MCAO神經(jīng)行為學評價(總12頁

1、MCAO造模后行為學評定小結(jié)MCAO后大鼠行為學評定方法比較在對MCAO后大鼠進行行為學評定(behavior test)的十分重要，對其評定的方法也頗多，但究竟那種方法更適用，目前未有人進行過比較。為了合理選擇MCAO后的評定方法，如下對目前常用的幾種行為學評定方法進行比較。行為學檢查方法:由3位參加試驗的人員分別以單盲法對試驗的大鼠進行打分和記錄.然后將3組的記分結(jié)果進行平均后的得分進行統(tǒng)計計算。（國內(nèi)）由一個對試驗實施過程不了解的觀察者對大鼠進行行為學評測。評測續(xù)貫進行。如果大鼠在一次評測中出現(xiàn)恰當?shù)男袨?，而以后卻未出現(xiàn)，按前者記分。(國外)1.神經(jīng)行為學檢查 Longa評分法2Berd

2、erson評分法 姿勢反射測驗（postural reflex test）3. 攀繩實驗4網(wǎng)屏測驗（screen test）5肢體放置測驗（limb-placement test）Elicited Forelimb Placing6開野試驗（OpenField法）測定行為7MNSS8.轉(zhuǎn)動桿測驗（rotating pole test）9Rotation10肢體對稱試驗評分法11. rotarod test12. adhesive-removal somatosensory test13Spontaneous Activity14Symmetry in the Movement of Four

3、Limbs15Forepaw Outstretching16Climbing17Body Proprioception18. Response to Vibrissae Touch改善記憶作用1.跳臺試驗2.避暗試驗3.穿梭箱試驗4.水迷宮試驗Motor behavior(1) observation of spontaneous ipsilateral circling,(2) contralateral hindlimb retraction,(3) beam walking ability,平衡木測驗（balance beam test）(4) bilateral forepaw gra

4、sp,Skilled forelimb function(1)staircase feeding apparatusnociception(1) plantar test1神經(jīng)行為學檢查:Longa評分法神經(jīng)學檢查分5個等級：0分：正常，無神經(jīng)功能缺損；1分：左側(cè)前爪不能完全伸展，輕度神經(jīng)功能缺損；2分：行走時，大鼠向左側(cè)（癱瘓側(cè)）轉(zhuǎn)圈，中度神經(jīng)功能缺損；3分：行走時，大鼠身體向左側(cè)（癱瘓側(cè)）傾倒。重度神經(jīng)功能缺損；4分：不能自發(fā)行走，有意識喪失。2Berderson評分法 姿勢反射測驗（postural reflex test） 提尾懸空試驗大鼠提尾懸空一米，觀察前肢屈曲情況。正常大鼠向地面

5、伸展雙上肢?？缮煺闺p上肢向地面并無其它神經(jīng)缺陷大鼠得0分。梗死后大鼠持續(xù)屈曲損傷半球?qū)y患肢；各種不同姿勢，從輕度腕屈曲、肩內(nèi)收、肘伸展到嚴重姿勢，完全腕、肘屈曲，肩內(nèi)旋轉(zhuǎn)并內(nèi)收。有任何持續(xù)前肢屈曲和其它不正常得1分。大鼠被放于大的軟墊子上，表面有大鼠可用爪子牢牢抓住的塑料壓膜紙。用手抓其尾巴，緩慢在鼠肩后給予側(cè)壓直至前肢滑動幾英寸。此手法在每個方向重復幾次。正常或輕度功能損傷大鼠兩側(cè)抵抗滑動力相同。嚴重功能損傷大鼠癱瘓側(cè)始終抵抗力下降，得2分。然后讓大鼠自由活動，觀察轉(zhuǎn)圈行為。大鼠持續(xù)向癱瘓側(cè)轉(zhuǎn)圈得3分。前肢屈曲帶有側(cè)推抵抗力下降經(jīng)常見到；雙上肢屈曲并側(cè)推抵抗力下降大鼠經(jīng)常有轉(zhuǎn)圈行為。神經(jīng)檢

6、查在35分鐘內(nèi)完成。正常0分未見行為缺陷中度1分前肢屈曲(即提尾懸空實驗陽性)重度2分側(cè)推抵抗力下降(即側(cè)向推力實驗陽性).伴前肢屈曲.無轉(zhuǎn)圈行為3分同2級行為.伴自發(fā)性旋轉(zhuǎn)。提起鼠尾.使其離地0. 33 m高.觀察前肢情況。正常大鼠兩前肢對稱向前伸開;如果有肩部內(nèi)旋、前肢內(nèi)收現(xiàn)象發(fā)生者.根據(jù)其嚴重程度.最高評分為4分.正常評分為0分。將動物置于平滑地板上.分別推其左、右肩向?qū)?cè)移動，此時檢查動物抵抗推動時的阻力，正常大鼠兩側(cè)推動的阻力明顯對稱。如一側(cè)阻力下降者.可根據(jù)下降程度評13分.正常評分為0分。觀察兩前肢肌力。將動物兩前肢置于一金屬網(wǎng)上.然后輕提起大鼠.檢查兩前肢肌力。正常左右對稱.如

7、有一側(cè)前肢肌力下降者.根據(jù)下降程度評為13分.正常評分為0分。根據(jù)上述行為檢查的情況.積分值為0一10分。積分越高說明動物行為障礙越嚴重。此測驗對皮層和紋狀體的損傷較敏感。3攀繩實驗:本實驗主要檢查大鼠雙前肢和雙后肢的協(xié)調(diào)配合能力。取直徑1. 5 cm的麻繩從高1 m、長和寬分別為15、50 cm的平臺上垂下，手術前1d訓練每只大鼠.使之能夠爬到繩子的上1/ 3。每個測試日測試3次。評分的標準如下:0分.不需任何刺激在10s內(nèi)爬上平臺;1分.不需任何刺激在15 s內(nèi)爬上平臺;2分.刺激次數(shù)小于5次.在30 s內(nèi)爬上平臺;3分.刺激次數(shù)小于5次.在60 s內(nèi)爬上平臺;4分.刺激次數(shù)大于5次.在6

8、0 s內(nèi)爬上平臺或是刺激小于5次.但爬上平臺時間超過60 s; 5分.大鼠不能爬上平臺。4網(wǎng)屏測驗（screen test）網(wǎng)屏訓練:網(wǎng)屏為50cm X 40cm網(wǎng)帶.網(wǎng)眼為lcm X lcm.網(wǎng)板的左右和上方都用25cm高的木板框邊.網(wǎng)屏距地面高度為80cm.下方鋪以12cm厚的海綿。先將網(wǎng)屏水平放置.將老鼠放在其上.然后緩緩地將其一端抬高.在2s內(nèi)將此屏風變成垂直位.保持5s.觀察大鼠是否會從網(wǎng)屏上下來或用前爪抓握住網(wǎng)屏，從而評價前爪抓握能力及肌力情況。網(wǎng)屏實驗:評分標準分為4個等級。0分:前爪握住網(wǎng)屏達5s之久，不會掉下來;1分:暫時握住網(wǎng)屏，滑落一段距離，但沒有掉下來;2分:在5s內(nèi)掉

9、下來;3分:網(wǎng)屏轉(zhuǎn)動時，鼠即刻掉下來。5肢體放置測驗（limb-placement test）Elicited Forelimb Placing肢體放置于三種獨立刺激（視覺、觸覺、本體感覺）以評測運動感覺完整性。參照等2采用的動物行為測試評分方法。具體實驗有:前肢放置實驗,包括(1)視覺亞實驗,即前方刺激實驗者將動物握于手中,使其前爪懸空,自桌面上方10處向桌面緩慢斜線靠近(此時桌子位于大鼠前方),大鼠正常反應為前肢即刻抓向桌面,損傷大鼠則表現(xiàn)為肢體反應延遲。0分動物肢體放置反應正常;1分反應延遲但不超過2;2分反應延遲且超過2。側(cè)方刺激,此時桌子位于動物側(cè)方,其余實驗方法及評分標準同前方刺激

10、。(2)觸覺亞實驗,將動物置于桶狀凹陷實驗器內(nèi),使其前爪懸空,此時大鼠應該既看不見,也不能用胡須觸及桌面,用其前爪背側(cè)輕觸桌面,刺激深度僅達皮膚和毛發(fā),動物反應及評分同視覺亞實驗,觸覺刺激同樣分前方及側(cè)方刺激。(3)本體覺亞實驗,操作及評分同觸覺亞實驗,僅刺激深度不同,本體覺亞實驗給予前爪較大壓力,刺激深達肌肉及關節(jié)。該亞實驗只有前方刺激。動物前肢放置實驗總分范圍為010分,功能損傷越重,得分越高。6開野試驗（OpenField法）測定行為方法：本實驗所用敞箱為正方形,長、寬各80cm,高40cm,底面由面積相等邊長16cm的正方形25塊組成,以動物穿越底面的塊數(shù)為水平活動(crossing)

11、得分,以直立次數(shù)為垂直活動(rearing)得分,每只動物共進行7次測定,每次3minOpen2Field法是測定抑郁癥大鼠行為學的經(jīng)典方法,分水平運動和垂直運動2個觀察面,以觀察得分為評分計算單位，記各組大鼠的水平運動評分及垂直運動評分。Elevated open field activity is characteristic of hippocampally damaged animals19 20 21 22 and has been observed in animals with striatal/cortical damage as well.23 Rats are placed

12、 in a novel environment (3x3 ft) with 3-inch-square grid markings on the base. The number of times a rat crosses a grid line is recorded for a 5-minute period.7肢體對稱試驗評分法將大鼠置于網(wǎng)中，網(wǎng)眼大小為2.3 x2.3cm,當大鼠在網(wǎng)絡中行走時，計數(shù)在2分鐘內(nèi)其前爪漏到網(wǎng)眼中的次數(shù)。計算公式為：（腦病變對側(cè)前爪的錯步數(shù)病變同側(cè)前爪的錯步數(shù)）/總步數(shù)。其分值如為正數(shù)表明腦病變對側(cè)功能缺損；如為負數(shù)表明腦病變同側(cè)功能缺損。8mNSSmNS

13、S體系的設計原理,評分越低表示功能越健全，0分表示功能完全健全的大鼠。modified Neurological Severity Scores見附件19.轉(zhuǎn)動桿測驗（rotating pole test）轉(zhuǎn)棒上行走測評:評分標準分為4個等級。0分:轉(zhuǎn)動過程中.鼠可在棒上行走;1分:轉(zhuǎn)動過程中.60s以上時間.鼠不會掉下來;2分:轉(zhuǎn)動開始后.鼠從棒上掉下來;3分:轉(zhuǎn)動開始前.鼠從棒上掉下來。10The detection was operated at 1 week after surgery by injecting 0. 5 mg/kg of apomorphine into abdomi

14、nal cavity for observation of the changes in The behaviours for once a week and continuous detected for 6 weeks. If constant right-side rotation occurred and The numbers of The circles rotated were more than 210 r per 30 minutes, those rats were considered as successful models: if no rotation occurr

15、ed or The direction of rotation was not constant or constant right-side or lell-side rotation but The speed was slower than 210 r per 30 minutes, those animals were considered as unsuccessful models. In additional to rotation, other companioned abnormal behaviours also should be observed, such as, t

16、remor, sluggish, grab, and smell exploring, etc.11. rotarod testrats were placed on an accelerating rotarod cylinder, and the time the animals remained on the rotarod was measured. The speed was slowly increased from 4 to 40 rpm within 5 minutes. A trial ended if the animal fell off the rungs or gri

17、pped the device and spun around for 2 consecutive revolutions without attempting to walk on the rungs. The animals were trained 3 days before MCAO. The mean duration (in seconds) on the device was recorded with 3 rotarod measurements 1 day before surgery. Motor test data are presented as percentage

18、of mean duration (3 trials) on the rotarod compared with the internal baseline control (before surgery).12. adhesive-removal somatosensory testsomatosensory deficit was measured both before and after surgery. All rats were familiarized with the testing environment. In the initial test, 2 small piece

19、s of adhesive-backed paper dots (of equal size, 113.1 mm2) were used as bilateral tactile stimuli occupying the distal-radial region on the wrist of each forelimb. The rat was then returned to its cage. The time to remove each stimulus from forelimbs was recorded on 5 trials per day. Individual tria

20、ls were separated by at least 5 minutes. Before surgery, the animals were trained for 3 days. Once the rats were able to remove the dots within 10 seconds, they were subjected to MCAO.13Spontaneous ActivityThe animal was observed for 5 minutes in its normal environment (cage). The rats activity was

21、assessed by its ability to approach all four walls of the cage. Scores indicate the following: 3, rat moved around, explored the environment, and approached at least three walls of the cage; 2, slightly affected rat moved about in the cage but did not approach all sides and hesitated to move, althou

22、gh it eventually reached at least one upper rim of the cage; 1, severely affected rat did not rise up at all and barely moved in the cage; and 0, rat did not move at all.14Symmetry in the Movement of Four LimbsThe rat was held in the air by the tail to observe symmetry in the movement of the four li

23、mbs. Scores indicate the following: 3, all four limbs extended symmetrically; 2, limbs on left side extended less or more slowly than those on the right; 1, limbs on left side showed minimal movement; and 0, forelimb on left side did not move at all.Julio H. Garcia, MD; Simone Wagner, MD; Kai-Feng L

24、iu, MD; Xiao-jiang Hu, MD, PhD。Neurological Deficit and Extent of Neuronal Necrosis Attributable to Middle Cerebral Artery Occlusion in Rats。Stroke. 1995;26:627-635。15Forepaw OutstretchingThe rat was brought up to the edge of the table and made to walk on forelimbs while being held by the tail. Symm

25、etry in the outstretching of both forelimbs was observed while the rat reached the table and the hindlimbs were kept in the air. Scores indicate the following: 3, both forelimbs were outstretched, and the rat walked symmetrically on forepaws; 2, left side outstretched less than the right, and forepa

26、w walking was impaired; 1, left forelimb moved minimally; and 0, left forelimb did not move.Julio H. Garcia, MD; Simone Wagner, MD; Kai-Feng Liu, MD; Xiao-jiang Hu, MD, PhD。Neurological Deficit and Extent of Neuronal Necrosis Attributable to Middle Cerebral Artery Occlusion in Rats。Stroke. 1995;26:6

27、27-635。16ClimbingThe rat was placed on the wall of a wire cage. Normally the rat uses all four limbs to climb up the wall. When the rat was removed from the wire cage by pulling it off by the tail, the strength of attachment was noted. Scores indicate the following: 3, rat climbed easily and gripped

28、 tightly to the wire; 2, left side was impaired while climbing or did not grip as hard as the right side; and 1, rat failed to climb or tended to circle instead of climbing17. Body ProprioceptionThe rat was touched with a blunt stick on each side of the body, and the reaction to the stimulus was obs

29、erved. Scores indicate the following: 3, rat reacted by turning head and was equally startled by the stimulus on both sides; 2, rat reacted slowly to stimulus on left side; and 1, rat did not respond to the stimulus placed on the left side.18. Response to Vibrissae TouchA blunt stick was brushed aga

30、inst the vibrissae on each side; the stick was moved toward the whiskers from the rear of the animal to avoid entering the visual fields. Scores indicate the following: 3, rat reacted by turning head or was equally startled by the stimulus on both sides; 2, rat reacted slowly to stimulus on left sid

31、e; and 1, rat did not respond to stimulus on the left side.改善記憶作用1跳臺試驗跳臺裝置為lOcm X 15cm X 60cm的被動條件反射箱，四周用黑色塑料板、底面鋪可通電的銅柵，每間內(nèi)放置一高4 .5cm，直徑4.5cm的橡皮墊作為動物回避電擊的安全區(qū)，先將動物放在反應箱內(nèi)的橡皮臺上適應3min，然后立即通以40V交流電，動物受到電擊后逃避反應為跳上橡皮臺。記錄5min內(nèi)大鼠受到電擊次數(shù)，稱為錯誤次數(shù)。24h后在底部銅柵通電情況下，直接將動物置于平臺上，記錄第1次跳下的潛伏期和5 min內(nèi)的錯誤次數(shù)。2避暗實驗 Avoidance

32、 response根據(jù)大鼠有嗜暗習性分設明暗兩室。明室上方20cm處懸一個60 W日光燈，在暗室的中后部分底部銅柵加40 V電壓，兩室之間設有一直徑為3cm大小的圓洞。實驗時將大鼠背對洞口放入明室，同時啟動計時表，動物自放入明室至進入暗室遭電擊所需時間為潛伏期，鼠受電擊后可自行逃出暗室。記錄潛伏期及5min內(nèi)動物受電擊次數(shù)，24h后復測。3.穿梭箱試驗4水迷宮試驗（Morris ）4.1 Morris水迷宮為圓形水池.直徑100 cm.深50 cm.水深30 cm.水溫(24士2).水而覆一層塑料泡沫屑.池壁上4個等距離點分水池為4個象限.在第三象限中央放置平臺.平臺無色透明.直徑6 cm.高

33、28 cm.平臺沒于水而下2 cm.水池周圍參照物保持不變。試驗包括:定位航行試驗(place navigation test):實驗歷時7d。將大鼠從入水面向池壁置入水槽中.記錄大鼠從入水到爬上平臺所需的時間.即逃避潛伏期。每只大鼠每人從4個不同象限入水點入水進行訓練各1次.取其平均值為其每人逃避潛伏期。訓練中.若大鼠在60 s內(nèi)找到平臺.讓其于平臺上站立lOs;若未找到.用棒將其引上平臺.并讓其站立10 s.潛伏期限記為60 s。將大鼠從平臺上拿卜來休息60 s之后.再按序由下一入水點入水進行下一次實驗。空間探索試驗(spatial probe test)第8天撤去平臺.將大鼠從第二象限入

34、水點放入水槽.記錄60 s內(nèi)其在平臺象限的滯留時間。4.2 水迷宮試驗主要用于測試大鼠對空間位置覺和方位覺的學習記憶能力5水迷宮試驗測試的一個重要指標是大鼠的平均逃避潛伏期平均逃避潛伏期指的是大鼠在放入迷宮后學會在5中內(nèi)找到平臺所需要的平均時間,主要與海馬功能有關,與大腦皮層也有一定的關系。Morris Water MazeThis is a task that measures learning and memory in an aqueous environment. Briefly, the rat was placed in a 4-ft-diameter water tank tha

35、t is visually separated into four quadrants. In the center of one quadrant, the goal quadrant, a platform is hidden 1 inch below the waterline. The rat was placed in the tank at one of four start locations (north, south, east, west) and swam for up to 60 seconds until it found the platform. Once the

36、 animal located the platform, it remained on it for 20 seconds. In the event that an animal did not locate the platform, the observer positioned the animal on the platform at the end of the 60-second swim period. During a 2- to 4-minute intertrial interval, the animals were kept warm by infrared hea

37、ting lamps. The temperature of the water maze was 200.5C. The latency and path length of the rat were measured by a video tracking system interfaced to a computer.Nancy E. Alexis, MS; W. Dalton Dietrich, PhD; Edward J. Green, PhD; Ricardo Prado, MD; Brant D. Watson, PhD. Nonocclusive Common Carotid

38、Artery Thrombosis in the Rat Results in Reversible Sensorimotor and Cognitive Behavioral Deficits. Stroke. 1995;26:2338-2346.4.3 Y迷宮學習記憶訓練及測試:Y型電迷宮臂長30cm.高15cm.寬15cm。每臂末端有15W信號燈示安全區(qū).箱底銅柵間隔4 mm。電流強度0. 7 mA.選用電壓38 V.電擊延時2s。設一臂為起步區(qū).按I- II- III-I臂順序輪流作為安全區(qū)。大鼠在起步區(qū)靜置3 m in.予以電擊致其逃至安全區(qū).燈光持續(xù)15 s.然后熄燈體急45 s.

39、開始下一次操作。學習訓練及測試:規(guī)定大鼠受電擊后從起步區(qū)直接逃至安全區(qū)為“正確反應”.以達到連續(xù)10次電擊均為正確反應(即10/10)前所需的電擊次數(shù)(即嘗試次數(shù))表示其學習獲得能力(acquisition)。若嘗試次數(shù)超過30次則不再測試.并以30次為最大值計數(shù)。記憶再現(xiàn)(retrieval)測試:選上述達到10/ 10標準的大鼠.休息24 h后再次同法測試.以達10/10標準前的嘗試次數(shù)表示記憶再現(xiàn)能力。Motor behavior(1) observation of spontaneous ipsilateral circling, graded from 0 (no circling)

40、 to 3 (continuous circling);(2) contralateral hindlimb retraction, which measured the ability of the animal to replace the hindlimb after it was displaced laterally by 2 to 3 cm, graded from 0 (immediate replacement) to 3 (replacement after minutes or no replacement);(3) beam walking ability, graded

41、 0 for a rat that readily traverses a 2.4-cm-wide, 80-cm-long beam to 3 for a rat unable to stay on the beam for 10 seconds;平衡木測驗（balance beam test）平衡木長80cm，寬2. 5 cm的方木棒，平放在距離地面l0cm處，讓大鼠在其上行走。評分標準為6.個等級。0分:能跳上平衡木，在上面行走不會跌倒;T分:能跳上平衡木，在上面行走跌倒機會小于50；2分：能跳上平衡木，在上面行走跌倒機會大于50；3分：在健側(cè)后肢幫助下能跳上平衡木，但受累癱瘓側(cè)后肢不能幫

42、助向前移動；4分：在平衡木上不能行走，但可坐在上面；5分：將大鼠放在平衡木上會掉下來。Beam balance performance of animals was assessed on a 6-point scale17: 0=balances with steady posture; 1=grasps side of beam; 2=hugs beam and 1 limb, falls down from beam; 3=hugs beam and 2 limbs, falls down from beam, or spins on beam 60 seconds; 4=attempts to balance on beam b