Study On The Expression Of S100B And NSE In The Brain Injury And The Mechanism Of Nerve Repair

Objective: Shu and He points acupuncture intervene brain injury model causedby hypoxia-ischemia, premature delivery, lipopolysaccharide infection inneonatal mice. Through neuroethology, I observe expression of S100B, NSE,Nogo-A,MAG, OMgp, caspase3, HIF-1to explore protein expression changes in brain injurymodel and its mechanism.Materials and Methods:1Establish hypoxia ischemia, lipopolysaccharide infection and preterm braininjury model in mice, we use neuroethology and ELISA method to judge degree ofcerebral injury model by evaluating content of S100B, NSE.1.1Preparation of hypoxia ischemia model in mice: Four days after birth, rightjugular vein of mice are ligated. And then put these mice into250ml sealedcontainer within8%oxygen and92%nitrogen mixed gas, then using anhydrouscalcium chloride and sodium lime to remove the influence of CO2and water. Whenconditions as activity decrease, tachypnea, limbs twitching, skin cyanosisappear, neonatal mice are taken back to their mothers to keep feeding.1.2Preparation of LPS model:3,4,5,6,7days after birth, mice areintraperitoneal injected continually with LPS (30,30,60,60,120ug/kg) toprevent drug resistance.1.3Preparation of preterm model: we put male and female mice with2:1into cageat18:00pm daily, using vaginal plug to detect pregnancy at8:00a.m. next day.The pregnant rats were randomly divided into a digital random table cesareangroup and LPS groups of6pregnant rats. The pregnant mice are delivered bycesarean at21days of pregnancy. Female mice were sacrificed by cervical tothe supine position on the operating panel, disinfection abdominal skin, openabdominal, uterine exposure, extrusion newborn rats, placed in37°C water bath tank warm. Remove all the time does not exceed neonatal rats2min. Thenewborn pups with milk replacer fed mice were observed situation.2. Acupuncture point acupuncture intervention group and intervention group, andnormal control group, three days after the start, to identify the neuroethology,respectively in7,14,21days the laboratory tests, take the rats had discussedacupuncture effect on brain injury seed protein expression in mouse organization,observation combined with acupuncture point losing in brain damage repairmechanism.Results1Experimental ischemia and hypoxia group six pregnant rats, birth pups59,three days after the death of six pups, and the remaining53as ischemia andhypoxia in the experimental group, the53neonatal rats were randomly wererandomly divided into intervention group (n=26) and non-intervention group(n=27), the intervention group died during the experiment three, died eightnon-intervention group.The experimental group of LPS pups60neonatal rats were randomly divided intoLPS group (n=40) and control group (n=20) in which the group was injectedwith lipopolysaccharide LPS, the CCP died during injection pups6, the remaining34pups as the experimental group were randomly assigned to the interventiongroup (n=17) and non-intervention group (n=17), in the intervention groupdied during the experiment three pups, pups died of non-intervention group3.The experimental group consisted of pregnant rats LPS six pregnant rats, sixpregnant rats at17days gestation to receive intraperitoneal injection of LPS,3days after injection, there are two pregnant rats were killed and threepregnant rats at22days after birth pups9, within three days of9pups alldied, a pregnant rats at23days gestation birth, does not comply with pretermremoved. The experimental group had six preterm pregnant rats at22days gestationcesarean section, a total of65pups childbirth, surgery three days after thedeath of14pups, and the remaining51as preterm group were randomly assignedto the intervention group (n=25) and the non-intervention group (n=27),the experimental process without a rat died.2neonatal mice weightBy comparing each group of7th and neonatal rats weighing21days, the resultsshow: the intervention group and each group is not in the intervention groupdid not differ (P>0.05) compared to7,14days hypoxic-ischemic interventiongroup, hypoxic-ischemic intervention group did not, the intervention group LPS,lip polysaccharide is not intervention group, the intervention group preterm,premature birth weight group did not interfere with the normal comparison groupwere significantly different (P<0.01), the first21days, the weight of eachgroup no longer statistically significant difference (P>0.05).3Mice on the14th day of liver, spleen, kidney comparison Through the first14days after birth, newborn rats in each group, liver, spleen, left brain weighedcomparison, among the group of liver, there is no difference in the weight ofthe left hemisphere P>0.05, spleen weight were significantly different P <0.01,multiple comparison analysis within the group, LPS group spleen weighthypoxic-ischemic group and the preterm group were significantly different P<0.01, and the normal group differences P <0.05.4Newborn mice developmental test: After neonatal rat nerve growth anddevelopment of the test found that the plane righting test, cliff avoidancetest, shock reflection test items, there are differences (p<0.05) between thetwo groups, there were significant forelimb placing test between the two groupsdifference (p<0.01), there was no other difference (p>0.05)5Analysis of S100B，NSERat S100B, NSE is closely related to the degree of expression and brain damage. Model group S100B, NSE expression was significantly higher than the control group(p <0.01). And acupuncture intervention after28days, the results show: thebrain S100B, NSE expression in acupuncture intervention group and interventiongroup was lower than not.6Comparasion of Nogo-A expression in brainNogo-A protein expression of the average optical density value, the resultsshowed that each group intervention, intervention group not brain tissue at eachtime point Nogo-A blank expression of albumin were higher than control group,the intervention group at7,14,21d three time points on brain Nogo-A proteinexpression of both lower than their not intervention group; Prematureintervention group at various time points brain tissue Nogo-A proteinexpression is lower than other groups (p <0.01).7Comparasion of MAG expression in brain: Comparison between groups with thenewborn rat postnatal brain tissue slices at each time point MAG proteinexpression of the average optical density value, the results showed: theintervention group at7,14,21days brain MAG protein expression of three timepoints are higher than their respective not intervention group; Prematureintervention group at various time points on brain MAG protein expression ishigher than other groups, and brain tissue at each time point MAG proteinexpression is close to blank control group, but there are differences betweenthe statistical analysis; Each group intervention, the intervention group braintissue at each time point MAG protein expression were lower than the blank controlgroup (p <0.01).8Comparasion of OMgp expression in brain: Comparison between groups with thenewborn rat postnatal brain tissue slices at each time point OMgp proteinexpression of the average optical density value, the results showed: theintervention group at7,14,21d three time points on brain OMgp proteinexpression were lower than their non-intervention group; Premature intervention group at various time points on brain OMgp protein expression islower than other groups, brain tissue OMgp protein expression at each time pointclose to the blank control group, but there are differences between thestatistical analysis; Each intervention, the intervention group brain tissueat each time point blank OMgp protein expression were higher than control group(p<0.01).9Comparison between groups with the newborn mice brain tissue slice Nogo-21days after he was born A, MAG, OMgp positive cells in the average optical densityvalue: Building groups were higher than the average optical density value ofblank control group, the difference between groups was statistically significant(P<0.01), the LPS intervention group, hypoxic ischemia intervention group andthe value of the preterm group were lower than the value of the interventiongroup, the difference between the assets with statistical significance (P<0.01),compares two, Nogo-A LPS, MAG, OMgp average optical density value notintervention group, hypoxic ischemia not there was no statistically significantdifference between intervention group (P>0.05). In cell apoptosis rate showed:blank group, the LPS group and LPS intervention not intervention group, hypoxicischemia, intervention group, hypoxic ischemia without intervention group andpremature birth was not significant differences between intervention group(P<0.01), but with premature birth there was no statistically significantdifference between the intervention group(P>0.05).10Comparation of Nogo-A and OMgp expression in immunohistochemic: the resultsshowed that glial cells positive nuclei are deep and tan, small nucleus, morefor solid, fusiform, round or rods. The model group compared with blank controlgroup, positive cells was significantly greater than the blank control group,lose combined with acupuncture point acupuncture intervention group positivecells was less than not intervention group.11Comparation of MAG expression in immunohistochemic: the results showed that glial cells positive nuclei are deep and tan, nucleus of smaller, more for solid,fusiform, round or rods. The model group compared with blank control group,positive cells was significantly less than the blank control group, lose combinedwith acupuncture point acupuncture intervention group positive cells was moreintervention group.12Comparasion of caspase3mRNA expression and HIF-1: comparison between groupswith the newborn rat brain tissue slice caspase321days after birth, HIF-1mRNA level. The result shows: the building level of each group were higher thanblank control group, and the blank control group difference was statisticallysignificant (P <0.01), intervention group caspase3premature, HIF-1mRNA levelcompared with blank control group, difference was not statistically significant(P>0.05); Compares two, premature intervention group caspase3, HIF-1mRNAlevel and LPS intervention group and the hypoxia ischemia intervention groupdifference was statistically significant (P <0.01). Blank control groupexpression content is relatively less, electrophoresis banding dark; Each modelgroup expressed relative content increases, the electrophoresis bands is bright,the model group did not express content is relatively higher than that of theintervention group, intervention group electrophoresis banding is bright.13Comparasion of apoptosis: mice had apoptosis cells homogeneously deep dyedblack or brown, nucleus disappear, the cell membrane is smooth, narrow cell body.The model group compared with blank control group, positive cells wassignificantly greater than the blank control group, lose combined withacupuncture point acupuncture intervention group positive cells was less thannot intervention group.14Comparasion of caspase3and HIF-1protein expression: comparison betweengroups with the newborn rat brain tissue slice caspase321days after birth,HIF-1mRNA level. The result shows: the building level of each group were higherthan blank control group, and the blank control group difference was statistically significant (P <0.01), intervention group caspase3premature,HIF-1mrna level compared with blank control group, difference was notstatistically significant (P>0.05); Compares two, premature intervention groupcaspase3, HIF-1mRNA level and LPS intervention group and the hypoxia ischemiaintervention group difference was statistically significant (P <0.01). Blankcontrol group expression content is relatively less, electrophoresis bandingis fine; Each model group expressed relative content increases, the enlargementof electrophoretic bands, each model group did not express content is relativelyhigher than that of the intervention group, intervention group electrophoresisbanding coarser.Conclusion:Conclusion1. By identifying neuroethology and testing protein expression we can find that “Shu and Hepoints acupuncture”method can enhance the effect of nerve regeneration of MAG in cerebraldamaged rats, and can also lessen the inhibitory effect of Nogo-A，OMgp, in order to promotethe protection of nerve cells.2. By identifying neuroethology and testing protein expression we can find that “Shu and Hepoints acupuncture” method is clinically effective.3Shu and He points acupuncture method can inhibit the apoptosis of the damaged nervecells.4The expression of S100B，NSE，Caspase-3、HIF-1a plays an importnat role in cerebraldamage; In each model, oligodendrocytes myelin related protein expression in newborn larvaeof mice shows various forms at different stages, which is also relevant to the degree ofcerebral damage.