These methods can help answer key questions in the field of neurology and ophthalmology, and help to evaluate a large variety of retinopathies and retinal manifestations in various rodent models. The main advantage of these techniques is that noninvasive longitudinal investigations can be performed, reducing variability and cutting down the number of animals needed for the experiments. These methods allow quantitative in vivo investigation of structure and function which can be used to investigate the pathological conditions, or to evaluate the potential utility of novel therapeutics.
However, especially the application of optical coherence tomography technology in rodent models is still challenging, mainly because of the small eye size. Demonstrating the procedures will be Michael Dietrich, a PhD student from our laboratory. After anesthetizing the rodent, apply one drop of Phenylephrine Tropicamide on each eye for pupillary dilation.
Wipe off any excess eye droplet wet after one minute. And lubricate the eyes methylcellulose based ophthalmic gel to avoid drying out the eye and creating a turbid cornea. Then use forceps to place a custom contact lens on the eye.
Press the Start button in the right corner of the control panel display to start the acquisition mode. Next, to image the left eye, position the holder to ensure that the left eye bulb of the rodent faces the camera. Set the filter level to R, and select BR and OCT for blue reflections fundus imaging and B-scan acquisition in the software.
Next, set the focus distance to approximately 38 diopters, using the focus knob on the back of the camera. And zoom in on the retina, until the OCT scan is visible on the screen. Then to ensure a beam path through the middle of the pupil, with an orthogonal angle of the retina in all planes, position the optic disc in the middle of the illuminated field.
And adjust the horizontal and vertical line B-scans to a horizontal level by rotating the holder. On the software screen select the volume scan mode, and set it to 25 B-scans in high resolution mode at 50 automatic real time tracking. Center the middle of the volume scan grid on the optic disc and start acquisition by pressing the black sensitivity knob, and then Acquire on the control panel.
After the acquisition is complete, set the filter level to A.Select Blue Autofluorescence on the control panel. And adjust image brightness with the sensitivity knob. Press the sensitivity knob and then Acquire to image fluorescent cells or autofluorescent deposits.
For analysis of the volume scans use the automated segmentation of the OCT device's software by right-clicking on the scan. And select Segmentation, then All Layers. Perform manual correction of the layers by double-clicking on the desired scan.
Select Thickness Profile. And click on Edit Layer Segmentations. Select one layer at a time.
And if necessary correct the green line by moving the red dots by dragging and dropping to the correct position. Next select the tab Thickness Map. Choose the 1, 2, 3 millimeter ETDRS grid.
Center the inner circle on the optic disc. Finally, calculate the thickness of retinal layers from the thickness values provided by the software for the different retinal sectors of interest. To compute the main thickness values from volume scans, use the whole 1, 2, 3, millimeter ETDRS grid which covers an angle of approximately 25 degrees, excluding the inner one millimeter circle which contains the optic disc.
Begin by selecting a platform to measure the rodents. Open the Presettings window by double-clicking on the software. Select New Group and choose the group name, the number of subjects, the species, and strains.
Select a variable stimulus, spatiotemporal frequency, contrast sensitivity, speed or orientation in the dropdown menu and press Create New Group. Focus on the platform by manipulating the focus ring of the camera on top of the chamber. And calibrate the system by aligning the red circle around the black circle on the platform.
Next, place the rodent on the platform and let it adapt to the environment for about five minutes. Then start the measurement by selecting the left arrow for yes or the square for no, if the animal tracks or does not track respectively. Manually select the step size of the stimulus by clicking on the Up and Down Arrows next to the variable stimulus.
Finally, select the Summary tab and click on the File, Export Table or Graph to export the desired data set. Results indicate that the degeneration of the inner retinal layers is reduced. And a clinical EAE score is attenuated during the EAE course when substance 1 was administered.
Further, OKR reveals an improved visual acuity of animals treated with substance 1 compared to untreated MOG EAE mice measured by spatial frequency threshold testing over a period of 120 days. When performing OCT measurements, A critical step is the orthogonal orientation of the laser beam in all planes in relation to the retina to ensure the quality and reproducibility of the thickness values. For the OKR technique, distinguishing between tracking and normal behavioral movements of the animal, requires some training of the investigator.
It is important to be blinded for the experimental group. Following these investigations of structural and functional damage occurring in the context of experimental Autoimmune Encephalomyelitis, the procedure may also be helpful in other models involving the visual system, including but not limited to the models of retinopathies, or optic nerve injury. These techniques pave the way for in vivo research in the fields of neurology and ophthalmology and are easily transferable to clinical trials.
