Ординатура / Офтальмология / Английские материалы / Age-Related Changes of the Human Eye_Cavallotti, Cerulli_2008
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Fig. 2.10 Morphological features of mitochondria with type I crystals. The basic structural unit of this type appears in thin sections as a rectangular unit—about 32nm wide and of varying length— always located within the intracrystal space or between the inner and outer membrane (x70K 71y)
Fig 2.11 Morphological features of a mitochondria with type II crystals. This structure is surrounded by the membranes of cristea—i.e., they are located in the intermembrane space but not in the mitochondrial matrix (x70 K 74y)
with specific antibodies against mitochondrial creatine kinase.31,32 Mitochondrial creatine kinase is located in the intermembrane compartment and is functionally coupled to oxidative phosphorylation. It shuttles high-energy phosphates, formed in the mitochondria, to the cytosol where they are utilized. Recent concepts suggest that mitochondrial creatine kinase has the dual role of a) functioning as a key enzyme in
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energy metabolism, and b) as a structural protein inducing the formation of mitochondrial contact sites between inner and outer membaranes. This leads to membrane cross-linking and an increased stability of the mitochondrial membrane architecture, thereby contributing to the organization of the entire organelle.33
These structural changes in mitochondria were associated with functional changes because it was demonstrated with enzyme-histochemical studies. Succinodehydrogenase activity showed a particular pattern with aging—some of the fibers or some areas in these fibers showed decreased activity (see Fig. 2.12).
This irregular distribution of oxidative enzymes and focal decrease of activity has been described as target-fiber, targetoid-fiber, moth eaten-fiber, central core disease, and multi-core disease in various muscular diseases, myopathies, dystrophies, neurogenic atrophy, and hormonal diseases—all in which the above described ultrastructural alterations were observed. These morphological and enzyme-activity alterations together suggest an age-related decline in metabolic activity.
From a pathophysiologic point of view, these findings can hardly be interpreted. We suppose that an increase in number of mitochondria, enlargement of mitochondria, and proliferation of cristae are morphologic manifestations of a hypermetabolic state, in which myofiber structure is usually well preserved, while loss of cristae and appearance of osmiophilic or paracrystalline inclusions are signs of mitochondrial dysfunctions, which is more frequently associated with myofiber abnormalities.
Early electron microscopic studies of skeletal muscle cells of young and old humans showed the cristae of mitochondria became irregularly spaced, disrupted, and replaced by lamellar, myelin-like structures. Giant mitochondria were often visible. They contained lipofuscin in the myofibrils, too, which was often in close relationship with the damaged mitochondria.34 A substantial fall in mitochondrial oxidative capacity was also observed in ageing muscles.35
Fig. 2.12 Irregular distribution of the succino-dehydrogenase activity in aged muscle in the transversal section (71 yrs, x200)
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Biopsies of skeletal muscle from 2- to 39-year old rhesus monkeys showed that the number of individual fibers containing electron transport chain abnormalities (predominately negative for cytochrome c oxidase activity and/or hyper-reactive for succinate dehydrogenase) increased with age. These alterations associated with the deletions of the mitochondrial genome were observed in 89 percent of these electron transport chain abnormal fibers.36 The correlation between these two findings, however, remains unclear. Mitochondrial volume density was significantly lower in elderly compared with adult muscle, and these alterations were accompanied by a 50 percent reduction in oxidative capacity in the elderly vs. the adult group. In addition,. elderly subjects had nearly 50 percent lower oxidative capacity per volume of muscle than adult subjects. The cellular basis of this drop was a reduction in mitochondrial content, as well as a lower oxidative capacity of the mitochondria with age.37
In sections of orbicular muscle from aged patients, intramitochondrial inclusions of different sizes can be seen in addition to numerous morphologically abnormal and enlarged mitochondria. The formation of inclusions is always preceded by marked changes in cristae membrane disposition, with these membranes often taking a concentric arrangement. The question as to whether lipid peroxidation—pre- sumed to be increased in patients with mitochondrial myopathies—is involved in this reorganization of the membrane system remains to be answered.
Sarcoplasma
Age-related alterations of the orbicular muscle also comprise accumulation of tubular structures called tubular aggregates (TA) in the subsarcolemmal region (see Figs. 2.13 and 2.14).
These densely packed tubular or tubulo-vesicular structures were apparently derived from the sarcoplasmic reticulum (SR) because it was clearly seen in some of our electron microscopic pictures, thus confirming the previous observations coming from other laboratories.
The SR is an internal membrane system of the striated muscle. SR is a type of smooth endoplasmic reticulum specially adapted to surround the myofibrils, and it forms triads with invaginations of the plasma membrane called T-tubules. The sarcoplasmic reticulum contains large stores of calcium that it sequesters and then releases when the cells become depolarized. This has the effect of triggering a muscle contraction.38 Activation of muscle contraction is a rapid event that is initiated by electrical activity in the surface membrane and T-tubules. This is followed by release of calcium from the SR. Relaxation is mediated by the transport of calcium into the lumen of SR by a Ca-ATPase. Calcium then binds to calsequestrin in the lumen of the SR. For the initiation of contraction, calcium is released through the calcium channels or ryanodine receptors, which are under regulation by junctin, triadin, and calsequestrin. Thus, the SR is the major regulator of Ca2+-handling and contractility in muscles.39 It was later found that all types of cells contain cell-specific forms and/or analogues of these three proteins that are responsible for handling calcium. In all cells, these
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Fig. 2.13 Tubular aggregates. Transversal section of muscle fibers (left) and tightly packed tubules in the subsarcolemmal area (right). They show nearly identical diameter. Glycogen particles appear as electrodense granules between myofibers and tubules (x32 K, 65 yrs)
Fig. 2.14 Tubular aggregates. Transversal section of tubules in the subsarcolemmal area with numerous glycogen particles between them. Mitochondria show severe alterations, loss and/or proliferation of cristae, as well as type II crystal inclusion in some of them (x27 K, 67 yrs)
proteins tend to be grouped together. In fact, the SR is an extensive and specialized form of endoplasmic reticulum (ER) in muscle cells. Conversely, all cells contain SR-like specialized domains, but in much smaller amounts.
Tubular aggregates are the most common alterations of the SR observed in various pathological conditions, but also in apparently healthy persons. Tubular aggregates
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are ultrastructural abnormalities characterized by the accumulation of densely packed tubules in skeletal muscle fibres, usually beneath the sarcoplasma membrane and rarely between the myofibers. In human skeletal muscle, they are especially rich in patients suffering from tubular aggregate myopathy. We found various forms of tubular aggregates in normal aged orbicular muscle.
Tubular aggregates are characterized as more or less densely packed aggregates of vesicular or tubular membranes of variable forms and sizes that may contain amorphous material, filaments, or inner tubules. Various types of tubular aggregates were reported—namely, proliferating terminal cisterns, vesicular membrane collections either with double-walled tubules or with single-walled tubules, aggregates of dilated tubules with inner tubules, aggregates of tubulo-filamentous structures, and filamentous tubules.40 We have also observed tubulo-reticular structures (see Fig. 2.15).
Tubular aggregates were immunopositive for the ryanodine receptor (RYR 1) of the SR, the SR Ca2+ pump (SERCA2-ATPase), and the intraluminal SR Ca2+ binding protein calsequestrin, indicating an SR origin of these aggregates. All of these proteins, calsequestrin, RyR, triadin, SERCAs, and sarcalumenin are involved in calcium uptake, storage, and release. These findings support the hypothesis that tubular aggregates form a tubular arrangement of a complete SR containing the junctional, cisternae, and longitudinal components of SR implicated in calcium homeostasis.41,42 They also showed decreased respiratory chain enzyme complex I and complex IV activity. These findings indicate a functional link between mitochondrial dysfunction and the presence of TAs originating from the SR.43 Lipid composition of TAs showed that the predominant lipid of the aggregates is an acetone and alcohol-soluble acidic phospholipid containing a high proportion of plasmalogens and unsaturated fatty acids—a pattern compatible with the lipid composition of SR and mitochondrial membranes in skeletal muscle.
Fig. 2.15 Tubulo-reticular aggregates. Honeycomb appearance of tubular aggregates and a severely altered mitochondria can be seen in the subsarcolemmal area (x50 K, 74 yrs)
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In mouse models of skeletal muscle aging, a persistent decrease in the expression of the calcium binding protein calreticulin, as well as a continuous increase in calseques- trin-like protein expression, both appear unrelated to the tubular aggregate formation.44 In vitro studies using SR-enriched membrane vesicles isolated from the slow-twitch soleus muscle, and the relatively fast-twitch gastrocnemius muscle isolated from adult and aged rats, showed muscle-specific impairment in SR Ca2+ pump function and skeletal muscle contractile properties, which may contribute to the age-associated slowing of relaxation in the soleus muscle.45 An impairment of the mechanisms controlling the release of calcium from internal stores (excitation-contraction coupling) has been proposed to contribute to the age-related decline of muscle performance that accompanies aging. Excitation-contraction coupling in muscle fibers occurs at the junctions between sarcoplasmic reticulum and transverse tubules, in structures called calcium release units. Recent studies showed significant alterations in the calcium release unit morphology and cellular disposition, and a significant decrease in their frequency between control and aged samples. These data indicate that in aging humans, the excitation relaxation coupling apparatus undergoes a partial disarrangement and a spatial reorganization that could interfere with an efficient delivery of Ca2+ response to the contractile proteins.46
TAs should be seen as dynamic structures that commence and cease, progress and retreat, and change their structure, functionality, and composition under multifactorial, yet not well-defined influences. The structural and functional development of tubular aggregates remains also unknown. Tubular aggregates frequently occur with mitochondrial alterations supported by growing evidence of participation of mitochondria in the development of TAs. Factors affecting formation of TAs in skeletal muscle fibers may, however, have different structural and/or functional influences on other cell types.
Sarcoplasmic inclusions were also observed in the sarcoplasma of orbicular muscle. They showed filamentary, paracrystalline, or fingerprint structure and usually located beneath the plasma membrane.(see Figs. 2.16, 2.17, 2.18, and 2.19).
The origin and pathological significance of these sarcolemmal inclusions are mostly unknown. They may come from abnormal SR and mitochondria, or from both. They may also be signs of degeneration or regeneration, or both. Most probably, they are identical with the desmin-containing sarcolemmal inclusions described in other instances.
Desmin is an intermediate filament protein that, in striated muscle, is normally located at Z-line, beneath the sarcolemma, and prominently at neuromuscular junctions. It is abundant during myogenesis and in regenerating fibers, but decreases in amount with maturation. Desmin is coexpressed with vimentin in regenerating and denervated muscle fibers. Aggregates of desmin occur as nonspecific cytoplasmic bodies similar to the aggregates of keratin filaments in Mallory bodies, or the neurofilament aggregates in Lewy bodies. There are now increasing numbers of neuromuscular disorders in which abnormal amounts of desmin are in myopathic muscle fibers.47 Myofibrillar, or desmin-related, myopathies encompass neuromuscular disorders with abnormal deposits of desmin and myofibrillar alterations. In a recent case report on three unrelated patients presenting with proximal and distal myopathy, muscle biopsies shared sarco-
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Fig. 2.16 Fingerprint inclusion. Typical electron microscopic feature of a fingerprint inclusion in the subsarcolemmal area. Note the paucity of other organelles (x22 K, 77 yrs)
Fig. 2.17 Filamentary sarcoplasmic inclusion. This inclusion is located in the subsarcolemmal area, formed by numerous thin filaments without any apparent substructure, and surrounded by numerous mitochondria with well-preserved cristae. However, some electodense granules—pre- sumably lipofuscin—can be seen (x22 K, 71 yrs)
plasmic inclusions—either plaque-like or amorphous, strongly immunoreacted on dystrophin, and variably for desmin, alphaB crystalline, and ubiquitin. In addition, cyclin-dependent kinases were overexpressed in affected fibers.
In conclusion, myofibrillar destruction occurs in heterogeneous conditions and may overlap with features of inclusion body myopathy and mitochondrial myopathy
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Fig. 2.18 Paracrystalline sarcoplasmic inclusion. This inclusion is located in the unusually empty subsarcolemmal area, formed by tightly packed, electrodense filaments showing paracrystalline substructure, and surrounded by fine granular material. Some mitochondria with normal cristae can also be seen (x22 Km 67 yrs)
Fig. 2.19 Nuclear inclusions. Two intranuclear inclusions are surrounded by nuclear membrane, suggesting their location in the invaginations of the nucleus (i.e., pseudoinclusions). They contain heterogeneous materials (amorphous, granular, fibrillary, and membraneous). Similar materials can also be seen in the surrounding subsarcolemmal area (x26 K, 65 yrs)
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and with tubulo-filamentous inclusions and sarcolemmal inclusions.48,49 Now, we may also add aging, which may be associated with similar alterations of myofibers, mitochondria, and sarcolemma. We considered alterations of the muscle caused by various genetic and acquired factors as elementary.
Connective Tissue Changes
The extracellular matrix (ECM) consists of a variety of substances, of which collagen fibrils and proteoglycans (PG) are ubiquitous. In addition to the PG, the hydrophilic ECM includes a variety of other proteins such as noncollagen glycoproteins and lipids. It is known that the force transmission of the muscle-tendon complex is dependent on the structural integrity between individual muscle fibers and the ECM as well as the fibrillar arrangement of the tendon and its allowance for absorption and loading of con- traction-generated energy. Furthermore, it is well-described that the tensile strength of the matrix is based on intraand intermolecular crosslinks, and the orientation, density, and length of both the collagen fibrils and fibers. The signals triggering the connective tissue cells in response to mechanical loading, however, and the subsequent expression, synthesis, and turnover of specific ECM components—as well as its coupling to the mechanical function of the tissue—are only partly described.
Intramuscular connective tissue accounts for one to ten percent of the skeletal muscle and varies quite substantially between muscles. Based on their localization and organization, three types are distinguished: 1) endomysium encloses each individual muscle fiber with a random arrangement of collagen fibrils to allow for movement during contraction, 2) the multisheet-layered perimysium is multisheetlayered and runs transversely to fibers and holds groups of fibers in place, and finally 3) epimysium is formed from two layers of wavy collagen fibrils to form a sheet-like structure at the surface of the tendon.
Intramuscular connective tissue has several functions: a) it provides a basic mechanical support for vessels and nerves, b) the connective tissue ensures the passive elastic response of muscle, and c) it contribute to the force transmission from the muscle fibers to tendons and subsequently to bone. The perimysium is especially capable of transmitting tensile force.
Up to seven collagen types have been identified in intramuscular connective tissue. The fibrillar collagen type I (from 30% and up to 97% of total collagen) and III (and to some extent type V) dominates the epi-, peri-, and endomysium, but type IV dominates the basement membrane adjacent to the plasma membrane of the sarcolemma.50
In orbicular muscle, an age-related decrease in the diameter of muscle fibers seen in electron microscopy was accompanied with a significant increase in density of interstitial connective tissue. This was particularly evident in transversal sections of orbicular muscle (see Fig. 2.20).
Detailed histo-chemical analysis of connective tissue changes by means of polarization microscopy revealed two types of changes: 1) an increase of collagen fibers,
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Fig. 2.20 Transversal section of the interstitial connective tissue of orbicular muscle stained with phenol and seen in polarization microscopy. Thinner septa in a 61-year old patient (A) is compared to those of a 80-year old patient (B) (x200)
and 2) an increase of glycosaminoglycan (GAG) content of fibers. In other words, more glycated fibers accumulated in the interstitial connective tissue (see Fig. 2.21).
These observations on the aging of intramuscular connective tissue in orbicular muscle are in full accordance with the previous data that—with aging the nonspecific cross-linking mediated by condensation of a reducing sugar with an amino group—result in accumulation of advanced glycation end products (AGEs) in the connective tissue.51 The accumulation of AGEs with aging thus indicates a stiffer and more load-resistant tendon and intramuscular ECM structure. On the other hand, it reduces the ability to adapt to altered loading because the turnover rate of collagen is markedly reduced. Furthermore, AGEs upregulate connective tissue growth factors in fibroblasts that therefore favor the formation of fibrosis over time in elderly individuals and patients with diabetes.52 Besides reduced physical activity, diet with low albumin concentration may be a risk factor for both muscular loss and connective tissue changes.53 These age-related alterations of the connective tissue may also contribute to the age-related malposition of the eyelid, particularly in senile entropion and ectropion. In these diseases, muscular alterations were always associated with relaxation of ligaments and other connective tissue structures.
There is increasing evidence that changes in quantity and quality of intermuscular connective tissue due to aging may influence at least two different types of muscle function. First, partial replacement of contractile muscle fibers with connective tissue essentially modifies muscle contraction-relaxation. This alone may explain