Erythrocyte aging: a more than superficial resemblance to apoptosis?

Contains fulltext : 47441.pdf (publisher's version ) (Open Access)In physiological circumstances, erythrocyte aging leads to binding of autologous IgG followed by recognition and removal through phagocytosis, mainly by Kupffer cells in the liver. This process is triggered by the appearance of a senescent erythrocyte-specific antigen. The functional and structural characteristics of senescent erythrocytes strongly suggest that this antigen originates on band 3, probably by calcium-induced proteolysis. Generation of vesicles enriched in denatured hemoglobin is an integral part of the erythrocyte aging process. These vesicles are also removed by Kupffer cells, with a major role for exposure of phosphatidylserine. Moreover, senescent erythrocyte-specific antigens are present on vesicles. Thus, vesicles and senescent erythrocytes may be recognized and removed through the same signals. These and other, recent data support the theory that erythrocyte aging is a form of apoptosis that is concentrated in the cell membrane, and provide the context for future studies on initiation and regulation of the erythrocyte aging process. Insight into the normal aging mechanism is essential for understanding the fate of erythrocytes in pathological circumstances and the survival of donor erythrocytes after transfusion

Membrane architecture as a function of lens fibre maturation: A freeze fracture and scanning electron microscopic study in the human lens

The ultrastructure of fibre membranes in human lenses, varying in age from premature to 40 years, was investigated using a strict protocol regarding their localization within the lens. The ultrastructural approaches used were scanning electron microscopy (SEM), and transmission electron microscopy (TEM) of ultrathin sections and freeze-fracture replicas. Irrespective of the age of the lens, superficial fibre membranes are characterized by a high density of intramembrane particles (IMPs) and numerous gap junctions (GJs). In contrast deep cortical fibres, at the SEM-level characterized by grooves and ridges, are largely free of IMPs but still contain numerous GJs. In between these regions a transitional zone was observed. At the SEM-level the transitional fibres are characterized by wrinkled membranes and formation of grooves and ridges. In freeze-fracture replicas the presence of numerous square arrays (SAs) associated with GJs is most remarkable. It is concluded that at all ages studied, the maturation and compaction of lens fibres results in a transformation of membrane architecture leading to clear-cut ultrastructural differences between superficial and deep cortical membranes. It is argued that this ultrastructural heterogeneity parallels the gradients observed biochemically for intrinsic membrane proteins and cholesterol:phospholipid ratios. The observations confirm the electrophysiological view that superficial membranes have an 'average' permeability and that deep cortical membranes are 'degenerate' or 'non-leaky'

Microplasmin: ex vivo characterization of its activity

PURPOSE. Microplasmin is a recombinant protein limited to the enzymatic moiety of plasmin without any of its cringle domains. Its enzymatic activity is similar to that of plasmin enzyme. The present study characterizes in a porcine eye model the vitreolytic ability of microplasmin. METHOD. Freshly harvested porcine eyes were used in these trials. Eyes were injected with escalating doses of microplasmin (62.5, 125, 250, 400 g) for 1 hour or with 125 g microplasmin with increasing time exposures (15, 30, 60, 120 minutes). Eyes were fixed by a very slow dehydration process to preserve the integrity of the vitreous retinal interface. They were examined by light microscopy to determine the degree of posterior vitreous detachment and by scanning electron microscopy (SEM) to study structural changes. RESULTS. Effective separation of the posterior hyaloid appeared to be dose dependent. After 1 hour, the posterior pole was detached in 100% of porcine eyes exposed to 125 g microplasmin and in the midperiphery to 250 g microplasmin. Vitreous at the ora did not detach. At 120 minutes of exposure, midperipheral detachment was observed with 125 g microplasmin. A smooth retinal surface was seen where the enzyme caused posterior vitreous detachment. There was also significant change to the integrity of the vitreous without any obvious structural alterations to the retina by histology or scanning electron microscopy. CONCLUSIONS. Microplasmin caused vitreolysis and posterior vitreous separation in an ex vivo porcine eye model in an apparent dose-and time-dependent fashion. In this model system, the minimal effective dose appeared to be 125 g. 1-3 A few decades ago, reports appeared suggesting that spontaneous posterior vitreous detachment (PVD) would decrease the risk for or lead to the resolution of proliferative disease. 4 -6 Surgically producing an atraumatic separation of the posterior vitreous face achieves the same goals but may be difficult to induce. Incomplete separation can lead to disease progression, whereas firm adhesions between the hyaloidal face and the underlying retina can lead to damage of the underlying structures. 7 Several compounds, both enzymatic and nonenzymatic, have been tried as vitreolytic agents. 8 -14 One of the most promising and extensively tested in humans has been the serine protease plasmin. Its efficacy in producing PVD has been documented in a number of studies. Microplasmin is a recombinant protein, a truncated form of human plasmin that lacks all five kringle domains but retains the protease activity. 20 Produced in Pichia pastoris, the recombinant protein is stabilized in a dilute citrate buffer at pH 3.1 and is lyophilized. Its half-life in blood is several times higher than in plasmin, whereas its biological activity in models of peripheral arterial occlusion and ischemic stroke are similar to that in intact plasmin enzyme. Early studies in cat and rabbit models have shown its safety in animal eyes. 14,21 The present study was designed to characterize the enzymatic activity of recombinant microplasmin on the vitreous interface in an ex vivo porcine model system in which artifactual separation of the vitreous from the retinal surface was minimized. In this system, dose and time exposure were evaluated by light microscopy (LM) and electron microscopy (EM). METHODS Enzyme Preparation and Injection Procedure Microplasmin was provided by ThromboGenics Ltd. (Dublin, Ireland). The solution was stored at Ϫ20°C until needed. Just before use, the solution was thawed, diluted to the appropriate concentration under sterile conditions with balanced salt solution plus (BSSϩ; Alcon Laboratories, Fort Worth, TX), and kept on ice until use. The injections were given with a maximal delay of 15 minutes, after which a new vial of microplasmin was prepared. In all experiments, fresh cadaver pig eyes were obtained from a local slaughterhouse. To ensure freshness, the eyes were obtained directly after the animal was killed and were cleaned and gutted. The procedure never took longer than 10 minutes. One hundred microliters of the appropriate concentration of microplasmin or physiologic saline was injected into the eye through the pars plana 3 mm posterior to the limbus. The site of injection was marked on the surface of the sclera with indelible ink. Eyes were kept at ambient temperature (20°C-25°C) for the duration of the incubation period and were then processed as described

CYCLOCRYOAPPLICATION EXPERIMENTALE

By means of an experimental study on 82 pigmented rabbits, the authors analyse the different parameters which influence the cryoapplication intensity. They have determined the best conditions for a permanent lowering of the intraocular pressure without provoking phtisis bulbi.SCOPUS: NotDefined.jinfo:eu-repo/semantics/publishe

Morphology of age-related cuneiform cortical cataracts: The case for mechanical stress

AbstractWe evaluated the gross morphology, location, and fiber cell architecture of equatorial cortical opacities in the aging human lens. Using dark-field stereomicroscopy, we photographed donor lenses in toto and as thick slices. In addition, we investigated the details of the fiber cell architecture using fluorescent staining for membranes and by scanning electron microscopy. We then combined our data with data from recent studies on lens viscoelasticity. We found that small cortical and cuneiform opacities are accompanied by changes in fiber structure and architecture mainly in the equatorial border zone between the lens nucleus and cortex. Because the lens cortex and nucleus have different viscoelastic properties in young and old lenses, we hypothesize that external forces during accommodation cause shear stress predominantly in this border zone. The location of the described changes suggests that these mechanical forces may cause fiber disorganization, small cortical opacities, and ultimately, cuneiform cataracts