论文部分内容阅读
BACKGROUND: Studies have confirmed that neuronal nitric oxide synthase (nNOS) mediates neurotoxic effects during the early stages of hypoxia-ischemia,while inducible nitric oxide synthase (iNOS) mediates delayed neurotoxicity during advanced stages of hypoxia-ischemia. OBJECTIVE: This study was designed to observe neuronal apoptosis and the expressions of nNOS,iNOS,p38 mitogen-activated protein kinase (MAPK),and caspase-3 mRNA following transfection of recombinant adeno-associated viral vectors separately expressing nNOS and iNOS antisense (rAAV-AsnNOS and rAVV-AsiNOS,respectively) into rat brains subjected to cerebral ischemia; to analyze mechanisms underlying elevated neuronal tolerance to hypoxia-ischemia. DESIGN: A randomized controlled in vivo experiment. SETTING: Fujian Institute of Neurosurgery & Department of Neurosurgery,Union Hospital,Fujian Medical University. MATERIALS: Eighty healthy adult male Sprague Dawley rats of clean grade were provided by the Zhejiang Laboratory Animal Center,China. The protocol was performed in accordance with ethical guidelines for the use and care of animals. The following vectors,rAAV-AsnNOS,rAAV-AsiNOS,and rAAV expressing the β-galactosidase gene (rAAV-LacZ),were successfully constructed by Fujian Institute of Neurosurgery. Rabbit anti-mouse nitrotyrosine (NT) monoclonal antibody (Zhongshan Jinqiao Biotechnology Co.,Ltd.,Beijing,China) and reverse transcription-polymerase chain reaction (RT-PCR) kit (two-step method) (Promega Company,USA) were used in this study. METHODS: This study was performed at the Fujian Institute of Neurosurgery in December 2003. Sixty rats were randomly divided into 3 groups,with 20 rats in each group: rAAV-AsnNOS group,rAAV-AsiNOS group,and rAAV-LacZ group. The remaining 20 rats served as controls. Pre-treated viral vectors (rAAV-AsnNOS,rAAV-AsiNOS,and rAAV-LacZ,respectively; each 50 μL,virus titer of 2×109 viral particles/mL) were transfected into the cerebral cortex of the targeted. Phosphate buffer saline (50 μL) was perfused identically into the rat cerebral cortex of the control group. All rats were subjected to cerebral ischemia by occluding the right middle cerebral artery with suture. Five time points (0,1,6,24,72 hours of ischemia,4 rats for each time point) were allotted to each group. Ischemic brain tissue specimens were prepared for index measurement. MAIN OUTCOME MEASURES: The percentage of NT-positive cells and apoptotic cells in the rat ischemic brain tissue specimens were measured by flow cytometry. The expressions of nNOS,iNOS,p38MAPK,and caspase-3 mRNA were measured by RT-PCR. RESULTS: During the early stages of ischemia (at 1 and 6 hours) with rAAV-AsnNOS transfection,the percentage of NT-positive cells,apoptotic cells,and the expressions of nNOS,p38MAPK and caspase-3 mRNA in the brain nerve cells were remarkably reduced compared to the control,rAAV-LacZ,and rAAV-AsiNOS groups. During the advanced stages of ischemia (at 24 and 72 hours) with rAAV-AsiNOS transfection,the above-mentioned indices were significantly reduced compared to control,rAAV-LacZ,and rAAV-AsnNOS groups. CONCLUSION: Following rAAV transfection,neuronal cells resisted ischemic injury in the MCAO ischemic rat model. Neurons transfected with rAAV-AsnNOS inhibited expression of nNOS,p38MAPK,and caspase-3 during the early stages of ischemia,and those transfected by rAAV-AsiNOS inhibited above-mentioned index expression during the advanced stages of ischemia. These results clearly demonstrate that neuronal apoptosis can be inhibited through the use of nNOS antisense.
BACKGROUND: Studies have confirmed that neuronal nitric oxide synthase (nNOS) mediates neurotoxic effects during the early stages of hypoxia-ischemia, while inducible nitric oxide synthase (iNOS) mediates delayed neurotoxicity during advanced stages of hypoxia-ischemia. OBJECTIVE: This study was designed following observe neuronal apoptosis and the expressions of nNOS, iNOS, p38 mitogen-activated protein kinase (MAPK), and caspase-3 mRNA following transfection of recombinant adeno-associated viral vectors separately expressing nNOS and iNOS antisense (rAAV- AsnNOS and rAVV- AsiNOS to analyze the underlying underlying neurological tolerance to hypoxia-ischemia. [0013] SETTING: Fujian Institute of Neurosurgery & Department of Neurosurgery, Union Hospital, Fujian Medical University. MATERIALS: Eighty healthy adult male Sprague Dawley rats of clean grade were provided by the Zhejiang Laboratory Ani The protocol was performed in accordance with ethical guidelines for the use and care of animals. The following vectors, rAAV-AsnNOS, rAAV-AsiNOS, and rAAV expressing the β-galactosidase gene (rAAV-LacZ), were successfully constructed by Fujian Institute of Neurosurgery. Rabbit anti-mouse nitrotyrosine (NT) monoclonal antibody (Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, China) and reverse transcription-polymerase chain reaction (two-step method) METHODS: This study was performed at the Fujian Institute of Neurosurgery in December 2003. Sixty rats were randomly divided into 3 groups with 20 rats in each group: rAAV-AsnNOS group, rAAV (Promega Company, USA) were used in this study. The remaining 20 rats served as controls. Pre-treated viral vectors (rAAV-AsnNOS, rAAV-AsiNOS, and rAAV-LacZ, respectively; each 50 μL, virus titer of 2 × 10 9 viral particles / mL) were transfected into the cerebral cortex of the targeted. Phosphate b uffFive rats were subjected to cerebral ischemia by occluding the right middle cerebral artery with suture. Five time points (0, 1, 6, 24, 72 hours of ischemia, 4 rats for each time point) were allotted to each group. Ischemic brain tissue specimens were prepared for index measurement. MAIN OUTCOME MEASURES: The percentage of NT-positive cells and apoptotic cells in the rat ischemic brain tissue specimens were measured by RESULTS: During the early stages of ischemia (at 1 and 6 hours) with rAAV-AsnNOS transfection, the percentage of NT- positive cells, apoptotic cells, and the expressions of nNOS, p38MAPK and caspase-3 mRNA were remarkably reduced compared to the control, rAAV-LacZ, and rAAV-AsiNOS groups. During the advanced stages of ischemia and 72 hours) with rAAV-AsiNOS transf ection, the above-incorporated indices were significantly reduced compared to control, rAAV-LacZ, and rAAV-AsnNOS groups. CONCLUSION: Following rAAV transfection, neuronal cells resisted ischemic injury in the MCAO ischemic rat model. Neurons transfected with rAAV- of nNOS, p38MAPK, and caspase-3 during the early stages of ischemia, and those transfected by rAAV-AsiNOS as above-mentioned index expression during the advanced stages of ischemia. These results clearly demonstrate that neuronal apoptosis can be inhibited through the use of nNOS antisense.