Name: assessment of acute wound healing using the dorsal subcutaneous polyvinyl alcohol sponge implantation and excisional tail skin wound models.
Uploaded: 2020-03-25T12:30:00
Description: Watch this Scientific Journal Video about Assessment of Acute Wound Healing using the Dorsal Subcutaneous Polyvinyl Alcohol Sponge Implantation and Excisional Tail Skin Wound Models. at JoVE.com

作者感谢布朗大学流动细胞测量和分拣基金的凯文·卡尔森在流细胞学实验方面的咨询和帮助。图 1B 和 C 中的图像是使用 BioRender 创建的。凯拉·李和格雷戈里·塞尔帕感谢他们的摄影帮助。这项工作得到了以下资助：国防高级研究计划局（DARPA）YFAA15 D15AP00100、院长新兴科学奖（布朗大学）、国家心肺血液研究所（NHLBI）1R01HL126887-01A1，国家环境科学研究所（NIES）T32-ES7272（环境病理学培训）和布朗大学研究种子奖。

这里描述的所有动物研究都得到布朗大学机构动物护理和使用委员会的批准，并根据《国家卫生研究院动物护理和使用指南》进行。 注：在视频中，手术窗帘被省略用于演示目的。
1. PVA海绵的皮下植入
<ol><li>使用剪刀将 PVA 海绵片切割成 8 毫米 x 8 毫米 x 4 毫米件。通过将 PVA 海绵碎片浸入无菌的 1x PBS 中，将其浸入烧杯中，使这些海绵重新补充水分。</li>
	<li>在 1x PBS 中高压...

<ol>
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本文介绍了两种可处理的鼠伤模型，可用于评估急性伤口愈合反应。第一种方法涉及在背皮皮空间中植入PVA海绵。这种方法比基于活检的伤口模型具有明显的优势，用于研究细胞伤口愈合反应，因为从分离海绵中获得的细胞数量和伤口液体数量众多。为了成功实施这一程序，通过彻底清洁切口周围的皮肤来维持无菌手术场势在必行，因为细菌在伤口中的易位将大大改变愈合过程。使用此处描述的?...

有许多鼠模型系统可用于检查伤口愈合过程，每个系统具有特定的优点和限制1，,2。以下方法提出了两种鼠伤模型，每个模型都涉及伤口愈合反应的特定方面，可用于识别损伤反应中扰动的因因和效果。伤口愈合的过程发生在不同的阶段。第一阶段是炎症，其特点是血小板、嗜中性粒细胞和单核细胞/巨噬细胞的迅速涌入，以及产生亲炎细胞因子和化学因子。炎症的解决后，环境过渡到一种更具弹性的状态，引入亲纤维和血管生成细胞因子和生长因子。造血组织沉积，新血管形成与肌纤维细胞、成纤维细胞、上皮细胞和内皮细胞的迁移。在最后阶段，临时细胞外基质进行改造，疤痕形成和伤口封闭进行232、3、4、5、6、7、8。6,7,84,5,,,
没有一个鼠模型提供一个系统来研究伤口愈合的所有阶段2。在这里，描述了两个手术伤口模型：一个阐明急性细胞和细胞因子伤口愈合反应，另一个允许评估伤口闭合以及组织分析。这两种方法可以以补充的方式用于评估扰动或合并症对伤口愈合反应不同方面的影响。聚苯乙烯醇（PVA）海绵的背皮下植入是一个系统，几十年来一直用于啮齿动物模型，阐明细胞和造粒组织反应的诸多方面9,109、10、11、12、13、14、15、16、17、18、19、20,11,12,13,14,15,16,17,18,19,20 ，21，,22，,23，,24.这种方法允许检索富含细胞因子的伤口液体和细胞渗透。在此模型中，1 厘米 x 1 厘米 x 0.5 厘米的 PVA 海绵通过后背背中线做出的 2 厘米切口放入皮下口袋。切口用手术夹闭合，以后可以取回海绵，用于细胞和流体隔离。分离海绵的细胞和细胞因子环境反映了急性伤口愈合的正常阶段，在植入后长达14天左右。在以后的时间点，该模型更有利于研究造粒组织的形成和异物反应1。有了这个系统，可以分离>106细胞，这为球形和功能测定和RNA分离提供了明显的优势，而不是从其他活检方法11，22，23，25，2622,23,25分离26细胞。
使用尾皮切除模型确定伤口闭合率。在这个模型中，如Falanga等人最初描述，并由其他人报告27，28，29，30，28,29一个1厘米x0.3厘米全厚的尾皮部分被去除附近的尾巴底部。,3027伤口区域易于可视化，并可随时间测量。或者，尾组织可以分离进行组织分析。这种方法可以用作替代或结合成熟的背孔活检方法。这两种型号的主要区别是伤口闭合率、毛皮的存在或缺失以及皮肤结构2,2、31、32。,32尾皮伤口提供了一个较长的时间框架来评估伤口关闭，因为它大约需要21天，完全关闭发生。这与未溅的背孔活检相反，这种活检愈合得更快（+7~10天），主要通过收缩引起的泛素性腺作用。溅条背孔活检愈合较慢，减少收缩愈合的影响，但依靠一个外国机构的存在来限制基于收缩的机制11，2，27，30，31，33。,2,27,30,31,33
所述伤口模型对于了解在无扰动的情况下正常伤口愈合过程的信息量很大。虽然啮齿动物皮肤的愈合与人类皮肤有非常显著的差异，包括结构松散、依赖收缩愈合和其他解剖学差异，但鼠系为机械和筛选研究提供了某些优势。其中最重要的是近亲繁殖菌株和遗传突变体的可用性、遗传可得性和更低的成本。从鼠学研究中获得的机械学见解可以转化为更复杂的动物模型，更密切地模仿人类皮肤愈合，如猪系统2,2，31。
除了检查伤口愈合反应在稳定状态，这些模型可以结合合并条件，以了解伤口愈合缺陷在细胞，细胞因子和毛组织水平的基础。正是在这个特殊环境中，两种模型可以协同评估特定合并症（如术后肺炎）对急性细胞伤口愈合反应和伤口闭合率的影响。

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			<td height="21" style="height:21px;width:317px;">10x Phosphate Buffered Saline</td>
			<td style="width:121px;">Fisher Scientific</td>
			<td style="width:104px;">BP3991</td>
			<td style="width:167px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:317px;">15 mL centrifuge tubes, Olympus</td>
			<td style="width:121px;">Genesee</td>
			<td style="width:104px;">28-103</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:317px;">1x HBSS (+Calcium, +Magnesium, &#8211;Phenol Red)</td>
			<td style="width:121px;">ThermoFisher Scientific</td>
			<td style="width:104px;">14025076</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:317px;">5ml Syringe</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:317px;">Anti-mouse CD45.2-APC Fire750</td>
			<td style="width:121px;">BioLegend</td>
			<td style="width:104px;">109852</td>
			<td>Clone 104</td>
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			<td height="42" style="height:42px;width:317px;">Anti-mouse F4/80-eFluor660</td>
			<td style="width:121px;">ThermoFisher Scientific</td>
			<td style="width:104px;">50-4801-82</td>
			<td>Clone BM8</td>
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			<td height="21" style="height:21px;width:317px;">Anti-mouse Ly6C-FITC</td>
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			<td style="width:104px;">553104</td>
			<td>Clone AL-21</td>
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			<td height="42" style="height:42px;width:317px;">Anti-mouse Ly6G-PerCP-eFluor710</td>
			<td style="width:121px;">ThermoFisher Scientific</td>
			<td style="width:104px;">46-9668-82</td>
			<td>Clone 1A8-Ly6g</td>
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			<td style="width:104px;">565183</td>
			<td>Clone E50-2440</td>
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			<td height="42" style="height:42px;width:317px;">Autoclip Stainless Steel Wound Clip Applier</td>
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			<td style="width:104px;">NC9021392</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:317px;">Autoclip Stainless Steel Wound Clips, 9mm</td>
			<td style="width:121px;">Braintree Scientific</td>
			<td style="width:104px;">NC9334081</td>
			<td></td>
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			<td height="21" style="height:21px;width:317px;">Blender Bag, 80mL</td>
			<td style="width:121px;">Fisher Scientific</td>
			<td style="width:104px;">14258201</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:317px;">Culture Tube, 16mL, 17x100</td>
			<td style="width:121px;">Genesee Scientific</td>
			<td style="width:104px;">21-130</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:317px;">Fetal Bovine Serum - Standard</td>
			<td style="width:121px;">ThermoFisher Scientific</td>
			<td style="width:104px;">10437028</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:317px;">Fixable Viability Dye eFluor506</td>
			<td style="width:121px;">ThermoFisher Scientific</td>
			<td style="width:104px;">65-0866-14</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:317px;">Hepes Solution, 1M</td>
			<td style="width:121px;">Genesee Scientific</td>
			<td style="width:104px;">25-534</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:317px;">ImageJ Software</td>
			<td style="width:121px;">NIH</td>
			<td style="width:104px;"></td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:317px;">Penicillin-Streptomycin (5000 U/mL)</td>
			<td style="width:121px;">ThermoFisher Scientific</td>
			<td style="width:104px;">15070-063</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:317px;">Polyvinyl alcohol sponge - large pore size</td>
			<td style="width:121px;">Ivalon/PVA Unlimited</td>
			<td style="width:104px;"></td>
			<td>www.sponge-pva.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">Povidone-iodine solution, 10%</td>
			<td style="width:121px;">Fisher Scientific</td>
			<td>3955-16</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:317px;">Spray barrier film, Cavilon</td>
			<td style="width:121px;">3M</td>
			<td style="width:104px;">3346E</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:317px;">Stomacher 80 Biomaster, 110V</td>
			<td style="width:121px;">Seward</td>
			<td style="width:104px;">0080/000/AJ</td>
			<td></td>
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assessment of acute wound healing using the dorsal subcutaneous polyvinyl alcohol sponge implantation and excisional tail skin wound models.

伤口愈合是一个复杂的过程，需要炎症、造粒组织形成、纤维化和解决的有序进展。毛林模型为这些过程提供了宝贵的机械洞察力;然而，没有一个模型完全解决伤口愈合反应的所有方面。相反，最好使用多个模型来解决伤口愈合的不同方面。在这里，描述了两种不同的方法来处理伤口愈合反应的不同方面。在第一个模型中，聚乙烯醇海绵被皮下植入鼠标多糖。在海绵回收后，细胞可以通过机械中断进行分离，流体可以通过离心提取，从而能够详细描述急性伤口环境中的细胞和细胞因子反应。此模型的一个局限性是无法评估伤口闭合率。为此，使用了尾皮肤切除模型。在此模型中，沿着尾部底部附近的背表面切除了 10 mm x 3 mm 矩形尾皮。该模型可以很容易地拍摄平面分析，以确定愈合率，并可以切除组织学分析。这两种描述的方法可用于基因改变的小鼠菌株，或与合并条件模型（如糖尿病、衰老或继发感染）结合使用，以阐明伤口愈合机制。

PVA海绵植入后的全身炎症反应 PVA海绵植入手术产生了全身炎症反应，如伤伤1天后血浆中IL-6的诱导（图2A）。其他亲炎细胞因子，包括TNF-α和IL-1+，以及包括CCL2和CXCL1在内的一系列化学因子在PVA海绵植入后的前7天被系统诱导，并在其他地方描述了26，30。26,
从PVA海绵伤口分离细胞和液体</str...

Watch this Scientific Journal Video about Assessment of Acute Wound Healing using the Dorsal Subcutaneous Polyvinyl Alcohol Sponge Implantation and Excisional Tail Skin Wound Models. at JoVE.com

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

这里描述了两个鼠伤愈合模型，一个旨在评估细胞和细胞因子伤口愈合反应，另一个用于量化伤口愈合率。这些方法可用于复杂的疾病模型，如糖尿病，以确定伤口愈合不良的各个方面的机制。

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 ...

这些模型可以实施，以评估伤口愈合反应的各个方面，包括细胞和细胞因子动力学和伤口闭合。PVA 海绵模型允许恢复数百万个伤口白细胞进行表型和功能分析，而尾皮肤切除模型允许缓慢愈合的伤口的轻松可视化。PVA 海绵和切除尾伤模型可与合并疾病（如糖尿病或肺炎）结合使用，以了解其对伤口愈合的影响。与梅雷迪斯·克莱恩一起演示这些程序的将是比尔·亨利，一个来自我实验室的研究助理。在开始实验之前，用剪刀将PVA海绵切成八分之八到四毫米的碎片。在洗脱PBS中的海绵片中再加水10分钟，然后对PBS中的海绵进行消毒。当海绵冷却后，将浸入PBS中的海绵储存在摄氏四度。在手术当天，将每个实验动物的六块海绵放入无菌层流罩中的无菌培养皿中，并确认在麻醉的8至12周大、雄性C57-Black/6小鼠中对头趾捏的缺乏反应。有一个助理使用剪子去除头发沿多苏姆和无菌纱布应用波维酮碘溶液剃光区域两次，然后一个单一的70%乙醇应用。然后，助手应将鼠标转移到放置在加热垫上的无菌手术窗帘上。戴上无菌手套，用钳子将背皮从底层组织中拉离，并使用无菌手术剪刀沿着背中线沿着背侧中线切口，在尾部前约两厘米处进行切口。用无菌钳子将切口打开，使用无菌、弯曲、钝尖的手术剪刀，在图中所示的位置之一沿多苏姆形成皮下口袋。插入剪刀后，打开并关闭刀尖两次，形成足够大的口袋，可以容纳插入的海绵。当口袋被创建时，使用无菌手术剪刀轻轻挤压培养皿中的一块PVA海绵，以去除多余的PBS。接下来，将海绵举到一角，用剪刀抓住的角落，将海绵放入皮下口袋。当所有六块海绵都放置到证明时，使用无菌钳子将切口的后体皮肤捏在一起，并使用两个不锈钢伤口夹关闭切口。对于PVA海绵液隔离，植入后1至14天，取出手术钉，用牙钳打开切口。使用剪刀沿后沿中线延伸切口，并使用钳子从皮下口袋中提取一块海绵。将海绵放入嵌套在冰上16毫升培养管中的5毫升注射器桶中，并尽可能用剪刀将粘在海绵表面的任何结缔组织。当所有海绵都提取并转移时，将培养管离心以收集伤口液。为了将细胞从PVA海绵中分离，在收集海绵后，请将它们放入含有5毫升HSS收集介质的15毫升圆锥管中。收集所有海绵后，将海绵悬浮液放入 80 毫升搅拌机袋中，并将袋子从桨搅拌机的舱口挂上，以便桨能够撞击海绵和介质。将桨式搅拌机设置为高运行 60 秒，然后按"启动"。当搅拌机停止时，挤压袋中的海绵以完全释放介质，并使用移液器将介质从搅拌袋移回 15 毫升管中。调整桨式搅拌机的设置，在高位置运行 30 秒，并在搅拌袋中添加 5 毫升介质。在离心收集的介质管之前，再重复胃部过程两次。在锥形管底部应可见细胞和红血球的红粒。将 900 微升蒸馏水与细胞混合三到五秒钟，然后用 100 微升 10x PBS 和 4 毫升 1x PBS 中和中和。通过离心收集细胞，并在适当的介质中重新暂停白细胞颗粒，用于下游分析。为了制造尾皮伤口，在确认对麻醉中对头趾捏缺乏反应后，8至12周大，雄性，C57-Black/6小鼠，使用无菌纱布两次将波维酮碘溶液涂抹到手术现场，随后应用70%乙醇。使用永久标记和预制模板，在尾部的后面上跟踪 10 到 3 毫米的截面，从尾部底部追踪 10 毫米，然后将鼠标放在无菌手术窗帘上。戴上无菌手术手套，使用无菌手术刀刀片，沿着伤口区域的右、下、左边缘进行全厚切口。使用无菌钳子，将切除的皮肤从尾部剥离，并使用无菌手术剪刀切开伤口区域的上边缘。使用无菌纱布对伤口施加压力以阻止出血，并在伤口床上涂抹喷雾阻隔膜。然后，以固定的时间间隔从固定距离拍摄伤口，通过平面分析分析照片以确定伤口面积测量结果。PVA海绵植入手术产生全身炎症反应，在受伤一天后在血浆中诱导IL-6就证明了这一点。可从PVA海绵伤口中恢复的细胞数量随着时间的推移而增加，其中嗜中性粒细胞、单核细胞和巨噬细胞是海绵内主要细胞渗透种群。中性粒细胞被确定为Ly6G阳性，西格尔克-F-阴性细胞。西格尔克-F阳性细胞主要是嗜酸性细胞。在Ly6G阴性、西格尔克-F阴性细胞上进行加注，可以识别F4/80阳性的单细胞和巨噬细胞。F4/80阳性细胞可以通过其Ly6C表达进一步分化，以区分Ly6C高炎症单细胞和Ly6C低单核细胞衍生巨噬细胞。切除尾皮肤模型提供了一种替代的后体皮肤冲孔活检方法，研究伤口闭合在缺乏密集毛皮的坚定的皮肤。伤口闭合可以通过测量伤口床的面积来量化。通过H和E和马森的三色染色组织学分析，也可以在横截面观察尾皮伤口。在这些图像中，切除皮肤的横向边距由尾部的支面上的箭头指示。为了了解稳定状态伤口愈合，治疗化合物或干预也可以测试通过他们直接注射到PVA海绵或通过交付到尾部伤口床。

Research

JoVE Journal

Immunology and Infection

Cutaneous Leishmaniasis in the Dorsal Skin of Hamsters: a Useful Model for the Screening of Antileishmanial Drugs

在仓鼠的背部皮肤：皮肤利什曼病Antileishmanial药物筛选的一个有用的模型

Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of ...

科研

免疫与感染

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

尾部皮肤来研究异基因CD4 + T细胞应答的小鼠移植

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

对于检测磁力搅拌器方法<em&gt;旋毛虫</em&gt;幼虫肌肉样品

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode ...

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

一种新的体外创面愈合试验评价细胞迁移

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to ...

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

在 vivo小鼠创面模型中活性氧的成像

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various ...

Chronic, Acute, and Reactivated HIV Infection in Humanized Immunodeficient Mouse Models

人类免疫缺陷小鼠模型中的慢性、急性和再激活HIV感染

Humanized NOD/SCID/IL-2 receptor &#947;-chainnull mice recapitulate some features of human immunity, which can be exploited in basic and pre-clinical ...

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

慢性假单位病伤口感染的延迟接种模型

Pseudomonas aeruginosa (P. aeruginosa) is a major nosocomial pathogen of increasing relevance to human health and disease, particularly in the setting of ...

Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells

Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells

香烟烟雾对肺 癫痫细胞 伪多多多细胞的影响研究

Cigarette smoking is the major etiological cause for lung emphysema and chronic obstructive pulmonary disease (COPD). Cigarette smoking also promotes ...

Live Imaging of Chemokine Receptors in Zebrafish Neutrophils During Wound Responses

在伤口反应期间斑马鱼中性粒细胞中趋化因子受体的实时成像

Leukocyte guidance by chemical gradients is essential for immune responses. Neutrophils are the first cells to be recruited to sites of tissue damage ...

A Novel High-Throughput Ex Vivo Ovine Skin Wound Model for Testing Emerging Antibiotics

A Novel High-Throughput Ex Vivo Ovine Skin Wound Model for Testing Emerging Antibiotics

用于测试新兴抗生素的新型高通量 离体 绵羊皮肤伤口模型

The development of antimicrobials is an expensive process with increasingly low success rates, which makes further investment in antimicrobial discovery ...