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Gene Expression Profiling of Cutaneous Injured and Non-Injured Nociceptors in SNI Animal Model of Neuropathic Pain.

ABSTRACT: Nociceptors are a particular subtype of dorsal root ganglion (DRG) neurons that detect noxious stimuli and elicit pain. Although recent efforts have been made to reveal the molecular profile of nociceptors in normal conditions, little is known about how this profile changes in pathological conditions. In this study we exploited laser capture microdissection to specifically collect individual injured and non-injured nociceptive DRG neurons and to define their gene profiling in rat spared nerve injury (SNI) model of neuropathic pain. We found minimal transcriptional changes in non-injured neurons at 7 days after SNI. In contrast, several novel transcripts were altered in injured nociceptors, and the global signature of these LCM-captured neurons differed markedly from that the gene expression patterns found previously using whole DRG tissue following SNI. Pathway analysis of the transcriptomic profile of the injured nociceptors revealed oxidative stress as a key biological process. We validated the increase of caspase-6 (CASP6) in small-sized DRG neurons and its functional role in SNI- and paclitaxel-induced neuropathic pain. Our results demonstrate that the identification of gene regulation in a specific population of DRG neurons (e.g., nociceptors) is an effective strategy to reveal new mechanisms and therapeutic targets for neuropathic pain from different origins.

SUBMITTER: Berta T

PROVIDER: S-EPMC5570923 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Traumatic spinal cord injury (SCI) has devastating implications for patients, including a high predisposition for developing chronic pain distal to the site of injury. Chronic pain develops weeks to months after injury, consequently, patients are treated after irreparable changes have occurred. Nociceptors are central to chronic pain; however, the diversity of this cellular population presents challenges to understanding mechanisms and attributing pain modalities to specific cell types. To begin to address how peripheral sensory neurons below the injury level may contribute to the below-level pain reported by SCI patients, we examined SCI-induced changes in gene expression in lumbar dorsal root ganglia (DRG) below the site of injury. SCI was performed at the T10 vertebral level, with injury produced by a vessel clip with a closing pressure of 15 g for 1 min. Alterations in gene expression produce long-term sensory changes, therefore, we were interested in studying SCI-induced transcripts before the onset of chronic pain, which may trigger changes in downstream signaling pathways and ultimately facilitate the transmission of pain. To examine changes in the nociceptor subpopulation in DRG distal to the site of injury, we retrograde labeled sensory neurons projecting to the hairy hindpaw skin with fluorescent dye and collected the corresponding lumbar (L2-L6) DRG 4 days post-injury. Following dissociation, labeled neurons were purified by fluorescence-activated cell sorting (FACS). RNA was extracted from sorted sensory neurons of naïve, sham, or SCI mice and sequenced. Transcript abundances validated that the desired population of nociceptors were isolated. Cross-comparisons to data sets from similar studies confirmed, we were able to isolate our cells of interest and identify a unique pattern of gene expression within a subpopulation of neurons projecting to the hairy hindpaw skin. Differential gene expression analysis showed high expression levels and significant transcript changes 4 days post-injury in SCI cell populations relevant to the onset of chronic pain. Regulatory interrelationships predicted by pathway analysis implicated changes within the synaptogenesis signaling pathway as well as networks related to inflammatory signaling mechanisms, suggesting a role for synaptic plasticity and a correlation with pro-inflammatory signaling in the transition from acute to chronic pain.

Project description:Objective: Chronic pain is a major problem in patients with spinal cord injury (SCI). According to different studies, approximately 70% of patients with SCI experience persistent pain. Although previous gene expression profiling studies have been conducted in animal models, gene expression profiling study in human whole blood has not been reported yet. The objective of this study was to define the gene expression profile of neuropathic pain in spinal cord injured patients. Materials and Methods: We performed gene expression analysis including 20.000 genes using Affymetrix GeneChip® Primeview™ Human Gene Expression Arrays. For confirmation of expression levels of selected genes, we performed real time polymerase chain reaction. We gathered samples from periferic blood mononuclear cells. Data of twelve patients with intractable neuropathic pain was compared with thirteen patients who don’t have pain. All patients have complete injuries with a level of injury above T5. Results: A total of 16 differentially expressed genes including 9 up-regulated and 7 down-regulated were obtained with a cut-off degree at 4 fold change. EIF1AY, RPS4Y1, DDX3Y, RPL31, ETS1, KLF3, JUN, CYorf15B, and ERAP2 were in up-regulated and HLA-C, XIST, C4BPA, FAM118A, ZNF506, OVOS2, and C1D were in down-regulated group. KEGG pathway database showed that 14.6% of genes were enriched in the nodes of immune system. Real time polymerase chain reaction analyses did not increase to statistically significant levels for confirmation of gene expression analyze results. Conclusions: Considering these data, findings of this first gene expression study in humans with SCI might provide valuable information for future targets for understanding neuropathic pain pathogenesis. The results of gene expression analyses were not confirmed by real time polymerase chain reaction. Studies with larger sample size are needed for confirmation. This is a cross-sectional study with a control group. Patients were divided into two groups according to intractable neuropathic pain existence.There were 12 patients in group A (Patients with pain) and 13 patients in control group.

Project description:Vincristine (VCR) is a widely used chemotherapy drug that induced peripheral painful neuropathy. Yet, it still lacks an ideal therapeutic strategy. The transient receptor potential (TRP) channels, purinergic receptor (P2Y), and mitogen-activated protein kinase (MAPK) signaling play a crucial role in the pathogenesis of neuropathic pain. Withametelin (WMT), a potential Phytosteroid isolated from datura innoxa, exhibits remarkable neuroprotective properties. The present investigation was designed to explore the effect of withametelin on VCR-induced neuropathic pain and its underlying molecular mechanism. Initially, the neuroprotective potential of WMT was confirmed against hydrogen peroxide (H2O2)-induced PC12 cells. To develop potential candidates for neuropathic pain treatment, a VCR-induced neuropathic pain model was established. Vincristine (75 μg/kg) was administered intraperitoneally (i.p.) for 10 consecutive days (day 1-10) for the induction of neuropathic pain. Gabapentin (GBP) (60 mg/kg, i.p.) and withametelin (0.1 and 1 mg/kg i.p.) treatments were given after the completion of VCR injection on the 11th day up to 21 days. The results revealed that WMT significantly reduced VCR-induced pain hypersensitivity, including mechanical allodynia, cold allodynia, and thermal hyperalgesia. It reversed the VCR-induced histopathological changes in the brain, spinal cord, and sciatic nerve. It inhibited VCR-induced changes in the biochemical composition of the myelin sheath of the sciatic nerve. It markedly downregulated the expression levels of TRPV1 (transient receptor potential vanilloid 1); TRPM8 (Transient receptor potential melastatin 8); and P2Y nociceptors and MAPKs signaling, including ERK (Extracellular Signal-Regulated Kinase), JNK (c-Jun N-terminal kinase), and p-38 in the spinal cord. It suppressed apoptosis by regulating Bax (Bcl2-associated X-protein), Bcl-2 (B-cell-lymphoma-2), and Caspase-3 expression. It considerably attenuated inflammatory cytokines, oxidative stress, and genotoxicity. This study suggests that WMT treatment suppressed vincristine-induced neuropathic pain by targeting the TRPV1/TRPM8/P2Y nociceptors and MAPK signaling.

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Gene Expression Profiling of Cutaneous Injured and Non-Injured Nociceptors in SNI Animal Model of Neuropathic Pain.

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