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.

Treatment of viral diseases of the cornea and external eye.

Ocular virus infections remain an important cause of corneal and external disease. Herpes simplex, the most important, is easily treated when it is confined to the epithelium. New studies indicate that herpetic stromal disease and iritis are effectively treated with a combination of corticosteroid and antiviral without additional risk. Recurrences of ocular herpetic disease can be reduced with acyclovir given orally; the benefit seems to be greatest in patients who have had at least one episode of stromal keratitis. Herpes zoster can be treated with either acyclovir or famciclovir, but to be effective, treatment must be initiated within 72 hours of onset. Early treatment reduces the risk of post-herpetic neuralgia and may reduce the risk of ocular complications. Adenovirus infection (epidemic keratoconjunctivitis) is often spread by the ophthalmologist. New medications such as cidofovir appear to be effective against the adenoviruses in non-human systems and may have some effect in man, although previously, drugs that appeared to have an effect in vitro have proven to be ineffective in the clinical setting.

Reactivation of herpes virus after lamellar keratoplasty.

PURPOSE: To determine if lamellar keratoplasty in rabbits latently infected with herpes simplex virus type 1 (HSV-1) would stimulate graft recipients to shed virus and induce viral-specific corneal lesions. METHODS: Rabbits latently infected with HSV-1 received lamellar allografts in one eye from normal uninfected rabbits and the contralateral eyes served as unoperated controls. Normal rabbits received lamellar grafts from rabbits latently infected with HSV-1. For 1 week after surgery, slit-lamp examination and ocular swab sampling were performed daily to assess viral reactivation. RESULTS: The occurrence of positive swab cultures and corneal epithelial lesions after lamellar keratoplasty was significantly higher in operated eyes of latently infected rabbits when compared to the control eyes. Ocular shedding or recurrent lesions were not observed in the normal rabbits receiving corneal grafts from latently infected donors. CONCLUSIONS: These results indicated that lamellar keratoplasty induces HSV-1 shedding and recurrent epithelial lesions in the eyes of rabbits latently infected with HSV-1, which received lamellar grafts, but not in the eyes of normal rabbits given lamellar grafts from HSV-1 latently infected rabbits. It seems that the site of viral latency is not the anterior corneal stroma or the epithelium.

Characterization of epithelial downgrowth by confocal microscopy.

A 43-year-old white woman with a history of multiple ocular surgeries, including 4 penetrating keratoplasties, developed a concentric retrocorneal membrane at the graft periphery in the right eye. A white-light, tandem, scanning confocal microscope using a 24x/0.60 contact objective was used to examine the right eye in vivo. At the endothelial layer, confocal microscopic images similar to corneal epithelial cells were detected at the graft periphery. Unlike normal endothelial cells, the imaged cells demonstrated easily recognizable nuclei.

Confocal microscopy in the iridocorneal endothelial syndrome.

AIMS: To report the appearances of iridocorneal endothelial (ICE) syndrome from real time, white light confocal microscopy. METHODS: Three consecutive patients, each with ICE syndrome, were examined prospectively. Corneal specular and confocal microscopic examinations were performed in all three patients. In the first patient, a penetrating keratoplasty was performed and the cornea was examined by light and scanning electron microscopy. No surgery was performed in the remaining two patients. RESULTS: In the first patient corneal oedema prevented endothelial specular microscopy. Confocal microscopy performed before penetrating keratoplasty successfully revealed abnormal epithelial-like endothelial cells. Histological examinations of the cornea following penetrating keratoplasty revealed the presence of multilayered endothelial cells with epithelial features (microvilli). In the remaining two patients, specular microscopy showed the presence of ICE cells with typical dark/light reversal. Confocal microscopy demonstrated groups of endothelial cells with epitheloid appearances. In all three patients, the contralateral endothelial appearance was normal by specular and confocal microscopy, except for moderate endothelial polymegathism in one patient. Epithelial-like endothelial cells were characterised by prominent nuclei on confocal microscopy. CONCLUSIONS: The application of confocal microscopy indicates that the ICE syndrome is characterised by epitheloid changes in the endothelium. Confocal microscopy may be used to diagnose the ICE syndrome by demonstrating epithelial-like endothelial cells with hyperreflective nuclei. This technique is especially of value in cases of corneal oedema, since specular microscopy may fail to image the endothelium in such cases.

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.

Characterization of fibrous retrocorneal membrane by confocal microscopy.

PURPOSE: To study the appearance of a fibrous retrocorneal membrane as seen by confocal microscopy. METHODS: A 67-year-old white woman with a history of multiple ocular surgeries, including repeated penetrating keratoplasties for aphakic bullous keratopathy, developed a retrocorneal membrane in the right eye. The membrane was first noticed 3 years after the last corneal transplant and remained stable subsequently. The patient was examined by in vivo white light tandem-scanning confocal microscopy. RESULTS: At the level of the retrocorneal membrane, confocal microscopy disclosed the presence of a hyperreflective fibrous-appearing layer. Normal endothelial cells could not be found. Anterior to the hyperreflective layer, activated keratocytes were identified. CONCLUSION: Confocal microscopy may allow noninvasive diagnosis of fibrous retrocorneal membrane. Additionally, our data suggest that the posterior keratocytes might play a role in the production and deposition of fibrous tissue.

Real-time confocal microscopy of keratocyte activity in wound healing after cryoablation in rabbit corneas.

A modified tandem scanning confocal microscope was used for real-time in vivo examination of the rabbit cornea following a cryogenic injury. The corneas of New Zealand white rabbits were frozen with a probe that had been cooled by immersion in liquid nitrogen, effectively destroying keratocytes in a central 5 mm diameter zone throughout the total thickness of the cornea. In these eyes, keratocyte repopulation and corneal stromal wound healing proceeded similarly to that which occurs after epikeratophakia, a refractive surgical procedure designed to change the curvature and optical power of the cornea. In epikeratophakia, a cryolathed donor corneal stroma lenticule is sutured onto the bare stroma of the recipient cornea. The collagen tissue lenticule is repopulated by keratocytes (corneal fibroblasts) that migrate in from the host cornea. In our study, the confocal microscope permitted sequential, noninvasive examination of the corneal stroma in the treated animals. Necrosis of the keratocytes, followed by activation of the remaining viable cells in the corneal periphery, was observed in the first 2 to 3 days after cryo injury. A fine stromal fibrous network was seen to develop; in three eyes, this network progressed to the development of a retrocorneal fibrous membrane and dense stromal fibrosis, both of which resulted in significant loss of corneal clarity. Our results suggest that the confocal microscope may be a valuable tool to provide much needed information on wound healing processes at the cellular level after corneal surgery and injury.

Quantitative histological studies of primate corneas after excimer laser photorefractive keratectomy.

OBJECTIVE: To evaluate histological changes in the primate cornea after excimer laser photorefractive keratectomy (PRK) and to correlate them with clinical observations. METHODS: Sixteen African green monkey (Cercopithecus aethiops sabaeus) corneas were examined by light and transmission electron microscopy 6 weeks to 18 months after 1.5- or 3-diopter PRK. RESULTS: All specimens had a smooth stromal surface surrounded by a smooth, centrally tapered Bowman's layer. Epithelial thickness appeared to peak 12 months after PRK. The trend was for the epithelium to be thickest in the central-treated area; this phenomenon was more obvious in the 3-diopter-treated corneas. The numbers of activated keratocytes beneath the treated zone peaked at 4 months and decreased thereafter, while the numbers in the untreated areas decreased in the first 2 months after surgery, increased by 4 months, and did not change thereafter. Regenerated basal lamina averaged 86% intact over ablated areas; thickness was normal and no duplications were seen. Overall, the density of hemidesmosomes was significantly less in ablated areas compared with unablated areas. CONCLUSIONS: These findings support the relationship between clinical observations of corneal haze after PRK, reestablishment of the epithelial cell layer, and the potential for stromal remodeling by active fibroblastic keratocytes beneath the ablation zone. Overall, quantification of several morphological parameters indicated that the values for the treated zone tended, with time, to approach those of the untreated cornea after PRK.

An adjustable double running suture technique for keratoplasty.

In a retrospective analysis, we examined 30 consecutive cases of penetrating keratoplasty in which a double running 10-0/11-0 nylon suture technique was used and the 10-0 suture was adjusted early in the postoperative period to reduce astigmatism. When the response to suture adjustment was inadequate, the presence of the 11-0 suture allowed for early (ten to 18 weeks) removal of the 10-0 suture. Rapid visual recovery (12.3 +/- 0.95 weeks; mean +/- standard error) and low levels of final astigmatism (2.66 +/- 0.24 diopters) were achieved. Visual acuity was 20/20 to 20/40 in 25 of the 28 patients (89%) who were visually rehabilitated. In these 28 patients, visual acuity remained stable for the remainder of the study. All patients had a minimum of six months' follow-up from the time of surgery; mean follow-up was 10.6 +/- 1.70 months.

Visual disability resulting from a dislocated intraocular lens.

Complications may arise from the failure to remove a dislocated intraocular lens, even when it appears to rest in a benign position and pose no problems. We examined a patient with a second intraocular lens left in the inferior vitreous cavity. Shifting the loose lens resulted in eventual complications and visual disability; removing the dislocated lens and replacing the functional intraocular lens yielded good results.

Optisol corneal storage medium.

We compared the safety and efficacy of Optisol (Chiron Ophthalmics, Irvine, California), a new corneal storage medium, with McCarey-Kaufman and Dexsol corneal storage media (Chiron Ophthalmics, Irvine, California) and K-Sol corneal storage medium (Cilco, Huntington, West Virginia). Optisol contains dextran, 2.5% chondroitin sulfate, vitamins, and precursors of adenosine triphosphate (adenosine, inosine, and adenine). In in vitro studies, rabbit and human corneas stored in Optisol were thinner after 12 to 14 days at 4 C than tissue stored in other media. Thymidine uptake showed increased mitotic activity in human corneal endothelial cells cultured in Optisol, compared to Dexsol. Specular microscopic fields showed larger are-as of visibly intact endothelial cells and ultrastructural examination disclosed fewer structural changes in endothelial cells stored in Optisol, compared to tissue stored in Dexsol. In vivo, no clinical signs of epithelial toxicity or histologic evidence of intraocular inflammation were observed in rabbit eyes in which Optisol drops were instilled four times a day for 14 days. Finally, 51 patients undergoing penetrating keratoplasty with tissue stored in Optisol for one to six days (mean, 3.6 days) were enrolled in an uncontrolled, open-label clinical study. The percentage of clear grafts (93%, 41 of 44 patients examined at three months; and 98%, 42 of 43 patients examined at six months) and endothelial cell loss (5.0% and 11.5% at three and six months, respectively) were comparable to data from previous studies that used tissue stored in other short-term and intermediate-term media. The results suggest that Optisol storage preserves corneal endothelial cells for up to two weeks at 4 C, thereby permitting flexibility in the use of donor tissue for corneal transplantation, and that Optisol storage yields thinner tissue, which may allow for more accurate evaluation and more effective surgical manipulation.

Early diagnosis of infectious keratitis with in vivo real time confocal microscopy.

The tandem scanning confocal microscope (TSM) was adapted for in vivo examination of the cornea in rabbits with experimental bacterial and fungal keratitis. Compared to slit lamp biomicroscopy, the TSM provides superior lateral and axial resolution and serial optical sectioning capability, which may be useful for identification of corneal pathogens in the early stages of infection. We used the TSM to examine normal rabbit eyes infected with bacteria (Bacillus cereus) and a filamentous fungus (Aspergillus). We also examined a human cornea removed by penetrating keratoplasty after a clinical diagnosis of amoebic keratitis. In the early stages of bacterial infection, slit lamp examination revealed a nonspecific minimal stromal haze and limbal injection indistinguishable from sterile ulcers and epithelial defects. With the TSM, bacteria were visible as highly refractile bodies in the epithelium and superficial stroma. Branching fungal hyphae were also easily identified by the TSM, as were Acanthamoeba cysts and parasites in the subepithelial stroma. Our results indicate that this technique may provide a new modality for quickly and accurately identifying the agent of corneal infection, thereby facilitating prompt and appropriate treatment.

Keratotomy model of pseudomonas keratitis: gentamicin chemotherapy.

BACKGROUND: Chemotherapy of bacterial keratitis requires frequent application of antibiotic drops. Collagen shields containing antibiotics could reduce the need for frequent antibiotic application. To determine the effect of gentamicin-containing collagen shields and gentamicin drops on Pseudomonas keratitis, a new keratotomy model of infection was employed. METHODS: Model--contact lenses (58% water content) presoaked in 1% bovine serum albumin and exposed to 10(8) colony forming units per mL of Pseudomonas aeruginosa strain 27853, were found to reproducibly retain 5.9 (log base 10) colony-forming units. Rabbit corneas were scarified centrally with two perpendicular intersecting diamond knife cuts (5 mm x 5 mm x 0.2 mm), and bacteria-impregnated contact lenses were positioned and held in place for 24 hours with partial tarsorrhaphies. Treatment--Fourteen hours after lens removal (38 hours after infection), corneas were treated for 8 hours with collagen shields hydrated in saline (control), or shields impregnated with 800 micrograms gentamicin during manufacture, or one drop every 30 minutes of fortified gentamicin drops (14 mg/mL). The rabbits were killed and corneas collected for bacterial enumeration after 8 hours of treatment (46 hours after infection). RESULTS: Model--Slit-lamp examination and microbiologic confirmation showed uniformity of keratitis in all eyes. Treatment--Corneas treated with saline (controls) contained 6.4 (log base 10) Pseudomonas. Corneas treated with gentamicin-impregnated collagen shields (total drug = 800 micrograms) and fortified gentamicin drops (total drug = 21 mg) showed a reduction in viable bacteria of 2 logs and 6 logs, respectively, relative to the control. CONCLUSIONS: In this new model of Pseudomonas keratitis, the amount of gentamicin introduced into collagen shields during manufacture effectively reduced bacterial growth in infected rabbit corneas. However, larger amounts of drug applied as fortified drops on a frequent dosing schedule were more effective by a factor of three. Treatment of keratitis with antibiotic-impregnated collagen shields may reduce the need for very frequent application of topical drops, but may be more effective with topical drop supplementation to increase the amount of drug available over the course of therapy.

Central photorefractive keratectomy for myopia. Partially sighted and normally sighted eyes.

Ten partially sighted and 19 normally sighted eyes underwent excimer laser photorefractive keratectomy for the correction of myopia. Nine of the partially sighted and 17 of the normally sighted eyes had 12 months of follow-up. Epithelial healing was complete in all eyes by day 6. None of the eyes had recurrent erosions, infections, or other medical complications. An increase in corneal haze after surgery was followed by a slow trend toward clearing. Average uncorrected visual acuity in the 7 normally sighted eyes with attempted corrections of 5 diopters (D) or less was 20/40 from month 2 on; the eyes with greater than 5 D attempted corrections had an average of 20/80--at month 2, which declined to 20/200--by month 6. Best spectacle-corrected visual acuity was within +/- 1 Snellen line of preoperative values in 14 of the normally sighted eyes, improved 2 or more lines in 2 eyes, and worsened two or more lines in two eyes. Hard contact lens overcorrection restored all of the two-line loss in 1 eye and 1 line of the 3-line loss in the other. Refraction and keratometry indicated corneal flattening without induced astigmatism.

Changes in corneal topography after excimer laser photorefractive keratectomy for myopia.

Computer-assisted analysis of corneal topography was performed in 17 normally sighted human eyes during the first year after excimer laser photorefractive keratectomy (PRK) for myopia. Laser ablation of the central cornea produced an optical zone with a smooth power transition to the peripheral cornea. Decentration of the ablation was noted in some eyes (less than 0.5 mm in 3 eyes, 0.5 to 1.0 mm in 10 eyes, 1 to 1.5 mm in 3 eyes, and 2.1 mm in 1 eye), suggesting that careful alignment of the laser beam is critical. Improved methods to align the ablation within the center of the entrance pupil are needed. In 12 of 17 eyes, the topographic pattern appeared to stabilize between 3 and 7 months after PRK. In the remaining five eyes, central ablation power changed by more than 0.5 diopters (D) between the 6- and 12-month examinations. Regression was more common and more pronounced in eyes with intended corrections more than 5 D, whereas the majority of eyes with intended corrections of 5 D or less showed good correspondence between the final change in central ablation power and the attempted correction. Two eyes had a loss of at least two lines of best spectacle-corrected visual acuity that was attributable to irregular astigmatism, decentration of the ablation, and/or corneal opacification.

An adjustable single running suture technique to reduce postkeratoplasty astigmatism. A preliminary report.

The authors compared postkeratoplasty astigmatism over a 4-month period after surgery in a randomized, prospective study of two groups of patients (total N = 18) who received two different suture techniques. The test group N = 8) had a single running suture with postoperative suture adjustment; on the basis of computer-assisted topographic analysis, the suture was tightened in the flatter meridian and loosened in the steeper meridian in the first month after surgery. The control group (N = 10) had a standard double running suture procedure with no postoperative adjustment; the single running 10-0 nylon suture was removed 3 months after surgery. Four months after penetrating keratoplasty, mean (+/- standard deviation) astigmatism in the test group was 1.7 +/- 0.7 diopters (D), and all patients had less than 2.6 D of astigmatism. In the control group, mean astigmatism was significantly higher (5.4 +/- 2.4 D; range, 0.7-9.0 D; P less than 0.01). The results suggest that postkeratoplasty astigmatism can be reduced with the single running suture technique accompanied by postoperative suture adjustment.