各种神经行为学评价

大鼠行为学实验评价汇总

大鼠行为学评定方法比较

大鼠进行行为学评定(behavior test)的十分重要，对其评定的方法也颇多，但究竟那种方法更适用，目前未有人进行过比较。为了合理选择MCAO后的评定方法，如下对目前常用的几种行为学评定方法进行比较。

行为学检查方法:由3位参加试验的人员分别以单盲法对试验的大鼠进行打分和记录.然后将3组的记分结果进行平均后的得分进行统计计算。（国内）

由一个对试验实施过程不了解的观察者对大鼠进行行为学评测。评测续贯进行。如果大鼠在一次评测中出现恰当的行为，而以后却未出现，按前者记分。(国外) 1. 神经行为学检查 Longa评分法

2．Berderson评分法 姿势反射测验（postural reflex test） 3. 攀绳实验

4．网屏测验（screen test）

5．肢体放置测验（limb-placement test）Elicited Forelimb Placing 6．开野试验（Open－Field法）测定行为 7．MNSS

8.转动杆测验（rotating pole test） 9．Rotation

10．肢体对称试验评分法 11. rotarod test

12. adhesive-removal somatosensory test 13．Spontaneous Activity

14．Symmetry in the Movement of Four Limbs 15．Forepaw Outstretching 16．Climbing

17．Body Proprioception

18. Response to Vibrissae Touch 改善记忆作用 1.跳台试验 2.避暗试验 3.穿梭箱试验 4.水迷宫试验

Motor behavior

(1) observation of spontaneous ipsilateral circling, (2) contralateral hindlimb retraction,

(3) beam walking ability,平衡木测验（balance beam test） (4) bilateral forepaw grasp, Skilled forelimb function (1)staircase feeding apparatus nociception (1) plantar test

1．神经行为学检查: Longa评分法

神经学检查分5个等级：

0分：正常，无神经功能缺损；

1分：左侧前爪不能完全伸展，轻度神经功能缺损；

2分：行走时，大鼠向左侧（瘫痪侧）转圈，中度神经功能缺损； 3分：行走时，大鼠身体向左侧（瘫痪侧）倾倒。重度神经功能缺损； 4分：不能自发行走，有意识丧失。

2．Berderson评分法 姿势反射测验（postural reflex test） 提尾悬空试验

大鼠提尾悬空一米，观察前肢屈曲情况。正常大鼠向地面伸展双上肢。可伸展双上肢向地面并无其它神经缺陷大鼠得0分。梗死后大鼠持续屈曲损伤半球对测患肢；各种不同姿势，从轻度腕屈曲、肩内收、肘伸展到严重姿势，完全腕、肘屈曲，肩内旋转并内收。有任何持续前肢屈曲和其它不正常得1分。大鼠被放于大的软垫子上，表面有大鼠可用爪子牢牢抓住的塑料压膜纸。用手抓其尾巴，缓慢在鼠肩后给予侧压直至前肢滑动几英寸。此手法在每个方向重复几次。正常或轻度功能损伤大鼠两侧抵抗滑动力相同。严重功能损伤大鼠瘫痪侧始终抵抗力下降，得2分。然后让大鼠自由活动，观察转圈行为。大鼠持续向瘫痪侧转圈得3分。前肢屈曲带有侧推抵抗力下降经常见到；双上肢屈曲并侧推抵抗力下降大鼠经常有转圈行为。神经检查在3－5分钟内完成。 正常 0分 未见行为缺陷

中度 1分 前肢屈曲(即提尾悬空实验阳性)

重度 2分 侧推抵抗力下降(即侧向推力实验阳性).伴前肢屈曲.无转圈行为 3分 同2级行为.伴自发性旋转。

提起鼠尾.使其离地0. 33 m高.观察前肢情况。正常大鼠两前肢对称向前伸开;如果有肩部内旋、前肢内收现象发生者.根据其严重程度.最高评分为4分.正常评分为0分。将动物置于平滑地板上.分别推其左、右肩向对侧移动，此时检查动物抵抗推动时的阻力，正常大鼠两侧推动的阻力明显对称。如一侧阻力下降者.可根据下降程度评1－3分.正常评分为0分。观察两前肢肌力。将动物两前肢置于一金属网上.然后轻提起大鼠.检查两前肢肌力。正常左右对称.如有一侧前肢肌力下降者.根据下降程度评为1－3分.正常评分为0分。根据上述行为检查的情况.积分值为0一10分。积分越高说明动物行为障碍越严重。 此测验对皮层和纹状体的损伤较敏感。

3攀绳实验:

本实验主要检查大鼠双前肢和双后肢的协调配合能力。取直径1. 5 cm的麻绳从高1 m、长

和宽分别为15、50 cm的平台上垂下，手术前1d训练每只大鼠.使之能够爬到绳子的上1/ 3。每个测试日测试3次。评分的标准如下:0分.不需任何刺激在10s内爬上平台;1分.不需任何刺激在15 s内爬上平台;2分.刺激次数小于5次.在30 s内爬上平台;3分.刺激次数小于5次.在60 s内爬上平台;4分.刺激次数大于5次.在60 s内爬上平台或是刺激小于5次.但爬上平台时间超过60 s; 5分.大鼠不能爬上平台。

4．网屏测验（screen test）

网屏训练:网屏为50cm X 40cm网带.网眼为lcm X lcm.网板的左右和上方都用25cm高的木板框边.网屏距地面高度为80cm.下方铺以12cm厚的海绵。先将网屏水平放置.将老鼠放在其上.然后缓缓地将其一端抬高.在2s内将此屏风变成垂直位.保持5s.观察大鼠是否会从网屏上下来或用前爪抓握住网屏，从而评价前爪抓握能力及肌力情况。 网屏实验:评分标准分为4个等级。0分:前爪握住网屏达5s之久，不会掉下来;1分:暂时握住网屏，滑落一段距离，但没有掉下来;2分:在5s内掉下来;3分:网屏转动时，鼠即刻掉下来。

5．肢体放置测验（limb-placement test）Elicited Forelimb Placing

肢体放置于三种独立刺激（视觉、触觉、本体感觉）以评测运动感觉完整性。参照Ｋａｗａｍａｔａ等[2]采用的动物行为测试评分方法。具体实验有:前肢放置实验,包括(1)视觉亚实验,即前方刺激———实验者将动物握于手中,使其前爪悬空,自桌面上方10ｃｍ处向桌面缓慢斜线靠近(此时桌子位于大鼠前方),大鼠正常反应为前肢即刻抓向桌面,损伤大鼠则表现为肢体反应延迟。0分———动物肢体放置反应正常;1分———反应延迟但不超过2ｓ;2分———反应延迟且超过2ｓ。侧方刺激,此时桌子位于动物侧方,其余实验方法及评分标准同前方刺激。(2)触觉亚实验,将动物置于桶状凹陷实验器内,使其前爪悬空,此时大鼠应该既看不见,也不能用胡须触及桌面,用其前爪背侧轻触桌面,刺激深度仅达皮肤和毛发,动物反应及评分同视觉亚实验,触觉刺激同样分前方及侧方刺激。(3)本体觉亚实验,操作及评分同触觉亚实验,仅刺激深度不同,本体觉亚实验给予前爪较大压力,刺激深达肌肉及关节。该亚实验只有前方刺激。动物前肢放置实验总分范围为0～10分,功能损伤越重,得分越高。 6．开野试验（Open－Field法）测定行为

方法：本实验所用敞箱为正方形,长、宽各80cm,高40cm,底面由面积相等边长16cm的正方形25块组成,以动物穿越底面的块数为水平活动(crossing)得分,以直立次数为垂直活动(rearing)得分,每只动物共进行7次测定,每次3min

Open2Field法是测定抑郁症大鼠行为学的经典方法,分水平运动和垂直运动2个观察面,以观察得分为评分计算单位，记各组大鼠的水平运动评分及垂直运动评分。

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 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．肢体对称试验评分法 将大鼠置于网中，网眼大小为2.3 x2.3cm,当大鼠在网络中行走时，计数在2分钟内其前爪漏到网眼中的次数。计算公式为：（脑病变对侧前爪的错步数－病变同侧前爪的错步数）/总步数。其分值如为正数表明脑病变对侧功能缺损；如为负数表明脑病变同侧功能缺损。 8．mNSS

mNSS体系的设计原理,评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

见附件1

9.转动杆测验（rotating pole test）

转棒上行走测评:评分标准分为4个等级。0分:转动过程中.鼠可在棒上行走;1分:转动过程中.60s以上时间.鼠不会掉下来;2分:转动开始后.鼠从棒上掉下来;3分:转动开始前.鼠从棒上掉下来。

10．The detection was operated at 1 week after surgery by injecting 0. 5 mg/kg of apomorphine into abdominal 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 occurred 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, tremor, sluggish, grab, and smell exploring, etc. 11. rotarod test

rats 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 gripped 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 of mean duration (3 trials) on the rotarod compared with the internal baseline control (before surgery). 12. adhesive-removal somatosensory test

somatosensory deficit was measured both before and after surgery. All rats were familiarized with the testing environment. In the initial test, 2 small pieces 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 trials 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. 13．Spontaneous Activity

The animal was observed for 5 minutes in its normal environment (cage). The rat's activity was 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, although 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. 14．Symmetry in the Movement of Four Limbs

The rat was held in the air by the tail to observe symmetry in the movement of the four limbs. 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 Liu, MD; Xiao-jiang Hu, MD, PhD。Neurological Deficit and Extent of Neuronal Necrosis Attributable to Middle Cerebral Artery Occlusion in Rats。Stroke. 1995;26:627-635。

15．Forepaw Outstretching

The rat was brought up to the edge of the table and made to walk on forelimbs while being held by the tail. Symmetry 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 forepaw 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;26:627-635。 16．Climbing

The 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 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 climbing 17. Body Proprioception

The rat was touched with a blunt stick on each side of the body, and the reaction to the stimulus was observed. 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 Touch

A blunt stick was brushed against 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 side; and 1, rat did not respond to stimulus on the left side. 改善记忆作用 1．跳台试验

跳台装置为lOcm X 15cm X 60cm的被动条件反射箱，四周用黑色塑料板、底面铺可通电的铜栅，每间内放置一高4 .5cm，直径4.5cm的橡皮垫作为动物回避电击的安全区，先将动物放在反应箱内的橡皮台上适应3min，然后立即通以40V交流电，动物受到电击后逃避反应为跳上橡皮台。记录5min内大鼠受到电击次数，称为错误次数。24h后在底部铜栅通电情况下，直接将动物置于平台上，记录第1次跳下的潜伏期和5 min内的错误次数。 2．避暗实验 Avoidance response

根据大鼠有嗜暗习性分设明暗两室。明室上方20cm处悬一个60 W日光灯，在暗室的中后部分底部铜栅加40 V电压，两室之间设有一直径为3cm大小的圆洞。实验时将大鼠背对洞口放入明室，同时启动计时表，动物自放入明室至进入暗室遭电击所需时间为潜伏期，鼠受电击后可自行逃出暗室。记录潜伏期及5min内动物受电击次数，24h后复测。 3.穿梭箱试验

4．水迷宫试验（Morris ）

4.1 Morris水迷宫为圆形水池.直径100 cm.深50 cm.水深30 cm.水温(24士2)℃.水而覆一层塑料泡沫屑.池壁上4个等距离点分水池为4个象限.在第三象限中央放置平台.平台无色透明.直径6 cm.高28 cm.平台没于水而下2 cm.水池周围参照物保持不变。

试验包括:①定位航行试验(place navigation test):实验历时7d。将大鼠从入水面向池壁置入水

槽中.记录大鼠从入水到爬上平台所需的时间.即逃避潜伏期。每只大鼠每人从4个不同象限入水点入水进行训练各1次.取其平均值为其每人逃避潜伏期。训练中.若大鼠在60 s内找到平台.让其于平台上站立lOs;若未找到.用棒将其引上平台.并让其站立10 s.潜伏期限记为60 s。将大鼠从平台上拿卜来休息60 s之后.再按序由下一入水点入水进行下一次实验。②空间探索试验(spatial probe test)第8天撤去平台.将大鼠从第二象限入水点放入水槽.记录60 s内其在平台象限的滞留时间。

4.2 水迷宫试验主要用于测试大鼠对空间位置觉和方位觉的学习记忆能力[5]\水迷宫试验测试的一个重要指标是大鼠的平均逃避潜伏期\平均逃避潜伏期指的是大鼠在放入迷宫后学会在5中内找到平台所需要的平均时间,主要与海马功能有关,与大脑皮层也有一定的关系。 Morris Water Maze

This is a task that measures learning and memory in an aqueous environment. Briefly, the rat was placed in a 4-ft-diameter water tank that 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 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 heating lamps. The temperature of the water maze was 20±0.5°C. 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 Artery Thrombosis in the Rat Results in Reversible Sensorimotor and Cognitive Behavioral Deficits. Stroke. 1995;26:2338-2346. 4.3 Y迷宫学习记忆训练及测试:Y型电迷宫臂长30cm.高15cm.宽15cm。每臂末端有15W信号灯示安全区.箱底铜栅间隔4 mm。电流强度0. 7 mA.选用电压38 V.电击延时2s。设一臂为起步区.按I- II- III-I臂顺序轮流作为安全区。大鼠在起步区静置3 m in.予以电击致其逃至安全区.灯光持续15 s.然后熄灯体急45 s.开始下一次操作。

学习训练及测试:规定大鼠受电击后从起步区直接逃至安全区为“正确反应”.以达到连续10次电击均为正确反应(即10/10)前所需的电击次数(即尝试次数)表示其学习获得能力(acquisition)。若尝试次数超过30次则不再测试.并以30次为最大值计数。

记忆再现(retrieval)测试:选上述达到10/ 10标准的大鼠.休息24 h后再次同法测试.以达10/10标准前的尝试次数表示记忆再现能力。 Motor behavior

(1) observation of spontaneous ipsilateral circling, graded from 0 (no circling) 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 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; 平衡木测验（balance beam test）

平衡木长80cm，宽2. 5 cm的方木棒，平放在距离地面l0cm处，让大鼠在其上行走。评分标准为6.个等级。0分:能跳上平衡木，在上面行走不会跌倒;T分:能跳上平衡木，在上面行走跌倒机会小于50％；2分：能跳上平衡木，在上面行走跌倒机会大于50％；3分：在健侧

后肢帮助下能跳上平衡木，但受累瘫痪侧后肢不能帮助向前移动；4分：在平衡木上不能行走，但可坐在上面；5分：将大鼠放在平衡木上会掉下来。

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 but falls off >40 seconds; 5=attempts to balance on beam but falls off >20 seconds; and 6=falls off, no attempt to balance or hang onto beam <20 seconds

This is a test of sensorimotor integration specifically examining hindlimb function. The testing apparatus and rating procedures were adapted from Feeney and colleagues.16 A 1-inch-wide beam, 4 ft in length, is suspended 3 ft above the floor in a dimly lit room. At the far end of the beam is a darkened goal box with a narrow entryway. At equal distances along the beam, four 3-inch metal screws are positioned, angling away from the beam's center. A white noise generator and bright light source at the start of the beam motivate the animal to traverse the beam and enter the goal box. Once inside the goal box, the stimuli are terminated. The rat's latency to reach the goal box (in seconds) and hindlimb performance as he traversed the beam (based on a 1 to 7 rating scale) are recorded. A score of 7 indicates normal beam walking with less than 2 foot slips, and a score of 1 indicates that the rat is unable to traverse the beam in less than 80 seconds. Each rat is trained for 3 days before surgery to acquire the task and to achieve normal performance (a score of 7) on three consecutive trials. Three baseline trials are collected 24 hours before surgery, and three testing trials are recorded daily thereafter. Mean values of latency and score for each day were computed. Beam Balance

Beam balance is sensitive to motor cortical insults.16 17 This task is used to assess gross vestibulomotor function by requiring a rat to balance steadily on a narrow beam. The test involves three 60-second training trials 24 hours before surgery to acquire baseline data. The apparatus consists of a 3/4-inch-wide beam, 10 inches in length, suspended 1 ft above a table top. The rat is positioned on the beam and must maintain steady posture with all limbs on top of the beam for 60 seconds. The animal's performance was rated with the scale of Clifton and colleagues,18 which ranges from 1 to 6, with a score of 1 being normal and a score of 6 indicating that the animal was unable to support itself on the beam. Beam Walking

This is a test of sensorimotor integration specifically examining hindlimb function. The testing apparatus and rating procedures were adapted from Feeney and colleagues.16 A 1-inch-wide beam, 4 ft in length, is suspended 3 ft above the floor in a dimly lit room. At the far end of the beam is a darkened goal box with a narrow entryway. At equal distances along the beam, four 3-inch metal screws are positioned, angling away from the beam's center. A white noise generator and bright light source at the start of the beam motivate the animal to traverse the beam and enter the goal box. Once inside the goal box, the stimuli are terminated. The rat's latency to reach the goal box (in seconds) and hindlimb performance as he traversed the beam (based on a 1 to 7 rating scale) are recorded. A score of 7 indicates normal beam walking with less than 2 foot slips, and a score of 1 indicates that the rat is unable to traverse the beam in less than 80 seconds. Each rat is trained for 3 days before surgery to acquire the task and to achieve normal performance (a score of 7) on three consecutive trials. Three baseline trials are collected 24 hours before surgery, and three testing trials are recorded daily thereafter. Mean values of latency and score for each day were

computed.

(4) bilateral forepaw grasp, which measures the ability to hold onto a 2-mm-diameter steel rod, graded 0 for a rat with normal forepaw grasping behavior to 3 for a rat unable to grasp with the forepaws. The scores from all 4 tests, which were done over a period of about 15 minutes on each assessment day, were added to give a motor deficit score (maximum possible score, 12). Skilled forelimb function (1)staircase feeding apparatus

This required pretraining before induction of ICH. Rats had free access to food and water during first 2 days after arrival from the supplier. The rats were housed in pairs in standard plastic boxes with a 12-hour day/night cycle. During the following 7 days, the rats were fed 8 to 15 g/d of standard laboratory chow to reduce their body weight to 85% to 90% of the initial weight. Hunger was the incentive to perform in the testing apparatus. The staircase pretraining was performed twice per day, with a time interval of at least 4 hours between trials. The rat was placed in a clear plastic box with a food-baited staircase on either side. Each staircase had 7 steps, each with a well containing 3 45-mg pellets (P.J. Noyes Co Inc). The number of food pellets reached and eaten in 20 minutes was counted. When a plateau was reached, the top well was no longer baited with pellets, because these can be reached with the tongue. An additional 4 to 6 trials were used to calculate mean pretraining number of pellets eaten from each side. If the side-to-side difference was >4 on the final trials, the side on which rat collected more pellets was designated its \After pretraining, the rats were allowed free access to food for 2 days before surgery and during the 4 weeks after ICH. Beginning 28 days after ICH, the rats were fed 10 to 12 g/d standard laboratory chow to decrease the body weight to 90% of the free feeding level. They were then evaluated daily in the staircase apparatus for 3 weeks. The top well of the staircase apparatus was not baited. The number of food pellets eaten in 20 minutes on each side was counted (maximum possible 18 per side). nociception (1) plantar test

Rats were placed in a clear plastic chamber (18x29x13 cm, 2.2-mm thickness). Acclimation of the animals to the environment occurred 20 minutes before baseline testing for 3 consecutive days with random activation of heating source to accommodate the animals to the pain stimulus. The heat source was then positioned under the glass floor beneath the right or left forepaw. Paw withdrawal automatically activated the timer with a latency of 0.1 second. Measurements were taken 5 times at 15-minute intervals and are given as averaged paw-withdrawal latency.

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