The Dynamic Study Of Brain Tissue Caspase-3and VEGF In Premature Rabbits With Germinal Matrix-Intraventricular Hemorrhage

Objective: Germinal matrix-intraventricular hemorrhage(GMH-IVH)and neurological sequelae of GMH-IVH are the common cause of death inpremature infants and nervous system dysfunction in children period. Inaddition, the incidence of preterm infants with GMH-IVH is significantlyhigher in our country than in developed countries, the incidence is56.6%,with severe hemorrhage16.3%,and about35%of the survivors left behindchronic neurological disorders, such as cerebral palsy, epilepsy, learningdifficulties and cognitive abnormalities. But the mechanism of GMH-IVHsecondary brain injury is complex, involving in many factors, such asinflammation, oxidative stress, microglia proliferation and so on. Whetherthere are some relations with these factors, and which is the final route ofGMH-IVH secondary brain injury still unclear. Studies have reported thatacute traumatic brain injury in adult rat is often accompanied by neuronalapoptosis, and has obvious consistency between neuronal apoptosis andCysteine Protein-ase-3(Caspase-3) expression. Therefore, some scholarsproposed that apoptosis may be the ultimate way of brain injury followingtraumatic brain injury. But in newborn animals, especially in newborn pretermanimals, whether Caspase-3involved in neuronal apoptosis after GMH-IVH isstill unclear, there is rare literature to report it. It has been confirmed in manyadult animals that vascular endothelial growth factor (VEGF) played animportant role in promoting angiogenesis, anti-apoptosis and the protection ofischemic brain tissue. But in preterm animals, especially in newborn pretermanimals, whether VEGF involved in secondary brain injury after GMH-IVH isalso unclear. The purpose of this experiment is by monitoring the expressionof Caspase-3and VEGF in preterm rabbits with GMH-IVH to see whether the Caspase-3and VEGF are involved in the secondary brain injury afterGMH-IVH and hope to provide some new ideas for the treatment ofpremature infants with GMH-IVH.Methods:1. Premature rabbits were born by cesarean section and given incubated,milk powder feeding after born. Then the premature rabbits were randomlydivided into two groups, experimental group and control group, each groupeighteen. Three hours later, the premature rabbits in experimental group weregiven intraperitoneally injection of50%glycerol (10ml/kg), the pretermrabbits in control group were given intraperitoneally injected with the samedose of saline.2. Cranial ultrasound scan was given to check the presence of GMH-IVHin preterm rabbits at24h after born.15pups were recognized havingGMH-IVH in experimental group (GMH-IVH model group) and an equalnumber of normal rabbits were also chosen from control group. Within eachgroup,15pups rabbits were randomly divided into three sub-groups: everytime period had5pups (at24h,48h and72h after born). All the pretermrabbits were given euthanasia and then the periventricular zone tissue wererapidly stripped into wax block, and sliced them up for immunohistochemicalstudy.3. The expression of Caspase-3and VEGF of periventricular zone tissuewere detected by immunohistochemical method and detected changes inneuronal apoptosis were detected by TUNEL apoptosis kit. Both the detectionof immunohistochemical method and apoptosis kit used horseradishperoxidase enzyme-labeled streptavidin colar, the cells with cytoplasm stainedbrown were positive-cells.4. The calculation of mean percentage of positive cells: At highmagnification,, an image analysis system(named imagepro-plus6.0) was usedto measure the percentage of positive cells in each slice at24h,48h,72h afterborn, the mean percentage was finally calculated. 5. Statistical analysis SPSS13.0statistics software and Microsoft Excel2003were used to carry on statistics processing, Chi-Square was used tocompare categorical variables. P-value<0.05(double side) had statisticalsignificance, highly significant when p-value<0.01.Results:1. A preterm rabbit was born by cesarean section(Fig1); The Specimenshowed the bilateral intraventricular hemorrhage with dilation of ventricles inpreterm rabbit(Fig2)2. The expression of Caspase-3of brain tissue: In GMH-IVH group, themean percentage of positive-cell of Caspase-3at each time was significantlyhigher than that in control group, all had significant difference(P-value<0.01);within GMH-IVH group, the mean percentage of positive-cell of Caspase-3atdifferent time also had significant difference(P-value<0.01), the highest meanpercentage of positive-cell was at48h after born, followed by24h,the last72hafter born, but within control group, the mean percentage of positive-cell ofCaspase-3at different time had no difference(P-value﹥0.05)3. The expression of VEGF of brain tissue: In GMH-IVH group, themean percentage of positive-cell of VEGF at each time was higher than that incontrol group, all had difference (P-value<0.01-0.05); within GMH-IVHgroup, the mean percentage of positive-cell of VEGF at different time also haddifference(P-value<0.01-0.05), the highest mean percentage of positive-cellwas at24h after born, followed by48h, the last72h after born, but withincontrol group, the mean percentage of positive-cell of VEGF at different timehad no difference(P-value﹥0.05).4The expression of apoptosis cell of brain tissue: In GMH-IVH group,the mean percentage of apoptosis cells was higher than that in control group at24h and at48h after born, and the difference was significant(P-value<0.01),but at72h after born, there was no difference betweenGMH-IVH group and control group(P-value﹥0.05); within GMH-IVH group,there was no difference between24h and72h after born (P-value﹥0.05),butthere was significant difference of the mean percentage of apoptosis cell among at24h,48h and72h after born (P-value<0.01), within control group,the mean percentage of apoptosis cell at different time had no difference(P-value﹥0.05).Conclusions:1.When premature rabbits suffered from GMH-IVH, the number ofapoptosis cells in brain tissue were increased than those in normal prematurerabbits, and the trend of Caspase-3expression was consistent with theapoptosis of neural cell, it indicated that Caspase-3participated in thesecondary brain injury after GMH-IVH.2. When premature rabbits suffered from GMH-IVH, the expression levelof VEGF was obviously higher than that in normal premature rabbits, itindicated that VEGF participated in in the secondary brain injury afterGMH-IVH and possible played the role of cerebral protection.