The Correlation Of Vascular Endothelial Growth Factor(VEGF) Expression And Angiogenesis In Brain Tissue Of Neonatal Rat Hypoxic-ischemic Brain Damage

IntroductionHypoxia - ischemia is a main reason for brain and nerve tissue injury after HIBD. As prenatal asphyxia, brain tissue strive a pathological and physiological course of hypoxia - ischemia - reperfusion injury or lasting hypo - perfusion, which effect brain function and structure and lead to brain injury, and so, brain blood supply improvement is close relation to the neuron function recovering after hypoxia -ischemia. Vascular endothelial growth factor (VEGF) is a newly discovered angiogenesis factor, which consists of VEGF - A,VEGF - B,VEGF -C,VEGF - D,VEGF - E and placenta growth factor (PlGF). Recent work done over the last few years has elucidated the important role of VEGF, which participates in the regulation of normal ( physiological or therapeutic) and pathological angiogenesis ( VEGF - A, VEGF - B ) and lymph - angiogenesis ( VEGF - C, VEGF - D). VEGF also possesses neurotrophic and neuroprotective activity both in the peripheral and in the central nervous system, exerting a direct action on neurons, Schwann cells, astrocytes, neural stem cells, and microglia. In addition, VEGF also regulates neurogenesis. There are a lot of evidences for VEGF that plays an important role on the brain vessel disease in adult. But the research on VEGF expression and distribution in normal newborn brain and HIBD and then the formation of new cerebral blood vessels is rare. The aim of the present research is to investigate not only the distribution and expression of VEGF protein but also angiogenesis in the brain of normal postnatal 7 - day - old rats and HIBD rats in different after HI.Materials and Methods1. Establishment of animal model;Choosing the 176 healthy postnatal 7 -day - old Wistar neonate rats was employed. These rats were divided into two groups at random. Every group in different time was 8. (1) The model group: The HIBD animal model produced by the traditional Rice' s method model . Briefly, 7 - day - old Wistar rats were subjected to the right carotid artery (RCA) ligation followed by a hypoxic (5% oxygen, 95% nitrogen) experience for 20minutes. (2) The control group:RCA was isolated only and the rats without experiencing hypoxic procedure. (3)The rats of the two groups at the same time were sacrificed at the 0N4N8N 12^24 v48 N72hours and 7^14^21 ^28days after HI. The cerebral samples were paraffin - embedded and cut into 5 - micron coronal sections.2. Methods;(1) The sections were stained by hemolytic and eosin.(2) Immunohistochemical staining was performed to examine the expression of VEGF protein in the brain of normal postnatal 7 - day - rats and the HIBD rats at different time.(3) Paraffin sections of the brain were stairafovith FVIfl - Rag by using single immunohistochemical to examine angiogenesis.3. Rat cerebral tissue slices were observed under microscopy at 400 fold. ( 1) The expression of VEGF protein: VEGF protein positive cells were stained brown - yellow and the nuclei of positive cells were blue;(2 ) Angiogenesis expression;positive productions were stained brown - yellow and localized in cy-tolymph of vascular endothelial cell;the nuclei of positive cell were blue. The following quantification of neovascularization, were produced by Weidner methods . The pictures were taken by the figure - camera of OLYMPUS BX51 TF made in Japan.4. Statistics by the calculator diagram resembles the analysis system and quantification of neovascularization, the date express by x ± s and statistically analyzed with t test,rank correlation.Results1. Brain pathology observation: Control rats did not show morphological signs of HIBD;There is hemorrhage in cerebral cortical at Oh after HI, edema in experimental brain nerve cell and interval at 12h, micro - vascular dilated at the lesion site at 12 -24h, malacia exist at 72h, glial cells hyperplasia, glial cells modes formed at 72h, necrosis still exist post HI 7d.2. For normal postnatal 7 - day Wistar rats, VEGF was expressed in the cytoplasm of many brain tissue cell, VEGF positive expression were mainly detected at cerebral cortical and hippocampus, and were also detected at ependy-maN glia ^choroids plesus and capillary vascular endothelial cell. The expression peaked at the postnatal 9 days, and then decreased;it was sustained until 35 days.3. The distribution of VEGF protein expression in model brain is nearly the same as normal postnatal rats. After HI, VEGF protein was observed at Oh and increased from 4h, with a peak at 48h, and then decayed, and remained until 28d. There are statistical meanings at the 4h - 14d after HI.4. Micro — vessel in the control rats cerebral cortical was found 3 — 8/HP at the every time points. The density of proliferating brain capillary on day 3 to day 28 of the model group was significantly higher than those of control. Angiogene-sis peaked at the post HI 3 -7 days.5. There is a significant positive correlation of VEGF expression and angio-genesis at the 0 -72h after HI.Conclusions1. The expression of VEGF protein is detected in brain of normal postnatal 7 - 35 days Wistar rat.2. Hypoxic - ischemic can up - regulated the expression level of VEGF protein. In the brain of normal postnatal 7 - day Wistar rat and Model group, VEGF protein are expressed at the same regions, but in the later, the expressionlevel is significantly higher and lasts a long time.3. Angiogenesis exists in the brain tissue of neonatal rat HIBD model and the peaks at the post HI 3 -7 days.4. There is a significant positive correlation of VEGF expression and angiogenesis at 0 - 72h after HI.