P2X7 receptors belong to a family of membrane bound ion channels which are activated by extracellular ATP, resulting in the opening of a non-selective cation channel. After prolonged or repeated exposure to agonist, functional and cellular changes can occur, including the formation of a large pore, cell lysis and the release of mature, biologically active interleukin-1β. It is this diversity of functions that underlies the significance of this receptor in pain processing. P2X7 receptors are expressed on microglia, which when activated, release a host of mediators which contribute to central sensitisation, a phenomenon associated with neuropathic pain. The role of P2X7 receptors in the activation of microglia is less well established and is the main subject of this thesis. Before considering the interaction between P2X7 receptors and microglia, the first aim was to establish whether P2X7 receptors played a role in a pathological process known to be associated with microglial activation. An additional aim was to establish whether the site of action was in the central nervous system (CNS), where microglia are located. These aims were accomplished using a surgery-based rat model of neuropathic pain, the chronic constriction injury (CCI) model, and by comparing the effects of different P2X7 receptor antagonists when dosed peripherally or directly into the spinal cord. The results indicated that P2X7 receptor antagonists produced efficacy in the CCI model via a mechanism located in the CNS. To further investigate the association between P2X7 receptors and microglia, a different experimental paradigm was explored. Chronically dosed morphine is known to activate microglia, the consequence of which is thought to underlie morphine tolerance and reduced morphine analgesia. By administering a P2X7 receptor antagonist to CCI-operated rats treated with chronic morphine, the interaction between the P2X7 receptor and morphine tolerance and analgesia was explored. The results showed that P2X7 receptor antagonism delayed morphine tolerance and increased the efficacy of low doses of morphine, suggesting an association between P2X7 receptors and microglia. It was intended to confirm the interaction between a P2X7 receptor antagonist and morphine in another neuropathic pain model, namely varicella zoster virus-induced neuropathy. However due to a lack of reproducibility, this model was not used for pharmacological studies. Having demonstrated an association between P2X7 receptor antagonist and morphine in a chronic pain setting, studies were initiated to explore whether this interaction occurred in other morphine-related behaviours. The effect on body weight, motor coordination and single dosed morphine-induced analgesia was assessed in rats co-administered with P2X7 receptor antagonist and morphine. Results demonstrated that the blockade of P2X7 receptors enhanced morphine acute dose-induced analgesia, but had no influence on motor-impairment and body weight. The final part of the thesis used immunohistochemical and molecular techniques to confirm that microglia played a role in established allodynia induced by CCI-surgery and that P2X7 receptors directly influenced microglia-activation. In conclusion, the data in this thesis has illustrated an association between centrally activated P2X7 receptors and microglia, as well as an association between the P2X7 receptor and morphine-induced tolerance and analgesia. It is possible that co-administration of a P2X7 receptor antagonist with morphine could reduce the effective dose of morphine clinically, thereby reducing the side effects of this commonly used analgesic.

Chronic pain is a major health problem worldwide. We have recently demonstrated the analgesic effect of the nitroxyl donor, Angeli's salt (AS) in models of inflammatory pain. In the present study, the acute and chronic analgesic effects of AS was investigated in chronic constriction injury of the sciatic nerve (CCI)-induced neuropathic pain in mice. Acute (7th day after CCI) AS treatment (1 and 3 mg/kg; s.c.) reduced CCI-induced mechanical, but not thermal hyperalgesia. The acute analgesic effect of AS was prevented by treatment with 1H-[1,2, 4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, a soluble guanylate cyclase inhibitor), KT5823 (an inhibitor of protein kinase G [PKG]) or glibenclamide (GLB, an ATP-sensitive potassium channel blocker). Chronic (7-14 days after CCI) treatment with AS (3 mg/kg, s.c.) promoted a sustained reduction of CCI-induced mechanical and thermal hyperalgesia. Acute AS treatment reduced CCI-induced spinal cord allograft inflammatory factor 1 (known as Iba-1), interleukin-1β (IL-1β), and ST2 receptor mRNA expression. Chronic AS treatment reduced CCI-induced spinal cord glial fibrillary acidic protein (GFAP), Iba-1, IL-1β, tumor necrosis factor-α (TNF-α), interleukin-33 (IL-33) and ST2 mRNA expression. Chronic treatment with AS (3 mg/kg, s.c.) did not alter aspartate aminotransferase, alanine aminotransferase, urea or creatinine plasma levels. Together, these results suggest that the acute analgesic effect of AS depends on activating the cGMP/PKG/ATP-sensitive potassium channel signaling pathway. Moreover, chronic AS diminishes CCI-induced mechanical and thermal hyperalgesia by reducing the activation of spinal cord microglia and astrocytes, decreasing TNF-α, IL-1β and IL-33 cytokines expression. This spinal cord immune modulation was more prominent in the chronic treatment with AS. Thus, nitroxyl limits CCI-induced neuropathic pain by reducing spinal cord glial cells activation.

Craniofacial nerve injury occasionally causes spread of mechanical hypersensitivity in humans. We modeled this abnormality by transecting the infraorbital nerve (IONX) in male and female mice of the 23 AXB-BXA recombinant inbred lines and their progenitor strains, comparing their responsivity to 7 applications of a 0.2 gram Von Frey filament to the ears, paws and tail. When normalizing their mechano-responsivity on postoperative days 14 and 21 by the preoperative values, subtracting data of sham-operated from IONX mice, highly contrasting line/strain-specific differences were demonstrated. Similar line/strain-specific variability in the spread of mechano-allodynia to the paws post-IONX was demonstrated in our novel 3 minute place-avoidance paradigm, assessing parameters of mobility on a smooth surface versus a pro-allodynic granular surface. These genetically-controlled, widespread changes in mechano-sensitivity caused by IONX were minimally sexually dimorphic and mapped to intervals on chromosomes 5, 9, and 13. Further analysis is needed to identify the causative genes.

Craniofacial nerve injury occasionally causes spread of mechanical hypersensitivity in humans. We modeled this abnormality by transecting the infraorbital nerve (IONX) in male and female mice of the 23 AXB-BXA recombinant inbred lines and their progenitor strains, comparing their responsivity to 7 applications of a 0.2 gram Von Frey filament to the ears, paws and tail. When normalizing their mechano-responsivity on postoperative days 14 and 21 by the preoperative values, subtracting data of sham-operated from IONX mice, highly contrasting line/strain-specific differences were demonstrated. Similar line/strain-specific variability in the spread of mechano-allodynia to the paws post-IONX was demonstrated in our novel 3 minute place-avoidance paradigm, assessing parameters of mobility on a smooth surface versus a pro-allodynic granular surface. These genetically-controlled, widespread changes in mechano-sensitivity caused by IONX were minimally sexually dimorphic and mapped to intervals on chromosomes 5, 9, and 13. Further analysis is needed to identify the causative genes.

Inflammation is an ubiquitous response of the body to cellular damage and injury. It not only leads to increased production of inflammatory molecules such as prostaglandins, bradykinins, histamine, and nerve growth factor (NGF) that sensitize the neurons, but is also associated with acidosis due to a local physiological decrease in extracellular pH, to as low as 5.3. The addition of protons to the sensitized area results in increased membrane conductance and depolarization in sensory neurons involving the acid sensing ion channels (ASICs), the capsaicin sensitive transient receptor potential vanilloid type 1 receptor (TRPV1), and blockade of background potassium conductance. Collectively, this combination heightens the pain state. As expression and activation of the NGF receptor tropomyosin-related kinase A (TrkA) are critically linked to inflammation-associated nociceptor sensitization, this led us to hypothesize that decreased pH may contribute to this process by depolarizing the neuron and mobilizing more TrkA to the cell membrane. This hypothesis is premised by previous experiments in other neuronal populations demonstrating that increased neuronal activity mobilizes more of the neurotrophin receptor TrkB to the membrane. We explored this question utilizing an in vitro model of acidosis akin to that observed with inflammation. Primary sensory neurons were grown on coverslips at a physiological pH of 7.4. The pH of the media was decreased to an acidic pH of 6.5 in half of the cultures for 30 minutes, followed by fixation under permeablizing or non-permeablizing conditions. Immunocytochemical analysis revealed a significant increase in the mobilization of TrkA to the plasma membrane in response to acidosis (confirmed using a biotinylation assay) and an enhanced level of TrkA activation in response to brief NGF challenge. This rapid mobilization of TrkA was attenuated with the addition of proton-sensitive channel blockers capsazepine and amiloride, for the TRPV and ASIC channels respectively. Unexpectedly, the amount of activated TrkA was also increased at pH 6.5 in the absence of NGF challenge. Taken together, the data suggests that sensory neurons can be rapidly sensitized to NGF in response to a decrease in pH and as such likely plays a role in the sensitization and hyperalgesia associated with an inflammatory state.

Inflammation is an ubiquitous response of the body to cellular damage and injury. It not only leads to increased production of inflammatory molecules such as prostaglandins, bradykinins, histamine, and nerve growth factor (NGF) that sensitize the neurons, but is also associated with acidosis due to a local physiological decrease in extracellular pH, to as low as 5.3. The addition of protons to the sensitized area results in increased membrane conductance and depolarization in sensory neurons involving the acid sensing ion channels (ASICs), the capsaicin sensitive transient receptor potential vanilloid type 1 receptor (TRPV1), and blockade of background potassium conductance. Collectively, this combination heightens the pain state. As expression and activation of the NGF receptor tropomyosin-related kinase A (TrkA) are critically linked to inflammation-associated nociceptor sensitization, this led us to hypothesize that decreased pH may contribute to this process by depolarizing the neuron and mobilizing more TrkA to the cell membrane. This hypothesis is premised by previous experiments in other neuronal populations demonstrating that increased neuronal activity mobilizes more of the neurotrophin receptor TrkB to the membrane. We explored this question utilizing an in vitro model of acidosis akin to that observed with inflammation. Primary sensory neurons were grown on coverslips at a physiological pH of 7.4. The pH of the media was decreased to an acidic pH of 6.5 in half of the cultures for 30 minutes, followed by fixation under permeablizing or non-permeablizing conditions. Immunocytochemical analysis revealed a significant increase in the mobilization of TrkA to the plasma membrane in response to acidosis (confirmed using a biotinylation assay) and an enhanced level of TrkA activation in response to brief NGF challenge. This rapid mobilization of TrkA was attenuated with the addition of proton-sensitive channel blockers capsazepine and amiloride, for the TRPV and ASIC channels respectively. Unexpectedly, the amount of activated TrkA was also increased at pH 6.5 in the absence of NGF challenge. Taken together, the data suggests that sensory neurons can be rapidly sensitized to NGF in response to a decrease in pH and as such likely plays a role in the sensitization and hyperalgesia associated with an inflammatory state.

The bed nucleus of the stria terminalis (BST) is a basal forebrain cluster of several distinct nuclei. It has been proposed that the BST coordinates autonomic, neuroendocrine, and behavioral functions through the integration and organization of homeostatic responses necessary for survival. Dysfunction of the BST contributes to pathophysiological states such as addiction, anxiety and aggression. Based on anatomical and behavioral studies, the BST could be a key contributor to descending modulation of nociception as well as the physiological responses related to the affective aspect of the pain experience. The objective of the present study was to further understand the neurophysiological bases underlying the involvement of the 7 anterior nuclei of the BST in pain. Using c-Fos as an indicator of neuronal activation, the results demonstrate that acute noxious stimulation produced an increase in the number of c-Fos immunoreactive cells (c-Fos-IR) in the dorsal anteromedial (dAM) and fusiform (FU) nuclei, while non-noxious stimulation did not increase c-Fos-IR in any of the nuclei examined. Chronic neuropathic pain induced by chronic constriction injury (CCI) did not alter basal c-Fos-IR in the FU or dAM. Unlike in the naïve condition, the number of c-fos-IR cells in the FU induced by acute noxious stimulation was attenuated in animals with either CCI or sham surgery compared to naive rats. In contrast, c-Fos-IR induced by acute noxious stimulation in the dAM was not affected by CCI or sham surgery. Acute noxious stimulation in animals that received CCI exhibited increased c-Fos expression in the ventromedial (vAM) nucleus of the BST, a finding not evident in naïve or sham control groups. Finally, there was an increase in c-Fos-IR in the oval (OV) nucleus of sham-operated, but not naive or CCI rats. This study reveals for the first time that pain induces neuronal activity in the BST in a nuclei- and condition-specific way. Given the efferent projection patterns from the BST, this system may relay supraspinal information to the periphery to produce physiological responses related to the affective pain experience. / Thesis (Master, Neuroscience Studies) -- Queen's University, 2007-09-28 14:36:55.632

This study investigated the utility of using a neurodynamic test, the Slump test by itself and with qualifiers to identify neuropathic pain (NeP). The study utilized a control group and a low back pain group. The low back pain group was pre-diagnosed as NeP or non neuropathic pain (NNP) by an experienced clinician using an accepted diagnostic examination. A slump test was performed recording knee ROM, pain location and verbal pain descriptors followed by Quantitative Sensory Testing (QST). Various versions of the slump test were compared to the pre test diagnosis. Sensitivity, specificity and likelihood ratios were calculated. The conventional slump test was shown to be a sensitive and moderately specific screening test for NeP. Including whether pain extended below the knee dramatically increased specificity. QST revealed localized cold sensation hyposensitivity, widespread cold pain hyposensitivity and suggestions of increased thresholds of pressure pain levels.

This study investigated the utility of using a neurodynamic test, the Slump test by itself and with qualifiers to identify neuropathic pain (NeP). The study utilized a control group and a low back pain group. The low back pain group was pre-diagnosed as NeP or non neuropathic pain (NNP) by an experienced clinician using an accepted diagnostic examination. A slump test was performed recording knee ROM, pain location and verbal pain descriptors followed by Quantitative Sensory Testing (QST). Various versions of the slump test were compared to the pre test diagnosis. Sensitivity, specificity and likelihood ratios were calculated. The conventional slump test was shown to be a sensitive and moderately specific screening test for NeP. Including whether pain extended below the knee dramatically increased specificity. QST revealed localized cold sensation hyposensitivity, widespread cold pain hyposensitivity and suggestions of increased thresholds of pressure pain levels.

Neuropathic pain is a common problem in clinical practice, affecting patients physically, emotionally, financially, and socially. Current treatment includes antidepressants, antiepileptics, and opioid analgesics. Bupropion is a specific inhibitor of neuronal noradrenaline reuptake and a weak inhibitor of dopamine reuptake, which shows some promise in the treatment of neuropathic pain.