A Study Of Haptoglobin Inhibition Of The Formation In Brain Edema After Intracerebral Hemorrhage

objective:This subject observed Hb degradation products’ content andhematoma surrounding brain tissue water content, blood-brain barrier changes，inflammatory cytokines and free radical formation in experimental intracerebralhemorrhage model hematoma by removing or plusing Hp in the SD rat brain studiesof autologous blood， it purposed to investigate Hp’s inhibitory effects andmechanism about the formation of brain edema after ICH．Methods:1.To divide SDrats into groups：100SD male rats were randomly divided into four groups（25pergroup）：named sham operation group, ICH control group,Hp removal group and Hpintervention group。Each group had five observations set：1d，3d，7d，14d，21d afteroperation．There were five animals at each observations set．2．Models：（1）TheICH control group:Autologous blood was injected in the right caudate nucleus of ratsby the solid directional instrument．（2）sham operation group： it had the sameprocess of operation as the ICH group，but autologous blood wasn’t injected，andneedle was kept on for about10minutes．（3）Hp removal group:100μl tails to takeblood，affinity chromatography removal of Hp．Stereotactic rat caudate nucleusinjection to remove50μl production after Hp autologous whole blood．（4）Hpintervention group：tail blood100μl by adding the appropriate amount the100μlrequired for Hp Lyophilized Powder stereotactic caudate nucleus of rats injected plus Hp autologous whole blood50μl production．3．Neurobehavioral scores would beestimated at each observation time point．4．observation of the content of Hb and Hbdegradation products (hemoglobin，CO，iron，bilirubin) in the four groups at differenttimes hematoma changed by Immunohistochemical analysis andspectrophotometry．5.Water contents of brain tissue circum-hematoma would be detected by dry-wet weighmethod at each observation time point．6. The expression of SOD activity and MDAcontent were measured respectively by xanthine oxidation(XTO)and thiobarbituricacid(TBA) methods．7. Use radiation immunity method to detect IL-1β，IL-6andTNF-a level at each observation time point．8. pathological changes of brain tissuecircum-hematoma would be detected by HE staining pathology method at eachobservation time point．9．Results were analyzed statistically by SPSS13.0．Results:1.Neurobehavioral scores at various stages in sham operation group，ICH controlgroup，Hp removal group and Hp intervention group（1）Comparison among thegroups at the same time point：Neurobehavioral scores at1d～14d in ICH controlgroup，Hp removal group and Hp intervention group were significantly lower thanthose at corresponding time points in sham operation group (P＜0.05)．Neurobehavioral scores at3d～14d in Hp removal group were significantlylower than those at corresponding time points in ICH control group(P＜0.05)；Neurobehavioral scores at3d～14d in Hp intervention group were significantly lowerthan those at corresponding time points in sham operation group (P＜0.05)。Neurobehavioral scores at3d～14d in Hp intervention group were significantlyhigher than those at corresponding time points in ICH control group (P＜0.05);（2） Comparison in each group at different time points: Neurobehavioral scores atdifferent time points in sham operation group did not differ significantly （P＞0.05)；Neurobehavioral scores at3d were significantly lower than those at other time pointsin ICH control group (P＜0.05)，Neurobehavioral scores at7d were significantlylower than those at other time points in Hp removal group and Hp intervention group(P＜0.05)．2. Water contents of brain tissue ciucum-hematoma at various stages insham operation group，ICH control group，Hp removal group and Hp interventiongroup（1）Comparison among the groups at the same time point：Water contents ofbrain tissue circum-hematoma at1d～7d in ICH control group，Hp removal groupand Hp intervention group were significantly higher than those at corresponding timepoints in sham operation group (P＜0.05)；Water contents of brain tissue at3d～14din Hp removal group were significantly higher than those at corresponding timepoints in ICH control group(P＜0.05)； Water contents of brain tissuecircum-hematoma at3d～14d in Hp intervention group were significantly lower thanthose at corresponding time points in ICH control group (P＜0.05)．（2）Different timepoints among the four groups：Water contents of brain tissue at different time pointsin sham operation group did not differ significantly（P＞0.05)；Water contents ofbrain tissue circum-hematoma at3d were significantly higher than those at other timepoints in ICH control group(P＜0.05)； Water contents of brain tissuecircum-hematoma at7d were significantly higher than those at other time points inHp removal group and Hp intervention group (P＜0.05)．3. Hb degradation productschanges in sham operation group，ICH control group，Hp removal group and Hp intervention group:(1) Comparison among the groups at the same time point：hemoglobin content， iron content，UCB content and CO content in the hematomaorganization at3d～21d in the ICH control group，Hp removal group and Hpintervention group were significantly higher than the corresponding time points in thesham operation group.；hemoglobin content，iron content，UCB content and COcontent in the hematoma organization at3d～21d in the Hp removal group weresignificantly higher than the corresponding time points in the ICH control group (P＜0.05)；hemoglobin content，iron content，UCB content and CO content in thehematoma organization at3d～21d in the Hp intervention group were lower than thecorresponding time points in the ICH control group(P＜0.05);(2) at different timepoints among the three groups: hemoglobin content，iron content， UCB content andCO content were no significant differences at different time points after hematomaorganization in sham operation group（P＞0.05)；the hemoglobin content，ironcontent，UCB content and CO content of the hematoma within the organization inICH control group，Hp remove group and Hp intervention group at7d weresignificantly higher than those at other time points；4．Free radical changes atvarious stages in sham operation group、ICH control group、Hp removal group andHp intervention group4.1SOD activity at various stages in sham operationgroup， ICH control group， Hp removal group and Hp interventiongroup．(1)Comparison among the groups at the same time point：SOD activity andat1d～7d in ICH control group，Hp removal group and Hp intervention group weresignificantly lower than those at corresponding time points in sham operation group(P＜0.05)；SOD activity at3d～14d in Hp removal group were significantlylower than those at corresponding time points in sham operation group(P＜0.05)；SOD activity at3d～14d in Hp intervention group were significantly higher thanthose at corresponding time points in ICH control group(P＜0.05)．(2) Comparison ineach group at different time points：SOD activity were no significant differences atdifferent time points after operation in sham operation group(P＞0.05)；SOD activityat3d were significantly lower than those at other time points in ICH control group (P＜0.05)，SOD activity at7d were significantly lower than those at other time points inHp removal group and Hp intervention group(P＜0.05)．4.2MDA level at variousstages in sham operation group，ICH control group，Hp removal group and Hpintervention group (1)Comparison among the groups at the same time point：MDAlevel at1d～7d in ICH control group，Hp removal group and Hp intervention groupwere significantly higher than those at corresponding time points in sham operationgroup（P <0.05)．MDA level at3d～14d in Hp removal group were significantlyhigher than those at corresponding time points in ICH control group(P＜0.05)．MDAlevel at3d～14d in Hp intervention group were significantly lower than those atcorresponding time points in ICH control group (P＜0.05)．(2)Comparison in eachgroup at different time points：MDA level were no significant differences at differenttime points after operation in sham operation group(P＞0.05)；MDA level at3d weresignificantly higher than those at other time points in ICH control group (P＜0.05)，MDA level at7d were significantly higher than those at other time points in Hpremoval group and Hp intervention group(P＜0.05)．5．Inflammatory factors change at various stages in sham operation group，ICH control group， Hp removal groupand Hp intervention group（1）Comparison among the groups at the same timepoint：IL-1β level、IL-6level and TNF-a level at1d～7d in ICH control group，Hp removal group and Hp intervention group were significantly higher than those atcorresponding time points in sham operation group(P＜0.05)；IL-1β level，IL-6leveland TNF-a level at3d～14d in Hp removal group were significantly higher thanthose at corresponding time points in ICH control group(P＜0.05)；IL-1β level，IL-6level and TNF-a level at3d～14d in Hp intervention group were significantly lowerthan those at corresponding time points in ICH control group (P＜0.05)．（2）Comparison in each group at different time points：IL-1β level，IL-6level andTNF-a level were no significant differences at different time points after operation insham operation group(P＞0.05)；IL-1β level，IL-6level and TNF-a level at3d weresignificantly higher than those at other time points in ICH control group(P＜0.05)，IL-1β level，IL-6level and TNF-a level at7d were significantly higher than those atother time points in Hp removal group and Hp intervention group (P＜0.05)．6.shamoperation group， the ICH control group， Hp removal group and Hp interventiongroup， HE staining observed by light microscopy results of light microscopy showsthat: brain tissue sections of the sham operation group was no significant pathologicalchanges after ICH. In ICH control group，Hp removal group and Hp interventiongroup。brain tissue sections were obviously visible to loose brain tissue, neurons, glialcells, vascular endothelial cell swelling，neurons and glial cells around the gapwidened surrounding brain tissue infiltration of lymphocytes，proliferation of glial cells, but also shows a small amount of blood vessels to dilate．7. CorrelationAnalysis：（1） Hb degradation product content（heme， iron， UCB， CO） ateach time point in ICH control group，Hp removal group and Hp intervention groupwas significantly positively correlated with water content of brain tissuecircum-hematoma, IL-1β levels，IL-6levels，TNF-a content and MDA content．Butsignificantly negatively correlated with SOD activity and neurobehavioral scores．（2）water content of brain tissue circum-hematoma was significantly positively correlatedwith IL-1β levels，IL-6levels，TNF-a content and MDA content。But significantlynegatively correlated with SOD activity．（3）neurobehavioral scores of brain tissuecircum-hematoma was significantly negatively correlated with water content of braintissue，IL-1β levels，IL-6levels，TNF-a content and MDA content．But significantlypositively correlated with SOD activity．Conclusion:1.The rat models with ICHwere made by autologous blood，the way was simple and made easily，he changes ofpathophysiology of rat models were almost the same as the patients with ICH，themodel could be used generally．2. The perihematoma showed obviouspathophysiological damage after ICH．Hemolysate from red blood cell lysis afterintracerebral hemorrhage contributes to significantly neurological deficits andinflammatory response，and the increase of brain water content，MDA content andlevels of inflammatory factor and the decrease of SOD activity inperihematoma．3.Red blood cell lysis contributes to brain edema formation afterintracerebral hemorrhage，and brain edema peaked at3days after intracerebralhemorrhage． Hb metabolites (heme， iron， UCB， CO) continued to rise significantly, It played an important role in delayed cerebral edema．4. Hp had theinhibition of brain damage after ICH and can Mitigate cerebral edema，reducesecondary brain damage and improve neurological function.5.Inhibition mechanismwhich Hp had inhibition of brain damage after ICH may be by reducing the release ofinflammatory cytokines, reducing the infiltration of inflammatory cells, inhibit freeradical production， form non-covalent complexes with Hb and inhibit furtherdegradation of Hb and thus play a Neuroprotective effects on brain injury after ICH．...