The powerful neuroprotective action of C1-inhibitor on brain ischemia-reperfusion injury does not require C1q

Abstract

C1-inhibitor (C1-INH) is a major regulator of the complement classical pathway. Besides this action, it may also inhibit other related inflammatory systems. We have studied the effect of C1-INH in C57BL/6 mice with focal transient brain ischemia induced by 30 minutes of occlusion of the middle cerebral artery. C1-INH induced a dose-dependent reduction of ischemic volume that, with the dose of 15 U/mouse, reached 10.8% of the volume of saline-treated mice. Four days after ischemia the treated mice had significantly lower general and focal neurological deficit scores. Fluoro-Jade staining, a marker for neuronal degeneration, showed that C1-INH-treated mice had a lower number of degenerating cells. Leukocyte infiltration, as assessed by CD45 immunostaining, was also markedly decreased. We then investigated the response to ischemia in C1q(-/-) mice. There was a slight, nonsignificant decrease in infarct volume in C1q(-/-) mice (reduction to 72.3%) compared to wild types. Administration of C1-INH to these mice was still able to reduce the ischemic volume to 31.4%. The study shows that C1-INH has a strong neuroprotective effect on brain ischemia/reperfusion injury and that its action is independent from C1q-mediated activation of classical pathway.

Figures

Figure 1

Infarct volume assessed 24 hours after ischemia in mice receiving saline (SAL), different doses of C1-INH at the beginning of ischemia (pre), or 15 U at reperfusion (post). Data are expressed as mean ± SEM (n = 6 to 9 mice per group). **, P < 0.01 versus saline, Dunnett test.

Figure 2

General and focal neurological deficit score evaluation (0 to 28) assessed daily in ischemic mice treated with 15 U of C1-INH at the beginning of ischemia (black dots, n = 8) or with saline (white dots, n = 9). Data are expressed as median and 25th to 75th percentiles. Differences between curves were evaluated by two-way analysis of variance for repeated measures. Differences between scores at a given time were evaluated by Fisher’s exact test. **, P < 0.01.

Figure 3

Fluoro-Jade labeling of degenerating neurons in different brain areas of representative ischemic mice 4 days after ischemia. Clusters of Fluoro-Jade-positive neurons amid a necrotic tissue can be observed in the cortex of saline-treated mice (A), whereas only a few scattered fluorescent cells can be found in the same brain area of mice treated with C1-INH (B). Several groups of positive cells can be observed in dentate gyrus of saline-treated (C), but not C1-INH-treated mice (D). Scale bars, 100 μm.

Figure 4

Leukocyte infiltrating the hypothalamic area in ischemic mice treated with saline (A) or C1-INH (B) as in Figure 2, 24 hours after ischemia. Scale bar, 100 μm. Insets show CD45-positive cells at higher magnification.

Figure 5

Infarct volume assessed 24 hours after ischemia in C1q−/− mice receiving saline (SAL) or 15 U of C1-INH at the beginning of ischemia. For comparison the ischemic volume of wild types (C57BL/6 mice) is also reported (black column, see Figure 1). Data are expressed as mean ± SEM (n = 7 to 10 mice per group). **, P < 0.01 versus wild types (black column); ○, P < 0.05 versus C1q−/− (gray column), Tukey-Kramer test.