这项工作是支持青少年糖尿病研究基金会国际（批17-2008-1031）和美国国立卫生研究院（授予1R01DK077903 - 01A1）。

简介： 周围神经病变的准确定量是很重要的定义处于危险的患者，预期恶化，并评估新的治疗方法。常规方法评估神经功能缺损和电生理和定量感觉测试的量化功能的改变，检测神经病变。然而，最早的损害，出现细小纤维，但这些测试主要是评估大纤维功能障碍，表现出的再生和修复能力有限。只允许直接检查的​​无髓神经纤维的损伤和修复技术，电子显微镜和皮肤打孔活检腓肠神经活检。然而，无论是侵入性的程序和需要长时间的实验程序和相当的专业知识。角膜共焦显微镜是一种非侵入性的临床技术，它提供了角膜神经纤维在体内成像。 HRT的III -罗斯托克角膜模块（RCM） 操作手册 1。相机的制备在考试的最初步骤，使用HRT的物镜尖的准备。 <ol><li>首先，在激光扫描相机的客观管，设置12屈光度的折射，然后相机调整到最低位置。 </li><li>套用一个大的同质性，泡沫自由，豌豆大小，镜头上的尖端，涉及的下降。 </li><li>从无菌容器中取出一个TomoCap，它安装在镜头一角，凝胶，形成了一个半月板之间的物镜和第。尽可能在持有人可能推。小心不要触摸TomoCap前表面在安装过程中。 <img src="/files/ftp_upload/2194/2194fig1.jpg" alt="图1" /> 图1。物镜尖（顶）申请viscotear凝胶（底部）应用TomoCap准备。 </li><li>移动激光扫描相机尽量向后摄像机安装可能。 </li><li> TomoCap的内外表面都出现一个明亮的激光反射。应调整焦平面调整轮，直到光明的反映，是观察，表明该镜头集中在前面的第的。在重点位置显示显示深度值现在应该-150微米，150微米之间。 </li><li>将深度设置重置为零。 <img src="/files/ftp_upload/2194/2194fig2.jpg" alt="图2" /> 图2。焦平面调整。 </li></ol> 2。准备病人拍摄主体的眼睛是麻醉使用下降0.4％benoxinate盐酸，“活视康”人造眼泪水润滑眼睛的前面。 坐在舒适的主题是，与下巴的下巴上休息，并要求坚决按他们的额头对额头栏。 <img src="/files/ftp_upload/2194/2194fig3.jpg" alt="图3" /> 图3准备病人。 3。对齐相机它使考试更容易，如果您指示病人与没有被检查眼外固定光修复。显微镜头小巧的尺寸意味着主体的对侧眼不遮挡，使使用距离目标。 <img src="/files/ftp_upload/2194/2194fig4.jpg" alt="图4" /> 图4。角膜模块的物镜。 <ol><li> CCD相机等，其光轴垂直的激光扫描摄像机的光轴的位置。相机应该对病人的右侧时，成像的右眼，反之亦然。 在这个位置上，你应该看到TomoCap前表面的CCD相机实时图像的中心。 </li><li>移动的激光扫描对病人的相机，直到病人的角膜在一个约5至10毫米的TomoCap距离是移动的激光扫描相机上/下和左/右使用摄像机安装上的黑色旋钮，直至在TomoCap是在患者的角膜的中心位置。在这个阶段，您可以检查红色激光灯是在角膜中心和微调，观察角膜，这是在CCD摄像机的图像中可见的激光束的反射。这种反思必须完全发生在角膜前极。 <img src="/files/ftp_upload/2194/2194fig5.jpg" alt="图5" /> 图5。对准捕捉角膜中央图像角膜的前极的激光束。 </li><li>让患者尽可能广泛的他/她的眼睛打开。慢慢地向前移动的激光扫描相机对病人直到TomoCap接触病人的角膜。如果它是必需正确的激光扫描摄像机的位置，然后移动相机远离病人第一，做了校正，然后将它再次接触角膜。 <img src="/files/ftp_upload/2194/2194fig6.jpg" alt="图6" /> 图6。TomoCap和角膜之间的薄露桥的示范。 </li><li>相机和角膜之间的最小的接触是不够的。在优化调整，薄露TomoCap和角膜之间的桥梁上的CCD相机实时图像是可见的。 </li></ol>如果按TomoCap或移动相机太接近角膜，然后你会看到一个通过CCD相机实时图像的外观扁平角膜在角膜上的压力。也得到了线的图像显示会出现的压力，因此并不适用于角膜的压力太大。一旦接触已经建立，不要移动headrestto避免角膜上TomoCap滑动的位置。 <img src="/files/ftp_upload/2194/2194fig7.jpg" alt="图7" /> 图7（左）一个straie由于对角膜的TomoCap压力过大应用程序的外观;（右）与正常的外观没有多余的压力形象。 4。检查病人视野已被选定后，被打开的HRT III激光相机和图像采集窗口出现在屏幕上。使用CCD图像TomoCap和对病人的角膜激光灯的位置。然后可以开始使用所选择的模式的考试。 <img src="/files/ftp_upload/2194/2194fig8.jpg" alt="图8" /> 图8。查看监视器上的图像采集过程中的模式。 采集模式在部分模式中，一个单一的形象收购和储存您每次按下脚踏开关。我们使用这种模式下，捕获图像从整个角膜层包括鲍曼的一层角膜中心。然后捕获足够数量的图像后，可以尝试其他模式，包括顺序和数量。 <img src="/files/ftp_upload/2194/2194fig9.jpg" alt="图9" /> 图9显示不同的图像采集方式与选择。 与序列扫描，可以获取一个可调节的帧速率高达100张图像的序列。您可以选择之间的帧速率30帧和1帧每秒（fps）和基于这种模式，它可以让你获得为期3至100秒的影片。 在容积扫描模式下，相机会自动获得一个系列40个连续的焦平面图像。一个85微米的总深度，可以扫描“视野400微米”场镜和连续两个图像之间的焦距，将约2.1微米。 经过几次扫描镜头聚焦平面是可以改变的，相对的角膜上皮细胞层，将成为关注的焦点，然后按一下复位按钮深度值设置为0表面的角膜细胞层的深度来衡量的。 对于我们目前研究的目的，我们得到从不同深度的角膜中央的图像。因此，我们不走动角膜激光扫描相机的水平或垂直的，我们只是向前或向后移动，并捕获在同一角膜点鲍曼的层深处的图像。每幅图像之间的距离大约是1微米，因此，如果鲍曼的深度为10μm，我们将有10张。然而，归根结底，我们选择约2-3微米它们之间的距离的图像。 检查后完成收购窗口中按一下相机的电源按钮关闭相机。使病人意识到，他或她不应该擦他或她的眼睛，直到局部麻醉消失。作为一项预防措施，你不妨在考试后执行一个病人的角膜裂隙灯检查。 从相机中取出TomoCap，处理它，然后用棉花清洁显微镜镜头棉签蘸蒸馏水。 5。考试分析 A.选择帧审查和分析现有的考试，选择合适的病人记录（S）必须在数据库窗口。图像浏览窗口，提供了一个现有的角膜检查概述选定的病人。在不同的日子进行的考试都存储在单独的访问选项卡。三种不同的扫描类型，代表三种不同的图标，在HRT三角膜访问“选项卡，喜的图像浏览窗口delberg眼科资源管理器。 调查角膜图像，单击相应的图像图标和“显示”功能打开下面的窗口： <img src="/files/ftp_upload/2194/2194fig10.jpg" alt="图10" /> 图10显示拍摄的图像后，检查所有。 <img src="/files/ftp_upload/2194/2194fig11.jpg" alt="图11" /> 图11出口前眼与CCD相机视图显示正确对齐选定的图像显示。 要导出相应的图像作进一步的分析，点击工具栏上的“导出”，然后选择你要保存的图像文件夹。 我们获取和为每一个病人的角膜层捕获的图像，但这项研究的主要焦点是鲍曼的层和角膜神经，只有这一层的图像进行分析。我们选择从鲍曼的每个科目在不同深度的角膜层5-6帧。 <img src="/files/ftp_upload/2194/2194fig12.jpg" alt="图12" /> 图12显示，从一个健康的主题所有角膜层。 在鲍曼的角膜中心层在不同深度的平均5-6帧是随机选取的定量测量和分析。 B.分析图像鲍曼的角膜中央与HRT的第三层，从拍摄的图像进行分析，我们使用专门的软件称为“CCM的图像分析工具v 0.6”本集团内已开发的。 角膜神经的四个主要参数的测量，用这个软件： <ul><li>神经纤维密度（NFD），这是定义为主要神经/帧的数量。 </li><li>神经分支密度（NBD）的主要分支/帧。 </li><li>神经纤维长度（NFL），这是所有神经/帧的总长度。 </li><li>神经纤维迂曲（NFT）的主要神经/帧。 </li></ul>要打开CCM的形象，去文件&gt;打开。 <ul><li>一旦加载图像，其大小和类型显示在控制面板的左上角手端的图像信息框。 </li><li>默认的规模是显示在编辑行的图像信息。如果规模是不同的，修改在编辑行的数量。 </li><li>要开始分析的图像，选择你想测量从在控制面板的右上角方公制中的参数。 </li><li>在结果框在页面的NFD的底部，NBD的实际数量，NFL作为一个曲折系数（TC）毫米和迂曲。 </li><li>然而，之后的结果导出到Excel工作表，结果将没有/ NFD的NBD和毫米/ 毫米 2为NFL 2毫米。 </li><li>为此，整理后的测量点击添加测量，然后转到文件&gt;另存为，然后用Excel打开保存的结果。 </li></ul><img src="/files/ftp_upload/2194/2194fig13.jpg" alt="图13" /> 图13。角膜神经图像显示与跟踪分析工具使用“CCM图像分析工具”。 6。代表性的成果： 我们已经证明了一个渐进的角膜神经变性日益增加1和糖尿病神经病变在糖尿病早期神经损伤，先在皮肤活检表皮内神经纤维的损失 2 以下 3胰腺移植对糖尿病患者神经性的严重程度和修复分层的严重性。我们也证明了神经损伤特发性小纤维神经病4 Fabry病5。 <img src="/files/ftp_upload/2194/2194fig14.jpg" alt="图14" /> 图14。HRT的角膜鲍曼的第三层（一）控制主体（b）与神经病变和严重的神经损伤患者相比角膜共聚焦显微镜图像。

<ol>
	<li>Abbott, C. A., Kallinikos, P., Marshall, A., Finnigan, J., Morgan, P., Efron, N., Boulton, A. J. M., Malik, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+Confocal+Microscopy:+A+Novel+Non-invasive+Test+to+Diagnose+and+Assess+Progression+of+Human+Diabetic+Neuropathy.">Corneal Confocal Microscopy: A Novel Non-invasive Test to Diagnose and Assess Progression of Human Diabetic Neuropathy.</a> Diabetes Care. 33, 1792-1797 (2010).</li><li>Quattrini, C., Tavakoli, M., Jeziorska, M., Kallinikos, P., Tesfaye, S., Marshall, A., Finnigan, J., Boulton, A. J. M., Efron, N., Malik, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surrogate+Markers+of+Small+Fiber+Damage+in+Human+Diabetic+Neuropathy.">Surrogate Markers of Small Fiber Damage in Human Diabetic Neuropathy.</a> Diabetes. 56, 2148-2154 (2007).</li><li>Mehra, S., Tavakoli, M., Efron, N., Boulton, A. J. M., Augustine, T., Malik, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+Confocal+Microscopy+Detects+Early+Nerve+Regeneration+After+Pancreas+Transplantation+in+Patients+with+Type+1+Diabetes.">Corneal Confocal Microscopy Detects Early Nerve Regeneration After Pancreas Transplantation in Patients with Type 1 Diabetes.</a> Diabetes Care. 30, 2608-2612 (2007).</li><li>Tavakoli, M., Marshall, A., Pitceathly, R., Fadavi, H., Gow, D., Roberts, M. E., Efron, N., Boulton, A. J. M., Malik, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+confocal+microscopy:+A+novel+means+to+detect+nerve+fibre+damage+in+patients+with+impaired+glucose+tolerance+and+idiopathic+small+fibre+neuropathy.">Corneal confocal microscopy: A novel means to detect nerve fibre damage in patients with impaired glucose tolerance and idiopathic small fibre neuropathy.</a> Experimental Neurology. 223, 245-250 (2010).</li><li>Tavakoli, M., Marshall, A., Thompson, L., Kenny, M., Waldek, S., Efron, N., Malik, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+confocal+microscopy:+A+novel+technique+to+detect+small-fibre+neuropathy+in+patients+with+Fabry+disease.">Corneal confocal microscopy: A novel technique to detect small-fibre neuropathy in patients with Fabry disease.</a> Muscle &#38; Nerve. 40, 976-984 (2009).</li></ol>

角膜共聚焦显微镜（CCM）是一种无创的临床技术，可用于检测早期神经损伤小纤维病变和周围神经病变，包括糖尿病，Fabry病和特发性小纤维神经病（ISFN）量化。眼前的这个技术的临床影响，在我们的能力诊断，后续的进展和评估糖尿病的神经病变和其他周围神经病变患者的治疗反应和患者的治疗效果将有一个直接的影响方面的一个巨大的飞跃。

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<td>HRT III CCM Machine - The Rostock Cornea Module</td>
<td>&nbsp;</td>
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<td> Heidelberg Engineering</td>
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<td>CCM image analysis software v0.6 </td>
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<td>University of Manchester</td>
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corneal confocal microscopy: a novel non-invasive technique to quantify small fibre pathology in peripheral neuropathies

周围神经病变的准确定量是很重要的定义处于危险的患者，预期恶化，并评估新的治疗方法。常规方法评估神经功能缺损和电生理和定量感觉测试的量化功能的改变，检测神经病变。然而，最早的损害，出现细小纤维，但这些测试主要是评估大纤维功能障碍，表现出的再生和修复能力有限。只允许直接检查的​​无髓神经纤维的损伤和修复技术，电子显微镜和皮肤打孔活检腓肠神经活检。然而，无论是侵入性的程序和需要长时间的实验程序和相当的专业知识。角膜共焦显微镜是一种非侵入性的临床技术，它提供了角膜神经纤维在体内成像。我们已经证明了早期神经损伤，先于皮肤活检表皮内的神经纤维与神经性的严重程度和修复以下胰腺移植对糖尿病患者分层损失。我们也证明了在特发性小纤维神经病变和法布里病的神经损害。

Watch this Scientific Journal Video about Corneal Confocal Microscopy: A Novel Non-invasive Technique to Quantify Small Fibre Pathology in Peripheral Neuropathies at JoVE.com

Corneal Confocal Microscopy: A Novel Non-invasive Technique to Quantify Small Fibre Pathology in Peripheral Neuropathies

角膜共焦显微镜是一种非侵入性的临床技术，可用于量化C纤维损伤与神经性的严重程度增加患者的诊断和分层。

角膜共聚焦显微镜：一种新型的非侵入性技术来量化小纤维病变周围神经病变

The accurate quantification of peripheral neuropathy is important to define at risk patients, anticipate deterioration, and assess new therapies. ...

corneal-confocal-microscopy-novel-non-invasive-technique-to-quantify

Research

JoVE Journal

Medicine

26.3K 次观看.  University of Manchester. 角膜共焦显微镜是一种非侵入性的临床技术，可用于量化C纤维损伤与神经性的严重程度增加患者的诊断和分层。

视频: Corneal Confocal Microscopy: A Novel Non-invasive Technique to Quantify Small Fibre Pathology in Peripheral Neuropathies

A Novel Capsulorhexis Technique Using Shearing Forces with Cystotome

Purpose:
To demonstrate a capsulorhexis technique using predominantly shearing forces with a cystotome on a virtual reality simulator and on a human eye.
Method:
Our technique involves creating the initial anterior capsular tear with a cystotome to raise a flap. The flap left unfolded on the lens surface. The cystotome tip is tilted horizontally and is engaged on the flap near the leading edge of the tear. The cystotome is moved in a circular fashion to direct the vector forces. The loose flap is constantly swept towards the centre so that it does not obscure the view on the tearing edge.
Results:
Our technique has the advantage of reducing corneal wound distortion and subsequent anterior chamber collapse. The capsulorhexis flap is moved away from the tear leading edge allowing better visualisation of the direction of tear. This technique offers superior control of the capsulorhexis by allowing the surgeon to change the direction of the tear to achieve the desired capsulorhexis size.
Conclusions:
The EYESI Surgical Simulator is a realistic training platform for surgeons to practice complex capsulorhexis techniques. The shearing forces technique is a suitable alternative and in some cases a far better technique in achieving the desired capsulorhexis.

This video demonstrates a novel capsulorhexis technique in modern cataract surgery. Capsulorhexis is the most difficult and important part of phacoemulsification surgery.

一种新型撕囊技术，使用剪切力与Cystotome

Purpose:
To demonstrate a capsulorhexis technique using predominantly shearing forces with a cystotome on a virtual reality simulator and on a human eye....

科研

医学

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

Muscle strains are one of the most common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and physical exam alone, however the clinical presentation can vary greatly depending on the extent of injury, the patient's pain tolerance, etc. In patients with muscle injury or muscle disease, assessment of muscle damage is typically limited to clinical signs, such as tenderness, strength, range of motion, and more recently, imaging studies. Biological markers, such as serum creatine kinase levels, are typically elevated with muscle injury, but their levels do not always correlate with the loss of force production. This is even true of histological findings from animals, which provide a "direct measure" of damage, but do not account for all the loss of function. Some have argued that the most comprehensive measure of the overall health of the muscle in contractile force. Because muscle injury is a random event that occurs under a variety of biomechanical conditions, it is difficult to study. Here, we describe an in vivo animal model to measure torque and to produce a reliable muscle injury. We also describe our model for measurement of force from an isolated muscle in situ. Furthermore, we describe our small animal MRI procedure.

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

An in vivo animal model of injury is described. The method takes advantage of the subcutaneous position of the fibular nerve. Velocity, timing of muscle activation, and arc of motion are all pre-determined and synchronized using commercial software. Post injury changes are monitored in vivo using MR imaging/spectroscopy.

一个在体内收缩引起的损伤，非侵入性的监测恢复啮齿动物模型

Muscle strains are one of the most  common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and  physical ...

Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants

Perinatal brain injury remains a significant cause of infant mortality and morbidity, but there is not yet an effective bedside tool that can accurately screen for brain injury, monitor injury evolution, or assess response to therapy. The energy used by neurons is derived largely from tissue oxidative metabolism, and neural hyperactivity and cell death are reflected by corresponding changes in cerebral oxygen metabolism (CMRO2). Thus, measures of CMRO2 are reflective of neuronal viability and provide critical diagnostic information, making CMRO2 an ideal target for bedside measurement of brain health.
Brain-imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) yield measures of cerebral glucose and oxygen metabolism, but these techniques require the administration of radionucleotides, so they are used in only the most acute cases.
Continuous-wave near-infrared spectroscopy (CWNIRS) provides non-invasive and non-ionizing radiation measures of hemoglobin oxygen saturation (SO2) as a surrogate for cerebral oxygen consumption. However, SO2 is less than ideal as a surrogate for cerebral oxygen metabolism as it is influenced by both oxygen delivery and consumption. Furthermore, measurements of SO2 are not sensitive enough to detect brain injury hours after the insult 1,2, because oxygen consumption and delivery reach equilibrium after acute transients 3. We investigated the possibility of using more sophisticated NIRS optical methods to quantify cerebral oxygen metabolism at the bedside in healthy and brain-injured newborns. More specifically, we combined the frequency-domain NIRS (FDNIRS) measure of SO2 with the diffuse correlation spectroscopy (DCS) measure of blood flow index (CBFi) to yield an index of CMRO2 (CMRO2i) 4,5.
With the combined FDNIRS/DCS system we are able to quantify cerebral metabolism and hemodynamics. This represents an improvement over CWNIRS for detecting brain health, brain development, and response to therapy in neonates. Moreover, this method adheres to all neonatal intensive care unit (NICU) policies on infection control and institutional policies on laser safety. Future work will seek to integrate the two instruments to reduce acquisition time at the bedside and to implement real-time feedback on data quality to reduce the rate of data rejection.

We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain.

婴幼儿的脑代谢和血流动力学的非侵入式光学测量

Perinatal brain injury remains a significant cause of infant mortality  and morbidity, but there is not yet an effective bedside tool that can  accurately ...

A Methodological Approach to Non-invasive Assessments of Vascular Function and Morphology

The endothelium is the innermost lining of the vasculature and is involved in the maintenance of vascular homeostasis. Damage to the endothelium may predispose the vessel to atherosclerosis and increase the risk for cardiovascular disease. Assessments of peripheral endothelial function are good indicators of early abnormalities in the vascular wall and correlate well with assessments of coronary endothelial function. The present manuscript details the important methodological steps necessary for the assessment of microvascular endothelial function using laser Doppler imaging with iontophoresis, large vessel endothelial function using flow-mediated dilatation, and carotid atherosclerosis using carotid artery ultrasound. A discussion on the methodological considerations for each of the techniques is also presented, and recommendations are made for future research.

The present article describes the methodological considerations for several non-invasive assessments of vascular function and morphology that are commonly used in medical research to assess different stages of atherosclerosis.

方法论方法血管功能和形态的非侵入性评估

The endothelium is the innermost lining of the vasculature and is involved in the maintenance of vascular homeostasis.  Damage to the endothelium may ...

Using a Laminating Technique to Perform Confocal Microscopy of the Human Sclera

The sclera is a dense connective tissue that covers and protects the eye. It mainly consists of dense collagen bundles (types I, III, IV, V, VI, and VII). Due to its autofluorescence, opaqueness, and thickness, it has not been found suitable for confocal microscopy. An alternative approach to the one presented here, which uses formalin-fixed sclera embedded in paraffin for immunohistochemistry, has technical challenges, especially when preheating the tissue for antigen retrieval. Since the sclera is relatively poor in both cells and vessels, the use of larger tissue samples was explored to help prevent overlooking cells and to understand their localization in relation to vessels and other anatomical sites. To allow for the analysis of larger tissue samples under the confocal microscope, a laminating technique was performed to create thin layers from the sclera. Following the analysis of results of CD31 blood vessels and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1) positive cells, for which approval for scientific examination was obtained, the advantages and limitations of this method are discussed.

Human sclera tissue is mainly collagen; therefore, it is not easily usable for immunohistochemistry. To achieve the goal of performing immunohistochemistry for confocal microscopy of scleral tissue, a laminating technique was used.

使用层压技术来执行人类巩膜共聚焦显微镜


The sclera is a dense connective tissue that covers and protects the eye. It mainly consists of dense collagen bundles (types I, III, IV, V, VI, and ...

Minimally Invasive Muscle Embedding (MIME) - A Novel Experimental Technique to Facilitate Donor-Cell-Mediated Myogenesis

Skeletal muscle possesses regenerative capacity due to tissue-resident, muscle-fiber-generating (myogenic) satellite cells (SCs), which can form new muscle fibers under the right conditions. Although SCs can be harvested from muscle tissue and cultured in vitro, the resulting myoblast cells are not very effective in promoting myogenesis when transplanted into host muscle. Surgically exposing the host muscle and grafting segments of donor muscle tissue, or the isolated muscle fibers with their SCs onto host muscle, promotes better myogenesis compared to myoblast transplantation. We have developed a novel technique that we call Minimally Invasive Muscle Embedding (MIME). MIME involves passing a surgical needle through the host muscle, drawing a piece of donor muscle tissue through the needle track, and then leaving the donor tissue embedded in the host muscle so that it may act as a source of SCs for the host muscle. Here we describe in detail the steps involved in performing MIME in an immunodeficient mouse model that expresses a green fluorescent protein (GFP) in all of its cells. Immunodeficiency in the host mouse reduces the risk of immune rejection of the donor tissue, and GFP expression enables easy identification of the host muscle fibers (GFP+) and donor-cell-derived muscle fibers (GFP-). Our pilot data suggest that MIME can be used to implant an extensor digitorum longus (EDL) muscle from a donor mouse into the tibialis anterior (TA) muscle of a host mouse. Our data also suggest that when a myotoxin (barium chloride, BaCl2) is injected into the host muscle after MIME, there is evidence of donor-cell-derived myogenesis in the host muscle, with approximately 5%, 26%, 26% and 43% of the fibers in a single host TA muscle showing no host contribution, minimal host contribution, moderate host contribution, and maximal host contribution, respectively.

Minimally Invasive Muscle Embedding MIME - A Novel Experimental Technique to Facilitate Donor-Cell-Mediated Myogenesis

We describe a novel experimental technique that we call Minimally Invasive Muscle Embedding (MIME), which is based on the evidence that skeletal muscle tissue contains viable myogenic cells that can facilitate donor-cell-mediated myogenesis when implanted into a host muscle.

微创肌肉植入 (MIME)--一种促进供体细胞介导的肌的新实验技术

Skeletal muscle possesses regenerative capacity due to tissue-resident, muscle-fiber-generating (myogenic) satellite cells (SCs), which can form new ...

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking (TXL) for Myopia

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy include photochemical cross-linking and tissue cross-linking (TXL) methods. Such methods for inducing mechanical tissue property changes are being employed to the cornea in corneal thinning (mechanically weakened) disorders such as keratoconus as well as the sclera in progressive myopia, where thinning and weakening of the posterior sclera occurs and likely contributes to axial elongation. The primary target proteins for such tissue strengthening are fibrillar collagens which constitute the great majority of dry weight proteins in the cornea and sclera. Fortuitously, fibrillar collagens are the main source of second harmonic generation signals in the tissue extracellular space. Therefore, modifications of the collagen proteins, such as those induced through cross-linking therapies, could potentially be detected and quantitated through the use of second harmonic generation microscopy (SHGM). Monitoring SHGM signals through the use of a laser scanning microscopy system coupled with an infrared excitation light source is an exciting modern imaging method that is enjoying widespread usage in the biomedical sciences. Thus, the present study was undertaken in order to evaluate the use of SHGM microscopy as a means to measure induced cross-linking effects in ex vivo rabbit sclera, following an injection of a chemical cross-linking agent into the sub-Tenon&#39;s space (sT), an injection approach that is standard practice for causing ocular anesthesia during ophthalmologic clinical procedures. The chemical cross-linking agent, sodium hydroxymethylglycinate (SMG), is from a class of cosmetic preservatives known as formaldehyde releasing agents (FARs). Scleral changes following reaction with SMG resulted in increases in SHG signals and correlated with shifts in thermal denaturation temperature, a standard method for evaluating induced tissue cross-linking effects.

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking TXL for Myopia

This protocol describes techniques for evaluating chemical cross-linking of the rabbit sclera using&#160;second harmonic generation imaging and differential scanning calorimetry.

兔巩膜二次谐波信号作为治疗组织交叉连接 (txl-) 评价近视的工具

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy ...

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 Novel Non-invasive Method for the Detection of Elevated Intra-compartmental Pressures of the Leg

Acute Compartment Syndrome is a devastating consequence of musculoskeletal trauma. Currently the diagnosis is based on clinical signs and symptoms, and while adjuncts such as invasive intra-compartmental pressure measurements are often used to corroborate the physical exam findings, there remains no reliable objective test to aid in the decision to perform a decompressive fasciotomy. In a cadaver model of compartment syndrome, an ultrasound (US) based method has been shown to be a reliable measurement of increased intra-compartmental pressure. An absolute pressure of &#62;100 mbar or a difference of 50 mbar in the CFFP between the legs can be considered pathologic. Using an ultrasound transducer, coupled with a pressure sensor, the pressure needed to flatten the superficial fascia of the anterior compartment of lower legs (Compartment Fascia Flattening Pressure [CFFP]) can be measured. The CFFP of the injured leg is compared to the CFFP of the uninjured leg. This US measured index can then serve as an adjunct to the physical exam in evaluating injured lower extremities and assessing the need for decompressive fasciotomy. The advantages of this protocol include: being a non-invasive method and an easily reproducible technique.

An ultrasound probe coupled with a pressure sensor is used to assess the intra-compartmental pressure of the leg by directly measuring the compartment fascia flattening pressure (CFFP). This non-invasive protocol will provide reliable assessment of the pressure inside the anterior muscular compartment of the lower leg.

一种检测腿部高室内压力的无创检测方法

Acute Compartment Syndrome is a devastating consequence of musculoskeletal trauma. Currently the diagnosis is based on clinical signs and symptoms, and ...

Characterization of a Novel Human Organotypic Retinal Culture Technique

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal pigment epithelium-choroid (RPE-choroid) and sclera, the integrity of the fragile retina can easily be compromised. The aim of this study was to develop a novel HORC model that contains the retina, RPE-choroid and sclera to maintain retinal integrity when culturing retinal explants.
After cutting circumferentially along the limbus to remove iris and lens, four deep incisions were made to flatten the eyecup. In contrast to previous HORC protocols, a trephine was used to cut through not only the retina but also the RPE-choroid and sclera. The resultant triple-layered explants were cultured for 72 h. Hematoxylin and Eosin staining (H&#38;E) was used to assess anatomical structures and retinal explants were further characterized by immunohistochemistry (IHC) for apoptosis, M&#252;ller cell integrity and retinal inflammation. To confirm the possibility of disease induction, explants were exposed to high glucose (HG) and pro-inflammatory cytokines (Cyt), to mimic diabetic retinopathy (DR). The Luminex magnetic bead assay was used to measure DR-related cytokines released into the culture medium.
H&#38;E staining revealed distinct retinal lamellae and compact nuclei in retinal explants with the underlying RPE-choroid and sclera, while retinas without the underlying structures exhibited reduced thickness and severe nuclei loss. IHC results indicated absence of apoptosis and retinal inflammation as well as preserved M&#252;ller cell integrity. The Luminex&#160;assays showed significantly increased secretion of DR-associated pro-inflammatory cytokines in retinal explants exposed to HG + Cyt relative to baseline levels at 24 h.
We successfully developed and characterized a novel HORC protocol in which retinal integrity was preserved without apoptosis or retinal inflammation. Moreover, the induced secretion of DR-associated pro-inflammatory biomarkers when exposing retinal explants to HG + Cyt suggests that this model could be used for clinically translatable retinal disease studies.

This study aims to develop a novel human organotypic retinal culture (HORC) model that prevents compromising retinal integrity during explant handling. This is achieved by culturing the retina with the overlying vitreous and the underlying retinal pigment&#160;epithelium-choroid (RPE-choroid) and sclera.

新型人类组织特征视网膜培养技术的特征

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal ...