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。
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 10x Phosphate Buffered Saline | Fisher Scientific | BP3991 | |
| 15 mL centrifuge tubes, Olympus | Genesee | 28-103 | |
| 1x HBSS (+Calcium, +Magnesium, –Phenol Red) | ThermoFisher Scientific | 14025076 | |
| 5ml Syringe | BD | 309646 | |
| Anti-mouse CD45.2-APC Fire750 | BioLegend | 109852 | Clone 104 |
| Anti-mouse F4/80-eFluor660 | ThermoFisher Scientific | 50-4801-82 | Clone BM8 |
| Anti-mouse Ly6C-FITC | BD Biosciences | 553104 | Clone AL-21 |
| Anti-mouse Ly6G-PerCP-eFluor710 | ThermoFisher Scientific | 46-9668-82 | Clone 1A8-Ly6g |
| Anti-mouse Siglec-F-APC-R700 | BD Biosciences | 565183 | Clone E50-2440 |
| Autoclip Stainless Steel Wound Clip Applier | Braintree Scientific | NC9021392 | |
| Autoclip Stainless Steel Wound Clips, 9mm | Braintree Scientific | NC9334081 | |
| Blender Bag, 80mL | Fisher Scientific | 14258201 | |
| Culture Tube, 16mL, 17×100 | Genesee Scientific | 21-130 | |
| Fetal Bovine Serum – Standard | ThermoFisher Scientific | 10437028 | |
| Fixable Viability Dye eFluor506 | ThermoFisher Scientific | 65-0866-14 | |
| Hepes Solution, 1M | Genesee Scientific | 25-534 | |
| ImageJ Software | NIH | ||
| Penicillin-Streptomycin (5000 U/mL) | ThermoFisher Scientific | 15070-063 | |
| Polyvinyl alcohol sponge – large pore size | Ivalon/PVA Unlimited | www.sponge-pva.com | |
| Povidone-iodine solution, 10% | Fisher Scientific | 3955-16 | |
| Spray barrier film, Cavilon | 3M | 3346E | |
| Stomacher 80 Biomaster, 110V | Seward | 0080/000/AJ |
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