In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Summary

Microglia activation and microgliosis are key responses to chronic neurodegeneration. Here, we present methods for in vivo, long-term visualization of retinal CX3CR1-GFP+ microglial cells by confocal ophthalmoscopy, and for threshold and morphometric analyses to identify and quantify their activation. We monitor microglial changes during early stages of age-related glaucoma.

Abstract

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with distinct functions and gene expression profiles. The roles of microglial activation in health, injury and disease remain incompletely understood due to their dynamic and complex regulation in response to changes in their microenvironment. Thus, it is critical to non-invasively monitor and analyze changes in microglial activation over time in the intact organism. In vivo studies of microglial activation have been delayed by technical limitations to tracking microglial behavior without altering the CNS environment. This has been particularly challenging during chronic neurodegeneration, where long-term changes must be tracked. The retina, a CNS organ amenable to non-invasive live imaging, offers a powerful system to visualize and characterize the dynamics of microglia activation during chronic disorders.

This protocol outlines methods for long-term, in vivo imaging of retinal microglia, using confocal ophthalmoscopy (cSLO) and CX3CR1^GFP/+ reporter mice, to visualize microglia with cellular resolution. Also, we describe methods to quantify monthly changes in cell activation and density in large cell subsets (200-300 cells per retina). We confirm the use of somal area as a useful metric for live tracking of microglial activation in the retina by applying automated threshold-based morphometric analysis of in vivo images. We use these live image acquisition and analyses strategies to monitor the dynamic changes in microglial activation and microgliosis during early stages of retinal neurodegeneration in a mouse model of chronic glaucoma. This approach should be useful to investigate the contributions of microglia to neuronal and axonal decline in chronic CNS disorders that affect the retina and optic nerve.

Introduction

Microglia are neuroimmune cells that exclusively reside in the central nervous system (CNS) since early embryonic development and throughout adulthood. Equipped with a complex repertoire of receptors, microglial activity and regional heterogeneity are regulated by their bidirectional interplay with neighboring neurons, glia, blood-brain barrier and infiltrating neuroinflammatory cells^1,2. Basal microglial functions contribute to physiological maintenance and repair, as they sample their territory for perturbations in homeostasis^3,4. During CNS injury or disease, microglia are the first responders to neuronal signals that then trigger their transi....

Protocol

In vivo imaging is performed in pathogen-free facilities using protocols approved by the Institutional Animal Care and Use Committee at the University of Utah.
NOTE: This imaging protocol is used for reporter mice in which retinal microglia and infiltrating monocytes/macrophages express green-fluorescent protein (GFP) under the control of the fractalkine receptor locus (CX3CR1).

1. In Vivo Imaging of Retinal GFP+ Microglia by Confocal Scanning Laser Ophthalmoscopy (cSLO)

1. Turn on the water-controlled heating system, set to stabilize mouse temperature between 35-37 ºC during procedure, and connect two ....

Results

Our recent in vivo studies used these live image acquisition and analysis methods to visualize and track the kinetics and patterns of ONH and retinal microglial changes during early stages of chronic glaucoma and their relationship to late neurodegeneration⁵⁹. Here we illustrate a cSLO image acquisition protocol to visualize microglial cells across a large area of the central retina in individual young heterozygous Cx3CR1-GFP DBA/2J retinas (Figure 5). Based on the high cell resolutio.......

Discussion

Live monitoring of microglial cell number and morphological activation during a neurodegenerative disease requires the use of non-invasive imaging methods that allow the detailed visualization of cell features. After imaging, microglial cells must be isolated (segmented) for morphometric analysis by use of multiple threshold steps to assess somal size and/or process complexity as readouts for microglia activation. In this protocol, we describe methods for live image acquisition using cSLO, and quantitative analysis of mi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DBA/2J and CX3CR1-GFP/+ mice	The Jackson Laboratory, Bar Harbor, ME	000671 and 00582	Mice are bred in house, introducing new breeders every 3 - 4 generations to prevent genetic drift.
30½ G needle and 1 ml tuberculin slip-tip syringe	BD, East Rutherford, NJ	305106 and 309659
2,2,2-tribromoethanol, tert-Amyl alcohol 99% and phosphate buffer saline tablets	Sigma-Aldrich, St. Louis, MO	T48402, 152463 and P4417	Avertin solution (pH 7.3, sterile filtered) must be freshly made solution or stored at 4 °C for up to 1 week.
Heat therapy T/Pump	Gaymar Industries, Orchard Park, NY	TP-650
Cotton-tipped applicators	Fisher Scientific, Pittsburgh, PA	23-400-100
Tropicamide 1%	Bausch & Lomb, Rochester, NY	NDC 24208-585-64
PMMA contact lenses, 1.70 base curve, 3.2 mm total diameter, 0.40 mm thick center	Cantor & Nissel Ltd., Northamptonshire, UK	G003709	Lenses are rinsed with sterile PBS and stored in polypropylene boxes.
HRA/Spectralis confocal scanning laser ophthalmoscope and Eye Explorer software	Heidelberg Engineering GmbH, Carlsbad, CA	Version 1.7.1.0
PowerPoint	Microsoft, Redmond, WA	Version 14.4.3
Adobe Photoshop	Adobe, San Jose, CA	Version CS3
FluoRender	Scientific Computing and Imaging Institute, University of Utah	Version 2.13	Freeware. http://www.sci.utah.edu/software/13-software/127-fluorender.html
NIS-Elements C	Nikon, Melville, NY	Version 4.30.01