Name: a novel high-throughput ex vivo ovine skin wound model for testing emerging antibiotics
Uploaded: 2022-09-16T13:20:00
Description: Watch this Scientific Journal Video about A Novel High-Throughput Ex Vivo Ovine Skin Wound Model for Testing Emerging Antibiotics at JoVE.com

作者要感谢EPSRC（EP/R513313/1）的资助。作者还要感谢切斯特菲尔德卡洛的R.B Elliot和Son屠宰场提供羊头并在项目的早期阶段如此包容，感谢Kasia Emery在整个协议开发过程中的支持，以及谢菲尔德大学感染，免疫和心血管疾病系的Fiona Wright处理组织学样本，并在整个项目中提供了令人难以置信的帮助。

来自R.B Elliott和Son屠宰场的羔羊头被用作该项目的皮肤样本来源。所有的羔羊都被宰杀作为食物食用。这些没有丢弃头部，而是被重新用于研究。不需要道德批准，因为组织来自屠宰场丢弃的废物。
1. 灭菌
<ol><li>在收集头部之前对镊子进行消毒，方法是取干净的镊子并在200°C的烤箱中进行干热灭菌1小时。使用前将所有玻璃器皿在121°C高压灭菌15分钟。</li>
	<li>在...

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抗菌剂的开发是一项重要但昂贵的冒险，估计耗资约10亿美元，大约需要15年才能完成。超过90%的抗菌药物发现和抗菌药物疗效的临床前研究是由学术研究人员和通常少于50名员工的中小型公司进行的22。这些团队在财务上非常拮据，这使得铅分子在转化研究后期阶段的失败是灾难性的。抗微生物药物耐药性的上升速度超过了新型抗菌药物的发展速度，这进一步加剧了负责任地管?...

皮肤感染是一个重要的全球性问题，给世界各地的医疗保健提供者带来了巨大的经济成本。病原体对多重耐药性和生物膜形成的发展在伤口不愈合的流行中起着关键作用1，2，3，4。因此，皮肤和软组织感染是延长住院和随后再次入院的更常见原因之一5。伤口愈合的延迟对患者和医疗保健提供者来说都是代价高昂的，据估计，美国每年约有 650 万患者受到影响。在英国，皮肤感染和相关并发症每年导致约75，000人死亡2，4，6。
金黄色葡萄球菌（S. aureus） 是一种强大的伤口病原体，经常从患者伤口中分离出来2，7。多重耐药性的出现率在2000年代急剧增加。在此期间，大约 60% 的急性细菌性皮肤和皮肤结构感染对耐甲氧西林 金黄色葡萄球菌1 呈培养阳性。在过去20年中， 葡萄球菌和其他病原体中耐多药菌株的数量不断增加，这表明迫切需要快速开发具有克服耐药性的新作用模式的抗生素。
然而，自 2000 年代初以来，抗生素发现计划一直以较长的开发时间和低成功率为主，只有 17% 的新型抗生素进入美国临床试验获得市场批准8.这表明新兴抗生素的 体外 检测结果与其临床结果之间存在差异。可以争辩说，这种差异主要是由于 体内 感染期间细菌生理学的差异以及在 体外 临床前阶段测试抗生素功效时常规微生物学方法的差异。因此，需要更能代表感染期间细菌生理学的新型实验室方法，以提高抗生素发现计划的成功率。
目前研究皮肤感染的方法包括活体动物（例如小鼠）、离体皮肤模型（例如猪）和3D组织工程皮肤模型（例如人类）的研究9，10，11，12。对活体动物的研究受到严格监管，通量相对较低。在动物模型中，伤害和感染会给动物带来巨大的痛苦，并引起伦理问题。人体皮肤模型，离体或组织工程，需要伦理批准，遵守当地和全球立法（人体组织法，赫尔辛基宣言），并且难以获得组织，有些要求需要数年才能满足13，14。这两种模型类型都是劳动密集型的，需要大量的专业知识才能确保实验成功。目前一些离体皮肤感染模型需要预先接种的椎间盘和伤口床添加剂才能使感染;尽管这些模型非常有用，但在感染过程中存在局限性，因为添加剂限制了伤口床作为营养来源的利用10，15，16，17。本研究中描述的模型不对伤口床使用添加剂，这确保了感染的病理学和活细胞计数是直接利用伤口床作为唯一营养来源的结果。
鉴于对新实验室方法的需求，已经开发出一种用于评估新兴抗生素疗效的新型高通量离 体 羊皮肤感染模型。皮肤感染研究面临许多挑战 - 高成本，伦理问题和无法显示全貌的模型20，21。 离 体模型和3D外植体模型可以更好地可视化疾病过程以及更具临床相关性的模型对治疗的影响。这里描述了一种新的绵羊皮肤模型的建立，该模型简单，可重复，临床相关且具有高通量。选择绵羊皮肤是因为绵羊是通常用于模拟 体内感染反应的大型哺乳动物之一。此外，它们很容易从屠宰场获得，确保为研究提供稳定的皮肤供应，并且它们的尸体不会被烫伤，确保良好的组织质量。这项研究使用 金黄色葡萄球菌 作为典型病原体;但是，该模型适用于其他微生物。

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			<td>24 Well Companion Plate</td>
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			<td>Labtech International</td>
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			<td>Not applicable</td>
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			<td>15373212&#160;</td>
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			<td>Fisher Scientific</td>
			<td>15575809</td>
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			<td>Homogenizer 220, plastic blending cones</td>
			<td>Fisher Scientific&#160;</td>
			<td>15585819</td>
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			<td>Insert Individual 24 well 0.4um membrane</td>
			<td>VWR International</td>
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			<td>91077C-1G</td>
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			<td>Medium 199 (MK media)</td>
			<td>Sigma</td>
			<td>M0393</td>
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			<td>Fisher Scientific</td>
			<td>10687551</td>
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			<td>Sigma&#160;</td>
			<td>P4417-100TAB</td>
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			<td>SLS&#160;</td>
			<td>P0781</td>
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			<td>Fisher Scientific</td>
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			<td>Fisher Scientific</td>
			<td>1284804</td>
			<td>Class II microbiology safety cabinet</td>
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			<td>Scalpel blade number 15</td>
			<td>Fisher Scientific</td>
			<td>O305</td>
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			<td>Scalpel Swann Morton</td>
			<td>Fisher Scientific</td>
			<td>11849002</td>
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			<td>Sigma</td>
			<td>S5761-1KG</td>
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			<td>Toothed Allis Tissue Forceps</td>
			<td>Rocialle</td>
			<td>RSPU500-322</td>
			<td></td>
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			<td>Tryptic Soy Agar</td>
			<td>Merck Life Science UK Limited</td>
			<td>14432-500G-F</td>
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			<td>Merck Life Science UK Limited</td>
			<td>41298-500G-F</td>
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			<td>Vimoba Tablets</td>
			<td>Quip Labs</td>
			<td>VMTAB75BX</td>
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a novel high-throughput ex vivo ovine skin wound model for testing emerging antibiotics

抗菌药物的开发是一个昂贵的过程，成功率越来越低，这使得对抗菌发现研究的进一步投资变得不那么有吸引力。如果在先导物优化阶段可以实施快速失败和廉价失败的方法，那么抗菌药物的发现和随后的商业化可以变得更加有利可图，研究人员可以更好地控制药物设计和配方。本文介绍了一种感染金黄色葡萄球菌的离体绵羊损伤皮肤模型的建立，该模型简单、经济高效、通量高且可重复。模型中的细菌生理学模拟了感染期间细菌增殖取决于病原体破坏组织的能力。伤口感染的建立通过与接种物相比活细菌计数的增加来验证。该模型可用作在先导物优化阶段测试新兴抗菌剂功效的平台。可以说，该模型的可用性将为开发抗菌剂的研究人员提供快速失败和失败便宜的模型，这将有助于提高后续动物试验的成功率。该模型还将促进减少和改进动物用于研究，并最终能够更快，更具成本效益地将用于皮肤和软组织感染的新型抗菌剂转化为临床。

在建立伤口感染模型之前确定对皮肤进行消毒的途径具有挑战性。挑战在于在不损害不同皮肤层的情况下对皮肤进行消毒，这可能会继续对感染结果产生意想不到的后果。为了确定适当的灭菌方案，如 表1所示，在不同的时间内尝试了不同的处理方法。污染记录为用于维持皮肤样品的MK培养基中48小时后浑浊的发展。通过组织学监测组织完整性，然后在处理后立即用苏木精和伊红（H&E?...

Watch this Scientific Journal Video about A Novel High-Throughput Ex Vivo Ovine Skin Wound Model for Testing Emerging Antibiotics at JoVE.com

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

该协议描述了一种逐步建立感染金黄色葡萄球菌的离体绵羊受伤皮肤模型的方法。与传统微生物学技术相比，这种高通量模型更好地模拟体内感染，并为研究人员提供了一个生理相关的平台来测试新兴抗菌剂的功效。

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 ...

该模型将使处于关键临床前阶段的研究人员能够更准确地确定新配方的抗菌功效，并使研究人员能够更好地控制药物设计和配方。该模型可在具有代表性的伤口环境中快速重现结果。它减少了对专门饲养的动物的需求，并有助于更快地将伤口感染的局部抗菌剂翻译成临床。主要挑战是污染和灵活性。严格的无菌技术和耐心的组织处理是必不可少的;这种方法的成功需要精确和有目的的皮瓣去除。首先对镊子进行消毒，将其置于200摄氏度的干热灭菌中一小时。使用前将所有玻璃器皿在 121 摄氏度下高压灭菌 15 分钟。通过将大约 100 毫升 200 PPM 二氧化氯溶液倒入样品区域来消毒灯的前额部分。使用电动剪剃掉额头部分，然后用 200 毫升 200 PPM 的二氧化氯溶液清洗。使用乙醇和蓝色卷擦拭该区域，并用脱毛膏覆盖样品区域。35分钟后，使用刮刀工具轻轻刮掉脱毛膏并评估样品区域。如果留下大量毛发，请重复脱毛过程。用200毫升二氧化氯溶液冲洗干净的样品区域，然后用乙醇和蓝色卷冲洗。使用无菌的 8 毫米活检打孔器从准备好的区域切出 8 毫米的皮肤样本。然后使用无菌镊子和 15 号刀片手术刀取出样品，确保去除所有皮肤脂肪。将样品放入装有无菌PBS的无菌0.5升罐中。然后将它们转移到装有50毫升200 PPM二氧化氯溶液的无菌50毫升管中。将试管倒置两次，使其消毒 30 分钟。将样品转移到装有40毫升无菌PBS的50毫升管中。PBS洗涤后，将每个皮肤样品放入24孔板的单独孔中。在孔中加入350微升预热的培养基，同时将样品保持在气液界面。用透气板密封24孔板，然后将板置于5%二氧化碳和37摄氏度的加湿组织培养箱中孵育24小时。孵育后，除去培养基并在500微升无菌PBS中冲洗样品。用5%二氧化碳和37摄氏度的无抗生素培养基处理样品中的残留抗生素24小时，从样品中去除残留的抗生素。如果在无抗生素培养基中出现浑浊或真菌感染，请丢弃样品。接下来，准备一个 50 毫升的管子和 10 毫升无菌胰蛋白酶大豆汤。然后将几个金黄色葡萄球菌菌落从新鲜的金黄色葡萄球菌平板转移到肉汤中，并将试管在 37 摄氏度和 150 RPM 下孵育 18 小时。将试管以4， 000G离心三分钟后，除去上清液并将细胞沉淀重悬于10毫升无菌PBS中。在使用无菌PBS将接种物调节至0.6纳米波长的600光密度之前，重复PBS洗涤两次。通过手动活板计数确认接种量。为了感染皮肤样品，用400微升预热的无抗生素培养基准备一个新的24孔板，并使用无菌镊子添加24孔插入物。从皮肤样品中取出培养基。用500微升无菌PBS清洗它们并去除洗涤液。使用无菌镊子将样品轻轻地固定在孔底部。使用四毫米的打孔活检将皮肤样本刺穿至一到两毫米的粗略深度，并制作中央伤口皮瓣。然后使用15号刀片手术刀和无菌齿Allis组织钳去除伤口瓣的顶层。一旦所有样品都被伤伤，使用无菌镊子将它们转移到24孔插入物中。将15微升细菌接种物移入伤口床中，然后在加湿的组织培养箱中以37摄氏度孵育24小时。对于更长的孵育期，取出培养基，每24小时用新鲜培养基更换一次，并在相同条件下孵育板。要确定细菌负荷，请从孔底部取出培养基。使用无菌镊子，将每个样品转移到装有一毫升无菌PBS的单独50毫升管中。然后，通过使用细尖均质器均质样品表面，将细菌从伤口床表面分离。确保伤口床与均质机的尖端直接接触。处理完所有样品后，涡旋每个样品，然后将20微升匀浆转移到含有180微升无菌PBS的96孔板的相应孔中。将每个样品匀浆连续稀释至1乘以10至负7，并将10微升稀释的匀浆移液到胰蛋白酶大豆琼脂平板上一式三份。将胰蛋白酶大豆琼脂平板在 37 摄氏度下孵育，18 小时后，计数菌落数以确定每个样品的菌落形成单位或 CFU。确定了适当的皮肤灭菌方案，使用不同的治疗方法，时间长短不一。通过组织学监测组织完整性，然后在处理后立即用苏木精和伊红染色。用二氧化氯进行30分钟的治疗是最有效的治疗方法，在保持组织完整性的同时，可重复地对皮肤组织进行消毒。感染后48小时，伤口床上的白色薄膜在组织上很明显。尽管划痕模型在 24 小时后的平均 CFU 数量更高，但皮瓣去除损伤技术产生了更一致的结果。48小时后，与接种物相比，从受伤组织中回收的细菌CFU大约多100倍，表明感染成功。感染组织的革兰染色组织学切片表明伤口床中存在金黄色葡萄球菌细胞，而未感染组织切片中不存在。在此过程之后，可以进行免疫组织化学以研究组织反应，修饰革兰染色以可视化细菌定位，以及抗菌暴露后的活板计数以测试抗菌功效。对于开发新型抗菌剂的研究人员来说，该模型将更好地控制先导物优化，减少对昂贵且严格监管的动物试验的依赖，并使抗菌剂能够快速转化为临床。

Research

JoVE Journal

Immunology and Infection

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

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

一个96孔微孔板为基础的方法监测的形成和抗真菌的药敏试验白色念珠菌生物膜

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third ...

科研

免疫与感染

High-throughput Detection Method for Influenza Virus

流感病毒的高通量检测方法

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

从表型分析和人体皮肤淋巴细胞分离<em&gt;离体</em&gt;细胞培养

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

高通量，实时，细胞内的抗菌活性的双读出测试和真核细胞的细胞毒性

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require ...

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 ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

沙门氏菌筛选的高通量平台.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

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 ...

A High-throughput Shigella-specific Bactericidal Assay

A High-throughput Shigella-specific Bactericidal Assay

一种高通量志贺氏菌特异性杀菌试验

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...

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

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 ...

Establishing a Porcine Ex Vivo Cornea Model for Studying Drug Treatments against Bacterial Keratitis

建立猪外维沃角膜模型研究药物治疗细菌性角膜炎

When developing novel antimicrobials, the success of animal trials is dependent on accurate extrapolation of antimicrobial efficacy from in vitro tests to ...

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

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