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

Summary

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.

Abstract

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.

Introduction

Maternal immune activation (MIA) has been described as one of the most critical independent risk factors for premature birth, fetal death, and lifelong cognitive and behavioral impairments in the progeny^1,2,3,4. Much of the existing preclinical research about the role of gestational inflammation on placental and developmental outcomes uses pathogen components, such as lipopolysaccharide (LPS) from E. coli and the synthetic analog of viral double-stranded RNA, polyinosinic: polycytidylic acid (Poly[I: C]), that mimic viral infections....

Protocol

All experiments were approved by the Research Institute of McGill University Health Centre (RI-MUHC). All experiments were performed according to the Canadian Council on Animal Care.

1. Pregnant Lewis rats

1. Obtain Lewis rats from commercial sources at gestational day (G)14. House them in an appropriate animal facility (RI-MUHC animal facility) in a controlled environment at 20-23 °C with a 12 h light/dark cycle, and access to water and food ad libitum<.......

Discussion

Critical steps in the protocol
Several steps of the protocol are critical and require some quality controls. For instance, there is a risk of contamination of the GBS stock by other pathogens. This can be rapidly identified using the appropriate technique of GBS microbial identification such as colony aspect on BHI agar (e.g., size, shape, color), plating in duplicate the β-hemolytic GBS dose on Columbia blood agar with 5% sheep blood medium and on CHROMID Strepto B agar, a selective chromogen.......

Materials

Name	Company	Catalog Number	Comments
5 mL sterile tube	BD Biosciences
50 ml falcon tubes	Thermo Fisher	339652
Blade or scalpel	BD Medical	371716
Brain Heart Infusion Broth	Criterion (Hardy diagnostics)	C5141
CHROMID Strepto B agar plate	BioMerieux, Saint-Laurent	43461
Columbia blood agar 5 % with sheep blood medium	Thermo Scientific	R01215
Forward primer	5' - TAC AGC CTG AGG ACA TAT TA3'	Sigma
Insulin syringe	Becton, Dickinson and Co(BD)	324702
Lewis rats	Charles River Laboratories
Methylbutan	Sigma Aldrich	M32631
Microtainer blood collection tubes	Becton, Dickinson and Co(BD)	365965
Reverse primer	5' - GCA CTT TAA CCC TTC GAT GA -3'	Sigma
Serological Pipettes 1 ML	Thermo Fisher	170353N
Serological Pipettes 10 ML	Thermo Fisher	170356N
Serological Pipettes 25 ML	Thermo Fisher	170357N
Serological Pipettes 5 ML	Thermo Fisher	170355N
Superfrost Plus Micro Slide, Premium	VWR	CA48311-703