The role of nitroxyl in the development of neuropathic pain

Abstract

Neuropathic pain is a debilitating persistent (chronic) pain condition which affects 2% of the total population, characterised by spontaneous pain (stimulus independent), allodynia (pain generated from non-noxious stimuli) and hyperalgesia (heightened sense of pain to noxious stimuli). Unlike other types of pain such as nociceptive or inflammatory, neuropathic pain is maladaptive and therefore neither protects or supports healing or repair. It is defined as “pain caused by a lesion or disease of the somatosensory nervous system” and can develop following an array of aetiologies such as peripheral or central nerve lesions, diabetes, herpes zoster, HIV and cancer, to name a few. However, resolution of the underlying disease and/or healing of the injury often does not alleviate the associated neuropathic pain symptoms suggesting that central maladaptive plasticity may occur in people with neuropathic pain. Compounding this situation, this maladaptive plasticity often renders traditional analgesics used for nociceptive and inflammatory pain ineffective, thus reducing the treatment options available for neuropathic pain sufferers. The spinal mechanisms which lead to persistent pain development have yet to be fully elucidated. It is well understood that adaptations in the reactivity of spinal glial cells (microglia and astrocytes) may also contribute to central neuronal plasticity, by releasing inflammatory mediators such as nitric oxide and other reactive nitrogen species, that enhance excitatory and/or reduce inhibitory neuronal signalling (also referred to as neuro-immune signalling). Previous limitations in methodology have limited our understanding of longitudinal changes in spinal glial during critical developmental stages in persistent pain pathology. Whether there is a correlation between glial reactivity and neuropathic pain severity during the development of the disease model, has yet to be established. Therefore, the initial aim of this thesis was to determine if reactivity characteristics of spinal microglia may correlate with peripheral injury severity and subsequent neuropathic pain symptoms, in mouse models of persistent pain (Chapter 5). Studies suggest that following peripheral injury, there may be alternative reactive nitrogen species, other than nitric oxide, released by highly reactive glial cells which may facilitate neural plasticity within the spinal cord. The recent development of novel fluorescent tools for measuring reactive nitrogen species, such as nitroxyl, have yet to be used to identify the endogenous presence of this reactive nitrogen species in neuropathic pain development. Therefore, the second aim of this thesis was to validate the use of a novel fluorescent probe for the detection of endogenous nitroxyl in mouse models of persistent pain (Chapter 3). The role of nitroxyl in persistent pain development, has been complicated by recent reports whereby exogenous application of high concentration of this reactive nitrogen species, can act as therapeutic agent for persistent pain. The mechanism of action has yet to be fully elucidated, however nitroxyl is highly reactive towards thiols and metalloproteases which have been implicated in various persistent pain pathways. This led to the subsequent aim of this thesis, which was to determine whether the exogenous nitroxyl donor (Angelis’s salt) may reduce allodynia via its ability to cleave active cysteine residues on lysosomal proteasomes and thus reduce their enzyme function (such as Cathepsin B) in persistent pain mouse models (Chapter 4). The studies offered herein demonstrate that: both the onset time post-injury, and level of microglial reactivity is closely correlated with the severity of peripheral injury and subsequent allodynia; endogenous nitroxyl is produced in models of persistent pain (and other diseases) and can be detected in multiple imaging platforms using novel fluorescent probes; and exogenous nitroxyl donor can reduce both Cathepsin B enzyme activity and allodynia, however Cathepsin B inactivation does not directly account for the reduced allodynia and may not be the pathway involved in this phenomenon. Collectively, these results highlight that there is a correlation between microglial reactivity and the severity of injury and subsequent allodynia which may suggest that physicians should consider the severity of the injury when prescribing treatment and at which timepoint post-injury to best intervene. In addition, novel tools developed at the ARC Centre of Excellence for Nanoscale Biophotonics, University of Adelaide, have provided a way to demonstrate that stimuli used in persistent pain models can generate endogenous nitroxyl which can be semi-quantitatively measured. Furthermore, exogenous nitroxyl donors may reduce allodynia via the in-activation of key thiols and metalloproteases which are critical to persistent pain development. With future research, these novel fluorescent probes may be used in vivo to measure the endogenous nitroxyl output in central glial cells in relation to peripheral injury severity. Furthermore, future work exploring the mechanisms by which exogenous nitroxyl is able to reduce allodynia, could provide a safe therapeutic tool for treating symptoms in neuropathic pain patients