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

Table 6.2 TEM Pictures: quantitative analysis of images of the trabecular meshwork in young and old humans

Note: Results are expressed as CU ± SEM (see Material and Methods section).

Table 6.3 Histo-chemical staining of the trabecular meshwork, quantitative analysis of images in young and old humans

Note: Results are expressed as CU ± SEM (see Material and Methods section).

Table 6.4 Measurement of GAGs with quantitative analysis of images in the trabecular meshwork of young and old humans

Note: Results are expressed as CU ± SEM (see Material and Methods section).

increase (Table 6.4). Therefore, hyaluronic acid corresponds to the fine fibrilgranular material, while the other substances correspond to the dense fibril-granular material that increases with age in correlation to the basal membrane.

Discussion

Our results show that the major morphological age-related changes of the trabecular meshwork are an increase of extra-cellular material and/or an increase in its electron density. The most abundant extracellular material in young eyes is a fine granular or fibril material. However, with age increase, the fibrillo-granular material decreases, while an electron-dense material becomes prominent. Increased amounts of electron-dense material deposition were found also in the juxta-canalicular zone.

Stroma of the Trabecular Meshwork

The stroma is formed by loosely arranged collagen fibers that are condensed around the vessels. The inter-fibril spaces are rich in glycosaminoglycans. Cells are mostly fibroblasts and melanocytes that often form a plexus and are arranged around the adventitia of a blood vessel. Many moving cells, macrophages, lymphocytes, and clump cells can also be observed in the stroma.

Morphological Changes of the Trabecular Meshwork

TC are embedded in various types of extracellular material. The major extracellular material in young eyes is a fine granular or fibril material. However, with age increase, the fibril-granular material occupies less space in the cribriform layer, and an electron-dense material (plaques) becomes prominent.29,31 The corneoscleral meshwork is composed of numerous flattened and perforated sheets. Two types of cells can be distinguished in the trabecular meshwork—TC and trabecular endothelial cells.

Description of Extracellular Matrix

We found four components in the extracellular matrix of the human trabecular meshwork: i) elastic fibers located in the trabecular sheets, corresponding to elastic parts of the ciliary muscles; ii) collagen fibers that surround the elastic fibers composed of type I collagen (normal periodicity collagen) with the so-called longspacing collagen in between; iii) basal membrane material (type IV collagen) just beneath TC; iv) fine fibril-granular material located between the trabecular sheets. This material is formed histo-chemically by proteoglycans, and proteoglycans are biochemically composed of hyaluronic acid (29%), chondroitin (14.1%), dermatan (21.5%), keratan (20.3%) and heparan sulphate (15%).32 This material represents the filter responsible for possible outflow resistance to aqueous humor.

Conclusions

GAGs of the human trabecular meshwork undergo age-related changes, as demonstrated by our morphological, histo-chemical and morphometric results. Our findings demonstrated the following age-related changes:

●Deposition of fibrous granular material in the trabecular meshwork

●Increased electron density of the structures

●Strong decrease of hyaluronic acid content

●Increase of sulphated proteoglycans

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●As support elements of neurons, give form and structure to nervous tissue

●Two types of glial cells (oligodendrocytes and Schwann cells) produce myelin

●Some glial cells have phagocytic function

●Some glial elements can re-uptake neurotransmitters released from neurons

●During the development of the nervous system, some types of the glial cells guide the migration of neurons and direct the growth of their axons

●Some glial cells actively operate in the regulation of the properties of the pre-synaptic terminations

●Some glial cells (astrocytes) form an impermeable barrier around cerebral capillaries and micro-venules, giving origin to the haemato-encephalic barrier2

The glial cells of the nervous system of vertebrates (including the glial cells of the eye) can be divided into two principle classes: Macroglia and Microglia.

The Macroglia, of neuroectodermic origin, includes olygodendrocytes, Schwann cells, and astrocytes, while the cells of Microglia are also called Del Rio Hortega cells for the name of the author who first recognized them. These cells are morphologically and embryologically different from other glial cells. They derive from the mesoderma and are present in the central nervous system towards the end of fetal life, when the vessels of the meningal sheets penetrate and develop into nervous tissue. In this period, it is easy to observe near the vessels lots of small cells. These cells migrate with amoebic movements to the nervous substance spreading in both white and grey substance.3 The cells of the microglia are small elements characterized by two or more fine, but short, extensions that are poorly branched and can be only seen with particular methods of silver impregnation. The nucleus is elongated and small with dense chromatin that is not uniform. The primary function of the microglia is phagocytosis. These cells contain lysosomes and vesicles characteristic of macrophages. Microglial cells can exist at rest or activated like macrophages. Not much is known of the function of microglial cells in the rest mode, but the components are activated during infection or following lesions. Once activated, these operate as phagocytes—they swell up and are called bitter cells. These cells present more sturdy extensions that are more branched than the nonactivated cells. They also have a richer variety of antigens, which might indicate that they represent elements with higher antigen properties of the nervous system. The reactions of the microglia are thus classified: proliferation, hypertrophy, lipid phagocytosis, neuronophagia (phagocytosis of neurons in necrosis) and dendrophagia (phagocytosis of astrocytes prolongation in degeneration).4,5,6,7,8 Macroglia contains cells that regulate the neuronal metabolism and modulate neuronal functions. Moreover, macroglia also regulates the eye blood vessel functions. In the eye bulb, two cell types can be found as part of the macroglia: Müller cells and astrocytes. The Müller cells cross the thickness of the retina from the retinal pigmented epithelium to the inner limiting membrane. The bodies of the Muller cells are located in the inner nuclear layer of the retina. These cells are the regulatory cells for the metabolism of glutamate, ion balance, and neuron function. The Müller cell prolongations form an extended net that sustains and surrounds all of the nervous cells. In addition, these prolongations help to form the inner and outer limiting membranes of the retina.

The astrocytes, on the contrary, are limited to the nervous cells layers and envelope the blood vessels and ganglion cells with their cellular protrusions. The

branched protrusions of these cells occur at right angles with respect to the Müller cells prolongations. These two structures are not linked. The astrocytes are starshaped cells, with round nuclei and numerous thin protrusions. They are horizontally placed and surround the blood vessels with a dense net of fibers. They form an arched, honeycomb structure that surrounds and sustains the axons of the ganglion cells. They are firmly anchored to the walls of the blood vessels.9

Microglia

The microglial cells are similar to the tissue macrophages.10 These cells are normally resting, but are sensitive to the pathological changes in the homeostasis of the various components of the eye. When the eye tissues undergo the pathological changes, the microglial cells rapidly change into phagocytes capable of mobility.

Moreover, the eye contains some types of cells, nonstructurally connected with the other, adjacent cells by mean of junctions, capability of migration, mobility, production of cytochines and phagocytosis. These cells are named mobile or floating cells. Finally, we also find endothelial cells and pericytes in the eye, which flank the nerve cells and glial cells or arrange themselves around the blood vessel walls. Pericytes are modified smooth muscle cells that regulate the vascular flow through dilation and/or contraction of the diameters of the vessels. The endothelial cells regulate the local homeostatic function and form the blood-retinal barrier.11,12

Aging of the Macroglia (Müller Cells and Astrocytes)

The functional weakening of the CNS that occurs with aging and age-related neurodegenerative disorders has been partially attributed to a decline in mitochondrial function. In particular, it has been demonstrated that oxidative damage occurs to mitochondrial DNA in elderly human brains. Recently, it has been shown that mitochondrial DNA is particularly sensitive to damage that accumulates due to the loss of protective histones, the reduction in repair systems, and the vicinity of the internal mitochondrial membrane to active oxygen species. The hypothesis that free radicals are involved in the weakening of the mitochondrial function has been confirmed by recent discoveries—i.e., the fact the administration of free radical scavengers, such as extract of ginkgo biloba (Egb761), improves the function of the brain and liver in elderly animals. Astrocytes are interconnecting cells between the neurons and the surrounding connective tissue (fibroblasts, mesenchymal cells, and endothelial cells). Changes to these cells induce modifications to the intercellular relationships and, eventually, to the nervous function. It has been shown that astrocytes are resistant to oxidative stress due to their high antioxidant content and their ability to regenerate glutathione and ascorbate. Astrocytes therefore act as neuronal protectors, defending the neurons against free radicals.

Astrocytes in Elderly Individuals

These astrocytes have large cell bodies and robust protrusions; particularly those found in the neurofilaments (NFL). In a group of subjects aged over 60 years, the astrocytes showed a higher glial fibrillar acidic protein (GFAP) immunoreactivity with respect to the younger subjects, particularly in the NFL. This observation has been confirmed by electron microscopy, which showed a higher density of glia filaments (formed by GFAP) in the astrocyte cytoplasm of the elderly group. In the NFL, the lack of GFAP(+) signal between the astrocyte bundles indicates that in this layer the astrocytes lose their protrusions. Occasionally, GFAP(+) organelles are found in the glial cells (CGL) and NFL—these correspond to decayed astrocytes. The perivasal astrocytes have few thin protrusions and form a thinner astroglial sheath than in younger individuals. Sporadically, reactive astrocytes are found. The honeycomb structure is not easily distinguished in the CGL, and the gaps in the astroglial plexus have variable forms (circular, square, rectangular). The dimensions are, however, larger than those in younger subjects in both zone A (nearer to the optic disk) and in zone B (closer to the periphery). The number of gaps in the astroglial honeycomb plexus in the CGL is lower in the 60-89 age group. This signifies that the gaps are larger due to the disappearance of astrocytes from the vessel walls and the astroglial protrusions that divide the gaps. The reduction in the number of astrocytes increases with age, as is shown by comparing people between 60 and 79 and those over 80. The comparison between young and elderly retinas has again shown that aging causes numerous changes to the retinal astrocytes. There is an increase in the number of intracytoplasmatic organelles (mitochondria, ribosomes, polyribosomes, wrinkled endoplasmatic reticulum) due to higher cellular activity, an increase in lysosomes and dense bodies (which increase also inside the Müller cells), an increase of the glial intermediate filaments, a thickening of the inner limiting membrane whose constituents are less homogeneous, and an increase in the space between the glial protrusions and the basal membrane of the inner limiting membrane.

Glial Cells in Elderly Individuals

All glial cells have numerous lysosomes in their cytoplasm. The prolongations of the glial cells contain dense bodies, formed from incompletely digested myelin that causes cellular swelling. In some elderly retinas, astrocytes of large dimensions are found that have very elevated cellular activity and a higher density of intermediate filaments. This type of astrocytes is called reactive astrocytes. The function of the reactive astrocytes is to protect the neurons (in this case, the ganglion cells) from ischemia-producing neurotrophic factors, increasing the expression of antioxidant substances (i.e., glutathione, vitamin C) and increasing the production and transport of glucose. However, it has been observed that astrocytes are more vulnerable

to oxidative damage during aging. In fact, as the years roll, the reactive astrocytes cause changes in the geometry and volume of the extracellular space that slows the diffusion of neuroactive substances. The extracellular space is not only the microenvironment of the nerve cells, but is also an important channel of communication between the neurons and astrocytes. The changes in the diffusion parameters, which arrive with aging, can bring on a disappearance of the transmission signals and increase the sensitivity of the nervous tissue to ischemia. The ischemia is due to an increase in the extra cellular acidity with an accumulation of potassium and other toxic substances (similar to glutamate) that damage the neurons.

Reactive Astrocytes

Reactive astrocytes have an elevated number of organelles (secondary lysosomes and lipofuscin) and an elevated cellular activity. If exposed to visible light (400700 nm) at a high concentration of oxygen (70 mm Hg)—i.e. conditions ideal for the formation of free radicals— the reactive astrocytes (and therefore the lipofuscin contained within) can cause damage to the cellular proteins and the membrane lipids. The reactive oxygen species can cause damage to cellular and nuclear elements. The presence of high concentrations of toxic substances (glutamate) in the extracellular space (coming from the reactive astrocytes), causes a massive increase of hydrogen and potassium with an increased permeability of the cell membranes, worsened by free radicals, which makes the cell swell. This cellular edema causes the breakage of the intermediate filaments of the astrocytes and, therefore, the loss of GFAP immunoreactivity and finally cell death. This fact explains why a reduction of the number of astrocytes in the CGL is observed, and why a disappearance of the protrusions of the NFL astrocytes has been observed in the elderly. In elderly people, it has also been observed that the basal membrane of the inner limiting membrane is thicker than in the younger group. The increase in thickness impedes the interchange of substances between the retina and the vitreous humor that represents a reserve of glucose, amino acids, potassium and glutathione, and so on for the retina, and a deposit for degradation products.

Hypertrophic Astrocytes

Madigan et al.11 have described that in retinas with age related macular degeneration (AMD), there are distended hypertrophic astrocytes on the internal surface of the retina. Many studies have been performed on the neovascular membrane that is found in AMD, produced by the migration of endothelial choroidal cells across the Bruch’s membrane in the subretinal space. None, however, have talked about epiretinal glial membranes. Many studies have shown that the epiretinal membranes may derive from inflammatory processes, retinal ruptures, or retinal vascular occlusions.

The extended retinal ischemia in AMD causes the astrocytes to migrate into the vitreous humor, where they can find metabolic reserves. In this way, the vitreous humor guarantees the nutrition of the remaining astrocytes in the inner retina while the intercellular junctions between the astrocytes remain intact. It is not known what factors cause the astrocytes and Müller cells to migrate into the vitreous humor in AMD. It is not known if these membranes are dangerous—i.e., if they can, after traction, cause a detachment of the retina.

Aging of the Microglia

The retinal microglia originate from the hematopoietic cells and enter the retina from the retinal margin and the optic disk through the blood vessels of the ciliary bodies and iris, and of the retina, respectively. The microglial precursors, which are found on the retina before the arrival of the vessels, are positive to some specific immune staining and express the CD45 marker, but are not positive for specific markers of the macrophages. A second category of microglial precursors, which express typical macrophage markers, migrate into the retina together with vascular precursors. These are localized around the blood vessels in the adult retina and are similar to macrophages or mononuclear phagocytes. The microglial cells are found in the outer plexiform layer, the external nuclear layer, the internal plexiform layer, the gangliar layer, and the nervous fiber layer of the retina in humans. The retinal macrophages are involved in the defense against viral, bacterial, and parasitic infection, in immunoregulation, in tissue repair, in the catabolism of neurotransmitters and hormones, and in the lipid turnover of nervous tissue. The microglias play an important role in the defense against microorganisms, in immune regulation, and in tissue repair. The occurrence of degenerative phenomena, but also normal aging, causes the conversion of the microglia from resting to reactive. Reactive microglias have the responsibility of consuming the debris and facilitating the regenerative processes. The morphology and localization of the microglia is not the same for all age groups. It has been seen that in newborn mice, the microglia cells are round and ameboid, with thick, squat psuedo-polipoid protrusions distributed in the ganglion and nervous fiber layers, while their morphology shows some age-related changes.

Lymphocytes

Lymphocytes are cells responsible for acquired immune response. These are rather small cells with a poor cytoplasm. Two functional types of antigen-specific lymphocytes can be distinguished: B and T lymphocytes. Lymphocytes are mainly localized in bone marrow, in peripheral lymphoid organs, in mucous surfaces, and in the thymus. They can also be found in blood and lymph nodes. Both B and T