Name: cecal ligation and puncture-induced sepsis as a model to study autophagy in mice
Uploaded: 2014-02-09T22:00:00
Description: Watch this Scientific Journal Video about Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice at JoVE.com

这项工作是由美国国立卫生研究院资助P01 HL108801，R01-HL60234，R01-HL55330，R01-HL079904，以AMK财支持。 S. Ryter收到工资支持，从洛夫莱斯呼吸研究所。

注：该机构动物护理和使用委员会在Brigham和妇女医院/哈佛医学院区通过了以下程序。 1。盲肠结扎穿孔使用相同背景的小鼠（C57BL / 6），男，8〜10周龄。雌性小鼠比对败血症引起的致死的男性更耐。年长超过8周的小鼠产生比年轻小鼠在CLP后生存期少变的结果。约N = 10小鼠相比，每群应该用于存活分析。 N = 3-5老鼠是足够的自体吞噬分析。 ...

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中电的主要优点是，它使研究人员能够（从低到中，高档IE）探讨不同严重程度的败血症。诱导的败血症的严重程度是受盲肠结扎的长度（这是最重要的决定因素），用于穿刺针的尺寸和孔的数量进行15。此外，小鼠品系和性别可以对脓毒症的严重程度影响;几株比其他人更容易与男性通常比女性19,20更容易。一起上述因素决定了电致死亡。 根据我们的经...

脓毒症，全身炎症反应感染，代表死亡的危重病患者1的首要原因。腹腔内感染，常常导致多种微生物脓毒症，占脓毒症病例的20％，其中有高达60％的2大量死亡。脓毒症相关的死亡率主要来自多器官功能障碍会导致随后的器官衰竭3,4。其他调查这种疾病的致病机理是目前急需推动新的和更有效的疗法的发展。 盲肠结扎穿孔（CLP）法是一种常用的方法， 在体内建模败血症。如盲肠是充分的细菌，其穿刺结果在多种微生物腹膜炎，易位细菌进入血液（菌血症），脓毒性休克，多器官功能障碍，并最终死亡5。人们普遍认为中电反映临床现实比以前的技 ​​术，例如注射内毒素或什至纯化的细菌进入啮齿动物更准确，因此，电被认为是金标准（尽管并非没有局限性）6对实验诱导，因此，脓毒症的发病机理的研究。在这本专着中，我们描述了旨在评估脓毒症的发病机制是否包括自噬协议。 吞噬，进化上保守的细胞的过程中，有利于受损的蛋白质和细胞器如线粒体的营业额，并播放在细胞内病原体包括细菌7,8的间隙中起重要作用。在自体吞噬，细胞质蛋白或细胞器螯合成双层膜结合的囊泡称为自噬体，其随后被输送到溶酶体降解9。一些蛋白质已被确定为自噬相关基因的哺乳动物同系物（署理）最初确定在酵母中，从而调节自噬过程。微管相关蛋白1轻链3B（LC3B）（的Atg8的同源物）从LC3B-I（自由格式）转换为LC3B-Ⅱ（磷脂酰乙醇胺结合的形式）表示在自噬体的形成9迈出的重要一步。自噬功能障碍与衰老和人类疾病包括癌症和神经退行性疾病相关的10。此外，自噬影响固有免疫和适应性免疫，如抗原提呈，淋巴细胞发育和细胞因子分泌免疫细胞8。因此，它似乎是合理的自噬作用也可能会在全身炎症反应感染（ 如败血症）发挥作用。 迄今为止的几种方法已经被描述，以评估自噬在组织损伤体内的作用。这些技术包括利用绿色荧光蛋白（GFP）-LC3表达的小鼠和自动的量化在组织的电子显微镜吞噬体（这两种方法都在本专着中描述）。另外的方法包括在组织匀浆自噬蛋白表达的定量，和自噬通量的分析（如在别处所述）11-13。本次审查的目的是评估在实验败血症的情况下自噬在体内提供当前协议。

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >GFP-LC3 Transgenic Mice</td>
			<td >Riken (Japan)</td>
			<td >RBRC00806</td>
			<td >GFP-LC3#53</td>
		</tr>
		<tr >
			<td >Xylazine</td>
			<td >Henry-Schein</td>
			<td >568-0606</td>
			<td>Xylazine HCl Injection Vet</td>
		</tr>
		<tr >
			<td >Ketamine</td>
			<td >Henry-Schein</td>
			<td >995-2949</td>
			<td>Ketaset Inj 100 mg/ml</td>
		</tr>
		<tr >
			<td >EtOH</td>
			<td >Fisher</td>
			<td>A405-20</td>
			<td>Histology Grade</td>
		</tr>
		<tr >
			<td >EtOH</td>
			<td >Fisher</td>
			<td>A407-1</td>
			<td>For Sterilizatiion</td>
		</tr>
		<tr >
			<td >silk surgical sutures 6-0</td>
			<td >Owens &#38; Minor</td>
			<td >2300-0078OG, 017624</td>
			<td></td>
		</tr>
		<tr >
			<td >buprenorphine-HCl</td>
			<td >Henry-Schein</td>
			<td >614-5157</td>
			<td>Buprenex Ampules</td>
		</tr>
		<tr >
			<td >paraformaldehyde (37%) solution</td>
			<td >JT Baker</td>
			<td >S898-09</td>
			<td></td>
		</tr>
		<tr >
			<td >xylenes</td>
			<td >Fisher</td>
			<td>X3P-1GAL </td>
			<td></td>
		</tr>
		<tr >
			<td >anti-GFP monoclonal antibody</td>
			<td>Life Technologies</td>
			<td>G10362</td>
			<td></td>
		</tr>
		<tr >
			<td >Hoescht</td>
			<td >Sigma</td>
			<td>944403</td>
			<td></td>
		</tr>
		<tr >
			<td >DAPI</td>
			<td >Invitrogen</td>
			<td >D1306</td>
			<td></td>
		</tr>
		<tr >
			<td >OCT</td>
			<td >VWR scientific</td>
			<td >25608-930</td>
			<td></td>
		</tr>
		<tr >
			<td >Sudan Black</td>
			<td >Santa Cruz</td>
			<td >sc-203760</td>
			<td ></td>
		</tr>
		<tr >
			<td >EM grade Glutaraldehyde 2.5% in sodium cacodylate</td>
			<td >Electron microscopy Sciences</td>
			<td>15960</td>
			<td></td>
		</tr>
		<tr >
			<td >propylene oxide</td>
			<td >Sigma</td>
			<td>240397</td>
			<td></td>
		</tr>
		<tr >
			<td >Agar 100 resin</td>
			<td >Agar scientific</td>
			<td > R1045</td>
			<td></td>
		</tr>
		<tr >
			<td >dodecenylsuccinic anhydride</td>
			<td >Sigma</td>
			<td>46346</td>
			<td></td>
		</tr>
		<tr >
			<td >methylnadic anhydride</td>
			<td >Sigma</td>
			<td>45359</td>
			<td></td>
		</tr>
		<tr >
			<td >N-benzyldimethylamine</td>
			<td >Sigma</td>
			<td>185582</td>
			<td></td>
		</tr>
	</tbody>
</table>

cecal ligation and puncture-induced sepsis as a model to study autophagy in mice

败血症实验中可以使用的小鼠盲肠结扎穿孔（CLP）方法，这会导致多种微生物败血症诱发。在这里，一个协议提供了使用中电技术诱发不同程度的败血症小鼠。自噬是一个根本性的组织应激反应和病原体的入侵。二是目前的协议，以评估在实验败血症的情况下自噬在体内也这里。 （Ⅰ）表达绿色荧光蛋白（GFP）-LC3融合蛋白的转基因小鼠进行CLP。 GFP信号（泪点）的局部增强，因为无论是通过免疫组织化学或共聚焦分析测定，可用于检测增强的自噬体的形成，并因此改变了自噬途径的活化。 （II）的增强型自噬泡（自噬体）的每单位面积的组织的形成（如吞噬刺激的标记物）可以用电子显微镜进行量化。自噬反应败血症的研究是一个重要的排版onent理解由感染组织响应的机制。在这方面的研究成果可能对理解脓毒症的发病机制，这代表在危重病医学的一大难题，最终贡献。

菌血症存在于小鼠早中电诱发败血症14后6小时。败血症的临床症状（包括寒战，呼吸急促和运动障碍的活动）的程序后，出现约12小时。老鼠受到电开始诱导性腹膜炎后死在大约18小时。更严重的是脓毒症越增加的杀伤力15。具体而言，高档败血症导致在2-3天内死亡率为100％，而中档败血症导致大约60％的死亡率在CLP后7天（超出此时间限制，无死亡病例预计）（ 图1）。</strong...

Watch this Scientific Journal Video about Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice at JoVE.com

Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice

实验性败血症可以在小鼠体内利用盲肠结扎穿孔（CLP）方法来诱导。当前的协议，以评估在CLP诱导的脓毒症的上下文中的自噬体内这里提出：一个协议，用于使用（GFP）-LC3的小鼠测定自噬和协议，用于测量自噬体形成通过电子显微镜。

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The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

A Controlled Mouse Model for Neonatal Polymicrobial Sepsis

新生儿多药脓毒症的对照小鼠模型

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse ...

Design of Cecal Ligation and Puncture and Intranasal Infection Dual Model of Sepsis-Induced Immunosuppression

脓毒症诱导免疫抑制的切卡结扎和穿刺和鼻内感染双重模型的设计

Sepsis, a severe and complicated life-threatening infection, is characterized by an imbalance between pro- and anti-inflammatory responses in multiple ...