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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>21G needle</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3,3&#8217;,5,5&#8217;-tetramethylbenzidine&#160;</td>
			<td>R&#38;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 &#38; 6-0)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>surgical instruments&#160;</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...

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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 ...

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Immunology and Infection

3.2K 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

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 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 ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and consequently poses an urgent threat to public health. Recurrence of a C. difficile infection (CDI) after successful treatment with antibiotics is high, occurring in 20-30% of patients, thus necessitating the discovery of novel therapeutics against this pathogen. Current animal models of CDI result in high mortality rates and thus do not approximate the chronic, insidious disease manifestations seen in humans with CDI. To evaluate therapeutics against C. difficile, a mouse model approximating human disease utilizing a clinically-relevant strain is needed. This protocol outlines the cefoperazone mouse model of CDI using a clinically-relevant and genetically-tractable strain, R20291. Techniques for clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol. Compared to other mouse models of CDI, this model is not uniformly lethal at the dose administered, allowing for the observation of a prolonged clinical course of infection concordant with the human disease. Therefore, this cefoperazone mouse model of CDI proves a valuable experimental platform to assess the effects of novel therapeutics on the amelioration of clinical disease and on the restoration of colonization resistance against C. difficile.

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

This protocol outlines the cefoperazone mouse model of Clostridium difficile infection (CDI) using a clinically-relevant and genetically-tractable strain, R20291. Emphasis on clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol.

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and ...

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate hosts, respectively. While pathogenic symptoms of leishmaniasis can vary widely, from benign cutaneous lesions to highly fatal visceral disease forms depending on the infective species, all Leishmania species reside inside host macrophages during the vertebrate stage of their lifecycle. Leishmania infectivity is therefore directly related to its ability to invade, survive and replicate within parasitophorous vacuoles (PVs) inside macrophages. Thus, assessing the parasite&#39;s ability to replicate intracellularly serves as a dependable method for determining virulence. Studying leishmaniasis development using animal models is time-consuming, tedious and often difficult, particularly with the pathogenically important visceral forms. We describe here a methodology to follow the intracellular development of Leishmania in bone marrow-derived macrophages (BMMs). Intracellular parasite numbers are determined at 24 h intervals for 72 - 96 h following infection. This method allows for a reliable determination of the effects of various genetic factors on Leishmania virulence. As an example, we show how a single allele deletion of the Leishmania Mitochondrial Iron Transporter gene (LMIT1) impairs the ability of the Leishmania amazonensis mutant strain LMIT1/&#916;Lmit1 to grow inside BMMs, reflecting a drastic reduction in virulence compared to wild-type. This assay also allows precise control of experimental conditions, which can be individually manipulated to analyze the influence of various factors (nutrients, reactive oxygen species, etc.) on the host-pathogen interaction. Therefore, the appropriate execution and quantification of BMM infection studies provide a non-invasive, rapid, economical, safe and reliable alternative to conventional animal model studies.

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

All pathogenic Leishmania species reside and replicate inside macrophages of their vertebrate hosts. Here, we present a protocol to infect murine bone marrow-derived macrophages in culture with Leishmania, followed by precise quantification of intracellular growth kinetics. This method is useful for studying individual factors influencing host-pathogen interaction and Leishmania virulence.

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate ...

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 ...

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 ...

Isolation and Quantitative Evaluation of Brush Cells from Mouse Tracheas

Tracheal brush cells are cholinergic chemosensory epithelial cells poised to transmit signals from the airway lumen to the immune and nervous systems. They are part of a family of chemosensory epithelial cells which include tuft cells in the intestinal mucosa, brush cells in the trachea, and solitary chemosensory and microvillous cells in the nasal mucosa. Chemosensory cells in different epithelial compartments share key intracellular markers and a core transcriptional signature, but also display significant transcriptional heterogeneity, likely reflective of the local tissue environment. Isolation of tracheal brush cells from single cell suspensions is required to define the function of these rare epithelial cells in detail, but their isolation is challenging, potentially due to the close interaction between tracheal brush cells and nerve endings or due to airway-specific composition of tight and adherens junctions. Here, we describe a procedure for isolation of brush cells from mouse tracheal epithelium. The method is based on an initial separation of tracheal epithelium from the submucosa, allowing for a subsequent shorter incubation of the epithelial sheet with papain. This procedure offers a rapid and convenient solution for flow cytometric sorting and functional analysis of viable tracheal brush cells.

Brush cells are rare cholinergic chemosensory epithelial cells found in the na&#239;ve mouse trachea. Due to their limited numbers, ex vivo evaluation of their functional role in airway immunity and remodeling is challenging. We describe a method for isolation of tracheal brush cells by flow cytometry.

Tracheal brush cells are cholinergic chemosensory epithelial cells poised to transmit signals from the airway lumen to the immune and nervous systems. ...

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 ...

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

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Influenza A Virus Studies in a Mouse Model of Infection

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

A Controlled Mouse Model for Neonatal Polymicrobial Sepsis

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

Isolation and Quantitative Evaluation of Brush Cells from Mouse Tracheas

A Neonatal Imaging Model of Gram-Negative Bacterial Sepsis