Measurement Of Brain Microglial Proliferation Rates In Vivo: A Biomarker For Discovering Novel Anti‐Neuroinflammatory Agents

The objective of this study was to screen drugs in vivo for inhibition of neuroinflammation, using a recently developed stable isotope method. Microglia (MG), the resident immune cells of the brain, mediates neuroinflammation and contributes to the pathogenesis of many diseases, including Alzheimer’s, multiple sclerosis and stroke. MG proliferation was measured by incorporation of 2 H from heavy water ( 2 H 2 O) into the deoxyribose moiety of DNA, after administration of lipopolysaccharide (LPS, 1 mg/kg, i.p). Follow‐up studies were in the experimental autoimmune encephalomyelitis (EAE) model. MG were isolated from the brain, as CD11b + cells (LPS model) or as F4/80 + /CD45 low cells by flow cytometry (EAE model). Male mice were treated with drugs of different classes for 7 days while receiving 2 H 2 O (8% in drinking water). Minocycline (45 mg/kg, i.p.) and dexamethasone (5 mg/kg) inhibited MG proliferation. In the initial screen of drugs, treatment with 13‐cis‐retinoic acid (Accutane ® , 40 mg/kg) significantly decreased LPS‐induced MG proliferation. Follow‐up studies in EAE confirmed inhibition of MG proliferation. Etiocholanedione combined with low‐dose minocycline (30 mg/kg) inhibited MG proliferation. No effects were seen with aspirin, calcitriol, clofibrate, enalapril, ibuprofen, imipramine, ketoconazole or rosiglitazone. In summary, 2 H 2 O labeling of DNA is a high‐throughput, quantitative and highly reproducible method for measuring MG proliferation, and is useful for screening and discovering novel anti‐neuroinflammatory drugs. Support: KineMed, Inc.