Repair of retinal pigment epithelium and choriocapillaries after laser photocoagulation: correlations between scanning electron, transmission electron and light microscopy.

Cell-to-cell interactions between retinal pigment epithelial (RPE) cells and vascular endothelium were examined by scanning electron microscopy, which demonstrates many facets of surface topography. The repair process after laser photocoagulation was characterized by a morphologically heterogeneous population of regenerated RPE cells, including normal-looking cells, macrophage-like cells and large elongated RPE cells. The macrophage-like and normal-looking RPE cells were predominantly seen adjacent to growing new choroidal vessels. Large cells appeared mainly in the vicinity of the extracellular matrix, where no viable new vessels were seen. The observed association between a specific phenotype of regenerated RPE cells and neovascularization may further support the hypothesis that regenerated RPE cells have a dual function, exerting both stimulatory and inhibitory effects on endothelial cells.

Reorganization of actin microfilaments and microtubules in regenerating retinal pigment epithelium.

Retinal pigment epithelium (RPE) regenerating after experimental damage in rabbits exhibits major changes in cell shape, polarity and junctions--features that depend on the cytoskeleton. This report correlates these changes with the redistribution of actin microfilaments and microtubules, using electron microscopy and confocal laser scanning microscopy. We compare immature cells with the more mature cells that form the new epithelial monolayer. Two populations of immature RPE cells are interspersed at the edge of the regenerating RPE sheet. One population of immature cells makes few junctions with their neighbors or the basement membrane. They form pseudopodia and exhibit a prominent network of actin microfilaments beneath the plasma membrane. These cells are probably motile and advance the epithelial sheet. Another population of immature cells contains numerous stress fibers that insert into large basement membrane attachments. The cells make focal adhesions with their neighbors, rather than the junctional complexes characteristic of mature RPE cells. These cells are probably not motile and mature into the cells forming the new monolayer--cuboidal cells with numerous basal folds and apical villi and a complete belt of intercellular junctions. Stress fibers are lost as the circumferential bundle associated with the zonula adherens re-forms. Microtubules, which form prominent longitudinal bundles running through the processes of immature cells, take on the meshwork organization characteristic of mature RPE as the immature cells differentiate.

Hyaluronate distribution in the regenerating retinal pigment epithelium of the rabbit: a study using confocal laser scanning microscopy.

The distribution of hyaluronate (HA) in regenerating retinal pigment epithelium (RPE) of the rabbit was examined using immunohistochemistry and confocal laser scanning microscopy. The goal was to determine if there is a correlation between differentiation and HA expression, like that seen in developing tissues, where HA accumulates and then disappears as the tissue matures. In normal RPE cells HA is associated mainly with the apical surface. In regenerating RPE (produced by i.v. injection of sodium iodate to damage the epithelium, regeneration arising from spared cells), HA exhibits a patchy distribution among the more immature cells and is especially prominent where they overlap or pile up on each other. Where cells are more mature and form a compact monolayer of cells, HA is expressed mainly on the apical surface, as in normal RPE. The accumulation of HA among the more immature cells in the regenerating epithelial sheet supports the hypothesis that HA influences differentiation by suppressing cell-cell associations until the proper time for their formation.

Carbonic anhydrase type II in regenerating retinal pigment epithelium. A histochemical study in the rabbit.

Type II carbonic anhydrase (CAII) in the cytoplasm of the retinal pigment epithelium (RPE) may contribute to the transport of water and solutes across the RPE. The activity of this enzyme in RPE during its response to damage, e.g., during regeneration, is therefore of interest in understanding retinal disease. Immuno-histochemistry was used to compare CAII activity of normal RPE and RPE experimentally induced to regenerate. In normal rabbits, the RPE stained intensely with a peroxidase-linked antibody specific for human CAII. Regenerating RPE stained less intensely. Within the regenerating epithelium, staining appeared more intense in mature cells than in immature ones, suggesting that CAII activity gradually returns during RPE regeneration.

Distribution of Na+ K(+)-ATPase in regenerating retinal pigment epithelium in the rabbit. A study by electron microscopic cytochemistry.

Electron microscopic cytochemistry was used to examine the distribution of the enzyme sodium potassium ATPase (Na+ K(+)-ATPase) on the plasma membrane of regenerating retinal pigment epithelium (RPE). Observations were made in rabbits that received intravenous sodium iodate, which elicits RPE degeneration followed by regeneration. A gradient in intensity and polarization of enzyme activity was observed from the least mature cells in the distal part of the regenerating epithelial sheet to more mature cells proximally. The least mature cells exhibited faint Na+ K(+)-ATPase activity over their entire plasma membrane except where it faced the basement membrane. As cells matured and re-expressed the cytologic characteristics of mature RPE, activity intensified and became localized to the apical plasma membrane, as in normal RPE. The observations show the ability of damaged RPE to recover its normal structural and functional polarity.

Restoration of the outer blood-retinal barrier after krypton laser photocoagulation.

The restoration of the outer blood-retinal barrier following krypton laser injury to the rat retina was studied at small and large laser lesions using intravenously injected sodium fluorescein, horseradish peroxidase, and catalase. The regenerated choriocapillaris and new blood vessels were permeable to fluorescein and peroxidase, but not to catalase. The regenerating retinal pigment epithelium gradually reformed a continuous sheet of cells covering all small laser sites and the periphery of large lesions. Zonulae occludens between the regenerated cells restored the outer retinal barrier and prevented diffusion of peroxidase into the retina. This occurred along Bruch's membrane and the new blood vessels that were covered by the regenerated pigment epithelial cells, but not in the center of the large lesion that was not relined by regenerated cells.

Changes in Müller cell plasma membrane specializations during subretinal scar formation in the rabbit.

The aim of this study was to identify changes in Müller cell plasma membrane specializations during experimentally induced subretinal gliosis in rabbits. When rabbits are dosed with sodium iodate, large expanses of retinal pigment epithelium and photoreceptors are destroyed. They are replaced by a subretinal scar consisting mainly of the ascending processes of Müller cells. These processes transform from the slender, highly polarized structures seen in normal animals into irregular processes that form a glia limitans along the basement membrane of the pigment epithelium, left bare following its degeneration. As the scar processes extend through the subretinal space and contract this basement membrane, they undergo dramatic changes in shape that are especially apparent in three-dimensional computer reconstructions of serial thick sections examined by high-voltage electron microscopy. Other changes involve the intercellular junctions and apical microvilli normally associated with the external limiting membrane. These structures become scattered over the surfaces of the ascending processes and are eventually lost. Loss of microvilli is associated with disappearance of immunostaining for a specific glycoconjugate normally associated with the microvillar plasma membrane. The observations document profound changes in Müller cell structural and functional polarity during subretinal scar formation.

Choroidal vascular repair: scanning and transmission electron microscopy.

Repair of the choroidal vasculature following laser photocoagulation in the rat was examined with vascular casts and correlated with observations on thin-sections. The regenerative process began at the periphery of the damaged area, starting from the surviving choriocapillaris and venules, and proceeding towards the center by means of recanalization of damaged vessels and growth of new ones. In small healed lesions the capillary bed was re-formed. It resembled the adjacent undamaged choriocapillaris morphologically but appeared to be less dense than the intact choriocapillaris when examined by scanning microscopy. In large lesions the capillary bed was re-formed at the periphery but not at the center. Also present at the edges of the large lesions were groups of new vessels which, when observed by scanning microscopy, appeared to extend in two directions; towards the subretinal space and towards the choroidal network. Another aspect of the repair process was the simultaneous occurrence of new vessel growth and capillary regression, which was observed both at the level of the choriocapillaris and at the foci of new vessels.

Vascular remodeling after laser photocoagulation: concurrent regeneration and atrophy.

Laser photocoagulation is associated with paradoxical results: it causes obliteration of vessels, but leads also to the formation of new ones. In an attempt to better understand this dual vascular response we conducted an ultrastructure study of the choroidal vascular repair following krypton laser injury in rats. Three processes were observed: recanalization, neovascularization, and atrophy of both recanalized and newly formed capillaries. Post-lasering repair of the choroidal vasculature can therefore be described as a remodeling process, characterized by both regeneration and involution. The latter appears to be a secondary process of atrophy, contributing to permanent vascular obliteration. These mechanisms might explain why, in spite of initial vascular regeneration, laser photocoagulation treatment has a beneficial effect on choroidal subretinal neovascularization.

Localization of alkaline phosphatase on basolateral plasma membrane of normal and regenerating retinal pigment epithelium. A cytochemical study in rabbits.

Ultrastructural cytochemistry was used to analyze the polarized distribution of alkaline phosphatase (AP) on the plasma membranes of normal and regenerating retinal pigment epithelium (RPE) and the ciliary body epithelium in rabbits. The AP activity was concentrated on the basolateral plasma membrane in normal RPE. In regenerating RPE (after intravenous administration of sodium iodate to damage the RPE), there was a differential expression of AP activity according to the site on the regenerating epithelial sheet. At the edge of the sheet, where cells were undifferentiated and immature (ie, without the polarized distribution of basolateral and apical plasma membrane specializations seen in normal RPE), no plasma membrane AP activity was observed. The AP activity was reexpressed more proximal in the regenerating sheet. It was first evident where contiguous cells formed junctional complexes and cytologic polarization again became apparent. New AP activity was restricted to the basolateral plasma membrane. Eventually, the polarized distribution of AP activity seen in normal RPE was reestablished. In the ciliary body, AP activity was localized to the basolateral plasma membranes of the outer, nonpigmented epithelial cells. It was concluded (1) that cytochemical activity for nonspecific AP is concentrated on the basolateral plasma membrane domain of normal RPE and the normal pattern of AP activity is lost initially and then reexpressed.

Microfibril-microvessel connections in the rabbit eye: correlative confocal and electron microscopy.

The connections between elastic tissue and microvessels (arterioles, capillaries, and venules) in the rabbit eye were examined by light and electron microscopy. In particular, confocal scanning laser microscopy of tissue stained with orcein and examined by fluorescence using a rhodamine filter was correlated with electron microscopic observations. The goal was an analysis of the way in which elastic tissue of the uvea (i.e., choroid, ciliary body, and iris) and the optic nerve of the eye connect to the microvessels in these structures. Confocal microscopy revealed these connections advantageously and showed how they link the elastic tissue meshwork in the perivascular tissue spaces with the wall of the blood vessels. Electron microscopy showed that the connections consist of bundles of 10-12 nm diameter microfilaments that insert into vascular basement membranes. These connections may contribute to the vascular response to changes in blood pressure or intraocular pressure in the eye.

Labelling of regenerating retinal pigment epithelium by colloidal iron oxide and ferritin conjugated to wheat germ agglutinin.

In order to determine if there are biochemical changes in plasma-membrane oligosaccharides of regenerating retinal pigment epithelium, the binding of colloidal iron oxide at low pH and ferritin-conjugated wheat germ agglutinin--probes of sialic acid and N-acetylglucosamine on the cell surface--was examined electron-microscopically. An animal model of retinal pigment epithelium regeneration--rabbits with sodium iodate induced retinopathy--was used. In this model, large expanses of regenerating pigment epithelium are present for comparison with zones of spared pigment epithelium in the same animals. In thin sections examined by transmission electron microscopy, ferritin-conjugated wheat germ agglutinin appeared to bind more intensely to the exposed plasma membrane of regenerating retinal pigment epithelium than to spared pigment epithelium, or that of normal rabbits. Morphometry verified this. Colloidal iron oxide intensely labelled the plasma membranes of regenerating, spared, and normal pigment epithelium, and was visibly reduced after exposure of tissue to neuraminidase. The observations indicate that the plasma membrane of regenerating retinal pigment epithelium bears sialic acid and N-acetylglucosamine residues as in normal retinal pigment epithelium. However, the amount of plasma membrane bearing exposed N-acetylglucosamine increases during regeneration.

Microfibril-microvessel connections in the uvea and optic nerve of the mammalian eye.

Light- and electron-microscopic examination of arterioles, venules and capillaries of the eyes of several mammalian species has shown that the microfibrils of ocular elastic tissue attach to microvascular basement membranes throughout the uvea (iris, ciliary body, choroid) and optic nerve. Although described sporadically in prior investigations, this report shows that the connections are a common feature of the mammalian eye. The connections appear most numerous at venules and capillaries and are sparse at arterioles. The connections may provide a mechanism by which perivascular elastic tissue influences microvascular function, e.g. the control of blood pressure in them or their response to changes in intraocular pressure.

Expression of plasma membrane alkaline phosphatase in normal and regenerating choriocapillaris in the rabbit.

Ultrastructural histochemistry for plasma membrane nonspecific alkaline phosphatase was performed on the normal and regenerating choriocapillaris (CC) of rabbits. In normal animals the CC endothelium expressed little or no staining, whereas in regenerating CC the endothelium exhibited staining. The staining was most intense at the unfenestrated plasma membrane. As the capillaries matured and the fenestrated plasma membrane became more extensive, the staining was reduced and eventually eliminated. Pericytes did not stain in normal or regenerating CC.

Repair of retinal pigment epithelium and its relationship with capillary endothelium after krypton laser photocoagulation.

The regeneration of the retinal pigment epithelial (RPE) cells and their relationship with the vascular endothelium were studied during the healing of laser burns produced by krypton laser photocoagulation in rats. During the healing process the epithelial sheet was reformed by a morphologically heterogeneous population of RPE cells, some of which resembled macrophages. There seems to be a correlation between the specific phenotype of the regenerated RPE cell and the state of vitality of the adjacent endothelium. This was particularly evident in the scattered foci of choroidal subretinal neovascularization surrounded by RPE cells. One possible explanation for this apparent correlation is that the cytologic variations of the RPE cells reflect the multiple functions of a single cell which is expressed differently in different situations and in the course of interaction with other cells. This presumed ability of RPE cells may explain an intriguing aspect of cell-cell interaction, namely, the dual stimulatory and inhibitory effects of RPE cells on the vascular endothelium.

Ultrastructural evidence of remodelling in the microvasculature of the normal rabbit and human eye.

Several investigators have recently presented ultrastructural evidence for remodelling in the mammalian, including human, choriocapillaris. This evidence consists of cytoplasmic processes off endothelium and pericytes that penetrate the basal lamina of the capillary and enter the pericapillary space and redundant layers of basal lamina interpreted as the result of secretory activity subsequent to cellular movement within the wall of the capillary. This report extends these observations to the remaining microvasculature of the choroid--its arterioles and venules--and to another part of the ocular microvasculature--the pars plana of the ciliary body--of the rabbit and human eye. Cytoplasmic processes and redundant basal laminae were observed in the microvasculature at both sites, most frequently associated with venules and capillaries. Cytoplasmic processes and redundant basal laminae are generally associated with cellular movement and vessel growth during ocular neovascularization. However, their presence in histologically normal eyes suggests that these phenomena occur in the absence of neovascularization, i.e. that the microvasculature is remodelled in the normal eye.

The elastic tissue of Bruch's membrane. Connections to choroidal elastic tissue and the ciliary epithelium of the rabbit and human eyes.

Ultrastructural observations on the elastic lamina of Bruch's membrane of the rabbit and human eyes revealed connections to the ciliary epithelium and choroidal elastic tissue. The connections to the ciliary epithelium are in the form of bundles of microfibrils that peel off the anterior extension of Bruch's membrane beyond the ora serrata and insert into the basement membrane of the pigmented epithelium of the pars plana. The connections to the choroidal elastic tissue are in the form of bundles of microfibrils (in the rabbit) or elastin and microfibrils (in the human) that cross the tissue space between the choriocapillaris and join bundles of choroidal elastic tissue. These connections suggest that the elasticity of Bruch's membrane exerts its influence distant from the membrane itself. The connections onto the pars plana of the ciliary body implicate Bruch's membrane in disaccommodation when the ciliary muscle relaxes.

Choriocapillaris regeneration in the rabbit. Ultrastructure of new endothelial tube formation.

After experimental destruction of the retinal pigment epithelium (RPE) by intravenous injection of sodium iodate, the rabbit choriocapillaris (CC) atrophies. This report studies the subsequent regeneration of the CC with the goal of identifying sources of new CC and mechanisms by which new endothelial tube is formed. Light and transmission electron microscopic methods were used. The CC appeared to regenerate from three sources: choroidal venules, remnant CC and newly produced cells. Whatever the source, new endothelial tube was formed by extensions of the endothelium that sealed off new lumenal space by junctional complexes. This process was always confined to the tissue space within remnant sleeves of basement membrane left behind by initial atrophy, and which served as a substrate for endothelial movement. The endothelium of new endothelial tube was initially thick and unfenestrated. It became thinner and fenestrated by a process of cavitation, in which cavities continuous with the lumen pushed into the thick endothelial cytoplasm towards its abluminal side. At this site new fenestrae were formed. Fenestrae were initially scattered about the endothelial tube, but with its maturation became localized on the side facing the regenerated RPE.