Hyperbilirubinemia and phototherapy differentially alter hippocampal transcriptomic patterns in a Gunn rat model of neonatal hyperbilirubinemia.

Neonatal hyperbilirubinemia (jaundice) is common in infants, with extremely preterm infants (EPT, <28 weeks gestational age) being at high risk for bilirubin-induced neurotoxicity, resulting in neurodevelopmental impairment. Hyperbilirubinemia is treated using phototherapy to lower unconjugated bilirubin levels. However, the benefits and risks of phototherapy in EPTs have not been well studied, and bilirubin at low levels may be protective as an antioxidant. Phototherapy is associated with markers of oxidative stress in the plasma, but the effects of phototherapy on the hippocampus (HPC) are not known. Bilirubin and insults associated with EPTs impair hippocampal development, a brain structure critical for cognitive function, but their underlying mechanisms remain unknown. The effects of hyperbilirubinemia and phototherapy on the HPC were studied using a Gunn rat model. Jaundiced (jj) and non-jaundiced (Nj) pups were subjected to phototherapy from postnatal day 4 (P4) through P6. The HPC was harvested and processed for RNA sequencing. Serum bilirubin levels were elevated in jj compared to Nj control rats. Phototherapy significantly lowered serum bilirubin levels in jj rats. Compared to Nj rats, 1294 genes were differentially expressed in the jj hippocampal transcriptome and mapped onto the nervous system development, inflammation, and ferroptosis signaling pathways. Phototherapy induces 3297 differentially-expressed genes (DEGs) in rat hippocampal transcriptome compared to untreated rats. These DEGs were annotated to pathways regulating synaptogenesis, long-term potentiation, and neurogenesis. Both hyperbilirubinemia and phototherapy altered expression of 407 genes, which mapped onto hippocampal plasticity functions, including neuritogenesis and long-term potentiation. Our study demonstrates a model for investigating molecular effects of hyperbilirubemia and phototherapy in an EPT-equivalent Gunn rat pup. Our data revealed the effects of hyperbilirubinemia and phototherapy on signaling pathways critical for hippocampal development and plasticity. Overall design: Gunn rat pups (non-jaundiced (Nj) controls and jaundiced (jj) pups (n=5/group ) were exposed to intermittent LED phototherapy from postnatal day 4 (P4) through P6. Phototherapy was provided for a total of 12 hours (3 hours on/3 hours off during daytime hours). Untreated Nj and jj (n=6) were used as controls. Rats were killed on P6 using sodium pentobarbital (100 mg/kg, i.p.). A blood sample (250 µL) was collected via cardiac puncture. Hippocampal lobes were dissected on an ice-cold metal block, flash-frozen using liquid nitrogen, and stored at -80°C until analysis. Total RNA was isolated from one hippocampal lobe using RNAqueous Isolation kit (Ambion). Five rats were used per experimental group (Nj+No PT, jj+No PT, Nj+PT, jj+PT). RNA was further processed for sequencing.

新生儿高胆红素血症（Neonatal hyperbilirubinemia，俗称黄疸，jaundice）在婴儿群体中极为常见，其中极早早产儿（extremely preterm infants, EPT，胎龄<28周）发生胆红素诱导神经毒性的风险极高，可引发神经发育损害。高胆红素血症的临床治疗手段为光疗（phototherapy），以降低未结合胆红素水平。但目前针对极早早产儿光疗的获益与风险尚未得到充分研究，且低水平胆红素作为抗氧化剂或具有保护作用。光疗已被证实与血浆氧化应激标志物水平变化相关，但光疗对海马体（hippocampus, HPC）的具体影响尚不明确。胆红素及极早早产儿相关的脑损伤会损害海马体发育——这一对认知功能至关重要的脑结构，但其潜在分子机制仍未阐明。本研究采用古恩大鼠（Gunn rat）模型，探究高胆红素血症与光疗对海马体的影响。将黄疸型（jj）与非黄疸型（Nj）幼鼠于出生后第4天（postnatal day 4, P4）至第6天（P6）接受光疗，采集海马体组织进行RNA测序（RNA sequencing）。结果显示，与Nj对照大鼠相比，jj组大鼠的血清胆红素水平显著升高，而光疗可有效降低jj组大鼠的血清胆红素水平。与Nj大鼠相比，jj组大鼠海马转录组中共鉴定出1294个差异表达基因，这些基因富集于神经系统发育、炎症反应及铁死亡信号通路。与未接受光疗的大鼠相比，光疗可诱导大鼠海马转录组中3297个差异表达基因（differentially-expressed genes, DEGs），这些差异基因被注释到突触发生、长时程增强及神经发生相关通路。高胆红素血症与光疗共同调控了407个基因的表达，这些基因的功能富集于海马体可塑性相关过程，包括神经突增生与长时程增强。本研究构建了一种可用于探究与极早早产儿等效的古恩幼鼠中高胆红素血症与光疗分子效应的实验模型。本研究数据揭示了高胆红素血症与光疗对海马体发育及可塑性关键信号通路的影响。实验整体设计：将古恩大鼠幼鼠分为非黄疸型（Nj）对照组与黄疸型（jj）组，每组各5只，于出生后第4天（P4）至第6天（P6）接受间歇性LED光疗，光疗总时长为12小时（日间采用3小时照射、3小时暂停的方案）。未接受光疗的Nj与jj组幼鼠（每组n=6）作为空白对照。于P6天使用戊巴比妥钠（100 mg/kg，腹腔注射，intraperitoneal, i.p.）处死大鼠，通过心脏穿刺采集250 μL血液样本。在冰浴金属块上分离海马叶，经液氮速冻后保存于-80℃直至后续分析。使用RNAqueous分离试剂盒（Ambion）从单侧海马叶中提取总RNA。每个实验组设置5只大鼠（Nj+无PT组、jj+无PT组、Nj+PT组、jj+PT组），随后对RNA进行测序处理。