Abstract: Focal intraretinal alterations have been studied to advance our understanding of the pathology of neurodegenerative diseases. The current literature involving focal alterations in the intraretinal layers was reviewed through PubMed using the search terms “focal alteration”, “region of interest”, “optical coherence tomography”, “glaucoma”, “multiple sclerosis”, “Alzheimer’s disease”, “Parkinson disease”, “neurodegenerative diseases” and other related items. It was found that focal alterations of intraretinal layers were different in various neurodegenerative diseases. The typical focal thinning might help differentiate various ocular and cerebral diseases, track disease progression, and evaluate the outcome of clinical trials. Advanced exploration of focal intraretinal alterations will help to further validate their clinical and research utility.
Background: Soft drusen and basal linear deposit (BLinD) are two forms of the same extracellular lipid rich material that together make up an Oil Spill on Bruch’s membrane (BrM). Drusen are focal and can be recognized clinically. In contrast BLinD is thin and diffusely distributed, and invisible clinically, even on highest resolution OCT, but has been detected on en face hyperspectral autofluorescence (AF) imaging ex vivo. We sought to optimize histologic hyperspectral AF imaging and image analysis for recognition of drusen and sub-RPE deposits (including BLinD and basal laminar deposit), for potential clinical application.
Methods: Twenty locations specifically with drusen and 12 additional locations specifically from fovea, perifovea and mid-periphery from RPE/BrM flatmounts from 4 AMD donors underwent hyperspectral AF imaging with 4 excitation wavelengths (λex 436, 450, 480 and 505 nm), and the resulting image cubes were simultaneously decomposed with our published non-negative matrix factorization (NMF). Rank 4 recovery of 4 emission spectra was chosen for each excitation wavelength.
Results: A composite emission spectrum, sensitive and specific for drusen and presumed sub-RPE deposits (the SDr spectrum) was recovered with peak at 510–520 nm in all tissues with drusen, with greatest amplitudes at excitations λex 436, 450 and 480 nm. The RPE spectra of combined sources Lipofuscin (LF)/Melanolipofuscin (MLF) were of comparable amplitude and consistently recapitulated the spectra S1, S2 and S3 previously reported from all tissues: tissues with drusen, foveal and extra-foveal locations.
Conclusions: A clinical hyperspectral AF camera, with properly chosen excitation wavelengths in the blue range and a hyperspectral AF detector, should be capable of detecting and quantifying drusen and sub-RPE deposits, the earliest known lesions of AMD, before any other currently available imaging modality.
Background: Cells of the retinal pigment epithelium (RPE) accumulate different kinds of granules (lipofuscin, melanolipofuscin, melanosomes) within their cell bodies, with lipofuscin and melanolipofuscin being autofluorescent after blue light excitation. High amounts of lipofuscin granules within the RPE have been associated with the development of RPE cell death and age-related macular degeneration (AMD); however, this has not been confirmed in histology so far. Here, based on our previous dataset of RPE granule characteristics, we report the characteristics of RPE cells from human donor eyes that show either high or low numbers of intracellular granules or high or low autofluorescence (AF) intensities.
Methods: RPE flatmounts of fifteen human donors were examined using high-resolution structured illumination microscopy (HR-SIM) and laser scanning microscopy (LSM). Autofluorescent granules were analyzed regarding AF phenotype and absolute number of granules. In addition, total AF intensity per cell and granule density (number of granules per cell area) were determined. For the final analysis, RPE cells with total granule number below 5th or above the 95th percentile, or a total AF intensity ± 1.5 standard deviations above or below the mean were included, and compared to the average RPE cell at the same location. Data are presented as mean ± standard deviation.
Results: Within 420 RPE cells examined, 42 cells were further analyzed due to extremes regarding total granule numbers. In addition, 20 RPE cells had AF 1.5 standard deviations below, 28 RPE cells above the mean local AF intensity. Melanolipofuscin granules predominate in RPE cells with low granule content and low AF intensity. RPE cells with high granule content have nearly twice (1.8 times) as many granules as an average RPE cell.
Conclusions: In normal eyes, outliers regarding autofluorescent granule load and AF intensity signals are rare among RPE cells, suggesting that granule deposition and subsequent AF follows intrinsic control mechanisms at a cellular level. The AF of a cell is related to the composition of intracellular granule types. Ongoing studies using AMD donor eyes will examine possible disease related changes in granule distribution and further put lipofuscin′s role in aging and AMD further into perspective.
Abstract: In the early days of deciphering the injured neuronal tissues led to the realization that contrast is necessary to discern the parts of the recovering tissues from the damaged ones. Early attempts relied on available (and often naturally occurring) staining substances. Incidentally, the active ingredients of most of them were small molecules. With the advent of time, the knowledge of chemistry helped identify compounds and conditions for staining. The staining reagents were even found to enhance the visibility of the organelles. Silver impregnation identification of Golgi bodies was discovered in owl optic nerve. Staining reagents since the late 1800s were widely used across all disciplines and for nerve tissue and became a key contributor to advancement in nerve-related research. The use of these reagents provided insight into the organization of the neuronal tissues and helped distinguish nerve degeneration from regeneration. The neuronal staining reagents have played a fundamental role in the clinical research facilitating the identification of biological mechanisms underlying eye and neuropsychiatric diseases. We found a lack of systematic description of all staining reagents, whether they had been used historically or currently used. There is a lack of readily available information for optimal staining of different neuronal tissues for a given purpose. We present here a grouping of the reagents based on their target location: (I) the central nervous system (CNS), (II) the peripheral nervous system (PNS), or (III) both. The biochemical reactions of most of the staining reagents is based on acidic or basic pH and specific reaction partners such as organelle or biomolecules that exists within the given tissue type. We present here a summary of the chemical composition, optimal staining condition, use for given neuronal tissue and, where possible, historic usage. Several biomolecules such as lipids and metabolites lack specific antibodies. Despite being non-specific the reagents enhance contrast and provide corroboration about the microenvironment. In future, these reagents in combination with emerging techniques such as imaging mass spectrometry and kinetic histochemistry will validate or expand our understanding of localization of molecules within tissues or cells that are important for ophthalmology and vision science.
