这项工作是由美国国立卫生研究院资助R01 GM087609（以HPL），从英格丽·莱和比尔舒荣誉安东尼舒（以HPL），美国国立卫生研究院资助U01 HL099776（以MTL）的礼物，在Hagey实验室拨款部分支持小儿再生医学和橡树基金会（MTL来和HPL）。 GGW是由斯坦福大学医学院，斯坦福大学医学院科学家培训项目的支持，并NIGMS培训资助GM07365。 MSH是由CIRM临床研究员培训资助TG2-01159支持。 WXH是从萨尔诺夫心血管基金会的资助支持。

注:本文中所描述的所有程序都是根据斯坦福大学行政专家组对实验动物护理（APLAC）制定的准则进行。 1.定时怀孕 - 超数排卵技术（图1） 注:精确定时小鼠胚胎的孕周胎儿外科E16.5和E18.5是至关重要的。在这一节中我们详细介绍我们使用怀孕母马血清（PMS）和人绒毛膜促性腺激素（HCG）注射诱导超数排卵定时鼠标怀孕协议。 <ol><li>注射雌性小鼠（&lt;5％笼）腹腔（IP）与3.0-5.0 PMS国际单位（IU）的100微升PBS 1:00和1天的3:00 PM之间的音量。 </li><li> 12:00至2:00 PM 3天（45至47小时PMS注射后）的，注射雌性小鼠IP与3.0-5.0 IU HCG在100微升PBS体积。 注:HCG注射诱导<html>排卵期大约12小时后注射。 </li><li>紧接着HCG注射，交配女性，年龄在8-16周的男性。 注:我们通常把两女成单个男性的笼子。 </li><li>独立的女性，从第4天上午男性（6:00 - 10:00 AM），并记录为胎龄E0.5。 注:阴道塞可在此时进行检查;然而，观察阴道塞不保证怀孕，女性可以怀孕时没有插头观察。鉴于妊娠通常通过目测和/或触诊在胎龄E16.5和E18.5观察到的，检查在第4天上午阴道塞不是严格必需的。根据我们的经验，并根据不同的应变，超排卵的女性约30-50％的怀孕使用此处所描述的技术。 </li></ol>皮克"/&gt; 图1.原理图超数排卵技术，对小鼠超数排卵定时怀孕示意图，显示程序。 <a href="https://www.jove.com/files/ftp_upload/52297/52297fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 2.小​​鼠胎儿手术（背伤人）在E16.5和E18.5胚胎（图2） <ol><li>在手术前，清洁手术室和装备有70％的异丙醇的所有表面。此外，灭菌的所有外科用品和仪器将由高压灭菌它们被用于该过程。一些机构可能允许后续采用热杀菌珠。每个操作，使用无菌包，包括纱布，手术器械。 </li><li>在每分钟之后维持麻醉，每分钟1升2升诱导麻醉孕妇（胎儿年龄E16.5或E18.5）低于2.5％异氟醚/氧气混合物。 </li><li>昆仑坚定正确的麻醉，保证了鼠标的深踩油门反射受到抑制，然后将鼠标在俯卧位。 </li><li>应用兽医眼药膏，如Puralube以防止在手术过程中眼睛发炎或干燥。 </li><li>通过给予时间不超过30秒（ 图2A）的脱毛霜轻应用做准备的腹部。 </li><li>准备腹部无菌手术用聚维酮碘和醇（ 图2B和2C）。 </li><li>采用显微剪刀（ 图2D和2E）显微镜下进行中线剖腹探查术。 </li><li>轻轻地揭露子宫和胎儿选择手术（ 图2F）。 </li><li>使用一个钝头针头灌溉外科领域用温水（38℃）磷酸盐缓冲盐水（PBS）中 注意:一个可以通过仔细弯曲大口径针的尖端制成。 （ 图2G和2H）。 </li><li>的方式，使定位胎儿s的接入背部。 </li><li>通过用7-0尼龙线通过子宫覆打算背伤人（ 图2I）的网站荷包缝合。位置荷包在背部左侧或脊髓的右边的一个区域，并在子宫壁缺乏大血管的区域。 </li><li>使在荷包（ 图2J）的中心，通过子宫壁和羊膜囊3毫米的切口。 </li><li>冲洗切口部位用温水（38℃）PBS。 </li><li>采用显微剪刀，切割单个全层切除伤口，大约为1毫米的长度，在胎儿的背部。 </li><li>轻轻印迹切口部位用干棉尖涂药。 </li><li>注3微升量墨汁皮下伤口部位标记伤口的位置（ 图2K）。 </li><li>用温水（38℃），PBS冲洗，以确保墨水一直保留在伤口部位。 </li><li>手术有我的助手nject温暖（38°C）通过钝尖10个G注射器插入羊膜囊的钱袋PBS被关闭（ 图2L）。收回的注射器作为荷包封接近完成（ 图2M）。 </li><li>轻轻返回子宫进入腹腔（ 图2N）。 </li><li>埃弗特皮肤和腹膜。 </li><li>有手术助理灌溉腹腔用温水（38℃）PBS。 </li><li>迅速关闭腹部被装订的皮肤和腹膜封闭（ 图2O）。标准封盖在两层进行;腹膜和在一层，皮下组织和皮肤在第二层中的腹部肌肉。对于眼前的牺牲和收获的胎儿，我们的演示显示了一层封闭。 </li><li>放置在观察所述动物在温暖的培养箱设定为37℃，30分钟，或直至动物重新获得足够意识保持胸骨斜卧。 </li><li>不要沤瓮动物给其他动物的公司，直到它已完全从过程中回收。 </li><li>一旦从麻醉，并在随后的48小时的觉醒，辖皮下注射丁丙诺啡（0.05毫克/千克），每12小时为镇痛根据需要基于疼痛评估。根据需要，通过皮下注射管理卡洛芬（5毫克/千克）的额外手术后疼痛减轻。 </li><li>回到动物的笼子，并为他们提供食物和水自由采食。 </li><li>密切留意市民对疼痛的表现。 </li><li> 48小时后手术，牺牲孕妈妈与异氟醚和收获受伤胎儿的过量。为了做到这一点，调整异氟烷浓度为5％或更高，并维持曝光呼吸停止后1分钟。确认安乐死颈椎脱位。收获的未受伤胚胎为年龄匹配的对照。后期胚胎应具有安乐死与IACUC建议，如相一致的单独的方法去人头，颈椎脱位，或化学注射。 </li></ol><img alt="图2" src="/files/ftp_upload/52297/52297fig2highres.jpg" /> 鼠标腹部图2.原理图小鼠胎儿手术。一般步骤背伤人案E16.5和E18.5小鼠胚胎。（A）脱毛。（B和C）小鼠腹部的制备。（D）显微镜用于外科手术通过子宫的过程。（E）中线剖腹探查术。（F）曝光子宫。（G）创造钝尖针头。子宫用温盐水（H）灌溉。（I）创造了荷包缝合。（J）切口墙上1毫米全层切除伤口的产生。（K）皮下注射印度墨水。（L和M）封闭的荷包缝合。（N和O）关闭腹部。 <a href="https://www.jove.com/files/ftp_upload/52297/52297fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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The authors have nothing to disclose.

这里介绍的手术协议描述的小鼠胎儿无瘢痕愈合首次出版于2006年，由我们的实验室10的切除模式。除了 ​​切除伤人11等建立模型，对小鼠胎儿无瘢痕愈合的切口车型存在，以及12,13。胎儿无瘢痕愈合猴，羊，兔，负鼠，老鼠和调查的报道14-17。然而，小鼠表现为探索胎儿无瘢痕愈合的理想模型，由于其相对较低的按日补助笼的成本和良好的特点的基因组。此外，?...

胎儿皮肤伤口愈合迅速，scarlessly直到怀孕后期1。胎疤痕伤口修复的特征在于正常组织结​​构和功能的再生。从瘢痕瘢痕表型的转变发生在怀孕的人类和周围18天胚胎（E18）小鼠2,3孕晚期。相较于成人，胎儿伤口修复的特征在于快速上皮，结缔组织沉积和成纤维细胞的迁移。 在早期胎儿发育许多研究都提供了可能的解释为瘢痕伤口愈合的现象。炎症是成人创面修复的一个基本组成部分;然而，胎儿伤口的特征在于缺乏急性炎症4。这是否是免疫系统的过程中的胎儿阶段官能不成熟的结果仍不清楚。最近的一项研究表明，在丰富，垫差异urity，和肥大细胞在E15与E18的胎儿皮肤的功能可以负责从瘢痕表型的转变中，至少在小鼠3。其他的研究断定，在性能和丰富的胎儿和成人伤口巨噬细胞的差异是负责正常的细胞外基质（ECM）的过程中胎儿创伤修复5的改革。 胎儿和成人发育期间差异在环境因素也可能影响伤口修复。龙雅葛和他的同事发现，从胎儿伤口液具有较高水平的透明质酸刺激活性相比，无成人伤口流体6。因此，更高水平的透明质酸，糖胺聚糖促进有利于细胞运动和增殖的微环境，在胎儿的伤口环境的可负责的瘢痕的表型在早期胎儿发育看见。其他线路的证据指向的一个事实，即在羊升伤口环境相对低氧血症和淹没在无菌羊水丰富的生长因子，7。然而，没有明确的答案已提供用于胚胎发育过程中的关键事件或因素触发从瘢痕再生纤维变性修复的过渡。 理解的机制负责在胎儿无瘢痕愈合就必须精确和可再现的模型。在这里，我们详细胎儿无瘢痕愈合的E16.5（无疤痕）和E18.5（疤痕）的小鼠胚胎的背部可再现的模型。此外，该模型的微小变化可用于执行许多进一步的研究，如胎儿的伤口和皮肤8,9的基因表达分析。鉴于精确定时怀孕是此无瘢痕伤口愈合模式成功的关键再演，我们对超排定时怀孕也详细我们的协议。

<table border="0" cellpadding="0" cellspacing="0" width="665">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:219px;">Name of Material/Equipment</td>
			<td style="width:184px;">Company</td>
			<td style="width:108px;">Catalog Number</td>
			<td style="width:155px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">7-O MONOSOF Suture</td>
			<td>eSuture</td>
			<td>SN-1647G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical Forceps</td>
			<td>Kent Scientific</td>
			<td>INS650916</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micro-scissors</td>
			<td>Kent Scientific</td>
			<td>INS600127</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Autoclip 9mm</td>
			<td>Texas Scientific Instruments</td>
			<td>205060</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Insulin Syringe</td>
			<td>Thermo Fisher Scientific</td>
			<td>22-272-382</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Black Pigment</td>
			<td>AIMS</td>
			<td>242</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BD Safety-Lok 3ml Syringe</td>
			<td>BD Biosciences</td>
			<td>309596</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate Buffered Saline</td>
			<td>Life Technologies</td>
			<td>10010-049</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">OPMI-MD Surgical Microscope</td>
			<td>Carl Zeiss Surgical Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pregnant Mares Serum (PMS)</td>
			<td>Millipore</td>
			<td>367222</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Human Chorionic Gonadotropin (HCG)</td>
			<td>Sigma-Aldrich</td>
			<td>CG10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Povidone Iodine Prep Solution</td>
			<td>Dynarex</td>
			<td>1415</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nair (depilatory cream)</td>
			<td>Church and Dwight Co.</td>
			<td>22600267058</td>
			<td></td>
		</tr>
	</tbody>
</table>

a mouse fetal skin model of scarless wound repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

对于组织学分析，皮肤伤口在E16.5和E18.5小鼠胚胎的背部皮肤，应收获后48小时伤人，固定在4％PFA和石蜡包埋。在荧光的转基因模型中，冷冻保存与十月可能是适当的。有几种污渍可以被用于可视化的细胞和结缔组织结构。苏木精和曙红是一种两色色斑，即污迹细胞核蓝色和嗜酸性结构（ 即 ，细胞质和细胞外胶原）的红色，粉红色，橙色和各种色调。马洛里的三色是一个三色染色选自苯胺?...

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A Mouse Fetal Skin Model of Scarless Wound Repair

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

对无瘢痕修复鼠标胎儿皮肤模型

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

a-mouse-fetal-skin-model-of-scarless-wound-repair

Research

JoVE Journal

Medicine

12.2K 次观看.  Stanford University School of Medicine. During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

视频: A Mouse Fetal Skin Model of Scarless Wound Repair

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&beta; using a Whole Blood Stimulation Assay

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues as well as altered cytokine homeostasis. The innate immune system plays a crucial role in recognizing pathogens and other endogenous danger stimuli. One of the major cytokines released by innate immune cells is Interleukin (IL)-1. Therefore, we utilize a whole blood stimulation assay in order to measure the secretion of inflammatory cytokines and specifically of the pro-inflammatory cytokine IL-1&beta; 1, 2, 3.
Patients with genetic dysfunctions of the innate immune system causing autoinflammatory syndromes show an exaggerated release of mature IL-1&beta; upon stimulation with LPS alone. In order to evaluate the innate immune component of patients who present with inflammatory-associated pathologies, we use a specific immunoassay to detect cellular immune responses to pathogen-associated molecular patterns (PAMPs), such as the gram-negative bacterial endotoxin, lipopolysaccharide (LPS). These PAMPs are recognized by pathogen recognition receptors (PRRs), which are found on the cells of the innate immune system 4, 5, 6, 7. A primary signal, LPS, in conjunction with a secondary signal, ATP, is necessary for the activation of the inflammasome, a multiprotein complex that processes pro-IL-1&beta; to its mature, bioactive form 4, 5, 6, 8, 9, 10.
The whole blood assay requires minimal sample manipulation to assess cytokine production when compared to other methods that require labor intensive isolation and culturing of specific cell populations. This method differs from other whole blood stimulation assays; rather than diluting samples with a ratio of RPMI media, we perform a white blood cell count directly from diluted whole blood and therefore, stimulate a known number of white blood cells in culture 2. The results of this particular whole blood assay demonstrate a novel technique useful in elucidating patient cohorts presenting with autoinflammatory pathophysiologies.

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&#946; using a Whole Blood Stimulation Assay

We describe a simple immunoassay to measure the production of pro-inflammatory cytokines, such as IL-1 beta production, in patients presenting with autoinflammatory phenotypes. By activating cells in whole blood cultures with pathogen-associated molecular patterns, specifically with lipopolysaccharide, cytokine secretion can be conveniently evaluated in whole blood supernatants.

使用全血刺激试验的促炎性细胞因子IL -1β的精确和简单的测量

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues ...

科研

医学

A Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of spinal cord injury (SCI) in which the thoracic spinal cord is injured. Additionally, because most human cord injuries occur as the result of blunt, non-penetrating trauma (e.g. motor vehicle accident, sporting injury) where the spinal cord is violently struck by displaced bone or soft tissues, the majority of SCI researchers are of the opinion that the most clinically relevant injury models are those in which the spinal cord is rapidly contused.1 Therefore, an important step in the preclinical evaluation of novel treatments on their way to human translation is an assessment of their efficacy in a model of contusion SCI within the cervical spinal cord. Here, we describe the technical aspects and resultant anatomical and behavioral outcomes of an unilateral contusive model of cervical SCI that employs the Infinite Horizon spinal cord injury impactor.
Sprague Dawley rats underwent a left-sided unilateral laminectomy at C5. To optimize the reproducibility of the biomechanical, functional, and histological outcomes of the injury model, we contused the spinal cords using an impact force of 150 kdyn, an impact trajectory of 22.5&deg; (animals rotated at 22.5&deg;), and an impact location off of midline of 1.4 mm. Functional recovery was assessed using the cylinder rearing test, horizontal ladder test, grooming test and modified Montoya's staircase test for up to 6 weeks, after which the spinal cords were evaluated histologically for white and grey matter sparing.
The injury model presented here imparts consistent and reproducible biomechanical forces to the spinal cord, an important feature of any experimental SCI model. This results in discrete histological damage to the lateral half of the spinal cord which is largely contained to the ipsilateral side of injury. The injury is well tolerated by the animals, but does result in functional deficits of the forelimb that are significant and sustained in the weeks following injury. The cervical unilateral injury model presented here may be a resource to researchers who wish to evaluate potentially promising therapies prior to human translation.

A reliable and repeatable way to produce a cervical unilateral spinal cord injury using the Infinite Horizon impactor is described. The method takes advantage of a custom designed frame and clamp to stabilize the spine. The standardized procedure and biomechanical injury parameters result in sufficient and sustained injuries.

单侧颈脊髓损伤的挫伤模型，利用无限的地平线冲击

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of ...

A Simplified Technique for Producing an Ischemic Wound Model

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work well in non-ischemic wounds may not work in human diabetic wounds because vasculopathy is one major factor that hinders healing of these wounds. We published an article in 2007 describing a rabbit ear ischemic wound model created by a minimally invasive surgical technique. Since then, we have further simplified the procedure for easier operation. On one ear, three small skin incisions were made on the vascular pedicles, 1-2 cm from the ear base. The central artery was ligated and cut along with the nerve. The whole cranial bundle was cut and ligated, leaving only the caudal branch intact. A circumferential subcutaneous tunnel was made through the incisions, to cut subcutaneous tissues, muscles, nerves, and small vessels. The other ear was used as a non-ischemic control. Four wounds were made on the ventral side of each ear. This technique produces 4 ischemic wounds and 4 non-ischemic wounds in one animal for paired comparisons. After surgery, the ischemic ear was cool and cyanotic, and showed reduced movement and a lack of pulse in the ear artery. Skin temperature of the ischemic ear was 1-10 &deg;C lower than that on the normal ear and this difference was maintained for more than one month. Ear tissue high-energy phosphate contents were lower in the ischemic ear than the control ear. Wound healing times were longer in the ischemic ear than in the non-ischemic ear when the same treatment was used. The technique has now been used on more than 80 rabbits in which 23 were diabetic (diabetes time ranging from 2 weeks to 2 years). No single rabbit has developed any surgical complications such as bleeding, infection, or rupture in the skin incisions. The model has many advantages, such as little skin disruption, longer ischemic time, and higher success rate, when compared to many other models. It can be safely used in animals with reduced resistance, and can also be modified to meet different testing requirements.

We have developed a minimally invasive technique to create a rabbit ischemic ear wound model by dividing the central artery and nerve and the cranial neurovascular bundle. A subcutaneous tunnel then cuts all subcutaneous tissues. This procedure causes minimal skin disruption and can be safely used in diabetic animals.

一个生产缺血性损伤模型的简化技术

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work ...

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

在斑马鱼模型的胎儿酒精暴露评估致畸的变化

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

一种新的手术方法气管内注射生物活性剂在胎鼠模型

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

伤口愈合的小鼠模型

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

Fetal Echocardiography and Pulsed-wave Doppler Ultrasound in a Rabbit Model of Intrauterine Growth Restriction

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ultrasound and fetal echocardiography. Increasingly, these imaging modalities are being employed clinically to examine cardiac function and assess wellbeing in utero, thereby guiding timing of birth decisions. Here, we used a rabbit model of IUGR that allows analysis of cardiac function in a clinically relevant way. Using isoflurane induced anesthesia, IUGR is surgically created at gestational age day 25 by performing a laparotomy, exposing the bicornuate uterus and then ligating 40-50% of uteroplacental vessels supplying each gestational sac in a single uterine horn. The other horn in the rabbit bicornuate uterus serves as internal control fetuses. Then, after recovery at gestational age day 30 (full term), the same rabbit undergoes examination of fetal cardiac function. Anesthesia is induced with ketamine and xylazine intramuscularly, then maintained by a continuous intravenous infusion of ketamine and xylazine to minimize iatrogenic effects on fetal cardiac function. A repeat laparotomy is performed to expose each gestational sac and a microultrasound examination (VisualSonics VEVO 2100) of fetal cardiac function is performed. Placental insufficiency is evident by a raised pulsatility index or an absent or reversed end diastolic flow of the umbilical artery Doppler waveform. The ductus venosus and middle cerebral artery Doppler is then examined. Fetal echocardiography is performed by recording B mode, M mode and flow velocity waveforms in lateral and apical views. Offline calculations determine standard M-mode cardiac variables, tricuspid and mitral annular plane systolic excursion, speckle tracking and strain analysis, modified myocardial performance index and vascular flow velocity waveforms of interest. This small animal model of IUGR therefore affords examination of in utero cardiac function that is consistent with current clinical practice and is therefore useful in a translational research setting.

We describe examination of fetal cardiac function with contemporary functional fetal echocardiography and fetoplacental Doppler ultrasound using the VisualSonics VEVO 2100 microultrasound in a surgically induced model of intrauterine fetal growth restriction in a rabbit.

在家兔模型宫内生长受限胎儿超声心动图和多普勒超声脉冲波

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ...

A New Murine Model of Endovascular Aortic Aneurysm Repair

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of Abdominal aortic aneurysm (AAA) pathophysiology. There is no abdominal aortic aneurysm endograft exclusion model cheap and reproducible, which would allow deep investigations of AAA before and after treatment. We hereby describe how to induce, and then to exclude with a covered coronary stentgraft an abdominal aortic aneurysm in a rat. The well known elastase induced AAA model was first reported in 19901 in a rat, then described in mice2. Elastin degradation leads to dilation of the aorta with inflammatory infiltration of the abdominal wall and intra luminal thrombus, matching with human AAA. Endovascular exclusion with small covered stentgraft is then performed, excluding any interactions between circulating blood and the aneurysm thrombus. Appropriate exclusion and stentgraft patency is confirmed before euthanasia by an angiography thought the left carotid artery. Partial control of elastase diffusion makes aneurysm shape different for each animal. It is difficult to create an aneurysm, which will allow an appropriate length of aorta below the aneurysm for an easy stentgraft introduction, and with adequate proximal and distal neck to prevent endoleaks. Lots of failure can result to stentgraft introduction which sometimes lead to aorta tear with pain and troubles to stitch it, and endothelial damage with post op aorta thrombosis. Giving aspirin to rats before stentgraft implantation decreases failure rate without major hemorrhage. Clamping time activates neutrophils, endothelium and platelets, and may interfere with biological analysis.

Histological and biochemical modifications after aneurysm endograft exclusion are still unclear. We describe a new model of endograft implantation on a murine aneurysm. Through thrombus analysis with and without persistant circulating blood stream interface, and new endovascular biomaterials evaluation, this model will help to better understand endovascular aneurysm exclusion pathobiology.

一种新的小鼠模型主动脉瘤腔内修复

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of ...

A Surgical Procedure for Resecting the Mouse Rib: A Model for Large-Scale Long Bone Repair

This protocol introduces researchers to a new model for large-scale bone repair utilizing the mouse rib. The procedure details the following: preparation of the animal for surgery, opening the thoracic body wall, exposing the desired rib from the surrounding intercostal muscles, excising the desired section of rib without inducing a pneumothorax, and closing the incisions. Compared to the bones of the appendicular skeleton, the ribs are highly accessible. In addition, no internal or external fixator is necessary since the adjacent ribs provide a natural fixation. The surgery uses commercially available supplies, is straightforward to learn, and well-tolerated by the animal. The procedure can be carried out with or without removing the surrounding periosteum, and therefore the contribution of the periosteum to repair can be assessed. Results indicate that if the periosteum is retained, robust repair occurs in 1 - 2 months. We expect that use of this protocol will stimulate research into rib repair and that the findings will facilitate the development of new ways to stimulate bone repair in other locations around the body.

The overall goal of this procedure is to successfully resect a portion of bone from the rib of a mouse. The procedure was developed as a model to study large-scale long bone repair.

外科手术切除为鼠标肋骨:模型进行大规模的长骨修复

This protocol introduces researchers to a new model for large-scale bone repair utilizing the mouse rib. The procedure details the following: preparation ...

Demonstration of the Rat Ischemic Skin Wound Model

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and unrelieved pressure due to immobility. Animal models have been developed that attempt to mimic these conditions for the purpose of furthering our understanding of the complexity of chronic wounds. The model described herein is a rat ischemic skin flap model that permits a prolonged reduction of blood flow resulting in wounds that become ischemic and resemble a chronic wound phenotype (reduced vascularization, increased inflammation and delayed wound closure). It consists of a bipedicled dorsal flap with 2 ischemic wounds placed centrally and 2 non-ischemic wounds lateral to the flap as controls. A novel addition to this ischemic skin flap model is the placement of a silicone sheet beneath the flap that functions as a barrier and a splint to prevent revascularization and reduce contraction as the wounds heal. Despite the debate of using rats for wound healing studies due to their quite distinct anatomic and physiologic differences compared to humans (i.e., the presence of a panniculus carnosus muscle, short life-span, increased number of hair follicles, and their ability to heal infected wounds) the modifications employed in this model make it a valuable alternative to previously developed ischemic skin flap models.

The rat, due to its size, availability, and rather docile behavior, has been utilized as a research model for many years. The goal of this protocol is to utilize the rat as an ischemic skin wound healing model to provide valuable insight into the pathophysiology of chronic wounds.