Female C57BL/6J mice aged 10–12 weeks (Australian Biological Resources, Moss Vale, NSW, Australia) were used in all experiments. Mice were housed in individually ventilated cages with water and food ad libitum in groups of 3–5 and maintained on a 12-h light–dark cycle. The facility was kept at a constant room temperature (RT) and humidity, and the animals were monitored daily throughout experiments. All experiments were approved by the Animal Care and Ethics Committee of the University of New South Wales, Sydney, NSW, Australia.

Experimental autoimmune encephalomyelitis was induced by subcutaneous immunization with myelin oligodendrocyte glycoprotein 35–55 (MOG_35–55) emulsified in complete Freund’s adjuvant (CFA). Emulsions were purchased from Hooke Laboratories (Lawrence, MA, USA) as prefilled syringes, each containing ~1 mg/mL MOG_35–55 emulsified with 2–5 mg killed Mycobacterium tuberculosis H37Ra/mL in incomplete Freund’s adjuvant. Control mice were immunized with CFA alone (Hooke Laboratories; at the same concentration given to mice immunized with MOG_35–55/CFA). Immunizations were given under 3–5% isoflurane anesthesia in oxygen as 2 × 100 μL subcutaneous injections; one at the base of the tail and one on the upper back. An intraperitoneal injection of 200 ng Pertussis Toxin (Hooke Laboratories) in 100 μL Dulbecco’s Phosphate Buffered Saline (D-PBS; Life Technologies Pty Ltd., VIC, Australia) was given to all mice 2–6 h after subcutaneous immunization, and again 22–26 h later.

Post-induction, mice were monitored daily for body weight and EAE clinical scores according to a detailed EAE grading system supplied by Hooke Laboratories. Briefly, EAE clinical scores were assigned as follows: Grade 1 = limp tail; Grade 2 = limp tail and weakness of hind legs; Grade 3 = limp tail and complete paralysis of hind legs or limp tail with paralysis of one front and one hind leg; Grade 4 = limp tail and complete hind leg and partial front leg paralysis; Grade 5 = complete hind and complete front leg paralysis.

Hind paw mechanical allodynia was assessed by placing mice in 10 × 10 cm red-tinted chambers on an elevated wire mesh. Mice were habituated to the behavioral testing apparatus twice for 1 h each prior to baseline testing. Mechanical allodynia of the hind paws was assessed using a set of calibrated von Frey filaments. These were applied to the mid-plantar surface of the hind paw until bending and maintained for 3 s. Paw withdrawals were noted as swift, sharp responses to application of the filament, and testing was conducted blind to experimental groups using Dixon’s up-and-down method (18). A 50% paw withdrawal threshold (PWT) per animal was found by averaging the 50% PWT calculated for each paw.

Facial allodynia was assessed using a method previously described by Lyons et al. to measure facial pain in a model of trigeminal inflammatory compression injury (19). In the week prior to baseline behavioral testing, animals were handled daily using a cotton glove in order to gradually acclimatize the mice to being gently restrained in the experimenter’s hand. Prior to testing, the same experimenter gently restrained the mouse in their palm with the head exposed using the cotton glove until the mouse was acclimated and calm. During testing, a second experimenter blinded to experimental groups applied a 0.07 g von Frey filament to the whisker pad five times per side, with a 1-min interval between tests. Responses were recorded as head withdrawal, fore paw swiping, or facial flinching by a blinded experimenter, and a percentage of total responses over the five tests per side were calculated for each animal.

Mice were habituated for 15 min once prior to baseline testing in a 5 × 5 × 10 cm plastic arch with glass windows at each end placed on an elevated wire mesh. During testing, mice were placed in the same arch, with two Canon 500D cameras positioned at each end for high definition video recording. Mice were filmed for 11 min total, and screen grabs were later taken at each minute mark following beginning of recording for a total of 10 photos per mouse. These were taken as soon as a clear head shot could be observed and scored according to the criteria developed by Langford and colleagues (20) with the omission of the whisker change action unit as this was deemed difficult in C57BL/6J mice due to coat color (Figure 2A). Mean mouse grimace scale (MGS) difference scores from baseline were calculated in a blinded manner at time points tested post-EAE induction.

At designated end-points post-EAE induction (day 8 pre-onset, day 16 clinical peak, and day 32 chronic phase), mice were euthanized using 0.1 mL of Lethabarb (Virbac, NSW, Australia), injected i.p. and were transcardially perfused with heparinised 0.9% saline solution. The L3–5 dorsal root ganglia (DRG; left and right), SC, brain, and trigeminal tissue (nerve and ganglia; right) were then dissected and placed into PBS on ice. Tissues were coarsely chopped and incubated with 1 mL of Accutase (Sigma Aldrich, NSW, Australia) for 30 min at 37°C and 5% CO₂. Tissues were then mechanically ground through 70 μm cell strainers (BD Biosciences, Franklins Lakes, NJ, USA) in 10 mL of PBS. Cell suspensions were centrifuged for 5 min at 1000 × g at 4°C, before discarding the supernatant. 10 mL of 30% Percoll (GE Healthcare Australia Pty Ltd., NSW, Australia) in PBS was added to each sample, which were then centrifuged for 25 min at 600 × g at RT. The supernatant, including the myelin/cell debris layer, was carefully removed using a pipette, and the cell pellet was resuspended in 1 mL of autoMACS (magnetic-activated cell sorting; Miltenyi Biotec Australia Pty Ltd., NSW, Australia) running buffer. Samples were centrifuged for 5 min at 600 × g at 4°C, before removing as much of the supernatant from the pellet as possible using a pipette. Cells were counted and PBS added to give a final cell concentration of ~1 × 10⁷ cells per mL.

One hundred microliters of cells from each sample were divided into separate tubes, and 1 μL of Zombie Violet cell viability dye (Biolegend, CA, USA) was added to each tube, which were incubated in the dark for 15 min at RT. Cells were washed once in 1 mL of autoMACS running buffer by centrifuging for 5 min at 600 × g at 4°C, the supernatant was discarded and the residual volume was incubated with 1 μL anti-mouse CD16/CD32 Fc Block (eBioscience, CA, USA) for 5 min at RT. For analysis of premyelinating and myelinating oligodendrocytes, primary anti-galactocerebroside (GALC) and anti-MOG antibodies (Merck Millipore, VIC, Australia) were first conjugated to unique fluorophores using Lightning-Link Antibody Labeling kits (Novus Biologicals, Littleton, CO, USA) as per manufacturer’s instructions. Anti-GALC-PE and anti-MOG-FITC conjugated antibodies were then incorporated into a standard flow cytometry staining protocol as follows. Antibodies including anti-CD45-APC (eBioscience; 1:1000), anti-CD4-FITC (eBioscience; 1:1000), Anti-GALC-PE (1:200), and anti-MOG-FITC (1:200) diluted in 100 μL of autoMACS running buffer were added to each sample, and cells were incubated for 30 min at 4°C in the dark. Appropriate isotype and fluorescence minus one controls were included by staining 100 μL of pooled samples for each tissue analyzed. After staining, cells were washed three times in 1 mL of autoMACS running buffer before being resuspended in 0.2 mL of the same buffer for analysis on a BD FACS Canto II flow cytometer. A minimum of 50,000 events was acquired per sample, and data were analyzed using FlowJo software (FlowJo, OR, USA).

At designated end-points post-EAE induction (day 8 pre-onset and day 16 clinical peak), mice were euthanized and transcardially perfused with heparinised 0.09% saline solution followed by 10% formalin solution (Sigma Aldrich). The L3–5 DRG, L3–5 SC, brain (including brain stem), and trigeminal tissue was then dissected and post-fixed in formalin solution overnight at 4°C. The medulla was then dissected from the brain, and all tissues were transferred to 30% sucrose + 0.1% sodium azide solution and stored at 4°C until sectioning. Sections were cut using a cryostat (Leica Biosystems, Buffalo grove, IL, USA), with DRGs, and trigeminal tissue samples sectioned longitudinally at a thickness of 10 μm, and SC and medulla samples sectioned coronally at a thickness of 20 μm. All sections were cut sequentially such that each slide contained 4–6 serial sections representative of the entire thickness of the tissue. Sections were transferred directly to gelatin-coated glass slides, air dried overnight, and stored at −80^oC until staining.

Prior to staining, sections were fixed with ethanol for 10 min at RT. Sections were then washed twice with distilled water and once with PBS containing 0.05% Tween-20 (PBS-T). A blocking solution containing PBS with 5% donkey or goat serum, 0.2% Tween-20, and 0.3% Triton X-100 was applied to each slide for incubation at RT for 1 h in the dark. The blocking solution was drained, and sections were incubated with the following antibodies diluted in PBS containing 5% bovine serum albumin and 0.03% Triton-X: rat anti-mouse CD3 (T cells; 1:100; R&D systems, MN, USA), rabbit anti-mouse/rat IBA-1 (ionized calcium-binding adaptor-1; macrophages/microglia; 1:2000; Wako Chemicals USA, Richmond, VA, USA), mouse anti-mouse GFAP (glial fibrillary acidic protein; activated astrocytes; 1:2000; Chemicon, Temecula, CA, USA), rabbit anti-mouse ATF-3 (activating transcription factor-3; cell damage; 1:400; Santa Cruz Biotechnology Inc., TX, USA), goat anti-mouse CGRP (calcitonin gene-related peptide; peptidergic neurons; 1:1000; Abcam, VIC, Australia), anti-mouse IB4-FITC (isolectin B4; non-peptidergic neurons; 1:100; Sigma Aldrich), and rabbit anti-mouse NF200 (neurofilament 200; myelinated neurons; 1:1000; Sigma Aldrich). IBA-1 and GFAP primary antibody incubation was conducted at RT for 1 h, while all other primary antibodies were incubated overnight at 4°C. Sections were then washed four times with PBS-T for 10 min each, before adding secondary antibodies, which included: Alexa Fluor 488 conjugated donkey anti-mouse (1:1000; Life Technologies, Mulgrave, VIC, Australia), Alexa Fluor 546 conjugated donkey anti-rabbit (1:1000; Life Technologies), or Cy3 conjugated donkey anti-rabbit (1:1000; Jackson ImmunoResearch Laboratories Inc., PA, USA) in the same buffer as the primary antibody. Negative staining controls were incorporated whereby tissues were incubated with secondary antibody alone in the absence of the relevant primary antibody (to control for non-specific binding of the secondary antibody). Following a 1 h incubation at RT in the dark, sections were washed four times in PBS-T for 10 min each. Prolong gold anti-fade reagent with 4′, 6-diamidino-2-phenylindole (DAPI; Life Technologies) was applied before slides were cover slipped and stored at 4°C until viewing.

Analysis of images was conducted blind to experimental groups from which the tissue was derived.

All statistical analyses were conducted using GraphPad Prism 6 software. Since pain behavioral data sets involving the testing of mechanical allodynia proved to be not normally distributed using a Shapiro–Wilk normality test, a non-parametric Mann–Whitney test was used as the most appropriate statistical test for comparing two groups – EAE mice vs. control mice at each time point. The Holm–Bonferroni correction for multiple comparisons was then used to account for multiple testing. MGS data were analyzed using a repeated measures two-way ANOVA (treatment; time) with Tukey’s post hoc test. Immunohistochemistry data were analyzed using an unpaired student’s t-test (when comparing two groups; EAE mice vs. control mice) or a one-way ANOVA with Tukey’s post hoc test (when comparing greater than two groups in neuropeptide analyses) at individual time points. Multiple time point flow cytometry data were analyzed using two-way ANOVA (treatment; time) with Tukey’s post hoc test. Pain behavioral data sets analyzed using non-parametric statistical tests (hind paw and facial allodynia) are presented as box and whisker plots, where box limits show the first and third quartile, the center line is the median and the whiskers represent the minimum and maximum values. MGS, flow cytometry, and immunohistochemistry data are presented as arithmetic mean ± SEM. The level of significance was set as p < 0.05 for all analyses.

To investigate pain symptoms during the course of chronic EAE, mice were immunized with the MOG_35–55 peptide in CFA. Mice developed chronic EAE with typical disease onset between 10 and 13 days post-induction, a mean peak clinical score of 3 on day 16, and a gradual, partial recovery to a mean clinical score of 2 by day 32 post-induction. Control mice immunized with CFA alone did not develop any clinical signs of EAE over the 32-day monitoring period (Figure 1A). Clinical EAE was accompanied by a significant loss of body weight, which was most severe at time points corresponding to the EAE clinical peak (Figure 1B).

To assess stimulus-evoked pain, we first tested mechanical pain hypersensitivity in the hind paws (Figure 1C). Compared to controls, EAE mice showed reduced 50% PWTs during the pre-clinical stage of disease on days 8 and 10 post-induction (n = 6–15, p < 0.01). Corresponding to the onset of clinical EAE and accompanying hind limb motor impairment, the 50% PWTs of EAE mice showed a sharp rise on day 15 (clinical peak of disease) where PWTs rose above baseline levels and reached the imposed upper limit of von Frey testing in all animals (1.4 g; Figure 1C). Testing of mechanical pain hypersensitivity in the hind paws was terminated at disease peak due to paralysis. The restrictions imposed by hind limb motor confounds on continual testing of mechanical allodynia in clinical EAE prompted us to use a technique developed for testing mechanical pain in the face (19), which is not affected by motor deficits. We found that EAE mice were significantly more responsive to mechanical stimuli applied to the whisker pad than control mice over the entire course of chronic EAE, as seen on days 6, 8, 10, 17, and 30 (Figure 1D, n = 4–9, p < 0.05–0.001).

To assess spontaneous (as opposed to experimenter-evoked) pain in EAE, we utilized the MGS (Figure 2A), a standardized murine facial expression-based coding system (20). This allowed both continuous testing of pain behaviors during paralytic clinical disease, and the assessment of spontaneous pain in EAE, which has not yet been explored in the model. No change in mean MGS scores were seen in the preclinical period (day 8); however, the mean MGS scores in EAE mice were higher at day 16 (n = 7–8, p < 0.01) and day 30 (n = 7–8, p < 0.01) post-EAE induction as compared to control mice (Figure 2B).

Given the fact that oligodendrocyte damage in the absence of an innate or adaptive immune response is sufficient to confer central pain behaviors in mice (21), we next investigated whether oligodendrocyte changes are associated with the pain behaviors observed in our chronic EAE model. Flow cytometry was used to quantify changes in CNS myelination, as previously described (22). CD45^lowGALC + MOG^low premyelinating and CD45^lowGALC + MOG^high myelinating oligodendrocyte populations in the brain and SC were assessed over the course of chronic EAE. Mononuclear cells were first gated, followed by selection of live singlets (Figures 3A–C). CD45^low resident CNS cells positive for GALC were then gated (Figure 3D) and analyzed for changes in MOG positivity between control (Figure 3E) and EAE mice (Figure 3F).

No changes in myelination were found in the brain or SC at the preclinical stage of EAE (day 8 post-EAE induction, n = 5). At the clinical peak (day 16 post-induction), decreases in GALC+ MOG^high oligodendrocytes were seen, accompanied by a subsequent increase (n = 5, p < 0.0001) in the proportion of GALC+ MOG^low premyelinating oligodendrocytes in the brain (Figure 3G) and SC (Figure 3H). This was also apparent in the chronic phase of EAE (day 32 post-induction) in the brain (Figure 3G, n = 3, p < 0.01) and SC (Figure 3H, n = 3, p < 0.0001). Interestingly, despite the partial recovery typical of mice entering the chronic phase of EAE, this regain of hind limb movement was associated with progressive and continued loss of GALC+ MOG^high myelinating oligodendrocytes in the SC (Figure 3H, n = 3, p < 0.05).

The lack of a difference in oligodendrocyte subtypes in the preclinical stage suggests that CNS demyelination has not yet taken place at day 8 and does not correspond with the early emergence of allodynia in the sequelae of our EAE model.

To investigate changes in neuroinflammation, we first assessed infiltration of cells expressing CD4 (a marker expressed predominantly on the surface of helper T cells as well as minor populations of other immune cells) into the CNS and peripheral ganglia over the course of chronic EAE using flow cytometry. Mononuclear cells were gated (as in Figure 3A) and single cells selected (as in Figure 3B), before gating viable cells (as in Figure 3C) for inclusion in further analysis for CD4 positivity in control and EAE mice (Figures 4A,B, respectively). Results showed that CD4+ cell infiltration was not apparent in the preclinical period, yet was clearly seen at the EAE clinical peak in all tissues analyzed including the brain, SC, trigeminal tissue, and DRG (Figures 4C–F, n = 4–6). CD4+ cells were also increased in the brain and SC in the chronic phase of the disease in EAE mice compared to control animals (Figures 4C,D, n = 6). A trend for increased CD4+ cells was seen in the trigeminal tissue of EAE mice compared to control animals in the chronic phase of disease, although this failed to reach significance (Figure 4E, n = 6, p = 0.0559). In the brain and DRG, CD4+ cells were reduced in the chronic phase compared to the EAE clinical peak (Figures 4C,F, n = 5–6). The absence of significant CD4+ cell infiltration at day 8 suggests that the preclinical allodynia observed in EAE mice is unlikely to arise from a central adaptive immune response.

To determine whether an early response mediated by immune activation is associated with the preclinical allodynia observed in the hind paw and whisker pad, immunostaining for IBA-1 (macrophage/microglia marker) and GFAP (astrocyte marker) was carried out. Areas analyzed for IBA-1 included the dorsal (DHSC) and ventral (VHSC) horns of the L3–5 SC (Figure 5A), STN (Figure 5B), TG, TN, and TREZ (Figure 5C). There was no difference between EAE and control mice in IBA-1+ immunoreactivity in any of the tissue regions analyzed, except for an increase in IBA-1 expressing cells in the dorsal horn of the SC of EAE mice compared to control mice (Figures 5D,E,H, n = 4–5, p < 0.05). Analysis of GFAP expression in the dorsal and ventral horns of the SC revealed no differences in astrocyte activation between EAE and control mice (Figure 5I, n = 4–5).

Since flow cytometric analysis of the CNS and peripheral ganglia showed no increase in CD4+ cells at day 8 (Figure 4), immunostaining for CD3+ T cells was carried out with a focus on specific regions of the trigeminal afferent pathway (Figure 5C). This was done to ascertain whether the preclinical facial allodynia of EAE mice may be linked to a subtle early T cell infiltration into the peripheral trigeminal afferent pathway occurring prior to more overt central infiltration. A small but significant increase in CD3+ cells was found restricted to the TN (Figures 5F,G,J, n = 4–5, p < 0.01), and a similar trend, though not statistically significant, was seen in the TG (Figure 5J, n = 4–5).

Immunohistochemical analysis to characterize changes in activation of macrophages/microglia in the SC and trigeminal afferent pathway and astrocytes in the SC at the clinical peak of EAE was also conducted. A substantial increase (n = 4–5, p < 0.0001) in IBA-1 expressing cells in both the ventral (Figures 6A,B,Q) and dorsal horns (Figures 6C,D,Q) of the SC in EAE mice compared to control mice was found. An increase in IBA-1 immunoreactivity was also apparent in the TN (Figures 6E,F,Q, n = 4–5, p < 0.05) and TREZ (Figures 6G,H,Q, n = 4–5, p < 0.05). GFAP+ cells were similarly increased in the ventral (Figures 6I,J,R, n = 4–5, p < 0.001) and dorsal (Figures 6K,L,R, n = 4–5, p < 0.001) horns of the SC in EAE mice compared to control mice.

Since T cell infiltration into the CNS at the EAE clinical peak has been demonstrated by numerous studies and by our flow cytometric analysis (Figure 4), we carried out immunohistochemistry for CD3 in the trigeminal afferent pathway and L3–5 DRGs. We found CD3+ cells in increased numbers within the TG (Figures 6M,N,S, n = 4–5, p < 0.05) and TREZ (Figures 6O,P,S, n = 4–5, p < 0.001) in EAE mice compared to control mice. A trend for increased CD3+ cells in the TN was also found, although this failed to reach significance (Figure 6S, n = 4–5). Immunostaining for CD3+ cells in the DRG showed no significant difference in T cell numbers between EAE and control mice (Figure 6S, n = 4–5).

As significant changes in T cell infiltration were observed in the TG in clinical EAE (day 16), we next examined whether ATF-3 (a marker of neurons with damaged primary afferents) is induced in peptidergic (CGRP+) and non-peptidergic (IB4+) small diameter neurons, and NF200+ large diameter myelinated neurons using immunohistochemistry. This was of interest as injured DRG neurons have been linked to pain behaviors in mice with peripheral nerve injury (23) and rats with brachial plexus avulsion (24). We found a small, though significant, increase in the percentage of total ATF-3 immunopositive cell bodies in the TG of EAE (1% ± 0.2) compared to control (0.1% ± 0.05) mice (Unpaired Student’s t-test, n = 4–5, p < 0.01). Further analysis revealed ATF3+ neurons present in EAE mice (Figure 7B), but not controls (Figure 7A), predominantly co-expressed NF200. An increase in NF200+ATF-3+ cell bodies compared to CGRP+ATF-3+ (n = 4–5, p < 0.0001), and IB4+ATF-3+ (n = 4–5, p < 0.01) cell bodies was seen in the TG of mice at the clinical peak of EAE (Figure 7C) suggesting that neuronal damage is selective for myelinated NF200+ neurons.