Table_1_Transcriptomic Analysis of Mouse Brain After Traumatic Brain Injury Reveals That the Angiotensin Receptor Blocker Candesartan Acts Through Novel Pathways.xlsx

Traumatic brain injury (TBI) results in complex pathological reactions, where the initial lesion is followed by secondary inflammation and edema. Our laboratory and others have reported that angiotensin receptor blockers (ARBs) have efficacy in improving recovery from traumatic brain injury in mice. Treatment of mice with a subhypotensive dose of the ARB candesartan results in improved functional recovery, and reduced pathology (lesion volume, inflammation and gliosis). In order to gain a better understanding of the molecular mechanisms through which candesartan improves recovery after controlled cortical impact injury (CCI), we performed transcriptomic profiling on brain regions after injury and drug treatment. We examined RNA expression in the ipsilateral hippocampus, thalamus and hypothalamus at 3 or 29 days post injury (dpi) treated with either candesartan (0.1 mg/kg) or vehicle. RNA was isolated and analyzed by bulk mRNA-seq. Gene expression in injured and/or candesartan treated brain region was compared to that in sham vehicle treated mice in the same brain region to identify genes that were differentially expressed (DEGs) between groups. The most DEGs were expressed in the hippocampus at 3 dpi, and the number of DEGs reduced with distance and time from the lesion. Among pathways that were differentially expressed at 3 dpi after CCI, candesartan treatment altered genes involved in angiogenesis, interferon signaling, extracellular matrix regulation including integrins and chromosome maintenance and DNA replication. At 29 dpi, candesartan treatment reduced the expression of genes involved in the inflammatory response. Some changes in gene expression were confirmed in a separate cohort of animals by qPCR. Fewer DEGs were found in the thalamus, and only one in the hypothalamus at 3 dpi. Additionally, in the hippocampi of sham injured mice, 3 days of candesartan treatment led to the differential expression of 384 genes showing that candesartan in the absence of injury had a powerful impact on gene expression specifically in the hippocampus. Our results suggest that candesartan has broad actions in the brain after injury and affects different processes at acute and chronic times after injury. These data should assist in elucidating the beneficial effect of candesartan on recovery from TBI.

创伤性脑损伤（Traumatic brain injury, TBI）会引发复杂的病理级联反应：原发性损伤发生后，会继发炎症与脑水肿。本团队及其他学术研究团队均已证实，血管紧张素受体拮抗剂（angiotensin receptor blockers, ARBs）可改善小鼠创伤性脑损伤后的功能恢复。采用亚低血压剂量的血管紧张素受体拮抗剂类药物坎地沙坦（candesartan）处理小鼠，可提升其神经功能恢复效果，并减轻病理损伤（包括病灶体积、炎症反应与神经胶质增生）。为深入阐明坎地沙坦改善控制性皮质撞击损伤（controlled cortical impact injury, CCI）后小鼠恢复的分子机制，我们对损伤及给药后的脑区开展了转录组分析。我们分别在损伤后3天与29天（days post injury, dpi），对分别给予坎地沙坦（0.1 mg/kg）或赋形剂的小鼠的同侧海马、丘脑与下丘脑进行了RNA表达检测。通过批量mRNA测序（bulk mRNA-seq）分离并分析总RNA，将损伤组及/或坎地沙坦处理组脑区的基因表达水平，与同脑区给予赋形剂的假手术组小鼠进行比对，以筛选各组间的差异表达基因（differentially expressed genes, DEGs）。损伤后3天时，差异表达基因数量最多的脑区为海马，且差异表达基因的数量随距离病灶的距离增加与时间推移而逐渐减少。在控制性皮质撞击损伤后3天的差异表达通路中，坎地沙坦处理可调控血管生成、干扰素信号通路、包括整合素在内的细胞外基质调控通路，以及染色体维持与DNA复制相关基因的表达。损伤后29天时，坎地沙坦处理可下调炎症反应相关基因的表达水平。我们通过另一组独立动物队列的定量聚合酶链反应（qPCR）验证了部分基因表达变化。损伤后3天时，丘脑内的差异表达基因数量较少，下丘脑中仅发现1个差异表达基因。此外，在假手术小鼠的海马组织中，连续3天给予坎地沙坦即可导致384个基因出现差异表达，这表明在无颅脑损伤的情况下，坎地沙坦仍可对海马的基因表达产生显著调控作用。本研究结果表明，坎地沙坦可在创伤性脑损伤后的脑内发挥广泛的调控作用，并在损伤后的急性期与慢性期影响不同的生理过程。本数据集将有助于进一步阐明坎地沙坦改善创伤性脑损伤恢复的有益机制。