Measurement of Brain Microglial Proliferation Rates In Vivo in Response to Neuroinflammatory Stimuli: Application to Drug Discovery Mahalakshmi Shankaran, 1 * Michael E. Marino, 1 Robert Busch, 1 Carole Keim, 1 Chelsea King, 1 Jean Lee, 1 Salena Killion, 1 Mohamad Awada, 1 and Marc K. Hellerstein 2,3 1 Kinemed Inc., Emeryville, California 2 Department of Nutritional Sciences and Toxicology, University of California, Berkeley, California 3 Department of Medicine, San Francisco General Hospital, University of California at San Francisco, San Francisco, California Microglial activation is emerging as an important etio- logic factor and therapeutic target in neurodegenerative and neuroinflammatory diseases. Techniques have been lacking, however, for measuring the different compo- nents of microglial activation independently in vivo. We describe a method for measuring microglial proliferation rates in vivo using heavy water ( 2 H 2 O) labeling, and its application in screening for drugs that suppress neuro- inflammation. Brain microglia were isolated by flow cytometry as F4/80 þ , CD11b þ , CD45 low cells, and 2 H enrichment in DNA was analyzed by gas chromatogra- phy/mass spectrometry. Basal proliferation rate was 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. Min- ocycline (45 mg/kg/day, i.p.) inhibited resident micro- glial proliferation in both the LPS and EAE models. Thir- teen 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 2 H 2 O label for 7 days. Among the drugs screened, treatment with isotretinoin dose- dependently reduced LPS-induced microglial prolifera- tion, representing an action of retinoids unknown previ- ously. Follow-up studies in the EAE model confirmed that isotretinoin not only inhibited proliferation of micro- glia but also delayed the onset of clinical symptoms. In conclusion, 2 H 2 O labeling represents a relatively high- throughput, quantitative, and highly reproducible tech- nique for measuring microglial proliferation, and is use- ful for screening and discovering novel anti-neuroin- flammatory drugs. V V C 2007 Wiley-Liss, Inc. Key words: microglia; proliferation; neuroinflammation; EAE; LPS Microglia are brain-resident cells of myeloid hema- topoietic origin, which become activated in response to inflammatory stimuli. Microglial activation may cause neuronal damage by production of neuroinflammatory mediators (e.g., pro-inflammatory cytokines, nitric oxide, reactive oxygen species) or by autoimmune mechanisms (e.g., presentation of self and foreign antigens to T cells) (Jack et al., 2005; Kim and Joh, 2006). Microglia have been implicated in the progression or adverse outcomes of several neurological diseases, including Alzheimer’s dis- ease (McGeer and McGeer, 2002b), multiple sclerosis (Neumann, 2003), amyotrophic lateral sclerosis (McGeer and McGeer, 2002a), Parkinson’s disease (Wullner and Klockgether, 2003), stroke (Danton and Dietrich, 2003), and variant Creutzfeldt-Jacob disease (Riemer et al., 2002). Accordingly, microglia represent a therapeutic tar- get (Aldskogius, 2001; Liu and Hong, 2003; van Rossum and Hanisch, 2004) for the treatment of chronic neuroin- flammatory and neurodegenerative disorders. Minocy- cline, an inhibitor of microglial activation, is being tested in clinical trials (Blum et al., 2004). Microglial activation includes several dynamic com- ponents, however, including changes in cellular morphol- M. Shankaran and M.E. Marino contributed equally to this manuscript. Dr. M. Marino is currently at the GE Global Research Center, 1 Research Circle, Niskayuna, NY 12309. Dr. R. Busch is currently at the Department of Medicine, University of Cambridge, Box 157, Level 5, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 2QQ, United Kingdom. Contract grant sponsor: KineMed Inc.; Contract grant sponsor: University of California-Industry Discovery Program; Contract grant number: Bio04-10445. *Correspondence to: Mahalakshmi Shankaran, PhD, KineMed Inc., 5980 Horton St, Suite 400, Emeryville, CA 94608. E-mail: mshankaran@kinemed.com Received 8 February 2007; Revised 3 April 2007; Accepted 4 April 2007 Published online 5 June 2007 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/jnr.21389 Journal of Neuroscience Research 85:2374–2384 (2007) ' 2007 Wiley-Liss, Inc.