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 &mu;l 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 &le;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="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-&#945;: Pleiotrophic cytokine that plays a central role in inflammation and apoptosis. Produced by monocytes, macrophages, neutrophils, dendritic cells and fibroblasts.</li>
 <li>nterleukin-1&#946;: 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 na&#239;ve and memory phenotypes.</li>
 <li>Interferon-&#947;: 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 &le;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="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="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="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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+of+severe+sepsis+in+the+United+States:+analysis+of+incidence,+outcome,+and+associated+costs+of+care.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+enigma+of+sepsis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meta-analysis+of+acute+lung+injury+and+acute+respiratory+distress+syndrome+trials+testing+low+tidal+volumes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+sepsis:+setting+the+stage.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+sepsis+and+sepsis-induced+kidney+injury.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+background+determines+susceptibility+during+murine+septic+peritonitis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+determinants+influencing+the+response+to+injury,+inflammation,+and+sepsis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gender+differences+in+the+inflammatory+response+and+survival+following+haemorrhage+and+subsequent+sepsis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+age+on+mortality+and+antibiotic+efficacy+in+cecal+ligation+and+puncture.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethyl+pyruvate+decreases+sepsis-induced+acute+renal+failure+and+multiple+organ+damage+in+aged+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+exaggerated+inflammatory+response+after+CLP+correlates+with+a+negative+outcome.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AP214,+an+analogue+of+alpha-melanocyte-stimulating+hormone,+ameliorates+sepsis-induced+acute+kidney+injury+and+mortality.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluid+resuscitation+attenuates+early+cytokine+mRNA+expression+after+peritonitis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluid+resuscitation+influences+cardiovascular+performance+and+mortality+in+a+murine+model+of+sepsis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+tramadol+and+buprenorphine+on+select+immunologic+factors+in+a+cecal+ligation+and+puncture+model.">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 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.

<table border="1">
	<tbody>
		<tr>
			<th>Name of the reagent</th>
			<th>Company</th>
			<th>Catalogue number</th>
			<th>Comments</th>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Ketaset</td>
			<td>0856-2013-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>AnaSed</td>
			<td>NADA# 139-236</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Insulin syringe 29G</td>
			<td>Exel</td>
			<td>26028</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Silk suture, 6-0 PROLENE</td>
			<td>Ethicon</td>
			<td>8680G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>19G and 25G Needle</td>
			<td>BD</td>
			<td>305186</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Shaver</td>
			<td>General supply</td>
			<td>General supply</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Infrared Heating lamp</td>
			<td>General supply</td>
			<td>General supply</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Michel wound clips 7mm</td>
			<td>Roboz Surgical Instr</td>
			<td>RS-9270</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ear Loop Mask</td>
			<td>Fisher Scientific</td>
			<td>19-130-4181</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dissection scissors</td>
			<td>Roboz Surgical Instr</td>
			<td>RS-6702</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Betadine solution</td>
			<td>VWR</td>
			<td>63410-992</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Surgical forceps</td>
			<td>Roboz Surgical Instr</td>
			<td>RS-5135</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>70% Isopropyl alcohol pad</td>
			<td>Fisher Scientific</td>
			<td>22-031-350</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Bedford Labs</td>
			<td>55390-100-10</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tramadol</td>
			<td>Sigma-Aldrich</td>
			<td>42965</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

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.

Watch this Scientific Journal Video about Cecal Ligation Puncture Procedure at JoVE.com

Cecal Ligation Puncture Procedure

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 ...

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54.5K Views.  Temple University. The mouse model of cecal ligation and puncture as a valuable tool for the study of human sepsis.

Video: Cecal Ligation Puncture Procedure

Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo. With the advent of genetic modifications to the whole organism or the myocardium and an array of biological and/or synthetic materials, there is great potential for any combination of these to assuage the extent of acute ischemic injury and impede the onset of heart failure pursuant to myocardial remodeling. 
Here we present the methods and materials used to reliably perform this microsurgery and the modifications involved for temporary (with reperfusion) or permanent coronary artery occlusion studies as well as intramyocardial injections. The effects on the heart that can be seen during the procedure and at the termination of the experiment in addition to histological evaluation will verify efficacy. 
Briefly, surgical preparation involves anesthetizing the mice, removing the fur on the chest, and then disinfecting the surgical area. Intratracheal intubation is achieved by transesophageal illumination using a fiber optic light. The tubing is then connected to a ventilator. An incision made on the chest exposes the pectoral muscles which will be cut to view the ribs. For ischemia/reperfusion studies, a 1 cm piece of PE tubing placed over the heart is used to tie the ligature to so that occlusion/reperfusion can be customized. For intramyocardial injections, a Hamilton syringe with sterile 30gauge beveled needle is used. When the myocardial manipulations are complete, the rib cage, the pectoral muscles, and the skin are closed sequentially. Line block analgesia is effected by 0.25% marcaine in sterile saline which is applied to muscle layer prior to closure of the skin. The mice are given a subcutaneous injection of saline and placed in a warming chamber until they are sternally recumbent. They are then returned to the vivarium and housed under standard conditions until the time of tissue collection. At the time of sacrifice, the mice are anesthetized, the heart is arrested in diastole with KCl or BDM, rinsed with saline, and immersed in fixative. Subsequently, routine procedures for processing, embedding, sectioning, and histological staining are performed. 
Nonsurgical intubation of a mouse and the microsurgical manipulations described make this a technically challenging model to learn and achieve reproducibility. These procedures, combined with the difficulty in performing consistent manipulations of the ligature for timed occlusion(s) and reperfusion or intramyocardial injections, can also affect the survival rate so optimization and consistency are critical.

Numerous genetic manipulations and/or intramyocardial injections of genes, proteins, cells, and/or biomaterials are superimposed upon the dimension of time in studies of acute ischemia/ reperfusion injury and chronic remodeling in mice. This video illustrates the microsurgical procedures for ischemia/reperfusion, permanent coronary artery ligation, and intramyocardial injection studies.

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo.  With the advent of genetic modifications to ...

The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to develop suitable animal models to study the events that unfold during ischemic insult. These techniques include transient or permanent, focal or global ischemia models using many different animal models, with the most common being rodents.
The permanent MCA ligation method which is also referred as pMCAo in the literature is used extensively as a focal ischemia model in rodents 1-6. This method was originally described for rats by Tamura et al. in 1981 7. In this protocol a craniotomy was used to access the MCA and the proximal regions were occluded by electrocoagulation. The infarcts involve mostly cortical and sometimes striatal regions depending on the location of the occlusion. This technique is now well established and used in many laboratories 8-13. Early use of this technique led to the definition and description of &#x201C;infarct core&#x201D; and &#x201C;penumbra&#x201D; 14-16, and it is often used to evaluate potential neuroprotective compounds 10, 12, 13, 17. Although the initial studies were performed in rats, permanent MCA ligation has been used successfully in mice with slight modifications 18-20 .
This model yields reproducible infarcts and increased post-survival rates. Approximately 80% of the ischemic strokes in humans happen in the MCA area 21 and thus this is a very relevant model for stroke studies. Currently, there is a paucity of effective treatments available to stroke patients, and thus there is a need for good models to test potential pharmacological compounds and evaluate physiological outcomes. This method can also be used for studying intracellular hypoxia response mechanisms in vivo. 
Here, we present the MCA ligation surgery in a C57/BL6 mouse. We describe the pre-surgical preparation, MCA ligation surgery and 2,3,5 Triphenyltetrazolium chloride (TTC) staining for quantification of infarct volumes.

Middle cerebral artery (MCA) ligation is a technique to study focal cerebral ischemia in animal models. In this method, the middle cerebral artery is exposed by craniotomy and ligated by cauterization. This method gives highly reproducible infarct volumes and increased post-operative survival rates compared to other methods available.

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to ...

Modified Technique for Coronary Artery Ligation in Mice

Myocardial infarction (MI) is one of the most important causes of mortality in humans1-3. In order to improve morbidity and mortality in patients with MI we need better knowledge about pathophysiology of myocardial ischemia. This knowledge may be valuable to define new therapeutic targets for innovative cardiovascular therapies4. Experimental MI model in mice is an increasingly popular small-animal model in preclinical research in which MI is induced by means of permanent or temporary ligation of left coronary artery (LCA)5. In this video, we describe the step-by-step method of how to induce experimental MI in mice.
The animal is first anesthetized with 2% isoflurane. The unconscious mouse is then intubated and connected to a ventilator for artificial ventilation. The left chest is shaved and 1.5 cm incision along mid-axillary line is made in the skin. The left pectoralis major muscle is bluntly dissociated until the ribs are exposed. The muscle layers are pulled aside and fixed with an eyelid-retractor. After these preparations, left thoracotomy is performed between the third and fourth ribs in order to visualize the anterior surface of the heart and left lung. The proximal segment of LCA artery is then ligated with a 7-0 ethilon suture which typically induces an infarct size ~40% of left ventricle. At the end, the chest is closed and the animals receive postoperative analgesia (Temgesic, 0.3 mg/50 ml, ip). The animals are kept in a warm cage until spontaneous recovery.

The surgical procedure used to induce experimental myocardial infarction in mice begins with left thoracotomy between the third and the fourth ribs in order to visualize the anterior surface of the heart and left lung. The left coronary artery is ligated, the chest is closed and the mouse is allowed to recover spontaneously.

Myocardial infarction (MI) is one of the most important causes of mortality in humans1-3. In order to improve morbidity and mortality in patients with MI ...

Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well as to excrete bilirubin and other metabolic products. In the liver, the experimental obstruction of the extrahepatic biliary system initiates a complex cascade of pathological events that leads to cholestasis and inflammation resulting in a strong fibrotic reaction originating from the periportal fields. Therefore, surgical ligation of the common bile duct has become the most commonly used model to induce obstructive cholestatic injury in rodents and to study the molecular and cellular events that underlie these pathophysiological mechanisms induced by inappropriate bile flow. In recent years, different surgical techniques have been described that either allow reconnection or reanastomosis after bile duct ligation (BDL), e.g., partial BDL, or other microsurgical methods for specific research questions. However, the most frequently used model is the complete obstruction of the common bile duct that induces a strong fibrotic response after 21 to 28 days. The mortality rate can be high due to infectious complications or technical inaccuracies. Here we provide a detailed surgical procedure for the BDL model in mice that induce a highly reproducible fibrotic response in accordance to the 3R rule for animal welfare postulated by Russel and Burch in 1959.

Disruption of bile flow results in severe inflammatory cholestatic liver injury with a characteristic time-dependent sequence of morphological alterations. Here we present a protocol for the surgical ligation of the common bile duct in mice that allows to induce a strong fibrotic response after 21 to 28 days.

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well ...

Rat Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy (ALPPS) Procedure

Recent clinical data support an aggressive surgical approach to both primary and metastatic liver tumors. For some indications, like colorectal liver metastases, the amount of liver tissue left behind after liver resection has become the main limiting factor of resectability of large or multiple liver tumors. A minimal amount of functional tissue is required to avoid the severe complication of post-hepatectomy liver failure, which has high morbidity and mortality. Inducing liver growth of the prospective remnant prior to resection has become more established in liver surgery, either in the form of portal vein embolization by interventional radiologists or in the form of portal vein ligation several weeks prior to resection. Recently, it was shown that liver regeneration is more extensive and rapid, when the parenchymal transection is added to portal vein ligation in a first stage and then, after only one week of waiting, resection performed in a second stage (Associating Liver Partition and Portal vein ligation for Staged hepatectomy = ALPPS). ALPPS has rapidly become popular across the world, but has been criticized for its high perioperative mortality. The mechanism of accelerated and extensive growth induced by this procedure has not been well understood. Animal models have been developed to explore both the physiological and molecular mechanisms of accelerated liver regeneration in ALPPS. This protocol presents a rat model that allows mechanistic exploration of accelerated regeneration.

Rat Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy ALPPS Procedure

Inducing rapid liver hypertrophy using Associating Liver Partition and Portal vein ligation for a Staged hepatectomy (ALPPS) has been proposed for resection of borderline resectable liver tumors. This model may elucidate mechanisms involved in rapid hypertrophy and allows testing of drugs that promote or block the acceleration of regeneration.

Recent clinical data support an aggressive surgical approach to both primary and metastatic liver tumors. For some indications, like colorectal liver ...

Puncture-Induced Iris Neovascularization as a Mouse Model of Rubeosis Iridis

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant for targeting neovascular glaucoma, a sight-threatening complication of diabetic retinopathy. This method is based on the induction of iris vascular response by a series of self-sealing uveal punctures on BALB/c mice and takes advantage of the postpartum maturation of mouse ocular vasculature. Mouse pups undergo uveal punctures from postnatal day 12.5, when the pups naturally open their eyes, until postnatal day 24.5. Due to the transparency of the cornea, iris vasculature can be analyzed easily through time by noninvasive in vivo methods. Furthermore, the semitransparent iris of BALB/c mice can be flatmounted for detailed immunohistologic analysis with minimal non-specific background staining. In this model, angiogenesis is mainly driven by the inflammatory and plasminogen activating systems. The puncture-induced model is the first to induce iris neovascularization in small rodents, and has the advantage of allowing direct noninvasive in vivo analysis of the angiogenic process. Moreover, the model can be combined with angiogenic modulating substances, which highlights its potential in the study of angiogenesis with an in vivo perspective.

Iris neovascularization, a common complication of ischemic retinal disease, may lead to sight-threatening neovascular glaucoma. Here, we describe a murine protocol for inducing experimental iris neovascularization that may be used for noninvasive evaluation of angiogenesis-modulating substances.

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant ...

Subclavian Vein Puncture As an Alternative Method of Blood Sample Collection in Rats

Blood collection with enough blood volume is essential in animal experiments. Blood collection from the tail vein of rats is popular and less stressful compared to other more aggressive methods such as retro-orbital plexus sample collection. However, this blood collection method is sometimes limited by an unsatisfactory success rate. Here, we introduce a method for blood collection through the subclavian vein puncture. The subclavian vein is located just under the clavicle and this vein is large enough to fulfill the volume requirements of blood collection. Our results show that this method is safe and applicable for blood collection sampling with the required blood volume. Blood collection through the subclavian vein puncture could serve as an alternative blood collection method in case of failed tail vein blood sampling in rats.

Here, we present a protocol to collect blood samples from the rat subclavian vein.

Blood collection with enough blood volume is essential in animal experiments. Blood collection from the tail vein of rats is popular and less stressful ...

Blood Collection Through Subclavian Vein Puncture in Mice

The mouse is the foremost mammalian model for studying human disease and human health. However, blood sample collection from mice is challenging in research work. Tail blood collection is a popular method when a small amount of blood sample is needed. Orbital artery could be considered if a large amount of blood is needed but this blood collection method has ethical issues. Formerly, we demonstrated the feasibility and safety of blood sample collection through subclavian vein puncture in rats, and here we investigate whether this method could be used in mice. We report that this method is safe and practical for blood collection in mice. Blood collection through the subclavian vein puncture in mice can be a convenient method in daily research works.

Here, we present a protocol to take blood samples from the subclavian vein of mice.

The mouse is the foremost mammalian model for studying human disease and human health. However, blood sample collection from mice is challenging in ...

Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine models of MI with permanent ligation of left-anterior descending (LAD) coronary artery closely mimics MI in humans. Murine models benefit from the extensive genetic engineering available nowadays. Here we propose a reproducible murine surgical model of myocardial infarction by permanent LAD coronary ligation. Our technique comprises anesthesia with ketamine/xylazine that can be rapidly reversed by administration of an antagonist, intubation without tracheotomy for mechanical-assisted ventilation, ventilation with application of extrinsic positive end-expiratory pressure (PEEP) to avoid alveolar collapse, a thoracotomy method limiting to the minimum surgical lesions made to skeletal muscles, and lung inflation without thoracentesis. This method is sparsely invasive, reproducible and reduces post-surgery mortality and complications.

Herein we describe a surgical procedure showing how to achieve permanent ligation of the left-anterior descending coronary artery in mice. This model is of high relevance to investigate the pathophysiology of myocardial infarction and the concomitant biological processes.

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine ...

An Image Guided Transapical Mitral Valve Leaflet Puncture Model of Controlled Volume Overload from Mitral Regurgitation in the Rat

Mitral regurgitation (MR) is a widely prevalent heart valve lesion, which causes cardiac remodeling and leads to congestive heart failure. Though the risks of uncorrected MR and its poor prognosis are known, the longitudinal changes in cardiac function, structure and remodeling are incompletely understood. This knowledge gap has limited our understanding of the optimal timing for MR correction, and the benefit that early versus late MR correction may have on the left ventricle. To investigate the molecular mechanisms that underlie left ventricular remodeling in the setting of MR, animal models are necessary. Traditionally, the aorto-caval fistula model has been used to induce volume overload, which differs from clinically relevant lesions such as MR. MR represents a low-pressure volume overload hemodynamic stressor, which requires animal models that mimic this condition. Herein, we describe a rodent model of severe MR in which the anterior leaflet of the rat mitral valve is perforated with a 23G needle, in a beating heart, with echocardiographic image guidance. The severity of MR is assessed and confirmed with echocardiography, and the reproducibility of the model is reported.&#160;&#160;

A rodent model of left heart volume overload from mitral regurgitation is reported. Mitral regurgitation of controlled severity is induced by advancing a needle of defined dimensions into the anterior leaflet of the mitral valve, in a beating heart, with ultrasound guidance.&#160;

Mitral regurgitation (MR) is a widely prevalent heart valve lesion, which causes cardiac remodeling and leads to congestive heart failure. Though the ...