EphB2-dependent signaling promotes neuronal excitotoxicity and inflammation in the acute phase of ischemic stroke

Local cerebral hypoperfusion causes ischemic stroke while driving multiple cell-specific responses including inflammation, glutamate-NMDAR-mediated neurotoxicity, edema formation and angiogenesis. Despite the relevance of these pathophysiological mechanisms for disease progression and outcome, molecular determinants controlling the onset of these processes are only partially understood. In this context, our study intended to investigate the functional role of EphB2 – a receptor tyrosine kinase that is crucial for synapse function and binds to membrane-associated ephrin-B ligands. Cerebral ischemia was induced in EphB2-deficient mice by transient middle cerebral artery occlusion followed by different times (6, 12, 24 and 48 h) of reperfusion. Histological, neurofunctional and transcriptome analyses indicated an increase in EphB2 phosphorylation under these conditions and attenuated progression of stroke in EphB2-deficient mice. Moreover, while infiltration of microglia/macrophages and astrocytes into the peri-infarct region was not altered, expression of the pro-inflammatory mediators MCP-1 or IL-6 was decreased in these mice. In fact, in vitro analyses indicated that binding of EphB2 to astrocytic ephrin-B ligands stimulates NF-κB-mediated cytokine expression via the MAPK pathway. Further magnetic resonance imaging of the EphB2-deficient ischemic brain revealed a lower level of neuronal cytotoxic edema formation within 6 h upon onset of reperfusion. On the mechanistic level, absence of neuronal EphB2 decreased the mitochondrial Ca2+ load upon activation of NMDAR but not AMPAR. Moreover, activation of EphB2 by ephrin-B2 enhanced the NMDA-evoked excitotoxic neuronal cell death in vitro while neuron-specific loss of ephrin-B2 reduced the extent of cerebral tissue damage in the acute phase of ischemic stroke. Collectively, EphB2 may promote the immediate response to an ischemia-reperfusion event in the central nervous system by (i) pro-inflammatory activation of astrocytes via ephrin-B-dependent signalling and (ii) amplification of the NMDA-evoked neuronal excitotoxicity. 3 WT and 3 Ephb2-KO mice (with a C57Bl/6 background.) were subjected to 60 min MCAo followed by 48 h of reperfusion. Brains were harvested. Bulbi and cerebellum were removed, brains were separated into hemispheres (ipsilateral=ischemic and contralateral=normoxic control). RNA was extracted from tissue for hybridization on Affymetrix microarrays. We aimed at identifying (1) genes and gene sets that are up- or down-regulated upon ischemia by comparison of ipsi- and contralateral hemispheres of the same genotype, (2) genes and gene sets that are differentially regulated between the two genotypes upon ischemia by comparison of the ipsilateral hemispheres, and (3) genes and gene sets that are differentially regulated between the two genotypes at basal levels by comparison of the contralateral hemispheres.