Creatine in the fetal brain: A regional investigation of acute global hypoxia and creatine supplementation in a translational fetal sheep model

Abstract

BackgroundCreatine supplementation during pregnancy is a promising prophylactic treatment for perinatal hypoxic brain injury. Previously, in near-term sheep we have shown that fetal creatine supplementation reduces cerebral metabolic and oxidative stress induced by acute global hypoxia. This study investigated the effects of acute hypoxia with or without fetal creatine supplementation on neuropathology in multiple brain regions.MethodsNear-term fetal sheep were administered continuous intravenous infusion of either creatine (6 mg kg–1 h–1) or isovolumetric saline from 122 to 134 days gestational age (dGA; term is approx. 145 dGA). At 131 dGA, global hypoxia was induced by a 10 min umbilical cord occlusion (UCO). Fetuses were then recovered for 72 h at which time (134 dGA) cerebral tissue was collected for either RT-qPCR or immunohistochemistry analyses.ResultsUCO resulted in mild injury to the cortical gray matter, thalamus and hippocampus, with increased cell death and astrogliosis and downregulation of genes involved in regulating injury responses, vasculature development and mitochondrial integrity. Creatine supplementation reduced astrogliosis within the corpus callosum but did not ameliorate any other gene expression or histopathological changes induced by hypoxia. Of importance, effects of creatine supplementation on gene expression irrespective of hypoxia, including increased expression of anti-apoptotic (BCL-2) and pro-inflammatory (e.g., MPO, TNFa, IL-6, IL-1β) genes, particularly in the gray matter, hippocampus, and striatum were identified. Creatine treatment also effected oligodendrocyte maturation and myelination in white matter regions.ConclusionWhile supplementation did not rescue mild neuropathology caused by UCO, creatine did result in gene expression changes that may influence in utero cerebral development.

The synergistic effects of mechanical ventilation and intrauterine inflammation on cerebral inflammation in preterm fetal sheep

“…RNA extraction, cDNA preparation and analysis were conducted as described previously ( Tran et al, 2023 ). Briefly, RNA was extracted from frozen brain tissue (20–30 mg) using an RNA extraction kit (RNeasy Mini Kit, Qiagen, Germany) following the manufacturer’s instructions.…”

“…Immunohistochemical staining was conducted using primary antibodies: rabbit anti-neuronal nuclei (NeuN; 1:200; Abcam, UK; CAT#: 177487) for mature neurons; rabbit anti-ionized calcium binding adaptor molecule 1 (Iba-1; 1:250; Abcam, UK; CAT#: ab178846) for microglia; rabbit anti-glial fibrillary acidic protein (GFAP; 1:200; Abcam, UK; CAT#: ab68428) for astrocytes; rabbit anti-Olig-2 (1:200; Abcam, UK; CAT#:ab109189) for oligodendrocytes. Immunohistochemical staining protocol was conducted as described previously ( Tran et al, 2023 ). Briefly, tissue sections were subjected to antigen retrieval using citrate buffer (10 mM Tri-sodium citrate in dH 2 O, pH 6.0; Sigma Aldrich), PBS washes, endogenous peroxidase blocking, and overnight incubation in primary antibodies at 4°C in primary diluent of 3% normal goat serum in PBS.…”