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Background: Complement activation via the lectin activation pathway (LP) has been identified as the key mechanism behind post-ischemic tissue inflammation causing ischemia-reperfusion injury (IRI) which can significantly impact the clinical outcome of ischemic disease. This work defines the contributions of each of the three LP-associated enzymes-mannan-binding lectin-associated serine protease (MASP)-1, MASP-2, and MASP-3-to ischemic brain injury in experimental mouse models of stroke., Methods: Focal cerebral ischemia was induced in wild-type (WT) mice or mice deficient for defined complement components by transient middle cerebral artery occlusion (tMCAO) or three-vessel occlusion (3VO). The inhibitory MASP-2 antibody was administered systemically 7 and 3.5 days before and at reperfusion in WT mice in order to assure an effective MASP-2 inhibition throughout the study. Forty-eight hours after ischemia, neurological deficits and infarct volumes were assessed. C3 deposition and microglia/macrophage morphology were detected by immunohistochemical, immunofluorescence, and confocal analyses., Results: MASP-2-deficient mice (MASP-2(-/-)) and WT mice treated with an antibody that blocks MASP-2 activity had significantly reduced neurological deficits and histopathological damage after transient ischemia and reperfusion compared to WT or control-treated mice. Surprisingly, MASP-1/3(-/-) mice were not protected, while mice deficient in factor B (fB(-/-)) showed reduced neurological deficits compared to WT mice. Consistent with behavioral and histological data, MASP-2(-/-) had attenuated C3 deposition and presented with a significantly higher proportion of ramified, surveying microglia in contrast to the hypertrophic pro-inflammatory microglia/macrophage phenotype seen in the ischemic brain tissue of WT mice., Conclusions: This work demonstrates the essential role of the low-abundant MASP-2 in the mediation of cerebral ischemia-reperfusion injury and demonstrates that targeting MASP-2 by an inhibitory therapeutic antibody markedly improved the neurological and histopathological outcome after focal cerebral ischemia. These results contribute to identifying the key lectin pathway component driving brain tissue injury following cerebral ischemia and call for a revision of the presently widely accepted view that MASP-1 is an essential activator of the lectin pathway effector component MASP-2.

Stroke, the loss of neurons after ischemic insult to the brain, is one of the leading causes of death and disability worldwide. Despite its prevalence and severity, current therapy is extremely limited, highlighting the importance of further understanding the molecular events underlying ischemia-induced neuronal cell death. An ischemic area can be subdivided into two separate pathophysiological regions: the rapidly dying necrotic core, and the potentially salvageable apoptotic penumbra. Understanding molecular events occurring in the apoptotic ischemic penumbra may give greater insight into mechanisms controlling this salvageable tissue. miRNAs are known to have key roles in the regulation of gene expression in numerous pathological conditions, including the modulation of distinct pathways in stroke. However, previous studies have profiled miRNAs in the whole ischemic infarct, and do not differentiate between miRNA regulation in the necrotic core versus the apoptotic penumbra. We asked if there were unique miRNAs that are differentially regulated following ischemic insults in the salvageable apoptotic penumbra. miRNA expression profiles were compared in the whole infarct from in vivo stroke models, using the three vessel occlusion approach, to an in vitro model of the ischemic penumbra, prior to apoptotic induction. Multiple miRNAs were found to be differentially regulated following ischemic insults in each system. However, miR-19b, miR-29b-2* and miR-339-5p were significantly up-regulated in both model systems. Further, we confirmed these results in a neuroblastoma cell line subjected to a penumbra-like ischemic insult that induced the apoptotic cell death pathway. The data show that miR-19b, miR-29b-2* and miR-339-5p are up-regulated following ischemic insults and may be regulating gene expression to control important cellular pathways in the salvageable ischemic penumbra. Further investigation of their role and mRNA target identification may lead to new insights into the molecular mechanisms taking place in the salvageable apoptotic penumbra.