Ascorbic acid entry into cornea of rat and guinea pig.

The transport rates of radiolabeled ascorbic acid (AA), 3-O-methyl-D-glucose (mD-glu) and L-glucose (L-glu) from blood into the aqueous humor, corneal endothelium, and stroma compartments were studied in male Sprague-Dawley rats and English short-haired guinea pigs. In vivo pulse chase kinetic studies supplied transport entry rate constants, Ki (min-1), and used L-glu as a passive internal control. Results in the rat indicate that AA enters the aqueous humor at rates similar to L-glu and likely via simple passive diffusion. Rate constants for entry into endothelium for both L-glu and mD-glu were high, indicating a quick equilibrium with the aqueous humor. Endothelium AA levels continued to increase to levels higher than in the aqueous humor. Although all three test molecules quickly entered the endothelium, L-glu and mD-glu levels were soon found to decrease, whereas levels of AA or its metabolites remained high. AA entered the stroma of the rat cornea slower than either L-glu or mD-glu, suggesting that no special transport mechanism for AA is present. mD-glu moves into the stroma quickly, but high levels are not maintained. The guinea pig maintains AA levels in the cornea about 10 times higher than that of the rat by first accumulating AA in the aqueous humor and again by actively accumulating AA in the endothelium. Data at 24 h postbolus suggests that both the rat and guinea pig maintain high corneal AA levels for extended periods. Thus, whereas both glucose analogues enter and leave the cornea via the aqueous humor relatively quickly, AA appears to be actively taken up by the endothelium and thus maintained high in both species. Metabolic roles for ascorbate in the eye could conceivably be similar in both nocturnal rats and diurnal guinea pigs, even though the means of supply differ. Because the guinea pig does not produce AA, it conserves and stores a supply in the aqueous humor via active transport. The rat instead could rely on a steady supply of AA to meet intraocular needs.     

Langerhans cell alterations in the guinea pig cornea

Langerhans cells (LC) are found in virtually all epithelial surfaces, but are rare or absent in the central cornea. LC migration into the cornea can be induced by several different stimuli. The authors studied LC in guinea pig corneal epithelium after: (1) suture placement; (2) suture removal; (3) UV-A irradiation; and (4) UV-C irradiation. Corneal LC markedly increased after suture placement during a 35-day observation period, and decreased by one-half during a 21-day observation period when sutures were removed. Suture-induced corneal LC decreased after UV-A irradiation, but increased after UV-C irradiation. These results demonstrate that ocular LC can be altered by nonimmunologic perturbations of the cornea.     

Drug modification of angiogenesis in a rat cornea model

PURPOSE: To evaluate the influence of some widely used antiglaucoma agents on angiogenesis in a novel rat cornea model. METHODS: Angiogenesis was induced in 32 rats by slow-release polymer pellets containing basic fibroblast growth factor (bFGF) placed in a corneal micropocket. Angiogenesis was later measured and compared in groups of rats given one of four antiglaucoma drug therapies and one control group. The drugs were commercially available preparations of prostaglandins, beta-blockers, alpha-2 agonists, and carbonic anhydrase inhibitors given for 7 days in a manner similar to that used in humans. Growth was measured by calculating the maximum linear vessel growth divided by pellet-limbus distance. RESULTS: Biomicroscopic observation disclosed that all tested animals showed an induction of neovascular reactions in their corneal stroma. The growth index results for the control, latanoprost, dorzolamide, brimonidine, and timolol malate groups were 1.65 +/- 0.16, 1.98 +/- 0.18, 1.85 +/- 0.19, 2.03 +/- 0.38, and 1.65 +/- 0.14, respectively, confirming the hypothesis that topically delivered antiglaucoma drugs modify the normal angiogenic response. Of them, the prostaglandins showed the most prominent angiogenic stimulatory effect (P = 0.03). CONCLUSIONS: This modified micropocket assay of corneal angiogenesis in rats demonstrated the stimulatory effect of several widely used topically delivered antiglaucoma medications on the angiogenic process. The results indicate that the selection of drugs for treating different ophthalmic diseases should take into account their influence on angiogenic processes.     

3H-thymidine autoradiography of guinea pig cornea and skin after exposure to solar simulating radiation

In vitro autoradiography with tritiated thymidine was performed in guinea pig cornea and guinea pig skin after in vitro exposure to solar simulating radiation (UVB). UVA-irradiated and unirradiated specimens served as controls. Sparsely labelled nuclei, indicating unscheduled thymine dirtier repair DNA-synthesis (dark repair), were observed immediately after UVB exposure in the epidermis and upper dermis of the skin and in all cellular compartments of the cornea. Control samples did not exhibit dark repair. The ratio of sparsely labelled cells was similar in the epidermis and in the corneal epithelium, but was significantly higher in the corneal stroma and highest in the endothelium.

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Zusammenfassung Hornhaut und Haut von Meerschweinchen wurde nach in vitro-Bestrahlung mit einem Sonnensimulator (UVB) mittels Autoradiographie unter Verwendung von Tritium-markiertem Thymidin in vitro untersucht.UVA-exponierte und unbestrahlte Proben dienten als Kontrollen. Lokkere autoradiographische Markierung, ein Marker der unprogrammierten DNS-Reparatur-Synthese, fand sich sofort nach UVB-Exposition über Zellkernen der Epidermis und oberen Dermis der Haut, sowie in allen zellulären Schichten der Hornhaut. In Kontrollpräparaten fand sich keine lockere Markierung. Der Anteil locker markierter Zellkerne in der Epidermis und im Hornhautepithel war nahezu gleich, während sich im Hornhautstroma und besonders im Endothel eine signifikant höhere Anzahl an Zellen in DNS-Reparatur-Synthese befanden.

     

Calcitonin gene-related peptide (CGRP) immunoreactive sensory nerves in the human and guinea pig uvea and cornea

The presence of calcitonin gene-related peptide (CGRP) immunoreactive nerves in the uvea and cornea of human and guinea pig eyes was evaluated using immunohistochemical techniques. CGRP immunoreactivity was found in thin, varicose nerve fibers in both species. Most of the fibres were localized in the ciliary body, and were mainly associated with blood vessels. In the human ciliary body, a moderate number of CGRP immunoreactive nerves were also seen in the ciliary muscle. In the iris and cornea, CGRP immunoreactive fibres were relatively uncommon. In the iris, they were mostly found associated with blood vessels, while in the cornea they were seen sub-epithelially or as free nerve endings in the epithelium. In the trigeminal ganglion, small sized ganglion cells displayed CGRP immunoreactivity. About 40% of all ganglion cells were immunoreactive nerves in the guinea pig, while sympathetic denervation did not change the staining pattern of CGRP immunoreactivity. The present findings, together with previous physiological data, suggest that CGRP might play a role in the regulation of the blood flow, aqueous humour dynamics, and neurogenic inflammation, not only in experimental animals but also in man.     

Distribution of ascorbic acid in the ciliary body of albino rabbit, guinea pig and rat.

The distribution of ascorbic acid in the ciliary body has been demonstrated by means of a silver nitrate technique. It appeared that the basal cells in the pars plana region and in the valleys between the ciliary processes contain great amounts of ascorbic acid, in contrast to the basal cells over the processes proper which showed only traces. Rabbit and guinea pig revealed mainly the same findings, and quantitative differences occurred before and after intracardial injection of ascorbic acid. On the other hand, ascorbic acid was absent in the ciliary body of untreated rats, whereas only traces were found after intracardial injection of this substance. These species differences are in keeping with previous reports showing high concentration of ascorbic acid in the aqueous humour from rabbit/guinea pig, and no ascorbic acid at all in aqueous humour from rats.     

Effect of cyclosporin A, dexamethasone and various preservatives on epithelial wound healing in the denervated guinea pig cornea

In order to examine a possible connection between punctate keratopathy following keratoplasty and the various drugs used in postoperative treatment, denervation was performed in white Pirbright guinea pigs by corneal trepanation with a 6 mm trephine to a depth of 0.25 mm. One week later an iodine gas applicator was used to produce erosion in the cornea, which had healed in the meantime. Until the erosions healed, the corneas were photographed four times daily after fluorescein staining; the negatives were enlarged by projection and the erosions measured planimetrically. The linear regression method was used to calculate a compensating straight line and thus the hourly epithelial wound-healing surface (mm2/h) for each eye. This was 0.76 mm2/h in control eyes. Dexamethasone 0.1%, benzalconium chloride 0.01%, and chlorobutanol 0.2% did not significantly inhibit wound healing, while Spersadex (dexamethasone 0.1% + benzalconium chloride 0.01%) and Totocortin (dexamethasone 0.1 + chlorobutanol 0.2%) did. Polyvinyl alcohol (1.4% without preservative) significantly delayed epithelial wound healing. Cyclosporin A 2% (in castor oil) had no influence on epithelial wound healing.     

Evaluation of tumor angiogenesis factor with the rabbit cornea model.

Sequential histopathological observations were made of the rabbit corneas after an implantation of viable and nonviable tumor cells in the corneal stroma. They showed a nonspecific localized interstitial keratitis accompanied by inflammatory cells and new capillaries. We could not observe any significant clinical or histopathological differences between the corneas containing live or dead tumor implants, or between those with different tumor types (i.e., retinoblastoma and melanoma). Some variation in the severity of the inflammatory response was observed in different animals with the same tumor. In all cases, the extent of the corneal neovascularization correlated with the degree of inflammation. However, in rabbits made immune-deficient by radiation, there was negligible inflammation and vascularization when tumor was implanted.     

Inhibition of experimental angiogenesis of cornea by somatostatin

BACKGROUND. This study evaluated the inhibitory activity of somatostatin 14 on the angiogenesis of cornea in vivo. METHODS. Corneal neovascularization was induced with a pellet containing 90 ng of basic fibroblast growth factor (bFGF) in a rat corneal pocket model. Three kinds of pellets were made containing bFGF plus somatostatin (SST) 0 ng, 20 ng and 200 ng for the control group, group 1 and group 2, respectively. Neovascularization was observed biomicroscopically from day 4 to day 8, and the corneas were then examined for changes in histology. Quantitation of angiogenesis in the cornea was accomplished by caliper and image analysis. RESULTS. The 200-ng dose of SST showed significant inhibition of both length and area of neovascularization on day 7 (0.62+/-0.11 mm vs 1.29+/-0.16 mm, 0.50+/-0.16 mm2 vs 1.35+/-0.29mm2, group 2 vs control; P<0.05). The 20 ng of somatostatin did not demonstrate any significant inhibition of neovascularization compared with the control group. CONCLUSION. Our study demonstrated that SST 14 can reduce bFGF-induced corneal angiogenesis. This shows the potential value of somatostatin in the treatment of corneal neovascularization.     

CD8+ T cell-mediated delayed rejection of orthotopic guinea pig cornea grafts in mice deficient in CD4+ T cells

PURPOSE: To determine the immunopathogenesis of delayed orthotopic corneal xenograft rejection in mice deficient in the xenoreactive CD4+ T cells that mediate acute rejection. METHODS: CB.17 SCID and BALB/c mice were used as recipients of orthotopic cornea grafts obtained from strain 13 guinea pigs. Before transplantation, SCID recipients, which do not normally reject guinea pig cornea grafts, were reconstituted with spleen cells (whole, CD4-depleted, CD4/CD8-depleted) or purified CD8+ T cells from normal BALB/c donors. Graft survival was assessed by clinical examination, and median survival times (MST) were calculated. Lymphocytes from mice that rejected guinea pig cornea grafts were analyzed in vitro for their capacity to respond to guinea pig xenoantigens and to lyse guinea pig target cells. RESULTS: SCID mice reconstituted with whole spleen cells from BALB/c donors rejected guinea pig corneas with a vigor identical with that of normal BALB/c mice (MST = 15 and 14 days, respectively), whereas SCID mice reconstituted with CD4-depleted BALB/c spleen cells rejected guinea pig corneas in a delayed fashion (MST = 27 days), as did SCID mice reconstituted with purified CD8+ T cells from BALB/c donors. Although CD8+ T cells from rejector mice failed to lyse guinea pig target cells in vitro, the T cells proliferated and secreted IFN-gamma in response to in vitro stimulation with guinea pig xenoantigens. CONCLUSIONS: Guinea pig cornea xenografts that avoid acute rejection in CD4+ T cell-depleted mice are vulnerable to rejection by CD8+ T cells. Effector CD8+ T cells destroy corneal xenografts through release of proinflammatory mediators (IFN-gamma) rather than by cytotoxicity.     

Anti-inflammatory therapy and outcome in a guinea pig model of Pseudomonas keratitis.

Corneal scarring as a consequence of bacterial keratitis is an important cause of visual loss and a major indication for penetrating keratoplasty. Anti-inflammatory agents might be useful in this condition for limiting corneal damage, but benefit from adjunctive anti-inflammatory therapy has never been demonstrated. In this limited pilot study, we compared the effect on clinical outcome of treating Pseudomonas keratitis in guinea pigs with prednisolone (a corticosteroid), flurbiprofen (a cyclo-oxygenase inhibitor), nordihydroguaiaretic acid (a lipoxygenase inhibitor), and a leukotriene antagonist, SKF104353 [R-(R*, S*)]-beta-[(2-carboxyethyl) thio-alpha-hydroxy-2-(8-phenyloctyl) benzenepropanoic acid, zinc salt]. None of the anti-inflammatory agents prevented sterilization of ulcers with antibiotic (ofloxacin) therapy. Therapy with the leukotriene antagonist appeared to reduce infiltrate size more quickly and produce a more rapid reduction in lesion size, but the differences were not statistically significant. Sample size calculations suggest that very large numbers of animals would be required to prove efficacy. The role of anti-inflammatory agents in reducing the stromal destruction caused by bacterial keratitis remains controversial.