C57BL/6J (B6) mice were purchased from the Jackson Laboratory (Bar Harbor, ME). B6.129P-Hrh1tm1Wat (H1RKO) [[51]], B6.129P-Hrh2tm1Wat (H2RKO) [[52]], B6.129P2-Hrh3tm1Twl (H3RKO) [[53]], and B6.129P-Hrh4tm1Thr (H4RKO)

mice (generated by Lexicon Genetics, Woodlands Park, TX) [[54]] were maintained at the University of Vermont (Burlington, VT). All strains were backcrossed to the C57BL/6J background for at least 10 generations. Individual HRKO mice were interbred and the resulting F1 mice were intercrossed together to generate H1H2RKO and H3H4RKO mice. The experimental procedures used in this study were approved by the Animal Care and Use Committee of the University of Vermont. Mice were immunized for the induction of EAE using a 2× immunization protocol. The animals were injected subcutaneously in the posterior right and left flank with a sonicated phosphate-buffered saline (PBS)/oil emulsion containing 100 μg of MOG35–55 and

PS-341 molecular weight CFA (Sigma-Aldrich, St. Louis, MO) supplemented with 200 μg of Mycobacterium tuberculosis H37Ra (Difco Laboratories, Detroit, MI). One week later, all mice received an identical injection of MOG35–55-CFA [[31]]. Mice were ranked scored daily for clinical quantitative trait variables beginning at day 5 after injection as follows: 0, no clinical expression of disease; 1, flaccid tail without hind limb weakness; 2, hind limb weakness; 3, complete hind limb paralysis and floppy tail; 4, hind leg paralysis accompanied Silmitasertib by a floppy tail and urinary or fecal incontinence; 5, moribund. Assessments of clinical quantitative trait variables were performed as previously described [[31]].

Histopathological evaluations were done as previously described [[55]]. Briefly, brains and spinal cords were dissected on 30th day postimmunization, from calvaria and vertebral columns, respectively, and fixed by immersion in 10% phosphate-buffered formalin (pH 7.2). After fixation, trimmed and representative transverse section-embedded in paraffin and mounted on glass slides. Sections were stained with hematoxylin and eosin for routine evaluation and Luxol fast blue-periodic Carnitine palmitoyltransferase II acid-Schiff reagent for demyelination. Representative areas of the brain and spinal cords were selected for histopathological evaluation. The following components of the lesions were assessed: (i) severity and extent of the lesion; (ii) extent and degree of myelin loss and tissue injury (swollen axon sheaths, swollen axons, and reactive gliosis); (iii) severity of the acute inflammatory response (predominantly neutrophils); and (iv) severity of the chronic inflammatory response (lymphocytes/macrophages). Lesions in the brain and spinal cord (SC) were evaluated separately and assigned a numerical score based on a subjective scale ranging from 0 to 5. A score of 0 indicates no lesions; 1 indicates minimal; 2, mild; 3, moderate; 4, marked; and 5, severe lesions. BBB permeability was assessed as previously described [[56]].