Project description:In this experiment we compare the effect of tibial nerve transection on gene expression within the dorsal root ganglion (DRG) of rats.
Project description:This program addresses the gene signature associated with DRG in the Chung rat model for neuropathic pain. The Chung neuropathic pain profiling data was analyzed by identifying genes that were up- and down-regulated at selected p value and fold change in DRG of the Sprague Dawley rats following spinal nerve ligation compared to the sham-operated controls.
Project description:Microglia have been implicated in the pathophysiology of neuropathic pain. Here, we sought to investigate whether cerebrospinal fluid (CSF) might be used as a proxy-measure of microglial activation in human participants. For this, we preformed fluorescent-activated cell sorting (FACS) of CSF immune cell populations derived from individuals who experienced pain with neuropathic features. We sorted CD4+, CD8+ T cells and monocytes and analyzed their transcriptome using RNA sequencing. We also performed Cellular Indexing of Transcriptomes and Epitopes (CITE)-seq to characterize the expression of all CSF immune cells in a patient with postherpetic neuralgia and in a patient with neuropathic pain after failed back surgery. Immune cell numbers and phenotypes were not obviously different between individuals regardless of the etiology of their pain. This was true when examining our own dataset, as well as when comparing it to previously published single-cell RNA sequencing data of human CSF. In all instances, CSF monocytes showed expression of myeloid cell markers commonly associated with microglia (P2RY12, TMEM119 and OLFML3), which will make it difficult to ascertain the origin of CSF proteins: do they derive directly from circulating CSF monocytes or could some originate in spinal cord microglia in the parenchyma? We conclude that it will not be straightforward to use CSF as a biomarker for microglial function in humans.
Project description:Neuropathic pain is a major clinic probelm as it is very difficult to treat and mechanism remain unknown. Here, we investigated the differential expression of proteins in the central nuecleus of amygdala (CeA) in neuropathic pain moldel spinal nerve transection (SNT)in rats. CeA was excised from naive, sham, SNTmodels at days 3, 7, 14 and 21 rats. The aim was to quatify the differential proteins in CeA including memebrane proteins. We used gel- and mass spectrometry- based proteomics. For gel-proteomics, total tissue lysate proteins were separated by 2D-PAGE. The 2D gels from different SNT time points against Sham and control rats were compared using Progenesis SameSpot software. The spots with fold change greater then 2 excised for the proteins IDs by LC-MS/MS. Protein spots were digested using trypsin. Extracted peptides were injected on the nano C18 column and measured by a LTQ Orbitrap XL or a LTQ Orbitrap Velos mass spectrometers. For the identification of membrane proteins in CeA, we used 1-SDS-PAGE and cut the gel region of MW 80 kDa and higher for nano-LC-MS/MS analysis. We also quantified membrane proteins by utilising triplex stabel isotope dimethyl labelling at the peptide level. Sham, control and day 3, 7 and 21 after SNT surgery rats CeA membrane proteins were purified and digested by trypsin and labelled with "light", "medium" and "heavy" dimethyl labelling reagents. The resulting peptides were analysed by a nano LC connected to the Q Exactive mass spectrometer. Quantification of peptide was processed using Proteome Discoverer 1.3.
Project description:Peripheral nerve injury alters the expression of hundreds of proteins in dorsal root ganglia (DRG). Targeting some of these proteins has led to successful treatments for acute pain, but not for sustained postoperative neuropathic pain. The latter may require targeting multiple proteins. Since a single microRNA (miR) can affect the expression of multiple proteins, here, we describe an approach to identify chronic neuropathic pain-relevant miRs. We used two variants of the spared nerve injury (SNI): Sural-SNI and Tibial-SNI and found distinct pain phenotypes between the two. Both models induced strong mechanical allodynia, but only Sural-SNI rats maintained strong mechanical and cold allodynia, as previously reported. In contrast, we found that Tibial-SNI rats recovered from mechanical allodynia and never developed cold allodynia. Since both models involve nerve injury, we increased the probability of identifying differentially regulated miRs that correlated with the quality and magnitude of neuropathic pain and decreased the probability of detecting miRs that are solely involved in neuronal regeneration. We found seven such miRs in L3-L5 DRG. The expression of these miRs increased in Tibial-SNI. These miRs displayed a lower level of expression in Sural-SNI, with four having levels lower than those in sham animals. Bioinformatics analysis of how these miRs could affect the expression of some ion channels supports the view that, following a peripheral nerve injury, the increase of the 7 miRs may contribute to the recovery from neuropathic pain while the decrease of four of them may contribute to the development of chronic neuropathic pain. The approach used resulted in the identification of a small number of potentially neuropathic pain relevant miRs. Additional studies are required to investigate whether manipulating the expression of the identified miRs in primary sensory neurons can prevent or ameliorate chronic neuropathic pain following peripheral nerve injuries. To identify the miRs that were differentially dysregulated between Tibial-SNI and Sural-SNI, we first performed 12 microarrays in a limited number of samples (in four individual DRGs per group: Sham, Tibial-SNI and Sural-SNI; two L3-DRG and two L4-DRG). Then, miRs identified as having differential expression were corroborated with real time qRT-PCR in RNA isolated from individual DRGs (L3, L4 and L5) derived from 4 rats per group (not presented here, but in the manuscript).