The Study Of Hypoxia-Inducible Factor-1 α Expression And Its Role Following Experimental Traumatic Brain Injury

Vascular injury after traumatic brain injury is the key primary event. It is the key link in the process of TBI repairment. Successful revascularization can provide the key nerve vascular microenvironment for the regeneration and reshape of neurons. Its effective perfusion can remove damage factor, promote nerve repair and the transfer of growth factor as well as provide nutrition for the regeneration of neurons and synapses, which makes it the key in traumatic brain injury treatment. Therefore, in recent years the strategy to promote angiogenesis of TBI treatment has attracted growing attention.Hypoxia-inducible factor-1α is a transcriptional regulation factor which could regulate the transcription of target genes, such as VEGF, EPO and i NOS; so it is the regulation center of vascular response system in low oxygen conditions. Related studies have confirmed that HIF-1α could induce the formation of new blood vessels in rats is chemic myocardial injury and the brain injury model, while the expression and function of HIF-1α after TBI is unknown. This study mainly explores the expression and function of HIF-1α after TBI in rats.PartⅠ: The expression and its role of HIF-1α following experimental traumatic brain injury Objective To observe the dynamic expression of HIF-1α and explore its possible mechanism following experimental traumatic brain injury.Methods 56 male SD rats were randomly divided into sham group(n=8), control group(n=8) and the traumatic brain injury group(TBI, n=40)to establish TBI models by ameliorated Feeney method. According the time from injury to execution, TBI group was divided into 5 subgroups respectively TBI-6h, TBI- 24 h, TBI- 3d, TBI- 7d and TBI- 14 d.Each subgroup had 8 rats. The rats in the control group were only cut the skin and openedbone window to ensure the hard membrane integrity and no combat damage, while normal rats were used in the sham group. RT-PCR and Western blot technology were used to explore the expression of HIF-1α m RNA and protein as well as the expression of its upstream regulatory factors PHD2 after establishing the model. HE staining was used to observe the pathological changes of the lesions after TBI model of remaining four rats. SP method was used to detect the expression of HIF-1α protein of the epicenter and the surrounding brain tissue. Microscopic image analysis system was used to calculate positive cells.Result(1)RT-PCR The expression of HIF-1α m RNA was detected in all sham group, the control group and TBI group brain tissue. There was no significant difference between the three groups.(p>0.05). Contrast to sham group, the expression of HIF-1α m RNA in TBI-24 h, 3d were obviously higher(P<0.01), while there was no significant difference in TBI-6h, 7d, 14 d.(2)Western blot There was no expression of HIF-1α protein detected in the sham group and the control group while improved expression detected in the epicenter and the surrounding brain tissue after TBI. The expression of PHD2 protein in sham group and control group was normal and significant degradation in TBI group was detected. The expression of three groups was significantly different(P<0.05).(3)S-P Immunohistochemistry There was no expression of HIF-1α protein detected in the sham group and the control group while improved expression detected in the epicenter and the surrounding brain tissue after TBI. Positive expression results were mainly located in neurons in the core. Each subgroup of TBI group began to express at TBI-6h, reaching the peak at TBI-3d. It began to decline at TBI-7d and basically recovered to pre-injury level at TBI-14 d.Conclusion Various pathological process led to the lack of oxygen in brain ischemia area and induced the expression of HIF-1α after TBI. Its expression regulated mostly by the post-transcription mechanisms. PHD2 protein degradation is important for the increased expression of HIF-1α.Part Ⅱ The influence of 2-ME-2 on the expression of rat HIF-1α and its downstream angiogenesis-related factor Objective To explore the role of HIF-1 alpha in angiogenesis after experimental TBI by observing the expression of HIF-1α, VEGF, Ang-1, MMP-9 and CD31 after 2-ME-2intervention.Methods 80 healthy adult SD rats were randomly divided into TBI group(n=40) and TBI+2-ME-2 group(n=40) by adopting the TBI models by ameliorated Feeney method.The group were randomly divided into 5 subgroups: 6h, 24 h, 3d, 7d and 14 d and each subgroup had 8 rats. After the success of establishing of the model, subgroups 6h and 24 h of TBI+2-ME-2 group would be taken intraperitoneal injection of 2-ME-2(2.5 mg/kg)immediately. Subgroups 3d, 7d and 14 d got first injection after injury and got second injection after 2 days. At the same time, TBI group got intraperitoneal injection with an equal amount of DMSO. Four rats were from each subgroup randomly and collect the epicenter and the surrounding brain tissue were collected. Western blot was used to detect the expression of HIF-1α from two groups at different time point. RT-PCR was used to detect the expression of HIF-1α, VEGF, Ang-1, MMP-9 m RNA. HE staining was used to observe the pathological changes of the lesions after TBI model of remaining four rats. S-P method was used to detect the expression of CD31 protein. Microscopic image analysis system was used to calculate the MVD.Results(1)Western blot The expression of HIF-1α began to increase at 6h after TBI model successfully established. It reached the peak during 24 h to 3d and began to decline at 7d and basically recovered to pre-injury level at TBI-14 d. The expression of HIF-1αfrom TBI+2-ME-2 group was significantly lower than that from TBI group at each time point.(p<0.01,statistically significance.)(2)RT-PCR ①The expression of HIF-1αm RNA between two groups was not significantly changed(p>0.05);②The expression of VEGF m RNA obviously increased at TBI-6h/TBI-7d/TBI-14 d, and it obviously decreased after2-ME-2 intervention(p < 0.05, statistically significance); ③The expression of Ang-1m RNA was obviously increased at time point TBI-3d, and reached the peak at 7d、14d, and obviously decreased after 2-ME-2 intervention(p<0.01,statistically significance); ④The expression of MMP-9 m RNA began to increase at 6h, and reached the peak at 24 h, andthen declined after 3d. It obviously decreased after 2-ME-2 intervention(p < 0.05,statistically significance);(3) S-P Immunohistochemistry The expression of CD31 protein was increased obviously at time point TBI-7d and TBI-14 d, and obviously decreased after 2-ME-2 intervention(p<0.05,statistically significance).Conclusion The expression of HIF-1α was increased after TBI, which induced the expression of angiogenesis related genes VEGF and Ang-1, as well as promoted the repair and regeneration of damaged blood vessels. However, HIF-1α could also promote MMP-9expression, which could aggravate the brain edema.