The role of nitroxyl in the development of neuropathic pain

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 patientsThesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 201

Differential effect of morphine on gastrointestinal transit, colonic contractions and nerve-evoked relaxations in Toll-Like Receptor deficient mice

Abstract Toll-like receptors (TLRs) are expressed in enteric neurons, glia, gastrointestinal (GI) smooth muscle and mucosa, yet their functional roles in the GI tract are not fully understood. TLRs have been linked to many of the undesirable central effects of chronic opioid administration including hyperalgesia and dependence via activation of central microglia. Opioid-induced bowel dysfunction (OIBD) remains a primary reason for the reduction or withdrawal of opioid analgesics. Morphine-induced inhibition of colonic motility was assessed in vivo by GI transit studies and in vitro using isolated colons from wildtype (WT) and TLR deficient mice. Morphine slowed movement of ingested content in WT but this retardation effect was attenuated in TLR4 −/− and TLR2/4 −/− . In isolated colons, morphine reduced amplitude and frequency colonic migrating motor contractions in both WT and TLR2/4 −/− . Electrical field stimulation elicited distal colon relaxation that was potentiated by morphine in WT but not in TLR2/4 −/− . Inhibitory junction potentials were of similar amplitude and kinetics in WT and TLR2/4 −/− distal colon and not altered by morphine. Enteric nerve density and proportion of nitrergic nerves were similar in WT and TLR2/4 −/− distal colon. These data suggest an involvement of TLRs in opioid pharmacodynamics and thus a potential interventional target for OIBD

Hyperspectral imaging of endogenous fluorescent metabolic molecules to identify pain states in central nervous system tissue

Fluorescence-based bio-imaging methods have been extensively used to identify molecular changes occurring in biological samples in various pathological adaptations. Auto-fluorescence generated by endogenous fluorescent molecules within these samples can interfere with signal to background noise making positive antibody based fluorescent staining difficult to resolve. Hyperspectral imaging uses spectral and spatial imaging information for target detection and classification, and can be used to resolve changes in endogenous fluorescent molecules such as flavins, bound and free NADH and retinoids that are involved in cell metabolism. Hyperspectral auto-fluorescence imaging of spinal cord slices was used in this study to detect metabolic differences within pain processing regions of non-pain versus sciatic chronic constriction injury (CCI) animals, an established animal model of peripheral neuropathy. By using an endogenous source of contrast, subtle metabolic variations were detected between tissue samples, making it possible to distinguish between animals from non-injured and injured groups. Tissue maps of native fluorophores, flavins, bound and free NADH and retinoids unveiled subtle metabolic signatures and helped uncover significant tissue regions with compromised mitochondrial function. Taken together, our results demonstrate that hyperspectral imaging provides a new non-invasive method to investigate central changes of peripheral neuropathic injury and other neurodegenerative disease models, and paves the way for novel cellular characterisation in health, disease and during treatment, with proper account of intrinsic cellular heterogeneity.8 page(s

Chronic morphine-induced changes in signaling at the A3 adenosine receptor contribute to morphine-induced hyperalgesia, tolerance, and withdrawal

Treating chronic pain by using opioids, such as morphine, is hampered by the development of opioid-induced hyperalgesia (OIH; increased pain sensitivity), antinociceptive tolerance, and withdrawal, which can contribute to dependence and abuse. In the central nervous system, the purine nucleoside adenosine has been implicated in beneficial and detrimental actions of morphine, but the extent of their interaction remains poorly understood. Here, we demonstrate that morphine-induced OIH and antinociceptive tolerance in rats is associated with a twofold increase in adenosine kinase (ADK) expression in the dorsal horn of the spinal cord. Blocking ADK activity in the spinal cord provided greater than 90% attenuation of OIH and antinociceptive tolerance through A 3 adenosine receptor (A 3 AR) signaling. Supplementing adenosine signaling with selective A 3 AR agonists blocked OIH and antinociceptive tolerance in rodents of both sexes. Engagement of A 3 AR in the spinal cord with an ADK inhibitor or A 3 AR agonist was associated with reduced dorsal horn of the spinal cord expression of the NOD-like receptor pyrin domain-containing 3 (60%-75%), cleaved caspase 1 (40%-60%), interleukin (IL)-1 β (76%-80%), and tumor necrosis factor (50%-60%). In contrast, the neuroinhibitory and anti-inflammatory cytokine IL-10 increased twofold. In mice, A 3 AR agonists prevented the development of tolerance in a model of neuropathic pain and reduced naloxone-dependent withdrawal behaviors by greater than 50%. These findings suggest A 3 AR-dependent adenosine signaling is compromised during sustained morphine to allow the development of morphine-induced adverse effects. These findings raise the intriguing possibility that A 3 AR agonists may be useful adjunct to opioids to manage their unwanted effects. SIGNIFICANCE STATEMENT: The development of hyperalgesia and antinociceptive tolerance during prolonged opioid use are noteworthy opioid-induced adverse effects that reduce opioid efficacy for treating chronic pain and increase the risk of dependence and abuse. We report that in rodents, these adverse effects are due to reduced adenosine signaling at the A 3 AR, resulting in NOD-like receptor pyrin domain-containing 3-interleukin-1β neuroinflammation in spinal cord. These effects are attenuated by A 3 AR agonists, suggesting that A 3 AR may be a target for therapeutic intervention with selective A 3 AR agonist as opioid adjuncts

Hyperpolarization-activated cyclic-nucleotide gated 4 (HCN4) protein is expressed in a subset of rat dorsal root and trigeminal ganglion neurons

Abstract Hyperpolarization-activated cyclic nucleotidegated (HCN) cation channels are active at resting membrane potential and thus are likely to contribute to neuronal excitability. Four HCN channel subunits (HCN1–4) have previously been cloned. The aim of the current study was to investigate the immunoreactivity of HCN4 channel protein in rat trigeminal (TG) and dorsal root ganglion (DRG) sensory neurons. HCN4 was present in 9% of TG neurons and 4.7% of DRG neurons, it was distributed in a discrete population of small-diameter neurons in the TG but was located in cells of all sizes in the DRG. Approximately two thirds of HCN4-containing neurons in each ganglia were labelled with antisera raised against the 200-kDa neurofilament (NF200). The remaining HCN4-containing neurons were NF200-negative, were not labelled with antisera raised against calcitonin-gene related peptide (CGRP), and did not bind the isolectin B4 (IB4). HCN4-containing neurons made up more than half of the population of small-diameter primary afferent neurons that did not contain either NF200 or CGRP or bind IB4 in both TG and DRG. This population was not insignificant, comprising 5% of TG neurons and 2% of DRG neurons.Hyun-jung Cho, Vasiliki Staikopoulos, Jason J. Ivanusic and Ernest A. Jenning

Design and Assessment of a Potent Sodium Channel Blocking Derivative of Mexiletine for Minimizing Experimental Neuropathic Pain in Several Rat Models

Physical or chemical damage to peripheral nerves can result in neuropathic pain which is not easily alleviated by conventional analgesic drugs. Substantial evidence has demonstrated that voltage-gated Na+ channels in the membrane of damaged nerves play a key role in the establishment and maintenance of pathological neuronal excitability not only of these peripheral nerves but also in the second- and third-order neurons in the pain pathway to the cerebral cortex. Na+ channel blocking drugs have been used in treating neuropathic pain with limited success mainly because of a preponderance of side-effects. We have developed an analogue of mexiletine which is approximately 80 times more potent than mexiletine in competing with the radioligand, 3H-batrachotoxinin for binding to Na+ channels in rat brain membranes and also it is much more lipophilic than mexiletine which should enhance its uptake into the brain to block the increased expression of Na+ channels on second- and third-order neurons of the pain pathway. This analogue, HFI-1, has been tested in three different rat models of neuropathic pain (formalin paw model, ligated spinal nerve model and contusive spinal cord injury model) and found to be more effective in reducing pain behaviours than mexiletine.Robert M. Weston, Kamani R. Subasinghe, Vasiliki Staikopoulos, Bevyn Jarrot