Antenatal Glucocorticoid Exposure Induced Neurodegenerative Changes in the Fetal Ovine Hippocampal Transcriptome Independent of Pulmonary Immaturity
[Background] Antenatal steroid (ACS) therapy conveys lifesaving benefits to the preterm fetus. However, current treatments are associated with increased risk of growth, endocrine, and neurodevelopmental abnormalities. [Objectives] To inform ACS therapy optimisation we used a sheep model of pregnancy to test whether adverse fetal brain effects were independent of respiratory benefit and ACS pharmacokinetics. [Study Design] Ewes carrying a single fetus received intramuscular injections of either: i) 2 x 12mg  dexamethasone phosphate 12 hourly (DexP) (Dex High); ii) 4 x 1.5mg DexP 12 hourly (Dex Low); iii) 1 x 0.125mg/kg betamethasone acetate (Beta-Acetate); or iv) Saline (Negative Control). Lambs were delivered for ventilation after 48 hours.  Hippocampal and lung tissues were subjected to RNA sequencing. [Results] Dex High treatment did not mature the preterm ovine lung. Pulsatile (Dex High and Dex Low) treatments caused hippocampal RNA changes consistent with neuronal death. Constant, low-amplitude treatment (Beta Acetate) significantly improved lung function, with few adverse RNA transcriptional changes in the fetal hippocampus. [Conclusions] All ACS exposures caused significant alterations in fetal homeostasis. Pulsatile exposures caused dose-independent neurodegenerative changes in the fetal hippocampus. These data highlight the importance of considering magnitude, duration and stability of fetal glucocorticoid exposures in optimising ACS therapy. Overall design: Animal Work and Antenatal Corticosteroid Treatments These studies were reviewed and approved by the University of Western Australia's Animal Ethics Committee prior to work commencing (2021/ET000974). Date-mated Merino ewes with singleton fetuses received an intramuscular injection (IM) of 150?mg medroxyprogesterone acetate (Depo-Provera, Pfizer, New York, NY) on 117 ±?2 d of gestational age (dGA) to decrease the risk of steroid-induced premature labour. Antenatal progesterone has been previously reported not to impact fetal lung maturation in the sheep 128. Baseline maternal blood samples were collected for analysis at 0800, 24 - 48hrs prior to allocation to treatment group.  Animals were then allocated to one of four groups (Figure 6), with first treatment given at 122 ±?2 dGA (term = 150 dGA). First injections occurring at 08:00 to control for circadian effects:    i) Saline Control Group, ewes received 4 x 2 ml sterile normal saline maternal IM injections at 12?h intervals (n=11);  ii) Dex High Group, ewes received 2 x 12 mg Dex-PO4 (DBL Dexamethasone sodium phosphate 4mg/ml, Hospira NZ, New Zealand) maternal IM injections at 12?h intervals followed by 2 x 2ml IM sterile saline injections at 12?h intervals (n=12); iii) Dex Low Group, ewes received 4 x 1.5 mg Dex-PO4 (DBL Dexamethasone sodium phosphate 4mg/ml, Hospira NZ, New Zealand) maternal IM injections 12?h intervals (n=12); or  iv) Beta Acetate Group, ewes received 1 x 0.125mg/kg Betamethasone Acetate maternal IM injection followed by 3 x 2 ml IM sterile normal saline injections at 12?h intervals (n=16).    Serial maternal plasma samples were collected for analysis at 6, 12, 18, 24, and 36 h post first maternal injection.  All preterm lambs included in this study for ventilation analysis (122 - 125 d gestation) were surgically delivered 48 hours after first maternal injection at 122 – 125 dGA.  Eight animals in the Beta-Acetate Group were delivered at 24 hours as per the study design but not included in ventilation analysis. One ewe from the Saline Control group was removed from the analysis as it was found not to be pregnant at pre-delivery ultrasound. One lamb from the Beta-Acetate Group was removed from the ventilation analysis as was incorrectly intubated and not ventilated for 15 minutes. Hippocampal and Lung mRNA extraction and RNA bulk sequencing  Twenty-four animals (Six animals from each group -Saline Control, Dex High, Dex Low, Beta-Acetate) were selected for RNA bulk sequencing based on the highest maternal steroid plasma concentration prior to delivery. The same animals were used for both lung and hippocampal RNA extraction and analysis thereby allowing for matched analysis with ventilation outcomes and ACS responsiveness.  Delivery and Ventilatory Assessment Maternal plasma samples for analysis were taken immediately prior to delivery, 48 hours after first maternal injection. Fetal plasma samples for analysis were taken during operative delivery and immediately prior to ventilation commencing. Prior to delivery, ewes received an intravenous bolus of midazolam (0.5?mg/kg) and ketamine (10?mg/kg) for the deep induction of anaesthesia. A 3?ml injection of 2% (20?mg/ml) lidocaine was given at L6/L7 for spinal analgesia. The head of the fetus was delivered through abdominal and uterine incisions and a 4.5?mm endotracheal tube was secured by tracheostomy. The fetus was then delivered, and the ewe euthanized under anaesthesia with pentobarbital. The lamb was weighed, dried, and placed in a radiant warmer (Cozy Cot, Fisher & Paykel Healthcare, New Zealand) bed under a plastic insulating wrap (Neowrap, Fisher & Paykel, NZ). Mechanical ventilation (Fabian HFO, Accutronic Medical Systems AG, Switzerland) was immediately commenced with the following settings: peak inspiratory pressure (PIP) of 35 cmH2O, positive end expiratory pressure (PEEP) of 5 cmH2O, respiratory rate (RR) of 50 breaths per minute, inspiratory time (iT) of 0.5?s, using 100% heated and humidified oxygen. The use of 100% oxygen allows the comparison of oxygenation through the partial arterial pressure of oxygen among the groups.   Investigators operating the ventilators were blinded to the animal treatment groups. An umbilical artery was catheterized for blood sampling and administration of supplemental anaesthesia with ketamine (5?mg/kg) if necessary. The tidal volume (VT) was continuously measured, and the PIP was adjusted to keep the VT between 7.0 and 8.0?ml/kg but with PIP limited to 35 cmH2O. At 10, 20, and 30?min of ventilation we measured temperature, blood pressure, ventilator data (PIP, VT, and compliance), and performed blood gas measurements; pH, PCO2 (mmHg), PO2 (mmHg), O2 saturation (SO2, %), total haemoglobin (Hb, g/dL) levels (Siemens RapidPoint 500, Munich, Germany). Dynamic compliance (Cdyn, ml/cmH2O/kg) was recorded as measured by the ventilator. The ventilation efficiency index (VEI) was calculated using the formula VEI = 3,800 / (RR × (PIP - PEEP) × PCO2 (mm Hg))129.  The ACS Responder Group for RNA bulk sequencing (total 6 animals) consisted of three animals from the Dex Low and three animals from Beta-Acetate Groups; ACS non-responder subgroup (total 12 animals) consisted of six animals from the Dex High Group, three animals from the Dex Low Group and three animals from Beta-Acetate Group.  RNA was extracted from snap-frozen hippocampal and lung tissue using RNeasy Plus Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. The concentration of extracted RNA and RNA Integrity Number (RIN) was determined using Agilent Technologies RNA Nano Chip as per manufacturer's instructions. All RNA extracts were diluted in nuclease-free water to yield a final RNA concentration of 30 ng/µl. Hippocampal and lung tissue bulk RNA sequencing was performed by Novogene Singapore.  Bulk RNA-sequencing Data Processing and Quality Control 150-bp paired-end sequenced reads were processed using the nf-core/rnaseq v3.10.1 pipeline130  with nextflow v22.10.4131. Briefly, raw reads were trimmed using Trim Galore! v0.6.7132 to remove low-quality bases and adapters. Trimmed reads were aligned to the human reference genome, hg38 with STAR v2.6.1d aligner133.  Finally, Salmon v1.9.0134 was utilized for assigning reads. Principle component analysis was conducted for quality control assessment.  Differential Gene Expression and Gene Set Enrichment Analysis All downstream analyses were performed using R statistical software version 4.2.1135. Differential gene expression analysis was performed using the DESeq2 package v1.36.0136 . Genes with at least 1.5-fold change in expression and FDR < 0.05 were considered significant. The results were visualized using the EnhancedVolcano v1.14.0137 and ComplexHeatmap v2.12.1138. Gene set enrichment analysis require full gene list therefore, all genes were ranked by their signed log p-value for gene set enrichment analysis (GSEA) using clusterProfiler v4.9.043  and KEGG database44.