Animal Studies

Herpes simplex virus-type 1 (HSV-1) infection of sensory neurons may lead to a significant reduction in the expression of voltage-activated Na and Ca channels, which can disrupt the transmission of pain information. Viral infection also results in the secretion of various pro-inflammatory cytokines, including interleukin (IL)-6. In this work, we tested whether IL-6 regulates the expression of Na and Ca channels post HSV-1 infection in ND7/23 sensory-like neurons. Our results demonstrate that HSV-1 infection causes a significant decrease in the protein expression of the Cav3.2 T-type Ca channel subunit, despite increasing Cav3.2 mRNA synthesis. Neither Cav3.2 mRNA nor total protein content was affected by IL-6 treatment post HSV-1 infection. In ND7/23 cells, HSV-1 infection caused a significant reduction in the expression of Na and T-type Ca channels within 48 h. Exposure of ND7/23 cells to IL-6 for 24 h post infection reverses the effect of HSV-1, resulting in a significant increase in T-type Ca current density. However, Na currents were not restored by 24 h-treatment with IL-6 post HSV-1 infection of ND7/23 cells. The ability of IL-6 to increase the functional expression of T-type Ca channels on the membrane was blocked by inhibition of protein trafficking with brefeldin-A and ERK1/2 activation. These results indicate that IL-6 release following HSV-1 infection regulates the expression of T-type Ca channels, which may alter the transmission of pain information. This article is protected by copyright. All rights reserved.

The impact of vitamin D on sensory function, including pain processing, has been receiving increasing attention. Indeed, vitamin D deficiency is associated with various chronic pain conditions, and several lines of evidence indicate that vitamin D supplementation may trigger pain relief. However, the underlying mechanisms of action remain poorly understood. We used inflammatory and non-inflammatory rat models of chronic pain to evaluate the benefits of vitamin D (cholecalciferol) on pain symptoms. We found that cholecalciferol supplementation improved mechanical nociceptive thresholds in monoarthritic animals and reduced mechanical hyperalgesia and cold allodynia in a model of mononeuropathy. Transcriptomic analysis of cerebrum, dorsal root ganglia, and spinal cord tissues indicate that cholecalciferol supplementation induces a massive gene dysregulation which, in the cerebrum, is associated with opioid signaling (23 genes), nociception (14), and allodynia (8), and, in the dorsal root ganglia, with axonal guidance (37 genes) and nociception (17). Among the identified cerebral dysregulated nociception-, allodynia-, and opioid-associated genes, 21 can be associated with vitamin D metabolism. However, it appears that their expression is modulated by intermediate regulators such as diverse protein kinases and not, as expected, by the vitamin D receptor. Overall, several genes-Oxt, Pdyn, Penk, Pomc, Pth, Tac1, and Tgfb1-encoding for peptides/hormones stand out as top candidates to explain the therapeutic benefit of vitamin D supplementation. Further studies are now warranted to detail the precise mechanisms of action but also the most favorable doses and time windows for pain relief.

Nerve growth factor (NGF) and its receptors TrkA and p75 play a key role in the development and function of peripheral nociceptive neurons. Here, we describe novel technology to selectively photoablate TrkA-positive nociceptors through delivery of a phototoxic agent coupled to an engineered NGF ligand and subsequent near-infrared illumination. We demonstrate that this approach allows for on demand and localized reversal of pain behaviors in mouse models of acute, inflammatory, neuropathic, and joint pain. To target peripheral nociceptors, we generated a SNAP-tagged NGF derivative NGF that binds to TrkA/p75 receptors but does not provoke signaling in TrkA-positive cells or elicit pain behaviors in mice. NGF was coupled to the photosensitizer IRDye700DX phthalocyanine (IR700) and injected subcutaneously. After near-infrared illumination of the injected area, behavioral responses to nociceptive mechanical and sustained thermal stimuli, but not innocuous stimuli, were substantially reduced. Similarly, in models of inflammatory, osteoarthritic, and neuropathic pain, mechanical hypersensitivity was abolished for 3 weeks after a single treatment regime. We demonstrate that this loss of pain behavior coincides with the retraction of neurons from the skin which then reinnervate the epidermis after 3 weeks corresponding with the return of mechanical hypersensitivity. Thus NGF-mediated photoablation is a minimally invasive approach to reversibly silence nociceptor input from the periphery, and control pain and hypersensitivity to mechanical stimuli.

Irritable bowel syndrome patients frequently complain of pain in body regions somatotopically distinct from the gut, suggesting the involvement of an exaggerated signaling process in both visceral and somatic sensory pathways. Increasing evidence has shown that sprouting of tyrosine hydroxylase immunoreactive (TH-IR) fibers toward sensory neurons in dorsal root ganglia maintains and exacerbates the neuropathic and inflammatory pain, as well as colonic inflammation. The aim of the present study was to determine whether electroacupuncture could alleviate the visceral and secondary somatic hyperalgesia in colitis rats by suppressing the TH-IR expression in related dorsal root ganglia. After trinitrobenzene sulfonic acid irritation, rats developed inflammatory tissue damage in the distal colon, which was accompanied by visceral hypersensitivity and secondary hind paw hyperalgesia, as indicated by enhanced visceromotor response to colorectal distension and decreased mechanical and thermal withdrawal latency of the hind paw. Additionally, excessive TH-IR fibers sprouted toward calcitonin gene-related peptide immunoreactive sensory neurons, and TH-IR neurons also increased in the sixth lumbar dorsal root ganglia of colitis rats. Both electroacupuncture and guanethidine attenuated visceral and referred hind paw hyperalgesia by inhibiting the over-expression of TH-IR neurons and fibers in the sixth lumbar dorsal root ganglia. Moreover local inflammatory damage in the distal colon was restored after 7 days of electroacupuncture intervention. These results suggest that electroacupuncture relieved visceral and referred hind paw hypersensitivity in colitis rats by inhibiting TH expression in the sixth lumbar dorsal root ganglia.

Chronic pain is an unmet clinical problem with vast individual, societal and economic impact. Pathologic activity of the peripheral somatosensory afferents is one of the major drivers of chronic pain. This overexcitable state of somatosensory neurons is, in part, produced by the dysregulation of genes controlling neuronal excitability. Despite intense research, a unifying theory behind neuropathic remodelling is lacking. Here we show that transcriptional suppressor, repressor element 1-silencing transcription factor (REST, NRSF), is necessary and sufficient for the development of hyperalgesic state following chronic nerve injury or inflammation. Viral overexpression of REST in mouse DRG induced prominent mechanical and thermal hyperalgesia in vivo. Sensory neuron specific, inducible Rest knock-out prevented the development of such hyperalgesic state in three different chronic pain models. Genetic deletion of Rest reverted injury-induced hyperalgesia. Moreover, viral overexpression of REST in the same neurons in which its gene has been genetically deleted restored neuropathic hyperalgesia. Finally, sensory neuron specific Rest knockout prevented injury-induced downregulation of REST target genes in DRG neurons. This work identified REST as a major regulator of peripheral somatosensory neuron remodelling leading to chronic pain. The findings might help to develop a novel therapeutic approaches to combat chronic pain.This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (CC BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Neuropathic pain is one of most intense types of chronic pain. Numerous studies investigating neuropathic pain have described the critical involvement of microglia in the spinal cord. Previous studies have indicated that activation of large conductance Ca2+‑activated K+ (BK) channels contributes to microglial activation in the spinal dorsal horn (SDH) and the generation of neuropathic pain. However, the specific role of BK channels in spinal microglia in neuropathic pain has not been fully addressed in previous studies, as BK channel inhibitors were used to inhibit microglial BK channel based on their inhibitory kinetics. We previously identified that Ca2+‑activated K+ channel β3 auxiliary subunit (KCNMB3), which is an auxiliary subunit of BK channels and regulates gating properties of the channel, is exclusively expressed in microglia in the spinal cord. The present study analyzed the role of BK channels in spinal microglia in neuropathic pain using a spinal microglia‑specific BK channel knockdown method, with intrathecal injection of KCNMB3 small interfering RNA. Neuropathic pain was significantly attenuated in KCNMB3 knockdown mice. Increases in the number of microglia in the SDH following nerve injury were attenuated by KCNMB3 knockdown. Furthermore, increased levels of pain‑associated molecules in the SDH were attenuated in KCNMB3 knockdown mice. Attempts were also made to analyze the effects of KCNMB3 knockdown on chronic pain. KCNMB3 knockdown ameliorated chronic pain and inhibited the expression levels of pain‑associated molecules in the SDH. The results from the present study suggested that BK channels modulated the activation state of spinal microglia, and that KCNMB3 is a potential therapeutic target for neuropathic pain.

Acute and persistent pain are recognized consequences of TBI that can enhance suffering and significantly impair rehabilitative efforts. Both experimental models and clinical studies suggest that TBI may result in an imbalance between descending pain facilitatory and inhibitory pathways. The aim of this study was to assess the role of enhanced descending serotonin-mediated pain facilitation in a rat TBI model using selective spinal serotonergic fiber depletion with 5, 7-dihydroxytryptamine (DHT). We observed significant hindpaw allodynia in TBI rats that was reduced after DHT but not vehicle treatment. Immunohistochemical studies demonstrated profound spinal serotonin depletion in DHT-treated rats. Furthermore, lumbar intrathecal administration of the 5-HT receptor antagonist ondansetron at 7 days post-injury (DPI), when hindpaw allodynia was maximal, also attenuated nociceptive sensitization. Additional immunohistochemical analyses of the lumbar spinal cord at 7 DPI revealed a robust bilateral microglial response in the superficial dorsal horns that was significantly reduced with DHT treatment. Furthermore, serotonin depletion also prevented the TBI-induced bilateral increase in c-Fos positive cells within the Rexed laminae I and II of the dorsal horns. These results indicate that in the weeks following TBI, pain may be responsive to 5-HT receptor antagonists or other measures which rebalance descending pain modulation.

Chronic visceral hypersensitivity is an important type of chronic pain with unknown etiology and pathophysiology. Recent studies have shown that epigenetic regulation plays an important role in the development of chronic pain conditions. However, the role of miRNA-325-5p in chronic visceral pain remains unknown. The present study was designed to determine the roles and mechanism of miRNA-325-5p in a rat model of chronic visceral pain. This model was induced by neonatal colonic inflammation (NCI). In adulthood, NCI led to a significant reduction in the expression of miRNA-325-5p in colon-related dorsal root ganglia (DRGs), starting to decrease at the age of 4 weeks and being maintained to 8 weeks. Intrathecal administration of miRNA-325-5p agomir significantly enhanced the colorectal distention (CRD) threshold in a time-dependent manner. NCI also markedly increased the expression of CCL2 (C-C motif chemokine ligand 2) in colon-related DRGs at the mRNA and protein levels relative to age-matched control rats. The expression of CXCL12, IL33, SFRS7, and LGI1 was not significantly altered in NCI rats. CCL2 was co-expressed in NeuN-positive DRG neurons but not in glutamine synthetase-positive glial cells. Furthermore, CCL2 was mainly expressed in isolectin B4-binding- and calcitonin gene-related peptide-positive DRG neurons but in few NF-200-positive cells. More importantly, CCL2 was expressed in miR-325-5p-positive DRG neurons. Intrathecal injection of miRNA-325-5p agomir remarkably reduced the upregulation of CCL2 in NCI rats. Administration of Bindarit, an inhibitor of CCL2, markedly raised the CRD threshold in NCI rats in a dose- and time-dependent manner. These data suggest that NCI suppresses miRNA-325-5p expression and enhances CCL2 expression, thus contributing to visceral hypersensitivity in adult rats.