The present study performed a morphometric analysis of the retina in AD. We provide, for the first time, a quantitative thickness assessment of the inner (RNFL, RGCL, IPL) and outer layers (INL and ONL) of the retina using postmortem human tissues. These tissues were histopathologically confirmed for amyloid deposition and were derived from patients neuropathologically confirmed for severe AD. We found significant thinning for all assessed retinal layers and observed differences in thickness by layer and by region, supero-nasally and supero-temporally with respect to the optic nerve. Taken together, these findings expand our understanding of the retina as a potential surrogate biomarker for AD and propose an objective, quantifiable means of assessing this disease.
Histological examination of the RNFL revealed significant thinning closest to the optic nerve in the superior-temporal region. In the superior-nasal region, we found diffuse thinning throughout the RNFL, most pronounced closest to the optic nerve. This thickness profile matches the distribution of the retinal Aβ deposits in the mid- and far-periphery of the superior quadrants of these tissues as previously demonstrated by our laboratory.
20,40,41 Similarly, Koronyo and colleagues
23,42 described an extensive loss of neurons in the superior quadrant and the mid- and far-peripheries. Interestingly, the supero-nasal RNFL was markedly thinner overall compared to the supero-temporal RNFL. This is consistent with our previous findings where axonal loss predominantly affected the larger fibers and spared the smaller fibers, similarly observed by Koronyo and colleagues.
20,23,42
Our study also histologically assessed RGCL and IPL thickness. While thickness changes for these layers have been previously reported in vivo using OCT, these studies predominantly evaluated the RGCL and IPL as a combined thickness and exclusively in the macula.
28–33 Since AD in the eye has been characterized as a disease primarily of the ganglion cells, we expand upon these studies by assessing the RGCL and IPL separately rather than combined, and geographically beyond the macula. Supero-temporally to the optic nerve, RGCL thinning was most severe toward the macula. However, supero-nasally, RGCL thinning extended uniformly out to the peripheral retina. Similarly, significant thinning of the supero-temporal IPL was seen in the macula with uniform thinning out to the periphery supero-nasally. This IPL atrophy may likely contribute to deficiencies in motion perception and contrast sensitivity frequently seen in patients with AD since processing of these visual stimuli begins in this layer.
43,44 Intriguingly, the IPL exhibited roughly half the magnitude of thickness reduction in comparison with the RNFL and RGCL. This is in parallel with the distribution of Aβ seen in these tissues, whereby Aβ deposits were found to be more concentrated in the RGCL relative to the IPL.
20 This finding also suggests that atrophy of the IPL, comprised of ganglion cell synaptic connections, dendrites, and bipolar cells, may likely be secondarily involved and follow after ganglion cell and nerve fiber loss. Previous studies have suggested IPL changes may precede RGCL changes. Williams et al.
45 found IPL loss prior to RGCL loss in a mouse model, while Snyder et al.
46 showed IPL thickening correlating with amyloid deposition in preclinical AD patients. However, animal models are not always truly representative of human disease,
45 and the limitations of OCT studies may persist without histopathologically confirming fibrillar Aβ within the inclusion bodies seen on live imaging.
47 The present study was performed in postmortem human eyes in severe stages of AD.
Thickness assessment of the outer retina showed remarkable thinning of both the INL and ONL. Supero-temporally to the optic nerve, INL thinning was significantly apparent throughout all measured points and most severe toward the macula. Supero-nasally, INL thinning was also significantly thinner across all measured points and most severe toward the optic nerve. Similar to the INL, significant ONL thinning was observed throughout all measured points and most severe toward the macula supero-temporally relative to the optic nerve. Also resembling the INL, ONL thinning supero-nasally was significantly thinner across all measured points and most severe toward the optic nerve. Therefore, the INL and ONL shared similar thickness profiles in AD. Intriguingly, the magnitude of thickness reduction for the INL and ONL was notably less than that of the RNFL and RGCL. These thickness findings may be explained by the distribution of the retinal Aβ deposits, which were more concentrated in the RGCL and to a lesser extent in the INL and ONL.
20 Similarly, Koronyo-Hamaoui et al.
23 observed neuronal reduction in the INL and ONL accompanied by retinal Aβ pathology.
The observed pattern of retinal thinning in AD, whereby thinning was greatest for the inner layers of the retina followed by the outer layers, may likely be attributed to Aβ deposition and consequent neurotoxicity as suggested by previous studies.
20,23,42,47,48 Notably, however, the IPL was less severely thinned relative to the INL and ONL. The INL consists of the cell bodies of horizontal cells, bipolar cells, and amacrine cells, while the ONL consists of photoreceptor cell bodies.
43 In the context of severe AD, tissue loss in these layers may be associated with retrograde transsynaptic degeneration.
49 However, future studies assessing the morphological changes of the various cell types comprising these retinal layers would be necessary to support this hypothesis.
While our measurements may have been impacted by postmortem tissue swelling and tissue shrinkage as induced by fixation, both tissue groups were prepared under identical protocols and deviations would be equally distributed between both AD and controls, mitigating measurement bias. Despite its limited sample size, the current study represents the largest histological analysis of retinal thickness in AD. Including only one eye for each patient is a particular strength of our study as it avoids artificial sample size enhancement, which may have arisen from including both eyes. In addition, we used 16 measurements per layer to account for case-specific differences in retinal structure, further strengthening the significance of our findings. We did not assess the OPL and photoreceptor layer as these layers exhibited fragmentation artifacts following tissue preparation. Glaucoma is another common ocular disease that has been implicated as sharing commonalities with AD.
21 However, the relationship between these two disease entities remains controversial and additional studies are warranted to investigate their similarities.