This protocol describes how high-resolution imaging techniques such as spectral domain optical coherence tomography and scanning laser ophthalmoscopy can be utilized in small rodents, using an ophthalmic imaging platform system, to obtain information on retinal thickness and microglial cell distribution, respectively.
The overall goals of this procedure are to use spectral-domain optical coherence tomography and scanning laser ophthalmoscopy to obtain information on retinal thickness and microglial cell distribution, respectively. This method can help answer key questions in the experimental ophthalmology field about the correlation between retinal abnormalities and microglial cell accumulation. The main advantages of this technique are that this information can be obtained in real-time in a non-invasive manner.
After confirming a lack of response to corneal swab, place the mouse in the prone position on the left side of the platform, with the right orbit facing the lens. Apply a drop of hydroxypropyl methylcellulose in a plus four diopter, rigid, gas-permeable contact lens onto the right eye and start the acquisition module. For B scans, select the Infrared plus Optical Coherence Tomography option Under Application and Structure, select Retina and use the micro-manipulator to move the lens towards the mouse eye.
Before focusing on the retina, confirm that the oculus dexter indicator is selected, then use the focus knob to zoom into the retina until the big vessels are clearly visible in the fundus image on the left side of the monitor screen. Use the micro-manipulator to adjust the position of the camera, as necessary, turning the sensitivity knob to reduce or increase the brightness of the fundus image, as appropriate. Select the Line Scan from the Pattern menu and use the micro-manipulator to move the B scan between the top and bottom corners of the spectral-domain optical coherence tomography, or SD-OCT, scan window.
Set the Automatic Real Time value to at least nine to obtain a high image quality and click Acquire. When all of the images have been obtained, transfer the contact lens from the eye into fresh-balanced salt solution and hydrate the cornea with a fresh drop of hydroxypropyl methylcellulose. After the left eye has been imaged, rotate the standard 30 degree optic in the counter-clockwise direction to remove it and mount the 55 degree lens for a second round of imaging.
To assess the auto fluorescence, without moving the mouse, select Infrared on the control panel and focus on the big retinal vessels. Select auto fluorescence imaging and use the sensitivity knob to adjust the image brightness, as necessary. Press the sensitivity knob once and set the automatic real-time value to at least 67.
When the automatic real-time value has been reached, acquire the image and press the sensitivity knob a second time to stop the averaging. Adjust the focus to visualize the different retinal layers, as experimentally appropriate. When all of the images have been obtained, on to wide-field 102 degree lens onto the optic and image the auto fluorescence in each eye, as just demonstrated.
To measure the manual retinal thickness in each image, double-click on the name in the first experimental animal to open the OCT scan. Open a B scan obtained with the 30 or 55 degree lens and select the thickness profile. Click the Edit Layer Segmentations icon.
The software will automatically identify the inner limiting and base membranes. To manually correct the position of the membranes, select the layer to be modified, as well as the red circle option. Holding down the mouse button, move the circle to modify the line until the corresponding layer is correctly positioned and click Save and Close to exit the window.
Under the Layer option, select Retina and click on a different position on the diagram to view the retinal thickness for the selected position. Then measure the retinal thickness and the desired distance from the optic nerve head and export the values into a spreadsheet. In these representative SD-OCT single scans from a mouse homozygous for the expression of gfp under the Cx3cr1 promoter, the retinal architecture is clearly visualized in both the 30 degree and 55 degree lens images.
However, a high reflectivity of the choroid is observed in the scans obtained with the 30 degree lens. Following SD-OCT, scanning laser ophthalmoscopy allows the visualization of the individual gfp positive microglial cells in the retina using a 55 degree or a 102 degree lens with larger fundus area coverage obtained with the 102 degree lens. In the SD-OCT scans, after manual correction of the inner limiting and base membrane retinal boundaries, a good correlation of the retinal thickness measurement is typically observed between the 30 and 55 degree lenses when the same distance from the optic nerve head is measured.
Once mastered, this technique can be completed in less than fifty minutes if it's performed properly. After its development, this technique paved the way for researchers in the experimental ophthalmology field to explore retinal pathology in small rodents in vivo.
