Name: Cecal Ligation Puncture Procedure (Video) | JoVE Journal
Uploaded: 2011-05-07T14:00:00.000+00:00
Duration: 11 min 53 s
Description: Learn the cecal ligation and puncture procedure for sepsis research using C57BL/6 mice. This method induces polymicrobial infection to study immune responses and organ damage in vivo.

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5 minutes

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.

1. Cecal Ligation and Puncture as a Mouse Model for Human Sepsis
For this procedure C57BL/6 mice (7-9 weeks old) are used.
<ol>
 <li>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. </li>
 <li>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 alternative antiseptic. Optional: a sterile drape may be used to maintain the area clean.</li>
 <li>Under aseptic conditions, practice a 1 to 2 cm midline laparotomy and expose the cecum with adjoining intestine (2 and 3).</li>
 <li>The cecum is tightly ligated with a 6.0 silk suture (6-0 PROLENE, 8680G; Ethicon) at its base below the ileo-cecal valve, and is perforated once or twice with a 19-gauge needle (4 and 5) on the same side of the cecum. Please note that the length of ligated cecum defined as the distance from the distal end of cecum to ligation point will determine the degree of severity. A distance of &gt;1cm produces high grade sepsis while a distance of ≤1 cm produces mid-to-low grade sepsis. Also note that using the cecal perforation method shown here is different from the standard through and through puncture technique (introducing a needle through the cecum). Both methods reliably produce the same sepsis outcome. However, one disadvantage of the through and through technique is that it does not facilitate controlling the amount of feces extruded when the cecum is squeezed. </li>
 <li>The cecum is then gently squeezed to extrude a small amount of feces from the perforation sites (6). The cecum is returned to the peritoneal cavity and the 
 peritoneum is closed with 6.0 silk sutures. (7). </li>
 <li>The skin is closed with Reflex 7mm clips (RS-9258, Roboz Surgical Instruments) or Michel wound clips (7 mm, RS-9270) (8).</li>
 <li>Resuscitate mice by injecting subcutaneously 1 ml of pre-warmed 0.9% saline solution using a 25G needle. This fluid resuscitation measure will induce the 
 hyperdynamic phase of sepsis. This critical step is described further in the discussion section. </li>
 <li>Optional: Inject subcutaneously buprenorphine (0.05mg/ kg body weight) or tramadol (20mg/kg body weight) for post operative analgesia. Be advised that these opiates can suppress respiration and locomotion and these effects can be misinterpreted as signs of sepsis. </li>
 <li>The animals are placed temporarily on a heating pad or alternatively returned immediately to a cage with exposure to an infrared heating lamp of 150W until they recover from the anesthesia. The recovery time is from 30 min to 1 hour.</li>
 <li>Provide free access to food and water (hydrogel) placed on the bottom of the cage.</li>
 <li>Mice are monitored every 12 hours for survival for one to two weeks or euthanized at different time points for analysis of different parameters.</li>
</ol>
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.
<img src="/files/ftp_upload/2860/2860fig0.jpg" alt="Wound healing process in mice, surgical procedure sequence, biomedical research experiment." data-altupdatedat="1788235205000" data-alt="Figure 0">
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. 
<ol>
 <li>Mice survival.</li>
 <li>Cytokines and chemokines in serum, peritoneal cavity and organ extracts.
 <ol>
 <li>Interleukin-6: Produced and released by monocytes, dendritic cells, macrophages, B cells, T cells, granulocytes, mast cells and many more cell types. It plays an important role in acute phase reaction and inflammation.</li>
 <li>Tumor necrosis factor-α: Pleiotrophic cytokine that plays a central role in inflammation and apoptosis. Produced by monocytes, macrophages, neutrophils, dendritic cells and fibroblasts.</li>
 <li>nterleukin-1β: Produced by monocytes, NK cells, dendritic cells, B cells and T cells. Induces fever and acute phase protein synthesis.</li>
 <li>Interleukin 10: Promotes phagocytic uptake and Th2 responses but suppresses antigen presentation and Th1 proinflammatory responses.</li>
 <li>Interleukin 10: Promotes phagocytic uptake and Th2 responses but suppresses antigen presentation and Th1 proinflammatory responses.</li>
 <li>Monocyte chemotactic protein-1 (also known as CCL2): Recruits monocytes, memory T cells, and dendritic cells to sites of tissue injury and infection.</li>
 <li>KC (CXCL1): Produced by macrophages and endothelial cells. Mouse KC is a potent neutrophil attractant and activator.</li>
 <li>RANTES: A monocyte chemoattractant. It can chemoattract unstimulated CD4+/CD45RO+ memory T cells and stimulated CD4+ and CD8+ T cells with naïve and memory phenotypes.</li>
 <li>Interferon-γ: Secreted by Th1 cells, cytotoxic T cells, dendritic cells and natural killer (NK) cells. Increases antigen presentation and lytic activity in macrophages and suppresses Th2 cell activity. Promotes adhesion and binding required for leukocyte migration and promotes NK cell activity. </li>
 <li>High-mobility group B-1 protein: In monocytes is a nuclear factor kappa B activator by binding to the cellular receptor for advance glycation end products (RAGE), activating the release of proinflammatory mediators in the late phase of sepsis. </li>
 </ol>
 </li>
 <li>Myeloperoxidase determination in organs as a measure of neutrophil infiltration. Myeloperoxidase (MPO) is a peroxidase enzyme most abundantly present in neutrophil granulocytes. It is a lysosomal protein stored in azurophilic granules of the neutrophil. MPO has a heme pigment, which causes its green color in secretions rich in neutrophils, such as pus and some forms of mucus.</li>
 <li>Bacteria load in organs and blood stream, measure as the number of colony forming units per milliliter. </li>
</ol>
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.
<img src="/files/ftp_upload/2860/2860fig1.jpg" alt="Ligation length survival graph, C57BL/6 mice, comparing puncture sizes, 19G, age 7-8 weeks." data-altupdatedat="1788235205000" data-alt="Figure 1"> 
 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).
<img src="/files/ftp_upload/2860/2860fig2.jpg" alt="Survival analysis chart of mice; needle thickness impact, 19G vs 22G, over 10 days, two punctures." data-altupdatedat="1788235205000" data-alt="Figure 2"> 
 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). 
<img src="/files/ftp_upload/2860/2860fig3.jpg" alt="Mouse strain survival comparison graph; Kaplan-Meier plot; C57BL/6 vs 129SvJ over 10 days." data-altupdatedat="1788235205000" data-alt="Figure 3"> 
 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).

<ol><li>Angus, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epidemiology+of+severe+sepsis+in+the+United+States%3A+analysis+of+incidence%2C+outcome%2C+and+associated+costs+of+care%5BTitle%5D)+AND+%22Crit+Care+Med%22%5BJournal%5D)">Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care.</a> Crit Care Med. 29, 1303-1310 (2001).</li>
<li>Riedemann, N. C., Guo, R. F., Ward, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+enigma+of+sepsis%5BTitle%5D)+AND+%22J+Clin+Invest%22%5BJournal%5D)">The enigma of sepsis.</a> J Clin Invest. 112, 460-467 (2003).</li>
<li>Eichacker, P. Q., Gerstenberger, E. P., Banks, S. M., Cui, X., Natanson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Meta-analysis+of+acute+lung+injury+and+acute+respiratory+distress+syndrome+trials+testing+low+tidal+volumes%5BTitle%5D)+AND+%22Am+J+Respir+Crit+Care+Med%22%5BJournal%5D)">Meta-analysis of acute lung injury and acute respiratory distress syndrome trials testing low tidal volumes.</a> Am J Respir Crit Care Med. 166, 1510-1514 (2002).</li>
<li>Buras, J. A., Holzmann, B., Sitkovsky, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Animal+models+of+sepsis%3A+setting+the+stage%5BTitle%5D)+AND+%22Nat+Rev+Drug+Discov%22%5BJournal%5D)">Animal models of sepsis: setting the stage.</a> Nat Rev Drug Discov. 4, 854-865 (2005).</li>
<li>Doi, K., Leelahavanichkul, A., Yuen, P. S., Star, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Animal+models+of+sepsis+and+sepsis-induced+kidney+injury%5BTitle%5D)+AND+%22J+Clin+Invest%22%5BJournal%5D)">Animal models of sepsis and sepsis-induced kidney injury.</a> J Clin Invest. 119, 2868-2878 (2009).</li>
<li>Godshall, C. J., Scott, M. J., Peyton, J. C., Gardner, S. A., Cheadle, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+background+determines+susceptibility+during+murine+septic+peritonitis%5BTitle%5D)+AND+%22J+Surg+Res%22%5BJournal%5D)">Genetic background determines susceptibility during murine septic peritonitis.</a> J Surg Res. 102, 45-49 (2002).</li>
<li>De Maio, A., Torres, M. B., Reeves, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+determinants+influencing+the+response+to+injury%2C+inflammation%2C+and+sepsis%5BTitle%5D)+AND+%22Shock%22%5BJournal%5D)">Genetic determinants influencing the response to injury, inflammation, and sepsis.</a> Shock. 23, 11-17 (2005).</li>
<li>Diodato, M. D., Knoferl, M. W., Schwacha, M. G., Bland, K. I., Chaudry, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gender+differences+in+the+inflammatory+response+and+survival+following+haemorrhage+and+subsequent+sepsis%5BTitle%5D)+AND+%22Cytokine%22%5BJournal%5D)">Gender differences in the inflammatory response and survival following haemorrhage and subsequent sepsis.</a> Cytokine. 14, 162-169 (2001).</li>
<li>Turnbull, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+age+on+mortality+and+antibiotic+efficacy+in+cecal+ligation+and+puncture%5BTitle%5D)+AND+%22Shock%22%5BJournal%5D)">Effects of age on mortality and antibiotic efficacy in cecal ligation and puncture.</a> Shock. 19, 310-313 (2003).</li>
<li>Miyaji, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ethyl+pyruvate+decreases+sepsis-induced+acute+renal+failure+and+multiple+organ+damage+in+aged+mice%5BTitle%5D)+AND+%22Kidney+Int%22%5BJournal%5D)">Ethyl pyruvate decreases sepsis-induced acute renal failure and multiple organ damage in aged mice.</a> Kidney Int. 64, 1620-1631 (2003).</li>
<li>Torres, M. B., De Maio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+exaggerated+inflammatory+response+after+CLP+correlates+with+a+negative+outcome%5BTitle%5D)+AND+%22J+Surg+Res%22%5BJournal%5D)">An exaggerated inflammatory response after CLP correlates with a negative outcome.</a> J Surg Res. 125, 88-93 (2005).</li>
<li>Doi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((AP214%2C+an+analogue+of+alpha-melanocyte-stimulating+hormone%2C+ameliorates+sepsis-induced+acute+kidney+injury+and+mortality%5BTitle%5D)+AND+%22Kidney+Int%22%5BJournal%5D)">AP214, an analogue of alpha-melanocyte-stimulating hormone, ameliorates sepsis-induced acute kidney injury and mortality.</a> Kidney Int. 73, 1266-1274 (2008).</li>
<li>Wilson, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluid+resuscitation+attenuates+early+cytokine+mRNA+expression+after+peritonitis%5BTitle%5D)+AND+%22J+Trauma%22%5BJournal%5D)">Fluid resuscitation attenuates early cytokine mRNA expression after peritonitis.</a> J Trauma. 41, 622-627 (1996).</li>
<li>Zanotti-Cavazzoni, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluid+resuscitation+influences+cardiovascular+performance+and+mortality+in+a+murine+model+of+sepsis%5BTitle%5D)+AND+%22Intensive+Care+Med%22%5BJournal%5D)">Fluid resuscitation influences cardiovascular performance and mortality in a murine model of sepsis.</a> Intensive Care Med. 35, 748-754 (2009).</li>
<li>Hugunin, K. M., Fry, C., Shuster, K., Nemzek, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+tramadol+and+buprenorphine+on+select+immunologic+factors+in+a+cecal+ligation+and+puncture+model%5BTitle%5D)+AND+%22Shock%22%5BJournal%5D)">Effects of tramadol and buprenorphine on select immunologic factors in a cecal ligation and puncture model.</a> Shock. 34, 250-260 (2010).</li>
</ol>

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.

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...

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cecal ligation puncture procedure

Cecal Ligation Puncture Procedure

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.

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

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 ...

Hello, my name is Miguel Toscano from Dr.Dena's lab in the department of Microbiology and Hemology at Temple University. Today we are going to show you how to set up the iCal allegation and puncture model in the C 57 Black Seas Mouse strain. We use this model in our laboratory to mimic human sepsis.Basically, this model consists in a puncture of the seum that allows the release of fecal material into the peritoneal cavity to produce a polymicrobial sepsis, thus generating an exacerbated immune response. In this model, many organs and different cell types are irreversibly damaged. That in the end can cause the death of the animal.So let's get Started. Anesthetizing the mouse. The first step is anesthetizing the animal by injecting intraperitoneal.A mixture, one to one of Ketomine and xylazine. To avoid being bitten by the mouse, to avoid hurting him, we must grab the mouse from the tail with one hand. With the other hand, we presses back against the floor.Then we can grab the mouse from the back of the head and then turn the animal on his back to expose the abdomen. With an insulin syringe of 29 gauge, we inject 20 to 30 microliters of the anesthesia in the peritoneal cavity. Preparing the mouse for procedure, grab the back of the mouse and shave its abdomen By pressing the tail and or the leg of the mouse.We check that the mouse is not sensitive to pain, Then the abdomen is Disinfected. First by scrubbing with Betadine solution, followed by wiping with an isopropyl alcohol wipe For at least one minute. Practicing the laparotomy To practice a laparotomy, we lift the skin in the middle of the abdomen with the forceps and making a small incision of about one centimeter In length.Then Lift the perineum and practice another small incision to expose the peritoneal cavity. Using curved Forceps, hold the peritoneal membrane. Identify and expose the cecum by passing it through the peritoneal membrane.Place it over the abdomen. Now is the moment of the ligation. This is an important factor.In order to determine the severity of the model, we can modulate the severity of the procedure. By the extension of the ligation, mice will develop a milder sepsis. If ligation is made closer to the end of the cecum, a mid-grade sepsis if done in the middle and a severe sepsis if done closer to the ileocecal valve, the severity of the procedure will Be reflected by the percentage of mortality.Never ligate the ileocecal valve. Now make the ligation using silk suture with a double knot to make it tight, making the puncture Hold the cecum and carefully make a puncture close to the ligation site and make another puncture closer to the end of the cecum. The membrane of the secum is soft and it'll be perforated very easily.Using needles with different thicknesses and making one or two perforations, we also can modulate the severity of the model. The the single or double puncture method shown here is an alternate technique to the classical through and through sequel puncture. The advantage of using the single or double puncture method is that one can control better the amount of feces extruded when they seek em is squeezed.Hold the secum with both fingers and gently extrude the cecum to extract a very small amount of feces. Closing the laparotomy, Then lift the peritoneum and carefully place back the cecum. In the peritoneal cavity.Be extremely careful to avoid accidental pricks of the cecum With the forceps, the use of bigger forceps Is recommended. Now is the time To close the peritoneal cavity. Using silk suture, it is very important that you make sure it is very well closed.We start from one side of the wound where we make a knot. Keep making the suture until the other side by passing the silk under the loop of the Previous puncture. To close the skin, we can use Mitchell wound clips or reflex clips using the right tool to close the clips.Place as many clips as needed to make sure the wound is Closed. Resuscitate Mice by injecting subcutaneously, one milliliter of prewarm saline solution. Using a 25 gauge needle, this fluid resuscitation measure will induce the hyperdynamic phase of sepsis, finishing the procedure.Place the mouse under an infrared heating light, or alternatively over a heating pad Until the mouse recovers from the anesthesia. Provide food and water on the floor of the cage and supervise the animals every eight to 12 hours for signs of disease. Inject buprenorphine Subcutaneously for postoperative analgesia.A different analgesic to use is T.Tramadol. Signs of sepsis, symptoms of sepsis are fever, lethargy, pelo, erection, diarrhea, huddling, and malaise. Very sick animals show a severe decrease in body temperature and they barely move to avoid suffering.They should be euthanized at this stage H.So this is how we perform the sickle ligation and punctual model. Hopefully, you have enjoyed the demonstration of this procedure and found it very useful for your research before starting your animal study. We strongly recommend you using old or unused minds for training in order to establish the appropriate degree of severity for your experiments by modifying the length of the ligation, the thickness of the needle, and the number of punctures.With that said, goodbye.

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Cecal Ligation Puncture Procedure

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