290 - Hippocampal Neuronal Loss after Neonatal Hypoxic-Ischemic Brain Injury

Publication Number: 4285.290

Eesha Natarajan, University of California San Francisco, San Francisco, CA, United States; Donna Ferriero, University of California, San Francisco, School of Medicine, Stanford, CA, United States; Emin Maltepe, UCSF Benioff Children's Hospital San Francisco, San Francisco, CA, United States; JANA MIKE, University of California, San Francisco, School of Medicine, SAN FRANCISCO, CA, United States

Background: Hypoxic-ischemic encephalopathy remains a major cause of neonatal mortality and long-term neurological disability, with limited treatment options beyond therapeutic hypothermia. Advancing effective treatments requires a deeper understanding of the complex and dynamic biological responses to hypoxic-ischemic brain injury (HI).

Objective: We used Sequential Fluorescence In-Situ Hybridization (SeqFISH) to map spatial gene changes after neonatal HI in our murine model.

Design/Methods: Neonatal HI was induced at postnatal day 10 by permanent coagulation of the left common carotid artery (ischemia), 1-hour recovery, and 10% O₂/90% N₂ for 50 minutes at 37 °C (hypoxia). Brains were collected at multiple post-injury time points, sections from OCT-embedded frozen brains were processed on the Spatial Genomics Gene Positioning System (GenePS) using the Mouse Brain Mapper Panel supplemented with ARG1, ARG2, fibrosis-related (TGFB1, COL1A2) and efferocytosis-related genes (Rac1, STAT6).Images were analyzed using Spatial Genomics Navigator, followed by clustering and differential expression analysis in MATLAB.

Results: The hippocampal neurons were among the most significantly injured cells by HI insult. These neurons formed Cluster 10 and spatially located to CA1-3 of the hippocampus. At Day 1 HI, these cells display prominent Rac1/STAT6 induction, as well as COL1A2 compared to sham mice, indicating early activation of efferocytotic pathways and extracellular matrix remodeling. By Day 5 HI, there is increased expression of ARG1, IFNGR1, and TGFB1, suggestive of activation of pro-repair pathways, while sham mice expressed ARG2, suggesting different functions of arginase isoforms.

Conclusion(s): Our preliminary data suggest early activation of efferocytosis and extracellular matrix remodeling pathways in neonatal hippocampus after HI. Further studies are necessary to validate the mechanistic pathways identified in our study.

Temporal changes in proportion of left hippocampal glutamatergic neuron cluster 10 after HI
Box-and-whisker plots show the proportion of cells assigned to Cluster 10 on D1 vs D5 in HI animals vs sham controls. These glutamatergic neurons declined in abundance by D5 following HI, when compared with both HI D1 (p=7.46E-04) and sham D5 (p=5.5E-06), indicating a selective loss of neurons in response to HI.

Select gene expression in Cluster 10 
Dot plot shows average expression (color; red = higher, blue = lower) and percent of cells expressing each gene (dot size) across Day 1 and Day 5 HI versus time-matched shams. At Day 1 HI, these neurons display prominent Rac1 induction as well as increased COL1A2 expression compared to sham mice. By Day 5 HI, a broader remodeling/repair pathway emerged with increased ARG1, COL1A2, IFNGR2, IFNGR1, and TGFB1, persistent Rac1 and less suppressed STAT6 expression among the HI mice compared to shams.