Effects Of GCPⅡ Gene Knockout Against Oxidative Stress And Apoptosis Following TBI In Mice

BackgroundTraumatic brain injury（TBI）results in immediate irreversible primary brain tissue damage and secondary delayed loss of neurons and glia in the penumbra surrounding the injury site leading to progressive emotional,cognitive and behavioral impairment.Excessive release of glutamate following TBI and subsequent intracellular calcium(Ca²⁺)overload significantly contributes to second brain injury,including brain edema,cerebral ischemia,energy failure,mitochondrial dysfunction,abnormal oxidative phosphorylation,and neuronal death.To date,lots of drug therapies have been proved useful in animal TBI models.However,the translation of scientific research into clinical practice remains disappointing.Under this circumstance,developing an endogenous neuroprotective mechanism represents a novel target of TBI therapy.N-acetylaspartylglutamate（NAAG）is one of the most abundant transmitters in the mammalian nervous system,and serves as a potent agonist at the type 3metabotropic glutamate receptor（mGluR3）.Activating mGluR3 by NAAG reduces the synaptic glutamate release.Furthermore,elevated concentration of NAAG promotes glutamate uptake in astrocytes as a consequence of m Glu R3 activation.NAAG peptidase consists of glutamate carboxypeptidase（GCP）Ⅱ and Ⅲ,which hydrolyze NAAG into NAA and glutamate and deprive the neuroprotective function of NAAG.GCPⅡ represents the main component of NAAG peptidase,and inhibition of GCPⅡ has been proved to be neuroprotective in TBI,stroke,and ischemic models.Our previous study found that GCPⅡ gene knockout（KO）mice developed normally with no apparent differences compared with WT mice in terms of survival and standard neurological tests,but had lessened neuronal degeneration and astrocyte damage after TBI.However,the downstream signaling pathway of GCPⅡ KO remains unclear.In the present study,a GCPⅡ KO mouse strain has been used to assess the potential role of oxidative stress related pathways in the neuroprotective activity of NAAG.The current study consists three parts.Part one: establishment of graded controlled cortical impact（CCI）injury models in mice;Part two: effects of GCPⅡ KO on oxidative stress following TBI in mice;Part three: effects of GCPⅡ KO on cortical apoptosis following TBI in mice.Part Ⅰ Establishment of graded controlled cortical impact injury models in miceObjective To establish a stable TBI model using precision cortical impactor and make a gradation based on the histopathological findings.Methods 48 male C57/BL mice in ages 8-12 weeks,weighing 25-28 g were randomly divided into 4 groups（1 sham TBI group and 3 TBI groups,n=12/group）.A CCI device（Pin Point TM PCI3000,Hatteras Instruments Inc.,USA）was used to induce TBI.Injury was induced by a 3.0 mm rounded metal tip that was angled to have a vertical direction to the brain surface.The impact velocity was 3.0 m/s and the contusion duration was 180 ms.The deformation depths were 0.5 mm,1.0 mm and 1.5 mm.Vital signs,coma duration and neurological reflex were observed,and TUNEL staining was used to evaluate cortical apoptosis following TBI.Results The degree of injury deepened as the deformation depth increased.Mild TBI was induced when the deformation depth was within 0.5 mm;Moderate TBI was induced when the deformation depth was 1.0 mm;Severe TBI was induced when the deformation depth was 1.5 mm.Conclusion Graded TBI model can be induced by CCI,which may provide a platform for further research.Part Ⅱ Effects of GCPⅡ KO on oxidative stress following TBI in miceObjective To investigate the neuroprotective effects of GCPⅡ KO following TBI and the possible underlying mechanism.Methods A total number of 36 male GCPⅡ KO mice and 36 male littermates were divided into 4 groups（WT + TBI,WT + sham TBI,KO + TBI,and KO + sham TBI,n = 18/group）.A CCI device was applied to induce Moderate TBI.Mice were sacrificed at 24 h after TBI.The levels of malondialdehyde（MDA）and glutathione（GSH）,and the activities of superoxide dismutase（SOD）and glutathione peroxidase（GPx）in brain tissues were detected by biochemistry methods.Wet-dry method was used to evaluate the water content of brain samples.Evans blue was used to examine the damage of blood-brain barrier.Results TBI induced a decrease of GSH level and the activities of SOD and GPx,and an increase of MDA level（All p < 0.05）.However,these changes were moderated in GCPⅡ KO mice compared to WT mice（All p < 0.05）.Furthermore,brain edema and the damage of blood brain barrier following TBI were attenuated by GCPⅡ KO after TBI relative to their WT counterparts.Conclusions GCPⅡ KO exhibits its neuroprotective efficacy against TBI through reducing the oxidative stress.Part Ⅲ Effects of GCPⅡ KO on cortical apoptosis following TBI in miceObjective To investigate the neuroprotective effects of GCPⅡ KO against cortical cell apoptosis following TBI.Methods A total number of 24 male GCPⅡ KO mice and 24 male littermates were divided into 4 groups（WT + TBI,WT + sham TBI,KO + TBI,and KO + sham TBI,n = 12/group）.A CCI device was applied to induce Moderate TBI.Mice were sacrificed at 24 h following TBI.Brain sections were collected to evaluate apoptosis using TUNEL-staining.Western blotting was used to examine the levels of cytosolic cytochrome c,cleaved caspase-3,and mitochondrial Bcl-2 and Bax.Results GCPⅡ KO mice exhibited reduced numbers of TUNEL-positive nuclei in the contusion margin of the cerebral cortex relative to WT mice following CCI（p < 0.05）.Impact injury increased the levels of cytosolic cytochrome C,cleaved caspase-3 and mitochondrial Bax,while the level of mitochondrial Bcl-2 showed an opposite tendency（All p < 0.05）.However,each of these effects was attenuated in the GCPⅡ KO mice compared to their wild type littermates（p < 0.05）.Conclusion GCPⅡ KO is neuroprotective against TBI induced cortical apoptosis,and the preservation of mitochondrial intergrity may underlie these effects.