Spared nerve injury causes sexually dimorphic mechanical allodynia and differential gene expression in spinal cords and dorsal root ganglia in rats

Abstract

BACKGROUND Neuropathic pain (NP) is more prevalent in women. However, females are under-represented in animal experiments, and the mechanisms of sex differences remain inadequately understood. Using the spared nerve injury (SNI) model in rats, we characterized sex differences in behaviour, analysed dorsal root ganglion (DRG) and spinal cord (SC) tissues with transcriptomics and immunohistochemistry assays, and examined the proteome of cerebrospinal fluid (CSF). METHODS The study comprised two experiments, with male and female Sprague-Dawley rats subjected to SNI- and sham-surgeries. Mechanical and cold allodynias were assessed using von Frey filaments and the acetone test, respectively. Samples were extracted on Day 21 for CSF proteome and DRG and SC analysis for neuronal markers (IB4 and CGRP) and glial cell markers (IBA1 and GFAP), respectively, in Experiment I. Lumbar 4-5 DRGs and SC segments were collected for RNA-seq analysis on Day 7 in Experiment II. Differential gene expression in DRG and SC was calculated using DESeq2, and pathway analyses were conducted using iPathwayguide. RESULTS Females developed stronger mechanical allodynia in both experiments. Equivalent decreases in CGRP and IB-4 positive cell counts in DRG and increases in glial cells markers in the SC were seen in both sexes. No CSF protein showed change following SNI in any group. RNASeq of DRG and SC showed abundant changes in gene expression. Sexually dimorphic responses were found in genes related to T-cells (cd28, ctla4, cd274, cd4, prf1), other immunological responses (dpp4, c5a, cxcr2 and il1b), neuronal transmission (hrh3, thbs4, chrna4 and pdyn), plasticity (atf3, c1qc and reg3b), and others (bhlhe22, mcpt1l, trpv6). Analyses of biological processes revealed differences in T and B cell functions in DRG and neuronal processes in both DRG and SC. CONCLUSIONS We observed significantly stronger mechanical allodynia in females and many sexually dimorphic changes in gene expression, following SNI in rats. Several genes have previously been linked to NP, while some are novel. Our results suggest gene targets for further studies in the development of new, possibly sex-specific, therapies for NP and underline the importance of investigating NP in both sexes.