郝彥利,黃鑫焱,THOIDINGJAM Bidyarani Chanu,張 斌
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·論著·
24小時快速眼動睡眠剝奪對青少年期C57BL/6J小鼠空間學(xué)習(xí)記憶能力和海馬一氧化氮水平的影響研究
郝彥利,黃鑫焱,THOIDINGJAM Bidyarani Chanu,張 斌
目的探討24 h快速眼動睡眠剝奪(REMSD)對青少年期C57BL/6J小鼠空間學(xué)習(xí)記憶能力和海馬一氧化氮(NO)水平的影響。方法2014年1月—2015年1月選取45只青少年期(3~4周齡)SPF級雄性C57BL/6J小鼠,采用隨機(jī)數(shù)字表法將小鼠分為對照組、寬平臺組和REMSD組,各15只。將REMSD組小鼠置于水平臺睡眠剝奪箱建立REMSD模型,寬平臺組小鼠置于假水平臺睡眠剝奪箱建立快速眼動睡眠假剝奪模型。通過3輪Morris水迷宮定位航行實(shí)驗(yàn)測試小鼠學(xué)習(xí)能力,記錄逃避潛伏期;Morris水迷宮空間搜索實(shí)驗(yàn)測試小鼠記憶能力,記錄小鼠穿臺次數(shù),在靶象限停留時間以及在靶象限游動距離。頸椎脫臼處死小鼠,分離海馬,采用NO檢測試劑盒檢測NO水平。結(jié)果REMSD組小鼠出現(xiàn)多動,毛發(fā)潮濕凌亂且光澤度差,四肢充血,煩躁不安,易激惹,興奮性增強(qiáng)。各組小鼠3輪Morris水迷宮定位航行實(shí)驗(yàn)逃避潛伏期比較,差異均有統(tǒng)計(jì)學(xué)意義(P<0.05);其中,REMSD組逃避潛伏期均較對照組、寬平臺組縮短(P<0.05)。各組小鼠穿臺次數(shù)比較,差異有統(tǒng)計(jì)學(xué)意義(P<0.05);其中,REMSD組小鼠穿臺次數(shù)多于對照組和寬平臺組(P<0.05)。對照組、寬平臺組、REMSD組海馬NO水平分別為(43.6±8.5)、(45.8±9.1)、(54.2±5.8)μmol/L,差異有統(tǒng)計(jì)學(xué)意義(F=7.460,P=0.002);其中,REMSD組小鼠海馬NO水平高于對照組和寬平臺組(P<0.05)。結(jié)論青少年期小鼠REMSD 24 h后,學(xué)習(xí)記憶能力提高,可能與海馬NO水平升高有關(guān)。
睡眠,快速眼運(yùn)動;青少年;學(xué)習(xí);記憶;一氧化氮
郝彥利,黃鑫焱,THOIDINGJAM Bidyarani Chanu,等.24小時快速眼動睡眠剝奪對青少年期C57BL/6J小鼠空間學(xué)習(xí)記憶能力和海馬一氧化氮水平的影響研究[J].中國全科醫(yī)學(xué),2016,19(23):2798-2801.[www.chinagp.net]
HAO Y L,HUANG X Y,THOIDINGJAM B C,et al.Effect of 24 h rapid eye movement sleep deprivation on the spatial learning and memory ability and nitric oxide level of hippocampus in adolescent C57BL/6J mice[J].Chinese General Practice,2016,19(23):2798-2801.
ZHANGBin,DepartmentofPsychology,NanfangHospital,SouthernMedicalUniversity,Guangzhou510445,China;E-mail:zhang73bin@hotmail.com
青少年期是由幼年期向成年期過渡的特殊時期,與成年期相比,青少年期中樞神經(jīng)系統(tǒng)突觸結(jié)構(gòu)和功能發(fā)育有其獨(dú)特的特點(diǎn),如大腦快速發(fā)育,神經(jīng)突觸過度增長,然后快速剪除[1]。人類青少年期為12~18歲,此時正處于中學(xué)階段,面臨沉重的學(xué)習(xí)壓力,導(dǎo)致青少年短暫睡眠不足廣泛存在??焖傺蹌铀?REMS)是重要的睡眠階段[2],臨床和動物研究發(fā)現(xiàn),REMS可促進(jìn)機(jī)體的學(xué)習(xí)記憶[3-4]。與成年人相比,青少年REMS頻率高、持續(xù)時間長[5],因此REMS對青少年期腦功能的影響可能更明顯。海馬與前額葉皮質(zhì)、額葉皮質(zhì)、基底核等存在豐富的神經(jīng)環(huán)路聯(lián)系,因此海馬在機(jī)體學(xué)習(xí)和記憶過程中發(fā)揮了重要作用。一氧化氮(NO)是機(jī)體最小的氣態(tài)生物信使分子,也是一種活躍的生物自由基,其作為重要的神經(jīng)遞質(zhì),參與了海馬突觸傳遞的長時程增強(qiáng)(LTP)和學(xué)習(xí)記憶形成過程[6]。然而,REMS對青少年期學(xué)習(xí)記憶能力和海馬NO水平的影響相關(guān)研究較少。本研究通過水平臺方法建立REMS剝奪(REMSD)模型,觀察比較REMSD對青少年期C57BL/6J小鼠空間學(xué)習(xí)記憶能力和海馬NO水平的影響,以探討REMS對青少年期小鼠腦功能的作用及其可能機(jī)制。
1.1實(shí)驗(yàn)動物2014年1月—2015年1月選取45只青少年期(3~4周齡)SPF級雄性C57BL/6J小鼠(來源于廣東省實(shí)驗(yàn)動物中心),許可證號SCXK(粵)2008-0002。小鼠分籠飼養(yǎng),每籠4~5只,分別于8:00、20:00室內(nèi)明暗自動切換,照明時采用40 W日光燈,自由攝取標(biāo)準(zhǔn)水和食物,保持室內(nèi)相對濕度40%~70%,溫度22~24 ℃,避免噪聲。采用隨機(jī)數(shù)字表法將小鼠分為對照組、寬平臺組和REMSD組,各15只。
1.2方法
1.2.1模型建立將REMSD組小鼠置于水平臺睡眠剝奪箱。水平臺睡眠剝奪箱為開口水箱(70 cm×50 cm×50 cm),內(nèi)含9個圓形小平臺(高10 cm,直徑1.5 cm,高于水面以上0.5 cm),平臺均間隔4 cm;小鼠可自由從一個平臺跳躍到另一個平臺來實(shí)現(xiàn)自由走動,但不能同時跨立在兩個平臺上。當(dāng)小鼠站立在小平臺上開始進(jìn)入REMS時,小鼠出現(xiàn)全身肌肉張力降低及節(jié)律性垂頭,導(dǎo)致落水后驚醒,從而建立REMSD模型。寬平臺組為REMS假剝奪組,即將小鼠置于假水平臺睡眠剝奪箱。假水平臺睡眠剝奪箱為開口水箱(70 cm×50 cm×50 cm),內(nèi)含6個圓形大平臺(高10 cm,直徑12 cm,高于水面以上0.5 cm),平臺均間隔4 cm,小鼠可自由從一個平臺跳躍到另一個平臺來實(shí)現(xiàn)自由走動,但不能同時跨立在兩個平臺上。小鼠可以在大平臺上安然進(jìn)入REMS并不會掉落入水中,從而建立REMS假剝奪模型。模型建立于12:00開始,至次日12:00結(jié)束。期間,對照組小鼠活動不受任何干擾。
1.2.2Morris水迷宮實(shí)驗(yàn)?zāi)P徒⒑?,立即對各組小鼠通過Morris水迷宮實(shí)驗(yàn)測試學(xué)習(xí)和記憶能力。Morris水迷宮定位航行實(shí)驗(yàn):將小鼠面朝Morris水迷宮內(nèi)壁并放入任意一個象限中入水點(diǎn)進(jìn)行訓(xùn)練,計(jì)時60 s,視頻分析系統(tǒng)自動記錄小鼠運(yùn)動軌跡,如果小鼠在60 s內(nèi)找到并爬上隱藏在水下的平臺停留2 s則表示上臺成功,記錄其入水到上平臺時間,即逃避潛伏期;如果60 s內(nèi)小鼠未找到平臺,則終止實(shí)驗(yàn)后引導(dǎo)其上平臺,逃避潛伏期記錄為60 s。每只小鼠在4個象限中各訓(xùn)練一次,共4次記為一輪,取4次逃避潛伏期平均值為此輪最終數(shù)據(jù)。小鼠共經(jīng)過3輪測試,每輪間隔1 h。每輪結(jié)束后,小鼠用布擦干,并放回原環(huán)境。
Morris水迷宮空間搜索實(shí)驗(yàn):于Morris水迷宮定位航行實(shí)驗(yàn)結(jié)束3 h后開始空間搜索實(shí)驗(yàn),實(shí)驗(yàn)前先撤出Morris水迷宮中的水平臺,將小鼠面朝Morris水迷宮內(nèi)壁并放入任意一個象限的入水點(diǎn),計(jì)時60 s。觀察并記錄小鼠穿臺次數(shù),在靶象限停留時間以及在靶象限游動距離。
1.2.3海馬NO水平檢測Morris水迷宮實(shí)驗(yàn)結(jié)束后立即頸椎脫臼處死小鼠,冰皿上快速分離出海馬體并稱重,在冰水浴下將海馬體置于含苯甲基磺酰氟(PMSF)的組織裂解液(400∶1)中進(jìn)行快速間斷電動勻漿(25 000 r/min)10 min,4 ℃低溫下在低溫離心機(jī)中以12 000 r/min離心5 min(離心半徑為18 cm),取其上清液置于-80 ℃超低溫冰箱待用。使用BCA蛋白測定試劑盒(武漢博士德生物工程有限公司)按說明書步驟測定上清液蛋白水平,使用NO檢測試劑盒(武漢博士德生物工程有限公司)按說明書步驟分別加入50 Griess Reagent Ⅰ和Ⅱ,在波長570 nm處采用酶標(biāo)儀測定各孔吸光度值,繪制標(biāo)準(zhǔn)曲線。根據(jù)待測樣本吸光度值和標(biāo)準(zhǔn)曲線,計(jì)算待測樣本NO水平。
2.1基本情況各組小鼠無死亡及逃逸現(xiàn)象。對照組與寬平臺組小鼠行為未見明顯異常;REMSD組小鼠出現(xiàn)多動,毛發(fā)潮濕凌亂且光澤度差,四肢充血,煩躁不安,易激惹,興奮性增強(qiáng)。
2.2Morris水迷宮定位航行實(shí)驗(yàn)各組小鼠3輪Morris水迷宮定位航行實(shí)驗(yàn)逃避潛伏期比較,差異均有統(tǒng)計(jì)學(xué)意義(P<0.05);其中,REMSD組逃避潛伏期較對照組、寬平臺組縮短,差異均有統(tǒng)計(jì)學(xué)意義(P<0.05,見表1)。
Table 1Comparison of escape latency in Morris water maze place navigation test of mice among each group
組別只數(shù)第1輪第2輪第3輪對照組1544.4±4.543.9±2.941.2±3.4寬平臺組1547.3±2.945.8±3.943.2±3.7REMSD組1537.4±3.4ab34.2±3.6ab28.2±3.0abF值5.3076.0049.441P值0.0360.0270.002
注:與對照組比較,aP<0.05;與寬平臺組比較,bP<0.05;REMSD=快速眼動睡眠剝奪
2.3Morris水迷宮空間搜索實(shí)驗(yàn)各組小鼠靶象限停留時間、游動距離及其百分比比較,差異均無統(tǒng)計(jì)學(xué)意義(P>0.05)。各組小鼠穿臺次數(shù)比較,差異有統(tǒng)計(jì)學(xué)意義(P<0.05);其中,REMSD組小鼠穿臺次數(shù)多于對照組和寬平臺組,差異均有統(tǒng)計(jì)學(xué)意義(P<0.05,見表2)。
Table 2Comparison of spatial probe test results in Morris water maze of mice among each group
組別只數(shù)穿臺次數(shù)(次)靶象限停留時間(s)靶象限停留時間百分比(%)靶象限游動距離(cm)靶象限游動距離百分比(%)對照組150.6±0.318.1±5.430.4±6.5258±6333.4±6.3寬平臺組150.6±0.117.9±4.229.5±2.8252±7831.5±3.1REMSD組151.4±0.2ab14.7±6.025.2±5.0266±8427.2±4.2F值6.3072.1741.9370.8701.189P值0.0290.3880.2940.6040.372
注:與對照組比較,aP<0.05;與寬平臺組比較,bP<0.05
2.4海馬NO水平對照組、寬平臺組、REMSD組海馬NO水平分別為(43.6±8.5)、(45.8±9.1)、(54.2±5.8)μmol/L,差異有統(tǒng)計(jì)學(xué)意義(F=7.460,P=0.002);其中,REMSD組小鼠海馬NO水平高于對照組和寬平臺組,差異均有統(tǒng)計(jì)學(xué)意義(P<0.05)。
本研究發(fā)現(xiàn),REMSD組小鼠學(xué)習(xí)記憶能力增強(qiáng),海馬NO水平升高。CLéMENT等[7]研究發(fā)現(xiàn),REMSD 72 h后,海馬、前額葉皮質(zhì)、基底前腦等與學(xué)習(xí)記憶相關(guān)的腦區(qū)內(nèi)NO水平上升。RYTK?NEN等[8]通過觸摸方法進(jìn)行REMSD 3 h后發(fā)現(xiàn),小鼠基底核NO水平升高。調(diào)控NO合成的酶主要有神經(jīng)元型一氧化氮合酶(nNOS)、誘導(dǎo)型一氧化氮合酶(iNOS)、上皮型一氧化氮合酶(eNOS)。nNOS廣泛表達(dá)于前額葉皮質(zhì)、基底核、海馬、中腦等腦區(qū)。研究發(fā)現(xiàn),nNOS在REMS啟動和維持等方面發(fā)揮重要作用[9]。同時,REMS也影響了nNOS的表達(dá)和活性。有研究通過水平臺方法進(jìn)行REMSD 24 h后發(fā)現(xiàn),大鼠大腦皮質(zhì)、海馬等區(qū)域nNOS表達(dá)和活性增高[10-11]。iNOS是一種不依賴Ca2+的一氧化氮合酶。在正常神經(jīng)系統(tǒng)中,iNOS主要表達(dá)于神經(jīng)膠質(zhì)細(xì)胞內(nèi),引導(dǎo)神經(jīng)突觸的發(fā)育和神經(jīng)遞質(zhì)的傳遞[12]。在神經(jīng)系統(tǒng)受到損傷時,iNOS在神經(jīng)膠質(zhì)細(xì)胞高表達(dá),產(chǎn)生過量NO,加重神經(jīng)元損傷。CLéMENT等[13]對老年大鼠研究發(fā)現(xiàn),iNOS表達(dá)于中顱神經(jīng)元,并且參與了REMS的發(fā)動和維持。既往研究未發(fā)現(xiàn)iNOS在正常成年大鼠神經(jīng)元內(nèi)表達(dá),但是,REMSD后的大鼠基底前顱神經(jīng)元內(nèi)出現(xiàn)iNOS表達(dá),并且隨著REMSD時間的延長,表達(dá)iNOS神經(jīng)元數(shù)量增多[8]。由此可見,REMSD可能導(dǎo)致神經(jīng)元耗氧量增多,激活nNOS和iNOS的表達(dá)和活性,上調(diào)神經(jīng)元內(nèi)NO水平。
綜上所述,短時間REMSD可能通過促進(jìn)NO合成適度增加,促進(jìn)青少年期小鼠學(xué)習(xí)記憶能力。
作者貢獻(xiàn):郝彥利進(jìn)行課題設(shè)計(jì)與實(shí)施、資料收集整理、成文并對文章負(fù)責(zé);黃鑫焱和THOIDINGJAM Bidyarani Chanu進(jìn)行課題設(shè)計(jì)與實(shí)施、評估、資料收集整理;張斌進(jìn)行質(zhì)量控制及審校。
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(本文編輯:吳立波)
Effect of 24 h Rapid Eye Movement Sleep Deprivation on the Spatial Learning and Memory Ability and Nitric Oxide Level of Hippocampus in Adolescent C57BL/6J Mice
HAOYan-li,HUANGXin-yan,THOIDINGJAMBidyaraniChanu,ZHANGBin.
DepartmentofAnatomy,GuangzhouMedicalUniversity,Guangzhou511436,China
HAOYan-li,DepartmentofAnatomy,GuangzhouMedicalUniversity,Guangzhou511436,China;E-mail:haoyanli26@126.com
ObjectiveTo explore the effect of 24 h rapid eye movement sleep deprivation(REMSD) on spatial learning and memory ability and nitric oxide(NO) level of hippocampus in adolescent C57BL/6J mice.MethodsFrom January 2014 to January 2015,45 adolescent male C57BL/6J mice(3-4 weeks old) at SPF level were selected.Random number table method was used to divide the mice into control group,wide platform(WP) group and REMSD group with 15 in each group.Mice in REMSD group were put into sleep deprivation water tank to establish REMSD model,while mice in WP group were put into artificial water tank to establish REMS fake deprivation model.Three-round place navigation of Morris water maze was applied to test the learning ability and record the escape latent period;spatial probe test of Morris water maze was used to test the memory ability of mice,record times of mice crossing platforms,standing time and the moving distance in the target quadrant.Mice were sacrificed by cervical dislocation to separate hippocampus and test NO level by using NO detection kit.ResultsMice in REMSD group presented hyperactivity,wet and messy hair with poor glossiness,limbs congestion,agitated activity,irritability and enhanced excitability.There was significant difference in the escape latent period among mice in each group in the three-round Morris maze place navigation test(P<0.05);the escape latent period of REMSD group was significantly shorter than that of control group and WP group(P<0.05).There was significant difference in the times of mice crossing platforms in each group(P<0.05);the times of mice crossing platforms in REMSD group was significantly more than that of control group and WP group(P<0.05).NO level of hippocampus of control group,WP group and REMSD group was(43.6±8.5),(45.8±9.1),and(54.2±5.8) μmol/L respectively,showing significant differences(F=7.460,P=0.002);NO level in hippocampus of mice in REMSD group was significantly higher than that of control group and WP group(P<0.05).ConclusionThe ability of learning and memorization of mice in adolescent period has improved after 24 h REMSD,and this may be related with the increasing NO level of hippocampus.
Sleep,rapid eye movement;Adolescent;Learning;Memory;Nitric oxide
廣東省科技廳社會發(fā)展領(lǐng)域科技計(jì)劃項(xiàng)目(2013B021800045,2014A020212563)
511436廣東省廣州市,廣州醫(yī)科大學(xué)人體解剖教研室(郝彥利),留學(xué)生學(xué)院(THOIDINGJAM Bidyarani Chanu);廣州醫(yī)科大學(xué)附屬第六醫(yī)院神經(jīng)內(nèi)科(黃鑫焱);南方醫(yī)科大學(xué)南方醫(yī)院心理科(張斌)
郝彥利,511436廣東省廣州市,廣州醫(yī)科大學(xué)人體解剖教研室;E-mail:haoyanli26@126.com
張斌,510445廣東省廣州市,南方醫(yī)科大學(xué)南方醫(yī)院心理科;E-mail:zhang73bin@hotmail.com
R 339.141
A
10.3969/j.issn.1007-9572.2016.23.012
2016-03-17;
2016-06-20)