这项研究如果资助的健康研究的加拿大研究所和心脏及中风BC和育空地区的基础。我们要感谢Rayshad Gopaul先生和谢利McErlane博士在动物保健技术支持和专业知识。

雄性Wistar（HSD:WI的Wistar）大鼠7周龄，体重300-350克本实验中使用。所有大鼠维持12小时的光/暗周期，并获得标准的实验室大鼠饲料和水随意。所有实验程序符合由加拿大议会关于动物饲养建立先导，以护理和使用实验动物，并授予伦理委员会批准由英国哥伦比亚大学。手术和动物护理是根据我们实验室的标准程序（拉姆齐等，2010）17进行。 1.动物的制备:外科手术<ol><li> 遥测装置植入 注:协议被实施根据制造商说明和我们以前的与设备的注入经验:16 <ol><li>与恩诺沙星预治疗的动物，一旦一个连续3天每天（10毫克/千克，S.C。）手术前。 </li><li>在手术前从笼中取出固体食物9-13小时。在此期间可能会提供软食营养代餐。 </li><li>麻醉用4％异氟烷在感应腔中诱导剂量和使用贝恩电路约1.5％异氟烷（1-1.5升/分钟氧气）的维持剂量的动物。用脚趾捏反射和角膜反射来评估动物是否是在麻醉手术平面。呼吸必须监测以评估是否麻醉深度太高（ 即 ，非常缓慢呼吸速率和喘息，苍白粘膜）或过低（ 即，快速，浅呼吸指示动物可能不会在麻醉的手术平面）。在手术期间和麻醉大约每5分钟的外科平面的重复监测调整异氟烷剂量是必要的。 注:所有手术使用的无菌技术进行:具体而言，医生穿外科擦洗，帽，口罩和无菌手套。开配戴手套过程观察，高压灭菌含有手术器械使用无菌技术被打开手术组和动物开始手术之前叠加。手术工具是尖灭菌烫珠手术间（最多5次手术）。外科海绵和纱布使用前高压灭菌。在手术过程中，将动物保持在维持在37℃的循环水毯。 </li><li>辖丁丙诺啡（0.02毫克/千克，SC）立即手术前。此外，管理酮洛芬（5毫克/千克; SC）手术前，以提供多峰镇痛。辖布比卡因（5毫克/公斤，SC）立即手术前，以提供本地线路块。在剃须腹部，应用交替氯己定和70％的乙醇拭子（各三次），接着碘伏为至少1分钟，并用70％的乙醇（具有新开醇每次擦拭）除去。皮肤的擦拭在同心的方式制成在切口线的中心开始并向外盘旋（以便不通过"回/来回"运动污染的区域）。用无菌纱布拭子和各个应用程序之间丢弃。此过程开始动物手术前重复三次。虽然修剪和手术擦洗，保持对异氟醚下约1.5％维持剂量连接到贝恩非再呼吸电路的鼻锥动物。以维持所需要的动物在麻醉的手术计划调整异氟烷水平。 </li><li>做一个5厘米长的中线腹部切口，通过肌肉和腹膜露出肠子切割。 </li><li>缩回肠（这将暴露在后腹壁降主动脉），盖上无菌手术纱布肠道用2-3的林格溶液毫升润湿并保持湿润与视需要，以防止组织脱水通过过程的其他林格。 </li><li>钝解剖围绕主动脉和主动脉从利用虹膜铲刀的下腔静脉分离。 </li><li>地方结扎（8-0丝缝合线）围绕主动脉的最外膜层，只是远端肾动脉的喙和尾部端。 </li><li>解除结扎简要地阻塞血液流动，并在1-2毫米前的水平与一个25号针头弯曲在位于头90°髂分叉刺穿主动脉。 </li><li>使用20号弯曲针引导遥测装置的导管的尖端进入主动脉。推进吻侧，以确保该针尖仅仅是远端肾动脉<ol><li>利用组织粘合剂少量修复它。去除结扎并确保有由于导管或组织粘合剂无血流闭塞。 </li></ol></li><li>固定遥测装置的主体成使用4-0丝线缝合腹壁。使用4-0薇乔和4-0普理灵缝线关闭MUSC乐和皮肤，分别。 </li></ol></li><li> 遥测植入手术后护理 注意:对于前14天手术后的动物应每天至少3次检查中，除了一个详细监测评估每天一次。评估动物在7类:体重，体貌（疼痛是基于迹象，如增加眼睛，鼻子和前爪周围看到卟啉生产和面部鬼脸体征评估），行为/活动，排便，皮肤（包括中恒，立毛，切口愈合不良及皮下积液），水化和呼吸。 <ol><li>喂动物，水果，谷物品种，软食营养代餐，并提供水自由采食，遥测装置植入后6天。动物应该有扣缴第一天操作后定期固体啮齿动物饲料。注入动物温热乳酸林格溶液（5毫升）皮下第3天手术后，经外观脱水的症状。管理在早上和晚上检查（皮肤帐篷苍白耳/粘膜的迹象）林格溶液（5毫升）在必要时对动物进行评估。 </li><li>辖恩诺沙星（10毫克/千克，SC）和酮洛芬（5毫克/公斤，SC），每天一次，连续3天手术后。手术后的以下3天早晚（ 即，每12小时）期间管理丁丙诺啡（0.02毫克/公斤，SC）。如果疼痛/窘迫的迹象都指出丁丙诺啡中旬的一天剂量（0.02毫克/千克，SC）可施用。 </li></ol></li><li> 脊髓全横断 注:让动物从遥测植入手术T3完全离断术前恢复至少14天。 <ol><li>预对待动物的预防性恩诺沙星（10毫克/公斤，SC）为SCI手术前三天。 </li><li>如前所述麻醉4％异氟烷的诱导剂量下的动物。进一步，用注射盐酸氯胺酮（70毫克/千克，IP）和盐酸右美托咪（0.5毫克/千克，IP）的动物。 注意:对于完全离断手术，造成异氟醚血管扩张往往呈现止血的问题，一旦线切断。可以通过使用氯胺酮/右美托咪鸡尾酒来防止这个问题。动物应麻醉20-25分钟，并使用脊髓横断手术以下阿替美唑（1毫克/千克，SC）逆转。 </li><li>手术前施用皮下丁丙诺啡（0.02毫克/千克），和酮洛芬（5毫克/千克）。辖布比卡因（5毫克/公斤，SC）立即手术前，以提供本地线路块。在剃须腹部，交替氯己定和70％乙醇的刷子（各三次），接着碘伏应适用于至少1分钟，并用70％的乙醇（具有新开醇每次擦拭）中除去。刷子在同心的方式作出开始在切口线的中心，并且向外盘旋（以便不通过"回/来回"运动污染的区域）。用无菌纱布和各个应用程序之间丢弃。开始动物手术前重复该过程三次。虽然修剪和手术擦洗，保持动物的鼻锥下异氟醚的约1.5％，维持剂量（按要求调整，以保持麻醉手术平面）。在整个手术动物的持续时间被保持在保持在37℃的循环水毯。 </li><li>做一个背中线切口，2厘米长，在浅肌层覆C8-T3椎体。 </li><li>布朗特以揭示硬脑膜层解剖。 </li><li>刺穿与microscissors或用25号针头的尖端硬脑膜层。 </li><li>打开在T2-T3椎间隙硬脑膜和使用microscissors执行完全横断。打开microscissors但不足够的横向破裂椎arterieS和静脉。在迅速的运动横切脊髓和搜索在动物后肢夸张的抽搐。确认通过喙和尾部脊髓树桩目视间隔完全横断。 </li><li>确认后，将树桩之间明胶海绵，以达到止血。施加足够的明胶海绵止血和脊髓的树桩分开。 </li><li>使用4-0薇乔和4-0普理灵缝线关闭肌肉和皮肤，分别为。脊髓损伤手术后，给动物预温热乳酸林格氏溶液（5毫升，SC 30℃），并允许它们在温度受控的环境中恢复。 </li></ol></li><li> 脊髓横断术后护理 注意:对于前14天手术后的动物应每天3次检查中，除了每天一次一个详细的监测评估。 14天后详细的监测评估可能隔日重复。评估动物在7 catego里斯:体重，体貌（疼痛是基于迹象，如卟啉和面部鬼脸评估），行为/活动，排便，皮肤（包括中恒，竖毛，切口愈合不良及皮下积液），水化和呼吸。用于第一5-7天气囊应该检查并表示每天至少3次。 <ol><li>喂动物，水果，谷物品种，营养软食代餐，并提供水自由采食，脊髓横断手术后8-14天。注入用温热乳酸林格氏溶液（5毫升）皮下注射动物手术后的第3天，在脱水症状。管理的检查过程中林格溶液（5毫升）在必要时对动物进行评估。 注:脊髓横断后，大鼠表现出后肢瘫痪，并采用屈曲和前肢，这是在这个损伤模型保存扩展走动。老鼠缺乏躯干和尾巴支持。为了方便它们在笼子里的运动，橡胶格应置于下滚燕麦床上用品。这使得大鼠与前爪抓住网格，并在笼子容易移动到获取食物和水。床上用品应每天更换。在脊髓横断后第7天，燕麦床上用品应该由它们原来的木片床上用品所取代。因为给大鼠丁丙诺啡燕麦卷被褥使用可以开发PICA在他们吃的床上用品，这可能会导致食管impactions。燕麦与燕麦食管吸湿嵌塞尚未观察到。 </li><li>辖恩诺沙星（10毫克/千克，SC）和酮洛芬（5毫克/公斤，SC）每天一次连续3天手术后。手术后的以下3天早晚（ 即 ，每12小时）期间管理丁丙诺啡（0.02毫克/公斤，SC）。如果疼痛/窘迫的迹象都指出丁丙诺啡中旬的一天剂量可以。 </li></ol></li></ol> 2.遥测血流参数监测<ol><li>收集每天打逐拍动脉血压（ABP）和核心体温24小时，预先和后SCI。采样动脉血压和核心体温在使用遥测装置24小时的块1000赫兹。 </li><li>放置的SmartPad下动物笼换能器的无线再充电，并用于接收来自换能器的数字遥测信号。 注意:输出电压随后被传递到连接到计算机，该组织从每个通道的数据流的配置。该电压被转换为连续的动脉血压和温度记录。可视利用数据采集软件的采样数据，如LabChart。 </li><li>丢弃动物监测过程中收集的遥测数据，以及在10分钟之前立即监测和监测10分钟后。 </li></ol> 3.自发INCID评估自主神经异常反射的分配办法（AD） 注:频率，严重程度，和自发的广告事件的持续时间是用我们自己的小说AD检测 JAVA平台软件（ 图4）开发了一种算法评估。新算法已经发展到自动根据之前和SCI利用图2指定的参数后24小时SBP和HR遥测录音自发的广告事件。 <ol><li>在提取使用采集软件的计息期限从原材料遥测数据SBP和HR值。 注意:采集软件检测并确定从连续ABP录音SBP峰的值。心脏速率是通过确定相邻收缩压峰之间的时间距离外推。 </li><li>上传遥测录音在A列interbeat区间，在B列SBP值，列C MAP值和时间的CSV文件我N个列D（图4A）。 </li><li>使用软件（图4B-D），指定相关生理HR范围（即 R到R的间隔）180 BPM和625 BPM之间的SBP录音。指定下心率和最小 心率最大面板的软件中的相关人力资源范围。 </li><li>通过240秒的移动平均窗口中创建了SBP和HR的阈值。表明移动平均阈值在广告持续时间阈值面板上的分析窗口长度。 </li><li>在20毫米汞柱均线基线上方设置一个SBP换位门槛。表明，在移动平均阈值换位BP面板换位值。 </li><li>隔离收缩压峰簇超出换位阈用峰 - 峰间隔少于2秒和大于10秒的持续时间。指定峰使用峰间间隔BP和杜峰间隔使用峰丛间隔BP面板比间隔。 </li><li>组收缩压峰簇是彼此的120秒内。 注意:在彼此的120秒内检测到的电位的AD事件将被分组为单个事件。指定连续SBP集群之间可以区分的广告持续时间阈值面板独立的广告事件的最长持续时间。 </li><li>确认是否收缩压峰簇群通过检测在40 BPM或更大的人​​力资源的下降与潜在自发的AD事件相关联。 <ol><li>在潜在事件的发病人力资源值的平均值的10％（指定要在上心脏速率这个百分比下降平均范围面板）。从电势事件结束的心脏速率值的平均值的75％（指定要在下部这个百分比心脏率下降平均范围面板）。 </li><li>减去从较高心脏速率阈下心脏速率的阈值，以确保一个correspon40 BPM或更大的下降鼎。使用指定的心脏率下降限制面板的心脏率下降限制。 </li></ol></li><li>一旦面板已经排满，按确定 。检测出的AD事件的图形表示呈现，包括收缩压的尖峰和相关联的人力资源数据（ 图4C-D）。一个输出excel文件也与升压反应（毫米汞柱），持续时间（秒），最大值收缩压（毫米汞柱），最小的HR（BPM），每个检测出的AD事件的心率下降（BPM）中产生。 </li></ol> 4.结直肠扩张有意引导学生AD 注:AD诱发的严重程度可以通过结直肠扩张（CRD），模仿肠例行3,18,19临床相关的刺激来确定。 <ol><li>验证弗伦奇导管的囊（直的，10法语，将3ml 35厘米，游离胶乳）不被用2ml空气充气它泄漏。 </li><li>第约束通过包裹在毛巾，让血压和心率稳定了半个小时ê动物。 </li><li>放置润滑剂在导管的尖端。放置一个标记在导管用永久性标记，2厘米从气囊的中间。导管经由肛门插入结肠插入直到标记在开口刚刚可见。固定导管与外科胶带的尾部。重新放置在他的家笼中的动物包裹放在接收器匹配的SmartPad在动物体内植入的传感器。允许动物的血压正常化10分钟。 </li><li>导管的插入和相关的适应期（ 即，10分钟）后，注入2毫升的空气的气球在10秒内，使血压和心率稳定。保持扩张1分钟。确保在导管的尖端保持在直肠内。重要的是不要过度膨胀球囊（ 即，将 3ml +），以避免对结肠的破裂。 注意:腹胀为1分钟的持续时间与＃160;允许升压反应的发展和与AD的感应情节相关联的压力感受器介导的HR降适当的时间。此外，此1分钟内还提供了足够的时间，心血管指数的正常化。 </li><li>重复扩张每次​​试验的3倍，试验间10分钟的最小间隔每日重复试验。一旦评估完成，从动物取出导尿管。 </li><li>平均节拍由拍数据超过1秒的间隔和报告SBP在多个试验最高涨幅和最大跌幅人力资源。 </li></ol>

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The authors have nothing to disclose.

该协议描述了详细的实施这将与一个遥测装置相结合，在SCI-动物（ 图1B）ABP的长期深入分析的新的JAVA平台的AD检测软件。这是第一个软件，它允许为ABP图案表征，因为它们偶尔发生在一天的持续时间，以检测自发广告事件。充分表征T3 SCI动物模型可以说明检测自发AD的频率，升压反应和持续的软件功能的能力。随着检测自发广告事件，该软件可以分析ABP振荡和辨别血流动力学参数的日变化。 小说AD检测软件的高效，可靠，准确地检测和特征的急性和慢性脊髓损伤动物自发广告事件。广告事件被认为对增加S中出现的20毫米汞柱或更高（ 图2A）和BP通常与明显的心动过缓9相关联。在这个特定的研究中，收缩压波动被认为要dysreflexic插曲的外观的主指示器，虽然软件具有也使用图应研究者愿望的能力。该软件具有通过检测用户确定量的经AD事件（图2B）的发病的HR降辨别明显心动过缓的能力。 Rabchevsky 等 。利用10 BPM的特性下降作为在其上高血压事件被分类为自发的AD事件的阈值。这必须被质疑，因为鼠类具有非常高的静止心脏速率;因此，10 BPM很可能不足以确定心动过缓。 HR压降限制可以由用户进行修改，以不仅确定在人力资源下降，但根据不同的脊髓损伤的水平和完整性也增加心率。 之前这个软件，CRD诱发AD被认为是引起AD的健壮方法，既实验可行和充分表征（ 图3A，3B）15,20。在大鼠动物模型具有较高的SCI AD的重复感应最初发现模仿高胸SCI 4相关的广告的自发和频繁发作。 CRD为大鼠高胸椎的SCI 14的AD一种有效，无创性刺激它将模拟一些AD的最常见的原因临床如便秘和粪便嵌塞3。 CRD诱发AD不占多种传入刺激也可能唤起这些反射介导的高血压事件。在检测和自发AD表征一起这样CRD诱发AD提供了理想的范围中，我们可以研究AD的时间表型。 遥测是清醒监测生理功能一个国家的最先进的方法和自由活动的动物，同时尽量减少应激相关的文物，如苦恼，处理和麻醉21。在此情况下，使用由轻质biosilicone材料的固态压力传感器。相对于流体基于导管的低压反应，固态压力传感器可以监视在心血管参数的细微变化。这些传感器可以品尝连续的，由心跳的血压和体温的频率一掌至2 kHz。固态传感器也具有防止在流体基于导管通常遇到的运动伪影的益处。虽然遥测设备是侵入性和昂贵的，它准确地监测血流动力学参数15,16,21,22的昼夜节律。植入后恢复时间为动物的存活极为重要的，因为机械性梗阻到由植入物本身的腹主动脉的血流量，可能会导致血液供应不足的后肢博DY 15。 自发的AD遥测检测，除了CRD诱发AD，考虑与这些威胁生命的事件的发生相关的刺激的宽光谱。因此，检测和表征广告事件是考虑脊髓损伤患者的治疗至关重要。目前可用的人类，使慢性，由跳动，血液动力学参数的监测击败没有可行的无创技术。动态血压监测不足由于低时间分辨率。动物模型是必要的，以便精确地检测和表征利用新颖的AD检测软件这些自发广告事件的发作。在新的连续的血液动力学监测技术的出现，该软件可作为重要工具临床监测这些事件的发生可以应用。与AD检测软件结合使用遥测设备的可能是一个有用的未来战略临床上动态监测急性AD的。

自主神经异常反射（AD）是在颈部或高胸节急性或慢性脊髓损伤（SCI）后的个体威胁生命的紧急情况和通常的特征在于持续性高血压和心动过缓1情节。 AD主要是由脊髓下行通道，通常要控制交感神经活性和血管张力1-4脊髓交感神经节前神经元提供了从脊髓上输入中心的破坏引起的。高达300毫米汞柱的AD发作的特征在于收缩压（SBP）尖峰并且如果不治疗会导致癫痫发作，颅内出血，心肌梗塞，甚至死亡5-8。各种有害和非伤害性刺激的作为AD的触发，包括肠和膀胱膨胀，痉挛，压疮，膀胱导尿或医源性程序9-12。 AD的RESP的时间发展ONSE以SCI已经在人类9和动物模型13,14了研究。通常这些研究已经使用了"诱导AD"的方法（ 即尿流动力学，在人类或动物的结直肠扩张阴茎vibrostimulations）确定AD的时间发展。这种方法由于需要在分离的时间点重复评估可能排除AD的时间发展的一个精确确定的限制。在人类中使用的24小时的血压监测器在预定的时间间隔进行串行血压测量。这种技术最近已用于监测慢性SCI自发产生的AD。在动物模型中，固态压力传感器正在越来越多地用于长期监控打逐击败动脉血压。近日，Rabchesvky 等 。 （2012），开发出了提取平均动脉压的二分之一的平均值的算法（MAP）的一个对一个移动平均阈值15相比Ð。基于MAP的峰是10毫米汞柱或更大的阈值以上的同时用10 BPM或更大的HR一滴自发公元事件进行了表征。 在这里，有一个建于公元检测算法一种新型的JAVA软件呈现。这个算法通过检测动脉血压（ABP）和心脏速率（HR），该指示一个自然发生​​的AD事件的预先确定的图案。用户能够手动调整所有输入变量的软件，使得&#39;检测算法"可以容易地定制，以所关注的具体参数。该软件还能够二分法ABP和HR成一个给定的时期，使得血流动力学参数的昼夜节律可以分析16。在本手稿，详细说明中给出，用于植入遥测设备和进行SCI手术手术技术。前还提供了相对于所述的AD检测软件的后处理能力和功能如何心血管amples被改变后的SCI。为了比较的目的，从被称为结直肠扩张诱导的AD的方法得到的方法和结果（CRD）也示出。

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</table>

development of an algorithm to perform a comprehensive study of autonomic dysreflexia in animals with high spinal cord injury using a telemetry device

Spinal cord injury (SCI) is a debilitating neurological condition characterized by somatic and autonomic dysfunctions. In particular, SCI above the mid-thoracic level can lead to a potentially life-threatening hypertensive condition called autonomic dysreflexia (AD) that is often triggered by noxious or non-noxious somatic or visceral stimuli below the level of injury. One of the most common triggers of AD is the distension of pelvic viscera, such as during bladder and bowel distension or evacuation. This protocol presents a novel pattern recognition algorithm developed for a JAVA platform software to study the fluctuations of cardiovascular parameters as well as the number, severity and duration of spontaneously occurring AD events. The software is able to apply a pattern recognition algorithm on hemodynamic data such as systolic blood pressure (SBP) and heart rate (HR) extracted from telemetry recordings of conscious and unrestrained animals before and after thoracic (T3) complete transection. With this software, hemodynamic parameters and episodes of AD are able to be detected and analyzed with minimal experimenter bias.

使用遥测技术，动脉血压是在1000赫兹的频率连续24小时取样。使用LabChart动脉血压（ABP）的说明性记录示于图1B。样品ABP被插入降主动脉固态压力传感器监测。小说JAVA平台AD检测软件能够提取相关收缩压（毫米汞柱）峰（ 图1C）。我们还可以提取来自相邻SBP峰（ 图1D）之间的时间间隔的心率（BPM）。 SBP和HR通过在1000赫兹采样节拍ABP从节拍提取被选作为AD检测软件的输入变量。该软件的增加收缩压大于20毫米汞柱伴随40 BPM或更大的人力资源的减少，如在图2中表征的自发广告事件，这些事件被初步确定利用240秒收缩压移动AVE愤怒基线垂直换位20毫米汞柱。该 AD检测软件，确保检测出的AD事件是通过检查40 BPM或以上相对于一个HR下降到AD事件的发生"真实"。软件检测和表征由节拍从连续击败自发的广告事件，24小时SBP和HR遥测录音。随着日子的这些广告活动的时间和频率，AD检测软件提供了以下表格信息:最大SBP，升压反应，在自发AD事件发生的广告事件的，最小的人力资源和人力资源下降的持续时间。 收缩压的代表图和图3本的HR CRD中的严重程度和引起的AD发作的持续时间。作为导管的气囊被充气时，有一个快速和持续的增加收缩压，伴随着明显的心动过缓。有心血管参数的逐步趋稳为腹胀的进展，和诺玛血流动力学参数小儿导尿管的补肾中药放气。导尿管的插入后，收缩压和HR指数的平均值为60秒的充气之前，以产生一个基线。从基线，在收缩压（毫米汞柱）的尖峰相关联的严重性或升压反应和人力资源（BPM）下降可以被确定。 <img alt="图1" src="/files/ftp_upload/52809/52809fig1.jpg" /> 图1.遥测装置植入动脉血压的代表性跟踪（ABP）。与定向到闭塞降主动脉导管的尖端和遥测设备的固定到腹壁体内遥测传感器植入的示意图的原理图和 16。 ABP的实施例数据在从遥测设备1000赫兹为一个24小时期间取样。从该记录，能够提取心血管指数，如systoliÇ血压（收缩压，毫米汞柱; 图1B）和心脏速率（HR，BPM; 图1C）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52809/52809fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52809/52809fig2.jpg" /> 图2.自主神经异常反射（AD）的脊髓损伤动物自发集检测到收缩压24小时遥测录音自发广告事件新颖的JAVA平台软件。（SBP; 图2A）和心脏速率（HR; 图2B ）。 20毫米汞柱以上收缩压连同后收缩压穗发病40 BPM或以上的人力资源下降的增加被认为是AD的事件。 "上HR门槛"于POTEN发病包括人力资源的平均值10％TiAl基合金的事件。 "低级HR门槛"包括从潜在事件结束的心脏速率值的平均值的75％。从"更高的HR门槛"，以确保40 BPM或更大的相应压降减去"低级HR门限"。 <img alt="图3" src="/files/ftp_upload/52809/52809fig3.jpg" /> 图3.诱导自主神经异常反射（AD）由结直肠扩张（CRD）脊髓代表跟踪受伤的动物。当CRD诱导，有人力资源伴有明显下降SBP（ 图3A）突然持续上升（ 图3B）。 <img alt="图4" src="/files/ftp_upload/52809/52809fig4.jpg" /> 图4. JAVA平台自发自主神经异常反射（AD）检测软件。 </st荣&gt;为CSV格式的连续动脉血压擅长与interbeat间隔（IBI），收缩压（SBP）文件，平均动脉压（MAP）和时间（ 图4A）被上传到该程序并指定了AD检测参数（ 图4B）在协议中所概述。该方案提出，这些自然发生的广告事件（ 图4C-D）的时间的表型。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52809/52809fig4highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Development of an Algorithm to Perform a Comprehensive Study of Autonomic Dysreflexia in Animals with High Spinal Cord Injury Using a Telemetry Device at JoVE

Development of an Algorithm to Perform a Comprehensive Study of Autonomic Dysreflexia in Animals with High Spinal Cord Injury Using a Telemetry Device

The catheter of a telemetry device is implanted into the abdominal aorta in order to continuously collect beat-by-beat hemodynamic data from animals pre and post-high thoracic spinal cord transection. A novel JAVA software was employed to analyze hemodynamic parameters as well as frequency and intensity of spontaneous episodes of autonomic dysreflexia.

一个算法的开发与高位脊髓损伤使用遥测装置进行自主神经异常反射的动物的综合研究

Spinal cord injury (SCI) is a debilitating neurological condition characterized by somatic and autonomic dysfunctions. In particular, SCI above the ...

development-an-algorithm-to-perform-comprehensive-study-autonomic

Nanyang Technological University

Research

JoVE Journal

Medicine

7.8K 次观看.  Faculty of Medicine, University of British Columbia. The catheter of a telemetry device is implanted into the abdominal aorta in order to continuously collect beat-by-beat hemodynamic data from animals pre and post-high thoracic spinal cord transection. A novel JAVA software was employed to analyze hemodynamic parameters as well as frequency and intensity of spontaneous episodes of autonomic dysreflexia.

视频: Development of an Algorithm to Perform a Comprehensive Study of Autonomic Dysreflexia in Animals with High Spinal Cord Injury Using a Telemetry Device

Acute and Chronic Tactile Sensory Testing after Spinal Cord Injury in Rats

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory testing in rat SCI models is needed. However, until recently, no single testing approach had been validated for SCI so that standardized methods have not been implemented across labs. Additionally, available testing methods could not be implemented acutely or when severe motor impairments existed, preventing studies of the development of SCI-induced allodynia3. Here we present two validated sensory testing methods using von Frey Hair (VFH) monofilaments which quantify changes in tactile sensory thresholds after SCI4-5. One test is the well-established Up-Down test which demonstrates high sensitivity and specificity across different SCI severities when tested chronically5. The other test is a newly-developed dorsal VFH test that can be applied acutely after SCI when allodynia develops, prior to motor recovery4-5. Each VFH monofilament applies a calibrated force when touched to the skin of the hind paw until it bends. In the up-down method, alternating VFHs of higher or lower forces are used on the plantar L5 dermatome to delineate flexor withdrawal thresholds. Successively higher forces are applied until withdrawal occurs then lower force VFHs are used until withdrawal ceases. The tactile threshold reflects the force required to elicit withdrawal in 50% of the stimuli. For the new test, each VFH is applied to the dorsal L5 dermatome of the paw while the rat is supported by the examiner. The VFH stimulation occurs in ascending order of force until at least 2 of 3 applications at a given force produces paw withdrawal. Tactile sensory threshold is the lowest force to elicit withdrawal 66% of the time. Acclimation, testing and scoring procedures are described. Aberrant trials that require a retest and typical trials are defined. Animal use was approved by Ohio State University Animal Care and Use Committee.

We describe two tactile sensory testing methods for acute or chronic periods of spinal cord injury in rats. These validated procedures can detect the development and maintenance of allodynia-like sensations.

在大鼠脊髓损伤后的急性和慢性触觉测试

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory ...

科研

医学

A Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of spinal cord injury (SCI) in which the thoracic spinal cord is injured. Additionally, because most human cord injuries occur as the result of blunt, non-penetrating trauma (e.g. motor vehicle accident, sporting injury) where the spinal cord is violently struck by displaced bone or soft tissues, the majority of SCI researchers are of the opinion that the most clinically relevant injury models are those in which the spinal cord is rapidly contused.1 Therefore, an important step in the preclinical evaluation of novel treatments on their way to human translation is an assessment of their efficacy in a model of contusion SCI within the cervical spinal cord. Here, we describe the technical aspects and resultant anatomical and behavioral outcomes of an unilateral contusive model of cervical SCI that employs the Infinite Horizon spinal cord injury impactor.
Sprague Dawley rats underwent a left-sided unilateral laminectomy at C5. To optimize the reproducibility of the biomechanical, functional, and histological outcomes of the injury model, we contused the spinal cords using an impact force of 150 kdyn, an impact trajectory of 22.5&deg; (animals rotated at 22.5&deg;), and an impact location off of midline of 1.4 mm. Functional recovery was assessed using the cylinder rearing test, horizontal ladder test, grooming test and modified Montoya's staircase test for up to 6 weeks, after which the spinal cords were evaluated histologically for white and grey matter sparing.
The injury model presented here imparts consistent and reproducible biomechanical forces to the spinal cord, an important feature of any experimental SCI model. This results in discrete histological damage to the lateral half of the spinal cord which is largely contained to the ipsilateral side of injury. The injury is well tolerated by the animals, but does result in functional deficits of the forelimb that are significant and sustained in the weeks following injury. The cervical unilateral injury model presented here may be a resource to researchers who wish to evaluate potentially promising therapies prior to human translation.

A reliable and repeatable way to produce a cervical unilateral spinal cord injury using the Infinite Horizon impactor is described. The method takes advantage of a custom designed frame and clamp to stabilize the spine. The standardized procedure and biomechanical injury parameters result in sufficient and sustained injuries.

单侧颈脊髓损伤的挫伤模型，利用无限的地平线冲击

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of ...

A Contusion Model of Severe Spinal Cord Injury in Rats

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is described to obtain a consistent model of severe SCI. Use of a stereotactic frame and computer controlled impactor allows for creation of reproducible injury. Hypothermia and urinary tract infection pose significant challenges in the post-operative period. Careful monitoring of animals with daily weight recording and bladder expression allows for early detection of post-operative complications. The functional results of this contusion model are equivalent to transection models. The contusion model can be utilized to evaluate the efficacy of both neuroprotective and neuroregenerative approaches.

A contusion model of severe spinal cord injury is described. Detailed pre-operative, operative and post-operative steps are described to obtain a consistent model.

挫伤模型脊髓严重损伤的保护作用

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is ...

Evaluation of Respiratory Muscle Activation Using Respiratory Motor Control Assessment (RMCA) in Individuals with Chronic Spinal Cord Injury

During breathing, activation of respiratory muscles is coordinated by integrated input from the brain, brainstem, and spinal cord. When this coordination is disrupted by spinal cord injury (SCI), control of respiratory muscles innervated below the injury level is compromised1,2 leading to respiratory muscle dysfunction and pulmonary complications. These conditions are among the leading causes of death in patients with SCI3. Standard pulmonary function tests that assess respiratory motor function include spirometrical and maximum airway pressure outcomes: Forced Vital Capacity (FVC), Forced Expiratory Volume in one second (FEV1), Maximal Inspiratory Pressure (PImax) and Maximal Expiratory Pressure (PEmax)4,5. These values provide indirect measurements of respiratory muscle performance6. In clinical practice and research, a surface electromyography (sEMG) recorded from respiratory muscles can be used to assess respiratory motor function and help to diagnose neuromuscular pathology. However, variability in the sEMG amplitude inhibits efforts to develop objective and direct measures of respiratory motor function6. Based on a multi-muscle sEMG approach to characterize motor control of limb muscles7, known as the voluntary response index (VRI)8, we developed an analytical tool to characterize respiratory motor control directly from sEMG data recorded from multiple respiratory muscles during the voluntary respiratory tasks. We have termed this the Respiratory Motor Control Assessment (RMCA)9. This vector analysis method quantifies the amount and distribution of activity across muscles and presents it in the form of an index that relates the degree to which sEMG output within a test-subject resembles that from a group of healthy (non-injured) controls. The resulting index value has been shown to have high face validity, sensitivity and specificity9-11. We showed previously9 that the RMCA outcomes significantly correlate with levels of SCI and pulmonary function measures. We are presenting here the method to quantitatively compare post-spinal cord injury respiratory multi-muscle activation patterns to those of healthy individuals.

Evaluation of Respiratory Muscle Activation Using Respiratory Motor Control Assessment RMCA in Individuals with Chronic Spinal Cord Injury

The purpose of this publication is to present our original work on a multi-muscle surface electromyographic approach to quantitatively characterize respiratory muscle activation patterns in individuals with chronic spinal cord injury using vector-based analysis.

评价呼吸肌慢性脊髓损伤个人使用呼吸电机控制评估（RMCA）

During breathing, activation of respiratory muscles is coordinated by integrated input from the brain, brainstem, and spinal cord. When this coordination ...

A Murine Model of Cervical Spinal Cord Injury to Study Post-lesional Respiratory Neuroplasticity

A cervical spinal cord injury induces permanent paralysis, and often leads to respiratory distress. To date, no efficient therapeutics have been developed to improve/ameliorate the respiratory failure following high cervical spinal cord injury (SCI). Here we propose a murine pre-clinical model of high SCI at the cervical 2 (C2) metameric level to study diverse post-lesional respiratory neuroplasticity. The technique consists of a surgical partial injury at the C2 level, which will induce a hemiparalysis of the diaphragm due to a deafferentation of the phrenic motoneurons from the respiratory centers located in the brainstem. The contralateral side of the injury remains intact and allows the animal recovery. Unlike other SCIs which affect the locomotor function (at the thoracic and lumbar level), the respiratory function does not require animal motivation and the quantification of the deficit/recovery can be easily performed (diaphragm and phrenic nerve recordings, whole body ventilation). This pre-clinical C2 SCI model is a powerful, useful, and reliable pre-clinical model to study various respiratory and non-respiratory neuroplasticity events at different levels (molecular to physiology) and to test diverse putative therapeutic strategies which might improve the respiration in SCI patients.

Respiratory failure is the leading cause of death following a cervical spinal cord injury. Having a reproducible, quantifiable, and reliable pre-clinical animal model of respiratory failure induced by a partial cervical injury will help to understand the subsequent respiratory and non-respiratory neuroplasticity and allow testing putative repair strategies.

颈脊髓损伤的小鼠模型研究后皮损呼吸神经可塑性

A cervical spinal cord injury induces permanent paralysis, and often leads to respiratory distress. To date, no efficient therapeutics have been developed ...

Controlled Cortical Impact Model for Traumatic Brain Injury

Every year over a million Americans suffer a traumatic brain injury (TBI). Combined with the incidence of TBIs worldwide, the physical, emotional, social, and economical effects are staggering. Therefore, further research into the effects of TBI and effective treatments is necessary. The controlled cortical impact (CCI) model induces traumatic brain injuries ranging from mild to severe. This method uses a rigid impactor to deliver mechanical energy to an intact dura exposed following a craniectomy. Impact is made under precise parameters at a set velocity to achieve a pre-determined deformation depth. Although other TBI models, such as weight drop and fluid percussion, exist, CCI is more accurate, easier to control, and most importantly, produces traumatic brain injuries similar to those seen in humans. However, no TBI model is currently able to reproduce pathological changes identical to those seen in human patients. The CCI model allows investigation into the short-term and long-term effects of TBI, such as neuronal death, memory deficits, and cerebral edema, as well as potential therapeutic treatments for TBI.

Traumatic brain injuries (TBIs) remain a serious health problem. Using the controlled cortical impact surgery model, research on the effects of TBI and possible treatment methods may be performed.

控制皮层影响模型创伤性脑损伤

Every year over a million Americans suffer a traumatic brain injury (TBI).  Combined with the incidence of TBIs worldwide, the physical, emotional, ...

A Radio-telemetric System to Monitor Cardiovascular Function in Rats with Spinal Cord Transection and Embryonic Neural Stem Cell Grafts

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter connected to a radio transmitter into the femoral artery of rats that underwent a T4 spinal cord transection with or without grafting of embryonic brainstem-derived neural stem cells expressing green fluorescent protein. Compared to other methods such as cannula insertion or tail-cuff, telemetry is advantageous to continuously monitor blood pressure and heart rate in freely moving animals. It is also capable of long term multiple data acquisitions. In spinal cord injured rats, basal cardiovascular data under unrestrained condition and autonomic dysreflexia in response to colorectal distension were successfully recorded. In addition, cardiovascular parameters before and after SCI can be compared in the same rat if a transmitter is implanted before a spinal cord transection. One limitation of the described telemetry procedure is that implantation in the femoral artery may influence the blood supply to the ipsilateral hindlimb.

We present a protocol for using a radio-telemetric system to record cardiovascular parameters in T4 spinal cord transected rats eight weeks after embryonic brainstem neural stem cell grafting into the lesion site. Telemetry is an advanced technique to accurately evaluate cardiovascular function in conscious freely moving spinal cord injured rats.

无线电遥测系统，以监测大鼠脊髓横断和胚胎神经干细胞移植在心血管功能

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter ...

Synergetic Use of Neural Precursor Cells and Self-assembling Peptides in Experimental Cervical Spinal Cord Injury

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. Clinically, more than 50% of SCI affect the cervical spine1. As a consequence of the primary injury, a cascade of secondary mechanisms including inflammation, apoptosis, and demyelination occur finally leading to tissue scarring and development of intramedullary cavities2,3. Both represent physical and chemical barriers to cell transplantation, integration, and regeneration. Therefore, shaping the inhibitory environment and bridging cavities to create a supportive milieu for cell transplantation and regeneration is a promising therapeutic target4. Here, a contusion/compression model of cervical SCI using an aneurysm clip is described. This model is more clinically relevant than other experimental models, since complete transection or ruptures of the cord are rare. Also in comparison to the weight drop model, which in particular damage the dorsum columns, circumferential compression of the spinal cord appears advantageous. Clip closing force and duration can be adjusted to achieve different injury severity. A ring spring facilitates precise calibration and constancy of clip force. Under physiological conditions, synthetic self-assembling peptides (SAP) self-assemble into nanofibers and thus, are appealing for application in SCI5. They can be injected directly into the lesion minimizing damage to the cord. SAPs are biocompatible structures erecting scaffolds to bridge intramedullary cavities and thus, equip the damaged cord for regenerative treatments. K2(QL)6K2 (QL6) is a novel SAP introduced by Dong et al.6 In comparison to other peptides, QL6 self-assembles into &#946;-sheets at neutral pH6.14 days after SCI, after the acute stage, SAPs are injected into the center of the lesion and neural precursor cells (NPC) are injected into adjacent dorsal columns. In order to support cell survival, transplantation is combined with continuous subdural administration of growth factors by osmotic micro pumps for 7 days.

Treating cervical spinal cord injury with both self-assembling peptides (SAP) and neural precursor cells (NPC), together with growth factors, is a promising approach to promote regeneration and recovery. A contusion/compression aneurysm clip rat model of cervical SCI and combined treatment involving SAP injection and NPC transplantation is established.

在实验颈脊髓损伤协同使用神经前体细胞，并自组装肽的

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. ...

Calibrated Forceps Model of Spinal Cord Compression Injury

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The calibrated forceps model of compression injury is a convenient, low cost, and very reproducible animal model for SCI. We used a pair of modified forceps in accordance with the method published by Plemel et al. (2008) to laterally compress the spinal cord to a distance of 0.35 mm. In this video, we will demonstrate a dorsal laminectomy to expose the spinal cord, followed by compression of the spinal cord with the modified forceps. In the video, we will also address issues related to the care of paraplegic laboratory animals. This injury model produces mice that exhibit impairment in sensation, as well as impaired hindlimb locomotor function. Furthermore, this method of injury produces consistent aberrations in the pathology of the SCI, as determined by immunohistochemical methods. After watching this video, viewers should be able to determine the necessary supplies and methods for producing SCI of various severities in the mouse for studies on SCI and/or treatments designed to mitigate impairment after injury.

Spinal cord injury models should be highly reproducible. We demonstrate that the calibrated forceps compression model of spinal cord injury is an easy to use surgical method for generating reproducible injuries to the murine spinal cord.

脊髓压迫损伤的校准钳型

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The ...

Facial Nerve Axotomy in Mice: A Model to Study Motoneuron Response to Injury

The goal of this surgical protocol is to expose the facial nerve, which innervates the facial musculature, at its exit from the stylomastoid foramen and either cut or crush it to induce peripheral nerve injury. Advantages of this surgery are its simplicity, high reproducibility, and the lack of effect on vital functions or mobility from the subsequent facial paralysis, thus resulting in a relatively mild surgical outcome compared to other nerve injury models. A major advantage of using a cranial nerve injury model is that the motoneurons reside in a relatively homogenous population in the facial motor nucleus in the pons, simplifying the study of the motoneuron cell bodies. Because of the symmetrical nature of facial nerve innervation and the lack of crosstalk between the facial motor nuclei, the operation can be performed unilaterally with the unaxotomized side serving as a paired internal control. A variety of analyses can be performed postoperatively to assess the physiologic response, details of which are beyond the scope of this article. For example, recovery of muscle function can serve as a behavioral marker for reinnervation, or the motoneurons can be quantified to measure cell survival. Additionally, the motoneurons can be accurately captured using laser microdissection for molecular analysis. Because the facial nerve axotomy is minimally invasive and well tolerated, it can be utilized on a wide variety of genetically modified mice. Also, this surgery model can be used to analyze the effectiveness of peripheral nerve injury treatments. Facial nerve injury provides a means for investigating not only motoneurons, but also the responses of the central and peripheral glial microenvironment, immune system, and target musculature. The facial nerve injury model is a widely accepted peripheral nerve injury model that serves as a powerful tool for studying nerve injury and regeneration.

We present a surgical protocol detailing how to perform a cut or crush axotomy on the facial nerve in the mouse. The facial nerve axotomy can be employed to study the physiological response to nerve injury and test therapeutic techniques.

面神经干切断小鼠:模型，研究运动神经元损伤反应

The goal of this surgical protocol is to expose the facial nerve, which innervates the facial musculature, at its exit from the stylomastoid foramen and ...