Spared Nerve Injury Causes Sexually Dimorphic Mechanical Allodynia and Differential Gene Expression in Spinal Cords and Dorsal Root Ganglia in Rats

Abstract

Neuropathic pain is more prevalent in women. However, females are under-represented in animal experiments, and the mechanisms of sex differences remain inadequately understood. We used the spared nerve injury (SNI) model in rats to characterize sex differences in pain behaviour, unbiased RNA-Seq and proteomics to study the mechanisms. Male and female rats were subjected to SNI- and sham-surgery. Mechanical and cold allodynia were assessed. Ipsilateral lumbar dorsal root ganglia (DRG) and spinal cord (SC) segments were collected for RNA-seq analysis with DESeq2 on Day 7. Cerebrospinal fluid (CSF) samples for proteomic analysis and DRGs and SCs for analysis of IB-4 and CGRP, and IBA1 and GFAP, respectively, were collected on Day 21. Females developed stronger mechanical allodynia. There were no differences between the sexes in CGRP and IB-4 in the DRG or glial cell markers in the SC. No CSF protein showed change following SNI. 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). We observed significantly stronger mechanical allodynia in females and numerous 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.

Sexual Dimorphism of Early Transcriptional Reprogramming in Dorsal Root Ganglia After Peripheral Nerve Injury

“…Other male-prevalent RAGs included the AP-1 component c-Fos ( Fos ). RAGs were consistently upregulated at later stages of PNI, including sciatic nerve chronic constriction injury (CCI; Stephens et al, 2019 ; Ahlström et al, 2021 ). We suggest that their early activation and sustained long-term expression are indispensable for a robust response to injury.…”

“…Additional considerations were given to the timing of sexually dimorphic changes, which directly relate to the temporal coordination of biological and behavioral processes. Generally, male DRG displayed a significantly higher number of differentially expressed genes, both up- and downregulated, in response to PNI within 24 h, than on day 7 ( Ahlström et al, 2021 ) and day 14 PNI timepoints ( Stephens et al, 2019 ). By comparing the most prominent sexually dimorphic transcripts at the early and later stages of PNI response, we observed consistent regulation of genes related to immunological response, neuronal transmission, regeneration, and plasticity obtained on day 7 ( Ahlström et al, 2021 ) and day 14 PNI timepoints in rats ( Stephens et al, 2019 ).…”

“…Generally, male DRG displayed a significantly higher number of differentially expressed genes, both up- and downregulated, in response to PNI within 24 h, than on day 7 ( Ahlström et al, 2021 ) and day 14 PNI timepoints ( Stephens et al, 2019 ). By comparing the most prominent sexually dimorphic transcripts at the early and later stages of PNI response, we observed consistent regulation of genes related to immunological response, neuronal transmission, regeneration, and plasticity obtained on day 7 ( Ahlström et al, 2021 ) and day 14 PNI timepoints in rats ( Stephens et al, 2019 ). However, as outlined in the present study, many sexually dimorphic and monomorphic transcripts were specific for the early stages of PNI response.…”

“…Transcriptome RNA-sequencing (RNA-seq)-based analyses of human DRG at baseline ( Ray et al, 2019 ) and in patients with radicular/neuropathic pain ( North et al, 2019 ) highlighted DRG’s central role in sex-specific gene regulation. Likewise, emerging research has revealed sexual dimorphism in rodent DRG transcriptomes in response to peripheral nerve injury ( Stephens et al, 2019 ; Ahlström et al, 2021 ), hyperalgesic priming by interleukin 6 (Il-6; Paige et al, 2020 ), sciatic nerve injection of myelin basic protein (MBP) derived peptides ( Chernov et al, 2020 ), and other stimuli. Transcriptome differences in DRG have been implicated in female-prevalent hypersensitivity partly due to the activity of prostaglandin signaling and neuroendocrine mechanisms involving prolactin receptors ( North et al, 2019 ; Ray et al, 2019 ; Chernov et al, 2020 ; Mecklenburg et al, 2020 ; Paige et al, 2020 ; Tavares-Ferreira et al, 2020 ).…”

Sex Differences in CGRP Regulation and Function in the Amygdala in a Rat Model of Neuropathic Pain

“…In the periphery, naïve female rats were shown to have fewer CGRP-immunoreactive dorsal root ganglion (DRG) neurons than their male counterparts, though ovariectomy produced a significant increase in immunoreactive neurons (Yang et al, 1998 ). A recent study reported no significant sex differences in CGRP-immunostaining cells in the DRG 7 days after the induction of neuropathic pain using the spared nerve injury (SNI) model (Ahlström et al, 2021 ). To our knowledge, this study is the first to report neuropathic pain stage-specific sex differences in mRNA expression levels of CGRP and its receptor components within the brain.…”