这项研究的一部分是由优异奖 #B1495 R (Capadona) 和总统早期职业奖的科学家和工程师 (美国青年, Capadona) 从美国 (美国) 退伍军人事务部的康复研究和开发服务。此外, 这项工作还部分由主管卫生事务的助理国防部长办公室通过经同侪审查的医学研究计划获得支持。W81XWH-15-1-0608。这些内容不代表美国退伍军人事务部或美国政府的意见。作者感谢博行荒川博士在 CWRU 啮齿动物行为核心中为他设计和测试啮齿动物行为协议提供指导。作者还要感谢 CWRU 机械和航空工程系的詹姆斯. 德雷克和凯文. 他在设计和制造啮齿动物阶梯试验方面的帮助。

所有的程序和动物护理实践都是根据路易斯. 克利夫兰退伍军人事务部医疗中心机构动物护理和使用委员会的批准和执行。
注: 为了教育研究人员关于使用刺伤模型作为控制的决定, 建议对波特等的工作进行回顾。21。
1. 微电极植入手术方法
<ol><li>
手术前动物准备
	<ol>
<li>使用异氟醚 (2-4%) 在感应腔内麻醉动物。在麻醉下, 用重要的测量系统持续监测动物的心率和血氧含量。</li>
		<li>将动物移到鼻锥上继续麻醉。皮下 (平方) 注射头孢菌素抗生素, 如头孢唑啉 (25 毫克/千克) 和非甾体抗炎, 如卡洛芬 (5 毫克/千克), 以防止感染和管理疼痛, 分别。</li>
		<li>在动物的眼睛上随意涂抹眼药膏以防止它们干燥。</li>
		<li>使用小动物指甲钳, 修剪脚趾甲, 以防止动物在伤口愈合过程中刮缝线。确保指甲不修剪太短, 因为这可能会导致疼痛和出血的动物。</li>
		<li>把动物的头从耳朵后面彻底剃掉, 用电动剃须刀修剪。</li>
		<li>提供局部镇痛, 以一平方注射布比卡因 (0.3 毫升0.125% 布比卡因稀释从库存溶液) 在动物的头部在切口区域的顶部。</li>
		<li>在立体定向的框架上安装动物, 用耳棒防止头部在手术过程中移动。在动物下放置循环水加热垫, 以维持动物的内部温度。</li>
		<li>应用不育的悬垂, 例如, 在体制上批准的无菌塑料包装, 以隔离手术领域。</li>
		<li>用交替 betadine 溶液和异丙醇磨砂擦洗手术部位。</li>
		<li>根据机构协议执行脚趾夹, 确保动物在手术平面下。</li>
	</ol>
</li>
	<li>
准备植入动物
	<ol>
<li>创建一个切口约1在下中线揭露头骨使用10号手术刀刀片。用棉尖的涂抹器直截了当地取出骨膜, 用纱布垫止血。用鳄鱼夹子收回周围的组织, 用过氧化氢清洁和脱水头骨。</li>
		<li>在暴露的头骨上放置几滴氰的组织粘合剂, 以改善牙齿水泥在以后的步骤中的粘合。</li>
		<li>在所选择的半球, 标记与前爪运动相关的运动皮层区域, 在骨骼中创建一个尼克, 其侧向中线和2毫米的 bregma。</li>
		<li>用1.75 毫米圆尖牙钻取出头骨的一部分, 特别考虑不要钻得太快或太深, 并支持一只手在立体定向的框架上。该钻头应间歇性地应用于颅骨, 以避免过热31。</li>
		<li>用细钩的45°硬脑膜来反射硬脑膜。</li>
		<li>用棉签和生理盐水清洗任何出血, 注意不要直接接触大脑表面。</li>
	</ol>
</li>
	<li>
微电极在运动皮层中的插入
	<ol>
<li>小心地安装在立体定位架上的万能支架上的灭菌微电极, 小心不要撞到电极的小腿。确保电极的 headstage 接口连接器牢固地持有。 注: 在这里, 一个非功能的密歇根风格的硅柄电极测量2毫米 x 123 µm x 15 µm 使用, 和小腿插入使用细钳。</li>
		<li>使用并联在立体定向的框架上, 小心地将电极尖端定位在打开的开颅手术上。</li>
		<li>用并联作为测量指南 (根据电极的选择, 用控制速率自动插入), 将电极大约2毫米轻轻地降低到大脑中。小心, 尽可能避免任何可见的血管。一旦电极到位, 小心地释放连接器从万能持有人和收回插入臂。</li>
		<li>用棉签和生理盐水仔细清洗电极周围的任何出血。</li>
		<li>使用硅胶弹性体在植入电极周围封闭开颅手术。</li>
		<li>用牙科水泥把电极固定在头骨上。</li>
		<li>一旦水泥完全干燥, 把切口的边缘一起在水泥 headcap 的正面和后面, 并且缝合他们闭合。</li>
	</ol>
</li>
	<li>
术后护理
	<ol>
<li>允许动物在循环水加热垫上恢复, 同时继续监测其生命体征。避免使用热灯, 因为灯具的温度更难以控制, 动物也会过热。</li>
		<li>一旦动物完全清醒, 把动物移到一个干净的笼子里, 方便地获得食物和水。</li>
		<li>在术后 1-3 天, 为动物提供的平方头孢菌素抗生素 (25 毫克/千克) 和非甾体抗炎 (5 毫克/千克), 以防止感染和管理他们的痛苦。</li>
		<li>每天监测动物的疼痛或不适, 出血, 体重变化, 或缝合问题的迹象, 至少在手术后5天。</li>
	</ol>
</li>
</ol>2. 行为测试
<ol><li>对于所有的行为测试, 在电极植入手术前一周测试动物 2x, 以计算其手术前基线评分。手术后, 允许动物休息1周前开始行为测试每周2x 每测试。在整个研究中都应使用一致的测试条件来进行前和术后检查, 以尽量减少压力对性能的影响, 这可能导致焦虑的测量。<ol>
<li>在每个测试阶段开始和每种动物之后, 用二氧化氯基消毒剂清洁所有测试设备。</li>
		<li>薄膜的开放领域网格和阶梯测试。这些测试需要一个摄像机 (1080p, 最小 15 fps, 78°对角线视野), 笔记本电脑, 和存储视频数据的空间。</li>
		<li>在每个测试日开始时, 将动物带到测试室, 并允许他们在开始测试前至少进行20分钟的适应。房间应轻, 温控, 同一人员应完成所有测试。理想情况下, 同一房间将用于所有的动物在整个测试过程中, 没有改变的房间。</li>
		<li>使用食物奖励鼓励动物完成任务, 特别是在阶梯训练期间。谷物或小块的香蕉薯片或饼干可以得到很好的回报。</li>
		<li>对每一个动物的手术前评分规范化所有每周测试表现 (等式 1)。 等式 1:<img alt="Equation 1" src="/files/ftp_upload/57829/57829eq1.jpg">
</li>
	</ol>
</li>
	<li>
开放域网格测试 注: 开放场网格测试建在内部, 并有一个运行表面1米2与大约40厘米高不透明侧壁。网格的底部运行表面使用磁带从底面分成9个相等的正方形 (图 1A)。记录摄像机将永久安装在脚手架上的网格中心上方。<ol>
<li>要开始打开字段网格测试, 请将该动物置于网格的中心, 使其远离测试人员。</li>
		<li>允许动物在录制视频时自由运行3分钟。</li>
		<li>当动物完成测试后, 从网格中取出动物并将其返回笼子。用二氧化氯基消毒剂彻底清洁网格。</li>
		<li>每测试日测试每只动物1x。</li>
		<li>使用视频跟踪软件分析交叉网格线的数目、总距离以及动物的最大速度作为总马达功能的度量。 注意: 这里提供的数据是由训练有素的研究人员手工量化的, 但最好使用最近开发的内部跟踪算法32。</li>
	</ol>
</li>
	<li>
梯子测试 注: 梯子测试是内置的, 包括2透明丙烯酸侧壁, 每1米长度, 连接3毫米直径的梯级间隔在2厘米 (图 2A)。梯子测试是一个熟练的测试, 因此需要1周的训练前记录手术前基线评分。培训和测试的协议是相同的。<ol>
<li>移动动物到一个临时的干净的保持笼子开始梯子测试。</li>
		<li>把梯子放上去, 这样它就能桥2笼了。梯子的开始端在一个干净的笼子上, 终点在动物的笼子里, 作为完成跑步的动力。</li>
		<li>在梯子中心的三脚架上放置相同 (或类似的) 摄像机。相机的位置应在高度, 并允许整个梯子被看到。</li>
		<li>随着摄像机的运行, 把动物放到梯子的起跑线上, 让他们的前爪触到第一个台阶。</li>
		<li>允许动物以自己的速度跨过梯子。在动物的爪子触及第一个梯级和终点线在第三到最后一阶的那一刻之间的时间流逝将决定动物的穿越时间。</li>
		<li>如果动物在梯子上转过身或在二十年代不动, 那就考虑一下动物跑不及格的情况。为每个失败的运行分配动物一个点球得分时间。在手术前测试中记录的最慢的表现, 确定惩罚时间27。</li>
		<li>允许每一个动物跨越梯子5x 每测试日与大约1分钟休息在每个奔跑之间。</li>
		<li>平均每天最快的3运行作为一个指标的精细马达功能。此外, 使用视频跟踪软件记录每一个前爪在横档上滑动的次数。 注意: 这里提供的数据是由训练有素的研究人员手工量化的, 但它更倾向于使用一个最近开发的内部跟踪算法 。32。</li>
	</ol>
</li>
	<li>
握杆强度测试
	<ol>
<li>在每个测试阶段前校准握杆强度表, 并测量克的强度。</li>
		<li>将握杆强度计放在计数器的边缘, 并将手柄把手伸到地板上。</li>
		<li>允许动物用两个前爪抓住把手, 同时按住动物的尾巴底部 (图 3A)。</li>
		<li>一旦动物牢牢抓住每只爪子, 把动物从米的底部拉走, 缓慢而稳定的力量。</li>
		<li>记录由握杆强度计显示在数字输出上的动物所施加的最大抓握强度。</li>
		<li>每个测试日测试每种动物 3x, 每项测试之间大约有2分钟的休息时间。</li>
		<li>作为精细运动功能的指标, 记录和平均3试验中每一次的最大握力强度。</li>
	</ol>
</li>
</ol>3. 行为后协议
<ol><li>经过所有的行为测试 (e. g, 植入后 8-16 周), 麻醉动物深度使用氯胺酮 (160 毫克/千克) 和甲苯噻嗪 (20 毫克/千克), transcardially 灌注他们, 收割他们的大脑和冷冻切片, 并染色组织使用免疫组化标记定量分析33、34、35、36、37、38植入部位周围细胞的反应。</li>
</ol>4. 统计分析
注: 对于试图回答特定研究问题的任何研究, 强烈建议进行前瞻性的权力分析。功率分析, 这表明需要为特定的研究设计达到统计学意义的动物数量, 应根据特定的研究假设, 实验的设计, 估计的效果大小和变异性预期的治疗, 以及达到临床或科学相关性所需的效果大小。
<ol><li>使用通用统计软件进行统计分析。</li>
	<li>对描述性统计进行制表, 并将其显示为标准错误。</li>
	<li>在每个每周时间点分析 [在开放域网格 (步骤 2.2)、阶梯 (步骤 2.3) 和握力测试 (步骤 2.4)] 中的行为性能, 用两个样本 t 检验来比较控制与植入组.每个每周时间点都要考虑一个独立的度量值。</li>
	<li>使用混合效应线性模型量化纵向性能。星期和小组是固定的因素, 并且实验动物被嵌套在小组里作为一个随机作用。采用方差分析法确定因子效应与p < 0.05 的意义水平。</li>
	<li>用线性回归分析方法比较阶梯性能与免疫球蛋白 G (IgG) 强度。用皮尔逊方法计算相关系数。</li>
</ol>

作者没有什么可透露的。

本文概述的协议已被用于有效和重现性测量的罚款和总马达缺陷模型的啮齿动物脑损伤。此外, 它允许在运动皮层微电极植入后, 精细运动行为与组织学结果的相关性。这些方法很容易遵循, 建立起来成本低廉, 可以修改以适应研究者的个人需求。此外, 行为测试不会给动物造成很大的压力或痛苦;相反, 研究人员认为, 这些动物生长的目的是享受测试带来的锻炼和奖赏。先前的研究表明, 运动皮层损伤会导致马达、记忆和功能损伤44,45。然而, 尽管有这方面的知识, 有有限的信息的功能影响的微电极植入在电机皮质27, 这可能会对临床结果产生负面影响的病人。
可以在整个协议中进行修改, 无论是在手术过程中还是在行为测试中。本议定书概述了在影响前爪的区域动物的运动皮层中植入电极的程序。这个程序可以很容易地适应改变种植体, 包括电刺激46或套管的电极为药物交付47, 或伤害的类型, 包括外伤模型48。对在野外网格测试中使用的评分指标, 以及阶梯测试仪, 可以进行进一步的修改。除了越过网格线的数量外, 总的距离, 以及动物所达到的最大速度, 停滞的时间和右、左转弯的次数也可以记录为电机性能的附加参数32.在梯子测试, 去除阶49或放置梯子在倾斜50可能增加困难, 虽然与当前植入物作者没有发现这必要在这个应用中取笑罚款马达缺乏。最后, 虽然这里提出的测试仪器被设计用来与大鼠, 单位可以放大或下降, 以用于各种大小的啮齿动物。重要的是要注意, 如果出现的问题, 如果动物不能持续完成手术前的测试, 动物应该从研究中删除。
与所有的行为测试一样, 在研究过程中尽可能保持一致是至关重要的。已经表明, 测试结果可以根据与动物工作的研究员51, 测试的地点进行52, 和环境因素, 包括动物住房和畜牧业程序53。此外, 研究显示, 在开颅手术过程中, 通过颅骨加热的方式产生脑损伤有很大的变异性, 其中包括体重下降模型54和受控皮质的机械变异31和模型。影响模型55。因此, 研究人员应特别注意保持手术过程、测试和住房条件的一致性, 以及测试人员等。
这些行为测试方法的未来方向可能会扩展到此处提供的测试, 以获得更彻底的结果。例如, 水迷宫试验或转子杆试验可纳入进一步提取焦虑56或总马达功能57赤字, 分别。此外, 未来的工作也可能旨在减少由设备插入大脑造成的组织损害。目前这一领域的工作重点是通过抗氧化剂治疗42,58, 机械兼容植入物41,59,60, 抑制炎症, 先天免疫信号通路14,15, 减少血管损伤在设备植入31,61。
最后, 必须认为, 目前的工作是使用健康的, 少年, 雄性大鼠, 并不一定体现的特点, 典型的病人接受大脑植入。需要进一步研究在特征性疾病模型中进一步进行精细和总的运动功能任务, 以批准这里提出的研究结果。在不同的疾病模型中, 植入和非植入性假动物之间的差异可能需要上述的修改来测试条件。

Intracortical 电极最初用于绘制大脑的电路, 并已发展成为一个有价值的工具, 使检测的马达意图, 可用于产生功能输出1。检测到的功能输出可以为患有脊髓损伤、脑瘫、肌萎缩侧索硬化 (ALS) 或其他运动限制条件的人提供计算机光标2、3或机器人的控制手臂4,5,6, 或恢复功能到自己的残疾肢体7。因此, intracortical 微电极技术已成为一个有希望和快速增长的领域8。
由于该领域的成功, 临床研究正在进行, 以改善和更好地了解 BMI 技术的可能性5,9,10。通过实现与大脑神经元的充分沟通潜能, 康复应用被认为是无限的8。虽然对 intracortical 微电极技术的未来有很大的乐观, 但也众所周知, 电极最终会失败11, 可能是由于植入后的急性 neuroinflammatory 反应。在大脑中植入异物会立即损害周围组织, 导致 neuroinflammatory 反应造成的进一步损害, 这取决于植入物12的特性。此外, 大脑中的植入物可能导致 microlesion 的效果: 由于设备插入13导致的急性水肿和出血, 糖代谢减少。此外, 无论动物模型11、14、15、16, 信号质量和有用信号记录的时间长短不一致。几项研究表明 neuroinflammation 和微电极性能之间的联系17,18,19。因此, 社区的共识是, 围绕电极的神经组织的炎症反应, 至少部分地损害了电极的可靠性。
许多研究已经检查了局部炎症 11,20,21,22或探索方法, 以减少对大脑的损害, 由插入 11,23, 24,25, 目标是提高记录性能随着时间的推移14,26。此外, 我们最近发现, 在大鼠的运动神经皮质中由微电极插入引起的医源性损伤会导致即刻和持久的精细马达缺陷27。因此, 这里提出的协议的目的是给研究人员一个定量的方法来评估可能的运动缺陷, 由于脑外伤后的植入和持续存在的 intracortical 设备 (电极在本手稿的情况下)。这里描述的行为测试旨在梳理毛和精运动功能损伤, 并可用于多种脑损伤模型。这些方法简单, 重现性好, 可以很容易地在啮齿动物模型中实现。此外, 这里提出的方法允许运动行为与组织学结果的相关性, 这一好处, 直到最近, 作者还没有看到在 BMI 领域发表。最后, 由于这些方法是为了测试精细电机功能28, 总马达功能29, 压力和焦虑行为29,30, 这里提出的方法也可以实现为各种头部损伤模型的研究人员希望排除 (或) 任何运动功能缺陷。

<table><tbody><tr><td>Sprague Dawley rats, male, 201-225g</td><td>Charles River</td><td>CD</td><td></td></tr><tr><td>Compac5 anesthesia system</td><td>Vetequip</td><td>901812</td><td></td></tr><tr><td>Electric trimmers</td><td>Wahl</td><td>9918-6171</td><td></td></tr><tr><td>Stereotaxic frame</td><td>David Kopf Instruments</td><td>1760</td><td></td></tr><tr><td>Gaymar heated water pad and pump</td><td>Braintree Scientific Inc</td><td>&amp;nbsp;TP-700</td><td></td></tr><tr><td>Vetbond tissue adhesive</td><td>3M</td><td>07-805-5031</td><td></td></tr><tr><td>Dental drill</td><td>Pearson Dental</td><td>O60-0045</td><td></td></tr><tr><td>Dura pick</td><td>Fine Science Tools</td><td>10064-14</td><td></td></tr><tr><td>Silicon shank microelectrode</td><td>Made in-house at Cleveland VA Medical Center</td><td>N/A</td><td></td></tr><tr><td>KwikCast silicone elastomer</td><td>World Precision Instruments</td><td>KWIK-CAST</td><td></td></tr><tr><td>Teets dental cement&amp;nbsp;</td><td>A-M Systems</td><td>525000</td><td></td></tr><tr><td>Webcam HD Pro c920</td><td>Logitec</td><td>960-000764</td><td></td></tr><tr><td>Grip strength meter</td><td>Harvard Apparatus</td><td>565084</td><td></td></tr><tr><td>Minitab 17 statistical software</td><td>Minitab Inc</td><td></td><td></td></tr><tr><td>Open field grid test</td><td>Made in-house at Case Western Reserve University</td><td>N/A</td><td></td></tr><tr><td>Ladder test</td><td>Made in-house at Case Western Reserve University</td><td>N/A</td><td></td></tr><tr><td>Rabbit anti rat IgG antibody</td><td>Bio-Rad</td><td>618501</td><td></td></tr></tbody></table>

rodent behavioral testing to assess functional deficits caused by microelectrode implantation in the rat motor cortex

植入大脑的医疗器械拥有巨大的潜能。作为脑机接口 (BMI) 系统的一部分, intracortical 电极显示了从单个或小群神经元记录动作电位的能力。这种记录的信号已成功地被用来让病人与计算机、机器人四肢和四肢接触或控制。然而, 以前的动物研究表明, 大脑中的微电极植入不仅会损伤周围的组织, 还会导致功能缺陷。在这里, 我们讨论了一系列的行为测试, 以量化潜在的运动损伤后, 植入 intracortical 电极到运动皮层的大鼠。开放领域网格、阶梯交叉和抓握强度测试的方法为微电极植入产生的潜在并发症提供了有价值的信息。行为测试的结果与端点组织学相关, 提供了有关此过程对相邻组织的病理结果和影响的补充信息。

使用此处介绍的方法, 在39、40、41、42等既定程序完成后, 在运动皮层进行微电极植入手术, 然后进行野外网格测试。评估总马达功能和梯子和握杆强度测试, 以评估优良的马达功能27。在植入动物术后16周内, 每星期完成2x 的运动功能测试, 没有手术的非植入动物作为控制。所有手术后评分平均每周和规范化的每一个动物的前手术基线评分。所有错误都报告为平均值 (SEM) 的标准误差。
为了测量它们的总运动功能和应力行为, 允许动物在野外网格测试中自由运行3分钟 (图 1A)。这项测试的各种指标都可以记录下来, 包括网格线的交叉数, 总距离的移动, 以及动物所达到的最大速度。在先前报告的数据中, 交叉的网格线数显示为27。在恢复期后的第一周 (2 周 timepoint) 中, 2 组之间的开放域网格性能有显著差异。然而, 在整个研究的其余部分没有进一步的意义 (图 1B)。控制和微电极植入的动物在测试中同样得分, 在两组动物中, 性能的差异相对较高。在对两组动物的野外网格性能进行比较的整个实验时间中, 没有发现任何意义。由于2组动物的性能没有差异, 这一结果被解释为表明没有总马达亏缺或严重限制的压力由微电极植入在运动神经皮质27。在解释数据时, 越过的网格线数减少, 总距离移动, 或动物达到的最大速度表明其总马达功能下降 (表 1)。
为了测量协调的抓地和精细的运动功能, 动物参加了水平梯试验 (图 2a), 在那里, 动物穿越梯子的时间和爪滑的频率被记录下来。术后阶梯穿越时间为每一个动物的前手术评分规范化。因此, 正百分比与阶梯的时间缩短和性能的提高相吻合, 负百分比与梯子的时间推移和性能下降相吻合 (图 2B, 表 1)。
在先前报告的数据中, 控制动物在未接受植入物的情况下, 在恢复阶段27后的第一周内立即进行手术后测试, 显示出最慢的性能时间 (82.6 至 26.0%)。从手术后阶梯测试的第二周开始, 控制动物恢复了他们的基线表现时间, 并保持了与他们的基线得分相比, 在研究过程中的分数非常小的差异。
接受 intracortical 微电极的动物在手术后直接看到了性能的降低。在手术后的第一周, 这些动物的阶梯穿越时间比基线高出199.1 到61.4%。在研究期间, 植入的动物表现出的性能下降, 他们的表现没有回到基线得分。在最坏的情况下, 被植入的动物在11周的表现下降到平均 526.9 @ 139.4% 相比, 他们的基线表现。另外, 与对照动物相比, 植入的动物表现出更高的方差。在第一周的试验中, 控制和植入动物之间没有显著的差异。但是, 与基线时间相比, 在研究的所有后续周 (p < 0.05) 中, 各组之间的百分比变化有显著差异 (图 2B)。
在2组动物之间的前右爪的频率证明了细微运动损伤的进一步证据。前右爪的性能特别有趣, 因为电极被植入大脑的左半球, 该区域的运动皮层负责前爪控制。通过细致的视频分析, 爪滑被记录和量化 (图 2C)。虽然在左爪滑的频率上没有明显的差异, 但发现植入的动物比对照动物有明显的前右爪滑倒 (平均每周 0.54 0.07 左右的右爪滑被植入的动物相比, 平均每星期 0.32 @ 0.02 左右爪在控制动物每周滑动)。在解释数据时, 通过梯子的时间增加或爪子滑数的增加表明精细马达功能下降 (表 1)。
作为协调掌握和精细运动功能的次要措施, 动物完成了握杆强度测试 (图 3a), 其中记录了动物施加的最大握力强度。个别动物每周的抓握得分被规范化为他们的前手术基线握力强度。观察到, 在几乎每个手术后的时间点, 与控制动物相比, 植入动物的术后握持强度明显降低。(图 3B)。术前检查后, 控制动物的抓握强度有所提高, 可能是由于训练效果所致。此外, 在整个研究过程中, 控制动物的抓握强度明显大于基线 (p < 0.05)。有趣的是, 植入动物的握杆强度表现比基线 (p < 0.01) 在测试后的第一周的恢复阶段, 但慢慢恢复到他们的基线性能明显差。值得注意的是, 动物所达到的最大抓握强度的降低表明, 优良的马达功能下降 (表 1)。
各种组织学标记可用于可视化大脑植入物附近的微环境, 包括神经细胞核、星形胶质细胞和血脑屏障稳定性。在这里, 我们进行免疫组化染色的 IgG, 一种常见的血液蛋白不常见的大脑。早先的研究表明, IgG 是血脑屏障完整性的有用指标, 因为它是血液中发现的抗体, 通常不存在于大脑16,18, 因此在周围的脑组织中存在 igg。可与血脑屏障的完整性相关43。在这里, igg 荧光强度被规范化为背景脑组织和量化开始在电极 explantation 孔的边界和移动在同心箱, 直到 IgG 不再存在于组织中。与对照动物相比, 植入的动物在µm 附近的 IgG 强度明显增加了150。植入的动物的 IgG 强度逐渐恢复到背景强度的距离辐射从植入微电极孔。在控制动物, 从来没有植入微电极, 规范化的 IgG 强度没有在大量的背景强度以上, 因为血脑屏障没有损坏这些动物。
由于在阶梯表现和 IgG 强度上均有显著差异, 两者有相关性 (图 4)。在这一研究过程中, 每种动物的组织-电极界面0-50 µm 下的 IgG 区的规范化荧光强度与每种动物的阶梯性能的平均值相关。测定了0.90 的相关系数, 证明了优良的运动性能与血脑屏障损伤之间存在很强的相关性。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57829/57829fig1.jpg"> 图 1.代表开放域网格测试结果.(A) 本小组展示了一个开放领域网格测试 (用于总马达和焦虑测试) 的行为测试设置。开放场网格测试包括1米2丙烯酸板材与4个不透明的墙壁 40 cm 在高度, 并且正方形底部部分大约 33 cm 每个。(B) 本小组显示, 与基线性能相比, 通过跨越的网格线数衡量的总马达功能性能。在手术后2周 (p < 0.05), 对照组 (n = 10) 和植入 (n = 17) 小组的表现有显著差异。所有错误都报告为 SEM。这个数字是从瓦利等转载的。27以允许从自然出版小组。<a href="/files/ftp_upload/57829/57829fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57829/57829fig2.jpg"> 图 2.有代表性的梯子测试结果.(A) 此面板显示了阶梯测试的行为测试设置 (用于精细的马达功能测试)。梯子由2个清楚的丙烯酸边 1 m 在长度和 25 cm 在高度, 连接由不锈钢台阶间隔在 2 cm 与3毫米直径。(B) 本小组显示, 与基线性能相比, 通过梯子的时间测量的发动机性能良好。虚线下面的结果表明, 与基线性能相比, 性能下降。在手术后周 3-16 (** = p < 0.05, ** = p < 0.01) 和纵向贯穿整个研究中发现了对照 (n = 10) 和植入 (n = 17) 组之间的显著差异 (# = p < 0.05)。(C) 本小组展示了一个量化的右前爪滑的实例。比较对照组和植入群 (** p < 0.05) 时, 每周右前爪滑的发生有显著差异。(D) 这是一个爪滑的例子。所有错误都报告为 SEM。这个数字是从瓦利等转载的。27以允许从自然出版小组。<a href="/files/ftp_upload/57829/57829fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57829/57829fig3.jpg"> 图 3.具有代表性的握杆强度试验结果.(A) 本小组展示了握杆强度的行为测试设置 (用于精细电机功能测试)。握杆强度计由一个加权基座组成, 安装的强度计与手柄车把相连。(B) 本小组显示了优良的马达功能性能, 以与基线性能相比所施加的最大握力强度来衡量。虚线下面的结果表明, 与基线性能相比, 性能下降。在控制 (n = 5) 和植入 (n = 6) 动物几乎所有手术后周 (** = p < 0.05, ** = p < 0.01, *** = p < 0.001) 之间有显著差异。在控制动物每周和基线表现之间 (# = p < 0.05) 和植入动物的每周和基线表现之间也有进一步的意义 (# = p < 0.01)。控制和被植入的动物在整个研究中的纵向表现显著不同 (@@@ = p < 0.001)。<a href="/files/ftp_upload/57829/57829fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57829/57829fig4.jpg"> 图 4.IgG 与阶梯性能的相关性.植入部位周围的规范化 IgG 荧光强度与阶梯性能的变化相关, 发现0.901 的相关系数 (p < 0.001)。<a href="/files/ftp_upload/57829/57829fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Table 1" src="/files/ftp_upload/57829/57829table1.jpg"> 表 1.总体代表性行为数据显示, 与每个测试指标的基线评分相比, 性能的增加和降低.绿色盒子代表了一个改进的性能, 使电机赤字不太可能, 红色的盒子代表了降低性能, 使电机功能的赤字可能。

Watch this Scientific Journal Video about Rodent Behavioral Testing to Assess Functional Deficits Caused by Microelectrode Implantation in the Rat Motor Cortex at JoVE.com

Rodent Behavioral Testing to Assess Functional Deficits Caused by Microelectrode Implantation in the Rat Motor Cortex

我们已经表明, 在大鼠的运动皮层的微电极植入导致立即和持久的运动缺陷。本文提出的方法概述了微电极植入手术和三啮齿目动物行为任务, 以阐明由于植入造成的运动皮层损伤的细微或总运动功能的潜在变化。

大鼠运动皮质微电极植入所致功能缺损的啮齿动物行为检测

Medical devices implanted in the brain hold tremendous potential. As part of a Brain Machine Interface (BMI) system, intracortical microelectrodes ...

rodent-behavioral-testing-to-assess-functional-deficits-caused

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

8.8K 次观看.  Louis Stokes Cleveland Department of Veterans Affairs Medical Center. 我们已经表明, 在大鼠的运动皮层的微电极植入导致立即和持久的运动缺陷。本文提出的方法概述了微电极植入手术和三啮齿目动物行为任务, 以阐明由于植入造成的运动皮层损伤的细微或总运动功能的潜在变化。

视频: Rodent Behavioral Testing to Assess Functional Deficits Caused by Microelectrode Implantation in the Rat Motor Cortex

Simultaneous fMRI and Electrophysiology in the Rodent Brain

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in which functional MRI data and in vivo intracortical recording can be performed simultaneously. The combination of MRI and electrical recording is technically challenging because the electrodes used for recording distort the MRI images and the MRI acquisition induces noise in the electrical recording. To minimize the mutual interference of the two modalities, glass microelectrodes were used rather than metal and a noise removal algorithm was implemented for the electrophysiology data. In our studies, two microelectrodes were separately implanted in bilateral primary somatosensory cortices (SI) of the rat and fixed in place. One coronal slice covering the electrode tips was selected for functional MRI. Electrode shafts and fixation positions were not included in the image slice to avoid imaging artifacts. The removed scalp was replaced with toothpaste to reduce susceptibility mismatch and prevent Gibbs ringing artifacts in the images. The artifact structure induced in the electrical recordings by the rapidly-switching magnetic fields during image acquisition was characterized by averaging all cycles of scans for each run. The noise structure during imaging was then subtracted from original recordings. The denoised time courses were then used for further analysis in combination with the fMRI data. As an example, the simultaneous acquisition was used to determine the relationship between spontaneous fMRI BOLD signals and band-limited intracortical electrical activity. Simultaneous fMRI and electrophysiological recording in the rodent will provide a platform for many exciting applications in neuroscience in addition to elucidating the relationship between the fMRI BOLD signal and neuronal activity.

We have developed a method for simultaneous functional magnetic resonance imaging and electrophysiological recording in the rodent brain, providing a platform for the investigation of the relationship between neural activity and the blood oxygenation level dependent (BOLD) MRI signal.

同时在啮齿动物脑功能磁共振成像和电

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in ...

科研

神经科学

A Novel Approach to Assess Motor Outcome of Deep Brain Stimulation Effects in the Hemiparkinsonian Rat: Staircase and Cylinder Test

Deep brain stimulation of the subthalamic nucleus is an effective treatment option for Parkinson&#39;s disease. In our lab we established a protocol to screen different neurostimulation patterns in hemiparkinsonian (unilateral lesioned) rats. It consists of creating a unilateral Parkinson&#39;s lesion by injecting 6-hydroxydopamine (6-OHDA) into the right medial forebrain bundle, implanting chronic stimulation electrodes into the subthalamic nucleus and evaluating motor outcomes at the end of 24 hr periods of cable-bound external neurostimulation. The stimulation was conducted with constant current stimulation. The amplitude was set 20% below the individual threshold for side effects. The motor outcome evaluation was done by the assessment of spontaneous paw use in the cylinder test according to Shallert and by the assessment of skilled reaching in the staircase test according to Montoya. This protocol describes in detail the training in the staircase box, the cylinder test, as well as the use of both in hemiparkinsonian rats. The use of both tests is necessary, because the staircase test seems to be more sensitive for fine motor skill impairment and exhibits greater sensitivity to change during neurostimulation. The combination of the unilateral Parkinson model and the two behavioral tests allows the assessment of different stimulation parameters in a standardized way.

Deep brain stimulation (DBS) is an effective treatment option for Parkinson's disease. We established a study design to screen novel stimulation paradigms in rats. The protocol describes the use of the staircase test and cylinder test for motor outcome assessment in DBS treated hemiparkinsonian rats.

一种新的方法，以评估在帕金森病大鼠脑深部电刺激效应的运动结果:楼梯和缸测试

Deep brain stimulation of the subthalamic nucleus is an effective treatment option for Parkinson&#39;s disease. In our lab we established a protocol to ...

行为学

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, ...

A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats

In vivo electrophysiology is a powerful technique to investigate the relationship between brain activity and behavior at a millisecond and micrometer scale. However, current methods mostly rely on tethered cable recordings or only use unidirectional systems, allowing either recording or stimulation of neural activity, but not at the same time or same target. Here, a new wireless, bidirectional device for simultaneous multichannel recording and stimulation of neural activity in freely behaving rats is described. The system operates through a single portable head stage that both transmits recorded activity and can be targeted in real-time for brain stimulation using a telemetry-based multichannel software. The head stage is equipped with a preamplifier and a rechargeable battery, allowing stable long-term recordings or stimulation for up to 1 h. Importantly, the head stage is compact, weighs 12 g (including battery) and thus has minimal impact on the animal&#180;s behavioral repertoire, making the method applicable to a broad set of behavioral tasks. Moreover, the method has the major advantage that the effect of brain stimulation on neural activity and behavior can be measured simultaneously, providing a tool to assess the causal relationships between specific brain activation patterns and behavior. This feature makes the method particularly valuable for the field of deep brain stimulation, allowing precise assessment, monitoring, and adjustment of stimulation parameters during long-term behavioral experiments. The applicability of the system has been validated using the inferior colliculus as a model structure.

A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats

A wireless, bidirectional system for multi-channel neural recordings and stimulation in freely behaving rats is introduced. The system is light and compact, thus having minimal impact on the animal&#180;s behavioral repertoire. Moreover, this bidirectional system provides a sophisticated tool to assess causal relationships between brain activation patterns and behavior.

用于体内的无线双向接口记录和刺激自由行为大鼠的神经活动

In vivo electrophysiology is a powerful technique to investigate the relationship between brain activity and behavior at a millisecond and micrometer ...

A Novel Single Animal Motor Function Tracking System Using Simple, Readily Available Software

We have recently demonstrated that implanting intracortical microelectrodes in the motor corteces of rats&#160;results in immediate and lasting motor deficits. Motor impairments were manually quantified through an open field grid test to measure the gross motor function and through a ladder test to measure the fine motor function. Here, we discuss a technique for the automated quantification of the video-recorded tests using our custom Capadona Behavioral Video Analysis System: Grid and Ladder Test, or BVAS. Leveraging simple and readily available coding software (see the Table of Materials), this program allows for the tracking of a single animal on both the open field grid and the ladder tests. In open field grid tracking, the code thresholds the video for intensity, tracks the position of the rat over the 3 min duration of the grid test, and analyzes the path. It then computes and returns measurements for the total distance traveled, the maximum velocity achieved, the number of left- and right-handed turns, and the total number of grid lines crossed by the rat. In ladder tracking, the code again thresholds the video for intensity, tracks the movement of the rat across the ladder, and returns calculated measurements including the time it took the rat to cross the ladder, the number of paw slips occurring below the plane of the ladder rungs, and the incidence of failures due to stagnation or reversals. We envision that the BVAS developed here can be employed for the analysis of motor function in a variety of applications, including many injury or disease models.

The current study aimed to automate the quantification of motor deficits in rats. The initial evaluation model assesses motor loss resulting from an intracortical microelectrode implantation in the motor cortex. We report on the development and use of a tracking algorithm using easily adaptable, simple, and readily available coding software.

一种新的单动物运动功能跟踪系统, 使用简单、易于使用的软件

We have recently demonstrated that implanting intracortical microelectrodes in the motor corteces of rats&#160;results in immediate and lasting motor ...

Simultaneous Monitoring of Wireless Electrophysiology and Memory Behavioral Test as a Tool to Study Hippocampal Neurogenesis

Brainwaves amplitude obtained from electroencephalography (EEG) has been well-recognized as a basis for cognitive capacity, memory, and learning on animals and humans. Adult neurogenesis mechanism is also linked to memory and learning improvement. Traditionally, researchers used to assess learning and memory parameters in rodent models by behavioral tasks. Therefore, the simultaneous monitoring of behavioral changes and EEG is particularly interesting in correlating data between brain activity and task-related behaviors. However, most of the equipment required to perform both studies are either complex, expensive, or uses a wired setup network that hinders the natural animals&#8217; movement. In this study, EEG was recorded with a wireless electrophysiology device along with the execution of a novel object recognition task (NORT). The animal&#8217;s behavior was monitored simultaneously by a video tracking system. Both recordings were analyzed offline by their timestamps which were synched to link EEG signals with the animal&#8217;s actions. Subjects consist of adult Wistar rats after medium-term environmental enrichment treatment. Six skull screw electrodes were fixed in pairs on both hemispheres over frontal, central, and parietal regions and were referenced to an electrode located posterior of the nasal bone. NORT protocol consists of exposing the animal to two identical objects for 10 min. After 2 h and 24 h, one of the objects was replaced with a novel one. Exploration time for each object was monitored by a behavioral tracking software (BTS) and EEG data recording. The analysis of the EEG synced with behavioral data consists of estimations of alpha and beta relative band power and comparisons between novel object recognition versus familiar object exploration, between three experimental stages. In this manuscript, we have discussed electrodes manufacturing process, epidural electrodes implantation surgery, environmental enrichment protocol, NORT protocol, BTS setup, EEG &#8211; BTS coupling for simultaneous monitoring in real-time, and EEG data analysis based on automatic events detection.

The protocol presented here provides information on the simultaneous electroencephalography (EEG) and behavioral assessment in real-time. We have discussed all steps involved in this protocol as an attractive solution for researchers in many fields of neuroscience, particularly in learning and memory areas.

同时监测无线电生理学和记忆行为测试作为研究海马神经发生的工具

Brainwaves amplitude obtained from electroencephalography (EEG) has been well-recognized as a basis for cognitive capacity, memory, and learning on ...

Gathering Self-Initiated Rat Behavioral Data to Characterize Post-Stroke Deficits

Behavioral testing in rat models is frequently utilized for diverse purposes, including psychological, biomedical, and behavioral research. Many traditional approaches involve individual, one-on-one testing sessions between a single researcher and each animal in an experiment. This setup can be very time consuming for the researcher, and their presence may impact the behavioral data in unwanted ways. Additionally, traditional caging for rat research imposes a lack of enrichment, exercise, and socialization that would normally be typical for the species, and this context may also skew the results of behavioral data. Overcoming these limitations may be worthwhile for several research applications, including the study of acquired brain injury. Here, an example method is presented for automatically training and testing individual rat behavior in a colony cage without the presence of humans. Radio frequency identification can be utilized to tailor sessions to the individual rat. The validation of this system occurred in the example context of measuring skilled forelimb motor performance before and after stroke. Traditional characteristics of post-stroke behavioral impairments and novel measures enabled by the system are measured, including success rate, various aspects of pull force, bout analysis, initiation rate and patterns, session duration, and circadian patterns. These variables can be collected automatically with few limitations; though the apparatus removes experimental control of exposure, timing and practice, the validation produced reasonable consistency in these variables from animal to animal.

A system for acquiring data from self-initiated individual behavior sessions within a social colony cage setting is presented. The efficacy of this system is demonstrated using an automated skilled reach assessment, enabling the characterization of post-stroke motor impairments, potential behavioral alterations related to motivation, circadian variations, and other innovative dependent variables.

收集自发的大鼠行为数据以表征中风后缺陷

Behavioral testing in rat models is frequently utilized for diverse purposes, including psychological, biomedical, and behavioral research. Many ...

Microelectrode Guided Implantation of Electrodes into the Subthalamic Nucleus of Rats for Long-term Deep Brain Stimulation

Deep brain stimulation (DBS) is a widely used and effective therapy for several neurologic disorders, such as idiopathic Parkinson&#8217;s disease, dystonia or tremor. DBS is based on the delivery of electrical stimuli to specific deep anatomic structures of the central nervous system. However, the mechanisms underlying the effect of DBS remain enigmatic. This has led to an interest in investigating the impact of DBS in animal models, especially in rats. As DBS is a long-term therapy, research should be focused on molecular-genetic changes of neural circuits that occur several weeks after DBS. Long-term DBS in rats is challenging because the rats move around in their cage, which causes problems in keeping in place the wire leading from the head of the animal to the stimulator. Furthermore, target structures for stimulation in the rat brain are small and therefore electrodes cannot easily be placed at the required position. Thus, a set-up for long-lasting stimulation of rats using platinum/iridium electrodes with an impedance of about 1 M&#937; was developed for this study. An electrode with these specifications allows for not only adequate stimulation but also recording of deep brain structures to identify the target area for DBS. In our set-up, an electrode with a plug for the wire was embedded in dental cement with four anchoring screws secured onto the skull. The wire from the plug to the stimulator was protected by a stainless-steel spring. A swivel was connected to the circuit to prevent the wire from becoming tangled. Overall, this stimulation set-up offers a high degree of free mobility for the rat and enables the head plug, as well as the wire connection between the plug and the stimulator, to retain long-lasting strength.

A method for implanting electrodes into the subthalamic nucleus (STN) of rats is described. Better localization of the STN was achieved by using a microrecording system. Furthermore, a stimulation set-up is presented that is characterized by long-lasting connections between the head of the animal and the stimulator.

微电极引导电极植入到大鼠丘脑底核的长期深部脑刺激

Deep brain stimulation (DBS) is a widely used and effective therapy for several neurologic disorders, such as idiopathic Parkinson&#8217;s disease, ...

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a closed-loop event-triggered manner. Although many studies are focusing on the possible benefits and side-effects of TES in healthy and pathologic brains, there are still many fundamental open questions regarding the mechanism of action of the stimulation. Therefore, there is a clear need for a robust and reproducible method to test the acute and the chronic effects of TES in rodents. TES can be combined with regular behavioral, electrophysiological, and imaging techniques to investigate neuronal networks in vivo. The implantation of transcranial stimulation electrodes does not impose extra constraints on the experimental design while it offers a versatile, flexible tool to manipulate brain activity. Here we provide a detailed, step-by-step protocol to fabricate and implant transcranial stimulation electrodes to influence brain activity in a temporally constrained manner for months.

This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving rats.

大鼠慢性经颅电刺激及 Intracortical 记录

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

Establishment of Acute Pontine Infarction in Rats by Electrical Stimulation

Pontine infarction is the most common stroke subtype in the posterior circulation, while there lacks a rodent model mimicking pontine infarction. Provided here is a protocol for successfully establishing a rat model of acute pontine infarction. Rats weighing about 250 g are used, and a probe with an insulated sheath is injected into the pons using a stereotaxic apparatus. A lesion is produced by the electrical stimulation with a single pulse. The Longa score, Berderson score, and beam balance test are used to assess neurological deficits. Additionally, the adhesive-removal somatosensory test is used to determine sensorimotor function, and the limb placement test is used to evaluate proprioception. MRI scans are then used to assess the infarction in vivo, and TTC staining is used to confirm the infarction in vitro. Here, a successful infarction is identified that is located in the anterolateral basis of the rostral pons. In conclusion, a new method is described to establish an acute pontine infarction rat model.

Presented here is a protocol for establishing acute pontine infarction in a rat model via electrical stimulation with a single pulse.

电刺激在大鼠中建立急性庞廷梗死

Pontine infarction is the most common stroke subtype in the posterior circulation, while there lacks a rodent model mimicking pontine infarction. Provided ...