Effects of cyclic AMP and Sephadex fractions of chick embryo extract on cloned retinal pigmented epithelium in tissue culture.

The effects of dibutyryl cyclic 3',5'-adenosine monophosphate (BcAMP) and Sephadex G-25 fractions of chick embryo extract on the growth rate, morphology, and pigmentation of normal chick retinal pigmented epithelium (PE) were investigated. Seven cloned PE cell lines were each grown in modified Ham's F-12 medium alone (F-12), or in F-12 supplemented with either high molecular weight (H) or low molecular weight (L) fractions of chick embryo extract. Cells grown in F-12 alone or in L medium formed compact epithelial sheets, whereas cells grown in H had a fibrocytic appearance and formed poorly organized monolayers. In H plus BcAMP, cell morphology was more epithelioid than in H alone, and generally the monolayers appeared more differentiated. Under each of these three culture conditions, 2 x 10(-4) M BCAMP retarded the increase in cell number and decreased the final number of cells per culture dish, but had little effect on plating efficiency. BcAMP also increased the rate of cell adhesion to a plastic substratum. Pigmentation was marked in cultures grown in F-12 or in L alone, but the addition of BcAMP dramatically reduced visible pigmentation. This effect was reversed when BcAMP was removed from the culture medium. Thus BcAMP modifies cell and colonial morphology, rate of cell accumulation, adhesive properties, and pigmentation of normal PE cells.

Ocular manifestations of keratitis-ichthyosis-deafness (KID) syndrome.

OBJECTIVE: Keratitis-ichthyosis-deafness (KID) syndrome is a rare congenital ectodermal dysplasia characterized by the association of hyperkeratotic skin lesions, moderate to profound sensorineural hearing loss and vascularizing keratitis. Mutations in the GJB2 gene coding for connexin 26, a component of gap junctions in epithelial cells, have been observed in several KID patients. Variable ocular manifestations of the disease in 3 patients with molecular genetically confirmed KID syndrome are reported. DESIGN: Retrospective case series. METHODS: Clinical examination and molecular genetic analysis for mutations in the GJB2 gene were performed in 3 patients with KID syndrome ages 5, 13, and 41 years. RESULTS: Visual acuity ranged from normal to severe visual loss. The ocular signs included loss of eyebrows and lashes, thickened and keratinized lids, trichiasis, recurrent corneal epithelial defects, superficial and deep corneal stromal vascularization with scarring, keratoconjunctivitis sicca, and, in one patient, presumed limbal insufficiency. Whereas ocular surface integrity could be maintained with artificial tears in one patient, and an epithelial defect healed under conservative treatment in the second patient, multiple surgical procedures including superficial keratectomies, limbal allograft transplantation with systemic immunosuppression, amniotic membrane transplantation, lateral tarsorrhaphies, and lamellar keratoplasty could not preserve useful vision in the third patient. CONCLUSIONS: KID syndrome may affect the ocular adnexae and surface with variable severity independent of the age of the patient. Lid abnormalities, corneal surface instability, limbal stem cell deficiency with resulting corneal complications, and dry eye are the main ocular manifestations.

Paraphenylenediamine: a new method for tracing human visual pathways.

Morphologic investigations of the human visual pathways have been limited by the infeasibility of modern neuroanatomical approaches. Although contemporary methods for tracing axon pathways (such as tracer injections and electrophysiology) have elucidated the visual system in experimental animals, these techniques cannot be similarly applied in humans. Thus, the present view of the neuroanatomy of the human visual system is based largely on experimental animal studies, classical simple observations of gross human brains, and clinical inference. We demonstrate use of the stain, paraphenylenediamine (PPD), in conjunction with standard methods of tissue preparation for transmission electron microscopy. Osmium precipitates on degenerating neural processes, resulting in a dark profile when examined by electron microscopy. PPD chelates the osmium in osmium tetroxide-fixed tissue, and thus becomes a light-opaque marker of degenerating processes. This technique allows the identification and tracing of degenerating or degenerated axons. Postmortem studies of six patients, four with documented optic nerve lesions, are presented. The degenerated retinal ganglion nerve fibers are followed with PPD and confirmed with electron microscopy. Previously proposed, primary visual projections are confirmed and new retinofugal pathways are demonstrated in the human brain.

Noninvasive measurements of pyridine nucleotide and flavoprotein in the lens.

Abnormalities in glucose metabolism are thought to be among the main causes of cataract formation. The authors have made noninvasive biochemical measurements of the lens that provide information concerning glucose metabolism in the lens epithelium. The autofluorescence of reduced pyridine nucleotides (PN) and oxidized flavoproteins (Fp) within the rabbit lens were noninvasively measured as a function of depth using redox fluorometry. The peak of the autofluorescence at 440 nm (excited at 360 nm) and 540 nm (excited at 460 nm) were determined at the lens epithelium. When 8 mM sodium pentobarbital, a known inhibitor of mitochondrial respiration, was applied to the lens, the autofluorescence peak at 440 nm increased and that at 540 nm decreased. The 440 nm autofluorescence is thought to be from reduced pyridine nucleotides, whereas the 540 nm autofluorescence is from the oxidized flavoprotein. Blocking lens respiration with pentobarbital caused an increase in the PN/Fp ratio by a factor of 3 within 3.5 hr after pentobarbital application.

Studies on the source and release of collagenase in thermally burned corneas of vitamin A-deficient and control rats.

In previous studies we found that a mild thermal burn of the vitamin A-deficient rat cornea caused collagenase release into the medium of corneas placed in culture media 72 hr after applying the burn. Collagenase was released only on day 1 of culture and was not released from identically burned corneas of control rats. We now demonstrate that in deficient corneas, this collagenase release on day 1 of culture increased gradually with increasing time between burn and sacrifice, reaching a maximum at 16 hr after burning a remaining high up to 72 hr. In control rats day-1 collagenase release also increased to a maximum at 16 hr after the burn but then declined to almost zero at 72 hr. Trypsin treatment of day-1 media from both control and deficient corneas, taken at 72 hr after the burn, showed an almost complete absence of latent (inhibited) collagenase. Histologic observations revealed a close correlation between the presence of infiltrating polymorphonuclear neutrophils (PMNs) and the ulcerative lesions seen in burned, deficient corneas. When PMN infiltration was blocked by application of a tissue adhesive, no ulceration occurred and collagenase activity in the day-1 media dropped to almost zero. If burned and unburned areas of deficient corneas were separated and cultured separately, the burned area (containing most of the PMNs) was found to have 10 times the collagenase activity of the unburned area. In the controls, PMNs and some collagenase activity was detectable only 16 hr after the burn. We concluded that in the burned, vitamin A-deficient cornea there is increased attraction of PMNs to the lesion, resulting in collagenase release by these and possibly other cells, and ultimately resulting in ulceration.

Effect of fibronectin on corneal epithelial wound healing in the vitamin A-deficient rat.

The authors investigated the in vitro and in vivo effects of fibronectin (Fn) on the migration of corneal epithelium of vitamin A-deficient (A-) and pair-fed control (A+) rats. Groups treated with 50 micrograms/ml Fn showed accelerated healing of epithelium in vitro (P less than 0.05) compared with control groups of A- and A+ rats. However, when 100 micrograms/ml Fn eye drops were administered 14 times over 20 hr, they had no significant effect on A+ rats in vivo, but increased the healing in A- rats (P less than 0.05). In this model, Fn promoted the healing of corneal epithelium under A- conditions where decreased endogenous Fn is seen, whereas A+ corneas in vivo, which have sufficient Fn over the wound surface, did not benefit from topical Fn administration.

Morphogenesis of rat conjunctival goblet cells.

Conjunctival flat-mount preparations stained with alcian blue and PAS were used to study the development and mucin differentiation of conjunctival goblet cells in Sprague-Dawley rats from newborn to 17 months of age. In the neonatal conjunctiva, only single goblet cells containing primarily acidic mucin (alcian-blue positive) were noted. With the increase of age, the rat conjunctival goblet cells were in clusters with heterogeneous mucin contents, which differ from the nonclustered, single goblet cells of human and rabbit conjunctiva. Topographical analysis revealed that the density and size of goblet cell clusters were highest in the forniceal zone, with gradual decrease towards the bulbar and orbital zones. and were absent in the limbal and tarsal conjunctiva in all ages. The goblet cells in the forniceal zone contained predominantly acidic mucin in all ages, except that selective loss of acidic mucin with vacuolation of the clusters was noted in the 17-month-old rats. Despite the fact that the size of the goblet cell clusters increased with age, the overall density of the clusters remained rather constant throughout the ages studied. These results indicate that each goblet cell cluster may derive from a single progenitor (stem) cell and represents a glandular primordium, and that goblet cell development and mucin differentiation are modulated by the aging process.

Plasminogen activator activity in vitamin A-deficient rat corneas.

Plasminogen activator (PA) activity during epithelial wound healing in vitamin A-deficient rats was investigated to determine whether a relationship exists between corneal defect formation and PA activity. Uniform, central 3 mm diameter corneal epithelial wounds were made by scalpel debridement in vitamin A-deficient and in pair-fed control rats. Cryostat sections of such corneas, taken at various times post-scrape, were overlaid with fibrin films containing plasminogen to examine the distribution of PA activity; and antibodies to tissue plasminogen activator (tPA) or to urokinase-like activator (uPA) were incorporated into the films so that the immunochemical natures of detected PA activities could be determined. Corneas from pair-fed controls showed tPA-dependent lysis in association with the regenerating epithelium as well as in the defect region during epithelial wound healing. Corneas from vitamin A-deficient rats also demonstrated tPA activity in association with corneal epithelium post-scrape but showed no detectable tPA activity in the defect region. Histological examination of the vitamin A-deficient corneas demonstrated that a pseudomembrane composed of PMNs, cell debris, and fibrinous exudate had formed on the scrape-debrided stromal surface. The migrating edge of regenerating epithelium overlaid this membrane and was, therefore, not in contact with the stromal surface. The formation of the pseudomembrane, which delays reepithelialization, might have resulted from the absence of PA activity in the defect region. Both excessive and inadequate levels of PA activity may result in impaired epithelial wound healing.

Pathologic changes in the exorbital lacrimal gland of the vitamin A-deficient rat.

Histologic changes in lacrimal glands of vitamin A-deficient (A-) and pair-fed control rats were compared. In A- lacrimal glands, secretory granules were strikingly diminished, and rough endoplasmic reticulum appeared somewhat atrophic. Nuclei of acinar cells were hyperchromatic and pleomorphic. Using alcian blue-PAS, no positive staining was present in acini of A- lacrimal glands, whereas in controls apical portions of acini were intensely stained. Thus, lacrimal tissues of A- rats were thought to be poorly differentiated as a glandular epithelium. When A- rats were supplemented with retinyl acetate, secretory granules reappeared, rough endoplasmic reticulum cisternae greatly dilated, and mitochondria proliferated, indicating accelerated secretory activity. Resupply of vitamin A can induce glandular differentiation in A- lacrimal tissues. Tear volume was not decreased in A- rats compared with pair-fed controls. Regression of secretory organelles in A- lacrimal tissues may lead to a decrease in protein and mucoprotein secretion and subsequent changes in tear composition.

Paracellular permeability of corneal and conjunctival epithelia.

The paracellular permeability of normal rabbit cornea and conjunctiva was studied in vivo and in vitro. After intravenous administration, horseradish peroxidase was found to percolate to the intercellular space of conjunctival epithelia and was restricted by the tight junctions of the superficial epithelium. Only minimal tracer was present in the limbus and cornea. The difference between corneal and conjunctival paracellular pathways was further compared in vitro by tissue perfusion studies using various tracers from subepithelial space to apical surface. The intact full-thickness cornea was permeable to mannitol (MW 182) but not to inulin or dextran. The conjunctiva was permeable to mannitol, inulin and FITC-dextran (MW 20,000). The quantitative permeability to 3H-mannitol (X10(-8) cm/sec) of adult rabbit cornea was 0.12 +/- 0.02, which is about 55-fold and 50-fold lower than that of conjunctiva (6.78 +/- 0.21) and peritoneum (6.12 +/- 0.63), respectively. Removal of the corneal epithelium increased the permeability 40-fold; however, removal of the endothelium had little effect on the solute permeation. When both corneal epithelium and endothelium were debrided, the bare stroma became edematous and the permeability increased 70-fold. The permeability of 1-week-old rabbit cornea was 1.32 +/- 0.18, which decreased to 0.46 +/- 0.06 in 2-week-old rabbits, and became similar to the adult level at 4 weeks of age. When Tenon's capsule was included in the perfusion, the conjunctival permeability decreased 2.5-fold. With the apposition of bare corneal stroma to the conjunctiva and Tenon's capsule, the permeability decreased further (4-fold).(ABSTRACT TRUNCATED AT 250 WORDS)

Standard models of corneal injury using alkali-immersed filter discs.

Central corneal alkali burn injuries were induced in rabbits by applying five NaOH concentrations on uniformly soaked 7 mm filter paper discs. Clinical parameters were evaluated daily by microscopic examination and photography, and corneal myeloperoxidase levels were measured periodically. Satisfactory alkali burn models of corneal inflammation, vascularization and ulceration were developed by manipulating the alkali concentration.

Alteration of epithelial paracellular permeability during corneal epithelial wound healing.

We studied the paracellular permeability to mannitol of corneas with epithelium of corneal, limbal, or conjunctival origin. Corneas with epithelial defects reepithelialized by corneal or limbal epithelium were nonvascularized; the corneal permeability was initially increased and returned to normal 3 days later. When epithelial defects extended beyond the limbus, they were healed by conjunctival epithelium. If corneas remained avascular or minimally vascularized, the conjunctiva-derived epithelium underwent a transdifferentiation process into a cornealike morphology in which the corneal permeability was initially increased upon complete reepithelialization, and gradually decreased to a level similar to that of normal cornea, 4 weeks after healing. However, when corneas became vascularized, the conjunctiva-derived epithelium retained its original phenotype, and corneal permeability remained increased throughout the 8-month period of study. The deranged barrier functions noted in the above vascularized cornea were demonstrated further by horseradish peroxidase tracer, which was found in the intercellular spaces of conjunctiva-derived epithelium of vascularized corneas but not in the avascular corneas with epithelia of corneal or limbal origin, or transdifferentiated conjunctival epithelium. To study further the effect of subsequent ocular surface trauma, conjunctival biopsy was performed on transdifferentiated avascular corneas 3 months after initial wounding. The biopsy resulted in extensive vascularization in three of eight previously nonvascularized corneas. Two weeks later, the corneal permeability was increased to a level similar to that of conjunctiva. These results indicate that corneal epithelial paracellular permeability correlates well with the status of the epithelial phenotype.

Expression of transforming growth factor-beta in wound healing of vitamin A-deficient rat corneas.

Transforming growth factor-beta (TGF-beta) is a multifunctional peptide that modulates cell proliferation and differentiation in many cell types and accelerates tissue repair response. In this study, expression of TGF-beta was investigated immunohistochemically in the healing of central 3 mm epithelial wounds of vitamin A-deficient (A-) rat and pair-fed controls. In control rat corneas, a positive reaction to TGF-beta was not evident during wound healing. In A- rat corneas at 4 hr post-abrasion, acute inflammatory cells showing high positivity to TGF-beta appeared in the peripheral stroma and gradually spread to the central cornea. By 24 hr, these cells accumulated and formed a pseudomembrane in the epithelial defect, which also showed an intense positivity to TGF-beta, suggesting that the peptide participates even in the acute inflammatory response. From about 16 hr post-injury, many fibroblasts revealing intense positivity to TGF-beta infiltrated the entire stroma and were part of the healing process. Reepithelialization occurred over the pseudomembrane and was completed by 48 hr. The central cornea showed remodeling of collagen structure and neovascularization. Fibroblasts containing TGF-beta were seen in the stroma, indicating that TGF-beta plays an important role in corneal wound healing. Indeed, in the absence of vitamin A, a common modulator of cell differentiation, TGF-beta may play a more important role in wound healing than in the normal state.

Change of paracellular permeability of ocular surface epithelium by vitamin A deficiency.

Dietary vitamin A deficiency in young rabbits caused advanced squamous metaplasia with keratinization of conjunctival epithelium and concomitant reduced paracellular permeability to 3H-mannitol. Both morphologic and permeability changes were reversed with systemic administration of vitamin A. In adult rabbits, vitamin A deficiency caused milder changes of goblet cell loss and increased cellular stratification in conjunction with reduced permeability in the conjunctiva-like epithelium that covers the vascularized cornea after chemical injury with n-heptanol. Topically applied retinoid (tretinoin 0.1%) did not affect the morphology and permeability of the normal corneal or conjunctival epithelium of rabbits that were not vitamin A deficient. These studies showed that altered permeability is associated with the epithelial abnormality during vitamin A deficiency and helped clarify the physiologic function of retinoids in the ocular surface epithelia in the nondeficient state.

Epithelial abrasion precipitates stromal ulceration in the vitamin A--deficient rat cornea.

Although the role of vitamin A deficiency in the development of xerophthalmia is well established, there is still some question as to whether the deficiency alone is sufficient cause for the development of keratomalacia. This article describes the clinical, histologic, and microbiologic changes occurring in eyes of vitamin A-deficient rats when keratomalacia-like stromal ulceration is induced by epithelial injury alone. The corneal epithelia of 21 severely vitamin A-deficient rats and 11 pair-fed controls were totally removed either by scraping or by n-heptanol. At 96 hr after epithelial removal, 93% of the deficient animals showed extensive epithelial defects and stromal ulceration. Histologically, an intense acute inflammatory response and abundant bacterial forms were consistently evident. Staphylococcus aureus and Streptococcus fecalis were the most frequent pathogens cultured from these ulcerating eyes. In contrast, the control corneas showed essentially complete re-epithelialization, with no ulceration, minimal inflammatory reaction, and an absence of morphologically demonstrable bacteria. Bacterial cultures from the control eyes showed abundant Pasteurella, with pathogens also present. These observations suggest that abnormal epithelial recovery, acute inflammation, and bacterial infection may be important factors for the development of keratomalacia-like corneal ulceration in experimental vitamin A deficiency.

Prevention of stromal ulceration in the alkali-burned rabbit cornea by glued-on contact lens. Evidence for the role of polymorphonuclear leukocytes in collagen degradation.

Stromal ulceration of the alkali-burned rabbit cornea was found to be associated invariably with phagocytically active polymorphonuclear leukocytes (PMNs). A glued-on methylmethacrylate lens applied to corneas soon after burning, however, prevented re-epithelialization and also prevented PMN infiltration of the stroma and stromal ulceration. Subsequent partial detachment or complete removal of the lens resulted in epithelial resurfacing of the stroma, PMN infiltration, and stromal ulceration. Glued-on lenses applied to already ulcerating corneas arrested further ulceration by prohibiting additional PMN infiltration. Either surface debridement or glued-on methylmethacrylate rings also prevented re-epithelialization and ulceration in stromas not infiltrated by PMNs, but neither treatment was sufficient to prevent ulceration in corneas already containing numerous PMNs. The data suggest the possibility that the epithelium stimulates infiltration of the stroma by PMNs which then participate in stromal matrix degradation. Although no claim is made that only PMNs mediate matrix destruction in corneal ulceration, the efficacy of the lens would seem to be due to exclusion of the epithelium and the consequent prevention of stromal infiltration by PMNs.

Cleavage and activation of corneal matrix metalloproteases by Pseudomonas aeruginosa proteases.

PURPOSE: To examine the effect of Pseudomonas aeruginosa on the expression of corneal matrix metalloproteases and the effect of its proteases on activation of corneal matrix metalloproteases in vitro. METHODS: Rat corneas and human corneal fibroblasts were co-cultivated with two different strains (RPS & 599A) of P. aeruginosa and one strain of Staphylococcus aureus, and the conditioned media were analyzed for proteolytic activity by gelatin and casein zymography. Human corneal fibroblast-conditioned medium was incubated with that from either strain of P. aeruginosa and was analyzed in a similar manner. RESULTS: Normal rat corneas in organ culture produce a 65 kDa gelatinase (inactive matrix metalloprotease-2), whereas thermally injured rat corneas additionally produce gelatinases with molecular masses of 92 kDa (inactive matrix metalloproteases-9) and > 200 kDa. Matrix metalloprotease-2 is also detected in human corneal fibroblast-conditioned medium. Although these matrix metalloproteases are no longer detectable when rat corneas or human corneal fibroblasts are co-cultured with two strains of P. aeruginosa for 48 hr, a 58 kDa gelatinase fragment appears in earlier stages of co-culture. In contrast, S. aureus does not affect matrix metalloprotease-2. The 58 kDa fragment is also evident by incubating human corneal fibroblast-conditioned medium with that from either strain of P. aeruginosa. Conditioned medium from the RPS strain, which produces both elastase and alkaline protease, is more effective in cleaving matrix metalloprotease-2 than that from the 599A strain, which expresses mainly alkaline protease. CONCLUSION: The secreted inactive corneal matrix metalloprotease-2 is activated through limited proteolysis by pseudomonal proteases.

Regeneration of corneal epithelial basement membrane following thermal cauterization.

Thermal cauterization of the corneas of nine rhesus monkeys was performed by multiple thermokeratophore applications at temperatures of 90 degrees or 120 degrees C. Significant clinical observations included the resteepening of corneal curvature, delayed epithelial healing, stromal haze or scarring, and peripheral neovascularization. Reestablishment of tight adhesion of the regenerated epithelium to Bowman's layer required approximately 6 weeks. By transmission and scanning electron microscopy, the original basement membrane seemed relatively intact immediately following cauterization but disappeared within 1 week. The regeneration of new basement membrane complexes began at 1 week with the appearance of short discontinuous segments of basement material with hemidesmosomes and similarly required approximately 6 weeks for complete restoration.

Tear film osmolarity and ocular surface disease in two rabbit models for keratoconjunctivitis sicca.

We report the natural history of keratoconjunctivitis sicca (KCS) in two rabbit models. The first one (full KCS model) was created by closing the lacrimal gland excretory duct, and removing the nictitating membrane and harderian gland. We created the second one (lacrimal gland duct only [LGDO]-KCS model) by closing the lacrimal gland excretory duct. Although tear film osmolarity was abnormally high in both models, it was higher in the full KCS model. Decreases in corneal epithelial glycogen and in conjunctival goblet cell density, and morphological abnormalities of the conjunctiva correlated with increases in tear film osmolarity and duration of disease.

Cellular migration and morphology in corneal endothelial wound repair.

After a mechanical denudation of rabbit corneal endothelial cells, the healing process was followed with wide-field specular microscopy. Individual cell migration and morphologic changes were analyzed by computer-assisted morphometry. The cells surrounding the wound migrated to cover the defect without producing intercellular gaps. The greatest cellular migration and morphologic alterations occurred close to the wound edge. As the cells migrated toward the wound, they elongated and increased their surface area in the direction of the migration. As the healing proceeded, the cells lost their original hexagonal pattern, which returned after coverage was complete. The wound was covered completely by large, irregularly shaped cells showing mitotic figures between 24 and 48 hr. During this period, cellular migration decreased and normal cellular morphology began to recover. When mitosis decreased, the normal cellular pattern rearranged towards a more hexagonal shape. During the healing process, the degree and direction of cellular migration varied from cell to cell. Additionally, changes in cell-to-cell contact (positional changes of neighboring cells) occurred in one-third of migrating cells. Such cellular migration can account for monolayered cells sliding without producing gaps between individual cells.