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We describe two differentiation protocols to derive sensory spinal interneurons (INs) from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). In protocol 1, we use retinoic acid (RA) to induce pain, itch, and heat mediating dI4/dI6 interneurons, and in protocol 2, RA with bone morphogenetic protein 4 (RA+BMP4) is used to induce proprioceptive dI1s and mechanosensory dI3s in hPSC cultures. These protocols provide an important step toward developing therapies for regaining sensation in spinal cord injury patients. For complete details on the use and execution of this protocol, please refer to Gupta et al. (2018).
Learn More >In this protocol, we provide step-by-step instructions for dissection and culture of primary murine dorsal root ganglia (DRG), which provide an opportunity to study the functional properties of peripheral sensory neurons . Further, we describe the analysis of neuropeptide release by ELISA as a possible downstream application. In addition, isolated DRGs can be used directly for immunofluorescence, flow cytometry, RNA sequencing or proteomic approaches, electrophysiology, and calcium imaging. For complete details on the use and execution of this protocol, please refer to Perner et al. (2020).
Learn More >CGRP, a neuropeptide involved in migraine pathophysiology, is also known to play a role in the respiratory system and in immunological conditions such as sepsis. We analyzed the impact of the use of CGRP antagonists in patients with migraine during the COVID-19 pandemic, caused by the SARS-CoV-2 coronavirus.
Learn More >Spinal cord injury (SCI) is one of the main causes leading to neuropathic pain. Here, we aim to explore the molecular mechanism and function of lncRNA PVT1 in neuropathic pain induced by SCI. The expression of lncRNA PVT1, microRNA (miR) - 186-5p was measured via quantitative reverse transcription PCR (qRT-PCR), and the activation of astrocytes (labeled by GFAP) was detected by immunohistochemistry. Western blot was conducted to detect the expression of chemokine ligand 13 (CXCL13), chemokine receptor 5 (CXCR5), cyclooxygenase-2 (COX2), inducible nitric oxide synthase (iNOS) and glial fibrillary acidic protein (GFAP) in spinal cord injury lesions. The levels of inflammatory cytokines (including IL-1β and IL-6) and MDA in tissues were examined via Enzyme-linked immunosorbent assay (ELISA). In vitro experiments were also conducted in primary cultured astrocyte to explore the response of astrocyte to lipopolysaccharide (LPS). What's more, the PVT1-miR-186-5p interaction was verified via the dual luciferase activity assay and RNA immunoprecipitation (RIP) assay. The results demonstrated that the levels of PVT1, CXCL13 and CXCR5 were upregulated, while miR-186-5p were decreased in SCI rats' spinal cord and LPS-mediated astrocytes. In the SCI model, PVT1 depletion significantly alleviated neuropathic pain, astrocytic activation and reduced the expression of neuroinflammatory factors and proteins. The relevant mechanism studies confirmed that PVT1 is a competitive endogenous RNA (ceRNA) of miR-186-5p, targets and inhibits its expression and promotes the expression of CXCL13/CXCR5, while miR-186-5p targets CXCL13. In conclusion, inhibition of lncRNA PVT1 alleviates neuropathic pain in SCI rats by upregulating miR-186-5p and down-regulating CXCL13/CXCR5. The PVT1/miR-186-5p/CXCL13/CXCR5 axis can be used as a new therapeutic target for neuropathic pain.
Learn More >Unlike motor symptoms, the effects of deep brain stimulation (DBS) on non-motor symptoms associated with dystonia remain unknown.
Learn More >Spinal microglia are highly responsive to peripheral nerve injury and are known to be a key player in pain. However, there has not been any direct evidence showing that selective microglial activation in vivo is sufficient to induce chronic pain. Here, we used optogenetic approaches in microglia to address this question employing CX3CR1creER/+: R26LSL-ReaChR/+ transgenic mice, in which red-activated channelrhodopsin (ReaChR) is inducibly and specifically expressed in microglia. We found that activation of ReaChR by red light in spinal microglia evoked reliable inward currents and membrane depolarization. In vivo optogenetic activation of microglial ReaChR in the spinal cord triggered chronic pain hypersensitivity in both male and female mice. In addition, activation of microglial ReaChR up-regulated neuronal c-Fos expression and enhanced C-fiber responses. Mechanistically, ReaChR activation led to a reactive microglial phenotype with increased interleukin (IL)-1β production, which is likely mediated by inflammasome activation and calcium elevation. IL-1 receptor antagonist (IL-1ra) was able to reverse the pain hypersensitivity and neuronal hyperactivity induced by microglial ReaChR activation. Therefore, our work demonstrates that optogenetic activation of spinal microglia is sufficient to trigger chronic pain phenotypes by increasing neuronal activity via IL-1 signaling.
Learn More >To evaluate gender differences in clinical characteristics of migraine by examining presence and severity of cutaneous allodynia, migraine-related disability, neck pain and its associated disability, passive mobility of the upper cervical spine, and performance of the deep neck flexor muscles.
Learn More >Cognitive impairment and chronic pain are amongst the most prevalent neurological sequelae of HIV infection, yet little is understood about the potential bidirectional relationship between the two conditions. Cognitive dysfunction can occur in chronic pain populations whilst those with cognitive impairment can display modified responses to experimentally induced painful stimuli. To date, this has not been explored in HIV cohorts.This study aimed to identify any contribution of chronic pain to cognitive impairment in HIV and to determine differences in pain characteristics between those with and without cognitive dysfunction.This was an observational cohort study involving people living with HIV ( = 148) in the United Kingdom. Participants underwent validated questionnaire-based measurement of pain severity, interference and symptom quality as well as conditioned pain modulation and quantitative sensory testing. All participants completed a computer-based cognitive function assessment.Fifty-seven participants met the criteria for cognitive impairment and 73 for chronic pain. The cognitive impairment group had a higher prevalence of chronic pain ( = 0.004) and reported more neuropathic symptoms ( = 0.001). Those with chronic pain performed less well in emotional recognition and verbal learning domains. The interaction identified between chronic pain and cognitive dysfunction warrants further exploration to identify causal links or shared pathology.
Learn More >Chronic pain is not well understood in opioid-dependent populations. We report the prevalence of chronic pain and pain characteristics in an opioid-dependent population by treatment type and gender.
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