Sex differences in the inflammatory response of the mouse DRG and its connection to pain in Multiple Sclerosis 

Sex differences in the inflammatory response of the mouse DRG and its connection to pain in Multiple Sclerosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Sex differences in the inflammatory response of the mouse DRG and its connection to pain in Multiple Sclerosis Aislinn D Maguire, Timothy N Friedman, Dania N Villarreal Andrade, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1800909/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Multiple Sclerosis (MS) is an autoimmune disease with notable sex differences. Women are not only more likely to develop MS but are also more likely than men to experience neuropathic pain in the disease. It has been postulated that neuropathic pain in MS can originate in the peripheral nervous system at the level of the dorsal root ganglia (DRG), which houses primary pain sensing neurons (nociceptors). These nociceptors become hyperexcitable in response to inflammation, leading to peripheral sensitization and eventually central sensitization, which maintains pain long-term. The mouse model experimental autoimmune encephalomyelitis (EAE) is a good model for human MS as it replicates classic MS symptoms including pain. Using EAE mice as well as primary mouse DRG neurons cultured in vitro , we sought to characterize the sex differences specifically in peripheral sensory neurons which may underlie the disparities in MS pain. We found sex differences in the inflammatory profile of the EAE DRG, and in the TNFα signaling pathways activated intracellularly in cultured nociceptors. Given that TNFα signaling has been shown to impact on mitochondrial function, this led us to investigate sex differences in the mitochondria’s response to TNFα. Our results demonstrate that male sensory neurons are more sensitive to mitochondrial stress, making them prone to neuronal injury. In contrast, female sensory neurons appear to be more resistant to mitochondrial stress and exhibit an inflammatory and regenerative phenotype that may underlie greater nociceptor hyperexcitability and pain. Understanding these sex differences at the level of the primary sensory neuron is an important first step in our eventual goal of developing sex-specific treatments to halt pain development in the periphery before central sensitization is established. Multiple Sclerosis Pain Experimental Autoimmune Encephalomyelitis Sex Differences Tumour Necrosis Factor α Mitochondria Neuropathy Plasticity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Multiple sclerosis (MS) is an autoimmune disease characterized by widespread inflammation, immune cell activation, and demyelinating lesions of the central nervous system (CNS) [ 1 ]. The symptoms of MS are extensive, affecting sensory, motor, and cognitive functions [ 2 , 3 ]. One of the most debilitating symptoms, which affects over half of patients, is pain [ 4 ]. Adding another level of complexity to this condition, women are more likely than men to experience pain in MS [ 4 – 6 ]. Neuropathic pain is caused by injury or disease of the nervous system, producing pain in response to non-noxious stimuli (allodynia), increased pain in response to noxious stimuli (hyperalgesia), and spontaneous pain which occurs without a stimulus. Neuropathic pain generally originates in the peripheral nervous system (PNS) within the dorsal root ganglia (DRG), which house the cell bodies of primary pain-sensing neurons called nociceptors [ 7 , 8 ]. The hypothesis for MS pain, developed in animal research, is that inflammation and immune cell activation in the DRG cause nociceptors to become hyperexcitable, meaning they respond more readily and intensely to painful stimuli [ 9 , 10 ]. Peripheral sensitization of nociceptors triggers similar mechanisms in the spinal cord and brain, leading to central sensitization which maintains pain persistently, regardless of disease progression [ 7 , 8 ]. This may help explain why conventional pain treatments are ineffective in the treatment of neuropathic pain in MS [ 4 , 11 ]. To further investigate the mechanisms of peripheral sensitization and potential sex differences in MS, we used the animal model, experimental autoimmune encephalomyelitis (EAE) [ 12 , 13 ]. Not only does EAE replicate the inflammation and demyelination found in MS, but most importantly for our purposes, EAE animals exhibit classic signs of neuropathic pain [ 14 – 17 ]. We also used a simplified cell culture model to specifically study primary DRG neurons from naïve mice exposed to the inflammatory cytokine TNFα. We chose TNFα as our stimulus because it is upregulated both peripherally and centrally in MS and EAE and is involved in the development of many other neuropathic pain conditions [ 18 – 20 ]. Past work from our lab has also shown upregulation of TNFα from circulating immune cells in EAE [ 21 ]. A major focus of this study was on mitochondrial responses to TNFα between the sexes. Mitochondrial dysfunction has been linked to neuropathic pain, and is known to be regulated by TNFα effectors, c-jun N-terminal kinase (JNK) and P38 mitogen-activated protein kinases (MAPKs), as well as nuclear factor κB (NFκB) [ 22 – 25 ]. In this study, we reveal sex differences in the inflammatory response to disease, TNFα signaling, mitochondrial function, neuronal injury, and plasticity in the DRG in vivo and in vitro. These differences suggest that males and females engage distinct, sex-specific pathways at the level of the DRG that may have important implications when considering strategies to treat pain in the disease. 2. Materials And Methods 2.1 Experimental Autoimmune Encephalomyelitis As previously described [ 26 ], we induced EAE in both male and female C57BL/6 mice (8–10 weeks old, Charles River) by subcutaneous injection of 50µg of myelin oligodendrocyte glycoprotein (MOG 35 − 55 ) emulsified in complete Freund’s adjuvant (CFA, 1.5mg/mL). On the same day as induction, and 48h later, mice were also inoculated with 300ng of pertussis toxin. Animals were euthanized and perfused with cold saline on the first day of EAE symptom onset alongside their “CFA controls”, which received only the adjuvants, CFA and Pertussis, but not MOG 35 − 55 . Tissue was dissected and snap frozen in liquid nitrogen, then stored at -80°C. All animal experiments were performed according to the Canadian Council on Animal Care's Guidelines and Policies with approval from the University of Alberta Health Sciences Animal Care and Use Committee (protocol 0000274). 2.2 Immunohistochemistry (IHC) Fresh frozen DRG tissue was cryo-sectioned at 10µm thickness. Tissue slides were then immersed in 4% paraformaldehyde (PFA) and fixed for 5 minutes at room temperature. Next, they were immersed in antigen retrieval solution (1.92g anhydrous citric acid and 0.5mL Tween20 in 1L H 2 O, pH 6.0) for 10 minutes before 3x 10-minute washes in PBS. Tissue was blocked for one hour at room temperature in 10% normal donkey serum (NDS) in 0.2% triton X-100 in PBS (PBS TX ). Primary antibody was incubated overnight at 4°C in 2% NGS and 2% bovine serum albumin (BSA) in PBS TX . Primary antibodies included CD45 (1:200, BD Pharmigen 550539), Iba1 (1:500 Cellular Dynamics 019-19741), CD3 (1:200, Bio-Rad MCA1477), cleaved caspase 3 (1:100, Cell Signaling #9661), ATF3 (1:200, Abcam ab207434), and pCREB (1:500, Cell Signaling #9198). Slides were washed the next day 3x 10 minutes in PBS before incubation with secondary antibodies (Jackson Immunoresearch) at a 1:200 dilution for 45 minutes. Slides were again washed 3x 10 minutes in PBS before mounting with fluoromount G™ Mounting Medium (Invitrogen 00-4958-02). Slides were imaged at 20x for analysis using a Zeiss Axio Observer Z1. Representative images were taken at 40x using a Leica TCS SPE Confocal. 2.3 Immunopanned Primary DRG Cultures Immunopanning protocol was adapted from Zuchero 2014 [ 27 ]. All DRGs were collected from 2–4 male and 2–4 female 8–10 week old C57BL/6 mice, and digested with 2mg/mL Stemxyme I (Worthington LS004106) for 1hr at 37°C. Cells were triturated gently and spun for 10 minutes at 300xg. They were next resuspended and strained through a 70µm filter then spun through a 3mL 15% BSA cushion at 300xg to remove myelin debris. After the BSA cushion, cells were allowed to rest for 30 minutes in a 10% CO 2 incubator at 37°C for antigen retrieval. Immunopanning dishes were coated with secondary anti-rat, rabbit, and mouse antibodies (1:500, Jackson 112-005-167, 111-005-003, and 115-005-020 respectively) overnight at 4°C, then washed and coated with rat CD45 (10µg, BD Pharmigen 550539), rabbit PDGFRβ (10µg, Abcam ab32570), and mouse O4 (1:2.5, O4 hybridoma [ 28 , 29 ]) primary antibodies for 2 hours at room temperature. Cells were panned in 5mL of panning buffer (0.02% BSA and 0.008% DNAse in D-PBS) in each of the 3 dishes for 20 minutes, shaking gently at 10 minutes. Cells were spun down, counted with a haemocytometer, and pre-plated for 30min in 25µL of panning buffer prior to flooding wells with media (1:1 DMEM:Neurobasal, supplemented with SATO, N-acetyl-cysteine, insulin, penicillin/streptomycin, B27+, glutamate, and sodium pyruvate). Cells for protein collection (ELISA and Western Blotting) were plated in 24 well clear plastic plates (Falcon 353047) at 2000 cells per well. Cells for imaging were plated in 24 or 96 well black glass-bottom plates (Cellvis P24-1.5H-N, and Falcon 353219 respectively) at 500 cells per well. Cells were allowed to rest 24h in culture before treatment with 1000pg/mL TNFα for 1, 6, or 24h respectively. 2.4 Western Blotting Protein lysates from cultured cells were scraped collected with RIPA lysis buffer (Thermofisher 89900) with cOmplete EDTA-free, (Roche 04693159001) and PhoSTOP (Roche 04906837001) added. Lysates were stored at -80°C. Prior to blotting, protein was precipitated with 4 volumes of cold acetone for one hour at 4°C, then centrifuged a 10 000xg for 10min before resuspension in a smaller volume. Lysates were then diluted with 4x Bio-Rad laemmli sample buffer and 50mM dithiothreitol and boiled for 10min at 95°C. Samples were loaded into 4–20% Mini-PROTEAN TGX Stain-free gels (Bio-Rad 4568096), and run at 120mV for 1-1.5 hours. After running, the total protein stain in the gels was activated using the Bio-Rad ChemiDoc XRS+. Gels were then transferred onto low-fluorescence PVDF membranes (Bio-Rad BioRad 1620264) using extra thick blot paper (Bio-Rad 1703965) on the Bio-Rad Trans-Blot Turbo system V1.02 for 30min at 25V and 1.0A. Total protein was then imaged with the ChemiDoc. Membranes were blocked for one hour at room temperature in 5% BSA dissolved in 0.5% TBS-T. Primary antibody was incubated overnight at 4°C in 1% BSA in TBS-T. Primary antibodies included phospho-P65 (1:500, Millipore MAB3026), P38 (1:500, Cell Signaling #8690S), phospho-P38 (1:500, Cell Signaling #4631S), JNK (1:500, Cell Signaling #9252S), and phospho-JNK (1:500, Cell Signaling #4668S). Membranes were washed 3x 10 minutes in TBS-T before incubation with secondary antibody, Goat anti-Rabbit HRP (1:10 000, Jackson Laboratories 111-035-144) for one hour at room temperature. Membranes were again washed 3x 10 minutes in TBS-T, then for 10min in TBS before imaging with the ChemiDoc after 5min incubation with ECL Prime (Amersham RPN2232). Membrane stripping was performed according to the mild stripping protocol from abcam. 2.5 Mitochondrial Live Staining For morphological characterization, live cells were stained with MitoTracker™ Deep Red FM at 1:10 000 for 30 minutes, and NucBlue™ Live ReadyProbes™ reagent at 1:200 for 10min. Cells were then fixed in 4% PFA for 15 minutes at room temperature and washed with PBS before imaging. Footprint analysis was performed with Fiji MiNA software from the Stuart lab at Brock University [ 30 ]. Morphology analysis was adapted from Leabeau et al. 2018 [ 31 ]. Mitochondrial superoxide was stained with MitoSOX™ red dye (Invitrogen M36008) at 1:1000 concentration and NucBlue™ Live ReadyProbe (1:200 Invitrogen R37605). Cells were imaged live in an environment controlled Molecular Devices ImageXpress Micro high content screening system. 2.6 Immunocytochemistry Cells were fixed in 4% PFA for 15 minutes at room temperature, then washed 3x with PBS and blocked in 10% NDS in PBS TX . Cleaved caspase 3 primary antibody (1:250, Cell Signaling #9661) was diluted in 2% NGS and 2% BSA in PBS TX at 4°C overnight. Cells were washed 3x in D-PBS before incubation with secondary antibody (1:500 goat ant-rabbit 488, Invitrogen A11008) and DAPI (1:2000, Invitrogen D1306) at room temperature for 30 minutes. Cells were imaged in a Molecular Devices ImageXpress Micro high content screening system. 2.7 Cytochrome C ELISA Cytochrome C ELISA was performed on cell lysates from immunopanned cultures described in section 2.3, which were scraped and collected in 0.5% PBS TX . The protocol for the kit (R&D systems MCTC0) was followed with no variations. 2.8 Data Analysis Statistical analyses were performed using GraphPad Prism 9, apart from Fig. 3 G, the statistics for which were performed by Dr. Karen Buro of MacEwan University using Fisher exact tests with Bonferroni’s multiple comparison. Otherwise, all data was expressed as fold change of sex-respective controls and analyzed by regular two-way ANOVA. Significance was set at P < 0.05. Immunohistochemistry data represents manually counted positive cells normalized to tissue area. P38 and JNK western blot data was calculated as the intensity of phosphorylated over total antibody staining, normalized to total protein. NFκB data was calculated as phosphorylated P65 antibody staining normalized to total protein due to lack of a sufficient total P65 antibody. Mitochondrial footprint data represents the total area of mitochondrial staining, normalized to cell diameter. 3. Results 3.1 Sex differences in EAE DRG inflammation Previous work from our lab has revealed inflammation in the DRGs of post-mortem female MS patients [ 32 ]. Given the prominent sex differences in neuropathic pain in MS [ 33 – 35 ], we sought to determine if there were underlying sex differences in the inflammatory signatures of mouse EAE DRGs. Using immunohistochemistry, we found significant increases in staining for the immune cell marker CD45 and T cell marker CD3 in both male and female EAE DRGs compared to their CFA controls (Fig. 1 A-F, F(1, 16) = 7.113, P = 0.0169 and F(1, 15) = 13.34, P = 0.0258 respectively, Two-way ANOVA), with no apparent sex differences (F(1, 16) = 0.2351, P = 0.6344 and F(1, 15) = 0.04575, P = 0.8335 respectively, Two-way ANOVA). We also observed an increase in Iba1 staining, indicative of an increased macrophage and monocyte presence, in both male and female EAE DRGS (Fig. 1 G-I, F(1, 15) = 53.62, P < 0.0001, Two-way ANOVA). However, there was more Iba1 staining in males than females (F(1, 15) = 12.65, P = 0.0029, Two-way ANOVA). Dectin-1 is a pattern-recognition receptor which facilitates cytokine production, phagocytosis, and respiratory burst (rapid release of reactive oxygen species) [ 36 ]. Given this link to inflammation, we investigated co-labelling of Dectin-1 and Iba1 in the mouse EAE DRG (Fig. 2 A-C). We found increased Dectin-1/ Iba1 co-labelled cells in EAE animals compared to their CFA controls (F(1, 14) = 16.45, P = 0.0012), with a significant sex difference as male EAE animals demonstrated more co-labelling than females (F(1, 14) = 11.62, P = 0.0042). This suggests that not only is there an increased macrophage/ monocyte presence in the male EAE DRG (Fig. 1 G-I), but these cells may have a different inflammatory phenotype than females. Over all these results reveal sex differences in the DRG of EAE animals, which may contribute to sex differences in pain. 3.2 Sex differences in TNFα signaling in vitro TNFα is a pleiotropic cytokine that has been linked to various neuropathic pain conditions and is present both peripherally and centrally in MS [ 19 , 37 ]. Our lab has shown previously that TNFα is increased peripherally in circulating immune cells in EAE [ 21 ]. We investigated TNFα levels in the male and female EAE DRG by polymerase chain reaction (PCR) using previously described methods [ 38 ], and found TNFα mRNA is elevated in both sexes in EAE ( Supplementary Fig. 1 ). TNFα is secreted by macrophages, monocytes, and lymphocytes, and we know that there is an increased presence of these inflammatory cell types in the DRG with EAE [ 39 ]. For these reasons, we selected TNFα as an in vitro stimulus to understand how it might contribute to peripheral sensitization of sensory neurons in the EAE model. We immunopanned male and female DRGs to reduce the contribution of non-neuronal cells in the culture, then treated them with TNFα (1ng/mL) for 1, 6, or 24 hours. Cells were collected and western blotted for TNFα signaling effectors, JNK, P38, and NFκB to look for sex differences in the TNFα signalling pathway (Fig. 3 A). There was no sex difference evident in JNK activation with TNFα treatment (Fig. 3 B, F(1, 20) = 0.01980, P = 0.8895, Two-way ANOVA). However, male sensory neurons exhibited greater P38 activation (Fig. 3 C, F(1, 19) = 4.900 P = 0.0393, Two-way ANOVA). In contrast, female sensory neurons had greater NFκB activation (Fig. 3 D, F(1, 19) = 18.55, P = 0.0004, Two-way ANOVA). 3.3 Mitochondrial morphology is affected by TNFα treatment We determined that male sensory neurons preferentially activate P38 while female neurons preferentially engage the NFκB pathway in response to TNFα treatment in vitro (Fig. 3 ). Since TNFα signaling through both NFκB and P38 has been linked to mitochondrial changes [ 22 – 25 ], we next sought to characterize whether exposure to TNFα leads to changes in mitochondrial morphology in male and female nociceptors (DRG neurons ≤ 30µm in diameter). The mitochondrial footprint in these cells was significantly increased with 24h of TNFα treatment compared to control, (Fig. 4 A-C, F(1,78) = 3.648, P = 0.0598, Two-way ANOVA). Specifically, there was a greater increase in the footprint from females when compared to their respective controls (P = 0.0197 and 0.9757 respectively, Sidak’s multiple comparisons test). This result prompted us to further investigate the specific morphological changes of these mitochondrial networks in response to TNFα treatment (Fig. 4 D-G). In male neurons, we observed a change in mitochondrial network morphology after 24 hours of TNFα treatment compared to control, wherein the proportion of fragmented morphology decreased and the tubular morphology increased (P = 0.00062 and 0.0031 respectively, Fisher exact test, Bonferroni’s multiple comparison). However, female neurons exhibited changes in mitochondrial morphology at all three timepoints. At one hour of TNFα treatment, fragmented morphology increased, and tubular morphology decreased compared to control (P = 2.2e-16 for both morphologies, Fisher exact test, Bonferroni’s multiple comparison). At 6 hours and 24 hours of TNFα treatment there was a shift in how the females responded to TNFα treatment. Like the male 24-hour treatment, female neurons exhibited decreased fragmented morphology (P = 0.00012 and 3.3e-11 respectively), and increased tubular morphology compared to control (P = 0.000027 and 8.2e-10 respectively, Fisher exact test, Bonferroni’s multiple comparison) at both 6 and 24 hours of TNFα treatment. Taken together, the increased mitochondrial footprint in female neurons, along with a concomitant increase in tubular morphology at 6 and 24 hours of TNFα treatment, may reflect a protective mechanism engaged by sensory neurons undergoing persistent inflammation, which appears to be more effective in females than in males. 3.5 Mitochondrial function is altered in male sensory neurons with TNFα treatment To determine if adaptive mitochondrial changes are more effective in females than males, we next explored mitochondrial superoxide levels with Mitosox live cell staining (Fig. 5 A-C). Male cells produced significantly more superoxide in response to TNFα than female cells (F(1,42) = 16.40, P = 0.0002, Two-way ANOVA). To further understand the extent of male mitochondrial dysfunction we assessed levels of cytochrome C release from mitochondria in cell lysates by ELISA (Fig. 5 D). Male cells released significantly more cytochrome C than female cells (F(1,48)-13.63, P = 0.0006, Two-way ANOVA). While both P38 and NFκB are linked to mitochondrial changes, P38 in particular can mediate intrinsic apoptosis through cytochrome c release [ 40 ]. These results suggest that male DRG neurons are more susceptible to mitochondrial damage in response to TNFα-mediated inflammation, potentially due to their preferential activation of P38. 3.6 Male DRGs are more susceptible to neuronal damage in vitro and in vivo Cytochrome C release is a marker of intrinsic apoptosis, one effector of which is caspase 3 [ 41 ]. Since we found increased P38 activation and cytochrome C release in male sensory neurons, we sought to determine whether this led to increased caspase 3 cleavage. Using immunocytochemistry to asses our TNFα-treated sensory neurons, we found there was significantly more caspase 3 cleavage in males than females (Fig. 6 A-C, F(1,42) = 15.29, P = 0.0003, Two-way ANOVA). To validate our in vitro model, we next determined if this result occurred in vivo in EAE. In DRG sections from mice with EAE, there was indeed greater caspase 3 cleavage in males than in females (Fig. 6 D-F, F(1, 15) = 14.97, P = 0.0015, Two-way ANOVA). These results suggest that male DRG sensory neuron activation of p38 in response to TNFα may trigger structural and functional mitochondrial changes, which result in increased neuronal damage through caspase 3. 3.7 Plasticity markers are increased in female EAE DRGs To gain an understanding of what adaptive mechanisms may be present in female EAE DRGs to protect them from mitochondrial damage and neuronal injury we first investigated activating transcription factor 3 (ATF3), an injury and plasticity-associated factor, by IHC (Fig. 7 A-C). There was a significant increase in ATF3 staining in EAE DRGs compared to their CFA controls (F(1, 14) = 17.51, P = 0.0011, Two-way ANOVA, Sidak’s multiple comparisons test), with a significantly greater increase in females than males (F(1, 14) = 6.060, P = 0.0274, Two-way ANOVA). Finally, we stained EAE DRGs for phosphorylated cAMP response element-binding protein (pCREB). CREB is a transcription factor strongly linked to neuroplasticity and pro-survival gene transcription as well as neuropathic pain [ 42 , 43 ]. We found a significant increase in pCREB staining in EAE DRGs compared to their CFA controls, normalized to naïve mice (Fig. 8 A-C, F(1, 16) = 7.3, P = 0.0155, Two-way ANOVA), with a strong sex difference (F(1, 16) = 7.9, P = 0.0124, Two-Way ANOVA) where EAE females in particular demonstrated increased pCREB staining (P = 0.0135, Sidak’s multiple comparisons test). This female-dominated increase in ATF3 and pCREB staining in the EAE DRG suggests that females may have a stronger plastic and/or regenerative response to inflammation, which could explain their protection from mitochondrial dysfunction and neuronal damage, as well as contribute to their elevated hyperexcitability. 4. Discussion Our results reveal sex differences in the DRG in both the EAE mouse model of MS, and our TNFα-treated culture model. Both male and female EAE DRGs exhibit increased staining for inflammatory markers, but notably, males have greater macrophage/ monocyte staining than females at the onset of EAE. In addition, male macrophages and monocytes have more Dectin-1 staining in male EAE mice, suggestive of greater inflammatory activation. Using an in vitro preparation of DRG neurons stimulated with the inflammatory cytokine TNFα, we observed that males preferentially activated P38 MAPK, and presented with disrupted mitochondrial morphology, elevated mitochondrial superoxide production, and cytochrome c release. This mitochondrial pathology is likely responsible for the increased caspase 3 cleavage we observed in the male DRG both in vitro and in vivo . Females, however, preferentially activated the transcription factor NFκB rather than P38, and while their mitochondrial morphology was also altered, they did not elevate superoxide production, cytochrome c release, or caspase 3 cleavage. Rather, we show that female EAE DRGs have increased levels of the injury and plasticity-associated factors, ATF3 and pCREB in vivo . These observations have significant implications for our understanding of neuropathy and pain in chronic neuroinflammatory disorders such as MS. First, we found a significant increase in global inflammation and immune cell infiltration indicated by CD45, CD3, and Iba1 staining at the level of the DRG in the EAE model in both sexes. Many studies of neuropathic pain using peripheral nerve injury models have focused on the spinal cord and report that females have a T-cell-mediated pain phenotype [ 44 , 45 ]. This, however, may not be true for the onset of peripheral sensitization in the EAE DRG, as we saw no sex differences in CD3 T cell infiltration between the sexes. However, the indication that microglia/macrophages are responsible for pain in the male spinal cord [ 44 – 47 ] may be analogous to the EAE DRG, as evidenced by our finding that more cells were stained for Iba1 in males, and that a greater number of these Iba1-positive cells were co-labelled with the inflammatory marker Dectin-1. DRG macrophages have already been identified as important players in peripheral sensitization and neuropathic pain in a multitude of studies [ 48 ]. For example, their secretion of pro-inflammatory mediators, including TNFα, can directly increase sensory neuron excitability [ 49 – 51 ]. We found increased TNFα mRNA in both male and female EAE DRGs, and our lab has previously confirmed that TNFα is increased peripherally in EAE [ 21 ]. This motivated us to use TNFα as our in vitro stimulus to further elucidate sex differences in intracellular signaling in DRG neurons. To determine if the inflammatory profile we identified in EAE DRGs has consequences on intracellular neuronal processes, we moved in vitro , where we observed sex differences in the downstream signaling pathways engaged by TNFα stimulation. Our finding that male neurons are biased towards activation of P38 in response to TNFα is supported by studies which have shown that males activate P38 in the spinal cord in mouse models of neuropathic pain [ 52 , 53 ]. Additionally, we observed that female neurons preferentially activate NFκB with TNFα stimulation. This aligns with reports indicating that females preferentially activate NFκB in endothelial cells and inflamed lung tissue [ 54 , 55 ]. Given the strong links of both P38 and NFκB to functional changes in the mitochondria [ 22 – 25 ], we went on to conduct a surface-level investigation of the changes that occur in mitochondrial morphology in response to TNFα. We found that female neurons increased their mitochondrial footprint in response to 24 hours of TNFα treatment, and that their mitochondrial morphology responded more quickly to treatment than males. We observed more fragmented mitochondria, and less tubular mitochondria in females after one hour of TNFα treatment compared to control, which may indicate a maladaptive phenotype such as impaired mitochondrial fission. However, at 6 and 24 hours of TNFα treatment, female mitochondrial networks appeared to shift their response to TNFα treatment, as they exhibited less fragmented mitochondria and more tubular mitochondria compared to control. Male neurons also exhibited less fragmentation and more tubular networks at 24 hours of treatment but showed no significant changes at earlier timepoints. We hypothesize that the decreased fragmentation and increased tubular mitochondrial networks we observed after TNFα treatment may be evidence of an adaptive response to inflammation, which appears be more effective in females than in males. To further investigate this mechanism, we turned to functional readouts of mitochondrial health in our TNFα-stimulated culture, superoxide production and cytochrome C release. We found that male DRG neurons produce more superoxide and release more cytochrome C than females in response to TNFα. This led us to speculate that DRG neurons initiate an adaptive response to inflammation that is more effective in females than in males, potentially due to their differential activation of the TNFα signaling pathways. The dramatic increase in cytochrome C release from male neurons lead us to investigate caspase 3 cleavage. We found that both in vitro and in vivo , male DRGs exhibit more cleaved caspase 3 staining than females. This finding aligns with TNFα signaling literature as P38 can cause cytochrome C release and eventually, intrinsic apoptosis through B-cell lymphoma (Bcl) protein regulation [ 40 ]. In addition, previous work from our lab has investigated sex differences in the mouse EAE spinal cord and found significantly greater increases in the levels of amyloid precursor protein (APP) and non-phosphorylated neurofilament (SMI32) in males, suggesting that male spinal neurons are more sensitive to axonal injury and degeneration in EAE [ 56 ]. These results support our interpretation that male EAE DRG neurons are more prone to neuronal injury than females. Neuronal injury in males may also explain previous work from our lab which has shown that while female EAE DRG neurons are hyperexcitable, males are not [ 21 ]. We hypothesize that while female pain is driven by hyper-excitability in the periphery, this is not the source of pain in males. Instead, male pain in EAE may be mediated by a central mechanism. Finally, we examined mouse EAE DRGs for the expression of the injury and plasticity-associated proteins ATF3 and pCREB. Females had significantly more ATF3-positive cells than males, suggesting that females engage an adaptive mechanism in response to inflammation. NFκB is known to induce ATF3 signaling, acting in a negative feedback loop to dampen its own pro-inflammatory signaling, as ATF3 can bind and inhibit the P65 subunit of NFκB [ 57 – 59 ]. There was also significantly more pCREB staining in the female EAE DRG than in males, which provides further evidence of a regenerative phenotype. Much like ATF3, CREB can directly inhibit NFκB and pro-inflammatory signaling [ 60 ]. It has also been linked to aberrant plasticity, neuronal hyperexcitability, and pain in spinal cord injury [ 42 , 43 ]. While activation of these transcription factors in the female EAE DRG may protect neurons from inflammation, mitochondrial pathology, and neuropathy, they may also be responsible for neuronal hyperexcitability and pain via maladaptive plasticity. Thus, we propose that a regenerative mechanism is induced by preferential NFκB signaling in the female EAE DRG (Fig. 9 .1 ). While this mechanism prevents neuropathy, it may be either insufficient to prevent neuronal hyperexcitability and pain or may even contribute to pain through aberrant plasticity [ 61 , 62 ]. Males, however, preferentially activate P38, which leads to mitochondrial dysfunction and caspase 3 cleavage (Fig. 9 .2 ) This pathway may cause male DRGs to become more prone to neuropathy. These studies reveal that there are distinct pathological changes that occur at the level of the DRG between the sexes. There is a need for future therapeutic strategies to treat neuropathic pain to be tailored in a sex specific manner. Our findings suggest that targeting processes of axonal degeneration and neuropathy will be more efficacious in males while modulating inflammation and aberrant plasticity will be more beneficial in females. Declarations Ethics Approval and Consent to Participate All animal experiments were performed according to the Canadian Council on Animal Care's Guidelines and Policies with approval from the University of Alberta Health Sciences Animal Care and Use Committee (protocol 0000274). Consent for Publication Not applicable. Availability of Data and Materials Applicable data and materials available upon reasonable request. Competing Interests The authors declare that they have no competing interests. Funding This work was supported by a Project Grant from the Canadian Institutes of Health Research (FRN-162434) and a Discovery Grant from the MS Society of Canada (EGID-3761). Author Contributions D.M Wrote and edited the manuscript prepared the figures (including final analysis and graphing) Prepared and stained all primary cell cultures and performed western blots, ELISAs, and immunocytochemistry Performed and analyzed immunohistochemistry (IHC) for figures 6 and 7 N.F assisted with dissections for all EAE tissue and primary cultures V.A performed PCRs for supplementary figure 1 H imaged and analyzed cells for figure 4 D. performed and analyzed IHC for figures 1&2 P performed and analyzed IHC for figure 8 T. sliced tissue for all IHC and edited figures using adobe illustrator B performed statistical analysis for figure 4 and advised on statistical reporting for all figures R.P shared equipment and expertise, and reviewed the manuscript J.K is the principal investigator (supervised all the above activities and reviewed/ edited the manuscript) Acknowledgments The authors would like to thank Dr. Sun and the Cross Cancer Institute for training and use of the ImageXpress system. 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Catuneanu A, Paylor JW, Winship I, Colbourne F, Kerr BJ: Sex differences in central nervous system plasticity and pain in experimental autoimmune encephalomyelitis. Pain 2019, 160: 1037-1049. Ku HC, Cheng CF: Master Regulator Activating Transcription Factor 3 (ATF3) in Metabolic Homeostasis and Cancer. Front Endocrinol (Lausanne) 2020, 11: 556. Park SH, Do KH, Choi HJ, Kim J, Kim KH, Park J, Oh CG, Moon Y: Novel regulatory action of ribosomal inactivation on epithelial Nod2-linked proinflammatory signals in two convergent ATF3-associated pathways. J Immunol 2013, 191: 5170-5181. Kwon JW, Kwon HK, Shin HJ, Choi YM, Anwar MA, Choi S: Activating transcription factor 3 represses inflammatory responses by binding to the p65 subunit of NF-kappaB. Sci Rep 2015, 5: 14470. Wen AY, Sakamoto KM, Miller LS: The role of the transcription factor CREB in immune function. J Immunol 2010, 185: 6413-6419. Hirai T, Mulpuri Y, Cheng Y, Xia Z, Li W, Ruangsri S, Spigelman I, Nishimura I: Aberrant plasticity of peripheral sensory axons in a painful neuropathy. Sci Rep 2017, 7: 3407. Thapa T, Graven-Nielsen T, Schabrun SM: Aberrant plasticity in musculoskeletal pain: a failure of homeostatic control? Exp Brain Res 2021, 239: 1317-1326. Additional Declarations No competing interests reported. Supplementary Files S1.tiff Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1800909","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":117457998,"identity":"2aa2bf25-d51a-4841-94e6-d6715f02f31e","order_by":0,"name":"Aislinn D Maguire","email":"","orcid":"","institution":"University of Alberta","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Aislinn","middleName":"D","lastName":"Maguire","suffix":""},{"id":117457999,"identity":"7750c1cc-c527-44e5-9ced-439639179c0d","order_by":1,"name":"Timothy N Friedman","email":"","orcid":"","institution":"University of Alberta","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Timothy","middleName":"N","lastName":"Friedman","suffix":""},{"id":117458000,"identity":"ba429d02-ef13-4146-a25a-a78edacc4bd9","order_by":2,"name":"Dania N Villarreal Andrade","email":"","orcid":"","institution":"University of Alberta","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Dania","middleName":"N Villarreal","lastName":"Andrade","suffix":""},{"id":117458001,"identity":"fbc949cc-7d27-4064-a0bf-88b091d85856","order_by":3,"name":"Fajr Haq","email":"","orcid":"","institution":"University of Alberta","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Fajr","middleName":"","lastName":"Haq","suffix":""},{"id":117458003,"identity":"7f72f550-78fb-4e08-ae6c-d85185a1fd63","order_by":4,"name":"Jacob Dunn","email":"","orcid":"","institution":"University of Alberta","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jacob","middleName":"","lastName":"Dunn","suffix":""},{"id":117458005,"identity":"781a2dc4-6a65-4936-8f4b-e24d9ee15983","order_by":5,"name":"Keiana Pfeifle","email":"","orcid":"","institution":"University of Alberta","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Keiana","middleName":"","lastName":"Pfeifle","suffix":""},{"id":117458009,"identity":"aa32bd6e-8484-4409-9818-ca777575e32c","order_by":6,"name":"Gustavo Tenorio","email":"","orcid":"","institution":"University of Alberta","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Gustavo","middleName":"","lastName":"Tenorio","suffix":""},{"id":117458010,"identity":"9de8b1db-6051-4c58-a1b8-fb3f83db6360","order_by":7,"name":"Karen Buro","email":"","orcid":"","institution":"MacEwan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Karen","middleName":"","lastName":"Buro","suffix":""},{"id":117458011,"identity":"ba867cc0-2df3-430d-8864-82416d009bae","order_by":8,"name":"Jason R Plemel","email":"","orcid":"","institution":"University of Alberta","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jason","middleName":"R","lastName":"Plemel","suffix":""},{"id":117458012,"identity":"c7f08e67-4987-4731-8810-af50f45c3b25","order_by":9,"name":"Bradley J Kerr","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA20lEQVRIiWNgGAWjYBACA4ZjIMqGgYGdhzQtaQwMzCAtCURpYQNRh0nQYs54LPFx5Z7zif3NvAcfV/6wY+BvP4Bfi2XDscOGZ57dTpxxmC/Z8ExCMoPEGQJWGRw43ibZcOB2YsNhHjPJhgRmoFMJa2n/2XDgXOL8wzzmPxsS6hkM+B8Q0nLsGGPDgQOJG4C2MDYkHGYwkCBgC9AvyUCHJRtvBPpFsiHtOI/EDQK2mEscM/zYcMBOdt7x3oMfG2yq5fj7CdjCIHEAlU9EGuBvIKxmFIyCUTAKRjgAALhGSTEap1SMAAAAAElFTkSuQmCC","orcid":"","institution":"University of Alberta","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Bradley","middleName":"J","lastName":"Kerr","suffix":""}],"badges":[],"createdAt":"2022-06-27 18:14:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1800909/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1800909/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":23544599,"identity":"294639ad-d2e3-41e6-90c0-9e54e578095b","added_by":"auto","created_at":"2022-07-06 17:18:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1962477,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eInflammatory signature of mouse EAE DRGs in vivo.\u003c/em\u003e \u003c/strong\u003eLumbar DRG tissue was collected from male and female EAE mice at disease onset, as well as from their respective control mice (referred to here as CFA). CD45 (A-C), CD3 (D-F), and Iba1 (G-I) staining increases in both sexes at EAE onset compared to CFA controls, however, Iba1 staining exhibits a greater increase in male DRGs than females. Bars indicate mean ± standard error mean (SEM). #p\u0026lt;0.05, ##p\u0026lt;0.01, ####p\u0026lt;0.0001 treatment factor, ** p\u0026lt;0.01 sex factor. Two-way ANOVA.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure1..png","url":"https://assets-eu.researchsquare.com/files/rs-1800909/v1/6d447d15b782da1bcc8f8a02.png"},{"id":23544596,"identity":"c9c51bef-6e28-42c6-97b5-afcfd5faead1","added_by":"auto","created_at":"2022-07-06 17:18:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":348241,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDectin-1 staining in Iba1-positive cells increases in male EAE DRGs in vivo.\u003c/em\u003e \u003c/strong\u003eMale DRGs exhibit increased Dectin-1 and Iba1 co-labelling in EAE DRGs compared to females (A-C). Bars indicate mean ± standard error mean (SEM). ###p\u0026lt;0.001 treatment factor, **p\u0026lt;0.01 sex factor, two-way ANOVA.\u003c/p\u003e","description":"","filename":"Figure2..png","url":"https://assets-eu.researchsquare.com/files/rs-1800909/v1/db965a0104f8f863a8cb9f4e.png"},{"id":23544597,"identity":"e114cd8b-ab78-43f2-9314-7d73de89006b","added_by":"auto","created_at":"2022-07-06 17:18:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":191553,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSex specific activation of the TNFα signaling pathway in vitro. \u003c/em\u003e\u003c/strong\u003e(A) DRGs were harvested from male and female mice, immunopanned to enrich the proportion neuronal cells, treated for 1, 6, or 24 hours with 1ng/mL TNFα, then collected for western blotting. (B) There were no sex differences evident in JNK phosphorylation. (C) Male cells have greater P38 phosphorylation (D) Female cells have greater NFkB (P65 subunit) phosphorylation. Bars indicate mean ± standard error mean (SEM). *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, sex factor, two-way ANOVA.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure3..png","url":"https://assets-eu.researchsquare.com/files/rs-1800909/v1/cbc19491adb80ba5ca5367a9.png"},{"id":23545155,"identity":"34b986a5-de51-4705-a320-ff98cab5c0a6","added_by":"auto","created_at":"2022-07-06 17:28:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":455218,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMitochondrial morphology changes in response to TNFα treatment in vitro.\u003c/em\u003e\u003c/strong\u003e (A-C) Female cells increased their mitochondrial footprint compared to males with 24 hours of TNFα treatment, determined using Fiji MiNA software. (D-F) Cells were manually scored into three categories of mitochondrial morphology; elongated, tubular, and fragmented. Bars indicate mean ± standard error mean (SEM). #p\u0026lt;0.05, two-way ANOVA, Sidak’s multiple comaprisons test. (E) The morphology breakdown changed in males with 24 hours of TNFα treatment, and in females with 1,6, and 24 hours of treatment. Bars indicate mean fraction of cells ± standard error mean (SEM). #p\u0026lt;0.0083, Fisher exact test, Bonferroni’s multiple comparison.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure4..png","url":"https://assets-eu.researchsquare.com/files/rs-1800909/v1/c5073cfe3974e22e2d02046f.png"},{"id":23544602,"identity":"fd917145-70bf-43ff-a094-07753741f837","added_by":"auto","created_at":"2022-07-06 17:18:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1169566,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunctional alterations to mitochondria in male cells with TNFα treatment in vitro.\u003c/em\u003e\u003c/strong\u003e (A-C) Male DRGs increased live cell mitochondrial superoxide staining compared to females with TNFα treatment. (D) Male DRGs increased cytochrome C release compared to females, measured by ELISA. Bars indicate mean ± standard error mean (SEM). ***p\u0026lt;0.001, sex factor, two-way ANOVA.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure5..png","url":"https://assets-eu.researchsquare.com/files/rs-1800909/v1/369aeb2791128f73f8e5f0e3.png"},{"id":23545156,"identity":"732fe757-9ce0-421e-b8be-5a5c6ad9d7fe","added_by":"auto","created_at":"2022-07-06 17:28:41","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":963625,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCaspase 3 cleavage increases in male DRGs both in vitro and in vivo\u003c/em\u003e.\u003c/strong\u003e (A-C) Cleaved caspase 3 staining is increased in cultured male DRGs compared to females with TNFα treatment. (D-F) Cleaved caspase 3 staining is increased in male EAE DRGs compared to females. Bars indicate mean ± standard error mean (SEM). ###p\u0026lt;0.001 treatment factor, **p\u0026lt;0.01, ***p\u0026lt;0.001, sex factor, two-way ANOVA.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure6..png","url":"https://assets-eu.researchsquare.com/files/rs-1800909/v1/ffac4d1e566f9c6f79a49a1e.png"},{"id":23544604,"identity":"78e8d41e-cb69-49f3-832e-83c4d793789e","added_by":"auto","created_at":"2022-07-06 17:18:41","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":533289,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eATF3 staining increases in female EAE DRGs in vivo.\u003c/em\u003e\u003c/strong\u003e (A-C) ATF3 staining is increased in female EAE DRGs compared to males. Bars indicate mean ± standard error mean (SEM). ##p\u0026lt;0.01 treatment factor, *p\u0026lt;0.05 sex factor, two-way ANOVA.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure7..png","url":"https://assets-eu.researchsquare.com/files/rs-1800909/v1/f0a55ee1ef756f133a82a4ac.png"},{"id":23544605,"identity":"090d90b8-ef60-4e50-8572-294d619f3f29","added_by":"auto","created_at":"2022-07-06 17:18:41","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":484500,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePhospho-CREB staining increases in female EAE DRGs in vivo.\u003c/em\u003e \u003c/strong\u003e(A-C) pCREB staining is increased in female EAE DRGs compared to males. Bars indicate mean ± standard error mean (SEM). #p\u0026lt;0.05 treatment factor, *p\u0026lt;0.05 sex factor, two-way ANOVA.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure8..png","url":"https://assets-eu.researchsquare.com/files/rs-1800909/v1/8e408b5cf3167c8351085156.png"},{"id":23544939,"identity":"55e54214-259b-4149-85d8-141bf6d875a0","added_by":"auto","created_at":"2022-07-06 17:23:41","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":515585,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eVisual representation of sex differences in TNFα signaling\u003c/em\u003e.\u003c/strong\u003e (7.1 A-C) In female DRGs, inflammatory TNFα signaling activates NFkB, which affects mitochondrial morphology, activates ATF3 and pCREB, and may contribute to pain. (7.1 D, E) ATF3 is known to negatively regulate NFkB, and is also associated with plasticity which may also contribute to pain. (7.2 A-D) In male DRGs, TNFα activates P38 signaling which triggers mitochondrial dysfunction, cytochrome c release, and caspase 3 cleavage, which may contribute to neuropathy and degeneration.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-1800909/v1/a0e51bfba5f5f1d87f80059d.png"},{"id":25944400,"identity":"5440479c-ccc3-4dba-b8da-8b06c2b734f9","added_by":"auto","created_at":"2022-09-01 17:14:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6172607,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1800909/v1/7e85127e-e221-41ad-908a-20c90006d417.pdf"},{"id":23544942,"identity":"64bbe0be-ca3d-4ecc-bf42-ff3e2389e511","added_by":"auto","created_at":"2022-07-06 17:23:41","extension":"tiff","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":388966,"visible":true,"origin":"","legend":"","description":"","filename":"S1.tiff","url":"https://assets-eu.researchsquare.com/files/rs-1800909/v1/1595a4ebfb1d3d5c9d179b8d.tiff"}],"financialInterests":"No competing interests reported.","formattedTitle":"Sex differences in the inflammatory response of the mouse DRG and its connection to pain in Multiple Sclerosis ","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMultiple sclerosis (MS) is an autoimmune disease characterized by widespread inflammation, immune cell activation, and demyelinating lesions of the central nervous system (CNS) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The symptoms of MS are extensive, affecting sensory, motor, and cognitive functions [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. One of the most debilitating symptoms, which affects over half of patients, is pain [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Adding another level of complexity to this condition, women are more likely than men to experience pain in MS [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Neuropathic pain is caused by injury or disease of the nervous system, producing pain in response to non-noxious stimuli (allodynia), increased pain in response to noxious stimuli (hyperalgesia), and spontaneous pain which occurs without a stimulus. Neuropathic pain generally originates in the peripheral nervous system (PNS) within the dorsal root ganglia (DRG), which house the cell bodies of primary pain-sensing neurons called nociceptors [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The hypothesis for MS pain, developed in animal research, is that inflammation and immune cell activation in the DRG cause nociceptors to become hyperexcitable, meaning they respond more readily and intensely to painful stimuli [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Peripheral sensitization of nociceptors triggers similar mechanisms in the spinal cord and brain, leading to central sensitization which maintains pain persistently, regardless of disease progression [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. This may help explain why conventional pain treatments are ineffective in the treatment of neuropathic pain in MS [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo further investigate the mechanisms of peripheral sensitization and potential sex differences in MS, we used the animal model, experimental autoimmune encephalomyelitis (EAE) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Not only does EAE replicate the inflammation and demyelination found in MS, but most importantly for our purposes, EAE animals exhibit classic signs of neuropathic pain [\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. We also used a simplified cell culture model to specifically study primary DRG neurons from na\u0026iuml;ve mice exposed to the inflammatory cytokine TNFα. We chose TNFα as our stimulus because it is upregulated both peripherally and centrally in MS and EAE and is involved in the development of many other neuropathic pain conditions [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Past work from our lab has also shown upregulation of TNFα from circulating immune cells in EAE [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. A major focus of this study was on mitochondrial responses to TNFα between the sexes. Mitochondrial dysfunction has been linked to neuropathic pain, and is known to be regulated by TNFα effectors, c-jun N-terminal kinase (JNK) and P38 mitogen-activated protein kinases (MAPKs), as well as nuclear factor κB (NFκB) [\u003cspan additionalcitationids=\"CR23 CR24\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In this study, we reveal sex differences in the inflammatory response to disease, TNFα signaling, mitochondrial function, neuronal injury, and plasticity in the DRG \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro.\u003c/em\u003e These differences suggest that males and females engage distinct, sex-specific pathways at the level of the DRG that may have important implications when considering strategies to treat pain in the disease.\u003c/p\u003e"},{"header":"2. Materials And Methods","content":"\u003cdiv class=\"Section2\" id=\"Sec3\"\u003e\n \u003ch2\u003e2.1 Experimental Autoimmune Encephalomyelitis\u003c/h2\u003e\n \u003cp\u003eAs previously described [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e], we induced EAE in both male and female C57BL/6 mice (8\u0026ndash;10 weeks old, Charles River) by subcutaneous injection of 50\u0026micro;g of myelin oligodendrocyte glycoprotein (MOG\u003csub\u003e35\u0026thinsp;\u0026minus;\u0026thinsp;55\u003c/sub\u003e) emulsified in complete Freund\u0026rsquo;s adjuvant (CFA, 1.5mg/mL). On the same day as induction, and 48h later, mice were also inoculated with 300ng of pertussis toxin. Animals were euthanized and perfused with cold saline on the first day of EAE symptom onset alongside their \u0026ldquo;CFA controls\u0026rdquo;, which received only the adjuvants, CFA and Pertussis, but not MOG\u003csub\u003e35\u0026thinsp;\u0026minus;\u0026thinsp;55\u003c/sub\u003e. Tissue was dissected and snap frozen in liquid nitrogen, then stored at -80\u0026deg;C. All animal experiments were performed according to the Canadian Council on Animal Care\u0026apos;s Guidelines and Policies with approval from the University of Alberta Health Sciences Animal Care and Use Committee (protocol 0000274).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec4\"\u003e\n \u003ch2\u003e2.2 Immunohistochemistry (IHC)\u003c/h2\u003e\n \u003cp\u003eFresh frozen DRG tissue was cryo-sectioned at 10\u0026micro;m thickness. Tissue slides were then immersed in 4% paraformaldehyde (PFA) and fixed for 5 minutes at room temperature. Next, they were immersed in antigen retrieval solution (1.92g anhydrous citric acid and 0.5mL Tween20 in 1L H\u003csub\u003e2\u003c/sub\u003eO, pH 6.0) for 10 minutes before 3x 10-minute washes in PBS. Tissue was blocked for one hour at room temperature in 10% normal donkey serum (NDS) in 0.2% triton X-100 in PBS (PBS\u003csub\u003eTX\u003c/sub\u003e). Primary antibody was incubated overnight at 4\u0026deg;C in 2% NGS and 2% bovine serum albumin (BSA) in PBS\u003csub\u003eTX\u003c/sub\u003e. Primary antibodies included CD45 (1:200, BD Pharmigen 550539), Iba1 (1:500 Cellular Dynamics 019-19741), CD3 (1:200, Bio-Rad MCA1477), cleaved caspase 3 (1:100, Cell Signaling #9661), ATF3 (1:200, Abcam ab207434), and pCREB (1:500, Cell Signaling #9198).\u003c/p\u003e\n \u003cp\u003eSlides were washed the next day 3x 10 minutes in PBS before incubation with secondary antibodies (Jackson Immunoresearch) at a 1:200 dilution for 45 minutes. Slides were again washed 3x 10 minutes in PBS before mounting with fluoromount G\u0026trade; Mounting Medium (Invitrogen 00-4958-02). Slides were imaged at 20x for analysis using a Zeiss Axio Observer Z1. Representative images were taken at 40x using a Leica TCS SPE Confocal.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec5\"\u003e\n \u003ch2\u003e2.3 Immunopanned Primary DRG Cultures\u003c/h2\u003e\n \u003cp\u003eImmunopanning protocol was adapted from Zuchero 2014 [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e]. All DRGs were collected from 2\u0026ndash;4 male and 2\u0026ndash;4 female 8\u0026ndash;10 week old C57BL/6 mice, and digested with 2mg/mL Stemxyme I (Worthington LS004106) for 1hr at 37\u0026deg;C. Cells were triturated gently and spun for 10 minutes at 300xg. They were next resuspended and strained through a 70\u0026micro;m filter then spun through a 3mL 15% BSA cushion at 300xg to remove myelin debris. After the BSA cushion, cells were allowed to rest for 30 minutes in a 10% CO\u003csub\u003e2\u003c/sub\u003e incubator at 37\u0026deg;C for antigen retrieval.\u003c/p\u003e\n \u003cp\u003eImmunopanning dishes were coated with secondary anti-rat, rabbit, and mouse antibodies (1:500, Jackson 112-005-167, 111-005-003, and 115-005-020 respectively) overnight at 4\u0026deg;C, then washed and coated with rat CD45 (10\u0026micro;g, BD Pharmigen 550539), rabbit PDGFR\u0026beta; (10\u0026micro;g, Abcam ab32570), and mouse O4 (1:2.5, O4 hybridoma [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e]) primary antibodies for 2 hours at room temperature. Cells were panned in 5mL of panning buffer (0.02% BSA and 0.008% DNAse in D-PBS) in each of the 3 dishes for 20 minutes, shaking gently at 10 minutes. Cells were spun down, counted with a haemocytometer, and pre-plated for 30min in 25\u0026micro;L of panning buffer prior to flooding wells with media (1:1 DMEM:Neurobasal, supplemented with SATO, N-acetyl-cysteine, insulin, penicillin/streptomycin, B27+, glutamate, and sodium pyruvate). Cells for protein collection (ELISA and Western Blotting) were plated in 24 well clear plastic plates (Falcon 353047) at 2000 cells per well. Cells for imaging were plated in 24 or 96 well black glass-bottom plates (Cellvis P24-1.5H-N, and Falcon 353219 respectively) at 500 cells per well. Cells were allowed to rest 24h in culture before treatment with 1000pg/mL TNF\u0026alpha; for 1, 6, or 24h respectively.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec6\"\u003e\n \u003ch2\u003e2.4 Western Blotting\u003c/h2\u003e\n \u003cp\u003eProtein lysates from cultured cells were scraped collected with RIPA lysis buffer (Thermofisher 89900) with cOmplete EDTA-free, (Roche 04693159001) and PhoSTOP (Roche 04906837001) added. Lysates were stored at -80\u0026deg;C. Prior to blotting, protein was precipitated with 4 volumes of cold acetone for one hour at 4\u0026deg;C, then centrifuged a 10 000xg for 10min before resuspension in a smaller volume. Lysates were then diluted with 4x Bio-Rad laemmli sample buffer and 50mM dithiothreitol and boiled for 10min at 95\u0026deg;C.\u003c/p\u003e\n \u003cp\u003eSamples were loaded into 4\u0026ndash;20% Mini-PROTEAN TGX Stain-free gels (Bio-Rad 4568096), and run at 120mV for 1-1.5 hours. After running, the total protein stain in the gels was activated using the Bio-Rad ChemiDoc XRS+. Gels were then transferred onto low-fluorescence PVDF membranes (Bio-Rad BioRad 1620264) using extra thick blot paper (Bio-Rad 1703965) on the Bio-Rad Trans-Blot Turbo system V1.02 for 30min at 25V and 1.0A. Total protein was then imaged with the ChemiDoc.\u003c/p\u003e\n \u003cp\u003eMembranes were blocked for one hour at room temperature in 5% BSA dissolved in 0.5% TBS-T. Primary antibody was incubated overnight at 4\u0026deg;C in 1% BSA in TBS-T. Primary antibodies included phospho-P65 (1:500, Millipore MAB3026), P38 (1:500, Cell Signaling #8690S), phospho-P38 (1:500, Cell Signaling #4631S), JNK (1:500, Cell Signaling #9252S), and phospho-JNK (1:500, Cell Signaling #4668S). Membranes were washed 3x 10 minutes in TBS-T before incubation with secondary antibody, Goat anti-Rabbit HRP (1:10 000, Jackson Laboratories 111-035-144) for one hour at room temperature. Membranes were again washed 3x 10 minutes in TBS-T, then for 10min in TBS before imaging with the ChemiDoc after 5min incubation with ECL Prime (Amersham RPN2232). Membrane stripping was performed according to the mild stripping protocol from abcam.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec7\"\u003e\n \u003ch2\u003e2.5 Mitochondrial Live Staining\u003c/h2\u003e\n \u003cp\u003eFor morphological characterization, live cells were stained with MitoTracker\u0026trade; Deep Red FM at 1:10 000 for 30 minutes, and NucBlue\u0026trade; Live ReadyProbes\u0026trade; reagent at 1:200 for 10min. Cells were then fixed in 4% PFA for 15 minutes at room temperature and washed with PBS before imaging. Footprint analysis was performed with Fiji MiNA software from the Stuart lab at Brock University [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e]. Morphology analysis was adapted from Leabeau \u003cem\u003eet al.\u003c/em\u003e 2018 [\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003eMitochondrial superoxide was stained with MitoSOX\u0026trade; red dye (Invitrogen M36008) at 1:1000 concentration and NucBlue\u0026trade; Live ReadyProbe (1:200 Invitrogen R37605). Cells were imaged live in an environment controlled Molecular Devices ImageXpress Micro high content screening system.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec8\"\u003e\n \u003ch2\u003e2.6 Immunocytochemistry\u003c/h2\u003e\n \u003cp\u003eCells were fixed in 4% PFA for 15 minutes at room temperature, then washed 3x with PBS and blocked in 10% NDS in PBS\u003csub\u003eTX\u003c/sub\u003e. Cleaved caspase 3 primary antibody (1:250, Cell Signaling #9661) was diluted in 2% NGS and 2% BSA in PBS\u003csub\u003eTX\u003c/sub\u003e at 4\u0026deg;C overnight. Cells were washed 3x in D-PBS before incubation with secondary antibody (1:500 goat ant-rabbit 488, Invitrogen A11008) and DAPI (1:2000, Invitrogen D1306) at room temperature for 30 minutes. Cells were imaged in a Molecular Devices ImageXpress Micro high content screening system.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec9\"\u003e\n \u003ch2\u003e2.7 Cytochrome C ELISA\u003c/h2\u003e\n \u003cp\u003eCytochrome C ELISA was performed on cell lysates from immunopanned cultures described in section 2.3, which were scraped and collected in 0.5% PBS\u003csub\u003eTX\u003c/sub\u003e. The protocol for the kit (R\u0026amp;D systems MCTC0) was followed with no variations.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec10\"\u003e\n \u003ch2\u003e2.8 Data Analysis\u003c/h2\u003e\n \u003cp\u003eStatistical analyses were performed using GraphPad Prism 9, apart from Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eG, the statistics for which were performed by Dr. Karen Buro of MacEwan University using Fisher exact tests with Bonferroni\u0026rsquo;s multiple comparison. Otherwise, all data was expressed as fold change of sex-respective controls and analyzed by regular two-way ANOVA. Significance was set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Immunohistochemistry data represents manually counted positive cells normalized to tissue area. P38 and JNK western blot data was calculated as the intensity of phosphorylated over total antibody staining, normalized to total protein. NF\u0026kappa;B data was calculated as phosphorylated P65 antibody staining normalized to total protein due to lack of a sufficient total P65 antibody. Mitochondrial footprint data represents the total area of mitochondrial staining, normalized to cell diameter.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Sex differences in EAE DRG inflammation\u003c/h2\u003e \u003cp\u003ePrevious work from our lab has revealed inflammation in the DRGs of post-mortem female MS patients [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Given the prominent sex differences in neuropathic pain in MS [\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], we sought to determine if there were underlying sex differences in the inflammatory signatures of mouse EAE DRGs. Using immunohistochemistry, we found significant increases in staining for the immune cell marker CD45 and T cell marker CD3 in both male and female EAE DRGs compared to their CFA controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-F, F(1, 16)\u0026thinsp;=\u0026thinsp;7.113, P\u0026thinsp;=\u0026thinsp;0.0169 and F(1, 15)\u0026thinsp;=\u0026thinsp;13.34, P\u0026thinsp;=\u0026thinsp;0.0258 respectively, Two-way ANOVA), with no apparent sex differences (F(1, 16)\u0026thinsp;=\u0026thinsp;0.2351, P\u0026thinsp;=\u0026thinsp;0.6344 and F(1, 15)\u0026thinsp;=\u0026thinsp;0.04575, P\u0026thinsp;=\u0026thinsp;0.8335 respectively, Two-way ANOVA). We also observed an increase in Iba1 staining, indicative of an increased macrophage and monocyte presence, in both male and female EAE DRGS (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e1\u003c/span\u003eG-I, F(1, 15)\u0026thinsp;=\u0026thinsp;53.62, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Two-way ANOVA). However, there was more Iba1 staining in males than females (F(1, 15)\u0026thinsp;=\u0026thinsp;12.65, P\u0026thinsp;=\u0026thinsp;0.0029, Two-way ANOVA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDectin-1 is a pattern-recognition receptor which facilitates cytokine production, phagocytosis, and respiratory burst (rapid release of reactive oxygen species) [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Given this link to inflammation, we investigated co-labelling of Dectin-1 and Iba1 in the mouse EAE DRG (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-C). We found increased Dectin-1/ Iba1 co-labelled cells in EAE animals compared to their CFA controls (F(1, 14)\u0026thinsp;=\u0026thinsp;16.45, P\u0026thinsp;=\u0026thinsp;0.0012), with a significant sex difference as male EAE animals demonstrated more co-labelling than females (F(1, 14)\u0026thinsp;=\u0026thinsp;11.62, P\u0026thinsp;=\u0026thinsp;0.0042). This suggests that not only is there an increased macrophage/ monocyte presence in the male EAE DRG (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e1\u003c/span\u003eG-I), but these cells may have a different inflammatory phenotype than females. Over all these results reveal sex differences in the DRG of EAE animals, which may contribute to sex differences in pain.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Sex differences in TNFα signaling \u003cem\u003ein vitro\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eTNFα is a pleiotropic cytokine that has been linked to various neuropathic pain conditions and is present both peripherally and centrally in MS [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Our lab has shown previously that TNFα is increased peripherally in circulating immune cells in EAE [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. We investigated TNFα levels in the male and female EAE DRG by polymerase chain reaction (PCR) using previously described methods [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], and found TNFα mRNA is elevated in both sexes in EAE (\u003cb\u003eSupplementary Fig.\u0026nbsp;1\u003c/b\u003e). TNFα is secreted by macrophages, monocytes, and lymphocytes, and we know that there is an increased presence of these inflammatory cell types in the DRG with EAE [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. For these reasons, we selected TNFα as an \u003cem\u003ein vitro\u003c/em\u003e stimulus to understand how it might contribute to peripheral sensitization of sensory neurons in the EAE model. We immunopanned male and female DRGs to reduce the contribution of non-neuronal cells in the culture, then treated them with TNFα (1ng/mL) for 1, 6, or 24 hours. Cells were collected and western blotted for TNFα signaling effectors, JNK, P38, and NFκB to look for sex differences in the TNFα signalling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). There was no sex difference evident in JNK activation with TNFα treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, F(1, 20)\u0026thinsp;=\u0026thinsp;0.01980, P\u0026thinsp;=\u0026thinsp;0.8895, Two-way ANOVA). However, male sensory neurons exhibited greater P38 activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, F(1, 19)\u0026thinsp;=\u0026thinsp;4.900 P\u0026thinsp;=\u0026thinsp;0.0393, Two-way ANOVA). In contrast, female sensory neurons had greater NFκB activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, F(1, 19)\u0026thinsp;=\u0026thinsp;18.55, P\u0026thinsp;=\u0026thinsp;0.0004, Two-way ANOVA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Mitochondrial morphology is affected by TNFα treatment\u003c/h2\u003e \u003cp\u003eWe determined that male sensory neurons preferentially activate P38 while female neurons preferentially engage the NFκB pathway in response to TNFα treatment \u003cem\u003ein vitro\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Since TNFα signaling through both NFκB and P38 has been linked to mitochondrial changes [\u003cspan additionalcitationids=\"CR23 CR24\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], we next sought to characterize whether exposure to TNFα leads to changes in mitochondrial morphology in male and female nociceptors (DRG neurons\u0026thinsp;\u0026le;\u0026thinsp;30\u0026micro;m in diameter). The mitochondrial footprint in these cells was significantly increased with 24h of TNFα treatment compared to control, (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C, F(1,78)\u0026thinsp;=\u0026thinsp;3.648, P\u0026thinsp;=\u0026thinsp;0.0598, Two-way ANOVA). Specifically, there was a greater increase in the footprint from females when compared to their respective controls (P\u0026thinsp;=\u0026thinsp;0.0197 and 0.9757 respectively, Sidak\u0026rsquo;s multiple comparisons test). This result prompted us to further investigate the specific morphological changes of these mitochondrial networks in response to TNFα treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-G). In male neurons, we observed a change in mitochondrial network morphology after 24 hours of TNFα treatment compared to control, wherein the proportion of fragmented morphology decreased and the tubular morphology increased (P\u0026thinsp;=\u0026thinsp;0.00062 and 0.0031 respectively, Fisher exact test, Bonferroni\u0026rsquo;s multiple comparison). However, female neurons exhibited changes in mitochondrial morphology at all three timepoints. At one hour of TNFα treatment, fragmented morphology increased, and tubular morphology decreased compared to control (P\u0026thinsp;=\u0026thinsp;2.2e-16 for both morphologies, Fisher exact test, Bonferroni\u0026rsquo;s multiple comparison). At 6 hours and 24 hours of TNFα treatment there was a shift in how the females responded to TNFα treatment. Like the male 24-hour treatment, female neurons exhibited decreased fragmented morphology (P\u0026thinsp;=\u0026thinsp;0.00012 and 3.3e-11 respectively), and increased tubular morphology compared to control (P\u0026thinsp;=\u0026thinsp;0.000027 and 8.2e-10 respectively, Fisher exact test, Bonferroni\u0026rsquo;s multiple comparison) at both 6 and 24 hours of TNFα treatment. Taken together, the increased mitochondrial footprint in female neurons, along with a concomitant increase in tubular morphology at 6 and 24 hours of TNFα treatment, may reflect a protective mechanism engaged by sensory neurons undergoing persistent inflammation, which appears to be more effective in females than in males.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Mitochondrial function is altered in male sensory neurons with TNFα treatment\u003c/h2\u003e \u003cp\u003eTo determine if adaptive mitochondrial changes are more effective in females than males, we next explored mitochondrial superoxide levels with Mitosox live cell staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-C). Male cells produced significantly more superoxide in response to TNFα than female cells (F(1,42)\u0026thinsp;=\u0026thinsp;16.40, P\u0026thinsp;=\u0026thinsp;0.0002, Two-way ANOVA). To further understand the extent of male mitochondrial dysfunction we assessed levels of cytochrome C release from mitochondria in cell lysates by ELISA (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Male cells released significantly more cytochrome C than female cells (F(1,48)-13.63, P\u0026thinsp;=\u0026thinsp;0.0006, Two-way ANOVA). While both P38 and NFκB are linked to mitochondrial changes, P38 in particular can mediate intrinsic apoptosis through cytochrome c release [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. These results suggest that male DRG neurons are more susceptible to mitochondrial damage in response to TNFα-mediated inflammation, potentially due to their preferential activation of P38.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Male DRGs are more susceptible to neuronal damage \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eCytochrome C release is a marker of intrinsic apoptosis, one effector of which is caspase 3 [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Since we found increased P38 activation and cytochrome C release in male sensory neurons, we sought to determine whether this led to increased caspase 3 cleavage. Using immunocytochemistry to asses our TNFα-treated sensory neurons, we found there was significantly more caspase 3 cleavage in males than females (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-C, F(1,42)\u0026thinsp;=\u0026thinsp;15.29, P\u0026thinsp;=\u0026thinsp;0.0003, Two-way ANOVA). To validate our \u003cem\u003ein vitro\u003c/em\u003e model, we next determined if this result occurred \u003cem\u003ein vivo\u003c/em\u003e in EAE. In DRG sections from mice with EAE, there was indeed greater caspase 3 cleavage in males than in females (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e6\u003c/span\u003eD-F, F(1, 15)\u0026thinsp;=\u0026thinsp;14.97, P\u0026thinsp;=\u0026thinsp;0.0015, Two-way ANOVA). These results suggest that male DRG sensory neuron activation of p38 in response to TNFα may trigger structural and functional mitochondrial changes, which result in increased neuronal damage through caspase 3.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Plasticity markers are increased in female EAE DRGs\u003c/h2\u003e \u003cp\u003eTo gain an understanding of what adaptive mechanisms may be present in female EAE DRGs to protect them from mitochondrial damage and neuronal injury we first investigated activating transcription factor 3 (ATF3), an injury and plasticity-associated factor, by IHC (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-C). There was a significant increase in ATF3 staining in EAE DRGs compared to their CFA controls (F(1, 14)\u0026thinsp;=\u0026thinsp;17.51, P\u0026thinsp;=\u0026thinsp;0.0011, Two-way ANOVA, Sidak\u0026rsquo;s multiple comparisons test), with a significantly greater increase in females than males (F(1, 14)\u0026thinsp;=\u0026thinsp;6.060, P\u0026thinsp;=\u0026thinsp;0.0274, Two-way ANOVA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFinally, we stained EAE DRGs for phosphorylated cAMP response element-binding protein (pCREB). CREB is a transcription factor strongly linked to neuroplasticity and pro-survival gene transcription as well as neuropathic pain [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. We found a significant increase in pCREB staining in EAE DRGs compared to their CFA controls, normalized to na\u0026iuml;ve mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-C, F(1, 16)\u0026thinsp;=\u0026thinsp;7.3, P\u0026thinsp;=\u0026thinsp;0.0155, Two-way ANOVA), with a strong sex difference (F(1, 16)\u0026thinsp;=\u0026thinsp;7.9, P\u0026thinsp;=\u0026thinsp;0.0124, Two-Way ANOVA) where EAE females in particular demonstrated increased pCREB staining (P\u0026thinsp;=\u0026thinsp;0.0135, Sidak\u0026rsquo;s multiple comparisons test). This female-dominated increase in ATF3 and pCREB staining in the EAE DRG suggests that females may have a stronger plastic and/or regenerative response to inflammation, which could explain their protection from mitochondrial dysfunction and neuronal damage, as well as contribute to their elevated hyperexcitability.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eOur results reveal sex differences in the DRG in both the EAE mouse model of MS, and our TNFα-treated culture model. Both male and female EAE DRGs exhibit increased staining for inflammatory markers, but notably, males have greater macrophage/ monocyte staining than females at the onset of EAE. In addition, male macrophages and monocytes have more Dectin-1 staining in male EAE mice, suggestive of greater inflammatory activation. Using an \u003cem\u003ein vitro\u003c/em\u003e preparation of DRG neurons stimulated with the inflammatory cytokine TNFα, we observed that males preferentially activated P38 MAPK, and presented with disrupted mitochondrial morphology, elevated mitochondrial superoxide production, and cytochrome c release. This mitochondrial pathology is likely responsible for the increased caspase 3 cleavage we observed in the male DRG both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. Females, however, preferentially activated the transcription factor NFκB rather than P38, and while their mitochondrial morphology was also altered, they did not elevate superoxide production, cytochrome c release, or caspase 3 cleavage. Rather, we show that female EAE DRGs have increased levels of the injury and plasticity-associated factors, ATF3 and pCREB \u003cem\u003ein vivo\u003c/em\u003e. These observations have significant implications for our understanding of neuropathy and pain in chronic neuroinflammatory disorders such as MS.\u003c/p\u003e \u003cp\u003eFirst, we found a significant increase in global inflammation and immune cell infiltration indicated by CD45, CD3, and Iba1 staining at the level of the DRG in the EAE model in both sexes. Many studies of neuropathic pain using peripheral nerve injury models have focused on the spinal cord and report that females have a T-cell-mediated pain phenotype [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. This, however, may not be true for the onset of peripheral sensitization in the EAE DRG, as we saw no sex differences in CD3 T cell infiltration between the sexes. However, the indication that microglia/macrophages are responsible for pain in the male spinal cord [\u003cspan additionalcitationids=\"CR45 CR46\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] may be analogous to the EAE DRG, as evidenced by our finding that more cells were stained for Iba1 in males, and that a greater number of these Iba1-positive cells were co-labelled with the inflammatory marker Dectin-1. DRG macrophages have already been identified as important players in peripheral sensitization and neuropathic pain in a multitude of studies [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. For example, their secretion of pro-inflammatory mediators, including TNFα, can directly increase sensory neuron excitability [\u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. We found increased TNFα mRNA in both male and female EAE DRGs, and our lab has previously confirmed that TNFα is increased peripherally in EAE [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. This motivated us to use TNFα as our \u003cem\u003ein vitro\u003c/em\u003e stimulus to further elucidate sex differences in intracellular signaling in DRG neurons.\u003c/p\u003e \u003cp\u003eTo determine if the inflammatory profile we identified in EAE DRGs has consequences on intracellular neuronal processes, we moved \u003cem\u003ein vitro\u003c/em\u003e, where we observed sex differences in the downstream signaling pathways engaged by TNFα stimulation. Our finding that male neurons are biased towards activation of P38 in response to TNFα is supported by studies which have shown that males activate P38 in the spinal cord in mouse models of neuropathic pain [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Additionally, we observed that female neurons preferentially activate NFκB with TNFα stimulation. This aligns with reports indicating that females preferentially activate NFκB in endothelial cells and inflamed lung tissue [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGiven the strong links of both P38 and NFκB to functional changes in the mitochondria [\u003cspan additionalcitationids=\"CR23 CR24\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], we went on to conduct a surface-level investigation of the changes that occur in mitochondrial morphology in response to TNFα. We found that female neurons increased their mitochondrial footprint in response to 24 hours of TNFα treatment, and that their mitochondrial morphology responded more quickly to treatment than males. We observed more fragmented mitochondria, and less tubular mitochondria in females after one hour of TNFα treatment compared to control, which may indicate a maladaptive phenotype such as impaired mitochondrial fission. However, at 6 and 24 hours of TNFα treatment, female mitochondrial networks appeared to shift their response to TNFα treatment, as they exhibited less fragmented mitochondria and more tubular mitochondria compared to control. Male neurons also exhibited less fragmentation and more tubular networks at 24 hours of treatment but showed no significant changes at earlier timepoints.\u003c/p\u003e \u003cp\u003eWe hypothesize that the decreased fragmentation and increased tubular mitochondrial networks we observed after TNFα treatment may be evidence of an adaptive response to inflammation, which appears be more effective in females than in males. To further investigate this mechanism, we turned to functional readouts of mitochondrial health in our TNFα-stimulated culture, superoxide production and cytochrome C release. We found that male DRG neurons produce more superoxide and release more cytochrome C than females in response to TNFα. This led us to speculate that DRG neurons initiate an adaptive response to inflammation that is more effective in females than in males, potentially due to their differential activation of the TNFα signaling pathways.\u003c/p\u003e \u003cp\u003eThe dramatic increase in cytochrome C release from male neurons lead us to investigate caspase 3 cleavage. We found that both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e, male DRGs exhibit more cleaved caspase 3 staining than females. This finding aligns with TNFα signaling literature as P38 can cause cytochrome C release and eventually, intrinsic apoptosis through B-cell lymphoma (Bcl) protein regulation [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In addition, previous work from our lab has investigated sex differences in the mouse EAE spinal cord and found significantly greater increases in the levels of amyloid precursor protein (APP) and non-phosphorylated neurofilament (SMI32) in males, suggesting that male spinal neurons are more sensitive to axonal injury and degeneration in EAE [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. These results support our interpretation that male EAE DRG neurons are more prone to neuronal injury than females. Neuronal injury in males may also explain previous work from our lab which has shown that while female EAE DRG neurons are hyperexcitable, males are not [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. We hypothesize that while female pain is driven by hyper-excitability in the periphery, this is not the source of pain in males. Instead, male pain in EAE may be mediated by a central mechanism.\u003c/p\u003e \u003cp\u003eFinally, we examined mouse EAE DRGs for the expression of the injury and plasticity-associated proteins ATF3 and pCREB. Females had significantly more ATF3-positive cells than males, suggesting that females engage an adaptive mechanism in response to inflammation. NFκB is known to induce ATF3 signaling, acting in a negative feedback loop to dampen its own pro-inflammatory signaling, as ATF3 can bind and inhibit the P65 subunit of NFκB [\u003cspan additionalcitationids=\"CR58\" citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. There was also significantly more pCREB staining in the female EAE DRG than in males, which provides further evidence of a regenerative phenotype. Much like ATF3, CREB can directly inhibit NFκB and pro-inflammatory signaling [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. It has also been linked to aberrant plasticity, neuronal hyperexcitability, and pain in spinal cord injury [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. While activation of these transcription factors in the female EAE DRG may protect neurons from inflammation, mitochondrial pathology, and neuropathy, they may also be responsible for neuronal hyperexcitability and pain via maladaptive plasticity.\u003c/p\u003e \u003cp\u003eThus, we propose that a regenerative mechanism is induced by preferential NFκB signaling in the female EAE DRG (Fig.\u0026nbsp;9\u003cb\u003e.1\u003c/b\u003e). While this mechanism prevents neuropathy, it may be either insufficient to prevent neuronal hyperexcitability and pain or may even contribute to pain through aberrant plasticity [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Males, however, preferentially activate P38, which leads to mitochondrial dysfunction and caspase 3 cleavage (Fig.\u0026nbsp;9\u003cb\u003e.2\u003c/b\u003e) This pathway may cause male DRGs to become more prone to neuropathy. These studies reveal that there are distinct pathological changes that occur at the level of the DRG between the sexes. There is a need for future therapeutic strategies to treat neuropathic pain to be tailored in a sex specific manner. Our findings suggest that targeting processes of axonal degeneration and neuropathy will be more efficacious in males while modulating inflammation and aberrant plasticity will be more beneficial in females.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics Approval and Consent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were performed according to the Canadian Council on Animal Care\u0026apos;s Guidelines and Policies with approval from the University of Alberta Health Sciences Animal Care and Use Committee (protocol 0000274).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Data and Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eApplicable data and materials available upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by a Project Grant from the Canadian Institutes of Health Research (FRN-162434) and a Discovery Grant from the MS Society of Canada (EGID-3761).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003e\n \u003cp\u003eD.M\u003c/p\u003e\n \u003cul\u003e\n \u003cli\u003e\n \u003cp\u003eWrote and edited the manuscript\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eprepared the figures (including final analysis and graphing)\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003ePrepared and stained all primary cell cultures and performed western blots, ELISAs, and immunocytochemistry\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003ePerformed and analyzed immunohistochemistry (IHC) for figures 6 and 7\u003c/p\u003e\n \u003c/li\u003e\n \u003c/ul\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eN.F assisted with dissections for all EAE tissue and primary cultures\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eV.A performed PCRs for supplementary figure 1\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eH imaged and analyzed cells for figure 4\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eD. performed and analyzed IHC for figures 1\u0026amp;2\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eP performed and analyzed IHC for figure 8\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eT. sliced tissue for all IHC and edited figures using adobe illustrator\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eB performed statistical analysis for figure 4 and advised on statistical reporting for all figures\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eR.P shared equipment and expertise, and reviewed the manuscript\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eJ.K is the principal investigator (supervised all the above activities and reviewed/ edited the manuscript)\u003c/p\u003e\n \u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Dr. Sun and the Cross Cancer Institute for training and use of the ImageXpress system. The authors would also like to thank the University of Alberta SMART Network for use of their imaging core.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHemmer B, Kerschensteiner M, Korn T: \u003cstrong\u003eRole of the innate and adaptive immune responses in the course of multiple sclerosis.\u003c/strong\u003e \u003cem\u003eLancet Neurol \u003c/em\u003e2015, \u003cstrong\u003e14:\u003c/strong\u003e406-419.\u003c/li\u003e\n\u003cli\u003eFilippi M, Bar-Or A, Piehl F, Preziosa P, Solari A, Vukusic S, Rocca MA: \u003cstrong\u003eMultiple sclerosis.\u003c/strong\u003e \u003cem\u003eNat Rev Dis Primers \u003c/em\u003e2018, \u003cstrong\u003e4:\u003c/strong\u003e43.\u003c/li\u003e\n\u003cli\u003eReich DS, Lucchinetti CF, Calabresi PA: \u003cstrong\u003eMultiple Sclerosis.\u003c/strong\u003e \u003cem\u003eN Engl J Med \u003c/em\u003e2018, \u003cstrong\u003e378:\u003c/strong\u003e169-180.\u003c/li\u003e\n\u003cli\u003eMurphy KL, Bethea JR, Fischer R: \u003cstrong\u003eNeuropathic Pain in Multiple Sclerosis-Current Therapeutic Intervention and Future Treatment Perspectives.\u003c/strong\u003e In \u003cem\u003eMultiple Sclerosis: Perspectives in Treatment and Pathogenesis.\u003c/em\u003e Edited by Zagon IS, McLaughlin PJ. 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\u003cstrong\u003e239:\u003c/strong\u003e1317-1326.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Multiple Sclerosis, Pain, Experimental Autoimmune Encephalomyelitis, Sex Differences, Tumour Necrosis Factor α, Mitochondria, Neuropathy, Plasticity","lastPublishedDoi":"10.21203/rs.3.rs-1800909/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1800909/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMultiple Sclerosis (MS) is an autoimmune disease with notable sex differences. Women are not only more likely to develop MS but are also more likely than men to experience neuropathic pain in the disease. It has been postulated that neuropathic pain in MS can originate in the peripheral nervous system at the level of the dorsal root ganglia (DRG), which houses primary pain sensing neurons (nociceptors). These nociceptors become hyperexcitable in response to inflammation, leading to peripheral sensitization and eventually central sensitization, which maintains pain long-term. The mouse model experimental autoimmune encephalomyelitis (EAE) is a good model for human MS as it replicates classic MS symptoms including pain. Using EAE mice as well as primary mouse DRG neurons cultured \u003cem\u003ein vitro\u003c/em\u003e, we sought to characterize the sex differences specifically in peripheral sensory neurons which may underlie the disparities in MS pain. We found sex differences in the inflammatory profile of the EAE DRG, and in the TNFα signaling pathways activated intracellularly in cultured nociceptors. Given that TNFα signaling has been shown to impact on mitochondrial function, this led us to investigate sex differences in the mitochondria\u0026rsquo;s response to TNFα. Our results demonstrate that male sensory neurons are more sensitive to mitochondrial stress, making them prone to neuronal injury. In contrast, female sensory neurons appear to be more resistant to mitochondrial stress and exhibit an inflammatory and regenerative phenotype that may underlie greater nociceptor hyperexcitability and pain. Understanding these sex differences at the level of the primary sensory neuron is an important first step in our eventual goal of developing sex-specific treatments to halt pain development in the periphery before central sensitization is established.\u003c/p\u003e","manuscriptTitle":"Sex differences in the inflammatory response of the mouse DRG and its connection to pain in Multiple Sclerosis ","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-07-06 17:18:39","doi":"10.21203/rs.3.rs-1800909/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d5278bb6-0f23-4e9c-803b-709fbba3f7bb","owner":[],"postedDate":"July 6th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-09-01T17:14:22+00:00","versionOfRecord":[],"versionCreatedAt":"2022-07-06 17:18:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1800909","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1800909","identity":"rs-1800909","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}