The Effects Of Intraventricular Administration Of Insulin On Neuronal Apoptosis In Neonate Rat After Hypoxic-Ischemia Brain Damage

Introduction: Hypoxic-ischemic encephalopathy (HE) is a nervous system lesion of newborns caused by perinatal hypoxia and ischemia. It is a major contributor to morbidity and mortality in infants and children. There are no specific therapies proven to ameliorate long-term outcome for HIE in the clinical practice at present. Apoptosis which is one of the main patterns of neuronal death plays an important role in the delayed brain damage after HIE. So anti-apoptosis treatment may become one of important remedies for HIE by inhibiting or ameliorating delayed neuronal damage. Evidences have accrued that insulin is beneficial in both global ischemia and focal ischemia in adult rats. It is found that insulin rescues retinal neurons from apoptosis by the phosphatidylinositol 3-kinase /Akt-mediated mechanism. We injected insulin into brain ventricular after HIE in neonate rat and studied neuronal apoptosis in cortex and hippocampus 24 hours after HEE. The possible mechanism of anti-apoptosis of insulin was studied too.Methods: Part one: Sixteen Seven-day Sprague-Dawlay (SD) neonatal rats were used. We established HIE model based on Rice-Vanned method. Briefly, Rat pups were anesthetized with halothane, and the left common carotid artery was surgically exposed and permanently ligated. The incisions were sutured and the pups were return to the dam for a 2-h recovery. Pups were then placed in containers (37℃ water bath to maintain normothermia) through which humidified 8% oxygen (balance nitrogen) flowed for the next 2h. Upon completion of the H-I, the pups were returned to theirdam. Two rats died during hypoxia. Fourteen SD rats were randomly divided into insulin-treating group and control group. Low dose insulin or phosphate-buffered saline (PBS) was injected into cerebroventricules respectively in treating group and control group 2 hours after HIE. Apoptosis neurons in cortex and hippocampus were observed 24 hours after HIE by transmission electron microscope and Hematoxylin and eosin (HE) staining. Blood sugar was checked 90 minutes after intracerebroventricular injection by cutting tail.Part two: Forty Seven-day Sprague-Dawlay (SD) neonatal rats were used. We established HIE model based on Rice-Vanned method. Eight rats died during hypoxia. Thirty-two rats were randomly divided into four groups. Low-dose ( l.0IU/kg ), moderate-dose ( 2.5IU/kg) and high-dose ( 5.0IU/kg ) insulin were administered into cerebroventricules of treating groups respectively. PBS was injected into cerebroventricles of control group. Apoptosis neurons in cortex and hippocampus were counted by TUNEL (TdT mediated dUTP nick end labeling) method 24 hours after HE. Blood was obtained by cutting tail and blood sugar was checked 90 minutes after intracerebroventricular injection. The effect of different doses of insulin on neuronal apoptosis after HIE were evaluated .The relationship between anti-apoptosis and lowing blood sugar of insulin was investigated.Part three: Twenty-six Seven-day Sprague-Dawlay (SD) neonatal rats were used. We established HIE model based on Rice-Vanned method. Two pups died during hypoxia. Twenty-four pups were randomly divided into four groups. Low-dose (l.0IU/kg), moderate-dose (2.5IU/kg) and high-dose (5.0IU/kg) insulin were administered into cerebroventricles of treating groups respectively. PBS was injected into cerebroventricles of control group. In order to researching the mechanism of anti-apoptosis of insulin, fresh brain tissue was taken out and Caspase-3 P20 subunit was checked by Western-blot 24 hours after HIE. NIH2.0 Scion Image software was used to determine the absorbency of Caspase-3 P20 band. Results:1. It was observed clearly by HE staining and light microscope that there were many apoptosis neurons and apoptosis bodies besides necrotic neurons in the cortex andhippocampus of cerebral hemisphere of ligation side in PBS control group. Apoptosis neurons and apoptosis bodies in low insulin treating group distribute sparsely.2. We counted 3000 neurons in cortex and 1000 neurons...