We thank the Scientific Computing and Imaging Institute of the University of Utah for use of FluoRender software (R01GM09815). This work was supported by grants from the Glaucoma Research Foundation, Melsa M. and Frank Theodore Barr Foundation and the US National Institute of Health, (R01EY020878 and R01EY023621) to M.L.V., and (R01EY017182 and R01EY017950) to B.K.A.

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)<...

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...

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 cells1,2. Basal microglial functions contribute to physiological maintenance and repair, as they sample their territory for perturbations in homeostasis3,4. During CNS injury or disease, microglia are the first responders to neuronal signals that then trigger their transition to a reactive phenotype, termed &#8220;activated microglia2,5-7. Microglia activation involves an intricate cycle of gene and protein expression, which are coupled to cell soma and process resizing and remodeling6-9. Microglia activation, as well as cell redistribution and clustering, can be accompanied the local overall increase in the number of cells (termed microgliosis). This can result from cell proliferation and self-renewal, with or without recruitment of blood-derived monocytes3,4,7,10-14. In a broad range of age-dependent, chronic CNS diseases, sustained microgliosis and microglia activation parallel disease progression15-19. How microglia impact neurodegeneration remains unclear, mainly because they play both neuroprotective and deleterious roles that may have diverse contributions to disease onset and progression. Live imaging studies aimed at understanding chronic CNS disease have monitored microglial behavior in the damaged CNS of animal models and humans, and demonstrated that microglial alterations are detectable beginning at early disease stages15-17,19,20. Thus, it is critical to develop approaches to detect and monitor microglial activation in vivo.
Non-invasive detection of regional changes in brain microglia activation was established as an important in vivo indicator of neurodegenerative disease progression, using molecular imaging or bioluminescence and positron emission tomography or magnetic resonance imaging18,21,22. These highly quantitative and non-invasive molecular and nuclear imaging methods detect gliosis with regional resolution. Alternatively, two-photon confocal imaging in CX3CR1GFP/+ mice has allowed the observation of brain microglia with cellular resolution3,4,9,20,23-28. However, this approach limits long-term and repeated observation of chronic microglial alterations, given the potential risk of disturbing their behavior by even minimally invasive brain imaging procedures29. Alternatively, the retina offers optimal conditions for direct, in vivo visualization and repeated monitoring of microglia in their intact CNS niche throughout aging, following acute injury, and potentially during chronic neurodegenerative diseases. Thus, recent studies have proved the feasibility of high-resolution imaging of retinal microglia expressing GFP by adapting the confocal scanning laser ophthalmoscopy (cSLO) to image live CX3CR1GFP/+ mice. This has been used to track weekly changes in GFP+ cell numbers in individual mice for up to 10 weeks following acutely induced injury or ocular hypertension30-36.
We have extended this approach to perform long-term imaging over several months, and quantitatively track changes in microglia activation based on soma size using morphometric analysis. Somal size was defined as a useful metric of microglia activation in live imaging studies using two-photon confocal microscopy in cortical slices to perform in vivo imaging of CX3CR1-GFP+ microglia9. These and other studies also demonstrated the correlation between somal size and levels of Iba1 protein expression, which also increases with activation9,37. Thus, activated microglia can be identified in live mice, and their numbers and distribution monitored over time during CNS health and disease.
This protocol describes methods for cSLO live image acquisition and analysis to monitor microglial cell numbers, distribution and morphological activation during retinal ganglion cell (RGC) degeneration (Figure 1). Thus, this study uses: 1) a mouse model of inherited glaucoma (DBA/2J) that undergoes age-dependent optic nerve and retinal neurodegeneration and shows remarkable variability in disease progression between 5 and 10 months of age38,39, 2) monthly cSLO in vivo imaging for long-term visualization of GFP+ cells in the retina and unmyelinated optic nerve of heterozygous Cx3cr1GFP/+ DBA/2J mice aged 3-5 months, 3) live imaging analysis by segmentation and thresholding to isolate cell somata and measure their area. These strategies are applied to assess the kinetics of retinal microglial activation states during early stages of chronic glaucoma.

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			<td height="60" style="height:60px;width:617px;">DBA/2J and 
			CX3CR1-GFP/+ mice</td>
			<td>The Jackson Laboratory, Bar Harbor, ME</td>
			<td>000671 and 00582</td>
			<td>Mice are breed in-house, introducing new breeders every 3 to 4 generations to prevent genetic drift.</td>
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			<td height="20" style="height:20px;">301/2G needle and 1 ml tuberculin slip-tip syringe</td>
			<td>BD, East Rutherford, NJ</td>
			<td>305106 and 309659</td>
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			<td height="40" style="height:40px;width:617px;">2,2,2-tribromoethanol, tert-Amyl alcohol 99% and phosphate buffer saline tablets</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>T48402, 152463 and P4417</td>
			<td>Avertin solution (pH 7.3, sterile filtered) must be freshly made solution or stored at 4&#186;C for up to 1 week.</td>
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			<td height="20" style="height:20px;">Heat therapy T/Pump</td>
			<td>Gaymar Industries, Orchard Park, NY</td>
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			<td height="20" style="height:20px;">Cotton-tipped applicators</td>
			<td>Fisher Scientific, Pittsburgh, PA</td>
			<td>23-400-100</td>
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			<td height="20" style="height:20px;">Tropicamide 1%&#160;</td>
			<td>Bausch &#38; Lomb, Rochester, NY</td>
			<td>NDC 24208-585-64</td>
			<td></td>
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			<td height="20" style="height:20px;">PMMA contact lenses, 1.70-base curve, 3.2 mm total diameter, 0.40 mm-thick center</td>
			<td>Cantor &#38; Nissel Ltd., Northamptonshire, UK</td>
			<td>G003709</td>
			<td>Lenses are rinsed with sterile PBS and stored in polypropylene boxes.</td>
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			<td height="20" style="height:20px;">HRA/Spectralis confocal scanning laser ophthalmoscope and Eye Explorer software</td>
			<td>Heidelberg Engineering GmbH, Carlsbad, CA</td>
			<td>Version 1.7.1.0</td>
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			<td>Microsoft, Redmond, WA</td>
			<td>Version 14.4.3</td>
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			<td height="20" style="height:20px;">Adobe Photoshop</td>
			<td>Adobe, San Jose, CA</td>
			<td>Version CS3</td>
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			<td height="20" style="height:20px;">FluoRender</td>
			<td>Scientific Computing and Imaging Institute, University of Utah</td>
			<td>Version 2.13</td>
			<td>Freeware. http://www.sci.utah.edu/software/13-software/127-fluorender.html</td>
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			<td height="20" style="height:20px;">NIS-Elements C</td>
			<td>Nikon, Melville, NY</td>
			<td>Version 4.30.01</td>
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in vivo dynamics of retinal microglial activation during neurodegeneration: confocal ophthalmoscopic imaging and cell morphometry in mouse glaucoma

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 CX3CR1GFP/+ 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.

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...

Watch this Scientific Journal Video about In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma at JoVE.co

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

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.

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

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with ...

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10.5K vistas.  University of Utah.  La activación de la microglia y microgliosis son respuestas clave a la neurodegeneración crónica. A continuación, presentamos los métodos para in vivo, visualización a largo plazo de la retina células microgliales CX3CR1-GFP + por oftalmoscopía confocal y de umbral y los análisis morfométricos para identificar y cuantificar su activación. Hacemos un seguimiento de los cambios microgliales durante las primeras etapas del glaucoma relacionada con la eda...

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