M. A. Yakovleva

Eye Adaptation to Different Light Environments in Two Populations of Mysis relicta: A Comparative Study of Carotenoids and Retinoids

Abstract The content of carotenoids and retinoids was compared in the eyes of two Finnish populations of the opossum shrimp, Mysis relicta, which have been reproductively isolated for at least 9000 years: one from the deep, dark, Lake Pääjärvi, the other from the Baltic Sea (Pojoviken Bay). The eyes of the lake population (LP) are highly susceptible to light damage, while those of the sea population (SP) are more resistant. Carotenoids are known to act as antioxidants protecting cells against free radicals and reactive oxygen species; retinoids, on the contrary, may be phototoxic in certain conditions. Analyzed by spectrophotometry and HPLC, the carotenoid content was broadly similar in the eyes of the two populations as regards both total amount and relative proportions of more than 20 components. Noteworthy differences were found in only three of the major components, among these astaxanthin, which was two times higher in SP compared with LP. The most interesting finding was the 1.6-fold higher content of retinoids in LP compared with SP, with retinol as the dominant component (40% of total) in both populations. Retinol is a precursor of the visual-pigment chromophore retinal. The result is consistent with the idea that animals inhabiting extremely dim light environments, where very little photoregeneration of metarhodopsin to rhodopsin can occur, need a large store of chromophore (or precursors) for effective “dark” regeneration of visual pigment. We suggest that almost all the rhodopsin is then in the native state and massive pigment activation following exposure to stronger light may trigger photoreceptor damage. If such animals are handled without due light protection, e.g., when transferred to a new habitat or collected for biological experiments, their vision will be severely impaired.

Lake and Sea Populations of Mysis relicta (Crustacea,Mysida) with Different Visual-Pigment Absorbance Spectra Use the Same A1 Chromophore

Glacial-relict species of the genus Mysis (opossum shrimps) inhabiting both fresh-water lakes and brackish sea waters in northern Europe show a consistent lake/sea dichotomy in eye spectral sensitivity. The absorbance peak (λmax) recorded by microspectrophotometry in isolated rhabdoms is invariably 20–30 nm red-shifted in “lake” compared with “sea” populations. The dichotomy holds across species, major opsin lineages and light environments. Chromophore exchange from A1 to A2 (retinal → 3,4-didehydroretinal) is a well-known mechanism for red-shifting visual pigments depending on environmental conditions or stages of life history, present not only in fishes and amphibians, but in some crustaceans as well. We tested the hypothesis that the lake/sea dichotomy in Mysis is due to the use of different chromophores, focussing on two populations of M. relicta from, respectively, a Finnish lake and the Baltic Sea. They are genetically very similar, having been separated for less than 10 kyr, and their rhabdoms show a typical lake/sea difference in λmax (554 nm vs. 529 nm). Gene sequencing has revealed no differences translating into amino acid substitutions in the transmembrane parts of their opsins. We determined the chromophore identity (A1 or A2) in the eyes of these two populations by HPLC, using as standards pure chromophores A1 and A2 as well as extracts from bovine (A1) and goldfish (A2) retinas. We found that the visual-pigment chromophore in both populations is A1 exclusively. Thus the spectral difference between these two populations of M. relicta is not due to the use of different chromophores. We argue that this conclusion is likely to hold for all populations of M. relicta as well as its European sibling species.

Estimation of fluorescence lifetime of lipofuscin fluorophores contained in lipofuscin granules of retinal pigment epithelium of human cadaver eyes without signs of pathology

The fluorescence lifetimes of lipofuscin fluorophores contained in chloroform extracts from retinal pigment epithelium (RPE) of human cadaver eyes without signs of pathology were evaluated by single photon counting. The comparison of fluorescence lifetimes of N-retinylidene-N-retinylethanolamine (A2E) and its photooxidation and photodegradation products has been carried out. It was shown that the contribution of A2E to the total fluorescence of chloroform extract from lipofuscin granules is not major. The results are important for the improvement of noninvasive diagnostic method of degenerative diseases of the retina and RPE—fundus autofluorescence (FAF).

Lutein and its oxidized forms in eye structures throughout prenatal human development

&NA; The presence of carotenoids in the vitreous body, retina, lens, retinal pigment epithelium together with choroid (hereinafter RPE), and ciliary body and iris together with choroidal stroma (hereinafter CBI) was studied throughout the second trimester of prenatal development of the human eye. It has been found that the vitreous body, retina, and RPE contain lutein and its oxidized forms. Zeaxanthin was not found in the tissues studied. The presence of lutein in the vitreous body is transient and no longer detected after 28 weeks of gestation. Lutein was not detected in the lens and CBI, but its oxidized forms were found. The presence of carotenoids in different tissues of the eye in the course of normal eye development and the antioxidant role of carotenoids are discussed. Graphical abstract HPLC of chloroform extracts from different tissues of human fetal eyes. Figure. No caption avaialble. HighlightsUV spectroscopy, HPLC, and mass spectrometry were used to find carotenoids in eyes.Lutein content was measured in different tissues of human fetal eyes.Lutein oxidized forms were shown to be also present in all fetal eye tissues studied.

The fluorescence lifetime of lipofuscin granule fluorophores contained in the retinal pigment epithelium cells from human cadaver eyes in normal state and in the case of visualized pathology

A comparative analysis of fluorescence lifetime of lipofuscin granule fluorophores contained in the retinal pigment epithelium cells from human cadaver eyes in normal state and in the case of visualized pathology was carried out. Measurements of fluorescence lifetimes of bis-retinoids and their photooxidation and photodegradation products were carried out using the method of counting time-correlated photons. Comparative analysis showed that, in the case of visualized pathology, the contribution of photooxidation and photodegradation products of bis-retinoids to the total fluorescence of the retinal pigment epithelium cell suspension increases in comparison with the norm.

Time-of-flight secondary ion mass spectrometry to assess spatial distribution of A2E and its oxidized forms within lipofuscin granules isolated from human retinal pigment epithelium

Lipofuscin granules accumulate in the cells of retinal pigment epithelium with age, particularly in patients with hereditary diseases. These granules are heterogeneous, being composed of mixtures of proteins and lipids, including more than 21 different fluorescent compounds. Bisretinoids and their photo-oxidation and photodegradation products represent the main source of lipofuscin fluorescence and exhibit phototoxic properties. This study used time-of-flight secondary ion mass spectrometry (ToF–SIMS) with in-depth probing to assess the depth distribution of N-retinylidene-N-retinylethanolamine (A2E) and its singly and doubly oxidized forms (A2E-ox and A2E-2ox, respectively) within lipofuscin granules and in their surface layer (lipid membrane). ToF–SIMS showed that A2E and its oxidized forms were uniformly distributed throughout lipofuscin granules but were not present at the membrane surface layer. This finding is important for understanding the process involved in the formation of lipofuscin granules and in their toxicity.

Study of visual pigment rhodopsin supramolecular organization in photoreceptor membrane by small-angle neutron scattering method with contrast variation

Supramolecular organization of rhodopsin in the photoreceptor membrane was investigated by small-angle neutron scattering method. The experiments, which were performed with mixtures of heavy/light water as solvent (contrast variation method), were aimed at obtaining information about the lipid and protein components of the photoreceptor disc membrane separately. It was shown that the packaging density of the rhodopsin molecules in the photoreceptor membrane was unusually high: the distance between the centers of the molecules was approximately 56 Å. The probability of the monomeric state of rhodopsin molecules in the photoreceptor membrane, according to the data obtained, is rather high.

Spectral analysis of fundus autofluorescence pattern as a tool to detect early stages of degeneration in the retina and retinal pigment epithelium

PurposeThe aim of this work is the determination of quantitative diagnostic criteria based on the spectral characteristics of fundus autofluorescence to detect early stages of degeneration in the retina and retinal pigment epithelium (RPE).MethodsRPE cell suspension samples were obtained from the cadaver eyes with and without signs of age-related macular degeneration (AMD). Fluorescence analysis at an excitation wavelength of 488 nm was performed. The fluorescence lifetimes of lipofuscin-granule fluorophores were measured by counting time-correlated photon method.ResultsComparative analysis of fluorescence spectra of RPE cell suspensions from the cadaver eyes with and without signs of AMD showed a significant difference in fluorescence intensity at 530–580 nm in response to fluorescence excitation at 488 nm. It was notably higher in eyes with visual pathology than in normal eyes regardless of the age of the eye donor. Measurements of fluorescence lifetimes of lipofuscin fluorophores showed that the contribution of photooxidation and photodegradation products of bisretinoids to the total fluorescence at 530–580 nm of RPE cell suspensions was greater in eyes with visual pathology than in normal eyes.ConclusionBecause photooxidation and photodegradation products of bisretinoids are markers of photodestructive processes, which can cause RPE cell death and initiate degenerative processes in the retina, quantitative determination of increases in these bisretinoid products in lipofuscin granules may be used to establish quantitative diagnostic criteria for degenerative processes in the retina and RPE.