We would like to thank Dr. Harry Orr at the University of Minnesota for his generous gift of transgenic mice. We would also like to thank the following Skidmore College alum for their work performing the preceding experiments: Monica Villegas, Porter Hall, Mitchell Spring, Nicholas Toker, Jenny Zhang, Chloe Larson and Cheyanne Slocum. Furthermore, we would like to thank Skidmore College for funding the development of these methods.

该协议遵循的斯基德莫尔学院IACUC准则小鼠工作。 1.治疗水溶性化合物<ol><li>溶解琥珀酸在笼饮用水0.75毫克/毫升的浓度。注意，在所需的浓度感兴趣的任何水溶性化合物可以在这个阶段被取代。搅拌溶液，以确保该化合物完全溶解。 </li><li>小鼠达到治疗的所需岁以后，与在步骤1.1的溶液更换处理条件组的家笼的饮用水。 </li><li>测量每日两实验组水瓶的重量，以确保两个处理的和对照小鼠饮用的水的量相同。使用这些测量结果计算每笼总药物剂量。继续在整个实验和监测瓶重量的治疗。 </li></ol> 2.足迹分析<ol><li>跑道放置到地板或在密闭室的表。在对与床上用品和食物奖励两旁置箱小幅上升的角度（ 如含糖的谷物）设置了跑道。行跑道的长度与一张白纸。允许受试者对中置箱适应5分钟。 </li><li>除去从主箱主体和油漆左和右后腿，分别以蓝色和红色无毒水性涂料。油漆打印将被用来计算多个输出行为的测量。 </li><li>放置在跑道的开端的主体，并允许该受步行到家庭框。一旦这个问题已经达到了置箱，关闭活门，并允许受回到各自的家笼前呆在家里框内一分钟。清洗，用30％的乙醇在跑道和置箱并更换被褥，奖励食品，并为下一个主题跑道纸张。 </li><li>量化采取匝进展的数目，栅极宽度，步骤的角度，以及数通过根据先前6中所述的分析方法的主题。一个成功的足迹试验被定义为最小的被检体朝向目标框步行5顺序脚印没有转身或停止。 </li></ol> 3.束分析<ol><li>根据与以下6束先前描述6条截面规格设置束装置:梁1 =矩形28毫米宽;梁2 =圆形，直径28毫米;光束3 =矩形12毫米宽;梁4 =圆形，直径为12毫米;梁5 =长方形，6mm宽;梁6 =圆形，直径6毫米。在梁的一端，设置与奖励食物昏暗置箱。 </li><li>培养在梁主体，组装与射束3.装置轻轻地放置在光束相对置箱的端部的主体，并允许该主体在整个行走。允许每个主题最多三个2分钟的试验，以实现成功运行，其定义为穿越波束和在2分钟内进入最后框。在试验之间，让中置箱受试者休息2分钟。在主体之间，清洗，用30％的乙醇在梁和置箱并更换奖励食品中的下一个主题制备。 </li><li>重复步骤3.2，每天一次，共导致的连续3天的训练，未来两天。测试当天，第4天，应遵循顺序培训天。 </li><li>为了测试第4天的主题，第一个组装与梁1设备和步骤3.1的描述设置置箱。放置一个摄像机在装置的前部，并验证充分束装置可以在视频被清晰地捕捉。开始录制，并放置在横梁上对面的家框中的第一个问题，允许最多3 2分钟尝试成功完成审判。在试验间，让受休息中置箱2分钟。 </li><li>继续测试科目上的每个数字顺序六梁，从而开始下梁前须休息中置箱2分钟。主体之间，清洁每个波束并用30％的乙醇置箱和替换奖励食品中为每个主题制剂。 <ol><li>录像带每个试验并记录每束每个受试者试验取得成功的人数如下:"1"表示该主题是成功的第一个试验中，"2"表示主题是成功的第二个试验中，"3"表示主题是成功的第三个和最后一个试验中，和"4"表示对象没有成功的任何三个试验。 </li></ol></li><li>谁是失明的课题实验条件的得分手使用的试验测量footslips录像。定义一个footslip因为这是在下面穿过梁的长度的理论中线照相机浸渍的直接视图后足。从多个得分平均footslip分数。 </li><li>分析的位数uccessful试验和footslips数据的数目，计算出每个基因型和实验条件队列内的平均和标准误差。进行单向ANOVA和多重比较事后分析，以确定是否有治疗和基因型上成功的试验和footslips的数目的数目的效果。 </li></ol> （四）加快转棒<ol><li>适应科目之前，在审判开始试验室10分钟。在此适应期，准备旋转杆装置中，在清洁装置的各泳道，用30％的乙醇。 </li><li>打开转棒软件，并设置启动设置为4转速，加速度设置5分钟，最终速度设置，以40转。这些设置提供来自4的恒定加速度 - 40rpm下在一段5分钟。小鼠可以以40rpm保持在转棒以外的加速期为10分钟的最大游程长度的附加5分钟。 </li><li>后适应，地方主体Øn要在各自的车道旋转杆，而杆4转速进行旋转。一旦所有科目都在各自的车道，开始了第一次审判。记录落使用仪器软件的延迟;二次计时器可以用作备份。 </li><li>试用之后完成，让每一个受其余在各自的车道水库为前下试，防止过度疲劳十分钟。 </li><li>总共四个试验重复步骤4.3和4.4。 </li><li>重复步骤4.3 - 4.5，共连续四天，持续整个试验期间的待遇。完成后，量化延迟脱落旋转杆每科目6每次试验。 </li></ol> 5.小脑提取和固定<ol><li>根据机构IACUC指导原则通过二氧化碳窒息安乐死小鼠。放置受试者入二氧化碳室中，并释放二氧化碳进入腔室。看在步实施安乐死在任何时候都动物和不离开动物无人值守。 </li><li>一旦呼吸已经停止和主题显示了由后足压力没有剩余的疼痛感，去除室内主题，并通过从头骨中间的上皮组织卓越正中切口尾端头骨通过上皮组织的基础。 </li><li>用解剖剪，通过矢状缝到前囟门切断从基部头骨在脊髓。通过每个冠状缝用钝钳脱下额叶，顶叶和interparietal骨前横向切割。 </li><li>使用钝钳骨外侧脱落小脑。使用镊子小脑从丘和脑干中分离并从组织的其余部分将其挤出。分离小脑与镊子左，右半球。 </li><li> 立即将右小脑成液态氮保存HPLC ANA裂解和左小脑成预冷4％低聚甲醛（PFA），用于组织固定。切断主动脉和丢弃根据机构IACUC规则的尸体前收获任何其他感兴趣的组织。 注意:PFA是一种剧毒，易燃，腐蚀性。 </li><li>二十四小时后提取，放置组织成30％蔗糖于4℃多达洗前在磷酸盐缓冲盐水（PBS），其固定在PFA（在PBS中的4％）组织（三次，5分钟/洗涤） 2周。 </li></ol> 6.免疫病理分析<ol><li>附加雪橇切片机的管道到水槽水龙头和温度控制箱。使用温度控制箱，冷却切片机阶段至-25℃。切片厚度旋钮设置为50微米。 注:如果在雪橇切片表盘未达到50微米时，转盘设置为较薄的设置，并通过相应地移位设置相乘的厚度（例如设定对dIAL至10μm和切片为50微米 ）的总厚度之前移位五次。 </li><li>民建联最佳温度切割（OTC）解决方案的一个硬币大小的部分搬上了舞台。场外冻结之前，使用产钳，这样正中矢状切面朝下场外层定位半球。迅速工作，使得半球的横向前端尖朝向叶片，优于阶段轻轻定向钳组织。允许OTC完全冻结。 <ol><li>在图层，添加额外的OTC周围的组织，允许OTC添加到下一层之前完全冻结。在组织和冻结的顶部添加场外的薄层。 </li></ol></li><li>部的组织，等于体重散发到切割刀片而切片。收集使用精细的艺术家的画笔和地方部分为含有1x PBS适当标记的孔板。部分之间，重新设置在切割厚度雪橇切片表盘，如果需要的话，至50微米。 </li><li>在孔板用尿素溶液[0.01M的尿素的PBS]的每个孔取代的PBS并将板在冰上。准备就绪后，每一次揭露抗原表位的免疫标记去除冰和沸腾部分板块中的尿素溶液三次15秒。该步骤可以通过将板的加热块设定为沸腾温度上很容易地完成。每沸腾步骤之间，含凉冰的组织切片板。 注:如果在执行脲，阻断，初级抗体，二级抗体和DAPI步骤在96孔板，在整个使用100μL的体积。如果使用不同大小的孔，相应地调整试剂卷。代替加热块的，微波炉可用于煮沸组织样品中的尿素。在高功率设置了三个15秒的间隔微波炉样本。 </li><li>交的沸腾，洗部分三次，每次除去本解决方案，取代了溶液用1×PBS并搅拌10分钟。 </li><li>块部分用驴血清溶液（3％正常驴血清，0.3％的Triton X-100的PBS）通过在血清中，在室温下轻轻搅动温育组织为一小时。删除驴血清的解决方案。 </li><li>用0.2微克/毫升的抗钙结合蛋白和1标签部分:在驴血清溶液2000抗共济失调蛋白-1抗体（11NQ）16和孵育在4℃下温和搅拌72小时。 </li><li>在4℃（在驴血清溶液稀释500的Alexa Fluor-594抗兔抗体:500的Alexa Fluor-488抗山羊和1 1）培养在适当的二级抗体部分之前用PBS洗部分如在步骤6.5对温和搅拌48小时。 </li><li>洗切片用PBS三倍在步骤6.5之前用DAPI标记细胞核（4&#39;，6-二脒基-2-苯基吲）溶液稀释至0.5微克/毫升的在PBS中的终浓度。 DAPI潜伏期最长不超过10分钟。删除搜索解决方案n和用PBS洗如在步骤6.5。 </li><li>安装组织到2％明胶包被的载玻片用封固剂，以减少光漂白。盖玻片并用指甲油密封。 </li><li>用扫描共聚焦显微镜下找到明光小脑的主要裂缝。用10X UIS2目标，扫描并用DM488 / 559毫微米依次激发DAPI，AlexaFluor-488，并用405nm的二极管激光器，一个488nm的氩气体激光器和一个559纳米二极管激光器，分别AlexaFluor-594信道，在结合二色分光镜。单独使用SDM490和SDM560光束分离器和一个BA575-675带通滤波器，分别收集发射的光。 <ol><li>对于每个通道，构造对于z = 20微米，步长=0.1μm的初级裂隙内的z栈。使用设置有共焦显微镜的后处理软件，以在一个大小标记物加入到最终的图像。 注:另类激光器，过滤器和荧光团可以代替基于availabiliTY。如果AlexaFluor-488或AlexaFluor-594检测弱，磷化镓（磷砷化镓）高灵敏度的探测器可以使用（如果可用）来增强信号。 </li></ol></li></ol> 7.量化分子层的厚度和浦肯野细胞数<ol><li>使用ImageJ，打开彩色主裂的Z堆栈图像并从图像选项卡菜单栏，"颜色"和"分离通道"从颜色中选择"图像"每个图像成它的红色，蓝色和绿色通道分割标签。 </li><li>创建分裂每个信道的最大ž投影。当感兴趣的通道图像是开放的，从菜单栏中选择"图像"，从"节"选项卡中的图像选项卡和"Z-项目&#39;&#39;节&#39;。中的"Z-工程"命令菜单中，选择"最大投影ž"，然后单击"好"。重复每个通道。 </li><li>从日选择"插件"打开细胞计数插件Ë菜单栏，从插件选项卡"分析"，并从分析选项卡"细胞计数"。 注:ImageJ的和ImageJ的细胞计数插件可以在材料和设备的表中提供的网站上下载。 </li><li>通过计数共济失调蛋白-1标记的细胞核沿着一个预先确定的200微米拉伸主裂缝的前部和后部的长度位于量化浦肯野细胞的数目。从菜单栏中选择"图像"从图像选项卡和"门槛"从调整选项卡使用门槛地图，"调整"，使所关心的通道（红色的门槛地图中共济失调-1标有核的情况下， ）。 <ol><li>选择，可以在整个的量化（31-225像素例如）进行归一化的像素范围。从检查的阈值窗口中的"黑后台"框图像丢弃的噪音。图像信号可以被反向为便于计数。 </li></ol></li><li>量化分子层THICkness在使用"拆分频道"和"阈值地图的"工具如在步骤7.1和7.2的绿色通道。利用每个图像为指导的大小标记，随机测量中的每个的两个指定每个主裂缝图像为200微米绵延3分子层的长度。使在浦肯野细胞体到浦肯野树突的前端的基部从浦肯野树突的近端测量。 </li><li>记录浦肯野细胞计数和分子层厚度为平均值加或减标准误差。与多重比较进行单向ANOVA 事后分析，以测试的处理条件或基因型上细胞的数目和树突的长度的影响。 </li></ol> 8.小脑琥珀酸含量治疗后的HPLC分析<ol><li> 分析试样的制备 <ol><li>之前权衡每个收获右侧小脑半球在HPLC分析使用。剁碎半球半部逐个在预冷Dounce匀浆时间和添加的75:25 0.5毫升（按体积）水:甲醇溶液到每个均化半17。 </li><li>采用窄预冷匀浆，严重打破了组织5 DOUNCE招。继续与更广泛的均质精细讳言每个组织样本，运用20 DOUNCE招。 </li><li>转移匀浆预冷的微量离心管。每个样品后，冲洗用0.5mL冰冷的水的均化:甲醇溶液和漂洗添加到离心管中。 </li><li>离心匀浆样品在在25℃下1300×g离心30分钟，并收集在一个干净的微量离心管的上清液。 </li><li>通过安置与10千道尔顿（kDa）的标称分子量极限的再生纤维素过滤器的离心式过滤器单元进行过滤上清液。如果没有立即运行HPLC分析，储存在-80°过滤样本C。 </li></ol></li><li> HPLC仪器和条件 <ol><li>具有至少30厘米的长度和7μm的粒度到高性能液相色谱仪附加一个离子排斥色谱柱。 </li><li>制备和脱气调节到pH为5 1mM的乙酸缓冲液的足够量。 </li><li>设定的为0.8ml /分钟流动相速度的HPLC中。如果使用UV-Vis检测器，选择224纳米的检测器波长。在这些条件下典型的洗脱时间对于琥珀酸为10和12分钟之间。 </li></ol></li><li> 运行和分析的样品和标准 <ol><li>准备一组与0和250ppm的之间的琥珀酸浓度乙酸盐缓冲液流动相的标准。 </li><li>运行每个标准，并使用从每个运行测得的峰面积制成校正曲线。 </li><li>一旦获得准确的标准曲线，运行在乙酸盐缓冲液稀释预先制备的样品。用于测量的精确度更高，可对样品进行琥珀酸已知浓度已经在流动相中稀释被掺入。一式三份运行的所有样本。 </li><li>分析HPLC色谱通过使用校准曲线，然后通过样品体积乘以与由原始样品的质量除以以确定样品的琥珀酸浓度。 </li></ol></li></ol>使用氧电极控制单元小脑线粒体功能9. 离体分析<ol><li>通过首先施加氯化钾一滴创建使用市售系统Clark型电极（50％的KCl重量/体积）在铂阴极溶液，切割1.5厘米的2片商业烟草的卷烟纸和过轻轻放置纸铂阴极。 <ol><li>覆盖阴极和围绕阳极有2.5 平方厘米片聚四氟乙烯（PTFE）纸和紧贴地固定两个MEMBranes与O型圈，确保有膜内没有折叠或气泡。滴50％KCl溶液到周围的井。 </li></ol></li><li>固定预制电极进入试验室，并与空气饱和的水填充。加热腔室至30℃，添加0.8厘米搅拌棒到该溶液中，并使用仪器的软件以70rpm搅拌。通过建立零氧校准室中。这样做，串联还原剂连二亚硫酸钠的几晶体添加到该溶液中。 <ol><li>一旦校准完成，用70％乙醇洗室一次，然后使该腔室，以1.5毫升呼吸缓冲适应（5mM的MgCl 2的，60毫米氯化钾，100mM的甘露糖醇，10mM的前洗净，用去离子水六倍KH 2 PO 4，0.5毫的 Na 2 EDTA，60mM的Tris-盐酸[pH为7.4]）7。 注:连二亚硫酸钠的所有痕迹必须完全在洗涤步骤，尝试测量之前取出呼吸实验过程中的氧浓度。 </li></ol></li><li>轻轻均匀称量小脑半球于0.5mL匀浆缓冲液（0.25M蔗糖，0.5mM的EDTA，50mM的Tris-盐酸[pH为7.4]）。匀浆转移到一个干净的1.5毫升离心管中，并保持冷藏，直到开始试验。 </li><li>匀浆添加到腔室，达到2毫升的最终体积。通过加入毛地黄皂苷或皂苷（5毫克/毫升）的40微升透小脑匀浆;允许匀浆组织孵育10分钟。 注:毛地黄皂苷和皂苷是有毒的，并且温育时间应保持在最低限度。 </li><li>要开始录制的耗氧量，通过激活和使用分别为底物和抑制剂，氧化磷酸化链（每个基板3分钟录音）的抑制确定线粒体功能。 <ol><li>添加使用清洁针具如下基板:10微升2M的谷氨酸[终浓度（FC）0.01 M]。5μL的0.8M的苹果酸[FC 2毫]，20微升的0.5M腺苷二磷酸（ADP）FC的5mM]，1微升1mM的鱼藤酮[FC 0.5μM]，20μL的1M琥珀酸[10毫]，1微升5毫抗霉素A [FC 2.5μM]，1微升0.8M的抗坏血酸[FC 0.4毫]和5微升0.2M的四甲基 - 对苯二胺（TMPD）。 [FC 0.5毫米]在添加基材室要小心，不要引入气泡进入系统。 注:每个底物和抑制剂引起的功能性作用的详细信息在先前公布的协议7中描述。个体剂量 - 反应曲线，可能需要产生，以确定以前没有研究了制剂/组织/种类的底物和抑制剂的最佳浓度。同样地，该协议可以用于组织的先天代谢倾向被优化/物种被研究，例如作为底物偏好。 </li></ol></li><li>一旦数据采集完成后，轻轻一spirate小脑匀浆并清洗用乙醇和去离子水的腔室。一旦清洁，附加小脑匀浆样品可以在不附加的校准，只要在PTFE膜保持完整运行。最终完成，清洁电极用去离子水和乙醇和存储用硅胶或干燥剂后。 注:间同一天运行，填补了室内用去离子水确保膜不会干。 </li><li>使用仪器软件创建的数据表，开始2分钟呼吸导出数据的40分钟后加入底物到电子表格程序。 </li><li>计算每个底物或抑制剂的呼吸速率。由抗坏血酸TMPD衬底加入期间除以呼吸速率的总呼吸正常化的数据。 注:呼吸速率可进一步归一化到组织中的蛋白质含量可以是在神经变性的实验特别期望在该组织中的含量可以变化。这样做，保留50微升样品匀浆（来自步骤9.3）的用于在标准蛋白质测定中使用。 </li></ol>

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作者什么都没有透露。

如果如所述使用这些方法，它们是能够检测和小脑的神经变性疾病的小鼠模型减轻氧化磷酸化介导的线粒体功能障碍。将合并的生物化学和行为测定是用于确定对小脑的神经变性疾病的病理线粒体贡献程度多方面的方法。通过与琥珀酸治疗小鼠，刺激新陈代谢，促进线粒体功能，我们能够显示小脑行为缺陷和小脑变性的救援。 这里提出的方法可以用于测试多种水溶性代谢靶向化合物为小脑的神经变性疾病的潜在治疗。琥珀酸是在0.75毫克/毫升其允许简单递送经由笼饮用水和并不需要补充食物颗粒的创建易溶于水。其它水溶性化合物可以很容易地替换到协议。如果使用不是水溶性代谢靶向化合物，我们的方法可以容易地适于用补充食物颗粒11，12的治疗。当测试一个新的化合物，它是临界通过HPLC分析或其他合适的检测方法，在开始治疗前，以验证该药物的小脑渗透。 病理评估的电池在检测小脑的神经变性病症，由于选择了他们的报道功效。小脑的神经变性疾病包括各种管制协议，和弗里德赖希共济失调6，23，24的覆盖测定中，光束法和旋转杆试验被广泛使用的评估。这些特殊的行为评估与小脑变性很强的相关性。然而，其他behaviora升范例可以被取代或根据需要加入。 浦肯野细胞中与共济失调蛋白-1和钙结合蛋白SCA1小鼠标签被广泛用于检测SCA1模型5，20，21小脑变性。其他神经元选择性抗体和组织区域可被用作需要可视化的神经变性的减轻。 呼吸分析是从小脑组织个体的线粒体复合物内功能障碍或修理的特异性检测的有力工具。应注意，以尽量减少收获组织和运行检测，以实现可重复的结果之间的时间。此外，要注意的被分析的组织的均匀性是很重要的。具体来说，小脑组织绝大多数由颗粒神经元不表达在我们的模型中SCA1转基因的。因此，这是一个关键步骤协议是确定是否整个小脑呼吸是检测氧化复杂功能的一种可行的方法。在转基因小鼠SCA1小脑呼吸变化可能是微妙的，并可能需要的科目数量的增加达到统计上显著的效果。 在这个协议中详述的行为和神经病理学方法的组合提供SCA1疾病的不同量化，并且可以有力地与水溶性化合物检测处理时的改进。此协议的意义在于在其广泛的适应性，以各种治疗和小鼠模型的多样化。此外，许多在这个协议中使用的技术不需要昂贵的设备或复杂的技术，并且因此适合于本科研究设置。这个协议的未来应用将用于评估治疗功效的经化合物的年龄依赖性递送的程度。

线粒体是三磷酸腺苷（ATP），为细胞能量一种必需的辅酶的关键生产，大多数使用电子传递链通过氧化磷酸化（OXPHOS）产生的线粒体ATP。脑，鉴于其高代谢需求及对氧化磷酸化进行供电的神经活动的依赖，是高度敏感的线粒体功能障碍。其结果是，线粒体功能障碍是在老化过程中触发 1，并在多个神经变性疾病2，3，4的发病机制有关。因此，它遵循线粒体是神经变性吸引力的治疗目标。 在这个协议中，我们已经采用脊髓小脑性共济失调1型（SCA1）作为模型神经退行性疾病线粒体的研究升功能障碍和线粒体靶向疗法的开发。 SCA1是通过在共济失调蛋白-1基因产物触发小脑浦肯野神经元和神经元其他脑区域中的进行性变性一个多聚谷氨酰胺（的polyQ）重复扩张突变引起的。此处所用的转基因小鼠系（指定为SCA1小鼠），它表达了浦肯野细胞特异性启动子的控制下的polyQ突变共济失调蛋白-1基因，允许SCA1 5的浦肯野细胞成分的针对性的分析。 SCA1小鼠进行逐步浦肯野细胞变性和发展共济失调步态6。 线粒体复合物功能障碍和线粒体靶向治疗功效可与分子和行为分析的电池进行评价。线粒体复合物功能障碍是测定体外通过检测在小脑组织内改变氧消耗呼吸测定存在电子传递链底物和抑制剂7。呼吸检测先前已经用于透化组织，线粒体分离物，和全组织7，8，9。它们允许不同形态的数据收集方法，如透射电子显微镜或免疫荧光染色线粒体功能的直接评估。利用整个组织，而不是孤立的线粒体防止在分离过程7可能出现的健康线粒体的偏倚选择。当如图适于协议，所述呼吸检测是在小脑的神经变性疾病状态检测线粒体功能障碍的有价值的方法。 代谢的非特异性活化剂可以用来推断线粒体功能障碍的神经变性diseas的转基因小鼠模型e和在新疗法的发展援助。槲皮素，辅酶Q10和肌酸都被证明改善神经变性疾病的病理的患者，并在神经变性疾病10，11，12，13，14，15，16的动物模型。这里，我们提出了一种新的代谢活化剂，琥珀酸，刺激代谢，促进线粒体功能中的神经变性疾病。以确保该活化剂穿越血脑屏障，高效液相色谱法检测在处理的小鼠17递送至神经组织。 为了评价如琥珀酸代谢靶向水溶性化合物的治疗效果，可以使用行为范例和免疫病理研究的电池。杜E要在小脑的神经变性疾病，足迹跑道试验，梁法和加速旋转杆实验发现，运动协调障碍是用来检测行为病理6，18，19日抢救。这些措施是由一个定义的小脑组织6，20，21的小叶内评估分子层的厚度（定义为浦肯野细胞树突长度）和浦肯野细胞胞体计数补充有小脑细胞构筑的免疫病理评估。在这里，我们提出了诊断和治疗的线粒体功能障碍与代谢有针对性的水溶性化合物多的神经病理学和行为的方法。 我们使用线粒体呼吸体外分析来分析线粒体功能障碍在SCA1 TRANsgenic鼠标。此外，我们表明，疾病的症状和病理学由水溶性线粒体增压琥珀酸改进，还牵连于SCA1疾病进展的线粒体功能障碍。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adenosine diphosphate</td>
			<td>Sigma Aldrich</td>
			<td>A2754</td>
			<td>ADP</td>
		</tr>
		<tr>
			<td>Ascorbate</td>
			<td>Sigma Aldrich</td>
			<td>A7631</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma Aldrich</td>
			<td>A2153</td>
			<td>BSA</td>
		</tr>
		<tr>
			<td>4&#39;,6-Diamidino-2-phenylindole</td>
			<td>Sigma Aldrich</td>
			<td>D9542</td>
			<td>DAPI</td>
		</tr>
		<tr>
			<td>Digitonin</td>
			<td>Sigma Aldrich</td>
			<td>D141</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol</td>
			<td>Sigma Aldrich</td>
			<td>D0632</td>
			<td>DTT</td>
		</tr>
		<tr>
			<td>Donkey serum</td>
			<td>Sigma Aldrich</td>
			<td>D9663</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamate</td>
			<td>Sigma Aldrich</td>
			<td>1446600</td>
			<td></td>
		</tr>
		<tr>
			<td>Malate</td>
			<td>Sigma Aldrich</td>
			<td>6994</td>
			<td></td>
		</tr>
		<tr>
			<td>Mannitol</td>
			<td>Sigma Aldrich</td>
			<td>M4125</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium-lactobionate</td>
			<td>Bio-Sugars</td>
			<td>69313-67-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotenone</td>
			<td>Sigma Aldrich</td>
			<td>R8875</td>
			<td></td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Sigma Aldrich</td>
			<td>47036</td>
			<td></td>
		</tr>
		<tr>
			<td>Succinic Acid</td>
			<td>Sigma Aldrich</td>
			<td>S3674</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N,N&#8242;,N&#8242;-Tetramethyl-p-phenylenediamine</td>
			<td>Sigma Aldrich</td>
			<td>T7394</td>
			<td>TMPD</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>T9284</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Sigma Aldrich</td>
			<td>U0631</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield mounting medium</td>
			<td>Vector Labs</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>11NQ antibody (anti-ataxin-1 )</td>
			<td></td>
			<td></td>
			<td>Servadio, et al. 1995, PMID: 7647801</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 anti-mouse secondary antibody</td>
			<td>Life Technologies</td>
			<td>A-11015</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 anti-rabbit secondary antibody</td>
			<td>Life Technologies</td>
			<td>A-11012</td>
			<td></td>
		</tr>
		<tr>
			<td>Calbindin antibody (goat)</td>
			<td>Santa Cruz</td>
			<td>C-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Animals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Control transgenic mice</td>
			<td>Harry Orr, Ph.D.</td>
			<td>A02</td>
			<td>Burright, et al. 1997, PMID: 9217978</td>
		</tr>
		<tr>
			<td>SCA1 mice</td>
			<td>Harry Orr, Ph.D.</td>
			<td>B05</td>
			<td>Burright, et al. 1997, PMID: 9217978</td>
		</tr>
		<tr>
			<td>Wildtype mice</td>
			<td>The Jackson Laboratory</td>
			<td>001800</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ESM-100L microtome</td>
			<td>ERMA</td>
			<td></td>
			<td>Sledge microtome</td>
		</tr>
		<tr>
			<td>Fluoview FV1200 Confocal Microscope</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol-gelatin slides</td>
			<td>FD Neuro Technologies</td>
			<td>PO101</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe</td>
			<td>Sigma Aldrich</td>
			<td>VCAT 80465</td>
			<td></td>
		</tr>
		<tr>
			<td>OXYT1 Oxytherm Electrode Control Unit</td>
			<td>Hansatech Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>P.T.F.E. paper</td>
			<td>Cole-Parmer</td>
			<td>UX-08277-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotallion Rotarod</td>
			<td>PPP&#38;G</td>
			<td></td>
			<td>contact corresponding author for information</td>
		</tr>
		<tr>
			<td>Ultimate 3000 HPLC</td>
			<td>Dionex</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>National Institute of Health</td>
			<td></td>
			<td><a href="http://imagej.nih.gov/ij/">http://imagej.nih.gov/ij/</a></td>
		</tr>
		<tr>
			<td>Cell counter plugin (for ImageJ)</td>
			<td>National Institute of Health</td>
			<td></td>
			<td><a href="http://rsb.info.nih.gov/ij/plugins/cell-counter.html">http://rsb.info.nih.gov/ij/plugins/cell-counter.html</a></td>
		</tr>
		<tr>
			<td>3P&#38;G Rota-Rod v3.3.3 (rotarod software)</td>
			<td>PPP&#38;G</td>
			<td></td>
			<td>contact corresponding author for information</td>
		</tr>
		<tr>
			<td>Phidget21.dll (required for rotarod software)</td>
			<td>DLL-Files.com</td>
			<td></td>
			<td><a href="https://www.dll-files.com/phidget21.dll.html">https://www.dll-files.com/phidget21.dll.html</a></td>
		</tr>
	</tbody>
</table>

treating sca1 mice with water-soluble compounds to non-specifically boost mitochondrial function

线粒体功能障碍起着老化过程，并在神经退行性疾病，包括遗传性的几个小脑萎缩症和小脑进行性变性标记等运动障碍一个显著的作用。该协议的目的是评估在脊髓小脑性共济失调1型（SCA1）线粒体功能障碍，并通过该水溶性化合物琥珀酸评估代谢呼吸药理定位的功效减缓疾病进展。这种方法也适用于其他小脑疾病和可适应的水溶性治疗的宿主。 线粒体呼吸的离体分析被用于检测和量化在线粒体功能疾病相关的变化。遗传证据（未发表的数据），并在SCA1小鼠模型线粒体功能障碍蛋白质组学的证据，我们评价与水溶性代谢增压待遇的功效uccinic酸由该化合物直接溶解到笼子的饮用水。药物的通过血脑屏障的能力可使用高效液相色谱（HPLC）来推导。这些化合物的疗效然后可以使用多种行为范式，包括加速转棒，平衡木试验和足迹分析测试。小脑Cytoarchitectural完整性可以使用检测浦肯野细胞的细胞核和浦肯野细胞树突和胞体免疫荧光法进行评估。这些方法是用于确定线粒体功能障碍和治疗与小脑的神经变性疾病的水溶性化合物的功效健壮的技术。

通过与琥珀酸小脑线粒体的药理学靶向我们能够防止线粒体功能障碍在小脑的神经变性疾病SCA1的小鼠模型。琥珀酸脱氢酶，琥珀酸，所述的典型电子供体中治疗和神经病理学评估的第二周溶于SCA1小鼠家笼饮用水一个月，与行为评估开始以下处理（ 图 1A）。由足迹分析，梁法和加速旋转杆法测定琥珀酸治疗22 SCA1提高小鼠的表现。检测到的行为的改善表示增强运动协调和运动学习（ 图1B-D）。 行为表现救援音乐会被发现有保鲜的蜡膜bellar组织（ 图2）。琥珀酸治疗显著保存浦肯野细胞机构和相比，未经处理SCA1小鼠治疗SCA1小鼠小脑主要裂缝增大分子层厚度（指示浦肯野细胞树突延长）。浦肯野细胞树突长度和浦肯野细胞计数身体提供全面的小脑细胞构筑5强有力的措施。 HPLC是从处理的小鼠在小脑组织上进行验证溶解在笼饮用水琥珀酸成功越过血脑屏障并渗入小脑组织。剂量反应曲线不同治疗进一步的长度支持琥珀酸随意处理，到达小脑组织（ 图3A-B）的一种可行的方法。我们呼吸实验正在进行，我们的结果将在研究发表手稿。这里，我们表明，我们可以通过小脑氧化磷酸络合物在加入不同电子传递链底物和抑制剂（ 图3C）的成功记录呼吸的速率。最终，我们将使用呼吸法，以显示SCA1鼠标小脑氧化磷酸化缺陷和这些赤字由丁二酸处理的逆转。 <img alt="图1" src="/files/ftp_upload/53758/53758fig1.jpg" /> 图1: 从脚印分析，梁分析处理方案和代表性的数据，并加速旋转杆。 （A）的琥珀酸溶解于SCA1小鼠家笼饮用水四星期17周龄开始。 SCA1小鼠开始在5周龄显示共济失调表型。在示意图中未示出三种对照队列:丁二酸处理的野生型小鼠，已经hicle处理SCA1小鼠和媒介物处理的野生型小鼠。在治疗期间如下行为评估进行了17周时的足迹分析，18个星期内束法和19周期间加速旋转杆在20周龄时，处死小鼠小脑组织中收获免疫病理学检测，HPLC测定和呼吸检测。 （B）中从足迹测定的代表性数据示出的车辆处理的野生型步态（上），媒介物处理的SCA1步态（中）和琥珀酸处理SCA1步态（底部）。 （C）光束分析数据显示，与琥珀酸处理SCA1小鼠梁性能改进（如试验次数为衡量成功运行）。 （* P &lt;0.05）。 （四）丁二酸治疗显著改善加速SCA1 22小鼠的转棒的性能如由延迟增加下降（* P &lt;0.05）。在CD重新误差线flect平均值的标准误差。显着性通过单因素ANOVA和多重比较事后分析来确定。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53758/53758fig1large.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/53758/53758fig2.jpg" /> 图2:从浦肯野细胞计数与分子层厚度免疫病理学测定的代表性数据。 （A）中从浦肯野细胞计数测定法的代表性结果示出染色的载体处理的对照转基因小脑主裂隙（上），媒介物处理的SCA1小脑主裂隙（中）和琥珀酸处理SCA1小脑主裂隙（底部） ATXN1阳性细胞核（红色），并用DAPI（蓝色）复染色。白色线条勾勒出组织的200微米的区域指定用于细胞计数。对于这种特定的分析中，控制转基因小鼠过表达在一个特定的小区浦肯野启动子控制的非致病ATXN1基因代替野生型小鼠，因为转基因的对照功能容易核浦肯野ATXN1可检测水平和野生型小鼠才不是。控制转基因小鼠不显示共济失调型或小脑的神经变性。 （B）选自分子层厚度测定代表性的结果示出了溶媒处理的野生型小脑主裂隙（上），媒介物处理的SCA1小脑主裂隙（中）和琥珀酸处理SCA1小脑主裂隙（底部）染色钙结合蛋白，浦肯野细胞的标记（白色）。三个小脑细胞层的两个可以与钙结合蛋白可视化。 （由浦肯野细胞体）的中间浦肯野细胞层与最外分子层沿着可见光（由浦肯野CE的LL树突）。分子层出现在主裂的中间，由于组织的自然褶皱。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53758/53758fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53758/53758fig3.jpg" /> 图3: 从HPLC和呼吸测定代表性的成果。已知浓度的（A）的标准曲线高达250ppm的琥珀酸稀释在乙酸盐缓冲液和作图测得的HPLC峰下的面积。线的方程来计算从处理过的组织样品未知琥珀酸浓度。 （B）中从小鼠小脑组织治疗后的不同时间代表剂量响应琥珀酸峰（或0,1或5周）。在此条件下描述，琥珀酸典型地洗脱10和12分钟之间。 （C）从载体治疗的野生型小鼠小脑组织代表呼吸运行。蓝线描绘了氧气浓度的变化，红色虚线描绘了呼吸速率变化和红色实线描绘了虚线的平滑版本。 D =此外毛地黄皂苷，G / M =除了谷氨酸/苹果酸，A =此外ADP的，R =除了鱼藤酮的，S =此外琥珀酸，AA =除了抗霉素A和A / T =另外升序的/ TMPD。所有的衬底在图中所描绘的时间和在协议（步骤9.5）中描述的浓度加入。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53758/53758fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Treating SCA1 Mice with Water-Soluble Compounds to Non-Specifically Boost Mitochondrial Function at JoVE.com

用水溶性化合物治疗 SCA1 小鼠以非特异性增强线粒体功能医学|视频

Treating SCA1 Mice with Water-Soluble Compounds to Non-Specifically Boost Mitochondrial Function

We present a biochemical and behavioral protocol to evaluate the efficacy of mitochondria-targeted water-soluble compounds for the treatment of Spinocerebellar ataxia type 1 (SCA1) and other cerebellar neurodegenerative diseases.

治疗SCA1小鼠水溶性化合物，以非特异性升压线粒体功能

Mitochondrial dysfunction plays a significant role in the aging process and in neurodegenerative diseases including several hereditary spinocerebellar ...

treating-sca1-mice-with-water-soluble-compounds-to-non-specifically

Nanyang Technological University

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Cellular Respiration

SCIENTISTS IN ACTION

10.5K 次观看.  Skidmore College. We present a biochemical and behavioral protocol to evaluate the efficacy of mitochondria-targeted water-soluble compounds for the treatment of Spinocerebellar ataxia type 1 (SCA1) and other cerebellar neurodegenerative diseases. 

视频: Treating SCA1 Mice with Water-Soluble Compounds to Non-Specifically Boost Mitochondrial Function

Assessing Endothelial Vasodilator Function with the Endo-PAT 2000

The endothelium is a delicate monolayer of cells that lines all blood vessels, and which comprises the systemic and lymphatic capillaries. By virtue of the panoply of paracrine factors that it secretes, the endothelium regulates the contractile and proliferative state of the underlying vascular smooth muscle, as well as the interaction of the vessel wall with circulating blood elements. Because of its central role in mediating vessel tone and growth, its position as gateway to circulating immune cells, and its local regulation of hemostasis and coagulation, the the properly functioning endothelium is the key to cardiovascular health. Conversely, the earliest disorder in most vascular diseases is endothelial dysfunction.
In the arterial circulation, the healthy endothelium generally exerts a vasodilator influence on the vascular smooth muscle. There are a number of methods to assess endothelial vasodilator function. The Endo-PAT 2000 is a new device that is used to assess endothelial vasodilator function in a rapid and non-invasive fashion. Unlike the commonly used technique of duplex ultra-sonography to assess flow-mediated vasodilation, it is totally non-operator-dependent, and the equipment is an order of magnitude less expensive. The device records endothelium-mediated changes in the digital pulse waveform known as the PAT ( peripheral Arterial Tone) signal, measured with a pair of novel modified plethysmographic probes situated on the finger index of each hand. Endothelium-mediated changes in the PAT signal are elicited by creating a downstream hyperemic response. Hyperemia is induced by occluding blood flow through the brachial artery for 5 minutes using an inflatable cuff on one hand. The response to reactive hyperemia is calculated automatically by the system. A PAT ratio is created using the post and pre occlusion values. These values are normalized to measurements from the contra-lateral arm, which serves as control for non-endothelial dependent systemic effects. Most notably, this normalization controls for fluctuations in sympathetic nerve outflow that may induce changes in peripheral arterial tone that are superimposed on the hyperemic response.
In this video we demonstrate how to use the Endo-PAT 2000 to perform a clinically relevant assessment of endothelial vasodilator function.

A noninvasive procedure to assess endothelial function is demonstrated using the Endo-PAT 2000.

评估与远藤的PAT 2000内皮血管舒张功能

The endothelium is a delicate monolayer of cells that lines all blood vessels, and which comprises the systemic and lymphatic capillaries. By virtue of ...

科研

医学

Tumor Treating Field Therapy in Combination with Bevacizumab for the Treatment of Recurrent Glioblastoma

A novel device that employs TTF therapy has recently been developed and is currently in use for the treatment of recurrent glioblastoma (rGBM). It was FDA approved in April 2011 for the treatment of patients 22 years or older with rGBM. The device delivers alternating electric fields and is programmed to ensure maximal tumor cell kill1.
Glioblastoma is the most common type of glioma and has an estimated incidence of approximately 10,000 new cases per year in the United States alone2. This tumor is particularly resistant to treatment and is uniformly fatal especially in the recurrent setting3-5. Prior to the approval of the TTF System, the only FDA approved treatment for rGBM was bevacizumab6. Bevacizumab is a humanized monoclonal antibody targeted against the vascular endothelial growth factor (VEGF) protein that drives tumor angiogenesis7. By blocking the VEGF pathway, bevacizumab can result in a significant radiographic response (pseudoresponse), improve progression free survival and reduce corticosteroid requirements in rGBM patients8,9. Bevacizumab however failed to prolong overall survival in a recent phase III trial26. A pivotal phase III trial (EF-11) demonstrated comparable overall survival between physicians&#8217; choice chemotherapy and TTF Therapy but better quality of life were observed in the TTF arm10.
There is currently an unmet need to develop novel approaches designed to prolong overall survival and/or improve quality of life in this unfortunate patient population. One appealing approach would be to combine the two currently approved treatment modalities namely bevacizumab and TTF Therapy. These two treatments are currently approved as monotherapy11,12, but their combination has never been evaluated in a clinical trial. We have developed an approach for combining those two treatment modalities and treated 2 rGBM patients. Here we describe a detailed methodology outlining this novel treatment protocol and present representative data from one of the treated patients.

A novel methodology that is employed for the treatment of recurrent glioblastomas is described. This treatment approach employs the application of alternating electric tumor treating fields (TTFields), known as TTF Therapy in combination with bevacizumab, a targeted agent that is currently FDA approved as monotherapy.

肿瘤治疗领域的治疗与联合贝伐单抗治疗复发性胶质母细胞瘤的治疗

A novel device that employs TTF therapy has recently been developed and is currently in use for the treatment of recurrent glioblastoma (rGBM). It was FDA ...

Using Saccadometry with Deep Brain Stimulation to Study Normal and Pathological Brain Function

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including Parkinson's and Huntington's can disrupt it. People with Parkinson's disease, for example, tend to have increased saccadic latencies. Consequently, the quantitative measurement of saccadic eye movements has received considerable attention as a potential biomarker for neurodegenerative conditions. A lot more can be learned about the brain in both health and disease by observing what happens to eye movements when the function of specific brain areas is perturbed. Deep brain stimulation is a surgical intervention used for the management of a range of neurological conditions including Parkinson's disease, in which stimulating electrodes are placed in specific brain areas including several sites in the basal ganglia. Eye movement measurements can then be made with the stimulator systems both off and on and the results compared. With suitable experimental design, this approach can be used to study the pathophysiology of the disease being treated, the mechanism by which DBS exerts it beneficial effects, and even aspects of normal neurophysiology.

This paper describes the use of quantitative measurement of eye movements in conjunction with stimulation of focal areas of the deep brain in order to study physiology, pathophysiology, and the mechanisms of deep brain stimulation.

使用Saccadometry与深部脑刺激来研究正常和病理脑功能

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including ...

Assessment of Respiratory Function in Conscious Mice by Double-chamber Plethysmography

Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to non-invasively assess some features of the respiratory function in humans as well as in laboratory animals. The present article focuses on the application of the double-chamber plethysmography (DCP) in small animals. It provides background information on the methodology as well as a detailed step-by-step procedure to successfully assess respiratory function in conscious, spontaneously breathing animals in a non-invasive manner. The DCP can be used to monitor the respiratory function of multiple animals in parallel, as well as to identify changes induced by aerosolized substances over a chosen time period and in a repeated manner. Experiments on control and allergic mice are used herein to demonstrate the utility of the technique, explain the associated outcome parameters, as well as to discuss the related advantages and shortcomings. Overall, the DCP provides valid and theoretically sound readouts that can be trusted to evaluate the respiratory function of conscious small animals both at baseline and after challenges with aerosolized substances.

The objective of the present article is to provide a detailed description of the recommended procedures to evaluate respiratory function in conscious mice by double-chamber plethysmography.

双室 Plethysmography 对清醒小鼠呼吸功能的评价

Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to ...

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively affect skeletal muscle. This can result in a loss of muscle mass (atrophy) and/or loss of muscle quality (reduced force per unit of muscle mass), both of which are prevalent in chronic disease, muscle-specific disease, immobilization, and aging (sarcopenia). Skeletal muscle function in animals can be evaluated by a range of different tests. All tests have limitations related to the physiological testing environment, and the selection of a specific test often depends on the nature of the experiments. Here, we describe an in vivo, non-invasive technique involving a helpful and easy assessment of force frequency-curve (FFC) in mice that can be performed on the same animal over time. This permits monitoring of disease progression and/or efficacy&#160;of a potential therapeutic treatment.

Measurement of rodent skeletal muscle contractile function is a useful tool that can be used to track disease progression as well as efficacy of therapeutic intervention. We describe here the non-invasive, in vivo assessment of the dorsiflexor muscles that can be repeated over time in the same mouse.

小鼠肌功能的无创评价

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively ...

A Non-Invasive Method for Generating the Cyclic Loading-Induced Intra-Articular Cartilage Lesion Model of the Rat Knee

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely correlated with a history of articular cartilage damage and ligament avulsion. Surgical animal models of OA of the knee play an important role in understanding the onset and progression of post-traumatic OA and aid in the development of novel therapies for this disease. However, non-surgical models have been recently considered to avoid traumatic inflammation that could affect the evaluation of the intervention. 
In this study, an intra-articular cartilage lesion rat model induced by in vivo cyclic compressive loading was developed, which allowed researchers to (1) determine the optimal magnitude, speed, and duration of load that could cause focal cartilage damage; (2) assess post-traumatic spatiotemporal pathological changes in chondrocyte vitality; and (3) evaluate the histological expression of destructive or protective molecules that are involved in the adaptation and repair mechanisms against joint compressive loads. This report describes the experimental protocol for this novel cartilage lesion in a rat model.

Here, we present the cyclic loading-induced intra-articular cartilage lesion model of the rat knee, generated by 60 cyclic compressions over 20 N, resulting in damage to the femoral condylar cartilage in rats.

一种生成周期负荷诱导的大鼠膝关节内软骨病变模型的非侵入性方法

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely ...

通过高分辨率呼吸测定法测定人精子细胞中的呼吸

High-Resolution Respirometry to Assess Mitochondrial Function in Human Spermatozoa

Semen quality is often studied by routine semen analysis, which is descriptive and often inconclusive. Male infertility is associated with altered sperm mitochondrial activity, so the measurement of sperm mitochondrial function is an indicator of sperm quality. High-resolution respirometry is a method of measuring the oxygen consumption of cells or tissues in a closed-chamber system. This technique can be implemented to measure respiration in human sperm and provides information about the quality and integrity of the sperm mitochondria. High-resolution respirometry allows the cells to move freely, which is an a priori advantage in the case of sperm. This technique can be applied with intact or permeabilized spermatozoa and allows for the study of intact sperm mitochondrial function and the activity of individual respiratory chain complexes. The high-resolution oxygraph instrument uses sensors to measure the oxygen concentration coupled with sensitive software to calculate the oxygen consumption. The data are used to calculate respiratory indices based on the oxygen consumption ratios. Consequently, the indices are the proportions of two oxygen consumption rates and are internally normalized to the cell number or protein mass. The respiratory indices are an indicator of sperm mitochondrial function and dysfunction.

作者聚焦:通过揭示精子代谢和线粒体功能推进男性不育研究 

The analysis of sperm mitochondrial function by high-resolution respirometry permits the measurement of the oxygen consumption of freely moving spermatozoa in a closed-chamber system. The technique can be applied to measure respiration in human spermatozoa, which provides information on sperm mitochondrial characteristics and integrity.

高分辨率呼吸测定法评估人类精子的线粒体功能

Semen quality is often studied by routine semen analysis, which is descriptive and often inconclusive. Male infertility is associated with altered sperm ...

大鼠 DRG 压缩模型的标准化 Tuina 协议

Analgesic Effect of Tuina on Rat Models with Compression of the Dorsal Root Ganglion Pain

Neuropathic pain is a prevalent condition that affects 6.9%-10% of the population and results from nerve damage due to various etiologies, such as lumbar disc herniation, spinal canal stenosis, and intervertebral foramen stenosis. Although Tuina, a traditional Chinese manual therapy, has shown analgesic effects in clinical practice for the treatment of neuropathic pain, its underlying neurobiological mechanisms remain unclear. Animal models are essential for elucidating the basic principles of Tuina. In this study, we propose a standardized Tuina protocol for rats with compression of the dorsal root ganglion (DRG), which involves inducing DRG compression by inserting a stainless steel rod into the intervertebral foramen, performing Tuina manipulation with specific parameters of location, intensity, and frequency in a controlled environment, and assessing the behavioral and histopathological outcomes of Tuina treatment. This article also discusses the potential clinical implications and limitations of the study and suggests directions for future research on Tuina.

作者聚焦： 推拿对压迫背根神经节疼痛的大鼠模型的镇痛作用  

This article presents a manipulation for treating chronic compression of the dorsal root ganglion in rats using Tuina therapy, along with a method for evaluating its effectiveness based on pain behavior and histopathological results.

推拿对大鼠模型的镇痛作用，压迫背根神经节疼痛

Neuropathic pain is a prevalent condition that affects 6.9%-10% of the population and results from nerve damage due to various etiologies, such as lumbar ...