Ординатура / Офтальмология / Английские материалы / Age-Related Changes of the Human Eye_Cavallotti, Cerulli_2008

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References – Clinical Approach to Lens Modifications with Ageing

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through a series of morphologic and functional modifications of the human organs and tissues.2 Aging is characterized by a progressive decline of the organs and is accompanied with modifications of the connective tissue. Although it is not always possible to distinguish between aging and senility—the pathology versus the physiology in the morphological and/or functional changes of the organs3—the possible age-related changes without pathological modifications are, the following:

●Decrease of the mitoses and of the capacity of cellular increase with slowing down of the processes of repair of tissues and reduction of the ability to produce antibodies.

●Gradual dehydration and progressive cellular atrophy with processes of degeneration and of pigment accumulation.

●Lessening of the elasticity of the tissues, degenerative modifications of connective elastic tissue and fat infiltration of various parenchyma.

●Lessening of the oxidation processes and then of the basal metabolism.

●Deficit of the regulatory mechanisms of homeostasis.

●Lessening of the enzymatic activities.

●Slowing down of the neuromuscular reactions and weakening of the muscular force and reduction of the fatigue strength.

●Degeneration and atrophy of the nervous system.

●Weakening of the visual acuity, hearing, olfactory sensibility, memory, attention, ability to the intellectual job.

The anatomical features of these histological and/or physiological alterations are always an involution and/or an atrophy that hits, in various degrees, all organs and viscera, including eyes,4 The modifications of the connective tissue, and the increase of the intercellular cementing substance with an increase of the collagen and of the elastic tissue represent the characteristic aspects of aging and/or senility. Bogomoletz and Kavetzky5 think that senility is characterized by the differentiation of the histio-reticular tissue (the last residual of the embryo tissue.6,8 In senility, the cells of the parenchyma develop a progressive atrophy with an increase of the pigment—therefore, this phenomenon is called brown atrophy. The description of normal age-related eye changes is difficult for the frequency of numerous eye diseases in older humans.9,10

The human trabecular meshwork (Tm) contains hyaluronic acid, chondroitin sulphate and dermatan sulphate11 while, in adults, fine fibril-like components are also found. The presence of type IV collagen in the same structures shows the implication in the cell/extra-cell matrix interactions at this site, and its abnormal increase in aging eyes probably reflects a functional defect of Tm in these conditions.12,13 Histo-chemical studies with polarization microscopy showed—in aged eyes—a decrease of hyaluronic acid and an increase of sulphated GAGs.14 With age, collagen tissue becomes more prominent in Tm. At the same time, the hyaluronic acid content decreases and the sulphated GAGs increase.15 Francois16 suggested that an increased amount of GAGs might influence the functional properties of Tm. Quantitative biochemical studies showed a depletion of hyaluronic acid content and an increase of chondroitin sulphates in the trabecular meshwork, ciliary

processes, and the anterior sclera in age-matched, normal eyes.17,18,19 Sugahara et al 20,21 demonstrated the complex functions of GAGs, and Goes et al.22 characterized GAGs as component of rabbit eye. Rusova et al.23 described a method for sulphated GAGs measurement in tissues. Despite all these data, the actual ocular physiology of GAGs is still not well-known. The aim of the present work is to study the presence of GAGs in the extracellular matrix of Tm in young and older individuals.

Materials and Methods

We studied 24 human eyes. Samples of Tm (left eye) were harvested during autopsies. Because post-mortem phenomena may produce early morphologic modifications of the eye structures, our samples were harvested as earliest as possible after death (after 12-18 h). The Ethics Committees of the involved hospitals gave their approval, and the relatives of the dead humans gave their written informed consents. All experiments were performed according to the guidelines of the Declaration of Helsinki and in conformity with the ARVO Statement for the use of human samples in ophthalmic and vision research applied by all Ethics Committees.24

Some characteristics of the dead human eye-donors are reported in Table 6.1. Eight of these patients were classified as young (age range was 201.2 years), while sixteen eye donors were classified as old (age range was 721.6 years). In none of our donors, eyes showed either macroscopic or microscopic abnormalities. Small pieces of the Tm were dissected immediately (< 2 min). All samples were harvested from the same site.25

Light Microscopy

Our samples were immediately prefixed in 2 percent osmium tetroxide at pH 7.4 in veronal-acetate buffer for five minutes at 4 ° C. After fixation, the specimens were washed with veronal-acetate buffer (pH 7.4), dehydrated in a graded ethanol series and embedded in paraffin. Thin sections (about 4 m) were made for morphological staining with toluidine blue (0.05% for 1 minute). Lipids were stained by means of special histo-chemical techniques for light microscopic analysis. In order to

Table 6.1 Clinical data on individuals from which small fragments of trabecular meshwork were harvested

determine the composition and distribution of the lipids, three different stains were used: a) Bromine - Sudan black B, which stains all classes of lipids; b) Bromine - acetone - Sudan black B, which stains only phosphoric lipids, and c) Oil red O, which stains neutral lipids (especially esters of saturated and unsaturated fatty acids).

Transmission Electron Microscopy (TEM)

Samples were pre-fixed in buffered 2 percent gluta-raldehyde for 2 hours, washed in buffer and than post-fixed in buffered 2 percent osmium tetroxyde for 2 hours, dehydrated and embedded in araldite. Ultra thin sections were made with Reichert Ultra microtome. These sections were counterstained by uranyl acetate lead citrate and studied with Zeiss EM 109 electron microscope.26

Staining of Acidic Proteoglycans

Acidic proteoglycans are made by using a protein that bounds long and numerous heteropolysaccharidic chains, formed by hexosamine molecules, hexoses, uronic, sialic or sulphuric acid—the latter being usually external to the hydroxylic groups. The best fixative agent for acidic proteoglycans is calcic formalin, and they are PAS negative. The reasons for their PAS negativity are not yet clear. One explanation could be that their powerfully negative electric charge impedes contact with periodic acids. All staining methods for acidic proteoglycans are based on the presence of numerous acidic valences contained in such substances, and so they actually are aspecific methods. In animal tissues, acid groups are essentially represented by carboxylic groups (-COOH) of proteins, by acidic proteoglycans and glycoproteins, by phosphoric groups (==HPO4) of the nucleic acids, and by sulphuric groups (-HSO4) present in the sulphate. To attach themselves with electro polar connections to the basic staining agents (cations), such acidic groups must have a negative electric charge—i.e., must be dissociated in [-COO-] and [H+]. Their dissociation naturally depends on the solution pH the means in which they are placed. In fact, at pH 4, all the acid groups are dissociated; while at pH 2, only the phosphoric and sulphuric acid groups are dissociated; and, finally, at pH 1.8, only sulphuric acid groups are still dissociated and reactive.

ALCIAN-PAS Method

This method is realized first by staining with Alcian blue at pH 2.5, and then a normal PAS staining (after Step 3). Acidic proteoglycans appear blue-green, while PAS-positive substances (glycoprotein, glycogen, etc.) are red. The ALCIAN BLUE-CEC Method (Critical Electrolyte Concentration) was performed according to the Pearse method.26

Quantitative Analysis of Images

For a detailed evaluation of the effects of aging on RPE morphology, a quantitative analysis of images (QAI) was performed on slides using a Quantimet Analyzer (Leica) equipped with specific software.

Final values were statistically analyzed. The values reported in the current manuscript represent the values of staining for each age group, and are expressed in conventional units (CU) ± S.E.M. CU are arbitrary units furnished and printed directly by the Quantimet Analyzer.27

Statistics

Mean values, maximum and minimum limits (experimental values), variations, standard deviation (SD), standard error of the mean (SEM) and correlation coefficients were carried out.28 A correlative analysis of the morphological and histo-chemical data was performed by comparing the significant differences for each group with the corresponding values of the other homogenous groups. The significance of differences between age groups was assessed by the Duncan’s multiple range test.

Results

Our results are reported in Figures 6.1 through 6-8 and summarized in Tables 6.1 through 6.4. (The figures had been included in the paper Opthalmic Research 2004; 36:311–217, and have been reproduced with the permission of S. Karger AG, Basel.) The trabecular meshwork has two components: i) the corneoscleral (see Fig. 6.1), formed by trabecular cells (TC), elastic fibers, collagen fibers, and fine fibrilgranular material—the basal membrane is thin in young subjects; and ii) the uveoscleral (see Fig. 6.2), formed by numerous endothelial cells surrounded by other components in the corneoscleral meshwork. Observing a trabecular sheet, we can detect the central elastic fibers surrounded by collagen fibers. Many TC are in close relationships with the basal membrane (see Fig. 6.3). In one eye drawn from an old subject, we could observe the typical age-related changes consisting of a decrease of the fine fibril-granular material, substituted by gross fibril-granular material that causes an increased electron density (see Fig. 6.4). Fig. 6.5 shows other typical age-related changes of the trabecular meshwork of a 71-year old subject—an accumulation of electron-dense material with increased electron density of the whole sample. Other typical age-related changes of the trabecular meshwork are mitochondrial abnormalities in the TC, and swelling and loss of the mitochondrial crystal (see Fig. 6.6). Morphological results regarding the histo-chemical staining of GAGs, observed by means of polarization microscope, are reported in Figs. 6.7 and 6.8 (from a young and old subject, respectively). Schlemm’s canal is embedded

Fig. 6.1 TEM of a sample of the eye drawn from a young subject (20 years old). Typical structure of the corneoscleral trabecular meshwork formed by trabecular cells, elastic fibers, collagen fibers, fibril-granular material and basal membrane (Magnification 3000x) (Included in the paper Opthalmic Research 2004; 36:311–217, have been reproduced with the permission of S. Karger AG, Basel)

Fig. 6.2 TEM of a sample of the eye drawn from a young subject (21 years old). Typical structure of the uveal meshwork formed by elastic fibers, collagen fibers, basal membrane, fibril-granular material and trabecular cells. (Magnification 5000x) (Included in the paper Opthalmic Research 2004; 36:311–217, have been reproduced with the permission of S. Karger AG, Basel)

into a spongiform tissue rich in GAGs. A positive staining can also be observed in the uveal trabecular meshwork and in the anterior portion of the ciliary muscle. Typical age-related changes are the decrease of the lumen of Schlemn’s canal and the staining increase that corresponds to a GAGs increase.

Fig. 6.3 TEM of a sample of the eye drawn from a young subject (19 years old). Slanting section of a trabecular sheet: central elastic fibers, surrounded by collagen fibers, basal membrane material, and trabecular cells (Magnification 3000x) (Included in the paper Opthalmic Research 2004; 36:311–217, have been reproduced with the permission of S. Karger AG, Basel)

Fig. 6.4 TEM of a sample of the eye drawn from an older subject (70 years old).Typical age-related changes of corneoscleral trabecular meshwork: increased electron density of the collagen and decrease of fibril-granular material (Magnification 3000x) (Included in the paper Opthalmic Research 2004; 36:311–217, have been reproduced with the permission of S. Karger AG, Basel)

Table 6.1 reports the clinical data relevant in subjects from which the trabecular meshwork was harvested. Eight subjects were young (20 ± 1.2 years) and 16 were old (72 ± 1.6 years). All were male and the left eye was harvested in the same area by the same investigators (n=6). None of the subjects had any ocular disease. Table 6.2

Fig. 6.5 TEM of a sample of the eye drawn from an older subject (71 years old). Typical age-related changes of the trabecular meshwork: accumulation of electron-dense material, decrease of fibril-granular material, increased electron density (Magnification 5000x) (Included in the paper Opthalmic Research 2004; 36:311–217, have been reproduced with the permission of S. Karger AG, Basel)

Fig. 6.6 TEM of a sample of the eye drawn from an older subject (72 years old). Typical age-related changes of the trabecular meshwork: mitochondrial abnormalities in the trabecular cells as swelling and loss of cristae (Magnification 5000x) (Included in the paper Opthalmic Research 2004; 36:311–217, have been reproduced with the permission of S. Karger AG, Basel)

shows the amount of fine and dense fibril-granular material in the trabecular meshwork stroma in young and old humans. These results have been compared with the ones obtained by electron density from each sample. As can be seen, we found specific age-related changes, such as a decrease of fine fibril-granular material, an increase of dense fibril-granular material, and an increase in electron density. Table 6.3 shows that hyaluronic acid content decreases in old age, while proteoglycans sulphate content increases with aging.