Membrane attack complex inhibitor CD59a protects against focal cerebral ischemia in mice

Abstract

Background: The complement system is a crucial mediator of inflammation and cell lysis after cerebral ischemia. However, there is little information about the exact contribution of the membrane attack complex (MAC) and its inhibitor-protein CD59.

Methods: Transient focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) in young male and female CD59a knockout and wild-type mice. Two models of MCAO were applied: 60 min MCAO and 48 h reperfusion, as well as 30 min MCAO and 72 h reperfusion. CD59a knockout animals were compared to wild-type animals in terms of infarct size, edema, neurological deficit, and cell death.

Results and discussion: CD59a-deficiency in male mice caused significantly increased infarct volumes and brain swelling when compared to wild-type mice at 72 h after 30 min-occlusion time, whereas no significant difference was observed after 1 h-MCAO. Moreover, CD59a-deficient mice had impaired neurological function when compared to wild-type mice after 30 min MCAO.

Conclusion: We conclude that CD59a protects against ischemic brain damage, but depending on the gender and the stroke model used.

Figure 1

Comparison of infarct volume and brain swelling of CD59a-deficient and wild-type-mice (C57BL/6) after 60 min MCAO. Infarct volumes (A, C, E), and brain swelling (B, D, F), given as box-and-whisker plots, at 48 h of reperfusion after induction of ischemia are visualized for both, CD59a-deficient (CD59a-/-) and wild-type (WT) mice, of both genders (A, B) (n = 22_{CD59a-/- mix}; n = 16_{WT mix}) as well as male (C, D) (n_{CD59a-/- m} = 11; n_{WT m} = 6) and female (E, F) (n_{CD59a-/- f} = 11; n_{WT f} = 10) mice separately. Statistical analysis was performed using the Mann-Whitney-U-test. The indirect infarct volume was calculated as the volume of the difference between contralateral hemisphere and the non-infarcted volume of the ipsilateral hemisphere. There is no significant difference between infarct sizes of CD59-ko mice and wild-type mice. In all box plots, the top of the box represents the 75th percentile, the bottom of the box represents the 25th percentile, and the line in the middle represents the 50th percentile (median). The whiskers (the lines that extend out the top and bottom of the box) represent the highest and lowest values that are not outliers or extreme values.

Figure 2

Comparison of infarct volume and brain swelling of CD59a-deficient and wild-type-mice (C57BL/6) after 30 min MCAO. Infarct volumes (A, C, E), as well as volume of brain swelling (B, D, F) given as box-and-whisker-plots are visualized. Statistical analysis was performed with the Mann-Whitney-U-test (*: p ≤ 0.05). In contrast to 1 h MCAO-model (figure 1), after 30 min MCAO and 72 h of reperfusion there was a significant difference of infarct volumes and brain swelling between wild-type and CD59a-deficient mice in both, mixed gender and male mice: infarct volumes were larger and brain swelling increased in male CD59a knockout mice (n_{CD59a-/- m} = 22; n_{WT m} = 18), as well as in knockout mice of mixed gender (n = 25_{CD59a-/- mix}; n = 24_{WT mix}), when compared to age- and gender-matched wild-type control mice. No significant differences were seen for female mice. In all box plots, the top of the box represents the 75th percentile, the bottom of the box represents the 25th percentile, and the line in the middle represents the 50th percentile. The whiskers (the lines that extend out the top and bottom of the box) represent the highest and lowest values that are not outliers or extreme values.

Figure 3

TUNEL-positive cell count in post-ischemic mouse brain tissue after 30 min and 60 min MCAO. Absolute counts of TUNEL (apoptotic and necrotic)-positive cells in whole ischemic brain hemispheres of male wild-type and CD59a-deficient mice, determined in different brain sections. A: TUNEL-positive cell counts in MCAO-model 1 (60 min MCAO), B: cell counts in MCAO-model 2 (30 min MCAO). Distance to bregma: a, 5.3 mm; b, 3.9 mm; and c, 1.9 mm (*: p < 0.05, SD, unpaired t-test, n = 3 per group).

Figure 4

Representative images of TUNEL-positive cells in post-ischemic mouse brain tissue after 60 min MCAO and 48 h reperfusion. TUNEL (apoptotic and necrotic)-positive cells (B, D) in ischemic brain tissue of the infarct border zone (penumbra) of male wild-type (C, D) and CD59a-deficient mice (A, B). A,C: staining of cell nuclei using Hoechst 33258, which stains DNA (Invitrogen, Germany).

Figure 5

Comparison of neurological dysfunction of CD59a-deficient and wild-type-mice (C57BL/6) after 60 min MCAO. Either 24 h after reperfusion (A, C, E) or 48 h after reperfusion (B, D, F) there is no significant difference in neurological dysfunction, neither in male (C, D) nor in female (E, F) mice group when comparing CD59-deficient mice to wild-type (WT) mice. Statistical analysis was performed using the Mann-Whitney-U-test. Score of 0: no neurological dysfunctions; score 1: failure to extend right forepaw, score 2: circling to the contralateral side; score 3: loss of postural reflex and score 4: death. Number of animals: for CD59a-ko n = 24, WT n = 21. Both male and female animals were used in this study.

Figure 6

Comparison of neurological dysfunction of CD59a-deficient and wild-type-mice (C57BL/6) after 30 min MCAO. The CD59-ko mice in the mixed gender group show 24 h after reperfusion (A), 48 h after reperfusion (B) and 72 h after reperfusion (C) a significant increased neurological dysfunction (p < 0,05) compared to wild-type (WT) mice. In the male gender group (D, E, F) there is only a significant difference after 48 h (E) and 72 h (F). No increase was observable in the female group (G, H, I). Statistical analysis was performed using the Mann-Whitney-U-test. A score of 0 shows no neurological dysfunctions; score 1: failure to extend right forepaw, score 2: circling to the contralateral side; score 3: loss of postural reflex and score 4: death. Number of animals: for CD59a-ko n = 27, WT n = 27. Both male and female animals were used in this study.