Ketamine

Under anaesthesia (a mixture medetomidine 0.25 mg/kg [Pfizer] and ketamine 60 mg/kg [Boehringer-Ingelheim]), a small laminectomy was performed at the sixth thoracic vertebra and a flexible cannula was inserted under the dura mater and glued in place, such that the tip is rested at the lumbar enlargement of the spinal cord. The opposite end of the cannula was placed subcutaneously, and an osmotic minipump (ALZET, Charles River Laboratories) was connected to the cannula [9 (link), 11 (link)]. For the first pharmacological experiment, the P2X7 receptor antagonist A-438079 (50 μg/12 μL/day) or saline was delivered for 8 days (from 1 day before to 7 days post-immunization). For the second experiment, rats received the irreversible CatS inhibitor LHVS (30 nmol/12 μL/day) or vehicle (20 % Cremophor EL/saline) for 14 days beginning 1 day before and until day 13 post-immunization. Doses of A-438079 and LHVS were based on previous studies ([29 (link), 30 (link)], respectively).

At the end of the study period, EAE mice were deeply anesthetized with an i.p. injection of ketamine (10 mg ml⁻¹, Boehringer Ingelheim) plus xylazine (1.17 mg ml⁻¹, Bayer) and transcardially perfused with saline (0.9% NaCl + 5 M EDTA) for 5 min followed by 4% PFA for an additional 5 min. The spinal columns were isolated and post-fixed in 4% PFA at 4 °C overnight. The next day, the spinal cords were extracted, washed in 1× PBS and immersed in a 30% sucrose (in PBS) cryoprotectant solution for at least 72 h at 4 °C. The spinal cords were then embedded in optimum cutting temperature medium in a plastic mould and frozen on 2-methylbutane (Thermo Fisher Scientific) on dry ice. The tissue blocks were cryo-sectioned (30 μm for Cx3cr1-YFP^creERT2R26^tdTomato EAE mice, 10 μm for all of the other EAE experiments) using a cryostat (Leica, CM1850) with a microtome blade (Feather) onto Superfrost Plus slides (Thermo Fisher Scientific). The sections were stored at −80 °C until use.
Human tissue was obtained from the Multiple Sclerosis and Parkinson’s Tissue Bank (Imperial College London) under ethical approval (IRAS reference: 279989). Autopsy flash-frozen brain tissue from one case with secondary progressive MS (44 years old, male) was cryo-sectioned with a 10 μm thickness using a cryostat (CM1850, Leica) with a microtome blade (A35, Feather). The sections were stored at −80 °C until use.

The chocolate bars were elaborated with milk-chocolate pellets of 33% cocoa (Vanleer, Barry Callebaut, Zurich, CH). The fish oil (Omega Pure^®) was purchased from America Alimentos S.A. de C.V. (Zapopan, Jalisco, MX). Probiotic strains Lactobacillus plantarum 299v (DSMZ 9843) and Lactobacillus rhamnosus GG (ATCC 7469) were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, DE) and American Type Culture Collection (ATCC, Manassas, VA, US), respectively. Sodium alginate was purchased from DEIMAN (CDMX, MX) and maltodextrin food grade from Best Ingredients (Monterrey, NL, MX). MRS agar was obtained from BD Difco™ (NJ, US). For the euthanasia of rats, a mix of Ketamine (Boehringer Ingelheim, JC, MX) and Xylazine (PROCIN^®, PiSA, JC, MX.) were utilized.