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. However, even though Group B Streptococcus (GBS) is the most frequent cause of perinatal infection, few animal models have addressed its role in inflammatory mechanisms at play and the outcomes⁵.

GBS is an encapsulated gram-positive coccus that colonizes the lower genital tract in approximately 15%-30% of pregnant women⁶. It leads to placental infection/inflammation, termed chorioamnionitis^7,8. Of the ten GBS serotypes, the two most frequent serotypes Ia and III are major infectious determinants of injuries in maternofetal tissues^9,10. GBS infection has been shown to lead to a higher inflammatory response in fetal blood and placental deficiency, which are highly suspected to be involved in multiple neurodevelopmental disorders such as cerebral palsy (CP), attention deficit hyperactivity disorder (ADHD), and autism spectrum disorder (ASD)^5,11.

Over the past ten years, we have developed a rat model of GBS-induced chorioamnionitis that leads to various developmental impairments in the offspring¹². This preclinical model demonstrates the causal link between GBS-induced placental inflammation and a range of sex-specific neurodevelopmental impairments in the offspring^13,14,15. The goal of this article is to provide readers with insight into the design of a preclinical rat model of end-gestational infection and resulting neuro-behavioral impairments in the offspring. The present protocol aims to mimic the clinical reality of GBS-induced chorioamnionitis.

Results from this preclinical model show that end-gestational intra-peritoneal (IP) inoculation (Figure 1) of GBS leads to (i) placental infection and inflammation, fulfilling the diagnostic criteria of chorioamnionitis¹⁶; (ii) a massive upregulation of IL-1β and downstream inflammatory molecules from the IL-1-pathway, within the placenta¹²; (iii) neurodevelopmental impairments in the offspring¹²; (iv) sex differences in immune responses and subsequent neurobehavioral impairments, such as female offspring presenting adult ADHD-like traits while male offspring present early-onset and long-lasting ASD-like traits; (v) distinct neurobehavioral outcomes in the progeny depending on the GBS serotype used to induce chorioamnionitis^14,15. In line with these findings, the main next steps utilizing this model will be to test, firstly, the role of androgen in GBS-induced chorioamnionitis and, secondly, the placental- and neuro-protective role of molecules targeting specific inflammatory pathways, in the hope to bring some of these molecules to the threshold of therapeutic clinical trials.

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 libitum¹⁷.
2. Weigh dams daily to detect any illness behavior from G14 (i.e., the day of arrival) until G22 (i.e., the day of C-section)

2. Bacterial growth

1. On G18, prepare two sterile test tubes with 5 mL of sterile Brain Heart Infusion (BHI) broth. Take a small portion of frozen bacteria stock (β-hemolytic capsular serotype Ia in BHI and 15% glycerol¹⁴) from -80 °C and add it into 5 mL BHI tubes (Figure 2).
2. Place the tubes in the shaker (240 rpm) for 18 h at 37 °C.
3. On G19, prepare a 3% solution of GBS in sterile BHI broth by collecting 1.5 mL of the incubated solution into 48.5 mL sterile BHI broth.
4. Collect 1.5 mL of the 3% GBS plus BHI solution into a cuvette. Using a spectrophotometer, record the initial absorption as T₀ (optical density (OD)_{600 nm}).
   NOTE: A blank made with sterile BHI broth was used each time to balance the spectrophotometer.
5. Place the 3% solution in the incubator at 37 °C with 240 rpm shaking for approximately 2 h.Check the absorption every 20 min after 2 h until a measure of absorbance between 0.6 and 0.8 (OD_{600 nm}) has been reached.
6. After reaching the desired absorbance, collect 20 mL of 3% GBS plus BHI solution and add it to a 50 mL tube.
7. Centrifuge (1792 x g) the samples at 4 °C for 13 min and wash the precipitated GBS twice with 20 mL of 0.9% sterile saline.
8. Suspend the precipitated GBS in 2 mL of 0.9% sterile saline. Keep this aliquot on ice until the time of injection.
9. Inject (intraperitoneally) the control group with 100 µL of sterile 0.9% saline and the GBS group with 100 µL β-hemolytic serotype Ia GBS suspended in sterile 0.9% saline.
   NOTE: The injected dose was 10⁸ colony forming units (CFUs) of GBS or saline (for control). Inoculation of 10⁸ CFU has been well established as a model of human chorioamnionitis. Inoculation with a higher dose of GBS will likely cause dam mortality. Injecting less than the mentioned dose will not mimic the infection and inflammation.
10. Make dilutions between 10^-5 and 10^-10 and plate the dilutions in triplicate on BHI agar plates. To rule out contamination, make two negative controls (without any addition of substance), one on a BHI agar plate and the other on a CHROMID Strepto B agar plate. Make two positive controls by plating the prepared bacteria on the BHI agar plate and the CHROMID Strepto B agar plate. Place all plates in the incubator overnight at 37 °C (Figure 3).
    NOTE: CHROMID Strepto B agar plates are a selective medium for screening GBS on which the GBS colonies appear red.

3. Injection technique

1. On G19, remove the rat gently from the cage and place it onto a flat surface. Immobilize the rat by using a towel to cover the head and upper body. Lift the hind leg to allow easy access to the injection site.
   NOTE: Make sure the appropriate anatomical area for injection is in the lower right quadrant of the abdomen to avoid puncture of organs such as the urinary bladder and cecum (Figure 1).
2. Use a U-100 insulin syringe with a 29 G ½ inch needle. Insert the needle bevel facing up towards the head at a 40-45° angle to the horizontal, as shown in Figure 1. Perform GBS injections once for each dam. Make sure to perform injections every 1 h to avoid a time effect between inoculated dams.
   NOTE: Injections should be varied between the left and right sides on days when more than one injection per day is performed.

4. Dose determination

1. On G20, verify four controls (step 10.2) and count the bacterial colonies on each BHI agar plate.
2. Calculate the mean GBS colonies for each dilution factor (10^-5 to 10^-10) to determine the exact injected dose of GBS

5. C-section and tissue collection

1. Perform C-sections on G22 (72 h post-injection) and perform subsequent surgeries with 1 h between dams according to each dam's inoculation time.
2. Anesthetize the dam in a euthanasia chamber with 2% isoflurane and 1.5% O_2, for general anesthesia.
3. Place the dam on a heating pad covered with an appropriate surgical dressing and apply the ophthalmic ointment to the eye to avoid drying.
4. Prepare the surgical area by removing hair from the lower abdominal area using a blade or scalpel.
5. Clean the surgical area with sterile gauze soaked with disinfectant.
6. Using a sterile scalpel and fine-tipped scissors, make a horizontal incision in the lower abdomen of the rat. Make a vertical incision on either side of the abdomen to reveal underlying organs.
7. Separate placenta samples from fetuses. Record the weights of fetuses, placentas, and the fetus/placenta ratio.
8. Using a sterile scalpel, cut the placenta into two halves.
   1. Use 2-methylbutane to fast freeze one-half of the placenta and keep at -80 °C until needed for determination of protein levels using ELISA.
   2. Fix the other half of the placenta in 4% buffered formaldehyde for in situ analysis by immunohistochemistry (IHC) to study the expression of GBS and polymorphonuclear cells (PMNs) in collected placentas.
9. Decapitate to collect blood from live fetuses and transfer the blood to Lithium Heparin Gel Separator tubes.
10. Centrifuge (18,928 x g) blood samples at 4 °C to separate the plasma and store the plasma samples at -80 °C until further analysis.
    NOTE: The collected fetal blood plasma samples will be used for ELISA to check the protein levels of different cytokines in fetus blood.
11. Collect fetal tails to determine the sex of fetuses by amplification of a sequence within the SRY gene, using the following primers (forward primer: 5' - TAC AGC CTG AGG ACA TAT TA3'; reverse primer: 5' - GCA CTT TAA CCC TTC GAT GA -3') as described earlier¹⁸.
12. Using a 5 mL 23 G needle, collect blood from the dam by cardiac puncture to check and compare protein levels of different cytokines in the dam blood with those in fetal blood. Euthanize the dams by diaphragm puncture and decapitation method.
    NOTE: Between animals, clean all the used instruments with sterile tissue and sterile saline. To perform neuropathological and behavioral studies in the progeny, dams gave birth naturally on G23. After euthanizing offspring on postnatal day (PN) 80, brains were collected for molecular and histological studies.

Results

IP inoculation of GBS resulted in placental infection
Immunohistochemistry (IHC) (using polyclonal antibodies targeting GBS serotype Ia) staining showed that GBS infection reached the decidual compartment of the placenta. Infection also spread from the decidua to the labyrinth, chorionic plate, and in some instances, to fetuses leading to fetal death (5.8 ± 0.8 in GBS-exposed vs. 9.3 ± 0.6 pups in control (CTL) litters)¹⁸. Hence, the litter size was decreased at birth among GB...

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

List of materials used in this article

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