Effects of anisodamine on altered Ca^(2+)i and cerebral cortex ultrastructure following acute cerebral ischemia/reperfusion injury in rabbits

摘要

BACKGROUND: Calcium ion (Ca2+) overload plays an important role in cerebral ischemia/reperfusion injury. Anisodamine, a type of alkaloid, can protect the myocardium from ischemia and reperfusion injury by inhibiting intracellular calcium [Ca2+]i overload. OBJECTIVE: To investigate effects of anisodamine on [Ca2+]i concentration and cortex ultrastructure fol- lowing acute cerebral ischemia/reperfusion in rabbits. DESIGN, TIME AND SETTING: Randomized and controlled trial was performed at the Department of Emergency, Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology from September to December 2006. MATERIALS: Forty healthy rabbits were used to establish models of acute cerebral ischemia/reperfusion. Anisodamine was provided by Lianyungang Dongfeng Pharmaceutical Factory; Fura-2 was purchased from Nanjing Jiancheng Bioengineering Institute; dual-wave length fluorescent spectrophotometry system and DM-300 software were provided by Bio-Rad, USA; OPTON-EM10C transmission electron microscope was product of Siemens, Germany. METHODS: Forty rabbits were randomly divided into the following groups: sham operation, ischemia, ischemia/reperfusion, and anisodamine, with ten rabbits in each group. Models of complete cerebral ischemia injury were established. In addition, blood was collected from the femoral artery of rats in the ischemia/reperfusion and anisodamine groups to induce hypotension and establish reperfusion injury models. The bilateral common carotid artery clamp was removed from the anisodamine group 20 minutes after ischemia, and anisodamine (10 mg/kg body mass) was injected via the femoral vein. Rabbits in the sham operation group underwent only venous cannulation. MAIN OUTCOME MEASURES: [Ca2+]i concentration was determined using a dual-wave length fluorescent spectrophotometry system, and cortical ultrastructure was observed following uranyl-lead citrate staining. RESULTS: The levels of [Ca2+]i in the ischemia and ischemia/reperfusion groups were significantly in- creased, compared with the sham operation group (P < 0.01), and the levels of [Ca2+]i in the anisodamine group were remarkably less than the ischemia and ischemia/reperfusion groups (P < 0.01). Ultrastructural damage to the cortex was greatly aggravated with increasing levels of [Ca2+]i. In the ischemia group, cortical neuronal membranes were fragmentally damaged, including the mitochondria and endoplasmic reticulum, as well as neurite swelling, and slight chromatin margination. In the ischemia/reperfusion group, the cellular membrane was ruptured with aggravated mitochondrial swelling, increased chromatin margination, obscure neurite structure, and the disappearance of endoplasmic reticulum. However, in the anisodamine group, cel- lular damage was obviously alleviated. The appearance and structure of cortical neurons was relatively normal, with intact cells. There was slight swelling of the mitochondria and endoplasmic reticulum, as well as mild chromatin margination. CONCLUSION: Cerebral tissue injury was related to increased [Ca2+]i levels following ischemia/ reperfusion. Anisodamine exhibited a protective role on acute cerebral ischemia/reperfusion injury by inhibiting the increase in [Ca2+]i levels.