This work was supported by National Institutes of Health Grants R01 AI138203-01, AI109317-04, AI101973-01, and AI097532-01A1 to M. W. The University of North Dakota Core Facilities were supported by NIH grants (INBRE P20GM103442 and COBRE P20GM113123). This work was also supported by the Key Program of National Nature Science Foundation of China (81530063) to Jianxin Jiang. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank Marvin Leier (Center for Rural Health, University of North Dakota) for making the video.

All methods described here were performed in accordance with the National Institute of Health Guide for Care and Use of Laboratory Animals and approved by the University of North Dakota Institutional Animal Care and Use Committee (IACUC).
1. Cecal ligation and puncture
NOTE: Female C57BL/6 mice (weight, 18-22 g; age, 6-8 weeks) are randomly divided into six groups: control group (Ctrl), infection group (SA for S. aureus), two sham groups, and two CLP groups. Ctrl animals are left without surgery and secondary infection injuries. SA animals are subjected to S. aureus lung infection without the operation. Sham-operated animals undergo the same laparotomy with an exposition of the cecum (except the cecum is neither ligated nor punctured). Eight mice per group are used for survival analysis, and three to five mice are used for assessment of inflammation at various timepoints.
<ol>
	<li>Prior to surgery, sterilize all surgical instruments and materials by autoclaving for 20 min. Clean and disinfect the operating table with disinfectant wipes. To establish sterile conditions during the surgery, cover the entire operative fields with proper sterile surgical drapes, and wear a hair cover, surgical mask, protective eyewear, surgical gown, and sterile gloves.</li>
	<li>Weigh and anesthetize the mouse using 0.1 mL/mouse of ketamine (80 mg/kg), xylazine (10 mg/kg) mixture intraperitoneally administered. With the thumb and index finger of the left hand forming a U-shaped position, approach the neck of the mouse from behind smoothly and gently, then hold the neck immediately behind the ears so that the mouse head is highly restrained. Use a pinky to hold the mouse&#39;s right back leg and use the middle and ring fingers (all fingers from left hand) to hold the body.</li>
	<li>Check the intensity of anesthesia by toe pinch until no flexion of the extremity.</li>
	<li>Shave the lower quadrants of the abdomen using an electric razor and disinfect the area with iodine.</li>
	<li>Place the mouse onto the sterile surface in a supine position, with the head oriented away from the operator. Drape the mouse with sterile towel with hole over the planned surgical incision to maintain sterile fields.</li>
	<li>Make a longitudinal skin incision (about 0.5 cm long) with a scalpel in the left lower abdomen. Use small scissors to extend the incision (1-2 cm). 
	&#8203;CAUTION: Be careful not to penetrate the peritoneal cavity.</li>
	<li>Make the intramuscular incision to gain access into the peritoneal cavity and allow exposure of the cecum with the adjoining intestine.</li>
	<li>Isolate the cecum on the left side of the abdomen (in most cases, this is done by using blunt anatomical forceps) and remove it on a sterile drape, leaving the small and large intestines in the peritoneal cavity. 
	CAUTION: Avoid damaging the mesenteric blood vessels.</li>
	<li>Ligate the cecum at the distal 25% position to create a prolonged infection with relatively low mortality. 
	CAUTION: Make sure not to ligate the ileocecal valve.</li>
	<li>Perforate the cecum with a 21 G 1 &#189; needle by single through-and-through puncture (two holes) midway between the ligation and tip of the cecum at the least-vascularized area. 
	CAUTION: Make sure not to puncture blood vessels.</li>
	<li>Remove the needle. Gently extrude a small amount of feces from the penetration holes to ensure full thickness perforation. 
	CAUTION: Take care not to obstruct the bowel continuity during the procedure.</li>
	<li>Replace the cecum into the abdominal cavity.</li>
	<li>Close the abdomen in two layers with 4-0 nylon surgical sutures. Close the abdominal musculature by applying simple running sutures and close the skin by applying simple interrupted sutures. Clean the skin with Iodine.</li>
	<li>Inject 1 mL of prewarmed (37 &#176;C) 0.9% sterile normal saline (NS) solution on the back subcutaneously to replace third space loss and place it on a warm pad to recover.</li>
	<li>Return the mice in cage in a temperature-controlled room (22 &#176;C) with 12 h light/dark cycles and free access to food and water. Monitor the mice every 0.5 hours for at least 2 h, then every 6 h for at least 2 d, then every 12 h for 3 days, or euthanize them when moribund for survival analysis. 
	NOTE: Pre- and post-operative analgesia (buprenorphine, 50 &#181;g/kg subcutaneously every 12 h) are recommended for 24 h before surgery until 48 h after surgery9,10.</li>
</ol>
2. Secondary lung infection with S. aureus
NOTE: Except for the Ctrl and SA groups, surviving mice at 3 days post-CLP in the sham and CLP groups should be administered intranasally with 30 &#181;L of a bacterial suspension or NS, respectively. The surviving mice in the SA group should be instilled intranasally with the bacterial suspension.
<ol>
	<li>Anesthetize the mouse by intraperitoneally injecting 100 &#181;L solution of ketamine (45mg/kg) and xylazine (10 mg/kg) and wait until the mouse breaths slowly. The puncturing angle between the needle and abdominal wall is less than 30&#176; for slow injection.</li>
	<li>With the help of a micropipette, slowly and intranasally instill with 30 &#181;L of 1 x 107 colony forming unit (CFU) of S. aureus to be aspirated on inhalation. Holding the mouse by its ears in an upright position, slowly drop 2-3 &#181;L of bacterial suspension into both nostrils each time. Adjust the rate of release to allow the mouse to inhale without forming bubbles and dropping off.</li>
	<li>Raise the mouse up and down, with its head up and tail down, to help the bacteria enter the trachea. Move the mouse up quickly and strongly, while moving it down gently and slowly to increase the gravity. Repeat instillation about 15x until all bacteria are inhaled, which should take 10-15 min for each mouse.</li>
	<li>Lay the mouse on its back and head up on the angled bedding (about 35&#176;) and watch the mouse until its breathing returns to normal and it is completely recovered from the anesthesia and procedures.</li>
	<li>Place the mouse back in the cage in a temperature-controlled room (22 &#176;C) with 12 h light/dark cycles and unlimited access to food and water.</li>
	<li>Check mouse conditions every 2 h for the first day and every 12 h afterwards for 4 days after the surgery. Euthanize them when moribund for survival analysis or 24 h after infection for assessment of inflammation.</li>
</ol>
3. Analyzed parameters
<ol>
	<li>Mice survival
	<ol>
		<li>Monitor the mice for 7 days. Euthanize when moribund and generate survival curves using Kaplan-Meier methods11.</li>
	</ol>
	</li>
	<li>Bacterial counts
	<ol>
		<li>24 h after SA injection, harvest the blood, bronchoalveolar lavage fluid (BALF), and peritoneal lavage fluid (PLF) from the mice. Subsequently, add 100 &#181;L of the diluents to nutrient agar plates and culture them at 37 &#176;C for 24 h to determine the bacterial colony counts11,12,13.</li>
	</ol>
	</li>
	<li>Cytokines 
	&#8203;NOTE: Pro-inflammatory cytokine concentrations of IL-1&#946;, IL-6, and TNF&#945; in serum and BALF from mice should be assessed using ELISA kits according to the manufacturer&#39;s instructions.
	<ol>
		<li>Coat the 96 well ELISA plate with 100 &#181;L/well of different capture antibody working solution to run the standards in duplicate and samples in triplicate. Seal the plate and leave it overnight at 4 &#176;C.</li>
		<li>Wash the plate three times with 255 &#181;L/well wash buffer (1x PBS, 0.05% Tween-20).</li>
		<li>To block nonspecific binding, add 200 &#181;L/well diluent. Seal the plate and incubate at room temperature (RT) for 1 h.</li>
		<li>Prepare standard solution: Eight two-fold serial dilutions from 1000 pg/mL.</li>
		<li>Add 100 &#181;L/well of standards or samples to the appropriate wells and 100 &#181;L/well of diluent to the blank wells. Seal the plate and incubate at RT for 2 h or overnight at 4 &#176;C.</li>
		<li>Wash the plate 5x with 255 &#181;L/well wash buffer.</li>
		<li>Add 100 &#181;L/well of different detection antibody working solution to the appropriate wells. Seal the plate and incubate at RT for 1 h.</li>
		<li>Wash the plate 5x with 255 &#181;L/well wash buffer.</li>
		<li>Add 100 &#181;L/well of diluted HRP solution to all wells. Seal the plate and incubate at RT for 30 min.</li>
		<li>Wash the plate 5x with 255 &#181;L/well wash buffer.</li>
		<li>Add 100 &#181;L/well diluted TMB solution. Incubate the plate away from light at RT for 15 min.</li>
		<li>Add 50 &#181;L/well stop solution (1 M H3PO4). Read absorbance at 450 nm with a plate reader.</li>
	</ol>
	</li>
	<li>Flow cytometry
	<ol>
		<li>Obtain the total number of cells from BALF. Lyse the erythrocytes using ACK lysing buffer and wash 2x using PBS.</li>
		<li>Analyze then the single-cell suspensions by flow cytometry as previously described11. For surface staining, stain neutrophils (CD11b+GR-1+) with APC anti-mouse CD11b and anti-mouse Gr-1 (Ly-6G/Ly-6C) antibodies. Collect a minimum of 3 x 104 live, non-debris cells for analysis.</li>
	</ol>
	</li>
</ol>

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	<li>Singer, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Third+International+Consensus+Definitions+for+Sepsis+and+Septic+Shock+(Sepsis-3).">The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3).</a> JAMA. 315 (8), 801-810 (2016).</li><li>Delano, M. J., 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+immune+system's+role+in+sepsis+progression,+resolution,+and+long-term+outcome.">The immune system's role in sepsis progression, resolution, and long-term outcome.</a> Immunological Reviews. 274 (1), 330-353 (2016).</li><li>Mayr, F. B., Yende, S., 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.">Epidemiology of severe sepsis.</a> Virulence. 5 (1), 4-11 (2014).</li><li>Fleischmann, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+Global+Incidence+and+Mortality+of+Hospital-treated+Sepsis.+Current+Estimates+and+Limitations.">Assessment of Global Incidence and Mortality of Hospital-treated Sepsis. Current Estimates and Limitations.</a> American Journal of Respiratory and Critical Care Medicine. 193 (3), 259-272 (2016).</li><li>Reinhart, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognizing+Sepsis+as+a+Global+Health+Priority+-+A+WHO+Resolution.">Recognizing Sepsis as a Global Health Priority - A WHO Resolution.</a> The New England Journal of Medicine. 377 (5), 414-417 (2017).</li><li>Boomer, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunosuppression+in+patients+who+die+of+sepsis+and+multiple+organ+failure.">Immunosuppression in patients who die of sepsis and multiple organ failure.</a> JAMA. 306 (23), 2594-2605 (2011).</li><li>Hotchkiss, R. S., Monneret, G., Payen, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sepsis-induced+immunosuppression:+from+cellular+dysfunctions+to+immunotherapy.">Sepsis-induced immunosuppression: from cellular dysfunctions to immunotherapy.</a> Nature Reviews Immunology. 13 (12), 862-874 (2013).</li><li>Dejager, L., Pinheiro, I., Dejonckheere, E., Libert, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cecal+ligation+and+puncture:+the+gold+standard+model+for+polymicrobial+sepsis?.">Cecal ligation and puncture: the gold standard model for polymicrobial sepsis?.</a> Trends in Microbiology. 19 (4), 198-208 (2011).</li><li>Rittirsch, D., Huber-Lang, M. S., Flierl, M. A., 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=Immunodesign+of+experimental+sepsis+by+cecal+ligation+and+puncture.">Immunodesign of experimental sepsis by cecal ligation and puncture.</a> Nature Protocols. 4 (1), 31-36 (2009).</li><li>Hugunin, K. M. S., Fry, C., Shuster, K., Nemzek, J. 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Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atg7+Deficiency+Intensifies+Inflammasome+Activation+and+Pyroptosis+in+Pseudomonas+Sepsis.">Atg7 Deficiency Intensifies Inflammasome Activation and Pyroptosis in Pseudomonas Sepsis.</a> Journal of Immunology. 198 (8), 3205-3213 (2017).</li><li>Zanotti-Cavazzoni, S. 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The authors have no financial conflicts of interest.

As the gold standard model for sepsis research, CLP has a combination of three insults, including tissue trauma caused by the laparotomy, necrosis due to ligation of the cecum, and infection as a result of microbial leakage that causes peritonitis with translocation of bacteria into blood8. Therefore, CLP mimics the complexity of human sepsis better than many other models. However, a major limitation of current CLP model is the inability to reflect the more prolonged phase of sepsis seen in patien...

Sepsis initiates a complex interplay of host pro-inflammatory and anti-inflammatory processes and is characterized by a hyperinflammatory response and subsequent immune dysfunction1,2. Sepsis represents a global health priority and causes a high number of deaths in intensive care units (ICUs)3. The incidence of sepsis is estimated to exceed 30 million cases worldwide per year, with mortality rates as high as 30% despite advances in ICU management4,5. In 2017, the World Health Organization adopted a resolution to improve the prevention, diagnosis, and management of this deadly disease5. However, recent studies have illustrated that death does not result from primary infection in severe septic patients but rather from secondary nosocomial infection (particularly pneumonia) that caused by immunosuppression6,7. Therefore, understanding the mechanisms of why septic patients develop secondary infection and discovering more effective treatments are urgently required. Herein, a dual model, also known as a double-hit model, to study the immunosuppressive phenomenon occurring in patients with protracted sepsis is described. 
As the gold standard experimental model in research on polymicrobial sepsis, cecal ligation and puncture (CLP) is a surgery characterized by cecum ligation and perforation, which contributes to polymicrobial peritonitis and sepsis8,9. The pathophysiological process and cytokine profiles, along with the kinetics and magnitude, are similar to clinical sepsis. The position of the ligation, needle size used for the puncture, and number of cecal punctures are major factors that impact the mortality following CLP.
The nosocomial pneumonia is the leading cause of mortality among critically ill patients with sepsis. The major type of organisms causing severe sepsis includes Staphylococcus aureus (20.5%), Pseudomonas species (19.9%), Enterobacteriacae (mainly E. coli, 16.0%), and fungi (19%). Meanwhile, recent studies have suggested an increasing incidence of gram-positive organisms, which are now almost as common as gram-negative infections3.
The method described in this protocol involves CLP, performed as the "first hit" to induce sublethal polymicrobial peritonitis, which manifests an immunosuppression condition. The procedure also involves subsequent intranasal instillation of S. aureus as the "second hit" to provide a clinically relevant research platform.

<table><tbody><tr><td>21 G 1 &amp;frac12; Needle</td><td>BD</td><td>BD305167</td><td></td></tr><tr><td>ACK lysing buffer</td><td>Gibco</td><td>A10492-01</td><td></td></tr><tr><td>Anti-mouse CD11b antibody</td><td>Biolegend</td><td>101201</td><td></td></tr><tr><td>Anti-mouse Ly-6G/Ly-6C (Gr-1) antibody</td><td>Biolegend</td><td>108401</td><td></td></tr><tr><td>C57BL/6 mice&amp;nbsp;</td><td>Harlan (Indianapolis)</td><td>C57BL/6NHsd</td><td></td></tr><tr><td>Desk light</td><td>General Supply</td><td>General Supply</td><td></td></tr><tr><td>Disinfecting wipes</td><td>Clorox</td><td>B07NV5JMCS</td><td></td></tr><tr><td>Electric razor</td><td>General Supply</td><td>General Supply</td><td></td></tr><tr><td>ELISA kits (mouse IL-1&amp;beta;, IL-6 and TNF&amp;alpha;)</td><td>Invitrogen</td><td>88-7013, 88-7064, and 88-7324</td><td></td></tr><tr><td>Iodine</td><td>Dynarex</td><td>B003U463PY</td><td>PVP Iodine Wipes</td></tr><tr><td>Ketamine</td><td>FORT DODGE</td><td>NDC 0856-2013-01</td><td>Amine hydrochloride injection</td></tr><tr><td>Laboratory scale</td><td>General Supply</td><td>General Supply</td><td></td></tr><tr><td>LB Agar, Miller</td><td>Fisher Scientific</td><td>BP1425-500</td><td>Molecular genetics, powder</td></tr><tr><td>Micropipette</td><td>ErgoOne</td><td>7100-1100</td><td></td></tr><tr><td>Normal saline</td><td>General Supply</td><td>General Supply</td><td></td></tr><tr><td>Polylined towel</td><td>CardinalHealth, Convertors</td><td>3520</td><td>Surgical drape, sterile, for single use only</td></tr><tr><td>Silk suture, 4-0</td><td>DAVIS &amp;amp; GECK</td><td>1123-31</td><td></td></tr><tr><td>Small animal needle holder</td><td>General Supply</td><td>General Supply</td><td></td></tr><tr><td>Small animal surgery scissors</td><td>General Supply</td><td>General Supply</td><td></td></tr><tr><td>Small animal surgical forceps</td><td>General Supply</td><td>General Supply</td><td></td></tr><tr><td>&lt;em&gt;Staphylococcus aureus&lt;/em&gt;</td><td>ATCC</td><td>13301</td><td></td></tr><tr><td>Warm pad</td><td>General Supply</td><td>General Supply</td><td></td></tr><tr><td>Xylazine</td><td>Alfa Aesar</td><td>7361-61-7</td><td></td></tr></tbody></table>

design of cecal ligation and puncture and intranasal infection dual model of sepsis-induced immunosuppression

Sepsis, a severe and complicated life-threatening infection, is characterized by an imbalance between pro- and anti-inflammatory responses in multiple organs. With the development of therapies, most patients survive the hyperinflammatory phase but progress to an immunosuppressive phase, which increases the emergence of secondary infections. Therefore, improved understanding of the pathogenesis underlying secondary hospital-acquired infections in the immunosuppressive phase during sepsis is of tremendous importance. Reported here is a model to test infectious outcomes by creating double-hit infections in mice. A standard surgical procedure is used to induce polymicrobial peritonitis by cecal ligation and puncture (CLP) and followed by intranasal infection of Staphylococcus aureus to simulate pneumonia occurring in immune suppression that is frequently seen in septic patients. This dual model can reflect the immunosuppressive state occurring in patients with protracted sepsis and susceptibility to secondary infection from nosocomial pneumonia. Hence, this model provides a simple experimental approach to investigate the pathophysiology of sepsis-induced secondary bacterial pneumonia, which may be used for discovering novel treatments for sepsis and its complications.

Depending on the experimental design and procedures, C57BL/6 mice were subjected to CLP, and after 3 days, they were administered bacteria intranasally (Figure 1). As shown in Figure 2, the mice began to die at ~12 h after induction of peritonitis. Two mice in the CLP+SA group and three mice in the CLP+NS group died before intranasal S. aureus instillation. No mortality was detected in uninfected non- or sham-operated mi...

Watch this Scientific Journal Video about Design of Cecal Ligation and Puncture and Intranasal Infection Dual Model of Sepsis-Induced Immunosuppression at JoVE.com

Design of Cecal Ligation and Puncture and Intranasal Infection Dual Model of Sepsis-Induced Immunosuppression

This protocol describes techniques to measure infectious outcomes underlying secondary hospital-acquired infections in the immunosuppressive condition, first by establishing cecal ligation/puncture mice then challenging them with intranasal infection to create a clinically relevant model of immunosuppression sepsis.

Sepsis, a severe and complicated life-threatening infection, is characterized by an imbalance between pro- and anti-inflammatory responses in multiple ...

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Immunology and Infection

9.8K Views.  Sichuan University. This protocol describes techniques to measure infectious outcomes underlying secondary hospital-acquired infections in the immunosuppressive condition, first by establishing cecal ligation/puncture mice then challenging them with intranasal infection to create a clinically relevant model of immunosuppression sepsis. 

Video: Design of Cecal Ligation and Puncture and Intranasal Infection Dual Model of Sepsis-Induced Immunosuppression

Colon Ascendens Stent Peritonitis (CASP) - a Standardized Model for Polymicrobial Abdominal Sepsis

Sepsis remains a persistent problem on intensive care units all over the world. Understanding the complex mechanisms of sepsis is the precondition for establishing new therapeutic approaches in this field. Therefore, animal models are required that are able to closely mimic the human disease and also sufficiently deal with scientific questions. The Colon Ascendens Stent Peritonitis (CASP) is a highly standardized model for polymicrobial abdominal sepsis in rodents. In this model, a small stent is surgically inserted into the ascending colon of mice or rats leading to a continuous leakage of intestinal bacteria into the peritoneal cavity. The procedure results in peritonitis, systemic bacteraemia, organ infection by gut bacteria, and systemic but also local release of several pro- and anti-inflammatory cytokines. The lethality of CASP can be controlled by the diameter of the inserted stent. A variant of this model, the so-called CASP with intervention (CASPI), raises opportunity to remove the septic focus by a second operation according to common procedures in clinical practice. CASP is an easily learnable and highly reproducible model that closely mimics the clinical course of abdominal sepsis. It leads way to study on questions in several scientific fields e.g. immunology, infectiology, or surgery.

Colon Ascendens Stent Peritonitis CASP - a Standardized Model for Polymicrobial Abdominal Sepsis

The Colon Ascendens Stent Peritonitis (CASP) is a highly standardized model for polymicrobial abdominal sepsis in rodents. This article describes the surgical procedure of CASP. The CASP model and its variants allow the systematic investigation of various problems concerning the subject of sepsis.

Sepsis remains a persistent problem on intensive care units all over the world. Understanding the complex mechanisms of sepsis is the precondition for ...

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 ...

Medicine

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several techniques, including infusion of exogenous bacterial toxin (endotoxemia) or bacteria (bacteremia), as well as surgical perforation of the cecum by cecal ligation and puncture (CLP)1-3. CLP allows bacteria spillage and fecal contamination of the peritoneal cavity, mimicking the human clinical disease of perforated appendicitis or diverticulitis. The severity of sepsis, as reflected by the eventual mortality rates, can be controlled surgically by varying the size of the needle used for cecal puncture2. In animals, CLP induces similar, biphasic hemodynamic cardiovascular, metabolic, and immunological responses as observed during the clinical course of human sepsis3. Thus, the CLP model is considered as one of the most clinically relevant models for experimental sepsis1-3.
Various animal models have been used to elucidate the intricate mechanisms underlying the pathogenesis of experimental sepsis. The lethal consequence of sepsis is attributable partly to an excessive accumulation of early cytokines (such as TNF, IL-1 and IFN-&gamma;)4-6 and late proinflammatory mediators (e.g., HMGB1)7. Compared with early proinflammatory cytokines, late-acting mediators have a wider therapeutic window for clinical applications. For instance, delayed administration of HMGB1-neutralizing antibodies beginning 24 hours after CLP, still rescued mice from lethality8,9, establishing HMGB1 as a late mediator of lethal sepsis. The discovery of HMGB1 as a late-acting mediator has initiated a new field of investigation for the development of sepsis therapies using Traditional Chinese Herbal Medicine. In this paper, we describe a procedure of CLP-induced sepsis, and its usage in screening herbal medicine for HMGB1-targeting therapies.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection, and can be simulated by a surgical technique termed cecal ligation and puncture (CLP). Here we describe a method to use CLP-induced animal model to screen medicinal herbs for therapeutic agents.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several ...

Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is provided to induce sepsis of varying severity in mice using the CLP technique. Autophagy is a fundamental tissue response to stress and pathogen invasion. Two current protocols to assess autophagy in vivo in the context of experimental sepsis are also presented here. (I) Transgenic mice expressing green fluorescence protein (GFP)-LC3 fusion protein are subjected to CLP. Localized enhancement of GFP signal (puncta), as assayed either by immunohistochemical or confocal assays, can be used to detect enhanced autophagosome formation and, thus, altered activation of the autophagy pathway. (II) Enhanced autophagic vacuole (autophagosome) formation per unit tissue area (as a marker of autophagy stimulation) can be quantified using electron microscopy. The study of autophagic responses to sepsis is a critical component of understanding the mechanisms by which tissues respond to infection. Research findings in this area may ultimately contribute towards understanding the pathogenesis of sepsis, which represents a major problem in critical care medicine.

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method. Current protocols to assess autophagy in vivo in the context of CLP-induced sepsis are presented here: A protocol for measuring autophagy using (GFP)-LC3 mice, and a protocol for measuring autophagosome formation by electron microscopy.

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is ...

A Controlled Mouse Model for Neonatal Polymicrobial Sepsis

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse polymicrobial sepsis can be induced by injecting cecal slurry intraperitoneally into day of life 7 mice and monitoring them for the following week. Presented here are the detailed steps necessary for the implementation of this neonatal sepsis model. This includes making a homogeneous cecal slurry stock, diluting it to a weight- and litter-adjusted dose, an outline of the monitoring schedule, and a definition of observed health categories used to define humane endpoints. The generation of a homogeneous cecal slurry stock from pooled donors allows for the administration into many litters over time, reducing the variation between donors, and preventing the use of potentially toxic glycerol. The monitoring strategy used allows for the anticipation of survival outcome and the identification of mice that would later progress to death, allowing for an earlier identification of the humane endpoint. Two main behavioral features are used to define the health scores, namely, the ability of the neonatal mice to right themselves when placed on their back and their level of mobility. These criteria could potentially be applied to address humane endpoints in other studies of neonatal disease in mice, as long as a pilot study is performed to confirm accuracy. In conclusion, this approach provides a standardized method to model newborn sepsis in mice, while providing resources to assess animal welfare used to define early humane endpoints for challenged animals.

This protocol provides the necessary steps to establish and evaluate neonatal sepsis in 7-day-old mice.

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse ...

A Neonatal Imaging Model of Gram-Negative Bacterial Sepsis

Neonates are at an increased risk of bacterial sepsis due to the unique immune profile they display in the first months of life. We have established a protocol for studying the pathogenesis of E. coli O1:K1:H7, a serotype responsible for high mortality rates in neonates. Our method utilizes intravital imaging of neonatal pups at different time points during the progression of infection. This imaging, paralleled by measurement of bacteria in the blood, inflammatory profiling, and tissue histopathology, signifies a rigorous approach to understanding infection dynamics during sepsis. In the current report, we model two infectious inoculums for comparison of bacterial burdens and severity of disease. We find that subscapular infection leads to disseminated infection by 10 h post-infection. By 24 h, infection of luminescent E. coli was abundant in the blood, lungs, and other peripheral tissues. Expression of inflammatory cytokines in the lungs is significant at 24 h, and this is followed by cellular infiltration and evidence of tissue damage that increases with infectious dose. Intravital imaging does have some limitations. This includes a luminescent signal threshold and some complications that can arise with neonates during anesthesia. Despite some limitations, we find that our infection model offers an insight for understanding longitudinal infection dynamics during neonatal murine sepsis, that has not been thoroughly examined to date. We expect this model can also be adapted to study other critical bacterial infections during early life.

Infection of neonatal mice with bioluminescent E. coli O1:K1:H7 results in a septic infection with significant pulmonary inflammation and lung pathology. Here, we describe procedures to model and further study neonatal sepsis using longitudinal intravital imaging in parallel with enumeration of systemic bacterial burdens, inflammatory profiling, and lung histopathology.

Neonates are at an increased risk of bacterial sepsis due to the unique immune profile they display in the first months of life. We have established a ...

A Contemporary Warming/Restraining Device for Efficient Tail Vein Injections in a Murine Fungal Sepsis Model

In rodent models, tail vein injections are important methods for intravenous administration of experimental agents. Tail vein injections typically involve warming of the animal to promote vasodilation, which aids in both the identification of the blood vessels and positioning of the needle into the vessel lumen while securely restraining the animal. Although tail vein injections are common procedures in many protocols and are not considered highly technical if performed correctly, accurate and consistent injections are crucial to obtain reproducible results and minimize variability. Conventional methods for inducing vasodilation prior to tail vein injections generally depend on the use of a heat source such as a heat lamp, electrical/rechargeable heat pads, or pre-heated water at 37 &#176;C. Despite being readily accessible in a standard laboratory setting, these tools evidently suffer from poor/limited thermo-regulatory capacity. Similarly, although various forms of restraining devices are commercially available, they must be used carefully to avoid trauma to the animals. These limitations of the current methods create unnecessary variables in experiments or result in varying outcomes between experiments and/or laboratories.
In this article, we demonstrate an improved protocol using an innovative device that combines an independent, thermally regulated, warming device with an adjustable restraining unit into one system for efficient streamlined tail vein injection. The example we use is an intravenous model of fungal bloodstream infection that results in sepsis. The warming apparatus consists of a heat-reflective acrylic box installed with an adjustable automatic thermostat to maintain the internal temperature at a pre-set threshold. Likewise, the width and height of the cone restraining apparatus can be adjusted to safely accommodate various rodent sizes. With the advanced and versatile features of the device, the technique shown here could become a useful tool across a range of research areas involving rodent models that employ tail vein injections.

Here, we present an effective and efficient method for rodent tail vein injections using a uniquely designed warming/restraining device. By streamlining the initiation of vasodilation and restraining processes, this protocol allows accurate and timely intravenous injections of large groups of animals with minimal distress.

In rodent models, tail vein injections are important methods for intravenous administration of experimental agents. Tail vein injections typically involve ...

Characterizing Salmonella Typhimurium-induced Septic Peritonitis in Mice

Sepsis is a dysregulated host immune response to microbial invasion or tissue damage, leading to organ injury at a site distant from that of the infection or damage. Currently, the widely used mice models of sepsis include lipopolysaccharide (LPS)-induced endotoxemia, cecal ligation and puncture (CLP), and monobacterial infection model systems. This protocol describes a method to study the host responses during Salmonella Typhimurium infection-induced septic peritonitis in mice. S. Typhimurium, a Gram-negative intracellular pathogen, causes typhoid-like disease in mice.
This protocol elaborates the culture preparation, induction of septic peritonitis in mice through intraperitoneal injection, and methods to study systemic host responses. Furthermore, the assessment of bacterial burden in different organs and the flow cytometric analysis of increased neutrophil numbers in the peritoneal lavage is presented. Salmonella Typhimurium-induced sepsis in mice leads to an increase in proinflammatory cytokines and rapid infiltration of neutrophils in the peritoneal cavity, leading to lower survival.
Every step in this protocol has been optimized, resulting in high reproducibility of the pathogenesis of septic peritonitis. This model is useful for studying immunological responses during bacterial sepsis, the roles of different genes in disease progression, and the effects of drugs to attenuate sepsis.

Characterizing Salmonella Typhimurium-induced Septic Peritonitis in Mice

This protocol describes the induction of Gram-negative monobacterial sepsis in a mouse model system. The model is useful in investigating the inflammatory and lethal host responses during sepsis.

Sepsis is a dysregulated host immune response to microbial invasion or tissue damage, leading to organ injury at a site distant from that of the infection ...

Evaluation of a Reliable Biomarker in a Cecal Ligation and Puncture-Induced Mouse Model of Sepsis

Sepsis is a severe life-threatening and rapidly developing disease that causes millions of deaths annually worldwide. Researchers have made tremendous efforts to elucidate the pathophysiology of sepsis using various animal models; the mouse model of sepsis induced by cecal ligation and puncture (CLP) is widely used in laboratories. The three technical aspects that affect the severity and replicability of the CLP model are the percentage of cecum ligated, the size of the needle used for cecal puncture, and the volume of feces squeezed into the abdominal cavity. The rapid and specific diagnosis of sepsis is a crucial factor that affects the outcome. The gold standard for sepsis diagnosis is microbial culture; however, this process is time-consuming and sometimes inaccurate. The detection of sepsis-specific biomarkers is fast, but the existing biomarkers are unsatisfactory due to a short half-life, non-specificity, and insufficient sensitivity. Therefore, there is an urgent need for a reliable biomarker of sepsis in the early stages. Previous publications suggest that excessive neutrophil extracellular traps (NETs) occur in sepsis. Citrullinated histone H3 (CitH3), as a NET component, is elevated both in septic animals and patients, and the presence of CitH3 is a reliable diagnostic biomarker of sepsis. The present study aimed to describe a standardized mouse model of CLP-induced sepsis and establish a reliable blood biomarker of sepsis. Our work may contribute to the early and accurate diagnosis of sepsis in the future.

This protocol presents the operative details of cecal ligation and puncture (CLP) in a mouse model of sepsis. CLP is one of the most widely used techniques to create an animal model of sepsis. Therefore, a standardized CLP protocol is required for the attainment of reliable research results.

Sepsis is a severe life-threatening and rapidly developing disease that causes millions of deaths annually worldwide. Researchers have made tremendous ...

A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness

Streptococcus pneumoniae (pneumococcus) is an asymptomatic colonizer of the nasopharynx in most individuals but can progress to a pulmonary and systemic pathogen upon influenza A virus (IAV) infection. Advanced age enhances host susceptibility to secondary pneumococcal pneumonia and is associated with worsened disease outcomes. The host factors driving those processes are not well defined, in part due to a lack of animal models that reproduce the transition from asymptomatic colonization to severe clinical disease. 
This paper describes a novel mouse model that recreates the transition of pneumococci from asymptomatic carriage to disease upon viral infection. In this model, mice are first intranasally inoculated with biofilm-grown pneumococci to establish asymptomatic carriage, followed by IAV infection of both the nasopharynx and lungs. This results in bacterial dissemination to the lungs, pulmonary inflammation, and obvious signs of illness that can progress to lethality. The degree of disease is dependent on the bacterial strain and host factors. 
Importantly, this model reproduces the susceptibility of aging, because compared to young mice, old mice display more severe clinical illness and succumb to disease more frequently. By separating carriage and disease into distinct steps and providing the opportunity to analyze the genetic variants of both the pathogen and the host, this S. pneumoniae/IAV co-infection model permits the detailed examination of the interactions of an important pathobiont with the host at different phases of disease progression. This model can also serve as an important tool for identifying potential therapeutic targets against secondary pneumococcal pneumonia in susceptible hosts.

A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness

This paper describes a novel mouse model for the transition of pneumococcus from an asymptomatic colonizer to a disease-causing pathogen during viral infection. This model can be readily adapted to study polymicrobial and host-pathogen interactions during the different phases of disease progression and across various hosts.

Streptococcus pneumoniae (pneumococcus) is an asymptomatic colonizer of the nasopharynx in most individuals but can progress to a pulmonary and systemic ...

Design of Cecal Ligation and Puncture and Intranasal Infection Dual Model of Sepsis-Induced Immunosuppression

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Chapters in this video

Colon Ascendens Stent Peritonitis (CASP) - a Standardized Model for Polymicrobial Abdominal Sepsis

Cecal Ligation Puncture Procedure

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice

A Controlled Mouse Model for Neonatal Polymicrobial Sepsis

A Neonatal Imaging Model of Gram-Negative Bacterial Sepsis

A Contemporary Warming/Restraining Device for Efficient Tail Vein Injections in a Murine Fungal Sepsis Model

Characterizing Salmonella Typhimurium-induced Septic Peritonitis in Mice

Evaluation of a Reliable Biomarker in a Cecal Ligation and Puncture-Induced Mouse Model of Sepsis

A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness