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.

Keywords: Behaviour; Neuropathic pain; Proteomics; Rat; Sex-differences; Transcriptomics.

Fig. 1

Results from the von Frey assay. Conducted on the ipsilateral paw in Experiment I at baseline prior to SNI surgeries, on Days 7 and 21 and in Experiment II at baseline prior to surgeries and on Day 7. Mean responses and SEM are reported. Five trials with five von Frey filaments (0.4, 1, 2, 4 and 6g) were conducted. Positive responses in the experiments were summed and are shown in Fig. 1a and 1e, respectively. The maximum number of responses was 25. Positive responses for filaments 1g, 2g and 4g in the experiments are reported in Fig. 1b, c and d, and in Fig. 1f, g and h, respectively. n = 9 or 10 rats per group (Experiment I) and n = 12 rats per group (Experiment II). 2-way-ANOVA with Holm-Sidak correction, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Fig. 2

Results from the acetone test conducted on the ipsilateral paw in Experiment I (Fig. 2a) at baseline prior to spared nerve injury (SNI) surgeries, on Days 7 and 21 and II (Fig. 2b) at baseline prior to surgeries, and on Day 7. Mean responses and SEMs are reported. Five trials with acetone were conducted on each animal. Positive responses were summed. n = 9 or 10 rats per group (Experiment I) and n = 12 rats per group (Experiment II). 2-way-ANOVA with Holm-Sidak correction, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Fig. 3

Analysis of IBA1 expression in the L4 region of the spinal cord (SC). Sham- and SNI-operated male (M) and female (F) rats on day 21 day after baseline tests. Figure 3a and b shows representative images of IBA1-immunostained L4 SC section of a male (3a) and a female (3a) rat. The dotted line outlines the analysed areas. 3c shows the area covered by IBA1-positive cells in the dorsal horn (DH); 3d shows the number of IBA1-positive cells in the DH; 3e shows the area covered by IBA1-positive cells in the vental horn (VH); and 3f the number of IBA1-positive cells in VH. All data normalized to the size of the analysed area (in pixels) and expressed as percentage of the contralateral side. *** p < 0.001, **** p < 0.0001, Student’s t-test with Holm-Sidak correction for multiple comparisons. n = 8–10 rats per group. Contra. = contralateral side; Ipsi. = ipsilateral side; n of cells = number of cells

Fig. 4

L4 dorsal root ganglion (DRG) immunostaining for IB-4 (a) and CGRP (b) after SNI in male (M) and female (F) rats and representative images of staining in L4 (c). **** p < 0.001, ** p < 0.01 by Student’s t-test with Holm-Sidak correction for multiple comparisons and ANOVA. ## p < 0.01 by ANOVA. n = 10

Fig. 5

Venn diagram (a) and volcano plots (b) of RNA-Seq data in rat spinal cord (SC) following spared nerve injury (SNI). All genes included in the Venn diagram have a FDR < 0.05. Volcano plots report log₂ fold-change and negative logarithmic (base 10) False Discovery Rate (FDR). Dotted line at -log( FDR 0.05), red symbols -log(FDR<0.05). Ipsilateral L4-L5 SC segment on day 7 after SNI and Sham-surgeries. All SNI vs. All Sham: n = 14 (50% male and female), Male SNI vs. Male Sham and Female SNI vs. Female Sham: n = 7. DESeq2 utilized for DE analysis

Fig. 6

Venn diagram (a) and volcano plots (b) of RNA-Seq data in rat dorsal root ganglion (DRG) following spared nerve injury (SNI) and Sham surgery. All genes included in the Venn diagram have an FDR < 0.05. Volcano plots report log₂ fold-change and negative logarithmic (base 10) False Discovery Rate (FDR). Dotted line at -log( FDR 0.05), red symbols -log(FDR<0.05). Ipsilateral L4-L5 DRG segment on day 7 after SNI and Sham-surgeries. (n = 14: 50% male and female). DESeq2 utilized for DE analysis