Animal Studies

The high incidence of treatment-resistant pain calls for the urgent preclinical translation of new analgesics. Understanding the behavioral readout of pain in animals is crucial for efficacy evaluation when developing novel analgesics. Mas-related G protein-coupled receptor D-positive (Mrgprd) and transient receptor potential vanilloid 1-positive (TRPV1) sensory neurons are two major non-overlapping subpopulations of C-fiber nociceptors. Their activation has been reported to provoke diverse nocifensive behaviors. However, what kind of behavior reliably represents subjectively conscious pain perception needs to be revisited. Here, we generated transgenic mice in which Mrgprd or TRPV1 sensory neurons specifically express channelrhodopsin-2 (ChR2). Under physiological conditions, optogenetic activation of hindpaw Mrgprd afferents evoked reflexive behaviors (lifting, etc.), but failed to produce aversion. In contrast, TRPV1 afferents activation evoked marked reflexive behaviors and affective responses (licking, etc.), as well as robust aversion. Under neuropathic pain conditions induced by spared nerve injury (SNI), affective behaviors and avoidance can be elicited by Mrgprd afferents excitation. Mechanistically, spinal cord-lateral parabrachial nucleus (lPBN) projecting neurons in superficial layers (lamina I-II ) were activated by TRPV1 nociceptors in naïve conditions or by Mrgprd nociceptors after SNI, whereas only deep spinal cord neurons were activated by Mrgprd nociceptors in naïve conditions. Moreover, the excitatory inputs from Mrgprd afferents to neurons within inner lamina II (II ) are partially gated under normal conditions. Altogether, we conclude that optogenetic activation of the adult Mrgprd nociceptors drives non-pain-like reflexive behaviors the deep spinal cord pathway under physiological conditions and drives pain-like affective behaviors superficial spinal cord pathway under pathological conditions. The distinct spinal pathway transmitting different forms of nocifensive behaviors provides different therapeutic targets. Moreover, this study appeals to the rational evaluation of preclinical analgesic efficacy by using comprehensive and suitable behavioral assays, as well as by assessing neural activity in the two distinct pathways.

Neuropathic pain (NP) is chronic and associated with poor effects of general analgesia. It affects patients' health and quality of life. The apoptotic process of lipid peroxidation caused by iron overload is called ferroptosis, which may be associated with nervous system disease. A recent study has found that sirtuin 2 (SIRT2) achieves a neuroprotective effect by suppressing ferroptosis. Herein, we aimed to examine whether SIRT2 regulated spared nerve injury (SNI)-induced NP by suppressing ferroptosis in rats. A rat model of NP was induced in adult male Sprague-Dawley rats weighing 200-250 g. Mechanical allodynia was observed from the first day after SNI and continued for 14 days. Compared with age-matched control rats, the expression of SIRT2 and ferroportin 1 (FPN1) decreased in the L4-6 spinal cord of the SNI-induced NP rats. In addition, we observed that the levels of both iron and anti-acyl-coenzyme A synthetase long-chain family member 4 (ACSL4) were significantly increased in the spinal cord after SNI, while the expression of glutathione peroxidase 4 (GPX4) was decreased. Furthermore, an intrathecal injection of SIRT2 overexpressed recombinant adenovirus, which upregulated the expression of SIRT2, attenuated mechanical allodynia, enhanced the level of FPN1, inhibited intracellular iron accumulation, and reduced oxidant stress levels, thereby reversing the changes to ACSL4 and GPX4 expression in the SNI rats. This evidence suggests that SIRT2-targeted therapeutics may help relieve the symptoms of chronic NP.

Inflammatory bowel disease (IBD) results in chronic abdominal pain in patients due to the presence of inflammatory responses in the colon. Electroacupuncture (EA) is effective in alleviating visceral pain and colonic inflammation associated with IBD. Cannabinoid CB2 receptor agonists also reduce colonic inflammation in a mouse model of IBD. However, whether EA reduces visceral pain and colonic inflammation the CB2 receptor remains unknown. Here, we determined the mechanism of the antinociceptive effect of EA in a mouse model of IBD induced by rectal perfusion of 2,4,6-trinitrobenzenesulfonic acid solution (TNBS). EA or sham EA was performed at the bilateral Dachangshu (BL25) point for seven consecutive days. The von Frey and colorectal distension tests were performed to measure mechanical referred pain and visceral pain. Western blotting and immunohistochemistry assays were carried out to determine the expression of IL-1β and iNOS and activation of macrophages in the colon tissues. We found that EA, but not sham EA, attenuated visceral hypersensitivity and promoted activation of CB2 receptors, which in turn inhibited macrophage activation and the expression of IL-1β and iNOS. The effects of EA were blocked by AM630, a specific CB2 receptor antagonist, and by CB2 receptor knockout. Our findings suggest that EA attenuates mechanical allodynia and visceral hypersensitivity associated with IBD by activating CB2 receptors and subsequent inhibition of macrophage activation and expression of IL-1β and iNOS.

Joint pain is a complex phenomenon that involves multiple endogenous mediators and pathophysiological events. In addition to nociceptive and inflammatory pain, some patients report neuropathic-like pain symptoms. Examination of arthritic joints from humans and preclinical animal models have revealed axonal damage which is likely the source of the neuropathic pain. The mediators responsible for joint peripheral neuropathy are obscure, but lysophosphatidic acid (LPA) has emerged as a leading candidate target. In the present study, male and female Wistar rats received an intra-articular injection of LPA into the right knee and allowed to recover for 28 days. Joint pain was measured by von Frey hair algesiometry, while joint pathology was determined by scoring of histological sections. Both male and female rats showed comparable degenerative changes to the LPA-treated knee including chondrocyte death, focal bone erosion, and synovitis. Mechanical withdrawal thresholds decreased by 20-30% indicative of secondary allodynia in the affected limb; however, there was no significant difference in pain sensitivity between the sexes. Treatment of LPA animals with the neuropathic pain drug amitriptyline reduced joint pain for over 2 hours with no sex differences being observed. In summary, intra-articular injection of LPA causes joint degeneration and neuropathic pain thereby mimicking some of the characteristics of neuropathic osteoarthritis.

Immunotherapy is now considered as the new pillar in treatment of cancer patients. Dendritic cells (DCs) play an essential role in stimulating anti-tumor immune responses, as they are capable of cross-presenting exogenous tumor antigens in MHCI complexes to activate naïve CD8+ T cells. Analgesics, like non-steroid anti-inflammatory drugs (NSAIDs), are frequently given to cancer patients to help relieve pain, however little is known about their impact on DC function.

Satellite glial cells (SGCs) tightly surround and support primary sensory neurons in the peripheral nervous system and are increasingly recognized for their involvement in the development of neuropathic pain following nerve injury. SGCs are difficult to investigate due to their flattened shape and tight physical connection to neurons and their rapid changes in phenotype and protein expression when cultured . Consequently, several aspects of SGC function under normal conditions as well as after a nerve injury remain to be explored. The recent advance in single cell RNA sequencing (scRNAseq) technologies has enabled a new approach to investigate SGCs. In this study we used scRNAseq to investigate SGCs from mice subjected to sciatic nerve injury. We used a meta-analysis approach to compare the injury response with that found in other published datasets.  Furthermore, we also used scRNAseq to investigate how cells from the dorsal root ganglion (DRG) change after 3 days in culture. From our meta-analysis of the injured conditions, we find that SGCs share a common signature of 18 regulated genes following sciatic nerve crush or sciatic nerve ligation, involving transcriptional regulation of cholesterol biosynthesis. We also observed a considerable transcriptional change when culturing SGCs, suggesting that some differentiate into a specialised state while others start resembling Schwann cell-like precursors. By using integrated analyses of new and previously published scRNAseq datasets, this study provides a consensus view of which genes are most robustly changed in SGCs after injury. Our results are available via the Broad Institute Single Cell Portal, so that readers can explore and search for genes of interest.

To analyze the effect and mechanism of dexmedetomidine (DEX) analgesia pretreatment on functional chronic visceral pain in rats.