Characterizing the developmental, motor, and neuroimmunological impacts within a non-invasive two hit mouse model of neonatal hypoxic ischemic encephalopathy
Date
2025
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
Hypoxic ischemic encephalopathy (HIE) is a perinatal brain injury characterized by a lack of oxygen or blood flow to the fetal brain. HIE is one of the most serious causes of neurological deficits in children born at term and can lead to significant long-term deficits including cerebral palsy, epilepsy, learning deficits, and vision/hearing impairments. There are limits to therapeutic hypothermia, the only current treatment of HIE, as it must be initiated within six hours following birth, and up to 40% of treated infants still experience adverse neurodevelopmental outcomes. Other treatments have shown promise in animal models; however, none have been effective in clinical trials. This points to the need for additional animal models that better incorporate the risk factors and etiology of HIE to develop and test additional treatments. ☐ Maternal immune activation (MIA) is a significant risk factor for the development of HIE and cerebral palsy. MIA is hypothesized to have a priming effect on microglia, the immune cells of the brain, leading to an exacerbated proinflammatory response and worse outcomes following a later immune challenge. It is easy to include MIA in animal models through the peripheral injection of bacterial or mimetics into pregnant animals, additionally incorporating the maternal and fetal interaction that occurs in HIE. ☐ Our novel, non-invasive model of HIE pairs maternal immune activation via the administration of lipopolysaccharide (LPS) on gestational day 18 with a progressive hypoxia to 0% oxygen for 8 minutes on postnatal day 6 (P6). The aims of this dissertation are 1) to characterize developmental changes and long-term motor outcomes within our model of HIE, 2) to investigate areas of injury or altered microglia response within the brain at acute and long-term timepoints, and 3) to characterize the neuroimmune response to our model of HIE. ☐ We found that our model of HIE leads to significant developmental delays within the neonatal period in most, but not all, measures. In adulthood, HIE animals exhibited long-term motor deficits including reduced forelimb strength and gait disturbances but not altered social behavior. Anatomical MRI one day following the second hit of hypoxia identified that HIE animals trended towards a decrease in overall brain volume, but did not have a decrease in the volume of specific brain regions. In adulthood, MRI did not find any differences in overall brain volume. We utilized immunohistochemistry to quantify the density of apoptotic cells, neurons, and microglia in motor regions one day after hypoxia (P7). HIE animals had significantly increased microglial density in the cerebellum, indicating region-specific neuroinflammation. There were no differences in the density of apoptotic cells or neurons in either the dorsal striatum or the cerebellum on P7. We additionally analyzed neuronal density in adulthood in the dorsal striatum and found no decreases due to HIE. We utilized bulkRNAseq to characterize the initial transcriptional response of microglia to HIE. On P7, microglia exhibited an upregulation of pro-inflammatory, proliferation-related, and apoptotic gene sets. Single cell-RNAseq on P8/P10 revealed two microglia subclusters of interest following HIE. Pseudobulk analysis of this data revealed increased expression of genes in microglia related to mobility and localization of cells following HIE, as well as an upregulation of epigenetic machinery and neurodevelopmental genes in macrophages. ☐ Together these results support our model as a valid model of HIE as it produces motor deficits throughout the lifespan as well as an initial proinflammatory response in microglia following insult. We were additionally able to characterize the nuanced response of microglia and macrophages within our model. This model does not produce an overt focal histological injury or increase in apoptosis at the timepoints assessed and therefore represents a milder or global injury phenotype. HIE in humans is highly heterogeneous and varies in severity and outcome. Therefore, this novel model fills a niche which will contribute to further our understanding of the variable phenotype of HIE.
Description
Keywords
Maternal immune activation, Neonatal brain injury, Neuroimmunity