Mechanism Of The Protective Effect Of Insulin-like Growth Factor-1 On Focal Cerebral Ischemia-reperfusion In Rats

Ischemic cerebrovascular disease (ICVD) is one of the frequently occurring diseases of the nervous system, which is acute and severe, its rate of multilation and mortality is very high. The disability rates in these patients of ICVD roughly make up 75 %. It leads to heavy burden to patient family and society. According to recent medical material, people who were dead of ICVD are less than the people dead of malignant tumor, putting it in second spot in dead reason. Serious degree of nervous system injure depends on the time and area of cerebral ischemia. The most important purpose of curing ICVD is to protect and avoid more neurons to dead. Insulin-like growth factor-1 (IGF-1) is one of neurotrophic factor. IGF-1 has been caused attention, because it can promote increasing of nerve cells and brain tissues. Studies have shown that IGF-1 injected through lateral ventricle markedly decreased the infarct volume and neuron injure. But neuroprotection of IGF-1 via tail vein hasn't been known. The brain protective mechanism of IGF-1 is not clear. Cysteinyl asparate-specofic proteinase (Caspase-3) is key enzyme of neuronal apoptosis after stroke. Activatin of caspase-3 contributes to neuronal apoptosis. Caspase-3 plays important part in chronic neuronal injury. Neuronal nitric oxide synthase (nNOS) can produce nitric oxide (NO) during stroke and ischemia-reperfusion injury. It is clear that this kind of NO does harm to body. Some scholars think neuroprotection of IGF-1 is related to Caspase-3 and nNOS.By creating rat focal cerebral ischemia reperfusion model established with suture emboli method and injecting IGF-1 via tail vein, the change of rat cerebral infarct volume and nerve score and injure of nerve cell under light microscope were measured to estimate whether IGF-1 played a protective role. By using immunohistochemical techniques, the expression of Caspase-3 and nNOS were observed to estimate protecting mechanism of IGF-1. It can provide experimental proof for IGF-1 preventing and curing ICVD.Materials and Methods: (1) Thirty-six healthy SD rats weighing from 300-350 grams were randomly divided into three groups (twelve rats each group). Group A was sham-operated. Group B were ischemia reperfusion. Group C were ischemia reperfusion plus IGF-1 treatment. Each group was again randomly divided into group I and II. Group C injected IGF-1 5Mg via tail vein at one hour of reperfusion after left middle cerebral artery occlusion (MCAO) for two hours. IGF-1 was dissolved in lml saline. Group A and B injected lml saline via tail vein at the same time. (2) The rats of each group I were sacrificed by decapitation at twenty-four hours after MCAO for two hours. The brain tissue was removed and cut a coronal slice approximately 4mm thick centered with optic chiasma. The slice was embed in paraffin and made immunohistochemical sections (4Mm thick). By using HE staining and immunohistochemical SP methods, we detected the number of nNOS and Caspase-3 positive cell and examined injure of nerve cell under light microscope. (3) The rats in each group II were sacrificed by decapitation at one hundred twenty hours after operation. The brain tissue was cut five coronal slices after being cut brain stem and cerebellum, each slice approximately 2mm thick. The slices were stained for thirty minutes without light by being put in 2% 2,3,5-triphenyterazolium chloride (TTC) at 37°C. During the period of being stained, these brain slices were turned every seven or eight minutes. The red color zone of rat brain slices was normal region and the white zone was infarct region after TTC staining. The infarct regions were excavated and weighed after entire rat brain was weighed. The weigh proportion of infarct and entire region was their volume proportion. (4) The data was handled with SPSS 10.0 statistic software. The difference of two groups was compared withone-way analysis of variance, significant level is a =0.05.Results: (1) After the operation of ischemia reperfusion, all the animals of group B and C behaved low spirit, eat little, low ability of self-clearance, the myodynamia of left extremities decrease, especially front limb. Most rats occurred spin. But they recovered at twenty-four hours after MCAO for two hours. Group C recovered markedly. (2) The rats of group A had not nervous injured symptom. The behavior score of rats in group B and C at two hours after operation: 2.25 ±0.62; 2.08 + 0.67. They hadn't difference (P>0.05). The score of rats in group B and C at twenty-four hour and one hundred twenty hours after operation : 1.67 + 0.49, 0.83+0.41; 1.00± 0.60, 0.17+0.41. They had marked difference(P<0.05). (3) The result of pathological section with HE staining in every group: The rat cerebral section of group A hadn't obvious abnormal under light microscope. The cerebral section of group B showed normal neuron was less , deformation of neuron bodies and concentration of nucleus and bubble in cytoplasm were more, more inflammatory cells infiltrated. The section of group C showed the number of injured neuron and inflammatory cells was less than that of group B. (4) The cerebral infarct percentage: group B 0.246 + 0.003; group C 0.124 + 0.008. The difference between group B and C was significant (P<0.05). Infarct region hadn't been found in group A. (5) The mean of Caspase-3 positive cells of every group (neuron /every 200 neurons): group A 2.17 + 0.75; group B 67.17 + 6.34; group C 20.33 ±3.08. The difference between every two groups was significant (P<0.01). (6) The mean nNOS positive cells of every group (neuron /every 200 neurons): group A 15.00+1.41; group B 38.83 + 3.92; group C 28.33 ±2.58. The difference between every two groups was significant (P<0.01).Conclusion: (1) Middle cerebral artery occlusion method was adopted to make focal ischemic cerebrovascular model in this research. The model was similar to clinical cerebral ischemia. It can control accurately the time of Ischemia-reperfusion. It had the high rate of repeating. (2) In this experiment IGF-1 injected via tail vein can improve nervous function, effectively attenuate the pathological changes and infarction volumes, therefore, IGF-1 injected via tail vein can play protective role in cerebral ischemia reperfusion injury. The method that IGF-1 is injected by via tailvein is convenient and economical. (3) The nervous protective mechanism of IGF-1 acts by inhibiting apoptotic cascade, decreasing nNOS activity and production of NO. (4) The experiment can be served as theoretical testimony for IGF-1 curing ischemic cerebrovascular diseases in clinic.