Retinal Vascular Pathology in Alzheimer’s Disease
Cerebral amyloid angiopathy is defined as a cerebrovascular disease characterized by intense deposition of Aβ in the walls of cerebral arteries, arterioles, and capillaries, among other vascular damage, and is commonly found in the brains of AD patients (DeSimone et al., 2017). Aβ in CAA primarily consists of Aβ40 (Roher et al., 1993; Gravina et al., 1995). In macrovasculature, CAA is composed of Aβ deposition in tunica media and adventitia of leptomeningeal and cerebral parenchymal arteries (Tian et al., 2004). The Aβ in CAA will eventually affect all vascular layers and result in degeneration of smooth muscle cells (Keable et al., 2016). CAA is prevalent in the elderly who have developed lobar cerebral hemorrhage (ICH) (Keable et al., 2016). Although CAA remains a distinct clinical entity from dementia, previous studies have demonstrated that over 85% of AD patients have of CAA of varying severity (Arvanitakis et al., 2011; Viswanathan and Greenberg, 2011). The Aβ deposited in the vascular walls triggers several ischemia-induced pathogenic molecular pathways, such as oxidative stress, inflammation and increased blood-brain barrier (BBB) permeability, leading to further hemorrhagic complications (Ghiso et al., 2010). A study based on parametric analysis of neuropathological data from the National Alzheimer’s Coordinating Centers’ dataset suggested that CAA was facilitating early stage dementia and the transition to moderate dementia (Vidoni et al., 2016). In severe CAA cases, Aβ deposition is usually followed by microaneurysms in cerebral blood vessels (Vonsattel et al., 1991), a vascular pathology shared by retinal vascular diseases such as diabetic retinopathy, which can be easily examined by fundoscopy (Friberg et al., 1987; Hellstedt et al., 1996).
As the retina is a developmental outgrowth of the diencephalon (Purves, 2001; Erskine and Herrera, 2014), it shares many vascular morphological and physiological features with cerebral vessels (Patton et al., 2005; Crair and Mason, 2016). For instance, the retina has a highly selective blood retinal barrier (BRB), with many similar structural and functional properties as the BBB, which modulates the influx of ions, proteins and water, as well as limits the infiltration of circulating immune cells (Cunha-Vaz et al., 2011). The recent identification of an ocular glymphatic drainage system in rodents, clearing fluids and metabolites such as Aβ from the retina and vitreous via an aquaporin-4 (AQP4)-dependent pathway, is a sign of yet another structural and physiological similarity shared by the brain and retina (Wang et al., 2020). The same group also demonstrated that this clearance route may be impaired in ocular conditions associated with retinal damage such as glaucoma (Wang et al., 2020). Whether similar glymphatic drainage occurs in the human eye and the extent to which disruptions in this process contribute to pathological changes in neurological diseases such as AD remain to be investigated.
Numerous reports have broadly described vascular dysfunctions in the AD retina, including increased tortuosity, narrowed veins, decreased blood flow, microvascular network damage, and compromised branching complexity (Berisha et al., 2007; Frost et al., 2010, 2013; Cheung et al., 2014; Feke et al., 2015; Williams et al., 2015; Einarsdottir et al., 2016; Abbasi, 2017; Cabrera DeBuc et al., 2018; O’Bryhim et al., 2018), similar to earlier findings in the brain (Smith et al., 1999; Bookheimer et al., 2000; Kimbrough et al., 2015). Such studies have extensively focused on live imaging of retinal blood flow dynamics of AD patients, yet there is a significant lack of understanding regarding the precise molecular and cellular mediators involved in retinal vascular AD pathology, which could lead to the discovery of potential intervention points of treatments and guide the development of next-generation retinal imaging. Thus, more in-depth investigations of the pathogenic mechanisms of retinal vasculature in AD development are needed.
To this end, work by Koronyo-Hamaoui and colleagues demonstrated the existence of different alloforms of Aβ deposits in retinal vessels of MCI and AD patients in both perivascular and within vessel walls including within the tunica media and outside the basement membrane (Koronyo et al., 2017). These findings were consistent with what is known regarding cerebral vascular Aβ pathology (Vinters and Gilbert, 1983; Vinters, 1987; Bennett, 2001; Bakker et al., 2016; Engelhardt et al., 2016). TEM analysis confirmed the ultrastructures of retinal Aβ deposits, often in close proximity to or within blood vessels, similar to those found in the brain (Iadanza et al., 2016; Koronyo et al., 2017; Shi et al., 2020). Retinal Aβ42 fibrils near a blood basal membrane were approximately 10-15nm in width with typical anti-parallel β-sheets (Koronyo et al., 2017). In the same human study, the existence of retinal Aβ was verified by Congo red staining under polarized light, by which the investigators revealed positive Aβ fibrils along blood vessels (Koronyo et al., 2017). According to an animal study with APP-overexpressing mice, vascular amyloidosis resulted in increased rigidity of the blood vessels and decreased blood flow in the brain (Kimbrough et al., 2015). In addition, a recent clinical study has proposed that cerebral vascular changes may propel other abnormalities in AD pathogenesis (Besser et al., 2016).
Recent reports suggest that vascular pathology in AD brains occurs very early during disease progression (Nikolakopoulou et al., 2017). Yet, it is unclear how early vascular pathology can be detected via retinal imaging and whether it may specifically foretell AD development. Furthermore, the connections between vascular pathology and Aβ accumulation and clearance in both the brain and retina should be explored in future studies. To date, several studies imply the potential of utilizing retinal vascular biomarkers for early AD or pre-symptomatic stage screening as well as for predicting cognitive decline (Vidal and Mavet, 1989; Gharbiya et al., 2014; Bulut et al., 2016; Cunha et al., 2017; McGrory et al., 2017; Bulut et al., 2018; Jiang et al., 2018; O’Bryhim et al., 2018; Jung et al., 2019).
A recent investigation by Shi and colleagues of retinal vasculature in 62 AD and MCI patients and matched human controls revealed early and progressive PDGFRβ deficiency and pericyte loss along with intense retinal vascular Aβ accumulation in AD (Shi et al., 2020). Using a modified retinal vascular isolation technique (Figure 6A) in 12 AD patient and control donors, the authors report a significant increase in various types of Aβ in AD retinal microvasculature, and more importantly, accumulation of Aβ in retinal pericytes together with pericyte loss (Figure 6B–E’). The existence of Aβ in pericytes was validated by TEM, which showed intense Aβ42 deposition in retinal pericytes as well as in microvascular lumen and adjacent to microvasculature (Figure 6F). Further, accumulation of Aβ in arterial tunica media in the retina of several AD patients (Figures 6E,E’; Shi et al., 2020) has implications related to lymphatic Aβ clearance pathways in the human retina: a subject warranting future exploration.
FIGURE 6  Identification of early and progressive PDGFRβ/pericyte loss, associated with vascular amyloidosis in AD retina. (A) Schematic diagram of retinal vascular network isolation and immunofluorescence staining. (B,C) Representative fluorescent images showing Aβ42 (12F4 immunoreactivity in red), blood vessels (lectin in green) and nuclei (DAPI in blue) in isolated retinal microvasculature networks from a cognitively normal (CN) control subject (B) and AD patient (C), with higher Aβ42 deposits in AD retinal microvasculature and pericytes. (D) Higher magnification image of AD retina shows co-localization of Aβ42 and lectin-positive vascular walls in yellow. (E,E’) Retinal cross-section from an AD patient immunostained with anti-Aβ40 (JRF/cAβ40/28) mAb and DAB labeling and hematoxylin counterstain. Red arrow, also shown in higher magnification image (E’), points to vascular Aβ40 in tunica media, adventitia or intima. Scale bar: 20 μm. (F) TEM image of retinal cross-section from an AD patient immunostained with anti-Aβ42 mAb (12F4) and peroxidase-based DAB, revealing the localization and ultrastructure of vascular-associated deposits. Cytoplasmic Aβ42 deposits in pericytes are demarcated by yellow lines. Scale bar: 0.5 μm. Reproduced from Shi et al. (2020) under terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/). In the same study, histological analysis based on immunofluorescent staining or 3,3′-Diaminobenzidine staining and the use of specific monoclonal antibodies recognizing diverse Aβ epitopes revealed intense deposition of both Aβ42 and Aβ40 in AD retinal vasculature (n = 28–36 human samples). In addition, early loss of pericyte marker PDGFRβ was noted in longitudinal and vertical retinal vessels of MCI and AD patients compared to normal controls (n = 38). The dramatic loss of vascular PDGFRβ expression significantly correlated with CAA (n = 14) and Mini-Mental State Examination (MMSE) cognitive scores (n = 10) in a subset of patients, suggesting that retinal pericyte and PDGFRβ loss could predict the cerebral vascular disease and cognitive function. Further TUNEL and cleaved caspase-3 staining demonstrated that apoptosis may be the dominant pathway of retinal pericyte death in MCI and AD retina.
In the brain, the BBB plays an indispensable role in mediating clearance of Aβ through its efflux in the cerebral vascular network (Zlokovic et al., 1993; DeMattos et al., 2002; Banks et al., 2003; Do et al., 2015; Zhao et al., 2015; Sweeney et al., 2018). The BBB is established by endothelial cells forming vessel walls, astrocyte end-feet, and pericytes in the basement membrane. In comparison, the BRB is composed of tight junctions of retinal endothelial and epithelial cells together with supporting pericytes. Despite their organ-specific functions, the BBB and BRB display very similar functions in transport and permeation characteristics (Steuer et al., 2005). A recent study has established a correlation among BBB, ApoE ε4 and cognitive decline regardless of AD pathology (Montagne et al., 2020). The recent findings implicated pericytes in AD pathogenesis and future investigations may shed light on the role of the BRB in AD.
Permeability of the BRB may be measured in live laboratory animal models of retinal diseases by the extent of leakage using fluorescent dyes with predefined molecular weight such as fluorescein (Do carmo et al., 1998), Evans blue (Xu et al., 2001) and others (Ivanova et al., 2019). In the clinical setting, fundus fluorescein angiography (FFA) utilizing a fluorescent dye and fundus camera has been an established method for examining retinal vascular circulation and BRB damage (Marmor and Ravin, 2011). Recently, a newly modified OCT method was developed, the OCT-Leakage. By using a proprietary algorithm to identify sites of decreased optical reflectivity, the system quantifies and detects the correlation of retinal extracellular space with degrees of retinal edema (Cunha-Vaz et al., 2016; Cunha-Vaz, 2017). A live imaging study in 28 patients has demonstrated agreement between OCT-Leakage and FFA in identifying sites of impaired BRB in diabetes (Cunha-Vaz et al., 2017), providing a new noninvasive low-cost alternative method to detect and quantify BRB leakage.
Given that retinal vascular amyloidosis has been detected in AD (Koronyo et al., 2017; Shi et al., 2020), future studies should aim to examine if BRB permeability is also altered by disease, and if this is a cause or effect of retinal vascular amyloidosis. Indeed, a study using OCT angiography in patients revealed increased fovea avascular zone and decreased foveal thickness in eyes of AD patients (O’Bryhim et al., 2018), implying extensive retinal microvascular damage in the AD retina. Accordingly, recent human studies have demonstrated that retinal vascular abnormalities can predict cognitive decline (Baker et al., 2007; Cabrera DeBuc et al., 2018; Deal et al., 2018). Recent progress in retinal amyloid imaging (Koronyo et al., 2017), pericyte imaging by adaptive optics (Schallek et al., 2013) together with FFA and the recently developed OCT-Leakage (Cunha-Vaz et al., 2016) should allow for a comprehensive assessment of retinal Aβ pathology and BRB damage, potentially revolutionizing AD screening techniques.