Effects Of VEGF In Radiation-induced Brain Injury And First Exploration Of Recombinant Human Endostatin Injection In The Treatment Of Radiation-induced Brain Injury

Part1:Establishment of the rats models of radiation-induced brain injury (RBI) and the effects of VEGF protein in RBIPurpose:To establish the RBI rats model and research the pathologic mechanism of RBI. To investigate the expression of VEGF protein in brain capillary endothelial cells (BEC), gliacyte and neuron (G&N) and the biological function of vascular endothelial growth factor (VEGF) protein in process of RBI.Method:Sprague-Dawley rats were randomly divided into two groups, sham group with sham-irradiation, and irradiation group in which the whole brains of rats were administered by electron beam (6MeV) to establish the animal model of RBI with the single fractionated dose of20Gy. All rats were sacrificed at1,3,7,14,28,42,56day post-irradiation respectively. The pathological changes were observed with light microscopies and transmission electron microscopy (TEM). Brain water contents (BWC) were determined by wet/dry weight method. The VEGF protein in BEC and G&N was observed via immunohistochemistry (IHC). The integrated optical density (IOD) of VEGF protein in BEC, G&N were measured via immunohistoehemistry with computerized image processing.Results:(1) Enlarged perivascular spaces and the damage of vascular intima was appeared1day post-irradiation. The peak time of thrombosis formation in IR group were3r and28th day respectively. The pathological process in rats models of RBI includes brain endothelial cell damage, brain edema, thrombosis formation and ablation, revascularization and angiogenesis.(2) The BWC in IR group has elevated since the14th day, which was increased significantly than that of Sham group14,28day post-irradiation respectively.(3) There are positive cells by VEGF staining in BEC, G&N from the first day after irradiation to the end of observation via IHC, both of which reached the peak at the14th day.(4) The IOD of VEGF protein in G&N showed a significant rise1,14,42,56day post-irradiation, however which in BEC was increased significantly at the1,14,42day after irradiation.(5) There was a partly positive correlation between BWC and the IOD of VEGF protein in G&N.Conclusion:(1) Radiation induced the damage of blood-brain-barrier (BBB) and the formation of brain edema, which participated in the process of RBI.(2) It lead us to suspect that the course of thrombosis formation and ablation would induce the change of brain blood perfusion and dual-phase of BBB open.(3) It was observed that VEGF protein was involved in the inducement of brain edema and the formation of thrombosis at the acute RBI phase, however it participated in the progression of angiogenesis and thrombosis ablation at the early-delayed RBI phase.Part2:Alterations of VEGF mRNA and protein in RBI rats model and molecular mechnisms of interaction of VEGF, NF-κB and ICAM-1Purpose:To investigate the change of expression of VEGF mRNA and protein and the regulation among VEGF, nuclear factor κB (NF-κB) and intercellular adhesion molecule-1(ICAM-1). To choose the optimal opportunity in anti-VEGF treatment.Method:Real-time quantitative reverse transcriptase-PCR was used to measure the level of VEGF/NF-κB/ICAM-1mRNA in the brain tissue. The expression of VEGF/NF-κB/ICAM-1protein in whole brain tissue was detected with Western blot.Results:(1) The expression of VEGF mRNA in IR group was declined significantly than that of sham group1,3,7,28,42day post-irradiation respectively.(2) The up-regulation of VEGF protein expression in whole brain tissue was observed for1and7day after irradiation, however which was down regulated3,14,28,42,56day post-irradiation.(3) The expression of NF-κB mRNA in IR group was decreased significantly than that of sham group3,28day post-irradiation respectively. (4) The overexpression of NF-κB protein was observed1,7,14,42,56day post-irradiation, which reached the peak at the7th day. It reduced to the normal level at the3rd and28th day.(5) The expression of ICAM-1mRNA in IR group was increased significantly than that of sham group14day post-irradiation, however which down-regulated at the3rd and28th day respectively.(6) The expression of ICAM-1protein in IR group was declined than that of Sham group at the1st,7th,56th day, but it turned to up-regulated3,14,28,42day post-irradiation, which peaked at the28th day.(7) There were positive correlations between each pair of the expression of VEGF, NF-κB and ICAM-1mRNA significantly.Conclusion:(1) VEGF gene transcription was inhibited relatively in RBI rats models. The expression of VEGF protein in whole brain tissue was up-regulated only at the early stage of RBI. Feedback inhibition might play some part in the opposite changes between mRNA and protein of VEGF in the early term of irradiation.(2) The balance between apoptosis and antiapoptosis in RBI rats models might be highly correlated with suppression of NF-κB gene transcription. The level of NF-κB protein expression fluctuated during the observation period and disagreed with the change of NF-κB mRNA expression, which indicated the presence of post-translational regulation.(3) The change of ICAM-1protein expression lagged behind that of ICAM-1mRNA, both of those had the same varying trend.(4) These results suggested that there might be a positive feedback loop among VEGF, NF-κB and ICAM-1genes.Part3:First exploration of Recombinant Human Endostatin Injection in the treatment of RBIPurpose:To determine the role of Human Recombinant Endostatin Injection (Endostar) in the treatment of RBI and assess the feasibility of Endostar treatment in RBI.Method:We research the pathogenesis change of brain injury via light microscopies, the expression of VEGF mRNA and protein in rat brain via QRT-PCR, western-blot and IHC respectively among Sham, Irradiation (IR) and Irradiation+Endostar (IR+EN) groups. We assess the feasibility of Endostar treatment in RBI through the measurements of body weight and BWC, and COX regression univariate analysis.Results:(1) Endostar treatment against RBI induced vasogenic brain edema at acute RBI, but the reverse occurred at early delayed RBI, along with an obviously broadened therapeutic window for thrombosis. However, the benefits of neurons were less significant compared with the treatment of RBI.(2) Endostar treatment against RBI down-regulated the mRNA expression of VEGF throughout the observation period. This expression increased on day7. However, the mRNA expression of VEGF in the IR+EN group was significantly higher than that in the IR group on days3,7, and28. And it down-regulated the protein expression of VEGF on days1,14,42, and56. However, this treatment resulted in the unexpected overexpression of the VEGF protein on days3,7, and28. Endostar treatment against RBI induced the VEGF protein expression of BEC, which peaked on day7. The VEGF protein expression of G&N was lower in the IR+EN group than in the IR group.(3) Endostar treatment against RBI aggravated brain edema mainly at acute RBI (days1to7post-irradiation). However, the reverse occurred with early delayed RBI (post-irradiation for weeks).(4) Endostar treatment against RBI significantly increased the risk of weight loss.(5) Endostar treatment against RBI elevated the survival rate only during the early stage of the observation period but significantly reduced the survival rate from day5until the endpoint.Conclusion:The anti-VEGF therapy for RBI using Endostar broadened the therapeutic window for thrombosis at the early stage without the controllable brain edema. However, this therapy also involved serious side effects, which were greatly influenced by the inhibition of VEGF at the early-delayed stage of RBI.