Molecular and spatial profiling of multiple sclerosis lesions identifies dysfunctional lipid efflux as a driver of chronic active inflammation

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8-week-old male Cx3cr1CreERT2/+; Abca1flox/flox; Abcg1flox/flox experimental animals (KO) and Cx3cr1CreERT2/+ controls (Ctrl) were used for EAE experiments. Myelin oligodendrocyte glycoprotein (MOG)-EAE was induced in mice purchased from Charles River Laboratories by subcutaneous immunization with 200 mg of MOG peptide 35–55 (MOG35–55) in complete Freund’s adjuvant (Mycobacterium tuberculosis at 3.75 mg ml−1; BD) and intraperitoneal injection twice with 500 ng of pertussis toxin (Sigma). Animals were examined daily and scored for clinical signs of disease. If disease did not start within 15 d of induction or the clinical score rose above 4, animals were excluded from the analysis. Clinical scores were defined as: 0, normal; 0.5, loss of tail tip tone; 1, loss of tail tone; 1.5, ataxia and mild walking deficits (slip off the grid); 2, mild hind limb weakness, severe gait ataxia and twist of the tail causing rotation of the whole body; 2.5, moderate hind limb weakness and inability to grip the grid with the hind paw but ability to stay on an upright tilted grid; 3, mild paraparesis and falls from an upright tilted grid; 3.5, paraparesis of the hind limbs (legs strongly affected but move clearly); 4, paralysis of the hind limbs and weakness in the forelimbs; 4.5, forelimbs paralyzed; 5, moribund or dead. Tamoxifen administration commenced 4 weeks before immunization. Mice were deeply anesthetized and perfused with cold PBS. The spinal cord was removed from vertebrae and cervical and thoracal parts (C1-Th10) of the spinal cord were used for scRNA-seq experiments. Cells were isolated with a previously established isolation protocol using gentleMACS with the Papain Neural Tissue Dissociation Kit (Miltenyi Biotec) and actinomycin D (catalogue no. A1410, Sigma-Aldrich) at a final concentration of 45 mM. After dissociation, myelin debris was removed from the cell suspension using Myelin Removal Beads II (Miltenyi Biotec). Cells were resuspended in 0.04% BSA + PBS and counted using an automated cell counter (TC20, Bio-Rad).