We thank Katherine Clark for technical assistance. A.D.R.G. is funded in part by 5K01MH097957-03

成人（3-4个月）雄性小鼠的混合C57BL / 6背景是在这个协议中使用。动物保持在12小时光照/黑暗周期，并允许自由摄取食物和水。在此协议下的所有程序都按照批准的德雷克塞尔大学机构动物护理和使用委员会的协议进行的。 1.准备手术区<ol><li>消毒手术台上，用70％的乙醇，然后覆盖整个手术台上，吸收垫，并安排手术器械相邻立体。 </li><li>建立立体设备无机械手臂。安排上的立体定位的加热垫，并设置为37℃。避免通过将一小片纸巾或手术垫的动物和加热垫之间过热的动物。 </li><li>使用高压灭菌剪刀，切小块蒸压明胶海绵到含有0.9％无菌盐水的溶液，直到意图的无菌培养皿DY使用。 注：使用蒸压文书和无菌手术耗材保持无菌的工作​​环境。在过程或在通过浸渍到珠灭菌10-15秒，可以根据需要重新消毒的手术器械的动物之间。保持干净的手套在整个过程通过摩擦的手用70％的乙醇，根据需要，进行消毒。 </li></ol> 2.鼠标预学习外科手术<ol><li>从家笼中取出鼠标和重量（克）。 </li><li>鼠标放置到异氟醚诱导室，并设置氧气2升/分钟，异氟醚蒸发至5诱导麻醉手术平面，约3-5分钟。监测呼吸减慢和固定。检查鼠标使用的是脚趾捏反射完全镇静。 </li><li>当鼠标被完全镇静，放置在立体框架，将鼻的鼻锥，其中附有油管的异氟醚。插入耳棒插入耳道，并拧紧，保证了头部是稳定的。 </li><li>从耳刮头部的耳朵，并且从眼睛之间到耳朵后面。 </li><li>消毒用异丙醇和优碘碘溶液，每次3倍的交替湿巾皮肤。 </li><li>应用人工泪液来双眼，以防止它们在外科手术过程中变干。 </li></ol> 3.手术过程<ol><li>按捏脚趾或尾部监测麻醉深度。鼠标是在合适的手术飞机的时候没有反应，呼吸缓慢而均匀。 </li><li>使矢状窦旁皮肤切口从刚才的眼神几乎用11号手术刀片在一个单一的，坚定的运动两耳之间的后面。移动肌肤一边夹右侧有止血。 </li><li>覆盖膜使用11号手术刀和棉花的暗面颅骨清除涂药。或者，擦拭头骨棉签蘸0.9％生理盐水。允许完全干燥。 </li><li>我们荷兰国际集团的小尺，标记开颅在1毫米尾椎冠状缝，和开颅在1毫米左边缘的前缘横向于矢状缝（ 图1），用一个永久性标记。然后标记开颅的右侧和尾部的边界处从矢状面和冠状缝线，分别（ 图1）4毫米。 </li><li>使用0.5毫米钻头，开始钻慢慢继永久性标记轮廓进行开颅手术。一定不要通过头骨打破完全。轻轻按压在隔离片顶骨与5号钳，薄弱环节将让位给的压力。当薄骨是整个周长足够弱，骨片是准备拆除。 注意：如果调查的经验困难除去骨在一块，这表明骨没有充分钻孔期间变薄。考虑在随后的动物facilita进一步钻探头骨忒容易去除的骨片。 </li><li>使用10毫升注射器配有23政针，将少量0.9％盐水浸泡分离骨和钻孔区域。 </li><li>附加操纵臂的立体定位设备。附加一个新的第11号解剖刀刀片的探针架座具有面向吻侧刀片的锋利边。 注意：虽然喙和尾部组织遇到刀片的锋利和钝边，分别引起的穿透性损伤的机械损伤是整个病变的程度相当。我们观察到反应神经胶质增生的主要特征包括上调GFAP表达或扩散，喙和尾部部分之间没有明显的差异。 </li><li>保持机械臂出的方式，小心抬起使用5/45角度的钳分离骨。插入镊子进入分离骨和升降机的一侧的前端，使用杠杆拉断的骨片在一个FUL留下升运动。 注意：要小心，不要刺伤大脑或干扰颅骨下方的硬脑膜。 </li><li>取一小块浸泡吸收凝胶泡沫，并放置在裸露的大脑，以防止其干燥和吸收血液中任何可能存在的。 </li><li>一旦凝胶泡沫就位，摆动操纵臂到位并调整刀片开颅中心在凝胶泡沫。取下凝胶泡沫，降低叶片直至尖端接触硬膜，而不刺穿硬脑膜。马克背/使用游标刻度上的立体的纵臂腹坐标。 </li><li>使用机械臂，慢慢放下刀精确3毫米进入大脑。这是通过使用游标刻度标记上的操纵臂实现。允许刀片留在原地5-10秒。移动立体手臂插脚喙移动到opposi前尾鳍三次使刀片到达开颅手术的喙和尾部的边界TE结束。 注：硬膜之前不插入刀片拆除。与此相反的大鼠硬脑膜，这是〜80微米厚7，鼠标硬脑膜是相当薄（只有几个细胞层厚度）和插入过程中不会产生对解剖刀片的明显的阻力。使用一个新的手术刀刀片每只小鼠以确保每个动物接受了一致的损伤。 </li><li>慢慢抬高手臂立体，去除脑部的刀片。去除叶片后，立即放在脑表面另一块明胶海绵来吸收多余的血液或体液。 </li><li>同时，除去立体手臂和处置的第11号手术刀刀片。一旦出血停止后，取出明胶海绵。 </li><li>通过缝合与非吸收缝线，如ethilon或普理灵皮肤闭合伤口。下列手术缝线应去掉9-10天。 </li><li>返回鼠标其家笼，并让鼠标在加热系统缓慢复苏摹垫和监测遇险的任何迹象。回收异氟烷诱导麻醉通常去除的异氟烷后发生在2-5分钟。不要让动物无人看管，直到它已经恢复胸骨recumbancy。 </li><li>辖0.5-1毫升乳酸林格氏液皮下的，以确保水合作用。 </li></ol> 4.手术后护理<ol><li>回到殖民地之前密切监测动物手术后，直到恢复从麻醉。 <ol><li>为了减轻疼痛和不适术后，管理0.05-0.1毫克/公斤丁丙诺啡腹腔注射立刻过程如下。 </li><li>动物观察2-3天手术后遇险严重迹象，如限制运动，缺乏疏导，或体重减轻。安乐死的动物展示任何痛苦的这些迹象，并从研究中删除。 </li></ol></li><li>为了检查受伤组织的病理学，由Stan动物安乐死准心内灌注。 <ol><li>简言之，麻醉动物用氯胺酮/赛拉嗪的过量，则心内灌注15-20毫升的0.9％的NaCl，或直到肝清除血液，随后60毫升4％多聚甲醛在实验结束时。 </li></ol></li><li>转移到30％的蔗糖溶液之前，解剖的大脑和定位后2-4小时在4％多聚甲醛。或如加西亚8,9-描述部分的大脑上低温恒温器在40-60微米，并处理通过标准组织学或免疫组织化学程序。 </li></ol>

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The authors have nothing to disclose.

该颅骨或底层硬脑膜的钻井过程中不被损坏，这一点至关重要。使用轻的压力，同时钻探，以保证头骨不刺破。另外，应注意同时抬起头骨片，以确保硬脑膜不解除了与骨。 这里的前脑锐器伤描述模型穿透伤到中枢神经系统。虽然比TBI模型如FPI或CCI少临床翻译，前脑病变刺模型充当了广泛的研究，旨在通过调查离散的中枢神经系统损伤引发的各种生化，细胞，分子或事件的?...

A major challenge for developing successful therapies to treat the injured CNS is an incomplete understanding of the complex multicellular events that are triggered by the trauma. Reactive astrocytes are gaining increasing recognition as a promising target for novel therapies1. Though historically regarded as hostile to neural repair, reactive astrocytes are now recognized as critical components of a complex, multicellular neuroprotective response that includes attenuation of inflammatory processes and limiting secondary damage and neurodegeneration2-6. Although the neuropathological characteristics of reactive gliosis have long been well defined, the cellular and molecular mechanisms regulating reactive gliosis, and the diverse array of downstream consequences remain poorly understood. Understanding the mechanisms that drive reactive gliosis, as well as the subsequent cellular and molecular events, is an important step towards developing strategies aimed at promoting the neuroprotective properties of reactive gliosis, while attenuating the detrimental effects. 
Here we demonstrate a method to induce severe reactive astrogliosis in the forebrain of adult mice using a stab injury. In contrast to other traumatic brain injury (TBI) models, such as controlled cortical impact (CCI) or fluid percussion injury (FPI), which require specialized equipment to produce an injury, the forebrain stab requires only a stereotaxic device to stabilize the head and a No. 11 scalpel blade. Thus the forebrain stab lesion model is more broadly accessible to a wide range of laboratories that do not have access to the specialized devices necessary for creating an FPI or CCI injury. The method described here enables investigators to reliably and reproducibly trigger a robust gliosis response to investigate subsequent cellular and molecular events. Once recovered from surgery, animals that have received a forebrain stab injury can survive for prolonged periods without the need for specialized care and can be returned to the colony for acute, intermediate, or chronic studies. Though less clinically translatable than FPI or CCI models of TBI, a forebrain lesion produced by a stab injury serves as a simple yet useful experimental model to investigate basic biological mechanisms underlying reactive gliosis and other neuropathological events following trauma to the CNS.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Stereotax</td>
			<td>Harvard Apparatus</td>
			<td>726049</td>
			<td></td>
		</tr>
		<tr>
			<td>High speed micro drill</td>
			<td>Harvard Apparatus</td>
			<td>724950</td>
			<td></td>
		</tr>
		<tr>
			<td>stainless steel scalpel blade, #11</td>
			<td>MedVet</td>
			<td>JOR581S</td>
			<td></td>
		</tr>
		<tr>
			<td>5/45 angled forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-35</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelfoam sponge 12cmx7mm</td>
			<td>Fisher</td>
			<td>NC9841478</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies and other reagents</td>
			<td>Manufacturer</td>
			<td>Catalog Number</td>
			<td>Dilution (brightfield) 
			Dilution (fluorescence)</td>
		</tr>
		<tr>
			<td>Rb anti-GFAP</td>
			<td>DAKO</td>
			<td>&#160;Z033429-2</td>
			<td>1:20k 
			1:1k</td>
		</tr>
		<tr>
			<td>Shp anti-BrdU</td>
			<td>Abcam</td>
			<td>ab1893</td>
			<td>1:2k 
			1:500</td>
		</tr>
		<tr>
			<td>Biotinylated goat anti-rabbit</td>
			<td>Vector Laboratories</td>
			<td>BA-1000&#160;</td>
			<td>1:400</td>
		</tr>
		<tr>
			<td>Biotinylated rabbit anti-sheep</td>
			<td>Vector Laboratories</td>
			<td>BA-6000</td>
			<td>1:400</td>
		</tr>
		<tr>
			<td>Alexafluor 488 goat anti-rabbit</td>
			<td>Life Technologies</td>
			<td>A-11008</td>
			<td>1:400</td>
		</tr>
		<tr>
			<td>Alexafluor 568 donkey anti-sheep</td>
			<td>Life Technologies</td>
			<td>A-21099</td>
			<td>1:1000</td>
		</tr>
		<tr>
			<td>DAPI Nucleic Acid Stain</td>
			<td>Life Technologies</td>
			<td>D3571</td>
			<td>1:1000</td>
		</tr>
		<tr>
			<td>Cresyl Violet Acetate</td>
			<td>Sigma Aldrich</td>
			<td>C5042-10G</td>
			<td>1%</td>
		</tr>
	</tbody>
</table>

triggering reactive gliosis in vivo by a forebrain stab injury

Following injury to the CNS, astrocytes undergo a broad range of biochemical, morphological, and molecular changes collectively referred to as reactive astrogliosis. Reactive astrocytes exert both inflammatory and protective effects that inhibit and promote, respectively, neural repair. The mechanisms underlying the diverse functional properties of reactive astrogliosis are not well understood. Achieving a greater understanding of these mechanisms is critical to developing therapeutic strategies to treat the injured CNS. Here we demonstrate a method to trigger reactive astrogliosis in the adult mouse forebrain using a forebrain stab lesion. This lesion model is simple, reliable, and requires only a stereotaxic device and a scalpel blade to produce the injury. The use of stab lesions as an injury model in the forebrain is well established and amenable to studies addressing a broad range of neuropathological outcomes, such as neuronal degeneration, neuroinflammation, and disruptions in the blood brain barrier (BBB). Thus, the forebrain stab injury model serves as a powerful tool that can be applied for a broad range of studies on the CNS response to trauma.

由于动物接受该方法不需要专门的术后护理，短期或长期的时间生存期很容易被纳入研究，根据不同的需要进行调查急性或慢性的病理损伤后。反应性神经胶质增生的主要功能，如GFAP和体细胞的肥大的上调，可以早在2-3天损伤后观察到的。增生的星形胶质细胞反应的高峰期是在受伤后10天3-5。如下所示的代表性的结果是从该接收一刀病变7天早期动物。 前脑的以下一?...

Watch this Scientific Journal Video about Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury at JoVE.com

Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

This article describes a detailed protocol to produce a forebrain stab injury in adult mice. The stab injury induces severe reactive gliosis and glial scar formation which can be subsequently examined by standard immunohistochemistry methods.

触发反应胶质细胞增生<em&gt;在体内</em&gt;由前脑锐器伤

Following injury to the CNS, astrocytes undergo a broad range of biochemical, morphological, and molecular changes collectively referred to as reactive ...

triggering-reactive-gliosis-in-vivo-by-a-forebrain-stab-injury

Research

JoVE Journal

Medicine

Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

10.4K 次观看.  Drexel University. This article describes a detailed protocol to produce a forebrain stab injury in adult mice. The stab injury induces severe reactive gliosis and glial scar formation which can be subsequently examined by standard immunohistochemistry methods. 

视频: Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

Muscle strains are one of the most common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and physical exam alone, however the clinical presentation can vary greatly depending on the extent of injury, the patient's pain tolerance, etc. In patients with muscle injury or muscle disease, assessment of muscle damage is typically limited to clinical signs, such as tenderness, strength, range of motion, and more recently, imaging studies. Biological markers, such as serum creatine kinase levels, are typically elevated with muscle injury, but their levels do not always correlate with the loss of force production. This is even true of histological findings from animals, which provide a "direct measure" of damage, but do not account for all the loss of function. Some have argued that the most comprehensive measure of the overall health of the muscle in contractile force. Because muscle injury is a random event that occurs under a variety of biomechanical conditions, it is difficult to study. Here, we describe an in vivo animal model to measure torque and to produce a reliable muscle injury. We also describe our model for measurement of force from an isolated muscle in situ. Furthermore, we describe our small animal MRI procedure.

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

An in vivo animal model of injury is described. The method takes advantage of the subcutaneous position of the fibular nerve. Velocity, timing of muscle activation, and arc of motion are all pre-determined and synchronized using commercial software. Post injury changes are monitored in vivo using MR imaging/spectroscopy.

一个在体内收缩引起的损伤，非侵入性的监测恢复啮齿动物模型

Muscle strains are one of the most  common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and  physical ...

科研

医学

Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce atherosclerosis (reviewed in 1 and 2). The role of shear stress has been extensively studied in vitro investigating the influence of flow dynamics on cultured endothelial cells 1,3,4 and in vivo in larger animals and humans 1,5,6,7,8. However, highly reproducible small animal models allowing systematic investigation of the influence of shear stress on plaque development are rare. Recently, Nam et al. 9 introduced a mouse model in which the ligation of branches of the carotid artery creates a region of low and oscillatory flow. Although this model causes endothelial dysfunction and rapid formation of atherosclerotic lesions in hyperlipidemic mice, it cannot be excluded that the observed inflammatory response is, at least in part, a consequence of endothelial and/or vessel damage due to ligation.
In order to avoid such limitations, a shear stress modifying cuff has been developed based upon calculated fluid dynamics, whose cone shaped inner lumen was selected to create defined regions of low, high and oscillatory shear stress within the common carotid artery 10. By applying this model in Apolipoprotein E (ApoE) knockout mice fed a high cholesterol western type diet, vascular lesions develop upstream and downstream from the cuff. Their phenotype is correlated with the regional flow dynamics 11 as confirmed by in vivo Magnetic Resonance Imaging (MRI) 12: Low and laminar shear stress upstream of the cuff causes the formation of extensive plaques of a more vulnerable phenotype, whereas oscillatory shear stress downstream of the cuff induces stable atherosclerotic lesions 11. In those regions of high shear stress and high laminar flow within the cuff, typically no atherosclerotic plaques are observed.
 
In conclusion, the shear stress-modifying cuff procedure is a reliable surgical approach to produce phenotypically different atherosclerotic lesions in ApoE-deficient mice.

The constricting cuff presented in this article is designed to induce atherosclerosis in the murine common carotid artery. Due to the conical shape of its inner lumen the implanted cuff generates well-defined regions of low, high and oscillatory shear stress triggering the development of atherosclerotic lesions of different inflammatory phenotypes.

植入小鼠触发剪应力致动脉粥样硬化的颈袖

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

在体内 19女性 MRI对细胞跟踪

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

宫内遥测来衡量鼠标收缩压力<em&gt;在体内</em

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death processes that are generally assumed to be intrinsically irreversible. Promoting anastasis could in principle rescue or preserve injured cells that are difficult to replace such as cardiomyocytes or neurons, thereby facilitating tissue recovery. Conversely, suppressing anastasis in cancer cells, undergoing apoptosis after anti-cancer therapies, may ensure cancer cell death and reduce the chances of recurrence. However, these studies have been hampered by the lack of tools for tracking the fate of cells that undergo anastasis in live animals. The challenge is to identify the cells that have reversed the cell death process despite their morphologically normal appearance after recovery. To overcome this difficulty, we have developed Drosophila and mammalian CaspaseTracker biosensor systems that can identify and permanently track the anastatic cells in vitro or in vivo. Here, we present in vivo protocols for the generation and use of the CaspaseTracker dual biosensor system to detect and track anastasis in Drosophila melanogaster after transient exposure to cell death stimuli. While conventional biosensors and protocols can label cells actively undergoing apoptotic cell death, the CaspaseTracker biosensor can permanently label cells that have recovered after caspase activation - a hallmark of late-stage apoptosis, and simultaneously identify active apoptotic processes. This biosensor can also track the recovery of the cells that attempted other forms of cell death that directly or indirectly involved caspase activity. Therefore, this protocol enables us to continuously track the fate of these cells and their progeny, facilitating future studies of the biological functions, molecular mechanisms, physiological and pathological consequences, and therapeutic implications of anastasis. We also discuss the appropriate controls to distinguish cells that undergo anastasis from those that display non-apoptotic caspase activity in vivo.

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis is technically challenging to detect in vivo because the cells that have reversed the cell death process can be morphologically indistinguishable from normal healthy cells. Here we describe protocols for detecting and tracking cells that undergo anastasis in live animals by using our newly developed in vivo CaspaseTracker biosensor system.

用 CaspaseTracker 生物传感器检测 Anastasis体内

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

从人类肿瘤异种移植的小鼠细胞的消耗显着改善靶细胞下游分析

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute respiratory distress syndrome (ARDS), characterized by neutrophilic alveolitis, and injury to both alveolar epithelium and vascular endothelium. Clinically, ARDS is defined by acute onset of hypoxemia, bilateral patchy pulmonary infiltrates and non-cardiogenic pulmonary edema. Human studies have provided us with valuable information about the physiological and inflammatory changes in the lung caused by ARDS, which has led to various hypotheses about the underling mechanisms. Unfortunately, difficulties determining the etiology of ARDS, as well as a wide range of pathophysiology have resulted in a lack of critical information that could be useful in developing therapeutic strategies.
Translational animal models are valuable when their pathogenesis and pathophysiology accurately reproduce a concept proven in both in vitro and clinical settings. Although large animal models (e.g., sheep) share characteristics of the anatomy of human trachea-bronchial tree, murine models provide a host of other advantages including: low cost; short reproductive cycle lending itself to greater data acquisition; a well understood immunologic system; and a well characterized genome leading to the availability of a variety of gene deletion and transgenic strains. A robust model of low pH induced ARDS requires a murine ALI that targets mainly the alveolar epithelium, secondarily the vascular endothelium, as well as the small airways leading to the alveoli. Furthermore, a reproducible injury with wide differences between different injurious and non-injurious insults is important.
The murine gastric acid aspiration model presented here using hydrochloric acid employs an open tracheostomy and recreates a pathogenic scenario that reproduces the low pH pneumonitis injury in humans. Additionally, this model can be used to examine interaction of a low pH insult with other pulmonary injurious entities (e.g., food particles, pathogenic bacteria).

This protocol induces acute lung injury in a mouse that has close fidelity to the pathogenesis of acid pneumonitis observed in humans. We generate a maximal acute nonlethal low pH lung injury and account for differences in rodent-human anatomic respiratory structure using an open tracheostomy coupled with circumferential pressure release.

急性肺损伤开放气管切开胃酸吸入小鼠模型结果在急性最大非致命性肺损伤

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute ...

Reproducible Arterial Denudation Injury by Infrarenal Abdominal Aortic Clamping in a Murine Model

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These complications can be attributed to impairments in re-endothelialization within the wound margins. Yet, the cellular and molecular mechanisms of re-endothelialization remain to be defined. While several animal models to study re-endothelialization after arterial denudation are available, few are performed in the mouse because of surgical limitations. This undermines the opportunity to exploit transgenic mouse lines and investigate the contribution of specific genes to the process of re-endothelialization. Here, we present a step-by-step protocol for creating a highly reproducible murine model of arterial denudation injury in the infrarenal abdominal aorta using external vascular clamping. Immunocytochemical staining of injured aortas for fibrinogen and &#946;-catenin demonstrate the exposure of a pro-thrombotic surface and the border of intact endothelium, respectively. The method presented here has the advantages of speed, excellent overall survival rate, and relative technical ease, creating a uniquely practical tool for imposing arterial denudation injury in transgenic mouse models. Using this method, investigators may elucidate the mechanisms of re-endothelialization under normal or pathological conditions.

Understanding the cellular and molecular mechanisms of re-endothelialization following arterial denudation injury is of paramount importance in preventing thrombosis and restenosis of arteries. Here we describe a protocol for reproducible arterial denudation injury of the infrarenal abdominal aorta. The procedure was developed to investigate the underlying mechanisms that regulate endothelial regeneration using mouse models.

重复性的动脉剥脱损伤腹主动脉阻断在小鼠模型

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These ...

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various intrinsic and extrinsic inhibitory factors involved in the restriction of axon regeneration have been identified. However, simply removing these inhibitory cues is insufficient for successful regeneration, indicating the existence of additional regulatory machinery. Drosophila melanogaster, the fruit fly, shares evolutionarily conserved genes and signaling pathways with vertebrates, including humans. Combining the powerful genetic toolbox of flies with two-photon laser axotomy/dendriotomy, we describe here the Drosophila sensory neuron &#8211; dendritic arborization (da) neuron injury model as a platform for systematically screening for novel regeneration regulators. Briefly, this paradigm includes a) the preparation of larvae, b) lesion induction to dendrite(s) or axon(s) using a two-photon laser, c) live confocal imaging post-injury and d) data analysis. Our model enables highly reproducible injury of single labeled neurons, axons, and dendrites of well-defined neuronal subtypes, in both the peripheral and central nervous system.

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

Here, we present a protocol using the Drosophila sensory neuron - dendritic arborization (da) neuron injury model, which combines in vivo live imaging, two-photon laser axotomy/dendriotomy, and the powerful fly genetic toolbox, as a platform for screening potential promoters and inhibitors of neuroregeneration.

一种研究外周和中枢神经系统 Neuroregeneration 的果蝇体内损伤模型

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various ...

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. Various approaches have been described, such as the inhalation of aerosolized LPS as well as nasal or intratracheal instillation. The presented protocol describes a detailed step-by-step procedure to induce ALI in mice by direct intratracheal LPS instillation and perform FACS analysis of blood samples, bronchoalveolar lavage (BAL) fluid, and lung tissue. After intraperitoneal sedation, the trachea is exposed and LPS is administered via a 22 G venous catheter. A robust and reproducible inflammatory reaction with leukocyte invasion, upregulation of proinflammatory cytokines, and disruption of the alveolo-capillary barrier is induced within hours to days, depending on the LPS dosage used. Collection of blood samples, BAL fluid, and lung harvesting, as well as the processing for FACS analysis, are described in detail in the protocol. Although the use of the sterile LPS is not suitable to study pharmacologic interventions in infectious diseases, the described approach offers minimal invasiveness, simple handling, and good reproducibility to answer mechanistic immunological questions. Furthermore, dose titration as well as the use of alternative LPS preparations or mouse strains allow modulation of the clinical effects, which can exhibit different degrees of ALI severity or early vs. late onset of disease symptoms.

Presented here is a step-by-step procedure to induce acute lung injury in mice by direct intratracheal lipopolysaccharide instillation and to perform FACS analysis of blood samples, bronchoalveolar lavage fluid, and lung tissue. Minimal invasiveness, simple handling, good reproducibility, and titration of disease severity are advantages of this approach.

通过直接内切内脂多糖灌输诱导小鼠急性肺损伤

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. ...