We thank Faekah Gohar for proofreading the manuscript. We thank Sonja Dufentester and Elke Weber for expert technical assistance.

所有的动物实验是由根据德国动物保护法的地区伦理委员会（Landesamt献给NATUR，UMWELT UND Verbraucherschutz，LANUV，德国）的批准。明斯特大学的当地伦理委员会批准的组织学和显微镜分析使用人体组织。 1.动物与材料<ol><li>根据当地的动物保护立法，使用女性或体重20〜25克所需的DSS敏感菌株的雄性小鼠和房子。啮齿动物提供特殊的食物和饮用水灭菌水随意 。 </li><li>在高压灭菌自来水5天:通过给予3％w / v的葡聚糖硫酸钠（36,000-50,000达DSS，分子量）诱发急性 DSS结肠炎。 注:DSS的效力是高度可变取决于制造商和批次。第一个测试你的供应商提供的DSS疾病活动的诱导，为此，达ILY体重是一个可靠和客观的指标。 </li><li>结肠组织样本的组织学评价，在实验结束时安乐死用CO 2吹入（或由国家和机构准则指定）的小鼠。 </li></ol> 2.实验设置为DSS-结肠炎和体外伤口愈合测定<ol><li>细胞培养和建立伤口愈合测定。 <ol><li>生长的Caco-2细胞在95％湿度和5％CO 2的环境中，在37℃。使用RPMI培养基含有10％胎牛血清（FBS）和1％青霉素/链霉素。 </li><li>种子Caco-2细胞以4×10 5个细胞/ cm 2的在35 mm培养皿高培养插入的密度（参见图4A）。 注意:插入生成与待分析代表受伤区域中的规定的小单元的自由空间分离的两个小区覆盖的区域。 </li><li> seedin两天后变介质G。通过首先通过使用自动移液管抽吸以细胞碎片残留介质执行，冲洗用100μl磷酸盐缓冲盐水（PBS）中，并添加新鲜RPMI培养基或血清剥夺介质。 </li><li>培养的细胞在补充有20纳克的EGF /毫升血清或2微克丝裂霉素C /毫升血清，以检测在伤口愈合的行为改变血清剥夺培养基（0.1％FBS）24小时。加正常RPMI培养基对照细胞24小时。 </li><li>在培养24小时，取出，如步骤3.4中所述丢弃培养插入并执行DHM。 </li></ol></li><li>急性的DSS诱发结肠炎<ol><li>溶解在100毫升高压灭菌水以获得3％（​​重量/体积）的DSS溶液3克DSS的。提供到位饮用水的小鼠随意持续7天的该溶液中。计算溶于5ml每小鼠/天DSS溶液。无DSS的对照组小鼠自由采食提供高压灭菌的水。 </li></ol></li><li>鼠和人结肠的低温薄样切片部分的制备<ol><li>在实验结束时安乐死的CO 2吹入的小鼠。 </li><li>开腹23解剖动物的腹部。除去全结肠仔细使用镊子和手术用剪刀剪回肠和直肠末端。从盲肠到直肠末用剪刀剪结肠纵向打开的结肠。使用镊子，然后用PBS洗涤24试样取出所有的粪便。 </li><li>通过卷起从盲肠纵向芽直肠结束与向内弯曲粘膜棉花全结肠准备瑞士卷。在最佳切割温度（OCT）化合物嵌入结肠样品并在-80℃下直至进一步使用保持冷冻。 </li><li>从在最佳切割温度OCT化合物手术标本嵌入人结肠组织和在-80℃直至进一步使用保持冷冻。 </li><li> 7微米厚的OCT-化合物包埋标本的切断面与cryoto的帮助我只是之前的检查。 注:最佳样品厚度取决于持久性和所研究的组织类型的散射特性。对于使用结肠组织，层厚在噪声&gt;10μm的原因显著增加由于在定量DHM相衬图像的光散射所描述的实验中，而厚度的样品&lt;5微米显示用于从深冷切削工艺损伤诱导的伪影的风险较高。 </li><li>转移到一个玻璃对象载体滑动部分。 </li></ol></li></ol> 3.技术设备，软件和程序的数字全息图的采集和评估<ol><li>定量细胞和组织成像数字全息显微镜<ol><li>使用离轴马赫-策德尔数字全息显微术系统，用于活细胞成像25， 如图1。确保在显微镜配备有10X显微镜透镜，与玻璃对象载体幻灯片和培养皿的直径为35毫米的支架显微镜阶段，加热室保持在37℃的生理温度和软件进行定量相成像25。 注意:例如，如在肯珀等人 26和Langehanenberg 等 27描述。 注:另外，使用类似的系统，该系统能够执行亮视野显微镜和活细胞的定量相成像和解剖组织幻灯片。 </li><li>干净的显微镜透镜，用镜头清洁纸和推荐的显微镜的制造商，以除去灰尘或其它污染物的清洗剂（ 例如，乙醇）冷凝器。 </li><li>启动DHM显微镜的图像采集软件，选择"开"白光照明"明场"成像模式和切换。确保科勒 - 光量所推荐的显微镜的制造商同时观察在图像采集软件（或者标准图像采集软件可以在这个步骤中使用）的实时成像窗口的样品的样品的通货膨胀。 注:图像强度应均匀地分布在视场分布和样本位置不应该与显微镜的聚焦驱动光再聚焦过程中移动。 </li><li>选择"DHM"摄像模式时，关闭"关闭"的白光照明和开关"接通"的激光。检查用激光照射是均匀的（ 即 ，光强均匀分布在DHM显微镜的成像采集软件的实时成像窗口），并观察到离轴载波干涉条纹图案出现在适当的对比拍摄的图像（数字全息图）。 </li></ol></li><li>成像冰冻切片的制备与DHM <ol><li>取样品（低温恒温器部分的玻璃物体的载体上，厚度为7μm，如在2.3中描述）从冷冻的。解冻在室温和常压下约5分钟的样品。 </li><li>添加50 - 100微升磷酸盐缓冲盐水（PBS），为包埋介质上使用移液管，直到它完全与缓冲覆盖的组织切片。用干净的玻璃盖玻片（玻璃厚度170微米）的样品。 注:过干燥可诱导和折射率的显著变化试样的散射特性。 </li><li>确保玻璃载体和盖玻片的底部从灰尘和其他污染可诱导的光散射清洗。样品是准备与明场显微镜和DHM调查。 </li></ol></li><li>与DHM组织切片的定量相成像<ol><li>交换机上的数字全息显微镜，选择成像10X显微镜镜头。开始我在DHM显微镜法师采集软件，选择明亮的文件成像模式。 </li><li>放置组织滑如在显微​​镜载玻片支架在2中所描述），朝向显微镜物镜盖玻片。 </li><li>开关"关于"DHM显微镜明视场照明。将样品与显微镜载物台的位置，并确保感兴趣的组织区域是在实时监控窗口可见。提高使用显微镜的聚焦驱动的图像的清晰度。 注意:除了感兴趣的区域，滑动的无组织的区域也应存在于视场中。 </li><li>捕获使用图像采集软件的重点突出样品的亮场图像。 </li><li>选择"DHM"摄像模式时，关闭"关闭"的白光照明和转"上"的激光照射。选择低于3毫秒的"曝光时间"，全息记录，观察holographic离轴干涉条纹出现与在成像采集软件的实时成像窗口足够的对比度和捕获的数字全息图。 </li><li>直到亮场图像和不同的样本区域的数字全息图的足够数量的已经记录3.3.5 - 重复步骤3.3.3。全息收购现已完成。 </li></ol></li><li>伤口愈合测定为DHM成像的制备<ol><li>打开约1 DHM显微镜的培养皿加热腔 - 在实验开始前3小时，以确保在DHM测量稳定的温度条件。 </li><li>备的工作台上所需要的设备（培养皿中2.3中描述了伤口愈合测定）:移液管，镊，玻璃盖为培养皿，4-（2-羟乙基）-1-哌嗪乙磺酸（HEPES）缓冲的细胞培养基用生理温度（37℃）在无菌环境中的样品制备。 注意: </stro纳克&gt; Dulbecco氏改良的Eagle培养基（DMEM）是由10％胎牛犊血清（FCS），20毫米的HEPES（4-（2-羟乙基）-1-哌嗪乙磺酸），和850毫克/升碳酸氢钠的。 </li><li>从培养皿中取出的塑料盖并用移液器除去细胞培养基。删除用镊子培养皿底部插入。 </li><li>用1ml的HEPES缓冲的细胞培养液2次，以去除死细胞，并在伤口区域剩余细胞组分（ 例如 ，血清） -洗样品1。加2ml HEPES缓冲的细胞培养液中，并盖上培养皿的玻璃盖。 </li><li>确保玻璃盖和培养皿底部已经从灰尘和其他污染清洗。样品是准备与DHM定时观察。 </li></ol></li><li>伤口愈合的连续多峰监测在体外用DHM <ol><li>交换机上的DHM显微镜1的培养皿加热室 - 前3小时在实验的开始，以确保在DHM测量稳定的温度条件。 </li><li>开关"的"数字全息显微镜，选择成像10X显微镜镜头。启动DHM显微镜的图像采集软件，选择"亮场"成像模式。确保，培养皿加热室是经营生理温度（37℃）。 </li><li>放置培养皿与伤口愈合测定法，如在4所述制备），在DHM显微镜的加热室。 </li><li>选择明场成像模式以及同时在DHM显微镜的图像采集软件的实时监控窗口观察它的样品与显微镜阶段的位置。观察样品的所需区域在白光照明下出现清晰地聚焦。 </li><li>捕捉白下样品的不同区域的亮场图像（伤口面积和融合细胞周边地区）光照射与图像采集软件和文件的外观，细胞密度和均匀性。 </li><li>选择DHM显微镜"明场"成像模式。选择下与DHM显微镜的图像采集软件实​​时监测窗口白光照明的合适的伤口面积。确保卷绕区域是从死细胞和无血清遗体自由，并确保双方包括单个同质细胞层，优选具有直边界。 </li><li>捕获与DHM显微镜的图像采集软件明场成像模式下的初始伤口面积的白色光图像。 </li><li>关闭"关闭"白光照明，选择"对"激光照排"DHM"模式，切换。选择低于3毫秒，全息记录的曝光时间（观察到全息离轴干涉条纹出现在T的图像采集软件的实时成像窗口足够的对比他DHM显微镜），捕捉数字全息图。 </li><li>捕捉DHM模式与图像采集软件样品全息图和重建的DHM显微镜的重建软件定量相位图像，以检查图像质量。 </li><li>选择一个合适的时间延迟-时间推移全息采集与DHM显微镜的图像采集软件（ 例如 ，3 5分钟）。 </li><li>选择该图像采集软件，其中样品与激光全息采集期间仅照亮的时间推移采集模式。 </li><li>启动伤口愈合测定法的时间推移DHM观察。 </li><li>停止时间推移收购预期的时间后，选择明场成像模式和记录样品在白光下成像的最终外观。 </li></ol></li><li>重建解剖组织的数字全息图，并确定平均折射率作为参数量化组织密度<ol><li>从与DHM显微镜， 例如软件解剖组织的数字全息重建量化相位的图像，如在肯珀等人 26和Langehanenberg 等人 27所述。 </li><li>确定在利益（投资回报）19选择适当的区域不同的组织层（上皮，粘膜下层，基质）的平均相差Δφ。 </li><li>通过使用折射计或者可选地通过使用从文献适当的值确定所述灌封介质的折射率。 （为典型的嵌入媒体的折射率值:N 水 = 1.334 28，正磷酸盐缓冲盐水（PBS）= 1.337 29中，n 细胞培养基 = 1.337-1.339 29,30）。 </li><li>计算不同组织的层的折射率从平均相衬值19 <img alt="式（1）"SRC ="/文件/ ftp_upload / 54460 / 54460eq1.jpg"/&gt;（1） 注意:在公式。 1λ是激光光（这里:λ= 532纳米）的波长，D的解剖组织的厚度（这里:7微米）和正 介质是包埋介质（此处的折射率:N 介质 = 正 PBS = 1.337，由阿贝-折射测定）。 </li></ol></li><li>重建并从时间推移伤口愈合系列观察评估数字全息图<ol><li>从与DHM显微镜软件26,27伤口愈合的观察过程中获得的时间间隔全息系列重建定量相图像。 </li><li>每个系列的定量相图像的规范化与最大相衬图像。 </li><li>确定面积S C，它是由细胞中由图像分割定量DHM相位图像所覆盖，其可以是每使用免费的软件单元探查形成（www.cellprofiler.org 31）。 </li><li>计算面积S c中的细胞Δφ 细胞的平均相衬。 </li><li>检索来自平均相差Δφ 细胞的细胞干块DM中的面积S C 15 <img alt="公式（2）" src="/files/ftp_upload/54460/54460eq2.jpg" /> （2） 注:在等式2中的DM表示蜂窝干质量，S C为是受细胞和α= 0.002米2 /千克的占有面积。 </li><li>确定积分蜂窝折射率n 细胞和细胞培养基Ñ 介质的折射率。从悬浮细胞确定ñ 细胞实验分别在30描述测量用折射ñ 网上平台 。爱特纳tively使用文献值n个 单元 30和N 中 29,30。 </li><li>从λ，Δφ 细胞 中，n 细胞和n 介质 26,32计算平均电池厚度d 细胞 <img alt="公式3" src="/files/ftp_upload/54460/54460eq3.jpg" /> （3） 注意:在公式。 3参数d 小区是平均单元厚度，λ表示激光的光的波长，Δφ 细胞表示平均相位相反，且n 细胞和n 介质是积分蜂窝折射率和周围介质的折射率。 </li></ol></li></ol>

The authors have nothing to disclose.

我们证明DHM提供了在小鼠模型结肠炎组织损伤和人体结肠组织样本体外的准确评估。此外，我们还显示DHM可连续监测上皮伤口愈合，而同时提供有关细胞改变多式联运信息。在DHM，以数字32进行捕获数字全息图的重建。因此，在比较明亮的视野显微镜，泽尼克相差和差分干涉相差显微术，DHM提供任选的随后数值焦点校正（多聚焦成像）定量相衬27。定量相位成像是基于光路长度变化的确定，从而是高度独立使用的测量装置的。这简化了与不同的仪器获得的测量数据的比较。另外，DHM需要OBJE仅低光强克拉照明。这使得未染色解剖组织的微创分析并最大限度地减少在活细胞的长期的时间推移观测所需样品的相互作用的量。 鸡传染性法氏囊病的治疗一套设备在两个过去几十年显著扩大。除了 ​​引进抗TNF-α疗法，它是有效的UC和CD，并具有可接受的副作用轮廓，新颖和定位整合最近已引入临床实践34-37的特异性抗体。几个其它有希望的药物，目前正在评估在IBD 38-40它们的治疗功效。然而，之前在人类临床上使用，这些潜在的疗法的有效性和安全性已在动物研究中被证明。所述DSS诱导的结肠炎是最经常使用的小鼠结肠炎模型之一，由于它的高再现性和低的成本23。此外，这种模式可以人所以被应用到基因改造的小鼠品系41。而全身标记， 例如，C反应蛋白，是在动物模型中高度可变的，在结肠的组织学评估代表最有效的方法来评估疾病严重程度42,43。尽管如此，根据评分系统的组织学炎症的定量评价是高度依赖于研究者的专业知识和经验。通过客观指标尽可能与DHM自动评估和打分克服了这些限制，此外还节省时间，避免了染色19的需要。此外，DHM可以用于检查外科结肠标本以及内窥镜期间获得的粘膜样品。 上皮再生和伤口愈合是在一些病理过程，包括胃肠道溃疡或吻合口瘘的关键机制。虽然是有希望的方法来评估V中的上皮伤口愈合龙腾光电44，这些技术需要复杂的检查工具和目前尚未得到广泛应用。因此，目前上皮愈合 ​​通常是由划痕试验在体外研究9。这些分析让伤口愈合有效的检查，但通常首先要求细胞染色，然后可防止重复评价10,45。此外，任何蜂窝变更数据可以在平行与该实验装置获得的。然而，该信息可以是显著重要的，因为它可以用于细胞凋亡和坏死46之间进行区分，并评估细胞增殖和迁移46。因此，有可能以确定细胞的厚度，干重或组织密度同时通过DHM检查监测伤口愈合的是黏膜的研究具有很高的价值。 人类IBD患者的治疗目标已不断变化，在过去几十年&lt;SUP&gt; 47。虽然炎症最初的控制被认为是首要的，后来无类固醇缓解现在粘膜愈合治疗是48的主要目标。最近，有人推测病理缓解甚至可能优于单49粘膜愈合。然而，尽管有可用于人类IBD几个组织学评分系统中，观察者间的变异仍然很高50。因此，需要一种用于组织学结肠样品的评价的标准化方法。 DHM可能有助于这一目标的实现，应在前瞻性临床试验与人类IBD患者进行进一步评估。 在呈现的实验程序，将样品照明和成像使用高度相干的激光。因此，在DHM分辨率主要是由所引起的样品照明的未对准光散射和衍射效应的限制，厚的样品，灰尘或其他杂质，例如在光路中的冷凝水。为了从定量DHM相衬图像，DHM设置的精心准备和校准和样品实现高度精确的测量数据在协议中最重要的步骤。彻底清洗光学成像元件，特别是在显微镜聚光器和显微镜物镜，需要以减少噪声。还样品载体幻灯片，培养皿盖和底部，以及所利用的细胞培养基应该是从颗粒状杂质。这是通过仔细清洗施加液体的所有组件和足够的滤波来实现的。 更详细的故障排除提示如下:如果数字全息图和/或解剖组织的定量DHM相位图像是嘈杂，玻璃载体和盖玻片可与灰尘污染。如果是这样，清洁玻璃载体和盖玻片用酒精（如纯乙醇）。如果厚解剖组织的岬过大（&gt; 10微米），从而导致光散射，尝试使用较薄的组织切片。在与激光光线的照射是不均匀分布的情况下，对准经由显微镜聚光器物体照明。如果在玻璃盖的底部的冷凝水引起的散射的影响，检查加热室和室内湿度的温度。在情况下，细胞密度是在一个以上的层过高或细胞的生长，减少制备伤口愈合测定法期间，细胞密度。最后，如​​DHM是基于干涉的原理，该方法是对振动或其他机械干扰，根据个人的实验室环境中变化敏感。然而，这种影响通常是可以通过适当地选择曝光时间（通常在毫秒范围或以下）为全息记​​录实验装置的制备过程中最小化。 综上所述，我们的结果表明，DHM持有超过明场和现有相衬成像显微技术如泽尼克相差和DIC主要优点，因为它能够定量评估几乎自动的方式组织学炎症体外能力。此外DHM允许体外上皮伤口愈合与样品最小相互作用的无标记的连续多峰的评价。这铺平了道路，以自动化的组织学检查在&#39;数字病理&#39;，并进一步扩展研究的角度来探讨IBD患者DHM的转化潜力。另外，DHM提供新型体外的机会，定量研究细胞迁移，形态和增殖。 DHM的到临床实践的翻译具有改善IBD诊断的潜力。作为DHM允许通过不同程度的肠道炎症的量化像密度的变化，这种病的在一个"数字病理学"的意义上的目标和更精确的评估物理参数似乎可能的。客观评估可以对IBD患者的临床管理的一个重要的影响。

炎性肠病（IBD）， 即，溃疡性结肠炎（UC）和克罗恩氏病（CD）是胃肠道1的特发性炎性病症。研究IBD的病理生理学和潜在的新药物或新的诊断方法评价特别重要性。在这两个基础研究和IBD患者的临床管理，肠黏膜已成为人们关注2,3的焦点。粘膜表示解剖边界，在该共生细菌，上皮细胞和肠道免疫系统的各种细胞成分之间的交互编排肠道动态平衡4,5。然而，在IBD患者，不受控制的和持久性肠炎症导致粘膜损伤，如溃疡或狭窄，其可以在上皮的屏障功能，它本身加剧局部炎症6的击穿终于达到高潮检测的。 <p类="jove_content"&gt;上皮伤口愈合因此，以下炎症至关重要的上皮再生，但也是胃肠道溃疡或吻合口瘘的胃肠道手术后7愈合的核心需求。上皮伤口愈合可以在体外伤口进行模拟愈合测定法和在肠道炎症8,9的小鼠模型在体外 以及 体内的方法有缺点，它限制了实验评估的准确性， 在体外测定，如古典划痕测定法，需要旷日持久的染色步骤或转染荧光发色团。他们往往可以通过细胞增殖和迁移不能被自动化10的不连续的监测的限制。 在体内模型，例如硫酸葡聚糖钠（DSS）诱发的结肠炎，经常由于看到的显著变化缺乏鲁棒读奏，在部分在实验室指标，MA不当国王这样的标记，以评估严重结肠炎11,12。发炎粘膜的组织学分析是目前仍然确定结肠炎的严重性的最有效的方法，但是这一点，像在体外上皮伤口愈合测定法，要求染色和依赖于研究者的专门知识13。 最近的数字全息显微术（DHM），定量相显微镜14的变型中，被确定为在体外和体内 15上皮伤口愈合的评价有用的工具。DHM允许组织密度的评估通过测量光路长度的延迟（OPD） ，其前景新型癌症诊断和16-18炎症相关组织改建19的定量。此外，DHM允许细胞形态动力学监测通过确定单元厚度，细胞覆盖的表面区域和细胞内（蛋白质）含量的数量<sup&gt; 15,20。 在体外试验中，DHM还使生理过程， 例如，通过评估在细胞体积和厚度21,22更改蜂窝透水性的分析。此外，DHM测量可以自动其防止研究者相关样本偏差。 在这里，我们证明在肠道炎症的小鼠模型中使用DHM的，并且也适用DHM到人体组织样品的分析为定量监测伤口愈合的作为无标记的体外测定。首先，我们评估在从与IBD的人类结肠炎小鼠组织切片的不同结肠壁层的炎症性变化。描述DHM定量相成像过程后，我们提供了使用显微镜成分，组织切片的准备和还描述了获得定量的相位图像的评价的详细说明。 下一步，我们表明，DHM可以UTIlized 体外连续多监测上皮伤口愈合，并描述像细胞层的厚度，干重和细胞体积的细胞特征的分析深入了解药物引起和生理细胞改变。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Azoxymethane (AOM)</td>
			<td>Sigma - Aldrich, Deisenhofen, Germany</td>
			<td>A5486</td>
		</tr>
		<tr>
			<td>Cell Culture Flask</td>
			<td>Greiner Bio-One, Frickenhausen, Germany</td>
			<td>658170</td>
		</tr>
		<tr>
			<td>Costar Stripette</td>
			<td>Corning Inc., New York, USA</td>
			<td>4488</td>
		</tr>
		<tr>
			<td>Dextran sulphate sodium (DSS)</td>
			<td>TdB Consulatancy, Uppsala, Sweden</td>
			<td>DB001</td>
		</tr>
		<tr>
			<td>DMEM/Ham&#180;s F12</td>
			<td>PAA Laboratories - Pasching - Austria</td>
			<td>E15-813</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Sigma - Aldrich, Deisenhofen, Germany</td>
			<td>SPR3196</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>Sigma - Aldrich, Deisenhofen, Germany</td>
			<td>E 9884</td>
		</tr>
		<tr>
			<td>Falcon Tube 50ml</td>
			<td>BD Biosciences, Erembodegem, Belgium</td>
			<td>352070</td>
		</tr>
		<tr>
			<td>Isopentane (2- Methylbutane)</td>
			<td>Sigma - Aldrich, Deisenhofen, Germany</td>
			<td>M32631-1L</td>
		</tr>
		<tr>
			<td>Methylene blue</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1159430025</td>
		</tr>
		<tr>
			<td>Mitomycin C</td>
			<td>Sigma - Aldrich, Deisenhofen, Germany</td>
			<td>M4287</td>
		</tr>
		<tr>
			<td>Microscope Slides</td>
			<td>G. Menzel, Braunschweig, Germany</td>
			<td>J1800AMNZ</td>
		</tr>
		<tr>
			<td>O.C.T. Tissue Tek compound&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>Sakura, Zoeterwonde, Netherlands</td>
			<td>4583</td>
		</tr>
		<tr>
			<td>Pen/Strep/Amphotericin B</td>
			<td>Lonza, Verviers, Belgium</td>
			<td>1558</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline, PBS</td>
			<td>Lonza, Verviers, Belgium</td>
			<td>4629</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Lonza, Verviers, Belgium</td>
			<td>3626</td>
		</tr>
		<tr>
			<td>Sodium Chloride 0,9%</td>
			<td>Braun, Melsungen, Germany</td>
			<td>5/12211095/0411</td>
		</tr>
		<tr>
			<td>Standard diet</td>
			<td>Altromin, Lage, Germany</td>
			<td>1320</td>
		</tr>
		<tr>
			<td>Tissue-Tek Cryomold</td>
			<td>Sakura, Leiden, Netherlands</td>
			<td>4566</td>
		</tr>
		<tr>
			<td>Trypsin EDTA</td>
			<td>Lonza, Verviers, Belgium</td>
			<td>7815</td>
		</tr>
		<tr>
			<td>Vitro &#8211; Clud&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>R. Langenbrinck, Teningen, Germany</td>
			<td>04-0002&#160;</td>
		</tr>
		<tr>
			<td>&#160;&#181;-Dish 35 mm with Culture-Insert, high</td>
			<td>ibidi GmbH, Munich, Germany</td>
			<td>81176</td>
		</tr>
		<tr>
			<td>DIC Lid for &#181;-Dishes, with a glass insert</td>
			<td>ibidi GmbH, Munich, Germany</td>
			<td>80050</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MICROM HM550</td>
			<td>Thermo Fisher Scientific, Inc., Waltham, USA</td>
			<td>46320</td>
		</tr>
		<tr>
			<td>Digital holographic microscope</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Model</td>
			<td>Company</td>
		</tr>
		<tr>
			<td>Inverted Microscope</td>
			<td>iMIC</td>
			<td>Till Photonics, Graefelfing, Germany</td>
		</tr>
		<tr>
			<td>Laser</td>
			<td>Compass 315M</td>
			<td>Coherent GmbH, Luebeck, Germany</td>
		</tr>
		<tr>
			<td>Microscope lens</td>
			<td>Zeiss EC Plan Neofluar 10x/0.3</td>
			<td>Zeiss, Goettingen, Germany</td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>DMK 41BF02</td>
			<td>The Imaging Source, Bremen, Germany</td>
		</tr>
	</tbody>
</table>

multimodal quantitative phase imaging with digital holographic microscopy accurately assesses intestinal inflammation and epithelial wound healing

炎性肠疾病， 即的发病率，克罗恩病和溃疡性结肠炎，已显著在过去十年中增加。 IBD的病因仍然是未知的和当前的治疗策略是基于对免疫系统的非特异性抑制。中专门针对肠道炎症和上皮伤口愈合能显著改善IBD的管理治疗的发展，但是这需要的炎性改变精确检测。目前，潜在候选药物所使用的动物模型在体内或在体外细胞培养为基础的技术，通常进行评价。组织学检查通常需要的细胞或感兴趣的组织被染色，其可以改变样本特征，此外，研究结果的解释可以通过调查专门知识而异。数字全息显微镜（DHM），根据该检测光路长度的延迟，使得染色无定量相衬成像。这使得结果与绝对生物物理参数直接相关。我们展示了如何在与DHM组织密度的变化的测定中，根据测定折射率，可以量化炎症改变，而不染色，在从小鼠和人类与结肠炎结肠组织标本的不同的层。此外，我们表现出体外 ，可能通过简单的自动测定受伤面积和形态学参数的同时测定使用上皮DHM伤口愈合的连续多无标签的监测，如干燥质量和迁移细胞层的厚度。总之，DHM表示用于肠道炎症与参数可能绝对值的评估有价值的，新的和定量的工具， 在体外上皮创伤愈合的简化量化，因此具有用于平移诊断ù高电位本身。

典型设置为DHM成像数字全息显微镜（DHM） 执行明场成像和定量DHM相衬成像，我们应用倒置显微镜如在图 1B中描绘。该系统是通过将一个DHM模块改性，如前面25所描述。通过光照生成具有倍频Nd的光的样品的数字全息图:在马赫-曾德尔配置（ 图 1 的 ）YAG激光λ= 532纳米）。解剖组织进行了分析体外玻璃 ​​载体幻灯片。在特殊的培养皿，观察观察伤口愈合活细胞培养物如示于图 1℃。将样品在透射照明经由10X英里成像croscope透镜（NA = 0.3）和管透镜。电荷耦合器件照相机是用来记录与略微倾斜的参考波叠加样本图像（对象波）产生干涉条纹（数字离轴全息图）。该数字方式获取全息图是由空间相结合移重建可选自动对焦全息重建26,33。 小鼠DSS诱导结肠炎DHM评估 急性结肠炎小鼠通过的3％的管理饮用水5天诱导w / v的葡聚糖硫酸钠（DSS）。的表现形式和结肠炎的过程之后通过监测对DSS处理的组中的进行性消瘦相比于对照组。 （ 图 2A改编自19）。胃镜，中度至重度大肠杆菌观察那朵由结肠壁增厚所反映，血管图案（ 例如，自发出血， 红色箭头 ），纤维蛋白渗出（ 白色箭头 ）和粘膜表面（ 图2B）的粒度的改变。苏木精和曙红的常规组织学评价（HE）染色的结肠切片中的节的对照动物，主要位于粘膜下层（ 图 2 C 灰色箭头 ）DSS处理的结肠壁揭示明显水肿。对应由公式1检索定量DHM相衬图像和相关的折射率的地图，也示出的上述组织的改变。此外，折射率，编码为256个灰度级，表明在组织层中的密度差异，包括天然的，未染色的组织样品中的上皮细胞（ 白色箭头 ）和基质（ 黑色箭头 ）（ 图 2 </st荣&gt; C）。折射率在上皮细胞，以及在相对 ​​于健康对照（ 图 2 E）的结肠炎小鼠的粘膜下层和间质中显著降低。此外，临床参数（体重的相对损失）和绝对物理参数（折射率）之间的显著相关性，可以观察到（R 2线性= 0.64; 图 2 D，改编自19）。 由DHM人类克罗恩病的评估 克罗恩病往往是由肠壁炎症显着改变，通过消化道内镜检测鉴定。特性宏观特征包括粘膜表面的粒度，纵向溃疡纤维蛋白exudat离子（也被称为"蜗牛创新"）和自发性出血（ 图 3 A）。从患者积极的CD的组织样本进行HE染色组织学检查往往揭示结肠壁及粘膜下水肿。对应由公式1检索定量DHM相衬图像和相关折射率地图还检测出的结肠壁的炎症介导的增强/肿胀，在这里看到的上皮（ 图 3 的B）。另外，折射率为绝对参数，也显著结肠组织样品中的所有壁的层（上皮，粘膜下层和基质）减少患者活动性CD节的那些缓解（ 图 3℃）。 伤口愈合 体外 的多模式监视 </eM&gt; Caco-2细胞伤口愈合使用一种特殊的培养皿提供标准化的条件进行测定。菜配备有培养插管形成两个不同的腔室，其通过为500μm（ 图 4A）的间隙隔开。以模拟不同的生理环境中，细胞进行了检查或未经处理，由表皮生长因子（EGF）刺激或用丝裂霉素C抑制。在DHM设置允许伤口愈合随时间过程中，经过0，22.5和45小时通过代表定量DHM相衬图像（编码为256个灰度级）这里所示的实验开始（ 图 4 B）之后的连续监测。 在图 5 中对应的假彩色编码伪三维图说明在三个二细胞层厚度的空间发展mensions。基于该光路长度的延迟，蜂窝折射率和方程2和3，DHM允许通过的细胞特征，例如细胞覆盖的表面面积，平均单元厚度和细胞干重（时间监测的迁移，增殖和形态的同时动态评估图 4℃）。总之，在图 4中的数据说明，表皮生长因子的模拟结果中的细胞更快迁移到伤口区域，以增加的细胞平均厚度和在较高的干质量中的视场与对照细胞相比。与此相反，丝裂霉素C抑制细胞覆盖略高于对照细胞表面积的实验过程中降低，也显示出稍低的干质量增加。然而，相对于两者的平均单元厚度出现明显减少，刺激和对照细胞。 图1。 已用离轴设置为数字全息显微镜（DHM）的DHM工作站（A）的示意图设置。对应照片描绘了显微镜的设置（显示器，实时图像和控制软件（1），离轴设置数码显微镜（2），（B）中，在显微镜（ 绿色虚线 ）和样品的光路（由红色指示箭头 ;（C））。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54460/54460fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54460/54460fig2.jpg" /> 图 2。小鼠DSS诱导结肠炎DHM的评估。结肠炎的课程由每天我相对体重和反复内镜随访检查asurement。从第4天DSS管理（改编自19）（A）开始后相对体重的大量流失是伴随着建立结肠炎内镜迹象包括联络溃疡和粘膜脆弱性（B）。常规HE染色切片低温薄样切片分析显示粘膜下水肿，肌层和粘膜有明显的粘膜炎性浸润增厚。对应由公式1检索定量DHM相衬图像和相关折射率映射（编码为256个灰度级）确证了粘膜下层中炎症过程的水肿的变化（白色箭头指示上皮，灰色粘膜下层和黑色基质）（℃）。体重的相对损失（一个临床参数）与折射率（绝对物理参数中，R 2线性= 0.6显著相关4; （D）中 ，改编自19），这在上皮以及在粘膜下层和比健康对照的结肠炎小鼠的间质被显著减少（平均±SEM *** P &lt;0.001; E） <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54460/54460fig2large.jpg" target="_blank">，请此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54460/54460fig3.jpg" /> 图 3. 克罗恩病的评估人类通过DHM。从患者的胃镜和病理证实克罗恩病结肠组织样本进行了检查（A）。 HE染色显示粘膜隐窝延长和炎性细胞的浸润标记。此外，检测到粘膜下水肿和固有肌层的放大图。科尔分析esponding由公式检索定量DHM相衬图像和相关的折射率的地图（1）（编码为256个灰度级）也揭示了在上皮（B）的水肿的变化（由白和黑星这里表示）。从CD患者积极耀斑（ 清晰条 ）或缓解（ 黑网吧 ）样品检测平均折射率显著的差异。这些差异在每个组织类型可见（平均值±SEM，*** P &lt;0.001; C）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54460/54460fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54460/54460fig4.jpg" /> 图 4.白光显微镜和DHM上皮伤口愈合的监测。培养细胞移植sferred入培养皿特殊插入物（ 红色箭头 ）。前培养插入物除去，将细胞用或EGF或丝裂霉素C处理24小时，或者用单独的培养基（未刺激的对照）（A）处理。去除培养插入后，伤口愈合连续通过相衬成像随着时间的推移监测由DHM。细胞轮廓清晰可认。代表量化DHM相衬图像在实验开始示出，后22.5小时，并在测量的后45小时（B）的末端。面板（C）显示了细胞表面覆盖（S C）对应的时间变化，平均电池厚度（d 细胞 ）和细胞干重（DM）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54460/54460fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图 5。通过 定量DHM相位对比图像的 假彩色编码的伪 三维地块 细胞层形态变化示意图 。 图4B在t = 0定量DHM相衬图像的代表性假彩色编码的伪3D绘图，经过22.5小时，在后45小时测量结束时，说明细胞层的厚度控制-为空间发展，丝裂霉素C抑制和3D EGF刺激的细胞。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54460/54460fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Multimodal Quantitative Phase Imaging with Digital Holographic Microscopy Accurately Assesses Intestinal Inflammation and Epithelial Wound Healing at JoVE.com

Multimodal Quantitative Phase Imaging with Digital Holographic Microscopy Accurately Assesses Intestinal Inflammation and Epithelial Wound Healing

Accurate assessment of anti-inflammatory effects is of utmost importance for the evaluation of potential new drugs for the treatment of inflammatory bowel disease. Digital holographic microscopy provides assessment of inflammation in murine and human colonic tissue samples as well as automated multimodal evaluation of epithelial wound healing in vitro.

多式联运定量相成像数字全息显微准确评估肠道炎症和上皮伤口愈合

The incidence of inflammatory bowel disease, i.e., Crohn&#39;s disease and Ulcerative colitis, has significantly increased over the last decade. The ...

multimodal-quantitative-phase-imaging-with-digital-holographic

Nanyang Technological University

Research

JoVE Journal

Medicine

8.3K 次观看.  University Hospital Münster. Accurate assessment of anti-inflammatory effects is of utmost importance for the evaluation of potential new drugs for the treatment of inflammatory bowel disease. Digital holographic microscopy provides assessment of inflammation in murine and human colonic tissue samples as well as automated multimodal evaluation of epithelial wound healing in vitro. 

视频: Multimodal Quantitative Phase Imaging with Digital Holographic Microscopy Accurately Assesses Intestinal Inflammation and Epithelial Wound Healing

Functional Assessment of Intestinal Motility and Gut Wall Inflammation in Rodents: Analyses in a Standardized Model of Intestinal Manipulation

Inflammation of the gastrointestinal tract is a common reason for a variety of human diseases. Animal research models are critical in investigating the complex cellular and molecular of intestinal pathology. Although the tunica mucosa is often the organ of interest in many inflammatory diseases, recent works demonstrated that the muscularis externa (ME) is also a highly immunocompetent organ that harbours a dense network of resident immunocytes.1,2 These works were performed within the standardized model of intestinal manipulation (IM) that leads to inflammation of the bowel wall, mainly limited to the ME. Clinically this inflammation leads to prolonged intestinal dysmotility, known as postoperative ileus (POI) which is a frequent and unavoidable complication after abdominal surgery.3 The inflammation is characterized by liberation of proinflammatory mediators such as IL-64 or IL-1&beta; or inhibitory neurotransmitters like nitric oxide (NO).5 Subsequently, tremendous numbers of immunocytes extravasate into the ME, dominated by polymorphonuclear neutrophils (PMN) and monocytes and finally maintain POI.2 Lasting for days, this intestinal paralysis leads to an increased risk of aspiration, bacterial translocation and infectious complications up to sepsis and multi organ failure and causes a high economic burden.6
In this manuscript we demonstrate the standardized model of IM and in vivo assessment of gastrointestinal transit (GIT) and colonic transit. Furthermore we demonstrate a method for separation of the ME from the tunica mucosa followed by immunological analysis, which is crucial to distinguish between the inflammatory responses in these both highly immunoactive bowel wall compartments. All analyses are easily transferable to any other research models, affecting gastrointestinal function.

Postoperative ileus (POI) is a complication of abdominal surgery leading to increased morbidity and a prolonged hospital stay. Because prophylactic or therapeutic strategies are lacking intensified research is necessary. Therefore we established a standardized and feasible mouse model to investigate the pathophysiology of POI and to study potential therapeutic options.

在啮齿类动物的肠动力及胃肠壁的炎症分析功能评价一个标准化的模式肠道操作

Inflammation of the gastrointestinal tract is a common reason for a variety of human diseases. Animal research models are critical in investigating the ...

科研

医学

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

伤口愈合的小鼠模型

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions preclinically. However, valid assessment of pathological alterations often requires histological analysis, and when performed ex vivo, necessitates death of the animal. Therefore in conventional experimental settings, intra-individual follow-up examinations are rarely possible. Thus, development of murine endoscopy in live mice enables investigators for the first time to both directly visualize the gastrointestinal mucosa and also repeat the procedure to monitor for alterations. Numerous applications for in vivo murine endoscopy exist, including studying intestinal inflammation or wound healing, obtaining mucosal biopsies repeatedly, and to locally administer diagnostic or therapeutic agents using miniature injection catheters. Most recently, molecular imaging has extended diagnostic imaging modalities allowing specific detection of distinct target molecules using specific photoprobes. In conclusion, murine endoscopy has emerged as a novel cutting-edge technology for diagnostic experimental in vivo imaging and may significantly impact on preclinical research in various fields.

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Small animal imaging techniques allow serial diagnostic examinations and therapeutic interventions in vivo. Recently, the scope of applications has significantly widened and currently includes assessment of colonic tumor development, wound healing and monitoring of inflammation. This protocol illustrates these diverse potential applications of murine endoscopy.

鼠内镜<em&gt;体内</em&gt;发生和肠道的评价多模式成像伤口愈合和炎症

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions ...

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 ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

通过轻型显微镜对小鼠急性心肌炎模型中白细胞浸润的定量可视化

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not routinely employed in animal models. Conventional experimental readouts often rely on post mortem and ex vivo analyses, which prevent intra-individual follow-up examinations and increase the number of study animals needed. Fluorescence-mediated tomography enables the non-invasive, repetitive, quantitative, three-dimensional assessment of fluorescent probes. It is highly sensitive and permits the use of molecular makers, which allows for the specific detection and characterization of distinct molecular targets. In particular, targeted probes represent an innovative tool for analyzing gene activation and protein expression in inflammation, autoimmune disease, infection, vascular disease, cell migration, tumorigenesis, etc. In this article, we provide step-by-step instructions on this sophisticated imaging technology for the in vivo detection and characterization of inflammation (i.e., F4/80-positive macrophage infiltration) in a widely used murine model of intestinal inflammation. This technique might also be used in other research areas, such as immune cell or stem cell tracking.

Target-specific probes represent an innovative tool for analyzing molecular mechanisms, such as protein expression in various types of disease (e.g., inflammation, infection, and tumorigenesis). In this study, we describe a quantitative three-dimensional tomographic assessment of intestinal macrophage infiltration in a murine model of colitis using F4/80-specific fluorescence-mediated tomography.

荧光介导的断层扫描对巨噬细胞相关小鼠肠道炎症的检测和定量

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not ...

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During In Vitro Wound Healing

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated experimental procedures, while potentially lacking in precision. In vitro culture of epithelial cell lines provides a suitable platform for researching the cell migratory behavior in wound healing and the impact of treatments on these cells. The physiology of epithelial cells is often studied in non-confluent conditions; however, this approach may not resemble natural wound healing conditions. Disrupting the epithelium integrity by mechanical means generates a realistic model, but may impede the application of molecular techniques. Consequently, microscopy based techniques are optimal for studying epithelial cell migration in vitro. Here we detail two specific methods, the artificial wound scratch assay and the artificial migration front assay, that can obtain quantitative and qualitative data, respectively, on the migratory performance of epithelial cells.

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During  In Vitro Wound Healing

This manuscript describes how incision-like lesions made on cultured epithelial cell monolayers conveniently model wound healing in vitro, allowing for imaging by confocal or laser scanning microscopy, and which can provide high-quality quantitative and qualitative data for studying both cell behavior and the mechanisms involved in migration.

以显微镜为基础的方法评估上皮细胞迁移过程中的体外创面愈合

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated ...

Phase Contrast Magnetic Resonance Imaging in the Rat Common Carotid Artery

Phase contrast magnetic resonance imaging (PC-MRI) is a noninvasive approach that can quantify flow-related parameters such as blood flow. Previous studies have shown that abnormal blood flow may be associated with systemic vascular risk. Thus, PC-MRI can facilitate the translation of data obtained from animal models of cardiovascular diseases to pertinent clinical investigations. In this report, we describe the procedure for measuring blood flow in the common carotid artery (CCA) of rats using cine-gated PC-MRI and discuss relevant analysis methods. This procedure can be performed in a live, anesthetized animal and does not require euthanasia after the procedure. The proposed scanning parameters yield repeatable measurements for blood flow, indicating excellent reproducibility of the results. The PC-MRI procedure described in this article can be used for pharmacological testing, pathophysiological assessment, and cerebral hemodynamics evaluation.

The overall goal of this procedure is to measure the blood flow in the rat common carotid artery by using noninvasive phase contrast magnetic resonance imaging.

大鼠颈总动脉的相位对比磁共振成像

Phase contrast magnetic resonance imaging (PC-MRI) is a noninvasive approach that can quantify flow-related parameters such as blood flow. Previous ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

眼毛刺角膜上皮磨损与角膜创面愈合的模型研究

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.

A protocol for an ex vivo corneal organ culture model useful for wound healing studies is described. This model system can be used to assess the effects of agents to promote regenerative healing or drug toxicity in an organized 3D multicellular environment.