gms | German Medical Science

Introduction: Oxidative stress caused by reactive oxygen species (ROS) is known to be a major factor promoting neuronal damage in multiple sclerosis (MS) and in experimental autoimmune encephalomyelitis (EAE). Additionally, certain subunits of NADPH oxidase (NOX), the main catalyst of ROS production, are highly expressed in MS lesions.

Objectives: The cellular source and the dynamics of ROS remain elusive, due to the lack of appropriate detection methods. Since freely diffusing ROS molecules cannot be localized, while their production requires the assembly and not the mere expression of NOX subunits, this question can be answered only by detecting NOX during its catalyst function. We want to quantify the ROS production and cellular source in correlation with the neuronal damage in vivo.

Material & methods: We designed a unique NAD(P)H fluorescence lifetime imaging technique to identify functional NOX in vivo. Thereby we are able to capture the very moment and the site of the biochemical reaction: NADPH being oxidized to NADP+, O2 being reduced to O2-, catalyzed by NOX. The advantage of this method is the unique possibility to identify the real source (in time and space) of ROS under in vivo and in vitro conditions, both in human and in mouse samples and organisms, as the method relies only on endogenous molecules – the probe being NADPH itself.

Results: We identified inflammatory monocytes, microglia and astrocytes as the main cellular source of oxidative stress in the central nervous system of mice affected by EAE. This directly affects neuronal function in vivo, even in the absence of overt inflammation. Interestingly, the systemic dimension of the disease was mirrored by NOX overactivation in peripheral CD11b+ cells both in MS and EAE. This effect was antagonized by systemic intake of the anti-oxidant epigallocatechin-3-gallate.

Conclusion: This study provides new insights regarding the pathological mechanisms in chronic neuroinflammation. Our approach with functional microscopy could become a new powerful tool for neuropathology in research and diagnostics.