The authors would like to thank Beatrice Beck-Schimmer (MD) and Erik Schadde (MD) for their critical examination and their valuable contribution for this manuscript.

All experiments presented in this protocol were approved by the Veterinary Authorities of the Canton Zurich, Switzerland (approval numbers 134/2014 and ZH088/19). Moreover, all steps performed in this experiment were in accordance with the Guidelines on Experiments with Animals by the Swiss Academy of Medial Sciences (SAMS) and Guidelines of the Federation of European Laboratory Animal Science Associations (FELASA).
1. Anesthesia induction and animal monitoring
<ol>
	<li>Keep male Wistar rats with a weight of 250-300 gram (g) in ventilated cages under pathogen-free conditions. Provide a 12-12-hour light/dark cycle at an ambient temperature of 22 &#177; 1 &#176;C, and free access to food and water.</li>
	<li>Induce general anesthesia either by volatile induction with isoflurane (concentration of 3-5%) in an anesthesia induction box for 30 seconds (Figure 1A) or alternatively, induce anesthesia with a single-shot injection of ketamine/xylazine (10/1 milligram (mg) per 100 g body weight).</li>
	<li>Transfer the animal to a working place and lay the animal on a heating-mat throughout the entire experiment. Keep the body-temperature between 36.5 and 37 &#176;C.</li>
	<li>Use a nosecone to provide oxygen (600 mL/minute). Add isoflurane 2-3% if volatile anesthesia was chosen for anesthesia maintenance. Make sure that the animal is spontaneously breathing.</li>
	<li>Confirm the level of anesthesia by the absence&#160;of the toe-pinch reflex prior to the installation of tracheostomy and arterial and venous catheters.</li>
	<li>Verify a sufficient oxygenation by peripheral oxygen saturation monitoring (normal oxygen saturation 98 - 100%).</li>
	<li>Use an ointment (Vitamin A ointment) to protect the eyes.</li>
	<li>Prepare sterile surgical instruments and catheters on a side table as displayed in Figure 1B.</li>
	<li>Additionally, prepare pressure and oxygen saturation monitoring as displayed in Figure 1C.</li>
</ol>
2. Intravenous access
<ol>
	<li>Apply a tourniquet at the rat&#39;s proximal tail to facilitate venous access (Figure 2A).</li>
	<li>Disinfect the tail 3 times with alcohol.</li>
	<li>Induce a G26 intravenous catheter into one of the two lateral tail veins. 
	NOTE: From our experience it is easier to place the intravenous access at the distal part of the rat&#39;s tail, because the vein here is located closer to the skin. In addition, in case of a failed cannulation, there is enough space to move proximally.</li>
	<li>Strictly avoid air injection.</li>
	<li>Untie the tourniquet after placing the intravenous catheter.</li>
	<li>Fix the intravenous catheter in place with adhesive tape (Figure 2B).</li>
	<li>Connect syringe-pumps to the intravenous access for continuous fluid and drug application.</li>
	<li>Use 3-way stopcocks for bolus fluid, drug application, and venous blood sampling.</li>
</ol>
3. Tracheostomy
<ol>
	<li>Shave the animal&#39;s anterior neck-area.</li>
	<li>Disinfect the shaved skin 3 times with providone-iodine solution.</li>
	<li>Perform a circa 2 cm longitudinal incision using a scalpel (with a blade number 10).</li>
	<li>Retract the skin with 2-0 silk sutures.</li>
	<li>Bluntly prepare the larynx and the trachea with surgical scissors (Figure 3A).</li>
	<li>Make sure to open the trachea with surgical micro&#160;scissors at the 3-5th tracheal clasp.</li>
	<li>Insert a sterile tracheal cannula into the trachea. Be careful, do not insert the cannula too deeply in order to avoid unilateral ventilation.</li>
	<li>Fix the cannula in place using a 2-0 silk suture.</li>
	<li>Connect the cannula to a ventilator for pressure or volume-controlled ventilation (Figure 3B).</li>
</ol>
4. Arterial access
<ol>
	<li>Disinfect the rat tail 3 times with povidone-iodine solution.</li>
	<li>Cut the skin using a scalpel (with a blade number 10) circa 1 cm longitudinally at the ventral side.</li>
	<li>Take care, do not cut too deeply to avoid an injury of the tail artery.</li>
	<li>Use a surgical microscope to expose the artery carefully. Cut the fascia surrounding the artery with surgical micro scissors.</li>
	<li>Ligate the distal part of the artery using a 6-0 silk suture.</li>
	<li>Prepare a proximal 6-0 silk suture but do not tighten the silk (Figure 4A).</li>
	<li>Introduce a G-26 catheter into the artery between the distal and proximal silk suture.</li>
	<li>Once the catheter is in the artery, tighten the proximal silk suture and fix the catheter in place (Figure 4B).</li>
	<li>Connect the catheter to a pressure transducer to provide continuous arterial pressure measurement (normal mean arterial pressure: 60 - 100 mmHg) (Figure 4C).</li>
	<li>Additionally, place a 3-way stopcock between the catheter connected to the pressure transducer and the G-26 catheter for arterial blood sampling.</li>
</ol>
5. Baseline measurement, sepsis induction and follow-up measurements
<ol>
	<li>After the animal reached a steady state, inject the LPS.</li>
	<li>Collect blood samples when a steady state is reached (usually after 15-30 minutes).</li>
	<li>Replace fluid loss from blood samples by Ringer&#39;s solution in a ratio of 1:4.</li>
	<li>To induce sepsis, inject the LPS as a bolus or as a continuous LPS application.</li>
	<li>For the bolus application, inject 1 mg of LPS/kilogram body weight (kg) dissolved in phosphate buffered saline (PBS) at a concentration of 1 mg/mL.</li>
	<li>For continuous application, inject 300 &#181;g of LPS/kg/hour throughout the entire experiment using a syringe pump (stock solution of LPS: 1 mg/mL in PBS).</li>
	<li>Avoid air-injection at all times in order to prevent air embolism.</li>
	<li>Define fluid replacement protocols, vasoconstrictor application protocols, and abortion criteria (for example hypotension defined as a mean arterial blood pressure below 50 mmHg for more than 30 minutes despite fluid replacement) before setting up the experiment. 
	NOTE: We suggest a continuous infusion of Ringer&#39;s solution at a rate of 10 mL/kg/hour.</li>
	<li>Subtract any continuous administration of fluids (e.g., for continuous LPS application) from the amount infused so that the results are comparable with those of the control groups. 
	NOTE: At the end of the experiment, and prior to harvesting any organs such as liver, kidney or spleen for further analyses such as histological or biochemical examination animals can be euthanized by an incision of the inferior cava vein. The recommended method of euthanasia is to bring the animals to a surgical plane of anesthesia prior to incision of the inferior vena cava and injection of ice-cold saline into the left heart, especially if inflammatory markers in organs are to be assessed. Ensure to comply with the legal requirements and local guidelines. To verify sepsis-related organ failure, pro-apoptosis marker like caspase-3 may be analyzed as well as &#945;1-microglobuline to verify tubular damage in the kidneys. The organ specific analysis&#160;of markers like CINC-1, MCP-1 and IL-6 may also provide information about the organ specific inflammatory response.</li>
</ol>

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L., 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+the+worldwide+burden+of+critical+illness:+the+intensive+care+over+nations+(ICON)+audit.">Assessment of the worldwide burden of critical illness: the intensive care over nations (ICON) audit.</a> Lancet Respiratory Medicine. 2 (5), 380-386 (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. 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The authors have no conflicts of interests with regard to the presented study. Martin Schl&#228;pfer has submitted a patent to mitigate the negative effects of surgery and/or anesthesia for patients using medical gases, particularly oxygen (O2) and carbon dioxide (CO2). He has received unrestricted research grants from Sedana Medical, Sweden, and from Roche, Switzerland, not related to this work.

The protocol described here allows for a highly reproducible, yet simple to learn sepsis model, which can be adapted according the research question. Essential in vivo data referring to organ function such as heart rate, blood pressure, and peripheral arterial oxygen saturation may be collected continuously, and blood sampling may be performed repetitively throughout the experiment. In addition, modifications with regard to fluid replacement protocols and vasopressor support can be installed. Given the hemodynamic stabil...

Sepsis and its more severe form, septic shock, are syndromes on the ground of an infection, resulting in an overshooting inflammatory reaction with the release of cytokines, leading to physiological and biochemical changes with a suppressed immune defense and fatal results1,2. This unbalanced inflammatory reaction results in organ dysfunction and organ failure in various vital organs such as lung, kidney, and liver. With 37%3, sepsis is one of the most common reasons for a patient to be admitted to an intensive care unit (ICU). Mortality of sepsis currently ranges around 20-30%4. Early and effective antibiotic treatment is of utmost importance5. Fluid and vasopressor resuscitation need to be installed early, other than that, treatment is purely supportive6.
Sepsis is defined as a proven or suspected infection with bacteria, fungi, viruses, or parasites, which is accompanied by organ dysfunction. Septic shock criteria are met when a further cardiovascular collapse irresponsive to fluid treatment alone, and a lactate level of more than 2 millimole/liter is present2. Sepsis related organ failure may occur in any organ, but is very common in the cardiovascular system, the brain, the kidney, the liver, and the lung. Most patients suffering from sepsis require endotracheal intubation to secure the patient&#39;s airway, to protect from aspiration, and to apply positive end expiratory ventilation with a high fraction of inspired oxygen to prevent or overcome hypoxia. In order to tolerate a tracheal tube and mechanical ventilation, patients usually require sedation.
Endotoxins, such as lipopolysaccharides (LPS) as a component of the membrane of gram negative bacteria induce a strong inflammatory reaction via the toll-like receptor (TLR) 47. Activation of a defined pathway ensures a stable inflammatory reaction. Cytokines like cytokine induced neutrophil chemoattractant protein 1 (CINC-1), monocyte chemoattractant protein 1 (MCP-1), and interleukin 6 (IL-6) are known as prognostic factors for severity and outcome in this model8. Intravenous LPS application has been successfully used to study various aspects of sepsis in rats8,9.
Treatment of sepsis is still a challenge, particularly due to the lack of predictive animal models. If endotoxemia with activation of systemic inflammation is an adequate model for the development of pharmacological therapies is debatable. However, with the well-known LPS-induced TLR 4 pathway important knowledge can be gained.

<table><tbody><tr><td>2-0 silk sutures</td><td>Ethicon, Sommerville, NJ</td><td>K833</td><td>Standard surgical</td></tr><tr><td>26 intravenous catheter</td><td>Becton Dickinson, Franklin Lakes, NJ</td><td>391349</td><td>Standard anesthesia equipment</td></tr><tr><td>6-0 LOOK black braided silk</td><td>Surgical Specalities Corporation, Wyomissing, PA</td><td>SP114</td><td>Standard surgical</td></tr><tr><td>Alaris Syringe Pump</td><td>Bencton Dickinson</td><td></td><td></td></tr><tr><td>Betadine</td><td>Mundipharma, Basel, Switzerland</td><td>7.68034E+12</td><td>GTIN-number</td></tr><tr><td>Curved fine tips microforceps</td><td>World precision instruments (WPI), Sarasota, FL</td><td>504513</td><td>Facilitates vascular preparation</td></tr><tr><td>Fine tips microforceps</td><td>World precision instruments (WPI), Sarasota, FL</td><td>501976</td><td>Tips need to be polished regularly</td></tr><tr><td>Infinity Delta XL Anesthesia monitoring</td><td>Draeger, L&amp;uuml;beck, Germany</td><td></td><td></td></tr><tr><td>Isoflurane, 250 mL bottles</td><td>Attane, Piramal, Mumbai, India</td><td>LDNI 22098</td><td>Standard vet. equipment</td></tr><tr><td>Ketamine (Ketalar)</td><td>Pfitzer, New York, NY</td><td></td><td></td></tr><tr><td>Lipopolysaccharide (LPS) from &lt;em&gt;Escherichia coli&lt;/em&gt;, serotype 055:B5</td><td>Sigma, Buchs, Switzerland</td><td></td><td></td></tr><tr><td>Q-tips small</td><td>Carl Roth GmbH, Karlsruhe, Germany</td><td>EH11.1</td><td>Standard surgical</td></tr><tr><td>Ringerfundin</td><td>Bbraun, Melsungen, Germany</td><td></td><td></td></tr><tr><td>Tec-3 Isofluorane Vaporizer</td><td>Ohmeda, GE-Healthcare, Chicago, IL</td><td>not available anymore</td><td>Standard vet. equipment</td></tr><tr><td>Xylazine (Xylazin Streuli)</td><td>Streuli AG, Uznach, Switzerland</td><td></td><td></td></tr></tbody></table>

a reproducible intensive care unit-oriented endotoxin model in rats

Sepsis and septic shock remain the leading cause of death in intensive care units. Despite significant improvements in sepsis management, mortality still ranges between 20 and 30%. Novel treatment approaches in order to reduce sepsis-related multiorgan failure and death are urgently needed. Robust animal models allow for one or multiple treatment approaches as well as for testing their effect on physiological and molecular parameters. In this article, a simple animal model is presented.
First, general anesthesia is induced in animals either with the use of volatile or by intraperitoneal anesthesia. After placement of an intravenous catheter (tail vein), tracheostomy, and insertion of an intraarterial catheter (tail artery), mechanical ventilation is started. Baseline values of mean arterial blood pressure, arterial blood oxygen saturation, and heart rate are recorded.
The injection of lipopolysaccharides (1 milligram/kilogram body weight) dissolved in phosphate-buffered saline induces a strong and reproducible inflammatory response via the toll-like receptor 4. Fluid corrections as well as the application of norepinephrine are performed based on well-established protocols.
The animal model presented in this article is easy to learn and strongly oriented towards clinical sepsis treatment in an intensive care unit with sedation, mechanical ventilation, continuous blood pressure monitoring, and repetitive blood sampling. Also, the model is reliable, allowing for reproducible data with a limited number of animals in accordance with the 3R (reduce, replace, refine) principles of animal research. While animal experiments in sepsis research cannot easily replaced, repetitive measurements allow for a reduction of animals and keeping septic animals anesthetized diminishes suffering.

The system presented allows for endotoxemia with hemodynamically stable animals as reported previously9. While the mean arterial pressure remains stable in animals with, and without LPS stimulation LPS treated animal develop characteristics of sepsis such as a negative base excess and a strong inflammatory reaction measured by plasma cytokines (6 hours after application) such as CINC-1 (867 ng/mL), MCP-1 (5027 ng/mL), and IL-6 (867 ng/mL)8, Figure 5</st...

Watch this Scientific Journal Video about A Reproducible Intensive Care Unit-Oriented Endotoxin Model in Rats at JoVE.com

A Reproducible Intensive Care Unit-Oriented Endotoxin Model in Rats

Here, we present a reproducible intensive care unit-oriented endotoxin model in rats.

Sepsis and septic shock remain the leading cause of death in intensive care units. Despite significant improvements in sepsis management, mortality still ...

a-reproducible-intensive-care-unit-oriented-endotoxin-model-in-rats

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2.0K Views.  University of Zurich. Dit protocol is een robuust en reproduceerbaar model van endotoxemie waarin fysiologische en moleculaire parameters herhaaldelijk kunnen worden beoordeeld. Deze aanpak kan helpen om vragen in sepsisonderzoek te beantwoorden. Hoewel ons model geen dierproeven vervangt, verfijnt het sepsismodellen door continue sedatie en vermindert het het aantal dieren door herhaalde bemonstering.Beginners kunnen moeite hebben met het inbrengen van arteriële en veneuze katheters, maar er is slechts be...

Video: A Reproducible Intensive Care Unit-Oriented Endotoxin Model in Rats

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus (MRSA) in Rat

Endovascular infections, including endocarditis, are life-threatening infectious syndromes1-3. Staphylococcus aureus is the most common world-wide cause of such syndromes with unacceptably high morbidity and mortality even with appropriate antimicrobial agent treatments4-6. The increase in infections due to methicillin-resistant S. aureus (MRSA), the high rates of vancomycin clinical treatment failures and growing problems of linezolid and daptomycin resistance have all further complicated the management of patients with such infections, and led to high healthcare costs7, 8. In addition, it should be emphasized that most recent studies with antibiotic treatment outcomes have been based in clinical settings, and thus might well be influenced by host factors varying from patient-to-patient. Therefore, a relevant animal model of endovascular infection in which host factors are similar from animal-to-animal is more crucial to investigate microbial pathogenesis, as well as the efficacy of novel antimicrobial agents. Endocarditis in rat is a well-established experimental animal model that closely approximates human native valve endocarditis. This model has been used to examine the role of particular staphylococcal virulence factors and the efficacy of antibiotic treatment regimens for staphylococcal endocarditis. In this report, we describe the experimental endocarditis model due to MRSA that could be used to investigate bacterial pathogenesis and response to antibiotic treatment.

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus MRSA in Rat

Experimental rat endocarditis model due to methicillin-resistant S. aureus.

Endovascular infections, including endocarditis, are life-threatening infectious syndromes1-3. Staphylococcus aureus is the most common world-wide cause ...

Immunology and Infection

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

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. ...

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or A. baumannii

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, preferably in models which mimic the intended clinical indication. A method for inducing robust lung infections in immunocompetent rats and mice is described which allows for the assessment of treatments in a model of serious pneumonia caused by S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae or A. baumannii. Animals are anesthetized, and an agar-based inoculum is deposited deep into the lung via nonsurgical intratracheal intubation. The resulting infection is consistent, reproducible, and stable for at least 48 h and up to 96 h for most isolates. Studies with marketed antibacterials have demonstrated good correlation between in vivo efficacy and in vitro susceptibility, and concordance between pharmacokinetic/pharmacodynamic targets determined in this model and clinically accepted targets has been observed. Although there is an initial time investment when learning the technique, it can be performed quickly and efficiently once proficiency is achieved. Benefits of the model include elimination of the neutropenic requirement, increased robustness and reproducibility, ability to study more pathogens and isolates, improved flexibility in study design and establishment of a challenging infection in an immunocompetent host.

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or  A. baumannii

A method for establishing lung infections in immunocompetent rodents is described. With proficiency, this method can be performed quickly and easily to induce stable infection with many isolates of S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae and A. baumannii.

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, ...

Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best advance translational objectives and uncover new therapeutic strategies for brain injury associated with preterm birth, preclinical models of EoP must include similar mechanisms of prenatal global injury observed in humans and involve multiple components of the maternal-placental-fetal system. Ideally, models should produce a similar spectrum of functional deficits in the mature animal and recapitulate multiple aspects of the pathophysiology. To mimic human systemic placental perfusion defects, placental underperfusion and/or chorioamnionitis associated with pathogen-induced inflammation in early preterm birth, we developed a model of prenatal transient systemic hypoxia-ischemia (TSHI) combined with intra-amniotic lipopolysaccharide (LPS). In pregnant Sprague Dawley rats, TSHI via uterine artery occlusion on embryonic day 18 (E18) induces a graded placental underperfusion defect associated with increasing CNS damage in the fetus. When combined with intra-amniotic LPS injections, placental inflammation is increased and CNS damage is compounded with associated white matter, gait and imaging abnormalities. Prenatal TSHI and TSHI+LPS prenatal insults meet several of the criteria of an EoP model including recapitulating the intrauterine insult, causing loss of neurons, oligodendrocytes and axons, loss of subplate, and functional deficits in adult animals that mimic those observed in children born extremely preterm. Moreover, this model allows for the dissection of inflammation induced by divergent injury types.

Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best ...

Intravenous Endotoxin Challenge in Healthy Humans: An Experimental Platform to Investigate and Modulate Systemic Inflammation

Activation of inflammatory pathways represents a central mechanism in multiple disease states both acute and chronic. Triggered via either pathogen or tissue damage-associated molecular motifs, common biochemical pathways lead to conserved yet variable physiological and immunological alterations. Dissection and delineation of the determinants and mechanisms underlying phenotypic variance in response is expected to yield novel therapeutic advances.
Intravenous (IV) administration of endotoxin (gram-negative bacterial lipopolysaccharide), a specific Toll-like receptor 4 agonist, represents an in vivo model of systemic inflammation in man. National Institutes for Health Clinical Center Reference Endotoxin (CCRE, Escherichia coli O:113:H10:K negative) is employed to reliably and reproducibly generate vascular, hematological, endocrine, immunological and organ-specific functional effects that parallel, to varying degrees, those seen in the early stages of pathological states. Alteration of dose (0.06 - 4 ng/kg) and time-scale of exposure (bolus vs. infusion) allows replication of either acute or chronic inflammation and a range of severity to be elicited, with higher doses (2 - 4 ng/kg) frequently being used to create a &#39;sepsis-like&#39; state. Established and novel medicinal compounds may additionally be administered prior to or post endotoxin exposure to appreciate their effect on the inflammatory cascade. Despite limitations in scope and generalizability, human IV endotoxin challenge offers a unique platform to gain mechanistic insights into inducible physiological responses and inflammatory pathways. Rationally employed it may aid translation of this knowledge into therapeutic innovations.

Intravenous administration of endotoxin reliably elicits dose-dependent physiological and immunological alterations consistent with several pathological states. This reductionist approach permits the investigation, modeling and experimental modification of systemic inflammation and its downstream effects in man.

Activation of inflammatory pathways represents a central mechanism in multiple disease states both acute and chronic. Triggered via either pathogen or ...

Intrarenal Injection of Escherichia coli in a Rat Model of Pyelonephritis

Pyelonephritis is a bacterial infection of the kidney and is most commonly caused by Escherichia coli. Recurrent infections can cause significant renal inflammation and fibrosis ultimately resulting in declining kidney function. Before improved clinical management and prevention of pyelonephritis can be instituted, a reliable animal model must be established in order to study the mechanisms of progression, recurrence, and therapeutic efficacy. The transurethral infection model closely mimics human pyelonephritis but exhibits considerable variation due to its reliance on urethral reflux to transport the bacteria to the kidney. Herein, a detailed surgical protocol for performing bacterial injections into the rat renal pelvis is provided and confirmed by non-invasive Magnetic Resonance Imaging (MRI). Using this protocol, animals receive direct exposure to a desired concentration of E. coli bacteria and can fully recover from the surgical procedure with adequate post-operative care. This facilitates subsequent longitudinal MRI assessments of the experimental animal models for comparison with saline (sham) controls. Using this direct delivery approach, the severity of infection is controllable and applicable for mechanistic studies of progression as well as development of novel treatment strategies.

Intrarenal Injection of Escherichia coli in a Rat Model of Pyelonephritis

This manuscript describes a rat surgical model of pyelonephritis using direct intra-renal infection by Escherichia coli into the renal pelvis. The experimental procedure can be utilized to study the pathogenesis of pyelonephritis as well as the associated inflammation and fibrosis.

Pyelonephritis is a bacterial infection of the kidney and is most commonly caused by Escherichia coli. Recurrent infections can cause significant renal ...

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 ...

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 Reproducible Intensive Care Unit-Oriented Endotoxin Model in Rats

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