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.

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.

Neuropathic (NP) pain is a debilitating chronic pain disorder that is a challenge to diagnose and an even greater challenge to treat. Commonly described as burning or shock-like, NP pain is characteristically resistant to traditional analgesic therapy. This thesis project aimed to investigate the potential therapeutic benefit of delta opioid receptor (δOR)-selective agonists in the management of NP pain. In the current experiments, rats that underwent unilateral sciatic nerve injury displayed characteristic behavioural manifestations including cold and thermal hyperalgesia as well as tactile allodynia in the ipsilateral hind paw. The spinal administration of DLT, a δOR-selective agonist, dose-dependently reversed tactile allodynia in NP rats and attenuated cold and thermal hypersensitivities. Moreover, DLT produced greater antinociceptive effects in NP rats compared with controls in the cold water paw withdrawal, hot water tail flick, and thermal plantar box tests. Nerve injury-induced augmentation in δOR function was dependent on nociceptive afferents, since the effect was absent in NP rats that received neonatal treatment with capsaicin. Furthermore, it was not due to increased δOR biosynthesis as western blots and immunohistochemistry revealed no change in spinal δOR protein. We hypothesized that an alternative mechanism, such as redistribution of receptors within the neuron, may underlie δOR function changes. Using immunogold electron microscopy, we showed that nerve injury indeed increased the cell surface expression of δORs within dendritic profiles of the dorsal horn via redistribution of existing receptors. Interestingly, this event was observed bilaterally in the deep dorsal horn, with no effect in the superficial laminae. The mechanisms underlying nerve injury-induced δOR trafficking remain unclear however we may take cues from other δOR trafficking events. We showed that concomitant treatment of rats with morphine and a glial inhibitor prevented both the activation of spinal glia and the changes in δOR agonist effects observed with morphine alone, suggesting that glial activity contributes to morphine-induced δOR trafficking in vivo and may provide insight into the mechanisms underlying nerve injury-induced δOR trafficking. Collectively, these studies reveal an important role of δORs in modulating pain symptoms associated with nerve injury, supporting further exploration of δORs as novel therapeutic targets in the treatment of NP pain. / Thesis (Ph.D, Pharmacology & Toxicology) -- Queen's University, 2009-04-20 14:46:29.83

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States of abnormal pain induced by injuries to peripheral nerves share common features with opioid antinociceptive tolerance including mechanical and thermal hypersensitivity. Sustained administration of morphine in humans and in animals induces a state of abnormal pain (i.e., hyperalgesia) and may be associated with the development opioid antinociceptive tolerance. Persistent neuropathic pain states and opioid induced abnormal pain require descending facilitation arising from the rostral ventromedial medulla (RVM). Cholecystokinin (CCK), a pronociceptive peptide, may be up-regulated following opioid treatment and nerve injury in the brain and spinal cord. Therefore, it is hypothesized that CCK in the RVM may be up-regulated by sustained opioid administration and my consequently drive descending pain facilitatory mechanisms to produce hypersensitivity and antinociceptive tolerance.Acute systemic morphine administration produced a potentiation of CCK release in the RVM as measured using microdialysis techniques. Sustained systemic morphine administration sufficient to produce thermal and tactile hypersensitivity resulted in a significant increase in basal CCK release in the RVM. Spinal nerve ligation (SNL) produces similar behavioral hypersensitivity. CCK levels in the RVM also increased following SNL. These findings suggest that endogenous CCK released in the RVM drives descending facilitatory pathways to produce hypersensitivity following sustained morphine administration and neuropathic pain.Disease states such as neuropathic pain offer special challenges in drug design due to system changes that accompany these diseases. Here, novel peptides with agonist binding affinity and bioactivity at δ and μ opioid receptors and simultaneous antagonist activity at CCK receptors have been developed. Using in vivo behavioral measures, it was shown that intrathecal (i.th.) administration of these compounds suppresses the thermal and tactile hypersensitivity caused by spinal nerve ligation (SNL).These studies support the hypothesis that endogenous CCK drives descending pain facilitatory pathways and that bi-functional compounds that act as opioid agonists and CCK antagonists are effective for the treatment of neuropathic pain.

The impact of microRNA (miRNA) as key players in the regulation of immune and neuronal gene expression and their role as master switches in the pathophysiology of neuropathic pain is increasingly recognized. miR-21 is a promising candidate that could be linked to the immune and the nociceptive system. To further investigate the pathophysiological role of miR-21 in neuropathic pain, we assesed mice deficient of B7 homolog 1 (B7-H1 ko), a protein with suppressive effect on inflammatory responses. B7-H1 ko mice and wildtype littermates (WT) of three different age-groups, young (8 weeks), middle-aged (6 months), and old (12 months) received a spared nerve injury (SNI). Thermal withdrawal latencies and mechanical withdrawal thresholds were determined. Further, we investigated anxiety-, depression-like and cognitive behavior. Quantitative real time PCR was used to determine miR-21 relative expression in peripheral nerves, dorsal root ganglia and white blood cells (WBC) at distinct time points after SNI. Naïve B7-H1 ko mice showed mechanical hyposensitivity with increasing age. Young and middle-aged B7-H1 ko mice displayed lower mechanical withdrawal thresholds compared to WT mice. From day three after SNI both genotypes developed mechanical and heat hypersensitivity, without intergroup differences. As supported by the results of three behavioral tests, no relevant differences were found for anxiety-like behavior after SNI in B7-H1 ko and WT mice. Also, there was no indication of depression-like behavior after SNI or any effect of SNI on cognition in both genotypes. The injured nerves of B7-H1 ko and WT mice showed higher miR-21 expression and invasion of macrophages and T cells 7 days after SNI without intergroup differences. Perineurial miR-21 inhibitor injection reversed SNI-induced mechanical and heat hypersensitivity in old B7-H1 ko and WT mice. This study reveals that reduced mechanical thresholds and heat withdrawal latencies are associated with miR-21 induction in the tibial and common peroneal nerve after SNI, which can be reversed by perineurial injection of a miR-21 inhibitor. Contrary to expectations, miR-21 expression levels were not higher in B7-H1 ko compared to WT mice. Thus, the B7-H1 ko mouse may be of minor importance for the study of miR-21 related pain. However, these results spot the contribution of miR-21 in the pathophysiology of neuropathic pain and emphasize the crucial role of miRNA in the regulation of neuronal and immune circuits that contribute to neuropathic pain. / Die Beteiligung von microRNA (miRNA) an der Genregulation immunologischer und neuronaler Prozesse und deren Rolle als Schlüsselelement in der Pathophysiologie von neuropathischem Schmerz gewinnt zunehmend an Bedeutung. miR-21 ist ein vielversprechender Kandidat, der sowohl das Immunsystem, als auch das nozizeptive System beeinflusst. Um die pathophysiologische Rolle von miR-21 bei neuropathischem Schmerz besser zu verstehen wurden Mäuse mit B7 homolog 1 Defizienz (B7-H1 ko), einem immunsupprimierendem Protein, untersucht. Eine frühere Studie zeigte eine Hochregulierung von miR-21 in murinen Lymphozyten. Junge (8 Wochen), mittelalte (6 Monate) und alte (12 Monate) B7-H1 ko Mäuse und Wildtypwurfgeschwister (WT) erhielten eine spared nerve injury (SNI) als neuropathischem Schmerzmodell. Es wurden thermische Rückzugslatenzen und mechanische Rückzugsschwellen bestimmt. Des weiteren wurde sowohl das Angstverhalten, das depressive Verhalten, als auch das kognitive Verhalten untersucht. Um die relative Expression von miR-21 in den peripheren Nerven, den Spinalganglien und in den weißen Blutzellen zu verschiedenen Zeitpunkten zu bestimmen, wurde die quantitative real time PCR angewandt. Naive B7-H1 ko Mäuse zeigten mit zunehmendem Alter eine mechanische Hyposensitivität. Bereits 3 Tage nach SNI entwickelten beide Genotypen eine Überempfindlichkeit gegenüber Hitze und mechanischer Stimulation. In drei durchgeführten Verhaltenstests konnten keine relevanten Unterschiede im Angstverhalten nach SNI von B7-H1 ko und WT Mäusen festgestellt werden. Bei beiden Genotypen gab es weder Hinweise auf depressives Verhalten nach SNI, noch wurde das kognitive Verhalten durch SNI beeinträchtigt. Die verletzen Nerven der B7-H1 ko und WT Mäuse zeigten 7 Tage nach SNI eine höhere miR-21 Expression und eine Invasion durch Makrophagen und T-Zellen ohne Gruppenunterschiede. Die perineurale Injektion eines miR-21 Inhibitors konnte die durch SNI induzierte mechanische und thermische Hypersensitivität lindern. Diese Studie zeigt, dass der Anstieg von miR-21 im N. tibialis und N. peroneus communis mit reduzierten Rückzugsschwellen gegen mechanische Reize und verkürzten Wegzugslatenzen bei Hitzestimulation einhergeht, welche durch perineurale Injektion eines miR-21 Inhibitors verringert werden können. Entgegen der Erwartungen zeigten B7-H1 ko Mäuse im Vergleich zu WT Mäusen keine erhöhte miR-21 Expression und sind daher möglicherweise von geringer Bedeutung für die Untersuchung von miR-21 assoziiertem Schmerz. Jedoch bekräftigen diese Ergebnisse eine Beteiligung von miR-21 an der Pathophysiologie von neuropathischem Schmerz und bestätigen die wichtige Rolle von miRNA bei der Regulation von neuronalen und immunologischen Prozessen, die zu neuropathischem Schmerz beitragen.