A Neonatal Imaging Model of Gram-Negative Bacterial Sepsis

Summary

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.

Abstract

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.

Introduction

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 sep....

Protocol

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

1. 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 luciferase and carries kanamycin resistance³. Incubate overnight at 37 °C.
2. The following day allow Luria broth (L....

Results

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.......

Discussion

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.......

Materials

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