Supplementary Material for: Biomarker Discovery by Mass Spectrometry in Cerebrospinal Fluid and Plasma after Global Hypoxia-Ischemia in Newborn Piglets

Background: Biomarkers may qualify diagnosis, treatment allocation, and prognostication in neonatal encephalopathy. Biomarker development is challenged by competing etiologies, inter-individual genetic variability, and a lack of specific neonatal markers. To address these challenges, we used a standardized neonatal hypoxic-ischemic (HI) encephalopathy model with pre- and post-HI sampling of cerebrospinal fluid (CSF) and plasma. Objectives: The study aimed to identify novel candidate protein biomarkers of HI encephalopathy in a newborn piglet model in CSF and plasma. Methods: FiO2 was lowered to 4% in 6 newborn piglets, then adjusted over a 45-min period keeping the amplitude integrated-EEG < 7 µV to induce HI encephalopathy. CSF and plasma was sampled pre-HI and 2 h after HI, protein levels were then analyzed by mass spectrometry. Results: Protein levels after HI changed significantly for 18 CSF proteins and 37 plasma proteins. CSF and plasma data showed distinct information, although peptidyl-prolyl cis-trans isomerase A had elevated levels in both fluids. HI regulation involved functional groups such as the antioxidant system, cell proliferation, cell structure, and apoptosis. S100-A8, which increased the most in CSF (9.5 fold), is known to be involved in inflammatory and immune response and to be highly regulated during injury. In plasma, increased proteins included FABP1 (31.8 fold) and proteins with antioxidant (SOD1, GPX3) and lectin function (REG3A, LGALS3). Conclusions: In this exploratory study, we have identified candidate biomarkers for HI in CSF and plasma, many not previously associated with HI. Identified proteins are promising candidates for further validation in time series experiments and clinical studies.

背景：生物标志物可为新生儿脑病的诊断、治疗方案选择及预后评估提供依据。然而，生物标志物的开发面临诸多挑战，包括多重竞争病因、个体间遗传异质性，以及缺乏特异性新生儿标志物。为应对上述挑战，本研究采用标准化新生仔猪缺氧缺血性脑病（hypoxic-ischemic, HI）模型，于缺氧缺血造模前后采集脑脊液（cerebrospinal fluid, CSF）与血浆样本。
研究目标：本研究旨在通过新生仔猪模型，筛选脑脊液与血浆中缺氧缺血性脑病候选新型蛋白生物标志物。
方法：对6只新生仔猪将吸入氧浓度（fraction of inspired oxygen, FiO2）降至4%，随后在45分钟内调整参数，维持振幅整合脑电图（amplitude integrated electroencephalogram, aEEG）低于7 µV，以诱导缺氧缺血性脑病造模。分别于造模前及造模后2小时采集脑脊液与血浆样本，随后通过质谱法分析蛋白水平。
结果：缺氧缺血造模后，脑脊液中18种蛋白、血浆中37种蛋白的水平发生显著变化。尽管肽基脯氨酰顺反异构酶A（peptidyl-prolyl cis-trans isomerase A）在两种体液中均呈表达上调，但脑脊液与血浆的蛋白组数据呈现出独特的信息特征。缺氧缺血调控的蛋白涉及多个功能类群，包括抗氧化系统、细胞增殖、细胞结构及细胞凋亡。其中，脑脊液中上调幅度最大的蛋白为S100-A8（上调9.5倍），已知其参与炎症与免疫应答，且在损伤过程中呈现显著表达调控。血浆中上调的蛋白包括脂肪酸结合蛋白1（fatty acid-binding protein 1, FABP1，上调31.8倍），以及具有抗氧化功能的超氧化物歧化酶1（superoxide dismutase 1, SOD1）、谷胱甘肽过氧化物酶3（glutathione peroxidase 3, GPX3）和具有凝集素功能的再生源蛋白3A（regenerating islet-derived protein 3 alpha, REG3A）、半乳糖凝集素3（galectin 3, LGALS3）。
结论：本探索性研究成功筛选出脑脊液与血浆中缺氧缺血性脑病候选生物标志物，其中多数此前未被报道与缺氧缺血性脑病相关。本次鉴定的蛋白有望作为候选标志物，在后续时序实验与临床研究中开展进一步验证。