Investigating the Contribution of the Inflammasome to Persistent Pain States
Neuropathic pain, a debilitating condition, occurs as a consequence of nerve injury with symptoms such as spontaneous pain, hyperalgesia, and allodynia commonly reported. In animal models of peripheral nerve injury, there is accumulation and proliferation of microglial cells in the injured spinal cord and macrophages within the injured nerve and dorsal root ganglion (DRG), contributing to the development of pain-like behaviours. The pro-inflammatory cytokine interleukin-1β (IL-1β) plays a key role in acute and chronic inflammation. It causes potent mechanical and thermal hyperalgesia when injected into peripheral tissues, and increased expression of IL-1β in the spinal cord, DRG, and injured nerve is seen in several animal models of inflammatory and neuropathic pain. A key step in the release of active IL-1β is the cleavage of pro-IL-1β by active caspase-1, generating mature IL-1β. Activation of this enzyme requires assembly of the inflammasome, a multi-protein complex. The active complex contains a central scaffold protein (eg nod-like receptor-1 (NLRP1), NLRP3), the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC), and pro-caspase-1. It is known to assemble in response to a variety of exogenous and endogenous danger signals (eg adenosine-5’-triphosphate (ATP), monosodium urate (MSU) crystals). Data presented in this thesis demonstrates that, following L5 Spinal Nerve Transection (SNT), an animal model of neuropathic pain, inflammasome components NLRP3, ASC, and caspase-1 are upregulated in the injured DRG and the ipsilateral lumbar dorsal horn. Immunohistochemical analysis reveals that ASC and caspase-1 are both highly co-localised with macrophage and microglial marker ionised calcium-binding adaptor molecule-1 (IBA1), but not with astrocytic marker glial fibrillary acid protein (GFAP) or neuronal marker NeuN. While daily intrathecal administration of Ac-YVAD-CMK, a cell-permeant caspase-1 inhibitor, significantly attenuated established behavioural hypersensitivity following L5 SNT, ASC-/- and NLRP3-/- mice developed behavioural hypersensitivity normally in the Partial Sciatic Nerve Ligation (PSNL) model of neuropathic pain, indicating an inflammasome-independent role of caspase-1 in neuropathic pain. In a model of central inflammatory pain, 2 pharmacological inhibition of caspase-1 prevented the development of hindpaw mechanical hypersensitivity following intrathecal administration of lipopolysaccharide (LPS). ASC-/- mice had a significantly reduced mechanical hindpaw hypersensitivity following intrathecal administration of LPS. However NLRP3-/- mice developed hindpaw hypersensitivity normally following intrathecal administration of LPS, indicating a NLRP3-independent role for ASC and caspase-1 activation in this animal model of central inflammatory pain. Finally, although ASC appears to contribute to the development of hypersensitivity in a model of central inflammatory pain, ASC-/- mice developed behavioural hypersensitivity normally following intraplantar administration of complete Freund’s adjuvant (CFA) in an animal model of persistent peripheral inflammatory pain. Thus data presented in this thesis reveal a complex role for the inflammasome in animal models of pain, with differential contribution of inflammasome components to behavioural hypersensitivity in animal models of neuropathic and central and peripheral inflammatory pain.
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