Possible mechanisms of Cyclosporin A ameliorated the ischemic microenvironment and inhibited mitochondria stress in tree shrews’ hippocampus

Abstract

Objective: The ischemic brain damage is always accompanied by the significant accumulation of glutamate and calcium ions (Ca 2+). Our objectives were to observe the effects of glutamate and Ca 2+ overloading in tree shrew's hippocampal microenvironment on mitochondrial stress resulting in cytochrome C release and caspase apoptotic gene activation, and to explore the possible mechanism of Cyclosporin A (CsA) inhibiting mitochondrial stress. Methods: The thrombotic focal cerebral ischemia was induced by photochemical reaction in tree shrews. The extracellular contents of amino acidic neurotransmitters and Ca 2+ were determined, respectively, with high performance liquid chromatography (HPLC) and atomic absorption spectrophotometry at 4, 24 and 72 h after cerebral ischemia. The glutamate–calcium chloride solutions were microperfused into hippocampus by a kind of single-pumped push–pull perfusion (SPPP) system under three-dimensional orientation instrument in tree shrews. At 24 h, the expression of cytochrome C was observed in perfused lateral hippocampus by immunochemistry. Also, the hippocampus was removed, then mitochondria and cytoplasmic fragment were divided by low temperature centrifugation and the distribution of cytochrome C was assessed through Western blot. Real time fluorescence polymerase chain reaction was used to evaluate the relative amounts of caspase-3 and caspase-9 mRNA. In the treated group, CsA (40 mg/kg) was intravenously injected at 6 h after the microperfuse or cerebral ischemia. The glutamate–calcium solutions were perfused into the hippocampus and inspected the above-mentioned items at 24 h. Data were compared between the two groups (ischemia group vs. sham group, or ischemia group vs. CsA group). Results: Thrombotic cerebral ischemia led to significant increase in extracellular glutamate and Ca 2+ level of hippocampus ( P < 0.01). The cerebral ischemia group and the microperfusion group, which cytochrome C immunoreactivity increased and Western blot analysis demonstrated that the cytochrome C content in the mitochondria of hippocampal cells decreased ( P < 0.01), but the cytochrome C in the cytosol increased ( P < 0.01). When CsA was intravenously injected at 6 h after the microperfusion or cerebral ischemia, the cytochrome C expression weakened and its release was diminished to a lesser extent. By real time PCR, in relation to the control group, the caspase-3 and caspase-9 mRNA was higher in the glutamate–calcium chloride solution perfused group. CsA treatment cut down the contents of caspase-3 mRNA and caspase-9 mRNA ( P < 0.01). Conclusions: It is a primary factor that glutamate and Ca 2+ accumulate in hippocampal microenvironment, which results in proapoptotic protein cytochrome C release from mitochondria into cytoplasm and caspase cascade activation, and finally mitochondria stress and neuronal secondary injury appear. The neuroprotection of CsA is in relation to inhibiting glutamate receptor overactivation and reducing the Ca 2+ influx, which can decrease cytochrome C release and caspase mRNA transition.