卡瓦略医生的工作是对妇女的健康和国立儿童健康和人类发展的美国国立卫生研究院（5K12 HD043452）研究办公室的研究经费支持。焦虑医生收到I-流量（加利福尼亚州Lake Forest）的电源（ON-Q PainBuster术后疼痛缓解系统）和资金的生化检测。

1。研究协议<ul><li>获得机构审查委员会的批准，并签署知情同意书。 </li><li>选择适当的手术人群研究的行为。这种方法只适用于剖宫产的伤口，但大多数外科伤口将是适当的。 </li><li>考虑您的参与研究的纳入和排除标准。病人和外科伤口感染率高的程序，可能不适合。 </li><li>规范麻醉及术后管理，对所有患者，以避免潜在的混杂因素。 </li></ul> 2。伤害性刺激和炎症的生化调解收藏<ul><li>到皮下组织层，将ON-Q SilverSoaker（I-流，森林湖，CA）的ON-Q PainBuster的前伤口愈合的其他组织层（可以考虑）。 ON-Q PainBuster疼痛救助制度不断提供生理盐水2毫升/小时的速度。载体可以是梳状独立非执行董事或作为局部麻醉剂和非甾体类消炎药等药物。 </li><li>进入这个系统包括一个标准的三通阀，允许愿望，伤口渗出液中，在指定的时间点。连续输注凝血防止导管，并提高了系统的可靠性，以产生渗出液样品。如果渗出物的愿望是困难的（约5％的情况下），轻轻推上面的伤口，改变对象的位置（ 例如 ，坐或卧的病人他们平），用0.5-1毫升生理盐水冲洗，或者撤销导管1-2厘米。 </li><li>在协议规定的时间点（ 例如 ，1，4，6，24，和48小时后，剖宫产），吸撤出伤口渗出液1毫升。渗出液转移到含有30μl的蛋白酶抑制剂的聚乙烯杯中。我们的目的，我们使用完整的蛋白酶抑制剂（罗氏生物科学，帕洛阿尔托，加利福尼亚州）。 </li><li在相同的时间间隔的研究协议规定，收集10毫升血液。我们已经使用了含有肝素锂的血液收集管，在我们的机构，这是一个绿色的平顶的玻璃管。添加一个额外的300微升蛋白酶抑制剂进入血液样本。 </li><li>收集在1小时内，把样品在冰和离心机以3,000 rpm离心10分钟。 </li><li>删除血清和伤口的上清液和地方在一个标准的离心管中，储存在-20°C </li></ul> 3。含量分析<ul><li>一旦所有的研究渗出液和血清样品的收集和存储，解冻和分析的样品在相同的时间。 </li><li>我们测量了使用多重免疫细胞因子和趋化因子。多重免疫检测技术，使我们能够分析物分析众多小体积样品（50微升）。复用产生的结果与那些获得与ELISA。我们已经使用了17复杂生物PlexTM的系统（BioRad公司，Herc并且ULES，CA）为我们分析。此测定是能够测量的肿瘤坏死因子α（TNFα），干扰素α（INFα），白细胞介素1β（IL-1β），白细胞介素2（IL-2），白细胞介素4（IL-4），白细胞介素5（IL-5。 ），白细胞介素6（IL-6），白细胞介素7（IL-7），白细胞介素8（IL-8），白细胞介素（IL-10），白细胞介素12（IL-12），白细胞介素13（IL-13），白细胞介素17（IL-17），粒细胞集落刺激因子（G-CSF），粒细胞 - 巨噬细胞集落刺激因子（GM-CSF），单核细胞趋化蛋白1（MCP-1）和巨噬细胞炎性蛋白-1（MIP-1β）。神经生长因子与NGF DY256抗体测定用的Bio-Plex的胺偶联试剂盒，通过将其添加到17复杂的板。 </li><li>为了保证良好的质量分析，每次测量应一式两份。每个分析物的标准曲线所产生的使用由制造商提供的参考分析物（浓度为0.20，0.78，3.13，12.5，50，200，800，3200皮克/毫升加零标准生理盐水）。标准曲线包括在每次运行中，样品浓度的计算生物-Plex的Manager软件。 </li><li> P物质和前列腺素E2的测定采用高灵敏度ELISA试剂盒。我们已经使用含量设计公司（密歇根州Ann Arbor）这一分析。下进行分析，一式两份，根据制造商的说明书。 </li></ul>

<ol>
	<li>Carvalho, B., Clark, D. J., Angst, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+and+Systemic+Release+of+Cytokines,+Nerve+Growth+Factor,+Prostaglandin+E2,+and+Substance+P+in+Incisional+Wounds+and+Serum+Following+Cesarean+Delivery.">Local and Systemic Release of Cytokines, Nerve Growth Factor, Prostaglandin E2, and Substance P in Incisional Wounds and Serum Following Cesarean Delivery.</a> J. Pain. 9, 650-657 (2008).</li><li>Carvalho, B., Clark, D. J., Yeomans, D. C., Angst, M. 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没有利益冲突的声明。

上文所述的方法，被认为是敏感，可以探测生化伤口渗出液中的炎症和疼痛介质。在不同时间点的采样也让我们能够观察到这些介质的变化。1,2随着时间的推移，我们不知道任何其他人的手术伤口分析，可以连续测量手术伤口渗出液中的炎症和疼痛介质的释放曲线。从我们的方法从手术切口部位的伤口渗出液收集的结果是不同研究利用水渠从髋关节，7,8这表明，皮下伤口关节内或其他体腔生化介质的释放介质的释放可能会有所不同。此外，在水渠的结果可能会被混淆到伤口失血，因此，他们可能无法准确地反映当地的生化介质的释放。 之间的相关性较差确定特定地点的释放，在研究手术伤口时，伤口和血清演示的重要性。本地更改可能不会反映系统的原因是多方面的，包括在手术创伤和压力的差全身和局部的生产。虽然我们已经选择测量的特定选择的亲和抗炎性细胞因子和趋化因子，以及NGF，SP和PG-E2从剖腹产伤口渗出液，1,2，这方法可以应用到其他的手术模型和许多其他生化介质。 手术伤口的局部麻醉剂滴入改善术后疼痛是一种常用的技术，应用的方法，我们已经能够证明，手术伤口的局部麻醉剂滴入不影响伤口的生物化学。2下降，IL-10和P物质增加布比卡因灌注伤口渗出液中建议中断Øf，消炎的调制。外周静脉给药的局部麻醉剂，从而影响炎症过程，10,11可能的部分原因引起的关节软骨溶解在局部麻醉后手术。 ON-Q PainBuster疼痛的救济制度是美国食品和药物管理局（FDA）批准的设备自1998年以来，用于交付的局部麻醉剂的手术伤口部位，以减轻疼痛，手术后。连续伤口灌注利用这一点，其他的导管会增加伤口感染。9,13 ON-Q PainBuster ON-Q SilverSoaker注入药物，而不是反复品尝伤口渗出液中。虽然我们有没有伤口感染，用这种方法，我们强调无菌技术，从伤口渗出液中，一边品尝这个系统。我们所提出的修改，以方便取样应保留用于研究目的，而不是常规的临床ü本身。我们还建议，导管保持在原位的时间被限制在48-72小时。 这种手术部位的生物测定技术提供了一个很好的模型，以了解在手术后的炎症和疼痛的生化介质之间的关系。本地管理的非甾体类抗炎药和阿片类药物的研究，我们将在今后的研究中使用的方法。技术本身也可能有助于伤口愈合，过手术疤痕和伤口感染的调查研究。

手术切口后，居住和迁移炎症细胞释放的亲和抗炎性细胞因子，前列腺素，神经肽和趋化因子是重要的起始和维持疼痛。3,4收集站点特定的生化标志物的能力是至关重要的决定血清及手术伤口的水平之间的关系，了解介质的释放和手术的痛苦之间的复杂关系，并能够手术伤口生物化学的效果进行评估的全身和周边药品监督管理局。研究都集中在全身的生化介质的测量，并没有考虑特定地点的释放。5,6血清细胞因子和趋化因子的测量结果可能并不能反映局部手术伤口的生化事件，只能说明一个全球性的全身反应，手术损伤。本文提供了一个详细的和独特的视觉描述，这种方法是杉木ST在2008年引用的疼痛期刊。此朱庇特的文章的目的是提供一个独特的视觉效果的描述方法。

collecting and measuring wound exudate biochemical mediators in surgical wounds

我们描述了一种方法，使我们能够收集并测量生化在手术伤口部位的炎症和疼痛介质。收集站点特定的生化标志物，重要的是要理解之间的关系血清及手术伤口的水平，确定任何介质的释放，疼痛，止痛药使用和其他成果之间的关联，并评估全身和周边药品监督管理局对手术伤口的效果生物化学。这种方法已被应用于健康与脊髓麻醉的择期行剖宫产的妇女。我们测量在相同的时间间隔为病人的疼痛评分和止痛剂的消费量高达48小时后剖宫产的伤口渗出液和血清介质。使用这种方法，我们已经能够检测各种生化介质，包括神经生长因子（NGF），前列腺素E2（PG-E2），P物质，IL-1β，IL-2，IL-4，IL-6，IL-7， IL-8，IL-10，IL-12，IL-13，IL-17，TNFα，INFγ，G-CSF，GM-CSF，MCP-1和MIP-1β。应用此人的手术伤口生物测定的研究都没有发现之间的相关性伤口和血清细胞因子或释放（J疼痛，2008，9（7）:650-7）。我们也记录了实用的技术，识别药物介导，伤口细胞因子含量（2010年麻醉; 111:1452-9）。

使用这种方法，我们已经能够检测到所有的生化介质上面概述与伤口渗出液和血浆中的IL-5的异常。1,2一些额外的血浆中未检测到细胞因子（IL-4，IL-17，TNFα，INFγ的，GM-CSF）的时间过程中测量的细胞因子和趋化因子的研究报道是一致的，达到了高原的前几个小时内，持续24小时的观察期内1,2然而，NGF稳步增长24小时采集周期，而PGE2在1小时内达到高峰，然后逐步下降。特别值得一提的结果是显 ​​着降低的IL-10水平和P物质水平的提高，伤口灌输与局部麻醉剂布比卡因，兼容整体亲抗炎作用的药物（ 图1）。在不同的研究中，我们表明，剖宫产术后的镇痛药的消耗量涂装手术呈负相关，与伤口渗出液中IL-6，IL-1β，和G-CSF的浓度（ 图2）。 <img alt="图1" fo:content-width="3in" fo:src="/files/ftp_upload/50133/50133fig1highres.jpg" src="/files/ftp_upload/50133/50133fig1.jpg" /> 图1。白细胞介素10（IL-10）和P物质的伤口渗出物水平水平（皮克/毫升）测量1，3，5，7，和24小时后，剖腹产手术。生理盐水组的灰色钻石，布比卡因组，用黑色方块。 *显着性差异（P &lt;0.005）。 <img alt="图2" src="/files/ftp_upload/50133/50133fig2.jpg" /> 图2。白细胞介素6（皮克/毫升）和吗啡用量在第一个24小时后，剖宫产的相关性。

Watch this Scientific Journal Video about Collecting And Measuring Wound Exudate Biochemical Mediators In Surgical Wounds at JoVE.com

Collecting And Measuring Wound Exudate Biochemical Mediators In Surgical Wounds

本文提供了一个详细和视觉的描述的生化在剖宫产手术伤口部位的炎症和疼痛介质收集和测量方法的。已被用于这种人类生物测定，以确定伤口和血清中的细胞因子的浓度和伤口的细胞因子，趋化因子和神经肽的药物介导的变化之间的相关性。

手术伤口伤口渗出液生化调解员在收集和测量

We describe a methodology by which we are able to collect and measure biochemical inflammatory and nociceptive mediators at the surgical wound site. ...

collecting-measuring-wound-exudate-biochemical-mediators-surgical

Nanyang Technological University

Research

JoVE Journal

Medicine

11.9K 次观看.  Stanford University School of Medicine. 本文提供了一个详细和视觉的描述的生化在剖宫产手术伤口部位的炎症和疼痛介质收集和测量方法的。已被用于这种人类生物测定，以确定伤口和血清中的细胞因子的浓度和伤口的细胞因子，趋化因子和神经肽的药物介导的变化之间的相关性。

视频: Collecting And Measuring Wound Exudate Biochemical Mediators In Surgical Wounds

Collecting and Measuring Nociceptive and Inflammatory Mediators in Surgical Wounds 

We describe a methodology by which we are able to collect and measure inflammatory and nociceptive biochemical mediators at the surgical wound site. Collecting site-specific biochemical markers allows us to evaluate the relationship between surgical wound and serum levels; determine any associations between mediator release, pain and analgesic consumption; and evaluate the effect of systemic and peripheral drug administration on surgical wound biochemistry.
This methodology has been applied to healthy women undergoing elective cesarean delivery with spinal anesthesia. Wound exudate and serum mediators, in conjunction with pain scores and analgesics consumption were measured at 1, 6, 24, and 48 hours post-cesarean delivery. Biochemical mediators that were detected included IL-1&beta;, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, TNF&alpha;, INF&gamma;, G-CSF, GM-CSF, MCP-1 and MIP-1&beta;, nerve growth factor (NGF), prostaglandin E2 (PG-E2) and substance P. We found no correlations between wound and serum cytokines concentrations or time-release profiles (J Pain. 2008 Jul 9(7):650-7). This article describes and demonstrates the feasibility of collecting and assaying nociceptive and inflammatory mediators in surgical wounds at specific time points. The lack of significant correlations between serum and wound levels shows the importance of determining site-specific release if surgical wounds and localized pathologies are to be studied.

Collecting and Measuring Nociceptive and Inflammatory Mediators in Surgical Wounds

A technique to collect and measure surgical wound biochemical mediators at specific time points.

在手术伤口的痛觉及炎症介质的收集和测量

We describe a methodology by which we are able to collect and measure inflammatory and nociceptive biochemical mediators at the surgical wound site. ...

科研

生物学

Murine Excisional Wound Healing Model and Histological Morphometric Wound Analysis

The murine excisional wound model has been used extensively to study each of the sequentially overlapping phases of wound healing: inflammation, proliferation and remodeling. Murine wounds have a histologically well-defined and easily recognizable wound bed over which these different phases of the healing process are measurable. Within the field, it is common to use an arbitrarily defined &#8220;middle&#8221; of the wound for histological analyses. However, wounds are a three-dimensional entity and often not histologically symmetrical, supporting the need for a well-defined and robust method of quantification to detect morphometric defects with a small effect size. In this protocol, we describe the procedure for creating bilateral, full-thickness excisional wounds in mice as well as a detailed instruction on how to measure morphometric parameters using an image processing program on select serial sections. The two-dimension measurements of wound length, epidermal length, epidermal area, and wound area are used in combination with the known distance between sections to extrapolate the three-dimension epidermal area covering the wound, overall wound area, epidermal volume and wound volume. Although this detailed histological analysis is more time and resource consuming than conventional analyses, its rigor increases the likelihood of detecting novel phenotypes in an inherently complex wound healing process.

This protocol describes how to generate bilateral, full-thickness excisional wounds in mice and how to subsequently monitor, harvest, and prepare the wounds for morphometric analysis. Included is an in-depth description of how to use serial histological sections to define, precisely quantify and detect morphometric defects.

穆林切除伤口愈合模型与组织学形态性伤口分析

The murine excisional wound model has been used extensively to study each of the sequentially overlapping phases of wound healing: inflammation, ...

发育生物学

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC2010 birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e.&#160;from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

黏膜衬里流体为体内上呼吸道免疫介质水平量化的无创性取样

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

免疫与感染

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. Chronic mouse wounds models have been used to understand the underlying interactions between the microorganisms and the host. The models developed to date rely on the use of haired animals and terminal collection of wound tissue for determination of viable bacteria. While significant insight has been gained with these models, this experimental procedure requires a large number of animals and sampling is time consuming. We have developed a novel murine model that incorporates several optimal innovations to evaluate biofilm progression in chronic wounds: a) it utilizes hairless mice, eliminating the need for hair removal; b) applies pre-formed biofilms to the wounds allowing for the immediate evaluation of persistence and effect of these communities on host; c) monitors biofilm progression by quantifying light production by a genetically engineered bioluminescent strain of Pseudomonas aeruginosa, allowing real-time monitoring of the infection thus reducing the number of animals required per study. In this model, a single full-depth wound is produced on the back of STZ-induced diabetic hairless mice and inoculated with biofilms of the P. aeruginosa bioluminescent strain Xen 41. Light output from the wounds is recorded daily in an in vivo imaging system, allowing for in vivo and in situ rapid biofilm visualization and localization of biofilm bacteria within the wounds. This novel method is flexible as it can be used to study other microorganisms, including genetically engineered species and multi-species biofilms, and may be of special value in testing anti-biofilm strategies including antimicrobial occlusive dressings.

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

Here we describe a novel diabetic murine model utilizing hairless mice for real-time, non-invasive, monitoring of biofilm wound infections of bioluminescent Pseudomonas aeruginosa. This method can be adapted to evaluate infection of other bacterial species and genetically modified microorganisms, including multi-species biofilms, and test the efficacy of antibiofilm strategies.

来到光明的一面: 体内糖尿病无毛小鼠慢性伤口生物膜感染的监测

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. ...

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

Wound healing is a complex process that requires the orderly progression of inflammation, granulation tissue formation, fibrosis, and resolution. Murine models provide valuable mechanistic insight into these processes; however, no single model fully addresses all aspects of the wound healing response. Instead, it is ideal to use multiple models to address the different aspects of wound healing. Here, two different methods that address diverse aspects of the wound healing response are described. In the first model, polyvinyl alcohol sponges are subcutaneously implanted along the mouse dorsum. Following sponge retrieval, cells can be isolated by mechanical disruption, and fluids can be extracted by centrifugation, thus allowing for a detailed characterization of cellular and cytokine responses in the acute wound environment. A limitation of this model is the inability to assess the rate of wound closure. For this, a tail skin excision model is utilized. In this model, a 10 mm x 3 mm rectangular piece of tail skin is excised along the dorsal surface, near the base of the tail. This model can be easily photographed for planimetric analysis to determine healing rates and can be excised for histological analysis. Both described methods can be utilized in genetically altered mouse strains, or in conjunction with models of comorbid conditions, such as diabetes, aging, or secondary infection, in order to elucidate wound healing mechanisms.

Here, two murine wound healing models are described, one designed to assess cellular and cytokine wound healing responses and the other to quantify the rate of wound closure. These methods can be used with complex disease models such as diabetes to determine mechanisms of various aspects of poor wound healing.

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 ...

Digital Planimetry for Assessing Wound Closure Kinetics in a Mouse Model

Chronic wounds, due to their high prevalence, are a serious global health concern. Effective therapeutic strategies can significantly accelerate healing, thereby reducing the risk of complications and alleviating the economic burden on healthcare systems. Although numerous experimental studies have investigated wound healing, most rely on qualitative observations or quantitative direct measurements. The objective of this study was to standardize an indirect wound measurement method using digital planimetry, incorporating digital scaling and segmentation. This approach addresses the lack of detailed, step-by-step methodologies for accurate wound assessment. A photodocumentation booth was designed and constructed, and computer-assisted digital planimetry tools were employed to minimize variability in measurements of the wound area, perimeter, and the distance from the wound center to its edges. A circular traumatic wound (5 mm in diameter) was created on the dorsal midline at the shoulder blade level of male CD1 mice (n = 4, 10 weeks old, 30-35 g). Wound evolution was photodocumented for 14 days using the custom-designed photo booth, which controlled lighting conditions, focal distance, and subject positioning. Scaling and wound measurements were performed using segmentation in ImageJ software, and statistical analysis was conducted using statistical analysis software. The kinetics of wound closure showed a slight increase in wound size and perimeter between day 0 and day 2, followed by a gradual decrease until complete closure by day 14. The photodocumentation booth and computer-assisted digital planimetry enabled quantitative measurements with minimal variability. In conclusion, these tools provide a reliable and reproducible method for evaluating wound closure kinetics in pre-clinical models.

Wounds represent a global health challenge. This study developed a standardized photo booth utilizing digital planimetry to minimize wound measurement variability. Monitoring wounds in mice over 14 days revealed an initial increase in wound area and perimeter, followed by gradual closure. This methodology may aid in evaluating wound closure kinetics in pre-clinical models.

用于评估小鼠模型中伤口闭合动力学的数字平面测量法

Chronic wounds, due to their high prevalence, are a serious global health concern. Effective therapeutic strategies can significantly accelerate healing, ...

生物工程

Laser Capture Microdissection on Surgical Tissues to Identify Aberrant Gene Expression in Impaired Wound Healing in Type 2 Diabetes

The global prevalence Type 2 diabetes mellitus (T2DM) is escalating at a rapid rate. Patients with T2DM suffer from a multitude of complications and one of these is impaired wound healing. This can lead to the development of non-healing sores or foot ulcers and ultimately to amputation. In healthy individuals, wound healing follows a controlled and overlapping sequence of events encompassing inflammation, proliferation, and remodelling. In T2DM, one or more of these steps becomes dysfunctional. Current models to study impaired wound healing in T2DM include in vitro scratch wound assays, skin equivalents, or animal models to examine molecular mechanisms underpinning wound healing and/or potential therapeutic options. However, these do not fully recapitulate the complex wound healing process in T2DM patients, and ex vivo human skin tests are problematic due to the ethics of taking punch biopsies from patients where it is known they will heal poorly. Here, a technique is described whereby expression profiles of the specific cells involved in the (dys)functional wound healing response in T2DM patients can be examined using surplus tissue discarded following amputation or elective cosmetic surgery. In this protocol samples of donated skin are collected, wounded, cultured ex vivo in the air liquid interface, fixed at different time points and sectioned. Specific cell types involved in wound healing (e.g., epidermal keratinocytes, dermal fibroblasts (papillary and reticular), the vasculature) are isolated using laser capture microdissection and differences in gene expression analyzed by sequencing or microarray, with genes of interest further validated by qPCR. This protocol can be used to identify inherent differences in gene expression between both poorly healing and intact skin, in patients with or without diabetes, using tissue ordinarily discarded following surgery. It will yield greater understanding of the molecular mechanisms contributing to T2DM chronic wounds and lower limb loss.

This technique provides a guide to inflicting ex vivo wounds, performing laser capture microdissection and quantifying changes in gene expression related to poor wound healing processes in diabetes using clinically relevant human tissue.

The global prevalence Type 2 diabetes mellitus (T2DM) is escalating at a rapid rate. Patients with T2DM suffer from a multitude of complications and one ...

Monitoring Neutrophil Recruitment Dynamics and Bacterial Burden at the Infection Site in a Murine Model

Source: Anderson, L. S., et al. <a href="https://app.jove.com/v/59015">A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection.</a> J. Vis. Exp. (2019).
This video demonstrates in vivo imaging to study the neutrophil recruitment dynamics at an infection site in an immunocompromised, transgenic mouse with GFP-expressing neutrophils. It involves injecting luminescent bacteria to cause an infection. This leads to neutrophil recruitment to the infection site, whose tracking is done by imaging.

在小鼠模型中监测感染部位的中性粒细胞募集动力学和细菌负荷

Source: Anderson, L. S., et al. A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection. J. Vis. Exp. (2019).
This video ...

JoVE Encyclopedia of Experiments

Immunology

Immune Response

Host Defense Mechanism

Quantification of Inflammatory Mediators in Infected Human Tissue-Derived Cells

Source: Dousha, L. et al., <a href="https://app.jove.com/v/62572">Assessing Respiratory Immune Responses to Haemophilus Influenzae.</a>&#160;J. Vis. Exp.&#160;(2021)
This video demonstrates the quantification of inflammatory mediators during infection by incubating human lung-tissue-derived immune cells with Haemophilus influenzae. Flow cytometry is used to measure cytokine fluorescence in labeled lymphocytes, correlating with the production of pro-inflammatory cytokines in response to bacterial infection.

感染的人体组织来源细胞中炎症介质的定量

Source: Dousha, L. et al., Assessing Respiratory Immune Responses to Haemophilus Influenzae.&#160;J. Vis. Exp.&#160;(2021)
This video demonstrates the ...

Colonoscopic Guided Murine Wound Model: A Method to Isolate Biopsy Induced Wound Bed from Colon to Understand the Healing Process

Source: Montrose, D. C., et al. <a href="https://www.jove.com/v/60949">Performing Colonoscopic-Guided Pinch Biopsies in Mice and Evaluating Subsequent Tissue Changes.</a> J. Vis. Exp. (2021).
In this video, we describe a procedure to isolate the colonoscopic-induced wound bed at an appropriate time point for detailed molecular analysis. This analysis helps to understand the wound healing process in inflammatory conditions of the GI tract.

结肠镜引导小鼠伤口模型:一种将活检诱导的伤口床与结肠分离以了解愈合过程的方法

Source: Montrose, D. C., et al. Performing Colonoscopic-Guided Pinch Biopsies in Mice and Evaluating Subsequent Tissue Changes. J. Vis. Exp. (2021).
In ...