Participants
This study was approved by the Cedars-Sinai Institutional Review Board. All subjects older than 40 years of age, presenting to our Neurology clinic with subjective cognitive decline and interested in undergoing retinal fluorescence imaging were included in this cohort (Table 1). All subjects provided written informed consent prior to the commencement of the study.
Retinal Imaging
After ocular dilation, retinal imaging was performed with a confocal scanning ophthalmoscope (RetiaTM, CenterVue SpA) that utilizes blue light for excitation of curcumin emission to obtain fluorescent images of the retina, following a study design described in prior reports (Figure 1A) (32, 33). The investigators conducting the retinal image processing and quantifications were blinded to the patients’ clinical characteristics.
Retinal amyloid quantification
Retinal images were processed using an automated retinal fluorescence measurement software system (NeuroVision Imaging, Inc.). A common region of interest (ROI) in the supero-temporal quadrant was applied with a field of view of 50 degrees positioned on the image center, using fovea and optic nerve head centers as reference points to correct for eye rotation, with a zone around the fovea and optic nerve head masked, as previously reported (33). The ROI was further divided into three subregions: posterior pole, proximal mid-periphery and distal mid-periphery (Figure 1B). Retinal amyloid count was quantified in the target ROI and the three specified subregions.
Retinal vascular quantification
From the same retinal fundus images, a ROI was defined within a circumpapillary region centered on optic nerve head (ONH) and extended between 1.5 and 4 ONH radii (51) (Figure 1C). Retinal vessels within the ROI were segmented using Frangi vesselness filter to generate a binary image (52). The vessels were classified into arteries and veins by a human observer based on the facts that retinal arteries are brighter in color and thinner in width compared to veins (53). For each vessel segment on the binary image, vessel endpoints were selected, and distance transformation was used to extract the vessel centerline. The extracted centerlines were smoothed using a cubic spline with a regularization parameter of 3 x 10-5. For each centerline, several geometric features including vessel tortuosity index (VTI), vessel inflection index, and branching angle were non-automatically quantified. VTI was calculated for each centerline based on combination of local and global centerline geometric variables as explained previously (54). Equation (1) shows formula for VTI,
where is standard deviation of angle difference between lines tangent to each centerline pixel and a reference axis (i.e. x-axis). M is average ratio of centerline length to its chord length between pairs of inflection points including centerline endpoints. N is number of critical points where the first derivative of the centerline vanishes. LA and Lc are the length of the vessel and its chord length, respectively. VTI is shown to provide good correspondence with human perception of tortuosity and is invariant to rigid transformations. Similar to other measures of tortuosity, VTI is unitless. Its minimum value is zero while it has no theoretical maximum as it can increase with twistedness of a vessel. Vessel inflexion index was determined based on number of inflection points along the vessels. Mathematically, these are pixels where the second derivative of centerline vanishes. Vessel inflexion index represents local changes in tortuosity of vessels and was found to be robust for ranking tortuosity of vessels with similar length (55). Branching angle of the vessels was calculated interactively using an open source tool GIMP 2.8.
Cognitive evaluation
All participants underwent a standard battery of neuropsychometric testing performed by a licensed neuropsychologist (DS). Standard neuropsychological testing included assessment of the Montreal Cognitive Assessment (MOCA), global Clinical Dementia Rating (CDR), general cognitive (ACS-Test of Premorbid Functioning) and specific cognitive domains: attention and concentration [Wechsler Adult Intelligence Scale (WAIS)-IV]; verbal memory [California Verbal Learning Test (CVLT) II, Wechsler Memory Scale (WMS)-IV Logical Memory II]; non-verbal memory [Rey Complex Figure Test and Recall (RCFT) 30 min, Brief Visuo-Spatial Memory Test Revised (BVMT-R) Delayed Recall]; language [Fluency-Letter (FAS), Fluency-Category (Animals)]; visuo-spatial ability [Rey Complex Figure Test and Recognition Trial (RCFT) Copy]; speed of information processing (Trails A and B); symptom validity and functional status [SF-36 Physical Component Score (PCS) and Mental Component Score (MCS)]. We also evaluated the subject’s emotional status using Beck Depression Inventory II, Geriatric Depression Scale, and Profile of Mood State Total Mood Disturbance.
Statistical Analysis
Descriptive statistics were calculated for patient demographics and clinical characteristics. Unless otherwise stated, data are expressed as mean ± standard deviation. Subjects were partitioned into three groups according to Clinical Dementia Rating (CDR; 0.5, questionable impairment; 1, mild cognitive impairment; 2, moderate cognitive impairment) (56). The subjects were also partitioned into groups according to the neuropsychometric diagnosis (normal cognition versus impaired cognition).
To produce combined indices of retinal vascular and amyloid measures, each variable was first inspected for normality; any non-normal variables were then log transformed to produce a normal distribution. Each normalized variable was then standardized to a mean of 0 and a standard deviation of 1. While higher amyloid count was associated with worse cognitive function, higher venous vascular tortuosity index (VTI) values were associated with better cognitive function. To account for this inverted scale, the standardized values of venous VTI were multiplied by -1. Standardized variables were then summed to produce exploratory, combined index measures of retinal amyloid and retinal vascular features.
Differences in continuous variables between levels of CDR were assessed through one-way analysis of variance (ANOVA). Differences in continuous variables between diagnostic scores were assessed using Student’s t-test. Linear regression was performed to assess the relationship between retinal vascular and retinal amyloid measures, as well as the relationship between combined retinal vascular and amyloid counts and cognitive parameters. All statistical analyses were performed using STATA v15.1 (StataCorp, College Station, TX) with an a priori significance level of 0.05.