Role Of Gut Microbial Translocation-induced Monocyte Activation In Experimental Cerebral Ischemia/Reperfusion Injury

Objective: LPS is the component of intestinal gram-negative bacterial cell wall. LPS could trigger an immune response by activating monocytes in circulation. It has been suggested that monocytes play an important role of in the pathogenesis of cerebral ischemia/reperfusion injury, especially when a occluded vessel is recanalized by thrombolysis. Ly6 Chi monocyte in mice has been considered a proinflammatory subset. Recent studies have shown that acute stress could induce the dysfunction in gut barrier and consequently the increased level of bacterial and/or its byproduct in the circulation, which is called “gut microbial translocation”. We hypothesized that cerebral ischemia/reperfusion injury, as a severe form of stress, can lead to gut microbial translocation and a transient increase of gut permeability, which contributes to increased circulating levels of intestinal bacterial products(i.e., LPS and D-lactate) and commitment activation of circulating monocyte; these changes will exacerbate cerebral ischemic lesion; accordingly, interventions that target bacterial product or gut permeability will ultimately reduce cerebral ischemia/reperfusion injury.Method:(1) Intestinal permeability and circulating monocyte subsets after cerebral I/R injury: we used FITC-labeled dextran(FD4)-based method to evaluate intestinal permeability after cerebral I/R injury. The dynamic chages of circulating monocyte subsets(Ly6Chi, Ly6 Cint, Ly6Clo) were examined by flow cytometry on day 1, 2 and 3. At the same time, D-lactate and LPS was detected as intestinal permeability marker. The severity of cerebral I/R injury was evaluated by 2,3,5-Triphenyltetrazolium chloride(TTC) staining. The structural changes gut barrier was evaluated by H&E staining, immunofluoresscent staining and transmission electron microscopy. Superior mesenteric artery blood flow was examined by Doppler-based method.(2) Intestinal permeability increase as an intervention target after cerebral I/R injury: LPS scavenger-PMB and intestinal mucosa protectant agent- alanyl-glutamine(GLN) was used to evaluate their effect on microbial translocation, monocyte activation and cerebral I/R injury.Result:(1) Intestinal permeability and circulating monocyte subsets after cerebral I/R injury: the maximal level of circulating FD4 was observed on day 2 after I/R injury, and significantly higher that that measured in sham group. Correlation analysis showed that significant correlation between FD4 concentration and cerebral infarct area; the results of different intestinal segments’ permeability shows that permeability of colon increased about 4.1-fold compare with other intestinal segments.Colonic mucosa underwent the most significantly structural alterations after cerebral I/R injury, mucosal inflammation cells infiltration, villi shape irregularity, discontinued mucocal protein distribution, as well as tight junction damage. Ly6 Chi monocytes was significantly increased on day 1 in MCAO group, and reaching the peak level on day 2, followed by a decreasing trend from day 3. The Pearson correlation analysis showed that the percentage of infarction area and m NSS score were all significant correlated with Ly6 Chi monocyte percent. D-lactate and LPS concentration significantly increased after 1 d in MCAO group.(2) Intestinal permeability increase an intervention target for cerebral I/R injury: TTC staining showed that the infarct area was significant decreased in MCAO+GLN group compare with MCAO group, infarct area was also decreased in MCAO+PMB group. The proportion of Ly6 Chi monocyte in MCAO+PMB group on day 2 and day 3 was significantly decreased. The proportion of Ly6 Chi monocyte was significantly decreased after GLN treatment as well.Conclusion: Our work for the first time demonstrates an increased gut microbial translocation after cerebral I/R injury in mice, which is accompanied by Ly6 Chi monocytosis. Accordingly, strategies targeting this pathophysiological phenomenon by using LPS scavenger and gut mucosa protective agent can improve cerebral I/R injury. Thus, gut microbial translocation may serve as a novel therapeutic target for cerebral I/R injury.