Confocal microscopy of Aspergillus fumigatus keratitis.

AIM: To use a confocal microscope to characterise the treated and untreated courses of fungal keratitis. METHODS: In the first experiment, Aspergillus fumigatus stromal keratitis was produced in both eyes of seven New Zealand white rabbits. In the second experiment, keratitis was induced in right eyes of 20 rabbits. Group 1 rabbits were treated with topical fluconazole, group 2 rabbits received oral fluconazole, and group 3 rabbits were used as controls. The rabbits were examined with a slit lamp and confocal microscope 2, 6, 10, 14, and 20 days after inoculation. The corneal cultures were taken on days 2, 14, and 20 and biopsies were taken on days 2 and 22. RESULTS: On days 14 and 22 confocal microscopy was more sensitive than culture technique in both treated and untreated animals, since not all cases of fungal keratitis can be cultured. CONCLUSION: This study indicates that confocal microscopy is a rapid and sensitive diagnostic tool for both the early diagnosis and non-invasive follow up of fungal keratitis

Murine keratocytes function as antigen-presenting cells.

Keratocytes express MHC class I molecules constitutively, and keratocytes stimulated with IFN-gamma express MHC class II molecules. Unstimulated keratocytes constitutively express B7-1 and ICAM-1, as well as low levels of CD40 and 4-1BBL. These findings indicate that keratocytes may deliver both antigen-specific and costimulatory signals to CD4(+) and CD8(+) T cells. To demonstrate that keratocytes expressing B7-1 provide a costimulatory signal to T cells, CD4(+) or CD8(+) mouse T cells were incubated with anti-CD3 mAb and irradiated keratocytes. Enhanced proliferation of both CD4(+) and CD8(+) T cells occurred, and could be inhibited by anti-B7-1 mAb, indicating T cell costimulatory activity by B7-1 on the keratocytes. To demonstrate that keratocytes can deliver an antigen-specific signal, CD4(+) and CD8(+) T cells from herpes-infected mice were incubated with HSV-1-infected, irradiated keratocytes. The resulting T cell proliferation and production of Th1 cytokines (IL-2, IFN-gamma) indicated T cell activation by antigens presented by the infected keratocytes. These results show that keratocytes in the corneal stroma of the mouse can function as antigen-presenting cells and, thus, may play a role in immune-mediated stromal inflammation such as herpetic stromal keratitis.

Effects of topical unoprostone and latanoprost on acute and recurrent herpetic keratitis in the rabbit.

PURPOSE: To determine the effect of the topical ocular hypotensive drug, isopropyl unoprostone, a docosanoid molecule with very weak prostaglandin activity, on herpes keratitis in the rabbit eye. METHODS: For acute disease, rabbit corneas inoculated with the corticosteroid-sensitive F(MP)E strain of herpes simplex virus type 1 were treated with various combinations of 0.12% isopropyl unoprostone, latanoprost, trifluridine, benzalkonium chloride 0.02%, dexamethasone sodium phosphate, ketorolac tromethamine, or saline solution beginning 1 day after infection. Severity of keratitis was evaluated in a masked manner. For recurrent disease, rabbit corneas infected with McKrae strain herpes simplex virus type 1 were treated with unoprostone or saline solution on postinfection days 25 to 42, and the presence or absence of lesions was recorded. RESULTS: Eyes treated with unoprostone showed significantly less severe disease than saline-treated or latanoprost-treated eyes during acute infection. Unoprostone-treated and saline-treated eyes showed no significant difference in the frequency of recurrent lesions. Eyes treated with latanoprost and/or dexamethasone, separately or in combination, showed increased severity of acute herpes simplex virus keratitis, whereas benzalkonium chloride 0.02%--treated eyes showed no significant difference, compared with saline treatment. Trifluridine resulted in rapid healing. CONCLUSIONS: Unoprostone did not increase the severity or recurrence rate of herpes simplex virus keratitis. Unoprostone requires twice-a-day administration, compared with once-a-day for latanoprost, and unoprostone lowers intraocular pressure less than latanoprost. Nevertheless, unoprostone's superior safety profile may make its use advantageous. Benzalkonium chloride alone did not make the keratitis worse.

Cidofovir and experimental herpetic stromal disease.

OBJECTIVE: To compare topical cidofovir with topical trifluridine for the prevention and treatment of herpes simplex type 1 stromal keratitis in rabbits. METHODS: The RE strain of herpes simplex virus 1 was injected into the central stroma of both eyes of New Zealand white rabbits. Two to 3 days after virus inoculation, the rabbits were randomized to treatment groups of 10 each and treated with 1% trifluridine administered 5 or 7 times a day, 1%, 0.5%, or 0.2% cidofovir administered twice a day, fluorometholone administered twice a day, or balanced salt solution (BSS) administered twice a day (control) until day 21 after injection. The treated corneas were examined 3 times a week and the severity of stromal keratitis was graded in a masked fashion. To evaluate the ability of cidofovir to treat established stromal disease, groups of 10 rabbits each were inoculated with herpes simplex virus and treated with 1% cidofovir twice a day, 1% trifluridine 5 times a day, fluorometholone twice a day, or BSS twice a day beginning on day 7 after virus inoculation through day 21. RESULTS: Treatment with 0.2% cidofovir twice a day was not effective in preventing the appearance of stromal disease (P = .89), whereas treatment with 0.5% (P<.001) or 1% (P<.001) cidofovir twice a day or 1% trifluridine 5 times a day (P<.001) or 7 times a day (P = .006) significantly reduced the appearance of stromal keratitis on the 8 evaluation days, compared with BSS treatment (F test analysis of variance). There was no difference between the eyes treated with 0.5% cidofovir twice a day and those treated with 1% trifluridine 5 times a day. Treatment with 1% cidofovir was not effective in treating established stromal disease. CONCLUSIONS: Cidofovir and trifluridine are highly effective in preventing the appearance of herpetic stromal disease. Cidofovir is as effective as, but no more effective than, trifluridine in this model. Neither cidofovir nor trifluridine benefits established stromal disease, however. CLINICAL RELEVANCE: Cidofovir is a new, potent antiviral that seems similar in efficacy to trifluridine and is effective in the prevention of the development of stromal herpes, but is not effective in the treatment of established stromal disease in which hypersensitivity predominates.

Latanoprost increases the severity and recurrence of herpetic keratitis in the rabbit.

PURPOSE: To determine whether topically applied latanoprost increases the severity of acute herpes simplex keratitis, the rate of recurrence of herpes keratitis, or both, in the rabbit. METHODS: To determine the effect on severity of acute herpetic keratitis, the corneas of New Zealand white rabbits were infected with either the less-corticosteroid-sensitive McKrae strain or the corticosteroid-sensitive F(MP)E strain of herpes simplex virus type 1. Rabbits were randomly assigned to twice-a-day treatment with latanoprost 0.005%, dexamethasone sodium phosphate 0.1%, or balanced saline solution within 3 days of infection and evaluated daily for up to 13 days after infection. The severity of keratitis was graded in a masked manner. To determine the effect on recurrences of herpetic keratitis, animals infected with McKrae strain herpes simplex virus type 1 that survived to day 32 after infection were randomized to treatment with latanoprost 0.005% or balanced saline solution and evaluated for the presence of corneal lesions from postinfection day 32 to day 47. RESULTS: In the severity studies, treatment of F(MP)E-infected corneas with latanoprost or dexamethasone significantly worsened herpetic keratitis; by postinfection day 5, F(MP)E-infected eyes treated with dexamethasone or latanoprost demonstrated significantly higher severity scores than the eyes treated with balanced saline solution (P = .0001 and .008, respectively). Scores of McKrae-infected corneas treated with latanoprost or dexamethasone were not significantly different from scores of balanced saline solution-treated corneas. In the recurrence study, treatment with latanoprost significantly increased the appearance of clinical recurrences in McKrae-infected eyes, compared with balanced saline solution treatment (P = .0064). CONCLUSION: Latanoprost may worsen acute herpetic keratitis in the rabbit eye and increase the risk of recurrences in latently infected animals.

Protection of corneal allografts by CTLA4-Ig.

PURPOSE: CTLA4, a high-affinity ligand of B7, can, in soluble form, prevent antigen-driven T-cell activation by blocking CD28-B7 interaction and can thereby prevent immune graft rejection. In this study, we tested the capacity of soluble CTLA4-Ig alone or in combination with UV-B irradiation to suppress corneal allograft rejection in rabbits. METHODS: Corneas from Dutch belted rabbits were incubated in corneal storage medium containing 0, 1, 10, 25, or 250 microg/ml of CTLA4-Ig for 18 h and were then transplanted into the vascularized or nonvascularized corneas of New Zealand White rabbit recipients. A series of donor corneas were exposed to UV-B irradiation alone or a combination of irradiation and CTLA4-Ig to determine if these two treatments would have an additive effect in prolonging graft survival. The fate and clinical condition of the allografts were evaluated by slit-lamp photomicroscopic observation and corneal-thickness measurements. Grafts that were rejected were processed for histopathologic and immunohistochemical analysis to determine the characteristics of cells infiltrating the grafts. RESULTS: Grafts placed in nonvascularized corneas showed no differences in survival times, regardless of treatment. Among the grafts placed in vascularized corneas, those incubated with CTLA4-Ig at a concentration of 250 microg/ml failed within 7-14 days. Histopathologic and immunocytochemical examination revealed a dense accumulation of immune inflammatory cells, especially class II major histocompatibility complex (MHC)-expressing, antigen-presenting cells, in the failed grafts. Grafts incubated with CTLA4-Ig at concentrations of 1 and 10 microg/ml had mean survival times greater than the control, untreated corneal allografts. Some of the grafts in these two treatment groups survived for the 100-day observation period, whereas none of the grafts in the other treatment groups survived to this end point. UV-B irradiated grafts incubated with CTLA4-Ig at a concentration of 1 microg/ml appeared to have longer survival times and fewer rejections compared with control, untreated grafts and grafts treated with UV-B or CTLA4-Ig alone. CONCLUSION: The results show that the CTLA4-Ig coreceptor blocking agent can prolong corneal allograft survival in vascularized graft sites and that UV-B irradiation followed by incubation in CTLA4-Ig may prolong graft survival better than either treatment alone. These results suggest that agents that prevent second-signal interaction between antigen-presenting cells and T lymphocytes may be useful for inhibiting corneal allograft rejection.

Prolonged survival of corneal allografts incubated in alloantibody fragments.

BACKGROUND: In this study, we determined the binding characteristics of F(ab')2 alloantibody fragments to corneal antigens and assessed the capacity of these antibody fragments to protect corneal allografts from immune attack. METHODS: Goat anti-rabbit alloantibodies were pepsin-digested and labeled with 125I, and the time course of association and dissociation of the F(ab')2 fragments was determined. Corneal allografts were incubated in unlabeled F(ab')2 fragments and transplanted into allogeneic recipients, and the graft survival times were recorded. RESULTS: Binding of radiolabeled F(ab')2 fragments to rabbit cornea cells reached a maximum at 12 hr. At 32 degrees C (rabbit corneal temperature), the radiolabel eluted rapidly from the cornea, reaching baseline at 72 hr. At 4 degrees C (corneal graft storage temperature), significant amounts remained associated with the cornea at 96 hr. Mean survival time for grafts incubated in F(ab')2 anti-rabbit fragments was significantly greater than that of grafts incubated in nonimmune F(ab')2 fragments. Three of the corneal allografts incubated in goat F(ab')2 anti-rabbit fragments survived for 100 days, whereas the longest surviving control allograft incubated in goat F(ab')2 nonimmune fragments was rejected on day 24. Preincubation of corneas in unlabeled, immune F(ab')2 fragments followed by incubation in radiolabeled, immune F(ab')2 fragments suggested that antigen masking was not a factor in the prolongation of graft survival. CONCLUSION: Based on the binding and release kinetics and the graft survival times, it appears that the protective effect of immune F(ab')2 fragments extends well beyond the binding interval of the antibody fragments to corneal cell membranes.

Trifluridine, cidofovir, and penciclovir in the treatment of experimental herpetic keratitis.

OBJECTIVE: To compare trifluridine eyedrops, cidofovir eyedrops, and penciclovir ophthalmic ointment for the treatment of herpes simplex virus type 1 keratitis. METHODS: New Zealand white rabbits were infected with the McKrae strain of herpes simplex virus type 1. Three days after viral inoculation, the rabbits were randomly assigned to treatment with 1% trifluridine, 0.2% cidofovir, 3% penciclovir ointment, or phosphate-buffered saline (for control) on various schedules. The severity of keratitis was graded in a masked manner. RESULTS: Treatment with any of the antiviral drugs resulted in significantly less severe keratitis than treatment with phosphate-buffered saline. There was no statistically significant difference between eyes given trifluridine 2, 4, or 7 times a day and eyes given cidofovir 2 times a day (P=.06, P=.43, and P=.19, respectively, using the F test of the analysis of variance). Cidofovir given twice a day was significantly more effective than penciclovir given either 2 or 4 times a day (P<.001 and P=.002, respectively). Even with once-a-day dosage, all 3 drugs were significantly more effective than phosphate-buffered saline (P<.001 for all). There was no significant difference between once-a-day trifluridine and cidofovir treatments (P=.17). Trifluridine administered 5 times a day was as effective as 1% cidofovir. A similar degree of punctate keratitis was seen after 4 to 5 days in eyes treated with trifluridine at the highest frequency, 1% cidofovir, or penciclovir ointment. CONCLUSION: Trifluridine treatment was highly effective in this rabbit model, even when given only once a day. Treatment with cidofovir was as effective as that with trifluridine. CLINICAL RELEVANCE: Cidofovir and penciclovir treatments may prove to be effective against epithelial keratitis. Clinical trials of trifluridine, cidofovir, and penciclovir with lower treatment frequencies appear to be warranted.

A 437-base-pair deletion at the beginning of the latency-associated transcript promoter significantly reduced adrenergically induced herpes simplex virus type 1 ocular reactivation in latently infected rabbits.

In this study we used a herpes simplex virus type 1 (HSV-1) deletion mutant to identify a segment of the genome necessary for epinephrine-induced reactivation in the rabbit eye model of herpetic recurrent disease. In HSV-1 latently infected neural tissue, the only abundant viral products are the latency-associated transcripts (LATs). At least one promoter of LAT has been identified, and mutations in the LAT domain have been used to investigate HSV-1 reactivation. We used an ocular rabbit model of epinephrine-induced HSV-1 reactivation to study the effects of deleting a 437-bp region beginning 796 bp upstream of the LAT CAP site. Specifically, the 437-bp deletion is located between genomic positions 118006 and 118443 of the parent 17Syn+, and the construct is designated 17 delta S/N. This region also controls a portion of the genome encoding two transcripts (1.1 and 1.8 kb) from the LAT domain. A rescuant, 17 delta S/N-Res, was constructed from 17 delta S/N. Following ocular infection, all three viruses produced similar acute dendritic lesions in rabbits. Five weeks after infection, rabbits received transcorneal iontophoresis of epinephrine. The parent, 17Syn+, and the rescuant, 17 delta S/N-Res, underwent a high frequency of HSV-1 ocular reactivation as determined by recovery of infectious virus in the tear film. Rabbits infected with 17 delta S/N had a significantly lower frequency of ocular reactivation. Analysis of the trigeminal ganglia from all three groups of latently infected rabbits revealed (i) similar amounts of HSV DNA (genomic equivalents), (ii) accumulation of 2.0- and 1.45-kb LATs, and (iii) explant reactivation at the same high frequency. Therefore, these studies indicate that the 437-bp deleted region in 17 delta S/N is essential for epinephrine-induced reactivation and could implicate the 1.1- and 1.8-kb transcripts in the mechanisms controlling HSV-1 reactivation.

Effect of 9-(4-hydroxybutyl)-N2-phenylguanine (HBPG), a thymidine kinase inhibitor, on clinical recurrences of ocular herpetic keratitis in squirrel monkeys.

9-(4-Hydroxybutyl)-N2-phenylguanine (HBPG) is a new viral thymidine kinase inhibitor that we tested for the ability to prevent recurrences of herpetic keratitis. Eighteen squirrel monkeys (Saimiri scuireus) were infected in both corneas with the Rodanus strain of herpes simplex virus type 1 (HSV-1). All corneas showed typical dendritic keratitis 3 days after infection, followed by spontaneous healing. On day 21, the monkeys were randomized into two coded groups and ocular examinations were begun. One group received intraperitoneal (i.p.) injections of HBPG, 150 mg/kg, in a corn oil suspension every 8 h, and the other group received i.p. injections of the corn oil vehicle only. On day 22, recurrences were induced by reducing the temperature of the room in the late afternoon so that a low of 18 degrees C was achieved during the night. After the morning treatment, room temperature was raised to the normal ambient temperature (24-27 degrees C), and treatment was discontinued. Treatment was reinstituted on day 27, the room temperature was lowered again on day 28, and treatment was again discontinued as before. Third and fourth cycles of treatment and cold stress were begun on days 34 and 69. Ocular examinations were continued until day 73, at which point the code was broken. We found that the HBPG treatment significantly reduced the number of corneas with recurrences during the treatment periods, compared with recurrences in untreated, cold-stressed animals (P = 0.01).

Propranolol suppression of ocular HSV-1 recurrence and associated corneal lesions following spontaneous reactivation in the rabbit.

PURPOSE: Hyperthermia has been shown to induce HSV-1 ocular shedding in mice. Systemic administration of propranolol significantly reduced the recovery of infectious virus in the tears, cornea, and trigeminal ganglia of mice subjected to hyperthermia. The present study was performed to determine the effects of systemic propranolol on ocular shedding and recurrent corneal epithelial lesions in the rabbit model. METHODS: New Zealand white rabbits were infected with HSV-1 strain McKrae or 17Syn+. After latency was established, the animals were treated with systemic propranolol or saline (control) and examined by slit lamp biomicroscopy for corneal lesion. Tear film swabs were cultured to determine the frequency and duration of viral shedding. RESULTS: Propranolol caused a significant reduction in the frequency and duration of ocular HSV-1 shedding and a reduction in the frequency of recurrent corneal epithelia disease, compared with saline treatment. CONCLUSIONS: These results suggest that beta-adrenergic receptor blockers such as propranolol could be useful in suppressing HSV-1 ocular recurrences and corneal disease.

Cold stress-induced recurrences of herpetic keratitis in the squirrel monkey.

PURPOSE: Models of recurrent herpetic keratitis that depend on tissue damage or immunosuppression have been described. The authors report a model that depends only on minimal temperature stress to produce clinical recurrences in a small primate. METHODS: Squirrel monkeys (Saimiri sciureus) infected by the ocular route with the Rodanus strain of herpesvirus type 1 (HSV-1) were exposed to temperatures approximately 5 degrees C lower than the usual ambient temperature for periods as short as 12 hours. RESULTS: The corneas showed more or larger lesions typical of recurrent herpetic keratitis than are usually seen in these animals under normal conditions. Statistical analysis showed that there were significantly higher frequencies of epithelial keratitis at 18 degrees C and 20 degrees C (P < 0.0001). CONCLUSIONS: A minimal temperature change produced significant recurrences in this small animal within a short time. Tissues were not damaged to produce the recurrences. This approach may provide an efficient primate model for rapid testing of drugs to prevent clinical recurrence of ocular herpetic keratitis.

Cyclosporine in collagen particles: corneal penetration and suppression of allograft rejection.

The purpose of this study was to determine the pharmacokinetics of cyclosporine in the cornea and aqueous humor following topical application in collagen particles and to determine the capacity of cyclosporine in collagen particles to suppress corneal allograft rejection. Cyclosporine was prepared for topical application in three vehicles: collagen particles suspended in methylcellulose, collagen shields, and corn oil. Rabbit eyes were treated with a single application of one formulation. Corneas, aqueous humor samples, and blood samples were collected at 2, 4, 8, 12, and 24 hours. Peak concentrations of drug were reached between 2 and 4 hours in the cornea and between 4 and 8 hours in the aqueous humor. No drug was found in the blood. Collagen particles and shields produced significantly higher concentrations of cyclosporine in the aqueous humor and cornea, compared with corn oil drops, at virtually all time points. In the second part of the study, rabbits with vascularized corneas underwent corneal transplantation and were treated with one of the formulations beginning either at the time of transplantation (prophylactically) or at the first sign of an allograft immune reaction (delayed treatment). Cyclosporine delivered in collagen particles was effective in preventing primary corneal allograft rejection in nearly all of the grafts (96%) treated prophylactically, and in 67% of the grafts treated when signs of the first stage (grade 1) of immune graft reaction were observed. Grafts treated with cyclosporine in collagen shields showed similar survival rates (87% and 64%, respectively), whereas only 40% of the grafts treated prophylactically and only one (18%) receiving delayed treatment with the corn oil preparation survived. These results demonstrate the efficacy of collagen particles for delivery of hydrophobic drugs to the ocular surface and the potential for preventing corneal graft rejection with topically applied cyclosporine in a collagen-particle vehicle.

Collagen-based drug delivery and artificial tears.

For patients with conditions requiring chronic rather than acute therapy, the advantages of collagen shields in providing high and sustained levels of drugs and/or lubricants to the cornea are outweighed by the difficulty of insertion of the shield and the problem of blurred vision. We have developed a delivery system in which collagen pieces suspended in a viscous vehicle can be instilled into the lower forniceal space, thereby simplifying application and reducing blurring of vision. The collagen pieces (Collasomes) can be formulated with various constituents such as antibiotics or cyclosporine, or with chemical alterations such as the inclusion of a lipid (Lacrisomes) for the treatment of dry eyes. In the normal eyes of volunteers, Collasomes hydrated in a solution of sodium fluorescein and suspended in a methylcellulose vehicle as a model for delivery of water-soluble drugs produced fluorescein concentrations 17 to 42 times higher in the cornea and 6 to 8 times higher in the aqueous humor, compared with fluorescein-containing vehicle alone. In a preliminary controlled study, 76% of patients with moderately severe keratoconjunctivitis sicca (KCS) preferred Lacrisomes to the vehicle control because of a more soothing effect and longer duration of comfort. All preparations were well tolerated by all study subjects. Current studies involve improving drug delivery by chemically modifying the collagen molecule to slow diffusion of the drug from the Collasome matrix, as well as varying the amount of cetyl alcohol and combining it with modified collagen in Lacrisomes to maximize comfort in patients with dry eyes.

Vancomycin-enriched corneal storage medium.

Antibiotics in a corneal preservation solution probably have little effect during storage at 4 C, but are effective as the tissue is warmed. The tissue acts as a sponge, soaking up the antibiotic from the solution and releasing it into the eye, where the bactericidal effect is achieved. Currently, high concentrations of gentamicin (relative to the minimal inhibitory concentration) are used in the preserving solution for this purpose. Presumably, proportionately high concentrations of any proposed new antibiotic added to supplement the bactericidal effect of gentamicin, such as the vancomycin used in this study, would be required. However, neither the ability of donor tissue to tolerate high concentrations of vancomycin nor the stability of vancomycin at neutral pH in appropriate storage media has been documented. We evaluated the addition of vancomycin (100 micrograms/ml) to two corneal storage media that contained gentamicin in terms of stability of the antibiotic in solution and the effect on the endothelial cells of donor tissue stored for two weeks at 4 C. Vancomycin was stable in solution at neutral pH (7.2) during the five-month period of the study; the concentration exceeded 90 micrograms/ml for the first five weeks. The endothelial cells from donor tissue stored in the vancomycin-enriched media showed no notable differences from those stored in the same media without vancomycin in terms of cell shape, cell borders, cell swelling, and apical holes. The stability of vancomycin in storage and the absence of endothelial toxicity in vitro support the potential use of this antibiotic as a supplement to gentamicin for the prevention of endophthalmitis in patients receiving corneal transplants.

Suppression of ocular herpes recurrences by a thymidine kinase inhibitor in squirrel monkeys.

5'-Ethynylthymidine, an inhibitor of viral thymidine kinase (TK), was given intraperitoneally to squirrel monkeys previously infected by the ocular route with Rodanus strain herpes simplex virus. Spontaneous ocular recurrences were reduced during therapy, compared to saline-treated controls. This is the first in vivo demonstration that a viral TK inhibitor can reduce recurrences of HSV-1. Similar benefit would be expected for HSV-2 and perhaps VZV (varicella zoster virus).

Optisol corneal storage medium.

Optisol is an investigational, intermediate-term corneal storage medium containing chondroitin sulfate and dextran to enhance corneal dehydration during storage. We used scanning electron microscopy to grade endothelial cell morphologic characteristics in terms of cell shape, cell borders, cell swelling, and apical holes in pairs of corneas stored in Optisol and Dexsol. Optisolstored corneas showed significantly fewer morphologic changes after 14 days at 4 degrees C than did Dexsol-stored corneas. No significant differences were seen after 1 to 4 days at 26 degrees C. Temperature-reversal analysis showed no significant change in corneal thickness with warming after 2-week storage at 4 degrees C in either medium, although Optisol-stored corneas were significantly thinner than those stored in Dexsol at all times. The results of scanning electron microscopy suggest that preservation at refrigerator temperature for 2 weeks in Optisol is superior to preservation in Dexsol. Both media may be useful in preserving endothelial structure for limited periods at room temperature, which could provide a measure of safety in shipping or storage where refrigeration is unreliable.

Endothelial cell damage in human and rabbit corneas stored in K-Sol without antioxidants.

Human and rabbit corneas were stored at 4 degrees C in K-Sol with and without antioxidants (ascorbic acid, reduced glutathione, alpha-tocopherol, and retinol acetate) for two to three weeks. All the corneas were then examined visually and by scanning electron microscopy. They appeared clear and slightly oedematous. Scanning electron micrographs were used to grade corneal endothelial cell morphology in a masked manner in terms of cell shape, cell borders, cell swelling, and apical holes. Corneas stored in K-Sol without antioxidants showed changes in cell shape, cell borders, and apical holes. Human corneas showed more morphological changes than rabbit corneas. The results suggest that antioxidants in K-Sol have an important role in the preservation of endothelial cell morphology.

K-Sol corneal preservation at room temperature.

K-Sol, a recently developed corneal storage medium that contains purified chondroitin sulphate in tissue culture medium (TC 199), is capable of preserving corneal tissue for 14 days at 4 degrees C. To study the effect of tissue storage in K-Sol at room temperature we preserved rabbit corneas in K-Sol and M-K medium for three or seven days at 25 degrees C. All of the corneal endothelial sheets were intact after three days. At seven days the change in pH of the K-Sol medium was less than that of M-K medium. Corneas preserved in M-K medium showed swelling of mitochondria and a decrease in the number of cytoplasmic organelles. Corneas preserved in K-Sol had organised cytoplasmic organelles and nuclei. Scanning electron micrographs revealed well preserved endothelial sheets. Corneas stored in the two media showed no significant difference in thickness. A pair of human corneas preserved in K-Sol at room temperature for six days maintained about 95% of the endothelial sheet in good condition. Small separations were observed between some of the endothelial cells. However, even in these areas, the cytoplasmic organelles were well preserved. It appeared that K-Sol is more stable than M-K medium at room temperature, and that both rabbit and human corneas can be preserved in good condition in K-Sol for at least six days at 25 degrees C.

Histological study of corneas preserved in two new media.

A new corneal preserving medium (K-Sol), developed by Kaufman and others, contains purified chondroitin sulphate, TC 199, HEPES buffer, and gentamicin. Another new medium (JM) containing bicarbonate-free glucose-phosphate Ringer solution and dextran 70 has been developed in Japan. New Zealand white rabbit corneas with scleral rims were stored in each medium at 4 degrees C for one or two weeks. The condition of the endothelium was evaluated histologically. Corneas preserved in both media were in good condition at the end of one week. Corneas preserved in K-Sol for two weeks showed fewer endothelial changes than similar tissue stored in JM for two weeks. Corneal swelling was also less in corneas preserved in K-Sol, than in corneas preserved in JM.