Animal Studies

Peripheral nerve injury can lead to remodeling of brain circuits, and this can cause chronification of pain. We have recently reported that male mice subjected to spared injury of the sciatic nerve undergo changes in the function of the medial prefrontal cortex (mPFC) that culminate in reduced output of layer 5 pyramidal cells. More recently, we have shown that this is mediated by alterations in synaptic inputs from the basolateral amygdala (BLA) into GABAergic interneurons in the mPFC. Optogenetic inhibition of these inputs reversed mechanical allodynia and thermal hyperalgesia in male mice. It is known that the processing of pain signals can exhibit marked sex differences. We therefore tested whether the dysregulation of BLA to mPFC signaling is equally altered in female mice. Injection of AAV-Arch3.0 constructs into the BLA followed by implantation of a fiberoptic cannula into the mPFC in sham and SNI operated female mice was carried out, and pain behavioral responses were measured in response to yellow light mediated activation of this inhibitory opsin. Our data reveal that Arch3.0 activation leads to a marked increase in paw withdrawal thresholds and latencies in response to mechanical and thermal stimuli, respectively. However, we did not observe nerve injury-induced changes in mPFC layer 5 pyramidal cell output in female mice. Hence, the observed light-induced analgesic effects may be due to compensation for dysregulated neuronal circuits downstream of the mPFC.

Neuropathic pain is difficult to treat and remains a major clinical challenge worldwide. While the mechanisms which underlie the development of neuropathic pain are incompletely understood, interferon signaling by the immune system is known to play a role. Here, we demonstrate a role for IFNβ in attenuating mechanical allodynia induced by the spared nerve injury in mice. The results show that intrathecal administration of IFNβ (dosages up to 5000U) produces significant, transient, and dose-dependent attenuation of mechanical allodynia without observable effects on motor activity or feeding behavior, as is common with IFN administration. This analgesic effect is mediated by the ubiquitin-like protein ISG15, which is potently induced within the spinal cord following intrathecal delivery of IFNβ. Both free and conjugated ISG15 are elevated following IFNβ treatment, and this effect is increased in UBP43 mice lacking a key deconjugating enzyme. The IFNβ-mediated analgesia reduces MAPK signaling activation following nerve injury, and this effect requires induction of ISG15. These findings highlight a new role for IFNβ, ISG15 and MAPK signaling in immunomodulation of neuropathic pain and may lead to new therapeutic possibilities. Perspective: Neuropathic pain is frequently intractable in a clinical setting, and new treatment options are needed. Characterizing the anti-nociceptive potential of IFNβ and the associated downstream signaling pathways in preclinical models may lead to the development of new therapeutic options for debilitating neuropathies.

Inflammatory bowel disease (IBD), mainly comprising Crohn's disease and ulcerative colitis, is characterized by chronic inflammation in the digestive tract. Approximately 60% of the patients experience abdominal pain during acute IBD episodes, which severely impairs their quality of life. Both peripheral and central mechanisms are thought to be involved in such abdominal pain in IBD. Although much attention has been paid to peripheral mechanisms of abdominal pain in IBD pathophysiology, the involvement of supraspinal mechanisms remains poorly understood. To address this issue, we investigated regional brain activity in response to colorectal distension (CRD) in normal and IBD model rats using voxel-based statistical analysis of 2-deoxy-2-[18F]fluoro-D-glucose (FDG) PET imaging. The rat IBD model was generated by colorectal administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS), a chemical compound widely used to generate colitis. Tissue damage and inflammation were induced and dynamically changed with time after TNBS injection, while CRD-induced visceromotor response showed corresponding temporal changes. We found that characteristic brain activations were observed in response to visceral innocuous and noxious CRD and supraspinal nociception shared some physiological sensory pathway. Moreover, widespread brain regions were activated, and the functional coupling between the central medial thalamic nucleus (CMT) and anterior cingulate cortex (ACC) was enhanced after noxious CRD in IBD model of rats. Increased brain activity in the anterior insular cortex (aINS) and ACC positively correlated with noxious CRD-induced pain severity in normal and IBD rats respectively. These findings suggest that the pain matrix was shifted following persistent colonic inflammation, and thalamocortical sensitization in the pathway from CMT to ACC might be a central mechanism of the visceral hyperalgesia in IBD pathophysiology.

Endogenous Pain Modulation (EPM) impairment is a significant contributor to chronic pain. Conditioned pain modulation (CPM) testing assesses EPM function. Osteoarthritic (OA) dogs are good translational models, but CPM has not been explored. Our aim was to assess EPM impairment in OA dogs compared to controls using CPM. We hypothesized that CPM testing would demonstrate EPM impairment in OA dogs compared to controls. Dogs with stifle/hip OA and demographically-matched controls were recruited. The pre-conditioning test stimulus, using mechanical/thermal quantitative sensory testing (MQST or TQST), were performed at the metatarsus. A 22N blunt probe (conditioning stimulus) was applied to the contralateral antebrachium for 2 minutes, followed by MQST or TQST (post-conditioning test stimulus). The threshold changes from pre to post-conditioning (∆MQST and ∆TQST) were compared between OA and control dogs. Twenty-four client-owned dogs (OA, n = 11; controls, n = 13) were recruited. The ∆MQST(p < 0.001) and ∆TQST(p < 0.001) increased in control dogs but not OA dogs (∆MQST p = 0.65; ∆TQST p = 0.76). Both ∆MQST(p < 0.001) and ∆TQST(p < 0.001) were different between the OA and control groups. These are the first data showing that EPM impairment is associated with canine OA pain. The spontaneous OA dog model may be used to test drugs that normalize EPM function.

Descending control from supraspinal neuronal networks onto spinal cord neurons can modulate nociceptionEndogenous opioids in these brain circuits participate in pain modulationA differential opioidergic role for brain nuclei involved in supraspinal pain modulation has not been previously reported WHAT THIS ARTICLE TELLS US THAT IS NEW: In vivo electrophysiologic recordings from the dorsal horn of the spinal cord in male rats reveal differential effects of morphine at the anterior cingulate cortex, right amygdala, and the ventromedial medulla on evoked pain responsesThese data differentiate supraspinal opioid circuit regulation of spinal nociceptive processing and suggest that the regulation of sensory and affective components of pain are likely separate BACKGROUND:: The anterior cingulate cortex and central nucleus of the amygdala connect widely with brainstem nuclei involved in descending modulation, including the rostral ventromedial medulla. Endogenous opioids in these circuits participate in pain modulation. The hypothesis was that a differential opioidergic role for the brain nuclei listed in regulation of spinal neuronal responses because separable effects on pain behaviors in awake animals were previously observed.

C-LTMRs are known to convey affective aspects of touch and to modulate injury-induced pain in humans and mice. However, a role for these neurons in temperature sensation has been suggested, but not fully demonstrated. Here, we report that deletion of C-low-threshold mechanoreceptor (C-LTMR)-expressed bhlha9 causes impaired thermotaxis behavior and exacerbated formalin-evoked pain in male, but not female, mice. Positive modulators of GABA receptors failed to relieve inflammatory formalin pain and failed to decrease the frequency of spontaneous excitatory post-synaptic currents (sEPSCs) selectively in bhlha9 knockout (KO) males. This could be explained by a drastic change in the GABA content of lamina II inner inhibitory interneurons contacting C-LTMR central terminals. Finally, C-LTMR-specific deep RNA sequencing revealed more genes differentially expressed in male than in female bhlha9 KO C-LTMRs. Our data consolidate the role of C-LTMRs in modulation of formalin pain and provide in vivo evidence of their role in the discriminative aspects of temperature sensation.

Glycosphingolipids (GSLs) are abundant, ceramide-containing lipids in the nervous system that play key functional roles in pain and inflammation. We measured gene expression (Ugcg, St3gal5, St8sia1, B4galNT1, Ugt8a, and Gal3st1) of glycosyltransferases involved in GSL synthesis in murine dorsal root ganglion (DRG) and spinal cord after complete Freund's adjuvant (CFA)-induced unilateral hind-paw inflammation (1 day vs. 15 days). Chronic inflammation (15 days) sensitized both ipsilateral and contralateral paws to pain. One day of induced unilateral hind-paw inflammation (1d-IUHI) increased Ugcg, St8sia1, B4galnt1, and Gal3st1 expression in ipsilateral cord, suggesting that sulfatide and b-series gangliosides were also elevated. In addition, 1d-IUHI increased Ugcg, st3gal5 and B4galnt1 expression in contralateral cord, suggesting that sulfatide and a-/b-series gangliosides were elevated. By contrast, 1d-IUHI decreased Ugcg, St3gal5, and St8sia1 expression bilaterally in the DRG, suggesting that b-series gangliosides were depressed. Since intrathecal injection of b-series ganglioside induced mechanical allodynia in naïve mice, it seems reasonable that b-series gangliosides synthesized from upregulated St8sia1 in the ipsilateral spinal cord are involved in mechanical allodynia. By contrast, chronic inflammation led to a decrease of Ugcg, St3gal5, B4galnt1, and Gal3st1 expression in spinal cord bilaterally and an increase of St8sia1 expression in the ipsilateral DRG, suggesting that a-/b-series gangliosides in the spinal cord decreased and b-series gangliosides in ipsilateral DRG increased. These changes in glycosyltransferase gene expression in the DRG and the spinal cord may contribute to the modification of pain sensitivity in both inflamed and non-inflamed tissues and the transition from early to chronic inflammatory pain.