Name: a neonatal imaging model of gram-negative bacterial sepsis
Uploaded: 2020-08-12T13:00:00
Description: Watch this Scientific Journal Video about A Neonatal Imaging Model of Gram-Negative Bacterial Sepsis at JoVE.com

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

All procedures were approved by the West Virginia Institutional Animal Care and Use Committees and conducted in accordance with the recommendations from the Guide for the Care and Use of Laboratory Animals by the National Research Council18.
1. Preparation of Bacterial Inoculum
<ol>
	<li>Streak a Tryptic soy agar (TSA) plate with an inoculating loop for isolation of a single colony from a freezer stock of E. coli O1:K1:H7-lux that stably expresses lu...

<ol>
	<li>Qazi, S. A., Stoll, B. J. <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:+a+major+global+public+health+challenge.">Neonatal sepsis: a major global public health challenge.</a> Pediatr Infect Dis J. 28, 1-2 (2009).</li><li>Simonsen, K. A., Anderson-Berry, A. L., Delair, S. F., Davies, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early-onset+neonatal+sepsis.">Early-onset neonatal sepsis.</a> Clinical Microbiology Reviews. 27 (1), 21-47 (2014).</li><li>Seman, B. G., 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=Elevated+levels+of+interleukin-27+in+early+life+compromise+protective+immunity+in+a+mouse+model+of+Gram-negative+neonatal+sepsis.">Elevated levels of interleukin-27 in early life compromise protective immunity in a mouse model of Gram-negative neonatal sepsis.</a> Infections and Immunity. , (2019).</li><li>Schrag, S. J., 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=Epidemiology+of+Invasive+Early-Onset+Neonatal+Sepsis,+2005+to+2014.">Epidemiology of Invasive Early-Onset Neonatal Sepsis, 2005 to 2014.</a> Pediatrics. 138 (6), 20162013 (2016).</li><li>Stoll, B. J., 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+onset+neonatal+sepsis:+the+burden+of+group+B+Streptococcal+and+E.+coli+disease+continues.">Early onset neonatal sepsis: the burden of group B Streptococcal and E. coli disease continues.</a> Pediatrics. 127 (5), 817-826 (2011).</li><li>Weston, E. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+burden+of+invasive+early-onset+neonatal+sepsis+in+the+United+States,+2005-2008.">The burden of invasive early-onset neonatal sepsis in the United States, 2005-2008.</a> Pediatrics and Infectious Disease Journal. 30 (11), 937-941 (2011).</li><li>Hornik, C. 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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. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+antigen-presenting+cell+function+in+human+neonates.">Defective antigen-presenting cell function in human neonates.</a> Clinical Immunology. 121 (3), 251-259 (2006).</li><li>Le Garff-Tavernier, 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=Human+NK+cells+display+major+phenotypic+and+functional+changes+over+the+life+span.">Human NK cells display major phenotypic and functional changes over the life span.</a> Aging Cell. 9 (4), 527-535 (2010).</li><li>Weinberger, B., 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=Mechanisms+underlying+reduced+responsiveness+of+neonatal+neutrophils+to+distinct+chemoattractants.">Mechanisms underlying reduced responsiveness of neonatal neutrophils to distinct chemoattractants.</a> Journal of Leukocyte Biology. 70 (6), 969-976 (2001).</li><li>Gabrilovich, D. I., Nagaraj, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloid-derived+suppressor+cells+as+regulators+of+the+immune+system.">Myeloid-derived suppressor cells as regulators of the immune system.</a> Nature Reviewss Immunology. 9 (3), 162-174 (2009).</li><li>National Research Council. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guide for the care and use of laboratory animals, 8th ed. , (2011).</li><li>Tucker, D. K., Foley, J. F., Bouknight, S. A., Fenton, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sectioning+Mammary+Gland+Whole+Mounts+for+Lesion+Identification.">Sectioning Mammary Gland Whole Mounts for Lesion Identification.</a> Journal of Visualized Experiments. (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. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescent+Imaging+Reveals+Novel+Patterns+of+Colonization+and+Invasion+in+Systemic+Escherichia+coli+K1+Experimental+Infection+in+the+Neonatal+Rat.">Bioluminescent Imaging Reveals Novel Patterns of Colonization and Invasion in Systemic Escherichia coli K1 Experimental Infection in the Neonatal Rat.</a> Infection and Immunity. 83 (12), 4528 (2015).</li><li>Singh, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inter-alpha+inhibitor+protein+administration+improves+survival+from+neonatal+sepsis+in+mice.">Inter-alpha inhibitor protein administration improves survival from neonatal sepsis in mice.</a> Pediatric Research. 68 (3), 242-247 (2010).</li><li>Pluschke, G., Pelkonen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+factors+in+the+resistance+of+newborn+mice+to+K1+Escherichia+coli+infection.">Host factors in the resistance of newborn mice to K1 Escherichia coli infection.</a> Microb. Patho. , 93-102 (1988).</li><li>Mancuso, G., 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=Role+of+interleukin+12+in+experimental+neonatal+sepsis+caused+by+group+B+streptococci.">Role of interleukin 12 in experimental neonatal sepsis caused by group B streptococci.</a> Infections and Immunity. 65 (9), 3731-3735 (1997).</li><li>Thammavongsa, V., Rauch, S., Kim, H. K., Missiakas, D. M., Schneewind, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+A-neutralizing+monoclonal+antibody+protects+neonatal+mice+against+Staphylococcus+aureus.">Protein A-neutralizing monoclonal antibody protects neonatal mice against Staphylococcus aureus.</a> Vaccine. 33 (4), 523-526 (2015).</li><li>Andrade, E. 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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>Escherichia coli O1:K1:H7</td>
			<td>ATCC</td>
			<td>11775</td>
			<td></td>
		</tr>
		<tr>
			<td>Escherichia coli O1:K1:H7-lux (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...

Watch this Scientific Journal Video about A Neonatal Imaging Model of Gram-Negative Bacterial Sepsis at JoVE.com

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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