Evaluation of inner retinal layers as biomarkers in mild cognitive impairment to moderate Alzheimer’s disease

<div><p>Inner retina in Alzheimer's Disease (AD) may experience neuroinflammation resulting in atrophy. The objective of our study was to determine whether retinal GCIPL (ganglion cell-inner plexiform layer) or nerve fiber layer (NFL) thickness may serve as noninvasive biomarkers to diagnose AD. This cross-sectional case-control study enrolled 15 mild cognitive impairment (MCI) patients, 15 mild-moderate AD patients, and 18 cognitively normal adults. NFL and GCIPL thicknesses on optical coherence tomography (OCT) were measured using Duke Optical Coherence Tomography Retinal Analysis Program (DOCTRAP) and Spectralis software. We demonstrated that regional thicknesses of NFL or GCIPL on macular or nerve OCTs did not differ between groups. However, a multi-variate regression analysis identified macular areas with a significant thickening or thinning in NFL and GCIPL in MCI and AD patients. Our primary findings controvert previous reports of thinner NFL in moderate-to-severe AD. The areas of thickening of GCIPL and NFL in the macula adjacent to areas of thinning, as revealed by a more complex statistical model, suggest that NFL and GCIPL may undergo dynamic changes during AD progression.</p></div

NFL thicknesses obtained using the automated Heidelberg software in various regions surrounding the optic nerve.

<p>NFL thicknesses obtained using the automated Heidelberg software in various regions surrounding the optic nerve.</p

Global GCIPL and NFL thicknesses obtained using the DOCTRAP software in OCT volume scans of the macula and nerve.

<p>Global GCIPL and NFL thicknesses obtained using the DOCTRAP software in OCT volume scans of the macula and nerve.</p

Results of the multi-variate regression analysis with quasi-least squares, adjusted for multiple comparisons.

<p>Results of the multi-variate regression analysis with quasi-least squares, adjusted for multiple comparisons.</p

Results of a multi-variate regression analysis with quasi-least squares, without correction for multiple comparisons.

<p>This analysis identified areas in the macula that were statistically significantly thinner (red) or thicker (green) in NFL and GCIPL in MCI and AD patients as compared to controls, or in AD compared to MCI.</p

Distribution of MoCA scores among the three study groups.

<p>Distribution of MoCA scores among the three study groups.</p

Representative 2D MS analyses of TAG species in a human plasma lipid extract.

<p>Neutral loss scans (NLS) of all naturally-occurring aliphatic chains (i.e. the building blocks of TAGs) of a human plasma lipid extract were used to determine the identities of each lithiated molecular ion, deconvolute isomeric species, and quantify individual TAG species by comparisons with a selected internal standard (i.e., T17:1 TAG, shown in NLS268). Collision activation was performed with collision energy of 32 eV and gas pressure of 1 mT on a triple quadrupole mass spectrometer (TSQ Quantum Ultra Plus, Thermo Fisher Scientific, San Jose, CA, USA). All displayed mass spectral traces are normalized to the base peak in each trace.</p

Rank of the changed mean mass levels of individual sphingomyelin and ceramide molecular species in all AD patients relative to the normal controls (panel A) and the differences in sphingomyelins and ceramides between patients with moderate Alzheimer's disease (MMSE<20) and patients with mild Alzheimer's disease (MMSE> = 20) (panel B).

<p>The Y axes show the standardized differences in lipid level: lipid levels were standardized to a mean of 0 and standard deviation of 1 across all samples before ranking and plotting. For Alzheimer's disease versus controls, P = 1.6×10⁻⁹ for sphingomyelins and P = 0.00034 for ceramides (Wilcoxon signed rank test two sided on means over the standardized data). For moderate versus mild Alzheimer's disease, P = 7.1×10⁻⁸ for sphingomyelins and P = 0.10 for ceramides.</p

Clinical and demographic characteristics of Alzheimer's subjects and controls^a.

a<p>WT, Wilcoxon rank-sum test, two-sided; FET, Fisher's exact test, two-sided; SD, standard deviation; MAD, median absolute deviation; MMSE, Mini-Mental State Examination.</p

Lipid classes, species and their mass levels in Alzheimer and control subjects^a.

a<p>SD denotes standard deviation. P values were computed by Welch two-sample t-test. All the results are obtained by shotgun lipidomics as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021643#s2" target="_blank">Materials and Methods</a> and presented in unit of nmol/mg protein. See text for abbreviations.</p