这项工作是由美国国立卫生研究院资助R01 GM087609（以HPL），从英格丽·莱和比尔舒荣誉安东尼舒（以HPL），美国国立卫生研究院资助U01 HL099776（以MTL）的礼物，在Hagey实验室拨款部分支持小儿再生医学和橡树基金会（MTL来和HPL）。 GGW是由斯坦福大学医学院，斯坦福大学医学院科学家培训项目的支持，并NIGMS培训资助GM07365。 MSH是由CIRM临床研究员培训资助TG2-01159支持。 WXH是从萨尔诺夫心血管基金会的资助支持。

注:本文中所描述的所有程序都是根据斯坦福大学行政专家组对实验动物护理（APLAC）制定的准则进行。 1.定时怀孕 - 超数排卵技术（图1） 注:精确定时小鼠胚胎的孕周胎儿外科E16.5和E18.5是至关重要的。在这一节中我们详细介绍我们使用怀孕母马血清（PMS）和人绒毛膜促性腺激素（HCG）注射诱导超数排卵定时鼠标怀孕协议。 <ol><li>注射雌性小鼠（&lt;5％笼）腹腔（IP）与3.0-5.0 PMS国际单位（IU）的100微升PBS 1:00和1天的3:00 PM之间的音量。 </li><li> 12:00至2:00 PM 3天（45至47小时PMS注射后）的，注射雌性小鼠IP与3.0-5.0 IU HCG在100微升PBS体积。 注:HCG注射诱导<html>排卵期大约12小时后注射。 </li><li>紧接着HCG注射，交配女性，年龄在8-16周的男性。 注:我们通常把两女成单个男性的笼子。 </li><li>独立的女性，从第4天上午男性（6:00 - 10:00 AM），并记录为胎龄E0.5。 注:阴道塞可在此时进行检查;然而，观察阴道塞不保证怀孕，女性可以怀孕时没有插头观察。鉴于妊娠通常通过目测和/或触诊在胎龄E16.5和E18.5观察到的，检查在第4天上午阴道塞不是严格必需的。根据我们的经验，并根据不同的应变，超排卵的女性约30-50％的怀孕使用此处所描述的技术。 </li></ol>皮克"/&gt; 图1.原理图超数排卵技术，对小鼠超数排卵定时怀孕示意图，显示程序。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52297/52297fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 2.小​​鼠胎儿手术（背伤人）在E16.5和E18.5胚胎（图2） <ol><li>在手术前，清洁手术室和装备有70％的异丙醇的所有表面。此外，灭菌的所有外科用品和仪器将由高压灭菌它们被用于该过程。一些机构可能允许后续采用热杀菌珠。每个操作，使用无菌包，包括纱布，手术器械。 </li><li>在每分钟之后维持麻醉，每分钟1升2升诱导麻醉孕妇（胎儿年龄E16.5或E18.5）低于2.5％异氟醚/氧气混合物。 </li><li>昆仑坚定正确的麻醉，保证了鼠标的深踩油门反射受到抑制，然后将鼠标在俯卧位。 </li><li>应用兽医眼药膏，如Puralube以防止在手术过程中眼睛发炎或干燥。 </li><li>通过给予时间不超过30秒（ 图2A）的脱毛霜轻应用做准备的腹部。 </li><li>准备腹部无菌手术用聚维酮碘和醇（ 图2B和2C）。 </li><li>采用显微剪刀（ 图2D和2E）显微镜下进行中线剖腹探查术。 </li><li>轻轻地揭露子宫和胎儿选择手术（ 图2F）。 </li><li>使用一个钝头针头灌溉外科领域用温水（38℃）磷酸盐缓冲盐水（PBS）中 注意:一个可以通过仔细弯曲大口径针的尖端制成。 （ 图2G和2H）。 </li><li>的方式，使定位胎儿s的接入背部。 </li><li>通过用7-0尼龙线通过子宫覆打算背伤人（ 图2I）的网站荷包缝合。位置荷包在背部左侧或脊髓的右边的一个区域，并在子宫壁缺乏大血管的区域。 </li><li>使在荷包（ 图2J）的中心，通过子宫壁和羊膜囊3毫米的切口。 </li><li>冲洗切口部位用温水（38℃）PBS。 </li><li>采用显微剪刀，切割单个全层切除伤口，大约为1毫米的长度，在胎儿的背部。 </li><li>轻轻印迹切口部位用干棉尖涂药。 </li><li>注3微升量墨汁皮下伤口部位标记伤口的位置（ 图2K）。 </li><li>用温水（38℃），PBS冲洗，以确保墨水一直保留在伤口部位。 </li><li>手术有我的助手nject温暖（38°C）通过钝尖10个G注射器插入羊膜囊的钱袋PBS被关闭（ 图2L）。收回的注射器作为荷包封接近完成（ 图2M）。 </li><li>轻轻返回子宫进入腹腔（ 图2N）。 </li><li>埃弗特皮肤和腹膜。 </li><li>有手术助理灌溉腹腔用温水（38℃）PBS。 </li><li>迅速关闭腹部被装订的皮肤和腹膜封闭（ 图2O）。标准封盖在两层进行;腹膜和在一层，皮下组织和皮肤在第二层中的腹部肌肉。对于眼前的牺牲和收获的胎儿，我们的演示显示了一层封闭。 </li><li>放置在观察所述动物在温暖的培养箱设定为37℃，30分钟，或直至动物重新获得足够意识保持胸骨斜卧。 </li><li>不要沤瓮动物给其他动物的公司，直到它已完全从过程中回收。 </li><li>一旦从麻醉，并在随后的48小时的觉醒，辖皮下注射丁丙诺啡（0.05毫克/千克），每12小时为镇痛根据需要基于疼痛评估。根据需要，通过皮下注射管理卡洛芬（5毫克/千克）的额外手术后疼痛减轻。 </li><li>回到动物的笼子，并为他们提供食物和水自由采食。 </li><li>密切留意市民对疼痛的表现。 </li><li> 48小时后手术，牺牲孕妈妈与异氟醚和收获受伤胎儿的过量。为了做到这一点，调整异氟烷浓度为5％或更高，并维持曝光呼吸停止后1分钟。确认安乐死颈椎脱位。收获的未受伤胚胎为年龄匹配的对照。后期胚胎应具有安乐死与IACUC建议，如相一致的单独的方法去人头，颈椎脱位，或化学注射。 </li></ol><img alt="图2" src="/files/ftp_upload/52297/52297fig2highres.jpg" /> 鼠标腹部图2.原理图小鼠胎儿手术。一般步骤背伤人案E16.5和E18.5小鼠胚胎。（A）脱毛。（B和C）小鼠腹部的制备。（D）显微镜用于外科手术通过子宫的过程。（E）中线剖腹探查术。（F）曝光子宫。（G）创造钝尖针头。子宫用温盐水（H）灌溉。（I）创造了荷包缝合。（J）切口墙上1毫米全层切除伤口的产生。（K）皮下注射印度墨水。（L和M）封闭的荷包缝合。（N和O）关闭腹部。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52297/52297fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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The authors have nothing to disclose.

这里介绍的手术协议描述的小鼠胎儿无瘢痕愈合首次出版于2006年，由我们的实验室10的切除模式。除了 ​​切除伤人11等建立模型，对小鼠胎儿无瘢痕愈合的切口车型存在，以及12,13。胎儿无瘢痕愈合猴，羊，兔，负鼠，老鼠和调查的报道14-17。然而，小鼠表现为探索胎儿无瘢痕愈合的理想模型，由于其相对较低的按日补助笼的成本和良好的特点的基因组。此外，可在胚胎发育过程中使用转基因小鼠提供系统的机会，正是破译疤痕修复的机制来实现功能时空遗传损失/收益。 尽管有这些优势，目前小鼠在胎儿手术一定的技术挑战，由于其体积小。鉴于麻醉对孕妈妈的压力，应十分注意采取监视异氟烷/氧气被管理的水平。 2.5％异氟醚/氧气每分钟之后维持麻醉，每分钟1 L 2 L A混合物应认真遵守。在这个关键因素的偏差或波动显著影响发病率。妥善安置了荷包缝合代表手术的技术上最具挑战性的方面。如果没有正确放置此针会造成羊水和PBS后关闭泄漏。流体在子宫的不足量将导致创伤的胚胎，并可能诱发过早的子宫收缩。出于这个原因，置换羊水后开口子宫丢失的也是这种手术模型的关键步骤。必须小心，以不断地喷射温PBS作为荷包被关闭。要求这些因素显著故障排除和我们强调的是注意这些细节会增加手术成功的可能性。然而，即使是那些经验丰富的外科手应该计划的约40％在E​​16.5一个胎儿发病率。 了解胎儿无瘢痕愈合的持有价值，转化医学在发展，成人阶段的目的是在体内调制纤维化行为。相较于成人皮肤，胎儿皮肤具有显影真皮，降低抗拉强度，新生毛囊，和不同的ECM组分2,4。胎儿皮肤具有升高比率III型胶原的相比，成人皮肤4,9到I型相比，成人皮肤，更少的和更小的成熟肥大细胞，角蛋白8和19，和更高水平的透明质酸表达。通过破译基本无瘢痕愈合胎儿的机制，我们就可以开始转向负责纤维化对再生型成人在分子和细胞通路。出现Ø˚F谱系追踪和其他遗传工具在鼠标打开了充满希望的新途径无瘢痕伤口愈合的调查。鉴于发病率和胎儿手术相关过早的子宫收缩的固有的高利率，对胎儿无瘢痕皮肤的调查伤口愈合体内需要精确和可重复的手术模式。在这里，我们详细胎儿无瘢痕愈合的E16.5（无疤痕）和E18.5（疤痕）的小鼠胚胎的背部可再现的模型。

胎儿皮肤伤口愈合迅速，scarlessly直到怀孕后期1。胎疤痕伤口修复的特征在于正常组织结​​构和功能的再生。从瘢痕瘢痕表型的转变发生在怀孕的人类和周围18天胚胎（E18）小鼠2,3孕晚期。相较于成人，胎儿伤口修复的特征在于快速上皮，结缔组织沉积和成纤维细胞的迁移。 在早期胎儿发育许多研究都提供了可能的解释为瘢痕伤口愈合的现象。炎症是成人创面修复的一个基本组成部分;然而，胎儿伤口的特征在于缺乏急性炎症4。这是否是免疫系统的过程中的胎儿阶段官能不成熟的结果仍不清楚。最近的一项研究表明，在丰富，垫差异urity，和肥大细胞在E15与E18的胎儿皮肤的功能可以负责从瘢痕表型的转变中，至少在小鼠3。其他的研究断定，在性能和丰富的胎儿和成人伤口巨噬细胞的差异是负责正常的细胞外基质（ECM）的过程中胎儿创伤修复5的改革。 胎儿和成人发育期间差异在环境因素也可能影响伤口修复。龙雅葛和他的同事发现，从胎儿伤口液具有较高水平的透明质酸刺激活性相比，无成人伤口流体6。因此，更高水平的透明质酸，糖胺聚糖促进有利于细胞运动和增殖的微环境，在胎儿的伤口环境的可负责的瘢痕的表型在早期胎儿发育看见。其他线路的证据指向的一个事实，即在羊升伤口环境相对低氧血症和淹没在无菌羊水丰富的生长因子，7。然而，没有明确的答案已提供用于胚胎发育过程中的关键事件或因素触发从瘢痕再生纤维变性修复的过渡。 理解的机制负责在胎儿无瘢痕愈合就必须精确和可再现的模型。在这里，我们详细胎儿无瘢痕愈合的E16.5（无疤痕）和E18.5（疤痕）的小鼠胚胎的背部可再现的模型。此外，该模型的微小变化可用于执行许多进一步的研究，如胎儿的伤口和皮肤8,9的基因表达分析。鉴于精确定时怀孕是此无瘢痕伤口愈合模式成功的关键再演，我们对超排定时怀孕也详细我们的协议。

<table><tbody><tr><td>7-O MONOSOF Suture</td><td>eSuture</td><td>SN-1647G</td><td></td></tr><tr><td>Surgical Forceps</td><td>Kent Scientific</td><td>INS650916</td><td></td></tr><tr><td>Micro-scissors</td><td>Kent Scientific</td><td>INS600127</td><td></td></tr><tr><td>Autoclip 9 mm</td><td>Texas Scientific Instruments</td><td>205060</td><td></td></tr><tr><td>Insulin Syringe</td><td>Thermo Fisher Scientific</td><td>22-272-382</td><td></td></tr><tr><td>Black Pigment</td><td>AIMS</td><td>242</td><td></td></tr><tr><td>BD Safety-Lok 3 ml Syringe</td><td>BD Biosciences</td><td>309596</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>Life Technologies</td><td>10010-049</td><td></td></tr><tr><td>OPMI-MD Surgical Microscope</td><td>Carl Zeiss Surgical Inc</td><td></td><td></td></tr><tr><td>Pregnant Mares Serum (PMS)</td><td>Millipore</td><td>367222</td><td></td></tr><tr><td>Human Chorionic Gonadotropin (HCG)</td><td>Sigma-Aldrich</td><td>CG10</td><td></td></tr><tr><td>Povidone Iodine Prep Solution</td><td>Dynarex</td><td>1415</td><td></td></tr><tr><td>Nair (depilatory cream)</td><td>Church and Dwight Co.</td><td>22600267058</td><td></td></tr></tbody></table>

a mouse fetal skin model of scarless wound repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

对于组织学分析，皮肤伤口在E16.5和E18.5小鼠胚胎的背部皮肤，应收获后48小时伤人，固定在4％PFA和石蜡包埋。在荧光的转基因模型中，冷冻保存与十月可能是适当的。有几种污渍可以被用于可视化的细胞和结缔组织结构。苏木精和曙红是一种两色色斑，即污迹细胞核蓝色和嗜酸性结构（ 即 ，细胞质和细胞外胶原）的红色，粉红色，橙色和各种色调。马洛里的三色是一个三色染色选自苯胺蓝，酸性品红和橙G，最适合于从周围结缔组织区分细胞。 <img alt="图3" src="/files/ftp_upload/52297/52297fig3highres.jpg" /> 图3.无疤痕E16.5胎儿伤口组织学。 （A），HE染色染色再veals完整表皮细胞再生和炎症细胞的数目轻微上升存在（箭头）。（100倍;巴= 100微米）（B）的曙红染色显示印度油墨（箭头）围绕再生毛囊（箭头）（400×;巴= 25 。微米）（C）马洛里的三色染色揭示了毛囊（400X的存在细小网状真皮层胶原蛋白的图案;酒吧= 25微米）。从科尔韦尔等 10授权转载<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52297/52297fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 如果背切除伤口的适当大小（1毫米）和深度（全层）的​​，苏木精和曙红染色将揭示E16.5皮肤愈合与最小的疤痕，表皮细胞再生完整的，只有一个小的增​​加的炎性数细胞（ 图3A）。此外，这些wounDS应愈合用约正常皮肤结构和包含的损伤部位内再生毛囊（箭头; 图3B）。正确应用墨汁到新创建的伤口应导致油墨沉积在损伤部位（箭头头; 图3B）。最后，三色染色应揭露别来无恙真皮（ 图3C）的细小网状真皮层胶原蛋白的图案特征。 <img alt="图4" src="/files/ftp_upload/52297/52297fig4highres.jpg" /> 图4.瘢痕E18.5胎儿伤口组织学。 （A）苏木和HE染色显示增加伊红 ​​染色在真皮的损伤部位（箭头）。（200X，酒吧= 50微米），（B）马洛里的三色染色显示密集的真皮层胶原蛋白（箭头）（400X;巴= 25微米）。转载自科尔韦尔等人的许可。10 相较于在E16.5，苏木和在E18.5制成并收获后48小时伤人应揭示的致密瘢痕与正常皮肤结构的损失在损伤（ 图4A）的部位背侧伤口伊红染色制成的伤口。同样，三色染色揭示了杂乱无章的胶原沉积密集模式（箭头; 图4B）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52297/52297fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about A Mouse Fetal Skin Model of Scarless Wound Repair at JoVE.com

A Mouse Fetal Skin Model of Scarless Wound Repair

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

对无瘢痕修复鼠标胎儿皮肤模型

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

a-mouse-fetal-skin-model-of-scarless-wound-repair

Nanyang Technological University

Research

JoVE Journal

Medicine

12.2K 次观看.  Stanford University School of Medicine. During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

视频: A Mouse Fetal Skin Model of Scarless Wound Repair

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

三维人体皮肤重建模型：一种工具来研究正常皮肤恶性黑色素瘤进展

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

科研

生物工程

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

一个完整的皮肤缺损模型，以评估生物材料的血管化<em&gt;体内</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

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. ...

医学

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

三维全层皮肤的生成等效和自动伤人

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are still occurring, particularly in reconstructive flap surgery. Multiple experimental flap models have been developed to analyze underlying causes and mechanisms and to investigate treatment strategies to prevent ischemic complications. The limiting factor of most models is the lacking possibility to directly and repetitively visualize microvascular architecture and hemodynamics. The goal of the protocol was to present a well-established mouse model affiliating these before mentioned lacking elements. Harder et al. have developed a model of a musculocutaneous flap with a random perfusion pattern that undergoes acute persistent ischemia and results in ~50% necrosis after 10 days if kept untreated. With the aid of intravital epi-fluorescence microscopy, this chamber model allows repetitive visualization of morphology and hemodynamics in different regions of interest over time. Associated processes such as apoptosis, inflammation, microvascular leakage and angiogenesis can be investigated and correlated to immunohistochemical and molecular protein assays. To date, the model has proven feasibility and reproducibility in several published experimental studies investigating the effect of pre-, peri- and postconditioning of ischemically challenged tissue.

The window of the murine dorsal skinfold chamber presented visualizes a zone of acute persistent ischemia of a musculocutaneous flap. Intravital epi-fluorescence microscopy permits for direct and repetitive assessment of the microvasculature and quantification of hemodynamics. Morphologic and hemodynamic results can further be correlated with histological and molecular analyses.

在背皮褶商会鼠标的缺血性组织损伤:一个皮瓣模型，探讨急性缺血持续

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are ...

Assessment of Acute Wound Healing using the Dorsal Subcutaneous Polyvinyl Alcohol Sponge Implantation and Excisional Tail Skin Wound Models.

Wound healing is a complex process that requires the orderly progression of inflammation, granulation tissue formation, fibrosis, and resolution. Murine models provide valuable mechanistic insight into these processes; however, no single model fully addresses all aspects of the wound healing response. Instead, it is ideal to use multiple models to address the different aspects of wound healing. Here, two different methods that address diverse aspects of the wound healing response are described. In the first model, polyvinyl alcohol sponges are subcutaneously implanted along the mouse dorsum. Following sponge retrieval, cells can be isolated by mechanical disruption, and fluids can be extracted by centrifugation, thus allowing for a detailed characterization of cellular and cytokine responses in the acute wound environment. A limitation of this model is the inability to assess the rate of wound closure. For this, a tail skin excision model is utilized. In this model, a 10 mm x 3 mm rectangular piece of tail skin is excised along the dorsal surface, near the base of the tail. This model can be easily photographed for planimetric analysis to determine healing rates and can be excised for histological analysis. Both described methods can be utilized in genetically altered mouse strains, or in conjunction with models of comorbid conditions, such as diabetes, aging, or secondary infection, in order to elucidate wound healing mechanisms.

Here, two murine wound healing models are described, one designed to assess cellular and cytokine wound healing responses and the other to quantify the rate of wound closure. These methods can be used with complex disease models such as diabetes to determine mechanisms of various aspects of poor wound healing.

使用多盐皮下聚乙烯醇海绵植入和外向尾皮肤伤口模型进行急性伤口愈合评估。

Wound healing is a complex process that requires the orderly progression of inflammation, granulation tissue formation, fibrosis, and resolution. Murine ...

免疫与感染

A Mouse Model of Mechanotransduction-driven, Human-like Hypertrophic Scarring

Hypertrophic scarring (HTS) is an abnormal process of wound healing that results in excessive scar tissue formation. Over the past decade, we have demonstrated that mechanotransduction&#8212;the conversion of mechanical stimuli into cellular responses&#8212;drives excessive fibrotic scar healing. A mouse model to assess human-like hypertrophic scarring would be an essential tool for examining various therapeutics and their ability to reduce scarring and improve healing. Specifically, our laboratory has developed a murine wound model that increases mechanical strain to promote human-like HTS. This&#160;protocol utilizes biomechanical loading devices, made from modified 13 mm palatal expanders, whose arms are placed on either side of the incision and distracted incrementally apart in order to apply continuous tension across the wound bed during healing. Over nearly two decades of use, this model has been significantly advanced to improve efficacy and reproducibility. Using the murine HTS model, significant dermal fibrotic scars can be induced to be histologically comparable to human hypertrophic scars. This murine model provides an environment to develop biologics involved in the treatment of HTS and mechanotransduction-related conditions such as foreign body response.

This protocol will explain how to establish a hypertrophic scarring murine model that increases mechanotransduction signaling to simulate human-like scarring. This method involves increasing mechanical tension across a healing incision in a mouse and using a specialized device to create reproducible, excessive scar tissue for detailed histological and bioinformatic analyses.

机械转导驱动的类人增生性瘢痕形成小鼠模型

Hypertrophic scarring (HTS) is an abnormal process of wound healing that results in excessive scar tissue formation. Over the past decade, we have ...

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, ...

A Murine Model of a Burn Wound Reconstructed with an Allogeneic Skin Graft

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and require surgical debridement and skin grafting. Successful integration of the donor graft into a recipient wound bed depends on timely recruitment of immune cells, robust angiogenic response and new extracellular matrix formation. The development of novel therapeutic agents, which target some key processes involved in wound healing, are hindered by the lack of reliable preclinical models with optimized objective assessment of wound closure. Here, we describe an inexpensive and reproducible model of experimental full thickness burn wound reconstructed with an allogeneic skin graft. The wound is induced on the dorsum surface of anaesthetized inbred wild type mice from the BALB/C and SKH1-Hrhr backgrounds. The burn is produced using a brass template measuring 10 mm in diameter, which is preheated to 80 &#176;C and delivered at a constant pressure for 20 s. Burn eschar is excised 24 hours after the injury and replaced with a full thickness graft harvested from the tail of a genetically similar donor mouse. No specialized equipment is required for the procedure and surgical techniques are straightforward to follow. The method may be effortlessly implemented and reproduced in most research settings. Certain limitations are associated with the model. Due to technical difficulties, the harvest of thinner split thickness skin grafts is not possible. The surgical method we describe here allows for the reconstruction of burn wounds using full thickness skin grafts. It may be used to carry out preclinical therapeutic testing.

The aim of this study was to develop a murine model of burn wound healing. A thermal burn was induced on the dorsal skin of mice using a preheated brass template. Burned tissue was debrided and overlaid with a skin graft harvested from the tail of a genetically similar donor mouse.

用异体皮肤移植重建的烧伤伤口的穆林模型

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and ...

生物学

Optimized Protocol for Mouse Wound Healing Model with Stem Cell-Derived Exosomes

Mouse Wound Models and Preparation of Single-Cell Suspensions 

The management of acute full-thickness skin injuries presents a considerable challenge in clinical practice. The complexity of wound healing, involving diverse cell populations and the intricate wound microenvironment, complicates the assessment of treatment effects and their interactions using traditional experimental approaches. Single-cell transcriptome sequencing technology has revolutionized the understanding of cellular functions and mechanisms during wound healing. However, the variability in methodologies for creating mouse wound models introduces confounding factors that can impact experimental results. Furthermore, the process of dissociating mouse skin tissues presents significant technical hurdles, highlighting the critical need for high-quality single-cell suspensions for accurate transcriptome sequencing. Therefore, this study aims to optimize the construction of mouse wound models to accurately assess changes in wound closure while eliminating variables that could influence the subsequent sequencing result. Additionally, the preparation of single-cell suspensions was performed using both enzyme preparation protocols and test kit protocols to optimize cost and effectiveness.

Mouse Wound Models and Preparation of Single-Cell Suspensions

This study presents two crucial experimental techniques: the construction of a murine wound model for assessing wound closure and the preparation of single-cell suspensions from murine skin to preserve cellular viability.

The management of acute full-thickness skin injuries presents a considerable challenge in clinical practice. The complexity of wound healing, involving ...

对无瘢痕修复鼠标胎儿皮肤模型

摘要

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Mouse Wound Models and Preparation of Single-Cell Suspensions