This work was supported by NIH grants P01 HL108801, R01-HL60234, R01-HL55330, R01-HL079904, to A. M. K. Choi. S. Ryter received salary support from the Lovelace Respiratory Research Institute.

Note:&#160; The Institutional Animal Care and Use Committee at Brigham and Women&#39;s Hospital/ Harvard Medical School Area approved the following procedures.
1. Cecal Ligation and Puncture
Use mice of the same background (C57Bl/6), male, 8-10 weeks old. Female mice are more resistant than males against sepsis-induced lethality. Mice older than 8 weeks produce less variable results than younger mice in terms of survival after CLP. Approximately N=10 ...

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The major advantage of CLP is that it allows researchers to investigate sepsis of different severities (i.e.&#160;from low- to mid- and high-grade). Severity of induced sepsis is affected by the length of cecum ligated (which the most important determinant), the size of needle used for puncture and the number of holes performed15. In addition, the mouse strain and gender can impact on the severity of sepsis; several strains are more susceptible than others and males are generally more susceptible than...

Sepsis, a systemic inflammatory response to infection, represents a leading cause of death in critically-ill patients1. Intra-abdominal infections, often leading to polymicrobial sepsis, account for 20% of sepsis cases, which have substantial mortality of up to 60%2. Sepsis-associated mortality primarily results from multi-organ dysfunction with subsequent organ failure3,4. Additional investigation into the pathogenic mechanism of this disease is urgently needed to promote the development of novel and more effective therapies.
The cecal ligation and puncture (CLP) method is a commonly used procedure for modeling sepsis in vivo. As the cecum is full of bacteria, its puncture results in polymicrobial peritonitis, translocation of bacteria into the blood (bacteremia), septic shock, multi-organ dysfunction and, ultimately, death5. It is generally accepted that CLP reflects clinical reality more accurately than previous techniques, such as injection of endotoxin or even purified bacteria into rodents, Thus, CLP is considered the gold standard (albeit not without limitations)6 for the experimental induction and, hence, the investigation of the pathogenesis of sepsis. In this monograph, we describe protocols designed to assess whether pathogenic mechanisms of sepsis include autophagy.
Autophagy, an evolutionarily conserved cellular process, facilitates the turnover of damaged proteins and organelles such as mitochondria and plays an important role in the clearance of intracellular pathogens including bacteria7,8. During autophagy, cytosolic proteins or organelles are sequestered into double membrane-bound vesicles called autophagosomes, which are subsequently delivered to the lysosomes for degradation9. A number of proteins have been identified as the mammalian homologues of autophagy-related genes (Atg), originally identified in yeast, which regulate the process of autophagy. The conversion of microtubule associated protein-1 light chain 3B (LC3B) (homologue of Atg8) from LC3B-I (free form) to LC3B-II (phosphatidylethanolamine-conjugated form) represents a major step in autophagosome formation9. Autophagic dysfunction is associated with aging and human diseases including cancer and neurodegenerative disorders10. Moreover, autophagy affects innate and adaptive immunity such as antigen presentation, lymphocyte development and cytokine secretion by immune cells8. Thus, it seems reasonable that autophagy might also play a role in the systemic inflammatory response to infection (i.e.&#160;in sepsis).
To date several methods have been described to assess the role of autophagy in tissue injury in vivo. These include the use of green fluorescence protein (GFP)-LC3 expressing mice&#160;and the quantification of autophagosomes in tissue by electron microscopy&#160;(these two methods are described in this monograph). Additional methods include the quantification of autophagic protein expression in tissue homogenates, and the analysis of autophagic flux (as described elsewhere)11-13. The goal of this review is to provide current protocols for assessing autophagy in vivo in the context of experimental sepsis.

<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

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is provided to induce sepsis of varying severity in mice using the CLP technique. Autophagy is a fundamental tissue response to stress and pathogen invasion. Two current protocols to assess autophagy in vivo in the context of experimental sepsis are also presented here. (I) Transgenic mice expressing green fluorescence protein (GFP)-LC3 fusion protein are subjected to CLP. Localized enhancement of GFP signal (puncta), as assayed either by immunohistochemical or confocal assays, can be used to detect enhanced autophagosome formation and, thus, altered activation of the autophagy pathway. (II) Enhanced autophagic vacuole (autophagosome) formation per unit tissue area (as a marker of autophagy stimulation) can be quantified using electron microscopy. The study of autophagic responses to sepsis is a critical component of understanding the mechanisms by which tissues respond to infection. Research findings in this area may ultimately contribute towards understanding the pathogenesis of sepsis, which represents a major problem in critical care medicine.

Bacteremia is present in mice as early as 6 hr after CLP-induced sepsis14. Clinical signs of sepsis (including chills, tachypnoea and impaired motor activity) appear approximately 12 hr after the procedure. Mice subjected to CLP begin to die at around 18 hr after induction of peritonitis. The more severe is the sepsis the more increased is the lethality15. In detail, high-grade sepsis causes 100% mortality within 2-3 days, while mid-grade sepsis results in around 60% mortality at 7 days after CLP (b...

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

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method. Current protocols to assess autophagy in vivo in the context of CLP-induced sepsis are presented here: A protocol for measuring autophagy using (GFP)-LC3 mice, and a protocol for measuring autophagosome formation by electron microscopy.

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is ...

cecal-ligation-puncture-induced-sepsis-as-model-to-study-autophagy

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Immunology and Infection

28.1K visualizações.  Brigham and Women's Hospital.  Sepse experimental pode ser induzida em camundongos usando a ligadura e punção do ceco método (CLP). Protocolos correntes para avaliar a autofagia in vivo no contexto de sepsia induzida por CLP são aqui apresentados: Um protocolo para medir autofagia utilizando ratinhos (GFP)-LC3, e um protocolo para medir a formação de autophagosome por microscopia electrónica. 