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  • In rodents Wang et al Wang

    2020-11-14

    In rodents (Wang et al., 2003, Wang and Tsirka, 2005b, Zhu et al., 2012) and humans (Wang et al., 2011), the major forms of cell death after ICH are necrosis and apoptosis. In the perihematomal region of rodents, the number of necrotic and apoptotic cells peaks at 72h post-ICH (Matsushita et al., 2000, Zhu et al., 2012). The attenuation of cell and neuronal degeneration conferred by EP1R inhibition, as evidenced by reductions in PI staining, FJB staining, and cleaved caspase-3, is consistent with our in vitro study showing that SC51089 decreases the death of neurons exposed to hemoglobin. These data suggest that EP1R inhibition has a direct protective effect on neurons. Similar results were reported in a recent in vitro study in which primary neurons were treated with toxic levels of hemin (Mohan et al., 2013). Interestingly, contrary to their in vitro data, the same group reported that EP1 deletion exacerbates ICH outcomes in vivo, potentially by impairing microglial phagocytosis (Singh et al., 2013). A prior study showed that EP1R is expressed in neurons but not in microglia in the ischemic Cy5 hydrazide (non-sulfonated) (Kawano et al., 2006). In the ICH brain, we found that EP1R was expressed primarily in neurons and axons, less frequently in round CD11b+ cells, and rarely in typical process-bearing CD11b+ microglia. Based on the fact that CD11b is a marker for both microglia and blood-borne myeloid cells and that all microglia are GFP+ in Cx3cr1GFP/+ mice (Cardona et al., 2006), we further confirmed that resting and reactive Cx3cr1+ microglia in the perihematomal region rarely express EP1R. This finding supports the possibility that EP1R might be expressed mostly in amoeboid CD11b+ myeloid cells, such as macrophages, mast cells, neutrophils, and dendritic cells, but not in microglia. Therefore, our data do not support the idea that EP1R can directly affect microglial phagocytosis in vivo after ICH. It will be important to determine what blood-borne myeloid cells express EP1R and the effects of EP1R deletion or inhibition on the function of EP1R-expressing myeloid cells, which might play an important role in ICH pathology. Cellular inflammatory responses, including overactivation of microglia and astrocytes and infiltration of leukocytes and macrophages that release proinflammatory cytokines, chemokines, ROS, and other toxic mediators, contribute to ICH-induced secondary brain injury (Wang, 2010, Wang and Dore, 2007b). Consistent with this notion, we have shown previously that inhibition of microglial activation before or 2h after ICH improves histologic and functional outcomes (Wang et al., 2003, Wang and Tsirka, 2005b). Others have shown that leukocyte depletion reduces blood–brain barrier disruption, axon injury, and inflammation after ICH (Moxon-Emre and Schlichter, 2011). In this study, we showed that EP1R activation exacerbates cellular inflammation on day 3 after ICH, whereas EP1R inhibition mitigates this inflammation; thus EP1R may target the signals that mediate cellular inflammatory response. Finally, the reduction in inflammation that we observed after EP1R inhibition was associated with reductions in ROS production and oxidative brain damage. This finding is important because reducing ROS with free radical scavengers, or by genetic deletion of the ROS-generating enzyme NADPH oxidase, reduces ICH-induced brain damage in mice (Nakamura et al., 2008, Tang et al., 2005). To minimize the concern that increases or decreases in cell death, cellular inflammatory responses, and ROS production are due to differences in lesion volume, we performed profile-based cell counting in vehicle and treatment groups using brain sections with similar-sized hematomas. Increased Src kinase activity contributes to ischemic stroke injury (Paul et al., 2001, Zan et al., 2011, Zan et al., 2014) and thrombin-induced cell death (Ardizzone et al., 2007, Liu et al., 2010, Liu et al., 2008). However, the link between EP1R and Src kinases has not been established in ICH models. It is well known that Src and other Src family kinases are abundant in neurons (Kalia et al., 2004). We showed here that EP1R is present mostly in neurons in the ICH brain. Therefore, the interaction between EP1R and Src should also occur in the neurons. The phosphorylation state of Src kinase is altered by EP1R activation or inhibition, suggesting that Src could be a downstream target of EP1R. The fact that EP1R antagonist SC51089 and Src inhibitor PP2 did not have an additive effect further supports the sequential pathway of EP1R and Src signaling. In this regard, the increase in lesion volume in EP1R knockout mice after ICH (Singh et al., 2013) might be caused by chronic inhibition of Src signaling, which has the opposite effect of acute inhibition (Liu and Sharp, 2011). These opposing effects could also explain the contradictory findings from EP1R knockout mice (Singh et al., 2013) and mice with acute inhibition of EP1R, as we report here.