Caroline Machado Kopruszinski

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• Fiatcoski, F., Jesus, C., de Melo Turnes, J., Chichorro, J., & Kopruszinski, C. (2024). Sex differences in descending control of nociception (DCN) responses after chronic orofacial pain induction in rats and the contribution of kappa opioid receptors. Behavioural Brain Research, 459. doi:10.1016/j.bbr.2023.114789
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  Descending control of nociception (DCN), a measure of efficiency of descending pain inhibition, can be assessed in animals by the combined application of test and conditioning noxious stimuli. Evidence from pre-clinical and clinical studies indicates that this mechanism of pain control may differ between sexes and might be impaired in many chronic pain states. However, little is known about sex differences in DCN efficiency in models of acute and chronic orofacial pain. Herein, we first evaluated DCN responses in male and female rats by the applying formalin into the upper lip or capsaicin into the forepaw as the conditioning stimulus, followed by mechanical stimulation (Randall-Selitto) of the hind paw as the test stimulus. The same protocol (i.e., capsaicin in the forepaw followed by mechanical stimulation of the hind paw) was evaluated in male and female rats on day 3 after intraoral incision and on day 15 and 30 after chronic constriction injury of the infraorbital nerve (CCI-ION). Additionally, we assessed the effect of the kappa opioid receptor (KOR) antagonist Norbinaltorphimine (nor-BNI) on DCN responses of female nerve-injured rats. This study shows that naïve female rats exhibit less efficient DCN compared to males. Postoperative pain did not alter DCN responses in female and male rats, but CCI-ION induced loss of DCN responses in females but not in males. Systemic pretreatment with nor-BNI prevented the loss of DCN induced by CCI-ION in female rats. The results reveal sex differences in DCN responses and female-specific impairment of DCN following chronic orofacial pain. Moreover, the findings suggest that, at least for females, blocking KOR could be a promising therapeutic approach to prevent maladaptive changes in chronic orofacial pain.
• Kopruszinski, C., Lee, G., Martin, L., Barber, K., Moutal, A., Dodick, D., Navratilova, E., & Porreca, F. (2024). A male-specific mechanism of meningeal nociceptor sensitization promoting migraine headache. Cephalalgia, 44(9). doi:10.1177/03331024241281493
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  Background: We wished to explore possible sexual dimorphism in mechanisms sensitizing or activating meningeal nociceptors that can promote the headache phase of migraine. Methods: Male and female C57BL6J mice received either supradural orexin B and an inflammatory mediator cocktail (IM) with migraine-like pain behaviors and photophobia recorded. Expression of orexin 2 receptor (OX2R) in trigeminal ganglion (TG) and phosphorylated extracellular signal-regulated kinases (ERK) levels in trigeminal nucleus caudalis (TNC) were evaluated. Orexin B-induced excitability of TG cells was assessed with patch-clamp electrophysiology. Intranasal delivery of CRISPR/Cas9 plasmids was used to edit the expression of OX2R in the TG. Results: Supradural orexin B induced migraine-like pain behaviors, photophobia and increased TNC ERK phosphorylation exclusively in males. Blockade of orexin signaling with supradural suvorexant, a dual orexin receptor antagonist, prevented, but did not reverse, migraine-like pain in males induced by supradural IM cocktail. OX2R expression was higher in male TG and orexin B increased TG neuron excitability in males. Intranasal OX2R CRISPR/Cas9 reduced TG receptor expression and orexin B-induced TNC ERK phosphorylation and prevented migraine-like pain induced by supradural orexin B in males. Conclusions: Our studies reveal a male-specific mechanism of TG nociceptor sensitization and migraine-like pain behavior mediated by orexin B/OX2R signaling. Sexually dimorphic mechanisms of trigeminal nociceptor sensitization and activation offer opportunities to improve patient outcomes by considering patient sex and may influence clinical trial design and interpretation.
• Lillo Vizin, R., Kopruszinski, C., Redman, P., Ito, H., Rau, J., Dodick, D., Navratilova, E., & Porreca, F. (2024). Unraveling the directional relationship of sleep and migraine-like pain. Brain Communications, 6(2). doi:10.1093/braincomms/fcae051
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  Migraine and sleep disorders are common co-morbidities. Patients frequently link their sleep to migraine attacks suggesting a potential causal relationship between these conditions. However, whether migraine pain promotes or disrupts sleep or whether sleep disruption can increase the risk of migraine remains unknown. We assessed the potential impact of periorbital allodynia, a measure consistent with migraine-like pain, from multiple preclinical models on sleep quantity and quality. Additionally, we evaluated the possible consequences of sleep deprivation in promoting susceptibility to migraine-like pain. Following the implantation of electroencephalogram/ electromyography electrodes to record sleep, mice were treated with either single or repeated systemic injections of nitroglycerin at the onset of their active phase (i.e. nocturnal awake period). Neither single nor repeated nitroglycerin affected the total sleep time, non-rapid eye movement sleep, rapid eye movement sleep, sleep depth or other measures of sleep architecture. To account for the possible disruptive effects of the surgical implantation of electroencephalogram/electromyography electrodes, we used immobility recordings as a non-invasive method for assessing sleep-wake behaviour. Neither single nor repeated nitroglycerin administration during either the mouse sleep (i.e. daylight) or active (i.e. night) periods influenced immobility-defined sleep time. Administration of an inflammatory mediator mixture onto the dura mater at either sleep or active phases also did not affect immobility-defined sleep time. Additionally, inhalational umbellulone-induced migraine-like pain in restraint-stressed primed mice did not alter immobility-defined sleep time. The possible influence of sleep disruption on susceptibility to migraine-like pain was evaluated by depriving female mice of sleep over 6 h with novel objects, a method that does not increase circulating stress hormones. Migraine-like pain was not observed following acute sleep deprivation. However, in sleep-deprived mice, subthreshold doses of systemic nitroglycerin or dural calcitonin gene-related peptide induced periorbital cutaneous allodynia consistent with migraine-like pain. Our data reveal that while migraine-like pain does not significantly disrupt sleep, sleep disruption increases vulnerability to migraine-like pain suggesting that a therapeutic strategy focused on improving sleep may diminish migraine attacks.
• Ito, H., Navratilova, E., Vagnerova, B., Watanabe, M., Kopruszinski, C., Moreira De Souza, L., Yue, X., Ikegami, D., Moutal, A., Patwardhan, A., Khanna, R., Yamazaki, M., Guerrero, M., Rosen, H., Roberts, E., Neugebauer, V., Dodick, D., & Porreca, F. (2023). Chronic pain recruits hypothalamic dynorphin/kappa opioid receptor signalling to promote wakefulness and vigilance. Brain, 146(3). doi:10.1093/brain/awac153
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  Increased vigilance in settings of potential threats or in states of vulnerability related to pain is important for survival. Pain disrupts sleep and conversely, sleep disruption enhances pain, but the underlying mechanisms remain unknown. Chronic pain engages brain stress circuits and increases secretion of dynorphin, an endogenous ligand of the kappa opioid receptor (KOR). We therefore hypothesized that hypothalamic dynorphin/KOR signalling may be a previously unknown mechanism that is recruited in pathological conditions requiring increased vigilance. We investigated the role of KOR in wakefulness, non-rapid eye movement (NREM) sleep and rapid eye movement (REM) sleep in freely moving naïve mice and in mice with neuropathic pain induced by partial sciatic nerve ligation using EEG/EMG recordings. Systemic continuous administration of U69,593, a KOR agonist, over 5 days through an osmotic minipump decreased the amount of NREM and REM sleep and increased sleep fragmentation in naïve mice throughout the light-dark sleep cycle. We used KORcre mice to selectively express a Gi-coupled designer receptor activated by designer drugs (Gi-DREADD) in KORcre neurons of the hypothalamic paraventricular nucleus, a key node of the hypothalamic-pituitary-adrenal stress response. Sustained activation of Gi-DREADD with clozapine-N-oxide delivered in drinking water over 4 days, disrupted sleep in these mice in a similar way as systemic U69,593. Mice with chronic neuropathic pain also showed disrupted NREM and total sleep that was normalized by systemic administration of two structurally different KOR antagonists, norbinaltorphimine and NMRA-140, currently in phase II clinical development, or by CRISPR/Cas9 editing of paraventricular nucleus KOR, consistent with endogenous KOR activation disrupting sleep in chronic pain. Unexpectedly, REM sleep was diminished by either systemic KOR antagonist or by CRISPR/Cas9 editing of paraventricular nucleus KOR in sham-operated mice. Our findings reveal previously unknown physiological and pathophysiological roles of dynorphin/KOR in eliciting arousal. Physiologically, dynorphin/KOR signalling affects transitions between sleep stages that promote REM sleep. Furthermore, while KOR antagonists do not promote somnolence in the absence of pain, they normalized disrupted sleep in chronic pain, revealing a pathophysiological role of KOR signalling that is selectively recruited to promote vigilance, increasing chances of survival. Notably, while this mechanism is likely beneficial in the short-term, disruption of the homeostatic need for sleep over longer periods may become maladaptive resulting in sustained pain chronicity. A novel approach for treatment of chronic pain may thus result from normalization of chronic pain-related sleep disruption by KOR antagonism.
• Kopruszinski, C., Watanabe, M., Martinez, A., De Souza, L., Dodick, D., Moutal, A., Neugebauer, V., Porreca, F., & Navratilova, E. (2023). Kappa opioid receptor agonists produce sexually dimorphic and prolactin-dependent hyperalgesic priming. Pain, 164(6). doi:10.1097/j.pain.0000000000002835
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  Repeated stress produces hyperalgesic priming in preclinical models, but underlying mechanisms remain uncertain. As stress engages kappa opioid receptors (KORs), we hypothesized that repeated administration of KOR agonists might mimic, in part, stress-induced hyperalgesic priming. The potential contribution of circulating prolactin (PRL) and dysregulation of the expression of PRL receptor (PRLR) isoforms in sensory neurons after KOR agonist administration was also investigated. Mice received 3 daily doses of U-69593 or nalfurafine as a "first-hit" stimulus followed by assessment of periorbital tactile allodynia. Sixteen days after the first KOR agonist administration, animals received a subthreshold dose of inhalational umbellulone, a TRPA1 agonist, as the second-hit stimulus and periorbital allodynia was assessed. Cabergoline, a dopamine D2 receptor agonist, was used to inhibit circulating PRL in additional cohorts. Prolactin receptor isoforms were quantified in the V1 region of the trigeminal ganglion after repeated doses of U-69593. In both sexes, KOR agonists increased circulating PRL and produced allodynia that resolved within 14 days. Hyperalgesic priming, revealed by umbellulone-induced allodynia in animals previously treated with the KOR agonists, also occurred in both sexes. However, repeated U-69593 downregulated the PRLR long isoform in trigeminal neurons only in female mice. Umbellulone-induced allodynia was prevented by cabergoline co-treatment during priming with KOR agonists in female, but not male, mice. Hyperalgesic priming therefore occurs in both sexes after either biased or nonbiased KOR agonists. However, a PRL/PRLR-dependence is observed only in female nociceptors possibly contributing to pain in stress-related pain disorders in females.
• Rudolph, M., Kopruszinski, C., Wu, C., Navratilova, E., Schwedt, T., Dodick, D., Porreca, F., & Anderson, T. (2023). Identification of brain areas in mice with peak neural activity across the acute and persistent phases of post-traumatic headache. Cephalalgia, 43(11). doi:10.1177/03331024231217469
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  Background: Post-traumatic headache is very common after a mild traumatic brain injury. Post-traumatic headache may persist for months to years after an injury in a substantial proportion of people. The pathophysiology underlying post-traumatic headache remains unknown but is likely distinct from other headache disorders. Identification of brain areas activated in acute and persistent phases of post-traumatic headache can provide insights into the underlying circuits mediating headache pain. We used an animal model of mild traumatic brain injury-induced post-traumatic headache and c-fos immunohistochemistry to identify brain regions with peak activity levels across the acute and persistent phases of post-traumatic headache. Methods: Male and female C57BL/6 J mice were briefly anesthetized and subjected to a sham procedure or a weight drop closed-head mild traumatic brain injury. Cutaneous allodynia was assessed in the periorbital and hindpaw regions using von Frey filaments. Immunohistochemical c-fos based neural activity mapping was then performed on sections from whole brain across the development of post-traumatic headache (i.e. peak of the acute phase at 2 days post- mild traumatic brain injury), start of the persistent phase (i.e. >14 days post-mild traumatic brain injury) or after provocation with stress (bright light). Brain areas with consistent and peak levels of c-fos expression across mild traumatic brain injury induced post-traumatic headache were identified and included for further analysis. Results: Following mild traumatic brain injury, periorbital and hindpaw allodynia was observed in both male and female mice. This allodynia was transient and subsided within the first 14 days post-mild traumatic brain injury and is representative of acute post-traumatic headache. After this acute post-traumatic headache phase, exposure of mild traumatic brain injury mice to a bright light stress reinstated periorbital and hindpaw allodynia for several hours – indicative of the development of persistent post-traumatic headache. Acute post-traumatic headache was coincident with an increase in neuronal c-fos labeling in the spinal nucleus of the trigeminal caudalis, primary somatosensory cortex, and the nucleus accumbens. Neuronal activation returned to baseline levels by the persistent post-traumatic headache phase in the spinal nucleus of the trigeminal caudalis and primary somatosensory cortex but remained elevated in the nucleus accumbens. In the persistent post-traumatic headache phase, coincident with allodynia observed following bright light stress, we observed bright light stress-induced c-fos neural activation in the spinal nucleus of the trigeminal caudalis, primary somatosensory cortex, and nucleus accumbens. Conclusion: Examination of mild traumatic brain injury-induced changes in peak c-fos expression revealed brain regions with significantly increased neural activity across the acute and persistent phases of post-traumatic headache. Our findings suggest mild traumatic brain injury-induced post-traumatic headache produces neural activation along pain relevant pathways at time-points matching post-traumatic headache-like pain behaviors. These observations suggest that the spinal nucleus of the trigeminal caudalis, primary somatosensory cortex, and nucleus accumbens may contribute to both the induction and maintenance of post-traumatic headache.
• Ikegami, D., Navratilova, E., Yue, X., Moutal, A., Kopruszinski, C., Khanna, R., Patwardhan, A., Dodick, D., & Porreca, F. (2022). A prolactin-dependent sexually dimorphic mechanism of migraine chronification. Cephalalgia, 42(3). doi:10.1177/03331024211039813
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  Objective: Determination of possible sex differences in mechanisms promoting migraine progression and the contribution of prolactin and the prolactin long (PRLR-L) and short (PRLR-S) receptor isoforms. Background: The majority of patients with chronic migraine and medication overuse headache are female. Prolactin is present at higher levels in women and increases migraine. Prolactin signaling at the PRLR-S selectively sensitizes nociceptors in female rodents, while expression of the PRLR-L is protective. Methods: Medication overuse headache was modeled by repeated sumatriptan administration in male and female mice. Periorbital and hindpaw cutaneous allodynia served as a surrogate of migraine-like pain. PRLR-L and PRLR-S isoforms were measured in the trigeminal ganglion with western blotting. Possible co-localization of PRLR with serotonin 5HT1B and 5HT1D receptors was determined with RNAscope. Cabergoline, a dopamine receptor agonist that inhibits circulating prolactin, was co-administered with sumatriptan. Nasal administration of CRISPR/Cas9 plasmid was used to edit expression of both PRLR isoforms. Results: PRLR was co-localized with 5HT1B or 5HT1D receptors in the ophthalmic region of female trigeminal ganglion. A single injection of sumatriptan increased serum PRL levels in female mice. Repeated sumatriptan promoted cutaneous allodynia in both sexes but down-regulated trigeminal ganglion PRLR-L, without altering PRLR-S, only in females. Co-administration of sumatriptan with cabergoline prevented allodynia and down-regulation of PRLR-L only in females. CRISPR/Cas9 editing of both PRLR isoforms in the trigeminal ganglion prevented sumatriptan-induced periorbital allodynia in females. Interpretation: We identified a sexually dimorphic mechanism of migraine chronification that involves down-regulation of PRLR-L and increased signaling of circulating prolactin at PRLR-S. These studies reveal a previously unrecognized neuroendocrine mechanism linking the hypothalamus to nociceptor sensitization that increases the risk of migraine pain in females and suggest opportunities for novel sex-specific therapies including gene editing through nasal delivery of CRISPR/Cas9 constructs.
• Kopruszinski, C., Porreca, F., & Chichorro, J. (2022). Editorial: Chronic orofacial pain. Frontiers in Pain Research, 3. doi:10.3389/fpain.2022.1086256
• Kopruszinski, C., Vizin, R., Watanabe, M., Martinez, A., de Souza, L., Dodick, D., Porreca, F., & Navratilova, E. (2022). Exploring the neurobiology of the premonitory phase of migraine preclinically – a role for hypothalamic kappa opioid receptors?. Journal of Headache and Pain, 23(1). doi:10.1186/s10194-022-01497-7
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  Background: The migraine premonitory phase is characterized in part by increased thirst, urination and yawning. Imaging studies show that the hypothalamus is activated in the premonitory phase. Stress is a well know migraine initiation factor which was demonstrated to engage dynorphin/kappa opioid receptors (KOR) signaling in several brain regions, including the hypothalamus. This study proposes the exploration of the possible link between hypothalamic KOR and migraine premonitory symptoms in rodent models. Methods: Rats were treated systemically with the KOR agonist U-69,593 followed by yawning and urination monitoring. Apomorphine, a dopamine D1/2 agonist, was used as a positive control for yawning behaviors. Urination and water consumption following systemic administration of U-69,593 was also assessed. To examine if KOR activation specifically in the hypothalamus can promote premonitory symptoms, AAV8-hSyn-DIO-hM4Di (Gi-DREADD)-mCherry viral vector was microinjected into the right arcuate nucleus (ARC) of female and male KORCRE or KORWT mice. Four weeks after the injection, clozapine N-oxide (CNO) was administered systemically followed by the assessment of urination, water consumption and tactile sensory response. Results: Systemic administration of U-69,593 increased urination but did not produce yawning in rats. Systemic KOR agonist also increased urination in mice as well as water consumption. Cell specific Gi-DREADD activation (i.e., inhibition through Gi-coupled signaling) of KORCRE neurons in the ARC also increased water consumption and the total volume of urine in mice but did not affect tactile sensory responses. Conclusion: Our studies in rodents identified the KOR in a hypothalamic region as a mechanism that promotes behaviors consistent with clinically-observed premonitory symptoms of migraine, including increased thirst and urination but not yawning. Importantly, these behaviors occurred in the absence of pain responses, consistent with the emergence of the premonitory phase before the headache phase. Early intervention for preventive treatment even before the headache phase may be achievable by targeting the hypothalamic KOR.
• Watanabe, M., Kopruszinski, C., Moutal, A., Ikegami, D., Khanna, R., Chen, Y., Ross, S., MacKenzie, K., Stratton, J., Dodick, D., Navratilova, E., & Porreca, F. (2022). Dysregulation of serum prolactin links the hypothalamus with female nociceptors to promote migraine. Brain, 145(8). doi:10.1093/brain/awac104
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  Migraine headache results from activation of meningeal nociceptors, however, the hypothalamus is activated many hours before the emergence of pain. How hypothalamic neural mechanisms may influence trigeminal nociceptor function remains unknown. Stress is a common migraine trigger that engages hypothalamic dynorphin/kappa opioid receptor (KOR) signalling and increases circulating prolactin. Prolactin acts at both long and short prolactin receptor isoforms that are expressed in trigeminal afferents. Following downregulation of the prolactin receptor long isoform, prolactin signalling at the prolactin receptor short isoform sensitizes nociceptors selectively in females. We hypothesized that stress may activate the kappa opioid receptor on tuberoinfundibular dopaminergic neurons to increase circulating prolactin leading to female-selective sensitization of trigeminal nociceptors through dysregulation of prolactin receptor isoforms. A mouse two-hit hyperalgesic priming model of migraine was used. Repeated restraint stress promoted vulnerability (i.e. first-hit priming) to a subsequent subthreshold (i.e. second-hit) stimulus from inhalational umbellulone, a TRPA1 agonist. Periorbital cutaneous allodynia served as a surrogate of migraine-like pain. Female and male KORCre; R26lsl-Sun1-GFP mice showed a high percentage of KORCre labelled neurons co-localized in tyrosine hydroxylase-positive cells in the hypothalamic arcuate nucleus. Restraint stress increased circulating prolactin to a greater degree in females. Stress-primed, but not control, mice of both sexes developed periorbital allodynia following inhalational umbellulone. Gi-DREADD activation (i.e. inhibition through Gi-coupled signalling) in KORCre neurons in the arcuate nucleus also increased circulating prolactin and repeated chemogenetic manipulation of these neurons primed mice of both sexes to umbellulone. Clustered regularly interspaced short palindromic repeats-Cas9 deletion of the arcuate nucleus KOR prevented restraint stress-induced prolactin release in female mice and priming from repeated stress episodes in both sexes. Inhibition of circulating prolactin occurred with systemic cabergoline, a dopamine D2 receptor agonist, blocked priming selectively in females. Repeated restraint stress downregulated the prolactin receptor long isoform in the trigeminal ganglia of female mice. Deletion of prolactin receptor in trigeminal ganglia by nasal clustered regularly interspaced short palindromic repeats-Cas9 targeting both prolactin receptor isoforms prevented stress-induced priming in female mice. Stress-induced activation of hypothalamic KOR increases circulating prolactin resulting in trigeminal downregulation of prolactin receptor long and pain responses to a normally innocuous TRPA1 stimulus. These are the first data that provide a mechanistic link between stress-induced hypothalamic activation and the trigeminal nociceptor effectors that produce trigeminal sensitization and migraine-like pain. This sexually dimorphic mechanism may help to explain female prevalence of migraine. KOR antagonists, currently in phase II clinical trials, may be useful as migraine preventives in both sexes, while dopamine agonists and prolactin/ prolactin receptor antibodies may improve therapy for migraine, and other stress-related neurological disorders, in females.
• Kopruszinski, C., Navratilova, E., Swiokla, J., Dodick, D., Chessell, I., & Porreca, F. (2021). A novel, injury-free rodent model of vulnerability for assessment of acute and preventive therapies reveals temporal contributions of CGRP-receptor activation in migraine-like pain. Cephalalgia, 41(3). doi:10.1177/0333102420959794
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  Aim: Development and characterization of a novel injury-free preclinical model of migraine-like pain allowing mechanistic assessment of both acute and preventive treatments. Methods: A “two-hit” hyperalgesic priming strategy was used to induce vulnerability to a normally subthreshold challenge with umbellulone, a transient receptor potential ankyrin 1 (TRPA1) activator, in uninjured female and male C57BL/6 mice. Priming (i.e. the first hit) was induced by three consecutive daily episodes of restraint stress; repeated umbellulone was also evaluated for potential priming effects. Sixteen days after the first restraint stress, mice received inhalational umbellulone (i.e. the second hit) to elicit migraine-like pain. Medications currently used for acute or preventive migraine therapy including propranolol (a beta blocker) and sumatriptan (5HT1B/D agonist), as well as olcegepant, an experimental calcitonin gene related peptide (CGRP) receptor antagonist and nor-Binaltorphimine (nor-BNI), an experimental long-acting kappa opioid receptor (KOR) antagonist, were investigated for their efficacy to block priming and prevent or reverse umbellulone-induced allodynia in primed animals. To assess migraine-like pain, cutaneous allodynia was determined by responses to periorbital or hindpaw probing with von Frey filaments. Results: Repeated restraint stress, but not umbellulone exposure, produced transient cutaneous allodynia that resolved within 16 d. Restraint stress produced long-lasting priming that persisted beyond 16 d, as demonstrated by reinstatement of cutaneous allodynia following inhalational umbellulone challenge. Pretreatment with propranolol or nor-BNI prior to restraint stress prevented both transient cutaneous allodynia and priming, demonstrated by a lack of umbellulone-induced cutaneous allodynia. Following establishment of restraint stress priming, olcegepant, but not propranolol or nor-BNI, prevented umbellulone-induced cutaneous allodynia. When administered 1 h after umbellulone, sumatriptan, but not olcegepant, reversed umbellulone-induced cutaneous allodynia in restraint stress-primed rats. Conclusion: We have developed a novel injury-free model with translational relevance that can be used to study mechanisms relevant to migraine-like pain and to evaluate novel acute or preventive treatments. Restraint stress priming induced a state of vulnerability to a subthreshold stimulus that has been referred to as “latent sensitization”. The development of latent sensitization could be prevented by blockade of stress pathways with propranolol or with a kappa opioid receptor antagonist. Following establishment of latent sensitization, subthreshold stimulation with umbellulone reinstated cutaneous allodynia, likely from activation of meningeal TRPA1-expressing nociceptors. Accordingly, in restraint stress-primed animals, sumatriptan reversed umbellulone-induced cutaneous allodynia, supporting peripheral sites of action, while propranolol and nor-BNI were not effective. Surprisingly, olcegepant was effective in mice with latent sensitization when given prior to, but not after, umbellulone challenge, suggesting time-dependent contributions of calcitonin gene-related peptide receptor signaling in promoting migraine-like pain in this model. Activation of the calcitonin gene-related peptide receptor participates in initiating, but has a more limited role in maintaining, pain responses, supporting the efficacy of small molecule calcitonin gene-related peptide antagonists as preventive medications. Additionally, the effectiveness of sumatriptan in reversal of established pain thus suggests modulation of additional, non-calcitonin gene-related peptide receptor-mediated nociceptive mechanisms. Kappa opioid receptor antagonists may represent a novel preventive therapy for stress-related migraine.
• Kopruszinski, C., Swiokla, J., Lee, Y., Navratilova, E., VanderVeen, L., Yang, M., Liu, Y., Miyazaki, T., Schmidt, W., Zalevsky, J., & Porreca, F. (2021). Preclinical Assessment of the Analgesic Pharmacology of NKTR-181 in Rodents. Cellular and Molecular Neurobiology, 41(5). doi:10.1007/s10571-020-00816-3
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  Objective: Pharmacological evaluation of the mu-opioid receptor (MOR) agonist properties of NKTR-181 in rodent models. Methods: Graded noxious stimulus intensities were used in rats to establish the antinociceptive potency and efficacy of NKTR-181 relative to morphine, fentanyl, and oxycodone. Characteristics of MOR agonist actions, as measured by antinociceptive tolerance and cross-tolerance, as well as opioid-induced hyperalgesia (OIH) and naloxone-precipitated withdrawal in NKTR-181- and morphine-dependent in mice, were compared. Results: NKTR-181 showed dose- and time-related antinociception with similar maximal effects to morphine in the rat and mouse hot-water tail-flick test. No sex or species differences were observed in NKTR-181 or morphine antinociception. Rats treated with NKTR-181 and morphine exhibited decreases in both potency and maximal efficacy as nociceptive stimulus intensity was increased from a water temperature of 50 °C to 54 °C. Evaluation of antinociception at a high stimulus intensity revealed that oxycodone and fentanyl exhibited greater efficacy than either NKTR-181 or morphine. The relative potency difference between NKTR-181 and morphine across all tail-flick studies was determined to be 7.6-fold (90% confidence interval, 2.6, 21.5). The peak antinociceptive effect of NKTR-181 was delayed compared to that of the other opioids and cumulative drug effects were not observed. Repeated treatment with escalating, approximately equi-analgesic doses of NKTR-181 or morphine, produced antinociceptive tolerance and cross-tolerance. Under these pharmacological conditions, OIH and naloxone-precipitated physical dependence were similar for NKTR-181 and morphine. Conclusions: NKTR-181 had a slower onset, but similar efficacy, to morphine in the models studied supporting reduced abuse potential while maintaining analgesic effect in comparison with current opioids.
• Kopruszinski, C., Turnes, J., Swiokla, J., Weinstein, T., Schwedt, T., Dodick, D., Anderson, T., Navratilova, E., & Porreca, F. (2021). CGRP monoclonal antibody prevents the loss of diffuse noxious inhibitory controls (DNIC) in a mouse model of post-traumatic headache. Cephalalgia, 41(6). doi:10.1177/0333102420981688
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  Aim: Determine the role of calcitonin-gene related peptide in promoting post-traumatic headache and dysregulation of central pain modulation induced by mild traumatic brain injury in mice. Methods: Mild traumatic brain injury was induced in lightly anesthetized male C57BL/6J mice by a weight drop onto a closed and unfixed skull, which allowed free head rotation after the impact. We first determined possible alterations in the diffuse noxious inhibitory controls, a measure of net descending pain inhibition called conditioned pain modulation in humans at day 2 following mild traumatic brain injury. Diffuse noxious inhibitory control was assessed as the latency to a thermally induced tail-flick that served as the test stimulus in the presence of right forepaw capsaicin injection that provided the conditioning stimulus. Post-traumatic headache-like behaviors were assessed by the development of cutaneous allodynia in the periorbital and hindpaw regions after mild traumatic brain injury. We then determined if intraperitoneal fremanezumab, an anti-calcitonin-gene related peptide monoclonal antibody or vehicle administered 2 h after sham or mild traumatic brain injury induction could alter cutaneous allodynia or diffuse noxious inhibitory control responses on day 2 post mild traumatic brain injury. Results: In naïve and sham mice, capsaicin injection into the forepaw elevated the latency to tail-flick, reflecting the antinociceptive diffuse noxious inhibitory control response. Periorbital and hindpaw cutaneous allodynia, as well as a loss of diffuse noxious inhibitory control, was observed in mice 2 days after mild traumatic brain injury. Systemic treatment with fremanezumab blocked mild traumatic brain injury-induced cutaneous allodynia and prevented the loss of diffuse noxious inhibitory controls in mice subjected to a mild traumatic brain injury. Interpretation: Sequestration of calcitonin-gene related peptide in the initial stages following mild traumatic brain injury blocked the acute allodynia that may reflect mild traumatic brain injury-related post-traumatic headache and, additionally, prevented the loss of net descending inhibition within central pain modulation pathways. As loss of conditioned pain modulation has been linked to multiple persistent pain conditions, dysregulation of descending modulatory pathways may contribute to the persistence of post-traumatic headache. Additionally, evaluation of the conditioned pain modulation/diffuse noxious inhibitory controls response may serve as a biomarker of vulnerability for chronic/persistent pain. These findings suggest that early anti-calcitonin-gene related peptide intervention has the potential to be effective both for the treatment of mild traumatic brain injury-induced post-traumatic headache, as well as inhibiting mechanisms that may promote post-traumatic headache persistence.
• Chen, Y., Moutal, A., Navratilova, E., Kopruszinski, C., Yue, X., Ikegami, M., Chow, M., Kanazawa, I., Bellampalli, S., Xie, J., Patwardhan, A., Rice, K., Fields, H., Akopian, A., Neugebauer, V., Dodick, D., Khanna, R., & Porreca, F. (2020). The prolactin receptor long isoform regulates nociceptor sensitization and opioid-induced hyperalgesia selectively in females. Science Translational Medicine, 12(529). doi:10.1126/scitranslmed.aay7550
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  Pain is more prevalent in women for reasons that remain unclear. We have identified a mechanism of injury-free nociceptor sensitization and opioid-induced hyperalgesia (OIH) promoted by prolactin (PRL) in females. PRL signals through mutually inhibitory long (PRLR-L) and short (PRLR-S) receptor isoforms, and PRLR-S activation induces neuronal excitability. PRL and PRLR expression were higher in females. CRISPR-mediated editing of PRLR-L promoted nociceptor sensitization and allodynia in naïve, uninjured female mice that depended on circulating PRL. Opioids, but not trauma-induced nerve injury, decreased PRLR-L promoting OIH through activation of PRLR-S in female mice. Deletion of both PRLR-L and PRLR-S (total PRLR) prevented, whereas PRLR-L overexpression rescued established OIH selectively in females. Inhibition of circulating PRL with cabergoline, a dopamine D2 agonist, up-regulated PRLR-L and prevented OIH only in females. The PRLR-L isoform therefore confers protection against PRL-promoted pain in females. Limiting PRL/PRLR-S signaling pharmacologically or with gene therapies targeting the PRLR may be effective for reducing pain in a female-selective manner.
• Kopruszinski, C., Navratilova, E., Vagnerova, B., Swiokla, J., Patwardhan, A., Dodick, D., & Porreca, F. (2020). Cannabinoids induce latent sensitization in a preclinical model of medication overuse headache. Cephalalgia, 40(1). doi:10.1177/0333102419865252
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  Aim: Evaluation of cannabinoid receptor agonists in a preclinical model of medication overuse headache. Methods: Female Sprague Dawley rats received graded intraperitoneal doses of WIN55,212-2 or Δ-9-tetrahydrocannabinol (Δ-9-THC). Antinociception (tail-flick test), catalepsy and hypomotility (open field test) and impairment of motor function (rotarod test) were assessed to establish effective dosing. Rats were then treated twice daily with equianalgesic doses of WIN55,212-2 or Δ-9-THC, or vehicle, for 7 days and cutaneous tactile sensory thresholds were evaluated during and three weeks following drug discontinuation. Rats then received a one-hour period of bright light stress (BLS) on two consecutive days and tactile sensory thresholds were re-assessed. Results: WIN55,212-2 and Δ-9-THC produced antinociception as well as hypomotility, catalepsy and motor impairment. Repeated administration of WIN55,212-2 and Δ-9-THC induced generalized periorbital and hindpaw allodynia that resolved within 3 weeks after discontinuation of drug. Two episodes of BLS produced delayed and long-lasting periorbital and hindpaw allodynia selectively in rats previously treated with WIN55,212-2, and Δ-9-THC. Interpretation: Cannabinoid receptor agonists including Δ-9-THC produce a state of latent sensitization characterized by increased sensitivity to stress, a presumed migraine trigger. Overuse of cannabinoids including cannabis may increase the risk of medication overuse headache in vulnerable individuals.
• Kopruszinski, C., Thornton, P., Arnold, J., Newton, P., Lowne, D., Navratilova, E., Swiokla, J., Dodick, D., Dobson, C., Gurrell, I., Chessell, I., & Porreca, F. (2020). Characterization and preclinical evaluation of a protease activated receptor 2 (PAR2) monoclonal antibody as a preventive therapy for migraine. Cephalalgia, 40(14). doi:10.1177/0333102420966581
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  Aim: Migraine pain is thought to result from activation of meningeal nociceptors that might involve dural mast cell degranulation and release of proteases and pronociceptive mediators. Tryptase, the most abundant dural mast cell protease, has been demonstrated to stimulate dural mast cells, as well as trigeminal nociceptors by activating the protease activated receptor 2. Mast cell or neuronal protease activated receptors 2 may therefore represent a novel target for migraine treatment. In this study, we characterized and evaluated a novel protease activated receptor 2 monoclonal antibody as a preventive anti-migraine pain therapy in preclinical models. Methods: Flow cytometry, immunocytochemistry, calcium imaging, Homogeneous Time Resolved Technology (HTRF) epitope competition assay and serum pharmacokinetic (PK) assay in rats were performed to confirm the activity, specificity and in vivo stability of PAR650097, a novel anti- protease activated receptor 2 monoclonal antibody. In vivo assessment was performed in female C57BL/6J mice by evaluation of PAR650097 in preventing cutaneous allodynia elicited by (a) supradural injection of the protease activated receptor 2 agonist, Ser-Leu-Ile-Gly-Arg-Leu-amide trifluoroacetate (SLIGRL), or calcitonin gene-related (CGRP) peptide, and (b) induction of latent sensitization by priming with three daily episodes of restraint stress followed by challenge with a subthreshold inhalational exposure to umbellulone (UMB), a transient receptor potential ankyrin 1 (TRPA1) agonist. PAR650097 was administered as a pretreatment prior to the first restraint stress, umbellulone exposure, SLIGRL or calcitonin gene-related peptide injection. Additionally, fremanezumab, a calcitonin gene-related peptide antibody was administered as pre-treatment prior to supradural administration of calcitonin gene-related peptide or SLIGRL. Results: In vitro, PAR650097 demonstrated rapid interaction with protease activated receptor 2, enabling it to fully inhibit protease-induced protease activated receptor 2 activation, in human and mouse cells, with high potency. Furthermore, PAR650097 was highly selective for protease activated receptor 2, demonstrating no affinity for protease activated receptor 1 protein and no functional effect on the activation of cellular protease activated receptor 1 with thrombin. In addition, PAR650097 had an acceptable PK profile, compatible with testing the effects of selective protease activated receptor 2 inhibition in vivo. In vivo, PAR650097 blocked cutaneous allodynia induced by either supradural SLIGRL or calcitonin gene-related peptide. Fremanezumab abolished cutaneous allodynia induced by supradural CGRP, and partially attenuated cutaneous allodynia induced by SLIGRL. Administration of PAR650097, before the first restraint stress episode, did not prevent the acute stress-induced cutaneous allodynia or restraint stress priming revealed by cutaneous allodynia induced by inhalational umbellulone. In contrast, PAR650097 prevented expression of cutaneous allodynia when given before the umbellulone challenge in restraint stress-primed animals. Conclusion: PAR650097 specifically inhibits endogenously expressed protease activated receptor 2 in human and mouse cells with high potency. This antibody has an acceptable PK profile in rodents and effectively blocked SLIGR-induced cutaneous allodynia. PAR650097 additionally prevented cutaneous allodynia induced by supradural calcitonin gene-related peptide, indicating that the protease activated receptor 2 receptor is a downstream consequence of calcitonin gene-related peptide actions. Fremanezumab effectively blocked calcitonin gene-related peptide-induced cutaneous allodynia and only partially reduced cutaneous allodynia induced by a protease activated receptor 2 activator, suggesting both calcitonin gene-related peptide-dependent and -independent mechanisms in promoting migraine pain. While PAR650097 did not prevent stress-induced cutaneous allodynia or priming, it effectively prevented cutaneous allodynia induced by a TRPA1 agonist in animals with latent sensitization. Activation of protease activated receptor 2, therefore, contributes to both calcitonin gene-related peptide-dependent and -independent mechanisms in promoting migraine-like pain. Therapeutic targeting of protease activated receptor 2 receptors may represent an anti-migraine pain strategy with a potentially broad efficacy profile.
• dos Reis, R., Kopruszinski, C., Nones, C., Aguiar, D., & Chichorro, J. (2020). The opposing contribution of neurotrophin-3 and nerve growth factor to orofacial heat hyperalgesia in rats. Behavioural Pharmacology, 31(1). doi:10.1097/FBP.0000000000000503
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  It has been proposed that neurotrophin-3 acts in a manner that is opposed to nerve growth factor, especially in the modulation of heat hyperalgesia. Injury to the constriction of the infraorbital nerve (CION) is a well-established model of trigeminal neuropathic pain that leads to robust heat, cold, and mechanical hyperalgesia. Here, we assessed the effect of local neurotrophin-3 treatment on CION-induced hyperalgesia, and we examined some mechanisms related to the effect of neurotrophin-3. Neurotrophin-3 (1 µg/50 µl) injected into the upper lip of CION rats caused a significant and long-lasting reduction of CION-induced heat hyperalgesia, but failed to affect cold and mechanical hyperalgesia. Increased levels of neurotrophin-3 were detected in the injured nerve at the time point that represents the peak of heat hyperalgesia. The anti-hyperalgesic effect of neurotrophin-3 was markedly reduced in the presence of an antagonist of TrkA receptors (K-252a, 1 µg/50 µl). Moreover, association of lower doses of neurotrophin-3 with an antibody anti-nerve growth factor resulted in a synergistic anti-hyperalgesic effect in CION rats. Local injection of nerve growth factor (3 µg/50 µl) or the TRPV1 agonist capsaicin (1 µg/50 µl), but not neurotrophin-3 injection (1 µg/50 µl), resulted in long-lasting facial heat hyperalgesia, which was both significantly reduced by previous neurotrophin-3 local treatment. In conclusion, we suggest that neurotrophin-3 is a potent modulator of facial heat hyperalgesia, which may exert an inhibitory influence on the trkA pathway. Neurotrophin-3 treatment may represent a promising approach, especially in pain conditions associated with increased levels of nerve growth factor.
• Kopruszinski, C., dos Reis, R., Rae, G., & Chichorro, J. (2019). Blockade of peripheral endothelin receptors abolishes heat hyperalgesia and spontaneous nociceptive behavior in a rat model of facial cancer. Archives of Oral Biology, 97. doi:10.1016/j.archoralbio.2018.10.038
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  Objective: To improve understanding of the pathophysiology of cancer-induced facial nociception, by evaluating the contribution of peripheral endothelin receptors in tumor-induced facial heat hyperalgesia, increased spontaneous grooming, as well as ongoing nociception in a rat model of facial cancer. Design: The study was conducted using 396 rats. Facial cancer was induced by inoculating a suspension of Walker-256 cells into the rats’ right vibrissal pad. Facial heat hyperalgesia and spontaneous grooming were assessed on day 6, while the conditioned place preference (CPP) test was performed on days 3–6 after tumor cells inoculation. Rats received local injections of the non-peptidic dual ET A /ET B endothelin receptors antagonist, bosentan (10 and 30 μg/50 μL), single or combined injections of peptidic ET A and ET B endothelin receptors antagonists (BQ-123 and BQ-788, at 20 ug/50 μL, each), or of lidocaine (1 mg/50 μl) and morphine (30 μg/50 μL). Results: Bosentan, lidocaine and morphine local treatment all attenuated tumor-induced heat hyperalgesia (p < 0.05) and spontaneous facial grooming (p < 0.05). However, BQ-123 and BQ-788 did not modify tumor-induced heat hyperalgesia or the spontaneous facial grooming (p > 0.05). Whether this difference in effectiveness is due to receptor affinity or to pharmacokinetic factors still needs to be explored. Local injection of bosentan, lidocaine or morphine failed to control ongoing nociception, as evidenced by the absence of CPP in tumor-bearing rats (p > 0.05). Conclusion: Endothelins, acting through peripheral ET A and ET B receptors, may play a significant role on the development of heat hyperalgesia and increased spontaneous grooming associated to facial cancer in rats.
• Gambeta, E., Batista, M., Maschio, G., Turnes, J., Araya, E., & Chichorro, J. (2018). Anxiety- but not depressive-like behaviors are related to facial hyperalgesia in a model of trigeminal neuropathic pain in rats. Physiol Behav, 191. doi:10.1016/j.physbeh.2018.04.025
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  Trigeminal neuralgia (TN) is a painful condition characterized by excruciating facial pain, which has a serious impact on quality of life. Depression and anxiety have been commonly associated with TN, but clinical studies report that these comorbidities are frequently underdiagnosed and undertreated in TN patients. Herein it was investigated if rats submitted to the infraorbital nerve constriction (CION), a model of trigeminal neuropathic pain, would display anxiety- and depressive-like behaviors in addition to the facial sensory changes in different time points after the nerve injury. CION rats developed facial heat hyperalgesia on day 5 after the nerve injury, but at this time point the time spent and the number of entries on open arms in the elevated plus maze (EPM) and the time spent on the lit compartment of light-dark transition test (LDT) was not statistically significant between SHAM and CION groups, suggesting that 5 days after CION animals do not display anxiety-like behavior. On the other hand, around 50% of CION rats developed mechanical allodynia on day 15 postsurgery and the analysis of the time spent and the number of entries on open arms on EPM and the time spent on lit compartment of LDT revealed that only CION-allodynic animals displayed anxiety-like behavior when compared to the SHAM group. The depressive-like behavior was assessed by measuring the time of immobility on the forced swim test (FST) and sucrose preference (SP) in rats previously tested for heat (day 5) and mechanical allodynia (days 15, 30 and 45) induced by CION. The evaluation of immobility time on FST and sucrose preference consumption revealed that both CION rats did not displayed depressive- and anhedonic-like behavior at any time point evaluated. Altogether, these results demonstrate that trigeminal neuropathic pain in rats leads to the development of anxiety-, but not depressive-like behavior, suggesting that the CION model represents a methodology that allows the study of drugs targeting both pain and anxiety.
• Kopruszinski, C., dos Reis, R., Gambeta, E., Acco, A., Rae, G., King, T., & Chichorro, J. (2018). Blockade of endothelin receptors reduces tumor-induced ongoing pain and evoked hypersensitivity in a rat model of facial carcinoma induced pain. European Journal of Pharmacology, 818. doi:10.1016/j.ejphar.2017.10.045
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  Pain reported by patients with head and neck cancer is characterized as persistent pain with mechanical allodynia. Pain management is inadequate for many patients, highlighting the need for improved therapies. We examined the hypothesis that the mixed endothelin ETA and ETB receptor antagonist, bosentan, reduces tumor-induced ongoing pain and evoked hypersensitivity in a rat model of facial cancer pain. Facial cancer was induced by inoculating a suspension of Walker-256 cells into the rat's right vibrissal pad. Tumor-bearing rats developed heat and tactile hypersensitivity along with increased spontaneous grooming behavior. Systemic morphine (2.5 mg/kg, s.c.) blocked tumor-induced thermal and tactile hypersensitivity, with a lower dose (0.625 mg/kg, s.c.) effective only against thermal hypersensitivity. Systemic bosentan blocked tumor-induced thermal hypersensitivity only at a high (300 mg/kg, p.o.) dose, but failed to modify tactile hypersensitivity. Co-administration of the low doses of bosentan and morphine resulted in improved reduction of the tumor-induced heat and tactile hypersensitivity compared to either dose alone. Bosentan (100 mg/kg, p.o.) reduced spontaneous grooming and induced conditioned place preference (CPP) selectively in tumor-bearing rats, suggesting that bosentan reduces tumor-induced ongoing pain at a lower dose than required to block tumor-induced hypersensitivity. This study provides evidence that endothelins may mediate tumor-induced ongoing pain and thermal hypersensitivity. In addition, bosentan enhanced morphine's effects on blocking tumor-induced heat and tactile hypersensitivity indicating that endothelin antagonists may be beneficial therapeutic targets that can be used to manage cancer-induced facial pain with opioid-sparing effects.
• Araya, E., Nones, C., Ferreira, L., Kopruszinski, C., Cunha, J., & Chichorro, J. (2017). Role of peripheral and central TRPV1 receptors in facial heat hyperalgesia in streptozotocin-induced diabetic rats. Brain Research, 1670. doi:10.1016/j.brainres.2017.06.004
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  There is increasing evidence that diabetes may be related to sensory changes in the trigeminal system. Long lasting facial heat hyperalgesia has been described in diabetic rats, but the mechanisms remain to be elucidated. Herein, the contribution of peripheral and central TRPV1 receptors to facial heat hyperalgesia in diabeticrats was investigated. Diabetes was induced in male Wistar rats by streptozotocin (60 mg/kg, i.p) and facial heat hyperalgesia was assessed once a week up to four weeks. The role of TRPV1 receptors in the heat hyperalgesia in diabetic rats was evaluated through: 1) the ablation of TRPV1 receptors by resiniferatoxin (RTX) treatment and 2) injection of the TRPV1 antagonist, capsazepine, into the upper lip, trigeminal ganglion or medullary subarachnoid space, at doses that completed prevented the heat hyperalgesia induced by capsaicin in naïve rats. Western blot was used to estimate the changes in TRPV1 expression in diabetic rats. Diabetic rats exhibited facial heat hyperalgesia from the first up to the fourth week after streptozotocin injection, which was prevented by insulin treatment. Ablation of TRPV1-expressing fibers prevented facial hyperalgesia in diabetic rats. Capsazepine injection in all sites resulted in significant reduction of facial heat hyperalgesia in diabetic rats. Diabetic rats exhibited a significant decrease in TRPV1 expression in the trigeminal nerve, increased expression in the trigeminal ganglion and no changes in subnucleus caudalis when compared to normoglycemic ones. In conclusion, our results suggest that facial heat hyperalgesia in diabetic rats is maintained by peripheral and central TRPV1 receptors activation.
• Gambeta, E., Kopruszinski, C., dos Reis, R., Zanoveli, J., & Chichorro, J. (2017). Facial pain and anxiety-like behavior are reduced by pregabalin in a model of facial carcinoma in rats. Neuropharmacology, 125. doi:10.1016/j.neuropharm.2017.07.035
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  Pain and anxiety are common symptoms in head and neck cancer patients. The anticonvulsant pregabalin has therapeutic indication for the treatment of pain and anxiety, and may represent a useful drug for both conditions. Thus, the aim of this study was to investigate the relationship between pain and anxiety in rats with facial carcinoma, as the influence of pregabalin treatment in both aspects. Facial carcinoma was induced by subcutaneous inoculation of Walker-256 tumor cells in the vibrissa pad of Wistar rats. On day 6 after inoculation spontaneous facial grooming and conditioned place preference were assessed as non-evoked pain measurements and facial mechanical hyperalgesia were assessed 3 and 6 days after tumor cells inoculation. Moreover, anxiety-like behavior was evaluated on the elevated plus maze and light-dark transition tests at the same time points. The effect of pregabalin treatment (30 mg/kg, p.o.) was evaluated in all tests. Our results demonstrated that pregabalin treatment reduced the spontaneous facial grooming and induced conditioned place preference 6 days post tumor inoculation. Tumor-bearing rats developed mechanical hyperalgesia starting 3 days post tumor induction, which was also significant on day 6, but the anxiety-like behavior was detected only in tumor-bearing rats that developed mechanical hyperalgesia and only six days after tumor cells inoculation. Both, the mechanical hyperalgesia and the anxiety–like behavior related to the tumor were significantly reduced by pregabalin treatment on day 6. Pregabalin treatment resulted in antinociceptive and anxiolytic-like effects on facial tumor-bearing rats and may represent a promising therapeutic option for cancer patients.
• Kopruszinski, C., Xie, J., Eyde, N., Remeniuk, B., Walter, S., Stratton, J., Bigal, M., Chichorro, J., Dodick, D., & Porreca, F. (2017). Prevention of stress- or nitric oxide donor-induced medication overuse headache by a calcitonin gene-related peptide antibody in rodents. Cephalalgia, 37(6). doi:10.1177/0333102416650702
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  Objective The objective of this study was the determination of the role of calcitonin gene-related peptide (CGRP) in the induction of medication overuse headache (MOH)-related migraine in an injury-free preclinical model. Methods Rats were primed by a 7-day period of exposure to acute migraine therapies including sumatriptan and morphine. After an additional 14-day drug-free period, rats were exposed to putative migraine triggers including bright light stress (BLS) or nitric oxide (NO) donor in the presence or absence of TEV48125, a fully humanized CGRP antibody. Cutaneous allodynia (CA) was used as an outcome measure and CGRP blood and cerebrospinal fluid (CSF) levels were measured. Results BLS and NO donor challenge evoked delayed, long-lasting CA selectively in rats that were previously treated with sumatriptan or morphine. BLS produced a significant increase in CGRP in the plasma, but not CSF, in animals that were previously exposed to sumatriptan compared to saline controls. TEV48125 did not modify baseline tactile thresholds or produce behavioral side effects, but significantly inhibited both BLS- and NO donor-induced CA in animals that were previously primed with sumatriptan or morphine; an isotype control protein that does not bind CGRP had no effect. Interpretation These data suggest that acute migraine medications may promote MOH in susceptible individuals through CGRP-dependent mechanisms and that anti-CGRP antibodies may be a useful clinical strategy for the treatment of MOH.
• Souza, R., Oliveira, L., Nones, C., dos Reis, R., Araya, E., Kopruszinski, C., Rae, G., & Chichorro, J. (2017). Mechanisms involved in facial heat hyperalgesia induced by endothelin-1 in female rats. Archives of Oral Biology, 83. doi:10.1016/j.archoralbio.2017.08.015
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  Objective Pronociceptive responses to endothelins in the trigeminal system seem to be mediated by ETA and ETB receptors, which have been shown to be expressed in neurons of the trigeminal ganglion of humans and rats. The present study aimed to evaluate the ability of endothelin-1 (ET-1) to induce facial heat hyperalgesia in female rats, the contribution of ETA and ETB receptors to this response, as well as the mechanisms underlying heat hyperalgesia induced by ET-1. Design ET-1 (100 pmol/50 μL) was injected into the upper lip and heat hyperalgesia was evaluated for up to 6 h. Facial heat hyperalgesia induced by ET-1 was assessed in rats pre-treated locally with BQ-123 or BQ-788 (selective ETA and ETB receptor antagonists, respectively, 30 nmol/50 μL); BCTC (TRPV1 receptor antagonist; 300 μg/50 μL); anti-NGF (3 μg/50 μL); K252a (TrkA inhibitor, 1 μg/50 μL); or in rats that received intraganglionar resiniferatoxin injection (RTX, 200 ng/10 μL) to promote C-fibers ablation. Results ET-1 induced facial heat hyperalgesia that persisted up to 6 h and was prevented by BQ-123, BQ-788 or by intraganglionar RTX injection. Likewise, local pre-treatment with BCTC abolished ET-1 induced facial heat hyperalgesia up to 3 h. Local pre-treatment with anti-NGF or K252a was effective to prevent ET-1 induced heat hyperalgesia. Conclusions In conclusion, ET-1 is able to induce heat hyperagelsia in trigeminal primary afferents of female rats, which is mediated by ETA and ETB receptors. Activation of TRPV1 receptors and NGF-signaling pathways may contribute to heat hyperalgesia induced by ET-1.
• Xie, J., De Felice, M., Kopruszinski, C., Eyde, N., Lavigne, J., Remeniuk, B., Hernandez, P., Yue, X., Goshima, N., Ossipov, M., King, T., Streicher, J., Navratilova, E., Dodick, D., Rosen, H., Roberts, E., & Porreca, F. (2017). Kappa opioid receptor antagonists: A possible new class of therapeutics for migraine prevention. Cephalalgia, 37(8). doi:10.1177/0333102417702120
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  Background Stress is the most commonly reported migraine trigger. Dynorphin, an endogenous opioid peptide acting preferentially at kappa opioid receptors (KORs), is a key mediator of stress responses. The aim of this study was to use an injury-free rat model of functional cephalic pain with features of migraine and medication overuse headache (MOH) to test the possible preventive benefit of KOR blockade on stress-induced cephalic pain. Methods Following sumatriptan priming to model MOH, rats were hyper-responsive to environmental stress, demonstrating delayed cephalic and extracephalic allodynia and increased levels of CGRP in the jugular blood, consistent with commonly observed clinical outcomes during migraine. Nor-binaltorphimine (nor-BNI), a long-acting KOR antagonist or CYM51317, a novel short-acting KOR antagonist, were given systemically either during sumatriptan priming or immediately before environmental stress challenge. The effects of KOR blockade in the amygdala on stress-induced allodynia was determined by administration of nor-BNI into the right or left central nucleus of the amygdala (CeA). Results KOR blockade prevented both stress-induced allodynia and increased plasma CGRP. Stress increased dynorphin content and phosphorylated KOR in both the left and right CeA in sumatriptan-primed rats. However, KOR blockade only in the right CeA prevented stress-induced cephalic allodynia as well as extracephalic allodynia, measured in either the right or left hindpaws. U69,593, a KOR agonist, given into the right, but not the left, CeA, produced allodynia selectively in sumatriptan-primed rats. Both stress and U69,593-induced allodynia were prevented by right CeA U0126, a mitogen-activated protein kinase inhibitor, presumably acting downstream of KOR. Conclusions Our data reveal a novel lateralized KOR circuit that mediated stress-induced cutaneous allodynia and increased plasma CGRP in an injury-free model of functional cephalic pain with features of migraine and medication overuse headache. Selective, small molecule, orally available, and reversible KOR antagonists are currently in development and may represent a novel class of preventive therapeutics for migraine.
• Dos Reis, R., Kopruszinski, C., Nones, C., & Chichorro, J. (2016). Nerve growth factor induces facial heat hyperalgesia and plays a role in trigeminal neuropathic pain in rats. Behavioural Pharmacology, 27(6). doi:10.1097/FBP.0000000000000246
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  There is preclinical evidence that nerve growth factor (NGF) contributes toward inflammatory hyperalgesia in the orofacial region, but the mechanisms underlying its hyperalgesic effect as well as its role in trigeminal neuropathic pain require further investigation. This study investigated the ability of NGF to induce facial heat hyperalgesia and the involvement of tyrosine kinase receptor A, transient receptor potential vanilloid 1, and mast cells in NGF pronociceptive effects. In addition, the role of NGF in heat hyperalgesia in a model of trigeminal neuropathic pain was evaluated. NGF injection into the upper lip of naive rats induced long-lasting heat hyperalgesia. Pretreatment with an antibody anti-NGF, antagonists of tyrosine kinase receptor A, and transient receptor potential vanilloid 1 receptors or compound 48/80, to induce mast-cell degranulation, all attenuated NGFinduced hyperalgesia. In a rat model of trigeminal neuropathic pain, local treatment with anti-NGF significantly reduced heat hyperalgesia. In addition, increased NGF levels were detected in the ipsilateral infraorbital nerve branch at the time point that represents the peak of heat hyperalgesia. The results suggest that NGF is a prominent hyperalgesic mediator in the trigeminal system and it may represent a potential therapeutic target for the management of painful orofacial conditions, including trigeminal neuropathic pain.
• Gambeta, E., Kopruszinski, C., dos Reis, R., Zanoveli, J., & Chichorro, J. (2016). Evaluation of heat hyperalgesia and anxiety like-behaviors in a rat model of orofacial cancer. Neuroscience Letters, 619. doi:10.1016/j.neulet.2016.03.001
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  Pain and anxiety are commonly experienced by cancer patients and both significantly impair their quality of life. Some authors claim that there is a relationship between pain and anxiety, while others suggest that there is not a direct association. In any case, there is indeed a consensus that anxiety impairs the pain condition beyond be under diagnosed and undertreated in cancer pain patients. Herein we investigated if rats presenting heat hyperalgesia induced by orofacial cancer cell inoculation would display anxiety-like behaviors. In addition, we evaluated if pain blockade would result in alleviation of anxiety behaviors, as well as, if blockade of anxiety would result in pain relief. Orofacial cancer was induced in male Wistar rats by inoculation of Walker-256 cells into the right vibrissal pad. Heat facial hyperalgesia was assessed on day 6 after the inoculation, and on this time point rats were submitted to the elevated plus maze and the light-dark transition tests. The influence of lidocaine and midazolam on heat hyperalgesia and anxiety-like behaviors was assessed. The peak of facial heat hyperalgesia was detected 6 days after cancer cells inoculation, and at this time point, rats exhibited increased anxiety-like behaviors. Local treatment with lidocaine (2%/50 μL) caused a marked reduction of heat hyperalgesia, but failed to affect the anxiety-like behaviors, while midazolam (0.5 mg/kg, i.p.) treatment failed to change the heat threshold, but induced an anxiolytic-like effect. Altogether, our data demonstrated that rats with orofacial cancer present pain- and anxiety-like behaviors, but brief heat hyperalgesia relief does not affect the anxiety-like behaviors, and vice-versa, in our experimental conditions.
• Kopruszinski, C. M., Reis, R. C., Bressan, E., & Reeh, P. W. (2015). Vitamin B complex attenuated heat hyperalgesia following infraorbital nerve constriction in rats and reduced capsaicin in vivo and in vitro effects. Eur J Pharmacol. doi:10.1016/j.ejphar.2015.05.063
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  Vitamins of the B complex attenuate some neuropathic pain sensory aspects in various animal models and in patients, but the mechanisms underlying their effects remain to be elucidated. Herein it was investigated if the treatment with a vitamin B complex (VBC) reduces heat hyperalgesia in rats submitted to infraorbital nerve constriction and the possibility that TRPV1 receptors represent a target for B vitamins. In the present study, the VBC refers to a combination of vitamins B1, B6 and B12 at low- (18, 18 and 1.8mg/kg, respectively) or high- (180, 180 and 18mg/kg, respectively) doses. Acute treatment of rats with either the low- or the high-doses combination reduced heat hyperalgesia after nerve injury, but the high-doses combination resulted in a long-lasting effect. Repeated treatment with the low-dose combination reduced heat hyperalgesia on day four after nerve injury and showed a synergist effect with a single injection of carbamazepine (3 or 10mg/kg), which per se failed to modify the heat threshold. In naïve rats, acute treatment with the high-dose of VBC or B1 and B12 vitamins independently reduced heat hyperalgesia evoked by capsaicin (3µg into the upper lip). Moreover, the VBC, as well as, each one of the B vitamins independently reduced the capsaicin-induced calcium responses in HEK 293 cells transiently transfected with the human TRPV1 channels. Altogether, these results indicate that B vitamins can be useful to control heat hyperalgesia associated with trigeminal neuropathic pain and that modulation of TRPV1 receptors may contribute to their anti-hyperalgesic effects.
• Martins, G., Stolf, A., Kopruszinski, C., Cardoso, C., Beltrame, O., Queiroz-Telles, J., Strapasson, R., Stefanello, M., Oude-Elferink, R., Acco, A., & Lívero, F. (2015). Sesquiterpene lactones of Moquiniastrum polymorphum subsp. floccosum have antineoplastic effects in Walker-256 tumor-bearing rats. Chemico-Biological Interactions, 228. doi:10.1016/j.cbi.2015.01.018
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  Background and aim: This study aimed to evaluate the in vivo antitumor actions and toxicity of the dichloromethane fraction (F1B) of Moquiniastrum polymorphum subsp. floccosum (formerly Gochnatia polymorpha ssp. floccosa), composed of sesquiterpene lactones, against Walker-256 carcinosarcoma in rats. Methods: Male Wistar rats received 100 mg kg-1 F1B per day orally for 16 days after subcutaneous inoculation of Walker-256 cells in the pelvic limb. The tumor progression was monitored, and after treatment, tumor weight, oxidative stress, plasma biochemistry, inflammatory parameters, gene expression and histology of tumor and/or liver were evaluated. The toxicity of F1B was analyzed through the relative weight of organs. Additionally, an LD50 test was performed in mice. Results: F1B treatment significantly reduced tumor volume and weight. There was no difference in oxidative stress in tumor tissue after treatment. F1B treatment modified hepatic glutathione and superoxide dismutase, and normalized plasma glucose, alkaline phosphatase, and amylase. F1B did not affect the activity of myeloperoxidase and N-acetylglucosaminidase or the nitric oxide levels in tumor tissue. However, F1B decreased the tumor necrosis factor (TNF)-α levels. Additionally, F1B increased apoptosis in the tumor, mediated by up-regulation of the p53 and Bax gene expression. No clinical signs of toxicity or death were observed in the rats treated with F1B. The LD50 calculated for mice was 1209 mg kg-1. Conclusions: F1B, which is rich in sesquiterpene lactones, showed antitumor activity against Walker-256 carcinosarcoma. This effect may be, at least in part, related to the induction of apoptosis and inhibition of TNF-α signaling.
• Cruz, L., Kopruszinski, C., & Chichorro, J. (2014). Intraganglionar resiniferatoxin prevents orofacial inflammatory and neuropathic hyperalgesia. Behavioural Pharmacology, 25(2). doi:10.1097/FBP.0000000000000024
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  Trigeminal ganglion C-fiber neurons bearing transient receptor potential vanilloid-1 (TRPV1) channels are selectively destroyed by resiniferatoxin (RTX), a potent capsaicin analogue. The current study assessed the effect of an RTX injection (200 ng/4 μl) into the trigeminal ganglion in inflammatory and neuropathic rat models of orofacial thermal hyperalgesia. Intraganglionar RTX injection resulted in trigeminal ganglion C-fiber deletion, which was confirmed by the capsaicin eye wipes test, performed 6 days after the injection. The nociceptive responses induced by 2.5% formalin injected into the orofacial region were unchanged by a previous intraganglionar RTX injection. However, orofacial heat and cold hyperalgesia, induced by carrageenan injected into the upper lip (50 μg/50 μl), was abolished by previous intraganglionar RTX treatment. In addition, the development of orofacial heat and cold hyperalgesia after constriction of the infraorbital nerve was prevented by previous RTX treatment. Thus, trigeminal ganglion neurons expressing TRPV1 are crucial for the development of orofacial inflammatory and neuropathic thermal hyperalgesia. Copyright © Lippincott Williams & Wilkins.
• Hummig, W., Kopruszinski, C., & Chichorro, J. (2014). Pregabalin reduces acute inflammatory and persistent pain associated with nerve injury and cancer in rat models of orofacial pain. Journal of Oral and Facial Pain and Headache, 28(4). doi:10.11607/ofph.1317
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  Aims: To assess the analgesic effect of pregabalin in orofacial models of acute inflammatory pain and of persistent pain associated with nerve injury and cancer, and so determine its effectiveness in controlling orofacial pains having different underlying mechanisms. Methods: Orofacial capsaicin and formalin tests were employed in male Wistar rats to assess the influence of pregabalin (or vehicle) pretreatment in acute pain models, and the results from these experiments were analyzed by one-way analysis of variance (ANOVA) followed by Newman Keuls post-hoc test. Pregabalin (or vehicle) treatment was also tested on the facial heat hyperalgesia that was evaluated in rats receiving injection of the inflammatory irritant carrageenan into the upper lip, as well as after constriction of the infraorbital nerve (a model of trigeminal neuropathic pain), or after inoculation of tumor cells into the facial vibrissal pad; two-way repeated measures ANOVA followed by Newman-Keuls post-hoc test was used to analyze data from these experiments. Results: Facial grooming induced by capsaicin was abolished by pretreatment with pregabalin at 10 and 30 mg/kg. However, pregabalin failed to modify the first phase of the formalin response, but reduced the second phase at both doses (10 and 30 mg/kg). In addition, treatment of rats with pregabalin reduced the heat hyperalgesia induced by carrageenan, as well as by nerve injury and facial cancer. Conclusion: Pregabalin produced a marked antinociceptive effect in rat models of facial inflammatory pain as well as in facial neuropathic and cancer pain models, suggesting that it may represent an important agent for the clinical control of orofacial pain.
• Reis, R. C., Nones, C. F., Kopruszinski, C. M., Hummig, W., & Chichorro, J. G. (2014). Clinical aspects and pharmacological treatment of trigeminal neuralgia. RSBO.
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  Introduction: Trigeminal neuralgia (TN) is defined as sudden, usually unilateral, severe and brief pain episodes within the distribution of one or more branc...
• Kopruszinski, C., Reis, R., & Chichorro, J. (2012). B vitamins relieve neuropathic pain behaviors induced by infraorbital nerve constriction in rats. Life Sciences, 91(23-24). doi:10.1016/j.lfs.2012.08.025
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  Aims: There is mounting evidence that use of B vitamins can help control neuropathic pain. This study investigated if treatment with B1, B6 and B12 vitamins, alone or in combination with carbamazepine, can ameliorate distinct nociceptive behaviors in a model of trigeminal neuropathic pain. Main methods: Male Wistar rats were submitted to infraorbital nerve constriction or sham surgery and received a 5-day treatment with one of the B vitamins, a single carbamazepine injection or the association of both treatments and were tested for facial thermal and mechanical hyperalgesia at different time intervals. Key findings: Repeated treatment with B1 (thiamine), B6 (pyridoxine) and B12 (cyanocobalamin) vitamins (at 180, 180 and 18 mg/kg/day, respectively, for 5 days) prevented the development of heat hyperalgesia after infraorbital nerve injury, but only B12 and B6 treatments attenuated cold and mechanical hyperalgesia, respectively. A single injection of carbamazepine (30 mg/kg) significantly reduced thermal, but not mechanical, hyperalgesia after nerve injury. Combinations of lower doses of each B vitamin (B1 and B6 at 18 mg/kg/day and B12 at 1.8 mg/kg/day for 5 days) with carbamazepine (10 mg/kg) markedly reduced heat hyperalgesia after infraorbital nerve injury. Treatment with B12 (1.8 mg/kg/day) combined with carbamazepine (10 mg/kg) also synergized to attenuate cold hyperalgesia at some time points, but combination of B6 (18 mg/kg/day) with carbamazepine (30 mg/kg) failed to modify mechanical hyperalgesia. Significance: We suggest that B vitamins might constitute a relevant adjuvant to control some aspects of the pain afflicting patients suffering from trigeminal neuropathic pain. © 2012 Elsevier Inc. All rights reserved.