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Orofacial Pain Operant Analysis with Concomitant Investigation of Peripherally Restricted Cannabinoids and Nav1.8 Axonal Accumulation for the Potential Treatment of Neuropathic Pain

  • Author(s): Mulpuri, Yatendra
  • Advisor(s): Spigelman, Igor
  • et al.
Abstract

The primary emphasis of this dissertation project was to study the pathogenesis of neuropathic pain, and to develop new treatment strategies targeting primary sensory neurons of the pain pathway. Chronic neuropathic pain is debilitating and is maladaptive. Current treatment options are moderately effective with serious side-effects. Translational progress of novel drugs is impeded by side-effects, which is partly due to reliance on inaccurate methods of pain evaluation in animals. Our first step was to develop a novel behavioral assay for quantitation of spontaneous pain in an animal model. Results from this study showed that rats with trigeminal neuropathic pain had reduced drinking efficiency in an orofacial operant assay, whereas control rats maintained normal levels throughout testing.

The second step of this project was to investigate novel peripherally restricted cannabinoids (PRCBs) for the effective treatment of neuropathic pain. Our initial testing in a rat model of sciatic nerve entrapment (SNE) revealed that, systemic administration of a PRCB (0.3 mg/kg) completely suppressed signs of mechanical allodynia between two and three hours. Based on this information, we conducted a preliminary pharmacokinetic study that showed peak plasma drug levels at 2 hrs after administration with negligible traces of drug in the brain and cerebro-spinal fluid (CSF). Subsequently, we tested these novel PRCBs for CNS side-effects due to central activation of cannabinoid receptor subtype 1 (CB1R). Systemic and oral administrations at therapeutic and higher doses showed complete lack of CNS side-effects. These side-effects prevailed after systemic administration of centrally acting cannabinoid, HU-210 in the same group of animals. Site specific administrations and further evaluation in orofacial operant assay confirmed that anti-allodynic actions of PRCB are mainly mediated at the periphery. Our in vitro data showed that PRCBs are full agonists at CB1R and partial agonists at CB2R. Co-administration of PRCB (1 mg/kg i.g.) with receptor specific antagonists revealed that anti-allodynic actions are largely mediated by CB1R. Since tolerance is of main concern with repeated use of chronic pain medications, we tested the anti-allodynic efficacy of PRCB during repeated oral administrations (1 mg/kg, i.g.) and found no appreciable tolerance after two weeks of testing. Western blot analysis showed stable levels of CB1R in the L4-L5 dorsal root ganglia (DRG) of these animals after repeated testing.

The last research aim of this project was to investigate the axonal translocation of Nav1.8 mRNA after peripheral nerve injury. The hyperexcitability of afferent nociceptors is induced by axonal accumulation of certain ion channels and the role of Nav1.8 sodium channel is highly implicated. Therefore, we investigated the axonal translocation of Nav1.8 mRNA. Our qPCR data showed abnormal accumulation of Nav1.8 mRNA along with Nav1.6 and Nav1.9 mRNAs in

the injured infraorbital nerve. Since the 3’ untranslated region (UTR) is essential for sub-cellular localization of mRNAs, we did cloning and sequencing of Nav1.8 mRNA 3’ UTR. Results showed previously annotated short 3’ UTR and an alternative long UTR that is produced as a result of alternative splicing and alternative polyadenylation.

The current research work demonstrated the use of orofacial operant assay for the evaluation of ongoing or spontaneous pain in a rat model of trigeminal neuropathic pain. In addition, our extensive pre-clinical testing of PRCBs for the evaluation of therapeutic efficacy, may aid in the development of these novel drugs as future therapeutics. In conjunction, the newly identified 3’ UTR variant of Nav1.8 mRNA may represent the pathological basis for abnormal axonal accumulation of Nav1.8 mRNA after peripheral nerve injury.

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