We thank the thousands of brain donors, patients, families, and neuroscientists around the world who, during the last two centuries and through their generous gifts and intellectual efforts, helped to discover how the human brain works, to understand devastating brain diseases, and to develop treatments thereof. We particularly thank Mrs. Cecilia V. Feltis for editing and reviewing this manuscript.

涉及死后人体组织程序已被机构审查委员会审查，并在45 CFR（联邦法规）豁免。 注:本协议描述了完成在人类神经病理学研究死后的大脑评估对称bihemispheric脑部切割程序。的装置，仪器，材料和必要执行人类大脑切割建筑材料的详细描述将被排除。对于脑解剖材料和用品在单研究者决定被选择并且是基于允许或在各研究机构批准尸检工具。集此过程所需的工具和材料最小的材料/设备表中描述。具体切割程序和疑似传染性脑疾病，如人类克雅氏病的预防措施，是这个手稿的目标之外，并从其他来源获得7。 1.对称双大脑半球切割注意:确保大脑已经接收了必要的组织固定（使用，例如，中性缓冲的10％福尔马林），为期两到三周的，这取决于periagonal，代谢（ 即，pH值），和组织保存（ 即，温度）的条件。然而，对于成像病理学相关研究，固定的周期较长（&gt;5.4周）已建议8。 <ol><li>放置在面向研究者平坦表面脑，与朝向相反的方向相对于所述研究者的正面极。 </li><li>将这样的方式大脑以允许所有皮质脑回和整个大脑（ 图1a）的沟的完整和清晰的可视化。 </li><li>先来说一下脑膜异常，宏观半球不对称性（焦点，叶，或萎缩的广义半球现象可能的指标），宏观tissutal病变（ 即吐沫RS或症），先天性畸形，脉管异常，以及任何其他可能的异常或脑表面的不寻常的演示。 注:有关如何评估一个人的大脑的详细说明，请参阅市售的神经病理学教科书和尸检手册9,10。 </li></ol> 2.协议序列<ol><li>面对正面极点从调查路程，半球（端脑）所面临的研究者肤浅的方面。多少就拿多少数码照片根据需要在每一个特定的情况下记录可能的宏观异常现象，并考虑到可能的临床 - 神经解剖和切割后的注意事项。有一个研究助理拿数码照片垂直于大脑捕捉整个皮质表面（ 图1a - C）。 </li><li>马克前和使用的墨水或彩色针中央后皮质脑回切割前脑（ 图1b </s仲&gt;）。 注:此过程有利于电机和切割后体感皮层主要的更直接的认识。 </li><li>旋转180度的脑，同时保持其朝向相同的方向（即从研究者背向正面极）。仔细检查大脑底部。要特别注意脑血管系统的条件（ 即，基底动脉和椎动脉和Willis环）在其脑干出口/入口水平和颅神经。管理嗅球和大片特别小心，以避免tissutal裂伤，由于他们的极端脆弱性。 <ol><li>多少就拿多少数码照片根据需要在每一个特定的情况下记录可能的宏观异常现象，并考虑到可能的临床 - 神经解剖和切割后的注意事项。有一个研究助理拿数码照片垂直于大脑捕捉到皮层和脑干表面的全部。 </l我&gt; </ol></li><li>朝向脑的碱和使用手术刀，剪在脑桥的上部的水平脑干横向（尽可能接近到大脑的碱）。仔细检查SN（ 即 ，为苍白）11和其他邻近结构12。需注意，可能相比于正常脑13用脑的任何不寻常的外观的音频记录装置中，。 </li><li>再次旋转180度脑并用一把锋利的刀，通过集中切断胼胝体通过内侧纵裂缝，并遵循额枕部方向的两个半球分开。检查每个半球为可能的异常（ 例如，心室扩印，畸形，组织软化，肿瘤等 ）13的每一侧。参见图2a <ol><li>多少就拿多少数码照片根据需要在每一个特定的情况下记录可能的宏观异常和考虑到可能的临床 - 神经解剖和切割后的注意事项。有一个研究助理拿数码照片垂直于大脑捕捉整个皮质表面。注意到脑的任何不寻常的特点的相比于正常脑。 </li></ol></li><li>放置两个半球扁平，躺在其内侧方面，朝向从研究者远额叶， 如图2b所示 。将它们放置在这样一种方式，其中心触摸（也标记为半球不对称的情况下）。 </li><li>用一把锋利的刀，手动通过两个大脑半球切开，开始在额极，并通过半球的整个长度走向两极枕部移动。获得两个系列的脑组织1厘米厚的块（约18板为每个半球）。 </li><li>放置脑板坯在一个单独的平面上的解剖学组织（额枕骨方向）序列。使用白色surfa与拍摄时印有更好的对比度尺子CE。显示两个系列脑板坯在解剖学对称方式（额枕方向），确保它们的冠状表面是直接目视和数字摄影（ 图3a）中可见。使用两侧印刷毫米波网格切割表面来定位大脑结构，尺寸和可能的异常以更精确的方式。 <ol><li>多少就拿多少数码照片根据需要在每一个特定的情况下记录可能的宏观异常现象，并考虑到可能的临床 - 神经解剖和切割后的注意事项。有一个研究助理拿数码照片垂直于大脑捕捉整个皮质表面。记笔记相比于正常脑（可能使用音频记录器设备），大脑中的任何不寻常的方面。 </li></ol></li><li>用锋利的手术刀，解剖手动较小的矩形块脑组织中的每个建立脑区。按照表1所述建议的脑区域收集方案。 <ol><li>放入单独标示histocassettes每个组织块。 注:脑组织中的每块需要被切断，以适应，尽可能，标准histocassette最大体积（30×20×4mm的3）。 </li><li>标签使用每一种情况下的去识别代码和使用特定的神经解剖学标识符histocassettes（不使用随机字母或数字为不同的大脑;相反，始终使用相同的区域的解剖学的名称或相应的数字，如表1所示）。创建去识别码，例如，通过产生随机或半随机数为每一种情况下（ 即 ，BRC 130，其中B停留脑，R停留资源，C停留中心和130是一个渐进的加入或AD160001，其中，AD代表"阿尔茨海默氏症研究"，16是年时进行尸体解剖（2016），和0001逐行加入检体数）。 注意:这一步是为未来的研究非常有帮助的;保持一个传奇，并指定半球（L =左脑，R =右半球）。使用histocassettes两种不同颜色，建立为每个半球特定的颜色。 </li><li>多少就拿多少数码照片根据需要在每一个特定的情况下记录可能macroanomalies，并考虑到可能的临床 - 神经解剖和切割后的注意事项。有一个研究助理拿数码照片垂直于大脑捕捉整个皮质表面。注意到脑的任何不寻常的特点的相比于正常脑。 </li></ol></li><li>拍摄数字图片（多达必要或期望的）整个切脑和相关histocassettes的。 </li><li>冲床（ 例如，通过的Accu冲）小块组织的DNA提取和遗传分析。使用直径5毫米 - 2一记重拳。 注:对于其高的基因组材料的含量，小脑是首选;然而，任何其他区域是好的。 </li><li>再浸入含有相同类型的先前使用，直到组织处理的下一步骤固定液（ 例如，10％的中性缓冲的福尔马林）的脑组织块所有histocassettes。 </li><li>按照人类福尔马林固定，组织处理14的标准程序。 </li></ol> 3.特殊的方法:交替冷冻和固定对称Bihemispheric切割注:在第2描述的对称bihemispheric脑切削协议提供了从未定影，新鲜的大脑切割组织板坯在相同的系统和对称的方式的可能性（当可用时）。 <ol><li>将整个大脑清新倒挂（最好在半球碗状的塑料表面）8 - 10分钟，在-80℃冷冻硬化的脑组织withouŧ挑起生化损伤，以方便手动切割。 </li><li>用一把锋利的刀，切两半球在交替和连续的方式，按照第2节中描述的大脑切割协议，但冻结和修复每隔板（由两个半球和沿额枕部解剖方向）。 <ol><li>此时，不要试图削减各脑区域，如表1所述。切新鲜的特定脑区仅在必要时立即RNA或蛋白质的提取（ 即对基因组或蛋白质组学研究）15。 </li></ol></li><li>切割后，立即冻结，标签和数量每个新鲜组织。占据整个平板系列数码照片;装在一个塑料袋每个切片;收集在一个单独的，一个脑只容器中的板;和容器存放在专用-80℃冰箱中。 注:冰箱应致力于只有人脑组织。只是后来会唱根据需要为每个特定实验勒冷冻大脑区域进行切割。 </li><li>沉浸选择用于在含有固定剂的足够量（3/1体积固定剂/组织块的比例）分离袋固定（10％的中性缓冲的福尔马林或其他固定剂）每隔其它组织板坯。通过连续编号他们跟着一个额枕部序列标签每袋。密封每个袋，拍摄数字图片，并将它们存储在塑料容器中。 </li><li> 2周组织固定后打开含有固定剂袋和如表1中所述切割每个脑区。 </li></ol> 4. Histostain及免疫组化注:本套脑区的切基础上，提出方案（ 表1）足以满足大多数，如果不是全部，当前已建立共识为基础的病理标准AD 16，PD 17，路易体痴呆（DLB）18，额颞叶痴呆（FTD / MND） <suP&gt; 19，逐行Suprabulbar麻痹（PSP）20，多系统萎缩（MSA）21，慢性创伤性脑病（CTE）22， 等等 。 <ol><li>对于每一个脑区和两个半球，请执行下列最低限度histostains的:苏木和曙红（H＆E），甲酚紫（CV;如果形态计量学研究计划，例如），银染色（如"探索性"分析是需要）。 </li><li>对于每一个脑区和两个半球，请执行下列最低限度的免疫组化协议:β淀粉状蛋白（βA），磷酸化头（pTau），磷酸化α突触核蛋白（Pα-SYN）和磷酸化TDP43（pTDP43） ，如上所述14。 注:组织切片，以评估每个脑内此协议的总数为46（如果由两个半球各脑区都可用）。 </li></ol>

作者什么都没有透露。

这种脑切断方法可适于对每个神经病理学实验室的特定需求（例如，通过减少脑区域的数量，以评估每个半球），同时仍保留bihemispheric对称切割的步骤，其主要特征之一。此提出的协议可用于常规程序（研究型神经病理学中心）或只在必要时（具体临床取向研究）。它可以选择性地仅用于特定类型的调查（ 即，免疫组织化学）或分子分析（ 即 ，基因组或蛋白质组分析）。从技术角...

Neuropathologists有科学的特权，智力的荣誉和诊断义务评估人类大脑。几十年来，脑部疾病和重大努力的详细的临床描述，以个体化在人死后的大脑其可能neurohistological相关因素已经开展。从历史上看，这些努力所代表的最有成效的方式由医学科学和神经学尤其是在当今时代先进。由于以前的杰出neuropathologists和他们的奉献精神，决心，奖学金，惊人的能力，以正常和异常的脑组织（通常使用非常残留的工具）之间的区别，我们现在可以研究和目标疾病，如阿尔茨海默Perusini病（不公平只被称为阿尔茨海默氏症病; APD / AD）1，帕金森病（PD）2，克雅氏病（CJD）3，娄格里克病/肌萎缩侧索SclerosiS（ALS）4，和关岛病5，仅举几例。 神经影像学的先进的技术，如高清晰计算机断层扫描（ 即多层螺旋CT扫描，CT血管造影），功能和形态磁共振成像（ 即功能磁共振成像，弥散核磁共振成像，示踪-MRI 等 ），正电子发射断层扫描（PET），基于超声波的成像，等人，都肯定是我们修改关于如何诊断和治疗神经和精神病患者一般做法。尽管如此，虽然神经成像技术能够可视化的人的大脑时活着，他们不提供的机会，在发生的时刻，直接分析细胞，如神经元的高度复杂的细胞和亚细胞结构的;或可视化，标记和量化特定类型的细胞内病变;或精确显示他们的神经解剖学和次区域定位在circuital和分circuital解剖层次。例如，神经成像技术不能在嗅皮层识别或定位在黑质（SN），与帕金森病相关的一个共同的病理学特征，或神经原纤维缠结（NFT）的色素神经元Lewy小体（LB），AD的经典特征和其他脑病变。神经病理学的调查与先进的数码显微镜相结合仍然是不可替代的详细的临床相关性，因此，为明确诊断。 由于人脑的特有anatomo功能特性，特别是它的解剖定位（即，颅骨，天然保护系统，该系统不允许内容的直接检查内部），引入体内神经成像技术非常有帮助临床医生和调查人员发现最初的解答一些这种复杂组织的奥秘。然而，没有临床或neuroimagiNG的方法，可替换的独特的机会，脑组织尸检过程中直接分析。仅有组织收集，保存，和人脑的分类可允许神经元和非神经元细胞，亚细胞成分，细胞内和细胞外的病理病变直接与系统的调查，而大脑内的任何类型的异常进行确认，修改，或重新定义临床诊断和发现新的临床相关性。之一有关的大脑尸检评估表观限制一直是事实，这过程是一个剖方法论。总会有一个持续的神经病理过程（临床表现或不）和机会，如果有的话，在neurohistological级别来定义它之间的延迟。这主要是由于人的大脑没有能力再生本身。它目前还无法获得的脑组织在体内 ，而无需创建PErmanent伤害。因此，是不可能在纵向和神经病理学评估相同的大脑/人。然而，标准化的大脑银行程序和脑捐赠广大市民日益认识到可以极大地持续增加收集和分析案件的数量有助于脑尸检时序问题的解决。以这种方式，可以得到死后的大脑的更充分的数字，以定义病理起源和进展的恒定模式与每个人脑疾病相关的脑损伤的每个特定类型。这将需要从受任何神经精神障碍的患者在所有年龄捐献和尽可能多的大脑尽可能收集，以及来自健康对照受试者。一个可能的方法可以从一般和专门的医疗中心作为一个标准的程序中收集尽可能多的尸检大脑越好。需要对脑捐赠最近已表示那些谁研究老年痴呆症和正常老化6。相同的必要性应由神经字段作为整体来表示。 用于上述和其他原因，正在进行的脑切割程序的更新是必要的。此外，脑切割程序应普遍在不同的神经病理学的研究中心标准化在世界各地，也考虑帐户的可能性聘请当前和未来的生物技术，以更好地调查，并希望能以明确理解的原因和脑部疾病的机制人类。 在这里，主要用于研究目的，我们描述了死后的大脑在人类切对称方法。此过程提出收集比正常完成，并从两个大脑和小脑半球更多脑区。对称双大脑半球切割过程将符合我们当前的人类知识好得多神经解剖学，神经化学，神经生理学和。这种方法还允许的可能性神经病理学分析人类大脑的独特功能，诸如半球专业化和偏侧是与较高的认知和非认知功能一般或专门存在于我们的物种相关联。是否有半球专业化/偏侧和特定类型的脑病变之间的具体发病的关系，或一种特殊的神经精神病发病事件是否是最初，普遍地，或只与特定的半球关联和功能不是目前公知的。通过描述这种大脑对称切割程序，我们的目标是提出人类大脑解剖的更新方法，可以帮助更好地了解一个高度专业化的正常组织和病理条件下，大脑。该方法还考虑到，只存在于人类的那些吗啉官能半球方面。

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			<td>Copy of signed informed consent allowing autopsy and brain donation for research use.</td>
			<td></td>
			<td></td>
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			<td>Detailed clinical history of the subject which should include a detailed description of any neurologic and psychiatric symptoms and signs.</td>
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			<td>Medical or not-medical video-recordings when available (especially useful in movement disorders field). Next-of-kin&#8217;s consent required.</td>
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			<td>Neuroimaging, neurophysiology, neuropsychiatric and assessment or clinicometric scales.</td>
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			<td>Genetic and family history data. Genetic reports review, if neurogenetic diseases were diagnosed.</td>
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			<td>Histology Container</td>
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			<td>Disposable Blade Knife Handles</td>
			<td>ELECTRON MICROSCOPY SCIENCES</td>
			<td>72040-08</td>
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			<td>Scalpel Blades</td>
			<td>ELECTRON MICROSCOPY SCIENCES</td>
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			<td>Accu-Punch 2 mm</td>
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			<td>Polystyrene Containers &#8211; Sterile</td>
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symmetric bihemispheric postmortem brain cutting to study healthy and pathological brain conditions in humans

Neuropathologists，有时感到生成在人脑尸检要求的患者描述复杂的神经精神现象，明确的诊断所需要的知识量吓倒。尽管生物医学影像学和的进步已经彻底改变了神经精神领域，他们也产生了误导性的想法，大脑解剖只有一个确定的值。这个假想法在尸检速率的急剧减少，因此，降低的可能性进行更详细的和广泛的神经病理调查，这是必要的，以理解众多正常和病理方面未知的人脑。观察神经精神现象及其可能的相关因素neurohistological相应的本地化/特性之间的相关传统推理方法继续有一个不可否认的价值。在neuropsychi的上下文atric疾病，传统的临床方法仍是最好的可能的方法（通常是唯一可用的），以唯一的神经精神功能链接到其相应的神经病理学基材，因为它在脑组织中的直接身体评估特别依赖。死后大脑的评估是基于大脑切割跨越不同的神经病理学变化中心的程序。脑屑基于存在于每个机构的各种临床和学术突发相对广泛和系统的方式进行。更解剖学包容性和对称双向大脑半球切割的方法至少应该用于研究目的的人类神经病理学连贯调查，深入，与人脑（ 即半球专业化和偏侧特定的特殊性正常和病理条件功能）。这种方法将提供更全面的科尔神经病理良好的特点适用于当前和未来的生物科技和神经成像技术的大脑ction。我们描述了在人类大脑半球病变的差异进行调查并与目前使用的以及未来的生物分子/神经影像学技术的对称双大脑半球切割程序。

协议长度花为单个对称bihemispheric固定脑切削过程中的时间估计为1小时（不包括花费建立解剖台，工具和切割表面的时间;标签; 等 ）。为一个单一的对称双半球交替冻结和固定脑切割过程所需的时间估计需要2小时。它可以至少在4 - 6周获得明确的组织学诊断为单人脑/主题。后从头骨，组织固定的适当时期死后的大脑切除...

Watch this Scientific Journal Video about Symmetric Bihemispheric Postmortem Brain Cutting to Study Healthy and Pathological Brain Conditions in Humans at JoVE.com

Symmetric Bihemispheric Postmortem Brain Cutting to Study Healthy and Pathological Brain Conditions in Humans

有组织的脑切削程序是必要的，以与确定的神经病理学诊断特定神经精神现象相关。脑屑基于各种临床 - 学术突发不同的方式执行。本协议描述的对称bihemispheric脑部切割程序，调查人的大脑半球病变差异，以最大限度地提高当前和未来的生物分子/神经影像学技术。

对称Bihemispheric死后的大脑切割学习健康和病理条件大脑在人

Neuropathologists, at times, feel intimidated by the amount of knowledge needed to generate definitive diagnoses for complex neuropsychiatric phenomena ...

symmetric-bihemispheric-postmortem-brain-cutting-to-study-healthy

Research

JoVE Journal

Medicine

13.9K 次观看.  Biomedical Research Institute of New Jersey (BRInj). 有组织的脑切削程序是必要的，以与确定的神经病理学诊断特定神经精神现象相关。脑屑基于各种临床 - 学术突发不同的方式执行。本协议描述的对称bihemispheric脑部切割程序，调查人的大脑半球病变差异，以最大限度地提高当前和未来的生物分子/神经影像学技术。 

视频: Symmetric Bihemispheric Postmortem Brain Cutting to Study Healthy and Pathological Brain Conditions in Humans

Intra-Operative Behavioral Tasks in Awake Humans Undergoing Deep Brain Stimulation Surgery

Deep brain stimulation (DBS) is a surgical procedure that directs chronic, high frequency electrical stimulation to specific targets in the brain through implanted electrodes. Deep brain stimulation was first implemented as a therapeutic modality by Benabid et al. in the late 1980s, when he used this technique to stimulate the ventral intermediate nucleus of the thalamus for the treatment of tremor 1. Currently, the procedure is used to treat patients who fail to respond adequately to medical management for diseases such as Parkinson's, dystonia, and essential tremor. The efficacy of this procedure for the treatment of Parkinson's disease has been demonstrated in well-powered, randomized controlled trials 2. Presently, the U.S. Food and Drug Administration has approved DBS as a treatment for patients with medically refractory essential tremor, Parkinson's disease, and dystonia. Additionally, DBS is currently being evaluated for the treatment of other psychiatric and neurological disorders, such as obsessive compulsive disorder, major depressive disorder, and epilepsy. 
DBS has not only been shown to help people by improving their quality of life, it also provides researchers with the unique opportunity to study and understand the human brain. Microelectrode recordings are routinely performed during DBS surgery in order to enhance the precision of anatomical targeting. Firing patterns of individual neurons can therefore be recorded while the subject performs a behavioral task. Early studies using these data focused on descriptive aspects, including firing and burst rates, and frequency modulation 3. More recent studies have focused on cognitive aspects of behavior in relation to neuronal activity 4,5. This article will provide a description of the intra-operative methods used to perform behavioral tasks and record neuronal data with awake patients during DBS cases. Our exposition of the process of acquiring electrophysiological data will illuminate the current scope and limitations of intra-operative human experiments.

Deep brain stimulation surgery offers a unique opportunity to examine information encoding in the awake human brain. This article will describe intra-operative methods used to perform cognitive and behavioral tasks while simultaneously acquiring physiological data such as EMG, single-unit neuronal activity and/or local field potentials.

在接受深部脑刺激手术术中唤醒人类行为任务

Deep brain stimulation (DBS) is a surgical procedure that directs chronic, high frequency electrical stimulation to specific targets in the brain through ...

科研

医学

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being analyzed 1, 2. This classical banding technique, however, is not reliable in identifying complex chromosomal aberrations such as those associated with cancer. To overcome the limitations of the banding technique, Spectral Karyotyping (SKY) is introduced to provide much reliable information on chromosome abnormalities.
SKY is a multicolor fluorescence in-situ hybridization (FISH) technique to detect metaphase chromosomes with spectral microscope 3, 4. SKY has been proven to be a valuable tool for the cytogenetic analysis of a broad range of chromosome abnormalities associated with a large number of genetic diseases and malignancies 5, 6. SKY involves the use of multicolor fluorescently-labelled DNA probes prepared from the degenerate oligonucleotide primers by PCR. Thus, every chromosome has a unique spectral color after in-situ hybridization with probes, which are differentially labelled with a mixture of fluorescent dyes (Rhodamine, Texas Red, Cy5, FITC and Cy5.5). The probes used for SKY consist of up to 55 chromosome specific probes 7-10.
The procedure for SKY involves several steps (Figure 1). SKY requires the availability of cells with high mitotic index from normal or diseased tissue or blood. The chromosomes of a single cell from either a freshly isolated primary cell or a cell line are spread on a glass slide. This chromosome spread is labeled with a different combination of fluorescent dyes specific for each chromosome. For probe detection and image acquisition,the spectral imaging system consists of sagnac interferometer and a CCD camera. This allows measurement of the visible light spectrum emitted from the sample and to acquire a spectral image from individual chromosomes. HiSKY, the software used to analyze the results of the captured images, provides an easy identification of chromosome anomalies. The end result is a metaphase and a karyotype classification image, in which each pair of chromosomes has a distinct color (Figure 2). This allows easy identification of chromosome identities and translocations. For more details, please visit Applied Spectral Imaging website (<a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a>).
SKY was recently used for an identification of chromosome segregation defects and chromosome abnormalities in humans and mice with Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disease characterized by dysfunction in primary cilia 11-13. Using this technique, we demonstrated the presence of abnormal chromosome segregation and chromosomal defects in ADPKD patients and mouse models 14. Further analyses using SKY not only allowed us to identify chromosomal number and identity, but also to accurately detect very complex chromosomal aberrations such as chromosome deletions and translocations (Figure 2).

Spectral Karyotyping (SKY) is an advanced cytogenetics technique to identify genomic and chromosomal aberrations. This technique takes advantage of chromosome painting probes, which allow classification of all chromosomes. SKY can also identify complex chromosome aberrations and segregation defects in mice and humans with various diseases, including polycystic kidney disease.

光谱核型多囊肾病的研究在人类和小鼠的染色体异常

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being ...

Controlled Cervical Laceration Injury in Mice

Use of genetically modified mice enhances our understanding of molecular mechanisms underlying several neurological disorders such as a spinal cord injury (SCI). Freehand manual control used to produce a laceration model of SCI creates inconsistent injuries often associated with a crush or contusion component and, therefore, a novel technique was developed. Our model of cervical laceration SCI has resolved inherent difficulties with the freehand method by incorporating 1) cervical vertebral stabilization by vertebral facet fixation, 2) enhanced spinal cord exposure, and 3) creation of a reproducible laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm in depth without associated contusion. Compared to the standard methods of creating a SCI laceration such as freehand use of a scalpel or scissors, our method has produced a consistent lesion. This method is useful for studies on axonal regeneration of corticospinal, rubrospinal, and dorsal ascending tracts.

A novel technique to create a reproducible in vivo model of cervical spinal cord laceration injury in the mouse is described. This technique is based on spine stabilization by fixation of the cervical facets and laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm.

控制宫颈裂伤损伤小鼠

Use of genetically modified mice enhances our  understanding of molecular mechanisms underlying several neurological disorders  such as a spinal cord ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

一种新型的高分辨率<em&gt;在体内</em&gt;成像技术研究肿瘤生长及治疗颅内结构的动态响应

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

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 ...

A Multimodal Imaging- and Stimulation-based Method of Evaluating Connectivity-related Brain Excitability in Patients with Epilepsy

Resting-state functional connectivity MRI (rs-fcMRI) is a technique that identifies connectivity between different brain regions based on correlations over time in the blood-oxygenation level dependent signal. rs-fcMRI has been applied extensively to identify abnormalities in brain connectivity in different neurologic and psychiatric diseases. However, the relationship among rs-fcMRI connectivity abnormalities, brain electrophysiology and disease state is unknown, in part because the causal significance of alterations in functional connectivity in disease pathophysiology has not been established. Transcranial Magnetic Stimulation (TMS) is a technique that uses electromagnetic induction to noninvasively produce focal changes in cortical activity. When combined with electroencephalography (EEG), TMS can be used to assess the brain's response to external perturbations. Here we provide a protocol for combining rs-fcMRI, TMS and EEG to assess the physiologic significance of alterations in functional connectivity in patients with neuropsychiatric disease. We provide representative results from a previously published study in which rs-fcMRI was used to identify regions with abnormal connectivity in patients with epilepsy due to a malformation of cortical development, periventricular nodular heterotopia (PNH). Stimulation in patients with epilepsy resulted in abnormal TMS-evoked EEG activity relative to stimulation of the same sites in matched healthy control patients, with an abnormal increase in the late component of the TMS-evoked potential, consistent with cortical hyperexcitability. This abnormality was specific to regions with abnormal resting-state functional connectivity. Electrical source analysis in a subject with previously recorded seizures demonstrated that the origin of the abnormal TMS-evoked activity co-localized with the seizure-onset zone, suggesting the presence of an epileptogenic circuit. These results demonstrate how rs-fcMRI, TMS and EEG can be utilized together to identify and understand the physiological significance of abnormal brain connectivity in human diseases.

Resting-state functional-connectivity MRI has identified abnormalities in patients with a wide range of neuropsychiatric disorders, including epilepsy due to malformations of cortical development. Transcranial Magnetic Stimulation in combination with EEG can demonstrate that patients with epilepsy have cortical hyperexcitability in regions with abnormal connectivity.

评价中的连接有关的大脑兴奋患者癫痫多式联运Imaging-和激励为基础的方法

Resting-state functional connectivity MRI (rs-fcMRI) is a technique that identifies connectivity between different brain regions based on correlations ...

Intravenous Endotoxin Challenge in Healthy Humans: An Experimental Platform to Investigate and Modulate Systemic Inflammation

Activation of inflammatory pathways represents a central mechanism in multiple disease states both acute and chronic. Triggered via either pathogen or tissue damage-associated molecular motifs, common biochemical pathways lead to conserved yet variable physiological and immunological alterations. Dissection and delineation of the determinants and mechanisms underlying phenotypic variance in response is expected to yield novel therapeutic advances.
Intravenous (IV) administration of endotoxin (gram-negative bacterial lipopolysaccharide), a specific Toll-like receptor 4 agonist, represents an in vivo model of systemic inflammation in man. National Institutes for Health Clinical Center Reference Endotoxin (CCRE, Escherichia coli O:113:H10:K negative) is employed to reliably and reproducibly generate vascular, hematological, endocrine, immunological and organ-specific functional effects that parallel, to varying degrees, those seen in the early stages of pathological states. Alteration of dose (0.06 - 4 ng/kg) and time-scale of exposure (bolus vs. infusion) allows replication of either acute or chronic inflammation and a range of severity to be elicited, with higher doses (2 - 4 ng/kg) frequently being used to create a &#39;sepsis-like&#39; state. Established and novel medicinal compounds may additionally be administered prior to or post endotoxin exposure to appreciate their effect on the inflammatory cascade. Despite limitations in scope and generalizability, human IV endotoxin challenge offers a unique platform to gain mechanistic insights into inducible physiological responses and inflammatory pathways. Rationally employed it may aid translation of this knowledge into therapeutic innovations.

Intravenous administration of endotoxin reliably elicits dose-dependent physiological and immunological alterations consistent with several pathological states. This reductionist approach permits the investigation, modeling and experimental modification of systemic inflammation and its downstream effects in man.

静脉注射内毒素挑战人类健康的实验平台，研究并调制全身炎症

Activation of inflammatory pathways represents a central mechanism in multiple disease states both acute and chronic. Triggered via either pathogen or ...

Continuous IV Infusion is the Choice Treatment Route for Arginine-vasopressin Receptor Blocker Conivaptan in Mice to Study Stroke-evoked Brain Edema

Stroke is one of the major causes of morbidity and mortality in the world. Stroke is complicated by brain edema and other pathophysiological events. Among the most important players in the development and evolution of stroke-evoked brain edema is the hormone arginine-vasopressin and its receptors, V1a and V2. Recently, the V1a and V2 receptor blocker conivaptan has been attracting attention as a potential drug to reduce brain edema after stroke. However, animal models which involve conivaptan applications in stroke research need to be modified based on feasible routes of administration. Here the outcomes of 48 hr continuous intravenous (IV) are compared with intraperitoneal (IP) conivaptan treatments after experimental stroke in mice. We developed a protocol in which middle cerebral artery occlusion was combined with catheter installation into the jugular vein for IV treatment of conivaptan (0.2 mg) or vehicle. Different cohorts of animals were treated with 0.2 mg bolus of conivaptan or vehicle IP daily. Experimental stroke-evoked brain edema was evaluated in mice after continuous IV and IP treatments. Comparison of the results revealed that the continuous IV administration of conivaptan alleviates post-ischemic brain edema in mice, unlike the IP administration of conivaptan. We conclude that our model can be used for future studies of conivaptan applications in the context of stroke and brain edema.

Our studies have revealed that the beneficial effects of conivaptan are dependent on the method of delivery after experimental stroke in mice. We have developed a research protocol for delivery of the receptor blocker via IV catheter on stroke-evoked brain edema formation in mice.

持续静脉注射是精氨酸加压素受体拮抗剂考尼伐坦的选择治疗途径小鼠来研究中风诱发脑水肿

Stroke is one of the major causes of morbidity and mortality in the world. Stroke is complicated by brain edema and other pathophysiological events. Among ...

A Model to Simulate Clinically Relevant Hypoxia in Humans

In case of apnea, arterial partial pressure of oxygen (pO2) decreases, while partial pressure of carbon dioxide (pCO2) increases. To avoid damage to hypoxia sensitive organs such as the brain, compensatory circulatory mechanisms help to maintain an adequate oxygen supply. This is mainly achieved by increased cerebral blood flow. Intermittent hypoxia is a commonly seen phenomenon in patients with obstructive sleep apnea. Acute airway obstruction can also result in hypoxia and hypercapnia. Until now, no adequate model has been established to simulate these dynamics in humans. Previous investigations focusing on human hypoxia used inhaled hypoxic gas mixtures. However, the resulting hypoxia was combined with hyperventilation and is therefore more representative of high altitude environments than of apnea. Furthermore, the transferability of previously performed animal experiments to humans is limited and the pathophysiological background of apnea induced physiological changes is poorly understood. In this study, healthy human apneic divers were utilized to mimic clinically relevant hypoxia and hypercapnia during apnea. Additionally, pulse-oximetry and Near Infrared Spectroscopy (NIRS) were used to evaluate changes in cerebral and peripheral oxygen saturation before, during, and after apnea.

Hypoxia simulation in humans has usually been performed by inhaling hypoxic gas mixtures. For this study, apneic divers were used to simulate dynamic hypoxia in humans. Additionally, physiological changes in desaturation and re-saturation kinetics were evaluated with non-invasive tools such as Near-Infrared-Spectroscopy (NIRS) and peripheral oxygenation saturation (SpO2).

一个模型来模拟人类临床相关性缺氧

In case of apnea, arterial partial pressure of oxygen (pO2) decreases, while partial pressure of carbon dioxide (pCO2) increases. To avoid damage to ...

A Tissue Displacement-based Contusive Spinal Cord Injury Model in Mice

Producing a consistent and reproducible contusive spinal cord injury (SCI) is critical to minimizing behavioral and histological variabilities between experimental animals. Several contusive SCI models have been developed to produce injuries using different mechanisms. The severity of the SCI is based on the height that a given weight is dropped, the injury force, or the spinal cord displacement. In the current study, we introduce a novel mouse contusive SCI device, the Louisville Injury System Apparatus (LISA) impactor, which can create a displacement-based SCI with high injury velocity and accuracy. This system utilizes laser distance sensors combined with advanced software to produce graded and highly-reproducible injuries. We performed a contusive SCI at the 10th thoracic vertebral (T10) level in mice to demonstrate the step-by-step procedure. The model can also be applied to the cervical and lumbar spinal levels.

We introduce a tissue displacement-based contusive spinal cord injury model that can produce a consistent contusive spinal cord injury in adult mice.

基于组织位移的小鼠脊髓损伤模型

Producing a consistent and reproducible contusive spinal cord injury (SCI) is critical to minimizing behavioral and histological variabilities between ...