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 神经行为学检查 LongaLonga 评分法评分法 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 2 BerdersonBerderson 评分法评分法 姿势反射测验 姿势反射测验 posturalpostural reflexreflex testtest 提提 尾悬空试验尾悬空试验 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 3 攀绳实验攀绳实验 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 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 5 肢体放置测验 肢体放置测验 limb placementlimb placement testtest ElicitedElicited ForelimbForelimb PlacingPlacing 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 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 10 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 2 2 分 反应延迟且超过 2 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 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 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