Name: a new technique for quantitative analysis of hair loss in mice using grayscale analysis
Uploaded: 2015-03-09T13:00:00.000+00:00
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Description: Watch this Scientific Journal Video about A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis at JoVE.com

We would like to thank the Children&#8217;s Hospital at Montefiore, Department of Pediatrics, for providing support for these studies. We would like to thank the National Alopecia Areata Foundation for providing financial support for conducting studies with C3H/HeJ engrafted mice.

伦理学声明:所有涉及动物的研究必须由该机构IACUC批准（为此如下数据，协议批准了蒙蒂菲奥里IACUC，协议＃11-6-240和＃13-7-100）。动物提供了浅麻醉指示摄影过程中保持他们仍然是唯一的目的，有需要这种协议没有痛苦的过程。 1.采集照片的<ol><li>看设定对焦和现场使用的纸张与印刷文字凝胶成像仪。验证跨区域拍照光源均匀性。为了帮助确保均匀性，使用凝胶成像仪具有一个内置的光源，用于反射摄影。不要使用透照光源，因为这将创建一个不适合进一步的灰度分析动物的剪影。 注:这将将鼠标稍微失焦，其中跨过感兴趣区域（ROI）的区域提供了光学平均，并会有助于减少量子错误的利息非常小（ 即 &lt;10像素）的区域。感兴趣较大区域将不会影响此次要聚焦调整，也不会受到影响，在动物的大小差异。 </li><li>麻醉用氯胺酮（100毫克/毫升）/赛拉嗪（20毫克/毫升）的动物（2:1），因为这提供了快速麻醉效果和快速恢复和最适合拍摄多个动物。 注:麻醉证实当动物仍足以允许摄影。由于动物通常在10-15分钟这个浅麻醉恢复，兽医已经不推荐使用眼药膏的。 </li><li>放置麻醉动物在垂直取向凝胶成像仪（如接近平行越好）。 <ol><li>对于背照片，将动物的四肢伸展俯卧位。 </li><li>对于腹面照片，将动物在仰卧位，照顾这些动物不是横向旋转。 </li&gt; </ol></li><li>将在拍下区域灰度标准。 </li><li>关闭检修门。重要提示:环境光可以引入变化曝光。 </li><li>设置光圈的曝光哪个地方采集的线性范围内的感兴趣的区域（读出的F-停止）。 注意:大多数的系统将显示在图像饱和。 </li><li>拍摄照片。 </li><li>变化的F-停止向其中通过增加或由1减小光圈，这样，无论是标准的和感兴趣的区域保持在采集（参考的线性范围放置的线性范围内的感兴趣的区域的另一曝光设置F-停止）。 </li><li>拍摄照片。 </li><li>一旦拍摄完成后，将动物在加温台和监控，直到他们能保持胸骨斜卧。回到动物的笼子。返回组动物一次所有的动物完全恢复的动物饲养。 </li></ol> 2.定量光吸收的&lt;/ P&gt; <ol><li>使用提供的对动物的图像感兴趣区域标记软件用于凝胶成像仪。 <ol><li>对于整个动物背视图中，使用矩形或椭圆形的图像从上肢到下肢延伸，横向延伸尽可能使得盒的任何部分延伸超过该动物的背部， 如图1A所示 。 注意:一个也标记感兴趣的使用徒手画图工具的区域。 </li><li>对于整个动物腹面观中，使用2矩形:1覆盖骨盆区域，一个覆盖胸部区域， 如图1B所示 。 </li><li>对于感兴趣的， 即，给药位置，标记为适当的较小的区域。 </li></ol></li><li>对灰度吸收基准利率标记区域（S）。 </li><li>从每个感兴趣的标记区域的记录吸收。 </li></ol> 3.分析吸收水平obtained表示感兴趣的区域，可能需要进行归一化的背景标准照片比较。暴露于吸收的关系是双对数（ 即，该关系是log（曝光之间线性）和log（吸收））。使用这种关系，感兴趣的区域的吸收可以是归一化到一个标准的背景值，这将允许吸收直接不同的照片，其中包括那些在不同时间点（一个研究协议范围内，即，连续测量）拍摄之间进行比较。用于执行这些校正的过程详述在任选的步骤3.1和3.2。 <ol><li>日志（曝光）使用来自灰度吸收标准得到的数值（可选）绘制的曲线与日志（吸收）。 </li><li> （可选）调整的变化每张照片的标准如下: <ol><li>选择F-停止阅读和F-STOP（如步骤1.6确定），以供参考（如在下定决心TEP 1.8）。 </li><li>计算标准平均吸收所有的照片在阅读F-停止。这平均值为参考标准值（RSV）。 </li><li>计算在每个照片中的阅读和参考光圈设置之间的吸收差值为标准（Δ-S）和为每个定义的ROI（Δ-ROI-1，Δ-ROI-2 ... ..delta-ROI-X）的</li><li>计算校正吸收每个ROI如下:在阅读F-停止更正吸收率（ROI-X）=吸收率（ROI-X）+（RSV - 吸收的标准，在阅读F-停止）*（Δ-ROI-X / Δ-S） </li></ol></li><li>编译的实验数据，并用标准的统计方法对连续变量进行数据分析（ 即，方差分析，T检验）。 </li></ol>

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Authors have nothing to disclose.

在这份报告中的详细描述，提供了一种新技术，用于量化脱发在啮齿类动物。此技术利用的凝胶成像仪进行图像采集和分析，设备这是在大多数实验室容易获得。测量已被证明是健壮到微小变化在技术（ 图4 - 5），并且是公相关，在治疗模型视觉毛发损失（ 图6）的程度。这些技术已被成功地用于在实验性疗法的评价为脱发17。 定量工具，以评估头发生长在啮齿类动物可以在治疗推动研究脱发非常有价值的。许多小鼠模型脱发有固有变异性，这使得它难以立足的治疗性干预的主观评估的结果。同样地，一定量的评估，在形式提供的结果连续的测量可以总结为组的平均值和标准偏差/标准误差，并可以使用标准统计技术（方差分析，T检验）进行测试。这个定量评估可以利用在一系列时间点测量的响应，并且可以在产生用于试验化合物的剂量 - 响应曲线可以使用。定量分析从而允许疗法的疗效证据为基础的评估正在开发。 这种技术需要在许多小鼠品系的被毛和皮肤之间存在颜色差异的优势。对于小鼠，黑头发，头发的密度越大，则较大量的光吸收。该光吸收可使用凝胶成像装置，其被设计来评估光吸收在一个确定的感兴趣区域进行量化蛋白质的量在蛋白质印迹分析来定量。类似的免疫印迹分析，所产生的吸收测量可肛门yzed用标准的统计方法来比较小鼠组之间毛发密度（ 即，治疗与控制）。 一般上相比提供了图像上的标准被分析的单个图像进行Western印迹分析。但是，在该应用中，有必要使不同的图像，其可以采取不同的时间之间的比较。具有非常一致的摄影技术，照片之间的变化可被最小化。然而，对于实验性应用，这是有益的，如果在照片曝光之间的微小变化可以得到补偿。因此，重要的是一个灰度标准被包括在照片（ 即，蒂芬的灰度图表15）。然而，吸收和曝光间的关系的变化取决于目标（ 图2A）的光密度。因此，如果灰度标准具有从TA不同的光学密度rget，适当的修正可以是不同的。对数的校正是有帮助的，但不提供一个完美的解决这个问题的（ 图2B）。通过拍照2不同的F停止时，可以计算线性近似的灰度标准，并为在照片上的每个目标，并且因此提供了校正的各个目标的适当缩放。 图3示出了具有不同的光学如何目标密度将有不同的校正因子，这可以从在两个不同的F-站中每一个目标的吸收差异进行插值。因此，如果一个人希望正常化的标准为150的值，在光密度蒂芬19一个目标将是148，一个在蒂芬18将是140，一个在蒂芬17将是119，依此类推。而非图形解决这些校正，可以使用的斜坡的比率的灰度标准之间的目标之间的2个F停止计算FO校正R各自目标，如在所提供的例子说明（ 表1）。 像所有的技术，毛发的生长，通过灰度分析，定量分析有几个限制。首先，有在应用这种技术的显著计算复杂性。这导致一个事实，即在分析软件对凝胶成像仪被用于分析每个图作为一个单独的实验中进行了优化。因此，存在一个要求手动校正在图像之间的暴露的任何变化。线性近似这样的更正也有助于简化这一过程。一个例子是在表1中提供:2台的照片，在拍摄的模拟数据F-停止2.0和3.0，用通用的标准。每一组的照片具有2的ROI这就需要集之间进行比较。照相条件的微小变化造成的第一组照片，以具有比所述第二组稍大曝光，evideNT通过在共同标准的吸收细微的差别。 该示例示出了拍摄条件的微小差异如何可以创建在给定的ROI中的吸收明显的差异，但这些都可以使用参考标准，在此情况下，表明吸收在感兴趣区不的2套图像之间差异进行校正。 要注意在光吸收这种变化并不总是直接相关的毛发色素脱失或其他颜色的变化量可能被误认为是脱发或头发生长是重要的。不幸的是，这种技术的准确性不能被评估，因为目前还没有金标准与之比较。然而，在有明显的脱发，该技术确实产生预期的结果（ 图6）。最后，目前还没有数据，以评估是否使用这种技术的结果相关联，在潜在的治疗剂的临床试验结果。 &lt;P类="j​​ove_content"&gt;灰度分析定量脱发各种形式脱发是一个机会，完善和规范方法表征脱发的模型和测试应对潜在的治疗。这种技术的应用程序应该提高鉴定更有效的治疗脱发和发展。

脱发（脱发）可以是心理和情感上令人痛心的事件有多个原因。男性型脱发是脱发的最常见的原因，35岁1影响大约三分之二男性。脱发的一个类似的模式可以在女性多囊卵巢综合征被观察到。在这两种病症中，脱发是雄激素介导的。脱发也可发生自身免疫病，斑秃，从而影响人口2的1.7％。脱发可发生的一些医学治疗，如化疗3的副作用。化疗患者的高比例（65-85％）经历某种程度的脱发4,5。脱发的心理影响已经得到很好的研究，在化疗的设置。化疗引起的脱发会导致焦虑，抑郁，消极的身体形象，降低自尊和幸福6,7减少的感觉。高PE女性癌症患者rcentage（47-58％）认为脱发是化疗最痛苦的方面，以及高达8％的跌幅治疗恐惧脱发4,6。也有在雄激素性脱发的证据来支持治疗，以减少脱发8,9-心理甚至医学后果。同样，斑秃已报道有严重的心理后果2和脱发的斑片状性质可以导致更不愉快的美容效果比脱发的最其他原因。 虽然药物轻度抗雄激素效应（ 即，安体舒通）曾使用了有限的成功为治疗脱发，首先有效的药物治疗脱发的米诺地尔是10。这种降压具有引起毛发生长的观察副作用，现在用作局部治疗许多形式的脱发。然而，反应往往是不完整的，有些科目只显示缓慢荷兰国际集团的脱发，而不是实际的再生10。非那雄胺是一种竞争性拮抗剂II型5α还原酶其阻断睾酮转化为二氢睾酮，致使在局部全身雄激素阻断牺牲改善雄激素性脱发。应答率与长期（10年以上）的治疗是在50％左右11。总体而言，尽管相当多的研究在这方面，目前还没有适当的治疗脱发。 几十年来，科学家和临床医生已经检查测量的头皮头发生长的临床试验的方法。用的药物治疗脱发的发展，出现了用于测量头发的生长，并且具体地，对治疗的反应的可靠的，经济的和微创手段更加需要。图像分析技术对头发密度的患者的脱发症的准确定量，得到在过去的一致和有效的结果使用各种techniqu的ES，包括分析数字化图像12，每根毛发的图像分析和皮损13，和显微镜扫描量化毛传中定义的头皮区域14。 不幸的是，虽然上述的方法提供了效力的改进的评价对头发生长促进干预在临床试验中，这些方法还没有被应用到啮齿动物的研究中的临床前调查。我们的目标是开发一个一致的和可靠的方法来量化脱发小鼠，这将允许研究者能够更准确地评估和比较新的治疗方法对各种形式的脱发。我们已经开发使用的设备在大多数实验室，这将使头发密度的小鼠棕色或黑色的头发快速，可靠的量化一应俱全的方法。这种方法已在化疗诱导的脱发，斑秃，并从waxin脱发的小鼠模型被测试克。详细的协议提出了进行这些测量，包括C57BL / 6的验证数据和C3H /小鼠品系HeJ小鼠。作为这种技术依赖于从在毛干色素检测光的吸收，它不能被用于检测的毛发生长小白鼠或白化小鼠。

<table><tbody><tr><td>KODAK Gel Logic 100 Imaging System</td><td>Eastman Kodak Company, Rochester, NY, USA</td><td></td><td>Gel imager for obtaining and analyzing photographs, must have built in light source for reflective photography.</td></tr><tr><td>The Kodak/Tiffen Q-13 Gray Scale</td><td><a href="http://www.imatest.com/docs/q13/" target="_blank"><img src="/img/new_window.png" /> Imatest</a></td><td></td><td>Greyscale standard</td></tr><tr><td>C57BL/6J Mice</td><td>Jackson Laboratories, Bar Harbor, Maine</td><td></td><td>Mice for representative study</td></tr><tr><td>C3H/HeJ engrafted mouse</td><td>Jackson Laboratories, Bar Harbor, Maine</td><td></td><td>Mice for representative study</td></tr></tbody></table>

a new technique for quantitative analysis of hair loss in mice using grayscale analysis

脱发是它可以发生在许多不同的条件，包括男性型脱发，多囊性卵巢综合征和斑秃脱发的常见形式。脱发，也可能发生由于化疗的癌症患者的副作用。在这项研究中，我们的目标是开发一个一致的和可靠的方法来量化脱发小鼠，这将允许研究者准确地评估和比较新的治疗方法，这些不同形式的脱发。该方法利用一种标准的凝胶成像仪获取和处理的小鼠的图像，测量光吸收，这发生在粗糙成比例的黑色（或灰色）的头发上的鼠标的量。已量化以这种方式的数据然后可以使用标准的统计技术（ 即，方差分析，T检验）分析。这种方法是在化疗诱导的脱发，斑秃和脱发的小鼠模型从打蜡测试。在这份报告中，详细的协议是PRESented用于执行这些测量，其中包括来自C57BL / 6的验证数据和C3H /小鼠HeJ小鼠品系。这种新技术提供了许多优点，包括应用的相对简单性，依赖于设备这是在大多数研究实验室容易获得的，并且施加一个客观，定量评估这比主观评价更加健壮。改善头发生长的量化小鼠会改善脱发模型研究和促进有前途的新治疗方法的评价在临床前研究。

用的C3H / HeJ小鼠植入小鼠，脱发的小鼠模型斑秃16这种技术进行了验证。这些动物发展出进步逐渐随着时间的推移全球性脱发。然而，脱发发生在补丁，并且可以变化从一个鼠标到下，引入相当大的变化，并且会阻碍的定性评估。该模型提供了一个机会，在对小鼠的有效性进行评估的不同位置，以测试光密度的测量值之间的相关性。 如果使用的是第一个问题是措施不同的风险，不同的ROI划分，而投资回报率的非重叠区域上相同的鼠标呈正相关。有一个显著相关（p &lt;0.0001）看到比较投资回报由长方形与椭圆的标准鼠标（ 图4A）的背表面界定。同样的高相关性看出，在不同的网站上的动物和迪菲租暴露水平（未示出）。这表明在这些测量中的高度的精度。还有，在吸收之间的显著相关光圈2.0 与 1.2使用矩形的ROI后面（P &lt;0.0001， 图4B）的背水面。比较相同的鼠标（P &lt;0.0001， 图4C）的背部和腹部表面之间的吸收测量时，显著的相关性观察均匀。这些数据提供的证据表明从不同的网站上的相同的动物采取吸收测量是相关的，因为预期在C3H / HeJ小鼠植入小鼠模型，并且这些相关性保持在大范围的脱发线性。这有助于建立灰度测量的有效性和还建立这种技术的变型中的图像采集和分析技术的鲁棒性。 第二个问题是，如果有从取连续测量之间的一致性相同的动物在不同时间点。为了评估这一点，图像C3H获得/ HeJ小鼠移植的小鼠连续3周每周一次。在C3H / HeJ小鼠植入老鼠会预计在这段时间逐渐显示脱发。上腹面（胸部区域，盒形ROI）灰度分析表明这一预期脱发，从一个时间点一致的下降趋势下（ 图5）。值得注意的是，只有80％的C3H / HeJ小鼠植入小鼠显示脱发，和那些具有最高的灰度值不显示这种下降，而在整个实验期间表现出一致的结果。措施的这种一致性在动物中没有脱发也验证了在C57BL / 6小鼠19。上腹面的大腿区域和在背侧表面上进行相同的分析表明跨越时间类似的结果（未示出）。 从应用这种技术的这些令人鼓舞的结果C3H / HeJ小鼠植入动物，进一步调查，在不同的动物模型中，化疗引起的脱发之一进行。 C57BL / 6小鼠接受其中环磷酰胺3周的课程将可变制定全球的头发变薄和色素脱失。本文所描述的灰度分析技术被采用来量化这些变化，并评估响应治疗干预17。在这个模型中，灰度分析证明在提供的被毛化疗引起的变化的定量评估是一种强大的工具，并通过统计分析允许验证的治疗效果。在化疗脱发的另一种模式，还有人利用这种灰度分析了特殊的挑战。在此模型中，将动物蜡的背表面以同步毛囊，然后用单剂量的环磷酰胺9天以后，当毛囊进入生长期第六阶段，并且最大程度地敏感，以化疗药物治疗18。在打蜡区域是用于研究感兴趣的区域，但打蜡的面积可能是大小可变的。在打蜡区域内限制投资回报率使得双方的损失和头发再生环磷酰胺管理19后的量化。此外，该技术允许在一个实验性疗法的治疗反应的剂量依赖性的差异准确评估。本申请示出了限定在该技术在ROI具有灵活性的优点。 为了进一步说明该技术的应用，提供了一个持续的研究中的C57BL / 6利用每周环磷酰胺给药的协议的一个临时数据分析表明上面。 8周老小鼠用赋形剂或50毫克/公斤/周环磷酰胺治疗3周。化疗治疗的小鼠显示出视觉上明显的脱发后2个月，而载体处理的动物具有正常的头发涂层（ 图6A）。灰度分析是每形成为上面所指出的，表现出化疗的小鼠和媒介物对照（ 图6B）之间显著（P &lt;0.05）差异。 <img alt="图1" src="/files/ftp_upload/52185/52185fig1highres.jpg" /> 图1:对背侧和C3H的腹面头发变化总视图/ HeJ小鼠植入小鼠，斑秃的小鼠模型注意的动物发展出的进行随时间逐渐全局脱发（A）的背视图，具有代表性的划界。感兴趣（投资回报），用于灰度分析，椭圆形或所指示的矩形。（B）的腹侧与用于在胸灰度分析兴趣不同的区域，对应于ungroomed区域，或大腿，对应于一个整齐的区域视图的区域。 <img alt="图2" src="/files/ftp_upload/52185/52185fig2highres.jpg" wIDþ="450"/&gt; 图2:像素水平与蒂芬的数量， 对灰度分析与蒂芬的数量标准像素 A 级标准蒂芬的图表被拍到在凝胶成像仪在指定的F-站（A）图表显示预期的对数关系（B。 ）对数变换，在此范围内继续遵循一个非线性关系。 <img alt="图3" src="/files/ftp_upload/52185/52185fig3highres.jpg" width="450" /> 图3:在灰阶分析与 F-停止像素A级标准蒂芬的图表被拍到在凝胶成像仪在指定的F-停止。策划是看到了几个不同的蒂芬的标准与 F-停在像素水平上的变化。注意，该线不平行，这表明校正对准照像之间的标准PHS需要单独计算每个关心区域。 <img alt="图4" src="/files/ftp_upload/52185/52185fig4highres.jpg" width="450" /> 图4:以灰度分析不同的摄影技巧和感兴趣区域之间的相关性显示的是来自同一个C3H采取所指示的测量技术之间的相关性/ HeJ小鼠植入小鼠在相同的时间点的矩形的（A）的相关性对比的椭圆形区域。采用感兴趣矩形区域利息背视图。的F（B）的相关性停止2.0 与 1.2。从背，腹观点，感兴趣的矩形区域的灰度值的（C）的相关性。所有相关统计学显著如通过线性回归分析（P &lt;0.0001）测定。 <img alt="图5" src="/文件/ ftp_upload / 52185 / 52185fig5highres.jpg"WIDTH ="550"/&gt; 图5:使用C3H灰度分析变化的吸收时间当然/ HeJ小鼠移植的小鼠的颜色显示，从单个小鼠连续测量。注意，对于每个小鼠，从一个时间点到下一个的一致性。 <img alt="图6" src="/files/ftp_upload/52185/52185fig6highres.jpg" /> 图6:在化疗诱导的脱发 6周龄C57BL6 / J小鼠灰度分析，用载体处理的（N = 6），或3周剂量的环磷酰胺（50毫克/千克）（N = 6）（A）的 3只小鼠来自每个治疗组的代表组，化疗开始后拍摄2个月。（B）中收集的数据，从灰度级分析从各组中，显示出与环磷酰胺给药（P &lt;0.05）降低的吸收。注意可见头发之间的相关性和损失面板中的吸收统计学显著减少面板B. <table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always"><tbody><tr><td colspan="4">吸收值如下: </td></tr><tr><td>照片</td><td> F-STOP </td><td>投资回报率</td><td>吸收</td></tr><tr><td> 1 </td><td> 2 </td><td>标准</td><td> 90 </td></tr><tr><td> 1 </td><td> 2 </td><td> ROI-1 </td><td> 100 </td></tr><tr><td> 1 </td><td> 2 </td><td> ROI-2 </td><td> 200 </td></tr><tr><td> 1 </td><td> 3 </td><td>标准</td><td> 150 </td></tr><tr><td> 1 </td><td> 3 </td><td> ROI-1 </td><td> 160 </td></tr><tr><td> 1 </td><td> 3 </td><td> ROI-2 </td><td> 230 </td></tr><tr><td> 2 </td><td> 2 </td><td>标准</td><td> 110 </td></tr><tr><td> 2 </td><td> 2 </td><td> ROI-1 </td><td> 120 </td></tr><tr><td> 2 </td><td> 2 </td><td> ROI-2 </td><td> 210 </td></tr><tr><td> 2 </td><td> 3 </td><td>标准</td><td> 160 </td></tr><tr><td> 2 </td><td> 3 </td><td> ROI-1 </td><td> 170 </td></tr><tr><td> 2 </td><td> 3 </td><td> ROI-2 </td><td> 235 </td></tr><tr><td colspan="4">雷丁F-STOP评为2.0，参考光圈选择为3.0。 </td></tr><tr><td colspan="4">参考标准值（RSV）=从照片1和照片2的平均标准吸收光圈2.0，RSV =平均（90，110）= 100 </td></tr><tr><td colspan="4"></td></tr><tr><td colspan="4">对于照片1: </td></tr><tr><td colspan="4"> Δ-S = 150 - 90 = 60 </td></tr><tr><td colspan="4"> Δ-ROI-1 = 160 - 100 = 60 </td></tr><tr><td colspan="4"> Δ-ROI-2 = 230 - 200 = 30 </td></tr><tr><td colspan="4">更正吸收ROI-1 = 100 +（100 - 90）*（60分之60）= 110 </td></tr><tr><td cols泛="4"&gt;修正吸收ROI-2 = 200 +（100 - 90）*（六十○分之三十〇）= 205 </td></tr><tr><td colspan="4"></td></tr><tr><td colspan="4">对于照片2: </td></tr><tr><td colspan="4"> Δ-S = 160 - 110 = 50 </td></tr><tr><td colspan="4"> Δ-ROI-1 = 170 - 120 = 50 </td></tr><tr><td colspan="4"> Δ-ROI-2 = 235 - 210 = 25 </td></tr><tr><td colspan="4">更正吸收ROI-1 = 120 +（100 - 110）*（50/50）= 110 </td></tr><tr><td colspan="4">更正吸收ROI-2 = 210 +（100 - 110）*（25/50）= 205 </td></tr></tbody></table> 表1:样品的计算，用于校正在照片之间的曝光差异的表格示出了如何以补偿的照片之间的标准（可选步骤3.2）的吸收细微的差别的一个例子。

Watch this Scientific Journal Video about A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis at JoVE.com

A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis

Alopecia is a common form of hair loss which can occur in many different conditions, including as a side-effect of chemotherapy. We have developed a method to quantify hair loss in mice, utilizing a standard gel imager to perform a grayscale analysis, to facilitate study of promising new alopecia therapies.

新技术脱发的定量分析小鼠使用灰度分析

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and ...

a-new-technique-for-quantitative-analysis-hair-loss-mice-using

Universidad San Sebastian

Research

JoVE Journal

Biology

8.9K 次观看.  Children's Hospital at Montefiore. Alopecia is a common form of hair loss which can occur in many different conditions, including as a side-effect of chemotherapy. We have developed a method to quantify hair loss in mice, utilizing a standard gel imager to perform a grayscale analysis, to facilitate study of promising new alopecia therapies.

视频: A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis

Flat Mount Imaging of Mouse Skin and Its Application to the Analysis of Hair Follicle Patterning and Sensory Axon Morphology

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel cell clusters), sebaceous glands, nerves and nerve endings, and capillaries. The spatial arrangement of these structures is tightly controlled on a microscopic scale - as seen, for example, in the orderly arrangement of cell types within a single hair follicle - and on a macroscopic scale - as seen by the nearly identical orientations of thousands of hair follicles within a local region of skin. Visualizing these structures without physically sectioning the skin is possible because of the 2-dimensional geometry of this organ. In this protocol, we show that mouse skin can be dissected, fixed, permeabilized, stained, and clarified as an intact two dimensional object, a flat mount. The protocol allows for easy visualization of skin structures in their entirety through the full thickness of large areas of skin by optical sectioning and reconstruction. Images of these structures can also be integrated with information about position and orientation relative to the body axes.

Mammalian skin contains a diverse array of structures - such as hair follicles and nerve endings - that exhibit distinctive patterns of spatial organization. Analyzing skin as a flat mount takes advantage of the 2-dimensional geometry of this tissue to produce full-thickness high-resolution images of skin structures.

小鼠皮肤及其毛囊图案和感觉轴突形态分析中的应用平山成像

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel ...

科研

神经科学

Combined Recording of Mechanically Stimulated Afferent Output and Nerve Terminal Labelling in Mouse Hair Follicle Lanceolate Endings

A novel dissection and recording technique is described for monitoring afferent firing evoked by mechanical displacement of hairs in the mouse pinna. The technique is very cost-effective and easily undertaken with materials commonly found in most electrophysiology laboratories, or easily purchased. The dissection is simple and fast, with the mechanical displacement provided by a generic electroceramic wafer controlled by proprietary software. The same software also records and analyses the electroneurogram output. The recording of the evoked nerve activity is through a commercial differential amplifier connected to fire-polished standard glass microelectrodes. Helpful tips are given for improving the quality of the preparation, the stimulation and the recording conditions to optimize recording quality. The system is suitable for assaying the electrophysiological and optical properties of lanceolate terminals of palisade endings of hair follicles, as well as the outcomes from their pharmacological and/or genetic manipulation. An example of combining electrical recording with mechanical stimulation and labeling with a styryl pyridinium vital dye is given.

A simple and novel technique for recording afferent discharge due to mechanical stimulation of lanceolate terminals of palisade endings innervating mouse ear skin hair follicles is presented.

机械刺激传入输出和神经末梢标签联合录制在小鼠毛囊披针形结局

A novel dissection and recording technique is described for monitoring afferent firing evoked by mechanical displacement of hairs in the mouse pinna. The ...

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles in the skin of the mouse pinna. The preparation is simple and relatively fast, reliably yielding extensive regions of multiple labeled units of living nerve terminals to study uptake and release of styryl pyridinium dyes extensively used in studies of vesicle recycling. Subdividing the preparations before labeling allows test vs. control comparisons in the same ear from a single individual. Helpful tips are given for improving the quality of the preparation, the labeling and the imaging parameters. This new system is suitable for assaying pharmacologically and mechanically-induced uptake and release of these vital dyes in lanceolate terminals in both wild-type and genetically modified animals. Examples of modulatory influences on labeling intensity are given.

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

Here we describe a novel preparation for imaging live lanceolate sensory terminals of palisade endings that innervate mouse ear skin hair follicles during staining and destaining with styryl pyridinium dyes.

在毛囊披针形结局生活神经末端标记的光学监测<em&gt;离体</em&gt;鼠耳皮肤

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles ...

Bioluminescence-Based Tumor Quantification Method for Monitoring Tumor Progression and Treatment Effects in Mouse Lymphoma Models

Although bioluminescence imaging (BLI) shows promise for monitoring tumor burden in animal models of cancer, these analyses remain mostly qualitative. Here we describe a method for bioluminescence imaging to obtain a semi-quantitative analysis of tumor burden and treatment response. This method is based on the calculation of a luminoscore, a value that allows comparisons of two animals from the same or different experiments. Current BLI instruments enable the calculation of this luminoscore, which relies mainly on the acquisition conditions (back and front acquisitions) and the drawing of the region of interest (manual markup around the mouse). Using two previously described mouse lymphoma models based on cell engraftment, we show that the luminoscore method can serve as a noninvasive way to verify successful tumor cell inoculation, monitor tumor burden, and evaluate the effects of in situ cancer treatment (CpG-DNA). Finally, we show that this method suits different experimental designs. We suggest that this method be used for early estimates of treatment response in preclinical small-animal studies.

Bioluminescence imaging is a well-known tool for localizing tumors and metastases, but quantification of these images often requires complex calculations and particular instruments. We describe the easy-to-use luminoscore method, based on precise acquisition conditions, requiring no calculations, and enabling tumor burden and treatment response to be monitored in mouse models.

对小鼠淋巴瘤模型监测肿瘤进展和治疗效果生物发光基于肿瘤定量方法

Although bioluminescence imaging (BLI) shows promise for monitoring tumor burden in animal models of cancer, these analyses remain mostly qualitative. ...

医学

Analysis of Craniomaxillofacial Malformations in Mice Using Three-dimensional Microcomputed Tomography

To model craniofacial malformations caused by vitamin A deficiency (VAD), we expressed a dominant-negative retinoid receptor mutation in osteoblasts to specifically inhibit RAR transcriptional activity in mice. This approach allowed us to investigate the effects of VAD on cranial hypomineralization, mandibular deformity, and clavicular hypoplasia in clinical cases. In this study, microcomputed tomography (microCT) scanning of the craniomaxillofacial region of mice represented a valuable tool for studying the growth and development of this animal model. The manual estimation of images is both time-consuming and inaccurate. Hence, here, we present a straightforward, efficient, and accurate approach for segmenting and quantifying the microCT images of each craniomaxillofacial bone. MicroCT software was used to slice the mandible, frontal bone, parietal bone, nasal bone, premaxilla, maxilla, interparietal bone, and occipital bone of mice and measure their corresponding lengths and widths. This segmentation method can be applied to study growth and development in developmental biology, biomedicine, and other related sciences and allows researchers to analyze the effects of genetic mutations on individual craniofacial bones.

Here, we describe in depth a method, which is based on microcomputed tomography, to segment and measure 3D models of craniomaxillofacial bones in mice, for better assessment of craniomaxillofacial bone development in mice than what is possible with current methods.

使用三维显微计算机断层扫描分析小鼠颅颌面畸形

To model craniofacial malformations caused by vitamin A deficiency (VAD), we expressed a dominant-negative retinoid receptor mutation in osteoblasts to ...

发育生物学

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

在毛发再生的上皮 - 间充质信号的快速遗传分析

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

生物学

Digital Planimetry for Assessing Wound Closure Kinetics in a Mouse Model

Chronic wounds, due to their high prevalence, are a serious global health concern. Effective therapeutic strategies can significantly accelerate healing, thereby reducing the risk of complications and alleviating the economic burden on healthcare systems. Although numerous experimental studies have investigated wound healing, most rely on qualitative observations or quantitative direct measurements. The objective of this study was to standardize an indirect wound measurement method using digital planimetry, incorporating digital scaling and segmentation. This approach addresses the lack of detailed, step-by-step methodologies for accurate wound assessment. A photodocumentation booth was designed and constructed, and computer-assisted digital planimetry tools were employed to minimize variability in measurements of the wound area, perimeter, and the distance from the wound center to its edges. A circular traumatic wound (5 mm in diameter) was created on the dorsal midline at the shoulder blade level of male CD1 mice (n = 4, 10 weeks old, 30-35 g). Wound evolution was photodocumented for 14 days using the custom-designed photo booth, which controlled lighting conditions, focal distance, and subject positioning. Scaling and wound measurements were performed using segmentation in ImageJ software, and statistical analysis was conducted using statistical analysis software. The kinetics of wound closure showed a slight increase in wound size and perimeter between day 0 and day 2, followed by a gradual decrease until complete closure by day 14. The photodocumentation booth and computer-assisted digital planimetry enabled quantitative measurements with minimal variability. In conclusion, these tools provide a reliable and reproducible method for evaluating wound closure kinetics in pre-clinical models.

Wounds represent a global health challenge. This study developed a standardized photo booth utilizing digital planimetry to minimize wound measurement variability. Monitoring wounds in mice over 14 days revealed an initial increase in wound area and perimeter, followed by gradual closure. This methodology may aid in evaluating wound closure kinetics in pre-clinical models.

用于评估小鼠模型中伤口闭合动力学的数字平面测量法

Chronic wounds, due to their high prevalence, are a serious global health concern. Effective therapeutic strategies can significantly accelerate healing, ...

生物工程

Non-invasive Skeletal Muscle Quantification in Small Animals Using Micro-computed Tomography

Skeletal muscle size, mass, and composition are critical properties for studying metabolic and muscle-related diseases, as they directly impact the understanding of disease progression and treatment outcomes. Quantifying a live animal's lean, adipose, and skeletal mass is important in metabolic, physiology, pharmacologic, and geroscience studies. However, obtaining accurate body composition measurements, especially of lean mass, remains challenging due to the inherent limitations of conventional assessment techniques. Micro-computed tomography (micro-CT) is a non-invasive radiological technique that enables high-resolution visualization of internal structures in small animal models. A standardized micro-CT method can significantly enhance translational research with more reliable and impactful results, particularly during aging studies or at different time points within the same animal. Despite its potential, the lack of standardization in image acquisition and analysis methods significantly hinders the comparability of results across different studies. Herein, we present a comprehensive and detailed low-cost protocol for lean mass analysis using micro-CT to address these challenges and promote consistency in research involving small animal models.

This protocol uses micro-computed tomography to enable the cost-effective quantification of lean mass, including skeletal muscle and visceral tissue, adipose tissue, and skeletal tissue in small animals. It distinguishes between lean and adipose tissue, which offers significant advantages for biomedical research, particularly in translational research in small animals.

使用显微计算机断层扫描对小动物进行无创骨骼肌量化

Skeletal muscle size, mass, and composition are critical properties for studying metabolic and muscle-related diseases, as they directly impact the ...

Non-Invasive Visualization of Nailbed Microvascular Morphology in Mice Using Capillaroscopy

Imaging microcapillary networks of the skin in humans using nailfold capillaroscopy (NFC) has underscored the importance of microcirculation as a target organ system in critical systemic illnesses. Nailfold capillaroscopy is applied clinically to detect peripheral microvascular dysfunction and abnormalities in a range of systemic conditions, including rheumatic, cardiac, ocular (e.g., glaucoma), and endocrine disorders (e.g., hypertension and diabetes mellitus). NFC is useful not only in detecting peripheral systemic microvasculature disruption but also in assessing drug efficacy. However, translating clinical NFC findings to animal disease models can be challenging. Detecting microvascular dysfunction or abnormalities in animals is often invasive (e.g., endoscopic), carried out ex vivo (e.g., post-mortem imaging of tissues), or expensive, requiring specialized equipment such as those used in microcomputed tomography and photoacoustic imaging techniques. Developing quick, non-invasive, and inexpensive techniques to image peripheral microvasculature in animal models of disease is warranted to decrease research expenses and increase translatability to the clinic.
Capillaroscopy has previously been used to visualize the nailfold microvasculature in animal models, including in guinea pigs and mice, thus demonstrating the capability of capillaroscopy as a non-invasive imaging tool in animal models. This study provides a protocol that applies capillaroscopy to a mouse nailbed, allowing researchers to easily and inexpensively assess the morphology of its microvasculature. Representative images of typical nailbed microvascular architecture in wild-type mice using two commonly used laboratory strains, SV129/S6 and C57/B6J, are provided. Further studies using this method are essential for applying nailbed capillaroscopy to a wide range of mouse disease models with peripheral microvascular abnormalities.

Capillaroscopy is an accessible tool for direct, inexpensive, and non-invasive visualization of microvasculature. The goal of this protocol is to enable researchers to use capillaroscopy for the visualization of peripheral microvascular morphology in the nailbeds of mice.

使用毛细血管镜检查小鼠甲床微血管形态的无创可视化

Imaging microcapillary networks of the skin in humans using nailfold capillaroscopy (NFC) has underscored the importance of microcirculation as a target ...

A Technique for Human Skin Grafting in a Mouse Model

Source: Moss, M. I. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/64040">Xenograft Skin Model to Manipulate Human Immune Responses&#160;In Vivo.</a>&#160;J. Vis. Exp. (2022)
This video demonstrates a method for grafting human skin onto an immunodeficient mouse with a suppressed neutrophil response. The method involves applying human split-thickness skin to the mouse&#39;s graft bed, sealing it, and covering it for recovery. Eventually, the human skin adheres to the mouse&#39;s skin, establishing vascularization and circulation, resulting in a successful skin xenograft.

小鼠模型中的人体皮肤移植技术

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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