该项目得到河北省自然科学基金 (no。C2009001007、H2014406048)、河北省中医药管理局 (05027 号), 是河北省高等专科学校重点学科建设项目。

本程序符合国家科学技术委员会111988年10月31日颁布的《实验动物管理条例》。科学家应遵循其机构和国家动物管理组织制定和批准的准则。
注: 动物和摄政: 四月大的雄性大大鼠 (300-350 克) 提供了这个实验。所有老鼠都被安置在小组中 (每笼四或五), 温度为 23, 1 摄氏度, 有 12-小时的光-暗循环。食物和水是可利用的广告随意。在手术前7天, 大鼠适应了住房条件。Aβ25-35 在1% 亚砜盐水中溶解到1毫克/毫升, 在超声波振荡器中微气泡5分钟, 直到完全溶解。AlCl3和 RHTGF-β1分别在1% 和0.1 毫克/毫升的盐水中溶解。刚果红、硝酸银和其他化学品的分析等级, 并从普通商业来源购买。
1. 手术方法
注:20 只雄性大大鼠杏仁 Aβ到侧脑室和 anterodorsal 丘脑核, 并被指定为复合 Aβ治疗组。另有20只大鼠接受相同的手术, 但收到0.1% 亚砜生理盐水显微注射, 并被指定为假手术组。
<ol><li>麻醉100% 异氟醚的大鼠吸入, 然后抑制脑立体定向装置。</li>
	<li>用手术剪刀将头部顶点上的皮毛剃掉, 用碘伏消毒。然后, 在头上盖上一次性的手术巾。</li>
	<li>用手术 bistouries 和剪刀沿中位纵向颅骨对头部皮肤进行切口。</li>
	<li>将皮下组织和筋膜分开, 用无菌的干棉擦拭颅骨颅骨以止血, 并用记号笔标记 bregma。</li>
	<li>参考鼠脑立体定向图12;以 bregma 为起点, 标记三点, anterodorsal 丘脑核 (ad) (后 (P): 2.0 毫米到 bregma; 侧 (L): 1.4 毫米到中线) 注射 RHTGF-β1和固定一螺钉, 侧脑室 (LV) 区域 (后 (P): 0.8 毫米到 bregma;侧 (l): 2.0 毫米到中线) 为注射 Aβ25-35 和 AlCl3, 并且第二个螺丝固定的地方 (前面 (F): 2.0 毫米到 bregma; 侧 (l): 1.5 毫米到中线)。</li>
	<li>轻轻钻三1毫米直径孔与灵活的骨钻, 指定在上述三点头骨 (1.5. 步骤)。不要拧得比必要更深, 以免刺伤脑组织。</li>
	<li>用无菌干棉反复止血, 重复清理颅骨表面。</li>
	<li>插入一针连接到微注射泵, 到大脑在4.6 毫米深度和轻轻地注入 1 ul RHTGF-β1 (10 ng) 进入广告领域。注射后保持针2分钟, 慢慢拔出针 (补充文件 1)。</li>
	<li>将两个螺钉固定在头骨中, 指定在 ad 点和头骨的第二个螺钉固定位置 (1.5. 步骤) 用小螺丝刀。不要拧得比必要更深, 以免刺伤脑组织。</li>
	<li>装配套管植入系统 (补充文件 2), 在高压消毒后将假套管插入导套管内。</li>
	<li>通过颅骨孔 (补充文件 3), 在大鼠立体定向装置的套管支架上, 将不锈钢导管导套管插入到大脑中, 并将其4.6 毫米进入 LV 区域。</li>
	<li>将义齿基材与义齿基水混合, 以每1毫升1.5 克的比例, 将贴剂盖在导套管塑料底座和两个螺钉内, 固定引导套管, 直至覆盖整个皮肤切口以避免皮肤感染。</li>
	<li>在手术2天, 麻醉用小型动物麻醉机吸入异氟醚的大鼠。拔下假套管, 将内部套管插入导套管内, 拧紧固定螺钉, 使内套管不起。</li>
	<li>将微注射泵与内部套管连接的聚乙烯管道设置为 1 ul/分钟, 并将注射速度调节到 Microinject Aβ25-35 或 AlCl3。</li>
	<li>Microinject 4 微克 (1 ul) Aβ25-35 每日14天上午和 3 ul AlCl3 (1%) 每日5天下午在异氟醚麻醉下。</li>
	<li>完成注射后等待5分钟, 轻轻抽出内部套管, 再将假套管插入导套管内。</li>
	<li>15天后手术 (相当于 Aβ25-35 最后一次注射日) 拆除套管植入系统。用手术剪刀和镊子轻轻取出义齿基材, 拧开两个螺钉, 拔出导套管, 用 betadine 消毒伤口。用骨水泥填充颅骨孔, 用简单的间断缝合方法缝合皮肤。</li>
	<li>对假手术组和 microinject 0.1% 亚砜盐水进行相同的手术。</li>
	<li>
术后护理
	<ol>
<li>2只老鼠每笼手术后, 提供30天的食物。 注:18 只大鼠在假手术组生存 (90% 成功率的手术), 19 大鼠在复合治疗 Aβ组生存 (95% 成功率的手术)。</li>
	</ol>
</li>
</ol>2. 成功模型大鼠的筛选与莫里斯水迷宫对空间记忆的评价
<ol><li>
莫里斯水迷宫 注: 莫里斯水迷宫用于评估鼠空间记忆13。莫里斯水迷宫是不锈钢圆形罐, 直径为120厘米, 深度为50厘米。根据 j. 努涅斯14在行为神经科学中描述的 "金标准" 范式, 进行了水迷宫试验。<ol>
<li>用几滴食物着色, 使水池里的水发黑。</li>
		<li>保持水的深度在31.5 厘米和温度在 23 1 摄氏度。</li>
		<li>在水面下面设置一个1.5 厘米圆形透明有机玻璃平台。</li>
		<li>在水迷宫试验中保持水迷宫周围的所有空间信号是不变的。</li>
		<li>将池分成4个相等象限, 由假想的线条进行描述性数据收集。</li>
		<li>将隐藏平台放在水迷宫的第一象限 (Q1) 中。</li>
		<li>通过摄像机捕捉老鼠游泳行为 (通过潜伏期, 轨迹, 或交叉数), 在水迷宫链接到计算机的图形分析软件。</li>
	</ol>
</li>
	<li>
成功模型大鼠对复合 Aβ治疗组的筛选
	<ol>
<li>在45天的手术, 执行莫里斯水迷宫训练连续4天, 以筛选成功的模型大鼠记忆障碍和收集筛选率 (SR)。 注: SR 被定义为每复合 Aβ处理大鼠和假手术组的平均潜伏期, 在水迷宫训练4天的水面下寻找隐藏平台。"A" 是每个复合 Aβ鼠的平均潜伏期, 以找到隐藏的平台和 "B" 是假手术组大鼠的平均潜伏期, 以找到隐藏的平台, 在4天的水迷宫训练, 在以下等式: <img alt="Equation 1" src="/files/ftp_upload/56157/56157eq1.jpg"> 当 SR 大于0.2 的一个复合 Aβ处理大鼠, 大鼠被认为是一个成功的模型大鼠记忆受损的复合 Aβ治疗大鼠15。利用大鼠的平均值对2项试验数据进行了日内记忆性能的计算, 找出了隐藏平台。设计了莫里斯水迷宫试验的过程, 允许大鼠在水迷宫罐中游泳, 并在六十年代内搜索隐藏平台。如果一只老鼠在六十年代内没有发现隐藏的平台, 那么老鼠就用实验者的手放在平台上。当老鼠在隐藏的平台上 (独立或辅助) 到达时, 老鼠被放在那里二十年代。然后, 老鼠被从坦克中取出, 并允许在十年代的2试验之间恢复体力。</li>
	</ol>
</li>
</ol>3. 神经元检查
<ol><li>86天手术后, 异氟醚麻醉下, 弄死大鼠斩首 (图 1)。</li>
	<li>把大脑放在冰上, 轻轻地将两个半球分开。采取光学交叉的左半球和固定在4% 甲醛为光显微镜观察神经元苏木精和伊红 (他), 刚果红, 或硝酸银染色 (见4-6 节)。固定右半球海马 CA1 面积2.5% 戊二醛用于电子显微镜观察 (7 节)。</li>
	<li>处理大脑的光/电子显微镜样品准备, 如前所述16,17。</li>
</ol>4. 神经元 he 染色
<ol><li>Deparaffinize 每张幻灯片 (每个20分钟) 与梯度酒精 (100%, 95%, 90%, 80% 和70% 酒精) 到蒸馏水在油烟机罩。</li>
	<li>染色3分钟与苏木精 (0.5% 瓦特/v), 然后用自来水冲洗, 以消除未绑定染料从幻灯片。</li>
	<li>用0.1% 盐酸在酒精中冲洗1秒以去除无瑕核的颜色。</li>
	<li>浸泡在0.5% 氨溶液中2分钟, 直到背景变成浅蓝色。</li>
	<li>染色1分钟, 1%。</li>
	<li>用梯度酒精 (70%, 80%, 90%, 95% 和100% 酒精) 脱水5分钟。</li>
	<li>在二甲苯中清除并安装有树脂安装介质。</li>
	<li>观察和计数每0.125 毫米的他的活神经元在海马的中间 CA1 和每0.0352 毫米2在大脑皮层在放大400x 与光学显微镜由被失明对实验设计的人。</li>
</ol>5. 刚果红染色法测定神经元 Aβ负担
<ol><li>Deparaffinize 每张幻灯片 (20 分钟) 与梯度酒精 (100%, 95%, 90%, 80% 和70% 酒精) 到蒸馏水在油烟机罩。</li>
	<li>用刚果红工作溶液染色20分钟 (0.5 克刚果红, 80 毫升甲醇, 20 毫升 glycerinum)。</li>
	<li>用蒸馏水冲洗5分钟。</li>
	<li>用碱性80% 醇溶液 (0.2 克 alcohol/100 毫升氢氧化钾) 快速鉴别 3 s。</li>
	<li>漂洗两次, 每5分钟用蒸馏水。</li>
	<li>Counterstain 在鳃的苏木精3分钟。</li>
	<li>用自来水冲洗2分钟。</li>
	<li>在三十年代或直到部分变蓝之前, 在氨水中浸泡 (加入几滴氢氧化铵以自来水和混合)。</li>
	<li>用自来水冲洗5分钟。</li>
	<li>脱水与梯度酒精 (70%, 80%, 90%, 95% 和100% 酒精)。</li>
	<li>在二甲苯中清除并安装有树脂安装介质。</li>
	<li>观察和计数被染成刚果红色的细胞在400x 的放大倍数, 用光学显微镜被盲人的实验设计。</li>
</ol>6. 硝酸银染色法测定神经元测试形成
<ol><li>Deparaffinize 每张幻灯片 (20 分钟) 与梯度酒精 (100%, 95%, 90%, 80% 和70% 酒精) 到蒸馏水在油烟机罩。</li>
	<li>在黑暗中浸泡20% 硝酸银溶液和封盖剂20分钟。</li>
	<li>在蒸馏水中洗两次, 每次5分钟。</li>
	<li>浸泡在银氨溶液中。将氨水溶液放入20% 硝酸银溶液中, 加入到溶液从浊度到澄清。同时, 用玻璃棒搅拌溶液15分钟, 然后放入1% 稀释的氢氧化铵中2分钟。</li>
	<li>将幻灯片放到开发中的工作解决方案中3-7 分钟, 直到轴突中的黑块能被观察到。</li>
	<li>浸泡在0.1% 稀释氨溶液中1分钟, 然后用水冲洗1分钟。</li>
	<li>用5% 硫酸钠处理2分钟, 然后用水冲洗5分钟。</li>
	<li>脱水与梯度酒精 (70%, 80%, 90%, 95% 和100% 酒精)。</li>
	<li>在二甲苯中清除并安装有树脂安装介质。</li>
	<li>用光学显微镜观察和记录在400x 的硝酸银染色的细胞, 由一个被蒙蔽的人对实验设计。</li>
</ol>7. 海马神经元超微结构测量
<ol><li>将大鼠海马 CA1 切成几个立方体 (1 x 1 x 1 毫米3), 并将2.5% 戊二醛置于2摄氏度。</li>
	<li>用 PBS 冲洗立方体三次 (每一次的 pH 值为 7.2, 10 分钟)。</li>
	<li>固定在 1% osmic 酸的立方体为2小时在4摄氏度。</li>
	<li>用双蒸馏水 3x (每次10分钟) 冲洗立方体。</li>
	<li>脱水与梯度酒精 50%, 70% 和 90% (每个的10分钟), 100% 二次 (每时间15分钟)。</li>
	<li>用二氧化丙烯替换两次 (每次15分钟), 氧化丙烯: 树脂在 1:1 (每次室温时60分钟) 和丙烯氧化物: 树脂 1:4 (每次室温下60分钟)。浸泡在树脂 (120 分钟, 室温)。</li>
	<li>嵌入 EPON 812 和聚合 (5 h/35 °c, 5 h/60 °c, 5 h/80 °c)。</li>
	<li>切半薄切片部分 (1 µm), 染色的亚甲基蓝, 并在显微镜下本地化。</li>
	<li>将超薄切片 (50 nm) 与醋酸铀和柠檬酸铅染色。</li>
	<li>在 JEM-1400 电子显微镜下检查并收集图像。</li>
</ol>

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Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scutellaria+flavonoid+supplementation+reverses+ageing-related+cognitive+impairment+and+neuronal+changes+in+aged+rats.">Scutellaria flavonoid supplementation reverses ageing-related cognitive impairment and neuronal changes in aged rats.</a> Brain Inj. 23, 146-153 (2009).</li><li>Wang, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+RAS+inhibitors+Poricoic+Acid+ZG+and+Poricoic+Acid+ZH+attenuate+renal+fibrosis+via+a+Wnt/&#946;-Catenin+patheway+and+targeted+phosphorylation+of+smad3+signaling.">Novel RAS inhibitors Poricoic Acid ZG and Poricoic Acid ZH attenuate renal fibrosis via a Wnt/&#946;-Catenin patheway and targeted phosphorylation of smad3 signaling.</a> J Agric Food Chem. 66, 1828-1842 (2018).</li><li>Pini, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+atrophy+in+Alzheimer's+Disease+and+aging.">Brain atrophy in Alzheimer's Disease and aging.</a> Ageing Res. Rev. 30, 25-48 (2016).</li><li>Ubhi, K., Masliah, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+disease:+recent+advances+and+future+perspectives.">Alzheimer's disease: recent advances and future perspectives.</a> J. Alzheimers Dis. 33, 85-94 (2013).</li><li>Guo, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Scutellaria barbata flavonoids inhibite NFTs aggregation, tau protein phosphorylation and the regulated mechanism of related enzymes in rats induced by composited A&#946;. , (2016).</li><li>Shang, Y. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Effects and Mechanism of Scutellaria Barbata Flavonoids on Rat's Memory Impairment Induced by Compound A&#946;25-35. , (2013).</li><li>Zussy, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+disease+related+markers,+cellular+toxicity+and+behavioral+deficits+induced+six+weeks+after+oligomeric+amyloid-&#946;+peptide+injection+in+rats.">Alzheimer's disease related markers, cellular toxicity and behavioral deficits induced six weeks after oligomeric amyloid-&#946; peptide injection in rats.</a> PLoS One. 8, 1-20 (2013).</li><li>Walton, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aluminum+involvement+in+the+progression+of+Alzheimer's+disease.">Aluminum involvement in the progression of Alzheimer's disease.</a> J. Alzheimers Dis. 35, 7-43 (2013).</li><li>Neils-Strunjas, J., Groves-Wright, K., Mashima, P., Harnish, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysgraphia+in+Alzheimer's+disease:+a+review+for+clinical+and+research+purposes.">Dysgraphia in Alzheimer's disease: a review for clinical and research purposes.</a> J. Speech Lang Hear Res. 49, 1313-1330 (2006).</li><li>Morley, J. E., Farr, S. A., Kumar, V. B., Armbrecht, H. 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作者没有什么可透露的。

众所周知, 学习记忆的丧失是 AD 患者2的主要临床症状。本文所描述的过程是研究 AD 的体内方法;我们已经修改了以前发布的协议, 它测试了一种药物, 以减轻老鼠模型4中的记忆缺损和神经元损伤。我们的协议提供了重要的细节, 以获得有价值的数据, 以及成功地模型操作, 记忆缺陷, 神经元损伤, Aβ负担和测试沉积的动物的高存活率, 模仿广告 (在目前的实验中, 生存率成功的模型运行率超过 90%)。这些成功的模型大鼠被用来测量他们的空间记忆与莫里斯水迷宫试验。定位导航试验发现, 复合 Aβ可引起大鼠记忆捕获损伤;探针试验发现, 复合 Aβ可降低大鼠记忆保留率;逆转试验发现, 复合 Aβ可导致大鼠再学习损伤。这些莫里斯水迷宫试验数据表明, 复合 Aβ能诱发大鼠空间记忆。整体来说, 注射大鼠 intracerebroventricularly 与 Aβ25-35 结合 AlCl3和 TGF-β1为实验室创造了一个可行和可信的体内AD 样动物模型。
以前的研究表明, AD 患者的脑容量比健康个体少10%。在大脑半球可以通过视觉观察发现各种萎缩。皮质萎缩程度与19的记忆障碍呈正相关。在组织学上, 大量的神经元丢失和严重的形态学病理直接扰乱 AD 患者的记忆功能20。在本研究中, 光/电镜观察发现, 大鼠杏仁与复合 Aβ显示戏剧性的病理变化, 包括神经元丢失, 神经元和亚细胞结构中断。该结果证实了复合 Aβ诱导的大鼠空间记忆障碍, 与 AD 患者的状态相似。
众所周知, 脑 Aβ负担和测试聚集被认为是 AD 中最重要的 histopathogenic 特征。它们能破坏神经元结构, 扰乱神经信号, 扰乱神经元功能, 导致晚期痴呆17。目前的动物模型发现 Aβ负担和测试聚集在大脑, 这与 AD 病人的状态一致。因此, 采用复合 Aβ诱发大鼠神经元损伤, 可作为研究 AD 神经元病理和治疗策略的模型。
以下是在 AD 大鼠模型中筛选药物效应的例子: 赵等人, 报告说, 黄芩茎叶黄酮和黄芩莲 (SBF) 均可减轻大鼠记忆障碍和凋亡的作用。复合 Aβ8,9。郭等也报道说, SBF 可以抑制 Ser199、Ser214、Ser202、Ser404 和 Thr231 侧测试聚集和头蛋白过磷酸化, 降低复合 GSK-3β治疗大鼠的 CDK5、Aβ、蛋白和 mRNA 表达21.同时, 尚等人也报告说, SBF 可以抑制星形胶质细胞和小胶质细胞的增殖, 降低 Aβ1-40、Aβ1-42 和β部位应用裂解酶 1 (BACE1) mRNA 在脑内合成 Aβ大鼠22。在上述结果的基础上, 我们的动物模型优于其他类似于 AD 的模型, 它涉及更多的神经功能和结构紊乱。
对于其他类似 AD 的模型, 单脑室注射 Aβ对大鼠可能造成大鼠记忆缺损, 神经元丢失, neurogliocyte 增殖, 但可能或可能没有 Aβ和测试沉积23。暴露于高剂量 Al 的大鼠似乎有很高的成功率, 模拟广告, 和一个经济高效的动物模型, 与记忆障碍, 神经元丢失, neurogliocyte 增殖, 和老年斑块 (SP) 和测试聚集在大脑中。然而, 高剂量 Al 可能会导致大鼠肝损伤和厌食, 伴随着体重下降24。衰老的老鼠是另一种类似广告的模式。老年大鼠表现为记忆缺损、神经元结构/子组织病理改变、脂褐素沉积, 但无 Aβ负担和测试聚集。24月以上的大鼠被认为是老年人的模型, 因此需要较长的喂养时间, 因而成本更高17,25。SAMP8 和 APP 转基因小鼠是最接近广告的模仿, 它们是调查广告最理想的模型。但两种动物模型都价格较高, 仅限于实验室26、27中使用。与上述动物模型相比, 我们的复合 Aβ动物模型具有成本低、性能高的特性, 成为研究广告的理想工具。
总之, 脑室注射液 Aβ25-35 联合 AlCl3和 TGF-β1对大鼠提供了一个有价值的活体动物模型, 以更好地了解空间记忆障碍, 神经元损伤, Aβ负担, 测试沉积基础广告。该模型提供了一种快速、相对简单的实验方案, 其动物生存率高, 模型成功率高, 重复率高, 显示出更经济性。目前的动物模型是一种模拟 AD 的有效模型, 可以通过模拟各种其他疾病来进一步验证。

众所周知, AD 是一种慢性和渐进性神经变性疾病, 以逐渐记忆丧失为主要临床综合征。在一般病理学中, 有神经组织萎缩, 神经元和突触丢失, 以及神经元亚细胞结构和功能紊乱, 这些都参与了 AD1,2的发展和临床表现。据报道, 当动物被 intracerebroventricularly 注射 Aβ, 一些神经毒性事件发生在大脑涉及神经元丢失, 钙稳态干扰, 神经元凋亡, 和活性氧的过剩3。然而, 广告的发病机制涉及到多种因素, 因此建立一个较好的 ad 模型是十分必要的。
本文介绍了一种通过脑室注射 Aβ25-35 和 AlCl3, 结合 anterodorsal 丘脑核注射 RHTGF-β1大鼠体内模拟 AD 模型的详细协议。该鼠 modelhighly 模仿人的神经功能和 histopathogenesis 的 AD, 包括记忆障碍, 神经元丢失和结构损伤, 细胞凋亡, 细胞内 Aβ负担, 测试聚集4,5,6,7,8,9. AlCl3防止沉积 Aβ形成可溶性 Aβ, RHTGF-β1可促进沉积 Aβ生产, 促进 AD 发生10。这种从几个因素到神经元的攻击是符合 AD 的多病机的。
整个实验跨越了86天:图 1显示了实验设计的时间线, 有动物外科、动物模型筛选、动物空间记忆测试和样品准备。在手术的第一天, RHTGF-β1被杏仁到 anterodorsal 丘脑核。手术第二天, Aβ25-35 和 AlCl3每天早上连续14天杏仁到侧脑室, 下午连续5天。在手术后45天内, 所有老鼠都被允许康复。莫里斯水迷宫用于筛查成功的模型大鼠记忆障碍和评估大鼠的空间记忆。大鼠连续4天进行水迷宫训练, 每天进行2次试验, 在4天的训练中, 用莫里斯水迷宫对记忆障碍的表现进行评价。在动物模型筛选37天后, 所有的老鼠继续喂食。在莫里斯水迷宫中连续7天对大鼠的空间记忆进行了试验, 从79天到85日的手术后。所有的老鼠都是在86天被斩首, 为脑标本准备。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56157/56157fig1.jpg"> 图 1。实验设计的时间线。<a href="//cloudflare.jove.com/files/ftp_upload/56157/56157fig1large.jpg">请单击此处查看此图的较大版本.</a>

<table>
	<tbody>
		<tr>
			<td>Sprague-Dawley rat</td>
			<td>Beijing Vital River Laboratory Animal Technology Co., Ltd, China</td>
			<td>SCXK(Jing) 2012-0001</td>
			<td>300&#8211;350 g</td>
		</tr>
		<tr>
			<td>Morris water maze</td>
			<td>Chinese Academy of Medical Sciences and Peking Union Medical Research Institute, China</td>
			<td>No</td>
			<td></td>
		</tr>
		<tr>
			<td>Movable small animal anesthesi</td>
			<td>RWD Life Science Co., Ltd. China</td>
			<td>R580</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain Stereotaxic Apparatus</td>
			<td>RWD Life Science Co., Ltd. China</td>
			<td>68001</td>
			<td></td>
		</tr>
		<tr>
			<td>Flexible bone drill</td>
			<td>Shanghai Soft Long Technology Development Co., Ltd. China</td>
			<td>BW-sD908</td>
			<td></td>
		</tr>
		<tr>
			<td>Transmission electron microscope</td>
			<td>Japan Co., Ltd. Japan</td>
			<td>JEM-1400</td>
			<td></td>
		</tr>
		<tr>
			<td>Two channel microinjection pump</td>
			<td>RWD Life Science Co., Ltd. China</td>
			<td>RWD202</td>
			<td></td>
		</tr>
		<tr>
			<td>EM microtome</td>
			<td>Hitachi Co., Ltd. China</td>
			<td>H-7650</td>
			<td></td>
		</tr>
		<tr>
			<td>Dummy cannula</td>
			<td>RWD Life Science Co., Ltd. China</td>
			<td>62001</td>
			<td>0.D.0.64&#215;I.D.0.0.45mm/M3.5 
			http://www.rwdls.com/English/Product/3985102014.html</td>
		</tr>
		<tr>
			<td>Guide cannula</td>
			<td>RWD Life Science Co., Ltd. China</td>
			<td>62101</td>
			<td>0.D.0.40mm/M3.5</td>
		</tr>
		<tr>
			<td>Internal cannula</td>
			<td>RWD Life Science Co., Ltd. China</td>
			<td>62201</td>
			<td>0.D.0.41&#215;I.D.0.25mm/<img alt="Special" src="/files/ftp_upload/56157/special01.jpg" />M3.5</td>
		</tr>
		<tr>
			<td>Tighten the nut</td>
			<td>RWD Life Science Co., Ltd. China</td>
			<td>62501</td>
			<td>0.D.5.5mm/L7.5mm/M3.5</td>
		</tr>
		<tr>
			<td>Fixing screw</td>
			<td>RWD Life Science Co., Ltd. China</td>
			<td>62514</td>
			<td>M1.2&#215;L2.0mm(100BAO)</td>
		</tr>
		<tr>
			<td>The screwdriver</td>
			<td>RWD Life Science Co., Ltd. China</td>
			<td>62999</td>
			<td>45*1mm</td>
		</tr>
		<tr>
			<td>PE Tubing</td>
			<td>RWD Life Science Co., Ltd. China</td>
			<td>62302</td>
			<td></td>
		</tr>
		<tr>
			<td>Amyloid beta 25-35</td>
			<td>Sigma Aldrich Co. USA</td>
			<td>SCP0002-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human transforming growth factor-&#946;1</td>
			<td>PeproTech Inc. USA</td>
			<td>100-21</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminium trichloride</td>
			<td>Tianjin Kemiou Chemical Reagent Co., Ltd. China</td>
			<td>3011080</td>
			<td></td>
		</tr>
		<tr>
			<td>Congo red</td>
			<td>Tianjin Kemiou Chemical Reagent Co., Ltd. China</td>
			<td>3010016</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver nitrate</td>
			<td>Sinopharm Chcmical Reagent Co., Ltd. China</td>
			<td>20150720</td>
			<td></td>
		</tr>
		<tr>
			<td>Zinc phosphate dental cement</td>
			<td>Dental Material of Factory Shanghai Medical Instruments Co., Ltd. China</td>
			<td>201311</td>
			<td></td>
		</tr>
	</tbody>
</table>

establishment of a valuable mimic of alzheimer's disease in rat animal model by intracerebroventricular injection of composited amyloid beta protein

阿尔茨海默病 (AD) 是一种不可逆转的渐进性脑病, 它会慢慢地破坏记忆, 伴随着神经元的丢失和结构的改变。随着世界各地 AD 患者的增多, 疾病的病理和治疗已成为国际制药行业关注的焦点。因此, 在实验室中建立模仿广告的动物模型是非常重要的。
在这里, 我们描述了一个在大鼠动物模型中建立 AD 模拟的详细协议, 尽管脑室注射淀粉样β蛋白 25-35 (Aβ25-35) 与三氯化铝 (AlCl3) 和 anterodorsal 丘脑核相结合重组人转化生长 factor-β1 (RHTGF-β1) 对大鼠的注射。对 AD 的相关标记进行了测量, 包括: 空间记忆、神经元结构和子构造、神经元 Aβ和神经原纤维缠结 (测试) 生产。该模型显示了空间记忆障碍、神经元结构和子组织病理变化、神经元细胞内 Aβ负担和测试聚集, 并为神经结构和功能紊乱的临床研究提供了一个密切的模拟AD 病人。因此, 所提出的 ad 大鼠 modelprovides 一个有价值的体内工具来探索神经功能, 神经病理和药物筛选的 ad。

所有的数据都显示为平均值的电子扫描电镜. 用 SAS/统计包计算统计分析。利用方差分析和重复测量的方法, 分析了在记忆习得和记忆再学习试验中寻找隐藏平台的组滞后时间差异。采用单向方差分析法, 对邓肯的多范围试验进行了探针试验和神经元个数的组差异。p < 0.05 被考虑统计学上重要18。
复合 Aβ治疗组记忆障碍成功模型大鼠的筛选:
图 2AA1 和 2AA2的结果表明, 假手术组大鼠总是自由游动, 复合 Aβ治疗组大鼠 (图 2AB1, AB2) 总是游在泳池周围的自适应游泳在莫里斯水迷宫。在4天的记忆损伤模型大鼠的筛查中, 所有的老鼠都在逐渐下降, 以找到隐藏的平台 (潜伏期) (图 2B)。4天的莫里斯水迷宫训练, 如果 SR 超过 0.2 (这是基于每一个复合 Aβ处理大鼠的潜伏期和假操作的大鼠寻找隐藏平台), 那么复合 Aβ治疗大鼠被认为是成功的模型大鼠记忆障碍。18的19只老鼠 (94.70%) 幸存的手术通过了成功的模型筛选. 为下列实验选择了每组6只大鼠。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56157/56157fig2.jpg"> 图 2. 复合 Aβ治疗组记忆障碍模型大鼠的筛选使用莫里斯水迷宫训练.(A) 莫里斯水迷宫中大鼠的自适应游泳轨迹。(AA1-AA2)假手术组;(AB1-AB2)复合 Aβ治疗组。(B) 为假手术组和复合 Aβ治疗组在莫里斯水迷宫训练中连续4天找到隐藏平台的平均潜伏期。 图已从参考4修改。<a href="https://cloudflare.jove.com/files/ftp_upload/56157/56157fig2large.jpg">请单击此处查看此图的较大版本.</a>
复合 Aβ引起大鼠记忆习得和记忆重新学习障碍:
在莫里斯水迷宫试验1和2天的定位导航试验确定了大鼠记忆采集, 对应于79天和80后手术。在记忆采集试验的2天里, 所有的老鼠都呈现出逐渐下降的潜伏期, 以找到隐藏的平台。如图 3所示, 复合 Aβ治疗组寻找隐藏平台的潜伏期为360.67% 和 558.28% (F (1, 5) = 238.67, p < 0.01), 比在莫里斯水的1和2天的假操作组大。迷宫测试, 分别。这表明复合 Aβ可诱发大鼠记忆损伤。
在莫里斯水迷宫试验4、5和6天的逆转试验中, 对大鼠记忆重新学习进行了测定, 该实验对应于82、83和84后手术。如图 3所示, 复合 Aβ治疗组寻找隐藏平台的潜伏期为306.20%、650.16% 和 936.92%, 较假手术组的时间长 (F (1, 5) = 138.76, p < 0.01)。这说明复合 Aβ可以提高大鼠记忆再学习障碍 (图 3)。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56157/56157fig3.jpg"> 图 3.复合 Aβ引起大鼠记忆习得和记忆再学习障碍.定位导航试验用于评估在莫里斯水迷宫试验中, 1 和2天连续2天游泳成绩的记忆习得。这些是在79天和80的手术后进行的。逆转试验被用来评估在莫里斯水迷宫试验中, 在4、5和6天连续3天游泳成绩的记忆重新学习, 这对应于手术的82、83和84天。在莫里斯水迷宫试验中, 折线图图形显示了在1、2、4、5和6天为每个组寻找隐藏平台的平均滞后时间。采用双向方差分析 (日 x 组) 对数据进行重复测量。意思是 "扫描电镜" n = 6。**p < 0.01, 与假手术组。图已从参考4修改。<a href="https://cloudflare.jove.com/files/ftp_upload/56157/56157fig3large.jpg">请单击此处查看此图的较大版本.</a>
复合 Aβ引起大鼠记忆保留损伤:
在莫里斯水迷宫试验3天, 通过探针试验, 对大鼠记忆保留量进行了测量, 对应于81天的术后手术。在1天的记忆保留试验中, 复合 Aβ治疗组在六十年代内减少了游泳时间、游泳距离和交叉数 Q1, 分别对应于32.14%、30.11% 和 78.95% (p < 0.01), 而不是假操作组 (图 4A, 4B)。结果表明, 复合 Aβ可产生大鼠记忆保留损伤。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56157/56157fig4.jpg"> 图 4.复合 Aβ产生大鼠记忆保留损伤.探针试验被用来评估1天游泳成绩3天在莫里斯水迷宫试验, 这是在81天后手术进行的记忆保留。(A) 六十年代内 Q1 的游泳时间、游泳距离和过境人数 (无平台)。用单向方差分析法对数据进行多范围测试。意思是 "扫描电镜" n = 6。**p < 0.01, 与假经营的集团。(B) 大鼠在探针试验中的游泳轨迹。(A) 假手术组, 在目标象限内显示更大的游泳时间和距离 (Q1)。(B) 复合 Aβ治疗组, 在目标象限 (Q1) 中显示较少的游泳时间和距离。图已从参考4修改。<a href="https://cloudflare.jove.com/files/ftp_upload/56157/56157fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
复合 Aβ对大鼠游泳速度的影响:
在莫里斯水迷宫试验7天, 通过可见光平台试验计算了大鼠游泳速度, 对应于85天后手术。在大鼠游泳速度的基础上, 在计算游泳距离和时间步长的平台上, 各组在池中的差异没有显著性。因此, 在大鼠游泳速度的个体差异可以排除, 这表明动机和运动技能基本上是完整的所有老鼠 (图 5)。
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56157/56157fig5.jpg"> 图 5.复合 Aβ对大鼠游泳速度的影响。在85天的莫里斯水迷宫试验7天, 通过可见光平台试验计算了大鼠游泳速度。各组大鼠游泳速度无显著性差异。用单向方差分析法对数据进行多范围测试。意思是 "扫描电镜" n = 6。图已从参考4修改。<a href="https://cloudflare.jove.com/files/ftp_upload/56157/56157fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
复合 Aβ引起大鼠神经细胞形态学改变:
在86天的手术中, 所有的老鼠都被斩首致死。目视检查发现, 在几种复合 Aβ治疗大鼠中, 出现了黄色表面或薄或折叠的大脑皮层。与假手术组 (图 6AA2) 相比, Aβ治疗组经 he 染色的光学显微观察, 发现海马神经元病理改变, 如神经原纤维变性、neuronophagia、核固缩和核 margination (图 6AB2)。此外, 复合 Aβ治疗组的大脑皮层部分显示典型的 colliquative 坏死, 其特征是细胞膜紊乱, 细胞核分散, 炎症细胞浸润在坏死区 (图 6AB3)。这表明复合 Aβ可能导致大鼠神经细胞结构病理损伤。
除了神经结构的病理变化外, 与假手术组相比, 在海马和大脑皮层 (colliquative 坏死样本除外) 的神经元计数也显著降低。Aβ治疗组。神经元数目为 63.86% (p < 0.01), 低于在海马 CA1 节0.125 毫米和 55.46% (p < 0.01) 低的大脑皮质部分 0.0352 mm2 (图 6B) 的假手术组, 这表明复合 Aβ可能导致神经元数量减少。
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56157/56157fig6.jpg"> 图 6.复合 Aβ引起大鼠神经细胞形态学改变.(A) 有代表性的海马和大脑皮层神经元的图像与他的染色。(A1-B1)海马 40x;(A2-B2)海马 CA1 400x;(A3-B3)大脑皮层400x。(A1-A3)假手术组;(B1-B3)复合 Aβ治疗组;显示神经元标记丢失, 神经原纤维变性 (→), neuronophagia (←), 核固缩 (↗), 核 margination (↙) 在海马, 典型的 colliquative 坏死 (★), 扰乱细胞膜, 大量的炎症细胞浸润在大脑皮层部分的复合 Aβ治疗组。A1 的鳞片杆, B1 = 10 µm;鳞片酒吧A2, B2, A3, B3 = 100 µm. (B) 海马和大脑皮层有他染色的神经元数量, 在光显微镜下计算 (400x)。每卷代表了9个可视领域的3个独立的样本 (n = 3) 的平均的电子扫描电镜。**p < 0.01, 与假手术组。 图已从参考4修改。<a href="https://cloudflare.jove.com/files/ftp_upload/56157/56157fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
电镜观察海马神经元亚细胞超微结构。复合 Aβ治疗组 (图 7B1–B4) 海马神经元的子结构明显破坏, 显示线粒体肿胀和嵴断裂, 线粒体电子密度增加, 扩张粗糙与假手术组比较, 内质网, 降解核糖体和 polymicrotubules, 一些突触后密度 (PSD), 许多次生溶酶体, 和脂褐素沉积物 (图 7A1–A4)。核膜粗、凹陷, 染色质凝结变性, 髓鞘层松散或变性, 内轴突和纤维衰减。 结果表明, 复合 Aβ可产生大鼠神经元亚结构损伤。
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56157/56157fig7.jpg"> 图 7.用电镜观察评价海马神经元亚细胞结构。A1-A4: 假手术组。A1 的鳞片杆= 4 µm, 12,000x;A2的鳞片杆 = 3 µm, 15,000x;A3的鳞片杆 = 5 µm, 10,000x;A4的刻度条 = 1 µm, 35,000x。(B1-B4)复合 Aβ治疗组。(B1)神经元和核固缩 (←), 染色质凝结或变性 (#), 星形胶质细胞足肿胀 (*), 高电子密度线粒体 (▲), 髓鞘层松散或衰减 (→);(B2)更大的 GFAP, 高电子密度线粒体 (▲), 髓鞘层松散或衰减 (), 更大的次生溶酶体 (↑), 毛细血管固缩, 毛细血管染色质冷凝或变性 (☆), 星形胶质细胞足肿胀 (*), 高电子密度线粒体 (▲), 更多的脂褐素 (↓), 髓鞘层松散或衰减 (→)。B4: 更多兴奋性神经递质 (#), 高电子密度或损伤膜线粒体 (▲), 少联会。B1 的鳞片杆, B2 = 10 µm, 5,000x;B3的鳞片杆 = 5 µm, 8,000x;B4的刻度条 = 1 µm, 40,000x。图已从参考4修改。<a href="https://cloudflare.jove.com/files/ftp_upload/56157/56157fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
复合 Aβ引起大鼠神经元 Aβ负担:
刚果红染色用于检测神经元的 Aβ负担。结果表明, 复合 Aβ可显著诱发大鼠海马和大脑皮层细胞内 Aβ负担 (图 8A)。在复合 Aβ治疗组的海马和大脑皮层, Aβ红色的细胞阳性数为8.05 和4.09 倍 (p < 0.01) 大于假手术组 (图 8B)。这说明复合 Aβ能增加大鼠神经元 Aβ负担。
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/56157/56157fig8.jpg"> 图 8.复合 Aβ引起大鼠神经元 Aβ负担.(A) 刚果红在海马和大脑皮层染色的阳性 Aβ神经元的代表性图像。(A1-B1)海马 CA1 400x;(A2-B2)大脑皮层400x。(A1-A2)假手术组;(B1-B2)复合 Aβ治疗组, 显示更多的 Aβ阳性细胞染色的刚果红。刻度条 = 10 µm, 400x。(B) 由刚果红在海马和大脑皮层染色的 Aβ神经元的阳性数目, 在光显微镜下计算 (400x)。每卷代表了9个可视领域的3个独立的样本 (n = 3) 的平均的电子扫描电镜。**p < 0.01, 与假手术组。<a href="https://cloudflare.jove.com/files/ftp_upload/56157/56157fig8large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
复合 Aβ引起大鼠神经元测试沉积:
硝酸银染色用于检测神经元中的测试沉积。结果表明, 复合 Aβ可明显引起大鼠海马和大脑皮层细胞内测试沉积 (图 9A)。复合 Aβ治疗组海马和大脑皮层中硝酸银染测试褐色细胞阳性数为9.75 和4.82 倍 (p < 0.01) 大于假手术组 (图 9B).这表明, 复合 Aβ可以增加大鼠神经元测试聚集。
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/56157/56157fig9.jpg"> 图 9.复合 Aβ引起大鼠神经元测试聚集.(A) 海马和大脑皮层中硝酸银染色的阳性测试神经元的代表性图像。(A1-B1)海马 CA1 400x;(A2-B2)大脑皮层400x。(A1-A2)假手术组;(B1-B2)复合 Aβ治疗组。显示在复合处理组中硝酸银染色的测试阳性细胞越多。刻度条 = 10 µm, 400x。(B) 在光镜下 (400x) 计算的海马和大脑皮层中硝酸银染色的阳性测试神经元数。每卷代表了9个可视领域的3个独立的样本 (n = 3) 的平均的电子扫描电镜。**p < 0.01, 与假手术组。<a href="https://cloudflare.jove.com/files/ftp_upload/56157/56157fig9large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Establishment of a Valuable Mimic of Alzheimer's Disease in Rat Animal Model by Intracerebroventricular Injection of Composited Amyloid Beta Protein at JoVE.c

Establishment of a Valuable Mimic of Alzheimer's Disease in Rat Animal Model by Intracerebroventricular Injection of Composited Amyloid Beta Protein

通过对 Aβ25-35 联合注射诱导的空间记忆损伤、神经元病理变化、神经元淀粉样蛋白 (Aβ) 负担和神经原纤维缠结聚集的评价, 这是一种模拟阿尔茨海默病的协议。与三氯化铝和重组人转化生长 factor-β1。

脑室注射复合淀粉样蛋白β模型建立大鼠阿尔茨海默病的价值模拟

Alzheimer&#39;s disease (AD) is an irreversible, progressive brain disease that slowly destroys memory and is accompanied by neuron loss and structure ...

establishment-valuable-mimic-alzheimer-s-disease-rat-animal-model

Research

JoVE Journal

Behavior

11.7K 次观看.  Chengde Medical College. 通过对 Aβ25-35 联合注射诱导的空间记忆损伤、神经元病理变化、神经元淀粉样蛋白 (Aβ) 负担和神经原纤维缠结聚集的评价, 这是一种模拟阿尔茨海默病的协议。与三氯化铝和重组人转化生长 factor-β1。

视频: Establishment of a Valuable Mimic of Alzheimer's Disease in Rat Animal Model by Intracerebroventricular Injection of Composited Amyloid Beta Protein

Mouse Short- and Long-term Locomotor Activity Analyzed by Video Tracking Software

Locomotor activity (LMA) is a simple and easily performed measurement of behavior in mice and other rodents. Improvements in video tracking software (VTS) have allowed it to be coupled to LMA testing, dramatically improving specificity and sensitivity when compared to the line crossings method with manual scoring. In addition, VTS enables high-throughput experimentation. While similar to automated video tracking used for the open field test (OFT), LMA testing is unique in that it allows mice to remain in their home cage and does not utilize the anxiogenic stimulus of bright lighting during the active phase of the light-dark cycle. Traditionally, LMA has been used for short periods of time (mins), while longer movement studies (hrs-days) have often used implanted transmitters and biotelemetry. With the option of real-time tracking, long-, like short-term LMA testing, can now be conducted using videography. Long-term LMA testing requires a specialized, but easily constructed, cage so that food and water (which is usually positioned on the cage top) does not obstruct videography. Importantly, videography and VTS allows for the quantification of parameters, such as path of mouse movement, that are difficult or unfeasible to measure with line crossing and/or biotelemetry. In sum, LMA testing coupled to VTS affords a more complete description of mouse movement and the ability to examine locomotion over an extended period of time.

Locomotor activity (LMA) is a simple and easily performed measurement of behavior in mice. Coupling of video tracking software (VTS) and LMA allows for the improvement of specificity and sensitivity, especially when compared with the manual, line crossing method of LMA analysis. Additionally VTS allows long-term tracking of mouse LMA.

鼠标短期和长期活动性分析视频跟踪软件

Locomotor  activity (LMA) is a simple and easily performed measurement of behavior in mice  and other rodents. Improvements in video tracking software ...

科研

行为学

The Use of the Puzzle Box as a Means of Assessing the Efficacy of Environmental Enrichment

Environmental enrichment can dramatically influence the development and function of neural circuits. Further, enrichment has been shown to successfully delay the onset of symptoms in models of Huntington&#8217;s disease 1-4, suggesting environmental factors can evoke a neuroprotective effect against the progressive, cellular level damage observed in neurodegenerative disorders. The ways in which an animal can be environmentally enriched, however, can vary considerably. Further, there is no straightforward manner in which the effects of environmental enrichment can be assessed: most methods require either fairly complicated behavioral paradigms and/or postmortem anatomical/physiological analyses. This protocol describes the use of a simple and inexpensive behavioral assay, the Puzzle Box 5-7 as a robust means of determining the efficacy of increased social, sensory and motor stimulation on mice compared to cohorts raised in standard laboratory conditions. This simple problem solving task takes advantage of a rodent&#8217;s innate desire to avoid open enclosures by seeking shelter. Cognitive ability is assessed by adding increasingly complex impediments to the shelter&#8217;s entrance. The time a given subject takes to successfully remove the obstructions and enter the shelter serves as the primary metric for task performance. This method could provide a reliable means of rapidly assessing the efficacy of different enrichment protocols on cognitive function, thus paving the way for systematically determining the role specific environmental factors play in delaying the onset of neurodevelopmental and neurodegenerative disease.

Environmental enrichment provides a potential protective effect against neurodegenerative disorders. Currently, however, there is no easy way of determining the efficacy of enrichment procedures. This protocol describes a simple &#8220;Puzzle Box&#8221; method for assessing an animal&#8217;s cognitive function, in order to reveal the effectiveness of environmental enrichment.

该拼图方块的使用作为评价环境富集的功效的一种手段

Environmental enrichment can dramatically influence the development and function of neural circuits.  Further, enrichment has been shown to successfully ...

Signal Attenuation as a Rat Model of Obsessive Compulsive Disorder

In the signal attenuation rat model of obsessive-compulsive disorder (OCD), lever-pressing for food is followed by the presentation of a compound stimulus which serves as a feedback cue. This feedback is later attenuated by repeated presentations of the stimulus without food (without the rat emitting the lever-press response). In the next stage, lever-pressing is assessed under extinction conditions (i.e., no food is delivered). At this stage rats display two types of lever-presses, those that are followed by an attempt to collect a reward, and those that are not. The latter are the measure of compulsive-like behavior in the model. A control procedure in which rats do not experience the attenuation of the feedback cue serves to distinguish between the effects of signal attenuation and of extinction. The signal attenuation model is a highly validated model of OCD and differentiates between compulsive-like behaviors and behaviors that are repetitive but not compulsive. In addition the measures collected during the procedure eliminate alternative explanations for differences between the groups being tested, and are quantitative, unbiased and unaffected by inter-experimenter variability. The major disadvantages of this model are the costly equipment, the fact that it requires some technical know-how and the fact that it is time-consuming compared to other models of OCD (11 days). The model may be used for detecting the anti- or pro-compulsive effects of pharmacological and non-pharmacological manipulations and for studying the neural substrate of compulsive behavior.

The goal of the protocol described in this paper is to induce compulsive-like behavior in rats for the study of obsessive-compulsive disorder (OCD). This behavior is precipitated by attenuating a signal indicating that a lever-press response was effective in producing food.

信号衰减为强迫症的大鼠模型

In the signal attenuation rat model of obsessive-compulsive disorder (OCD), lever-pressing for food is followed by the presentation of a compound stimulus ...

Applying Stereotactic Injection Technique to Study Genetic Effects on Animal Behaviors

Stereotactic injection is a useful technique to deliver high titer lentiviruses to targeted brain areas in mice. Lentiviruses can either overexpress or knockdown gene expression in a relatively focused region without significant damage to the brain tissue. After recovery, the injected mouse can be tested on various behavioral tasks such as the Open Field Test (OFT) and the Forced Swim Test (FST). The OFT is designed to assess locomotion and the anxious phenotype in mice by measuring the amount of time that a mouse spends in the center of a novel open field. A more anxious mouse will spend significantly less time in the center of the novel field compared to controls. The FST assesses the anti-depressive phenotype by quantifying the amount of time that mice spend immobile when placed into a bucket of water. A mouse with an anti-depressive phenotype will spend significantly less time immobile compared to control animals. The goal of this protocol is to use the stereotactic injection of a lentivirus in conjunction with behavioral tests to assess how genetic factors modulate animal behaviors.

Stereotactic injection of lentiviruses expressing cDNAs or shRNAs can modulate gene expression in specific brain areas of mice. Here, we present a protocol to combine stereotactic injections with behavioral tasks, such as the Open Field Test (OFT) and the Forced Swim Test (FST).

应用立体定向注射技术在研究动物行为的遗传效应

Stereotactic injection is a useful technique to deliver high titer lentiviruses to targeted brain areas in mice. Lentiviruses can either overexpress or ...

The 4 Mountains Test: A Short Test of Spatial Memory with High Sensitivity for the Diagnosis of Pre-dementia Alzheimer's Disease

This protocol describes the administration of the 4 Mountains Test (4MT), a short test of spatial memory, in which memory for the topographical layout of four mountains within a computer-generated landscape is tested using a delayed match-to-sample paradigm. Allocentric spatial memory is assessed by altering the viewpoint, colors and textures between the initially presented and target images.
Allocentric spatial memory is a key function of the hippocampus, one of the earliest brain regions to be affected in Alzheimer&#39;s disease (AD) and impairment of hippocampal function predates the onset of dementia. It was hypothesized that performance on the 4MT would aid the diagnosis of predementia AD, which manifests clinically as Mild Cognitive Impairment (MCI).
The 4MT was applied to patients with MCI, stratified further based on cerebrospinal fluid (CSF) AD biomarker status (10 MCI biomarker positive, 9 MCI biomarker negative), and with mild AD dementia, as well as healthy controls. Comparator tests included tests of episodic memory and attention widely accepted as sensitive measures of early AD. Behavioral data were correlated with quantitative MRI measures of the hippocampus, precuneus and posterior cingulate gyrus.
4MT scores were significantly different between the two MCI groups (p = 0.001), with a test score of &#8804;8/15 associated with 100% sensitivity and 78% specificity for the classification of MCI with positive AD biomarkers, i.e., predementia AD. 4MT test scores correlated with hippocampal volume (r = 0.42) and cortical thickness of the precuneus (r = 0.55).
In conclusion, the 4MT is effective in identifying the early stages of AD. The short duration, easy application and scoring, and favorable psychometric properties of the 4MT fulfil the need for a simple but accurate diagnostic test for predementia AD.

This article describes the 4 Mountains Test (4MT), a hippocampus-dependent test of working allocentric spatial memory. The hippocampus is affected early in Alzheimer's disease (AD) and this article outlines the 4MT methodology and results of patient testing, which demonstrates the value of the 4MT in the diagnosis of pre-dementia AD.

四山测试:空间记忆的一个简短的测试与高灵敏度的预痴呆诊断阿尔茨海默氏病

This protocol describes the administration of the 4 Mountains Test (4MT), a short test of spatial memory, in which memory for the topographical layout of ...

Wheel Running and Environmental Complexity as a Therapeutic Intervention in an Animal Model of FASD

Aerobic exercise (e.g., wheel running (WR) extensively used in animal research) positively impacts many measures of neuroplastic potential in the brain, such as rates of adult neurogenesis, angiogenesis, and expression of neurotrophic factors in rodents. This intervention has also been shown to mitigate behavioral and neuroanatomical aspects of the negative impacts of teratogens (i.e., developmental exposure to alcohol) and age-related neurodegeneration in rodents. Environmental complexity (EC) has been shown to produce numerous neuroplastic benefits in cortical and subcortical structures and can be coupled with wheel running to increase the proliferation and survival of new cells in the adult hippocampus. The combination of these two interventions provides a robust &#34;superintervention&#34; (WR-EC) that can be implemented in a range of rodent models of neurological disorders. We will discuss the implementation of WR/EC and its constituent interventions for use as a more powerful therapeutic intervention in rats using the animal model of prenatal exposure to alcohol in humans. We will also discuss which elements of the procedures are absolutely necessary for the interventions and which ones may be altered depending on the experimenter&#39;s question or facilities.

Cardiovascular exercise and stimulating experiences in a complex environment have positive benefits on multiple measures of neuroplasticity within the rodent brain. This article will discuss the implementation of these interventions as a &#34;superintervention&#34; which combines wheel running and environmental complexity and will address the limitations of these interventions.

车轮运行环境的复杂性在胎儿酒精中毒综合症的动物模型干预治疗

Aerobic exercise (e.g., wheel running (WR) extensively used in animal research) positively impacts many measures of neuroplastic potential in the brain, ...

Noninvasive, High-throughput Determination of Sleep Duration in Rodents

Traditionally, sleep is monitored by an electroencephalogram (EEG). EEG studies in rodents require surgical implantation of the electrodes followed by a long recovery period. To perform an EEG recording, the animal is connected to a receiver, creating an unnatural tether to the head-mount. EEG monitoring is time consuming, carries risk to the animal, and is not a completely natural setting for the measurement of sleep. Alternative methods to detect sleep, particularly in a high-throughput fashion, would greatly advance the field of sleep research. Here, we describe a validated method for detecting sleep via activity-based home-cage monitoring. Previous studies have shown that sleep assessed via this method has a high degree of agreement with sleep defined by traditional EEG-based measures. Whereas this method is validated for total sleep time, it is important to note that sleep bout duration should be assessed by an EEG which has better temporal resolution. The EEG can also differentiate rapid eye movement (REM) and non-REM sleep, giving more detail about the exact nature of sleep. Nevertheless, activity-based sleep determination can be used to analyze multiple days of undisturbed sleep and to assess sleep as a response to an acute event (like stress). Here, we show the power of this system to detect the response of mice to daily intraperitoneal injections.

We describe a high-throughput method of measuring sleep by means of activity-based home-cage monitoring. This method offers advantages over traditional EEG-based methods. It is well validated for the determination of total sleep duration and can be a powerful tool to monitor sleep in rodent models of human disease.

无创高通量测定啮齿类动物睡眠时间

Traditionally, sleep is monitored by an electroencephalogram (EEG). EEG studies in rodents require surgical implantation of the electrodes followed by a ...

Automated, Long-term Behavioral Assay for Cognitive Functions in Multiple Genetic Models of Alzheimer's Disease, Using IntelliCage

Multiple factors&#8212;such as aging and genes&#8212;are frequently associated with cognitive decline. Genetically modified mouse models of cognitive decline, such as Alzheimer&#39;s disease (AD), have become a promising tool to elucidate the underlying mechanisms and promote the therapeutic advances. An important step is the validation and characterization of expected behavioral abnormality in the models, in the case of AD, cognitive decline. The long-term behavioral investigations of laboratory animals to study the effect of aging demand substantial efforts from researchers. The IntelliCage system is a high-throughput and cost-effective test battery for mice that eliminates the need for daily human handling. Here, we describe how the system is utilized in the long-term phenotyping of a genetic Alzheimer&#39;s disease model, specifically focusing on the cognitive functions. The experiment employs repeated battery of tests that assess spatial learning and executive functions. This cost-effective age-dependent phenotyping allows us to identify the transient and/or permanent effects of genes on various cognitive aspects.

This paper describes a protocol for cognitive assessments for genetic models of the Alzheimer&#39;s disease using the IntelliCage system, which is a high throughput automated behavioral monitoring system with operant conditioning.

基于 IntelliCage 的阿尔茨海默病多基因模型认知功能的自动、长期行为检测

Multiple factors&#8212;such as aging and genes&#8212;are frequently associated with cognitive decline. Genetically modified mouse models of cognitive ...

Chronic Sleep Deprivation in Mouse Pups by Means of Gentle Handling

Sleep is critical for proper development and neural plasticity. Moreover, abnormal sleep patterns are characteristic of many neurodevelopmental disorders. Studying how chronic sleep restriction during development can affect adult behavior may add to our understanding of the emergence of behavioral symptoms of neurodevelopmental disorders. While there are many methods that can be used to restrict sleep in rodents including forced locomotion, constant disruption, presentation of an aversive stimulus, or electric shock, many of these methods are very stressful and cannot be used in neonatal mice. Here, we describe gentle handling, a sleep deprivation technique that can be used chronically throughout development and into adulthood to achieve sleep restriction. Gentle handling involves close observation of the mice throughout the sleep deprivation period and requires the researcher to gently prod the animals whenever they are inactive or display behaviors associated with sleep. Coupled with EEG recordings, gentle handling could be used to selectively disrupt a specific phase of sleep such as rapid eye movement (REM) sleep. The technique of gentle handling is a powerful tool for the study of the effects of chronic sleep restriction even in neonatal mice that circumvents many of the more stressful procedures used for sleep deprivation.

We describe a sleep deprivation technique known as gentle handling where investigators gently prod mice any time sleep behavior is observed. This method is a powerful tool that allows researchers to study the effects that chronic sleep restriction throughout development can have on future brain physiology and behavior.

小鼠幼犬慢性睡眠剥夺的温和处理方法

Sleep is critical for proper development and neural plasticity. Moreover, abnormal sleep patterns are characteristic of many neurodevelopmental disorders. ...

Behavioral Assessments of Spontaneous Locomotion in a Murine MPTP-induced Parkinson's Disease Model

Parkinson&#8217;s disease (PD) is a common neurodegenerative disorder disease, causing the phenomenon of shaking, rigidity, slowness of movement and dementia. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) can lead to some Parkinson&#8217;s-like symptoms by destroying dopaminergic neurons in the substantia nigra of the brain. It has been thus used to establish PD models in various animal studies. Here, mice receive MPTP injections (20 mg/kg/day) for seven days and the behavioral tests are performed on the eighth day. This model is adapted efficiently in the study of PD. The behavioral tests here include the cylinder test and the open field test. The cylinder experiment is used to detect the animals&#8217; ability to lift their front paws when put into a different environment. As the PD model mice show arching&#8212;the mouse arches its back&#8212;the number of paw liftings decrease. This test is easy to execute. The open field test is used to detect the amount of time the mice spend on running, walking, and remaining immobile. We analyze animals&#8217; movements in open field using software and obtain data. Lastly, we use L-DOPA, one of the most commonly used PD drugs, as one example to show how to apply this model to the study of PD drugs. Our results indicate that MPTP neurotoxicity induces motor deficit which can be mitigated by L-DOPA.

We describe establishment of a murine model for Parkinson's disease using MPTP, and behavioral assessments using cylinder and open field tests to measure motor function. We then use L-DOPA as one example to show how to apply this model in the study of PD drugs.

小鼠 mptp 诱导的帕金森病模型中自发性运动的行为评估

Parkinson&#8217;s disease (PD) is a common neurodegenerative disorder disease, causing the phenomenon of shaking, rigidity, slowness of movement and ...