Microglia activation visualization via fluorescence lifetime imaging microscopy of intrinsically fluorescent metabolic cofactors.

Md Abdul K Sagar,Kevin P Cheng,Justin C Williams,Kevin W Eliceiri,Jyoti J Watters,Jonathan N Ouellette

doi:10.1117/1.nph.7.3.035003

Abstract

Significance: A major obstacle to studying resident microglia has been their similarity to infiltrating immune cell types and the lack of unique protein markers for identifying the functional state. Given the role of microglia in all neural diseases and insults, accurate tools for detecting their function beyond morphologic alterations are necessary.Aims: We hypothesized that microglia would have unique metabolic fluxes in reduced nicotinamide adenine dinucleotide (NADH) that would be detectable by relative changes in fluorescence lifetime imaging microscopy (FLIM) parameters, allowing for identification of their activation status. Fluorescence lifetime of NADH has been previously demonstrated to show differences in metabolic fluxes.Approach: Here, we investigate the use of the label-free method of FLIM-based detection of the endogenous metabolic cofactor NADH to identify microglia and characterize their activation status. To test whether microglial activation would also confer a unique NADH lifetime signature, murine primary microglial cultures and adult mice were treated with lipopolysaccharide (LPS).Results: We found that LPS-induced microglia activation correlates with detected changes in NADH lifetime and its free-bound ratio. This indicates that NADH lifetime can be used to monitor microglia activation in a label-free fashion. Moreover, we found that there is an LPS dose-dependent change associated with reactive microglia lifetime fluxes, which is also replicated over time after LPS treatment.Conclusion: We have demonstrated a label-free way of monitoring microglia activation via quantifying lifetime of endogenous metabolic coenzyme NADH. Upon LPS-induced activation, there is a significant change in the fluorescence lifetime following activation. Together, these results indicate that NADH FLIM approaches can be used as a method to characterize microglia activation state, both in vitro and ex vivo.

Highlights

Microglia have prominent roles in both central nervous system (CNS) injury/disease responses and in protection of the CNS.[1,2,3,4] Major obstacles in studying microglia have been the lack of specific protein markers that indicate their activation status in the absence of concomitant morphological changes and the difficulty in distinguishing them from related infiltrating myeloid cells
To study fluorescence lifetime imaging microscopy (FLIM) parameters in activated microglia, we investigated the relative change in NADH FLIM parameters in microglia stimulated with LPS.[39,40,41,42]
The underlying hypothesis of this experiment was that LPS treatment would initiate an inflammatory response resulting in a change in microglial metabolism, causing alterations in the metabolic cofactor NADH that could be assessed by FLIM

Summary

Introduction

Microglia have prominent roles in both central nervous system (CNS) injury/disease responses and in protection of the CNS.[1,2,3,4] Major obstacles in studying microglia have been the lack of specific protein markers that indicate their activation status in the absence of concomitant morphological changes and the difficulty in distinguishing them from related infiltrating myeloid cells. Other proteins such as the orphan receptor TMEM1199 and the lysosomal hexosaminidase enzyme B (HexB)[10] have been identified by transcriptomic analyses as unique to microglia, and are not expressed in macrophages,[11,12,13,14] but these cannot distinguish activation states either. Other studies have tried to characterize microglial activation using statistical changes in soma size and roundness.[18]

Methods

Results

Discussion

Conclusion