Complement-Dependent P-Selectin Expression and Injury following Ischemic Stroke

Abstract

The mechanisms that contribute to inflammatory damage following ischemic stroke are poorly characterized, but studies indicate a role for both complement and P-selectin. In this study, we show that compared with wild-type mice, C3-deficient mice showed significant improvement in survival, neurological deficit, and infarct size at 24 h after middle cerebral artery occlusion and reperfusion. Furthermore, P-selectin protein expression was undetectable in the cerebral microvasculature of C3-deficient mice following reperfusion, and there was reduced neutrophil influx, reduced microthrombus formation, and increased blood flow postreperfusion in C3-deficient mice. We further investigated the use of a novel complement inhibitory protein in a therapeutic paradigm. Complement receptor 2 (CR2)-Crry inhibits complement activation at the C3 stage and targets to sites of complement activation. Treatment of normal mice with CR2-Crry at 30 min postreperfusion resulted in a similar level of protection to that seen in C3-deficient mice in all of the above-measured parameters. The data demonstrate an important role for complement in cerebrovascular thrombosis, inflammation, and injury following ischemic stroke. P-selectin expression in the cerebrovasculature, which is also implicated in cerebral ischemia and reperfusion injury, was shown to be distal to and dependent on complement activation. Data also show that a CR2-targeted approach of complement inhibition provides appropriate bioavailability in cerebral injury to enable complement inhibition at a dose that does not significantly affect systemic levels of serum complement activity, a potential benefit for stroke patients where immunosuppression would be undesirable due to significantly increased susceptibility to lung infection.

Versatility of the complement system in neuroinflammation, neurodegeneration and brain homeostasis

“…To this end, studies concerning the inhibition of downstream-cascade complement proteins have been conducted to elucidate their specific contribution in mediating brain damage after ischemia. C3−/− ischemic mice showed better neurological scores, reduced ischemic volume, granulocyte infiltration and oxidative stress when compared to WT mice subjected to cerebral ischemia, confirming the deleterious effects of complement system activation in this condition (Atkinson et al, 2006; Mocco et al, 2006a). Furthermore, the expression of C3aR has been shown to increase after cerebral ischemia (Barnum et al, 2002) and its pharmacological inhibition with a C3aR antagonist conferred protection of anatomical damage associated with reduced endothelial ICAM-1 staining (Ducruet et al, 2008).…”

Pathogenic Natural Antibodies Propagate Cerebral Injury Following Ischemic Stroke in Mice

“…Briefly, a blunted 4-0 nylonsuture was passed through the internal carotid artery to occlude the middle cerebral artery. After 60 minutes ischemia, the suture was removed for a 24h period of reperfusion before sacrifice, as consistent with previous literature (3, 4, 21, 22). Blood pressure, temperature, and ipsilateral cerebral blood flow(measured by laser Doppler), were measured before, during and after ischemia as previously described (23, 24).…”

“…prior to performing middle cerebral artery occlusion (MCAO) as previously described (4). Briefly, a blunted 4-0 nylonsuture was passed through the internal carotid artery to occlude the middle cerebral artery.…”

“…Brains were then either processed to paraffin or immersed in 20% sucrose in paraformaldehyde and embedded in OCT medium for cryosectioning. Paraffin sections were stained with Luxol Fast Blue/Nissl stain for morphological analysis, as previously described (4). …”

“…Clinical and animal studies have established a causal role for complement in IRI of various organs and tissues(1), including the brain following ischemic stroke (2). Cleavage of complement component C3 is a central event in complement activation, and studies with C3 deficient (3) and inhibited (4) mice have revealed a key role for complement in murine ischemic stroke. Complement can be activated via the classical, lectin and alternative pathways, and recent data indicate a central role for the lectin pathway in ischemic stroke (5, 6).…”