title: Measurement of brain microglial proliferation rates in vivo in response to neuroinflammatory stimuli: application to drug discovery.
abstract: 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.
authors.affiliation: Kinemed Inc., Emeryville, California, USA.
authors.forename: Mahalakshmi
authors.initials: M
authors.lastname: Shankaran
authors.forename: Michael E
authors.initials: ME
authors.lastname: Marino
authors.forename: Robert
authors.initials: R
authors.lastname: Busch
authors.forename: Carole
authors.initials: C
authors.lastname: Keim
authors.forename: Chelsea
authors.initials: C
authors.lastname: King
authors.forename: Jean
authors.initials: J
authors.lastname: Lee
authors.forename: Salena
authors.initials: S
authors.lastname: Killion
authors.forename: Mohamad
authors.initials: M
authors.lastname: Awada
authors.forename: Marc K
authors.initials: MK
authors.lastname: Hellerstein
mesh_terms: D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology*; D001921:Brain / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects; D017666:Deuterium Oxide; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004681:Encephalomyelitis, Autoimmune, Experimental / Q000188:drug therapy; D005260:Female; D005434:Flow Cytometry; D007249:Inflammation / Q000188:drug therapy*; D008070:Lipopolysaccharides / Q000633:toxicity; D051379:Mice; D008810:Mice, Inbred C57BL; D017628:Microglia / Q000187:drug effects / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D000893:Anti-Inflammatory Agents; D008070:Lipopolysaccharides; D017666:Deuterium Oxide