Effect Of Oxidative Stress On Neuronic Apoptosis And C-myc Expression After Cerebral Hemorrhage

Cerebrovascular disease is common in clinics,with gradual increase of incidence,morbidity and mortality1.Relatively higher mortality existed for post-hemorrhage patients.Aged people are high risk population for cerebral hemorrhage.Major inducing factors include cerebral atherosclerosis,cerebral vascular deformation and hypertension.Currently the post-hemorrhage injury mechanism has not been fully illustrated,nor did effective treatment for improving patient's prognosis significantly.Post-hemorrhage neuron apoptosis is one important factor causing late-onset neuron injury.A complicated series of mechanism underlies secondary brain tissue injury after hemorrhage,involving inflammation,oxidative stress and cell apoptosis.Oxidative stress is closely correlated with post-hemorrhage neuron apoptosis.Under hypoxia condition,reactive oxygen species(ROS)and NO were activated in neurons,to induce mitochondrial apoptosis pathway.During pathological process of cerebral hemorrhage,abnormal structure/function of mitochondria,oxidative stress injury and increased production of free radicals works as intermediate bridge.Brain tissues are susceptible for ROS-induced injury due to their high oxidative metabolic rate and lower anti-oxidant levels.Inflammatory mediator induces cell adhesion and aggregation,causing blockade of micro-vessels,activating leukocytes for releasing inflammatory mediators and cytokines.Secondary neuron injury thus occurred in blood-brain barrier under the direction of ROS released from leukocytes.Nuclear factor(NF)-?B has pluripotent regulatory roles for mediating inflammatory factor,cytokine,chemotactic factor transcriptional levels such as IL-8 and IL-6,thus playing an important role in cell inflammation and apoptosis.Previous study showed the involvement of NF-?B in pathological process of brain hemorrhage,under which microglia was activated to produce neurotrophic factors and neurotoxic molecules,to release large amounts of free oxygen and cytokines,to activate NF-?B,and participates in neural injury and repair,inflammation,and oxidative stress.C-myc has dual roles of facilitating cell proliferation and apoptosis:under oxidative stress ROS activates NF-?B,which initiates apoptosis related gene c-myc to forming dimers with Max for further binding onto DNA core sequence for inducing neuron apoptosis.This study thus established rat brain hemorrhage model,on which the correlation between oxidative stress post-hemorrhage and neuron apoptosis was observed,in addition to gene expression effect of apoptosis related gene c-myc.Materials and methodsExperimental animal and groupingHealthy male SD rats(7 weeks old,body weight 220-240 g)were provided by Laboratory Animal Center,Shandong University(Certificate No.SYXK-2013-0025)and were kept in an SPF grade facility with food and water provided ad libitum.Animals were randomly assigned into sham,model and MPEG-SOD groups(N=28 each).Autoblood transfusion was performed to establish rat hemorrhage model,on which MPEG-SOD was applied via peritoneal injection.Rats in sham or model group received equal volume of saline.Rats were used for all experiments,and all procedures were approved by the Animal Ethics Committee of Shandong Provincial Hospital affiliated to Shandong University.Drug and reagentMalonaldehyde(MDA)and superoxide dismutase(SOD)were purchased from Jiancheng Bio(China).Chloral hydrate and paraformaldehyde were purchased from Kemiou Chem(China).Rabbit anti-NF-KB antibody was purchased from Boster Bio(China).Horseradish peroxidase(HRP)-labelled goat anti-rabbit secondary antibody was purchased from CST(US).C-myc antibody was purchased from Santa Cruz(US).DAB staining kit was purchased from Golden Bridge(China).TUNEL test kit was purchased from Roche(US).Animal modelNeurological deficit score(NDS)was measured 1 h before surgery to exclude those rats with primary neurological deficits.Brain hemorrhage model was generated by autoblood transfusion as previously described[13].In brief,rats were fasted 8h before surgery.After anesthesia in 10%hydrate chloral,rats were fixed in supine position,with head fixed in stereotaxic apparatus.Globus pallidus was located with reference to Rat brain atlas.A micro-loading needle was used to transfuse 50 ?L artery blood collected from venous blood vessel(at 25 ?L/min speed).With 10-min retention,the needle was slowly retracted,with bone wax to seal the drilling hole.The incision was sutured with sterilization.Equal volume of saline was applied in the same location in sham group.Drug deliveryMPEG-SOD(100U)was applied via intraperitoneal injection 20 min before model preparation.Equal volume of saline was given for sham and model group.Neurological deficit scoreLonga 5-grade system was used to evaluate rat neurological function.Successful generation of cerebral hemorrhage was deduced as grade ? or above after recovery from anesthesia.At 12 h,1 d,3 d and 7 d after surgery,NDS was re-evaluated by Garcia system,with a range from 3 to 18.MDA and SOD level assay and water content quantification in brain tissuesAt 12 h,1 d,3 d and 7 d after surgery,rats were sacrificed to extract brain tissues from hemorrhage side(N=7 each).Rat brain tissues were washed in pre-cold saline at 4?,and were weighted after removing surface water.10%cortical homogenate was prepared for 10 min centrifugation for collecting supernatants.Protein content was quantified using Coomassie brilliant blue colorimetric method.MDA and SOD levels were quantified using test kit following manual instruction and spectrometry method.Tissue samples(1 mm3)were collected from posterior and anterior side of hematoma puncture site for calculating water content,which was defined as(wet weight-dry weight)/wet weight×100%.HE stainingRats were fixed in 4%paraformaldehyde and decapitated for extracting brains.Cerebellum,olfactory bulb and lower brain stem were all removed,with remaining brain tissues immersed in paraformaldehyde.Coronal sections were prepared 1.4 mm posterior of Bregma from paraffin block.Hematoxylin-eosin staining was performed for observation under light field microscope.Immunohistochemistry(IHC)staining for NF-?B p65 protein expressionRats were sacrificed at all time points to extract the brain,in which cerebellum and olfactory bulb were removed,followed by immersion in paraformaldehyde.Paraffin section(8 ?m)was prepared.After de-waxed in paraffin,tissue sections were rinsed in PBS pH7.4 for three times(3 min each),antigen retrieval was performed,followed by 3%H2O2 incubation for 10 min to block endogenous activity of peroxidase.Primary antibody(1:1000 dilution)was added for 1 h room temperature incubation,followed by polymer enhancer(20 min room temperature incubation).Enzyme labelled anti-mouse/rabbit polymer was added for 30 min room temperature incubation.Freshly prepared DAB buffer was added for 5 min development under microscopic observation.Hematoxylin was used for counterstaining and 0.1%HCl differentiation.Tap water was used to rinse slides,which were then dehydrated in gradient ethanol.Xylene was added,followed by resin mounting.Image-pro plus system(Media Cybernetics,IPP imaging analysis software corp,US)was use to analyze results observed under light field microscopy.A total of five different fields were selected around hematoma under 40×magnification.Numbers of positive expressed cells were calculated for average.Western blotting for brain expression of c-myc proteinRat brain tissues around hematoma were lysed in lysis buffer to collect solution via centrifugation.Protein quantification was measured by BCA kit.Protein samples were separated by SDS-PAGE(8 ?L loading volume,75 V for 25 min,followed by 110 V electrophoresis until bromophenol blue reached bottom of separation gel).,and were transferred to PVDF membrane(110 V for 45 min).The membrane was blocked in blocking buffer for 1 h,followed by primary antibody(anti-c-myc at 1:100,or anti-beta-actin at 1:1000)incubation at 4? overnight and TBST washing.Secondary antibody(1:2000)was added for 1 h incubation.After TBST rinsing for three times,development reagent was added for exposure in dark.Quantity One image analysis software(BioRad,US)was used to analyze colored bands,whose relative expressions were determined as the ratio of absorbance values of target protein bands against internal reference proteins.TUNEL assay for neural ttissue apoptosisParaffin sections were de-waxed and rehydrated.Proteinase K was added for digestion at room temperature,followed by H2O2-methanol blocking.Tissue slides were rinsed in PBS twice,and in neutral buffers(1 h at room temperature).Hydrogen peroxidase was used for room temperature incubation for 30 min,followed by DAB development and methyl-green counter-staining.Tissue sections were dehydrated,and were observed under light field microscope.Image-pro plus system(Media Cybernetics,IPP imaging analysis software corp,US)was use to analyze total number of positive cells observed under light field microscopy(10 randomly selected fields,400× magnification).Statistical methodsSPSS20.0 software was used for statistical analysis.Measurement data were firstly tested for normality.Those fitted normal distribution were presented as mean ?standard deviation(SD).One-way analysis of variance(ANOVA)was used to compare means among multiple groups,followed by LSD test in paired comparison.A statistical significance was defined when P<0.05.ResultsGeneral information of NDSNo limb immobility was observed in sham group,which had sensitive response for pain stimuli in limb terminus,thus having NDS at grade 0.Both model and treatment group had paralysis of contralateral limbs,with circled gait toward the hematoma side.Significant round-shaped hematoma can be observed when extracting brain tissues.4 rats died from infection or subarachnoid hemorrhage plus 4 rats with grade 0 NDS after surgery were excluded from the test cohort.No significant behavioral change was found in sham group,while model rats had neurological deficits.Most severe deficits were found at 3 d post-surgery,with statistically significant difference of NDS between model and treatment groups at 12 h,1 d,3 d and 7 d post-surgery(P<0.05).Tissue hematoma after surgeryNo significant tissue hematoma was observed in brain tissues of sham group.Model group had brain hematoma after surgery,with reaching the peak at 3 d and gradual decrease.MPEG-SOD treatment group had significantly lower water content of brain tissues compared to model group at all time points(P<0.05).Tissue morphology of rat brainUsing HE staining and light-field microscopic observation,no significant change was observed in sham group,which had regular arrangement and intact morphology,along with abundant cell number and normal structure.Model group at all time points had irregular neuron arrangement,cell swelling at different grades,accompanied with inflammatory cell infiltration and gill proliferation.The most severe pathological damage occurred at 3 d after surgery.MPEG-SOD treatment alleviated brain damage at different time points.MDA and SOD levels of brain tissuesCompared to sham group,model rats had significantly elevated MDA at all time points after surgery,plus lowered SOD level(P<0.05).MPEG-SOD treatment remarkably depressed MDA content and increased SOD level compared to those in model group(P<0.05).NF-?B p65 protein expression assay by IHCPositive expression of NK-?B p65 protein locates in both nucleus and cytoplasm of cells,as shown by brown granules.Sham group had fewer positive expression of NK-?B p65 in brain tissues,while model group had elevated expression 12 h after surgery,and reached the peak at 3 d,with persistent positive expression until 7 d.MEPG-SOD treatment had lowered NK-?B p65 expression compared to model group at all time points.TUNEL for neuron apoptosisMost TUNEL-positive cells locate inside neuronal nucleus as shown by brown yellow color.Positive rate of TUNEL in sham group was relatively lower compared to model group.The ratio of TUNEL positive cell reached a peak at 3d after surgery,followed by gradual decrease.MPEG-SOD group had significantly lower TUNEL positive percentage compared to model group at all time points.Western blotting for c-myc protein expression in brain tissuesAt all time points after surgery,c-myc protein expression levels in rat brain tissues from model group were significantly higher than those of sham group(P<0.05).Model rats had peaked c-myc protein expression at 3 d post-surgery,whilst MPEG-SOD group had lower c-myc protein expression compared to model group at the same time point(P<0.05).Conclusion1.Model rats had significantly elevated MDA at all time points after surgery,plus lowered SOD level,suggesting that oxidative stress is significant after intracerebral hemorrhage.2.Positive expression of NK-?B p65 protein of Model group had elevated expression 12 h after surgery,and reached the peak at 3 d,with persistent positive expression until 7 d.Suggesting that oxidative stress-induced activation of NF-?B after hemorrhage,and a long duration,may be is an important part of secondary brain injury.TUNEL positive level reached the peak at 3 d after surgery,followed by gradual decrease.Expression of c-myc protein in brain tissues is consistent with TUNEL-positive cells.Neuron apoptosis induced by post-hemorrhage oxidative stress might be related with activation of NF-?B,and initiation of apoptosis related gene c-myc.3.MPEG-SOD can inhibit oxidative stress after intracerebral hemorrhage,reduce the expression of NF-?B and c-myc protein,decrease neuronal apoptosis and may provide a new treatment idea for reducing the secondary brain injury.