Cerebral hypoxia, microglial activation and altered brain development in lamb fetuses with left congenital diaphragmatic hernia

Abstract Congenital diaphragmatic hernia (CDH) is a developmental defect of the diaphragm leading to abdominal viscera herniation into the thoracic cavity. Children with CDH are at increased risk of neurodevelopmental disorders. Underlying alterations in cerebral blood flow secondary to the mechanical effects of left-sided CDH (L-CDH) have been suggested to be associated with prenatal brain injury. We aimed to characterize the regional neuroinflammatory, hypoxic, metabolic, and neurodevelopmental changes in a fetal L-CDH lamb model. Eight brain regions were examined using immunohistochemistry (GFAP: astrocyte density, IBA1: microglial density and morphology, NeuN: mature neurons, CD34: vascular density), in situ hybridization (HIF1A) and RNA sequencing (frontal lobe and thalamus samples). CDH lambs exhibited increased HIF1A expression in the hippocampus and elevated vascular density in the cerebral cortex, cerebellum, and brain stem, consistent with hypoxia-driven neoangiogenesis. Increased IBA1-positive microglial density accompanied by clustering patterns consistent with microglial activation was identified in the corpus callosum, cerebellum, cerebral cortex, and brain stem. Reduced astrocyte density was found in the corpus callosum and cerebellum, and anomalies in mature neuron density were found in the cerebral cortex and cerebellum. Histological findings were consistent with transcriptomic data from the frontal lobe and thalamus. These findings are consistent with the hypothesis that mechanical effects of L-CDH are associated with chronic cerebral hypoperfusion, triggering hypoxia-induced pathways and neuroinflammatory responses that may disrupt normal brain development. However, given the multifactorial nature of the experimental model, causal attribution requires further investigation.