A double-masked, randomized, 1-year study comparing the corneal effects of dorzolamide, timolol, and betaxolol. Dorzolamide Corneal Effects Study Group

OBJECTIVE: To compare the long-term effects of dorzolamide hydrochloride (Trusopt, Merck and Co Inc, White-house Station, NJ), timolol maleate, and betaxolol hydrochloride on corneal endothelial cell density and corneal thickness. METHODS: This 1-year multicenter study was conducted in 298 patients with ocular hypertension or open-angle glaucoma who had a baseline central corneal endothelial cell density greater than 1500 cells/mm2 and central corneal thickness less than 0.68 mm in each eye. Patients were randomized to 0.5% betaxolol twice daily, 0.5% timolol twice daily, or 2.0% dorzolamide 3 times daily. Specular microscopy and ultrasonic pachymetry of the central cornea was performed at baseline and 6 and 12 months following institution of therapy. Endothelial cell densities were determined by a single masked observer. RESULTS: The mean percent changes from baseline for both outcome measures were similar in all 3 treatment groups at both 6 and 12 months. After 1 year of treatment, the mean percent loss in endothelial cell density from baseline was 3.6%, 4.5%, and 4.2% for the dorzolamide, timolol, and betaxolol groups, respectively. The mean percent change from baseline for corneal thickness was 0.47%, -0.25%, and 0.39% for the dorzolamide, timolol, and betaxolol groups, respectively. CONCLUSIONS: Dorzolamide is equivalent to timolol and betaxolol in terms of the change in central endothelial cell density and thickness after 1 year of therapy. All 3 treatments exhibit good long-term corneal tolerability in patients with normal corneas at baseline.     

Influence of carbon monoxide, and its interaction with nitric oxide, on the adrenocorticotropin hormone response of the normal rat to a physico-emotional stress

BACKGROUND AND OBJECTIVES: To evaluate the corneal changes after Nd:YAG laser pupillary membranectomies (group A), iridotomies (group B), and capsulotomies (group C) in a follow-up period of 6 months. PATIENTS AND METHODS: The Nd:YAG laser was applied to 3 different groups of patients. Specular microscopy, including the counting of corneal thickness and cell density, the percentage of hexagonal cells, and the cell area, was performed preoperatively and postoperatively. RESULTS: Some dark areas were recorded in the corneal endothelium in groups A and B. A nonstatistically significant decrease of cell density was recorded by the end of the first month. A significant loss of endothelial cells during the sixth month was recorded in groups A and C. CONCLUSION: The appearance of dark areas in the corneal endothelium is mainly related to the total energy delivered. The YAG laser treatments do not provoke an immediate and conspicuous loss of the endothelial cells, but may act as a stimulus for an accelerated loss of endothelial cells.     

Corneal endothelial changes with long-term topical epinephrine therapy

Corneal thicknesses and endothelial cell densities were studied in nine patients with ocular hypertension receiving long-term unilateral topical epinephrine hydrochloride therapy. No difference was noted in corneal thickness in the treated eyes. There was a significant (P less than .02) reduction in endothelial cell count in the treated eyes compared to their untreated fellow controls. Those patients who had the most marked intraocular pressure response to epinephrine also had the greatest endothelial cell loss.     

Nasolabial fasciocutaneous free flap for cheek defects

Corneal metabolism is reduced after cataract extraction. PATIENTS AND METHODS: In a prospective study the corneal thickness and endothelial cell count of 48 patients were examined after phacoemulsification. Corneal thickness was measured with an ultrasound pachymeter and the endothelial cell count with a contact endothelial camera at the 12 o'clock position and in the corneal center before and 4 weeks, 4 months and 1 year after operation. RESULTS: One year after the operation, corneal thickness increased about 9% in the 12 o'clock position and about 12% in the corneal center. The endothelial cell count decreased about 27% in the 12 o'clock position and about 18% in the corneal center. We measured a significant correlation between cell loss and age at both points. Concerning the corneal thickness, no significant correlation was found. CONCLUSION: After cataract extraction corneal metabolism is reduced. The endothelial cell count or corneal thickness can be used as an indicator of the corneal trauma resulting from the operation.     

Autologous, hapten-modified vaccine as a treatment for human cancers

PURPOSE: To investigate the cellular dynamics of vessel formation during corneal neovascularization in the living eye by confocal microscopy. METHODS: Corneal neovascularization was initiated by placing a 7-0 silk suture through the corneal stroma 3 mm from the limbus at the 12 o'clock position in both eyes of 10 New Zealand white rabbits. The corneas were examined for vessel ingrowth at intervals from 1 to 15 days after suture placement using a tandem scanning confocal microscope with a 20X water immersion objective, as well as a slit-lamp biomicroscope. Changes in the limbal vessels were recorded on videotape for later analysis. As early vessel growth appeared to be associated with corneal nerves, the total number of sprouts and the number of sprouts along nerves were counted in confocal images, and the results analyzed for statistical significance. Vessel growth and the structural relationship between vascular buds and the deep stromal nerves were examined by light and transmission electron microscopy. RESULTS: The early events of cell migration from the limbal microvessels were found to be associated with the deep stromal nerves; although this association was easily visualized by confocal microscopy, it could not be documented by slit-lamp biomicroscopy. By 18 h after suture placement, the limbal vessels were dilated and the first vascular buds appeared as short, pointed, or flat-topped protrusions from the deep limbal capillaries. By 96 h, the capillary buds had increased in density and had begun to form lumens. Movement of red blood cells was established between 72 and 80 h after the first signs of bud formation, at the same time that cells of immune origin were seen. Confocal microscopy revealed and transmission electron microscopy verified that new bud formation began with the formation of vascular tubes by endothelial migration along the deep stromal nerves. The total number of sprouts and the number of sprouts associated with stromal nerves were similar on days 1 and 2 but differed on days 3-7, suggesting an association between sprouts and nerves in the early stages of neovascularization. CONCLUSION: Using real-time white light confocal microscopy, we were able, for the first time, to observe the process of corneal neovascularization in the living eye, from the earliest stages within hours after initiation to 2 weeks. The deep stromal nerves appear to serve as a focus for the growth of new vessels, by attracting and supporting vessel growth and/or by providing a potential space for movement of the endothelial cells. Confocal microscopy may provide a new approach to achieving a better understanding of the mechanisms involved in corneal neovascularization.     

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 pre-operative 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 microns 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.     

A review of 120 Becker permanent tissue expanders in reconstruction of the breast

For regular function the human cornea requires an intact endothelial cell layer with a sufficiently high cell density. One approach used to compensate endothelial cell loss is transplantation of cultured corneal endothelial cells. Using a previously described transplantation protocol, we observed topographic differences after transplantation of cultured human corneal endothelial cells to recipient corneas previously denuded of their own endothelium. The results presented in this paper suggest different interactions of the transplanted endothelial cells with the central or the peripheral part of the corneal matrix, respectively. Furthermore, cells isolated from the center of a human cornea differ from those isolated from the periphery in terms of their mitogenic capacity. The significance of these observations for corneal endothelial cell transplantation is discussed.     

Informing the patient

PURPOSE: To investigate the feasibility to use hydroxyethylstarch as an alternative deswelling additive in short-term preservation media. MATERIALS AND METHODS: Corneoscleral discs were prepared from pairs of eye balls of freshly slaughtered pigs. Corneas were stored in MEM-medium containing either 10% or 20% hydroxyethylstarch 450 000 at 4 degrees C in a refrigerator. Subsequently, the tissue was stored for 24 hours in organ culture at 37 degrees C in MEM-medium containing 10% fetal calf serum to detect latent endothelial cell damage. Mate corneas were treated the same except for being stored in Optisol GS during 4 degrees C storage. We determined corneal endothelial cell density, stromal thickness, and glucose concentration in the medium directly after preparation, after short-term storage at 4 degrees C, and after subsequent organ culture at 37 degrees C. Scanning electron microscopy of corneal endothelium was performed at each step during the experimental course. RESULTS: We did not observe any significant differences in endothelial-cell density between experimental groups and control groups. No decrease in endothelial-cell density was observed during the course of experiments. No increase in stromal thickness was determined in any group after short-term storage at 4 degrees C. Corneas stored in medium containing 20% hydroxyethylstarch showed a decrease in stromal thickness after short-term storage. After subsequent organ culture all corneas displayed a uniform stromal swelling. Glucose concentrations in the media decreased in all groups during the experiment. In scanning-electron microscopy we observed a reversible degeneration of cell borders after storage at 4 degrees C. Additionally, corneas stored in Optisol GS showed a reversible cobblestone appearance at this stage of the experiments. CONCLUSION: Hydroxyethylstarch appears to be an alternative to the use of dextran and chondroitin sulfate as a deswelling additive in corneal preservation media.     

Optimizing the review process of food additive and GRAS petitions. Overview of the workshop

PURPOSE: To assess endothelial barrier function, morphological appearance and corneal thickness three months after cataract surgery in order to evaluate intraoperative endothelial damage. METHODS: Endothelial permeability was examined by fluorophotometry, and contact specular microscopy and corneal pachymetry measurements were made in 40 patients (40 eyes) with senile, non-complicated cataracts one month before and three months after cataract surgery. Twenty eyes underwent uneventful phacoemulsification (Group 1) and 20 uneventful extracapsular cataract extraction (ECCE) with continuous curvilinear capsulotomy (Group 2). Results were analyzed using the two-tailed Student's t test, analysis of variance, and multifactorial and regression analysis. RESULTS: There was a significant postoperative increase in endothelial permeability in both groups (p < 0.001), but no real differences between the postoperative values (p = 0.07). Mean cell loss was 15.2% in ECCE and 18.3% in phacoemulsification (p = 0.4). There was a significant linear correlation between ultrasound time, cell loss and functional damage. Postoperative pachymetric measurements were not significant. CONCLUSIONS: Endothelial response showed no differences between the surgical techniques. Endothelial barrier function remained disturbed in spite of the apparent morphological stabilization. Corneal pachymetry is not useful for assessing postoperative endothelial changes.     

In vivo confocal microscopy of Fuchs' endothelial dystrophy

PURPOSE: The purpose of this study is to analyze in vivo confocal microscopic findings of corneas with Fuchs' endothelial dystrophy. METHODS: Central corneas of 17 eyes of 11 patients aged 41-86 years were examined using in vivo scanning slit confocal microscopy after being diagnosed with Fuchs' endothelial dystrophy. The cellular structure of the corneas was analyzed morphologically and quantitatively and compared to control results from 22 healthy corneas. RESULTS: Bullae were detected in the basal epithelial layer of one eye. Eight of 17 eyes (47%) exhibited an abnormal Bowman's layer: diffuse bright reflection and absence of nerves. Eleven eyes (65%) exhibited abnormal anterior stroma: lacunae and diffuse increased light reflection due to edema. In 12 eyes (71%), lacunae or dark bands 5-20 microm wide against increased background reflection were noted in the posterior stroma. Descemet's membrane was thickened in all eyes. Dark bands were detected in six eyes (35%). Guttae (137-1,231/mm2) 20-40 microm in diameter were found in every endothelial cell layer. The mean endothelial cell count was 1,202 +/- 850 (cells/mm2 +/- SD; range, 0-2,735). There was a positive correlation between endothelial cell counts obtained by specular microscopy and those obtained by confocal microscopy (r = 0.95). CONCLUSION: In vivo confocal microscopic findings of Fuchs' endothelial dystrophy are described for the first time in a series of cases. Pathological changes in Fuchs' dystrophy were detected in all corneal layers, more frequently in the posterior layers. Endothelial cell counts obtained with confocal microscopy were statistically similar to those obtained with standard specular microscopy.     

Mitotic inhibition of corneal endothelium in neonatal rats

PURPOSE: Corneal endothelium in humans does not divide to any significant extent after birth; therefore, with age there is a gradual loss of cells. When cell density is reduced to a critical level, the endothelium cannot function to maintain corneal clarity, and the cornea becomes permanently cloudy. Currently, the blindness that results can be treated only by corneal transplantation. The long-term goal is to find methods to stimulate corneal endothelial proliferation in a clinically relevant manner. The first step toward achieving this goal is to identify mechanisms responsible for the induction and maintenance of mitotic inhibition of the corneal endothelium in vivo. During corneal development, the endothelium is formed by migration and proliferation of mesenchymal cells from the ocular periphery. Soon after the monolayer is formed, proliferation ceases. In tissue culture, many cell types cease proliferating upon formation of stable cell-cell and cell-substrate attachments. The goal of the present studies was to determine whether establishment of stable contacts correlates with cessation of endothelial proliferation during corneal development in vivo. METHODS: Corneas from neonatal (days 1, 3, 7, 10, 13, 14, 17, 21, 28, and 42) and adult rats were used for immunolocalization of the following: bromodeoxyuridine (BrdU), an S-phase marker; p27kip1 and p21cip1, G1-phase inhibitors; connexin-43 and ZO-1, proteins associated with gap and tight junctions, respectively; Na+/K+-ATPase and beta3-integrin, markers of plasma membrane polarity; and fibronectin and collagen type IV, constituents of Descemet's membrane. Nuclei staining positively for BrdU were counted to determine the relative number of S-phase cells at various times after birth. Marker protein expression and localization were determined by conventional fluorescence microscopy and by confocal microscopy. RESULTS: The number of endothelial cells staining positively for BrdU gradually decreased between postnatal days 1 and 13. After postnatal day 13, positive BrdU staining was no longer detectable. During the first postnatal week, cells stained positively for the G1-phase inhibitor p27kip1 but not for p21cip1. Connexin-43 achieved its mature location by postnatal day 1. ZO-1, Na+/K+-ATPase, beta3-integrin, fibronectin, and collagen type IV achieved their mature localization patterns between postnatal days 14 and 21. CONCLUSIONS: In neonatal rat, corneal endothelial cells are still entering the cell cycle at birth, but cell cycle entry gradually decreases, so that by postnatal day 13 cells are no longer entering the S-phase. The G1-phase inhibitor p27kip1, but not p21cip1, may help mediate this inhibition. Stable cell-cell and cell-substrate contacts gradually form, and monolayer maturation is complete between postnatal days 14 and 21. The results lead to the hypothesis that, in developing rat cornea in vivo, the establishment of stable cell-cell and cell-substrate contacts initiates a cascade of events, mediated by p27kip1, which induces mitotic inhibition in the endothelial monolayer.     

Nuclear muscarinic acetylcholine receptors in corneal cells from rabbit

PURPOSE: Previous studies have indicated that muscarinic acetylcholine receptors (mAChR) may be present in an unexpected, unique location and play a singular role in cellular growth regulation of rabbit corneal epithelium that may be of general physiologic significance if found in other cells. The purpose of this study was to examine rabbit corneas and corneal cells in culture to determine mAChR location and tissue distribution. METHODS: Using [3H]-propylbenzilylcholine mustard ([3H]PrBChM), which binds covalently to the active site of mAChR, rabbit corneal cross-sections, cultured corneal keratocytes, epithelial and endothelial cells, as well as nuclei isolated from these cultured corneal cells were labeled, stained, and autoradiographed. Nuclei labeled with [3H]PrBChM were further analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. RESULTS: Direct visual confirmation of the localization of mAChRs was obtained. MAChR were found in epithelial and endothelial layers of fresh-frozen corneal cross-sections, in cultured rabbit epithelial and endothelial cells, and on isolated rabbit epithelial and endothelial cell nuclei. mAChR were not detectable in keratocytes with these techniques. When [3H]PrBChM-labeled nuclei from cultured corneal cells were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, epithelial and endothelial samples showed specific mAChR binding, whereas binding to keratocyte nuclei was not detectable. CONCLUSIONS: As a result of these findings, a revised hypothesis is suggested for the locations and possible functions of mAChR in regulation of growth in corneal and other cells.     

Human corneal studies with a vitrification solution containing dimethyl sulfoxide, formamide, and 1,2-propanediol

We tested the tolerance of human corneas to a vitrification solution, modified VS41A, containing 3.1 M dimethyl sulfoxide, 3.1 M formamide, and 2.2 M 1,2-propanediol in a carrier solution consisting of the corneal storage medium CPTES with 2.5% w/v chondroitin sulfate. Seven human corneas were exposed for 10 min each to graded concentrations of the solution at 0 degree C, remaining in the full-strength solution for 10 min. The corneas had significantly more endothelial cell damage (P < 0.05) than seven mated control corneas, but it was minimal (4.3% cell loss). Attempts at vitrification and rewarming of three corneas exposed to the solution by this protocol, however, resulted in ice formation in the peripheral corneal stroma and severe endothelial damage. Presumably, equilibration with the cryoprotectant in the thicker periphery of the human cornea had not occurred. Ice did not form on the center of one cornea, and substantial numbers of central endothelial cells survived after vitrification in this case. Immersion of the human corneas for 25 min in each of the four graded solutions at 0 degree C was required for sufficient penetration of the cryoprotectant to allow total corneal vitrification and rewarming without ice formation. This prolonged exposure to modified VS41A caused unacceptable damage to the corneal endothelium, however. Successful vitrification of human corneas with this solution will require a safe method for obtaining corneal equilibration with the cryoprotectant.     

Creating and curating a pediatric radiology digital library to make the internet a useful reference tool for the radiologist

BACKGROUND AND OBJECTIVE: The endothelial cell count after phacoemulsification serves as a sensitive indicator for the level of corneal damage caused by different phacoemulsification techniques. PATIENTS AND METHODS: In a prospective and randomized study, the "Reversed Tip and Snip" technique and the "Divide and Conquer" technique were performed in groups of 30 patients each. The corneal endothelial cell count was measured preoperatively as well as 4 weeks and 3 months postoperatively. RESULTS: The endothelial cell count showed significant (P < .001) reduction by approximately 10% after the "Reversed Tip and Snip" technique and by approximately 15% (P < .001) after the "Divide and Conquer" technique. The latter produced a significantly (P < .001) greater cell loss. CONCLUSIONS: The "Reversed Tip and Snip" phacoemulsification technique produces less endothelial cell loss than the "Divide and Conquer" technique.     

Corneal endothelial involvement in pseudoexfoliation syndrome

Information on corneal manifestation in pseudoexfoliation syndrome is restricted to specular microscopic studies. We investigated morphologic changes of the posterior cornea of two corneal buttons with Fuchs' endothelial dystrophy obtained at penetrating keratoplasty and of one enucleated glaucomatous eye from three patients with ocular pseudoexfoliation syndrome. By transmission electron microscopy, large clumps of typical pseudoexfoliation material were found adhering to the corneal endothelium and masses of pseudoexfoliation material were incorporated into the posterior Descemet's membrane. In the affected areas, the endothelial layer appeared irregular and discontinuous, with loosely adherent, degenerating cells producing pseudoexfoliation fibers and fibroblastic cells spreading to cover denuded Descemet's membrane. The present findings indicate that the pseudoexfoliation material is initially formed by degenerative endothelial cells and that it becomes subsequently buried by overgrowing cells. The corneal endothelial involvement may potentiate complications of cataract surgery and open angle glaucoma in patients with pseudoexfoliation syndrome.     

Central cloudy corneal dystrophy of Francois. A clinicopathologic study

Central cloudy corneal dystrophy of Francois was first described in 1955 by J. Francois; its pathophysiology remains unknown. An 80-year-old woman with bilateral central cloudy corneal dystrophy of Francois was examined after having undergone a combined penetrating keratoplasty and cataract extraction. The corneal button was obtained. Light microscopy revealed stromal staining for acid mucopolysaccharide. Transmission electron microscopy revealed extracellular vacuoles, some of which had fibrillogranular material and electron-dense deposits. Fibrillogranular material was present in and around some keratocytes. Numerous endothelial vacuoles contained light-staining fibrillogranular material and round electron-dense granules. Our findings suggest that the opacities in patients with central cloudy corneal dystrophy of Francois are due to the extracellular accumulation of mucopolysaccharide and lipidlike material. Further studies are needed to elucidate the nature of these deposits.     

The critical role of interleukin 4 but not interferon gamma in the pathogenesis of colitis in T-cell receptor alpha mutant mice

PURPOSE: To compare keratocyte density determined by using confocal microscopy with keratocyte density determined in the same corneas by histology. METHODS: Digital en face images of central corneas were recorded three times by using confocal microscopy in vivo in six New Zealand White rabbits. Bright objects (keratocyte nuclei) in the images were automatically identified by using a custom algorithm to estimate total and regional stromal keratocyte densities. The corneas were then excised, fixed, and sectioned in a sagittal plane for histology. Keratocyte nuclei were manually counted from digitized images of 50 histologic sections per cornea. Total and regional keratocyte densities were estimated from the histologic sections by using stereologic methods based on nuclei per unit area, mean nuclear diameter, and section thickness. Histologic cell densities were corrected for tissue shrinkage. RESULTS: By confocal microscopy, total keratocyte density was 39,000 +/- 1,200 cells/mm3 (mean +/- SE; n = 6); cell density was 47,100 +/- 1,300 cells/mm3 in the anterior stroma and decreased to 27,900 +/- 2,700 cells/mm3 in the posterior stroma (P = 0.004). Analysis of the three separate confocal images of each cornea produced repeatable total cell densities (mean coefficient of variation = 0.035). By histology, total keratocyte density was 37,800 +/- 1,100 cells/mm3, not significantly different from that estimated by confocal microscopy (P = 0.43); anterior cell density was 48,300 +/- 900 cells/mm3 and decreased to 29,400 +/- 900 cells/mm3 posteriorly (P < 0.001). CONCLUSIONS: Rabbit keratocyte density estimated by automated analysis of confocal microscopy images in vivo is repeatable and agrees with keratocyte density estimated from histologic sections.     

Nonsteroidal anti-inflammatory drugs and severe psychiatric side effects

In the systemic mucopolysaccharidoses (MPS) in animals, corneal clouding resulted from storage of glycosaminoglycans (GAG) in stromal keratocytes. The corneal epithelium was normal (MPS VI and VII) or minimally affected (MPS I), and stromal edema was not a feature even though the corneal endothelium demonstrated variable pathology. The MPS I (cat) cornea showed endothelial cells with large numbers of secondary lysosomal inclusions that were vacuolated or had a granular matrix. The endothelium was uniformly affected, but was not markedly hypertrophied. In contrast, the MPS VI (cat) cornea showed no endothelial cell disease. The MPS VII (dog) cornea had the most significant and dramatic endothelial pathology. The cells were massively hypertrophied and contained large numbers of vacuolated lysosomal inclusions. Regardless of the severity of the morphologic disease, the endothelial cells in these animal models functioned normally in maintaining the relative dehydration of the cornea. The corneal clouding was the result of storage in stromal keratocytes rather than corneal edema from endothelial dysfunction.