This work was supported by institutional funds to C.M.R.

<ol>
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P., 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=Early+and+late+onset+sepsis+in+very-low-birth-weight+infants+from+a+large+group+of+neonatal+intensive+care+units.">Early and late onset sepsis in very-low-birth-weight infants from a large group of neonatal intensive care units.</a> Early Human Development. , 69 (2012).</li><li>Vergnano, S., Sharland, M., Kazembe, P., Mwansambo, C., Heath, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+sepsis:+an+international+perspective.">Neonatal sepsis: an international perspective.</a> Archives of Disease in Childhood: Fetal and Neonatal Edition. 90 (3), 220-224 (2005).</li><li>Kraft, J. D., 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=Neonatal+macrophages+express+elevated+levels+of+interleukin-27+that+oppose+immune+responses.">Neonatal macrophages express elevated levels of interleukin-27 that oppose immune responses.</a> Immunology. 139 (4), 484-493 (2013).</li><li>Basha, S., Surendran, N., Pichichero, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+responses+in+neonates.">Immune responses in neonates.</a> Expert Reviews of Clinical Immunology. 10 (9), 1171-1184 (2014).</li><li>Gleave Parson, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+myeloid-derived+suppressor+cells+are+a+source+of+elevated+levels+of+interleukin-27+in+early+life+and+compromise+control+of+bacterial+infection.">Murine myeloid-derived suppressor cells are a source of elevated levels of interleukin-27 in early life and compromise control of bacterial infection.</a> Immunology and Cell Biology. 97 (5), 445-446 (2018).</li><li>Adkins, B., Leclerc, C., Marshall-Clarke, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+adaptive+immunity+comes+of+age.">Neonatal adaptive immunity comes of age.</a> Nature Reviews Immunology. 4 (7), 553-564 (2004).</li><li>Kim, S. K., Keeney, S. E., Alpard, S. K., Schmalstieg, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+L-selectin+and+CD11b+on+neutrophils+of+adults+and+neonates+during+the+first+month+of+life.">Comparison of L-selectin and CD11b on neutrophils of adults and neonates during the first month of life.</a> Pediatrics Research. 53 (1), 132-136 (2003).</li><li>Velilla, P. A., Rugeles, M. T., Chougnet, C. 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(125), e55796 (2017).</li><li>Bayarmagnai, B., Perrin, L., Esmaeili Pourfarhangi, K., Gligorijevic, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+Imaging+of+Tumor+Cell+Motility+in+the+Tumor+Microenvironment+Context.">Intravital Imaging of Tumor Cell Motility in the Tumor Microenvironment Context.</a> Methods in Molecular Biology. 1749, 175-193 (2018).</li><li>Beerling, E., Ritsma, L., Vrisekoop, N., Derksen, P. W., van Rheenen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy:+new+insights+into+metastasis+of+tumors.">Intravital microscopy: new insights into metastasis of tumors.</a> Journal of Cell Science. 124, 299-310 (2011).</li><li>Witcomb, L. A., Collins, J. W., McCarthy, A. J., Frankel, G., Taylor, P. 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The authors have no conflicts of interest to disclose.

Our subscapular infection model for inducing bacterial sepsis in neonatal mice is a novel method to study the longitudinal spread of bacterial pathogens in real time. Intravital imaging provides the opportunity to explore bacterial dissemination in real time in neonates. This is critical to understand the kinetics of bacterial dissemination and to further study the host response and damage at the appropriate phase of disease. Mouse pups are administered a subcutaneous, subscapular injection of bacterial inoculum. This in...

Bacterial sepsis is a significant concern for neonates that exhibit a unique immune profile in the first days of life that does not provide adequate protection from infection1. Neonatal sepsis continues to be a significant U.S. healthcare problem accounting for greater than 75,000 cases annually in the U.S alone2. To study these infections in depth, novel animal models that recapitulate aspects of human disease are required. We have established a neonatal mouse infection model using Escherichia coli, O1:K1:H73. E. coli is the second leading cause of neonatal sepsis in the U.S., but responsible for the majority of sepsis-associated mortality4,5. However, it is the leading cause when pre-term and very low birth-weight (VLBW)&#160;babies are considered independently5. The K1 serotype is most frequently associated with invasive bloodstream infections and meningitis in neonates6,7. Currently, there are no other treatment options beyond antibiotics and supportive care. Meanwhile, rates of antibiotic resistance continue to rise for many pathogenic bacteria, with some strains of E. coli resistant to a multitude of antibiotics commonly used in treatment8. Thus, it is imperative that we continue to generate methods to study the mechanisms of sepsis and the host response in neonates. These results can help to improve upon current treatments and infection outcomes.
The immune state of neonates is characterized by both phenotypic and functional differences compared to adults. For instance, elevated levels of anti-inflammatory and regulatory cytokines, such as interleukin (IL)-10 and IL-27, have been shown to be produced by cord blood-derived macrophages and are present at greater levels in the serum of murine neonates9,10,11. This is consistent with lower levels of IFN-&#945;, IFN-&#611;, IL-12, and TNF-&#945; that are frequently reported from neonatal cells compared with adult counterparts10. Additionally, the neonatal immune system is skewed toward a Th2 and regulatory T cell response as compared to adults12. Elevated numbers of neutrophils, T cells, B cells, NK cells, and monocytes are also present in neonates, but with significant functional impairments. This includes defects in expression of cell surface markers and antigen presentation that suggest immaturity13,14,15. Additionally, neonatal neutrophils are significantly deficient in their ability to migrate to chemotactic factors16. Myeloid-derived suppressor cells (MDSCs) are also found at elevated levels in neonates and recently shown to be a source of IL-2711. MDSCs are highly suppressive toward T cells17. Collectively, these data demonstrate limitations in neonatal immunity that lend to increased susceptibility to infection.
To study the progression of the bacterial burden and dissect protective host immune responses during neonatal sepsis, we have developed a novel infection model. Neonatal mice at days 3-4 of life are difficult to inject in the intraperitoneal space or tail vein. In our model, day 3 or 4 pups are administered the bacterial inoculum or PBS subcutaneously into the scapular region. A systemic infection develops and using luminescent E. coli O1:K1:H7, we can longitudinally image individual neonatal mice to follow the disseminated bacterial burden in peripheral tissues. This is the first reported model to utilize intravital imaging to understand the kinetics of dissemination of bacteria during sepsis in murine neonates3.
Here, we describe a protocol to induce septic E. coli infections in neonatal mice3. We describe how to prepare the bacterial inoculum for injection, and how to harvest tissue for assessment of pathology, measurement of inflammatory markers by gene expression analysis, and enumeration of the bacterial burden. In addition, the use of luminescent E. coli for intravital imaging of infected neonates and quantification of bacterial killing by neonatal immune cells is also described. These protocols may also be adapted to study other important bacterial infections in neonates. The data presented here represents an overall novel approach to understanding infection dynamics in a translatable neonatal sepsis model.

<table><tbody><tr><td>1 mL Insulin Syringe</td><td>Coviden</td><td>1188128012</td><td>Inoculum or PBS injection</td></tr><tr><td>10% Neutral Buffered Formalin</td><td>VWR</td><td>89370-094</td><td>Histopathology</td></tr><tr><td>ACK Lysis Buffer</td><td>Gibco</td><td>LSA1049201</td><td>Bacterial clearance assay</td></tr><tr><td>Animal Tattoo Ink Paste</td><td>Ketchum</td><td>KI1482039</td><td>Animal identification</td></tr><tr><td>Animal Tattoo Ink Green Paste</td><td>Ketchum</td><td>KI1471039</td><td>Animal identification</td></tr><tr><td>Anti-Ly-6B.2 Microbeads</td><td>Miltenyi Biotec</td><td>130-100-781</td><td>Cell isolation</td></tr><tr><td>&lt;em&gt;Escherichia coli&lt;/em&gt; O1:K1:H7</td><td>ATCC</td><td>11775</td><td></td></tr><tr><td>&lt;em&gt;Escherichia coli&lt;/em&gt; O1:K1:H7-&lt;em&gt;lux&lt;/em&gt; (expresses luciferase)</td><td>N/A</td><td>N/A</td><td>Constructed in-house at WVU</td></tr><tr><td>E.Z.N.A. HP Total Extraction RNA Kit</td><td>Omega Bio-tek</td><td>R6812</td><td>RNA extration</td></tr><tr><td>DPBS, 1X</td><td>Corning</td><td>21-031-CV</td><td></td></tr><tr><td>Difco Tryptic Soy Agar</td><td>Becton, Dickinson and Company</td><td>236950</td><td>Bacterial growth</td></tr><tr><td>IL-1 beta Primer/Probe (Mm00434228)</td><td>Thermo Fisher Scientific</td><td>4331182</td><td>Cytokine expression qPCR</td></tr><tr><td>IL-6 Primer/Probe (Mm00446190)</td><td>Thermo Fisher Scientific</td><td>4331182</td><td>Cytokine expression qPCR</td></tr><tr><td>iQ Supermix</td><td>Bio-Rad</td><td>1708860</td><td>Real-time quantitative PCR</td></tr><tr><td>iScript cDNA Synthesis Kit</td><td>Bio-Rad</td><td>1708891</td><td>cDNA synthesis</td></tr><tr><td>Isolation Buffer</td><td>Miltenyi Biotec</td><td>N/A</td><td>Bacterial clearance assay</td></tr><tr><td>IVIS Spectrum CT and Living Image 4.5 Software</td><td>Perkin Elmer</td><td>N/A</td><td>Intravital imaging</td></tr><tr><td>LB Broth, Lennox</td><td>Fisher BioReagents</td><td>BP1427-500</td><td>Bacterial growth</td></tr><tr><td>EASYstrainer (Nylon Basket)</td><td>Greiner Bio-one</td><td>542 040</td><td>Cell strainer</td></tr><tr><td>SpectraMax iD3</td><td>Molecular Devices</td><td>N/A</td><td>Plate reader</td></tr><tr><td>Pellet Pestle Motor</td><td>Grainger</td><td>6HAZ6</td><td>Tissue homogenization</td></tr><tr><td>Polypropylene Pellet Pestles</td><td>Grainger</td><td>6HAY5</td><td>Tissue homogenization</td></tr><tr><td>Prime Thermal Cycler</td><td>Techne</td><td>3PRIMEBASE/02</td><td>cDNA synthesis</td></tr><tr><td>TNF-alpha Primer/Probe (Mm00443258)</td><td>Thermo Fisher Scientific</td><td>4331182</td><td>Cytokine expression qPCR</td></tr><tr><td>TriReagent (GTCP)</td><td>Molecular Research Center</td><td>TR 118</td><td>RNA extration</td></tr></tbody></table>

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.

This protocol induced bacterial sepsis in neonatal mice, and we used longitudinal intravital imaging, enumeration of bacteria in the blood, histological assessments of pathology, and inflammatory cytokine expression profiles to study the course of disease. Signs of morbidity were observed in neonatal pups infected with both low (~2 x 106 CFUs) and high (~7 x 106 CFUs) inoculums of E.coli over time. Pups that received the greater inoculum displayed more prominent signs of distress that inclu...

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A Neonatal Imaging Model of Gram-Negative Bacterial Sepsis

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 ...

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Immunology and Infection

6.2K Views.  West Virginia University School of Medicine. 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.

Video: A Neonatal Imaging Model of Gram-Negative Bacterial Sepsis

Humanized Mouse Model to Study Bacterial Infections Targeting the Microvasculature

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood vessels resulting in vascular leak, the development of purpuric rashes and eventual tissue necrosis. Studying the pathogenesis of this infection was previously limited by the human specificity of the bacteria, which makes in vivo models difficult. In this protocol, we describe a humanized model for this infection in which human skin, containing dermal microvessels, is grafted onto immunocompromised mice. These vessels anastomose with the mouse circulation while maintaining their human characteristics. Once introduced into this model, N. meningitidis adhere exclusively to the human vessels, resulting in extensive vascular damage, inflammation and in some cases the development of purpuric rash. This protocol describes the grafting, infection and evaluation steps of this model in the context of N. meningitidis infection. The technique may be applied to numerous human specific pathogens that infect the blood stream.

Neisseria meningitidis is a human specific pathogen that infects blood vessels. In this protocol human microvessels are introduced into a mouse by grafting human skin onto immunocompromised mice. Bacteria adhere extensively to the human vessels, leading to vascular damage and development of the purpuric rash typically observed in human cases.

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood ...

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 ...

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the world&#8217;s 2.9 million neonatal deaths. Animal models of HIE have contributed to the understanding of the pathophysiology in HIE, and have highlighted the dynamic process that occur in brain injury due to perinatal asphyxia. Thus, animal studies have suggested a time-window for post-insult treatment strategies. Hypothermia has been tested as a treatment for HIE in pdiglet models and subsequently proven effective in clinical trials. Variations of the model have been applied in the study of adjunctive neuroprotective methods and piglet studies of xenon and melatonin have led to clinical phase I and II trials1,2. The piglet HIE model is further used for neonatal resuscitation- and hemodynamic studies as well as in investigations of cerebral hypoxia on a cellular level. However, it is a technically challenging model and variations in the protocol may result in either too mild or too severe brain injury. In this article, we demonstrate the technical procedures necessary for establishing a stable piglet model of neonatal HIE. First, the newborn piglet (&#60; 24 hr old, median weight 1500 g) is anesthetized, intubated, and monitored in a setup comparable to that found in a neonatal intensive care unit. Global hypoxia-ischemia is induced by lowering the inspiratory oxygen fraction to achieve global hypoxia, ischemia through hypotension and a flat trace amplitude integrated EEG (aEEG) indicative of cerebral hypoxia. Survival is promoted by adjusting oxygenation according to the aEEG response and blood pressure. Brain injury is quantified by histopathology and magnetic resonance imaging after 72 hr.

Hypoxic-ischemic encephalopathy following perinatal asphyxia can be studied using animal models. We demonstrate the procedures necessary for establishing a piglet model of neonatal hypoxic-ischemic encephalopathy.

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the ...

Medicine

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 ...

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 ...

C-section of Preclinical Animal Model of Chorioamnionitis Triggered by Group B Streptococcus (GBS)

Group B Streptococcus (GBS) is one of the most common bacteria isolated during human pregnancy. It is a leading cause of placental infection/inflammation, termed chorioamnionitis. Chorioamnionitis exposes the developing fetus to a high risk of organ injuries, perinatal morbidity, and mortality, as well as life-long neurobehavioral impairments and other non-neurological developmental issues. The two most frequent subtypes of GBS isolates from maternal and fetal tissues are serotypes Ia (13%-23%) and III (25%-53%). Our lab has developed and characterized a rat model of GBS-induced chorioamnionitis to study subsequent impacts on the central nervous system of the developing fetus and to understand underlying mechanistic aspects. This article presents the design as well as uses of the preclinical rat model, which closely reproduces the hallmark of GBS-induced chorioamnionitis in humans. This article aims to help scientists reproduce the experimental design as well as to provide support through examples of troubleshooting. The present model may also contribute to potential discoveries through uncovering causes, mechanisms, and novel therapeutic avenues, which remain unsettled in many developmental impairments arising from chorioamnionitis. Furthermore, the use of this model may be extended to the studies of perinatal non-neurological common and severe morbidities affecting, for instance, the retina, bowel, lung, and kidney. The main interest of this research is in the field of GBS-induced fetal neurodevelopmental impairments such as cerebral palsy (CP), attention deficit hyperactivity disorder (ADHD), and autism spectrum disorder (ASD). The rationale supporting this model is presented in this article, followed by procedures and results.

C-section of Preclinical Animal Model of Chorioamnionitis Triggered by Group B Streptococcus GBS

The goal of this protocol is to describe a preclinical animal model of Group B Streptococcus (GBS)-induced chorioamnionitis. The study is designed to investigate mechanistic processes, potential causal links with developmental impairments, and finally to develop translational anti-inflammatory placento- and neuro-protective treatments.

Group B Streptococcus (GBS) is one of the most common bacteria isolated during human pregnancy. It is a leading cause of placental infection/inflammation, ...

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 ...

A Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing enterocolitis (NEC). However, a mechanistic link between these two conditions remains yet to be discovered. We have adopted a murine model of chorioamnionitis involving lipopolysaccharide (LPS)-induced fetal exposure to maternal inflammation (FEMI). This model of FEMI induces a sterile maternal, placental, and fetal inflammatory cascade, which is also present in many cases of clinical chorioamnionitis. Although models exist that utilize live bacteria and more accurately mimic the pathophysiology of an ascending infection resulting in chorioamnionitis, these methods may cause indirect effects on development of the immature intestinal tract and the associated developing microbiome. Using this protocol, we have demonstrated that LPS-induced FEMI results in a dose-dependent increase in pregnancy loss and preterm birth, as well as disruption of normal intestinal development in offspring. Further, we have demonstrated that FEMI significantly increases intestinal injury and serum cytokines in offspring, while simultaneously decreasing goblet and Paneth cells, both of which provide a first line of innate immunity against intestinal inflammation. Although a similar model of LPS-induced FEMI has been used to model the association between chorioamnionitis and subsequent abnormalities of the central nervous system, to our knowledge, this protocol is the first to attempt to elucidate a mechanistic link between chorioamnionitis and later perturbations in intestinal development as a potential link between chorioamnionitis and NEC.

We developed a model of chorioamnionitis to simulate fetal exposure to maternal inflammation (FEMI) without complications of live organisms to examine the effects of FEMI on development of the offspring&#8217;s intestinal tract. This allows for study of mechanistic causes for development of intestinal injury following chorioamnionitis.

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing ...

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.

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 Neonatal BALB/c Mouse Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight ...

A Neonatal Imaging Model of Gram-Negative Bacterial Sepsis

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A Controlled Mouse Model for Neonatal Polymicrobial Sepsis

C-section of Preclinical Animal Model of Chorioamnionitis Triggered by Group B Streptococcus (GBS)

Characterizing Salmonella Typhimurium-induced Septic Peritonitis in Mice

A Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development

A Reproducible Intensive Care Unit-Oriented Endotoxin Model in Rats

A Neonatal BALB/c Mouse Model of Necrotizing Enterocolitis