in vivo imaging of retinal microglia in a mouse model of glaucoma

In Vivo Imaging of Retinal Microglia in a Mouse Model of Glaucoma

Start the imaging session by collecting a fundus view of the inner retina. For this, select the infrared mode, which corresponds to an excitation wavelength of 820 nanometers. Then, adjust the laser power to 100%, and the sensitivity to between 40% and 60%.Next, working at high speed, locate the eye using the joystick to orient the ophthalmoscope, and prepare to collect a fundus view of the optic disk and retinal vasculature. Inspect the cornea and lens for injury or opacity. Exclude from the study eyes with defects or injuries that may affect image acquisition. Now, by bringing the objective closer to the eye, locate the optic disk area, which is the surface of the optic nerve head, or ONH.Then, center the image on the ONH. Positioning the ONH correctly is key to obtaining an even focus and emission across the image. The next step is to visualize the inner planes of the retina, as well as the ONH. For a reference focal plane, use major blood vessels located on the vitriol surface of the retina, which corresponds to 59 and 60 diopters, or even deeper at 55 diopters in excavated optic disk areas.Next, adjust the image saturation, using the knob on the touch panel, until a white halo of illumination spans most of the fundus around the optic disk. Then, select the lower saturation that renders a uniform halo, indicative of an optimal contrast for that eye. It may be necessary to realign the camera if dark areas persist.Now, collect a high-resolution fundus image of the central retina by averaging 30 frames, taken in real time, to improve the signal-to-noise ratio. This corresponds to 4.7 frames per second normalized. Immediately, and at the same position, prepare to collect a fluorescence image of the GFP positive cells. Switch to fluorescence imaging mode in the touch screen panel, which selects the blue laser with 488-nanometer laser excitation, and a 460 to 490-nanometer barrier filter set. And set the acquisition to 100% laser power, and 100 to 125% sensitivity.Now, collect a single XY point bidimensional fluorescence image of the ONH, averaging 100x scans. Next, capture a multi-point image of the retina around the ONH. Select composite in the control panel, and then pan with the ophthalmoscope across the nasal temporal axis.The software automatically averages newly scanned areas and stitches them in real time. If the image quality of areas of the retina is insufficient to allow averaging, the green circle that identifies an area being scanned will become red. The resulting composite image covers an area of up to 1.7/4 millimeters.Care should be taken during acquisition of the composite image. Images at high resolution can be lost by moving the objective too quickly before completion of scan averaging and image stitching.When the anesthesia begins to wear off, the imaging session must end because the mouse starts to breathe heavily and imaging becomes impracticable.

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<figure class='table'><table class='ck-table-resized' style='border-collapse:collapse;font-family:Calibri;font-size:11pt;table-layout:fixed;' cellspacing='0' cellpadding='0' dir='ltr' border='1' data-sheets-root='1' data-sheets-baot='1'><colgroup><col style='width:25%;' width='333'><col style='width:25%;' width='293'><col style='width:25%;' width='192'><col style='width:25%;' width='343'></colgroup><tbody><tr style='height:19px;'><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>DBA/2J and CX3CR1-GFP/+ mice</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>The Jackson Laboratory, Bar Harbor, ME</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>000671 and 00582</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Mice are bred in house, introducing new breeders every 3 - 4 generations to prevent genetic drift.</td></tr><tr style='height:19px;'><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>30&#189; G needle and 1 ml tuberculin slip-tip syringe</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>BD, East Rutherford, NJ</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>305106 and 309659</td><td style='overflow:hidden;padding:0px 3px;vertical-align:middle;'>&#160;</td></tr><tr style='height:19px;'><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>2,2,2-tribromoethanol, tert-Amyl alcohol 99% and phosphate buffer saline tablets</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Sigma-Aldrich, St. Louis, MO</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>T48402, 152463 and P4417</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Avertin solution (pH 7.3, sterile filtered) must be freshly made solution or stored at 4 &#176;C for up to 1 week.</td></tr><tr style='height:19px;'><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Heat therapy T/Pump</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Gaymar Industries, Orchard Park, NY</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>TP-650</td><td style='overflow:hidden;padding:0px 3px;vertical-align:middle;'>&#160;</td></tr><tr style='height:19px;'><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Cotton-tipped applicators</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Fisher Scientific, Pittsburgh, PA</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>23-400-100</td><td style='overflow:hidden;padding:0px 3px;vertical-align:bottom;'>&#160;</td></tr><tr style='height:19px;'><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Tropicamide 1%</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Bausch &amp; Lomb, Rochester, NY</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>NDC 24208-585-64</td><td style='overflow:hidden;padding:0px 3px;vertical-align:bottom;'>&#160;</td></tr><tr style='height:19px;'><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>PMMA contact lenses, 1.70 base curve, 3.2 mm total diameter, 0.40 mm thick center</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Cantor &amp; Nissel Ltd., Northamptonshire, UK</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>G003709</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Lenses are rinsed with sterile PBS and stored in polypropylene boxes.</td></tr><tr style='height:19px;'><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>HRA/Spectralis confocal scanning laser ophthalmoscope and Eye Explorer software</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Heidelberg Engineering GmbH, Carlsbad, CA</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Version 1.7.1.0</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;'>&#160;</td></tr><tr style='height:19px;'><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>NIS-Elements C</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Nikon, Melville, NY</td><td style='overflow:hidden;padding:0px 3px;vertical-align:top;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Version 4.30.01</td><td style='overflow:hidden;padding:0px 3px;vertical-align:bottom;'>&#160;</td></tr></tbody></table></figure>

Source:&#160;<a href='https://appjove.unicartagenaproxy.elogim.com/v/52731/in-vivo-dynamics-of-retinal-microglial-activation-during-neurodegeneration-confocal-ophthalmoscopic-imaging-and-cell-morphometry-in-mouse-glaucoma'>Bosco, A.,&#160;et al. In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma. J. Vis. Exp. (2015)</a> &#160;This video demonstrates in vivo imaging of retinal microglia in a mouse model of glaucoma. A transgenic mouse with fluorescent protein-expressing retinal microglia is positioned under an ophthalmoscope. Infrared illumination is used to locate the optic nerve head (ONH), and the focal plane is established using retinal blood vessels as landmarks. Fluorescent imaging is then employed to visualize the fluorescent microglia. Activated microglia, characterized by enlarged cell bodies and reduced branching complexity, are assessed.

<figure class='image image-style-align-center image_resized' style='display:table;margin-left:auto;margin-right:auto;width:75%;'><img style='aspect-ratio:1771/2400;' src='https://cloudfront.jove.com/files/ftp_upload/23448/23448_Figure1_editor_23448.jpg' alt='23448_Figure1.jpg' /></figure>Figure 1: Live image acquisition controls in cSLO Spectralis.&#160;(A) Manual touch screen panel display on initiation. (B)&#160;cSLO software&#160;imaging window, showing the head of a live mouse. (C) Settings selected for infrared imaging (lit in blue). (D) Fundus image of the retina during real-time acquisition (left panel) and average (right panel). Normalization command indicated with a black circle. Arteries and veins radiating from the optic nerve head are visible (between brackets), as are the optic axonal bundles (arrows) between the blood vessels. (E) Settings selected for fluorescence imaging within a single&#160;xy&#160;point. (F) Single-point fluorescence (FA, 488 nm) imaging during acquisition (averaging). The smaller image (left) shows an individual scan, and the larger image (right) shows the progress of intensity averaging. (G) Settings were selected to acquire a multipoint (composite) fluorescence image. (H) Multipoint fluorescence imaging acquisition shows the scan in progress, which the software identifies with a green circle (or red when the acquisition is impossible). Scale bars represent ~5 mm (B) or 250 &#181;m (D-H)&#160;<figure class='image image-style-align-center image_resized' style='display:table;margin-left:auto;margin-right:auto;width:75%;'><img style='aspect-ratio:1017/2500;' src='https://cloudfront.jove.com/files/ftp_upload/23448/23448_Figure2_editor_23448.jpg' alt='23448_Figure2.jpg' /></figure>Figure 2: Key ocular defects and imaging errors that can prevent correct cSLO imaging of microglial cells.&#160;(A-C) Ocular damage or defects and image acquisition errors can prevent reliable imaging of GFP+ cells localized to the innermost planes of the retina.&#160;(A) Some gross eye defects are readily detectable in fundus images, including corneal or lens opacity or injury and deepening of the optic disc area, preventing clear imaging of GFP+ cells. (B) Focusing either above (the walls of the vessels become indistinguishable from the lumen) or below the retina's inner surface (the vessels are barely visible) reduces the detectability of GFP fluorescence emission from cells located at the nerve fiber and ganglion cell layers. (C) Saturation levels incorrectly or unevenly set can result in bright but oversaturated images (with cells lacking resolution) or images with obscured areas. The scale bar represents 250 &#181;m

Source:&#160;Bosco, A.,&#160;et al. In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell ...

Take an anesthetized transgenic mouse with glaucoma, a condition that damages the optic nerve and leads to vision loss.The microglia, resident immune cells in the eye's retina, express a fluorophore-tagged protein for visualization.Resting microglia display a ramified morphology with branched projections. Glaucoma-induced nerve damage activates microglia, which adopt an amoeboid shape with fewer projections.Place the mouse on an ophthalmoscope platform and align the objective lens with one eye.The eyes are covered with contact lenses to minimize dryness, and the pupils are dilated to expand the imaging field.Visualize the retina using infrared illumination, which reduces tissue damage.Identify the optic nerve head, where retinal ganglion cell axons converge to form the nerve.Utilize retinal blood vessels as landmarks to establish the focal plane.Switch to fluorescence mode to image the microglia, which appear as brightly fluorescent spots, and evaluate the presence of activated microglia comprising enlarged cell bodies and reduced branching complexity.

20 Views. In Vivo Imaging of Retinal Microglia in a Mouse Model of Glaucoma

Video: In Vivo Imaging of Retinal Microglia in a Mouse Model of Glaucoma - Experiment

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

Tomografia a coerenza ottica: Imaging Mouse ganglionari cellule In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

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Neuroscienze

In vivo Structural Assessments of Ocular Disease in Rodent Models using Optical Coherence Tomography

Spectral-domain optical coherence tomography (SD-OCT) is useful for visualizing retinal and ocular structures in vivo. In research, SD-OCT is a valuable tool to evaluate and characterize changes in a variety of retinal and ocular disease and injury models. In light induced retinal degeneration models, SD-OCT can be used to track thinning of the photoreceptor layer over time. In glaucoma models, SD-OCT can be used to monitor decreased retinal nerve fiber layer and total retinal thickness and to observe optic nerve cupping after inducing ocular hypertension. In diabetic rodents, SD-OCT has helped researchers observe decreased total retinal thickness as well as decreased thickness of specific retinal layers, particularly the retinal nerve fiber layer with disease progression. In mouse models of myopia, SD-OCT can be used to evaluate axial parameters, such as axial length changes. Advantages of SD-OCT include in vivo imaging of ocular structures, the ability to quantitatively track changes in ocular dimensions over time, and its rapid scanning speed and high resolution. Here, we detail the methods of SD-OCT and show examples of its use in our laboratory in models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia. Methods include anesthesia, SD-OCT imaging, and processing of the images for thickness measurements.

Here, we describe the use of spectral-domain optical coherence tomography (SD-OCT) to visualize retinal and ocular structures in vivo in models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia.

Valutazioni strutturali in vivo della malattia oculare in modelli di roditori mediante tomografia a coerenza ottica

Spectral-domain optical coherence tomography (SD-OCT) is useful for visualizing retinal and ocular structures in vivo. In research, SD-OCT is a valuable ...

Bioingegneria

In Vivo Methods to Assess Retinal Ganglion Cell and Optic Nerve Function and Structure in Large Animals

The optic nerve collects axons signals from the retinal ganglion cells and transmits visual signal to the brain. Large animal models of optic nerve injury are essential for translating novel therapeutic strategies from rodent models to clinical application due to their closer similarities to humans in size and anatomy. Here we describe some in vivo methods to evaluate the function and structure of the retinal ganglion cells (RGCs) and optic nerve (ON) in large animals, including visual evoked potential (VEP), pattern electroretinogram (PERG) and optical coherence tomography (OCT). Both goat and non-human primate were employed in this study. By presenting these in vivo methods step by step, we hope to increase experimental reproducibility among different labs and facilitate the usage of large animal models of optic neuropathies.

In Vivo Methods to Assess Retinal Ganglion Cell and Optic Nerve Function and Structure in Large Animals

Here we demostrate several in vivo tests (flash visual evoked potential, pattern electroretinogram and optic coherence tomography) in goat and rhesus macaque to understand the structure and function of the optic nerve and its neurons.

In vivo Metodi per valutare la funzione e la struttura delle cellule gangliari retiniche e del nervo ottico in animali di grandi dimensioni

The optic nerve collects axons signals from the retinal ganglion cells and transmits visual signal to the brain. Large animal models of optic nerve injury ...

In Vivo Imaging of Retinal Microglia in a Mouse Model of Glaucoma

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In Vivo Imaging of Retinal Microglia in a Mouse Model of Glaucoma