Shankaran Mahalakshmi, Marino Michael E, Busch Robert, Keim Carole, King Chelsea, Lee Jean, Killion Salena, Awada Mohamad, Hellerstein Marc K
Kinemed Inc., Emeryville, California, USA.
J Neurosci Res. 2007 Aug 15;85(11):2374-84. doi: 10.1002/jnr.21389.
Microglial activation is emerging as an important etiologic factor and therapeutic target in neurodegenerative and neuroinflammatory diseases. Techniques have been lacking, however, for measuring the different components of microglial activation independently in vivo. We describe a method for measuring microglial proliferation rates in vivo using heavy water (2H2O) labeling, and its application in screening for drugs that suppress neuro-inflammation. Brain microglia were isolated by flow cytometry as F4/80+, CD11b+, CD45(low) cells, and 2H enrichment in DNA was analyzed by gas chromatography/mass spectrometry. Basal proliferation rate was approximately 1%/week and systemic administration of bacterial lipopolysaccharide (LPS) markedly increased this rate in a dose-dependent manner. Induction of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice by MOG(35-55) peptide stimulated proliferation of CD45(low) microglia, which could be distinguished from the proliferation of CD45(high) infiltrating monocytes. Minocycline (45 mg/kg/day, i.p.) inhibited resident microglial proliferation in both the LPS and EAE models. Thirteen drugs were then screened for their ability to inhibit LPS-stimulated microglia proliferation. Female C57BL/6 mice were given LPS (1 mg/kg), and concomitant drug treatment while receiving 2H2O label for 7 days. Among the drugs screened, treatment with isotretinoin dose-dependently reduced LPS-induced microglial proliferation, representing an action of retinoids unknown previously. Follow-up studies in the EAE model confirmed that isotretinoin not only inhibited proliferation of microglia but also delayed the onset of clinical symptoms. In conclusion, 2H2O labeling represents a relatively high-throughput, quantitative, and highly reproducible technique for measuring microglial proliferation, and is useful for screening and discovering novel anti-neuroinflammatory drugs.
小胶质细胞激活正成为神经退行性疾病和神经炎症性疾病中的一个重要病因和治疗靶点。然而，一直缺乏在体内独立测量小胶质细胞激活不同成分的技术。我们描述了一种使用重水（2H2O）标记在体内测量小胶质细胞增殖率的方法，及其在筛选抑制神经炎症药物中的应用。通过流式细胞术将脑小胶质细胞分离为F4/80+、CD11b+、CD45（低）细胞，并通过气相色谱/质谱分析DNA中的2H富集情况。基础增殖率约为每周1%，全身给予细菌脂多糖（LPS）以剂量依赖方式显著提高了该增殖率。用MOG(35-55)肽诱导C57BL/6小鼠发生实验性自身免疫性脑脊髓炎（EAE）刺激了CD45（低）小胶质细胞的增殖，这可与CD45（高）浸润单核细胞的增殖区分开来。米诺环素（45mg/kg/天，腹腔注射）在LPS和EAE模型中均抑制了常驻小胶质细胞的增殖。然后筛选了13种药物抑制LPS刺激的小胶质细胞增殖的能力。给雌性C57BL/6小鼠注射LPS（1mg/kg），并在接受2H2O标记的7天内同时进行药物治疗。在所筛选的药物中，异维甲酸治疗剂量依赖性地降低了LPS诱导的小胶质细胞增殖，这代表了类视黄醇以前未知的作用。在EAE模型中的后续研究证实，异维甲酸不仅抑制小胶质细胞增殖，还延迟了临床症状的发作。总之，2H2O标记是一种用于测量小胶质细胞增殖的相对高通量、定量且高度可重复的技术，对于筛选和发现新型抗神经炎症药物很有用。
