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In This Article

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

Summary

This protocol describes the induction of Gram-negative monobacterial sepsis in a mouse model system. The model is useful in investigating the inflammatory and lethal host responses during sepsis.

Abstract

Sepsis is a dysregulated host immune response to microbial invasion or tissue damage, leading to organ injury at a site distant from that of the infection or damage. Currently, the widely used mice models of sepsis include lipopolysaccharide (LPS)-induced endotoxemia, cecal ligation and puncture (CLP), and monobacterial infection model systems. This protocol describes a method to study the host responses during Salmonella Typhimurium infection-induced septic peritonitis in mice. S. Typhimurium, a Gram-negative intracellular pathogen, causes typhoid-like disease in mice.

This protocol elaborates the culture preparation, induction of septic peritonitis in mice through intraperitoneal injection, and methods to study systemic host responses. Furthermore, the assessment of bacterial burden in different organs and the flow cytometric analysis of increased neutrophil numbers in the peritoneal lavage is presented. Salmonella Typhimurium-induced sepsis in mice leads to an increase in proinflammatory cytokines and rapid infiltration of neutrophils in the peritoneal cavity, leading to lower survival.

Every step in this protocol has been optimized, resulting in high reproducibility of the pathogenesis of septic peritonitis. This model is useful for studying immunological responses during bacterial sepsis, the roles of different genes in disease progression, and the effects of drugs to attenuate sepsis.

Introduction

Sepsis is defined as a dysregulated systemic inflammatory and immune response to microbial invasion or tissue damage, leading to organ injury distant from the site of infection or damage. Septic shock is a subset of sepsis characterized by hypotension persisting during volume resuscitation, with a substantially increased risk of mortality1. The general public has become more aware of this disorder during the COVID-19 pandemic. Despite its high associated mortality, comprehensive epidemiological data on the global burden of sepsis is lacking because of the complexity of its diagnosis. In 2017, there were 48.9 million sepsis incidences and 11 mil....

Protocol

All experiments using Salmonella Typhimurium were conducted in Bio Safety Level 2 (BSL-2) facilities. Care must be taken to use proper personal protective equipment (PPE), ensure safety, and follow standard BSL-2 biohazard disposal methods. All mice experiments were conducted following guidelines stated by the Institutional Animal Ethics Committee, IISc. Mice were bred and maintained at the Central Animal Facility of IISc (Registration number: 48/1999/CPCSEA, dated 1/3/1999), approved by the Ministry of Environm.......

Representative Results

A detailed characterization of the host immune response using this particular model is shown in previous publications8,9. A few representative results of the described protocol are depicted in this section. This model aims to induce systemic infection of S. Typhimurium by intraperitoneal injection of the bacterial culture to induce sepsis. To confirm the infection, the lysates of the liver and spleen from septic mice were spread on SS agar plates, and th.......

Discussion

This article describes a method of inducing a severe form of bacterial sepsis by intraperitoneal injection of Salmonella Typhimurium. This model is advantageous over others as Salmonella Typhimurium is an intracellular pathogen and, hence, highly pathogenic, mimicking the clinically relevant condition of Gram-negative sepsis. The outcome of peritonitis sepsis in this model is systemic, with 100% mortality within 96 h post infection. Therefore, this model is instrumental in studying the inflammatory and .......

Acknowledgements

We thank the Central Animal Facility, IISc for supplying us with mice for research. This study was funded by grants to DpN from the Department of Biotechnology and Science and Engineering Research Board, Government of India. The infrastructural support from the DBT-IISc program and DST-FIST grants are greatly acknowledged. We thank all previous and current members of the DpN lab for their support.

....

Materials

NameCompanyCatalog NumberComments
Consumables
1 mL Sterile Syringe with 26 G needleBeckton Dickinson, Singapore303060
1.5 mL Microcentrifuge TubeTarsons, USA500010
10 mL Sterile Syringe with 21 G needleBeckton Dickinson, Spain307758
50 mL Conical FlaskTarsons, USA441150
50 mL Graduated Centrifuge TubeTarsons, USA546041
50 mL Graduated Centrifuge TubeTarsons, USA546021
Cell spreaderVWR, USAVWRU60828-680
Dulbecco’s Phosphate Buffered SalineHiMedia, Mumbai, IndiaTS1006
EthanolMerck100983
FcR blockerBD Biosciences553142
Fetal Bovine SerumGibco10270-106
FITC Rat anti-mouse Ly6G (Clone 1A8)BD Pharmingen551460
GlycerolSigma-AldrichG9012
Hand based Homogenizer--
Hemocytometer (Neubauer counting chamber)Rohem, IndiaI.S. 10269
Luria Bertani BrothHiMedia, Mumbai, IndiaM1245
ParaformaldehydeSigma-Aldrich158127
PetriplatesTarsons, USA460091
RPMIHimedia, Mumbai, IndiaAT060-10X1L
Salmonella-Shigella AgarHiMedia, Mumbai, IndiaM108
Sodium azideSigma-AldrichS2002
Equipments
CentrifugeKubota
Flow cytometerBD FACSverse
IncubatorN-biotek
SpectrophotometerShimadzu
Weighing machineSartorius

References

  1. Hotchkiss, R. S., et al. Sepsis and septic shock. Nature Reviews Disease Primers. 2 (1), 1-21 (2016).
  2. Rudd, K. E., et al. regional, and national sepsis incidence and mortality, 1990-2017: Analysis for the Global Bur....

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