The Relation Between Heme Oxygenase And Cytoglobin Expression On The Hypoxia-ischemic Brain Damage And The Importance

Backgrounds and aimsNow how can the expression of CYGB improve after HIBD is unknown. As thenew generation of vertebrate heme protein, CYGB relates to the HO/CO systemgreatly.We had discovered that the NGB which had gene homology with CYGB, canbe induced by Hemin which is the accelerant of HO-1. But there is shortage ofthe data for CYGB. Here we used the accelerant Hemin and the inhibitor ZnPPto intervene 12 hours before hypoxic-ischemic, then observed the watercontent of the brain,changes of brain histopathology, HO-1 and CYGB proteinexpression in the brain after hypoxic-ischemic.Finally we assessed the longtermbehavioral outcomes by Morris water-maze after 4weeks. The aim of thisstudy was to discuss how can HO regulate CYGB while hypoxic-ischemic braindamage, to check the neuron protecting factor of CYGB, and to evaluatewhether Hemin treatment would reduce the brain damage against hypoxicischemicdamage.MethodTwo hundred and sixty wistar rats of 7-day old were divided into 5 groupsrandomly,including control group,sham-operated group,HIBD group, HIBD +Hemingroup,and HIBD+ZnPP group.The rats of HIBD+Hemin group, HIBD group andHIBD+ZnPP group were given Hemin(50mg/kg), NS(eqluivalent) or ZnPP 12 hoursbefore the operation.The rats of three last groups underwent left carotidartery ligation and followed by hypoxia(8%O2) for 2 hours.The sham-operatedgroup was just dissociated the left carotid artery but not ligated it.All thegroups was killed at 0h,24h,48h,72h after HIBD. At 0h,24h,48h after hypoxicischemic,observe HO-1 and CYGB protein expression in the brain after hypoxicischemic.Changes of brain histopathology were observed 48h after hypoxicischemic.The water content of the brain were observed 72h after HIBD. Long term behacioral outcomes was assissed by Morris water-maze(place nacigationtest and spatial probe test) after 4 weeks. Rats were killed 6days later,theextent of atrophy of the hypoxic-ischemic hemisphere and changes of brainhistopathology were observed.Result1. HIBD model: Many animals became cyanosis,with deep and quickrespiration,couldn't stand steady,and than became sleepy,even appearedconvulsion while hypoxic-ischemic brain damage. Changes of brainhistopathology 48h after hypoxic-ischemic were brain infarct and hemorrhagein HIBD group, HIBD+Hemin group and HIBD+ZnPP group. The sham-operated groupand control group didn't have these changes.The character below the lens wasthat ,the infarct range and hemorrhage of HIBD+ Hemin group was much smallerthan the HIBD+ZnPP group and HIBD group. 72 hours after HIBD,we checked thewater content of the brain .the water content of the HIBD group and HIBD+ZnPPgroup were all with great difference to the HIBD+Hemin group and the shamoperatedgroup. But the HIBD+Hemin group and the sham-operated group didn'thave difference.2. The expression of HO-1 and CYGB: the expression of HO-1 and CYGB inthe HIBD+Hemin group ,HIBD group and HIBD+ZnPP group were more than that insham-operated group and control group in 0h,24h,48h after hypoxic-ischemicbrain damage.(P<0.05 or P<0.01). And they increased by the time in the HIBDmodel groups.The average density of HO-1 protein expression in the hippocampiat 48h after HIBD was that:: HIBD+Hemin group was 124.92±6.49, HIBD groupwas 146±2.82, HIBD+ZnPP group was 165.72±4.28. The expression of theHIBD+Hemin group was highest,HIBD group was second,HIBD+ZnPP group was third.as well as the expression in the cortex .Comparison of the three groups iswith significant difference (P<0.01). The average density of CYGB proteinexpression in the hippocampi was that: HIBD+Hemin group was 124.69±3.78,HIBD group was 153.72±6.22, HIBD+ZnPP group was 162.43±5.33, sham-operatedgroup was 230.2±5.65, control group was 234.13±5.06. Comparison of the three groups is with significant difference(P<0.01). The expression of HO-1and CYGB had positive correlation in contex and hipppcampi in five groups.The relative factors r at 0h after HIBD were 0.687-0.916, 24h after HIBD were0.473-0.903,48h after were 0.659-0.962. The contex also had similar result.3. Morris water-maze: place navigation test showed that 5 days totalescape latency were: HIBD+Hemin group was 46.67±34s, HIBD group was 71.04±30.5s, HIBD+ZnPP group was 76.72±29.8s, sham-operated group was 38.32±30.3s,and control group was 36.72±30.8s, the first three groups were longer thanthe last two groups.but the EL of the HIBD+Hemin group was much shorter thanthe HIBD group and HIBD+ZnPP group(P<0.01); Spatial probe test showed thatHIBD ,HIBD+ZnPP groups, and normoxic groups were significant for the swimmingtime and distance in platform surrounding areaⅠ,but there was no differencebetween normoxic groups and HIBD+Hemin group(P>0.05).4. Specimen and histopathological evaluation discovered that HIBD ratsprobably had brain cavities,cerebromalacia and cerebral atrophy 34 days afterhypoxic-ischemic,successfully prepared HIBD model was confirmed.The cerebralatrophy rate of HIBD group and HIBD+ZnPP group were (29.73士6.53)% and(34.07士6.75)%, but for the HIBD+Hemin group was only (18.33士4.52)%,whichwas of great difference. we found that under the lens, the nerve cells ofHIBD group and HIBD+ZnPP group became thinner, many nerve cells lost. But inthe HIBD+Hemin group we only found a few nerve cells lost.Conclusion1. Animals appeared brain hypoxia symptom while making hyproxic-ischemicbrain damage model, and appeared typical brain infarct and hemorrhage 48hafter HIBD. The brain water content increased 72h after HIBD. These allshowed This experiment successfully set up the HIBD model. The level of braindamage in HIBD+Hemin group was lighter than in HIBD+ZnPP group, it showedthat Hemin intervention could protect brain while HIBD.2. Hemin which was the abduction of HO could increase the expression ofHO-1 in contex and hippocampi of HIBD+Hemin group. ZnPP which was the depressor of HO could decrease the expression of HO-1 of the HIBD+ZnPP group.The expression of HO-1 and CYGB was consistency, and they had clear positivecorrelation. It showed that HO-1 could control the expression of CYGB.3. The morris water-maze showed that: 5 days total escape latency andthe result of spatial probe test of HIBD+Hemin group were obvious better thanHIBD group and HIBD+ZnPP group. HIBD+ZnPP group was the worst. It proved thatthe positive correlation of CYGB by HO-1 could improve the long-termneurobehavioral achievements.4. the intervention of Hemin could decrease the atrophy of brain, thelost of nerve cell, and the forming of barren while HIBD. But theintervention of ZnPP would increase the changes of histopathology. It provedthat the positive correlation of CYGB by HO-1 could reduce the HIBD braindamage, and affect the neurobehavioral achievements.