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Critical Care

Volume 18 Supplement 2

Sepsis 2014

Open Access

Mice survival in a two-hit model of sepsis depends on intratracheal P. aeruginosa bacterial load

  • D Restagno1,
  • J-M Bonnet1,
  • A Kodjo1,
  • F Venet2,
  • C Paquet1,
  • L Freyburger1 and
  • V Louzier1
Critical Care201418(Suppl 2):P59

Published: 3 December 2014


Systemic Inflammatory Response SyndromeSecondary InfectionBacterial LoadC57Bl6 MouseMouse Susceptibility


Sepsis is a systemic reaction in the presence of an infection characterized by an early systemic inflammatory response syndrome followed by a compensatory anti-inflammatory response syndrome. The first side of this typical immune response is responsible for widespread damage organ whereas the second part leads to an immunoparalysis increasing patients' susceptibility to secondary infections. The goal of this study is to finalize a murine model of sepsis to understand the physiopathology of sepsis and eventually test new therapeutic approaches.


Male C57Bl6 mice (20 to 25 g, 6 to 8 weeks) were used. Sepsis is induced by a mild CLP (survival 80%). Cecum is isolated, ligated (1/3), and punctured twice (21G needle). A laparotomy is performed without ligation or puncture. Blood is collected 2 hours, 6 hours, 1, 2, 3, 5 and 13 days after the CLP. Plasmatic cytokines (TNFα, IL-6, IL-10) are evaluated via the Luminex technique. Five days after CLP, splenocytes are collected and used for immunological assays. Mice are intratracheally instilled with P. aeruginosa 5 days after CLP (5 × 106, 2 × 107 and 108 CFU) to evaluate survival.


Circulating TNFα, IL-6 and IL-10 levels increase 2 hours after CLP and remain high until the end of the experiment, as compared to Sham operated mice. Five days after CLP, total T cells (T-CD3+) population and proliferation significantly decrease as compared to the nonseptic condition whereas the T-reg cell (T-CD4+/CD25+) population significantly increases whereas it remains low in the Sham operated animals. TNFα induction by LPS in cultured splenocytes increases both for CLP and Sham operated mice but remains lower in septic mice. Survival following a secondary infection decreases while the quantity of instilled P. aeruginosa increases. No mortality is observed for 5 × 106 CFU, 50% of CLP die with 2 × 107 CFU and 100% of mortality is observed for both CLP and Sham with 108 CFU. Two days after instillation, P. aeruginosa is detected in the lungs of CLP and Sham but in the spleen and liver only for CLP mice.


This study demonstrates that immunosuppression following CLP increases mice susceptibility to secondary P. aeruginosa intratracheal infection. However, this susceptibility depends on the bacterial load.

Authors’ Affiliations

VetAgro sup, campus vétérinaire, Marcy l'Etoile, France
Groupement Hospitalier Edouard Heriot, Laboratoire d'immunologie cellulaire, Lyon, France


© Restagno et al.; licensee BioMed Central Ltd. 2014

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated.