellular tight junction (TJ) protein complexes of the brain microvasculature limit paracellular diffusion of substances from the blood into the brain. Hypoxia and reoxygenation (HR) is a central component to numerous disease states and pathologic conditions. We have previously shown that HR can influence the permeability of the BBB as well as the critical TJ protein occludin. During HR, free radicals are produced, which may lead to oxidative stress. Using the free radical scavenger tempol (200 mg/kg, intraperitoneal), we show that oxidative stress produced during HR (6% O(2) for 1 h, followed by room air for 20 min) mediates an increase in BBB permeability in vivo using in situ brain perfusion. We also show that these changes are associated with alterations in the structure and localization of occludin. Our data indicate that oxidative stress is associated with movement of occludin away from the TJ. Furthermore, subcellular fractionation of cerebral microvessels reveals alterations in occludin oligomeric assemblies in TJ associated with plasma membrane lipid rafts. Our data suggest that pharmacological inhibition of disease states with an HR component may help preserve BBB functional integrity.
• Davis, T., Willis, C. L., Meske, D. S., & Davis, T. P. (2010). Protein kinase C activation modulates reversible increase in cortical blood-brain barrier permeability and tight junction protein expression during hypoxia and posthypoxic reoxygenation. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism, 30(11).
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  Hypoxia (Hx) is a component of many disease states including stroke. Ischemic stroke occurs when there is a restriction of cerebral blood flow and oxygen to part of the brain. During the ischemic, and subsequent reperfusion phase of stroke, blood-brain barrier (BBB) integrity is lost with tight junction (TJ) protein disruption. However, the mechanisms of Hx and reoxygenation (HR)-induced loss of BBB integrity are not fully understood. We examined the role of protein kinase C (PKC) isozymes in modifying TJ protein expression in a rat model of global Hx. The Hx (6% O(2)) induced increased hippocampal and cortical vascular permeability to 4 and 10 kDa dextran fluorescein isothiocyanate (FITC) and endogenous rat-IgG. Cortical microvessels revealed morphologic changes in nPKC-θ distribution, increased nPKC-θ and aPKC-ζ protein expression, and activation by phosphorylation of nPKC-θ (Thr538) and aPKC-ζ (Thr410) residues after Hx treatment. Claudin-5, occludin, and ZO-1 showed disrupted organization at endothelial cell margins, whereas Western blot analysis showed increased TJ protein expression after Hx. The PKC inhibition with chelerythrine chloride (5 mg/kg intraperitoneally) attenuated Hx-induced hippocampal vascular permeability and claudin-5, PKC (θ and ζ) expression, and phosphorylation. This study supports the hypothesis that nPKC-θ and aPKC-ζ signaling mediates TJ protein disruption resulting in increased BBB permeability.
• Davis, T., McCaffrey, G., Willis, C. L., Staatz, W. D., Nametz, N., Quigley, C. A., Hom, S., Lochhead, J. J., & Davis, T. P. (2009). Occludin oligomeric assemblies at tight junctions of the blood-brain barrier are altered by hypoxia and reoxygenation stress. Journal of neurochemistry, 110(1).
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  Hypoxic (low oxygen) and reperfusion (post-hypoxic reoxygenation) phases of stroke promote an increase in microvascular permeability at tight junctions (TJs) of the blood-brain barrier (BBB) that may lead to cerebral edema. To investigate the effect of hypoxia (Hx) and reoxygenation on oligomeric assemblies of the transmembrane TJ protein occludin, rats were subjected to either normoxia (Nx, 21% O(2), 60 min), Hx (6% O(2), 60 min), or hypoxia/reoxygenation (H/R, 6% O(2), 60 min followed by 21% O(2), 10 min). After treatment, cerebral microvessels were isolated, fractionated by detergent-free density gradient centrifugation, and occludin oligomeric assemblies associated with plasma membrane lipid rafts were solubilized by perfluoro-octanoic acid (PFO) exclusively as high molecular weight protein complexes. Analysis by non-reducing and reducing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis/western blot of PFO-solubilized occludin revealed that occludin oligomeric assemblies co-localizing with 'TJ-associated' raft domains contained a high molecular weight 'structural core' that was resistant to disassembly by either SDS or a hydrophilic reducing agent ex vivo, and by Hx and H/R conditions in vivo. However, exposure of PFO-solubilized occludin oligomeric assemblies to SDS ex vivo revealed the non-covalent association of a significant amount of dimeric and monomeric occludin isoforms to the disulfide-bonded inner core, and dispersal of these non-covalently attached occludin subunits to lipid rafts of higher density in vivo was differentially promoted by Hx and H/R. Our data suggest a model of isoform interaction within occludin oligomeric assemblies at the BBB that enables occludin to simultaneously perform a structural role in inhibiting paracellular diffusion, and a signaling role involving interactions of dimeric and monomeric occludin isoforms with a variety of regulatory molecules within different plasma membrane lipid raft domains.
• Davis, T., Ronaldson, P. T., Demarco, K. M., Sanchez-Covarrubias, L., Solinsky, C. M., & Davis, T. P. (2009). Transforming growth factor-beta signaling alters substrate permeability and tight junction protein expression at the blood-brain barrier during inflammatory pain. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism, 29(6).
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  Our laboratory has shown that peripheral inflammatory pain induced by lambda-carrageenan (CIP) can increase blood-brain barrier (BBB) permeability and alter tight junction (TJ) protein expression leading to changes in BBB functional integrity. However, the intracellular signaling mechanisms involved in this pathophysiologic response have not been elucidated. Transforming growth factor (TGF)-beta signaling pathways are known to regulate vascular integrity and permeability. Therefore, we examined the function of TGF-beta signaling at the BBB in rats subjected to CIP. During CIP, serum TGF-beta1 and protein expression of the TGF-beta receptor activin receptor-like kinase-5 (ALK5) were reduced. Brain permeability to (14)C-sucrose was increased and expression of TJ proteins (i.e., claudin-5, occludin, zonula occluden (ZO-1)) were also altered after 3 h CIP. Pharmacological inhibition of ALK5 with the selective inhibitor SB431542 further enhanced brain uptake of (14)C-sucrose, increased TJ protein expression (i.e., claudin-3, claudin-5, occludin, ZO-1), and decreased nuclear expression of TGF-beta/ALK5 signaling molecules (i.e., Smad2, Smad3), which suggests a role for TGF-beta/ALK5 signaling in the regulation of BBB integrity. Interestingly, administration of exogenous TGF-beta1 before CIP activated the TGF-beta/ALK5 pathway and reduced BBB permeability to (14)C-sucrose. Taken together, our data show that TGF-beta/ALK5 signaling is, in part, involved in the regulation of BBB functional integrity.
• Ronaldson, P. T., Demarco, K. M., Sanchez-Covarrubias, L., Solinsky, C. M., & Davis, T. P. (2009). Transforming growth factor-beta (TGF-beta) signaling is involved in the regulation of blood-brain barrier (BBB) functional integrity during peripheral inflammatory pain. JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM, 29, S484-S484.
• Davis, T., Brooks, T. A., Nametz, N., Charles, R., & Davis, T. P. (2008). Diclofenac attenuates the regional effect of lambda-carrageenan on blood-brain barrier function and cytoarchitecture. The Journal of pharmacology and experimental therapeutics, 325(2).
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  The microenvironment of the brain requires tight regulation for proper neuronal function. Protecting the central nervous system (CNS) from the varying concentrations of ions, proteins, and toxins in the periphery is the dynamically regulated blood-brain barrier (BBB). Recent studies have demonstrated significant modulation of the BBB in a number of diseases and physiological states, including pain. This study expands on previous explorations of acute and chronic pain-induced effects on the function and molecular cytoarchitecture of the barrier. It describes the role of cyclooxygenase (COX) up-regulation by blocking with diclofenac (30 mg/kg, i.p.), and it examines the variation in BBB regulation through various brain regions. Edema and hyperalgesia were induced by lambda-carrageenan and attenuated by the additional administration of diclofenac. Examination of unidirectional [14C]sucrose permeability with multitime in situ perfusion studies demonstrated that lambda-carrageenan significantly increased cerebral permeability and decreased brainstem permeability. There were no significant changes in any of the other brain regions examined. These permeability changes correlated with up- and down-regulation of the tight junction (TJ) protein claudin-5 in the cerebrum and brainstem, respectively. Diclofenac administration attenuated the cerebral permeability uptake as well as the claudin-5 up-regulation. In addition, diclofenac reversed the lowered permeability in the brainstem, but it did not attenuate TJ protein expression. These studies demonstrate the complex regulation of the BBB occurring during inflammatory pain and the role of COX in this process. An understanding of BBB regulation during pain states is critically important for pharmacotherapy, and it holds great promise for new therapies to treat central nervous system pathologies.
• Davis, T., Campos, C. R., Ocheltree, S. M., Hom, S., Egleton, R. D., & Davis, T. P. (2008). Nociceptive inhibition prevents inflammatory pain induced changes in the blood-brain barrier. Brain research, 1221.
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  Previous studies by our group have shown that peripheral inflammatory insult, using the lambda-carrageenan inflammatory pain (CIP) model, induced alterations in the molecular and functional properties of the blood-brain barrier (BBB). The question remained whether these changes were mediated via an inflammatory and/or neuronal mechanism. In this study, we investigated the involvement of neuronal input from pain activity on alterations in BBB integrity by peripheral inhibition of nociceptive input. A perineural injection of 0.75% bupivacaine into the right hind leg prior to CIP was used for peripheral nerve block. Upon nerve block, there was a significant decrease in thermal allodynia induced by CIP, but no effect on edema formation 1 h post-CIP. BBB permeability was increased 1 h post-CIP treatment as determined by in situ brain perfusion of [(14)C] sucrose; bupivacaine nerve block of CIP caused an attenuation of [(14)C] sucrose permeability, back to saline control levels. Paralleling the changes in [(14)C] sucrose permeability, we also report increased expression of three tight junction (TJ) proteins, zonula occluden-1 (ZO-1), occludin and claudin-5 with CIP. Upon bupivacaine nerve block, changes in expression were prevented. These data show that the lambda-carrageenan-induced changes in [(14)C] sucrose permeability and protein expression of ZO-1, occludin and claudin-5 are prevented with inhibition of nociceptive input. Therefore, we suggest that nociceptive signaling is in part responsible for the alteration in BBB integrity under CIP.
• Davis, T., McCaffrey, G., Seelbach, M. J., Staatz, W. D., Nametz, N., Quigley, C., Campos, C. R., Brooks, T. A., & Davis, T. P. (2008). Occludin oligomeric assembly at tight junctions of the blood-brain barrier is disrupted by peripheral inflammatory hyperalgesia. Journal of neurochemistry, 106(6).
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  Tight junctions (TJs) at the blood-brain barrier (BBB) dynamically alter paracellular diffusion of blood-borne substances from the peripheral circulation to the CNS in response to external stressors, such as pain, inflammation, and hypoxia. In this study, we investigated the effect of lambda-carrageenan-induced peripheral inflammatory pain (i.e., hyperalgesia) on the oligomeric assembly of the key TJ transmembrane protein, occludin. Oligomerization of integral membrane proteins is a critical step in TJ complex assembly that enables the generation of tightly packed, large multiprotein complexes capable of physically obliterating the interendothelial space to inhibit paracellular diffusion. Intact microvessels isolated from rat brains were fractionated by detergent-free density gradient centrifugation, and gradient fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/ Western blot. Injection of lambda-carrageenan into the rat hind paw produced after 3 h a marked change in the relative amounts of oligomeric, dimeric, and monomeric occludin isoforms associated with different plasma membrane lipid raft domains and intracellular compartments in endothelial cells at the BBB. Our findings suggest that increased BBB permeability (i.e., leak) associated with lambda-carrageenan-induced peripheral inflammatory pain is promoted by the disruption of disulfide-bonded occludin oligomeric assemblies, which renders them incapable of forming an impermeant physical barrier to paracellular transport.
• Willis, C. L., & Davis, T. P. (2008). Chronic inflammatory pain and the neurovascular unit: A central role for glia in maintaining BBB integrity?. CURRENT PHARMACEUTICAL DESIGN, 14(16), 1625-1643.
• Davis, T., Hom, S., Fleegal, M. A., Egleton, R. D., Campos, C. R., Hawkins, B. T., & Davis, T. P. (2007). Comparative changes in the blood-brain barrier and cerebral infarction of SHR and WKY rats. American journal of physiology. Regulatory, integrative and comparative physiology, 292(5).
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  Hypertension is involved in the exacerbation of stroke. It is unclear how blood-brain barrier (BBB) tight-junction (TJ) and ion transporter proteins critical for maintaining brain homeostasis contribute to cerebral infarction during hypertension development. In the present study, we investigated cerebral infarct volume following permanent 4-h middle cerebral artery occlusion (MCAO) and characterized the expression of BBB TJ and ion transporter proteins in brain microvessels of spontaneously hypertensive rats (SHR) compared with age-matched Wistar-Kyoto (WKY) rats at 5 wk (prehypertension), 10 wk (early-stage hypertension), and 15 wk (later-stage hypertension) of age. Hypertensive SHR show increased infarct volume following MCAO compared with WKY control rats. BBB TJ and ion transporter proteins, known to contribute to edema and fluid volume changes in the brain, show differential protein expression patterns during hypertension development. Western blot analysis of TJ protein zonula occludens-2 (ZO-2) showed decreased expression, while ion transporter, Na(+)/H(+) exchanger 1 (NHE-1), was markedly increased in hypertensive SHR. Expression of TJ proteins ZO-1, occludin, actin, claudin-5, and Na(+)-K(+)-2Cl(-) cotransporter remain unaffected in SHR compared with control. Selective inhibition of NHE-1 using dimethylamiloride significantly attenuated ischemia-induced infarct volume in hypertensive SHR following MCAO, suggesting a novel role for NHE-1 in the brain in the regulation of ischemia-induced infarct volume in SHR.
• Davis, T., McCaffrey, G., Staatz, W. D., Quigley, C. A., Nametz, N., Seelbach, M. J., Campos, C. R., Brooks, T. A., Egleton, R. D., & Davis, T. P. (2007). Tight junctions contain oligomeric protein assembly critical for maintaining blood-brain barrier integrity in vivo. Journal of neurochemistry, 103(6).
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  Tight junctions (TJs) are major components of the blood-brain barrier (BBB) that physically obstruct the interendothelial space and restrict paracellular diffusion of blood-borne substances from the peripheral circulation to the CNS. TJs are dynamic structures whose intricate arrangement of oligomeric transmembrane and accessory proteins rapidly alters in response to external stressors to produce changes in BBB permeability. In this study, we investigate the constitutive trafficking of the TJ transmembrane proteins occludin and claudin-5 that are essential for forming the TJ seal between microvascular endothelial cells that inhibits paracellular diffusion. Using a novel, detergent-free OptiPrep density-gradient method to fractionate rat cerebral microvessels, we identify a plasma membrane lipid raft domain that contains oligomeric occludin and claudin-5. Our data suggest that oligomerization of occludin involves disulfide bond formation within transmembrane regions, and that assembly of the TJ oligomeric protein complex is facilitated by an oligomeric caveolin scaffold. This is the first time that distribution of oligomeric TJ transmembrane proteins within plasma membrane lipid rafts at the BBB has been examined in vivo. The findings reported in this study are critical to understand the mechanism of assembly of the TJ multiprotein complex that is essential for maintaining BBB integrity.
• Davis, T., Seelbach, M. J., Brooks, T. A., Egleton, R. D., & Davis, T. P. (2007). Peripheral inflammatory hyperalgesia modulates morphine delivery to the brain: a role for P-glycoprotein. Journal of neurochemistry, 102(5).
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  P-glycoprotein (Pgp, ABCB1) is a critical efflux transporter at the blood-brain barrier (BBB) where its luminal location and substrate promiscuity limit the brain distribution of numerous therapeutics. Moreover, Pgp is known to confer multi-drug resistance in cancer chemotherapy and brain diseases, such as epilepsy, and is highly regulated by inflammatory mediators. The involvement of inflammatory processes in neuropathological states has led us to investigate the effects of peripheral inflammatory hyperalgesia on transport properties at the BBB. In the present study, we examined the effects of lambda-carrageenan-induced inflammatory pain (CIP) on brain endothelium regulation of Pgp. Western blot analysis of enriched brain microvessel fractions showed increased Pgp expression 3 h post-CIP. In situ brain perfusion studies paralleled these findings with decreased brain uptake of the Pgp substrate and opiate analgesic, [(3)H] morphine. Cyclosporin A-mediated inhibition of Pgp enhanced the uptake of morphine in lambda-carrageenan and control animals. This indicates that the CIP induced decrease in morphine transport was the result of an increase in Pgp activity at the BBB. Furthermore, antinociception studies showed decreased morphine analgesia following CIP. The observation that CIP modulates Pgp at the BBB in vivo is critical to understanding BBB regulation during inflammatory disease states.
• Davis, T., Brooks, T. A., Ocheltree, S. M., Seelbach, M. J., Charles, R. A., Nametz, N., Egleton, R. D., & Davis, T. P. (2006). Biphasic cytoarchitecture and functional changes in the BBB induced by chronic inflammatory pain. Brain research, 1120(1).
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  The blood-brain barrier (BBB) is a dynamic system which maintains brain homeostasis and limits CNS penetration via interactions of transmembrane and intracellular proteins. Inflammatory pain (IP) is a condition underlying several diseases with known BBB perturbations, including stroke, Parkinson's, multiple sclerosis and Alzheimer's. Exploring the underlying pathology of chronic IP, we demonstrated alterations in BBB paracellular permeability with correlating changes in tight junction (TJ) proteins: occludin and claudin-5. The present study examines the IP-induced molecular changes leading to a loss in functional BBB integrity. IP was induced by injection of Complete Freund's Adjuvant (CFA) into the plantar surface of the right hindpaw of female Sprague-Dawley rats. Inflammation and hyperalgesia were confirmed, and BBB paracellular permeability was assessed by in situ brain perfusion of [14C]sucrose (paracellular diffusion marker). The permeability of the BBB was significantly increased at 24 and 72 h post-CFA. Analysis of the TJ proteins, which control the paracellular pathway, demonstrated decreased claudin-5 expression at 24 h, and an increase at 48 and 72 h post-injection. Occludin expression was significantly decreased 72 h post-CFA. Expression of junction adhesion molecule-1 (JAM-1) increased 48 h and decreased by 72 h post-CFA. Confocal microscopy demonstrated continuous expression of both occludin and JAM-1, each co-localizing with ZO-1. The increased claudin-5 expression was not limited to the junction. These results provide evidence that chronic IP causes dramatic alterations in specific cytoarchitectural proteins and demonstrate alterations in molecular properties during CFA, resulting in significant changes in BBB paracellular permeability.
• Davis, T., Brooks, T. A., Hawkins, B. T., Huber, J. D., Egleton, R. D., & Davis, T. P. (2005). Chronic inflammatory pain leads to increased blood-brain barrier permeability and tight junction protein alterations. American journal of physiology. Heart and circulatory physiology, 289(2).
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  The blood-brain barrier (BBB) maintains brain homeostasis by limiting entry of substances to the central nervous system through interaction of transmembrane and intracellular proteins that make up endothelial cell tight junctions (TJs). Recently it was shown that the BBB can be modulated by disease pathologies including inflammatory pain. This study examined the effects of chronic inflammatory pain on the functional and molecular integrity of the BBB. Inflammatory pain was induced by injection of complete Freund's adjuvant (CFA) into the right plantar hindpaw in female Sprague-Dawley rats under halothane anesthesia; control animals were injected with saline. Edema and hyperalgesia were assessed by plethysmography and infrared paw-withdrawal latency. At 72 h postinjection, significant edema formation and hyperalgesia were noted in the CFA-treated rats. Examination of permeability of the BBB by in situ perfusion of [14C]sucrose while rats were under pentobarbital anesthesia demonstrated that CFA treatment significantly increased brain sucrose uptake. Western blot analysis of BBB TJ proteins showed no change in expression of zonula occludens-1 (an accessory protein) or actin (a cytoskeletal protein) with CFA treatment. Expression of the transmembrane TJ proteins occludin and claudin-3 and -5 significantly changed with CFA treatment with a 60% decrease in occludin, a 450% increase in claudin-3, and a 615% increase in claudin-5 expression. This study demonstrates that during chronic inflammatory pain, alterations in BBB function are associated with changes in specific transmembrane TJ proteins.
• Davis, T., Egleton, R. D., & Davis, T. P. (2005). Development of neuropeptide drugs that cross the blood-brain barrier. NeuroRx : the journal of the American Society for Experimental NeuroTherapeutics, 2(1).
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  In recent years, there have been several important advancements in the development of neuropeptide therapeutics. Nevertheless, the targeting of peptide drugs to the CNS remains a formidable obstacle. Delivery of peptide drugs is limited by their poor bioavailability to the brain due to low metabolic stability, high clearance by the liver, and the presence of the blood brain barrier (BBB). Multiple strategies have been devised in an attempt to improve peptide drug delivery to the brain, with variable results. In this review, we discuss several of the strategies that have been used to improve both bioavailability and BBB transport, with an emphasis on antibody based vector delivery, useful for large peptides/small proteins, and glycosylation, useful for small peptides. Further development of these delivery methods may finally enable peptide drugs to be useful for the treatment of neurological disease states.
• Davis, T., Fleegal, M. A., Hom, S., Borg, L. K., & Davis, T. P. (2005). Activation of PKC modulates blood-brain barrier endothelial cell permeability changes induced by hypoxia and posthypoxic reoxygenation. American journal of physiology. Heart and circulatory physiology, 289(5).
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  The blood-brain barrier (BBB) is a metabolic and physiological barrier important for maintaining brain homeostasis. The aim of this study was to determine the role of PKC activation in BBB paracellular permeability changes induced by hypoxia and posthypoxic reoxygenation using in vitro and in vivo BBB models. In rat brain microvessel endothelial cells (RMECs) exposed to hypoxia (1% O2-99% N2; 24 h), a significant increase in total PKC activity was observed, and this was reduced by posthypoxic reoxygenation (95% room air-5% CO2) for 2 h. The expression of PKC-betaII, PKC-gamma, PKC-eta, PKC-mu, and PKC-lambda also increased following hypoxia (1% O2-99% N2; 24 h), and these protein levels remained elevated following posthypoxic reoxygenation (95% room air-5% CO2; 2 h). Increases in the expression of PKC-epsilon and PKC-zeta were also observed following posthypoxic reoxygenation (95% room air-5% CO2; 2 h). Moreover, inhibition of PKC with chelerythrine chloride (10 microM) attenuated the hypoxia-induced increases in [14C]sucrose permeability. Similar to what was observed in RMECs, total PKC activity was also stimulated in cerebral microvessels isolated from rats exposed to hypoxia (6% O2-94% N2; 1 h) and posthypoxic reoxygenation (room air; 10 min). In contrast, hypoxia (6% O2-94% N2; 1 h) and posthypoxic reoxygenation (room air; 10 min) significantly increased the expression levels of only PKC-gamma and PKC-theta in the in vivo hypoxia model. These data demonstrate that hypoxia-induced BBB paracellular permeability changes occur via a PKC-dependent mechanism, possibly by differentially regulating the protein expression of the 11 PKC isozymes.
• Davis, T., Hawkins, B. T., & Davis, T. P. (2005). The blood-brain barrier/neurovascular unit in health and disease. Pharmacological reviews, 57(2).
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  The blood-brain barrier (BBB) is the regulated interface between the peripheral circulation and the central nervous system (CNS). Although originally observed by Paul Ehrlich in 1885, the nature of the BBB was debated well into the 20th century. The anatomical substrate of the BBB is the cerebral microvascular endothelium, which, together with astrocytes, pericytes, neurons, and the extracellular matrix, constitute a "neurovascular unit" that is essential for the health and function of the CNS. Tight junctions (TJ) between endothelial cells of the BBB restrict paracellular diffusion of water-soluble substances from blood to brain. The TJ is an intricate complex of transmembrane (junctional adhesion molecule-1, occludin, and claudins) and cytoplasmic (zonula occludens-1 and -2, cingulin, AF-6, and 7H6) proteins linked to the actin cytoskeleton. The expression and subcellular localization of TJ proteins are modulated by several intrinsic signaling pathways, including those involving calcium, phosphorylation, and G-proteins. Disruption of BBB TJ by disease or drugs can lead to impaired BBB function and thus compromise the CNS. Therefore, understanding how BBB TJ might be affected by various factors holds significant promise for the prevention and treatment of neurological diseases.
• Davis, T., Hawkins, B. T., Egleton, R. D., & Davis, T. P. (2005). Modulation of cerebral microvascular permeability by endothelial nicotinic acetylcholine receptors. American journal of physiology. Heart and circulatory physiology, 289(1).
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  Nicotine increases the permeability of the blood-brain barrier in vivo. This implies a possible role for nicotinic acetylcholine receptors in the regulation of cerebral microvascular permeability. Expression of nicotinic acetylcholine receptor subunits in cerebral microvessels was investigated with immunofluorescence microscopy. Positive immunoreactivity was found for receptor subunits alpha3, alpha5, alpha7, and beta2, but not subunits alpha4, beta3, or beta4. Blood-brain barrier permeability was assessed via in situ brain perfusion with [14C]sucrose. Nicotine increased the rate of sucrose entry into the brain from 0.3 +/- 0.1 to 1.1 +/- 0.2 microl.g(-1).min(-1), as previously described. This nicotine-induced increase in blood-brain barrier permeability was significantly attenuated by both the blood-brain barrier-permeant nicotinic antagonist mecamylamine and the blood-brain barrier-impermeant nicotinic antagonist hexamethonium to 0.5 +/- 0.2 and 0.3 +/- 0.2 microl.g(-1).min(-1), respectively. These data suggest that nicotinic acetylcholine receptors expressed on the cerebral microvascular endothelium mediate nicotine-induced changes in blood-brain barrier permeability.
• Davis, T., Witt, K. A., Mark, K. S., Huber, J., & Davis, T. P. (2005). Hypoxia-inducible factor and nuclear factor kappa-B activation in blood-brain barrier endothelium under hypoxic/reoxygenation stress. Journal of neurochemistry, 92(1).
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  This investigation focuses on transcription factor involvement in blood-brain barrier (BBB) endothelial cell-induced alterations under conditions of hypoxia and post-hypoxia/reoxygenation (H/R), using established in vivo/ex vivo and in vitro BBB models. Protein/DNA array analyses revealed a correlation in key transcription factor activation during hypoxia and H/R, including NFkappaB and hypoxia-inducible factor (HIF)1. Electrophoretic mobility shift assays confirmed NFkappaB and HIF1 binding activity ex vivo and in vitro, under conditions of hypoxia and H/R. Hypoxia- and H/R-treated BBB endothelium showed increased HIF1alpha protein expression in both cytoplasmic and nuclear fractions, in ex vivo and in vitro models. Co-immunoprecipitation of HIF1alpha and HIF1beta was shown in the nuclear fraction under conditions of hypoxia and H/R in both models. Hypoxia- and H/R-treated BBB endothelium showed increased expression of NFkappaB-p65 protein in both cytoplasmic and nuclear fractions. Co-immunoprecipitation of NFkappaB-p65 with NFkappaB-p50 was shown in the nuclear fraction under conditions of hypoxia and H/R in the ex vivo model, and after H/R in the in vitro model. These data offer novel avenues in which to alter and/or investigate BBB activity across model systems and to further our understanding of upstream regulators during hypoxia and H/R.
• Davis, T., Brown, R. C., Egleton, R. D., & Davis, T. P. (2004). Mannitol opening of the blood-brain barrier: regional variation in the permeability of sucrose, but not 86Rb+ or albumin. Brain research, 1014(1-2).
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  Clinically, infusion of hyperosmolar solutions is used to enhance chemotherapeutic drug penetration of the blood-brain barrier (BBB) in patients with malignant brain tumors or metastases. We examined the effect of hyperosmolar BBB disruption on brain permeability of three compounds, 86Rb+, a marker for K+ permeability and transport, [14C]sucrose and Evans blue albumin, using a rat in situ perfusion model. 86Rb+ and [14C]sucrose had increased permeability 20 min after BBB disruption with 1.6 M mannitol. There was no change in Evans blue albumin permeability. Only [14C]sucrose showed regional variation in permeability after mannitol-induced BBB disruption, with the cortex and midbrain having higher sucrose permeability then either the cerebellum or brainstem. These data suggest that the clinical efficacy of hyperosmolar disruption therapy in conjunction with chemotherapeutic agents, of a similar molecular weight to sucrose, may be affected by the location of the tumor within the brain.
• Davis, T., Brown, R. C., Mark, K. S., Egleton, R. D., & Davis, T. P. (2004). Protection against hypoxia-induced blood-brain barrier disruption: changes in intracellular calcium. American journal of physiology. Cell physiology, 286(5).
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  Tissue damage after stroke is partly due to disruption of the blood-brain barrier (BBB). Little is known about the role of calcium in modulating BBB disruption. We investigated the effect of hypoxic and aglycemic stress on BBB function and intracellular calcium levels. Bovine brain microvessel endothelial cells were treated with A-23187 to increase intracellular calcium without hypoxia or treated with a calcium chelator (BAPTA) or calcium channel blockers (nifedipine or SKF-96365) and 6 h of hypoxia. A-23187 alone did not increase paracellular permeability. Hypoxia increased intracellular calcium, and hypoxia or hypoxia-aglycemia increased paracellular permeability. Treatment with nifedipine and SKF-96365 increased intracellular calcium under normoglycemic conditions, instead of blocking calcium influx, and was protective against hypoxia-induced BBB disruption under normoglycemia. Protection by nifedipine and SKF-96365 was not due to antioxidant properties of these compounds. These data indicate that increased intracellular calcium alone is not enough to disrupt the BBB. However, increased intracellular calcium after drug treatment and hypoxia suggests a potential mechanism for these drugs in BBB protection; nifedipine and SKF-96365 plus hypoxic stress may trigger calcium-mediated signaling cascades, altering BBB integrity.
• Davis, T., Hau, V. S., Huber, J. D., Campos, C. R., Davis, R. T., & Davis, T. P. (2004). Effect of lambda-carrageenan-induced inflammatory pain on brain uptake of codeine and antinociception. Brain research, 1018(2).
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  This study investigated the potential clinical implications of lambda-carrageenan-induced inflammatory pain on brain uptake of a commonly used analgesic, codeine, in relation to the fundamental properties of the blood-brain barrier (BBB) correlated to its antinociceptive profile over a 168-h time course. BBB uptake of [14C]sucrose (a membrane impermeant marker) and [3H]codeine were investigated using an in situ brain perfusion model in the rat. Results demonstrated a significantly increased brain uptake of [14C]sucrose at 1, 3, 6 and 48 h (139+/-9%, 166+/-19%, 138+/-13% and 146+/-7% compared with control, respectively) and [3H]codeine at 3 and 48 h (179+/-6% and 179+/-12% compared with control, respectively). Capillary depletion analyses ensured that increased radioisotope associated with the brain was due to increased uptake rather than trapping in the cerebral vasculature. Antinociception studies using a radiant-heat tail flick analgesia method demonstrated that lambda-carrageenan-induced inflammatory pain enhanced the in vivo antinociceptive profile of i.p.-administered codeine (7 mg/kg) at 3 and 48 h (144+/-11% and 155+/-9% compared with control, respectively). This study demonstrated that brain uptake and antinociception of codeine are increased during lambda-carrageenan-induced inflammatory pain, suggesting that the presence of inflammatory pain may be an important consideration in therapeutic drug dosing, potential adverse effects and/or neurotoxicity.
• Davis, T., Hawkins, B. T., Abbruscato, T. J., Egleton, R. D., Brown, R. C., Huber, J. D., Campos, C. R., & Davis, T. P. (2004). Nicotine increases in vivo blood-brain barrier permeability and alters cerebral microvascular tight junction protein distribution. Brain research, 1027(1-2).
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  The blood-brain barrier (BBB) is critical to the health of the central nervous system. The BBB is formed primarily by the presence of tight junctions (TJ) between cerebral microvessel endothelial cells. In light of the known effects of nicotine on endothelial cell biology, the specific effects of nicotine on the in vivo BBB were examined. Using in situ brain perfusion, it was found that continuous administration of nicotine (4.5 mg free base x kg(-1) x day(-1)) for 1 and 7 days led to increased permeability of the BBB to [14C]-sucrose without significant changes in its initial volume of distribution. The expression and distribution of the TJ-associated proteins actin, occludin, claudin-1, -3, and -5, and ZO-1 and -2 were analyzed by Western blot and immunofluorescence microscopy. Though no changes in total protein expression were observed, nicotine treatment was associated with altered cellular distribution of ZO-1 and diminished junctional immunoreactivity of claudin-3. It is proposed that nicotine leads to changes in BBB permeability via the modulation of TJ proteins.
• Davis, T., Mark, K. S., Burroughs, A. R., Brown, R. C., Huber, J. D., & Davis, T. P. (2004). Nitric oxide mediates hypoxia-induced changes in paracellular permeability of cerebral microvasculature. American journal of physiology. Heart and circulatory physiology, 286(1).
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  Ischemic stroke from a reduction in blood flow to the brain microvasculature results in a subsequent decreased delivery of oxygen (i.e., hypoxia) and vital nutrients to endothelial, neuronal, and glial cells. Hypoxia associated with stroke has been shown to increase paracellular permeability of the blood-brain barrier, leading to the release of cellular mediators and brain tissue injury. Whereas reperfusion does not occur in all ischemic strokes, increased permeability has been seen in posthypoxic reoxygenation. Currently, it is unknown whether these deleterious effects result from cellular mechanisms stimulated by decreased oxygen during stroke or posthypoxic reoxygenation stress. This study used primary bovine brain microvessel endothelial cells (BBMECs) to examine the involvement of nitric oxide (NO) as a mediator in hypoxia-induced permeability changes. Hypoxia-induced increased transport of [14C]sucrose across BBMEC monolayers compared with normoxia was attenuated by either posthypoxic reoxygenation or inhibition of NO synthase (NOS). The hypoxia-induced permeability effect was further reduced when NOS inhibition was combined with posthypoxic reoxygenation. Additionally, a significant increase in total NO was seen in BBMECs after hypoxic exposure. This correlation was supported by the increased [14C]sucrose permeability observed when BBMECs were exposed to the NO donor diethylenetriaamine NONOate. Western blot analyses of NOS isoforms showed a significant increase in the inducible isoform after hypoxic exposure with a subsequent reduction in expression on reoxygenation. Results from this study suggest that hypoxia-induced blood-brain barrier breakdown can be diminished by inhibition of NO synthesis, decreased concentration of NO metabolites, and/or reoxygenation.
• Davis, T., Brown, R. C., Mark, K. S., Egleton, R. D., Huber, J. D., Burroughs, A. R., & Davis, T. P. (2003). Protection against hypoxia-induced increase in blood-brain barrier permeability: role of tight junction proteins and NFkappaB. Journal of cell science, 116(Pt 4).
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  Co-culture with glial cells and glia-conditioned media can induce blood-brain barrier properties in microvessel endothelial cells and protect against hypoxia-induced blood-brain barrier breakdown. We examined the effect of two types of glia-conditioned media on brain microvessel endothelial cell permeability and tight junction protein expression, and studied potential mechanisms of action. We found that C6-glioma-conditioned media, but not rat astrocyte-conditioned media, protected against an increase in permeability induced by exposure to 1% oxygen for 24 hours. This hypoxic stress caused an increase in the expression of tight junction proteins claudin-1 and actin, particularly in cells treated with C6-conditioned media. We found that C6-conditioned media has a significantly higher level of both basic fibroblast growth factor and vascular endothelial growth factor. Treatment with C6-conditioned media for 1 or 3 days protects against hypoxia-induced permeability increases, and this protective effect may be mediated by signal transduction pathways terminating at the transcription factor NFkappaB.
• Davis, T., Egleton, R. D., Campos, C. C., Huber, J. D., Brown, R. C., & Davis, T. P. (2003). Differential effects of diabetes on rat choroid plexus ion transporter expression. Diabetes, 52(6).
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  Though diabetes is a disease with vascular complications, little is known about its effects on the blood-brain barrier or the blood-cerebrospinal fluid barrier (BCSFB). The BCSFB is situated at choroid plexuses located in the lateral, third, and fourth ventricles. Choroid plexuses are the primary site of cerebrospinal fluid (CSF) production and express numerous ion transporters. Previous studies have shown a perturbation of ion transport in the periphery and brain during diabetes. In this study, we investigated the effect of diabetes on ion transporters in the choroid plexuses of streptozotocin (STZ)-induced diabetic rats. Diabetes was induced in male Sprague-Dawley rats by intraperitoneal injection of STZ (60 mg/kg in citrate buffer, confirmed by glucose analysis: 601 +/- 22 mg/dl diabetic rats, 181 +/- 46 mg/dl age-matched controls); and at 28 days, rats were killed, choroid plexuses harvested, and protein extracted. Western blot analyses were carried out using antibodies for ion transporters, including Na(+)-K(+)-2Cl(-) cotransporter and the Na(+)-K(+)-ATPase alpha1-subunit. The efflux of the K(+) analog (86)Rb(+) from choroid plexus was also studied. Diabetic rats showed an increase in expression of the Na(+)-K(+)-2Cl(-) cotransporter and the Na(+)-K(+)-ATPase alpha1-subunit, as compared with age-matched controls, a decrease in Na(+)-H(+) exchanger expression, and no change in Na(+)-K(+)-ATPase beta1- or beta2-subunit. The net effect of these changes was a 66% increase in (86)Rb(+) efflux from diabetic choroid plexus compared with controls. These changes in expression may affect choroid plexus ion balance and thus significantly affect CSF production in diabetic rats.
• Davis, T., Huber, J. D., Campos, C. R., Egleton, R. D., Witt, K., Guo, L., Roberts, M. J., Bentley, M. D., & Davis, T. P. (2003). Conjugation of low molecular weight poly(ethylene glycol) to biphalin enhances antinociceptive profile. Journal of pharmaceutical sciences, 92(7).
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  The objectives of this study were to examine the effect of poly(ethylene glycol) (PEG) conjugation on the tyrosine residues of biphalin to determine the proper size PEG for optimal efficacy and investigate the antinociceptive profile of PEG-biphalin against biphalin via three routes of administration. All antinociception evaluations were made using a radiant-heat tail flick analgesia meter. (2 kDa)(2) PEG-biphalin was identified as the optimal size of PEG to enhance the antinociceptive profile following intravenous administration of 685 nmol kg(-1) of biphalin or PEG-biphalin [(1 kDa)(2), (2 kDa)(2), (5 kDa)(2), (12 kDa)(2), (20 kDa)(2)]. (2 kDa)(2) PEG-biphalin displayed an area under the curve (AUC) approximately 2.5 times that of biphalin with enhanced analgesia up to 300 min postinjection. (2 kDa)(2) PEG-biphalin was equipotent to biphalin following intracerebroventricular administration (0.4 nmol kg(-1)). Both biphalin and (2 kDa)(2) PEG-biphalin were effectively antagonized with naloxone (10 mg kg(-1)) and a partial antagonistic effect was seen following pretreatment with naltrindole (20 mg kg(-1)). (2 kDa)(2) PEG-biphalin showed significantly increased potency (A(50)) when administered intravenously and subcutaneously. Additionally, (2 kDa)(2) PEG-biphalin demonstrated a significantly enhanced antinociceptive profile (AUC) via all routes of administration tested. These findings indicate that PEG conjugation to biphalin retains opioid-mediated effects observed with biphalin and is a valuable tool for eliciting potent, sustained analgesia via parenteral routes of administration.
• Davis, T., Witt, K. A., Mark, K. S., Hom, S., & Davis, T. P. (2003). Effects of hypoxia-reoxygenation on rat blood-brain barrier permeability and tight junctional protein expression. American journal of physiology. Heart and circulatory physiology, 285(6).
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  Cerebral microvessel endothelial cells that form the blood-brain barrier (BBB) have tight junctions (TJs) that are critical for maintaining brain homeostasis. The effects of initial reoxygenation after a hypoxic insult (H/R) on functional and molecular properties of the BBB and TJs remain unclear. In situ brain perfusion and Western blot analyses were performed to assess in vivo BBB integrity on reoxygenation after a hypoxic insult of 6% O2 for 1 h. Model conditions [blood pressure, blood gas chemistries, cerebral blood flow (CBF), and brain ATP concentration] were also assessed to ensure consistent levels and criteria for insult. In situ brain perfusion revealed that initial reoxygenation (10 min) significantly increased the uptake of [14C]sucrose into brain parenchyma. Capillary depletion and CBF analyses indicated the perturbations were due to increased paracellular permeability rather than vascular volume changes. Hypoxia with reoxygenation (10 min) produced an increase in BBB permeability with associated alterations in tight junctional protein expression. These results suggest that H/R leads to reorganization of TJs and increased paracellular diffusion at the BBB, which is not a result of increased CBF, vascular volume change, or endothelial uptake of marker. Additionally, the tight junctional protein occludin had a shift in bands that correlated with functional changes (i.e., increased permeability) without significant change in expression of claudin-3, zonula occludens-1, or actin. H/R-induced changes in the BBB may result in edema and/or associated pathological outcomes.
• Davis, T., Wolka, A. M., Huber, J. D., & Davis, T. P. (2003). Pain and the blood-brain barrier: obstacles to drug delivery. Advanced drug delivery reviews, 55(8).
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  Delivery of drugs across the blood-brain barrier has been shown to be altered during pathological states involving pain. Pain is a complex phenomenon involving immune and centrally mediated responses, as well as activation of the hypothalamic-pituitary-adrenal axis. Mediators released in response to pain have been shown to affect the structure and function of the blood-brain barrier in vitro and in vivo. These alterations in blood-brain barrier permeability and cytoarchitecture have implications in terms of drug delivery to the central nervous system, since pain and inflammation have the capacity to alter drug uptake and efflux across the blood-brain barrier. An understanding of how blood-brain barrier and central nervous system drug delivery mechanisms are altered during pathological conditions involving pain and/or inflammation is important in designing effective therapeutic regimens to treat disease.
• Davis, T., Brown, R. C., & Davis, T. P. (2002). Calcium modulation of adherens and tight junction function: a potential mechanism for blood-brain barrier disruption after stroke. Stroke; a journal of cerebral circulation, 33(6).
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  This review deals with the role of calcium in endothelial cell junctions of the blood-brain barrier (BBB). Calcium is critical for adherens junction function, but it appears that calcium is also important in regulating tight junction function necessary for the barrier characteristics of cerebral microvessels.
• Davis, T., Hau, V. S., Huber, J. D., Campos, C. R., Lipkowski, A. W., Misicka, A., & Davis, T. P. (2002). Effect of guanidino modification and proline substitution on the in vitro stability and blood-brain barrier permeability of endomorphin II. Journal of pharmaceutical sciences, 91(10).
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  Endomorphin II (ENDII), an endogenous ligand for the mu-opioid receptor, was investigated as a possible analgesic with fewer side effects than morphine. To improve CNS entry of END II, structural modification was also examined to determine whether Pro(4) substitution and cationization affected physico-chemical characteristics, blood-brain barrier (BBB) transport, and analgesic profile. END II and its Pro(4)-substituted analog, Morphiceptin (MOR), were cationized by guanidino (GU)-addition. MOR was seven times less lipophilic than END II, whereas GU-addition decreased lipophilicity of both peptides. MOR did not affect in vitro BBB permeability; however, GU-addition increased permeability of MOR by 31%. MOR decreased protein binding by 23% compared to END II, whereas GU-addition increased protein binding of both peptides by 71 and 113%, respectively. MOR increased brain t(1/2) compared to END II. GU-addition significantly increased t(1/2) of MOR and END II in both brain (sixfold and 10-fold, respectively) and serum (over 10-fold). Pro(4)-substitution and GU-addition enhanced the in vivo analgesia profiles of i.v. administered END II and MOR, but decreased i.c.v. analgesia profiles. This study demonstrates Pro(4)-substitution decreases protein binding and enhances brain stability while cationization enhances both brain and serum stability with variable effects on BBB permeability. The analgesic profiles show that both Pro(4)-substitution and cationization enhance i.v. analgesia and thus, are promising structural modifications for the development of successful opioid drugs.
• Davis, T., Hawkins, B. T., Brown, R. C., & Davis, T. P. (2002). Smoking and ischemic stroke: a role for nicotine?. Trends in pharmacological sciences, 23(2).
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  Cigarette smoking is a preventable risk factor for ischemic stroke. The mechanisms by which smoking contributes to stroke are poorly understood and the role of nicotine in this process is controversial. Although nicotine administered transdermally and orally does not appear to have as many associated health risks as do cigarettes, nicotine does have acute vasoactive and mitogenic effects on vascular tissues. Nicotine might alter the function of the blood-brain barrier and disrupt normal endothelial cell function. Some of the detrimental effects of nicotine are prevented by nicotinic acetylcholine receptor antagonists. However, recent studies indicate that nicotine might also interact with intracellular signaling pathways that are independent of acetylcholine receptors. In light of these recent developments, the impact of nicotine on cerebrovascular pathology should not be dismissed.
• Davis, T., Huber, J. D., Hau, V. S., Mark, K. S., Brown, R. C., Campos, C. R., & Davis, T. P. (2002). Viability of microvascular endothelial cells to direct exposure of formalin, lambda-carrageenan, and complete Freund's adjuvant. European journal of pharmacology, 450(3).
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  We investigated three inflammatory agents to establish if these substances elicit a direct effect on the functional and structural integrity of the blood-brain barrier. Cellular cytotoxicity and paracellular permeability were assessed in vitro using primary bovine brain microvascular endothelial cells exposed to formalin, lambda-carrageenan, or complete Freund's adjuvant for 1, 3, or 72 h, respectively. Results showed that only the highest concentration (0.025%) of formalin produced a decrease in cell viability (approximately 34%) and a significant increase in cell permeability to [(14)C]sucrose at 120 min (approximately 137%). Brain perfusion using female Sprague-Dawley rats showed no difference in paracellular permeability to [(14)C]sucrose for any inflammatory agent. Western blot analyses were performed on isolated rat brain microvessels to assess the structural integrity of blood-brain barrier tight junctions. Results indicate that expression of zonula occludens-1, occludin, claudin-1, and actin remain unchanged following intravenous exposure to inflammatory agents. This study confirms that changes seen at the blood-brain barrier following a peripheral inflammation are due to physiological responses to the given inflammatory agent and not to any direct interaction between the inflammatory agent and the brain microvasculature.
• Davis, T., Mark, K. S., & Davis, T. P. (2002). Cerebral microvascular changes in permeability and tight junctions induced by hypoxia-reoxygenation. American journal of physiology. Heart and circulatory physiology, 282(4).
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  Cerebral microvessel endothelial cells that form the blood-brain barrier (BBB) have tight junctions (TJ) that are critical for maintaining brain homeostasis and low permeability. Both integral (claudin-1 and occludin) and membrane-associated zonula occluden-1 and -2 (ZO-1 and ZO-2) proteins combine to form these TJ complexes that are anchored to the cytoskeletal architecture (actin). Disruptions of the BBB have been attributed to hypoxic conditions that occur with ischemic stroke, pathologies of decreased perfusion, and high-altitude exposure. The effects of hypoxia and posthypoxic reoxygenation in cerebral microvasculature and corresponding cellular mechanisms involved in disrupting the BBB remain unclear. This study examined hypoxia and posthypoxic reoxygenation effects on paracellular permeability and changes in actin and TJ proteins using primary bovine brain microvessel endothelial cells (BBMEC). Hypoxia induced a 2.6-fold increase in [(14)C]sucrose, a marker of paracellular permeability. This effect was significantly reduced (~58%) with posthypoxic reoxygenation. After hypoxia and posthypoxic reoxygenation, actin expression was increased (1.4- and 2.3-fold, respectively). Whereas little change was observed in TJ protein expression immediately after hypoxia, a twofold increase in expression was seen with posthypoxic reoxygenation. Furthermore, immunofluorescence studies showed alterations in occludin, ZO-1, and ZO-2 protein localization during hypoxia and posthypoxic reoxygenation that correlate with the observed changes in BBMEC permeability. The results of this study show hypoxia-induced changes in paracellular permeability may be due to perturbation of TJ complexes and that posthypoxic reoxygenation reverses these effects.
• Davis, T., Witt, K. A., Huber, J. D., Egleton, R. D., & Davis, T. P. (2002). Pluronic p85 block copolymer enhances opioid peptide analgesia. The Journal of pharmacology and experimental therapeutics, 303(2).
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  Peptide-based drug development is a rapidly growing field within pharmaceutical research. Nevertheless, peptides have found limited clinical use due to several physiological and pathological factors. Pluronic block copolymers represent a growing technology with the potential to enhance efficacy of peptide therapeutics. This investigation assesses Pluronic P85 (P85) and its potential to enhance opioid peptide analgesia. Two opioid peptides, [D-Pen(2),D-Pen(5)]-enkephalin (DPDPE) and biphalin, were examined as to the benefits of P85 coadministration, above (1.0%) and below (0.01%) the critical micelle concentration, with morphine as a nonpeptide control. P85 was examined in vitro to assess blood-brain barrier uptake in association with P-glycoprotein effect, DPDPE and morphine being P-glycoprotein substrates. P85 coadministration with DPDPE and biphalin showed increased (p < 0.01) analgesia with both 0.01 and 1.0% P85. Morphine showed increased (p < 0.01) analgesia with 0.01% P85 only. This increase in analgesia is due to both an increase in peak effect, as well as a prolongation of effect. P85 increased cellular uptake of (125)I-DPDPE and [(3)H]morphine at 0.01% (p < 0.01) and 1.0% (p < 0.01 and p < 0.05, respectively). Cyclosporin-A coadministration with (125)I-DPDPE and [(3)H]morphine increased cellular uptake (p < 0.01 and p < 0.05, respectively). (125)I-DPDPE and [(3)H]morphine coadministered with 0.01% P85 and cyclosporin-A increased cellular uptake compared with control (p < 0.01) and compared with cyclosporin-A coadministration without P85 (p < 0.01 and p < 0.05, respectively). This indicates that, in addition to P-gp inhibition, 0.01% P85 increased (125)I-DPDPE and [(3)H]morphine uptake. In our examination, we determined that P85 enhanced the analgesic profile of biphalin, DPDPE, and morphine, both above and below the critical micelle concentration.

Poster Presentations

• Davis, T. P., Lochhead, J. J., & Ronaldson, P. T. (2020, February). Organic Anion Transporting Polypeptide (Oatp)-Mediated Transport is required for Statin-Induced Neuroprotection: A Role for Blood-Brain Barrier Transporters in Stroke Treatment. International Stroke Conference 2020. Los Angeles, California: American Heart Association.
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  Objectives: Treatment approaches for stroke include reperfusion therapies (i.e., recombinant tissue plasminogen activator, endovascular thrombectomy); however, many stroke patients still experience disability. This indicates a need to develop neuroprotective treatments that are effective in the setting of successful recanalization. Post-stroke outcomes are improved by treatment with 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (i.e., statins). We have shown that the endogenous blood-brain barrier (BBB) uptake transporter Oatp1a4 facilitates blood-to-brain transport of atorvastatin (ATV). The objective of this study was to show that Oatp-mediated transport at the BBB is an absolute requirement for ATV neuroprotective effectiveness in stroke.Methods: Male and female Sprague-Dawley rats (200-250 g) were subjected to transient middle cerebral artery occlusion (tMCAO) for 90 minutes followed by 22.5 h reperfusion. Sham-operated animals were used as controls. ATV (20 mg/kg, i.v.) was injected 2 h following reperfusion. The role of Oatp-mediated transport was determined using the Oatp transport inhibitor fexofenadine (FEX; 3.2 mg/kg, i.v.) injected at the same time as ATV. Following tMCAO, infarction volume and brain edema ratios were calculated from TTC-stained brain slices. Post-stroke outcomes were assessed via measurement of neurological deficit scores, by the adhesive removal test (i.e., sensorimotor function), and by the rotarod performance test (i.e., motor function). Results: In tMCAO animals, ATV reduced (p < 0.01) both infarction volume and brain edema ratio in both sexes. ATV improved neurological deficit scores and well as sensorimotor function and motor performance. In the presence of FEX, ATV had no effect on infarction volume or brain edema ratio. Similarly, positive effects of ATV on post-stroke outcomes were attenuated by FEX. Conclusions: Our data indicate that pharmacological inhibition of Oatp-mediated transport at the BBB prevents ATV from exerting neuroprotective effects in rats following tMCAO. Our results also suggest that i.v. ATV administered at an early time point following reperfusion (i.e., 2 h) can provide effective neuroprotection in male and female rats subjected to tMCAO.
• Lochhead, J. J., Williams, E. I., Betterton, R. D., Davis, T. P., & Ronaldson, P. T. (2020, February). Organic Anion Transporting Polypeptide 1a4: A Critical Determinant of Neuroprotective Drug Efficacy in Stroke. 5th Annual Arizona Biomedical Research Commission (ABRC)-Flinn Research Conference. Phoenix, Arizona: ABRC.
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  Background and Knowledge Gap: Stroke is the 5th leading cause of death in the United States. Despite significant advances in reperfusion therapies (i.e., thrombolytic drug therapy, mechanical endovascular thrombectomy), stroke patients still experience considerable neurological deficits despite these interventions. To date, drug discovery for stroke treatment has been challenging as indicated by poor translatability of compounds from preclinical studies to successful Phase III clinical trials. In contrast, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (i.e., statins) are routinely given to stroke patients because they are known to improve post-stroke outcomes; however, statin use in stroke patients is limited to patients that can swallow since statins are formulated for oral administration only. Indeed, neuroprotective effectiveness of statins requires efficient delivery across the blood-brain barrier (BBB). Our laboratory has shown, in vivo, that the endogenous BBB uptake transporter Oatp1a4 facilitates blood-to-brain transport of currently marketed statins (i.e., atorvastatin, pravastatin); however, little is known regarding the effects of this endogenous BBB transporter on CNS drug disposition in the setting of ischemic stroke, a significant knowledge gap.Hypothesis: We hypothesize that functional expression of Oatp1a4 at the BBB is a required mechanism that enables efficient statin delivery to the brain, thereby enabling these drugs to be effective neuroprotective agents. Methods: Male and female Sprague-Dawley rats (200-250 g) were subjected to transient middle cerebral artery occlusion (tMCAO) for 90 minutes followed by 22.5 h reperfusion. Sham-operated animals (i.e., controls) underwent the same surgical procedure except that the intraluminal suture was not inserted. Atorvastatin (20 mg/kg, i.v.) was injected 2 h following removal of the intraluminal suture (i.e., reperfusion). Oatp1a4 protein expression was determined by western blot analysis of isolated brain microvessels. The role of Oatp-mediated transport was determined using the pharmacological Oatp inhibitor fexofenadine (3.2 mg/kg, i.v.) injected at the same time as atorvastatin. Following tMCAO, infarction volume and brain edema ratios were calculated from TTC-stained brain tissue slices. Post-stroke outcomes were assessed after tMCAO via neurological deficit scores, the adhesive removal test (i.e., sensorimotor function), and rotorod analysis (i.e., motor function). Results: In tMCAO animals, Oatp1a4 protein expression was increased in microvessels from ischemic cortex (i.e., ipsilateral cortex) but not in contralateral cortex. Atorvastatin significantly reduced both infarction volume and the brain edema ratio. Atorvastatin also improved post-stroke outcomes as determined by neurological deficit scores, the adhesive removal test, and rotorod analysis. In the presence of fexofenadine, atorvastatin had no effect on infarction volume or the brain edema ratio. Similarly, positive effects of atorvastatin on post-stroke outcomes were attenuated by fexofenadine. Conclusions: Our data indicate that neuroprotective effects of atorvastatin in experimental stroke require functional expression of Oatp1a4 at the BBB. Of particular significance, our results suggest that intravenous atorvastatin administered at an early time point following reperfusion (i.e., 2 h) can provide effective neuroprotection in male and female Sprague-Dawley rats subjected to tMCAO. Studies are ongoing in the laboratory to rigorously study regulation and functional expression of Oatp isoforms at the BBB in the tMCAO model.
• Ronaldson, P. T., Davis, T. P., Reilly, B. G., Betterton, R. D., & Yang, J. (2018, September). Acetaminophen Modulates Transmembrane Tight Junction Proteins Claudin-5 and Occludin at the Blood-Brain Barrier. Mountain West Society for Toxicology Meeting. Phoenix, AZ: Society for Toxicology.
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  Opioids are effective as analgesics for treatment of chronic non-cancer pain; however, they cause clinically significant adverse events such as respiratory depression and development of tolerance. Acetaminophen (APAP) has been incorporated into many therapeutic products with opioids, or used in conjunction with opioids, in an effort to provide effective analgesia while reducing opioid dosages (i.e., opioid sparing effect). In 2011, the Food and Drug Administration (FDA) limited the dose of APAP that can be included in combination productions to 325 mg per tablet due to concerns related to liver injury; however, many patients who are prescribed combination products for management of moderate to severe non-cancer pain also consume APAP in excess of the maximum daily limit of 4000 mg/day. Overall use of opioids for chronic non-cancer pain has increased in the United States over the past two decades (Kaye et al. Pain Physician. 20: S93-S109, 2017). Additionally, prescription pain relievers are often used for non-medical purposes (i.e., opioid misuse), an established characteristic of the prescription drug abuse problem in the United States (Vowles et al. Pain. 156: 569-576, 2015). Of particular significance, there is a disproportionate increase in misuse of APAP-containing combination opioid products (Bond et al. Drug Saf. 35: 149-157, 2012). Therefore, it is essential to understand how high doses of APAP and/or high frequency of consumption of combination products containing APAP and opioids can cause injury to body systems other than the liver. Such knowledge is critical to inform development of dosing strategies to counteract misuse of analgesics and to produce safer medications that can be used for treatment of acute and chronic non-cancer pain.