Over-expression of nitric oxide and nitric oxide synthase in the rat brain following focal ischemia

BACKGROUND: In cerebral ischemia,over-expression of nitric oxide (NO) exerts neurotoxic effects. OBJECTIVE: This study was designed to measure NO and nitric oxide synthase (NOS) expression in rat brain tissue during cerebral ischemia,and to correlate expression levels of NO and NOS with ischemic time. DESIGN: A complete randomized grouping design,controlled animal experiment. SETTING: Fujian Institute of Neurosurgery & Department of Neurosurgery,Union Hospital Affiliated Fujian Medical University. MATERIALS: This study was performed at the Fujian Institute of Neurosurgery in December 2003. Eighty healthy adult male Sprague Dawley (SD) rats of clean grade were provided by the Zhejiang Laboratory Animal Center. The protocol was performed in accordance with ethical guidelines for the use and care of animals. Kits for measuring NO expression by method of nitrate reductase (Boster Company,Wuhan,China) and NOS activity (Jiancheng Bioengineering Co.,Ltd.,Nanjing,China),as well as a kit for reverse transcription-PCR (RT-PCR,Promega,USA) were used in the present study. METHODS: Sixty-eight rats underwent cerebral ischemia by occluding the middle cerebral artery by suture method. Forty-eight successfully lesioned rats were included in the study. Six rats were used for each length of ischemic event (30 minutes,1,3,6,12,24,and 72 hours,and 5 days of ischemia). Additionally,a normal control group (n = 6,normally raised) and a sham-operated group (n = 6,corresponding cerebral vessels exposed,without occlusion of middle cerebral artery) were included in the analysis. MAIN OUTCOME MEASURES: Neurological function deficits were scored according to methods of Longa,Bederson,and colleagues. NO levels and NOS activity in the brain tissue were measured via nitrate reductase and chemical colorimetry,respectively. The expression of nitrotyrosine (NT),an in vivo specific metabolite of NO,was quantitatively measured by flow cytometry. Expression of endothelial,neuronal,and inducible NOS mRNA (eNOS,nNOS,and iNOS mRNA,respectively) in the cerebral ischemic region were semi-quantitatively analyzed by RT-PCR. RESULTS: Forty-eight ischemic rats and six sham-operated rats were analyzed. Neurological functional deficits increased with ischemic times (r = 0.765,P < 0.05). There were no significant differences in NO levels,NOS activity,and the percentage of NT-positive cells between the sham-operated and the normal control groups (P > 0.05). NO levels,NOS activity,and the percentage of NT-positive cells in brain tissue positively correlated to ischemia times (r = 0.932,0.914,0.924,respectively,P < 0.05). The percentage of NT-positive cells began to noticeably increase within 0.5 hour of after ischemia (9.50 %). NOS activity began to increase within 0.5 hour of ischemia and reached its peak level 3 days after ischemia. RT-PCR semi-quantitative analysis demonstrated that in the early stages of ischemia (0.5-6 hours),the expression of both eNOS and nNOS mRNA increased with ischemic time (t = 28.482-100.459,P < 0.01),while iNOS mRNA levels were almost undetectable. In the middle and advanced stages of ischemia (6 hours-5 days),iNOS mRNA levels were significantly increased compared to the control group (t = 36.742-82.058,P < 0.01); however,eNOS and nNOS mRNA levels were markedly reduced. CONCLUSION: With the prolongation of ischemic time,NO levels increased in cerebral tissue due to activation of various NOS. These measurements correlated with an increase in NT-positive cells and behavioral deficits. In the early stages of ischemia,eNOS and nNOS activities were increased,while in the later stage of ischemia,iNOS activity was increased and eNOS and nNOS activities were reduced.