Supplementary Material for: Association between cold-inducible RNA binding motif 3 and hypothermia effect in murine hypoxia-ischemia model measured by metabolic MRI

Introduction: The efficacy of therapeutic hypothermia (TH) for neonatal hypoxic-ischemic encephalopathy (HIE) is inconsistent, and the cause remains unclear. This study aimed to explore the role of cold stress protein in the TH-induced neuroprotection following hypoxia-ischemia (HI) using metabolic MRI. Methods: Postnatal day 10 (P10) mice underwent unilateral HI followed by treatments with therapeutic hypothermia (TH) or normothermia (NT). HI and sham mice were scanned at 4 h and 22 h following TH after injection of hyperpolarized 13C-1 labeled pyruvate. The dynamic HP-13C MRSIs were acquired to examine the cerebral metabolic profile, i.e., the conversion rate from pyruvate to lactate (kPL) and the ratio of lactate to pyruvate (Lac/Pyr) in the injured hemisphere. T2-weighted images (T2WI) and diffusion MR images (dMRIs) were acquired to identify the anatomical structures and assess the injury. Mice brains were collected during and at 0 h, 4 h, 12 h, 18 h and 22 h after treatments for western blot to investigate the time course of the levels of the cold stress protein (RNA binding motif 3, RBM3) and cell death markers (spectrin 145/150 and spectrin 120) changes. The cerebral metabolic profile, RBM3 and spectrin levels, and injury size were compared across groups and between specific timepoints. The relationship between the cerebral metabolic profile and RBM3 levels in HI+TH group was also evaluated. Results: We observed the upregulation of RBM3 during TH at 4 h and 22 h after TH. The spectrin 145/150 and spectrin 120 were unchanged over time in HI+TH group, whereas they significantly increased at 18 h and 22 h in HI+NT group. Additionally, the injury size was noticeably larger at 22 h in HI+NT group. Lower kPL and Lac/Pyr were observed at 4 h and 22 h after TH, with a negative correlation to RBM3 levels in HI+TH group. Conclusion: This study demonstrates that RBM3 may be one of the key factors associated with TH-induced neuroprotection by reducing the anaerobic glycolysis process in HI mice, suggesting RBM3 upregulation may enhance the efficacy of TH for neonatal HIE.引言：治疗性低温（therapeutic hypothermia, TH）用于新生儿缺氧缺血性脑病（neonatal hypoxic-ischemic encephalopathy, HIE）的疗效尚存争议，其潜在机制尚未明确。本研究旨在借助代谢磁共振成像（metabolic MRI），探讨冷应激蛋白在缺氧缺血（hypoxia-ischemia, HI）后治疗性低温介导的神经保护中的作用。方法：选取出生后第10天（postnatal day 10, P10）的小鼠，实施单侧缺氧缺血造模后，分别予以治疗性低温（TH）或常温（normothermia, NT）干预。在超极化13C标记的丙酮酸注射后，分别于治疗后4小时和22小时对缺氧缺血小鼠及假手术小鼠进行扫描。采集动态超极化13C磁共振波谱成像（dynamic HP-¹³C MRSIs）以检测损伤侧半球的脑代谢特征，即丙酮酸向乳酸的转换速率（kPL）以及乳酸/丙酮酸比值（Lac/Pyr）。同时采集T2加权成像（T2WI）与弥散磁共振成像（dMRIs），用于识别脑部解剖结构并评估损伤程度。分别于干预期间及干预后0小时、4小时、12小时、18小时、22小时采集小鼠脑组织，通过蛋白质印迹法（western blot）检测冷应激蛋白RNA结合基序3（RNA binding motif 3, RBM3）及细胞死亡标志物血影蛋白145/150、血影蛋白120的水平随时间的变化趋势。对各组间、各时间点间的脑代谢特征、RBM3与血影蛋白水平以及损伤体积进行比较分析，并评估缺氧缺血+治疗性低温组（HI+TH）中脑代谢特征与RBM3水平的相关性。结果：本研究观察到，在治疗性低温干预期间，小鼠脑组织中RBM3的表达在治疗后4小时及22小时均出现上调。在HI+TH组中，血影蛋白145/150与血影蛋白120的水平随时间无明显变化；而在HI+NT组中，二者的水平在治疗后18小时、22小时显著升高。此外，HI+NT组小鼠在干预后22小时的损伤体积明显更大。在TH干预后4小时及22小时，小鼠的kPL与Lac/Pyr水平均较低，且在HI+TH组中二者与RBM3水平呈负相关。结论：本研究表明，RBM3可能通过降低缺氧缺血小鼠的无氧糖酵解过程，成为治疗性低温介导神经保护的关键调控因子之一，提示上调RBM3的表达或许可提升治疗性低温对新生儿缺氧缺血性脑病的疗效。