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

Summary

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.

Abstract

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.

Introduction

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 failure^3,4. 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 mode....

Protocol

Note:  The Institutional Animal Care and Use Committee at Brigham and Women'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 mice per compared group should be used for survival analysis. N=3-5 mice is adequate for autophagy assays.

1. Use sterile small surgical in....

Results

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.......

Discussion

The major advantage of CLP is that it allows researchers to investigate sepsis of different severities (i.e. 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.......

Materials

List of materials used in this article

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