笔者想承认资金支持，从国家健康和医学研究委员会（NHMRC），澳洲（项目格兰特ID:632512）。路易丝·邓恩由国家心脏基金会事业发展奖由NHMRC早期职业团契和克里斯蒂娜Bursill的支持。

1。夹板包扎敷料的制备<ol><li> 0.5毫米厚的硅薄膜和使用剪刀或活检冲床创建硅磁盘上，勾勒出10毫米的圆圈。 </li><li>中心一个5毫米的穿刺活检拳打在10 mm的圆圈中间，然后用力按压，创建一个孔洞，形成一个"甜甜圈"般的光盘将被用来作为夹板。 </li><li>纲要10毫米的圆圈，在一个透明的闭塞敷料，如Opsite和使用剪刀创建循环敷料。 </li></ol> 2。实验动物<ol><li>获取将要执行的所有实验动物伦理委员会批准。 </li><li>使用8周龄（22-26克）的雄性小鼠从商业饲养员（ 如杰克逊实验室）。 </li><li>养老鼠在标准条件下，21℃，12小时免费获得食物和水的光暗周期。 </li><li> （可选）:糖尿病可在6〜7周龄小鼠诱导由丸腹腔仁济CTION 165毫克/公斤菌素（柠檬酸缓冲液，pH值4.5），高血糖定期血糖监测（血糖仪AccuChek）确认。糖尿病小鼠有尿，所以他们的床上用品可能需要更频繁地更换，以消除潮湿，应密切监测它们的权重。 </li></ol> 3。麻醉和手术的准备<ol><li>诱导全身麻醉，用5％异氟醚在100％的氧气（流速1升/分钟），并用1-3％异氟烷维持麻醉。 </li><li>确保深踩油门反射被抑制的鼠标和鼠标放置在俯卧位。 </li><li>准备手术的区域，从基地的脖子，用指甲刀去除毛皮〜3厘米进一步回落的背部和两个肩胛骨之间。 </li><li>脱毛霜轻应用，可申请不超过2分钟。可以使用湿纱布，以确保所有的奶油，并除去剩余的毛皮。 </li><li>擦拭皮肤与酒精棉签和两个应用程序10％的聚维酮碘（优碘）和悬垂鼠标。 </li></ol> 4。伤口的切除和夹板<ol><li>在图1中图解表示的小鼠的伤口愈合模型。 </li><li>使用无菌4毫米活检冲床概述两个圆形型态的伤口上的鼠标的中线的两侧肩的水平（参见图2a-2b的 ）。 </li><li>使用锯齿钳，解除皮肤的轮廓和虹膜剪创建一个全层伤口穿过皮下组织（ 图2C-2D），包括脂膜肉质（ 图2e）在中间，和消费的一块圆形组织。 </li><li>重复该过程中线的另一侧（ 图2f）上的伤口。 </li><li>撕下的保护涂层从硅夹板的每一侧。 </li><li>应用cyanoacryla"特粘合剂（ 如强力胶或胶疯狂）一侧的硅酮夹板。 </li><li>中心夹板的伤口（ 图2g）和夹板固定中断6-0尼龙线缝合，以确保定位（ 图2H）。 </li><li>重复的夹板上的其他伤口的过程。 </li><li>如果需要在此时间点，适用于一个伤口（ 图2i），车辆控制的其他的治疗化合物以进行测试（100微升）。 </li><li>用透明闭塞敷料（例如，OpSite）（ 图2j的-2k的 ）覆盖伤口。 </li><li>一把尺子放在下面的夹板和采取的显微照片（ 图21）。 </li></ol> 6。术后管理<ol><li>卡洛芬（5毫克/公斤），每天服用一次通过皮下注射液用于手术后疼痛缓解。 </li><li>手术后动物笼和维护热米安非他明类兴奋剂，直到完全康复。 </li><li>监控动物每天两次，表现为疼痛和体重减轻。我们没有观察到毛的行为显示疼痛或减肥。 </li></ol> 7。伤口的测量和处理<ol><li>每天可以测量伤口。 </li><li>诱导全身麻醉，用5％异氟醚气体（流速为1升/分钟），然后确保鼠标深感觉反射被抑制使用1-3％异氟醚。 </li><li>轻轻剥开闭塞敷料钳。 </li><li>手术使用的游标卡尺来测量伤口的直径。沿X，Y和Z轴（ 图2米-2°），以及以供将来参考的显微照片（ 图2p）的三个测量值的平均值。 </li><li>可选:在这个时候，可以评估的动物的血液灌流，用激光多普勒成像仪（ 图2Q）。 </li><li>可以重新适用的治疗化合物的车辆控制的perforMED在这一点上。 </li><li>一个干净透明的包扎，然后再应用和动物保持温暖，直到完全康复。 </li><li>注:如果没有正确固定夹板，伤口会很容易收缩（2R图 ）。 </li></ol> 8。组织学分析<ol><li>安乐死过量的麻醉小鼠后10天。 </li><li>使用镊子和，手术刀刀片去除缝线，并仔细剥离夹板。 </li><li>使用虹膜剪创建一个宽，全切除伤口区域周围和下孵育组织中的4％多聚甲醛的磷酸盐缓冲盐水（PBS），在4℃下过夜。 </li><li>组织转移到17％蔗糖的PBS溶液中的另外24小时，在4℃，然后除去多余的溶液在-80℃下通过仔细地涂抹在OCT化合物中，冷冻的组织和地点</li><li>苏木精和伊红染色，可以使用可视化的卷绕结构和上皮缺口。 Neovascularisation免疫组化分析，可以评估的数量来确定的毛细血管（血管性血友病因子）。 </li></ol>

<ol>
	<li>Sen, C. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+skin+wounds:+a+major+and+snowballing+threat+to+public+health+and+the+economy.">Human skin wounds: a major and snowballing threat to public health and the economy.</a> Wound Repair. 17, 763-771 (2009).</li><li>Sen, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing+essentials:+let+there+be+oxygen.">Wound healing essentials: let there be oxygen.</a> Wound Repair Regen. 17, 1-18 (2009).</li><li>Gurtner, G. C., Werner, S., Barrandon, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+repair+and+regeneration.">Wound repair and regeneration.</a> Nature. 453, 314-321 (2008).</li><li>Lindblad, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Considerations+for+selecting+the+correct+animal+model+for+dermal+wound-healing+studies.">Considerations for selecting the correct animal model for dermal wound-healing studies.</a> J. Biomater. Sci. Polym. Ed. 19, 1087-1096 (2008).</li><li>Grose, R., Werner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound-healing+studies+in+transgenic+and+knockout+mice.">Wound-healing studies in transgenic and knockout mice.</a> Mol. Biotechnol. 28, 147-166 (2004).</li><li>Reid, R. R., Said, H. K., Mogford, J. E., Mustoe, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+future+of+wound+healing:+pursuing+surgical+models+in+transgenic+and+knockout+mice.">The future of wound healing: pursuing surgical models in transgenic and knockout mice.</a> J. Am. Coll. Surg. 199, 578-585 (2004).</li><li>Fang, R. C., Mustoe, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+wound+healing:+utility+in+transgenic+mice.">Animal models of wound healing: utility in transgenic mice.</a> J. Biomater. Sci. Polym. Ed. 19, 989-1005 (2008).</li><li>Wong, V. W., Sorkin, M., Glotzbach, J. P., Longaker, M. T., Gurtner, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+approaches+to+create+murine+models+of+human+wound+healing.">Surgical approaches to create murine models of human wound healing.</a> J. Biomed. Biotechnol. 2011, 969618 (2011).</li><li>Galiano, R. D., Michaels, J. t., Dobryansky, M., Levine, J. P., Gurtner, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+and+reproducible+murine+model+of+excisional+wound+healing.">Quantitative and reproducible murine model of excisional wound healing.</a> Wound Repair. 12, 485-492 (2004).</li><li>Galiano, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topical+vascular+endothelial+growth+factor+accelerates+diabetic+wound+healing+through+increased+angiogenesis+and+by+mobilizing+and+recruiting+bone+marrow-derived+cells.">Topical vascular endothelial growth factor accelerates diabetic wound healing through increased angiogenesis and by mobilizing and recruiting bone marrow-derived cells.</a> The American Journal of Pathology. 164, 1935-1947 (2004).</li><li>Thangarajah, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+basis+for+impaired+hypoxia-induced+VEGF+expression+in+diabetic+tissues.">The molecular basis for impaired hypoxia-induced VEGF expression in diabetic tissues.</a> Proceedings of the National Academy of Sciences of the United States of America. 106, 13505-13510 (2009).</li><li>Raza, A., Bayles, C., Biebel, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+Bacterial+Growth+and+Moisture+Handling+Properties+of+Transparent+Dressings:+3M+Tegaderm+Transparent+Dressing,+3M+Tegaderm+HP+Transparent+Dressing,+and+Opsite+IV3000+Transparent+Dressing.">Investigation of Bacterial Growth and Moisture Handling Properties of Transparent Dressings: 3M Tegaderm Transparent Dressing, 3M Tegaderm HP Transparent Dressing, and Opsite IV3000 Transparent Dressing.</a> Smith and Nephew Report. , (1998).</li><li>Chung, T. Y., Peplow, P. V., Baxter, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Testing+photobiomodulatory+effects+of+laser+irradiation+on+wound+healing:+development+of+an+improved+model+for+dressing+wounds+in+mice.">Testing photobiomodulatory effects of laser irradiation on wound healing: development of an improved model for dressing wounds in mice.</a> Photomed. Laser Surg. 28, 589-596 (2010).</li></ol>

什么都没有透露。

这是一个实验小鼠模型皮肤伤口愈合。在此模型中阿显着的特征是利用硅胶夹板，以防止伤口收缩，使再上皮化，形成新的组织，可能会发生，类似的过程发生在人类。此模型是通用的，可以用来评估在生理和病理（ 如糖尿病）的设置的伤口愈合。该模型也可使用在一种经济的环境中，以评估潜在的伤口愈合或血管生成疗法。每个鼠标作为自己的控制，动物数量最小化。此模型所需的外科...

伤口愈合的受损是负责重大的发病率和死亡率全球糖尿病患者1,2，这是尤其如此。在人类中，伤口愈合是一个连续的过程，其中有显着的重叠3。紧随伤人，炎症过程的开始。炎症细胞释放的因素，鼓励细胞增殖，迁移和血管生成的过程。再上皮化和新组织形成后重构的相位，既需要细胞凋亡及基质蛋白如胶原蛋白的重新组织。 复杂伤口愈合目前尚未在体外复制，这需要使用的动物模型。迄今为止，伤口愈合的研究已被限制到大型动物模型中，如猪，以确保愈合过程是等价的，与人类。但是，使用大阿尼玛LS这类研究可能是困难的房子，并总是不踏实4。该实验室的的鼠标代表经济动物模型，可以很容易地操纵基因机制研究5-7。然而，小鼠的伤口愈合不同的人类的，主要是由于收缩的过程中，8。这部分是由于广泛皮下横纹肌层脂膜肉质是主要缺席的人类。在小鼠中，该肌肉层使皮肤更深的肌肉独立移动，负责皮肤以下伤人的迅速收缩。 为了克服这个限制，可以适应小鼠的伤口愈合，用夹板固定（ 图1）8,9复制人类伤口愈合。在这段视频中，我们展示了夹板小鼠伤口模型，消除伤口收缩和更接近人类的过程中重新epithelialization和新组织的形成。在此模型中，两个全层切除，包括脂膜肉质上创建的背部，一个在中线的每一侧的鼠标。有机硅夹板放置在伤口周围与粘合剂的援助和夹板然后间断缝合固定。各小鼠作为它自己的控制，与接受治疗的伤口与其他车辆控制，从而降低动物数量。局部应用后，透明的闭塞敷料被应用。该敷料可以被删除，当需要用于进一步的局部应用和/或测定伤口面积10,11。通过免疫组化的实验，在完成伤口缝合，形态结构和新生血管的程度可以评估。这种经济和容易执行的模型也可以被用来评估糖尿病或其它病理生理学的上下文中的伤口愈合。

<table border="0">
<tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Press-to-seal silicone sheeting 0.5 mm thick</td>
 <td>Invitrogen</td>
 <td>P18178</td>
 <td>Cut into "donuts" with external diameter of 1cm external, 0.5 cm internal diameter</td>
 </tr>
 <tr>
 <td>Biopsy punch 5 mm</td>
 <td>Steifel</td>
 <td>BC-B1-0500</td>
 <td>To outline wound area to be excised</td>
 </tr>
 <tr>
 <td>Vannas scissors 8.5 cm curved</td>
 <td>World Precision Instruments </td>
 <td>501232</td>
 <td>For wound incision and excision</td>
 </tr>
 <tr>
 <td>Dumonte #7b forceps, 11 cm</td>
 <td>World Precision Instruments</td>
 <td>501302</td>
 <td>To grip skin when creating incision and excising skin </td>
 </tr>
 <tr>
 <td>Graefe forceps, serrated 10cm</td>
 <td>World Precision Instruments</td>
 <td>14142</td>
 <td>To help attach silicone splint to skin</td>
 </tr>
 <tr>
 <td>Needle holder, smooth jaws, curved, 12.5 cm</td>
 <td>World Precision Instruments</td>
 <td>14132</td>
 <td></td>
 </tr>
 <tr>
 <td>Malis forceps, smooth, straight, 12 cm</td>
 <td>Codman and Shurtleff, Inc (J&amp;J)</td>
 <td>80-1500</td>
 <td>To suture the silicon rings to the skin</td>
 </tr>
 <tr>
 <td>Ruler, 0.5 mm gradation</td>
 <td>n/a</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Calipers 0.25 mm gradation</td>
 <td>Duckworth and Kent</td>
 <td>9-653</td>
 <td>To measure wound area</td>
 </tr>
 <tr>
 <td>Opsite FlexiFix transparent adhesive film. 10 cm x 1 m</td>
 <td>Smith &amp; Nephew</td>
 <td>66030570</td>
 <td></td>
 </tr>
 <tr>
 <td>Rimadyl (Carprofen)</td>
 <td>Pfizer</td>
 <td>462986</td>
 <td></td>
 </tr>
 </tbody>
</table>

murine model of wound healing

伤口愈合和修复，是发生在人类生活中最复杂的生物过程。伤好后，多种生物途径被激活。伤口愈合受损，发生在糖尿病患者中，例如，可导致截肢等严重的不利后果。因此，有动力开发新的药物，促进伤口修复。检测这些已经被限制到大动物模型，如猪，这往往是不切实际的。老鼠代表了理想的临床前模型，因为它们是经济和适合的遗传操作，它允许进行机械的调查。然而，伤口愈合的小鼠，人类是根本不同的，因为它主要通过收缩发生。我们的小鼠模型，克服这将夹板在伤口周围。夹板伤口，然后修复过程依赖于上皮细胞增殖和血管生成，密切镜像人的伤口愈合的生物学过程。虽然这个小鼠模型需要一致性和护理，不涉及复杂的外科技术，并允许为可靠的测试有前途的代理，例如，可能会促进血管生成或抑制炎症。此外，各小鼠作为它自己的控制制备两个伤口，使应用程序的试验化合物，并在同一个动物的车辆控制。总之，我们展示了一个实用，易于学习，伤口愈合，这是与人类相媲美的强大的模型。

甲伤口闭合曲线确定伤口的平均直径，通过计算和表达的结果的百分比， 即 100 - （0天的直径/天×直径）。在这个实验中的治疗化合物（或车辆用控制）每天施加到伤口。的治疗化合物大大加速伤口闭合（ 图3）。重要的是要注意，夹板必须维持的持续时间的实验，去除夹板会导致伤口迅速收缩（ 图2R）和伤口愈合的人类中观察到的模式偏离。 <p class="jove_content"...

Watch this Scientific Journal Video about Murine Model of Wound Healing at JoVE.com

Murine Model of Wound Healing

甲小鼠模型的皮肤伤口愈合，可用于评估治疗化合物在生理和病理生理设置。

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. ...

Research

JoVE Journal

Medicine

63.6K 次观看.  The Heart Research Institute. 甲小鼠模型的皮肤伤口愈合，可用于评估治疗化合物在生理和病理生理设置。

视频: Murine Model of Wound Healing

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 ...

科研

医学

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.

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 ...

Demonstration of the Rat Ischemic Skin Wound Model

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and unrelieved pressure due to immobility. Animal models have been developed that attempt to mimic these conditions for the purpose of furthering our understanding of the complexity of chronic wounds. The model described herein is a rat ischemic skin flap model that permits a prolonged reduction of blood flow resulting in wounds that become ischemic and resemble a chronic wound phenotype (reduced vascularization, increased inflammation and delayed wound closure). It consists of a bipedicled dorsal flap with 2 ischemic wounds placed centrally and 2 non-ischemic wounds lateral to the flap as controls. A novel addition to this ischemic skin flap model is the placement of a silicone sheet beneath the flap that functions as a barrier and a splint to prevent revascularization and reduce contraction as the wounds heal. Despite the debate of using rats for wound healing studies due to their quite distinct anatomic and physiologic differences compared to humans (i.e., the presence of a panniculus carnosus muscle, short life-span, increased number of hair follicles, and their ability to heal infected wounds) the modifications employed in this model make it a valuable alternative to previously developed ischemic skin flap models.

The rat, due to its size, availability, and rather docile behavior, has been utilized as a research model for many years. The goal of this protocol is to utilize the rat as an ischemic skin wound healing model to provide valuable insight into the pathophysiology of chronic wounds.

在大鼠缺血性皮肤创伤模型的演示

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and ...

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

The incidence of inflammatory bowel disease, i.e., Crohn&#39;s disease and Ulcerative colitis, has significantly increased over the last decade. The etiology of IBD remains unknown and current therapeutic strategies are based on the unspecific suppression of the immune system. The development of treatments that specifically target intestinal inflammation and epithelial wound healing could significantly improve management of IBD, however this requires accurate detection of inflammatory changes. Currently, potential drug candidates are usually evaluated using animal models in vivo or with cell culture based techniques in vitro. Histological examination usually requires the cells or tissues of interest to be stained, which may alter the sample characteristics and furthermore, the interpretation of findings can vary by investigator expertise. Digital holographic microscopy (DHM), based on the detection of optical path length delay, allows stain-free quantitative phase contrast imaging. This allows the results to be directly correlated with absolute biophysical parameters. We demonstrate how measurement of changes in tissue density with DHM, based on refractive index measurement, can quantify inflammatory alterations, without staining, in different layers of colonic tissue specimens from mice and humans with colitis. Additionally, we demonstrate continuous multimodal label-free monitoring of epithelial wound healing in vitro, possible using DHM through the simple automated determination of the wounded area and simultaneous determination of morphological parameters such as dry mass and layer thickness of migrating cells. In conclusion, DHM represents a valuable, novel and quantitative tool for the assessment of intestinal inflammation with absolute values for parameters possible, simplified quantification of epithelial wound healing in vitro and therefore has high potential for translational diagnostic use.

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 ...

Creation and Transplantation of an Adipose-derived Stem Cell (ASC) Sheet in a Diabetic Wound-healing Model

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients with impeded blood flow or with large wounds might be prolonged. Cell-based therapies have appeared as a new technique for the treatment of diabetic ulcers, and cell-sheet engineering has improved the efficacy of cell transplantation. A number of reports have suggested that adipose-derived stem cells (ASCs), a type of mesenchymal stromal cell (MSC), exhibit therapeutic potential due to their relative abundance in adipose tissue and their accessibility for collection when compared to MSCs from other tissues. Therefore, ASCs appear to be a good source of stem cells for therapeutic use. In this study, ASC sheets from the epididymal adipose fat of normal Lewis rats were successfully created using temperature-responsive culture dishes and normal culture medium containing ascorbic acid. The ASC sheets were transplanted into Zucker diabetic fatty (ZDF) rats, a rat model of type 2 diabetes and obesity, that exhibit diminished wound healing. A wound was created on the posterior cranial surface, ASC sheets were transplanted into the wound, and a bilayer artificial skin was used to cover the sheets. ZDF rats that received ASC sheets had better wound healing than ZDF rats without the transplantation of ASC sheets. This approach was limited because ASC sheets are sensitive to dry conditions, requiring the maintenance of a moist wound environment. Therefore, artificial skin was used to cover the ASC sheet to prevent drying. The allogenic transplantation of ASC sheets in combination with artificial skin might also be applicable to other intractable ulcers or burns, such as those observed with peripheral arterial disease and collagen disease, and might be administered to patients who are undernourished or are using steroids. Thus, this treatment might be the first step towards improving the therapeutic options for diabetic wound healing.

Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model

Adipose-derived stem cells (ASCs) are easily isolated and harvested from the fat of normal rats. ASC sheets can be created using cell-sheet engineering and can be transplanted into Zucker diabetic fatty rats exhibiting full-thickness skin defects with exposed bone and then covered with a bilayer of artificial skin.

创作和移植的糖尿病伤口愈合模型中脂肪源性干细胞 (ASC) 表

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients ...

Development of a Direct Pulp-capping Model for the Evaluation of Pulpal Wound Healing and Reparative Dentin Formation in Mice

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is often capped with biocompatible materials in order to expedite pulpal wound healing. The ultimate goal is to regenerate reparative dentin, a physical barrier that functions as a "biological seal" and protects the underlying pulp tissue. Although this direct pulp-capping procedure has long been used in dentistry, the underlying molecular mechanism of pulpal wound healing and reparative dentin formation is still poorly understood. To induce reparative dentin, pulp capping has been performed experimentally in large animals, but less so in mice, presumably due to their small sizes and the ensuing technical difficulties. Here, we present a detailed, step-by-step method of performing a pulp-capping procedure in mice, including the preparation of a Class-I-like cavity, the placement of pulp-capping materials, and the restoration procedure using dental composite. Our pulp-capping mouse model will be instrumental in investigating the fundamental molecular mechanisms of pulpal wound healing in the context of reparative dentin in vivo by enabling the use of transgenic or knockout mice that are widely available in the research community.

We describe a step-by-step method of performing direct pulp capping on mice teeth for the evaluation of pulpal wound healing and reparative dentin formation in vivo.

伤口的牙髓评价一个直接盖髓模型的开发和治疗小鼠中修复性牙本质形成

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is ...

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 ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

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.