Comparative Analysis of Neonatal Hypomyelination Models Using Spatial Transcriptomics

Abstract

White matter injury (WMI) is a major cause of morbidity in premature infants, contributing to 5%-10% of cerebral palsy cases and up to 50% of cognitive and behavioral deficits in the United States. Two commonly used preclinical models, intermittent hypoxia (IH) and hypoxia-ischemia (HI) are widely employed to investigate the effects of WMI. The internal capsule (IC) and corpus callosum (CC) are major white matter tracts undergoing active myelination during the neonatal period, making them particularly vulnerable to hypoxic insults. This study aims to compare the effects of IH and HI models on myelination as well as the involvement of inflammatory cells in the IC and CC. We evaluated five oligodendrocyte (OL) subtypes, along with astrocytes, microglia, and activated microglia in IC and CC at postnatal day 12 (P12) and day 20 (P20) using spatial transcriptomics (CosMx, Novogene). For the HI model, C57BL/6 mice at P10 underwent permanent ligation of the left carotid artery followed by 45 minutes of hypoxia (8% O2 / 92% N2). For the IH model, P3 mice were exposed to 5% O2 / 95% N2, twice daily for five consecutive days. Animals were euthanized at P12 and P20, perfused transcardially, and brains were post-fixed in 4% paraformaldehyde, dehydrated in an ethanol series, embedded in paraffin, and coronally sectioned at 7 m. Slides were submitted for CosMx spatial transcriptomic analysis (NanoString Technologies), and data analysis was performed using the Seurat package in RStudio. Our results demonstrate that IH and HI models affect OL populations differently, and these effects vary by brain region. In the IC, the IH model caused earlier and more pronounced changes in OL differentiation-related gene expression compared to HI. In contrast, the CC was more affected by HI. Moreover, in the HI group, mature OL s in both regions showed reduced expression of myelination-associated genes. This was accompanied by greater activation of inflammatory cells and increased intercellular communication between these cells and mature OLs, potentially contributing to the observed hypomyelination. Overall, our study provides critical insights into how each model of neonatal hypoxia differentially impacts white matter development. This knowledge can help refine preclinical strategies and guide therapeutic research tailored to the underlying pathology of each model.