Synergistic neuroprotective effects of C3a and C5a receptor blockade following intracerebral hemorrhage

Abstract

Background: Intracerebral hemorrhage (ICH) is associated with neurological injury that may be ameliorated by a neuroprotective strategy targeting the complement cascade. We investigated the role of C5a-receptor antagonist (C5aRA) solely and in combination with C3a-receptor antagonist (C3aRA) following ICH in mice.

Methods: Adult male C57BL/6J mice were randomized to receive vehicle, C5aRA alone or C3aRA and C5aRA 6 and 12 h after ICH, and every 12 h thereafter. A double injection technique was used to infuse 30 microL of autologous whole blood into the right striatum. A final group of mice received a sham procedure consisting only of needle insertion followed by vehicle injections. Brain water content and flow cytometry analysis for leukocyte and microglia infiltration and activation in both hemispheres were measured on day 3 post ICH. Neurological dysfunction was assessed using a Morris water-maze (MWM), a 28-point scale, and a corner test at 6, 12, 24, 48 and 72 h after ICH induction.

Results: Neurological deficits were present and comparable in all three cohorts 6 h after ICH. Animals treated with C5aRA and animals treated with combined C3aRA/C5aRA demonstrated significant improvements in neurological function assessed by both the corner turn test and a 28-point neurological scale at 24, 48 and 72 h relative to vehicle-treated animals. Similarly, C5aRA and C3aRA/C5aRA-treated mice demonstrated better spatial memory retention in the Morris water-maze test compared with vehicle-treated animals (C3aRA/C5aRA: 23.4+/-2.0 s p< or =0.0001 versus vehicle: 10.0+/-1.7 s). Relative to vehicle-treated mice, the brain water content in C3aRA/C5aRA-treated mice was significantly decreased in the ipsilateral cortex and ipsilateral striatum (ipsilateral cortex: C3aRA/C5aRA: 0.755403+/-0.008 versus 0.773327+/-0.003 p=0.01 striatum: 0.752273+/-0.007 versus 0.771163+/-0.0036 p=0.02). C5aRA-treated mice and C3aRA/C5aRA-treated mice had a decreased ratio of granulocytes (CD45(+)/CD11b(+)/Ly-6G(+)) in the hemorrhagic versus non-hemorrhagic hemispheres relative to vehicle-treated animals (C5aRA: 1.78+/-0.36 p=0.02 C3aRA/C5aRA: 1.59+/-0.22 p=0.005 versus vehicle: 3.01).

Conclusions: While administration of C5aRA alone provided neuroprotection, combined C3aRA/C5aRA therapy led to synergistic improvements in neurofunctional outcome while reducing inflammatory cell infiltration and brain edema. The results of this study indicate that simultaneous blockade of the C3a and C5a receptors represents a promising neuroprotective strategy in hemorrhagic stroke.

Fig. 1

Corner test performance expressed by the laterality index in vehicle-treated (n = 17), C5aRA-treated (n = 13), C3aRA/C5aRA-treated (n = 16), and sham (n = 11) mice at 6, 12, 24, 48 and 72 h after intrastriatal infusion of 30 μL autologous blood. First injection was given at 6 h after hemorrhage. Values are expressed as mean±SEM. *Significantly different compared to vehicle by Kruskal–Wallis ANOVA on ranks (p<0.05).

Fig. 2

28-point neurological score in vehicle-treated (n = 17), C5aRA-treated (n = 13), C3aRA/C5aRA-treated (n = 16) and sham mice (n = 11) at 6, 12, 24, 48 and 72 h after intrastriatal infusion of 30 μL autologous blood. First injection was given at 6 h after hemorrhage. Values are expressed as mean±SEM. *Significantly different compared to vehicle by Kruskal–Wallis ANOVA on ranks (p<0.05).

Fig. 3

Morris water-maze performance in vehicle-treated (n = 10), C5aRA-treated (n = 8), C3aRA/C5aRA-treated (n = 9), and sham mice (n = 11) expressed as the time spent in the target quadrant 72 h after ICH. Values are expressed as mean±SEM. *Significantly different from vehicle by Kruskal–Wallis ANOVA on ranks (p<0.05).

Fig. 4

Brain water content at 72 h after ICH induction in vehicle-treated (n = 12), C5aRA-treated (n = 8), C3aRA/C5aRA-treated mice (n = 10) and sham (n = 6) mice. *Significantly different from vehicle by Kruskal–Wallis ANOVA on ranks (p<0.05).

Fig. 5

Hemorrhagic versus non-hemorrhagic hemisphere ratio of activated granulocytes in vehicle-treated (n = 7), C5aRA-treated (n = 8), C3aRA/C5aRA-treated (n = 8) and sham (n = 7) mice at 72 h after ICH. Values are expressed as mean±SEM. *Significantly different from vehicle by Kruskal–Wallis ANOVA on ranks (p<0.05).