The high concentration of glutamate release is the main cause for neuronal cell death. The relationship between glutamate level and apoptosis during ischemia/reperfusion injury is still unclear.
Objective
To observe the neuronal apoptosis at 24 and 72 hours following cerebral ischemia/reperfusion in rats, and analyze the possible influencing factors.
Design
A randomized controlled animal experiment.
Setting
School of Medicine, Southern Yangtze University.
Materials
Totally 30 male adult Sprague Dawley (SD) rats of clean grade, weighing 240–290 g, were obtained from Shanghai Experimental Animal Center, Chinese Academy of Sciences. The rats were randomly divided into sham-operated group (n=10) and model group (n=20). Each group was observed at 24 and 72 hours after ischemia/reperfusion, 5 rats at each time point in the sham-operated group, whereas 12 at 24 hours and 8 at 72 hours in the model group. Kits for determining apoptosis and Bcl-2 were bought from Wuhan Boster Biological Technology, Co., Ltd.; Kit for calcineurin from Nanjing Jiancheng Bioengineering Institute.
Methods
The experiment was carried out in the Functional Scientific Research Room of Southern Yangtze University from June to October in 2006. ![]()
Right middle cerebral artery was occluded by inserting a thread through internal carotid artery (ICA). The surgical process for the sham-operated rats was the same as that in the model group except a nylon suture inserted the ICA. According to Longa five-degree standard, the neurological deficit evaluation of rats was evaluated after surgery, and grades 1–3 were taken as successful model establishment. The blood was recirculated by withdrawing the nylon filament under anesthesia at 2 hours after ischemia in successful rat models. ![]()
After reperfusion, the brain tissue was quickly removed at 24 or 72 hours and the slices were obtained from optic chiasma to funnel manubrium. The changes of the number of apoptotic cells were observed using the terminal deoxynucleotidyl transferase mediated dUTP-biotin nick-end labeling method. The expressions of Bcl-2 protein were determined with immunohistochemical staining. The activity of calcineurin was determined by the inorganic phosphorus method. The content of excitatory amino acid was detected by high performance liquid chromatography.
Main Outcome Measures
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Glutanate content in brain tissue; ![]()
Conditions of apoptosis; ![]()
Calcineurin activity in brain tissue; ![]()
Bcl-2 expression in brain tissue.
Results
Totally 30 SD rats were used, 5 died and the other 25 were involved in the analysis of results. ![]()
Changes of apoptosis There were 0–3 apoptotic cells in the sham-operated group. In the model group, the numbers of apoptotic cells were obviously increased at 24 and 72 hours of reperfusion (P < 0.01), and it was markedly reduced at 72 hours as compared with 24 hours (P < 0.01). ![]()
Changes of glutanate content: The glutamate contents at 24 and 72 hours of reperfusion in the model group were obviously higher than those in the sham-operated group (P < 0.01); In the model group, it was obviously increased at 24 hours as compared with 72 hours (P < 0.01). ![]()
Changes of Bcl-2 protein: In the model group, the Bcl-2 protein expression had no obvious changes at 24 hours of reperfusion, and it was obviously enhanced at 72 hours, which was obviously different from that in the sham-operated group and that at 24 hours (P < 0.01). ![]()
Changes of calcinerin activity: In the model group, the activity of calcineurin in brain tissue had no obvious changes at 24 hours of reperfusion; The activity of calcineurin at 72 hours was obviously higher than that in the sham-operated group and that at 24 hours (P < 0.01).
Conclusion
The brain cyto-apoptosis action at different time points following reperfusion incompletely depends on the glutamate levels, while it depends on the interaction of some apoptosis related factors, such as amino acid, calcineurin, and Bcl-2, etc.
