Cecal Ligation Puncture Procedure

Summary

The mouse model of cecal ligation and puncture as a valuable tool for the study of human sepsis.

Abstract

Human sepsis is characterized by a set of systemic reactions in response to intensive and massive infection that failed to be locally contained by the host. Currently, sepsis ranks among the top ten causes of mortality in the USA intensive care units ¹. During sepsis there are two established haemodynamic phases that may overlap. The initial phase (hyperdynamic) is defined as a massive production of proinflammatory cytokines and reactive oxygen species by macrophages and neutrophils that affects vascular permeability (leading to hypotension), cardiac function and induces metabolic changes culminating in tissue necrosis and organ failure. Consequently, the most common cause of mortality is acute kidney injury. The second phase (hypodynamic) is an anti-inflammatory process involving altered monocyte antigen presentation, decreased lymphocyte proliferation and function and increased apoptosis. This state known as immunosuppression or immune depression sharply increases the risk of nocosomial infections and ultimately, death. The mechanisms of these pathophysiological processes are not well characterized. Because both phases of sepsis may cause irreversible and irreparable damage, it is essential to determine the immunological and physiological status of the patient. This is the main reason why many therapeutic drugs have failed. The same drug given at different stages of sepsis may be therapeutic or otherwise harmful or have no effect ^2,3. To understand sepsis at various levels it is crucial to have a suitable and comprehensive animal model that reproduces the clinical course of the disease. It is important to characterize the pathophysiological mechanisms occurring during sepsis and control the model conditions for testing potential therapeutic agents.

To study the etiology of human sepsis researchers have developed different animal models. The most widely used clinical model is cecal ligation and puncture (CLP). The CLP model consists of the perforation of the cecum allowing the release of fecal material into the peritoneal cavity to generate an exacerbated immune response induced by polymicrobial infection. This model fulfills the human condition that is clinically relevant. As in humans, mice that undergo CLP with fluid resuscitation show the first (early) hyperdynamic phase that in time progresses to the second (late) hypodynamic phase. In addition, the cytokine profile is similar to that seen in human sepsis where there is increased lymphocyte apoptosis (reviewed in ^4,5). Due to the multiple and overlapping mechanisms involved in sepsis, researchers need a suitable sepsis model of controlled severity in order to obtain consistent and reproducible results.

Protocol

1. Cecal Ligation and Puncture as a Mouse Model for Human Sepsis

For this procedure C57BL/6 mice (7-9 weeks old) are used.

1. Anesthetize the mouse by injecting intraperitoneally a solution of 1:1 ketamine (75mg/kg) and xylazine (15mg/kg). As a reference, inject 30 μl of the 1:1 solution into a mouse weighing 20 grams. Alternatively, mice can be anesthetized with inhaled isoflurane using an anesthetic vaporizer.
2. Shave the abdomen of the mouse and disinfect area by first applying betadine solution followed by wiping with a 70% alcohol swab (number 1 in the picture sequence below). Chlorhexidine can be used as an alterna....

Discussion

Here we show in detail how to perform the CLP model in mice and modulate the grade of severity.

Compared to other animal models of sepsis, CLP can be performed in any mouse strain of different age and sex. It is a relatively easy and inexpensive surgical procedure. In this model, the grade of severity has a direct impact on the percentage of survival. The length of the ligation, the thickness of the needle and the number of punctures are parameters that can be controlled to modulate the sev.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ketamine	Ketaset	0856-2013-01
Xylazine	AnaSed	NADA# 139-236
Insulin syringe 29G	Excel Scientific	26028
Silk suture, 6-0 PROLENE	Ethicon Inc.	8680G
19G and 25G Needle	BD Biosciences	305186
Shaver	General Supply	General supply
Infrared Heating lamp	General Supply	General supply
Michel wound clips 7mm	Roboz Surgical Instruments Co.	RS-9270
Ear Loop Mask	Fisher Scientific	19-130-4181
Dissection scissors	Roboz Surgical Instruments Co.	RS-6702
Betadine solution	VWR international	63410-992
Surgical forceps	Roboz Surgical Instruments Co.	RS-5135
70% Isopropyl alcohol pad	Fisher Scientific	22-031-350
Buprenorphine	Bedford Laboratories	55390-100-10
Tramadol	Sigma-Aldrich	42965