The overall goal of this protocol is to describe methods of quantifying vascular and neuronal structure in the retina using retinal imaging techniques which may reflect dementia related changes in the brain and potentially aid in diagnosis and risk assessment of dementia. The retina shares similar embryological origin, and anatomical features and physiological properties with the brain, hence offers a unique and accessible window to study dementia. Vascular and neuronal changes in the retina can now be imaged and quantified using advanced retinal imaging technology, which is non-invasive, affordable, easier to use, and widely available.
Current biomarkers for dementia, especially Alzheimer's disease, are limited. Developing new biomarkers or tests to allow prediction of future cognitive decline and onset of early dementia is a global research priority. Demonstrating the procedure will be Victor, Tiffany, and Fangyao from our research team.
To begin this procedure, dilate the pupils of the subject using mydriatic agents. Then, wait for at least 15 minutes to establish sufficient pupil dilation. Next, start the fundus camera and launch the image capturing program on the computer.
Rest the chin of the subject properly on the chin rest, with the forehead against the head strap. Afterward, move the control lever to align the light beam properly to the subject's pupils. Align the illumination points until they appear smallest on both sides in the viewfinder of the fundus camera.
Move the external fixation target to guide the subject's eyes until the optic disc is at the center of the viewfinder and the regions of interest are well within the limits of the image. Then, adjust the focusing knob to focus on the retina. Ask the subject to firmly look at the external fixation target and ensure the subject's eyes are not filled with tears.
Then depress the shutter release button to capture an image. Subsequently, save all the images in tiff format with gradable resolution. In order to obtain high quality retinal images, this is important to dilate the patient's pupils sufficiently, and encourage the patients to firmly look at the fixation target during the imaging.
For automatic tracing, open the images with the computer-assisted analysis program. Click the OD Center button on the left function panel. The mouse cursor will be replaced by a green circle.
Next, left click on the optic disc center to fix the green circle, then click the Find OD button to prompt the software to detect the optic disc rim and place a new measurement grid. Now, click the Process button to initiate the auto-tracing process of the vessels. Left click to select the vessels with incorrect vessel labels and click the Vessel Type button to change the vessel type.
Click the Add Segment button and extend the incomplete vessel tracings by using the cursor to click at the distal end of the incomplete vessel tracing. Afterward, left click at the points along the vessel to extend the vessel tracing. Stop the tracing at the outermost white circle if the distal part of the vessel falls outside the measurement grid.
Subsequently, click the Find Covers button to lay the vessel covers on all vessel segments automatically. Deactivate the vessel covers if they are not laid perpendicular to the vessel walls, the vessel is obscured under another vessel, or the vessel covers overestimate or underestimate the width of the internal lumen. The incorrect vessel tracings should be adjusted properly in order to obtain an accurate measurement of the retinal vascular parameters.
Mastering these steps may require repeated practice, as well as guidance from an experienced grader. Close the grading windows and click Send in the pop-up dialog to upload the graded image to the cloud-based server and record the automatically measured retinal vascular parameters, such as vessel caliber, fractal dimension, tortuosity, branching angle, and branching coefficient. To perform image acquisition, open the OCT program and select the macular cube scanning protocol to start a new macular scan.
Next, locate the pupils in the iris viewport by adjusting the chin rest. Click the AutoFocus button and then the Optimize button to improve the image quality, then click the Capture button to start the scan when the border surrounding the button becomes green. Perform another scan for the optic nerve head using the optic disc cube scanning protocol and save the scanning results.
After that, select the macular cube scan records of both eyes in the analysis interface. Click the ganglion cell OU analysis to initiate the automatic analysis algorithm to assess the GC IPL thickness of the captured image. Then, save the analysis printout in the pdf format.
Subsequently, select the optic disc cube scan records of both eyes in the analysis interface. Click the ONH and RNFL OU analysis to initiate the automatic analysis algorithm to assess the RNFL thickness of the captured image, and save the analysis printout in the pdf format. Fundus photograph can be used to assess retinal vasculature which in turn reflects the status of cerebral microcirculation.
These are the fundus photographs of a healthy subject, and an AD subject. When compared to the healthy subject, the AD subject showed narrower vessel caliber, smaller retinal vascular fractal dimension, and higher retinal vascular tortuosity. Optical coherence tomography can be used to assess retinal neuronal structure which in turn reflects the status of cerbral neuron.
The thickness maps of this normal subject demonstrate the normal thickness distribution of the RNFL and GC-IPL. The analysis algorithm also compares the measured thickness to the normative age-matched database, and generates a significance map. This subject is classified as normal, as all the sectors are either shown in green or white.
These two figures show the analysis output in an AD subject. As indicated by the blue regions in the thickness maps, this AD subject exhibits thinning in some regions of the RNFL and GC-IPL. The red and yellow sectors in the significance maps also indicate the thinning of GC-IPL, or RNFL, in the corresponding areas as abnormal when compared to the device's normative age-match database.
Once mastered, the whole procedure can be done in one hour. This method is non-invasive and done in confidence, and one can easily acquire repeated measurements in both short-term and long-term. As the retina allows non-invasive and direct imaging of the central nervous system, this paves the way for researchers to assess the effects of dementia on the microvasculature and the neural structure.
Changes in retinal microcirculation and neurologic structure have also been shown to be associated with dementia, especially Alzheimer's disease. Following these procedures, cognitive tests, and other neuroimaging modalities can also be performed to collect other information of dementia. After watching this video, you should have a good understanding of how to use retinal camera, optical coherence tomography, computer assisted analysis program, to obtain retinal images and quantify vascular and neuronal changes in the retina objectively.
