Animal Studies

Diminished synaptic inhibition in the superficial spinal dorsal horn contributes to exaggerated pain responses that accompany peripheral inflammation and neuropathy. α2GABA receptors (α2GABAR) constitute the most abundant GABAR subtype at this site and are the targets of recently identified antihyperalgesic compounds. Surprisingly, hoxb8-α2 mice that lack α2GABAR from the spinal cord and peripheral sensory neurons exhibit unaltered sensitivity to acute painful stimuli and develop normal inflammatory and neuropathic hyperalgesia. Here, we provide a comprehensive analysis of GABAergic neurotransmission, of behavioral phenotypes and of possible compensatory mechanisms in hoxb8-α2 mice. Our results confirm that hoxb8-α2 mice show significantly diminished GABAergic inhibitory postsynaptic currents (IPSCs) in the superficial dorsal horn but no hyperalgesic phenotype. We also confirm that the potentiation of dorsal horn GABAergic IPSCs by the α2-preferring GABAR modulator HZ-166 is reduced in hoxb8-α2 mice and that hoxb8-α2 mice are resistant to the analgesic effects of HZ-166. Tonic GABAergic currents, glycinergic IPSCs, and sensory afferent-evoked EPSCs did show significant changes in hoxb8-α2 mice rendering a compensatory up-regulation of other GABAR subtypes or of glycine receptors unlikely. Although expression of serotonin and of the serotonin producing enzyme tryptophan hydroxylase (TPH2) was significantly increased in the dorsal horn of hoxb8-α2 mice, ablation of serotonergic terminals from the lumbar spinal cord failed to unmask a nociceptive phenotype. Our results are consistent with an important contribution of α2GABAR to spinal nociceptive control but their ablation early in development appears to activate yet-to-be identified compensatory mechanisms that protect hoxb8-α2 mice from hyperalgesia.

The Sigma-1 receptor (σR) is highly expressed in the primary sensory neurons (PSNs) that are the critical site of initiation and maintenance of pain following peripheral nerve injury. By immunoblot and immunohistochemistry, we observed increased expression of both σR and σR-binding immunoglobulin protein (BiP) in the lumbar (L) dorsal root ganglia (DRG) ipsilateral to painful neuropathy induced by spared nerve injury (SNI). To evaluate the therapeutic potential of PSN-targeted σR inhibition at a selected segmental level, we designed a recombinant adeno-associated viral (AAV) vector expressing a small hairpin RNA (shRNA) against rat σR. Injection of this vector into the L4/L5 DRGs induced downregulation of σR in DRG neurons of all size groups, while expression of BiP was not affected. This was accompanied by attenuation of SNI-induced cutaneous mechanical and thermal hypersensitivity. Whole-cell current-clamp recordings of dissociated neurons showed that knockdown of σR suppressed neuronal excitability, suggesting that σR silencing attenuates pain by reversal of injury-induced neuronal hyperexcitability. These findings support a critical role of σR in modulating PSN nociceptive functions, and that the nerve injury-induced elevated σR activity in the PSNs can be a significant driver of neuropathic pain. Further understanding the role of PSN-σR in pain pathology may open routes to exploit this system for DRG-targeted pain therapy.

Joint pain is the major clinical symptom of arthritis that affects millions of people. Controlling the excitability of knee-innervating dorsal root ganglion (DRG) neurons (knee neurons) could potentially provide pain relief. Therefore, our objective was to evaluate whether the newly engineered adeno-associated virus (AAV) serotype, AAV-PHP.S, can deliver functional artificial receptors to control knee neuron excitability following intra-articular knee injection.

Sensory neurons respond to noxious stimuli by relaying information from the periphery to the central nervous system via action potentials driven by voltage-gated sodium channels, specifically Nav1.7 and Nav1.8. These channels play a key role in the manifestation of inflammatory pain. The ability to screen compounds that modulate voltage-gated sodium channels using cell-based assays assumes that key channels present in vivo is maintained in vitro. Prior electrophysiological work in vitro utilized acutely dissociated tissues, however, maintaining this preparation for long periods is difficult. A potential alternative involves multi-electrode arrays which permit long-term measurements of neural spike activity and are well suited for assessing persistent sensitization consistent with chronic pain. Here, we demonstrate that the addition of two inflammatory mediators associated with chronic inflammatory pain, nerve growth factor (NGF) and interleukin-6 (IL-6), to adult DRG neurons increases their firing rates on multi-electrode arrays in vitro. Nav1.7 and Nav1.8 proteins are readily detected in cultured neurons and contribute to evoked activity. The blockade of both Nav1.7 and Nav1.8, has a profound impact on thermally evoked firing after treatment with IL-6 and NGF. This work underscores the utility of multi-electrode arrays for pharmacological studies of sensory neurons and may facilitate the discovery and mechanistic analyses of anti-nociceptive compounds.

Substantial evidence from preclinical models of pain suggests that basal and noxious nociceptive sensitivity, as well as antinociceptive responses to drugs, show significant heritability. Individual differences to these responses have been observed across species from rodents to humans. The use of closely related C57BL/6 inbred mouse substrains can facilitate gene mapping of acute nociceptive behaviors in preclinical pain models. In this study, we investigated behavioral differences between C57BL/6 J (B6 J) and C57BL/6 N (B6 N) substrains in the formalin test, a widely used tonic inflammatory pain model, using a battery of pain-related phenotypes, including reflexive tests, nesting, voluntary wheel running, sucrose preference and anxiety-like behavior in the light/dark test at two different time points (1-h and 24-h). Our results show that these substrains did not differ in reflexive thermal and mechanical responses at the 1-h time point. However, B6 N substrain mice showed increased sensitivity to spontaneous pain-like behaviors. In addition, B6 N substrain continued to show higher levels of mechanical hypersensitivity compared to controls at 24-h. indicating that mechanical hypersensitivity is a more persistent pain-related phenotype induced by formalin. Finally, no sex differences were observed in our outcome measures. Our results provide a comprehensive behavioral testing paradigm in response to an inflammatory agent for future mouse genetic studies in pain.