1Department of Microbiology and Immunology School of Medicine, Temple University, 2Department of Biochemistry, School of Medicine, Temple University
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Toscano, M. G., Ganea, D., Gamero, A. M. Cecal Ligation Puncture Procedure. J. Vis. Exp. (51), e2860, doi:10.3791/2860 (2011).
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.
1. Cecal Ligation and Puncture as a Mouse Model for Human Sepsis
For this procedure C57BL/6 mice (7-9 weeks old) are used.
As control for the experimental design, sham animals would follow the laparotomy technique without ligation and puncture.
Six to 12 hours after the surgical procedure mice will become lethargic and develop fever, piloerection, diarrhea, huddling, and malaise; all the symptoms of sepsis. Mice with very severe sepsis can barely move prior to death and exhibit a dramatic decrease in body temperature. At this step mice should be euthanized to avoid prolonged pain and suffering.
2. Most Common Analyzed Parameters
To evaluate the outcome of the procedure, different parameters can be analyzed in organs, cell extracts or body fluids. Samples can be collected at different time points from 3 hours to one week after the surgical procedure.
3. Representative Results
The CLP procedure was initially performed in the C57BL/6 mouse strain. We tested several parameters to modulate the severity of sepsis by altering the length of the ligation and thickness of the needle as shown in Figures 1 and 2. Among these two factors, the length of the ligation seems to be more effective than the thickness of the needle to alter percent survival. As shown in Figure 1, increasing the length of the ligation by more than 1cm provokes an increase in mortality of 100% compared with mice having a ligation of ≤1cm. Increasing the thickness of the needle also decreased the percent survival from 100% (using a 22G needle) to 55% (using a 19G needle) with two punctures. We also tested the effect of CLP on C57BL/6 and 129SvJ mice to determine whether different mouse strains display similar or distinct susceptibility to CLP-induced sepsis. Figure 3 shows that under the same conditions, 129SvJ mice were more susceptible to infection than C57BL/6, indicated by an increased percent mortality.
Figure 1. Effect of ligation length of the cecum in animal survival. CLP was performed in C57BL/6 mice using two different lengths of cecal ligation. This single parameter dramatically affects animal survival since all of the animals in the group with a ligation area longer than 1 cm died in less than 3 days when compared with a group of animals having a ligation area of 1 cm approx. (n=8).
Figure 2. Influence of needle thickness on survival. CLP was performed in C57BL/6 mice using two different needle sizes, 19G and 22G. Mice with the cecum perforated using a 19G needle showed a 55-60% survival. In contrast, mice with the cecum perforated using a 22G needle showed 100% survival although they experienced typical symptoms of inflammation during the first 3-4 days; which disappeared after 4 days (n=8).
Figure 3. Comparison of sepsis susceptibility between two different mouse strains. The CLP model was performed in 129SvJ and C57BL/6 under the same conditions. At the end of the evaluation period the percent survival of the 129SvJ strain was 25% less than in the C57BL/6 strain, indicating a higher susceptibility to inflammation induced by polymicrobial infection. (n=6).
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 severity/mortality of CLP. Consequently, to obtain dramatic changes in CLP severity, the user can change the length of the ligation while slight modification in severity can be introduced by adjusting the thickness of the needle.
Other important parameters that could introduce a substantial variability in the CLP results are the mouse strain 6,7, sex8 and age of the animal9. In this regard we show that different mouse strains display distinct susceptibilities to the procedure when performed under the same conditions. Indeed, differences in plasma cytokine levels and tissue damage have been detected between different mouse strains10-12. Inclusion of fluid resuscitation and analgesic administration are factors that can introduce significant variability in the CLP model. For instance, fluid resuscitation increases survival rate possibly by diminishing the production of early pro-inflammatory cytokines11, 13, 14 thus masking the effects of potential therapeutic interventions. In contrast, narcotic analgesics can suppress respiration and locomotion in the animal and provoke an irreversible effect unrelated to sepsis. Their use, therefore, can influence survival rate and immune cell function15.
In conclusion, when performing the CLP model all of these conditions and the technical parameters must be carefully considered in order to obtain reproducible and consistent results. Therefore, a deep knowledge and mastering of the procedure is absolutely needed to perform the CLP model accurately.
Animal experiments were performed in accordance with the National Institute of Health Guide for Care and Use of Laboratory Animals, and approved by the animal care and use committee at Temple University.
This work was supported by a grant from the Pennsylvania Department of Health.
Dr. Miguel Garcia Toscano was a postdoctoral Fellow at Temple University, funded by the Alfonso Martin Escudero Foundation during this study.
We want to thank Iliya Yordanov and Kevin Kotredes for the making of the video.
|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|