The Correlation Between Hif-1αExpression And Apoptosis In Neonate Rats Brain After Undergoing Hypoxic-ischemic Injury And Intervention Study

Objective: To observe the correlation between hypoxia-inducible factor 1α(HIF-1α)expression and cell apoptosis after hypoxic-ischemic brain damage (HIBD) in a neonatalrat model with HIBD and intervention by HIF-1αinhibitor 2-Methoxyestradiol (2ME2), toexplor the effect and role of HIF-1αinhibitor (2ME2) in the HIBD. The experiment mayprovide an effective treatment for clinical therapy of neonatal HIE.Methods: 120 seven-day-old Wistar rats, female or male, with the birth weight of 10 to 15gram were divided into three groups randomly: sham-operated group (n=8),hypoxic-ischemic brain damage (HIBD) group (n=56) and 2ME2 group(n=56), the lattertwo groups are divided into seven subgroups (n=8 each) based on different time pointsafter HIBD (3 h, 6 h, 12 h, 1d, 2d, 3 d, 7 d). The hypoxic-ischemic brain injury (HIBD)model was established by Rice’s method: Rats in sham-operated group were given medianneck incision and free left common carotid artery, without hypoxic-ischemic, rats in HIBDmodel group and 2ME2 group were prepared by keeping rats into hypoxic environment of8% O2concentration and 92% nitrogen for 2 hours after freed and ligated left commoncarotid artery, and rats of 2ME2 group were instantly given single intraperitoneal injection2ME2 (10μg/g) after hypoxic-ischemic brain damage. Rats in three groups were sacrificedat different time points and brains tissue were collected. The general morphologic changesof brain tissue were obtained through macroscopic observation, the pathological changes ofleft brain tissue were observed under light microscope, and expressions of HIF-1αandcaspase-3 in left brain tissue were detected with immunohistochemistry. The apoptosisindex (AI) was determined by terminal-dexoy nucleotidyl transferase mediated nick endlabeling (TUNEL).Results:(1)Neonatal rats had various abnormal behaviors after HIBD.(2)There werevarious general morphologic changes of brain tissue of rats in HIBD model group atdifferent time points, but the general morphologic changes of brain tissue of rats in 2ME2group were reduced. (3) HE staining: Cellular layer of brain tissue in sham operation groupwas distinct and compact, cellular structure was normal and there were no obvious neuronloss. In HIBD model group, the brain cortical cells of left side edema at 3 to 6h after HIBD,neurons arrangement disorder, focal necrosis at 12 to 24h, nerve cells apoptosis increasedsignificantly at 2 to 3d, neuron loss and glial cells increased significantly at 7d.Compared with HIBD group, at each time point, the injury degree of brain tissues in 2ME2 group wassignificantly reduced, the number of survival nerve cell was more, and cell arrangementstill regular. The majority of nerve cells are normal of morphology. (4)Immunohistochemical staining: Positive staining mainly located in cerebral cortex, HIF-1αand Caspase-3 positive staining was brown, fine particle deposition. HIF-1αand Caspase-3in brain tissue showed no positive expression in sham operation group. The expression ofHIF-1αenhanced at 3h, reached the peak at 12h, and decreased gradually at 1 to 7d, butstill higher than normal level. Compared with sham operation group, the average grayvalue of HIF-1αpositive cells was lower in brain tissue in HIBD model group after HIBD,the differences at each time point had statistical significance (P<0.05). The expression ofCaspase-3 enhanced at 3h, increased gradually at 6 to 24h, reached the peak at 2d, anddecreased gradually at 3 to 7d, but still higher than normal level. Compared with shamoperation group, the average gray value of Caspase-3 positive cells was lower in braintissue in HIBD model group after HIBD, the differences at each time point had statisticalsignificance (P<0.05).The expression of HIF-1αand Caspase-3 positive cells in 2ME2group decreased at different time points compared with HIBD group, the average grayvalue of positive cells was higher, the difference was statistically significant (P<0.05). (5)TUNEL and AI: There was no significant apoptosis cell in sham operation group.Apoptosis cells were gradually increased at 3h to 7d after HIBD. Compared with HIBDgroup, apoptosis cells were decreased significantly at different time points in 2ME2 group,the difference was statistically significant (P<0.05).Conclusions:（1）Via observation of capacity of neonatal rats and pathological changes ofbrain tissues after hypoxic-ischemic, confirmed that the hypoxic-ischemic brain damage(HIBD) model was successfully established. 2ME2 was effective to reduce brain damageinduced by hypoxic-ischemic. (2) The expression of HIF-1αand Caspase-3 increasedsynchrony after HIBD, and apoptosis cells also increased. The expression of HIF-1αandCaspase-3 decreased at the same time after intervention by 2ME2. We consider thatHIF-1αand apoptosis has positive correlation. (3) HIF-1αinhibitors have neuroprotectiveeffect potentially. 2ME2 can restrain the elevation of HIF-1α, which has neuroprotectiveeffects on the brain tissues of HIBD. The experiment provided a new treatment idea forclinical treatment and prognosis improvement of neonatal hypoxic-ischemicencephalopathy.