The superiority and feasibility of 2,3,5-triphenyltetrazolium chloride-stained brain tissues for molecular biology experiments based on microglial properties

Background:TTC(2,3,5-triphenyltetrazolium chloride)staining is the most commonly used method in identifying and assessing cerebral infarct volumes in the transient middle cerebral artery occlusion model.Given that microglia exhibit different morphologies in different regions after ischemic stroke,we demonstrate the superiority and necessity of using TTC-stained brain tissue to analyze the expression of various proteins or genes in different regions based on microglia character.Methods:We compared brain tissue(left for 10 min on ice)from the improved TTC staining method with penumbra from the traditional sampling method.We identified the feasibility and necessity of the improved staining method using real time(RT)-PCR,Western blot,and immunofluorescence analysis.Results:There was no protein and RNA degradation in the TTC-stained brain tissue group.However,the TREM2 specifically expressed on the microglia showed a significant difference between two groups in the penumbra region.Conclusions:TTC-stained brain tissue can be used for molecular biology experiments without any restrictions.In addition,TTC-stained brain tissue shows greater superiority due to its precise positioning.

The protective effect of ginsenoside Rb1 pre-treatment on myocardial ischemia/reperfusion injury in Sprague-Dawley rats

Background Myocardial ischemia/reperfusion injury(MI/RI)during myocardial infarction worsens outcomes. It has been proved that ginsenoside Rb1 has a great impact on ischemia/reperfusion(I/R)injury. The aim of this study is to explore the protective effect of the pretreatment with ginsenoside Rb1 on MI/RI and investigate the underlying mechanisms about the preventive action. Methods A total of 27 healthy adult Sprague-Dawley(SD)rats were randomly divided into 3 groups(n=9 per group):A sham-operated group(Sham n=9);an ischemia 40 min/reperfusion 2 h(I/R n=9)of the cardiac muscle group;Rb1-treated group was divided into 3 subgroups(Rb1 10 mg/kg,20 mg/kg,40 mg/kg,n=3). Ginsenosides Rb1 at different concentrations were injected intraperitoneally for 3 days continuously before ligation. A model of MI/RI was constructed by ligation of the left coronary artery anterior descending branch in rats. Myocardial infarction area after I/R was measured bytriphenyltetrazolium chloride(TTC)staining of myocardial tissue. Hematoxylin and eosin(HE)staining and electrocardiogram indication were used to observe myocardial cell injury and ischemia,respectively. The expression of caspase-8 protein was observed by immunohistochemistry staining. Results The pretreatment of 40 mg/kg dose of ginsenoside Rb1 could decrease the expression of caspase-8 protein caused by I/R and the apoptosis of myocardial cells,improve myocardial ischemia,reduce the area of myocardial infarction and ameliorate MI/RI.Conclusions These results demonstrate that ginsenoside Rb1 has a significant protective effect on MI/RI through attenuating the apoptosis of myocardial cells and improving myocardial ischemia at appropriate dose,which provides new insights into the potential therapy of MI/RI.