Name: inhibition of wound epidermis formation via full skin flap surgery during axolotl limb regeneration
Uploaded: 2020-06-24T14:30:00
Duration: 4 min 24 s
Description: Watch this Scientific Journal Video about Inhibition of Wound Epidermis Formation via Full Skin Flap Surgery During Axolotl Limb Regeneration at JoVE.com

作者感谢Doug的不断鼓励和坚定不移的支持，以及Melton实验室成员对手稿的有益反馈和评论。作者还要感谢哈佛大学动物资源办公室（OAR）对动物的专注照顾。

所有动物实验均按照哈佛大学IACUC（协议编号：11-32）和AAALAC指南进行。
1. 准备麻醉和恢复的溶液和设置
<ol><li>准备新鲜的0.1%三卡因溶液用于麻醉和0.5%磺胺甲嘧啶钠盐溶液进行回收。根据相关研究机构批准的IACUC协议（例如，改进的Holtfreter解决方案），使用水制作适用于蝾螈畜牧业37 的解决方案。确保溶液充分混合，并准备足够的体积，以便淹没整个蝾螈。<ol>
<li>要制备0.1%三卡因溶液，将1克三卡因和1克碳酸氢钠与1升水混合。该解决方案可以根据此配方进行扩展。</li>
		<li>为了制备0.5%磺胺甲嘧啶钠盐溶液，将5g磺胺甲嘧啶钠盐与1L水混合。该解决方案可以根据此配方进行扩展。柳氨苄溶液是一种抗生素，可防止手术恢复过程中的细菌感染。</li>
	</ol>
</li>
	<li>通过用Clidox-S或70%乙醇喷洒手术区域来消毒手术区域。通过高压灭菌对手术工具（镊子，解剖剪刀，弹簧剪刀）进行消毒。如果进行多次手术，请确保在动物之间使用热珠灭菌器对手术工具进行消毒。</li>
	<li>要设置恢复区域，请将15厘米的培养皿或任何适合蝾螈的容器放在装满湿冰的桶顶部。用低水平的0.5%磺胺甲嘧啶钠盐溶液填充培养皿，足以使蝾螈醇不会完全浸没。手术后冰上的恢复将减缓动物的运动，同时从麻醉中醒来，使缝合区域相对不受干扰地愈合。 注意：此设置可由研究人员根据他们可用的材料进行自定义。</li>
</ol>2. 进行全皮瓣手术
<ol><li>通过将蝾螈浸没在0.1%三卡因溶液的容器中来麻醉蝾螈。这大约需要15-20分钟。通过进行尾巴捏合，确保蝾螈确实完全麻醉。如果蝾螈没有反应，请继续手术。 注意：使用较旧，较大的蝾螈进行此手术（至少15厘米大小）。确保蝾螈在整个手术过程中保持充足的水分，方法是使用塑料移液器定期用蝾螈系统水润湿皮肤。确保在手术前用无菌PBS冲洗该区域，无菌地准备手术部位。动物也应放置在无菌手术悬垂物上进行手术。</li>
	<li>使用解剖剪刀在角足骨骼元件的远端进行肢体截肢（图1.1）。</li>
	<li>使用弹簧剪刀，在皮肤的腹侧部分做一个小切口（约2毫米）（图1.2）。</li>
	<li>使用镊子，小心地将皮肤剥离到大约zeugopodial骨骼元件的中线，暴露下面的肢体组织（肌肉，骨骼等）。（图 1.3）。确保不要损伤皮肤。请参阅步骤 2.8 之后的说明。</li>
	<li>使用手术剪刀截肢Zeugopod中线暴露的下肢组织（图1.4）。</li>
	<li>用手术剪刀推回肌肉组织，修剪暴露的骨头。 注意：这是必要的，以确保改善愈合，并增加手术的成功率，因为突出的骨头可能会在缝合的皮瓣上锯齿状，并在以后破坏完整皮瓣的完整性。</li>
	<li>使用镊子，小心地将额外的全厚度皮肤拉过截肢平面，以覆盖暴露的下层组织，并通过与腹侧全厚度皮肤连接来缝合到位（图1.5）。</li>
	<li>将皮瓣的其余左右两侧缝合到完整皮肤的下腹侧部分。这可以通过以“交叉交叉”的方式缝合皮瓣的侧面（推荐）来完成（图1.6-1.9），或者只是直接缝合到腹侧皮肤中。使用镊子和弯曲的弹簧剪刀缝合。确保看不到暴露的下层组织，并且缝合线系紧（至少打结三次）。 注意：在步骤2.4，2.7-2.8中，完整的皮肤不会受损至关重要。我们发现，对全层皮肤的损伤与不成功的手术有关，因为损伤区域仍可能形成小的伤口表皮。如果可能的话，在处理全层皮瓣时，尝试使用一对较暗的镊子。</li>
	<li>在对侧肢体（可选动物内部控制）上进行截肢，方法是用手术剪刀在中角足水平截肢。用手术剪刀推回肌肉组织，修剪暴露的骨头。 注意：可以进行内部对侧肢体控制，以更好地评估同一动物在第4步期间手术的成功。然而，在单独的动物中截肢也可以用作对照。</li>
</ol>3. 术后恢复和护理
<ol><li>手术完成后，将Kimwipe或无菌纸巾放在容器或培养皿的底部以使其湿润。将动物放入湿冰上的容器中，轻轻地将Kimwipe或纸巾的暴露端包裹在动物的顶部，以保持其与磺胺甲嘧啶溶液的良好水合。在湿冰上静置 30 分钟至 1 小时，以确保在麻醉恢复期间活动最少。</li>
	<li>将动物放入含有0.5%磺胺甲嘧啶溶液的静态容器中。Axolotls必须在该溶液中保留前24小时，以防止感染。</li>
	<li>将蝾螈放入正常系统的水中，每天监测健康状况。确保每天没有缝合线脱落，因为这可能导致小伤口表皮形成，这将使结果混乱。 注意：确保外壳容器有足够的空间供蝾螈四处移动，并尽量减少蝾螈上缝合的肢体与容器侧面接触的机会。这将有助于确保缝合线保持在适当的位置，特别是在手术后的第一周。</li>
</ol>4. 在体视显微镜下评估手术的成功
注意：我们建议每周至少在体视显微镜下检查动物一次，以评估整个皮瓣的完整性和手术的成功。
<ol><li>如步骤2.1所示，在0.1%三卡因中麻醉蝾螈醇。确保容器中有足够的空间供蝾螈四处移动。</li>
	<li>如果在手术后的前两周内进行检查，请使用立体显微镜检查缝合的肢体，以确保没有缝合线弹出，并且任何地方都看不到清晰薄的伤口表皮。如果在手术后第三周或之后进行检查，请确保没有形成血浆水肿，并与正常对照截肢（来自同一动物或不同动物）在再生过程中的进展（即，是否形成血浆水肿）进行比较。</li>
	<li>完成后，将蝾螈恢复到正常的系统水和维护条件。</li>
</ol>

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F., 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=Application+and+optimization+of+CRISPR-Cas9-mediated+genome+engineering+in+axolotl+(Ambystoma+mexicanum).">Application and optimization of CRISPR-Cas9-mediated genome engineering in axolotl (Ambystoma mexicanum).</a> Nature Protocols. 13 (12), 2908-2943 (2018).</li><li>Flowers, G. P., Timberlake, A. T., McLean, K. C., Monaghan, J. R., Crews, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+targeted+mutagenesis+in+axolotl+using+Cas9+RNA-guided+nuclease.">Highly efficient targeted mutagenesis in axolotl using Cas9 RNA-guided nuclease.</a> Development. 141 (10), 2165-2171 (2014).</li><li>Khattak, S., 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=Optimized+axolotl+(Ambystoma+mexicanum)+husbandry,+breeding,+metamorphosis,+transgenesis+and+tamoxifen-mediated+recombination.">Optimized axolotl (Ambystoma mexicanum) husbandry, breeding, metamorphosis, transgenesis and tamoxifen-mediated recombination.</a> Nature Protocols. 9 (3), 529-540 (2014).</li><li>Joven, A., Elewa, A., Simon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Model+systems+for+regeneration:+salamanders.">Model systems for regeneration: salamanders.</a> Development. 146 (14), (2019).</li><li>Johnson, K., Bateman, J., DiTommaso, T., Wong, A. Y., Whited, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+cell+cycle+activation+is+induced+following+complex+tissue+injury+in+axolotl.">Systemic cell cycle activation is induced following complex tissue injury in axolotl.</a> Developmental Biology. 433 (2), 461-472 (2018).</li></ol>

作者没有什么可透露的。

本文介绍了在蝾螈四肢中进行全皮瓣手术以抑制伤口表皮形成的方案。虽然与其他抑制伤口表皮形成的方法相比，这种手术相对简单，技术上可重复，但有几个关键步骤可以影响手术的成功。首先，当将完整的全皮瓣拉过暴露的下层组织时，至关重要的是，全层皮肤不会以任何方式受损。对皮瓣的损伤仍可导致小伤口表皮的形成，从而导致小的胚泡样生长。其次，确保缝合线在术后护理期间不会脱落，因为这也可能导致小伤口表皮的形成。在这一点上，尽量减少缝合肢体与任何表面之间的潜在接触非常重要，特别是在手术后的第一周。预防这种情况的几种方法需要在足够大的容器中容纳和麻醉蝾螈，以便蝾螈有足够的空间在手术后四处移动。
这种手术也有几个局限性。也许最值得注意的是，手术的成功只能通过两种方式进行评估：在手术的前两周使用解剖范围寻找伤口表皮的缺失和/或检查3周内是否形成血涌水肿。虽然这些方法有效，但它们的吞吐量相对较低。未来用于伤口表皮特异性标志物的转基因报告蝾螈的开发可能有助于更快地筛查成功与失败的手术。此外，这种手术在年轻的动物身上更难进行，因为完整的皮肤更脆弱。因此，建议使用亚成人或成人蝾螈。
虽然这种手术最初是在N. viridiscens19中开发的，但它很容易适应蝾螈25，39，并且可能也适用于其他蝾螈物种。总而言之，将这种技术应用于未来的肢体再生研究将使研究人员能够开发更多工具来解决伤口表皮生物学问题，并确定驱动其启动卵涌水肿形成功能的潜在机制。

自从Lazzaro Spallanzani在17681年报道以来，蝾螈肢体再生一直是几个世纪以来一直迷恋生物学家的最充分研究的自然再生现象之一。成功的肢体再生取决于未分化细胞结构（称为胚芽肿）的形成、生长和随后的模式化。研究人员在了解卵涌水肿的细胞组成以及哪些支持组织和细胞类型是其形成所必需的方面取得了重大进展2，3，4，5，6，7，8，9，10，11，12，13.然而，导致启动原始细胞瘤形成的不同组织和细胞类型之间的协调信号传导机制仍然知之甚少。
成功形成和再生母细胞瘤的关键要求是伤口表皮，这是一种短暂的特化上皮，在截肢后12小时内覆盖截肢平面10。截肢后，从完整皮肤近端到损伤的上皮细胞迅速通过截肢平面迁移，形成薄薄的伤口上皮14。随着芽生菌在接下来的几周内形成，早期伤口表皮发展成更厚的上皮信号传导结构，称为顶端上皮帽（AEC）15。虽然正常的全层皮肤包含由基底层隔开的上皮层和真皮层，但伤口表皮/ AEC仅由上皮层组成，缺乏基底层16，17。基底层和真皮层的缺失允许伤口上皮细胞与下层组织之间直接接触，这有助于两个隔室之间的双向信号传导，这对芽生水肿的形成和维持至关重要17，18。
经典的实验研究设计了各种创新的手术方法，通过抑制伤口表皮/ AEC的功能和必要性来探测其形成。这些方法包括缝合19或将全层皮肤20，21移植到截肢平面上，立即将截肢缝合到体腔22中，以及连续每天移除或照射早期伤口表皮和AEC23，24。总而言之，这些实验不仅确定了伤口表皮/ AEC的重要性，而且还进一步确定了其在早期组织组织分解中的作用，以及在整个再生过程中维持祖细胞增殖和原始细胞生长13。
然而，这些先前的研究主要局限于组织学染色以及经三联胸腺嘧啶脉冲以跟踪细胞增殖。事实上，用现代测序技术和蝾螈的功能技术重新审视这些经典实验直到最近才完成，并导致发现伤口表皮在再生早期阶段调节炎症和ECM降解/沉积的其他作用25。随着各种蝾螈基因组和转录组序列的释放26，27，28，29，30，31，32，33，34，以及蝾螈物种中可用的功能方法数量的迅速增加11，35，36，37，38研究人员现在处于有利地位，可以开始解开驱动伤口表皮形成，功能和AEC发展的分子机制。
不幸的是，用于抑制伤口表皮形成的这些经典方法中的几种在技术上具有挑战性，在同一实验中，生物重复之间的可重复性存在困难。例如，维持皮肤移植物可能具有挑战性，因为移植物最终可能会从宿主肢体上脱落，并且每天切除伤口表皮/ AEC很难不损坏下面的组织。此外，将截肢缝合到体腔中具有挑战性，并且还需要在插入部位进行额外的伤害。另一方面，在截肢平面上立即缝合全层皮肤相对简单，技术上可重复，并且引入的组织损伤最小。这种全皮瓣（FSF）手术方法以前由Anthony Mescher于1976年在成年蝾螈（Notophthalmus viridiscens）中开发。他证明，FSF手术通过禁止上皮细胞在截肢平面上的迁移以及上皮细胞与下层组织之间的直接接触来抑制伤口表皮的形成和功能。
在这里，该外科手术使用蝾螈肢体逐步显示。结合现代分子和测序技术，这种技术可能有助于研究人员加深我们对伤口表皮/AEC形成和肢体再生过程中功能的理解。

<table>
	<tbody>
		<tr>
			<td>Curved spring scissors</td>
			<td>Fine Scientific Tools</td>
			<td>15009-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl 3-aminobenzoate methanesulfonate (Tricaine)</td>
			<td>Sigma-Aldrich</td>
			<td>886-86-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Scientific Tools</td>
			<td>11252-40</td>
			<td>Need two pairs</td>
		</tr>
		<tr>
			<td>Nylon monofilament sutures (9-0)</td>
			<td>Roboz</td>
			<td>SUT-1000-21</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>Leica</td>
			<td>MZ6</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfamerazine sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>127-58-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Fine Scientific Tools</td>
			<td>14002-14</td>
			<td></td>
		</tr>
	</tbody>
</table>

inhibition of wound epidermis formation via full skin flap surgery during axolotl limb regeneration

上个世纪蝾螈再生生物学的经典实验早已确定，伤口表皮是截肢后迅速形成的关键信号结构，是肢体再生所必需的。然而，在过去的几十年中，由于蝾螈模型系统中缺乏精确的功能技术和基因组信息，在分子水平上研究其精确功能的方法受到限制。令人兴奋的是，最近过多的测序技术加上各种蝾螈基因组的发布以及包括CRISPR在内的功能性基因检测方法的出现，使得以前所未有的分子分辨率重新审视这些基础实验成为可能。在这里，我描述了如何在成人蝾螈中进行经典开发的全皮瓣（FSF）手术，以抑制截肢后立即形成伤口表皮。伤口表皮通常通过皮肤近端上皮细胞的远端迁移到截肢平面形成，以将伤口与外部环境隔离开来。手术需要立即在截肢平面上缝合全层皮肤（包括表皮层和真皮层），以阻碍上皮细胞迁移和与潜在受损间充质组织的接触。成功的手术可抑制卵涌卵形成和肢体再生。通过将这种手术方法与当代下游分子和功能分析相结合，研究人员可以开始揭示肢体再生过程中伤口表皮功能和生物学的分子基础。

该手术方案将允许完全抑制伤口表皮的形成（图1），并最终抑制肢体再生。根据动物的大小，成功的手术在大约2-3周内不会形成胚芽肿，而对照再生肢体应正常形成胚芽肿。
研究人员应每2-3天用肉眼检查缝合的肢体，以确保缝合线没有弹出并且没有形成卵涌水肿。如果一个或多个缝合线弹出，伤口表皮仍可能形成，导致小或大的胚芽肿和不成功的手术（图2）。此外，研究人员应每周在立体显微镜下至少检查一次缝合的肢体，以确保截肢表面上的任何地方都不明显的薄伤口表皮。为了进行比较，研究人员还应该检查对照再生肢体，该肢体应该在截肢平面上有伤口表皮，并在2-3周内形成胚芽肿。伤口表皮将显得薄而清晰，而正常皮肤将分别在白血病，白化或野生型蝾螈中显得更不透明和淡粉红色（几乎是白色），浅黄色或深绿色。
如果研究人员希望在2-3周的胚芽肿形成阶段之前收集组织，他们应该在收集样本之前检查缝合的肢体，以确保缝合线保持在原位并且没有形成小的伤口表皮。此外，通过缝合的肢体组织矢状切片并在任何时间点进行组织学分析也可以从包围整个截肢平面的整个皮瓣中验证真皮的存在以及没有伤口表皮（图3）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61522/61522fig01.jpg"> 图1：全皮瓣手术的步骤示意图。 协议的步骤在这里编号和图表。虚线表示协议步骤1和3处的截肢平面。 <a href="https://www.jove.com/files/ftp_upload/61522/61522fig01large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61522/61522fig02.jpg"> 图2：成功和不成功的全皮瓣手术示例。 截肢后25天（dpa）成功手术（左）、手术不成功（右）和对照再生肢体（无手术）的具有代表性的明场图像。成功的手术有一个平坦的截肢平面，其中整个皮瓣被缝合，而不成功的手术有一个小的卵涌肿发展。箭头表示截肢平面和白色虚线有助于可视化成功手术中没有血涌水肿，以及不成功手术中胚泡的存在，并对照再生肢体。 <a href="https://www.jove.com/files/ftp_upload/61522/61522fig02large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61522/61522fig03.jpg"> 图3：正常再生和FSF缝合肢体的组织学染色。 （A-B'）在7 dpa下再生（A-A'）和缝合的蝾螈肢体（B-B'）的皮小球染色切片的代表性明场图像。 A 和 B 中的插图分别显示在 A' 和 B' 中。胶原蛋白较重的真皮层线条并覆盖缝合肢体的整个截肢平面。截肢平面在 A-B中用箭头表示。比例尺表示 500 μm。这个数字改编自Tsai et al.25。 <a href="https://www.jove.com/files/ftp_upload/61522/61522fig03large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>

Watch this Scientific Journal Video about Inhibition of Wound Epidermis Formation via Full Skin Flap Surgery During Axolotl Limb Regeneration at JoVE.com

Inhibition of Wound Epidermis Formation via Full Skin Flap Surgery During Axolotl Limb Regeneration

本文介绍了如何通过立即在截肢平面上缝合全层皮肤来执行手术方法，以抑制蝾螈肢体再生期间伤口表皮的形成。这种方法使研究人员能够研究伤口表皮在肢体再生早期阶段的功能作用。

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