Analysis of TIMP family mRNA levels during EAE. RNase protection assay was used to examine the expression of TIMP genes in samples of spinal cord from CFA-treated WT (black bars) or TIMP-1^−/− mice and during acute or chronic phases of MOG-induced EAE. The level of individual TIMP mRNAs was corrected for loading relative to the ribosomal gene L32, and the result presented as arbitrary units. Data are presented as mean + SEM of n = 3 per genotype per treatment for each gene. Statistical comparisons made using two-way analysis of variance (95% confidence), followed by Bonferroni posttests for significance (***P < 0.001 versus CFA, chronic, and all TIMP-1^−/− time points).

Analysis of MOG_35-55-specific T-cell responses during EAE. Scatterplot analysis of cellular infiltrates isolated from the spinal cords of CFA or MOG_35-55-immunized WT and TIMP-1^−/− mice. Ex vivo stimulation of pooled isolated cells with MOG_35-55 peptide identified antigen-specific CD4⁺ T-cell (x axis) responses by the evoked production of interferon-γ (y axis) as measured by flow cytometry. Data are representative of n = 3 to 4 per group.

Enhanced and persistent macrophage/microgliosis in spinal cords of TIMP-1^−/− mice after EAE. A: Immunohistochemical detection of the macrophage/microglial marker Iba-1 in the spinal cords of WT C57BL/6 mice or TIMP-1^−/− mice treated with CFA or during the acute or chronic phases of EAE after MOG_35-55 immunization, as indicated. During the acute phase of EAE, the boundary of infiltrating cell types is demarcated by the dotted black line relative to region of predominant macrophage/microglial reactivity. B: Flow cytofluorometric analysis of mononuclear leukocytes isolated from spinal cords of WT and TIMP-1^−/− mice reveal the dramatic expansion/infiltration of CD11b⁺/MHCII⁺ macrophage/microglial cells into the spinal cord during the acute and chronic phases of EAE. Note the enhanced proportion of CD11b⁺ cells remaining (top left quadrant, chronic phase) in the TIMP-1^−/− mice relative to EAE-WT mice.

EAE severity in TIMP-1^−/− and WT mice. A: Daily evaluation of EAE scores in MOG_35-55-immunized (EAE) or control-treated (CFA) mice revealed comparable progression of EAE in TIMP-1^−/− mice (n = 22) when compared with WT mice (n = 22). Mice treated with CFA (n = 8 per genotype) did not exhibit any signs of EAE. B: Analysis of peak EAE scores among MOG-immunized TIMP-1^−/− or WT mice indicated a similar overall severity of EAE within each group as indicated by the numbers of animals developing EAE at each clinical grade. C: Daily analysis of weight revealed weight loss coincident with inflammation among all MOG-immunized mice during the acute phase and recovery of this loss during the chronic phase of EAE. D: Comparison of the proportions of mortality, incidence-developed EAE (grade, >1), and animals that did not develop EAE (grade, <1) among all MOG_35-55-immunized mice of either WT or TIMP-1^−/− genotype. Data in A and B are presented as mean + SEM for each treatment group for each time point with statistical comparisons made using two-way analysis of variance (95% confidence), followed by Bonferroni posttests for intergroup determinations of significance; *P < 0.05, **P < 0.01, ***P < 0.001.

Evaluation of spinal cord myelination and myelin integrity during EAE. Spinal cord samples were analyzed by histochemical staining for LFB and immunohistochemistry for MBP in spinal cord cross sections of WT (A, C, E, G, I, K) or TIMP-1^−/− mice (B, D, F, H, J, L) that had been either CFA-treated or MOG_35-55-immunized (as indicated). A–F: Representative LFB staining for myelin in spinal cord sections from CFA or EAE-treated, WT, or TIMP-1^−/− mice revealed significant disruption of myelin in the anterior column area of the spinal cord during the acute phase of EAE. G–L: Immunohistochemistry for MBP validated the observed changes in spinal cord myelination evaluated by LFB staining and determined that during the chronic phase of EAE myelin in the spinal cords of TIMP-1^−/− mice was reduced (K, L) relative to WT mice. Nuclei, stained with 4,6-diamidino-2-phenylindole, also demonstrate in the TIMP-1^−/− mice an increase in the cellularity of the spinal cord during EAE (J, L). M: Quantification of MBP staining densities (as described in Materials and Methods) in WT and TIMP-1^−/− mice and during the acute and chronic phases of EAE. Statistical analyses made using analysis of variance, using evaluation of n = 3 per group CFA and acute EAE; n = 6 per group chronic EAE, followed by Bonferroni posttests for significance: *P < 0.05, CFA WT versus CFA TIMP-1^−/−; **P < 0.01; ***P < 0.001 versus CFA treatment of same genotype.

Enhanced dissemination of cellular infiltrates into brains of MOG_35-55-immunized TIMP-1^−/− mice. LFB staining of myelin in the cerebellum of WT and TIMP-1^−/− mice that were either treated with CFA or EAE. Although WT mice exhibited little myelin disruption within the brain during EAE (C), disorder of myelin in the brain of TIMP-1^−/− mice was apparent and remained throughout the 52-day experimental endpoint (ie, chronic phase) (D). A and B: No apparent differences were noted in the myelination of adult brains from CFA-treated WT or TIMP-1^−/− mice. Immunohistochemical detection of infiltrating T cells (αCD3⁺) in CFA-treated mice (E, F) revealed sparse cellular staining, whereas in WT mice, perivascular CD3⁺ cells were occasionally observed (G). In contrast, there were many CD3⁺ cells within the CNS parenchyma of TIMP-1^−/− mice 52 days after induction of EAE (H). I: Contour plots of flow cytometric analysis of antigen (MOG_35-55)-specific immune responses of cells isolated from the brains of mice. Data represent n = 3 per group per time point and demonstrate increased proportions of CD4⁺ T cells (x axis) during the acute phase of EAE in TIMP-1^−/− mice. Red text denotes percentage of lymphocytes isolated that were CD4⁺ cells. Ex vivo stimulation with MOG_35-55 peptide reveal a small proportion of antigen-specific T cells via production of IFN-γ (y axis) that was increased in TIMP-1^−/− mice.

Enhanced macrophage/microglial reactivity in brains of TIMP-1^−/− mice. Immunohistochemistry for the macrophage/microglial marker Iba-1 revealed scattered resting microglia in the cerebella of CFA-treated WT (A) or TIMP-1^−/− mice (B). During the chronic phase of EAE (52 days after MOG_35-55 immunization), clusters of Iba-1⁺ cells were noted in WT animals (C), whereas widespread intense Iba-1⁺ staining was observed in TIMP-1^−/− mice (D). Astrocyte labeling with GFAP revealed a band of strong GFAP⁺ cells surrounding the perimeters of the cerebellar commissure (dotted black line). During the chronic phase of EAE, widespread GFAP⁺ staining was observed in the cerebellar commissure of WT mice (G) but not TIMP-1^−/− mice (H). I–L: High-magnification images of GFAP staining in regions marked by red boxes in plates E–H. M: Cell sorting (FACS) analysis of CD11b⁺/MHCII⁺ macrophage/microglia isolated from the brains of CFA-treated or MOG_35-55-immunized mice during the acute or chronic phases of EAE, as indicated. Note the increased proportion of CD11b⁺ cells (top left quadrant, chronic phase) in TIMP-1^−/− during chronic EAE compared with WT mice at this same phase of disease. Analysis was performed on pooled samples, n = 3 to 4 per group per time point.