CN115517224A - 一种认知障碍动物模型的建立方法及应用 - Google Patents
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Abstract
本发明涉及动物模型构建技术领域,具体涉及一种认知障碍动物模型的建立方法及应用。本发明首次发现对小鼠采用乳酸钠腹腔注射给药可引起小鼠认知障碍,可以用于构筑具有认知障碍症状的小鼠动物模型,该方式操作简单、易行,可克服现有技术中的认知障碍模型在造模方面比较复杂或者繁琐的问题。
Description
技术领域
本发明涉及动物模型构建技术领域,具体涉及一种认知障碍动物模型的建立方法及应用。
背景技术
认知是机体认识和获取知识的智能加工过程,涉及学习、记忆、语言、思维、精神、情感等一系列随意、心理和社会行为。认知障碍指与上述学习记忆以及思维判断有关的大脑高级智能加工过程出现异常,从而引起严重学习、记忆障碍,同时伴有失语或失用或失认或失行等改变的病理过程。
目前常见的会导致认知障碍的模型有阿尔兹海默病、脑卒中等。但这些模型在造模方面比较复杂或者繁琐。例如阿尔兹海默病,由于其病因复杂,所以缺乏严格意义的体内外模型。目前国内外大部分体内、外实验只能反映阿尔兹海默病的某一部分病变。脑卒中分为缺血性脑卒中和出血性脑卒中,造模方法较为复杂,对于之前从未接触的人来说无法直接上手操作。
例如:专利CN201610873616.9所公开的一种非人哺乳动物认知障碍或其相关疾病动物模型的建立,其通过将非人哺乳动物的细胞中的Glce基因失活制备得到Glce基因失活的细胞,利用Glce基因失活的细胞制备得到Glce基因失活的认知障碍或其相关疾病动物模型。
发明内容
本发明的目的是为了克服现有技术存在的缺点和不足,而提供认知障碍动物模型的建立方法及应用。
本发明所采取的技术方案如下:一种认知障碍动物模型的建立方法,其包括以下步骤:向小鼠腹腔注射乳酸钠。
每天按1g/kg的计量进行注射乳酸钠,连续注射至少8周。
如上所述的认知障碍动物模型的建立方法所制备的动物认知障碍模型的用途,所述认知障碍动物模型用于筛选或鉴定能够缓解或治疗认知障碍的药物。
本发明的有益效果如下:本发明首次发现对小鼠采用乳酸钠腹腔注射给药可引起小鼠认知障碍,可以用于构筑具有认知障碍症状的小鼠动物模型,该方式操作简单、易行,可克服现有技术中的认知障碍模型在造模方面比较复杂或者繁琐的问题。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动性的前提下,根据这些附图获得其他的附图仍属于本发明的范畴。
图1 连续8周腹腔注射乳酸不影响小鼠运动能力。在最后一天给药结束后,测定了各组小鼠在5min内自主活动情况,分别记录(A)轨迹图,(B)活动次数,(C)活动总路程和(D)活动中平均速度。柱形图显示的是实验所得的平均值,每组n = 15;
图2 连续8周腹腔注射乳酸损伤小鼠工作记忆能力。在最后一天给药结束后第二天,记录各组小鼠6min内(A)在迷宫中a,b,c三个臂之间自主交替选择策略的情况,以(B)自主交替率和(C)总穿臂次数表示。柱形图显示的是实验所得的平均值,每组n = 15。与Lac组相比,* p < 0.05;
图3 三组水迷宫定位航行期潜伏时间和上台前游泳总距离。(A)动物实验流程图。(B)水迷宫定位航行试验4天动物寻找不可见平台潜伏时间(escape latency)和(C)上平台前游泳总距离。各时间点数据显示的是实验所得的平均值,每组n = 15。与Lac组相比,* p< 0.05,** p < 0.01;
图4 三组水迷宫去平台探索期(A)轨迹图,(B)穿台次数,(C)游泳总路程,(D)游泳速度和(E)各象限游泳时间百分比。各时间点数据显示的是实验所得的平均值,每组n =15。AL:Adjacent left,第一象限;OP:Opposite,第二象限;TQ:Target quadrant第三象限;AR:Adjacent right,第四象限。与Lac组相比,* p < 0.05,*** p < 0.001;
图5 长期高脑乳酸对突触可塑性基因表达的影响。8周腹腔注射药物后,取海马组织,提取总RNA,进行Q−PCR实验,测定脑源性神经营养因子(BDNF,A),C−Fos(B),活性调节细胞骨架相关蛋白(Arc,C),早期生长反应−1(Egr1,D),生长相关蛋白−43(GAP−43,E),突触素(synaptophysin,Syp,F),突触后致密物质93(PSD−93,G),突触后致密物质95(PSD−95,H)mRNA水平。柱形图显示的是实验所得的平均值(n = 5−8,三次独立实验)。使用one wayANOVA检验,对应的方差检验统计量,BDNF[F(2,16)= 0.033,p = 0.968],C−Fos[F(2,13)=4.860,p = 0.027],Arc[F(2,14)= 6.422,p = 0.010],Egr1[F(2,12)= 15.361,p <0.001],GPA−43[F(2,18)= 0.001,p = 0.999],Syp[F(2,20)= 0.182,p = 0.835],PSD−93[F(2,12)= 5.013,p = 0.026],PSD−95[F(2,16)= 6.393,p = 0.009]。pot hoc检验采用Scheffe方法,与对照组相比显著性,* p < 0.05,** p < 0.01,** p < 0.001;
图6长期高脑乳酸对突触可塑性相关蛋白及pCREB表达的影响。小鼠接受药物灌注并完成行为学后处理,取海马组织匀浆提蛋白,接着进行免疫印迹实验,(A)Veh组,Lac组和Pyr组三组测得蛋白免疫印迹条带。进行光密度分析,分别对(B)Arc,(C)C−Fos,(D)Egr1,(E)BDNF,(F)Syp,(G)PSD−93,(H)PSD−95定量,表达量相对Veh组作归一化。柱形图显示的是实验所得的平均值(n = 3),与Lac组相比,* p < 0.05,** p < 0.01。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面将结合附图对本发明作进一步地详细描述。
实施例1:
将试验小鼠进行适应性喂养1周后,每日给予乳酸钠(1 g/kg),腹腔注射持续8周,记为乳酸组(Lac)。8周后,进行检测认知能力的行为学实验。实验完成后,休息一天,解剖小鼠快速剥离出小鼠全脑后,分离皮质和海马,立即将它们在液氮中速冻,之后保存在−80°C,以备后用。
对比例1:
将试验小鼠进行适应性喂养1周后,每日给予无菌盐水(1 g/kg),腹腔注射持续8周,记为正常组(Veh)。8周后,进行检测认知能力的行为学实验。实验完成后,休息一天,解剖小鼠快速剥离出小鼠全脑后,分离皮质和海马,立即将它们在液氮中速冻,之后保存在−80°C,以备后用。
对比例2:
将试验小鼠进行适应性喂养1周后,每日给予丙酮酸钠(1 g/kg),腹腔注射持续8周,记为丙酮酸组(Pyr)。8周后,进行检测认知能力的行为学实验。实验完成后,休息一天,解剖小鼠快速剥离出小鼠全脑后,分离皮质和海马,立即将它们在液氮中速冻,之后保存在−80°C,以备后用。
以下内容为上述实施例1、对比例1和对比例2中的相关实验内容和实验结果对比说明(其中相关实验数据应用SPSS 23.0 软件进行统计分析,数据以平均值±标准差(mean ± SD) 表示,组间的统计差异采用单因素方差分析 (ANOVA),采用post hocDunnett’s two-sided检验进行两两比较。当且仅当P值小于0.05时,结果被认为是有统计显著性差异的)。
一、以下为检测认知能力的行为学实验内容,按实验的时间顺序依次为动物自主活动实验、Y迷宫实验和水迷宫实验,可以检测小鼠的运动能力和学习记忆能力。
(1)自主活动实验:通过将小鼠放在透明的有机玻璃(40 × 40 × 30 cm)开放场地的中心并让小鼠自由探索5min来评估其运动能力。在昏暗条件下,保持周围环境安静,正式实验前首先让小鼠在环境中熟悉2min。使用数字光学系统(上海欣软信息技术有限公司)对动物活动进行定量。记录三组运动次数(小鼠从停止到重新探索的次数),运动总路程(代表运动能力,cm),运动速度(cm/s)。
如图1A所示为三组小鼠自主活动轨迹图。所得结果显示,三组之间在5min内的运动次数[Reaning number,F(2,41)= 0.306,p = 0.738,图1B],运动总距离[Distances,F(2,42)= 1.848,p = 0.170,图1C]和平均速度[Average speed,F(2,42)= 0.306,p =0.738,图1D]均无明显差异。
(2)Y迷宫实验:Y迷宫装置由木板拼接成Y字形,伸出三臂(长:30 cm,宽:8 cm,高:16 cm)从中心延伸,每两臂之间夹角呈120°。迷宫内不设置标记,但在迷宫外环境中贴上不同的定位方向的标记。实验时将小鼠放入一条臂的末端,允许在迷宫中自由移动探索6min,动物运动轨迹用摄像机记录。每次Y迷宫结束后,用70%乙醇清洗迷宫装置内壁,干燥后进行后续实验。记录动物进入各臂的次序和总次数,当连续三次进入不同的臂时,这是一次正确交替反应,统计正确交替反应次数,计算自主交替率,计算公式如下:
自发交替反应率(%)= [正确交替反应次数/(穿壁总次数−2)] × 100。
三组小鼠在完成自主活动实验后第二天,接受Y迷宫实验,以测定动物工作记忆情况,实验装置如图2A所示。结果显示,各组之间6min内在Y迷宫中a,b,c三个臂之间自主交替率(spontaneous alterations,F(2,42)= 7.807,p < 0.01,图2B)有明显差异,其中Lac组较Veh组和Pyr组均明显下降(Veh vs. Lac与Pyr vs. Lac显著性分别为p = 0.000893和p= 0.00304,图2B)。另外,三组之间穿台次数无明显差异,如图2C所示。
(3)水迷宫实验:迷宫由一个直径100 cm,深50 cm内壁白色的水池组成,水池内注满水,水温控制在26 ± 2°C。相机采集到动物池中游泳轨迹信息连接到在线的XR−XM101分析系统上。分析系统中,将水池划分为四个象限,对应四个方向指定为游泳起始位置(即东、西、南、北方向)。水池和操作人员用屏风隔开,实验期间保持环境安静。池中另外放一个淹没的逃生平台(低于水面2 cm),平台实验期间固定在第三象限中心。水池用无毒白色颜料变浑浊,使从水面及水面上无法确定平台位置。实验进行5天,前4天为定位训练期,第5天为去平台空间探索期。定位训练期每天实验动物需要完成4个入水方向的游泳训练,每只动物每次实验间隔控制在20min以上,每天的入水方向按下方表格进行。规定动物水池内探索时间为60s,若规定时间内动物找到平台试验终止,同时继续让动物在平台上停留10s;若规定时间内动物并未找到平台,则引导动物上平台,另外让动物在平台停留20s。此时,记录动物寻找平台潜伏期,总游泳距离。去平台空间探索期(第5天)需要撤去平台,然后让动物从北方向入水,记录60s内动物穿台次数及游泳总距离。每天训练后,动物用毛巾擦干并用吹风机加快干燥,最后放回饲养笼内。
自主活动和Y迷宫实验结束后,动物休息一天,接着进行5天的水迷宫实验,期间继续灌注药物,动物实验一总流程见图3A所示。水迷宫定位航行实验期间,对潜伏时间和游泳总路程进行3×4双因素方差分析,对应折线图显示四天训练期三组不同时间点的平均值和标准误,如图3B,C所示。游泳潜伏时间结果显示,给药具有明显主效应,F(2,42)= 4.361,p= 0.019,而且时间也具有主效应,Sphericity Assumed的F(3,126)= 59.734,p < 0.001。对于给药效应,当给予乳酸后,从训练后第3天起潜伏时间较Veh组和Pyr组相比需要更多的时间寻找平台,Pyr组与Veh组潜伏时间折线图基本一致,有意思的是,Pyr组在训练第2天即能很快找到平台,但与Veh组无显著差异。简单效应检验的结果显示在训练后第4天Veh组和Pyr组寻找平台潜伏时间较Lac组均明显降低[F(2,42)= 6.077,p = 0.005,Veh vs. Lac,p= 0.016,Pyr vs. Lac,p = 0.0104],或者说连续学习过程中Lac组对平台位置的记忆能力明显不如Veh组和Pyr组好。游泳总路程结果显示,给药不具有主效应,F(2,42)= 0.111,p =0.895,但时间点具有主效应,Greenhouse−Geisser调整F(3,126)= 32.176,p < 0.001,且各组间随训练时间增加,找到平台的游泳总路程均逐渐减少,换句话说,给药与否对小鼠水迷宫实验中学习能力无显著性影响。从上面的结果可以得出,在定位航行实验中,药物因素不影响动物学习能力,但随着训练时间的延长,乳酸组小鼠记忆能力出现抑制现象,进一步对记忆能力考察需要继续对水迷宫去平台空间探索期结果进行分析。
从前面定位航行实验,我们发现在三组学习能力无差异的情况下,Lac组小鼠记忆能力较Veh组和Pyr组明显下降。进一步在水迷宫第5天,将第三象限平台撤去,以观察动物对平台位置记忆情况。小鼠在水池中60s内游泳轨迹如图4A所示,将穿台次数,游泳总路程,游泳速度和象限内游泳时间比作为指标。单因素方差分析显示,Lac组穿台次数较Veh组和Pyr组明显减少[F(2,42)= 9.885,p < 0.001,Veh(4.27 ± 1.79,SD)vs. Lac(1.87 ±1.26),p < 0.001,Pyr(3.53 ± 1.55)vs. Lac,p = 0.013,如图4B],提示乳酸给药后,伴随长时间脑乳酸升高小鼠长期记忆出现障碍。探索期游泳总路程由于不符合正态分布,使用单因素Kruskal–Wallis分析方法,结果显示三组之间游泳总路程无明显差异[H(2)=0.720,p = 0.698,如图4C],三组游泳速度也无明显差异[F(2,42)= 0.179,p = 0.837,如图4D],说明产生上述穿台次数差异,与动物本身运动能力差异无关。另外,Lac组目标象限游泳时间百分比较Veh组和Pyr组也明显降低[F(2,42)= 17.656,p < 0.001,Veh(34.15 ±6.98)vs. Lac(20.63 ± 5.82),p < 0.001,Pyr(35.92 ± 9.78)vs. Lac,p < 0.001,如图4E]。从水迷宫两个阶段结果可以得出,长期高脑乳酸小鼠学习能力出现障碍,并且对长期记忆有显著抑制作用。
二、以下为实时荧光定量聚合酶链式反应(Q−PCR)实验内容。
使用TRIzol试剂提取总RNA,详细操作详见说明书。取1 μg总RNA利用20 μl逆转录系统逆转录得到cDNA。混合TB Green™ Premix Ex Taq™ 预混悬液,对应不同突触可塑性基因引物对和之前的cDNA建立反应体系,在CFX96 Real−Time PCR模块上完成Q−PCR检测。基因的表达量使用2−△△Ct法计算。
取三组海马组织进行分子表征,测定了突触可塑性基因mRNA水平的表达情况。除BDNF基因,Lac组测定的其他ITF编码基因均较Veh组和Pyr组明显降低,如图5A−D所示。另外,代表突触前成分标记基因GAP−43和Syp,Lac组和其他两组无明显变化,而代表突触后成分标记基因PSD−93和PSD−95,Lac组较Veh组和Pyr组亦明显降低,如图5E−H所示。
三、以下为免疫印迹分析过程。
提取动物总蛋白后,对蛋白质样品进行SDS−PAGE凝胶电泳,凝胶电泳结束后进行蛋白转膜(采用Bio−Rad转膜槽),将分离开的蛋白质条带从凝胶中转移至PVDF膜上。将印有蛋白条带的PVDF膜在5%脱脂奶粉(伊利)中封闭1h。用TBST(含0.1% tween−20的TBS,同时含0.02%叠氮化钠)稀释一抗后与膜4°C孵育过夜,然后用TBST洗去非特异性结合的一抗。用辣根过氧化物酶标记的二抗结合1h,用TBST洗去非特异性结合的抗体。用EZ−ECL化学发光增强试剂盒显示目的蛋白条带。
如图6所示,与上述转录水平结果基本一致,Lac组BDNF和突触前成分Syp蛋白表达量较Veh组和Pyr组无明显变化,而C−Fos,Arc,Egr1经长期乳酸作用后在小鼠海马组织内表达水平较Veh组和Pyr组明显下降。并且乳酸长期刺激后PSD−95蛋白表达量较Veh组和Pyr组明显下降。另外,也考察了脑内参与突触相关蛋白表达的关键转录因子CREB活化形式pCREB的表达,Lac组pCREB相对tCREB表达量较Veh组和Pyr组均下降。
cAMP反应元件结合蛋白(CREB)是一种转录因子,其活性受细胞内cAMP和钙水平的调节。果蝇和小鼠等模式动物上的研究结果表明,CREB介导的转录是长期记忆所必需的。例如,将具有cAMP响应元件(CRE)的寡核苷酸注射到培养的Aplysia神经元中会选择性地阻断长期而非短期的反射作用,这是一种非联想记忆的神经元模型。此外,果蝇的嗅觉调节任务中,通过显性抑制CREB表达会破坏长期而非短期记忆。
本发明的实施例与对比例的结果对比,可以说明对小鼠乳酸钠腹腔注射给药能导致小鼠认知障碍,从而构建认知障碍动物模型。
以上所揭露的仅为本发明较佳实施例而已,当然不能以此来限定本发明之权利范围,因此依本发明权利要求所作的等同变化,仍属本发明所涵盖的范围。
Claims (3)
1.一种认知障碍动物模型的建立方法,其特征在于其包括以下步骤:向小鼠腹腔注射乳酸钠。
2.根据权利要求1所述的认知障碍动物模型的建立方法,其特征在于:每天按1g/kg的计量进行注射乳酸钠,连续注射至少8周。
3.如权利要求1或2所述的认知障碍动物模型的建立方法所制备的动物认知障碍模型的用途,其特征在于:所述认知障碍动物模型用于筛选或鉴定能够缓解或治疗认知障碍的药物。
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