Supplementary Material for: Neuroinflammation in Response to Intracerebral Injections of Different HMGB1 Redox Isoforms

<b><i>Background:</i></b> Neuroinflammation triggered by infection or trauma is the cause of central nervous system dysfunction. High-mobility group box 1 protein (HMGB1), released from stressed and dying brain cells, is a potent neuroinflammatory mediator. The proinflammatory functions of HMGB1 are tightly regulated by post-translational redox modifications, and we here investigated detailed neuroinflammatory responses induced by the individual redox isoforms. <b><i>Methods:</i></b> Male Dark Agouti rats received a stereotactic injection of saline, lipopolysaccharide, disulfide HMGB1, or fully reduced HMGB1, and were accessed for blood-brain barrier modifications using magnetic resonance imaging (MRI) and inflammatory responses by immunohistochemistry. <b><i>Results and Conclusions:</i></b> Significant blood-brain barrier disruption appeared 24 h after injection of lipopolysaccharide, disulfide HMGB1, or fully reduced HMGB1 compared to controls, as assessed in post-gadolinium T1-weighted MRI images and confirmed by increased uptake of FITC-conjugated dextran. Immunohistochemistry revealed that both HMGB1 isoforms also induced a local production of IL-1β. Additionally, disulfide HMGB1 increased major histocompatibility complex class II expression and apoptosis. Together, the results demonstrate that extracellular, cerebral HMGB1 causes significant blood-brain barrier disruption in a redox-independent manner and activates several components of neuroinflammation. Blocking HMGB1 might potentially improve clinical outcome in conditions such as stroke and traumatic brain injury.

**背景：** 感染或创伤诱发的神经炎症是中枢神经系统功能障碍的致病诱因。从应激状态及濒死脑细胞中释放的高迁移率族蛋白B1（HMGB1）是一种强效神经炎症介质。HMGB1的促炎功能受到翻译后氧化还原修饰的严格调控，本研究旨在探究不同氧化还原亚型HMGB1诱导的详细神经炎症反应。**方法：** 雄性Dark Agouti大鼠接受立体定向注射生理盐水、脂多糖、二硫键型HMGB1或完全还原型HMGB1，随后通过磁共振成像（MRI）评估血脑屏障变化，并采用免疫组织化学法检测炎症反应。**结果与结论：** 钆增强T1加权MRI图像分析及异硫氰酸荧光素标记葡聚糖摄取增强实验证实，与对照组相比，脂多糖、二硫键型HMGB1或完全还原型HMGB1注射后24小时均出现显著血脑屏障破坏。免疫组织化学结果显示，两种HMGB1亚型均可诱导局部白细胞介素1β（IL-1β）生成。此外，二硫键型HMGB1可上调主要组织相容性复合体II类分子表达并诱发细胞凋亡。综上，本研究结果表明，细胞外脑源性HMGB1可通过不依赖氧化还原的方式引发显著血脑屏障破坏，并激活神经炎症的多个通路。阻断HMGB1有望改善脑卒中、创伤性脑损伤等疾病的临床结局。