Concurrent OCT and OCT angiography of retinal neurovascular degeneration in the 5XFAD Alzheimer’s disease mice

Abstract. Significance: As one part of the central nervous system, the retina manifests neurovascular defects in Alzheimer’s disease (AD). Quantitative imaging of retinal neurovascular abnormalities may promise a new method for early diagnosis and treatment assessment of AD. Previous imaging studies of transgenic AD mouse models have been limited to the central part of the retina. Given that the pathological hallmarks of AD frequently appear in different peripheral quadrants, a comprehensive regional investigation is needed for a better understanding of the retinal degeneration associated with AD-like pathology. Aim: We aim to demonstrate concurrent optical coherence tomography (OCT) and OCT angiography (OCTA) of retinal neuronal and vascular abnormalities in the 5XFAD mouse model and to investigate region-specific retinal degeneration. Approach: A custom-built OCT system was used for retinal imaging. Retinal thickness, vessel width, and vessel density were quantitatively measured. The artery and vein (AV) were classified for differential AV analysis, and trilaminar vascular plexuses were segmented for depth-resolved density measurement. Results: It was observed that inner and outer retinal thicknesses were explicitly reduced in the dorsal and temporal quadrants, respectively, in 5XFAD mice. A significant arterial narrowing in 5XFAD mice was also observed. Moreover, overall capillary density consistently showed a decreasing trend in 5XFAD mice, but regional specificity was not identified. Conclusions: Quadrant- and layer-specific neurovascular degeneration was observed in 5XFAD mice. Concurrent OCT and OCTA promise a noninvasive method for quantitative monitoring of AD progression and treatment assessment.


Introduction
Alzheimer's disease (AD) is a progressive neurodegenerative disease and the fifth leading cause of death in Americans over 65 years. 1 The death rate resulting from AD soared 123% between 2000 and 2015, 1 and it is anticipated that one in every 85 people will be living with AD by 2050. 2 There is no cure for AD to date; thus, early detection and therapeutic interventions are imperative to prolong cognitive function in AD patients. 3,4 A definitive diagnosis of AD can be achieved by *Address all correspondence to Xincheng Yao, xcy@uic.edu

Imaging System
A custom-designed spectral-domain OCT system was used in this study. The system has been employed in multiple functional OCT/OCTA studies. [35][36][37] Briefly, a near-infrared superluminescent diode (λ ¼ 810 nm; Δλ ¼ 100 nm; D-810-HP, Superlum, Carrigtwohill, County Cork, Ireland) was used as a light source. A line CCD camera with 2048 pixels (AViiVA EM4; e2v Technologies, Chelmsford, United Kingdom) was used for recording OCT spectra in the custom-built OCT spectrometer. The frame rate of the camera was set to 50 kHz. The axial and lateral resolutions were theoretically estimated as 2.9 and 11 μm, respectively. The measured axial resolution from the scattering profile of the external limiting membrane (ELM) was 3.4 μm. The ELM is the thin line of junctional complexes between Müller cells and photoreceptors (<1 μm thickness), which can be well described by a Gaussian to confirm the axial point spread function. 38 1-mW power was illuminated on the mouse cornea.

Experimental Procedures
Anesthesia was intraperitoneally induced by a mixture of ketamine (100 mg∕kg body weight) and xylazine (5 mg∕kg body weight), and a drop of 1% tropicamide ophthalmic solution (Akorn, Lake Forest, Illinois) was applied to the imaging eye. Next, a cover glass (12-545-80; Microscope cover glass, Fisherbrand, Waltham, Massachusetts) with a drop of eye gel (Severe; GenTeal, Novartis, Basel, Switzerland) was placed on the imaging eye. After the mouse was fully anesthetized, the head was fixed by a bite bar and ear bar in the animal holder that provided five degrees of freedom (i.e., x, y, z, pitch, and roll). Volumetric raster scans were individually acquired from each retinal quadrant (dorsal, nasal, temporal, ventral quadrant, and the ONH). The ONH region was first captured, which can serve as a central point of the retina, and the dorsal/ventral quadrants were captured, followed by the nasal/temporal quadrants. Four repeated B-scans at each slow-scan position were collected for OCTA construction; thus, each OCT volume consisted of 4 × 600 × 600 A-scans. All animal experiments were approved by the local animal care and biosafety office and performed following the protocols approved by the Animal Care Committee (ACC) at the University of Illinois at Chicago (ACC Number: 19-044). This study followed the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.

OCTA Image Processing
The OCTA images were constructed by implementing intensity-based speckle variance processing. 39 Next, retinal flattening was performed by realigning each A-line. Three vascular layers were then manually segmented for vessel density analysis ( Fig. 1), i.e., the superficial vascular plexus (SVP), intermediate capillary plexus (ICP), and deep capillary plexus (DCP). 40 For each segmented layer, en face OCTA image was reconstructed by maximum intensity projection, and a Hessian-based vessel enhancement method proposed by Jerman was used to enhance vasculature in each en face image. 41 After that, binarization for the SVP was done by adaptive thresholding, and binarization for the ICP and DCP was done by ridge-based vessel detection method. 42 All binarized images were further processed by morphological opening operation to remove small particle noises. Image processing was performed on MATLAB R2016a (MathWorks, Natick, Massachusetts), in coordination with image processing package available in Fiji software. 43

Data and Statistical Analysis
Retinal thickness, vascular width, and vascular density were measured for quantitative comparison. For thickness measurement, flattened OCT-B scans were prepared, and large vessels were manually removed to measure the retinal thickness solely [ Fig. 1(k)]. Central 200 A-lines were averaged in the horizontal direction, returning one averaged intensity profile [ Fig. 1(l)]. The regions in where the vessels were manually removed [ Fig. 1(k)] were excluded from the averaging. Based on the average profile, the NFL, inner retinal layer, outer retinal layer, and total retinal thickness were manually measured [ Fig. 1(m)]. For vascular width measurement, the artery and vein (AV) around the ONH were first classified by referring vascular morphology on radially resliced OCT B-scans, 44 and the vessel width of all first branches was measured based on the circular profile, 300 μm away from the ONH [Figs. 1(n)-1(p)]. The full-width at halfmaximum (FWHM) was measured using the "findpeaks" function (MATLAB R2016a, MathWorks, Natick, Massachusetts) in determining the vessel diameter. The vessel density for each plexus was defined as the percentage of area occupied by vasculature in the binarized OCTA images. The two-sample t-test was performed for the statistical comparison between WT and 5XFAD, and a p-value < 0.05 was considered statistically significant. In this study, each quadrant measurement was treated as an independent sample based on the current understanding that AD-associated retinal degeneration appears in a specific retinal region, and this local effect does not affect measurements for other quadrant observations. 20,21 Statistical analysis was performed on Origin 2020b (OriginLab, Northampton, Massachusetts).  which is ∼61deg visual angle by the conversion factor of 36 μm per degree of visual angle. The conversion factor was estimated based on a recent work demonstrating the adult mouse eye model incorporated with a contact lens and gel thickness information. 45 This field of view can partially visualize network topologies of the mouse retina vasculature. Arteries and veins radially emanate from the ONH, whereas the capillary plexus reveals a circular mesh structure centered at the ONH. 46 Retinal abnormalities were not recognized between the WT and 5XFAD mouse through visual inspection, implying only subtle changes might occur in the 5XFAD mouse retina. Each quadrant OCT and OCTA volume was then individually processed and analyzed for the quantitative assessment.
We next investigated vascular changes. To quantify the vascular narrowing/widening effect, we measured arterial and venular width following the circular profile around the ONH with radii of 300 μm. AV classification was achieved by the pseudoradial scanning method. 44 Figures 1(o) and 1(p) show the radial OCT B-scans showing hyperreflective wall boundaries in retinal arteries, whereas these wall boundaries were absent in retinal veins. We found a significant arterial narrowing in 5XFAD mice (WT: 33.6 AE 4.7 μm; 5XFAD: 28.3 AE 4.8 μm; p < 0.0001 for average arterial width comparison, p ¼ 0.007 for average arterial width per mouse comparison, i.e., the average of the mean of each mouse dataset) [ Fig. 4(a)], and the venular width was comparable between WT and 5XFAD mice (WT: 33.9 AE 10.6 μm; 5XFAD: 31.1 AE 10.1 μm; p ¼ 0.309 for average venular width comparison, p ¼ 0.201 for average venular width per mouse comparison, i.e., the average of the mean of each mouse dataset) [ Fig. 4(b)]. The venular width exhibited more variations than the arterial width and did not show significant alterations between the two strains.  Data are expressed as mean AE standard deviation per group. A two-sample t -test was performed for intergroup comparison. P-value for statistical significance is indicated by an asterisk. *P < 0.05. N ¼ 6 for WT (wild-type) and N ¼ 5 for TG (transgenic 5XFAD).
Next vascular density was analyzed. The vasculature observed in OCTA reflects functional vessels. 47 To measure the layer-specific change, trilaminar vascular plexuses were segmented (Fig. 1). Figure 5 shows that the vessel density in the SVP was comparable between WT and 5XFAD mice (WT: 21.2 AE 1.8%; 5XFAD: 21.4 AE 1.3%; p ¼ 0.606), whereas the overall capillary density showed a decreasing trend in 5XFAD mice. Especially, the ICP density reduction in 5XFAD mice was close to being statistically significant (WT: 14.7 AE 1.5%; 5XFAD: 13.7 AE 1.6%; p ¼ 0.056) [ Fig. 5(b)]. We also found that capillary density consistently showed a decreasing trend in all retinal quadrants, although quadrant specificity tends to vary [Figs. 5(e) and 5(f)]. Collectively, the results demonstrate that the arterial narrowing effect was significant in 5XFAD mice, and functional capillary density in all quadrants was reduced in 5XFAD mice.

Discussion
In this study, region-specific neurovascular degeneration was found in 6-month-old 5XFAD mice. Significant inner retinal thinning was first noted in 5XFAD mice (Fig. 3), consistent with a recent finding of selective inner retinal deficits in 5XFAD mice at 6 months. 27 The retinal thinning generally indicates neuronal death, 48 and increasing evidence suggests that Aβ deposits in the inner retina may be associated with degeneration of retinal ganglion cells. 49 Especially, the 5XFAD mouse model was found with the highest concentration of Aβ peptides in the brain and retina among AD mouse models. 50 Although the exact role of Aβ in AD pathology remains an open question, it is believed that aggregation of extracellular Aβ can disrupt cellular communication and activities, which can damage synapses and ultimately induce neuronal cell death. 51 In a recent study, notable Aβ accumulation was found around degenerating retinal ganglion cells in AD subjects. 9 Also, we confirmed consistent outer retinal thinning in all quadrants [ Fig. 3(g)].
The reason can be attributed to aggressive Aβ deposition not only in the inner retina but also in the outer retina in 5XFAD mice. It was reported that Aβ was largely accumulated in the 5XFAD retina under the retinal pigment epithelium (RPE), which might cause inflammation and contribute to photoreceptor cell death. 52,53 Likewise, increased Aβ deposits around the rod photoreceptors were recently observed in AD subjects along with outer retinal thinning. 13,54 The presence of Aβ may cause defects in photoreceptor cell function and potentially contribute to visual impairments in AD subjects as reflected by the reduction of contrast sensitivity, visual acuity, color vision, and visual field. 55 Six-month-old 5XFAD mice also manifested vascular abnormalities in the retina. Arteries showed a significant narrowing in 5XFAD mice [ Fig. 4(a)], which might be a consequence of the capillary flow deficits. Although capillary density reduction did not reach statistical significance, the ICP and DCP consistently exhibited a decreasing trend in all quadrants of the 5XFAD mouse retina [Figs. 5(b) and 5(c)]. Hernández et al. 33 found that capillary stalling was elevated in 6-month-old 5XFAD mice and described that a small portion of capillary occlusion could dramatically decrease blood flow in up-and-down-stream vessels, resulting in the blood flow changes on cerebral blood flow. Nortley et al. also suggested that capillaries could be the most critical locus where Aβ can decrease cerebral blood flow, 56 and reduced blood flow was found to increase in Aβ deposition, suggesting that blood flow deficits can further worsen Aβ pathology. 57 However, there is an opposite pathophysiological basis that angiogenesis can occur in response to impaired cerebral perfusion and vascular injury. 58 Such inflammation due to Aβ accumulation can induce the retina to become hypoxic and trigger angiogenesis, 19 which can cause increased vessel density observed in OCTA. 59 A recent clinical study reported capillary density increase in AD subjects, 60 while others reported the opposite results. 61,62 We speculate that capillary density may increase at the initial stage of the Aβ accumulation. However, at the later stage, when neuronal cell loss occurs, capillary density may naturally decrease due to a reduction of metabolic demand and damaged endothelial cells. A further longitudinal study is required to verify this hypothesis.
Individual quadrant OCT/OCTA analysis suggested that neurovascular degeneration may occur in all retinal regions, but there seems to be a trend of regional specificity in terms of degeneration progress. Retinal thinning of the NFL and outer retina was distinguished in the dorsal and temporal quadrant, respectively, in 5XFAD mice (Figs. 3 and 5). Song et al. 26 also found different light scattering parameters only in the dorsal quadrant in AD mouse retinas. In fact, a region-specific degeneration is not only limited to the animal models but also appeared in human subjects with AD. 21 Previous studies demonstrated that retinal changes were predominantly focused on the superior and inferior regions of the NFL in AD subjects. 28,29 Querques et al. 12 reported that ganglion cell layer thickness was reduced in AD subjects only in the central and temporal retinal region. Koronyo et al. 13 found that Aβ plaques mainly appeared in the periphery of the superior quadrant, whereas some regional changes were discovered in each retinal layer differentially. 54 Such discrepancy requires further verification of region-specific changes in the retina associated with AD pathology.
This study has three limitations to be addressed. First, a relatively small sample size was used in this cross-sectional study, which naturally lowered the statistical power to draw a definitive conclusion. Prospective longitudinal monitoring with an increasing sample size would further elucidate retinal abnormalities associated with AD pathology in transgenic mouse models. Second, there was no conclusive biomarker confirmation. Postmortem histopathology would be helpful to confirm the presence of AD biomarkers, such as Aβ42, total tau (T-tau), and phosphorylated tau (P-tau), in different retinal regions to correlate the level of biomarkers with OCT/OCTA features. Third, it is important to be aware of anesthesia-related cardiovascular effects, including changes in blood pressures, cardiac output, and varying heart rhythms. The ketamine-xylazine mixture is known to have potent cardiodepressive effects that can potentially lower local vessel diameter and blood flow over time. 63,64 Since the effect is timedependent, in this study, the ONH region was always first captured in each mouse, and vessel width measurement was performed around the ONH [ Fig. 1(n)]. Monitoring vital signs during image acquisition would allow for better understanding of anesthesia-related cardiovascular effects on the mouse retina. Despite the limitations of this study, in vivo full quadrant imaging may provide a foundation for future detailed exploration of the relationship between retinal neurovascular defects and AD-associated retinal degeneration.

Conclusions
This study demonstrates in vivo OCT/OCTA monitoring of all retinal quadrants, which allowed examining region-specific neurovascular degeneration in the 5XFAD mouse retina.
Six-month-old 5XFAD mice revealed that inner and outer retinal degeneration were explicitly advanced in the dorsal and temporal quadrant, respectively. The arterial narrowing was observed in 5XFAD compared with wild-type mice. Overall decrease of measured capillary density was also observed in 5XFAD mice, suggesting a tight correlation between retinal neurons and vasculature. The proposed imaging strategy promises a noninvasive method for longitudinal monitoring of AD progression and treatment assessment in transgenic mouse models of AD.

Disclosures
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.