Name: evaluation of a reliable biomarker in a cecal ligation and puncture-induced mouse model of sepsis
Uploaded: 2022-12-09T12:30:00.000+00:00
Duration: 5 min 28 s
Description: Watch this Scientific Journal Video about Evaluation of a Reliable Biomarker in a Cecal Ligation and Puncture-Induced Mouse Model of Sepsis at JoVE.com

We thank Professor Wang Wei and Doctor Liu Shuai for helping with the experiments. This work was funded by grants from the Young Research Funding of Xiangya Hospital, Central South University (No. 2019Q10), From the National and Science Foundation of Hunan Province (No. 2020JJ4902), and from the National Natural Science Foundation of China (No. 82202394).

All animal experiments were performed in accordance with the guidelines approved by theAnimal Review Committee at Xiangya Hospital and Central South University (No. 202103149).
1. Preparation
<ol>
	<li>Select male C57BL/6J mice (weight: 20-25 g; age: 8-12 weeks) and house for 3 days before performing any procedures.</li>
	<li>Weigh the mouse.</li>
	<li>Anesthetize the mouse with 1.5% isoflurane by inhalation and pinch the toes to check the depth of anesthesia.</li>
	<li>Fix the mouse on the heating pad. Apply depilatory cream to the abdomen and leave for no longer than 1 min before removing the cream and hair. 
	&#8203;NOTE: Normal body temperature should be maintained by placing a warm pad under the anesthetized mouse during surgery.</li>
</ol>
2. Operation
<ol>
	<li>Prepare sterile surgical instruments suitable for rodent animal surgery. Wear sterile gloves, a face mask, and a surgical gown to ensure aseptic conditions.</li>
	<li>Disinfect the abdominal skin with iodine wipes at least three times. Cover the operative surgical area with sterile surgical drapes.</li>
	<li>Use sterile surgical scissors to make an approximately 2 cm incision in the abdominal wall along the linea alba. 
	NOTE: Do not damage the organs.</li>
	<li>Identify and isolate the cecum out of the peritoneal cavity with sterile tweezers.</li>
	<li>Ligate 75% of the volume of the cecum with 4-0 silk sutures. Do not ligate the mesenteric blood vessels. 
	NOTE: The percentage of the cecum that is ligated determines the severity of sepsis.</li>
	<li>Puncture the cecum by creating one through-and-through perforation (two holes) with a 21 G needle (from one side through the cecal wall to the opposite side) at the midpoint between the tail end and the ligation set.</li>
	<li>Discard the needle. Gently squeeze a small droplet of feces out of the cecum through the penetration holes. 
	&#8203;NOTE: The amount of feces squeezed into the peritoneal cavity should be consistent, as this also determines the severity of sepsis. Steps 2.5-2.7 should be skipped for sham mice.</li>
	<li>Gently replace the cecum into the abdominal cavity.</li>
	<li>Close the abdominal muscle and skin separately with 6-0 silk sutures.</li>
	<li>Disinfect the incision with iodine.</li>
	<li>Inject ketoprofen (5 mg/kg) in the lower left quadrant of the abdomen to avoid the cecum. Place the mouse on a warm pad until it has fully recovered from the anesthetic.</li>
	<li>Place the mouse in a cage in a temperature-controlled room (22 &#176;C) and give free access to food and water. Check the mouse every 6 h for the first week postoperatively.
	<ol>
		<li>Euthanize the mouse by carbon dioxide overdose when symptoms of sepsis meet the pre-defined endpoints.</li>
	</ol>
	</li>
</ol>
3. Treatment
<ol>
	<li>Randomly divide mice into the sham group, CLP group, CLP + YW3-56 group (Figure 2a), and CLP + Cl-amidine group (Figure 1). 
	NOTE: The sham group underwent the same procedures as the CLP groups except CLP. YW3-56 (5 mg/kg) or Cl-amidine (40 mg/kg) was administered via peritoneal injection 1 h after step 2.9 when suturing the muscle and skin layer.</li>
	<li>Sample harvest
	<ol>
		<li>Harvest peripheral blood from the retrobulbar plexus15 24 h after the operation.</li>
	</ol>
	</li>
	<li>Prepare the serum by centrifugation (1,000 x g, 5 min) and store at -80 &#176;C until use.</li>
	<li>Measure the CitH3 concentration using an indirect sandwich ELISA kit as previously described13.
	<ol>
		<li>Coat the anti-CitH3 monoclonal antibody on a 96-well plate to capture the CitH3 protein.</li>
		<li>Treat the serum with DNase I (150 unit/mL, 37&#176;for 1 h) and incubate in the wells (20 &#181;L, room temperature).</li>
		<li>Add anti-CitH3 polyclonal antibody (0.33 &#181;g/mL, 100 &#181;L, 2 h) to detect the captured CitH3 protein.</li>
		<li>Incubate the anti-rabbit peroxidase-labeled secondary antibody (0.02 &#181;g/mL, 100 &#181;l, 1 h) in the wells.</li>
		<li>After thorough washing, develop the wells with 3,3&#39;,5,5&#39;-tetramethylbenzidine (100 &#181;L, 20 min). 
		NOTE: Further protocol details of the ELISA kit are provided in previous publication13.</li>
	</ol>
	</li>
	<li>Statistical analysis
	<ol>
		<li>Perform one way ANOVA to analyze the differences among the three groups, followed by Bonferroni post hoc testing for multiple comparisons. Use a statistical software to perform the analysis. p values of 0.05 or less were considered significant.</li>
	</ol>
	</li>
</ol>

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No conflicts of interest declared.

CLP introduces pathogens into the abdomen to create a preclinical model of sepsis. When performing CLP, it is important to use sterile conditions to eliminate the interference of exogenous bacteria and to use accurate dosages of anesthetics16. The three technical aspects of CLP that affect the severity and replicability of the sepsis model are the percentage of the cecum ligated, the size of the needle used for cecal puncture, and the volume of feces squeezed into the abdominal cavity. Ligation of...

Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection1, and septic shock is the leading cause of death in severe cases of sepsis2. Sepsis and septic shock cause millions of deaths worldwide each year3. The key to improving the outcome of patients with sepsis is the prompt initiation of treatments such as antibiotics4. The gold standard method for the diagnosis of sepsis is microbial culture; however, microbial culture is time-consuming and can lead to false-positive and false-negative results, which greatly limit the clinical significance5. Thus, it is highly desirable to identify a blood biomarker of sepsis. Procalcitonin is recognized as an ideal sepsis biomarker but has limited diagnostic efficacy because it is unable to distinguish sepsis from sterile diseases6. 
Mouse cecal ligation and puncture (CLP) is commonly used to create a model of sepsis in scientific research. CLP is one of the most widely used sepsis models because it mimics polymicrobial peritonitis, activating both proinflammatory and anti-inflammatory immune responses7. It is well accepted that CLP creates a more clinically relevant sepsis model than alternative techniques, such as the injection of bacterial endotoxin. Therefore, CLP is considered the classical sepsis model for use in research8. However, a major disadvantage of CLP is its reproducibility, as the model severity is affected by several factors such as the percentage of cecum ligated, needle size, number of punctures, and laparotomy technique. Therefore, there is a need to standardize the CLP-induced sepsis model. The present study describes the protocol details of the CLP-induced sepsis model to show the standardized procedure and increase its reproducibility.
The inflammatory response occurs in the early stage of sepsis, with neutrophils releasing excessive amounts of oxidants and proteases that cause organ damage8. A key factor in the pathophysiology of sepsis is the formation of neutrophil extracellular traps (NETs), which release nuclear and cytosolic components such as DNA, citrullinated histones, and antimicrobial proteinases9. Recent studies suggest that excessive generation of NETs mediates the pathology of sepsis; meanwhile, a decrease of NETs, through enzymatic inhibition of peptidyl arginine deiminase (PAD) by chemicals like YW3-56 or Cl-amidine, exerts a pro-survival effect in mouse models of sepsis10,11. Citrullinated histone H3 (CitH3) was identified as a sepsis-specific protein in 201112, and subsequent publications have demonstrated that the circulating CitH3 concentration is a reliable diagnostic biomarker of sepsis13,14. CitH3 is considered a more sensitive and long-lasting biomarker than procalcitonin, and is more specific in distinguishing sepsis than inflammatory cytokines13.
In this study, we have evaluated a reliable diagnostic biomarker of sepsis in a CLP-induced mouse model of sepsis.

<table><tbody><tr><td>21G needle</td><td></td><td></td><td></td></tr><tr><td>3,3&amp;rsquo;,5,5&amp;rsquo;-tetramethylbenzidine&amp;nbsp;</td><td>R&amp;amp;D Systems Inc</td><td>DY999</td><td></td></tr><tr><td>anti-CitH3 monoclonal antibody</td><td>laboratory self developed</td><td></td><td></td></tr><tr><td>anti-CitH3 polyclonal antibody</td><td>Abcam</td><td>ab5103</td><td></td></tr><tr><td>anti-rabbit secondary antibody</td><td>Jackson ImmunoResearch</td><td>111-035-003</td><td></td></tr><tr><td>C57BL/6 mice</td><td>Xiangya School of Medicine, Central South University</td><td></td><td></td></tr><tr><td>Cl-amidine</td><td>Sigma Aldrich</td><td>SML2250</td><td></td></tr><tr><td>depilatory cream</td><td></td><td></td><td></td></tr><tr><td>Dnase I</td><td>Sigma Aldrich</td><td>11284932001</td><td></td></tr><tr><td>isoflurane</td><td>Sigma-Aldrich</td><td>26675-46-7</td><td></td></tr><tr><td>ketoprofen</td><td>Sigma Aldrich</td><td>PHR1375</td><td></td></tr><tr><td>silk sutures (4-0 &amp;amp; 6-0)</td><td></td><td></td><td></td></tr><tr><td>surgical instruments&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>YW3-56</td><td>GLPBIO</td><td>GC48263</td><td></td></tr></tbody></table>

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.

As shown in Figure 2A, no CitH3 was detected in the sham group by western blotting. The serum CitH3 concentration significantly increased after CLP, and this increase was blocked by the inhibition of NET formation via the administration of YW3-56, a PAD inhibitor10. Figure 2B shows the serumCitH3 concentrations determined by ELISA. At 24 h after CLP, the serum concentration of CitH3 was increased in the CLP groups compared with t...

Watch this Scientific Journal Video about Evaluation of a Reliable Biomarker in a Cecal Ligation and Puncture-Induced Mouse Model of Sepsis at JoVE.com

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

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 ...

evaluation-reliable-biomarker-cecal-ligation-puncture-induced-mouse

Research

JoVE Journal

Immunology and Infection

3.3K Views.  Central South University. 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.

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

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 ...

Standardized Colon Ascendens Stent Peritonitis in Rats - a Simple, Feasible Animal Model to Induce Septic Acute Kidney Injury

AKI in septic patients is associated with increased mortality and poor outcome despite major efforts to refine the understanding of its pathophysiology. Here, an in vivo model is presented that combines a standardized septic focus to induce AKI and an intensive care (ICU) setup to provide an advanced hemodynamic monitoring and therapy comparable in human sepsis. Sepsis is induced by standardized colon ascendens stent peritonitis (sCASP). AKI is investigated functionally by measurement of blood and urine samples as well as histologically by evaluation of histopathological scores. Furthermore, the advanced hemodynamic monitoring and the possibility of repetitive blood gas sampling enable a differentiated analysis of severity of induced sepsis.
The sCASP method is a standardized, reliable and reproducible method to induce septic AKI. The intensive care setup, continuous hemodynamic and gas exchange monitoring, low mortality rate as well as the opportunity of detailed analyses of kidney function and impairments are advantages of this setup. Therefore, the described method may serve as a new standard for experimental investigations of septic AKI.

Acute kidney injury (AKI) is a severe complication in critically ill patients and is related with an increased mortality. Here, we present a reliable and reproducible in vivo model to mimic AKI under inflammatory conditions that might contribute towards understanding the pathogenesis of septic AKI.

AKI in septic patients is associated with increased mortality and poor outcome despite major efforts to refine the understanding of its pathophysiology. ...

Injections of Lipopolysaccharide into Mice to Mimic Entrance of Microbial-derived Products After Intestinal Barrier Breach

The intestinal epithelial barrier separates the host from the microbiota that is normally tolerated or ignored. The breach of this barrier results in the entrance of bacteria or bacteria-derived products into the host, accessing the host circulation and inner organs leading to the uncontrolled inflammation as observed in patients with inflammatory bowel disease (IBD), that are characterized by an increased intestinal epithelial permeability.
To mimic the entrance of bacterial-derived compounds into the host, an endotoxemia model has been adopted in which lipopolysaccharide (LPS), a component of the outer cell wall of Gram-negative bacteria, were injected into mice. In this study, a sublethal dose of LPS was intraperitoneally injected and the mice were subsequently monitored for 8 h using a disease score. Furthermore, the expression levels of the inflammatory cytokines Il6, Il1b, and Tnfa were analyzed in the spleen, liver and colon by qPCR at different time points post LPS injection. This model could be useful for the studies involving investigation of immune responses after the invasion of microorganisms or bacterial-derived products caused by a barrier breach of body surfaces.

Here a protocol to mimic the entrance of bacterial-derived compounds after intestinal barrier breach is presented. A low sublethal dose of lipopolysaccharide was injected systemically into mice, which were monitored for 24 h post-injection. The expression of pro-inflammatory cytokines was determined at several time points in spleen, liver, and colon.

The intestinal epithelial barrier separates the host from the microbiota that is normally tolerated or ignored. The breach of this barrier results in the ...

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 ...

Use of a Central Venous Line for Fluids, Drugs and Nutrient Administration in a Mouse Model of Critical Illness

This protocol describes a centrally catheterized mouse model of prolonged critical illness. We combine the cecal ligation and puncture method to induce sepsis with the use of a central venous line for fluids, drugs and nutrient administration to mimic the human clinical setting. Critically ill patients require intensive medical support in order to survive. While the majority of patients will recover within a few days, about a quarter of the patients need prolonged intensive care and are at high risk of dying from non-resolving multiple organ failure. Furthermore, the prolonged phase of critical illness is hallmarked by profound muscle weakness, and endocrine and metabolic changes, of which the pathogenesis is currently incompletely understood. The most widely used animal model in critical care research is the cecal ligation and puncture model to induce sepsis. This is a very reproducible model, with acute inflammatory and hemodynamic changes similar to human sepsis, which is designed to study the acute phase of critical illness. However, this model is hallmarked by a high lethality, which is different from the clinical human situation, and is not developed to study the prolonged phase of critical illness. Therefore, we adapted the technique by placing a central venous catheter in the jugular vein allowing us to administer clinically relevant supportive care, to better mimic the human clinical situation of critical illness. This mouse model requires an extensive surgical procedure and daily intensive care of the animals, but it results in a relevant model of the acute and prolonged phase of critical illness.

This protocol describes a centrally catheterized mouse model of prolonged critical illness. We combine the cecal ligation and puncture method to induce sepsis with the use of a central venous line for fluids, drugs and nutrient administration to mimic the human clinical setting.

This protocol describes a centrally catheterized mouse model of prolonged critical illness. We combine the cecal ligation and puncture method to induce ...

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.

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 ...

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

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Injections of Lipopolysaccharide into Mice to Mimic Entrance of Microbial-derived Products After Intestinal Barrier Breach

A Controlled Mouse Model for Neonatal Polymicrobial Sepsis

Use of a Central Venous Line for Fluids, Drugs and Nutrient Administration in a Mouse Model of Critical Illness

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

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

Characterizing Salmonella Typhimurium-induced Septic Peritonitis in Mice