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Skin Wound Healing Model - Excisional Wounding and Assessment of Lesion Area
皮肤伤口愈合模型——切除性创伤和病变评估

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Abstract

This protocol focus on the most common surgical mouse model of cutaneous excisional wound healing used to study the cellular and molecular pathways involved in wound repair and regeneration as well as in translational applications such as the evaluation of new therapeutic modalities. This model allows the monitoring of the wound closure and the tissue collection for histological and molecular analyses. Briefly, full skin thickness excisional wounds are created on the dorsum of the mouse as the excision extends through the panniculosus carnosus. Wounds larger and minor diameters are then regularly measured and wound closure rate is calculated based on wound area relative to the original size.

Materials and Reagents

  1. 5 mm diameter circular biopsy punch (ABC, catalog number: 0418 )
  2. 10% Ketamine Hydrochloride (Agropecuária Tarumã, catalog number: 8565 )
  3. 2% Xylazine hydrochloride (Syntec)
  4. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
  5. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P0662 )
  6. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: 255793 )
  7. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888 )
  8. Saline solution (0.9% sodium chloride injectable solutio) (Equiplex)
  9. dH2O
  10. 70% alcohol (see Recipes)
  11. Phosphate buffered saline (PBS) (pH 7.2, 10x) (see Recipes)
  12. Phosphate buffered saline (PBS) (pH 7.2, 1x) (see Recipes)

Equipment

  1. Digital caliper (Mitutoyo, catalog number: 573-661 )
  2. Hair removal machine (Wahl and Toshico)

Procedure

  1. Creation of skin excisional wounds in mice
    1. Anesthetize mice as approved in your animal study proposal. We suggest intraperitoneally injecting a mixture of ketamine 100 mg/kg and xylazine 10 mg/kg, diluted in 100 µl of saline solution. It gives about twenty minutes of surgical anesthesia.
    2. Remove hair from the mice dorsum by using a hair removal machine.
    3. Prepare the surgical site with an appropriate skin disinfectant. We suggest 70% alcohol.
    4. Fold and raise the dorsal skin cranially and caudally at midline using the index fingers and thumbs to form a sandwiched skinfold (Figure 1A). Then, place the animal in a lateral position and press down the 5-mm diameter sterile biopsy punch to completely remove the two skin layers (Figure 1B) and create symmetrical full-thickness excisional wounds (Figure 1C).


      Figure 1. Stepwise skin excisional wounding surgery. Fold and raise the dorsal skin cranially and caudally at midline to form a sandwiched skinfold (A). Place the animal in a lateral position and punch through the folded skin (B) to create symmetrical full-thickness excisional wounds (C).

    5. After surgery, move the animal to a warm area and monitor its recovery from anesthesia. Return the fully recovered animal to its routine housing. Cage individually.

  2. Wound closure monitoring after surgery
    1. Animals are anesthetized as described in step A1 and the wound area is assessed every 2-3 days until full closure of the lesions.
    2. By using a digital caliper, measure the larger and minor diameters of the lesions (Figure 2) and determine the wound area by applying the following formula: (diameter A/2) x (diameter B/2) x π.
    3. Calculate the percentage of wound closure as follow: [(area of original wound-area of actual wound)/area of original wound] x 100.


      Figure 2. Assessment of the wound area. Measure the larger (A) and the minor (B) diameter of the lesion. Calculate the area as follows: (diameter A/2) x (diameter B/2) x π.

Notes

  1. Create wounds with approximately 5 mm apart.
  2. Analgesic drugs use depends on the experimental design and the approval by local committee for ethical conduct in the care and use of animals in scientific research. Preemptive use of analgesic drugs is recommended. We suggest Buprenorphine 0.05 mg/kg subcutaneously every 12 h for the first 24 h post-surgery.
  3. The actual surface area of a full-thickness cutaneous excisional wound becomes slightly larger than its initial size.
  4. For accuracy and reproducibility of the experiments, the area measurement must be performed by a single person throughout the experimental time-course.
  5. If the skin is going to be used for histological analysis, after harvesting the wound tissue, lay it on a sandwiched piece of filter paper to avoid tissue folding during the fixation process and place it in a histological cassette for fixation in 10% formalin solution for 24 h. Then, follow for conventional paraffin embedding processing. Otherwise, place the harvested tissue in 1.5 ml microtube and then immediately dip in liquid nitrogen to store the frozen tissue for further tissue analyzes (e.g., biochemical analysis, ELISA, qPCR, etc.). Please, see Cassini-Vieira et al. (2015) for details on how to harvesting wound tissues for analysis.

Recipes

  1. 70% alcohol solution
    700 ml of absolute alcohol
    Complete the volume with distillate water to 1,000 ml
  2. Phosphate buffered saline (PBS) (pH 7.2, 10x)
    NaCl 80 g
    Na2HPO4 11.05 g or Na2HPO4.12H2O 29 g
    KCl 2 g
    KH2PO4 2.1 g
    Add dH2O to 1,000 ml
  3. Phosphate buffered saline (PBS) (pH 7.2, 1x)
    50 ml PBS (10x)
    450 ml dH2O

Acknowledgments

This work was supported by Conselho Nacional de Pesquisa/CNPq, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior/CAPES, Fundação de Amparo à Pesquisa de Minas Gerais/FAPEMIG, and Pró-reitoria de Graduação PROGRAD-UFMG, Brazil. CFM holds a PROBIC-FAPEMIG Scientific Initiation scholarship. PCV holds a CAPES PhD scholarship. MSF holds a PROGRAD-UFMG undergraduate scholarship. LSB holds a CNPq Research Fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

  1. Canesso, M. C., Vieira, A. T., Castro, T. B., Schirmer, B. G., Cisalpino, D., Martins, F. S., Rachid, M. A., Nicoli, J. R., Teixeira, M. M. and Barcelos, L. S. (2014). Skin wound healing is accelerated and scarless in the absence of commensal microbiota. J Immunol 193(10): 5171-5180.
  2. Cassini-Vieira, P., Moreira, C. F., da Silva, M. F. and Barcelos, L. S. (2015). Estimation of wound tissue neutrophil and macrophage accumulation by measuring myeloperoxidase (MPO) and N-Acetyl-β-D-glucosaminidase (NAG) activities. Bio-protocol 5(22): e1662.

简介

该协议侧重于最常见的皮肤切除伤口愈合的外科小鼠模型,用于研究涉及伤口修复和再生以及翻译应用(例如评价新的治疗方式)的细胞和分子途径。 该模型允许监测伤口闭合和组织收集用于组织学和分子分析。 简而言之,当小鼠的背部延伸穿过肉芽肿时,在小鼠的背部产生完全皮肤厚度的切除伤口。 然后定期测量伤口的较大和较小直径,并基于相对于原始尺寸的伤口面积计算伤口闭合率。

材料和试剂

  1. 5mm直径圆形活检穿孔器(ABC,目录号:0418)
  2. 10%盐酸氯胺酮(AgropecuáriaTarum?,目录号:8565)
  3. 2%甲苯噻嗪盐酸盐(Syntec)
  4. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9541)
  5. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:P0662)
  6. 磷酸氢二钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:255793)
  7. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S9888)
  8. 盐水溶液(0.9%氯化钠注射液)(Equiplex)
  9. dH 2 2 O
  10. 70%酒精(见配方)
  11. 磷酸盐缓冲盐水(PBS)(pH 7.2,10x)(参见配方)
  12. 磷酸盐缓冲盐水(PBS)(pH 7.2,1×)(参见配方)

设备

  1. 数字卡尺(Mitutoyo,目录号:573-661)
  2. 脱毛机(Wahl和Toshico)

程序

  1. 在小鼠中创建皮肤切割伤口
    1. 麻醉小鼠批准在你的动物研究提案。我们建议 腹膜内注射氯胺酮100mg/kg和赛拉嗪的混合物 ?10mg/kg,在100μl盐水溶液中稀释。它给大约二十 分钟的手术麻醉
    2. 使用脱毛机从小鼠背部脱毛。
    3. 使用适当的皮??肤消毒剂准备手术部位。我们建议70%的酒精。
    4. 折叠和抬起背部皮肤头和尾在中线 使用食指和拇指形成夹层皮褶(图1) ?1A)。然后,将动物放在侧面位置,并按下 5毫米直径无菌活检穿孔完全去除两个皮肤 层(图1B),并创建对称的全厚度切除 伤口(图1C)。


      图1。 ?逐步皮肤切除创伤手术。 折叠并抬高背部 皮肤在中线处并且尾部形成夹层皮肤褶 (一个)。将动物放在侧面位置,穿透折叠 ?皮肤(B)以产生对称的全层切割伤口(C)
    5. 手术后,将动物移动温暖 并监测其从麻醉中的恢复。完全返回 恢复动物到其常规住房。单独笼。

  2. 手术后伤口闭合监测
    1. 如步骤A1中所述将动物麻醉,并且伤口面积为 每2-3天评估直到完全闭合病变
    2. 通过 使用数字卡尺,测量其较大和较小直径 损伤(图2),并通过应用确定伤口面积 (直径A/2)×(直径B/2)×π。
    3. 计算伤口闭合的百分比如下:[(原始面积) 实际伤口的伤口面积)/原始伤口的面积]×100

      图2.伤口面积的评估。测量较大的(A)和 次要(B)病变的直径。计算面积如下: (直径A/2)×(直径B/2)×π。

笔记

  1. 创建间隔约5 mm的伤口。
  2. 镇痛药的使用取决于实验设计和地方委员会批准在科学研究中保护和使用动物的伦理行为。推荐先用止痛药。我们建议在术后第一个24小时每12小时皮下注射丁丙诺啡0.05 mg/kg
  3. 全层皮肤切割伤口的实际表面积略大于其初始尺寸
  4. 为了实验的准确性和可重复性,面积测量必须由整个实验时间过程中的单个人进行。
  5. 如果皮肤将用于组织学分析,在收获伤口组织后,将其放置在夹层的滤纸上以避免在固定过程中的组织折叠,并将其置于组织盒中用于在10%福尔马林溶液中固定24小时。然后,按照常规石蜡包埋处理。否则,将收获的组织置于1.5ml微量管中,然后立即浸入液氮中以储存冷冻组织用于进一步的组织分析(例如生化分析,ELISA,qPCR,等)。 >)。有关如何收获伤口组织进行分析的详情,请参阅Cassini-Vieira等人(2015年)。

食谱

  1. 70%酒精溶液
    700毫升绝对酒精
    用蒸馏水将体积加到1000ml
  2. 磷酸盐缓冲盐水(PBS)(pH 7.2,10x)
    NaCl 80g
    Na 2 HPO 4 11.05g或Na 2 HPO 4 12.12H 2 O 29克
    KCl 2 g



    将dH <2> O添加至1,000 ml
  3. 磷酸盐缓冲盐水(PBS)(pH 7.2,1×) 50ml PBS(10x)
    450ml dH 2 O

致谢

这项工作得到了Consueho Nacional de Pesquisa/CNPq,Coordè??odeAperfei?oamentode Pessoal deNívelSuperior/CAPES,Funda??ode AmparoàPesquisa de Minas Gerais/FAPEMIG和Pró-reitoria deGradua??oPROGRAD-UFMG,Brazil的支持。 CFM拥有PROBIC-FAPEMIG科学启动奖学金。 PCV持有CAPES博士奖学金。无国界医生拥有PROGRAD-UFMG本科生奖学金。 LSB拥有CNPq研究奖学金。资助者在研究设计,数据收集和分析,决定发布或准备手稿方面没有任何作用。

参考文献

  1. Canesso,M.C.,Vieira,A.T.,Castro,T.B.,Schirmer,B.G.,Cisalpino,D.,Martins,F.S.Rachid,M.A.,Nicoli,J.R.,Teixeira,M.M.and Barcelos,L.S.(2014)。 在没有共生微生物群的情况下,皮肤伤口愈合加速,无疤痕。 J Immunol 193(10):5171-5180。
  2. Cassini-Vieira,P.,Moreira,C.F.,da Silva,M.F.and Barcelos,L.S.(2015)。 通过测量骨髓过氧化物酶(MPO)和N-乙酰基-β-D-氨基葡萄糖苷酶评估伤口组织嗜中性粒细胞和巨噬细胞累积NAG)活性。 生物协议 5(22):e1662。
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引用:Moreira, C. F., Cassini-Vieira, P., da Silva, M. F. and Barcelos, L. S. (2015). Skin Wound Healing Model - Excisional Wounding and Assessment of Lesion Area. Bio-protocol 5(22): e1661. DOI: 10.21769/BioProtoc.1661.
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