Immunologic tolerance to myelin basic protein decreases stroke size after transient focal cerebral ischemia

Abstract

Immune mechanisms contribute to cerebral ischemic injury. Therapeutic immunosuppressive options are limited due to systemic side effects. We attempted to achieve immunosuppression in the brain through oral tolerance to myelin basic protein (MBP). Lewis rats were fed low-dose bovine MBP or ovalbumin (1 mg, five times) before 3 h of middle cerebral artery occlusion (MCAO). A third group of animals was sensitized to MBP but did not survive the post-stroke period. Infarct size at 24 and 96 h after ischemia was significantly less in tolerized animals. Tolerance to MBP was confirmed in vivo by a decrease in delayed-type hypersensitivity to MBP. Systemic immune responses, characterized in vitro by spleen cell proliferation to Con A, lipopolysaccharide, and MBP, again confirmed antigen-specific immunologic tolerance. Immunohistochemistry revealed transforming growth factor beta1 production by T cells in the brains of tolerized but not control animals. Systemic transforming growth factor beta1 levels were equivalent in both groups. Corticosterone levels 24 h after surgery were elevated in all sham-operated animals and ischemic control animals but not in ischemic tolerized animals. These results demonstrate that antigen-specific modulation of the immune response decreases infarct size after focal cerebral ischemia and that sensitization to the same antigen may actually worsen outcome.

Figure 1

Delayed-type hypersensitivity reactions. The degree of ear swelling in MBP-immunized animals 48 h after rechallenge with MBP. Animals were orally tolerized to MBP (OT-MBP), orally tolerized to OVA, an irrelevant antigen (OT-OVA), or were not tolerized at all (control). ∗, significant difference from control and OT-OVA at P < 0.05 as determined by one way ANOVA with post-hoc Dunnett’s test.

Figure 2

Measurement of infarct size at five predetermined coronal levels 24 h after ischemia in OT-MBP (n = 7) and control (n = 6) animals as detected by triphenyl tetrazolium chloride staining. Groups differ from each other as determined by repeated measures ANOVA (P < 0.05).

Figure 3

Infarct size, measured on cresyl violet-stained sections, 96 h after MCAO. Infarct size, corrected for the presence of edema, is significantly less in OT-MBP animals (n = 8) as compared with control animals (n = 9). Groups differ from each other as determined by repeated measures ANOVA (P < 0.05).

Figure 4

Stimulation indices to MBP in animals 4 days after MCAO or sham surgery (OT-MBP, n = 5; control, n = 4). Experimental groups are compared with naive animals (n = 11) and significance set at P < 0.05 (∗) as determined by one-way ANOVA with post-hoc Dunnett’s test.

Figure 5

Corticosterone levels 24 h after MCAO (OT-MBP, n = 5; control, n = 8) or sham surgery (OT-MBP, n = 7; control, n = 6). Experimental groups are compared with naive animals (n = 6) and significance set at P < 0.05 (∗) as determined by one way ANOVA with post-hoc Dunnett’s test.

Figure 6

(A) T cells in orally tolerized animals (OT-MBP) are immunopositive for both OX-52, a pan T cell marker, and TGF-β1. OX-52 is detected with fluorescein isothiocyanate and TGF-β1 is detected with Texas red. Cells immunopositive for both markers fluoresce yellow (arrowhead). (B) T cells in control animals are not immunopositive for TGF-β1. There are distinct populations of OX-52-positive cells (fluorescein isothiocyanate, straight arrow) and TGF-β1-positive cells (Texas Red, arrowhead).