| In developed countries,thanks to advances in perinatal care,nearly90%ofpremature newborns survive.Many of these infants however develop perinatal braindamage leading to lifelong motor,cognitive and behavioral handicaps.Thisplethora of neurological deficits is encountered in acts and cerebralpalsy(CP).CP is a non-progressive condition characterized by a wide spectrumof motor deficits resulting from damages which mainly affect the supratentorialcorticospinal tracts and the basal ganglia.These damages to the developing braincould result in varying patterns of brain injuries depending on the developmentalstage,such as periventricular leukomalacia and selective neuronal necrosis ofthe vary preterm infant or parasagittal neocortical injuries in the term andnear-term infant.The incidence of CP remained stable for decades due to the lackof efficient therapeutic intervention that in turn reflected pathophysiologicaluncertainties.Perinatal hypoxia-ischema(H/I) has been considered for a longtime as the main--if not the only--mechanism causing perinatal braindamages.However,despite all improvements in perinatal care essentially designedto protect against direct H/I afflictions,the incidence of perinatal CP did notdecrease significantly.This observation raised the need to look for otheretiopathogenic factors explaining those neurological deficits.Evidence fromepidemiological,clinical and experimental studies suggested that the fetalinflammatory response(s) triggered by maternal infection, like subclinicalbacterial chorioamnionitis, contributes to perinatal braindamage.Infective-inflammatory mechanisms,alone or,most often,in combinationwith subsequent H/I,could thus underlie developmental brain injuries.Scientiststherefore desiged animal models to evaluate the effect of perinatalinfection/inflammation combined with H/I.However,in most of thesemodels,lipopolysaccharide(LPS) endotoxins and H/I combination was inflicted topostnatal day7(P7)rat pups which in terms of brain development is equivalent to a late human preterm gestational age of around32-34weeks.CP is a nonprogressive life-long disorder that is associated with white matterdamage (WMD) in the periventricular areas of the brain and results in a spectrumof nonprogressive motor and cognitive abnormalities.A model oflipopolysaccharide(LPS,also known as endotoxin) intracervical injection,whichresults in proinflammatory activation,has been shown to result inperiventricular WMD;however,without a motor or cognitive phenotype that wouldbe associated with CP.An inectious or inflammatory model is ideal as CP has beenstrongly linked to these mediators,yet the avaliable models have notdemonstrated a discernable phenotype that would allow testing of interventions.There is mounting clinical evidence that WMD may occur in the second and thirdtrimester resulting from subclinical chorioamnionitis or maternalinflammation.During this developmental stage,a fetal inflammatory response canresult in apoptosis of the preoligodendrocytes(pre-OLs),the myelin-producingcells,before the onset of myelinization and development of matureoligodendrocytes.In the rodent,the pre-OLs present in the first postnatalweek,which wquates to the second-third trimester in human pregnancy.Our goalwas to attempt to refine a model for CP by targeting the pre-OLs by using a chronicsubclinical inflammatory insult to induce WMD and the phenotypicalcharacteristics of CP.The research of expression of MAG and NgR in brain damage of LPS premature ratswhich were treated by acupuncture and manipulation at the Shu and He acupointsPurpose:Cerebral palsy(CP) is characterized by motor deficits.Our objective was todevelop an inflammatory model for CP based on chronic lipopolysaccharide (LPS)exposure with a recognizable phenotype in offspring. We use "Shu and Heacupoints" acupuncture treatments for newborn rat to observe this method isefficient. Material and method:Pregnant SD rats were obtained from Liaoning University of TraditionalChinese Medicine at embryonc day (E)15.The animals were acclimatized for48hoursand kept in a12-hour light/12-hour dark regimen with food available at all times.Infectious effect was produced by administrating lipopolysaccharide(LPS)intraperitoneally to pregnant rats starting on embryonic day17.Pregnant ratswere intracervically injected with200ug/kg/12h LPS or saline for controls.After birth, take the placenta do HE dyed, observation palace infectionstatus.The judgment standard for numerous neutrophils infiltrating.In order toreduce the error, the observer blinded to the way only randomly assigned eachrat with treatment (LPS/saline).This experiment will eliminate LPS full term are milking rats and thepremature newborn rat randomly divided into LPS intervention group and LPS theintervention group two groups. For3days of age LPS intervention group wereyoung acupuncture treatment, according to "lose close match point" theoryselection "QuChi","three rooms","foot three mile","trapped valley" handneedle treatment, not LiuZhen, once per day, intervention to3ZhouLing, allmilk daily neural behavior are rat testing.We record daily weight of newborn rat, and after its birth day LPS test of22intervention group and physiological saline groups the change of white mattermouse brains, observe brains, CNPase MBP expression and on the brain, liver,spleen weighing.Newborn rats were randomized into LPS intervention group andLPS nonintervention group.According to"Shu and He acupoints"theory,we selected"QuChi","Sanjian","Zu Sanli","Xiangu" to treat the rats.Evalution for motor and cognitive developmental milestones were performedon days PND1-21.Newborn rats were weighed and assessed for neonataldevelopmental milestones as previously described.Observers working inpairs,blinded to the treatment group with1member consistent throughout thetesting period assessed(1)surface righting(time for pups placed supine to returnto prone with all paws on the ground),(2)negative geotaxis(time for pups placedhead down on a45degree incline to turn180degrees and begin to crawl up the slope),(3)cliff aversion(time for pups positioned with forepaws and snout overthe edge of a shelf to turn and begin to crawl away from the edge),(4)rooting(head turns toward the side of the face being stroked with cottonswab),(5)Forelimb grasp(pups could remain suspended for at least1second aftergrasping thin rod with their forepaws),(6)audio startle(quick involuntary jumpafter a small metal object is dropped),(7)air righting(pups released upside-downfrom a height of approximately60cm,turn right-side up and land on paws on abed of shavings),(8)eye opening,(9)forelimb placement(grasps dowel strokedagainsr dorsal surface of paw),(10)open field activity assessment(time to moveoff a circle),and (11)ear twitch-first day ear twitches after stimulation withcotton swab.The behaviors measured all occur at differing stages throughout thefirst21days;therefore,they assess development throughout the neonatalperiod.All timed responses were limited to a maximum of30seconds andnonresponse was scored as30seconds.Responses were evalvated based on the firstday the skill was exhibited.On PND22,after neonatal tests,5animals per treatment were perfused and20um sections were cut,mounted,and frozen until use.After endogenousperoxidase was quenched,adjacent sections were incubated in either CNP orMBP.White matter damage was assessed with immunohistochemical Pre-OLmarkers(CNP,MBP).We use the immune imprinting methods detection MAG and NgR.The SPSS16.0statistical software package that newborn mice test neuraldevelopment in each kind of behavioral variables are used repeated measurementdata analysis of variance (ANOVA) method. Newborn rat compares the breasts weightmean±standard deviation (plus or minus s) said, use repeated measurementmethod analysis of variance, and in different time points are compared.Immunohistochemical data using the mean±standard deviation (plus or minuss) said average light density values (mean optical density, OD), with theanalysis of variance (ANOVA) method to compare the differences between the twogroups, with P <0.05says there are differences, P <0.01says there aresignificant differences. Results:1. The experimental group and control group LPS, salt water rat after birthday1,2days,21days there very significant weight difference (P <0.001).The third day after birth, day4both weight there was significant difference(P <0.01).5days after birth weight both existing difference (P <0.05).2. Through7day of birth, day14and21days two groups of milk brains, liver,spleen weighing carries on the comparison, found LPS intervention group andphysiological saline brain and liver in life of7days after the comparison (P<0.05), born14days after the comparison of the liver (P <0.01), born14daysafter the comparison of the spleen (P <0.05), born after21days the comparisonof the liver (P <0.05), born after21days LPS the intervention group liverand spleen and physiological saline compared (P <0.01).3. LPS treatment group in complete cliff avoid milk rat, fore place, foregrip, consternation reflex, several tests are late decreased activity in saltwater treatment group. But in the plane is, the air is turned over time and openyour eyes in salt water treatment early group. For newborn mice were nerve growthand development are testing found that plane turn is testing, cliffs, avoidtesting, consternation reflection test several, between the two groups isdifference (p <0.05), between the two groups placed fore test with significantdifference (p <0.01), other some of the more no difference (p>0.05).4. Suspension experiment and the wilderness experiment three groups therewere no difference (p>0.05). Group compared with physiological saline, LPSintervention group and the intervention group in the plane, cliffs, avoid andturn is consternation reflection (p <0.05). In the fore placed test, LPSinterention group (p <0.05), LPS the intervention group (p <0.01). LPSintervention group and the intervention group in the cliff avoid comparison testhave difference (p <0.05). However, the negative trend to test and ears twitchof test two not measure the results.5. Former oligodendrocytes markers in LPS processing milk CNP rat isincreasing number of group (p <0.01), the myelin alkaline protein (MBP) in LPS processing milk group is to reduce rat (p <0.01). LPS treatment group CNP averagelight density value of0.217+/-0.0060, salt water treatment group CNP averagelight density value of0.244+/-0.0052. LPS treatment group MBP average lightdensity value of0.380+/-0.0113, salt water treatment group MBP average lightdensity value of0.327+/-0.0087.6.Give pregnant mice after intraperitoneal injection of LPS, newborn ratafter birth are7d is the expression of the visible MAG,14days after birthis increasing gradually, was born after21days down gradually but still higherthan normal control group.7.The expression of a mature NgR with and gradually strengthened. Newbornrat is visible NgR be expression, P14to P21NgR expression graduallystrengthened.Conclusion:Pregnant mice exposure to LPS induced neonatal white matter damage,butthese are not find the same CP phenotype from a Moderate to severe to childrenwith cerebral palsy. Through the neural behavior identification,we found thatuse "Shu and He acupoints" acupuncture treatments was effective. The research of expression of CNP and MBP in brain damage of postnatal ratswhich were treated by LPS and Nerve growth testPurpose:Through the preparation of cerebral palsy rat model to evaluate damage ofmouse brains,Observe postnatal CP model to evaluate injury that would correlatewith presence of Pre-OL in human pregnancy.Material and method:On postnatal (P)days2,3,4,5and6, pups were treated with(lipopolysaccharide[LPS])(n=12;30,30,60,60,120ug/Kg) or saline(n=11).On PNDs2through6of the neonatal testing,animals on completion of the day’sassessment receives an injection of LPS or saline.None of the animals displayedsigns of clinical inflammation or illness before or after each injection.Therewas no difference in weight between the LPS-treated and saline treated groupsat PND1through21and all animals survived through PND21.Evalution for motor and cognitive developmental milestones were performedon days PND1-21.Newborn rats were weighed and assessed for neonataldevelopmental milestones as previously described.Observers working inpairs,blinded to the treatment group with1member consistent throughout thetesting period assessed(1)surface righting(time for pups placed supine to returnto prone with all paws on the ground),(2)negative geotaxis(time for pups placedhead down on a45degree incline to turn180degrees and begin to crawl up theslope),(3)cliff aversion(time for pups positioned with forepaws and snout overthe edge of a shelf to turn and begin to crawl away from the edge),(4)rooting(head turns toward the side of the face being stroked with cottonswab),(5)Forelimb grasp(pups could remain suspended for at least1second aftergrasping thin rod with their forepaws),(6)audio startle(quick involuntary jumpafter a small metal object is dropped),(7)air righting(pups released upside-downfrom a height of approximately60cm,turn right-side up and land on paws on abed of shavings),(8)eye opening,(9)forelimb placement(grasps dowel strokedagainsr dorsal surface of paw),(10)open field activity assessment(time to moveoff a circle),and (11)ear twitch-first day ear twitches after stimulation withcotton swab.The behaviors measured all occur at differing stages throughout thefirst21days;therefore,they assess development throughout the neonatalperiod.All timed responses were limited to a maximum of30seconds andnonresponse was scored as30seconds.Responses were evalvated based on the firstday the skill was exhibited.On PND22,after neonatal tests,5animals per treatment were perfused and20um sections were cut,mounted,and frozen until use.After endogenousperoxidase was quenched,adjacent sections were incubated in either CNP Or MBP.White matter damage was assessed with immunohistochemical Pre-OLmarkers(CNP,MBP).The SPSS16.0statistical software package that newborn mice test neuraldevelopment in each kind of behavioral variables are used repeated measurementdata analysis of variance (ANOVA) method. Newborn rat compares the breasts weightmean±standard deviation (plus or minus s) said, use repeated measurementmethod analysis of variance, and in different time points are compared.Immunohistochemical data using the mean±standard deviation (plus or minuss) said average light density values (mean optical density, OD), with theanalysis of variance (ANOVA) method to compare the differences between the twogroups, with P <0.05says there are differences, P <0.01says there aresignificant differences.Results:1. The experimental group and control group LPS, salt water rat from the thirdday after birth, day4,7day, day8,9days there very significant weightdifference (P <0.001).10days after birth weight both there was significantdifference (P <0.01).11days after birth to21days both weight withoutdifference (P>0.05).2. Through the day after the birth of the two groups are21mouse, liver,spleen weighing carries on the comparison, found that the weight of the spleenin LPS group compared with physiological saline difference (P <0.05).3. LPS treatment group in complete plane milk rat turned positive, foragingreflection, cliffs, withdrawal and fore to grasp several tests are late in saltwater treatment group. But in the fore place, consternation reflection, the airis decreased activity and open your eyes, turn over time in salt water treatmentearly group. For newborn mice were nerve growth and development are testing foundthat plane turn is testing, cliffs, avoid testing, fore grasping tests and openyour eyes time testing a few, between the two groups is difference (p <0.05),decreased activity test between the two groups with significant difference (p<0.01), other some of the more no difference (p>0.05). Suspension experiment two groups of comparisons no difference (p>0.05), the wilderness experimenttwo groups of comparisons has difference (p <0.05). However, the negative trendto test and ears twitch of test two not measure the results.4. Former oligodendrocytes markers in LPS processing milk CNP rat isincreasing number of group (p <0.01), the myelin alkaline protein (MBP) in LPSprocessing milk group is to reduce rat (p <0.01). LPS treatment group CNP averagelight density value of0.272+/-0.0099, salt water treatment group CNP averagelight density value of0.221+/-0.0086. LPS treatment group MBP average lightdensity value of0.207+/-0.0077, salt water treatment group MBP average lightdensity value of0.283+/-0.0083.Conclusion:Neonatal exposure to LPS induced white matter damage in the brain,but theseare similar to findings from a postnatal hypoxic model suggesting that in therodent,targeting the Pre-OL does not result in a CP phenotype. |
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