| Objective:Ischemic cerebrovascular disease is the common central nervous system disease and its rate of mortality and disability is very high.Ischemic cerebrovascular disease, heart disease and malignant tumor are the three diseases leading to disability in most countries and severely threaten humans'healthy. How to improve the cerebral blood flow of the ischemic brain damage and promote the repair and regeneration of the damaged neuron have become the focus and difficulty of many studies. Therefore, research on the nerve growth factors and vascular growth factors caused great extra attention. Currently, insulin-like growth factor-1 (IGF-1),vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are the mostly popular studied factors. Along with the continuous deepening of the study, endogenous IGF-1 has began to be used in the experiment of ischemic cerebrovascular disease, but it is still not clear about the relationship between IGF-1 and the expression of VEGF and bFGF and the exact neurotrophic protective mechanism of IGF-1 after cerebral ischemia so far.The rat model of middle cerebral artery occlusion (MCAO) and reperfusion was established with suture emboli method. After injecting IGF-1 via lateral cerebral ventricle, the IGF-1's effect on the expression of VEGF and bFGF both on gene and protein level was observed through RT-PCR and immunohisto- chemistry methods. In this study, we preliminarily discussed the possible molecular mechanism of IGF-1's protective effects towards cerebral ischemic damage and provided a new theoretical basis for IGF-1's further clinical application and a new way for the clinical treatment of cerebral infarction.Methods:1. The rat model of middle cerebral artery occlusion (MCAO) and reperfusion was established with suture emboli method.2. Animal experimental assignment: Seventy-two healthy SD adult rats, weighing 250-300g, either male or female, were randomly divided into three groups: (1) sham-operated group (n=24); (2) cerebral ischemia/reperfusion group(n=24);(3)IGF-1 treated group (n=24). Cerebral ischemia/reperfusion group and IGF-1 treated group were further randomly divided into 4 smaller groups (12h, 1d, 3d and 7d)according to the reperfusion time after being middle cerebral artery occlusion (MCAO) for two hours. Sham-operated control group was also randomly divided into 4 smaller groups at the same time point and each small group has six rats. The rats in IGF-1 treated group were established into middle cerebral artery occlusion (MCAO) model. Subsequently, IGF-1 treated group rats were slowly injected IGF-1 10μL (6μg) in the left lateral cerebral ventricle under stereotaxic apparatus, no less than 10 minutes. Cerebral ischemia/reperfusion group was treated with 0.01M phosphate-buffered saline (PBS) 10μL in the same way.The sham operated group was only treated with neck skin incision and vascular separation and wasn't established into MCAO,but it was injected PBS in the same volume.3. Within the time prescribed, the rats in each group were quickly sacrificed by decapitation and their brains were removed. The brain tissue was cut into four coronal slices from antinion to occipital lobe, which were A,B,C and D brain slices. A brain slice was abandonded. Within the time prescribed, the cerebral cortex tissue around the infarction region was stripped from B and C brain slices and the stripped tissue was about 100 mg. Rinsed it briefly in 1% DEPC water and put it into the frozen tube quickly and preserve in liquid nitrogen. Detect the content of VEGF-mRNA and bFGF mRNA of C brain slice by RT-PCR method. B brain slices were preserved for future use. B brain slice was used to detect brain-VEGF mRNA content,C brain slices was used to detect bFGF mRNA content, both by RT-PCR method.After being fixed in 4% paraformaldehyde for 24h, D brain slices were embedded in paraffin. Prepare the 5μm sections for HE staining, VEGF and bFGF immunohistochemi- cal staining.4. The evaluation of ethology:The evaluation of ethology was performed at 12h, 24h, 3d, 7d after ischemia-reperfusion by an investigator who was blinded to the experimental groups. The test was the Modified Neurological Severity Score (mNSS). Neurological function was graded on a scale of 0 to 18 (normal score, 0; maximal deficit score, 18). One point is awarded for inability to perform the tasks or for lack of a tested reflex:1–6, mild injury;7–12, moderate injury;13–18, severe injury. 5. Statistical analysis: All data were expressed as means±SE and analyzed statistically by one-way ANOVA and q test among groups,t-test beteew two groups .The significant level is fixed as a=0.05.Results:1. The rats in ischemia/reperfusion group and IGF-1 treated group showed Homer's sign in left side and their right front limbs were paralyzed after focal cerebral ischemia/reperfusion operation. The rats in sham-operation group only showed Homer's sign in left side. All the above indicated the succession of the MCAO model.2. The rats of sham-operated group had not nervous injured symptom. The mNSS of rats in IGF-1 treated group were lower than that in ischemia/reperfusion group at 12h,24h,3d,7d (P<0.05) after reperfusion.3. For ischemia/reperfusion group, the number of positive cells of VEGF protein in the area of semidarkness cortex reached highest level 24h after reperfusion and then decreased rapidly to a low level after 3d. 7d after reperfusion, it dropped to the baseline level. The expression of VEGF protein in IGF-1 treated group was higher than that of ischemia/reperfusion group from 12h to 7d after reperfusion. There was significant difference between the two groups (p<0.05).4. The expression of VEGF mRNA reached highest level at 12h, maintained to 24h after reperfusion and then decreased rapidly to a lowest level in 7d. Compared with the ischemia/reperfusion group, IGF-1 treated group showed higher expression level of VEGF mRNA at every time point. There was significant difference between the two groups at the corresponding time points (p<0.05).5. For ischemia/reperfusion group, the number of positive cells of bFGF protein in the cortex reached highest level 24h and then gradually decreased. There is still a little amount of expression of bFGF protein at 7d. For the IGF-1 treated group, the expression level of bFGF protein was higher than that of the ischemia/reperfusion group (p<0.05).6. The expression of bFGF mRNA reached its peak level 12h after reperfusion and then gradually decreased to a low level 7d after reperfusion. The expression of bFGF mRNA in IGF-1 treated group was higher than that of the ischemia/reperfusion group. The difference was significant between the two groups (p<0.05).7. HE staining showed that the rat's cerebral tissue structure of sham-operated group didn't have obvious abnormal signs under light microscope. 24h after reperfusion, in ischemia/ reperfusion group, the normal nerve cells decreased significantly and the vacuolar degeneration of the nerve cells increased. There was karyopyknosis, tissue looseness and increasing cell spaces. In IGF-1 treated group, the degree of injury was lighter than that of the ischemia/reperfusion group. There were less vacuolar degeneration in the cortical neurons, lighter edema in brain interstitial space and increased number of normal cells.Conclusions:1. In this experiment endogenous IGF-1 injected via lateral cerebral ventricle can improve nervous function, that indicated IGF-1plays an important protection role on ischemia/reperfusion injury .2. The expression of VEGF and bFGF both on gene and protein level was induced by cerebral ischemia damage. It is a reaction of self-protection mechanism.3. IGF-1 could increase the expression of VEGF and bFGF after reperfusion of the focal cerebral ischemia.4. IGF-1 could protect the injured brain tissue which resulted from reperfusion of the focal cerebral ischemia. This effect might be realized through increasing the expression of VEGF and bFGF both on gene and protein level.5. Our study approached the nervous protective effects of IGF-1 and the possible mechanism after the cerebral ischemia. This study provided theoretical basis for the clinical application of IGF-1 to cure cerebral ischemic injury.
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