Name: utilizing the modified t-maze to assess functional memory outcomes after cardiac arrest
Uploaded: 2018-01-05T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Utilizing the Modified T-Maze to Assess Functional Memory Outcomes After Cardiac Arrest at JoVE.com

这项工作得到了国家卫生研究院/国家神经紊乱和中风补助金 1R01NS096225-01A1, 美国心脏协会授予 AHA-13SDG1395001413, AHA-17GRNT33660336, AHA-17POST33660174,路易斯安那州立大学资助研究委员会, 马尔科姆费斯特心血管研究奖学金, 和伊夫林 F. 麦克奈特脑研究所。

所有实验程序都是按照国家卫生研究院的指导方针进行的, 并得到机构动物保育和使用委员会 (路易斯安那州立会健康科学中心-什里夫波特) 的批准, 用于雄性大大鼠的使用 (300-350 克, 9-10 周大)。大鼠在 ACA 手术前一夜禁食。
1. 迷宫设备的设计与设置
注: 在执事和罗的2006模型31上的 T 迷宫设计基础。
<ol><li>利用之32设计迷宫的3D 结构。要创建 T 型迷宫的3D 结构, 请构造一个外部长度为 200 mm、宽度为 165 mm 和高度 148 mm 的起始臂, 以适应3D 打印机的打印尺寸。使用壁厚5.5 毫米, 地板厚度为8毫米。</li>
	<li>使用3D 打印机打印迷宫 (请参阅材料表)32。如果实验室中没有3D 打印机, 请使用其他材料, 如木材、中密度纤维板或塑料 (即聚氯乙烯), 可以从家居改善商店购买。<ol>
<li>由于高度限制在打印区域, 构造迷宫的墙壁在二个分开的3D 印刷品并且一起在迷宫汇编 (即, 第二个墙壁高度被增加到迷宫部分增加高度由140毫米, 为总墙壁高度280毫米)。每个独立的3D 打印基座包含一个 "T" 形锁定机构, 其中一个部分连接到下一个。</li>
		<li>在起点胳膊的连接点与目标胳膊, 创造一个165毫米宽部分加入开始胳膊的宽度与目标胳膊的那。用类似的设计方法作为启动臂来构造目标臂;但是, 根据执事和罗的设计, 将手臂的宽度减小到100毫米。</li>
		<li>有关 T 形迷宫的详细示意图/维度, 请参见图 1 。</li>
		<li>在开始臂和目标臂的接合处包括一个中心分区到设计中。从 T 迷宫和200毫米的后壁扩展这个分区到开始胳膊划分目标胳膊。此分区还扩展了迷宫的高度 (图 1)。</li>
	</ol>
</li>
</ol>2. 窒息心脏骤停 (ACA)
<ol><li>高压釜手术工具 (121 ° c 为15分钟) 在开始手术之前。用70% 的乙醇消毒手术表15分钟, 在手术现场刮毛动物毛发。将控告溶液应用于皮肤表面进行外科手术。</li>
	<li>麻醉
	<ol>
<li>麻醉大鼠与4% 异氟醚和30:70 混合物 o2和 N2o (300 毫升/分钟 o 2 和700毫升/分钟 n 2O) 通过面具。</li>
		<li>给大鼠气管插管进行机械通气 (插管后, 大鼠与呼吸机连接)。</li>
		<li>通过将异氟醚降低4% 到 2%, 用30:70 混合物 o2和 N2来维持麻醉, 使用捏响应法确定麻醉深度。</li>
		<li>将药膏涂抹在眼部, 防止麻醉时干燥。Regulae 的体温由啮齿动物加热垫和肛门探针作为温度参考。</li>
	</ol></li>
	<li>气管插管
	<ol>
<li>把老鼠放在感应室里。麻醉4% 异氟醚和30:70 混合 o2和 N2o 的大鼠。</li>
		<li>从感应室取出老鼠将麻醉动物置于仰卧位, 将鼠脸朝向麻醉面罩。</li>
		<li>用左手拇指和食指轻轻地向动物的左边或右边移动舌头。</li>
		<li>滑行一个14口径的挠性静脉导管 (49 毫米长) 超过17口径钝尖移针 (93 毫米长, 在针的尖端10度角)。将17口径钝尖移针插入气管。</li>
		<li>从气管轻轻拉出17口径的移针。将14针导管中枢连接到呼吸器。调整呼吸机冲程容积为 0.67 mL/100 g, 呼吸率60呼吸/分钟。</li>
		<li>在整个过程中, 将头部和体温保持在37° c, 由啮齿动物加热垫以肛门探针作为温度参考。</li>
	</ol></li>
	<li>股动脉和静脉插管
	<ol>
<li>在腹股沟附近 (两侧) 剃毛, 并将控告到皮肤表面进行手术。</li>
		<li>把老鼠放在仰卧位。用手术剪刀在腹股沟区做一个切口 (10 毫米)。</li>
		<li>用钝尖钳分离结缔组织, 直至腹股沟韧带外露。用止血来抓腹股沟韧带。股动脉和静脉位于腹股沟韧带下方。</li>
		<li>使用钝尖端钳分离结缔组织, 直到股动脉和静脉暴露。</li>
		<li>用细尖钳将股神经沿股动脉轻轻分离。仔细分离股动脉和静脉作为一个单位通过细尖钳。</li>
		<li>用细尖钳将股动脉与静脉分离。</li>
		<li>在静脉下放置2件5-0 丝缝合线 (一条朝向腿部, 另一个朝向身体)。</li>
		<li>在身体旁边绑一个松散的结。使用止血来保持和拉缝合尽可能接近身体的两侧。</li>
		<li>在腿旁边系一个松散的结。通过止血将缝线拉向腿部, 使静脉充满血液。</li>
		<li>用微解剖剪刀 (45 °角) 在静脉 (大约0.1 毫米) 上做一个小切口。用消毒纱布浸泡任何血液。</li>
		<li>附上一个钝尖端针头注射器 (充满盐水与20ü/毫升肝素) 到一个 PE-50 导管。用生理盐水填充 PE-50 导管, 20 U/毫升肝素。切割 PE-50 导管与解剖剪刀在45°的角度, 以创建一个点或尖锐的结束。用钝尖钳夹住 PE-50 导管的末端。轻轻地将 PE-50 导管插入股静脉。</li>
		<li>在导管完全插入后, 慢慢地管理0.1 毫升的肝素/生理盐水, 以确保没有泄漏。扎牢固缝合结 (single-knot) 以稳定 PE-50 导管。保持 PE-50 导管持续静脉注射 (IV) 各种药物的注射。</li>
		<li>使用1毫升注射器连接23口存根适配器管理维库溴化 (0.67 毫克/千克, 管理每10分钟) 通过股静脉, 以固定在整个过程中的老鼠。</li>
		<li>在动脉下放置2件5-0 丝缝线 (一条朝向腿部, 另一根朝向身体)。</li>
		<li>在腿旁边系一个松散的结。使用止血来保持和拉缝合尽可能对腿部。</li>
		<li>在身体旁边绑一个松散的结。通过止血保持和拉动缝合体, 使动脉充满血液。</li>
		<li>用微解剖剪刀 (45 °角) 在动脉 (大约0.1 毫米) 上做一个小切口。</li>
		<li>附上一个钝尖端针头注射器 (充满盐水与20ü/毫升肝素) 到一个 PE-50 导管。用生理盐水填充 PE-50 导管, 20 U/毫升肝素。切割 PE-50 导管与解剖剪刀在45度角, 创造一个点或尖锐的结束。用钝尖钳夹住 PE-50 导管的末端。用钝尖钳夹住 PE-50 导管的末端。轻轻地将 PE-50 导管插入股动脉。</li>
		<li>在导管完全插入后, 慢慢地抽回注射器, 确保导管功能正常。扎牢固缝合结 (single-knot) 以稳定 PE-50 导管。保持 PE-50 导管连续记录动脉压力和血气。</li>
	</ol></li>
	<li>窒息心脏骤停 (ACA) 程序
	<ol>
<li>调整行程音量, o2或 N2o 级别, 根据需要调节生理参数 (如 pO2、不正常的2、血压和 pH 值)。使用这些参数的正常生理范围: pO2: 100 柱, 肺混浊,2: 35-40 柱, 血压: 100 柱, pH: 7.4。</li>
		<li>使用1毫升注射器连接23口存根适配器管理维库溴化铵 (0.67 毫克/千克, 静脉注射) 经股静脉, 并等待2分钟. 在执行 ACA 之前, 请确保血压在100柱左右。</li>
		<li>通过从呼吸机上拔下气管插管 (14 针导管中枢), 诱发窒息 (6 分钟)。通过1毫升注射器进一步阻断气管插管, 以确保完全窒息。 注意: 6 分钟的窒息时间被定义为呼吸机断开和复苏开始之间的时期。完整的心跳骤停被定义为平均动脉压低于10柱。</li>
		<li>在最后一分钟的呼吸暂停, 调整呼吸频率的呼吸机80呼吸/分钟, 并增加 O2到2升/分钟与 0% N2O。此操作将会将其余的异氟醚或 N2O 在呼吸机中残留。</li>
		<li>最小后呼吸暂停, 从气管插管中取出1毫升注射器。将气管插管重新连接到呼吸机。</li>
		<li>使用1毫升注射器连接23口的存根适配器管理肾上腺素 (0.005 毫克/千克, 静脉注射) 通过股静脉和管理手动胸部按压的拇指, 指数, 和中指在动物的胸部在 x 和轻循环运动z-axis (200/分钟) 直到自发循环返回 (平均动脉压力≥50柱)33,34,35。</li>
		<li>使用另1毫升注射器与23口的存根适配器连接, 以管理碳酸氢钠 (1 当量/千克, 静脉注射) 经股静脉后, 立即返回自发循环 (50 柱或更高)33,34,35以缓解呼吸道酸中毒。</li>
		<li>在复苏后10分钟测量血气, 以确定酸碱状态 (ACA 后的 pH 值应在7.35 到7.40 左右)</li>
		<li>用止血钳夹住股动脉和静脉。缓慢, 轻轻地用钝尖钳清除动脉和静脉导管。结扎股动脉/静脉5-0 丝缝合, 防止出血。用3-0 丝缝线缝合手术部位的皮肤。使用间断缝合技术, 以尽量减少伤口重新打开的机会。</li>
		<li>等待, 直到鼠呼吸本身 (通常是30分钟到60分钟后复苏), 断开从呼吸机的老鼠, 并轻轻地取出气管插管。</li>
		<li>将老鼠放在婴儿孵化器 (27 ° c, 50% 湿度) 过夜。将软化的食物 (通过浸泡在水中) 和水放入婴儿孵化器过夜。</li>
		<li>把老鼠转移到单独的笼子里, 然后把老鼠带到动物设施里, 用普通的食物和水。T 迷宫测试开始3天后 ACA。</li>
	</ol></li>
</ol>3. T 型迷宫
<ol><li>
动物制剂
	<ol>
<li>手术前一天 (假或 ACA), 处理每只老鼠5分钟。在处理 (图 2) 时, 切勿将老鼠从笼中提升 (480 mm x 250 mm x 200 mm, 塑料透明保持架)。</li>
		<li>在处理老鼠之后, 用一只手轻轻地把老鼠从尾巴上捡起来, 另一只手支撑着它的腿。让他们从手跳到笼子 (100 毫米高度) 5 次。把每只老鼠分成单独的笼子, 这样它们就不会在食物和/或战斗中占主导地位。</li>
		<li>假或 ACA 手术后三天 (图 2), 在第一次运行开始之前, 将笼子里的老鼠转移到一个安静、黑暗的房间里。只打开一个低功率的台灯, 并把它放在测试室的角落, 以保持最低照明。让老鼠适应黑暗10分钟。</li>
		<li>下午进行所有实验, 以避免日变化对大鼠的影响。不要建议操作者接受假或 ACA 手术。</li>
	</ol>
</li>
	<li>
自发交替
	<ol>
<li>铺一层薄薄的被褥 (〜10毫米厚) 覆盖整个地板的迷宫。然后将鼠放在开始臂 ("T" 的底部), 这是每一个运行的出发点, 并允许每只老鼠3分钟探索左右目标手臂。</li>
		<li>一旦老鼠提交到一个特定的目标手臂 (所有四只爪子的老鼠已经进入目标臂), 阻止 "T" 连接的开始臂和对方的目标手臂 (图 1), 以防止老鼠进入对方的目标手臂。把老鼠留在迷宫里30秒, 然后把老鼠捡起来放回笼子里, 时间最短 (约30秒)。然后从 t 形迷宫中取出 "t" 型接合块 (125 mm x 230 mm x 65 mm, 由3D 打印机制作)。</li>
		<li>把老鼠放在开始的手臂上, 重复3.2.2。交替定义为: 当鼠进入相反的手臂, 与以前的运行36。老鼠每天执行4次跑步, 如下所示: 1st运行 2nd运行 10分钟休息 3rd运行 4th运行</li>
		<li>改变在10分钟的休息和动物之间的床上用品, 以消除气味偏见。在每个实验日结束时, 用75% 乙醇和蒸馏水清洗 T 迷宫。</li>
		<li>重复步骤3.2.1。-3.2.4。两天 (总共运行 12), 如图 2所示。</li>
	</ol>
</li>
	<li>
交替率和边偏好率计算
	<ol>
<li>计算% 的替换和% 的边首选项, 其中 L: 老鼠选择左臂 R: 老鼠选择右臂 正确的选择: 2nd运行与给定集中的 1st不同 (每个集合包含两个运行) 不正确的选择: 大鼠选择相同的手臂类似于以前的运行 <img alt="Equation 1" src="/files/ftp_upload/56694/56694eq1.jpg"> <img alt="Equation 2" src="/files/ftp_upload/56694/56694eq2.jpg"> 例如: 1天: 升升/升 2天: 升升/升 R 3天: R 升/升 l 替换: 2 (正确的选择)/6 (执行的总集合) * 100 = 33.33% 边偏爱:10 (L, 优选的边)/12 (总奔跑执行) * 100 = 83.33%</li>
	</ol>
</li>
	<li>
术后护理:
	<ol>
<li>给老鼠丁丙诺啡 (0.01 毫克/千克 IP) 每12小时, 在手术后2天。观察大鼠心脏骤停后1小时。</li>
		<li>将大鼠附着在呼吸机和加热垫上, 直到它恢复足够的意识来维持胸骨卧床。为了保持动物的体温, 在手术后, 把老鼠放在一个婴儿孵化器 (设置在27° c, 50% 湿度)。</li>
		<li>在手术后的前24小时内, 向动物提供软化的 chow (浸泡在水中)。如果老鼠不喝水, 管理抑菌盐水 (100 毫升/公斤/天, ip), 直到动物恢复和饮用水自由。</li>
		<li>给大鼠外用抗生素止痛 (肽和利多卡因软膏) 在所有的伤口。在它们完全恢复后, 将老鼠移回动物设施。</li>
	</ol>
</li>
	<li>
安乐死法
	<ol>
<li>在实验结束时使用5% 异氟醚和 100% N2O 安乐动物。</li>
	</ol>
</li>
</ol>

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作者没有什么可透露的。

与执事和罗的协议31相比, 本研究做了一些修改。3D 打印机被用来建造 T 形迷宫。3的 d-印刷提供了可负担的和 cost-effective 的替代商业化 T 迷宫。为了减少实验过程中大鼠的焦虑, 在暗室中进行了最小光照的 T 迷宫。一旦鼠进入目标的武器之一, 我们轻轻地阻断对方的手臂。这避免了可能的压力, 从测试, 以及可能损害老鼠的尾巴, 而降低了断头台的门。任何不必要的压力都会抑制老...

根据美国心脏协会 (2017), 心脏骤停 (CA) 导致的死亡率每四分钟发生一次, 在美国每年影响超过40万人1。据记载, 由于血液灌注不足2,3,4, CA 可能导致神经元脑损伤。CA 诱发的脑损伤发生在海马的缺血敏感的 CA1 区5,6,7, 影响对学习和记忆至关重要的神经元8,9, 10、11、12。此外, 在海马 (即 CA1 神经元) 缺血条件下, 树突状脊柱密度的丧失在空间记忆损伤中起着重要作用13,14,15。由于这些病理变化的 CA, 行为障碍, 如: 焦虑, 抑郁症, 创伤后应激障碍和记忆丧失更普遍。尽管在医疗技术 (即有效的门诊服务) 方面取得了进步, 与提高的钙存活率相关, 但大多数的神经保护治疗 (低温除外) 在 ca 16 之后无法改善功能结果 ,17。CA 幸存者的生活质量通常很差, 并且背负着增加的医疗开支16。
通过行为测试对脑缺血的认知状态评估是重要的, 以确定药物疗效和最终发展成功的临床试验。在二十世纪四十年代, 爱德华托尔曼设计了第一个行为试验研究 hippocampus-based 空间记忆18。随后, 开发了不同的迷宫 (如莫里斯水迷宫、径向迷宫、T 或 Y 迷宫和巴恩斯迷宫) 来评估大鼠的 hippocampal-based 空间学习和记忆19,20,21,22 ,23。一个更广泛使用的行为测试是莫里斯水迷宫, 它检查空间学习和记忆在大鼠模型24。然而, 莫里斯水迷宫需要大鼠游泳和发挥充分的运动功能和控制。对于缺血实验, 如窒息心脏骤停 (ACA, 大鼠模型的钙) 模型, 需要插管股动脉/静脉, 以获得重要的血压, 血液气体和引进各种药物。由于股动脉/静脉插管可以抑制腿部移动, 使大鼠的游泳能力正常, 莫里斯水迷宫可能不是最适合测试认知损伤的 ACA。
巴恩斯迷宫是另一个被广泛使用的行为测试, 用来检查啮齿动物模型中的空间学习和记忆。巴恩斯迷宫不需要充分发挥马达功能和控制, 因此比莫里斯水迷宫的压力更小。在过去, 我们使用巴恩斯迷宫来进行实验, 以确定在控制或假与 ACA 诱导的大鼠之间是否发生功能性学习/记忆差异。由于巴恩斯迷宫广泛用于测量参考 (长期) 内存25,26, 而为巴恩斯迷宫所获得的数据并没有在轻度到中度 ACA 之后测试认知损伤的决心, 而aca 引起的认知缺陷与工作 (短期) 内存27、28、29、30有关, 这表明巴恩斯迷宫在我们的 aca 中评估内存功能是不可行的。模型.
因此, 我们开发了一个改进的 T 迷宫使用自发交替测试, 以评估工作 (短期) 记忆后 ACA。改进的 t 迷宫自发交替试验的主要优点是它的简单性和最小的压力, 与其他行为测试相比, 由于修改的 t 迷宫不需要事先的动物训练, 以及重计算根据莫里斯水迷宫和巴恩斯迷宫的要求, 分析或程序 (即大鼠的视频成像)。在这里我们表明, 改进的 T 迷宫自发交替试验是一个简单而高效的行为试验范例, 可以提供足够的分辨率, 以准确地检测和评价海马功能的疾病, 造成短期记忆丧失(即 ACA)。

<table><tbody><tr><td>3D Printer</td><td>MakerBot</td><td>Replicator</td><td>Fifth generation</td></tr><tr><td>3D Printer Filament</td><td>Hatchbox</td><td></td><td>PLA, 1.75 mm filament diameter</td></tr><tr><td>200 Proof Pure Ethanol</td><td>Koptec</td><td>V1005SG</td><td></td></tr><tr><td>Sani-Chips</td><td>PJ Murphy-Forest Products</td><td></td><td>Size: 8 to 20 mesh; 2.2 cubic foot/package; autoclavable bags</td></tr><tr><td>Rat</td><td>Charles River Laboratories</td><td>Sprague-Dawley</td><td></td></tr><tr><td>Vecuronium bromide</td><td>Sun Pharmaceutical</td><td>47335-931-40</td><td>10 mg</td></tr><tr><td>Epinephrine</td><td>Par Pharmaceutical</td><td>42023-103-01</td><td>Adrenalin Chloride Solution 1 mg/mL, 1:1000</td></tr><tr><td>Buprenorphine Hydrochloride Injection</td><td>Pfizer</td><td>00409-2012-32</td><td>0.3mg/mL</td></tr><tr><td>SketchUp</td><td>Trimble Inc.</td><td></td><td>3D modeling software</td></tr><tr><td>VentElite Small Animal Ventilator</td><td>Harvard Apparatus</td><td>55-7040</td><td>Animals raging in size from mouse to guinea pig (10g to 1kg)</td></tr><tr><td>PowerLab 8/35</td><td>Adinstruments</td><td>PL3508</td><td>8 analog input channels &amp;ndash; 4 of which can be used in differential mode.</td></tr><tr><td>Bio Amps</td><td>Adinstruments</td><td>FE132</td><td>The Bio Amp is a galvanically isolated, high-performance differential bio amplifier optimized for the measurement of a wide variety of biological signals such as ECG, EMG and EEG recordings.</td></tr><tr><td>Quad Bridge Amp</td><td>Adinstruments</td><td>FE224</td><td>A four-channel, non-isolated bridge amplifier designed to allow the PowerLab to connect to most DC bridge transducers.</td></tr><tr><td>LabChart 8</td><td>Adinstruments</td><td></td><td></td></tr><tr><td>ABL80 FLEX CO-OX blood gas analyzer</td><td>Radiometer</td><td></td><td>pH / &lt;em&gt;p&lt;/em&gt; CO&lt;sub&gt;2&lt;/sub&gt; / &lt;em&gt;p&lt;/em&gt; O&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>SURFLO Teflon I.V. Catheter</td><td>Terumo</td><td>sc-361556</td><td>Only use the flexible thin wall catheter (49-mm long)</td></tr><tr><td>Pipet/Infusion Needle</td><td>Hamilton</td><td>7748-03</td><td>17-gauge; 93-mm long; 10-degree angle</td></tr><tr><td>Classic T3 Vaporizer</td><td>SurgiVet</td><td>VCT302</td><td>Classic T3 Isoflurane Funnel Fill</td></tr><tr><td>ENVIRO-PURE Charcoal Canister</td><td>SurgiVet</td><td>32373B10</td><td>Designed to absorb waste anesthetic gas</td></tr><tr><td>O&lt;sub&gt;2&lt;/sub&gt; single flowmeter</td><td>SurgiVet</td><td>32375B1</td><td>0-1000 mL</td></tr><tr><td>N&lt;sub&gt;2&lt;/sub&gt;O Flowmeter</td><td>VetEquip</td><td>401721</td><td>0-4LPM</td></tr><tr><td>Clay Adams Intramedic Luer-Stub Adapter (Sterile)</td><td>Becton Dickinson</td><td>427565</td><td>23 gauge</td></tr><tr><td>Micro Forceps</td><td>Black and Black surgical</td><td>B3FRC-18 RM-8</td><td>7 1/4&quot; (18 cm), 8mm RH, counterweight w/ guide pin 2mm, platform 6 x .3 mm, curved.</td></tr><tr><td>Halstead Mosquito Forceps</td><td>Roboz</td><td>RS-7111</td><td>Curved; 5&quot; Length, 1.3 mm tip diameter, 2.1 mm jaw width</td></tr><tr><td>Mixter Forceps</td><td>Roboz</td><td>RS-7291</td><td>5.25&quot; Curved Extra Delicate, 1.1 mm tips</td></tr><tr><td>Castroviejo Micro Dissecting Spring Scissors</td><td>Roboz</td><td>RS-5650</td><td>Straight, Sharp Points; 9 mm Cutting Edge; 0.15 mm Tip Width; 3 1/2&quot; Overall Length</td></tr><tr><td>Mayo-Stille Scissors</td><td>Roboz</td><td>RS-6891</td><td>5.5&quot; Round Curved</td></tr><tr><td>Dumont #5 Forceps</td><td>Roboz</td><td>RS-5058</td><td>45 Deg Dumoxel Tip Size .10 x .06 mm</td></tr><tr><td>Olsen-Hegar Combination Scissor And Needle Holder</td><td>Roboz</td><td>RS-7884</td><td>Cross Serration Tip; 5.5&quot; Length</td></tr><tr><td>Moloney Forceps</td><td>Roboz</td><td>RS-8254</td><td>Serrated; Slight Curve; 4.5&quot; Length</td></tr></tbody></table>

utilizing the modified t-maze to assess functional memory outcomes after cardiac arrest

背景: 评估轻度到中度认知损害在全球脑缺血 (即心脏骤停) 模型可能是困难的, 因为在手术后运动不良。例如, 接受外科手术的大鼠和莫里斯水迷宫可能无法游泳, 从而使实验无效。
新方法: 建立了改进的行为自发交替 T 迷宫试验。修改后的 T 迷宫协议的主要优点是它的相对简单的设计, 是强大到足以评估功能学习/记忆后缺血。此外, 数据分析简单明了。我们使用 T 迷宫来确定在轻度到中度 (6 分钟) 窒息心跳骤停 (ACA) 时, 老鼠的学习/记忆力缺陷。大鼠有一个自然的勘探倾向, 并将探索在 T 迷宫的替代武器, 而海马损伤大鼠倾向于采取侧偏爱, 导致减少自发交替率, 揭示海马相关功能性学习/记忆在存在或缺席 ACA。
结果: ACA 组有较高的侧偏爱比和较低的交替对照。
与现有方法比较: 莫里斯水和巴恩斯迷宫在评估学习/记忆功能方面更为突出。然而, 莫里斯水迷宫比其他迷宫更有压力。巴恩斯迷宫被广泛用于测量参考 (长期) 记忆, 而 ACA 诱发的认知赤字则与工作 (短期) 记忆更密切相关。
结论: 我们已经制定了一个简单的, 但有效的策略来描绘工作 (短期) 记忆通过 T 迷宫在我们的全球脑缺血模型 (ACA)。

ACA (全球脑缺血) 主要导致工作 (短期) 记忆缺陷28,29。为了评估在 ACA 之后的学习和记忆功能, 我们使用了改进的自发交替测试来评估工作 (短期) 内存30。自发交替试验结果表明, ACA 组 (26.19 ± 4.96%) 连续三天的交替率明显低于对照组 (62.96 ± 6.07%)(* p0.05)35由于提交给 ACA 的大鼠与对照组相比 (82.14 ?...

Watch this Scientific Journal Video about Utilizing the Modified T-Maze to Assess Functional Memory Outcomes After Cardiac Arrest at JoVE.com

Utilizing the Modified T-Maze to Assess Functional Memory Outcomes After Cardiac Arrest

应用改良 T 迷宫评估心脏骤停后的功能记忆结果

本协议描述了使用一个改进的 T 迷宫评估功能学习/记忆在窒息心脏停搏诱发脑缺血。

Background: Evaluating mild to moderate cognitive impairment in a global cerebral ischemia (i.e. cardiac arrest) model can be difficult due to poor ...

utilizing-modified-t-maze-to-assess-functional-memory-outcomes-after

Research

JoVE Journal

Behavior

12.0K 次观看.  Louisiana State University Health Science Center. 本协议描述了使用一个改进的 T 迷宫评估功能学习/记忆在窒息心脏停搏诱发脑缺血。

视频: Utilizing the Modified T-Maze to Assess Functional Memory Outcomes After Cardiac Arrest

Use of an Eight-arm Radial Water Maze to Assess Working and Reference Memory Following Neonatal Brain Injury

Working and reference memory are commonly assessed using the land based radial arm maze.&#160;However, this paradigm requires pretraining, food deprivation, and may introduce scent cue confounds. The eight-arm radial water maze is designed to evaluate reference and working memory performance simultaneously by requiring subjects to use extra-maze cues to locate escape platforms and remedies the limitations observed in land based radial arm maze designs. Specifically, subjects are required to avoid the arms previously used for escape during each testing day (working memory) as well as avoid the fixed arms, which never contain escape platforms (reference memory). Re-entries into arms that have already been used for escape during a testing session (and thus the escape platform has been removed) and re-entries into reference memory arms are indicative of working memory deficits. Alternatively, first entries into reference memory arms are indicative of reference memory deficits. We used this maze to compare performance of rats with neonatal brain injury and sham controls following induction of hypoxia-ischemia and show significant deficits in both working and reference memory after eleven days of testing. This protocol could be easily modified to examine many other models of learning impairment.

The eight-arm radial water maze is designed to evaluate reference and working memory performance simultaneously by requiring subjects to use extra-maze cues to locate escape platforms and remedies the limitations observed in land based radial arm maze designs.

Working and reference memory are commonly assessed using the land based radial arm maze.&#160;However, this paradigm requires pretraining, food ...

科研

行为学

The Double-H Maze: A Robust Behavioral Test for Learning and Memory in Rodents

Spatial cognition research in rodents typically employs the use of maze tasks, whose attributes vary from one maze to the next. These tasks vary by their behavioral flexibility and required memory duration, the number of goals and pathways, and also the overall task complexity. A confounding feature in many of these tasks is the lack of control over the strategy employed by the rodents to reach the goal, e.g., allocentric (declarative-like) or egocentric (procedural) based strategies. The double-H maze is a novel water-escape memory task that addresses this issue, by allowing the experimenter to direct the type of strategy learned during the training period. The double-H maze is a transparent device, which consists of a central alleyway with three arms protruding on both sides, along with an escape platform submerged at the extremity of one of these arms.
Rats can be trained using an allocentric strategy by alternating the start position in the maze in an unpredictable manner (see protocol 1; &#167;4.7), thus requiring them to learn the location of the platform based on the available allothetic cues. Alternatively, an egocentric learning strategy (protocol 2; &#167;4.8) can be employed by releasing the rats from the same position during each trial, until they learn the procedural pattern required to reach the goal. This task has been proven to allow for the formation of stable memory traces.
Memory can be probed following the training period in a misleading probe trial, in which the starting position for the rats alternates. Following an egocentric learning paradigm, rats typically resort to an allocentric-based strategy, but only when their initial view on the extra-maze cues differs markedly from their original position. This task is ideally suited to explore the effects of drugs/perturbations on allocentric/egocentric memory performance, as well as the interactions between these two memory systems.

The goal of this protocol is to investigate spatial cognition in rodents. The double-H water maze is a novel test, which is particularly useful to elucidate the different components of learning, consolidation and memory, as well as the interplay of memory systems.

双-H迷宫:一个强大的行为测试学习和记忆的鼠害

Spatial cognition research in rodents typically employs the use of maze tasks, whose attributes vary from one maze to the next. These tasks vary by their ...

Quantifying Cognitive Decrements Caused by Cranial Radiotherapy

With the exception of survival, cognitive impairment stemming from the clinical management of cancer is a major factor dictating therapeutic outcome. For many patients afflicted with CNS and non-CNS malignancies, radiotherapy and chemotherapy offer the best options for disease control. These treatments however come at a cost, and nearly all cancer survivors (~11 million in the US alone as of 2006) incur some risk for developing cognitive dysfunction, with the most severe cases found in patients subjected to cranial radiotherapy (~200,000/yr) for the control of primary and metastatic brain tumors1. Particularly problematic are pediatric cases, whose long-term survival plagued with marked cognitive decrements results in significant socioeconomic burdens2. To date, there are still no satisfactory solutions to this significant clinical problem.
We have addressed this serious health concern using transplanted stem cells to combat radiation-induced cognitive decline in athymic rats subjected to cranial irradiation3. Details of the stereotaxic irradiation and the in vitro culturing and transplantation of human neural stem cells (hNSCs) can be found in our companion paper (Acharya et al., JoVE reference). Following irradiation and transplantation surgery, rats are then assessed for changes in cognition, grafted cell survival and expression of differentiation-specific markers 1 and 4-months after irradiation. To critically evaluate the success or failure of any potential intervention designed to ameliorate radiation-induced cognitive sequelae, a rigorous series of quantitative cognitive tasks must be performed. To accomplish this, we subject our animals to a suite of cognitive testing paradigms including novel place recognition, water maze, elevated plus maze and fear conditioning, in order to quantify hippocampal and non-hippocampal learning and memory. We have demonstrated the utility of these tests for quantifying specific types of cognitive decrements in irradiated animals, and used them to show that animals engrafted with hNSCs exhibit significant improvements in cognitive function3.
The cognitive benefits derived from engrafted human stem cells suggest that similar strategies may one day provide much needed clinical recourse to cancer survivors suffering from impaired cognition. Accordingly, we have provided written and visual documentation of the critical steps used in our cognitive testing paradigms to facilitate the translation of our promising results into the clinic.

Cognitive impairment resulting from the radiotherapeutic management of brain tumors represents a clinically intractable condition that adversely impacts quality of life. The capability to critically evaluate potential interventions for ameliorating radiation-induced cognitive decrements ultimately depends on the capability to undertake rigorous quantitative assessments of cognition.

量化颅放疗引起的认知递减

With the exception of survival, cognitive impairment stemming from the clinical management of cancer is a major factor dictating therapeutic outcome. For ...

医学

T-maze Forced Alternation and Left-right Discrimination Tasks for Assessing Working and Reference Memory in Mice

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in rodents. In our laboratory, we evaluated the two types of memory in more than 30 strains of genetically engineered mice using the automated version of this apparatus. Here, we present the modified T-maze apparatus operated by a computer with a video-tracking system and our protocols in a movie format. The T-maze apparatus consists of runways partitioned off by sliding doors that can automatically open downward, each with a start box, a T-shaped alley, two boxes with automatic pellet dispensers at one side of the box, and two L-shaped alleys. Each L-shaped alley is connected to the start box so that mice can return to the start box, which excludes the effects of experimenter handling on mouse behavior. This apparatus also has an advantage that in vivo microdialysis, in vivo electrophysiology, and optogenetics techniques can be performed during T-maze performance because the doors are designed to go down into the floor. In this movie article, we describe T-maze tasks using the automated apparatus and the T-maze performance of &alpha;-CaMKII+/- mice, which are reported to show working memory deficits in the eight-arm radial maze task. Our data indicated that &alpha;-CaMKII+/- mice showed a working memory deficit, but no impairment of reference memory, and are consistent with previous findings using the eight-arm radial maze task, which supports the validity of our protocol. In addition, our data indicate that mutants tended to exhibit reversal learning deficits, suggesting that &alpha;-CaMKII deficiency causes reduced behavioral flexibility. Thus, the T-maze test using the modified automatic apparatus is useful for assessing working and reference memory and behavioral flexibility in mice.

This article presents the protocol of T-maze tests using a modified automated apparatus for assessing the learning and memory functions in mice.

T-迷宫强迫交替和评估工作，并参考在小鼠记忆的左，右的歧视任务

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in ...

神经科学

Assessing Spatial Memory Impairment in a Mouse Model of Traumatic Brain Injury Using a Radial Water Tread Maze

Despite the recent increase in use of mouse models in scientific research, researchers continue to use cognitive tasks that were originally designed and validated for rat use. The Radial Water Tread (RWT) maze test of spatial memory (designed specifically for mice and requiring no swimming) has been shown previously to successfully distinguish between controlled cortical impact-induced TBI mice and sham controls. Here, a detailed protocol for this task is presented. The RWT maze capitalizes on the natural tendency of mice to avoid open areas in favor of hugging the sides of an apparatus (thigmotaxis). The walls of the maze are lined with nine escape holes placed above the floor of the apparatus, and mice are trained to use visual cues to locate the escape hole that leads out of the maze. The maze is filled with an inch of cold water, sufficient to motivate escape but not deep enough to require that the mouse swim. The acquisition period takes only four training days, with a test of memory retention on day five and a long-term memory test on day 12. The results reported here suggest that the RWT maze is a feasible alternative to rat-validated, swimming-based cognitive tests in the assessment of spatial memory deficits in mouse models of TBI.

Here we present a protocol for a mouse-specific test of cognition that does not require swimming. This test can be used to successfully distinguish controlled cortical impact-induced traumatic brain injury mice from sham controls.

使用径向水痕迷宫评估外伤性脑损伤小鼠模型中的空间记忆障碍

Despite the recent increase in use of mouse models in scientific research, researchers continue to use cognitive tasks that were originally designed and ...

Detecting Behavioral Deficits in Rats After Traumatic Brain Injury

With the increasing incidence of traumatic brain injury (TBI) in both civilian and military populations, TBI is now considered a chronic disease; however, few studies have investigated the long-term effects of injury in rodent models of TBI. Shown here are behavioral measures that are well-established in TBI research for times early after injury, such as two weeks, until two months. Some of these methods have previously been used at later times after injury, up to one year, but by very few laboratories. The methods demonstrated here are a short neurological assessment to test reflexes, a Beam-Balance to test balance, a Beam-Walk to test balance and motor coordination, and a working memory version of the Morris water maze that can be sensitive to deficits in reference memory. Male rats were handled and pre-trained to neurological, balance, and motor coordination tests prior to receiving parasagittal fluid percussion injury (FPI) or sham injury. Rats can be tested on the short neurological assessment (neuroscore), the beam-balance, and the Beam-Walk multiple times, while testing on the water maze can only be done once. This difference is because rats can remember the task, thus confounding the results if repeated testing is attempted in the same animal. When testing from one to three days after injury, significant differences are detected in all three non-cognitive tasks. However, differences in the Beam-Walk task were not detectable at later time points (after 3 months). Deficits were detected at 3 months in the Beam-Balance and at 6 months in the neuroscore. Deficits in working memory were detected out to 12 months after injury, and a deficit in a reference memory first appeared at 12 months. Thus, standard behavioral tests can be useful measures of persistent behavioral deficits after FPI.

The goal of the behavioral tests presented here is to detect functional deficits in rats after traumatic brain injury. Four specific tests are presented that detect deficits in behaviors to reflect the damage to specific brain areas at times extending to one year after injury.

颅脑损伤后大鼠行为缺陷的检测

With the increasing incidence of traumatic brain injury (TBI) in both civilian and military populations, TBI is now considered a chronic disease; however, ...

Motor and Hippocampal Dependent Spatial Learning and Reference Memory Assessment in a Transgenic Rat Model of Alzheimer's Disease with Stroke

Alzheimer&#39;s disease (AD) is a debilitating neurodegenerative disease that results in neurodegeneration and memory loss. While age is a major risk factor for AD, stroke has also been implicated as a risk factor and an exacerbating factor. The co-morbidity of stroke and AD results in worsened stroke-related motor control and AD-related cognitive deficits when compared to each condition alone. To model the combined condition of stroke and AD, a novel transgenic rat model of AD, with a mutated form of amyloid precursor protein (a key protein involved in the development of AD) incorporated into its DNA, is given a small unilateral striatal stroke.
For a model with the combination of both stroke and AD, behavioral tests that assess stroke-related motor control, locomotion and AD-related cognitive function must be implemented. The cylinder task involves a cost-efficient, multipurpose apparatus that assesses spontaneous forelimb motor use. In this task, a rat is placed in a cylindrical apparatus, where the rat will spontaneously rear and contact the wall of the cylinder with its forelimbs. These contacts are considered forelimb motor use and quantified during video analysis after testing. Another cost-efficient motor task implemented is the beam-walk task, which assesses forelimb control, hindlimb control and locomotion. This task involves a rat walking across a wooden beam allowing for the assessment of limb motor control through analysis of forelimb slips, hindlimb slips and falls. Assessment of learning and memory is completed with Morris water maze for this behavioral paradigm. The protocol starts with spatial learning, whereby the rat locates a stationary hidden platform. After spatial learning, the platform is removed and both short-term and long-term spatial reference memory is assessed. All three of these tasks are sensitive to behavioral differences and completed within 28 days for this model, making this paradigm time-efficient and cost-efficient.

To investigate the co-morbid Alzheimer&#39;s disease (AD) and stroke condition in a novel model, three behavior tasks are described that assess both motor control and cognitive behaviors. These tasks include the beam-walk task, cylinder task and Morris water maze.

汽车和海马依赖空间学习和记忆参考评估与中风阿尔茨海默氏病的转基因大鼠模型

Alzheimer&#39;s disease (AD) is a debilitating neurodegenerative disease that results in neurodegeneration and memory loss. While age is a major risk ...

A Metric Test for Assessing Spatial Working Memory in Adult Rats Following Traumatic Brain Injury

Impairments to sensory, short-term, and long-term memory are common side effects after traumatic brain injury (TBI). Due to the ethical limitations of human studies, animal models provide suitable alternatives to test treatment methods, and to study the mechanisms and related complications of the condition. Experimental rodent models have historically been the most widely used due to their accessibility, low cost, reproducibility, and validated approaches. A metric test, which tests the ability to recall the placement of two objects at various distances and angles from one another, is a technique to study impairment in spatial working memory (SWM) after TBI. The significant advantages of metric tasks include the possibility of dynamic observation, low cost, reproducibility, relative ease of implementation, and low stress environment. Here, we present a metric test protocol to measure impairment of SWM in adult rats after TBI. This test provides a feasible way to evaluate physiology and pathophysiology of brain function more effectively.

Traumatic brain injury (TBI) is commonly associated with memory impairment. Here, we present a protocol to assess spatial working memory after TBI via a metric task. A metric test is a useful tool to study spatial working memory impairment after TBI.

创伤性脑损伤后评估成年大鼠空间工作记忆的指标测试

Impairments to sensory, short-term, and long-term memory are common side effects after traumatic brain injury (TBI). Due to the ethical limitations of ...

A Needle Model for Vascular Cognitive Impairment Studies in Mice

A Mouse Model for Vascular Cognitive Impairment and Dementia Based on Needle-guided Asymmetric Bilateral Common Carotid Artery Stenosis

Vascular cognitive impairment and dementia (VCID) results from vascular brain injury. Given VCID&#39;s high incidence, which is expected to continue rising as the population ages, it is critical to establish a robust animal model for the disease. This paper presents a novel method of creating a mouse model of VCID that is based on asymmetric bilateral common carotid artery stenosis, which mimics human chronic cerebral hypoperfusion caused by carotid atherosclerosis.
Briefly, common carotid arteries (CCAs) are ligated to different gauge needles (32 G for the right CCA and 34 G for the left CCA) using 7-0 silk sutures followed by immediate needle removal. The remaining suture rings cause persistent blood flow reduction and long-term cognitive impairment associated with white matter injury, microinfarcts, and reactive gliosis, thus closely mimicking the pathogenesis of VCID. Importantly, in this needle model, the clinical representations do not revert with time, providing reliable long-term cognitive impairment. Moreover, the survival rate 24 weeks post surgery was 81.6%, which is higher compared to the other established models of VCID with a similar level of blood flow reduction.
Additional advantages include low material cost and compatibility with MRI to monitor brain injury in live animals since no metal is implanted. The main challenge in employing the needle model of VCID is the requirement for developing advanced surgical skills since mouse CCAs are less than 0.6 mm in diameter and are very fragile. High-quality visual representation of the surgery will thus help researchers to master this technique and advance our understanding of VCID, potentially leading to the development of novel therapeutic modalities to decrease the devastating cognitive decline associated with VCID.

The needle method of asymmetric bilateral common carotid artery stenosis is proposed to create a mouse model for vascular cognitive impairment and dementia. It results in longer-term outcomes compared to previously established models and is compatible with live MRI. Visual representation demonstrating the procedure provides guidance for mastering the surgery.

Vascular cognitive impairment and dementia (VCID) results from vascular brain injury. Given VCID&#39;s high incidence, which is expected to continue ...

Anterograde Amnesia

Source: Laboratories of Jonas T. Kaplan and Sarah I. Gimbel&#8212;University of Southern California
Anterograde amnesia is the loss of the ability to form new memories. This can be distinguished from retrograde amnesia, which is the loss of old memories. Anterograde amnesia can result from damage to structures in the brain that are involved in the formation of new memories. Patients who have damage to the structures of the medial temporal lobe, including the hippocampus, amygdala, and the surrounding cortices, often have severe deficits in the formation of certain kinds of memories. These cases can be informative as to how memory is organized in the brain, and how different systems support different kinds of memories. 
In this video, we will test a patient with medial temporal lobe damage on a series of memory tasks designed to distinguish between different forms of memory. First, we will test short-term or working memory, which is the process we use to keep information in mind temporarily. Next, we will test two different forms of long-term memory: explicit and implicit memory. Explicit memories are conscious and easy to verbalize. For example, memories of facts or episodes from our lives are explicit memories. We can easily tell someone what we ate for breakfast, or what city is the capital of France. Implicit memory involves knowledge we gain from experience but that is not easily expressible. For example, knowing how to do things, or becoming habituated to a stimulus are forms of implicit memory. 
These procedures are based in part on studies of the famous patient Henry Molaison, also known by his initials H.M., who had severe anterograde amnesia as a result of a surgery for intractable epilepsy in which parts of both temporal lobes were resected.1 We will perform a test of digit span, which measures short-term memory, a test of paired-associate learning, which measures explicit or declarative memory, and mirror-drawing, a test of implicit skill learning.2

Testing Short- and Long-term Memories in Patients with Brain Injuries

顺行性遗忘症

Source: Laboratories of Jonas T. Kaplan and Sarah I. Gimbel&#8212;University of Southern California
Anterograde amnesia is the loss of the ability to form ...