Research On The Neuroprotective Function Of Hemopexin Against Focal Cerebral Ischemia Injury In The Rat

BackgroundStroke has become the number one killer for human health and life safety because ofits high incidence，morbidity and mortality. Research on the stroke therapeutic strategieshas made great progress，but there still exist several problems to be overcome，for example，the narrow time window of treatment，unsatisfactory clinical efficacy and considerableside effects. Given all these problems，looking for effective new therapies has become anurgent and important scientific problem to be conquered.Hemopexin (HPX) is a plasma protein which exhibits the highest affinity to free Heme(<1Kd). The primary physiological function of HPX is to get rid of harmful free Heme. Inaddition，HPX also exert a protective effect in a series of physiological and pathologicalprocess，including free Heme mediated lung oxidative stress injury，the liverischemia-reperfusion(I/R) injury，as well as type I diabetes. In recent years，in vitro studiesand in vivo knockout animal models pointed that HPX might have a neuroprotective function. However，localization of HPX in the brain was not well elucidated and its role infocal cerebral I/R injury was also poorly studied. Furthermore, no direct data wasavailable on the neuroprotective effect of HPX on brain ischemia injury. This study was toobserve expression of HPX in normal rat brain tissue and its expressional alteration afterfocal cerebral ischemia，and further to explore its neuroprotective function againstischemia-reperfusion injury through central administration，in order to provide a newthought and theoretical foundation for treatment of cerebral ischemia and reperfusioninjury.Experiment1HPX localization in normal rat brain and its expressional alterationfollowing cerebral ischemia and reperfusionObjective: To examine localization of hemopexin in normal rat brain and to detectalteration of its protein level after ischemia-reperfusion injury.Methods:Part1Protein level and localization of HPX in normal rat brain.3male SD rats were given deep anesthesia before decapitated，brains were homogenizedand protein level of HPX was detected by Western blotting. Another3male SD rats weretranscardially perfused with saline after deep anesthesia，then brain tissues were removedand fixed. Corneal slices were prepared for double immunofluorescence staining.Colocalization of HPX with NeuN (a specific marker for neuron) and GFAP (a specificmarker for astroglial cells) were observed.Part2Protein level alteration of HPX in the ischemic penumbra after focalcerebral ischemia injury18male SD rats were randomly assigned to sham operation group (sham)(n=2) andmiddle cerebral artery occlusion group (MCAO)(n=16).Rats in MCAO group were againrandomly divided into four groups (n=4): transient cerebral ischemia was induced by aMCAO model. Western blotting of the HPX expression were performed in the rats ofsham group and MCAO operated group (at2h，12h and24h after I/R，respectively). Atdifferent time points post reperfusion，rats were decapitated under deep anesthesia，lateral penumbra brain tissues were homogenated and HPX expression was tested by Westernblotting analysis.Part3Alteration of HPX localization after transient focal cerebral ischemia6male SD rats were randomly assigned to sham group (n=3) and MCAO group (n=3).Animals in MCAO group were perfused transcardially under anesthesia at24h after I/R，then brain tissues were removed and fixed. Frozen slices were prepared for doubleimmunofluorescence staining of HPX with NeuN (neuron specific marker) and GFAP(astrocyte specific marker) to observe the HPX expression changes on neurons andastrocytes in lateral penumbra brain tissuesResults:(1) Western blotting: HPX positively expressed in normal rat brain; at2h，6h，12hfollowing I/R，expression of HPX in the ischemic penumbra did not change significantly.At24h following I/R，HPX protein level increased remarkably in comparison with shamgroup (P<0.05).(2) Immunofluorescence double label staining: HPX protein was mainlyexpressed in the cerebral vascular system in normal rat brain(especially in ependymalepithelial cells，choroid plexus cells), and in hippocampal neurons. In addition，HPX wasstrongly expressed in the plasma of a few astrocytes in the periphery of the ventricularvessels. At24h following I/R， HPX expression on neurons and astrocytes wassignificantly increased in the ischemic penumbra.Conclusion: HPX was mainly expressed in the vascular system and the hippocampusneurons in normal rat brain and was up-regulated after ischemia-repurfusion injury.Experiment2Protective function of HPX intraventricular administration in focalcerebral ischemia-reperfusion injuryObjective: To explore neuroprotective effect of different dosages of HPX centrally givenon focal cerebral ischemia injuryMethods:50male SD rats were randomly assigned to5groups (n=10): MCAO group(con); solvent comparison group (vehicle): intracerebroventricularly inject with5μl0.1%sodium azide; treatment groups [intracerebroventricularly inject with5μl differentconcentrations of HPX (dissolved in0.1%sodium azide):0.46mg/ml (low dose group)， 0.93mg/ml (middle dose group) and1.86mg/ml (high dose group)，respectively]. The ratsin control group and treatment group experienced middle cerebral artery occlusion(MCAO)， immediately after120minutes ischemia HPX or vehicle was injectedintracerebroventricularly.24hours later，neurobehavioral scores (NBS) were evaluatedbefore the rats were decapitatd, the brains were then removed and stained with TTC tocalculate the brain infarct volumes.Results:(1) Neurobehavioral scores: The neurobehavioral outcome of the high-dose (1.86mg/ml) and medium-dose (0.93mg/ml) HPX administered group was markedly improvedas compared with the control and vehicle-given group (P <0.011.86mg/ml group vs congroup; P <0.011.86mg/ml group vs vehicle group; P <0.050.93mg/ml group vs congroup; P <0.050.93mg/ml group vs vehicle group). There was no significant differenceon the neurobehavioral scores between other two groups.(2) Brain infarct volumes:Intracerebroventricular injection of high-dose (1.86mg/ml) and medium-dose (0.93mg/ml)HPX reduced the infarct size in the brain at24h post I/R(P <0.011.86mg/ml group vscon group; P <0.011.86mg/ml group vs vehicle group; P <0.050.93mg/ml group vscon group; P <0.050.93mg/ml group vs vehicle group). Further，the brain infarct volumepercent in the high-dose HPX group was also much lower than in the medium-dose group(P <0.05). There was no significant difference between other two groups.Conclusion: HPX dose dependently induced neuroprotection in the rat subjected to focalcerebral ischemia injuryExperiment3：Long-term neuroprotective effect of HPX on focal cerebral ischemiainjuryObjective: To assess the long-term neuroprotective effects of HPX centrallyadministration on focal cerebral ischemia injuryMethods：16rats were randomly assigned to a group receiving the carrier vehicle alone，or a group receiving the test agent HPX (n=8). All animals were subjected to MCAO. BothHPX (1.86mg/ml,5μl) and the carrier vehicle (0.1%sodium azide,5μl) wereintracerebroventricularly administered as soon as the initiation of reperfusion. The neurological behavior of rats was evaluated daily until day7after administration. Then therats were decapitated and the brain infarct volumes were assessed.Results：Neurological behavior scores of the rats in HPX group were much higher than inthe vehicle group in each corresponding time point (P <0.01). In addition, brain infarctsizes on the day7after reperfusion were significantly smaller in the HPX group (P <0.01).Conclusion: Neuroprotective effects of HPX could sustain for7days after focal cerebralischemia injurySummary1. HPX was mainly expressed in vascular system and hippocampus neurons，as well as ina few astrocytes adjacent to the vessels in normal rat brain and HPX was up-regulated inthe penumbra area at24h after ischemia-reperfusion.2. HPX dose-dependently induced neuroprotection in the rat subjected to transient focalcerebral ischemia injury.3. HPX could induce long-term neuroprotective effect on transient focal cerebral ischemiainjury in the rat.