Study On The Expression Of Receptors Of Vascular Endothelial Growth Factor In The Neonate Rat Brain With Hypoxic-ischemic Brain Damage

IntroductionHypoxic -ischemic encephalopathy(HIE) is fetal and neonatal brain damage resulted from all kinds of elements in perinatal period lead to cerebral hy-poxia and decrease or break cerebral blood flow. There are a series of syndrome in the postnatal. Hypoxia may produce cerebral acidosis, it can damage nervous cells directly and lead to nervous cells death. Cerebral ischemia influence supersession of Glucose and Energy is similar to hypoxia,but cerebral acidosis in ischemia is more serous than in hypoxia. That hypoxic - ischemia harm major cerebral region is vascular endothelial cells and cerebral nervous cells. Harm of vascular endothelial integrity can enhance vascular permeability and lead to cerebral edema, furtherly oppress cerebral vessel and aggravate damage of cerebral tissue. Precently, treatment of hypoxic - ischemic brain damage( HIBD) has attent-ted the importance of recover cerebral blood flow early to relief damage of cerebral function.Vascular endothelial growth factor( VEGF)is known as a regulator of blood vessels and vessel - permeability factor. it is a kind of mulfunctional cell factor that specially action vascular endothelial cells. It has been valued in recent research because its outstanding function to promote vascular endothelial cell reproduce and quicken newborn vessels to bread. The multiple biological functions of VEGF are mainly mediated via their specific receptors of VEGF. Known VEG-FRs contain:VEGFR - 1 (flt - 1, fms - like tyrosine kinase ) , VEGFR - 2 (flk - 1/KDR, fetal liver kinase - 1, in humans known as KDR, kinase insert domain receptor), VEGFR — 3(flt — 4) ,neuropilin - 1, neuropilin -2. multiple biological functions of VEGF/ VEGFR mainly show that it directly stimulate vascular endothelial cell to proliferate, polarizal, denatime and migrate, stick reciprocallybetween cells, and range into the right line along with the opening cavity type structure. Thereby promote neovascularization and increase vascular permeability. In addition, VEGF/ VEGFR play a major role in development of embryo and have neuroprotective effect.Present research about expression of VEGFR of neonate and HIBD is less. This research is based on the animal model of HIBD, dynamic observation of expression and distribution of VEGFR protein at different timepoints of the postnatal and HIBD. The aim of the present research is to investigate the function of VEGFR protein in the morbidity of HIBD.Methods and materials1. Establishment of animal model: choosing the healthy postnatal 7 - day -old Wistar neonate rats were employed. The HI brain injury animal model was produced by the traditional Rice s method model. Briefly,7 - day - old Wistar rats were subjected to the right common carotid artery ( RCA) ligation followed by a hypoxic(5% oxygen,95% nitrogen) experience for 20 minutes. The rat pups were sacriced at different timepoints after HI. The brains were taken on the ice board and finally embedded in paraffin. RCA was isolated only in the sham operated rats without experiencing hypoxic procedure, which were sacrificed at the different post - operation timepoints as mentioned above.2. Hematoxylin and eosin staining was done for general morphological evaluation of each case.3. Immunohistochemical staining was performed to examine the expression of VEGFR protein in the brain of normal ponstatal 7 - day - rats and the HIBD rats which undergo HI at different timepoints. The cerebral samples of paraffin -embedded were cut into 5 micron coronal sections. Antigen retrieve was performed by microwave heating in citric buffer (pH 6. 0) and were visualized by SABC method.4. Rat cerebral tissue slices were observed under microscopy at 400 fold and VEGFR positive cell were stained brown - yellow and the nuclei of positive cells were blue.5. The pictures were taken by the figure - camera of OLYMPUS BX51 TF made in Japan. The positive cells counting and mean gray value were taken. The data were expressed as(x ±s)and statistically analyzed with t test SPSS 10. 0 for Windows.Results1. Brain pathology examination: Control animals did not show morphological signs of hypoxic - ischemic brain injury. There is edema in experimental cell and interval at 24h. melted mess exist at 72h,glial cells were intensly. glial cells nodes were formed at 72h,still exist in postnatal 7.2. Expression of VEGFR protein by immunohistochemistied assay for normal Postnatal 7 - day Wistar rats' level of VEGFR protein is lower. Fit - 1/ Flk- 1 were mainly expressed in choroids piesus ^ capillary vascular endothelial cell and leptomeninges. Coital neurons expressed lowly. The positive cell percent of expression of protein of Fit - 1/ Flk - 1 reach the peak at 8d/9d postnatal(53. 45±19.04)/(61.09±10.73).3. HI can up - regulated the expression of Fit - 1/ Flk - 1 protein. Comparing with postnatal 7 - day Wistar rats,the distribution of Fit — 1/ Flk - 1 protein is nearly the same. Fit - 1/ Flk - 1 expressed both in ipsilateral cortel and hippocampus , but Flk - 2 more than Fit - 1. (1 ) The time course of expression of Fit-1 protein showed that it began to up -regulated at 2h post HI(29. 25 ±4. 42) ,and reached to the peak at 48h (76.91 ±6.25) after HI and to the normal in 7d(37. 60 ±7.01). There are statistical meaning at the timepoints of 2hA4hN 48h^72h. (2)The time course of expression of Flk - 1 protein showed that it began to up -regulated at 4h post HI(34. 96 ±6. 38) ,and reached to the peak at 24h after HI(79. 19 ± 12. 17) and reached to the normal in 14d(49. 15 ±5. 91). There are statistical meaning at the timepoints of 4h>8hN24h>72h.4. Investment of different timepoints gray value of Fit - 1/ Flk - 1 protein of postnatal 7 - day Wistar rats. The gray value of protein of Fit - 1/ Flk -1 reach to the peak at 8d/9d postnatal( 155.75 ± 12.91)/(138. 10 ±6.52). Comparing with up - group,the timepoints of Fit - 1 increased apparently at 4hN8hN7d( P < 0.05) ;the timepoints of Flk -1 increased apparently at 8h^24h J2hN14cU21cU28d(P<0.05).5. HI can down - regulated the gray value of Fit - 1/ Flk - 1 protein. Comparing with postnatal 7 - day Wistar rats, (1) The time course of expression of Fit - 1 protein showed that it began to up - regulated at 2h post HI(170. 10 ± 5. 82) ,and reached to the peak at 48h (131.40 ±5. 83) after HI and to the normal in 7d( 184.71 ±6. 82). the timepoints of 2hv4h.48h increased apparently very much( P < 0. 01) , the timepoints of 72h increased apparently ( P < 0. 05 ). Comparing with up - neighbor group, the timepoints of Fit - 1 increased apparently at 4h.8h J2h Jd( P <0.05). (2) The time course of expression of Flk -1 protein showed that it began to up - regulated at 4h post HI(160.20 ±7. 81) , and reached to the peak at 24h (119. 60 ±6. 41) after HI and to the normal in 7d( 151. 51 ± 10. 81). Comparing with control, the timepoints of 4h.24h increased apparently very much( P <0. 01) ,the timepoints of 8h^72h increased apparently(P <0. 05). Comparing with up - neighbor group,the timepoints of Flk - 1 increased apparently at 4hN24h .48h JdN21d,28d(P <0. 05).Conclusion1. The expression of VEGFRs protein exist in normal postnatal 7 days Wistar rat. especially in choroids plesus .capillary vascular endothelial cell and lep-tomeninges, also coital neurons expressed fixed quantity. Fit - 1 reach to the peak in postnatal 8 - day. Flk - 1 reach to the peak in postnatal 9 - day.2. HI can up - regulated the expression of VEGFRs protein. Fit - 1/Flk - 1 expressed both in ipsilateral hemisphere and hippocampus, Fit - 1 reach to the peak at 48h after HI and Flk - 1 reach to the peak at 24h after HI.3. The expression site of VEGFR protein is nearly the same. But the intensity of expression of VEGFR protein is up and the time is prolonged.