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

Abstract

Background: Creatine 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.

Methods: Near-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.

Results: UCO 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.

Conclusion: While supplementation did not rescue mild neuropathology caused by UCO, creatine did result in gene expression changes that may influence in utero cerebral development.

Keywords: creatine metabolism; hypoxia-ischemia encephalopathy; neuroprotection; perinatal asphyxia (PNA); perinatal brain injury.

FIGURE 1

Volcano plot visualizations of (A) umbilical cord occlusion (UCO) and (B) creatine main effects, and (C) SalUCO vs. CrUCO post-hoc comparisons. Volcano plots show fold change [log₂(fold change); i.e., log₂(fold change = 2) = 1; x-axis] and all gene expression changes [−log₂(adjusted P-value); y axis] in all brain regions: GM (●), WM (■), hippocampus (▲), striatum (◆) and thalamus (▼). Gray dashed line represents a P-value of 0.05 [−log₁₀(0.05)≈4.139]. Names of genes with P < 0.05 are included. Genes that were downregulated are presented in red and those upregulated in blue. (D) Heat map representation of gene expression determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) within saline control (SalCon; n = 5), creatine control (CrCon; n = 7), saline UCO (SalUCO; n = 7) and creatine UCO (CrUCO; n = 6) fetuses. Brain regions analysed include cortical grey matter (GM); white matter (WM); hippocampus (Hipp); striatum; and thalamus. Red indicates downregulation of mRNA expression; blue indicates upregulation relative to saline controls. Data are average log₂ transformed fold change.

FIGURE 2

(A) Representative images of TUNEL-positive cells (black arrows) indicating cell death in thalamus. (B) Representative images of sheep serum extravasation from blood vessels into the brain parenchyma (arrows), and sheep serum contained within the blood vessels (arrowhead) in the subcortical white matter (SCWM) and cortical grey matter (GM). Scale bar represents 100 μm.

FIGURE 3

(A) Representative images of NeuN-positive cells indicating mature neurons in the cortical grey matter (GM). GM counts were conducted in four bins roughly equivalent to cortical layer I (Bin 1), layer II and III (Bin 2), layer IV and V (Bin 3), and layer VI (Bin 4). No significant differences of bins were found between groups, therefore data were combined and presented as a total average for GM. (B) Representative images of IBA-1-positive cells indicating microglia in the subcortical white matter (SCWM). Insert demonstrates ramified microglia. (C) Representative images of glial fibrillary acidic protein (GFAP)-positive cells and processes indicating astrocytes in the dorsal hippocampal (Hipp) CA1-3, thalamus and corpus callosum (CC). Scale bar represents 100 μm.

FIGURE 4

(A) Representative images of Olig2-positive oligodendrocytes in the subcortical white matter (SCWM). (B) Representative images of CNPase-positive staining in the periventricular white matter (PVWM) and external capsule. (C) Representative images of MBP-positive oligodendrocytes and fibers indicating mature myelination in the rostral SCWM. Scale bar represents 100 μm.