Supplementary Material for: Exaggerated Increases in Microglia Proliferation, Brain Inflammatory Response and Sickness Behaviour upon Lipopolysaccharide Stimulation in Non-Obese Diabetic Mice

The non-obese diabetic (NOD) mouse, an established model for autoimmune diabetes, shows an exaggerated reaction of pancreas macrophages to inflammatory stimuli. NOD mice also display anxiety when immune-stimulated. Chronic mild brain inflammation and a pro-inflammatory microglial activation is critical in psychiatric behaviour. <b><i>Objective:</i></b> To explore brain/microglial activation and behaviour in NOD mice at steady state and after systemic lipopolysaccharide (LPS) injection. <b><i>Methods:</i></b> Affymetrix analysis on purified microglia of pre-diabetic NOD mice (8-10 weeks) and control mice (C57BL/6 and CD1 mice, the parental non-autoimmune strain) at steady state and after systemic LPS (100 μg/kg) administration. Quantitative PCR was performed on the hypothalamus for immune activation markers (IL-1β, IFNγ and TNFα) and growth factors (BDNF and PDGF). Behavioural profiling of NOD, CD1, BALB/c and C57BL/6 mice at steady state was conducted and sickness behaviour/anxiety in NOD and CD1 mice was monitored before and after LPS injection. <b><i>Results:</i></b> Genome analysis revealed cell cycle/cell death and survival aberrancies of NOD microglia, substantiated as higher proliferation on BrdU staining. Inflammation signs were absent. NOD mice had a hyper-reactive response to novel environments with some signs of anxiety. LPS injection induced a higher expression of microglial activation markers, a higher brain pro-inflammatory set point (IFNγ, IDO) and a reduced expression of BDNF and PDGF after immune stimulation in NOD mice. NOD mice displayed exaggerated and prolonged sickness behaviour after LPS administration. <b><i>Conclusion:</i></b> After stimulation with LPS, NOD mice display an increased microglial proliferation and an exaggerated inflammatory brain response with reduced BDNF and PDGF expression and increased sickness behaviour as compared to controls.

非肥胖糖尿病（non-obese diabetic, NOD）小鼠是自身免疫性糖尿病的经典造模动物，其胰腺巨噬细胞对炎症刺激的反应呈现过度激活状态。NOD小鼠在受到免疫刺激时还会表现出焦虑样行为。慢性轻度脑部炎症与促炎型小胶质细胞（microglia）激活，对精神行为的调控发挥关键作用。

【研究目的】探究稳态条件下，以及全身注射脂多糖（lipopolysaccharide, LPS）后，NOD小鼠的脑部/小胶质细胞激活状态与行为学特征。

【研究方法】对处于稳态下、以及经全身注射脂多糖（100 μg/kg）后的糖尿病前期NOD小鼠（8~10周龄）与对照小鼠（亲本非自身免疫品系C57BL/6及CD1小鼠）的纯化小胶质细胞进行Affymetrix基因芯片分析。针对下丘脑组织，通过定量聚合酶链式反应（quantitative PCR, qPCR）检测免疫激活标志物（IL-1β、IFNγ及TNFα）与生长因子（脑源性神经营养因子BDNF、血小板衍生生长因子PDGF）的表达水平。同时对稳态下的NOD、CD1、BALB/c及C57BL/6小鼠开展行为学表征分析，并监测NOD与CD1小鼠在脂多糖注射前后的疾病行为与焦虑样行为变化。

【研究结果】全基因组分析显示，NOD小鼠小胶质细胞存在细胞周期、细胞死亡与存活相关的异常，该结果通过溴脱氧尿苷（bromodeoxyuridine, BrdU）染色证实的更高增殖水平得到验证。基础状态下NOD小鼠未表现出明显炎症迹象，但对新奇环境呈现过度反应，并伴随部分焦虑样行为特征。脂多糖注射后，NOD小鼠的小胶质细胞激活标志物表达水平显著升高，脑部促炎设定阈值上调（IFNγ、吲哚胺2,3-双加氧酶IDO），且免疫刺激后BDNF与PDGF的表达水平显著降低。此外，NOD小鼠在脂多糖给药后表现出更为剧烈且持续时间更长的疾病行为。

【研究结论】与对照组相比，经脂多糖刺激后，NOD小鼠的小胶质细胞增殖水平升高，脑部炎症反应过度增强，同时伴随BDNF与PDGF表达下调，且疾病行为表现更为显著。