单侧迷路破坏后大鼠前庭神经内侧核区氨基酸含量的变化.pdf

71Acta Physiologica Sinica,February 25,2007,59(1):71-78http:/Research PaperReceived 2006-07-03 Accepted 2006-12-14This work was supported by the Prominent Youthful Science Foundation of Jilin Province(No.20040109).*Corresponding author.Tel:[phone];E-mail:y-z-Changes of amino acid concentrations in the rat medial vestibular nucleusfollowing unilateral labyrinthectomyYU Hai-Ling1,AN Ying2,JIANG Hai-Ying2,JIN Qing-Hua2,JIN Yuan-Zhe2,*1Department of Pharmacology;2Department of Physiology,Yanbian University College of Medicine,Key Laboratory of OrganismFunctional Factors of the Changbai Mountain,Ministry of Education,Yanji 133000,ChinaAbstract:To understand the neurochemical mechanisms underlying the vestibular compensation,we determined the levels of aminoacids such as aspartate,glutamate,glutamine,glycine,taurine,alanine in the medial vestibular nucleus(MVN)following unilaterallabyrinthectomy(UL),by using in vivo brain microdialysis and high-performance liquid chromatography technique.Rats were pre-treated by infusing 2%lidocaine 1.2 mL or 10 mg arsanilic acid into the tympanic cavity to obstruct uni-periphery vestibular organ,andthen the levels of amino acids were determined in MVN of normal control and ipsilateral or contralateral lesional(ipsi-/contra-lesional)rats.In the control experiment,the levels of aspartate,glutamate,glutamine,glycine,taurine,and alanine were(6.150.59),(18.131.21),(33.731.67),(9.260.65),(9.560.77)and(10.070.83)pmol/8 L sample,respectively.The concentrations of aspartate andglutamate decreased,while the concentration of taurine increased in the ipsi-lesional MVN of rats 10 min after infusing 2%lidocaineinto middle ear to obstruct uni-periphery vestibular organ.Whereas the concentration of glutamate increased,the concentrations ofglycine and alanine decreased in the contra-lesional MVN,accompanied by imbalances of glutamate,glycine and alanine in the bilateralnuclei.In contrast,the levels of glutamate and alanine decreased,the level of glutamine increased in the ipsi-lesional MVN,and the levelof glutamate decreased in the contra-lesional MVN of rats 2 weeks after infusing 10 mg arsanilic acid into the tympanic cavity toobstruct uni-periphery vestibular organ.Furthermore,the level of glutamine in the ipsi-lesional MVN was obviously higher than thatin the contra-lesional MVN.These results demonstrate that an imbalance of different amino acids appeared in bilateral MVN after UL,and this imbalance decreased after the development of vestibular compensation.Whereas the imbalance of glutamine release in bilateralnuclei appeared after vestibular compensation.Key words:amino acids;medial vestibular nucleus;unilateral labyrinthectomy;vestibular compensation单侧迷路破坏后大鼠前庭神经内侧核区氨基酸含量的变化单侧迷路破坏后大鼠前庭神经内侧核区氨基酸含量的变化于海玲1，安 英2，姜海英2，金清华2，金元哲2,*延边大学医学院 1药理学教研室；2生理学教研室，长白山生物功能因子省部共建教育部重点实验室，延吉 133000摘摘 要：要：本实验用脑部微量透析法和高效液相色谱法观察单侧迷路破坏(unilateral labyrinthectomy，经利多卡因或对氨基苯胂酸盐预处理以阻断单侧外周前庭器官)后大鼠同侧及对侧前庭神经内侧核(medial vestibular nucleus,MVN)区部分氨基酸(天冬氨酸、谷氨酸、谷氨酰胺、甘氨酸、牛磺酸和丙氨酸)含量的变化，以了解前庭代偿的部分神经化学机制。实验观察到，对照组大鼠MVN区天冬氨酸、谷氨酸、谷氨酰胺、甘氨酸、牛磺酸和丙氨酸浓度分别为(6.150.59)，(18.131.21)，(33.731.67)，(9.260.65)，(9.560.77)和(10.070.83)pmol/8 L透析样本。左侧中耳内灌注2%利多卡因后10 min，同侧MVN 区天冬氨酸、谷氨酸含量立即减少(P0.05)，牛磺酸含量增加(P0.05)；对侧MVN 区谷氨酸含量立即增加(P0.05)，甘氨酸和丙氨酸含量减少；双侧核团间谷氨酸、甘氨酸和丙氨酸含量失衡。而用对氨基苯胂酸盐永久阻断单侧前庭器官 2 周后，同侧 MVN 区谷氨酸和丙氨酸含量减少，谷氨酰胺含量增高；对侧MVN 区谷氨酸含量也减少；同侧MVN 区谷氨酰胺含量明显高于对侧MVN 区。结果提示，单侧迷路破坏后双侧 MVN 区氨基酸含量立即失去平衡，随着前庭代偿的进展，此差异减少，但是在前庭代偿后却出现双侧前庭核区谷氨酰氨的含量失衡，说明在前庭代偿过程中氨基酸含量变化起着重要作用。Acta Physiologica Sinica,February 25,2007,59(1):71-7872关键词：关键词：氨基酸；前庭内侧核；单侧迷路破坏；前庭代偿中图分类号：中图分类号：Q463；R338.2Unilateral labyrinthectomy(UL)produces vestibularsymptoms,including autonomic symptoms,oculomotorand postural asymmetry.Some of these symptoms gra-dually disappear over time1,known as vestibularcompensation,which might be considered as a functionalreassembly of the central vestibular system and used as anexperimental model of lesion-induced vestibular plasticityin the central nervous system(CNS)2,3.The medial vestibular nucleus(MVN),one of the mostimportant nuclei in the vestibular nucleus complex in thebrainstem4,is the main nucleus for transmission of infor-mation from peripheral vestibular input to the central path-ways and is mainly involved in monitoring the compensa-tory responses5,6.UL produces asymmetrical spatiotem-poral changes in the expressions of several inducible orconstitutive transcriptional factors in the vestibular nuclei(VN)7,8.Other phosphorylated proteins can also be de-tected in the MVN following UL5,9.Therefore,it is sug-gested that MVN mainly plays an important role in the pro-cess of vestibular compensation.Numerous previous studies suggest that amino acids arethe main neurotransmitters or neuromodulators in the CNS,including glutamate(Glu),aspartate(Asp),glycine(Gly),alanine(Ala),taurine(Tau),and-amino butyric acid(GABA)10-12.However,the detailed role of amino acids ofVN in vestibular compensation is still not clear.Therefore,we investigated how the actual release of amino acids inMVN changes during vestibular compensation in order todetermine the involvement of amino acids in the vestibularplasticity inducing the compensation following UL,by us-ing an in vivo microdialysis technique and high-perfor-mance liquid chromatography(HPLC)technique.1 MATERIALS AND METHODS1.1 AnimalsOne hundred and eleven male Wistar rats(Crj:Wistar;yanji,China),7-week old and weighing 200-300 g,were ran-domly divided into the lidocaine-UL group and the arsanilicacid-UL group including ipsilateral and contralateral lesional(ipsi-and contra-lesional)groups,n=15 and n=7 rats,respectively,and the normal control group(n=67 rats).1.2 Implantation of microdialysis probesThe rats were anesthetized with chloral hydrate(300 mg/kg,i.p.)and placed in a stereotaxic frame(Chengmo,Japan)with the incisor bar set at-3.3 mm.The skull from 1/2posterior to the lambdoid suture of the parietal bone to thegreat occipital foramen was removed by micro electro-rotor(Saeyang,Korea),and cerebellum was partly absorbedby electric suction apparatus(DEX-23D,China)to ex-pose vestibular area in the brainstem under an operatingmicroscope(AE 993330901,China),and a microdialysisprobe(the tip of the probe covered with a 1.5-mm lengthof hollow fibers,200-m outsider diameter,cellulose ace-tate membrane,48 kDa molecular cut-off;Terumo,Japan)was stereotaxically implanted into the left MVN(2.9-3.0mm anterior to the area postrema,0.7-0.8 mm lateral tothe midline,and 1.5-1.6 mm ventral to the surface)ac-cording to the atlas of Paxinos and Watson13.1.3 HemilabyrinthectomyTwenty-two rats were anesthetized with chloral hydrate(300 mg/kg,i.p.)and were right arm reclining,which wereinjected 0.1 mL arsanilic acid solution 100 mg/mL by add-ing arsanilic acid(Sigma)to 0.3 mol/L sodium carbonatesolution to rate the tympanic membrane through externalear into left middle ear.An absorbent cotton(diameter:2mm)was placed to prevent the medicament effuse.In thisway,the membranous labyrinth was destroyed chemically.After recovering from anesthesia,as the labyrinthectomywas completed,the animals showed severe symptoms ofunilateral vestibular loss(e.g.,spontaneous nystagmus,bodyrolling and head yaw).The animal models prepared wereused in the experiment after 2 weeks.Twenty-two rats were anesthetized with chloral hydrate(300 mg/kg,i.p.)and were dorsal position.The skin ofneck medisectted,left-lateral muscle intergroove of ster-nohyoid was stripped to expose the left tympanic bulla.Aneyehole was drilled by micro electro-rotor and a polyethy-lene catheter(caliber 0.5 mm)was inserted into the leftmiddle ear,another terminal was connected with a syringethat was filled with 2%lidocaine hydrochloric solution(0402131,Tianjin,China),the guide catheter was fitted bydental cement.Then,1.2 mL(divided into 5 times in 10min,0.4 mL for the first time)2%lidocaine was evenlypushed into the middle ear in the experimental course.Theimbalance of bilateral eyeball position appeared immedi-ately after lidocaine was injected.1.4 In vivo microdialysis studyThe MVN of each rat was perfused with modified Ringerssolution(147 mmol/L NaCl,4 mmol/L KCl,2.3 mmol/L73YU Hai-Ling et al:Changes of Amino Acid Concentrations in the Rat Medial Vestibular Nucleus Following Unilateral LabyrinthectomyCaCl2;pH 6.5)through the implanted microdialysis probe(exposed membrane 1.0 mm)at a constant rate of 1.5 L/minusing a microsyringe pump(ESP-64,Eicom,Japan).Thedialysate was collected in an Eppendorf tube using a frac-tion collector(EFC-82,Eicom,Japan)every 10 min.Themicrodialysis schedule is shown in Fig.1.As shown in ourprevious experiments,the concentration of amino acidsbecame stable after 90 min14.After a 90-min stabilizationperiod,samples of dialysate were collected.All samples ofcollected dialysates were kept at-80 oC for later analysis.1.5 Amino acid analysisAmino acid levels were measured by using HPLC with anelectrochemical detector(HPLC-ECD)system accordingto the precolumn derivatization method described by Jin etal15.In advance,2 mol/L standard solution by addingL-Asp13.31 mg,L-Glu 14.71 mg,L-glutamine(Gln)14.62mg,Gly 7.51 mg,Tau 12.51 mg,L-Ala 8.91 mg to 50 mLof 0.1 mol/L HCl,diluted in artificial cerebral spinal fluid(ACSF)1 000 times and 40 mmol/L o-phthalaldehyde(OPA)solution by adding 13.5 mg of OPA and 10 L2-mercaptoethanol to 2.5 mL of 0.1 mol/L K2CO3 buffer(pH 9.5)with 10%ethanol were prepared.The solutionwas then stored at 0-4 C and diluted in 0.1 mol/L K2CO3buffer to yield a 4 mmol/L OPA solution just beforedetection.The dialysate(12 L)was mixed with 3 L of 4mmol/L OPA solution and allowed to react for 2.5 min at25 C.After completing the reaction,10 L of the reactionmixture was manually injected into an HPLC system withan Eicompak MA-5 ODS column(4.6-mm inner diameter150 mm;Eicom,Japan)for assaying the amino acids.Detection was accomplished with an ECD(Eicom)with+700 mV Ag/AgCl electrodes.The mobile phase(0.1 mol/LNaH2PO42H2O,0.1 mol/L Na2HPO412H2O,30%MeOH,0.5 mmol/L EDTA2Na,adjusted to pH 6.0)was deliveredat 1.0 mL/min through an HPLC pump and the potential ofECD was set at+700 mV.1.6 HistologyAt the end of the experiment,the animals were sacrificedwith an overdose injection of chloral hydrate.The brainswere removed and fixed in 10%neutral phosphate-buf-fered formalin.After 3 d,the brains were placed in a 30%sucrose solution and cut into 50-m sections in order toascertain the location of the tip of dialysis probe using acryomicrotome(Wheel microtome,KD-1508,China).Sections were stained with neutral red and then examinedusing light microscopy.Only the data obtained from ani-mals in which the microdialysis probe was positioned cor-rectly in the appropriate dialysis site were processed andincluded in the results,and other samples were discarded.1.7 Statistical analysisThe area of each peak of the HPLC chromatograms repre-senting the sample content of amino acids was automati-cally integrated and compared with that of externalstandards.All data were expressed as meansSEM,anddata analysis was performed by a one-way analysis of vari-ance(ANOVA)for repeated measures,with treatment asmain factors,followed by the least-significant differencetest of multiple comparison Fishers least significant dif-ferences test(LSD)protected t-test.A probability level ofP0.05 was considered statistically significant.All statisti-cal procedures employed the Stat View version 11.5 soft-ware for SPSS(SPSS Inc.,Chicago,USA).2 RESULTSAnimals appeared immediately ocular asymmetries(descending in the ipsi-lesional eyeball and ascending inthe contra-lesional eyeball)after infusing lidocaine into theleft middle ear.In contrast,the vestibular symptoms,in-cluding oculomotor and postural asymmetry,were ob-served from 6 h to 168 h after infusing arsanilic acid intothe tympanic cavity.Some of these symptoms graduallydisappeared over time,but others continued.Frequency ofFig.1.The experimental protocols.Acta Physiologica Sinica,February 25,2007,59(1):71-7874nystagmus gradually enhanced from its appearance,the highincidence of nystagmus appeared at 24 h after UL,then re-duced and disappeared at 120 h.The head yaw graduallyaugmented,the peak time appeared at 120 h,and did not re-store until 168 h.The body rolling came forth two peak timesat 24 h and 96 h,respectively.The apex was 24 h,then re-duced and disappeared over 168 h after UL(Fig.2).The basal levels of Asp,Glu,Gln,Gly,Tau,and Ala in dialy-sis samples of the MVN were(6.150.59),(18.13 1.21),(33.731.67),(9.260.65),(9.560.77)and(10.07 0.83)pmol/8 L,respectively(n=67).The concentrations of Aspand Glu decreased,and the concentration of Tau increased inthe ipsi-lesional MVN of rats 10 min after infusing lidocaineinto middle ear to obstruct unilateral vestibular end-organ(P0.05).In contrast,the concentration of Glu increased,andthe concentrations of Gly and Ala decreased in the contra-lesional MVN,accompanied by imbalances of Glu,Gly andAla in the bilateral nuclei of rats(P0.05)(Fig.3 and 4).Decreased levels of Glu and Ala(P0.01,P0.05)but in-creased level of Gln(P0.05)were measured in the ipsi-Fig.3.Comparison of amino acid levels in the dialysate of bilateral MVN in the immediate post-UL rats that were pretreated by infusinglidocaine into the left middle ear.All values are presented as a percentage of the average of basal samples.meansSEM.*P0.05 compared withcontrol;#P0.05,#P0.01 compared with ipsi-lesional MVN.Fig.2.Changes after obstruct of unilateral vestibular end-organ by infusing arsanilic acid into the tympanic cavity in rats(n=10).Frequency ofspontaneous nystagmus gradually enhanced from its appearance.The high incidence of nystagmus appeared at 24 h after UL,then reduced anddisappeared at 120 h.The head yaw gradually augmented,the peak time appeared at 120 h,and did not restore until 168 h.The body rollingcame forth two peak times at 24 h and 96 h,respectively.The apex was 24 h,then reduced and disappeared over 168 h after UL.75YU Hai-Ling et al:Changes of Amino Acid Concentrations in the Rat Medial Vestibular Nucleus Following Unilateral LabyrinthectomyFig.4.Typical chromatogram of amino acid levels in the dialysate of bilateral MVN in the immediate post-UL rats that were pretreated byinfusing lidocaine into the left middle ear.A:Basal levels.B:Amino acid levels in the ipsi-lesional MVN after 1.2 mL lidocaine perfusion.C:Amino acid levels in the contra-lesional MVN after 1.2 mL lidocaine perfusion.Asp,aspartate;Glu,glutamate;Gln,glutamine;Gly,glycine;Tau,taurine;Ala,alanine.lesional MVN,and decreased level of Glu(P0.01)was ob-served in the contra-lesional MVN of rats 2 weeks after in-fusing 10 mg arsanilic acid into the tympanic cavity to ob-struct unilateral vestibular end-organ.In contrast,the level ofGln in the ipsi-lesional MVN increased more significantlythan that in the contra-lesional MVN(P0.05),and Gln lostits balance between ipsi-and contra-lesional MVN 2 weeksafter UL(Fig.5 and 6).Fig.5.Comparison of amino acid content in the dialysate of