Experimental Research On Correlations Between Expression Of Hif-1α, Vegf, Microvascular Dynamic Variation And Brain Edema After Traumatic Brain Injury

Objective:This study was designed to explore the process and regularity of brain edema through detecting water content of impact brain tissue in the models of local brain contusion and laceration in the rats referring to Feeney's method, and to probe the possible mechanism of brain edema following traumatic brain injury through investigating the associations between the expression of hypoxia-inducible factor-l alpha (HIF-lα), vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor-2 (VEGFR-2), the dynamic variation of microvessel density and brain edema with immunohistochemistry.Methods:1. Making the model of TBI and detecting the water content of brain tissues.Ninety healthy adult male Wistar rats were randomly divided into two groups: sham operation group (SO) and traumatic brain injury group (TBI). The subjects were sacrificed at 30 minutes, 1 hour, 3 hours, 6 hours, 12 hours, 1day, 3days, 7days and 14days after impact injury. Each group was divided into nine subgroups according to the nine time points. We made the models of local brain contusion and laceration in the rats referring to Feeney's method, and evaluated the model on the basis of their behaviors of balance, turning their bodies over, raising their head, running, elusion, and so on. Besides, we detect water content of impact brain tissue to explore the process and regularity of brain edema after trauma.2. Detecting the expression of HIF-1α, VEGF, and VEGFR-2 protein by IHC.On the basis of part one, the samples of injury brain tissues were collected to detect the expression of HIF-1α, VEGF, VEGFR-2 protein by immunohischemistry respectively following sever traumatic brain injury. Furthermore, their effects and the associations with brain edema in impact brain injury were explored based on correlations between their MOD and the water content of injury brain tissues. 3. Investigating the dynamic variation of microvessel densityOn the basis of part one and part two, further to color the microvessels with CD34 by immunohistochemistry and count amount and MVD, to investigate the association between the dynamic variation of microvessel density, the expression of HIF-1α, VEGF, VEGFR-2 and brain edema. The possible mechanism of brain edema was probed following traumatic brain injury.Result:1. The behavior variations of impact brain injury models: Rats breathed longly and deeply, and they had dysfunction such as motion and balance ability. The scales of 6h, 12h, 1d, 3d and 7d subgroups were significantly decreased compared with SO(P<0.05). The functions did not become normal until 14days after trauma(P>0.05).2. Water content of brain tissues: Water content was increased at the 3h(P<0.05), the peak value appeared at the 1~3d after TBI, and began to decline at 7d, went back to normal levels at the 14d subgroup compared with SO group(P>0.05).3. The expression of HIF-1αand VEGF protein and the association with brain edema: The expression of HIF-1αand VEGF protein were significantly increased within the injured cerebral cortex before the 12h subgroup compared with corresponding SO subgroup(P<0.05) and were in parallel with each other. The two factors had different expression tendency after the 12h subgroup: The expression of HIF-1αprotein began to reduce slowly and to be normal at 7d(P>0.05). Otherwise, the VEGF protein continued to increase, and maintain high at 7d compared with SO group(P<0.05). They all began normal at 14d compared with SO group(P>0.05).Data from the TBI group were collected to investigate the correlations between HIF-1α, VEGF and the water content of injury brain tissues. Linear trend each other was found from the scatter plot. Therefore, their associations were analyzed by Pearson product-moment correlation. The results show that correlation coefficient r=0.847(P<0.01) between HIF-1αand the water content of injury brain tissues, r=0.963(P<0.01) between VEGF and the water content of injury brain tissues, and r=0.786(P<0.01) between HIF-1αand VEGF. It indicated that they positively correlated with each other.4. The expression of VEGFR-2 protein: The expression of VEGFR-2 protein was very little in brain tissue of SO group. In TBI group the expression of VEGFR-2 protein didn't increase before 6h group compared with SO group(P>0.05). Until 12h group, the expression of VEGFR-2 protein was detected to increase apparently compared with corresponding SO subgroup(P<0.05) and to peak at 3d~7d. At the 14d subgroup the expression of VEGFR-2 protein had been different between SO group and TBI group. The expression of VEGFR-2 protein in TBI group was main in endothelial cells and very little in neurons and glial cells.5. Dynamic variation of microvessel density: Microvessel density (MOD) in normal brain cortex of rats was 14.9~17.2/HP. After traumatic brain injury, MOD decreased sharply(8.5±0.7/HP) in the 30min subgroup compared with SO group(P<0.05) and maintain to the 1d subgroup. And then MOD raised slowly(9.6±0.5/HP), and it recovered significantly at 3d but it was less than normal MOD(P<0.05). MOD began to surpass the SO's After injured 7d(18.2±0.5), and at 14d MOD was still high compared with SO group .But the difference had no statistical significance.Conclusion:1. It is successful that the animal model of free-fall impact brain injury imitates clinical traumatic brain injury in physiopathologic process.2. The peak of brain edema is about at 3days after experimental severe brain injury in rats. Brain edema may keep one week and is not conducive to function recovery.3. Ischemia and hypoxia because of brain edema make the expression of HIF-1αand VEGF protein increase. The two factors can protect cerebral cells through many pathways, such as improving hypoxia state, neurocyte regeneration, and so on. VEGF is regulated by HIF-1αprotein to increase, which regulates VEGF receptor-2 to rise in brain edema midanaphase that promotes angiogenesis and vasculogenesis to improve focal ischemia and hypoxia.4. During the early stage of brain edema, vasogenic element is not the key reason. Because microvessel has been spasmodic and its density has no variation till after injured 3d. It need more study that why VEGFR-2 is regulated to increase with VEGF rising in the morning of TBI. 5. In later period of TBI, water content of brain tissue increase with MOD rising, which indicate microcirculation change is important one of mechanisms of brain edema.