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

Summary

This protocol describes techniques to measure infectious outcomes underlying secondary hospital-acquired infections in the immunosuppressive condition, first by establishing cecal ligation/puncture mice then challenging them with intranasal infection to create a clinically relevant model of immunosuppression sepsis.

Abstract

Sepsis, a severe and complicated life-threatening infection, is characterized by an imbalance between pro- and anti-inflammatory responses in multiple organs. With the development of therapies, most patients survive the hyperinflammatory phase but progress to an immunosuppressive phase, which increases the emergence of secondary infections. Therefore, improved understanding of the pathogenesis underlying secondary hospital-acquired infections in the immunosuppressive phase during sepsis is of tremendous importance. Reported here is a model to test infectious outcomes by creating double-hit infections in mice. A standard surgical procedure is used to induce polymicrobial peritonitis by cecal ligation and puncture (CLP) and followed by intranasal infection of Staphylococcus aureus to simulate pneumonia occurring in immune suppression that is frequently seen in septic patients. This dual model can reflect the immunosuppressive state occurring in patients with protracted sepsis and susceptibility to secondary infection from nosocomial pneumonia. Hence, this model provides a simple experimental approach to investigate the pathophysiology of sepsis-induced secondary bacterial pneumonia, which may be used for discovering novel treatments for sepsis and its complications.

Introduction

Sepsis initiates a complex interplay of host pro-inflammatory and anti-inflammatory processes and is characterized by a hyperinflammatory response and subsequent immune dysfunction^1,2. Sepsis represents a global health priority and causes a high number of deaths in intensive care units (ICUs)³. The incidence of sepsis is estimated to exceed 30 million cases worldwide per year, with mortality rates as high as 30% despite advances in ICU management^4,5. In 2017, the World Health Organization adopted a resolution to improve the prev....

Protocol

All methods described here were performed in accordance with the National Institute of Health Guide for Care and Use of Laboratory Animals and approved by the University of North Dakota Institutional Animal Care and Use Committee (IACUC).

1. Cecal ligation and puncture

NOTE: Female C57BL/6 mice (weight, 18-22 g; age, 6-8 weeks) are randomly divided into six groups: control group (Ctrl), infection group (SA for S. aureus), two sham groups, and two CLP groups. .......

Discussion

As the gold standard model for sepsis research, CLP has a combination of three insults, including tissue trauma caused by the laparotomy, necrosis due to ligation of the cecum, and infection as a result of microbial leakage that causes peritonitis with translocation of bacteria into blood⁸. Therefore, CLP mimics the complexity of human sepsis better than many other models. However, a major limitation of current CLP model is the inability to reflect the more prolonged phase of sepsis seen in patien.......

Materials

Name	Company	Catalog Number	Comments
21 G 1 ½ Needle	BD	BD305167
ACK lysing buffer	Gibco	A10492-01
Anti-mouse CD11b antibody	Biolegend	101201
Anti-mouse Ly-6G/Ly-6C (Gr-1) antibody	Biolegend	108401
C57BL/6 mice	Harlan (Indianapolis)	C57BL/6NHsd
Desk light	General Supply	General Supply
Disinfecting wipes	Clorox	B07NV5JMCS
Electric razor	General Supply	General Supply
ELISA kits (mouse IL-1β, IL-6 and TNFα)	Invitrogen	88-7013, 88-7064, and 88-7324
Iodine	Dynarex	B003U463PY	PVP Iodine Wipes
Ketamine	FORT DODGE	NDC 0856-2013-01	Amine hydrochloride injection
Laboratory scale	General Supply	General Supply
LB Agar, Miller	Fisher Scientific	BP1425-500	Molecular genetics, powder
Micropipette	ErgoOne	7100-1100
Normal saline	General Supply	General Supply
Polylined towel	CardinalHealth, Convertors	3520	Surgical drape, sterile, for single use only
Silk suture, 4-0	DAVIS & GECK	1123-31
Small animal needle holder	General Supply	General Supply
Small animal surgery scissors	General Supply	General Supply
Small animal surgical forceps	General Supply	General Supply
Staphylococcus aureus	ATCC	13301
Warm pad	General Supply	General Supply
Xylazine	Alfa Aesar	7361-61-7