High resolution transcriptome analysis on a mouse model of hypoxic ischemic encephalopathy using single-nucleus RNA-seq

Hypoxic-ischemic encephalopathy (HIE) encompasses brain injuries resulting from reduced or interrupted oxygen or blood flow to the brain before, during, or immediately after birth. During the acute phase, neuronal damage is driven by excitotoxicity, with permanent injury manifesting over the subsequent hours. Current treatment options have limited efficacy, underscoring the need for deeper insights into HIE pathogenesis and the development of novel therapeutic strategies. Recent advances in single-cell RNA sequencing (snRNA-seq) have enabled molecular investigations of diverse diseases. However, the large size of neurons has posed challenges in studying conditions where neuronal damage is central. To address this, we employed snRNA-seq to evaluate region-specific neuronal damage in a mouse model of HIE. Our HIE model was established in postnatal day-7 mice, with cortical and hippocampal tissues harvested from the ipsilesional side one week post-injury. Analysis revealed pronounced changes in the hippocampus, especially in the CA1 region, with a significant reduction in neuronal populations in the affected hemisphere. These alterations were specific to combined hypoxic-ischemic conditions and were not observed with hypoxia or ischemia alone. In contrast, changes in the cerebral cortex were less prominent. Transcriptomic analysis showed similar gene expression patterns in the cortex and hippocampus of the HIE group, including the identification of genes related to viral response pathways, suggesting a potential link to interferon activation. These findings provide valuable insights into the molecular and anatomical impact of HIE and highlight the hippocampus as a critical focus for understanding disease mechanisms and therapeutic development. Overall design: HIE group (hypoxia exposure added to unilateral common carotid ligation), Hypoxia group, CL group (unilateral common carotid ligation), and Sham group(only sham surgery performed) mice were created on postnatal day7, and the cortex and hippocampus of the ipsilesional side were harvested for single-nucleus RNA sequencing one week later, respectively.A total of 4 mice, 2 males and 2 females per group, were included.缺氧缺血性脑病（Hypoxic-ischemic encephalopathy, HIE）指围产期（分娩前、分娩过程中及分娩即刻）因脑部血流或氧气供应减少、中断所引发的脑损伤。在急性期，神经元损伤由兴奋性毒性介导，永久性脑损伤会在后续数小时内逐步显现。目前临床治疗方案的疗效较为有限，凸显了深入阐明HIE发病机制、开发新型治疗策略的迫切需求。近年来，单细胞核RNA测序（single-nucleus RNA sequencing, snRNA-seq）技术的发展为多种疾病的分子机制研究提供了有力工具。然而，神经元体积较大的特性给以神经元损伤为核心的疾病研究带来了诸多挑战。为解决上述问题，本研究采用单细胞核RNA测序技术，对缺氧缺血性脑病小鼠模型的脑区特异性神经元损伤开展评估。本研究的HIE模型构建于出生后7天的小鼠，于造模后1周采集损伤侧的皮层与海马组织。分析结果显示，海马体尤其是CA1区出现显著损伤变化，受损半球的神经元数量显著减少。上述改变仅见于缺氧缺血联合损伤模型，单纯缺氧或单纯缺血模型均未出现此类改变。与之相比，大脑皮层的损伤变化相对较轻。转录组分析显示，HIE组小鼠的皮层与海马体呈现相似的基因表达谱，其中涵盖与病毒应答通路相关的基因，提示该过程可能与干扰素激活存在潜在关联。本研究结果为阐明HIE的分子与解剖学层面影响提供了重要参考，并凸显出海马体是阐释HIE发病机制、开发治疗策略的关键研究靶点。整体实验设计：本研究于出生后7天的小鼠中构建了四组模型，分别为HIE组（单侧颈总动脉结扎联合缺氧暴露）、单纯缺氧组、单纯结扎组（单侧颈总动脉结扎）以及假手术组（仅实施假手术）；造模后1周，分别采集各组小鼠损伤侧的皮层与海马体组织用于单细胞核RNA测序。每组纳入4只小鼠，雌雄各2只。