Fingolimod reduces neuropathic pain behaviors in a mouse model of multiple sclerosis by a sphingosine-1 phosphate receptor 1-dependent inhibition of central sensitization in the dorsal horn

Abstract

Multiple sclerosis (MS) is an autoimmune-inflammatory neurodegenerative disease that is often accompanied by a debilitating neuropathic pain. Disease-modifying agents slow down the progression of multiple sclerosis and prevent relapses, yet it remains unclear if they yield analgesia. We explored the analgesic potential of fingolimod (FTY720), an agonist and/or functional antagonist at the sphingosine-1-phosphate receptor 1 (S1PR1), because it reduces hyperalgesia in models of peripheral inflammatory and neuropathic pain. We used a myelin oligodendrocyte glycoprotein 35 to 55 (MOG35-55) mouse model of experimental autoimmune encephalomyelitis, modified to avoid frank paralysis, and thus, allow for assessment of withdrawal behaviors to somatosensory stimuli. Daily intraperitoneal fingolimod reduced behavioral signs of central neuropathic pain (mechanical and cold hypersensitivity) in a dose-dependent and reversible manner. Both autoimmune encephalomyelitis and fingolimod changed hyperalgesia before modifying motor function, suggesting that pain-related effects and clinical neurological deficits were modulated independently. Fingolimod also reduced cellular markers of central sensitization of neurons in the dorsal horn of the spinal cord: glutamate-evoked Ca signaling and stimulus-evoked phospho-extracellular signal-related kinase ERK (pERK) expression, as well as upregulation of astrocytes (GFAP) and macrophage/microglia (Iba1) immunoreactivity. The antihyperalgesic effects of fingolimod were prevented or reversed by the S1PR1 antagonist W146 (1 mg/kg daily, i.p.) and could be mimicked by either repeated or single injection of the S1PR1-selective agonist SEW2871. Fingolimod did not change spinal membrane S1PR1 content, arguing against a functional antagonist mechanism. We conclude that fingolimod behaves as an S1PR1 agonist to reduce pain in multiple sclerosis by reversing central sensitization of spinal nociceptive neurons.

Figure 1.. Pain-related hypersensitivity peaks before neurologic motor deficits in EAE mice.

Line graphs describing neuromuscular function (Clinical score) and pain-like behavioral responses at baseline (BL) and after sham injection of adjuvant (CFA and pertussis toxin) or after induction of EAE in C57BL/6 mice with MOG_35–55. (A) Neuromuscular deficits included hanging tail, impaired righting, and/or hindlimb paresis in MOG_35–55 (closed circles) but not sham (open circles) mice. (B) Mechanical sensitivity to hindlimb application of von Frey hairs in MOG_35–55 and sham mice. (C) Cold sensitivity to hindlimb application of acetone in MOG_35–55 and sham mice. Arrows indicate days of MOG injections. Data represent mean ± SEM from 5–7 mice per group. BL= Baseline. *P<0.05.

Figure 2.. Antihyperalgesic effects of fingolimod are reversible and repeatable.

(A) Mechanical and cold (B) hypersensitivity from baseline (BL) through 52 d post-EAE induction with MOG_35–55. Fingolimod (FTY720) was injected daily (i.p.) from D15 to 34 and from D45 to 52 (yellow). Arrows indicate days of MOG injections. Data represent mean ± SEM. N = 3–4 for vehicle and 0.1 mg/kg groups and 4–6 for 1 mg/kg group. *P<0.05, **P<0.01, †P<0.001 by t-test compared to vehicle.

Figure 3.. Fingolimod dose-dependently decreases hyperalgesia before it decreases motor deficits.

Line graphs describing (A) neuromuscular deficits (B) mechanical sensitivity and (C) cold sensitivity from baseline (BL) through 37 days post-EAE induction with MOG_35–55. The 2^nd MOG_35–55 injection on D2 is not shown. Fingolimod (FTY720) was injected daily (i.p.) from D 15 to 37 (yellow). Histograms describing effect of fingolimod doses on D21 (D, E, F) and D30 (G, H, I). Arrows indicate days of MOG injections. Data represent mean ± SEM from 4–7 mice per drug treatment group. *P<0.05, **P<0.01, ***P<0.001 compared to vehicle controls.

Figure 4.. Fingolimod prevents the EAE-induced potentiation of glutamate-evoked Ca²⁺ signaling.

(A) Concentration-dependence of glutamate-evoked Ca²⁺ responses in dorsal horn slices from MOG_35–55-treated and sham mice. Calcium level (expressed as ΔF340/F380 as a function of increasing glutamate concentration. (B) Glutamate-evoked Ca²⁺ responses in EAE mice treated with vehicle or fingolimod (1 mg/kg for 7–10 days). Data represent mean ± SEM. N = 7 in vehicle groups, 8 in MOG_35–55 + fingolimod group and 9 in MOG_35–55 and sham group. *P<0.05, **P<0.01.

Figure 5.. Fingolimod dose-dependently inhibits stimulus-evoked pERK in dorsal horn neurons via S1PR1 in EAE mice.

(A) Representative dorsal horn photomicrograph of NeuN and pERK immunoreactivity taken from a vehicle-treated EAE mouse ipsilateral to light touch stimulation. Enlarged images of (B) NeuN, (C) pERK and (D) NeuN/pERK merged. (E) Histogram describing the number of pERK-positive profiles at the side ipsilateral (I) and contralateral (C) to light touch hind-paw stimulation. Fingolimod (FTY720) was injected daily (i.p.) from D15–37 post-EAE induction at the doses shown and then processed for immunohistochemistry described in Methods. (F-G) Representative images of pERK immunoreactivity in ipsilateral dorsal horn from mice that received (F) vehicle or (G) 1 mg/kg fingolimod. (H) Histogram describing the number of pERK-positive profiles after daily administration of vehicle, fingolimod, W146 or fingolimod + W146. Representative images of pERK immunoreactivity from mice treated with (I) vehicle, (J) fingolimod, (K) W146 or (L) fingolimod + W146. Data represent mean ± SEM from 5–12 mice. *P<0.05, ***P<0.001. (M) Scatter plot shows detectable W146 concentrations in brain at 5 – 20 min after retro-orbital intravenous injection of W146. “Naïve-blank” mice did not receive W146, while “Naïve-W146” and “EAE-W146” mice did.

Figure 6.. Fingolimod decreases EAE-induced activation of astrocytes and microglia.

Fingolimod (FTY720) was injected daily (i.p.) from D16 to 37 post-EAE induction and then processed for immunohistochemistry described in Methods. Lumbar L4-L5 spinal cord sections were stained with primary antibodies against (A-C) GFAP and (D-F) Iba1. (A) GFAP immunoreactivity in sham controls or MOG_35–55-treated mice after daily administration of FT720 as indicated. Representative images show GFAP staining in either (B) vehicle, or (C) fingolimod-treated mice. (D) Iba1 immunoreactivity in sham controls or MOG_35–55-treated mice after daily administration of FT720 as indicated. Representative images show Iba1 staining in (E) vehicle or (F) fingolimod-treated mice. Data represent mean ± SEM from 4–6 mice per group. *P<0.05, **P<0.01 compared to MOG_35–55–treated animals that received vehicle (0 mg/kg fingolimod, red bar). Scale bars = 100 um.

Figure 7.. S1PR1 antagonist W146 prevents fingolimod-induced attenuation of hyperalgesia.

(A-B) Fingolimod (FTY720) and/or W146 were injected daily (i.p.) from D27 to 41 (yellow). (A) Mechanical and (B) cold hyperalgesia were monitored prior to and through 41 days post-EAE induction. n = 6–7. (C-D) fingolimod was injected daily (i.p.) from D14 to 34 (yellow); fingolimod and W146 were co-administered D35 to 44. (C) Mechanical and (D) cold hyperalgesia were monitored prior to and through 44 days post-EAE induction (n = 4–11). Arrows indicate days of MOG injections. Data represent mean ± SEM. *P<0.05 vehicle vs. fingolimod, †P<0.05 fingolimod alone vs. fingolimod + W146.

Figure 8.. Antihyperalgesic effects of repeated and single dosing of fingolimod are mimicked by the S1PR1-selective agonist, SEW2871.

(A) Motor function, (B) Mechanical and (C) cold hyperalgesia were monitored prior to and through 56 days post-EAE induction. Fingolimod (FTY720), SEW2871 or vehicle were injected daily (i.p.) from D16 to 43 (yellow; n = 5–6). On D44, drug administration was discontinued. Once behavioral scores returned to pre-treatment levels, mice were given a single injection as indicated and (D) Mechanical and (E) cold hyperalgesia were monitored acutely (n = 8–10). Data represent mean ± SEM. *P<0.05 fingolimod compared to MOG_35–55–treated animals that received vehicle. †P<0.05 SEW2871 compared to vehicle.

Figure 9.. S1PR1 content after fingolimod administration.

Representative blots of S1PR1 levels from (A) Naïve and (B) EAE mice. For vehicle and fingolimod-treated mice, 2–3 samples are shown for both S1PR1 and actin. Relative S1PR1 protein expression comparing vehicle to fingolimod-treated mice from naïve (left side) and EAE mice (right side) from (C-D) membrane, (E-F) cytosol and (G-H) total homogenates. Data represent mean ± SEM. *P<0.05 fingolimod compared to MOG_35–55–treated animals that received vehicle. Supplemental Digital Content 1, which demonstrates the lack of difference in responses in vehicle for fingolimod and W146.