The Effect Of HIF-1α To The Cellular Restoration Of Choroid Plexus Of Rats With Cerebral Ischemia Reperfusion Injury

Objective: The aim of the present study was to test the treatment effect of HIF-1αto the cellular structure restoration of choroid Plexus of rats with cerebral ischemia reperfusion injury based on the results from the experiment introduced. In this experiment, the vitro recombination technology of pcDNA3.1-HIF-1αwas used to prepare HIF-1αplasmid. We injected the vitro recombined HIF-1αplasmid into the middle cerebral artery occlusion (MCAO) reperfused rats through external carotid artery. Then we observed the microstructure changes of Choroid Plexus (CPs) cell and tight junction affects between endothelial cell in each group by Transmission Electron Microscope(TEM) at different time points 6h, 12h, 24h, 72h and 7d after ischemia reperfusion injury. The cellular structure restoration induced by HIF-1αis also what we need to evaluate .Under these experiment results, we finally find out the protection effect of transfecting vitro recombined gene—HIF-1αin nervous tissue during cerebral ischemia reperfusion injury.Methods:1 In vitro synthetic HIF-1αcDNA cloned into the T vector-pcDNA3.1 after gene sequencing, cloned sequence size: 2.5kb; identify the recombinant through restriction enzyme digestion, amplify in vitro according to the experiment need.2 The experiment model of cerebral ischemia reperfusion (MCAO) preparation: 10% chloral hydrate was injected into the abdominal cavity of male SD rats (weight 250-280g) for intraperitoneal anesthesia. Rats had a median neck incision. We separated the neck vessels to expose carotid artery furcation and inserted filament into the internal carotid through the external carotid artery. The filament was used to make a middle cerebral artery occlusion with end 1.8 cm away from the bifurcation of vessels, distal external carotid artery was ligated. We removed the filament and injected HIF-1αplasmid to the experimental groups from external carotid artery after 2 hours, while PBS was injected into control groups. Neurological disfunction scoring was graded by Zea Longa standard.3 Rats of experimental groups and control groups were decapitated at different time points: 6h, 12h, 24h, 72h and 7d . The brain tissue Specimens of corresponding time points were taken after brain death. We made serial sections(2mm thick) posterior optic chiasm ,the first slice was stained by TTC and the rest ones were embedded in paraffin and made into 5μm thick serial paraffin sections. The paraffin sections were then stained by HE.4 We took the choroid plexus of lateral ventricle of the rats'diseased side and looked into the changes in the microstructure of choroid plexus before and after treatment by TEM.5 Statistical analysis: Use SPSS15.0 statistical software to conduct statistical analysis of the experimental data. T test was used to compare measurement data between groups. P <0.05 indicated that there were statistically significant differences.Results:1 We identified pcDNA3.1-HIF-1αplasmid by restriction enzyme digestion. HIF-1αcDNA was amplified in vitro according to the experimental use . Cloned sequence size: 2.5kb .The sequencing results of RT-PCR were exactly the same with the Genebank records(GenBank ID:3091).2 The rats's neurological disfunction scoring was graded by Zea Longa standard 6h,12h,24h,72h and 7d after the MCAO. The differences of rats death rate between two groups external carotid artery injected with HIF-1αplasmid and those with PBS are no significant in time points 6h,12h,24h,72h and 7d. And the neurological disfunction scoring at corresponding time points of treatment groups and control groups are also not significant different.3 Infarct size in TTC stained brain slices of the experimental group and control group were no significant differences at early stage(6h—24h)of reperfusion.However, after 72h, the Infarct size of experimental group is smaller than control group.4 The affect of HIF-1αon choroid plexus tissue morphological changes (HE staining) of rats with cerebral ischemia- reperfusion injury: morphosis of the choroid plexus tissue was still intact in 6h and 12h after ischemia- reperfusion; epithelial cells had no significant cellular edema, lined up in order. Intercellular space is normal, local lymphocyte infiltration appeared on infarct zone. At this time point the choroid plexus tissue morphous of the treatment group and control group has no significant difference, macroscopic structure of choroid plexus was integrated. 24h after reperfusion: nerve tissue pathological changes had aggravated compared with the previous, choroid plexus had a loose structure with intercellular space expanding, choroid epithelium highly swelled. Apoptosis had appeared on a part of cells, vascular endothelial cell(VEC) had swelled. Part of the vascular endothelium fractured, the structural integrity of choroid plexus had damaged. In this time point we had observed that the microstructure damage of HIF-1αplasmid treatment group is lighter than those of control group PBS. 72h after reperfusion: apoptosis of choroid plexus epithelial cell had alleviated, and endotheliocyte damage mitigated but the edema did not reduce. Compared with the PBS control group, intercellular space of HIF-1αplasmid treatment group was not further widened. 72h after reperfusion: Choroid plexus restored the original structure, oedema of epithelial cell relieved, fibroplasia has been found in some areas.At this time point, compared with the previous, HIF-1αplasmid treatment group has restored more completely, the cell outline of vascular endotheliocyte was clearer, and intercellular space was more compact than the PBS group.5 The effect of HIF-1αto the microstructure of choroid Plexus of rats with cerebral infarction: Endothelial cell edema could be seen in both PBS control groups and HIF-1αinjection groups at 6h and 12h. There were loosening tight junctions between cells and were injuries in nuclear membrane and various organelles. And a few pinocytotic vesicles could be noticed. Differences were observed at 24h after treatment, the increase of pinocytosis vesicles in HIF-1αinjection groups was more obvious and brisement of tight junctions between cells is fewer compared with that in PBS control groups. After 72h of treatment, the tight junctions between cells began to appear obvious signs of repairing.The quantity of pinocytosis vesicles in HIF-1αinjection groups was more than that of PBS control groups, organelle edema were relieved obviously. Compared with HIF-1αinjection groups the intercellular space of PBS control groups was wider , tight junctions recovery was not obvious , intercellular space recovery was incomplete. After 7d of treatment, mitochondria and rough endoplasmic reticulum and other organelles in HIF-1αinjection groups returned to normal, and the endothelial cells entered the recovery process. Compared with HIF-1αinjection groups , recovery in organelles of PBS control groups was not obvious, particularly in the secretory organelles such as rough endoplasmic reticulum, Golgi apparatus and so on. Which demonstrated that the secretion function recovery of PBS control groups was not as good as the treatment group . At this time point, tight junctions of HIF-1αinjection groups were nearly common .While the intercellular space of PBS control groups was still not completely sealed .Conclusions:1 The recombinant pcDNA3.1-HIF-1αplasmid injected through the external carotid - internal carotid artery can be transfected into the ventricular system and nerve tissue.2 The external carotid—internal carotid artery injection of recombinant plasmid pcDNA3.1-HIF-1αplay a neuroprotective role in reducing infarct size of rats with cerebral ischemia-reperfusion injury3 Gene therapy such as HIF-1αplasmid transfection can reduce the injuries of vascular endothelial cells in the choroid plexus at the early stage of cerebral ischemia-reperfusion injury.4 The injection of recombination pcDNA3.1-HIF-1αplasmid through carotid artery could effectively repair the vascular endothelial injury induced by ischemia-reperfusion .