1Department of Internal Medicine, Beijing Mentougou Hospital, Beijing   102300, China

2Department of Neurology, Third Affiliated Hospital, Inner Mongolia Medical College, Baotou   014010, Nei Monggol Autonomous Region, China

3Department of Neurology, First Affiliated Hospital, Shanxi Medical University, Taiyuan   030001, Shanxi Province, China

Jianji Pei★, Master, Physician, Department of Internal Medicine, Beijing Mentougou Hospital, Beijing   102300, China Pei JJ, Liu LQ, Pang JP, Tian XH. Hyperlipidemia affects neuronal nitric oxide synthase expression in brains of focal cerebral ischemia rat model. Neural Regen Res 2008;3(6):642-6

Abstract

BACKGROUND: Hyperlipidemia, a risk factor for ischemic cerebrovascular disease, may mediate production of neuronal nitric oxide synthase (nNOS) to induce increased nitric oxide levels, resulting in brain neuronal injury.

OBJECTIVE: To investigate effects of hyperlipidemia on brain nNOS expression, and to verify changes in infarct volume and pathology during reperfusion, as well as neuronal injury following ischemia/reperfusion in a rat model of focal cerebral ischemia.

DESIGN, TIME AND SETTING: Complete, randomized grouping experiment was performed at the Laboratory of Physiology, Shanxi Medical University from March 2005 to March 2006.

MATERIALS: A total of 144 eight-week-old, male, Wistar rats, weighing 160-180 g, were selected. A rat model of middle cerebral artery occlusion was established by suture method after 4 weeks of formulated diet. Nitric oxide kit and rabbit anti-rat nNOS kit were respectively purchased from Nanjing Jiancheng Bioengineering Institute, China and Wuhan Boster Biological Technology, Ltd., China.

METHODS: The rats were equally and randomly divided into high-fat diet and a normal diet groups. Rats in the high-fat diet group were fed a high-fat diet, consisting of 10% egg yolk powder, 5% pork fat, and 0.5% pig bile salt combined with standard chow to create hyperlipidemia. Rats in the normal diet group were fed a  standard rat chow. A total of 72 rats in both groups were randomly divided into 6 subgroups: sham-operated, 4-hour ischemia, 4-hour ischemia/2-hour reperfusion, 4-hour ischemia/4-hour reperfusion, 4-hour ischemia/6-hour reperfusion, and 4-hour ischemia/12-hour reperfusion, with 12 rats in each subgroup.

MAIN OUTCOME MEASURES: nNOS expression was measured by immunohistochemistry, and pathomorphology changes were detected by hematoxylin-eosin staining. Infarct volume and nitric oxide levels were respectively measured using 2, 3, 5-triphenyltetrazolium chloride (TTC) and immunohistochemistry.

RESULTS: In the ischemic region, pathology changes were significant in the 4-hour ischemia/4-hour, 4-hour ischemia/6-hour reperfusion, and 4-hour ischemia/12-hour reperfusion subgroups fed on a high-fat diet compared to the same groups fed on a normal diet. In each ischemia subgroup, nNOS expression in brain tissues was higher than in the sham-operated subgroups fed on either the high-fat diet or normal diet (P < 0.01). At each ischemia/reperfusion time point, rats fed on a high-fat diet expressed higher levels of nNOS compared to rats fed on the normal diet (P < 0.05). When tissue was stained with TTC, a white infarction area was detected in the ischemic hemisphere, demonstrating that the infarct volume gradually increased with prolonged reperfusion time in each ischemia subgroup. At each ischemia/reperfusion time point, the infarct volume was larger in rats fed on a high-fat diet compared to those fed on a normal diet.

CONCLUSION: nNOS expression was greater in hyperlipidemia rats following ischemia/reperfusion. Cerebral ischemia/reperfusion injury is aggravated with prolonged reperfusion time.

Key Words: focal cerebral ischemia; hyperlipidemia; ischemia/reperfusion injury; neuronal nitric oxide synthase

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