The Safety Evaluation Of Analgesic Vaccine Targeting N-methyl-D-aspartate Receptor Subunit 2B In Rats

BackgroundNeuropathic pain is defined as pain initiated or caused by a primary lesion ordysfunction in the nervous system.The reasons responsible for neuropathic pain include trauma,ischaemic injury,infection/inflammation,cancer,drugs and compression etc.Patients withneuropathic pain do not respond to non-steroidal anti-inflammatory drugs and haveresistance or insensitivity to opiates.The current pharmacological mainstays of clinicalmanagement are tricyclic anti-depressants and certain anticonvulsants,but these onlyachieve clinical significant pain relief in less than 50% of patients and are associated withsuboptimal side effect profiles.Therefore,an important issue of pain research is to find anovel method of analgesia that is effective and safe.Peripheral nerve injury may result in apersistent pain due to abnormal activity in peripheral afferents and a sustained input to thespinal cord that leads to alterations in the activity and excitability of spinal cord neurons.These spinal changes involve central sensitization of dorsal horn neurons.N-methyl-D-aspartate (NMDA) receptor subunit 2B(NR2B) plays an important role in thefacilitation and maintenance of central sensitization.NMDA receptor antagonists such asketamine,dextromethorphan and MK-801 have been reported to produce symptomaticrelief in a number of neuropathies including postherpetic neuralgia,central pain caused byspinal cord injury and phantom limb pain.So,we constructed recombinant adenovirusserotype 5-mediated NR2B(rAd5/NR2B) by inserting NR2B gene into the adenovirus serotype 5.rAd5/NR2B vaccine targeting NR2B epitope could induce the fluidimmunological response in vivo,the NR2B-specific antibodies were produed,whichpenetrated the blood-brain barrier at the time of pain,reacted with NR2B protein todownregulate the activity of NR2B and fight against neuropathic pain.Over the last several years,an increasing number of reports have demonstrated theimportance of the NR2B subunit in determining the pharmacological and functionalproperties of the NMDA receptor.Consequently,NR2B has been implicated in modulatingfunctions such as learning,memory processing,and synaptic plasticity,as well as beinginvolved in a number of human disorders which include pain perception,hypoxia/ischemia,parkinson's disease,Alzheimer's disease,seizure disorder,diabetes,schizophrenia et al.So,the agents such as ketamine and MK801 induce unacceptable side effects at analgesicdoses including hallucinations,dysphoria and disturbances of cognitive and motor function,which exclude their widespread use.On the base of these researches,we detected whetherrAd5/NR2B targetting at NR2B produce any side effects accompanying the analgesic effect,to consider the safety of rAd5/NR2B.So our objective is to evaluate the safety of rAd5/NR2B in rat base on following 4parts.1.The influence of rAd5/NR2B on the primary neurons apoptosis induced by glutamicacid in vitro:Immuned serum was obtained from the rats immuned with rAd5/NR2B byintragastric gavage,then the primary neurons were separated and cultured from neonatalrats' hippocampus.After administrated with immuned serum,the intracellular calciumconcentration and apoptosis of primary neurons were detected.2.The influence of rAd5/NR2B on the neurons apoptosis in vivo:after immuned withrAd5/NR2B by intragastric gavage,the rats were stained by immunohistochemistry toexplore the apoptosis of neurons.3.The effect of rAd5/NR2B on the congnitive function of rats:the congnitive functionof rats immuned with rAd5/NR2B by intragastric gavage was detected through Morriswater maze testing,and the expression of NR2B was detected by immunohistochemistryand Western blot.4.The electrophysiological and molecular mechanism of rAd5/NR2B influencing on the cognitive function:the hippocampus sections were separated from rAd5/NR2Bimmuned rats,and long term potentiation(LTP) was observed by recording fEPSP inhippocampus sections,then the expression of phosphorylated tau protein was detected byWestern blot.Methods and Results1.The influence of rAd5/NR2B on the primary neurons apoptosisinduced by glutamic acid in vitroMethods Female SD rats (n=6) were choosed and immuned with rAd5/NR2B byintragastric gavage.Two weeks after the first gavage,we administrated this vaccine again.Two weeks later,NR2B-specific IgG titers in sera obtained from immuned rats weredetermined by ELISA,the normal serum was prepared and determined at the same time.Primary cultured neurons were separated from the hippocampus of neonatal rats in 24h,andcultured for 1 week,then neurons were stained with Fluo-4/AM and observed with confocallaser scaning to determine the effects of immuned serum,normal serum and glutamic acidon the intracellular calcium concentration of neurons.Following this,primary neurons wererandomly divided into 4 groups:Naive group,glutamic acid group(Glu group),immunedserum and glutamic acid group(Glu+rAd5/NR2B serum group),normal serum and glutamicacid group(Glu+Normal serum group),which were respectively administrated with saline,glutamic acid,immuned serum and glutamic acid,normal serum and glutamic acid.Aftercultured for more 3 days,the apoptosis percentage of primary neurons stained by AnnexinV-PI were explored with flow cytometer,and the neurons were stained byimrnunohistochemistry to detect the expression level of Caspase-3,then the apoptosis ofneurons was determined.Results①Following the primary immunization and the booster immunization,NR2Bspecific antibodies in serum was 12.7+2.8μg/ml.In contrast,NR2B specific antibodieswere not detected in normal serum from the norma rats.The intracellular calciumconcentration of neurons was increased after the stimulation of glutamic acid,thisincreasing effect of glutamic acid was reversed by rAd5/NR2B immuned serum,furthermore,rAd5/NR2B immuned serum decreased the concentration of intracellular calcium,and the concentration didn't increase obviously after injecting glutamic acid.But,normal serum hadn't this effect.②The results of flow cytometer showed:Compared with Glu group,the apoptosispercentage of primary neurons was increased in Glu+rAd5/NR2B group and Glu+Normalgroup(P＜0.05);Compared with Glu+Normal group,the apoptosis percentage wasdecreased in Glu+rAd5/NR2B group(P＜0.05).In the research about effect of rAd5/NR2Bimmuned serum on the Caspase-3 experssion,compared with Naive group,the expressionlevels were upregulated in the other 3 groups (P＜0.05).Compared with Glu group,expression levels of Caspase-3 were upregulated in Glu+rAd5/NR2B group andGlu+Normal group (P＜0.05).Compared with Glu+Normal group,expression level ofcaspase-3 in Immuned serum group was downregulated in Glu+rAd5/NR2B group(P＜0.05).2.The influence of rAd5/NR2B on the neurons apoptosis in vivoMethods 60 SD rats weighing about 200g were choosed and randomly divided into 6groups(n=10):Naive group,rAd5/NR2B group,Sham group,SNI model group(SNI group),SNI model treated with MK801 group(SNI+MK801 group),and SNI model treated withrAd5/NR2B group(SNI+rAd5/NR2B group).The rats were received saline in Naive groupand rAd5/NR2B in rAd5/NR2B group by intragastric gavage,the administration wererepeated 2 weeks later.After SNI model surgery,the rats were respectively injected withsaline and MK801 in SNI group and SNI+MK801 group.Rats of SNI+rAd5/NR2B groupwere administrated with rAd5/NR2B by intragastric gavage at first,then received SNImodel surgery.The sciatic nerves were separated and exposured,not ligated in Sham group.The mechanical withdrawal threshold(MWT) and thermal withdrawal duration(TWD) ofrats were detected after surgery.Two weeks after SNI model surgery,the spinal cords andhippocampuses of rats were obtained,then the expression level of caspase-3 and NR2Bproteins were detected by immunohistochemistry and Western blot to evaluate the apoptosisof neurons in vivo.Results Compared with SNI group,the MWT were increased,and the TWD wereshortened in SNI+rAd5/NR2B group.There was no difference on the the apoptosis of neurons in vivo between Naive group and rAd5/NR2B group (P＞0.05).Compared withSham group,apoptosis of neurons didn't increase obviously in SNI group andSNI+rAd5/NR2B group (P＞0.05),but increased in SNI+MK801 group (P＜0.05).Therewas no difference on the expression levels of NR2B in rats' spinal cord and hippocampusbetween Naive group and rAd5/NR2B group(P＞0.05).Compared with SNI group,theexpression of NR2B was decreased in SNI+rAd5/NR2B group and SNI+MK801 group(P＜0.05).3.The effect of rAd5/NR2B on the congnitive function of ratsMethods 60 SD rats weighing about 200g were choosed,divided and administrated asthe part 2.One week after SNI model surgery,all rats were detected with Morris watermaze testing.Training trials were given on the first 5 consecutive days,and rats underwentthree training trials each day.In eaeh training trial,if the rat found the platform,it wasallowed to remain there for 30s.If the rat was unable to find the platform within 60s,after itwas made to stay there for 30s.The time from diving to finding the hiden platform (escapetime) was recorded (escape time of the rat which was unable to find the platform wasrecorded 60s),swimming path length and the swimming velocity were recorded.On thenext day,each rat was exposed to an additive training trails test after the starting point waschanged to the adjacent left quadrant of the platform.The latency and distance werereeorded in the same way as the previous five training trials.Furthermore,rats performed aspatial probe test trial with the same starting point as in the five training trails.This trialconsisted of removing the platform from the pool and allowing the rat to swim for 60s insearch of it.The time spent in each of the four equal quadrants,and the times of throughingplatform was reeorded.After Morris water maze testing,the hippocampus segments of ratswere harvested,and the expression levels of NR2B were detected usingimmunohistochemistry and Western blot.Results There was no difference on the escape times between Naive group andrAd5/NR2B group(P＞0.05).Compared with Sham group,the escape time was prolongedin SNI group,SNI+rAd5/NR2B group and SNI+MK801 group(P＜0.05).Compared withSNI group,the escape time didn't change in SNI+rAd5/NR2B group(P＞0.05),however,it was prolonged in SNI+MK801 group(P＜0.05).There was no difference on the swimmngpath length and velocity among these rats of 6 groups.The expression level of NR2B in rats hippocampus didn't change between Naive groupand rAd5/NR2B group(P＞0.05).Compared with SNI group,the expression of NR2B wasdecreased in SNI+rAd5/NR2B group and SNI+MK801 group.4.The electrophysiological and molecular mechanism of rAd5/NR2Binfluencing on the cognitive functionMethods 60 SD rats weighing about 200g were choosed,divided and administrated asthe part 2.Two weeks after SNI model surgery,the rats' hippocampuses were seperated andslicinged into 400μm thickness sections.Incubated in ACSF,the hippocampal sections weredetected with electrophysiological method.In all experiments,base line synaptictransmission was monitored for 20 min before high frequency stimulation(HFS).Thestimulus intensity was set to evoke 50% of the maximal amplitude of PS.LTP was inducedusing HFS,which included 300 pluses at 100Hz,on Schaffer-collateral pathway.Theamplitude and slope of fEPSP was recorded from the CA1 region of rat hippocampal slices.If the fEPSP was increased more than 20% after HFS,and maintained more than 30min,itwas considered as a successful LTP induction.The influence of rAd5/NR2B on LTP wasevaluated by comparing the amplitude,slope of fEPSP and the achievement ratio of LTPinduction.Following electrophysiological research,the protein were extracted fromhippocampus,and the expression levels of phosphorylated tau protein and NR2B wereexplored using Western bolt.Results①Compared with Naive group,the slopes of fEPSP didn't change inrAd5/NR2B group,Sham group and SNI group(P＞0.05),but they were decreased inSNI+rAd5/NR2B group and SNI+MK801 group(P＜0.05).Compared with SNI group,theslopes of fEPSP were decreased in SNI+rAd5/NR2B group and SNI+MK801 group(P＜0.05).Compared with SNI group,they were also decreased in SNI+rAd5/NR2B groupand SNI+MK801 group(P＜0.05).②There was no difference on the expression levels of phosphorylated tau proteinamong these 6 groups(P＞0.05).Compared with SNI group,the expression level of NR2B was down-regulated in SNI+rAd5/NR2B group(P＜0.05),and the down-regulation wasmore obvious in SNI+MK801 group(P＜0.05.5.Statistical AnalysisAll of the analyses were performed by SPSS 13.0 software package.Measurement datawere presented as means+standard deviation (SD).The differences between multipleindividual means were analyzed by one-way ANOVA.P＜0.05 was considered statisticallysignificant in all tests.Conclusion1.rAd5/NR2B vaccine could induce the humoral immunological response andproduction of NR2B-specific antibody in sera.NR2B-specific antibody could react withNR2B subunit and inhibit or reverse the calcium overload induced by glutamic acid,furthermore,rAd5/NR2B immuned serum decreased the apoptosis of primary neuronsinduced by glutamic acid,provided partial protection for primary neurons.2.rAd5/NR2B vaccine couldn't influence the apoptosis of neurons in vivo,however,MK801 obviously increased the apoptosis of neurons.3.The administration of rAd5/NR2B didn't prolong the escape time of rats in Morriswater maze testing,and the frequency of throughing platform didn't increase.But theescape time was apparently extended after injecting of MK801,and the times of throughingplatform increased.The expression level of NR2B was down-regulated after immuned withrAd5/NR2B.4.After the rats were immuned with rAd5/NR2B vaccine,the LTP was inductedsuccessfully,but the amplitude and slope of fEPSP were lowered.This phenomenon wasmore obvious after administration of MK801.rAd5/NR2B vaccine didn't influence on theexpression level of phosphorylated tau protein in hippocampus,but decreased theexpression of NR2B simultaneously. In this study,the safety of rAd5/NR2B vaccine was explored in rats.We foundrAd5/NR2B vaccine could induce the humoral immunological response and production ofNR2B-specific antibody in serum,which could react with NR2B subunit and inhibit orreverse the calcium overload induced by glutamic acid.rAd5/NR2B immuned serum coulddecrease the apoptosis of primary neurons induced by glutamic acid compared with thenormal serum of rats,and provide partial protection for primary neurons,rAd5/NR2Bvaccine couldn't influence the apoptosis of neurons in vivo,however,MK801 obviouslyincreased the apoptosis of neurons.In Morris water maze testing,the administration ofrAd5/NR2B didn't impair the spatial learning and memory of rats.Furthermore,rAd5/NR2B vaccine was detected to inhibit the induction and maintance of LTP in rats,butdidn't upregulate the phosphorylation level of tau protein in hippocampus.The expressionlevels of NP2B were detected to explore this phenomenon.We found,though theexpression level of NR2B wasn't affected in normal rats immuned with rAd5/NR2B,theexpression were down-regulated after in SNI model rats immuned with rAd5/NR2B.Theseresults suggested:the antagonism and down-regulation of NR2B-specific antibody werepartial effective,it could lead to analgesic effects,but didn't obviously induced theapoptosis in vivo and impair the cognitive function of rats.So we concluded theadministration of of rAd5/NR2B vaccine was partly safe in the time of analgesia,whichproduced less side effects than MK801.