The Role Of Nrf2in Anti-neural Apoptosis After Fluid Percussion Brain Injury In Rats

Objective: To investigate the expression of nuclear factor E2-relatedfactor2（Nrf2）, related apoptotic proteins（Casepase-3、Casepase-12）andneural apoptosis in Nrf2gene knockout rats after fluid percussion brain injury,and explore the role of Nrf2in anti-neuronal apoptosis following fluidpercussion brain injury.Methods: The mice with Nrf2gene normal and knockout were employedfor the establishment of fluid percussion brain injury model. Nrf2+/+and Nrf2-/-each had16mice weighting28-32g. all the mice randomly divided into4groups: Nrf2+/+NC group(n=8)、 Nrf2+/+TBI group(n=8) and Nrf2-/-NCgroup(n=8)、 Nrf2-/-TBI group(n=8). NC group were only given generalcraniotomy without fluid percussion impact; TBI group animals were givengeneral craniotomy, and then received fluid percussion impact.After24h each group was valued with neurological severity scores atcorresponding points, and then all the animals were over-anesthesized with10%chloralhydrate and decapitated. Brains of mice were taken out. About50mg brain tissue taken from the front of the injury area was measured bydry-wet weight method, and the injury area were cut into approximately3mmthick coronal slices, fixed with paraformaldehyde, the examples were stainedwith HE for the observation of morphology；the expression of Caspase-3、12was detected by immunohistochemistry staining and neural apoptosis wasdetected by TUNEL; About100mg brain tissue taken from the back of theinjury was used to observe the expression of Caspase-3protein andCaspase-12protein by Western Blot.Results:1Neurological severity scoreCompared with those of the NC group, the neurological severity score of the each TBI group significantly increased at24h, the difference had statisticalsignificance（P<0.05）；but there were no statistical difference in NSSbetween Nrf2+/+group（5.25±0.89）and Nrf2-/-group（5.88±1.36）(P>0.05).2Histological observationsNrf2+/+NC group and Nrf2-/-NC group Naked eye: the brain surface issmooth, groove back clearly visible, the surface of brain and skull base nobruising, bleeding, congestion and other symptoms.Microscopic observation: the structural of brain tissue integrity, cellsarranged in order, nuclear membrane integrity and nucleolus light blue.Nrf2+/+TBI group and Nrf2-/-TBI group Naked eye：Visible the corticalof impact side was contusion, the brain sulcus hematocele, section observationshowed subcortical scattered spotting, severe cases intraventricularhemorrhage.Microscopic observation: the cells around contusion appeared edema andsignificant visibled, lightly stained cytoplasm was observed, someone hadvacuolar degeneration, accompanied by the proliferation of glial cells.3Brain water contentElliot formula was used to measure the brain water content. Comparedwith those of NC group, the brain water content of animals from each TBIgroup significantly higher; there were no statistical difference in brain watercontent between Nrf2+/+NC group（78.37±0.18）and Nrf2-/-NC group（78.32±0.13）(P>0.05); however, there were obvious statistical differencebetween Nrf2+/+TBI group（81.55±0.40）and Nrf2-/-TBI group（82.89±0.20）(P<0.05).4The expression of Caspase-3and Caspase-12by ImmunohistochemistryAnimals of Nrf2+/+NC group and Nrf2-/-NC group had almost no positivecells, but the expression of Caspase-3and Caspase-12in animals ofNrf2+/+TBI group and Nrf2-/-TBI group significantly increased. Comparedwith those of Nrf2+/+NC group, the positive cell of Caspase-3（28.892±5.865），Caspase-12（28.619±2.547） in animals of Nrf2+/+TBI group not onlyincreased higher, but also the color deepened(P<0.05). Compared with those of Nrf2-/-NC group, the positive cell of Nrf2-/-TBI group Caspase-3（56.884±5.819），Caspase-12（51.196±4.563）not only increased higher, butalso the color deepened(P<0.05). But compared with Nrf2+/+TBI group，thepositive cells increased significantly higher in animals of Nrf2-/-TBI group at24th hour(P<0.05).5Measure apoptotic cells by TUNELThere was almost no positive cells in animals of Nrf2+/+NC group andNrf2-/-NC group. After injury, the apoptotic cells of TBI group increasedhigher than those of NC group. Surprisingly, the number of positive cells fromNrf2-/-TBI group （59.30±9.90） was significantly higher than that fromNrf2+/+TBI group（31.11±5.31）, and there was the statistical significancebetween them(P<0.05).6The expression of Caspase-3and Caspase-12protein by Western BlotExpression of Caspase-3and Caspase-12protein was less in Animals ofNrf2+/+NC group and Nrf2-/-NC group, but increased in animals of Nrf2+/+TBIgroup and Nrf2-/-TBI group. Compared with those of Nrf2+/+NC group, theprotein expression of Caspase-3（0.8857±0.0226）， Caspase-12（0.6327±0.0080） in animals of Nrf2+/+TBI group increased, and thedifference between them had statistical significance (P<0.05). Compared withthose of Nrf2-/-NC group, the protein expression of Caspase-3（1.0894±0.2479），Caspase-12（1.0290±0.1421）in animals of Nrf2-/-TBIgroup also increased, equally the difference between them had statisticalsignificance (P<0.05). And compared with those of Nrf2+/+TBI group, theprotein expression of Caspase-3, Caspase-12became significantly higher inanimals of Nrf2-/-TBI group (P<0.05).Conclusions:1After fluid percussion brain injury, the protein of Caspase-3andCaspase-12are high expression in Nrf2+/+mice, and nerve cells apoptosisdeteriorated, implying that after fluid percussion brain injury the expression ofCaspase-3and Caspase-12is up-regulated, and induce nerve cells apoptosis.2After brain injury, the expression of Caspase-3, Caspase-12increase significantly higher in the Nrf2-/-group than that in Nrf2+/+group, indicatingthat Nrf2can play a important role in anti-apoptosis of nerve cell by inhibitingthe Caspase-dependent apoptosis pathway.