In this study, we set out to characterize sex differences in behaviour, immunohistochemical markers, protein content, and RNA expression in both male and female rats, following spared nerve injury. Compared with males, female rats demonstrated consistently greater and more rapidly developing mechanical allodynia. The decrease in the number of neurons positive for CGRP and IB-4 in the DRG was equal in both sexes, suggesting injury of similar magnitude in both sexes. The increase in staining of glial cell markers IBA1 and GFAP in the SC was fairly consistent and did not reveal convincing sexual dimorphism on Day 21 after injury. No changes to the proteome of the CSF due to SNI could be demonstrated at 21 days. Transcriptomics analyses revealed DE in several pain-related genes and others of interest in the two sexes at Day 7 after SNI in DRG and/or SC, among the most interesting being Ctla4, Cd274, Dpp4, Il1b, Cxcr2, Thbs4, Hrh3, Pdyn, and Bhlhe22.
In Experiment II, we set out to find transcripts, whose change in expression might explain the observed behavioural sex differences in NP on Day 7 after SNI, by analysing gene expression in both DRG and SC.
FCs in DRG were generally greater than those in the SC, reflecting the role of DRG neurons as a primary site of the production of proteins in response to PNI. In general, female DRG and SC showed more genes with DE after SNI compared with sham, than males. Altogether 2333 genes were sexually dimorphic in DRG in our study. A previous study reported 1513 sexually dimorphic genes in response to CCI, 14 days after injury, with more changes in males than in females , in contrast to our results. Our earlier time point of 7 days and behavioural data consistently showing greater mechanical allodynia in females imply a divergent change in transcription in DRG early in the course of development of NP.
In a meta-analysis of microarray studies for pain in rodents, the two most reliably upregulated genes in DRG after PNI were Reg3b and Ccl2 . These two genes were also upregulated in both male and female DRG in our data, with no sex differences.
To the best of our knowledge, no previous study has assessed sexually dimorphic gene expression changes in SC after PNI. As further support for the role of Reg3b and Ccl2, the genes were also upregulated in SC in our study but, interestingly, only in males (FDR < 0.05). In addition, the gene displaying the largest upregulation in SC, in both sexes, was Cxcl13, also shown to be upregulated following SNL and to be relevant for the activation of astrocytes and for driving NP . As discussed earlier, microgliosis occurred in both sexes on account of IBA1, the product of Aif1. However, the pathway analyses show alteration in several microglia-relevant pathways in females only in the DRG.
Pathway and biological process analysis revealed, in particular, several T cell and other immunology-related findings. Neuronal plasticity and related processes, such as apoptosis, may also be crucial in the development of pathological activity and sensitization in the sensory system. As we were particularly interested in genes that might explain the behavioural findings, we also focused on genes retrieved from the PainNetworks database. Many of these genes demonstrated significant sexually dimorphic responses in DRG, and/or lesser dimorphic responses in SC, after SNI. We also discuss other genes with a large that have been linked to pain, as well as other genes that are completely novel in this respect. We categorized our findings into several groups, aware that genes may fall into more than one category: genes related to T cells (Cd28, Ctla4, Cd274, Cd4, Prf1); other immunological responses, (Dpp4, C5ar1, Cxcr2 and Il1b); neuronal transmission (Hrh3, Thbs4, Chrna4 and Pdyn); plasticity (Atf3, C1qc and Reg3b); and other genes with significant sexually dimorphic changes (Bhlhe22, Mcpt1l, Trpv6).
Changes related to T cells
Several immune-related pathways and biological processes were altered in DRG and/or SC in both sexes. We identified numerous T-cell-related transcripts that showed altered expression. T cells are paramount regulators and effectors in immune responses, and they have been postulated to play a part in sex differences in pain, and to be of greater importance in females [9, 10].
One interesting T-cell-related gene was Cd274, which displayed male-only upregulation in DRG. The gene Cd274 is translated into the protein PD-L1, which downregulates effector CD8 + T-cell activity . Interestingly, PD-L1 is also produced in DRG neurons and it has been reported to have potent antinociceptive effects in mice through its receptor PD-1 . As PD-L1 may exert an antinociceptive effect, lack of its upregulation in females might contribute to sex differences in mechanical allodynia.
Other genes indicating a stronger female T-cell response in the DRG were Cd4, Prf1, and Dntt. The gene Cd4, the product of which is the prototypic marker for helper T cells, showed an increase in expression in females only (log2 FC 1.29). Upregulation of Prf1 also occurred in females only (log2 FC 2.47). The gene Prf1 codes for perforin, which forms cytolytic pores and is a key end-stage effector mechanism of CD8+- and NK-cells . Further, we demonstrate a female-only upregulation of Dntt, a lymphocyte-specific-DNA polymerase involved in adding nucleotides to T-cell receptors, which are essential for the function of T cells [51, 52].
Two additional T-cell-related transcripts of great interest that were upregulated in the DRG are Ctla4 (log2 FC = 2.6 upregulation in females) and Cd28 (log2 FC = 2.1 upregulation in males). Binding of ctla4 on T cells by CD80/86 on antigen-presenting cells (APC) arrests the cell cycle of T cells, while binding of cd28 on T-cells with the same CD80/86 on APC leads to increased proliferation and increases in effector mechanisms of T cells [53–56]. This finding initially seems to contradict our findings regarding Cd4, Prf1, and Dntt, which indicated a stronger T-cell response in females. However, ctla4 has been shown to be upregulated following CD8 + activation and, as such, the Ctla4 increase may be secondary to the prior activation of T cells . This hypothesis relies on the other T-cell findings described above. Interestingly, CTLA4-inhibitors are in clinical use as immunotherapeutics for their function of activating host immune response against tumours . Thus, the role of CTLA-4 inhibitors in the development of NP in cancer patients would be of interest.
To summarize, T-cell-related transcripts show large differences between the sexes, with several changes pointing to T cells having a greater importance in female than in male rats in NP, in line with the findings of Sorge et al .
Other changes to the immunological response
Other genes closely related to immunological processes were profoundly affected. Of these, Dpp4, C5ar1, Cxcr2, Il1b will be discussed.
The gene Dpp4 is a PainNetworks gene that displays a female-only upregulation following SNI and has been shown to be expressed in spinal astrocytes and microglia, as well as in T cells [59, 60]. Spinal application of DPP4-inhibitors, which are widely used clinically in the treatment of diabetes mellitus , decreases mechanical allodynia in inflammatory pain and shows a modest antihyperalgesic effect following partial SNL in male rats [60, 62]. Upregulation of Dpp4 might thus partly explain the observed sex difference in mechanical allodynia. This opens an opportunity to test the hypothesis that DPP-4-inhibitors might produce an antinociceptive effect in females.
Another prominent, female-specific, upregulated gene is Cxcr2, which encodes the receptor binding several chemokines, one of which being CXCL1 . Both Cxcr2 and Cxcl1 have been shown to be upregulated in pain states in DRG [64, 65]. The expression of Cxcl1 was not altered in our experiment but CXCR2-signalling has previously been linked to maintenance of inflammatory pain . Interestingly, CXCR2 antagonists have demonstrated an antinociceptive effect in the CCI model of NP . Therefore, the female-specific upregulation of cxcr2 suggests that CXCR2 antagonists might be more efficacious for NP in females.
The complement cascade genes C1qc and C3 were upregulated in the DRG of both sexes. Downstream in the complement cascade of the protein products of these genes is the anaphylotoxin C5a, which is the ligand for the receptor C5ar1. This receptor is coded by the gene C5ar1, which was upregulated in DRG only in females. C5a and its receptors have previously been shown to be increased in CCI-, SNL- and SNI-operated rats and inhibition of C5a has been shown to aggravate development of NP in male rats . What function this larger upregulation serves in females remains undetermined and it would be interesting to compare C5a responses in males and females.
Il1b, encoding the protein Interleukin-1β, was prominently upregulated in female DRG (FC = 6.31, FDR < 0.05) compared with males (FC = 1.41, FDR > 0.05). Il1b has been shown to be of importance in the development of NP [69, 70]. Notably, prior studies have mostly been performed in male rodents and we did not find a significant change in the upregulation of Il1b in male rats. This may be due, at least partly, to the strict statistical correction for multiple comparisons. The large upregulation in females might explain some of the observed difference in the pain behaviour between females and males. Several drugs target Il1b signalling. One of them is anakinra, an IL1 receptor antagonist. This offers a possibility for further studies of sex-specific indications for drugs in this group .
Several ion channels and neurotransmitter systems are important in the pathophysiology of NP and targets for its treatment. Cavα2δ1, for which Thrombospondin 4 is the endogenous ligand, α7 nicotinic receptor and histamine 3 receptors have been studied as target proteins for drugs for NP [72–74]. Interestingly, the biological process “neuronal action potential” was only altered in females while “regulation of synaptic transmission, GABAergic” showed a change only in males, and “positive regulation of synaptic transmission, glutamatergic” was altered in both sexes.
Interestingly, we observed an increase of Thbs4 in female, but not in male DRG. Thrombospondin 4 is an endogenous ligand of Cavα2δ1 and is implicated in central sensitization . Moreover, Cavα2δ1 is also the target protein for pregabalin and gabapentin, both of which are first-line drugs for NP . As a result of pregabalin or gabapentin binding to this receptor subunit, the calcium channel does not locate to the cell membrane, leading to inhibition of the release of presynaptic glutamate [76, 77]. As Thbs4 antibodies have been shown to exert antihyperalgesic effects, upregulation of Thbs4 in females might explain some of the observed increased mechanical allodynia in females. Prior studies have reported varying changes in expression of Thbs4, with both upregulation and downregulation being described in males, depending on the method used. Importantly, females have not been studied before [78–80]. We are not aware of any studies on gender differences in treating NP with pregabalin or gabapentin in humans.
The gene Chrna4, coding for the neuronal acetylcholine receptor subunit alpha-4 (nAChRα4), displayed significant downregulation in DRG in males only. Previously, CCI of the infraorbital nerve in rats showed greater mechanical allodynia and increased expression of Chrna4 in females . With an overall sex-dependent change in the same direction, and the fact that nicotinic receptors (of which nAChRα4 is one) mediate fast depolarisations at synapses , upregulation of Chrna4 might sensitize neurotransmission in a sex-specific manner.
The gene Hrh3, coding for the presynaptic inhibitory histamine 3 autoreceptor (H3 receptor) (listed in the PainNetworks database) shows dramatic downregulation exclusively in females. The evidence on the role of H3 receptors in NP is, however, conflicting. Most studies seem to suggest that H3 receptor antagonists and inverse agonists mediate antinociception [74, 82], so this finding might not directly explain the observed sex difference in mechanical allodynia. However, some studies have shown H3 agonists to have antinociceptive effects  and therefore, as one of the few PainNetworks genes displaying downregulation only in females, Hrh3 is of future interest.
The gene Pdyn, coding for prodynorphin, showed a male versus female sex FC ratio of 1.44, the most substantial change in expression amongst the PainNetworks genes in the SC. Prodynorphin is the precursor to dynorphin  and dynorphin-containing interneurons have been shown to inhibit excitatory somatostatin-containing interneurons in SC . These somatostatin-containing neurons have been suggested to mediate Aβ-fibre-mediated sensitization of projection neurons . In our study, both sexes displayed an increased expression of Pdyn in SC, although the increase was larger in males. Greater upregulation of prodynorphin in males would suggest stronger inhibition of projection neurons which could partly explain the observed behavioural finding.
Both functional and structural plasticity may be important for the development of NP [85, 86]. Although the exact mechanism of maladaptive structural remodelling and reorganization is not known, various genes related to apoptosis and neuronal remodelling may play an essential role in the formation of aberrant connections in the SC. C1q is primarily linked to complement cascade but may affect synaptic pruning . Expression of C1q subunits was upregulated at the spinal level in both males and females, supporting the idea of a role for C1q in SNI-induced plasticity/remodelling. The previously discussed thrombospondin 4 also promotes excitatory synaptogenesis and may therefore affect plasticity, especially in females. This in turn might explain the larger mechanical allodynia in these [73, 88]. In addition, the expression of many genes related to the biological process “Neuron apoptotic process” were found to be differentially expressed in SC tissue: Reg3b, Ccl2 and Grn (progranulin) were upregulated only in males, Coro1a only in females, and Cx3cr1 (the gene for CX3CR1, also known as fractalkine receptor), Ctsz and Tyrobp in both males and females. The gene Atf3, coding for the transcription factor ATF3, is regarded as a marker of neuronal injury, but also of neuronal regeneration [89, 90]. Previous studies have also found upregulation of Atf3 in both DRG and SC in males . The effect of male-only upregulation of Atf3 and Reg3b in SC on behavioural sex difference remains to be studied.
Genes displaying a sexually dimorphic response in the DRG were more plentiful and changes were larger than in the SC. The gene Mcpt1i1, displayed the largest change in females compared with males and the gene Trpv6 showed the largest male vs. female change in ratio in the DRG.
The gene Trpv6, encoding TRPV6 (a highly-selective Ca2+ channel), is expressed in epithelial cells [92, 93] but expression in neural tissue has also been reported . TRPV6 has been studied as a tumour marker and target  but its role in nociception and NP remains unknown.
The gene Mcpt1i1, coding for mast cell protease 1-like 1 displayed the largest female versus male upregulation in DRG. This enzyme and its functions are poorly understood and they have not been linked to NP. However, it is worth considering whether it could have a similar function to mast cell protease 1, which converts angiotensin 1 into angiotensin 2 . Angiotensin 2 and the angiotensin 2 receptor in DRG have been linked to NP. If the protein product of Mcpt1l1 had a similar function, it could explain some of the observed sex difference and would suggest a sex-specific efficacy for AT2-receptor blockers, which have been studied as drugs for NP [97, 98].
The gene Bhlhe22 displayed the largest change of all PainNetworks genes. It also showed a sexually dimorphic response in the DRG, with a 5-fold increase in males. The absence of its transcription factor (Bhlb5) causes loss of DH interneurons which normally inhibit itch modulated by dynorphin [99, 100]. However, the possible role of Bhlhe22 in the modulation of nociception/sensory systems at the DRG level remains to be studied.