The Expression And Effect Of CD47 After Intracerebral Hemorrhage

Spontaneous intracerebral hemorrhage (ICH) is a common and often fatal stroke subtype. Community based studies have indicated a mortality of more than 40%, and many survivors are left with significant neurological deficits. With the aging of society, urbanization accelerates, the disease showed an upward trend year by year. But meta-analysis of the literature shows that ICH remains a serious clinical problem lacking effective treatment.CD47 (originally named integr in-associated protein (IAP)) is a cell surface protein of the immunoglobulin (Ig) superfamily. Recently, CD47 has been found to play a critical role in the central nervous system. One study showed that viral overexpression of CD47 in neurons induced apoptosis. Another study showed that ligand-mediated activation of CD47 is neurotoxic. Activation of CD47 signaling induced cell death mechanisms in neurons. Therefore, in the context of stroke and brain injury, over activation of the CD47 may cause deleterious effects in brain. CD47 on erythrocytes inhibits phagocytosis through interaction with the inhibitory immunoreceptor SIRPα expressed by macrophages. Thus, the CD47-SIRPα interaction constitutes a negative signal for erythrocyte phagocytosis.In the present study, we examined the CD47 expression level in a porcine ICH model with vehicle or deferoxamine treatment. We did in vitro studies of erythrophagocytosis. And we investigated the different outcomes, such as hematoma volume, neuronal death, and neurological deficits after ICH in CD47 knockout mice and wild-type mice using blood injection models.Part 1 CD47 expression in a porcine model of intracerebral hemorrhage (ICH)Objective:To examine CD47 expression in a porcine model of intracerebral hemorrhage (ICH) with vehicle or deferoxamine treatment. Method:Pigs received an injection of autologous blood into the right frontal lobe. Deferoxamine (50 mg/kg, i. m.) or vehicle was given 2 hours after ICH and then every 12 hours up to 3 days or 7 days. Animals were killed at 4 hours,1 day,3 days,7 days or 14 days after ICH for immunohistochemistry, immunofluorescence, Western-Blot analysis. Results CD47 protein level increased at 4 hours and peaked at 3 days after ICH. CD47 protein level increased in ipsilateral white and gray matter after ICH, but not in contralateral white and gray matter. CD47 expresses on neuron, microglia/macrophage, and oligodendrocyte, but not on astrocyte. Deferoxamine treatment attenuated CD47 overexpression after ICH. CD47 expression on red blood cells decreased over time.Part 2 in vitro studies of erythrophagocytosisObjective:To observe the phenomenon of in vitro erythrophagocytosis and investigate the role of CD47 in erythrophagocytosis. Method:We cultured rat microglia/macrophage, labeled red blood cells(RBC) with PKH-26, and then added labeled RBC (young RBC and senescent RBC) to microglia/macrophage. Flow cytometric analysis was performed after microglia/macrophage was labeled with APC. Results:Decreased CD47 expression promotes erythrophagocytosis.Part 3 CD47 knockout mice and wild-type mice ICH modelObjective:To investigate the different outcomes, such as hematoma volume, neuronal death, and neurological deficits after ICH in CD47 knockout mice and wild-type mice using blood injection models. Method:CD47 knockout mice and wild-type mice received 30μL autologous whole blood infusion into the right basal ganglia. On day 1 and day 8, all mice underwent MRI. The neurological deficits were tested 1,3, and 8 days after ICH. Fluoro-Jade C staining was used to detect degenerating neuronal cells. Results:The MRI examination showed more rapid hematoma resolution in CD47 knockout mice compared with in wild-type mice 8 days after ICH. The wild-type mice showed more severe neurological deficits and delayed neurological recovering 8 days after ICH. There were more Fluoro-Jade C positive cells detected in the ipsilateral basal ganglia of wild-type mice 8 days after ICH.ConclusionA porcine model of intracerebral hemorrhage (ICH), in vitro erythrophagocytosis and comparing CD47 knockout mice with wild-type mice after ICH were done in the present study. It indicated:1. CD47 protein level increased in ipsilateral white and gray matter after ICH and deferoxamine treatment attenuated CD47 overexpression after ICH. So, CD47 can be an indicator of brain injury.2. The wild-type mice showed more severe neurological deficits and more degenerating neuronal cells comparing with CD47 knockout mice. So, CD47 may be one of the mechanisms of ICH induced brain injury.3. Decreased CD47 expression on red blood cells promotes erythrophagocytosis.In summary, CD47 is important in ICH and may be a potential target for stroke therapy.