This thesis examined the effects of long-term inhibition of excitatory amino acid transporter 2 (EAAT2) on the excitability of dorsal horn neurons in defined-medium organotypic slice cultures (DMOTCs). Previous reports suggest that inhibition of EAAT2 may be involved in development of neuropathic pain induced by brain-derived neurotrophic factor (BDNF). Experiments were carried out using confocal Ca2+ imaging to assess the excitability of dorsal horn neurons. Long-term treatment with EAAT2 blocker, dihydrokainate (DHK), prominently increased the neuronal excitability. Long-term exposure to DHK had a significant effect on NMDA, AMPA and metabotropic glutamate subtype 1 (mGluR1) receptors. Lastly, long-term treatment with BDNF and DHK increased activity of AMPA receptors but only DHK significantly increased activity of NMDA receptors. These findings suggest inhibition of EAAT2 and BDNF may have different pathways to promote neuropathic pain and modulating the activity of EAAT2 may be a novel therapeutic approach for neuropathic pain.

Thesis (Ph.D.)--Memorial University of Newfoundland, 2005. / Bibliography: leaves 122-146.

Previous research has demonstrated the impact of negative emotional states on pain modulation. The direction of this modulation has been shown to correspond to the arousal level and the valence of the emotional state, whether naturally occurring or induced in the laboratory. Other research has consistently linked written emotion disclosure of trauma to better long-term health outcomes among several populations. As most of these studies have focused on long-term health outcome effects of disclosure, little research has been done on the immediate effects of the paradigm on affective or physiological states. This study investigated the short-term effects of written disclosure of trauma on laboratory-induced pain, affective state, and other physiological measures of stress and arousal. Other goals of the study included investigating preexisting differences in pain sensitivity between participants corresponding to lifetime experience of trauma, and determining the degree to which baseline pain testing alters pain sensitivity after emotion induction by creating a conditioned, contextual fear. This is the first study to apply the written emotional disclosure paradigm to laboratory-induced pain.

Sciatic and saphenous neurectomy produces in mice a neuropathic pain-like behaviour (‘autotomy’). A/J mice express higher autotomy levels than C57BL6/J mice. A previous study used autotomy data for these strains and their 23 recombinant daughter inbred lines of the AXB-BXA set, to map a quantitative trait locus (QTL) for autotomy on chromosome 15. Since then, this QTL, named Pain1, was replicated twice. Since all three studies used a low-resolution genetic map based on microsatellites its confidence length precluded identification of candidate gene(s). To overcome this problem, I used a higher resolution SNP-based genetic map and refined Pain1’s peak location, identifying in it 80 candidate genes. But only Lynx1, Arc and Sharpin had sequence mismatches between A/J and C57BL6/J, known neural functions, and contrasting expression levels in DRGs and spinal cord of intact, sham-operated, and neurectomized mice of these lines. Meeting these criteria made them our best candidate genes for autotomy.

Background: An approach designed to characterize BDNF gene deletion within microglia of the dorsal horn of the spinal cord does not currently exist. Therefore, my goal was to develop methods to assess Cre- mediated BDNF deletion. To this end I designed and tested two different approaches focusing on the aspects of BDNF mRNA expression or genomic level gene deletion. Methods: Approach 1: BDNF messenger RNA was detected by in situ hybridization. Approach 2: BDNF gene deletion was detected by a positive signal semi-quantitative Polymerase Chain Reaction (PCR). Results: In situ hybridization detected spinal BDNF and regional changes in BDNF mRNA following PNI in wild-type mice. The BDNF PCR detected Cre-mediated BDNF deletions in transgenic animals. Conclusion: Two approaches have been developed and initial tests of these approaches show promising results and will provide valuable tools for researchers investigating BDNF deletion in transgenic animals.

Sciatic and saphenous neurectomy produces in mice a neuropathic pain-like behaviour (‘autotomy’). A/J mice express higher autotomy levels than C57BL6/J mice. A previous study used autotomy data for these strains and their 23 recombinant daughter inbred lines of the AXB-BXA set, to map a quantitative trait locus (QTL) for autotomy on chromosome 15. Since then, this QTL, named Pain1, was replicated twice. Since all three studies used a low-resolution genetic map based on microsatellites its confidence length precluded identification of candidate gene(s). To overcome this problem, I used a higher resolution SNP-based genetic map and refined Pain1’s peak location, identifying in it 80 candidate genes. But only Lynx1, Arc and Sharpin had sequence mismatches between A/J and C57BL6/J, known neural functions, and contrasting expression levels in DRGs and spinal cord of intact, sham-operated, and neurectomized mice of these lines. Meeting these criteria made them our best candidate genes for autotomy.

Background: An approach designed to characterize BDNF gene deletion within microglia of the dorsal horn of the spinal cord does not currently exist. Therefore, my goal was to develop methods to assess Cre- mediated BDNF deletion. To this end I designed and tested two different approaches focusing on the aspects of BDNF mRNA expression or genomic level gene deletion. Methods: Approach 1: BDNF messenger RNA was detected by in situ hybridization. Approach 2: BDNF gene deletion was detected by a positive signal semi-quantitative Polymerase Chain Reaction (PCR). Results: In situ hybridization detected spinal BDNF and regional changes in BDNF mRNA following PNI in wild-type mice. The BDNF PCR detected Cre-mediated BDNF deletions in transgenic animals. Conclusion: Two approaches have been developed and initial tests of these approaches show promising results and will provide valuable tools for researchers investigating BDNF deletion in transgenic animals.

There is a great need for new analgesics. The current problem in treatment of severe pain is that side effects limit the effectiveness of therapy. Glycine receptors are important in modulation of nociception, suggesting a novel class of analgesics. Previous studies in rats show that intrathecal administration of glycine agonists and amino acids structurally similar to glycine have antinociceptive effects. The effects of isovaline, a unique, non-proteogenic glycine-like aminoacid, have not been studied. Isovaline is absorbed from the gut and transported across the blood-brain-barrier. We examined the hypothesis that isovaline produces antinociception in mice. Administration of strychnine, an antagonist at glycine receptors, into the cisterna magna or lumbar intrathecal space resulted in allodynia, localized to the somatotopic distribution of the trigeminal and lumbar nerves. These findings provided a basis for models of lumbar and trigeminal neuralgia. Racemic isovaline blocked strychnine induced allodynia in both models without apparent side effects. We next investigated the antinociceptive effects of glycine-like amino acids in formalin foot assay, a conventional rodent model of acute and chronic pain. Antinociceptive effects were demonstrated on intrathecal administration of glycine, beta-alanine, and isovaline. Intravenous isovaline produced significant antinociceptive effects in the formalin foot model. The toxicity of isovaline and related amino acids were determined. Exploratory behavior, gait, and responses to stimuli were used to assess sedation. The rotarod test was used to examine central nervous system (CNS) and neuromuscular toxicities of intravenous isovaline. Lumbar administration of glycine and beta-alanine caused scratching and/or lower body weakness. Isovaline at 7-times intrathecal ED50 produced lower body weakness in some animals. None of the amino acids produced sedation comparable to morphine. At 6-times ED50, beta-alanine produced weakness. Both glycine (ED50) and beta-alanine (3x ED50) but not isovaline produced local nerve irritation. Intracisternal injection of glycine did not reverse allodynia and resulted in death. Neither R nor S enantiomers of isovaline impaired performance on the rotarod test. Isovaline has significant antinociceptive properties. Given the absence of apparent CNS or motor toxicity, isovaline has potential as a clinical analgesic.

Morphine is one of the most commonly prescribed medications for the relief of prolonged pain. Both basic science and clinical studies indicate that females require 2-3 times more morphine than males to achieve the same analgesic effect. To date, the mechanisms underlying sex differences in opiate responsiveness are unknown. Recent studies suggest that glial cells are potent modulators of morphine-based analgesia, and in particular, decrease the analgesic effect of opiates. Therefore, we tested the hypothesis that the sexually dimorphic effects of morphine were due to sex differences in glial cell activity. Our studies focused on the midbrain periaqueductal gray (PAG) as this region of the brain is critical for the analgesic effects of morphine. Adult male and female Sprague Dawley rats (250g- 400g) were procured from Charles River Laboratories, and were allowed 7 days to acclimate to the new facility. On the day of the experiment, animals received a subcutaneous injection of morphine (5mg/kg) or were handled in a similar manner. Thirty or 60 minutes after injections or handling, animals were perfused with a 4% paraformaldehyde and 2.5% acrolein tissue fixative solution. Brains were removed and stored in 20% sucrose until ready for sectioning. Brains were sectioned at 25mm using a freezing microtome, and immunohistochemical localization of markers for astrocyte glial cell activity was performed. Antibodies to glial fibrillary acidic protein (GFAP) were used to label activated astrocytes. If our hypothesis is correct, then females will have significantly greater density of the astrocyte cell activity marker GFAP as compared with males. Sex differences in PAG glial cell activity may provide the biological bases for the sexually dimorphic effect of morphine. This research may lead to better treatment for females experiencing prolonged chronic or neuropathic pain.

Background: Chronic pelvic pain (CPP) in women causes significant disability and distress. Like other chronic pain conditions, psychosocial variables likely play as key a role in the development and maintenance of CPP as physiological ones. The purposes of this study were to use the Biopsychosocial model to determine the predictors of pain and quality of life (QOL) and to specifically examine to effect of baseline catastrophizing on 12-month pain and QOL. Methods: Secondary analysis of baseline and 12-month data collected from women presenting for CPP treatment (n = 673) at a tertiary referral center was performed. Questionnaires assessed medical symptoms, physical and mental health, abuse, trauma, catastrophizing and the main outcome measures of pain reports (McGill Pain Questionnaire) and QOL scores (adapted version of the Irritable Bowel Syndrome QOL Questionnaire). Results: Of the 673 enrolled, 401 completed baseline questionnaires. These women were predominantly middle aged (M = 35.68, SD = 9.87), married (66%), Caucasian (78%), and educated (M = 14.83, SD = 2.55). Two hundred seventy-two women completed questionnaires at baseline and 12 months and were similar in most characteristics but reported fewer incidents of trauma and abuse, improved physical health and fewer medical symptoms. Women experienced a significant reduction in pain (t (261) = 11.23, p < .001) and improved QOL (t (257) = 6.78, p< .001). Baseline catastrophizing was a predictor of baseline pain (R2 = .42, pβ = .46, p < .001) and baseline QOL (R2 = .79, p< .001; β = .71 p < .001) with similar results at 12-month follow-up. While baseline catastrophizing contributed only 3% of the variance it remained a significant predictor of 12-month pain (R2 = .39, p < .001; β = .18, p = .003). Unexpectedly, abuse and trauma histories were not significant predictors of pain or QOL. Conclusions: These findings contribute to the existing body of literature by confirming the complex nature of CPP and suggest that psychological processes such as catastrophizing play a vital role in CPP. Future research in CPP will benefit from the exploration of the contribution of psychological processes to CPP and the application of research from other pain conditions to gynecologic pain disorders.