Semi-Automated Live Tracking of Microglial Activation in CX3CR1GFP Mice During Experimental Autoimmune Encephalomyelitis by Confocal Scanning Laser Ophthalmoscopy.

Moritz J Frenger,Andrea Issberner,Sven G Meuth,Hans-Peter Hartung,Philipp Albrecht,Mustafa Sindi,Christina Hecker,Michael Dietrich

doi:10.3389/fimmu.2021.761776

Abstract

Confocal scanning laser ophthalmoscopy (cSLO) is a non-invasive technique for real-time imaging of the retina. We developed a step-by-step protocol for the semi-automatic evaluation of myeloid cells in cSLO images from CX3CR1GFP mice, expressing green fluorescent protein (GFP) under control of the endogenous CX3C chemokine receptor 1 locus. We identified cSLO parameters allowing us to distinguish animals with experimental autoimmune encephalomyelitis (EAE) from sham-treated/naïve animals. Especially cell count (CC) and the total microglial area (SuA) turned out to be reliable parameters. Comparing the cSLO results with clinical parameters, we found significant correlations between the clinical EAE score and the SuA and of the inner retinal layer thickness, measured by optical coherence tomography, with the CC as well as the SuA. As a final step, we performed immunohistochemistry to confirm that the GFP-expressing cells visualized by the cSLO are Iba1 positive and validated the step-by-step protocol against manual counting. We present a semi-automatic step-by-step protocol with a balance between fast data evaluation and adequate accuracy, which is optimized by the option to manually adapt the contrast threshold. This protocol may be useful for numerous research questions on the role of microglial polarization in models of inflammatory and degenerating CNS diseases involving the retina.

Highlights

The chemokine receptor and chemokine ligand axis mediates chemotaxis of immune cells
The brightness parameters Average cell brightness (MVI) and Maximal cell brightness (MCB) did not reveal a significant difference between the experimental groups with the exception of the baseline measurements of the sham-treated compared to the MOG35-55 immunized mice (Figures 2B, C)
The Average cell area (AvA) reflected the results of the Cell count (CC) and Total microglial area (SuA), without reaching significant levels for naïve/sham-treated mice compared to EAE mice for the different timepoints (Figure 2F)

Summary

Introduction

The chemokine receptor and chemokine ligand axis mediates chemotaxis of immune cells. In the central nervous system (CNS), the CX3C chemokine receptor 1 (CX3CR1) is mainly expressed on the developmentally yolk sac-derived microglial cells or, in pathological conditions, on infiltrating monocytes and macrophages, whereas chemokine ligands are predominantly expressed on neurons [2, 3]. Microglial activity, with its reactive oxygen species-mediated phagocytosis, and with its cytokine- and chemokinemodulating function presents itself as a crucial aspect of the MS pathogenesis. It has been associated with blood-brain barrier permeability, dysregulated T-cell activation and modulating effects on B-cells [13,14,15,16,17,18,19]. Using non-invasive, in vivo confocal scanning laser ophthalmoscopy (cSLO), microglial activation can be assessed in the retina of CX3CR1GFP mice in the context of EAE [20]

Objectives

Methods

Results

Conclusion