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.

Regulation of APC development, immune response, and autoimmunity by Bach1/HO-1 pathway in mice

“…This was a consequence of both less infiltration of peripheral DCs ( Figure 2B) and fewer resident CNS microglia ( Figure 2C), which proliferate on EAE development. 35 In parallel to lower APCs, fewer infiltrating T and B cells were observed in the CNS of these animals ( Figure 2D-F). Thus, during pathogenesis of EAE, ablation of Bach1 partially protects the CNS from immune cell infiltration and disease development.…”

A gene expression network model of type 2 diabetes links cell cycle regulation in islets with diabetes susceptibility

“…The proliferation rate of islet cells was measured using a recently developed heavy water ( 2 H 2 O) labeling technique (Busch et al 2004;Shankaran et al 2006Shankaran et al , 2007. Briefly, the incorporation of deuterium ( 2 H) from 2 H 2 O into the deoxyribose moiety of deoxyribonucleotides in cells replicating their DNA is measured by gas chromatography/mass spectrometry (GC/MS).…”

Enhanced microglial pro‐inflammatory response to lipopolysaccharide correlates with brain infiltration and blood–brain barrier dysregulation in a mouse model of telomere shortening

“…Indeed, microglia telomere shortening has been associated with increasing age and dementia (Flanary et al ., 2007). The proliferative capacity of microglia has shown to be linked to its innate immune response (Shankaran et al ., 2007) and seems to be a critical component in the evolution of chronic neurodegeneration (Gomez‐Nicola et al ., 2013). In this study, we investigated whether telomere shortening could potentially alter the inflammatory reactivity of microglia in the brain.…”