Comparative study of three methods of evaluation of the corneal endothelium in pseudophakic patients: fluorophotometry, specular microscopy and pachymetry

Background: At present, three techniques, based on different methodological suppositions, are used indiscriminately for the study of the corneal endothelium. These are pachymetry, specular microscopy and fluorophotometry. The purpose of this work was to study the corneal endothelium of pseudophakic patients with the three techniques mentioned. On the basis of the results obtained, we discuss the relations between them and their practical clinical utility. Methods: One hundred and fourteen eyes of pseudophakic patients were studied using the three corneal endothelial evaluation techniques, both in the immediate preoperative period and 1 year after surgery. Results: Statistically significant relations exist between the number of endothelial cells and the fluorophotometric endothelial permeability coefficient (Kc.ca) 12 months after surgery, between the increase in corneal thickness in the first week after the operation and the Kc.ca 12 months after surgery, and between fluctuations of the corneal thickness greater than 30 m and both the endothelial cell count and the Kc.ca 12 months after surgery. There are no significant relationships among the pre-operative values obtained with the three methods. Conclusion: The results show how the parameters measured with the functional techniques (fluorophotometry, pachymetry) generally become normal after the surgical trauma, while the endothelial cell count remains irreversibly altered. The results obtained with different techniques are more closely related in more pathological endothelia, while no relation among them are shown in the study of normal endothelia. It is also shown how pachymetry is a good clinical predictor, in the immediate post-operative period, of the long-term status of the corneal endothelium.     

Effect of corticosteroids on healing of the corneal endothelium in cats

Background: Anterior segment surgery is frequently complicated by damage to the corneal endothelium. We examined the effects of corticosteroids, which are widely used for the suppression of postoperative inflammation, on the process of endothelial cell regeneration. Methods: The effect of corticosteroids on healing of the corneal endothelium was examined in 10 domestic cats. In both eyes a circular area, 8 mm in diameter, was scraped off at the center of the corneal endothelium without damaging Descemet's membrane. Immediately after scraping, as well as 2 and 5 days later, each animal received a unilateral retrobulbar injection of betamethasone sodium phosphate (2 mg). The other eye served as a control and received a retrobulbar injection of the vehicle only. Results: Evaluation of the corneal endothelium 2, 5 and 7 days after the trauma revealed that relative to the control contralateral eyes, the corticosteroid-treated eyes exhibited a higher mean coefficient of variation of the corneal endothelium cell area, fewer hexagonal cells, a larger number of polygonal cells with 3, 4, 7 and 8 cellular facets, thinner corneas and less inflammation. Conclusions: These findings suggest that corticosteroids unfavorably affect the regeneration of corneal endothelial cells after injury. As corticosteroids appear to have both positive and adverse effects on corneal function after trauma, they should be used with caution.     

Corneal neovascularization in rats as a model for photothrombotic therapy using bacteriochlorin a and an argon laser

Background: The presence of vessels has a negative influence on corneal transplant survival. Closure of such vessels prior to transplantation may improve the transplant results, and this might be achieved by irradiating the vessels with argon laser light after intravenous administration of a photosensitizer, e.g. bacteriochlorin a (BCA). A suture-induced corneal neovascularization model in rats was set up to test this hypothesis. Methods: Suture-induced vessels in the cornea of male Wistar rats were irradiated with argon laser light after intravenous administration of BCA. We applied irradiation of varying energy levels and duration and assessed the changes in the vessels by slit-lamp examination, fluorescein angiography and histology. Results: Suture-induced corneal vessels in the rat could be used effectively to study photothrombosis therapy. Intravenous administration of BCA prior to irradiation (=514.5 nm) of the corneal vessels led to vessel closure at lower energy levels and of longer duration than occurred with laser treatment alone. Conclusion: Suture-induced corneal neovascularization in the rat can be used as a model to study the efficacy of photothrombosis therapy. BCA can be used to enhance the rate and duration of vessel closure.     

Inhibitory effects of plasminogen fragment on experimentally induced neovascularization of rat corneas

Background: Corneal neovascularization plays an important role in the pathogenesis of a number of corneal disorders. Recently a polypeptide was demonstrated, generated by the primary tumor, that inhibited angiogenesis and growth in metastases. This polypeptide is similar to a 38-kDa plasminogen fragment. Methods: We surgically implanted into rat corneal stroma a slow-release ethylene-vinyl-acetate (EVA) copolymer pellet containing basic fibroblast growth factor (bFGF) to induce corneal neovascularization. Then we applied aqueous solution containing plasminogen fragment to the rat cornea in order to observe the degree of inhibition of angiogenesis. Results: In the eyes of control rats, neovascularization from the limbus to the pellet occurred, graded 4+ in all five animals. In plasminogen fragment-treated rats, there was virtually complete inhibition of the neovascular response to the pellet. Of five treated rats, three showed no neovascularization and two demonstrated grade 1+ neovascularization. The difference in the degree of neovascularization between control and plasminogen fragment treatment was statistically significant (P<0.05). Conclusion: Our studies provide the first direct evidence that rat corneal neovascularization is inhibited by instillation of plaminogen fragment. This agent may prove useful in the treatment of corneal angiogenic disorder.     

Intraocular irrigating solutions: a comparison of Hartmann's lactated Ringer's solution,BSS and BSS Plus

Background: We evaluated the effects of Hartmann's lactated Ringer's (HLR) solution, balanced salt solution (BSS) and BSS Plus on human corneal endothelium. Methods: Paired human corneas were mounted in the in vitro specular microscope for endothelial perfusion with HLR, BSS or BSS Plus for 15, 30, 60 and 120 min. Reversal experiments with BSS Plus after initial HLR perfusion were performed. At the end of the perfusions, electron microscopy, F-actin staining of the endothelial cytoskeleton and endothelial permeability measurements were carried out. Results: Longterm perfusion (120 min) with HLR resulted in a significantly higher swelling rate than in the paired controls perfused with BSS Plus. Short-term exposure to HLR for 15, 30 and 60 min after initial BSS Plus perfusion increased the swelling rates significantly. The increased corneal swelling after HLR perfusion for 60 min was reversed by BSS Plus perfusion. Ultrastructural changes in HLR-perfused corneas included endothelial cell edema, cytoplasmic vacuolation and mitochondrial swelling. F-actin staining showed overall cytoskeletal disorganization after perfusion with HLR. Corneal endothelial permeability was higher for BSS Plus-perfused corneas than with HLR solution. Conclusion: The results suggest that the clinically observed corneal clouding during irrigation with HLR is due to endothelial cell edema and decreased endothelial pump function. However, this increased corneal swelling is reversible by perfusion with BSS Plus.     

Electrolytes in the cornea: a therapeutic challenge

Background: Reported here are the results of electrolyte measurements in different layers of 70 apparently normal human corneas. Methods: Samples were examined by energy-dispersive X-ray analysis under calibrated conditions in a scanning electron microscope. The method allows the simultaneous quantitative analysis of, among others, sodium (Na), chloride (Cl), phosphorus (P) and potassium (K). The results are related to the dry weight of the analyzed samples. Four distinct layers, subepithelium, middle stroma, posterior stroma and Descemet's membrane, were analysed in each cornea. Results: In the middle stroma we found concentrations of: sodium 0.609 ± 0.13, chloride 0.557 ± 0.115, potassium 0.058 ± 0.02 and phosphorus 0.038 ± 0.01 (mmol/kg dry weight). Conclusion: The collation of normal electrolyte concentrations provides reference values for future studies on changes of the corneal electrolyte composition in diseased or injured eyes. The electrolyte composition of rinsing fluids or eye drops should be adjusted to that of the corneal stroma. Phosphate buffer, for example, is not a good vehicle for topical eye treatments and should be replaced by organic buffering systems.     

The spectrum of eye disease in children with AIDS due to vertically transmitted HIV disease: Clinical findings, virology and recommendations for surveillance

Background: Eye disease in children infected with HIV is uncommon, but there is little information on which children require screening or surveillance and the management of those with retinitis. Methods: We examined 12 children with symptomatic HIV disease (of a cohort of 98 HIV-positive children) using indirect ophthalmoscopy. When retinitis was found it was documented photographically at the time of diagnosis and on follow-up. Results: Four cases of infective retinitis were diagnosed: three with probable cytomegalo-virus retinitis and one with progressive outer retinal necrosis. Conclusion: Detection of reactivation is difficult and we recommend fundus photography under general anaesthesia wherever possible. Children with other end-organ disease, symptomatic eye disease and advanced HIV disease with severe immunosuppression require ophthalmological surveillance.     

Orthotopic corneal transplantation in the mouse — a new surgical technique with minimal endothelial cell loss

Background: The murine model of orthotopic perforating keratoplasty is important for studying the allograft reaction, but the small dimensions cause technical difficulties. Methods: The anterior chamber of the eye of the BALB/c mouse was measured with the confocal microscope and with histological methods. Ten C3H mouse donor corneas each were separated by the conventional technique and by the newly developed underwater technique, where the opened donor eye did not lose its shape under water. The corneal endothelium was stained with trypan blue and alizarin red S. Ten BALB/c (H-2d) mice received a corneal graft taken from a C3H (H-2k) mouse by the underwater technique. Results: The 3.7-mm eye of the BALB/c mouse has a corneal diameter of 3.5 mm. The cornea has a central thickness of 170 m, the epithelium comprising 30% and the stroma 70%. While none of the corneas separated by the new underwater technique evidenced endothelial damage, a 28 ± 17.0% defect of the endothelial surface was found with the conventional technique. All transplanted corneas were clear when the lids were opened on the 2 post-operative day and clouded between the 7th and 30th days (mean 16.5 days) due to an allograft reaction. Conclusion: The newly developed underwater technique is superior to the conventional technique, since floating of the very thin donor cornea during the separation procedure prevents endothelial defects by guarding against folds. By enabling reliable keratoplasty in the mouse, this technique facilitates studies on the experimental allograft reaction.     

The influence of the cutting procedure on the loss of corneal endothelial cells of the recipient eye in mechanically guided trephination

Background: Excessive damage to the endothelial monolayer during corneal trephination may contribute to transplant failure. For this reason, we performed several trephining experiments to determine the influence of various cutting conditions (sharpness, rotation rate, and cutting power) on the endothelial damage in the recipient bed. Methods: A mechanically guided motor trephine (Mikro-Keratron) was used for cutting experiments in fresh, enucleated porcine eyes. Eight different, reproducible combinations of cutting conditions were applied, and ten epithelial, full-thickness trephinations were performed for each combination. The endothelial defect was measured with a light microscope (60 × ). In addition, the bend radii of the porcine cornea were measured. Results: The mechanically guided Mikro-Keratron used with a sharp blade, high rotation rate (840 rev/min), and moderate cutting power (49 mN) caused a 31-m-wide band of damaged endothelium in the recipient bed. Decrease of the rotation rate to 30 rev/min resulted in an increase of the endothelial defect to 168%. A cutting power of 392 mN combined with a rotation rate of 840 rev/min increased the defect to 126%. The system allowed full-thickness 360° threphination. The corneal bend radii in porcine eyes measured 8.28 mm horizontally and 8.20 mm vertically. Conclusion: Our preliminary results indicate that the mechanically guided motor trephine, used under optimal conditions, is a suitable cutting device and may improve the outcome of penetrating keratoplasty; however, further investigation of this new approach is necessary.     

Integrins in human anterior chamber angle

Background: Integrins, which are composed of an and subunit, are capable of binding to a number of extracellular matrix proteins and, hence, affect cell adhesion and proliferation.Methods: The distribution of the integrin (₁, ₃-₅) and (_1–6 and _v) subunits in human anterior chamber angle was studied in eyes from subjects aged 9 months to 81 years using the indirect immunofluorescence technique.Results: Immunoreaction for the ₁ subunit was found throughout the trabecular meshwork (TM), in the cribriform layer, and in the endothelial lining of Schlemm's canal (SC). Labelling for the ₃ subunit was found in the TM and the cribriform layer only. In infant eyes the ₅ subunit was present in all three areas with the highest concentration in the cribriform layer, whereas no reaction was observed in adult eyes. The ₆ subunit was localized to the endothelium of SC only. Immunoreaction for the _v subunit was present in the TM and the cribriform layer of infants and young adults.Conclusion: The present results suggest the presence of several integrin heterodimers, acting as potential receptors for laminin, collagen, fibronectin, and vitronectin, in the anterior chamber angle.     

Corneal endothelial wound repair in normal and mitotically inhibited cultures

Background: The aim of the present study was to compare the morphology, proliferative activity and cytoskeletal organization of bovine corneal endothelial cells during wound healing under normal and mitotically inhibited conditions. Methods: Cell cultures were grown to confluency and incubated with the mitotic inhibitor 5-fluorouracil (5-FU; 2.5 g/ml) followed by a touch wound. Control cultures were maintained without 5-FU. Mitotic activity, F-actin, vinculin, vimentin and connexin 43 localization were evaluated before, during and after wound closure. Results: 5-FU inhibited irreversibly the mitotic activity of corneal endothelial cells during the whole wound healing process. In the presence of 5-FU, a high degree of polymegathism and delay in actin and vinculin redistribution to the cell borders after wound closure was observed. Vimentin and connexin 43 immunolabeling revealed only slight differences between 5-FU-treated and control cultures. Conclusion: Significant changes in cell geometry and cytoskeletal organization in the amitotic corneal endothelium became manifested only after wounding. These changes may influence cell-cell and cell-matrix interactions as well as functional restoration of the monolayer after wound closure.     

Therapeutic use of the 193-nm excimer laser in corneal pathologies

Purpose: To analyze the results of phototherapeutic keratectomy. Patients and methods: We performed 193-nm excimer laser phototherapeutic keratectomy (PTK) in 252 eyes of 216 patients suffering from pain and/or decrease in visual acuity. One hundred and three eyes had recurrent erosions of the cornea, 86 eyes underwent excimer laser smoothing of the cornea after pterygium surgery, 29 eyes had a bandlike keratopathy (25 rough, 4 smooth) and 34 eyes had other pathologic conditions such as amyloidosis of the cornea, anterior corneal dystrophies, scars after injuries, alkali burns, superficial stromal dystrophies and infections. Recurrent erosions and epithelial dystrophies were treated with 15–20 pulses (160–200 mJ/cm², 8 mm ablation zone) after mechanical abrasion of the epithelium. Removal of corneal opacities and scars required the use of a masking fluid (methylcellulose) in different concentrations and slit-lamp control (integrated in the delivery system of the excimer laser). Results: Some 91% of the eyes with recurrent erosions were recurrence-free. Fifty-two per cent of the eyes with pterygium had recurrences if the baresclera technique was used and 33% of the eyes if a free conjunctival graft was used. The difference was not significant. All of the patients with bandlike keratopathy were pain-free. In 88% of the eyes with special indications the treatment goal was achieved. No positive effect was seen after alkali burn, in a patient with anterior membrane dystrophy (Grayson-Wilbrandt corneal dystrophy) or in a patient with a corneal protuberance. In one patient with scleroperikeratitis a late recurrence of the opacity was observed 3 years after surgery. A loss of best corrected visual acuity was found only in one patient with bullous keratopathy in whom the treatment goal was the reduction of pain. All patients with smooth bandlike keratopathy had an improvement in best corrected visual acuity of at least one line. About 70% of patients with special indications improved by at least one line, up to nine lines. A possible hyperopic shift in all groups could be minimized using a large ablation zone. Conclusion: PTK with the 193-nm excimer laser is a safe and effective treatment for many superficial diseases of the cornea.Werner Förster is the Primary Investigator for Schwind Keratom     

Employment of bioluminescence for the quantification of adenosine phosphates in the human cornea

Background: Quantification of adenosine phosphates in human corneal extracts has been performed using spectrophotometry. We employed the bioluminescence technique to obtain a more sensitive assay for adenosine phosphates and to reduce the volume of the test sample. Methods: The bioluminescence assay for ATP, already known from sterility control, was modified and expanded. Standard curves were established using a standard solution with equimolar concentrations of ATP, ADP and AMP. To monitor the method, adenosine phosphates were measured in 35 human corneal extracts using both spectrophometry and bioluminescence. Results: Linear standard curves ranging from 1 to 45 pmol were established. The two methods yielded comparable results despite the use of a basic dilution of 1: 100 for the new technique. Conclusion: Bioluminescence provides a highly sensitive quantification of adenosine phosphates in the human cornea and facilitates an extremely detailed evaluation of the metabolic status of the cornea.Dedicated to Prof. Martin Reim on the occasion of his 65th birthday     

Unusual retinal pigment epitheliopathy and choroidopathy in carcinomatosis: A rare case of cancer-associated retinopathy

Background: Cancer-associated retinopathy is a syndrome causing ocular symptoms. It is a rare entity and only a few cases have been reported. Methods: A 67-year-old woman with small-cell endometrial carcinoma suffering from deterioration of visual acuity is presented. Results: The patient presented with extensive mottled changes of the retinal pigment epithelium, accompanied by diffuse subretinal fluid in the posterior pole and exudative retinal detachments inferior in both eyes. Conclusion: This patient suffered from a rare variety of cancer-associated retinopathy.     

Detection of herpes simplex virus after penetrating keratoplasty by polymerase chain reaction: correlation of clinical and laboratory findings

Background: The study was carried out to investigate the possible correlation of clinical findings, histopathologic features and detection of herpes simplex virus DNA in corneal buttons obtained after penetrating keratoplasty. Methods: We examined 47 consecutive corneal buttons sent for histopathologic examination by light microscopy and using the polymerase chain reaction for the detection of HSV1 and HSV2. Twenty-one corneal buttons from eyes with bullous keratopathy served as controls. Results: The 47 cases were graded from the clinical information available as unproven, suspected and clinically proven cases of herpetic keratitis. This grading did not correlate to specific histopathologic features or to the results of HSV1 DNA testing. None of the cases were positive for HSV2 DNA. Conclusion: HSV DNA was detected in some of the cases of clinically unsuspected herpetic keratitis. This technique of demonstrating the presence or absence of HSV in the cornea after keratoplasty is more reliable than clinical data or histopathologic findings and may be important in cases of recurrent inflammatory episodes involving grafts after keratoplasty.Presented in part at the meeting Frontiers in Penetrating Keratoplasty on 22 September 1994, Heidelberg, Germany     

Efficacy of argon laser trabeculoplasty: 3-year preliminary results of a prospective placebo-controlled study

Background: It was the purpose of this study to evaluate the efficacy of argon laser trabeculoplasty (ALT) in controlling intraocular pressure (IOP) and avoiding surgical intervention. We also investigated whether topically applied diclofenac sodium eye drops had an influence on the success rate of ALT compared with placebo eye drops. Methods: The indication for ALT was progressive glaucoma uncontrolled by maximal tolerated medical therapy. Thirty-nine of 41 patients were available for follow-up after 1122±239 days. Results: Twenty-one of 39 eyes failed during the first 3 years of follow-up for one or more reasons: 11 eyes required additional laser treatment and/or filtration surgery because of progressive visual field loss or unacceptably high IOP, 9 eyes failed to have a final IOP 21 mmHg, and 7 eyes failed because of an increase in the number of the medications. This yields a success rate for ALT of 46% for 3-year follow-up. There was no significant difference between diclofenac sodium- and placebotreated eyes concerning the success rate after 3 years (P>0.05). Conclusion: We conclude that the use of ALT for the treatment of glaucoma is best reserved for cases in which medical avenues of treatment have been exhausted.     

Role of protein tyrosine phosphorylation in rat corneal neovascularization

Background: Recent studies have suggested that tyrosine kinase pathways that are activated by angiogenic growth factors may play a role in corneal neovascularization. Methods: Corneal neovascularization was induced in rat corneas by chemical cauterization. At 6, 24, 48, 96, and 168 h after chemical cauterization the rat corneas without the corneal epithelium were prepared for gel electrophoresis. Total protein profiles of the corneal samples were examined by staining gels with Coomassie brilliant blue. Tyrosine-phosphorylated proteins, three angiogenic growth factors (basic fibroblast growth factor, vascular endothelial growth factor, and platelet-derived growth factor-B chain), and three intracellular signal proteins in the tyrosine kinase pathways (phospholipase C, SHC, and mitogen-activated protein kinase) in the corneal samples were examined by western blot analysis. A topical treatment of genistein eye drop (5 mg/ml) was used for inhibition of corneal neovascularization after chemical cauterization in rats. Results: In total protein profiles, three bands in the corneal samples were increased after cauterization. Overall tyrosine-phosphorylated proteins and all three angiogenic growth factors increased with progression of corneal neovascularization. The tyrosine-phosphorylated forms of three intracellular signal proteins were also increased after cauterization. Treatment with topical genistein was effective in inhibiting corneal neovascularization in rats. Conclusion: Protein tyrosine phosphorylation was involved in inflammation-induced corneal neovascularization. Tyrosine kinase inhibitors may have utility as inhibitors of corneal neovascularization.     

Susceptibility of human retinal pigment epithelial cells to different viruses

Background Different viruses have been reported to be involved in retinal diseases in animalsystems. In humans, herpes simplex virus and cytomegalovirus have been found to cause retinal disease. Most of the studied viruses are neurotropic. In this study, the in vitro susceptibility of human retinal pigment epithelial cells (RPEC) to representative members of different groups of human pathogenic viruses was investigated. Methods Early cultures of RPE C — after two or three passages — were infected with the following viruses: herpes simplex virus (HSV) type 1, human herpesvirus 6 (HHV-6), Epstein-Barr virus (EBV), cytomegalovirus (CMV), adenovirus types 1 and 7, measles virus, parainfluenza virus and coxsackie virus B3. Results Cultures of RPE C could be infected with neurotropic viruses like HSV or measles virus as well as with typical respiratory viruses like parainfluenza or adenoviruses. Coxsackievirus, an enterovirus, replicated as well as human CMV whereas EBV and HHV-6, two lymphotropic viruses, failed to infect RPE. Conclusion These findings suggest that a variety of viruses, including those causing rather common illnesses, might be capable of inducing retinal lesions under certain circumstances due to haematogenous spread during the course of viraemia.     

The effect of chemical stability and purification of perfluorocarbon liquids in experimental extended-term vitreous substitution

Background: To determine the importance of chemical stability and purification of perfluorocarbon liquids (PFCLs) in experimental retinal tolerance, we tested four different substances as long-term vitreous tamponade: purified and nonpurified perfluorodecalin (PFD) and perfluoro-octylbromide (PFOB) Method: After mechanical vitrectomy we replaced the vitreous of 65 rabbit eyes. Five groups were formed; four of them received the four PFCLs, while one served as control and received Ringer solution. The eyes were observed clinically every week and examined histologically after 1, 2, 4 and 8 weeks Results: After 1 week we observed foam cells and intraretinal macrophages in all eyes with PFCLs. Purified PFD caused retinal lesions in the photoreceptor, ganglion cell and outer nuclear layers after only 2 weeks in the lower part of the eyes. In eyes filled with purified PFOB we observed more pronounced damage of the same nature. Unpurified substances caused severe inflammation and retinal detachment Conclusion: Our study demonstrates that purification and chemical stability are important factors in retinal tolerance of PFCLs for vitreous replacement. Although purified PFD was tolerated by the rabbit eyes for 1 week, we cannot recommend this substance for short-term clinical use as a vitreous substitute.     

Long-term results of non-filtering surgery for the treatment of primary angle-closure glaucoma

Background: We previously reported the effectiveness of goniosynechialysis and trabeculotomy ab externo for adult-onset glaucoma. In this study, we performed non-filtering surgery on patients with primary angle-closure glaucoma and studied the long-term outcome of this treatment. Methods: Included in this study were 35 eyes of 25 patients with primary angle-closure glaucoma, each of which had an intraocular pressure greater than 20 mmHg with maximal tolerated antiglaucoma medication, even after laser iridotomy or surgical iridectomy. Of these 35 eyes, 22 underwent trabeculotomy and 13 underwent goniosynechialysis. All patients were followed up for at least 18 months. Results: In 21 (95%) of 22 eyes after trabeculotomy, and in 12 (92%) of 13 eyes after goniosynechialysis, intraocular pressures were well controlled at or below 21 mmHg at the final examination. However, in two of the 21 eyes in which trabeculotomy was a success, and in four of the 12 eyes in which goniosynechialysis was successful, the procedure had to be repeated before adequate control of pressure was achieved. Conclusion: Our results show that intraocular pressure in most cases of primary angle-closure glaucoma can be controlled by restructuring of the physiologic aqueous outflow route by means of goniosynechialysis or trabeculotomy, and that filtering surgery is not necessary.