Sign In

A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 dysfunction1,2. 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 management4,5. 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. .......

Representative Results

Depending on the experimental design and procedures, C57BL/6 mice were subjected to CLP, and after 3 days, they were administered bacteria intranasally (Figure 1). As shown in Figure 2, the mice began to die at ~12 h after induction of peritonitis. Two mice in the CLP+SA group and three mice in the CLP+NS group died before intranasal S. aureus instillation. No mortality was detected in uninfected non- or sham-operated mi.......

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.......

Acknowledgements

This work was supported by National Institutes of Health Grants R01 AI138203-01, AI109317-04, AI101973-01, and AI097532-01A1 to M. W. The University of North Dakota Core Facilities were supported by NIH grants (INBRE P20GM103442 and COBRE P20GM113123). This work was also supported by the Key Program of National Nature Science Foundation of China (81530063) to Jianxin Jiang. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank Marvin Leier (Center for Rural Health, University of North Dakota) for making the video.

....

Materials

NameCompanyCatalog NumberComments
21 G 1 ½ NeedleBDBD305167
ACK lysing bufferGibcoA10492-01
Anti-mouse CD11b antibodyBiolegend101201
Anti-mouse Ly-6G/Ly-6C (Gr-1) antibodyBiolegend108401
C57BL/6 mice Harlan (Indianapolis)C57BL/6NHsd
Desk lightGeneral SupplyGeneral Supply
Disinfecting wipesCloroxB07NV5JMCS
Electric razorGeneral SupplyGeneral Supply
ELISA kits (mouse IL-1β, IL-6 and TNFα)Invitrogen88-7013, 88-7064, and 88-7324
IodineDynarexB003U463PYPVP Iodine Wipes
KetamineFORT DODGENDC 0856-2013-01Amine hydrochloride injection
Laboratory scaleGeneral SupplyGeneral Supply
LB Agar, MillerFisher ScientificBP1425-500Molecular genetics, powder
MicropipetteErgoOne7100-1100
Normal salineGeneral SupplyGeneral Supply
Polylined towelCardinalHealth, Convertors3520Surgical drape, sterile, for single use only
Silk suture, 4-0DAVIS & GECK1123-31
Small animal needle holderGeneral SupplyGeneral Supply
Small animal surgery scissorsGeneral SupplyGeneral Supply
Small animal surgical forcepsGeneral SupplyGeneral Supply
Staphylococcus aureusATCC13301
Warm padGeneral SupplyGeneral Supply
XylazineAlfa Aesar7361-61-7

References

  1. Singer, M., et al. The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). JAMA. 315 (8), 801-810 (2016).
  2. Delano, M. J., Ward, P. A. The immune system's rol....

Explore More Articles

Cecal LigationCecal PunctureSepsisImmunosuppressionMouse ModelIntranasal InfectionStaphylococcus AureusPneumonia

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved