The Ultrastructure of the Nictitating Membrane of the Little Penguin (Eudyptula minor, Aves).

The ultrastructure of the nictitating membrane in the little penguin Eudyptula minor was studied using both scanning and transmission electron microscopy to improve our understanding of the function of ocular adnexa in diving birds. Following euthanasia, eyes were enucleated and immersion fixed in Karnovsky's fixative. The nictitating membrane and conjunctiva were embedded in araldite and semi- or ultra-thin sections were stained and photographed using compound and transmission electron microscopes, respectively. Ultrastructural dimensions were measured directly from digital photographs. Surface ultrastructure was examined using scanning electron microscopy. The transparent nictitating membrane consists of a dense stroma surrounded by epithelia on both the external (conjunctival) and internal (bulbar) surfaces. The conjunctival surface of the membrane near the leading edge is covered by microvilli, which transition to microplicae and finally to microridges in the periphery. Beneath the epithelial cells, there is a well-developed basement membrane. Scattered throughout this epithelium are a few goblet cells. The surface of the bulbar epithelium is covered by microvilli near the leading edge, which become denser peripherally. The stroma consists of densely-packed collagen fibrils, which are randomly oriented in bundles near the leading edge but are aligned in the same direction parallel with the epithelial and corneal surfaces and with the leading edge, when the membrane is extended. The ultrastructure of the nictitating membrane in the little penguin differs from other birds and its function is predominantly protective, while preserving clear vision in both water and air.

Topographic analysis of the retinal ganglion cell layer and optic nerve in the sandlance Limnichthyes fasciatus (Creeiidae, Perciformes).

The sandlance or tommy fish Limnichthyes fasciatus (Creeiidae, Perciformes) is a tiny species that lives beneath the sand with only its eyes protruding and is found throughout the Indopacific region. The retina of the sandlance possesses a deep convexiclivate fovea in the central fundus of its minute eye (1.04 mm in diameter). A Nissl-stained retinal whole mount in which the pigment epithelium had been removed by osmotic shock was used to examine the retinal topography of the ganglion cell layer. There was a foveal density of between 13.0 x 10(4) cells per mm2 (S.D. +/- 1.8 x 10(4) cells per mm2), counted in the retinal whole mount, and 15.0 x 10(4) cells per mm2, counted in transverse sections, which diminished to a peripheral density of 4.5 x 10(4) cells per mm2 (S.D. +/- 0.8 x 10(4) cells per mm2). The total population of axons within the optic nerve was assessed by electron microscopy. Optic axon densities ranged from 2 x 10(6) axons per mm2 in the caudal apex to over 16 x 10(6) axons per mm2 within a specialized region of unmyelinated axons in the rostral apex. The topography of the proportion of unmyelinated axon population (26%) follows closely that of the total population of optic nerve axons. There was a total of 104,452 axons within the optic nerve compared with 102,918 cells within the retinal ganglion cell layer. A close relationship is revealed between ganglion cell soma areas and axon areas where the organization in the optic nerve and retina may reflect some functional retinotopicity.

Basophils in vernal conjunctivitis in humans: an electron microscopic study.

The histopathological changes of vernal conjunctivitis comprise stromal infiltration by lymphocytes, plasma cells, and neutrophilic, eosinophilic, and basophilic leukocytes and epithelial invasion by mast cells and eosinophils. Blood vessels show swelling and death of endothelial cells and increased permeability with associated extravasation of erythrocytes and fibrin. The presence of basophils in ocular tissue has not been reported previously, and their occurrence in conjunction with the other pathological changes enables vernal conjunctivitis to be compared with, and classified as a manifestation of, delayed-type hypersensitivity of the cutaneous basophil type. Thus the mechanism is probably a mixture of both delayed and immediate immunological responses.

The fine structure of nuclear changes in superior limbic keratoconjunctivitis.

Superior limbic keratoconjunctivitis (SLK) is a condition of unknown etiology. Histological signs include acanthosis, dyskeratosis, keratinization, and ballon degeneration of the bulbar conjunctival epithelium. Ultrastructural examination of biopsy material from five eyes of patients with SLK shows numerous nuclear changes in the conjunctival epithelial cells. These include abnormal distribution and aggregation of nuclear chromatin, the presence of filaments within the nucleus, and dense accumulations of cytoplasmic filaments which surround the nucleus, resulting in "strangulation" and the formation of multilobed nuclei or multinucleated cells. These changes do not appear to have been described previously in any cell type.

Herpes simplex virus in human cornea, retrocorneal fibrous membrane, and vitreous.

A case of recurrent herpes simplex virus keratouveitis was studied using both electron microscopic and viral culture techniques. We describe a patient who had a particularly violent course, including three failed grafts, endophthalmitis, and wound leak. We report the identification of virus in a quiescent failed corneal graft and retrocorneal membrane; persistence of virus enabling its culture from the vitreous; and electron microscopic identification in the cornea removed at the third transplant. The identification of cases in which viral proliferation occurs is necessary for a greater understanding of the pathogenesis of herpetic corneal disease and for determining in which particular patient steroid therapy should be avoided.

Ultrastructure and organisation of the cornea, lens and iris in the pipefish, Corythoichthyes paxtoni (Syngnathidae, Teleostei).

The corneas of nine pipefish, Corythoichthyes paxtoni (Syngnathidae, Teleostei), five freshly fixed and four museum specimens, were examined using light and electron microscopy. In transverse section, the surface of the corneal epithelium is covered by a complex series of ridges or microplicae which extends over the conjunctiva. The cornea is considerably thicker in the centre (80 microns) than in the periphery (40 microns) and can be separated into two distinct zones. The anterior dermal cornea (23 microns) consists of two layers of epithelial cells, a thick basement membrane (0.75 micron) and numerous lamellae of collagen fibrils with a few scattered keratocytes. This layer is continuous with the conjunctiva which also contains two layers of epithelial cells and lamellae of collagen fibrils. In the juvenile, separating the two zones, is a lens-shaped (concavo-convex) region approximately 6 microns thick in the centre and about 175 microns in diameter containing a fine granular material. In the adult, this region contains both granular material and fibres. It overlies the posterior zone which consists of an anterior iridescent layer (21 microns thick) possessing numerous cell processes parallel with the corneal surface and a few collagen fibrils. The scleral cornea contains 33 lamellae of collagen fibrils without cells and a single layer of cells with several cell processes, similar in appearance to the anterior iridescent layer, which may represent a second or posterior iridescent layer. There is a thick (2 microns) Desçemet's membrane and a thin (1.5 microns) corneal endothelium. There is a spherical lens close to the posterior corneal surface and the iris contains guanine crystals anteriorly and pigment granules posteriorly.

The foveal photoreceptor mosaic in the pipefish, Corythoichthyes paxtoni (Syngnathidae, Teleostei).

The foveal and non-foveal retinal regions of the pipefish, Corythoichthyes paxtoni (Syngnathidae, Teleostei) are examined at the level of the light and electron microscopes. The pipefish possesses a deep, pit (convexiclivate) fovea which, although lacking the displacement of the inner retinal layers as described in other vertebrate foveae, is characterised by the exclusion of rods, a marked increase in the density of photoreceptors and a regular square mosaic of four double cones surrounding a central single cone. In the perifoveal and peripheral retinal regions, the photoreceptor mosaic is disrupted by the insertion of large numbers of rods, which reduce spatial resolving power but may uniformly increase sensitivity for off-axis rays. In addition to a temporal fovea subtending the frontal binocular field, there is also a central area centralis subtending the monocular visual field. Based on morphological comparisons with other foveate teleosts, four foveal types are characterised and foveal function discussed with respect to the theoretical advantage of a regular square mosaic.

Retinal and lenticular ultrastructure in the aestivating salamanderfish, Lepidogalaxias salamandroides (Galaxiidae, Teleostei) with special reference to a new type of photoreceptor mosaic.

The salamanderfish, Lepidogalaxias salamandroides (Galaxiidae, Teleostei) is endemic to southwestern Australia and inhabits shallow, freshwater pools which evaporate during the hot summer months. Burrowing into the substrate in response to falling water levels allows these fish to aestivate for extended periods of time while encapsulated in a mucous cocoon even when the pools contain no water. Only a few minutes after a major rainfall, these fish emerge into relatively clear water which subsequently becomes laden with tannin, turning the water black and reducing the pH to approximately 4.3. As part of a large study of the visual adaptations of this unique species, the retinal and lenticular morphology of the aestivating salamanderfish is examined at the level of the light and electron microscopes. The inner retina is highly vascularised by a complex system of vitreal blood vessels, while the outer retina receives a blood supply by diffusion from a choriocapillaris. This increased retinal blood supply may be an adaptation for reducing the oxygen tension during critical periods of aestivation. Large numbers of Müller cells traverse the thickness of the retina from the inner to the outer limiting membranes. The ganglion cells are arranged in two ill-defined layers, separated from a thick inner nuclear layer containing two layers of horizontal cells by a soma-free inner plexiform layer. The photoreceptors can be divided into three types typical of many early actinopterygian representatives; equal double cones, small single cones and large rods (2:1:1). These photoreceptors are arranged into a unique regular square mosaic comprising a large rod bordered by four equal double cones with a small single cone located at the corner of each repeating unit. The double cones may optimise perception of mobile prey which it tracks by flexion of its head and "neck" and the large rods may increase sensitivity in the dark tannin-rich waters in which it lives. Each single cone also possesses a dense collection of polysomes and glycogen (a paraboloid) beneath its ellipsoid, the first such finding in teleosts. The retinal pigment epithelium possesses melanosomes, phagocytes and a large number of mitochondria. The anatomy of the retina and the photoreceptor mosaic is discussed in relation to the primitive phylogeny of this species and its unique life history.

The deep-sea teleost cornea: a comparative study of gadiform fishes.

The corneal structure of three deep-sea species of teleosts (Gadiformes, Teleostei) from different depths (250-4000 m) and photic zones are examined at the level of the light and electron microscopes. Each species shows a similar but complex arrangement of layers with a cornea split into dermal and scleral components. The dermal cornea comprises an epithelium overlying a basement membrane and a dermal stroma with sutures and occasional keratocytes. Nezumia aequalis is the only species to possess a Bowman's layer, although it is not well-developed. The scleral cornea is separated from the dermal cornea by a mucoid layer and, in contrast to shallow-water species, is divided into three main layers; an anterior scleral stroma, a middle or iridescent layer and a posterior scleral stroma. The iridescent layer of collagen and intercalated cells or cellular processes is bounded by a layer of cells and the posterior scleral stroma overlies a Desçemet's membrane and an endothelium. In the relatively shallow-water Microgadus proximus, the keratocytes of the dermal stroma, the cells of the iridescent layer and the endothelial cells all contain aligned endoplasmic reticulum, which may elicit an iridescent reflex. No alignment of the endoplasmic reticulum was found in N. aequalis or Coryphanoides (Nematonurus) armatus. The relative differences between shallow-water and deep-sea corneas are discussed in relation to the constraints of light, depth and temperature.

Fine structure of the retina and pigment epithelium in the creek chub, Semotilus atromaculatus (Cyprinidae, Teleostei).

The structure of the light- and dark-adapted retina, the pigment epithelium and the choroid of the creek chub, Semotilus atromaculatus (Cyprinidae, Teleostei) is examined by light and electron microscopy. An extensive network of vitreal blood vessels emanating from the hyaloid artery enters the eye with the optic nerve and overlies the inner limiting membrane. This membrane closely apposes the fine protrusions of the Müller cell processes which traverse the entire retina, dividing the inner retina into alternating fascicles of ganglion cells and optic axons. The inner nuclear layer consists of bipolar, amacrine, Müller cell soma and two layers of horizontal cells. The outer plexiform layer possesses both rod spherules and cone pedicles. Each rod spherule consists of a single synaptic ribbon in either a triad or quadrad junctional arrangement within the invaginating terminal endings of the bipolar and horizontal cell processes. In contrast, cone pedicles possess multiple synaptic ribbons within their junctional complexes and, in the light-adapted state, the horizontal cell processes show spinule formation. Four photoreceptor types are identified on morphological criteria; unequal double cones, large single cones, small single cones and rods. All but the small single cones are capable of retinomotor responses. The rod to cone ratio is approximately 5:1 and the rods form two ill-defined rows in the light-adapted condition. The retinal pigment epithelium possesses two types of osmiophilic granules. These are bound within slender microvilli and migrate vitread to surround the photoreceptors in response to light. Bruch's membrane is trilaminar and the vascularised choroid consists of up to three layers of melanocytes. The endothelial borders of the choroidal blood vessels abutting the outer lamina of Bruch's membrane are fenestrated.

Ultrastructure and organisation of the retina and pigment epithelium in the cutlips minnow, Exoglossum maxillingua (Cyprinidae, Teleostei).

The structure of the light- and dark-adapted retina, pigment epithelium and choriocapillaris of the cutlips minnow, Exoglossum maxillingua (Cyprinidae, Teleostei) is examined by light and electron microscopy. A pronounced vitreal vascularisation overlies the inner retina where the blood vessel walls, the inner limiting membrane and the Müller cell endfeet are all closely apposed. The thick Müller cell processes divide the inner plexiform layer and nerve fibre layer into discrete compartments. The ganglion cells do not form fascicles and lie within both the ganglion cell and inner plexiform layers. The inner nuclear layer consists of amacrine, bipolar, Müller cell somata and two rows of horizontal cells. The photoreceptor terminals comprise either multiple (3-5 in cone pedicles) or single (rod spherules) synaptic ribbons. These photoreceptor terminals form either a triad (rods and cones) or a quadrad (cones) arrangement of contact with the invaginating processes of the inner nuclear layer cells. The horizontal cell processes of the cone photoreceptor terminals reveal spinule formation in the light-adapted condition. Five photoreceptor types are classified using morphological criteria; triple cones, unequal double cones, large single cones, small single cones and rods. The ratio of rods to cones is approximately 7:1. All photoreceptor types show retinomotor responses. Only the cones possess accessory outer segments but both rods (8-11) and cones (15-19) possess calycal processes. The retinal pigment epithelium displays retinomotor responses where pigment granules within fine apical processes move vitread to mask the rods in the light. The cells of the retinal pigment epithelium are joined by various types of junctions and contain numerous phagosomes, mitochondria and polysomes. Bruch's membrane or the complexus basalis is trilaminate with two types of collagen fibrils comprising the central layer. The endothelia of the blood vessels of the choriocapillaris, facing Bruch's membrane, are fenestrated. Two to three layers of melanocytes interspersed between large thin-walled capillaries and several layers of collagen fibrils comprise the choriocapillaris.

Recurrent keratoconus after keratoplasty.

A 35-year-old woman who had undergone bilateral penetrating corneal grafts for keratoconus had keratoconus in the eye that had received a graft 16 years earlier. A second penetrating keratoplasty was performed, and the excised button was examined with light and electron microscopy. Both the light and electron microscopic findings were consistent with the clinical diagnosis of keratoconus. Keratoconus in a graft for the same disease should be added to the list of late complications of successful keratoplasties.

Conjunctival resection treatment and ultrastructural histopathology of superior limbic keratoconjunctivitis.

Four patients with symptomatic superior limbic keratoconjunctivitis underwent resection of the superior bulbar conjunctiva. One of these patients also underwent a tarsal conjunctival resection in the other eye. Three of the patients had previously been treated by various regimens without resolution; the fourth had had no prior treatment. All four patients had immediate and continued relief of the ocular symptoms after the superior bulbar conjunctiva was excised. The patient who underwent tarsal conjunctival resection experienced only short-term relief. We studied the conjunctival tissue by light and transmission electron microscopy. Both techniques revealed abnormalities related to the bulbar conjunctival surface with keratinization of the epithelium, acanthosis, degeneration of the nuclei, and intracellular accumulation of glycogen. Inflammatory cells were minimally present. The tarsal conjunctiva appeared essentially normal.

The effects of hypertonic salicylate on neutrophil migration and vascularization in the rat cornea.

Thermal cauterization of the center of the rat cornea results in emigration of neutrophils into the extravascular limbal tissue and blood vessel growth into the cornea. In this study, 1.0 M sodium salicylate, 1.0 M sodium chloride, and ointment vehicle were administered to normal and cauterized rat corneas for periods of 6, 48, and 144 h. When applied to the normal cornea, salicylate resulted in a marked increase in neutrophils in the limbal tissue at 6 h, but an inhibition at 48 h. Similarly, for the cauterized corneas, administration of salicylate increased the extent of neutrophil emigration at 6 h, but this effect was not sustained at 48 h. Neither vehicle nor sodium chloride had any effect on the extravascular neutrophil population. After 6 days, administration of the vehicle resulted in a slight increase in vascular growth into the cornea, whereas sodium salicylate caused a decrease. These findings indicate that hypertonic (1 M) sodium salicylate does not inhibit the emigration of neutrophils from limbal vessels of cauterized rat corneas, but does appear to have a cytotoxic effect on the tissues and on blood vessel endothelial cells.

The limbal vascular response to corneal injury. An autoradiographic study.

Thermal cautery of the peripheral cornea in rats caused proliferation of the limbal vasculature and invasion of the cornea. Tritiated-thymidine was used to identify premitotic activity in a total of 53,192 limbal vascular cells in five categories, viz., arteriolar endothelial cells, venular endothelial cells, arteriolar perivascular cells, venular perivascular cells, and capillary cells. From normal values in the range of 0.29 to 1.37%, the 2 h labeling indices reached a maximum of 13 to 14% in both endothelial and perivascular cells of venules and capillaries. Of particular interest was the finding of 18% labeling in arteriolar perivascular cells, and 7% in arteriolar endothelial cells. The categories showed a staggered onset of DNA synthesis, ranging from 17 h postcautery for capillary cells to 36 h for arteriolar endothelium and both arteriolar and venular perivascular cells. The duration of increased DNA synthesis also varied. Endothelial cells of both arterioles and venules showed narrow labeling peaks (12 to 24 h), while the adjacent perivascular cells and cells of the small vessels labeled for some 60 to 70 h. These results suggest that more than one stimulus to angiogenesis may be involved, or that the various cell types respond differently to the same stimulus.

The cornea of the sand lance, Limnichthyes fasciatus (Creeiidae).

The sand lance, Limnichthyes fasciatus, is a small fish, 15-30 mm in length, found on the Great Barrier Reef. The eyes, which are not covered by a spectacle, are externally placed, move independently, and measure about 1 mm in diameter. The cornea, unusual even in the teleost world, is about 0.14 mm thick, which represents one-seventh of the length of the eye. The layers of the cornea include an epithelium with a complex pattern of surface microplicae, a grossly enlarged basal cell layer, and a thick basement membrane. Structures (iridophores), which may represent the vestigial remnants of a secondary spectacle, are present in the peripheral corneal epithelium. The stroma, which has no Bowman's layer, is composed of lamellae of collagen fibrils but contains no keratocytes. Posterior to the stroma is a thick (0.1 mm) cellular layer that may represent the autochthonous layer seen in some teleost species. An iridescent layer consisting of approximately 70 parallel cytoplasmic plates oriented at right angles to the visual axis lies anterior to a thick Descemet's membrane. There is a single layer of endothelial cells on the posterior surface.

The fine structure of the cornea of the salamanderfish, Lepidogalaxias salamandroides (Lepidogalaxiidae, Teleostei).

The salamanderfish, Lepidogalaxias salamandroides, is a small freshwater fish that is endemic to southwestern Australia. Its phylogeny is still uncertain, but it belongs to a monotypic family that is as primitive as any living teleost. Able to survive during periods of drought by burrowing into the substrate and estivating for many months, this species has evolved a unique visual system, which is highly adapted to its changing environment. Thought to use cutaneous respiration during periods of dormancy, it secretes a mucus sheath over the eyes and genital openings to inhibit desiccation. The cornea is split into dermal and scleral components, separated by an iridescent layer and a mucoid layer. The dermal cornea comprises an epithelium containing both goblet cells and mucus-secreting granules in the central region, which overlies a thick stroma of collagen lamellae. The dermal stroma possesses numerous sutural fibers and flattened keratocytes and collections of three types of pigment granulates in the periphery. Posterior to the dermal stroma lies an iridescent layer consisting of five flattened cell processes interspersed with membranous extensions of smooth endoplasmic reticula. The mucoid layer is composed of numerous membrane-bound structures that allow movement of the scleral cornea beneath the dermal cornea or secondary spectacle. The scleral stroma is thin (six to seven lamellae) contains keratocytes, and overlies a relatively thin and irregular Desçemet's membrane and a monolayered endothelium. In the iridocorneal angle and situated between Desçemet's membrane and the endothelium is an "annular ligament," which is composed of flattened cells with lobulated nuclei and a fine granular cytoplasm with a few mitochondria. The numerous corneal inclusions are discussed with respect to their phylogeny and function.

A comparative SEM study of the vertebrate corneal epithelium.

PURPOSE: The anterior surface of the cornea of mammals, including humans, has numerous folds in the anterior epithelial cell membranes in the form of microvilli and microplicae. The role of these surface irregularities may be to increase cell-surface area and therefore aid in intra- and extracellular movement of nutritional and waste products across the cell membranes in addition to stabilizing the corneal tear film. The aim of this study was to investigate and compare the nature of these corneal-surface features in various vertebrate classes residing in different environments. METHODS: The anterior corneal surfaces of various vertebrates were investigated by using field emission scanning electron microscopy. Cell areas were analyzed by using image-analysis software. RESULTS: Representative species were examined from all the vertebrate classes, with the exception of the Cephalaspidomorphi. The mean epithelial cell density of aquatic vertebrates (17,602 +/- 9,604 cells/mm2) is greater (p = 0.000018) than that of aerial and terrestrial vertebrate species, including amphibians (3,755 +/- 2,067 cells/ mm2). Similarly, the mean epithelial cell density for the marine vertebrates (22,553 +/- 8,878 cells/mm2) is greater (p = 0.0015) than that of the freshwater and estuarine species (10,529 +/- 5,341 cells/mm2). The anterior corneal surfaces of all species examined were found to show a variety of cell-surface structures. Microvilli are predominant in reptiles, birds, and mammals; microridges appear to be characteristic of the Osteichthyes; and microholes were observed only in the Chondrichthyes. CONCLUSION: The function of these morphologic variations in surface structure appear to be correlated with the range of ecologic environments (marine, aerial, and terrestrial) occupied by each species, corneal phylogeny, and the demands placed on the cornea to ensure clear vision.

The corneal endothelium in the blowfish (Torquigener pleurogramma).

PURPOSE: In vertebrates, a corneal endothelium is essential for the maintenance of corneal transparency in a variety of environments. Knowledge of the surface structure of the corneal endothelium may assist our understanding of this unique tissue and its evolutionary development. Although there have been many studies of the corneal endothelium of humans and some mammals, there have been few in other vertebrates. METHODS: The field emission scanning electron microscope was used to study the surface structure of the corneal endothelium in the blowfish, Torquigener pleurogramma (Tetraodontidae, Teleostei), and to examine cell density. Cell areas were measured by using image-analysis software. RESULTS: The endothelium is composed of a sheet of interdigitating hexagonal and pentagonal cells with a mean area of 154 microm2 and a density of 6,486 cells/mm2. Two types of surface features are identified; primary cilia and microvilli. The cilia are cylindrical, protrude from either a pore or circular indentation in the cell center, and possess a knob-like ending. The microvilli are button-like protrusions with a density of -3.5 x 105 microvilli/mm2 or 54 microvilli/cell in central cornea. CONCLUSION: The results show that the surface structure of teleost endothelial cells is similar to those described for other vertebrates and indicate that cell density varies across classes, with the presence of cilia a more widespread occurrence than previously believed.

Steroid inhibition of limbal blood and lymphatic vascular cell growth.

Steroids are widely used in the prevention of corneal neovascularization in a wide range of natural and experimental situations. However, no information is available on their effect on the growth of the individual limbal blood vascular cells or of lymphatic cells involved in corneal neovascularization. In addition, tritiated thymidine labelling index is commonly used as an indicator of cell population but doubt exists as to whether it truly represents cell growth. Remote thermal cautery of the rat cornea was used to elicit corneal neovascularization. New cell growth was measured by tritiated thymidine uptake and by the number of cell nuclei per section. Cells investigated were the arteriolar, venular, capillary and lymphatic endothelial cells as well as the arteriolar and venular perivascular cells. A total of 89,320 blood vascular endothelial and perivascular cell nuclei and 12,075 lymphatic nuclei were counted. Thermal cautery elicited a significant increase in labelling index and cell population of all limbal vascular cell types. Steroid application elicited a significant short term inhibition or delay for all six cell types although this was not apparent for venular endothelial cells using labelling index as a growth indicator. At six days only the lymphatic endothelial cell population showed a significant (p < 0.001) increase associated with steroid use.