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Excimer-laser-induced fluorescence of rabbit cornea: radiometric measurement through the cornea.

The laser-induced fluorescence spectrum of rabbit cornea irradiated at ablative intensities was measured. This system directly measured the radiant exposure of fluorescence transmitted through the cornea when the anterior surface of the cornea was irradiated by an ArF excimer laser. Evidence of changing spectral characteristics as a function of total laser dose suggests photochemical changes in the cornea may be occurring. Results are compared with previous data of laser-induced fluorescence in other models and detection schemes.

Animals

[Adrenochrome deposits in the cornea ("Black cornea"). A clinical-pathological case report].

A case of 'black cornea', occurring 6 years after commencement of a therapy with adrenaline derivative eye drops, is reported. Enucleation was performed because of suspected malignant melanoma. The eye was sent to our laboratory for histopathological examination. The clinical and histopathological findings are described, and the pathogenesis of the 'black cornea' is discussed.

Adrenochrome

(Na+K+)-activated ATPase in human cornea. Distribution within the cornea and properties of the enzyme from epithelial cells.

Distribution and principal characteristics of (Na+K+)-activated ATPase in human cornea were investigated. (Na+K+)-ATPase was present in both epithelium and endothelium, whereas the corneal stroma did not exhibit significant enzyme activity. In homogenates specific activity of the (Na+K+)-ATPase was 2.3-fold higher in endothelium than in epithelium. Calculation of total enzyme activity revealed a 6.1-fold higher content of (Na+K+)-ATPase in the epithelium. In the epithelium a 7-fold enrichment of (Na+K+)-ATPase compared to the homogenate was obtained in the 150-1500 X gav fraction. Maximum enrichment in the endothelium was 3.5-fold and was achieved in the 1500-2500 X gav fraction. Both fractions showed, however, the same specific activity. The pH-optimum of (Na+K+)-ATPase in the 150-1500 X gav fraction ranged from 8.0-8.2 in both epithelium and endothelium. In the epithelial 150-1500 X gav fraction the apparent Km-values were 4.0 mM for Na+, 2.8 mM for K+ and 0.12 mM for Mg2+ - ATP in equimolar concentrations. The inhibition constant of epithelial (Na+K+)-ATPase for ouabain was determined as Ki = 3.3 X 10(-7) M. The present data support the view that control of corneal hydration in man is a function of both endothelium and epithelium.

Adenosine Triphosphatases

Relationships between eye factors and lens-forming transformations in the cornea and pericorneal epidermis of larval Xenopus laevis.

Larval Xenopus laevis at stage 56 (Nieuwkoop and Faber, '56) were subjected to various types of lentectomy: (1) simple lentectomy, from the pupillary space after incision of outer and inner cornea; (2) lentectomy from the dorsal region of the eye; (3) lentectomy from the dorsal region of the eye and simultaneous incision of the outer cornea; (4) lentectomy from the dorsal region of the eye and simultaneous incision of the outer and inner cornea. The results obtained show that the outer cornea underwent lens-forming transformations only when the inner cornea had been incised, thus permitting outer cornea (Experiments I-IV). No lens regeneration occurred when the inner cornea was left intact (Experiments II, III). It was concluded that the factor(s) allowing the lens-forming transformations of the outer cornea is not an aspecific nutritional factor(s) but a more specific factor(s) that cannot reach the outer cornea when the inner cornea is intact. Therefore, the absence of the lens and sufficient nutrient available to the outer cornea are not enough to allow lens regeneration from the outer cornea. When lens removal was carried out through the dorsal part of the eye (Experiments III-IV) the lens regenerated from the pericorneal epidermis of this region in a large number of cases.

Animals

Critical role of corneal Langerhans cells in the CD4- but not CD8-mediated immunopathology in herpes simplex virus-1-infected mouse corneas.

Previous studies have revealed that the RE strain of HSV type 1 (HSV-1) induces a tissue-destructive inflammatory response in the mouse cornea that is mediated by CD4 T lymphocytes, whereas the KOS strain of HSV-1 preferentially activates CD8 T lymphocytes in the cornea. Langerhans cells (LC) normally reside only at the periphery of the cornea but can migrate centripetally after HSV-1 infection. We studied the relative contribution of LC to the corneal inflammation induced by the KOS and RE strains of HSV-1. Ten days after infection, the central one-third of RE HSV-1-infected corneas contained an average of 5.7 LC/high-power field compared with 0.6 LC/high-power field in KOS-infected corneas. We hypothesized that the increased density of LC in RE HSV-1-infected corneas at the time of T lymphocyte infiltration contributed to the preferential activation of CD4 T lymphocytes in these corneas. To test this hypothesis, we gave mice a low dose of UV-B corneal irradiation (150 mJ/cm2) 1 day before infection with HSV-1. UV-B irradiation effectively prevented the migration of LC into the central cornea when measured 10 or 21 days after corneal infection with either HSV-1 strain. UV-B corneal irradiation had no effect on the CTL response to HSV-1 Ag in the regional lymph nodes after corneal infection with KOS or RE HSV-1. The delayed-type hypersensitivity response induced by both strains of virus, when measured 8 and 14 days after corneal infection, was significantly reduced by UV-B irradiation. UV-B irradiation significantly reduced the incidence (p = 0.0023) and severity (p = 0.0008) of corneal stromal disease induced by RE HSV-1 but did not significantly affect the stromal disease induced by KOS HSV-1. To distinguish between the effect of UV-B treatment on the afferent and efferent arms of the Ir in mice, we administered UV-B treatment to one eye, followed 24 h later by RE HSV-1 infection of both eyes. These mice developed a normal delayed-type hypersensitivity response, and stromal inflammation developed normally in the untreated eye. However, stromal inflammation was significantly reduced in the treated eye. Our findings suggest that LC play a critical role in the activation of HSV-reactive CD4 T lymphocytes in the cornea. Moreover, the type of corneal inflammation induced by different strains of HSV-1 may reflect their differential capacity to induce LC migration into the central cornea.

Animals

Reducing intraocular pressure by intubation elicits precocious development and innervation of the embryonic chick cornea.

Growth of the embryonic chick cornea was directly related to, and coordinated with, overall eye growth. During normal development, the size of the embryonic chick cornea increased in three linear phases of diametric growth. Corneal diameter increased at a rate of 216 microns per day between embryonic day 4 (E4) and E7, 511 microns per day between E7 and E10, and 144 microns per day from E10 until after hatching. After the sustained release of intraocular pressure by intubation on E4, corneal diametric growth was reduced to a single phase of 122 microns per day. After intubation on E4, the mesenchyme surrounding the developing cornea was substantially thicker and the neural crest-derived corneal endothelium was established earlier. The primary corneal stroma of the intubated eye swelled and was precociously populated by neural crest-derived corneal fibroblasts. Thus, the timing of arrival of neural crest cells in the anterior segment and their contribution to the cornea were determined by the growth rate of the eye. Although the diameter of the cornea was substantially reduced after intubation, it was more densely populated by fibroblasts, resulting in a cornea that was substantially thicker than the control by E14. Prospective corneal nerves normally extend into the cornea proper on E11, concomitant with a decrease in its diametric growth rate. After intubation on E4, the perilimbal nerve ring was virtually complete by E5 and numerous nerves had extended throughout the E8 cornea. By E16, the cornea from the intubated eye contained a very high density of nerve fibers, possibly reflecting its reduced size. These data suggest that the primary corneal stroma does not permit nerve fiber extension and demonstrate that the timing of nerve fiber extension into the secondary corneal stroma is specified by the rate of oppositional diametric growth of the cornea.

Animals

Metabolic analysis of reepithelializing rabbit cornea using phosphorus-31 nuclear magnetic resonance spectroscopy.

To investigate metabolic differences between the central and peripheral cornea the latter including the limbal area, corneas were dissected and examined using phosphorus-31 (31P) nuclear magnetic resonance spectroscopy. Since most 31P signals originate from the epithelium, 31P spectra of the cornea primarily represent the metabolic state of the epithelium. The spectra of the peripheral cornea showed all phosphorus resonances detected in the whole cornea; in contrast, the central cornea showed no phosphocreatine and glycerophosrylethanolamine, and only low levels of ATP. These results indicate that there is a higher metabolic activity in the peripheral epithelium, especially in the limbal area, than in the central epithelium. To evaluate the metabolic state of corneal epithelium during regeneration, we also examined corneas reepithelializing after 7 mm of central epithelial tissue had been removed by mechanical scraping. Rabbits were killed 24 and 48 h after scraping. The reepithelializing corneas clearly showed an increase in ATP, phosphocreatine, and sugar phosphates with time, although phosphorylcholine remained depressed. These findings suggest that the reepithelializing cornea has an elevated level of energy production and that it may have reached a higher steady state, thereby indicating accelerated metabolism of the epithelium during regeneration.

Animals

Treatment of alkali-injured rabbit corneas with a synthetic inhibitor of matrix metalloproteinases.

Healing of corneal alkali injuries remains a severe clinical challenge. The authors evaluated the effect of a new synthetic inhibitor of matrix metalloproteinases (GM6001 or N-[2(R)-2-(hydroxamido carbonylmethyl)-4-methylpentanoyl]-L-tryptophane methylamide) on preventing ulceration of rabbit corneas after alkali injury. Topical treatment of corneas with severe alkali injuries with 400 micrograms/ml or 40 micrograms/ml GM6001 alone prevented ulceration for 28 days, although 8 of 10 corneas treated with vehicle perforated. Corneas treated with 4 micrograms/ml GM6001 had midstromal depth ulcers. Corneas treated with 400 micrograms/ml of GM6001 contained very few inflammatory cells and had significantly reduced vessel ingrowth compared with vehicle-treated corneas. Epithelial regeneration after moderate alkali injuries also was investigated. Persistent epithelial defects developed 4 days after moderate alkali injury in rabbit corneas treated with vehicle and progressively increased to an average of 20% of the original 6 mm diameter wound by 27 days after moderate alkali injury. By contrast, epithelial regeneration was complete and persisted for 21 days for corneas treated with a formulation containing GM6001 (400 micrograms/ml), epidermal growth factor (10 micrograms/ml), fibronectin (500 micrograms/ml), and aprotinin (400 micrograms/ml). Sporadic punctate staining developed in 20% of the corneas treated with the combination of agents between days 21-28 after moderate alkali injury. These results demonstrate that topical application of GM6001 prevented corneal ulceration after severe alkali injury and that a combination containing GM6001, epidermal growth factor, fibronectin, and aprotinin promoted stable regeneration of corneal epithelium after moderate alkali injury.

Alkalies

Effects of human epidermal growth factor on endothelial wound healing of human corneas.

Paired human donor corneas (age, 73 +/- 12 yr), preserved in organ culture medium, were used to evaluate the effect of human epidermal growth factor (hEGF) on endothelial wound closure rate (WCR), on morphometric parameters (cell size, shape, and density), and on cell division in the wound area. The endothelium of the corneas was mechanically wounded (area, 4.9 +/- 0.9 mm2). For each pair, one cornea was treated with 10 ng/ml hEGF, while the mate served as control. WCR was assessed by daily staining of the corneas with trypan blue. Morphometric data were obtained after alizarin staining. Mitotic activity was assessed using 3H-thymidine autoradiography. Addition of hEGF significantly increased the WCR compared to the control group. In the closed wound (between 4-9 d), the mean cell size in the center averaged 1940 microns2 in the control group and 1287 microns2 in the hEGF-treated group (P less than 0.01). Fifteen days after wounding, the mean cell sizes averaged 1910 microns2 and 1427 microns2 in the control and hEGF-treated group, respectively (P less than 0.01). All corneas exposed to hEGF had higher endothelial cell densities than the control corneas. In the early stages of wound closure, the cells in the transitional zone in hEGF-treated corneas had a somewhat more elongated shape. However, hEGF did not affect the final cell shape within the closed wound. Autoradiographic results revealed that hEGF accelerated DNA-synthesis, although only to a limited extent. The results indicate that, in human corneas, hEGF promotes endothelial wound healing predominantly by cell migration, at least in corneas from senior donors.

Adult

A microstructurally-based finite element model of the incised human cornea.

A mechanical model of the human cornea is proposed and employed in a finite element formulation for simulating the effects of surgical procedures, such as radial keratotomy, on the cornea. The model assumes that the structural behavior of the cornea is governed by the properties of the stroma. Arguments based on the microstructural organization and properties of the stroma lead to the conclusion that the human cornea exhibits flexural and shear rigidities which are negligible compared to its membrane rigidity. Accordingly, it is proposed that to a first approximation, the structural behavior of the cornea is that of a thick membrane shell. The tensile forces in the cornea are resisted by very fine collagen fibrils embedded in the ground substance of the stromal lamellae. When the collagen fibrils are cut, as in radial keratotomy, it is argued that they become relaxed since there is negligible transfer of load between adjacent fibrils due to the low shear modulus of the ground substance. The forces in the cornea are then resisted only by the remaining uncut fibrils. The cutting of fibrils induces an anisotropy and inhomogeneity in the membrane rigidity. By assuming a uniform angular distribution of stromal lamellae through the corneal thickness, geometric arguments lead to a quantitative representation for the anisotropy and inhomogeneity. All material behavior is assumed to be in the linear elastic regime and with no time-dependency. The resulting constitutive model for the incised cornea has been employed in a geometrically non-linear finite element membrane shell formulation for small strains with moderate rotations. A number of numerical examples are presented to illustrate the effectiveness of the proposed constitutive model and finite element formulation. The dependence of the outcome of radial keratotomy, measured in terms of the immediate postoperative shift in corneal power, on a number of important factors is investigated. These factors include the value of the elastic moduli of the stromal lamellae (dependent on the patient's age), the incision depth, the optic zone size, the number of incisions and their positions, and the intraocular pressure. Results have also been compared with expected surgical corrections predicted by three expert surgeons and show an excellent correspondence.

Collagen

Sympathetic nerves to the rat cornea.

The adrenergic innervation of the rat cornea was investigated by using the formaldehyde induced fluorescence (FIF) technique. The fluorescent nerves were observed mainly in the corneal stroma. Either cervical sympathectomy or pre-treatment with reserpine completely abolished the fluorescence of the adrenergic nerves of the cornea. After a stereotactic coagulation of the ophthalmic division of the trigeminal nerve, no adrenergic fibres were visible in the cornea and the number of the fluorescent iridic nerves was also reduced to a marked extent. On the other hand, ciliary ganglionectomy seemed to have no effect on the adrenergic fibres of the cornea. When the rats had been pre-treated with a monoamine oxidase inhibitors, nialamide, and noradrenaline, not only was there an increase in the intensity of the specific fluorescence, but also in the number of adrenergic nerves in the cornea. The epithelium was also shown to contain adrenergic nerves by administering high doses of nialamide combined with noradrenaline, both intravenously as well as topically on the cornea, under the protection of propranolol. It may be concluded that the rat cornea receives its adrenergic innervation along the posterior ciliary nerves. The short ciliary nerves do not appear to carry sympathetic nerves to the cornea.

Animals

Stromal degradation in vitamin A-deficient rat cornea. Comparison of epithelial abrasion and stromal incision.

Epithelial abrasions (3-mm diameter) and linear stromal incisions (50-75% depth) were made in vitamin A-deficient (A-) rat corneas to investigate what are contributory factors to the development of keratomalacia. Sixty-four rats were killed at various times after injury, and the corneas were histologically examined. In pair-fed control corneas, wounds healed without stromal degradation. Although abraded A- corneas were infiltrated by numerous polymorphonuclear neutrophil leukocytes (PMNs), reepithelialization eventually occurred and severe stromal degradation was not evident, suggesting that PMN infiltration alone cannot cause keratomalacia. In incised A- corneas, six (20%) exhibited marked stromal degradation, of which three (10%) were infected. Two other corneas studied shortly after incision were in the initial stages of infection. The results suggest that stromal injury importantly contributes to the development of keratomalacia. Bacterial infection might also be contributory. Cytochrome oxidase staining showed that metabolic functional levels of incised A- corneas were accelerated only in the wound zone, whereas abraded A- corneas were metabolically accelerated throughout.

Animals

A quantitative evaluation of Fourier components in transparent and opaque calf cornea.

Fourier transform methods were applied to STEM (scanning transmission electron microscopy) images to detect and quantify the subtle differences between the structure of normal transparent calf cornea and opaque calf cornea. In order for a tissue to be transparent, it can scatter or absorb only a small amount of light. Light scattering is minimized when the principal Fourier components of the spatial fluctuations in the index of refraction have wavelengths which are small relative to the wavelength of light (Benedek, 1971). Corneal opacity was produced as a result of high intraocular pressure (100-150 mmHg) when liquid was injected into calf eyes (0-2 weeks old). Pressurization created large structural defects and slight disruptions in the organization of the collagen fibers. Although the fiber organization appeared similar in the micrographs of both opaque and transparent corneas, Fourier analysis of STEM images collected at 50K magnification identified statistically significant differences. Far fewer Fourier components with wavelengths in the light scattering range (200-1100 nm) were observed in the transparent corneas than in the pressurized corneas as predicted by Benedek's theory. It was of interest that corneas treated with 100% glycerol prior to pressurization remained transparent at high intraocular pressures, possibly because glycerol stabilized the structure of the corneas and maintained a uniform index of refraction across the corneal stroma. The results demonstrate the effectiveness of Fourier analysis in detection and quantification of slight changes in structure at the electron microscopic level.

Animals

DNA damage is involved in the induction of opacification and neovascularization of the cornea by ultraviolet radiation.

Studies were conducted to examine ultraviolet radiation (UVR)-induced alterations of the cornea of the gray, short-tailed opossum. Monodelphis domestica, and the effect of post-UVR illumination to photoreactivation light (PRL, 320-500 nm). As photoreactivation treatment specifically monomerizes pyrimidine dimers, an amelioration of the UVR-induced biological end-point would implicate DNA as being a primary chromophore for induction of that end-point. Corneas of anesthetized, four-month-old, opossums were exposed to 250 J m-2 (0.025 J cm-2) from a Westinghouse FS20 sunlamp either one or three times a week for up to 13 exposures. The corneas of 4-5 animals received either: (a) 90 min of PRL immediately prior to UVR; (b) PRL immediately following UVR; (c) PRL alone; or (d) UVR alone. Eyes were examined with a slit lamp microscope 24 hr following each exposure and scored for the appearance of opacification and neovascularization of the cornea. In animals exposed to UVR alone, 2-5 exposures, depending on whether the exposures were given once or three times per week, were required to obtain opacification and neovascularization in 50% of the irradiated corneas. The onset of both opacification and neovascularization in 50% of the corneas required 8-11 exposures when the UVR was immediately followed by PRL. Based on the specificity of photoreactivation repair to act solely on pyrimidine dimers, these observations suggest that UVR-induced pyrimidine dimers in corneal DNA are involved in UVR-induced opacification and neovascularization of the cornea of Monodelphis domestica.

Animals

Donor cornea bacterial contamination.

The incidence of culture-positive cornea rims from 446 consecutive donor corneas cultured at the Doheny Eye Institute between 1986 and 1988 was determined. Both the identity and antibiotic sensitivities of the contaminating organisms were reviewed. Sixty-three (14.1%) of 446 cornea rims were culture-positive, but none of the 63 patients who received these contaminated donor corneas developed endophthalmitis. Streptococcus (26 of 63), Propionibacterium (15 of 63), Stphylococcus (14 of 63) species and diptheroids (8 of 63) were the most common cornea rim contaminants, and in most cases were resistant to gentamicin (i.e., 21 of 26 or 81%, 9 of 15 or 60%, 10 of 14 or 71%, 4 of 8 or 50%, respectively). Virtually all of the gentamicin-resistant bacteria isolated from cornea rims were found to be sensitive to vancomycin. Eye banks should consider the addition of other antibiotics to storage media to reduce donor cornea contamination. Surgeons performing corneal transplantation should also consider these results when selecting antibiotics for use at the time of surgery and in the postoperative period.

Anti-Bacterial Agents