Background and Purpose
Cerebral ischemia-reperfusion injury is associated with the development of inflammatory response including pathologic contributions by vascular leukocytes and endogenous microglia. Expression of Fc receptors (FcRs) on macrophages and microglia is thought to be involved in the inflammatory cascade. The present study assessed the role of FC gamma receptor in ischemia-reperfusion injury, using Fc gamma receptor knockout (FCgR−/−)mice and bone marrow chimera FCgR−/−mice, which express enhanced green fluorescent protein (EGFP).
Methods
Studies were conducted in 8-week old FCgR−/− and C57BL/6 (wild-type, WT) (n=50/group) mice of the same genetic background. Mice underwent occlusion of the middle cerebral artery for 60 minutes, followed by reperfusion. We generated the FcgR−/−/EGFP transgenic model by bone marrow transplantation of EGFP into FcgR−/− mice and induced transient cerebral ischemia in these animals 6 weeks later. At 6, 12, 24, 72 h, or 14 days post-reperfusion, the mice were anesthetized by intraperitoneal injection of 50 mg/kg pentobarbital (n=5/group) and decapitated. Infarct volume and mortality was calculated at several time points after ischemia. To clarify the function and distribution of microglia/macrophages, immunohistochemical staining and immunoblotting of ionized calcium-binding adapter molecule 1 (Iba-1), inducible nitric oxide synthase (iNOS), and nitrotyrosine were performed.
Results
FCgR−/−mice showed significantly reduced mortality (20%) and smaller infarcts (P < 0. 001) than wild-type (WT) mice at 72 hours post-reperfusion. FcgRI staining was detected in glial cells, which morphologically resembled microglia. However, no specific FcgRI staining was noted in the brain of FcgR−/− mice. In FcgR−/− mice, microglial activity in the transition area was weak compared with WT mice. In WT mice, induction of iNOS in microglia of peri-ischemia area reached a peak level at 48–72 h post-reperfusion. On the other hand, in FCgR−/− mice, iNOS was only detected in endothelial cells of the ischemic core area. Western blotting revealed that microglial activation (P < 0.002) and induction of iNOS (P < 0.005) was reduced in FCgR−/−mice compared with WT mice. At 7 days post-reperfusion, sections double-immunostained for EGFP and Iba-1 showed less activation and migration of EGFP-positive bone marrow-derived macrophages in FCgR−/− chimera mice than WT mice. In WT/EGFP chimera mice, induction of nitrotyrosine in microglia of transition area was detected and nitrotyrosine-staining was observed in the luminal surface of vessels at ischemic lesion, while there were few nitrotyrosine-positive cells in FCgR−/−/EGFP chimera mice. These nitrotyrosine-positive microglia did not stain for EGFP.
Conclusions
In this study, we demonstrated that FcgR deficiency decreased the inflammatory responses through microglial activation, iNOS induction and bone marrow-derived macrophage infiltration after transient focal cerebral ischemia/reperfusion. Therefore, Our results demonstrated that the neuroprotective effect of FcgR deficiency in our model might be primarily attributed to the suppression of activation and infiltration of inflammatory cells. Our data showed that anti-inflammatory therapy through the FcgR might be useful for neuroprotection after cerebral infarction.