Nerve growth factor downregulates c-jun mRNA and Caspase-3 in striate cortex of rats after transient global cerebral ischemia/reperfusion

Dacheng Jin; Tiemin Wang; Xiubin Fang

摘要：BACKGROUND: Immediate early gene (LEG) c-jun is a sensitive marker for functional status of nerve cells.Caspase-3 is a cysteine protease,which is a critical regulator of apoptosis. The effect of exogenous nerve growth factor (NGF) on the expression of c-jun Mrna and Caspase-3 protein in striate cortex of rats with transient global cerebral ischemia/reperfusion (IR) is unclear.OBJECTIVE: To study the protective effect of exogenous NGF on the brain of rats with transient global cerebral IR and its effecting pathway by observing the expression of c-jun Mrna and Caspase-3 protein.DESIGN: Randomized controlled animal trial.SETTING: Department of Neural Anatomy, Institute of Brain,China Medical University.MATERTALS:Eighteen healthy male SD rats of clean grade, aged 1 to 3 months, with body mass of 250 to 300 g, were involved in this study. NGF was provided by Dalian Svate Pharmaceutical Co.,Ltd, c-jun in situ hybridization detection kit, Caspase-3 antibody and SABC kit were purchased from Boster Biotechnology Co. ,Ltd.METHODS: This trial was carried out in the Department of Neural Anatomy, Institute of Brain, China Medical University during September 2003 to April 2005. ①Experimental animals were randomized into three groups with 6 in each: sham-operation group,IR group and NGF group. ②After the rats were anesthetized,the bilateral common carotid arteries and right external carotid arteries of rats were bluntly dissected and bilateral common carotid arteries were clamped for 30 minutes with bulldog clamps. Reperfusion began after buldog clamps were removed. Normal saline of 1mL and NGF (1×106 U/L) of 1 Ml was injected into the common carotid artery of rats via right external carotid arteries in the IR group and NGF group respectively.The injection was conducted within 30 minutes, and then the right external carotid arteries were ligated. In the sham-operation group, occlusion of bilateral common carotid arteries and administration of drugs were phosphate buffer solution (PBS, 0.1 mol/L) containing 40 g/L polyformaldehyde, their brains were quickly removed. The coronal section tissue mass containing striate cortex about 3 mm before line between two ears was taken and made into successive frozen sections.④The expression of c-jun Mrna and Caspase-3 protein in striate cortex of global cerebral ischemia rats were detected with in situ hybridization, immunohistochemistry and microscope image analysis. ⑤t test was used for comparing the difference of the measurement data.MATN OUTCOME MEASURES:Comparison of the expression of IEG c-jun Mrna and Caspase-3 protein in striate cortex of brain of rats in each group.RESULTS:All the 18 SD rats were involved in the analysis of results. The c-jun Mrna and Caspase-3 protein positive reaction cells were found brown yellow in the striate cortex of rats, and most of them were in lamellas Ⅱ and Ⅲ, mainly presenting round or oval. The expression of c-jun Mrna and Caspase-3 protein in sham-operation group was weak or negative. The average gray value of c-jun Mrna and Caspase-3 protein in the IR group was significantly lower than that in the sham-operation group (49.52±4.13 vs. 95.48±5.28; 74.73±4.29 vs. 162.38±9.16,P ＜ 0.01). The average gray value of c-jun Mrna and Caspase-3 protein in the NGF group was significantly higher than that in the IR group (63.96±4.25 vs.49.52±4.13; 83.98±4.13 vs. 74.73±4.29, P＜ 0.05).CONCLUSTON: NGF can protect ischemic neurons by down-regulating the expression of c-jun Mrna and Caspase-3 protein in striate cortex of global cerebral ischemia rats.