ABSTRACT
PURPOSE: Many studies of the cornea would benefit from a simple, objective method to measure corneal thickness. In this study, a new optical pachometer based on video and computer technology was designed and tested. METHODS: The slit beam of a photographic slit lamp was monitored with a video camera through one half of the biomicroscope. When the slit was properly aligned with the cornea, the operator triggered a flash, and one video frame that included the flash was captured. A custom software package detected epithelial and endothelial edges. Corneal thickness was calculated from the median corneal image width and image widths from similar measurements of contact lenses with known thicknesses. Corneal thickness was measured in 25 subjects by using this new instrument and was compared to thickness measured by using a conventional Haag-Streit pachometer. RESULTS: Corneal thickness in the 25 subjects measured on the new instrument was 512+/-20 microm and 515-/+21 microm in the right and left eyes, respectively (mean+/-SD). Thickness of the same corneas measured on the Haag-Streit pachometer was 530+/-22 microm and 534+/-20 microm in right and left eyes, respectively. The average SD of 10 consecutive measurements was 6.6 microm and 6.7 microm on the video and Haag-Streit pachometers, respectively (n = 50 corneas). CONCLUSIONS: The video pachometer provides a fast, objective means of measuring corneal thickness. It is simple to use and provides precision equal to that of the Haag-Streit pachometer.
Subject(s)
Cornea/anatomy & histology , Diagnostic Techniques, Ophthalmological/instrumentation , Video Recording , Anthropometry , Equipment Design , Humans , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
Seventy-two human corneas were maintained in a perfusion system at 37 degrees C and 18 mm Hg intracameral pressure for 1 to 3 weeks. Corneal thickness, which was initially greater than normal because the enucleated eyes were kept at 4 degrees C before excision of the corneas, decreased slowly during the period of incubation. Endothelial removal or perfusion with ouabain (10(-4) M) induced irreversible stromal swelling. Cooling to 4 degrees C for 8 hr during perfusion caused stromal swelling that disappeared after rewarming to 37 degrees C; less stromal swelling occurred with cooling after 3 weeks of perfusion than after 3 days. No enlargement of central endothelial cells was noted in most corneas by serial specular microscopy. Electron microscopy demonstrated reversal of postmortem changes and maintenance of normal intracellular ultrastructure for 3 weeks. This system for long-term corneal perfusion will allow controlled studies of the effects of new methods of corneal preservation and other perturbations upon the corneal endothelium in situ.
Subject(s)
Cornea , Perfusion/methods , Tissue Preservation , Animals , Cell Count , Cornea/anatomy & histology , Cornea/metabolism , Cornea/ultrastructure , Culture Media , Endothelium, Corneal/cytology , Epithelial Cells , Humans , Image Processing, Computer-Assisted , Organ Culture Techniques , Osmolar Concentration , Rabbits , Time FactorsABSTRACT
Measurements of corneal thickness, endothelial cell size, endothelial permeability to fluorescein, and intraocular pressure were made in two groups of human subjects: 21 persons with cornea guttata (early Fuchs dystrophy without epithelial edema) and 17 persons age- and sex-matched but with normal corneas. Statistically significant differences were found between the two groups in all four measured variables. The two groups did not differ with respect to the variability in endothelial cell sizes. There were statistically significant positive correlations between endothelial permeability to fluorescein, endothelial cell size, and corneal thickness. The endothelial pump rate was calculated for each group, and the difference was not statistically significant. Our results suggest that the earliest defect in Fuchs' dystrophy is solely a breakdown in barrier function and thus increased permeability, resulting in a thicker cornea.
Subject(s)
Cornea/pathology , Corneal Dystrophies, Hereditary/pathology , Fuchs' Endothelial Dystrophy/pathology , Adult , Aged , Cornea/cytology , Cornea/physiopathology , Endothelium/cytology , Endothelium/pathology , Endothelium/physiopathology , Female , Fluoresceins , Fuchs' Endothelial Dystrophy/physiopathology , Humans , Intraocular Pressure , Male , Middle Aged , PermeabilityABSTRACT
In order to determine which layers of the corneal stroma bear the stress of the intraocular pressure, 6.0-mm nonpenetrating trephine incisions were made centrally in one eye of each of 16 adult albino rabbits. After epithelial healing, the central corneal thickness was measured over 3 hr at 50 mmHg intraocular pressure in both eyes of the anesthetized rabbits. The animals were then killed and the uniformity and depth of trephine cut determined histologically. The mean differences in swelling rates between the cut and the opposite uncut eyes for trephine incisions of different depths were as follows: 1 +/- 2 micron/hr for 8-20% depth, 5 +/- 2 micron/hr for 21-40% depth, and 14 +/- 3 micron/hr for 41-60% depth (P less than 0.01 for all groups). These results indicate that the intraocular tension is probably distributed across all the corneal stromal lamellae rather than being borne primarily by the anterior or posterior layers.
Subject(s)
Cornea/anatomy & histology , Intraocular Pressure , Stress, Mechanical , Animals , Cornea/pathology , Cornea/physiology , Corneal Diseases/pathology , Corneal Diseases/physiopathology , Edema/pathology , Edema/physiopathology , RabbitsABSTRACT
We mechanically damaged the entire corneal endothelium of one eye of each of ten cats and then examined both eyes by fluorophotometry and specular microscopy for 5 months. Six weeks after damage, when the corneas had cleared sufficiently to make accurate measurements, the mean endothelial permeability to carboxyfluorescein was increased 11% (P = 0.02) and the mean central corneal thickness was increased 11% (P = 0.05) in the damaged eyes. The mean endothelial pump rate was decreased 29% (P = 0.05), indicating that the increase in permeability was insufficient to explain the increase in thickness. The permeability returned to normal by 3 months and the pump rate by 5 months. Six weeks after damage, the mean endothelial cell size was increased 89% (P less than 0.01), the mean coefficient of variation of cell size was increased 200% (P less than 0.01), and the mean percentage of hexagonal cells was decreased 34% (P less than 0.01). By 5 months, the mean cell size had changed very little, and none of the three morphologic measurements had returned to normal. As in rabbits, the endothelial barrier in cats recovers before the pump after wounding. Unlike in rabbits, functional recovery in cats requires at least several months. Such prolonged functional recovery after endothelial trauma might also be expected in humans who, like cats and unlike rabbits, have little capacity for endothelial mitosis during healing.
Subject(s)
Endothelium, Corneal/injuries , Wound Healing , Animals , Aqueous Humor/metabolism , Cats , Cell Count , Cell Membrane Permeability , Endothelium, Corneal/pathology , Fluorescent Dyes , Fluorometry , Intraocular Pressure , Mathematics , Rabbits , Time FactorsABSTRACT
Anterior segment fluorophotometry (topical) and central endothelial cell photography were performed on 40 long-term (2-23 years) contact lens wearers (four groups of ten each: hard, soft, gas permeable, and gas permeable plus prior lens usage) and 40 non-contact lens wearers of similar ages. Morphologically, the endothelial cells of contact lens wearers showed greater variability in size and shape compared to controls. The mean endothelial cell size in contact lens wearers (307 +/- 35 micron2) was smaller than that of controls (329 +/- 38 micron2, P less than 0.01). There was an increase in the coefficient of variation of cell size of the contact lens group (0.35 +/- 0.06 versus 0.25 +/- 0.04 for controls, P less than 0.0001). The endothelial cell mosaic contained a smaller percentage of hexagonal cells in contact lens wearers (66 +/- 8) compared to controls (71 +/- 7, P less than 0.01). There was a compensatory increase in five-sided cells. Functionally, there was no difference in corneal clarity, central corneal thickness or endothelial permeability to fluorescein (3.78 +/- 0.57 X 10(-4) cm/min versus 3.85 +/- 0.55 X 10(-4) cm/min for controls) between the two groups. Aqueous humor flow was increased 7% in contact lens wearers. We found no correlation between oxygen transmissibility, estimated underlying oxygen tension, or duration of wear of the contact lenses and any morphologic or functional variable. We also found no differences between the four groups of contact lens wearers except that the gas permeable lens wearers had more hexagonal and less pentagonal cells. Long-term contact lens wear induces morphologic changes in the corneal endothelium.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Contact Lenses/adverse effects , Endothelium, Corneal/pathology , Adolescent , Adult , Aqueous Humor/physiology , Endothelium, Corneal/metabolism , Endothelium, Corneal/physiopathology , Eye Proteins/metabolism , Humans , Permeability , Time FactorsABSTRACT
Thirteen patients with Fuchs' endothelial dystrophy were studied to measure the potential effects of topically applied dexamethasone on endothelial function. Endothelial permeability in the Fuchs' dystrophy patients was not different from that found in normal controls. One eye, chosen at random, was treated topically four times a day for 7 days with 0.1% dexamethasone phosphate. The contralateral eye was treated with a placebo of identical appearance. Prior to treatment, there were no statistically significant differences in the means of the intraocular pressure, corneal thickness, endothelial permeability, or endothelial pump rate between the dexamethasone- and placebo-treated groups. In the placebo-treated eyes, a significant decrease was observed in both endothelial permeability and endothelial pump rate over the course of the study. No statistically significant changes occurred in the dexamethasone-treated eyes over the same period. When the dexamethasone group was compared with the placebo group, there was a significant difference in the change in endothelial pump rate between the two groups, attributable in large part to the decrease in pump rate observed in the placebo group over the course of treatment. We interpret our data as lacking support for the concept that topical steroids are beneficial for the treatment of stromal edema in patients with Fuchs' dystrophy.
Subject(s)
Corneal Dystrophies, Hereditary/drug therapy , Dexamethasone/therapeutic use , Adult , Aged , Dexamethasone/pharmacology , Endothelium/drug effects , Humans , Middle AgedABSTRACT
Twenty-four normal human subjects were studied before and after one week of treatment with 0.1% topical dexamethasone. Intraocular pressure, corneal thickness and endothelial cell size were measured. The flow of aqueous humor and the endothelial permeability to fluorescein were determined using fluorophotometry. In addition, the relationship between the initial location of an iontophoretic depot of fluorescein and its kinetics was studied. There was a small and significant increase in intraocular pressure in the eyes treated with dexamethasone but no significant change in corneal thickness, endothelial permeability or the rate of aqueous humor flow. The elimination of fluorescein from the eye was slightly higher when the fluorescein depot was placed adjacent to the superior limbus than when the depot was placed in the central cornea. Smaller right to left differences were observed when fluorescein was placed peripherally than when it was placed centrally. The increased precision with peripheral placement is probably due to the improved signal-to-noise ratio for fluorescence measurements of the anterior chamber through the unstained cornea.
Subject(s)
Aqueous Humor/drug effects , Cell Membrane Permeability/drug effects , Dexamethasone/administration & dosage , Administration, Topical , Adolescent , Adult , Cornea/drug effects , Endothelium/drug effects , Fluoresceins/pharmacology , Humans , Intraocular Pressure/drug effects , KineticsABSTRACT
PURPOSE: To evaluate cell death in human donor corneas stored at 4 degrees C, to determine whether terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick-end labeling (TUNEL) discriminates between apoptosis and necrosis in corneas stored at 4 degrees C. METHODS: Ten human corneas were stored in Optisol (Chiron Ophthalmics, Irvine, CA) at 4 degrees C for periods ranging from 0 to 21 days and then fixed for histologic examination. Central corneal sections from each cornea were examined by transmission electron microscopy (TEM) and by the TUNEL assay. Electron micrographs of at least 15 keratocytes each from the anterior, middle, and posterior stroma were examined by three masked observers who graded each cell as normal, apoptotic, or necrotic. Central sections from the same corneas were processed by the TUNEL assay and evaluated with a laser scanning confocal microscope to determine the percentage of apoptotic cells. RESULTS: By TEM, apoptosis occurred in 23% of the keratocytes and necrosis in 12%. By TUNEL assay, apoptosis occurred in 11% of the keratocytes, with the results in individual corneas being similar to the findings by TEM for apoptosis, rather than for necrosis. By TUNEL assay, apoptosis occurred in 13% of the epithelial cells and in 8% of the endothelial cells. The percentage of apoptotic cells and storage time correlated significantly for the epithelium, but not for the keratocytes or endothelium in this small sample. CONCLUSIONS: Both apoptosis and necrosis occur in cells during corneal storage at 4 degrees C, with apoptosis appearing to predominate. The TUNEL assay identifies cells undergoing apoptosis, but not necrosis, in corneal tissue. Inhibition of apoptosis in corneas stored at 4 degrees C may prolong acceptable storage times.
Subject(s)
Apoptosis , Cornea , Cryopreservation , Organ Preservation , Adult , Child, Preschool , Chondroitin Sulfates , Complex Mixtures , Cornea/pathology , Cornea/ultrastructure , Cryopreservation/methods , Culture Media, Serum-Free , Dextrans , Gentamicins , Humans , In Situ Nick-End Labeling , Middle Aged , Necrosis , Organ Preservation/methodsABSTRACT
PURPOSE: To obtain longitudinal data to estimate long-term morphometric changes in normal human corneal endothelia. METHODS: Ten years after an initial study, the authors rephotographed the central corneal endothelium of 52 normal subjects with the same contact specular microscope. The findings for the 10 subjects younger than 18 years of age at the initial examination were considered separately. For the remaining 42 adult subjects, the time between examinations averaged 10.6 +/- 0.2 years (range, 10.1 to 11 years). At the recent examination, these subjects' ages averaged 59.5 +/- 16.8 years (range, 30 to 84 years). Outlines of 100 cells for each cornea were digitized. RESULTS: For the 42 adult subjects, the mean endothelial cell density decreased during the 10.6-year interval from 2715 +/- 301 cells/mm2 to 2539 +/- 284 cells/mm2 (P < 0.001). The calculated exponential cell loss rate over this interval was 0.6% +/- 0.5% per year. There was no statistically significant correlation between cell loss rate and age. During the 10.6-year interval, the coefficient of variation of cell area increased from 0.26 +/- 0.05 to 0.29 +/- 0.06 (P < 0.001), and the percentage of hexagonal cells decreased from 67% +/- 8% to 64% +/- 6% (P = 0.003). For the 10 subjects 5 to 15 years of age at the initial examination, the exponential cell loss rate was 1.1% +/- 0.8% per year. CONCLUSIONS: Human central endothelial cell density decreases at an average rate of approximately 0.6% per year in normal corneas throughout adult life, with gradual increases in polymegethism and pleomorphism.
Subject(s)
Endothelium, Corneal/cytology , Adult , Aged , Aged, 80 and over , Aging/physiology , Cell Count , Female , Humans , Longitudinal Studies , Male , Middle Aged , Reference ValuesABSTRACT
PURPOSE: To study the effects of long-term contact lens wear on morphologic and physiologic properties of corneal endothelial cells. METHODS: The endothelial permeability to fluorescein and the rate of corneal deswelling from hypoxia-induced edema were measured in 20 long-term (mean, 17+/-9 years; range, 5-33 years) contact lens wearers and 20 age-matched control subjects. From these data, the relative endothelial pump rate in each subject was estimated, based on the pump-leak hypothesis of corneal hydration control. Corneal autofluorescence and the aqueous humor flow rate were determined by fluorescein fluorophotometry. Images of corneal endothelial cells were recorded by using specular microscopy, and morphologic indices (cell density, coefficient of variation of cell area, percentage of hexagonal cells, and skewness) were determined. RESULTS: No statistically significant differences were found between the contact lens and control groups in endothelial permeability, corneal deswelling, relative endothelial pump rate ([mean +/- SD] 1.07+/-0.33 relative pump units versus 1.01+/-0.25 relative pump units; contact lens versus control; P = 0.57), and endothelial cell density. Contact lens wearers had a significantly higher aqueous humor flow rate (3.57+/-1.03 microl/min versus 2.77+/-0.51 microl/min; P = 0.005), coefficient of variation of cell area (0.35+/-0.09 versus 0.28+/-0.04; P = 0.006), and corneal autofluorescence (3.1+/-0.6 ng/ml versus 2.3+/-0.3 ng/ml fluorescein equivalents; P < 0.001) than did non-contact lens wearers. CONCLUSIONS: Despite the known effects of long-term contact lens wear on corneal endothelial morphometry, no effect on endothelial function was found.
Subject(s)
Contact Lenses , Endothelium, Corneal/physiology , Adult , Aqueous Humor/metabolism , Biological Transport, Active/physiology , Cell Count , Corneal Edema/etiology , Corneal Edema/metabolism , Fluorescein/metabolism , Fluorophotometry , Humans , Hypoxia/complications , Middle Aged , PermeabilityABSTRACT
PURPOSE: To assess the effects of diabetes mellitus on corneal structure and function. METHODS: The authors measured endothelial permeability to fluorescein and corneal deswelling for 7.5 hours after 2 hours of hypoxic contact lens wear in 20 patients with diabetes who had nonproliferative retinopathy and 21 age-matched control subjects. Central corneal endothelial photographs were also taken. Corneal deswelling rates, expressed as percent recovery per hour (PRPH), and open eye steady state (OESS) thickness were estimated by nonlinear regression techniques. RESULTS: The OESS thickness was greater in patients with diabetes than in controls (562 +/- 35 microns versus 539 +/- 24 microns, P = 0.02). During hypoxia, the diabetic corneas swelled less (7.7% +/- 1.8% versus 9.9% +/- 1.6%, P < 0.001) and had less endothelial permeability (3.55 +/- 0.83 x 10(-4) cm/min versus 4.14 +/- 0.68 x 10(-4) cm/min, P = 0.02) than the controls. During normoxia after contact lens removal, however, diabetic and control corneas had similar deswelling rates and permeabilities. Corneal autofluorescence was increased in the patients with diabetes (8.1 +/- 3.1 versus 6.0 +/- 1.9 ng/ml fluorescein equivalents, P = .005). The endothelial cells of the two groups were morphologically similar. Within the group with diabetes, however, those with moderate nonproliferative retinopathy had larger coefficients of variation of cell area and smaller percentages of hexagonal cells than those with mild nonproliferative retinopathy. CONCLUSIONS: Although the diabetic corneas were thicker and more autofluorescent than control corneas, during hypoxia they swelled less and had decreased endothelial permeability. During normoxia, however, no difference was found in endothelial permeability or deswelling rate. The effects of diabetes on endothelial cell morphologic features appear to be related to the severity of the diabetes.
Subject(s)
Cell Membrane Permeability/physiology , Diabetes Mellitus, Type 1/physiopathology , Diabetes Mellitus, Type 2/physiopathology , Endothelium, Corneal/physiology , Adult , Aged , Biological Transport, Active/physiology , Diabetic Retinopathy/physiopathology , Endothelium, Corneal/pathology , Female , Fluorescein , Fluoresceins , Fluorophotometry , Humans , Hypoxia/physiopathology , Male , Middle Aged , Water-Electrolyte Balance/physiologyABSTRACT
PURPOSE: To investigate the telomere hypothesis of cellular aging as the mechanism for cell cycle arrest in normal human corneal endothelium. METHODS: The corneal endothelium and epithelium from 21 human corneas from 13 donors 5 weeks to 84 years of age were dissected and frozen at -70 degrees C. Purified DNA, digested with the restriction enzyme, HinfI, was run on 0.7% agarose gels, probed with radiolabeled (AATCCC)4, and exposed to a phosphor screen. The length of the terminal restriction fragment (TRF) was determined by densitometry. RESULTS: The cells of the corneal endothelium had TRF lengths ranging from 11.0 to 14.0 kbp (mean, 12.2 +/- 0.9). Corneal epithelial specimens showed TRF lengths that were always less than (mean, 10.4 +/- 1.0; range 9.0-12.0) the corresponding endothelial TRF lengths. Human corneal endothelial cells, transformed with human papillomavirus type 16 oncogenes E6 and E7, showed decreasing TRF lengths from 11 kbp at population doubling level (PDL) 15 to 9.5 kbp at PDL 73. Neither the endothelial and epithelial cells from human donors nor the transformed pre-immortalized human endothelial cells showed evidence of telomerase activity. CONCLUSIONS: Human corneal endothelial cells have long telomeres throughout life. Their limited replicative ability does not appear to result from critically short telomere lengths.
Subject(s)
Aging/physiology , DNA/analysis , Endothelium, Corneal/physiology , Telomere/physiology , Adolescent , Adult , Aged , Aged, 80 and over , Cell Cycle/physiology , Cellular Senescence/physiology , Electrophoresis, Agar Gel , Epithelium, Corneal/physiology , Female , Humans , Infant , Male , Middle Aged , Telomere/genetics , Tissue DonorsABSTRACT
PURPOSE: To assess corneal structure and the effects of acute hyperglycemia on corneal function in subjects with type 1 diabetes. METHODS: Twenty-one diabetic and 21 nondiabetic volunteers of similar age were recruited. Baseline measurements of intraocular pressure (IOP), corneal thickness (CT), corneal autofluorescence (CAF), corneal sensitivity (CST), central and temporal endothelial cell density (DenC and DenT), and coefficient of variation in cell area (CVC and CVT) were taken. Corneal edema was induced, and the percent recovery per hour (PRPH) from hypoxic edema and endothelial permeability to fluorescein were determined. These procedures were done twice in the diabetic subjects under controlled euglycemic (EG) and hyperglycemic (HG) conditions, and once in control subjects while they were fasting. RESULTS: Substantial differences in baseline measurements were found for IOP, CT, CAF, CST, DenC, and CVT. The mean +/- SE corneal swelling in the HG diabetic subjects (51.6 +/- 2.3 microm) was less when compared to the swelling in the EG diabetic subjects (56.2 +/- 1.87 microm, P = 0.05) and the control subjects (58.9 +/- 1.56 microm, P = 0.011). During euglycemia, the mean +/- SE PRPH was less in diabetic subjects than in control subjects (65.0 +/- 3.20 versus 73.8 +/- 1.81%/hour, P = 0.02) but did not differ in diabetic subjects under EG and HG conditions (65.0 +/- 3.20 versus 67.7 +/- 3.1%/hour, P = 0.56). No significant differences were noted between groups in endothelial permeability. CONCLUSIONS: In addition to differences in baseline corneal structure, diabetic subjects showed less corneal swelling and reduced corneal recovery from hypoxia than did control subjects. During acute hyperglycemia, corneal swelling was less than during euglycemia in diabetic subjects, which suggests that hyperglycemia affected corneal hydration control.
Subject(s)
Blood Glucose , Cornea/physiopathology , Diabetes Mellitus, Type 1/physiopathology , Diabetic Retinopathy/physiopathology , Acute Disease , Adult , Blood Glucose/physiology , Cell Count , Cell Size , Cornea/metabolism , Cornea/pathology , Diabetes Mellitus, Type 1/blood , Diabetic Retinopathy/blood , Endothelium, Corneal/metabolism , Endothelium, Corneal/pathology , Female , Fluorophotometry , Humans , Hyperglycemia/blood , Hyperglycemia/physiopathology , Intraocular Pressure , Male , Middle Aged , Permeability , Sensation/physiologyABSTRACT
PURPOSE: To assess the effects of dorzolamide hydrochloride, a topical carbonic anhydrase inhibitor, on corneal endothelial function. METHODS: The authors measured the rate of corneal deswelling and the endothelial permeability to fluorescein after 2 hours of hypoxic contact lens wear in 19 normal human subjects. The study was double-masked; one eye of each subject was randomly assigned to receive 2% dorzolamide drops, and the other eye received placebo drops every 8 hours for 24 hours before the study day and twice during the study day. RESULTS: Dorzolamide-treated eyes were not significantly different from placebo-treated eyes in corneal deswelling rate, expressed as the percent recovery per hour (55.7% +/- 13.6% versus 59.6% +/- 14.5%; P > or = 0.10), open eye steady state thickness, swelling induced by hypoxia, and corneal autofluorescence. Endothelial permeability to fluorescein was increased in the dorzolamide eyes (4.40 +/- 0.84 x 10(-4) cm/minute versus 4.10 +/- 0.80 x 10(-4) cm/minute; P = 0.01). As expected, the intraocular pressure and aqueous humor flow rate were decreased in the dorzolamide eyes. CONCLUSIONS: Dorzolamide hydrochloride, when topically administered to normal human eyes for 24 hours, had no significant effect on the corneal deswelling rate after hypoxic stress. The corneal endothelial permeability to fluorescein, however, was increased by the drug, although this did not result in increased corneal thickness.
Subject(s)
Carbonic Anhydrase Inhibitors/pharmacology , Endothelium, Corneal/drug effects , Endothelium, Corneal/physiology , Sulfonamides/pharmacology , Thiophenes/pharmacology , Adult , Aqueous Humor/drug effects , Aqueous Humor/metabolism , Carbonic Anhydrase Inhibitors/administration & dosage , Corneal Edema/etiology , Corneal Edema/physiopathology , Double-Blind Method , Fluorescein/metabolism , Fluorophotometry , Humans , Hypoxia/complications , Hypoxia/physiopathology , Intraocular Pressure/drug effects , Intraocular Pressure/physiology , Microscopy , Middle Aged , Ophthalmic Solutions , Permeability , Sulfonamides/administration & dosage , Thiophenes/administration & dosageABSTRACT
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.
Subject(s)
Corneal Stroma/cytology , Microscopy, Confocal/methods , Algorithms , Animals , Cell Count , Fibroblasts/cytology , Image Processing, Computer-Assisted , Rabbits , Reproducibility of ResultsABSTRACT
PURPOSE: To measure the concentration of ascorbic acid in the human corneal epithelium. METHODS: Corneal epithelium was removed from postmortem eyes 4 to 16 hours after death and ascorbate measured by high-performance liquid chromatography. RESULTS: The concentration of ascorbate was 1.33 +/- 0.48 mg/gm wet weight (mean +/- SD), estimated to be 14 times its concentration in the aqueous humor. CONCLUSIONS: Ascorbate can protect the basal layer of the epithelium by absorption of incident ultraviolet radiation.
Subject(s)
Ascorbic Acid/analysis , Epithelium, Corneal/chemistry , Adult , Aged , Aged, 80 and over , Aqueous Humor/chemistry , Chromatography, High Pressure Liquid , Female , Humans , Male , Middle AgedABSTRACT
Several studies have suggested differences in aqueous humor composition in Fuchs' dystrophy, including elevations in fibrinogen-related factors, compared with controls. In the current study, aqueous humor was obtained at surgery from 10 uninflamed eyes with advanced Fuchs' dystrophy and cataracts and 11 control eyes with cataracts alone. Total fibrinogen-related antigen was measured in a masked manner using an enzyme-linked immunosorbant assay (ELISA). There was no statistically significant difference between the means of the two groups (0.281 +/- 0.292 (SD) and 0.176 +/- 0.090 mg/ml, respectively). Similarly, there was no difference in ELISA-determined aqueous humor small molecular weight fibrinogen-derived metabolites between the two groups, 623 +/- 141 and 550 +/- 55 fibrinogen equivalents, respectively. Also, no statistically significant difference was detected between Fuchs' dystrophy and control eyes in aqueous humor ascorbate, glucose, carbon dioxide, bicarbonate, and pH. Therefore, this study found no evidence of alterations in aqueous humor composition in Fuchs' dystrophy and supports the hypothesis that the disease is a primary disorder of the corneal endothelium.
Subject(s)
Aqueous Humor/analysis , Corneal Dystrophies, Hereditary/metabolism , Fuchs' Endothelial Dystrophy/metabolism , Antigens/analysis , Cataract/complications , Cataract/metabolism , Fuchs' Endothelial Dystrophy/complications , Fuchs' Endothelial Dystrophy/immunology , HumansABSTRACT
PURPOSE: (1) To test the hypothesis that corneas with enlarged endothelial cells (and thus less intercellular space) have decreased endothelial permeability to small polar solutes. (2) To measure corneal endothelial ouabain binding (Na+/K+ ATPase "pump site" density) and Descemet's membrane production after endothelial wounding. METHODS: Bilateral specular microscopy and anterior segment fluorophotometry were performed at 2-month intervals for 1 year in ten cats after mechanically damaging the corneal endothelium in one eye of each. The measurements were repeated at 2 years in four cats and at 3 years in two cats. Eighteen months after wounding, endothelial ouabain binding was measured in both eyes of six cats. Transmission electron micrographs of Descemet's membrane were analyzed in both eyes of six cats at 18 months, two cats at 2 years, and two cats at 3 years after wounding. RESULTS: From 6 to 12 months after wounding, the endothelial permeability to carboxyfluorescein was significantly decreased (P < 0.05), and the mean endothelial cell size was significantly increased (P < 0.001) in the damaged eyes. The enlarged endothelial cells persisted in the few cats observed 2 and 3 years after wounding. There was no significant difference in endothelial ouabain binding between the damaged and control corneas in six cats tested 18 months after wounding. On subsequent histologic examination, a layer of abnormal Descemet's membrane was present in all ten wounded eyes, with additional normal Descemet's membrane posterior to it, between the abnormal layer and the endothelial cells. CONCLUSIONS: The results are consistent with the hypothesis that corneal endothelial permeability to small polar solutes varies directly with the amount of intercellular space available for diffusion across the monolayer. The results also confirm clinical reports of decreased endothelial permeability in corneas with enlarged endothelial cells. In histopathologic specimens, a layer of abnormal Descemet's membrane can be a historical marker for a period of endothelial damage and corneal decompensation.
Subject(s)
Endothelium, Corneal/pathology , Eye Injuries/pathology , Wound Healing/physiology , Animals , Cats , Cell Count , Cell Membrane Permeability , Descemet Membrane/ultrastructure , Endothelium, Corneal/injuries , Endothelium, Corneal/physiopathology , Eye Injuries/physiopathology , Fluoresceins/metabolism , Fluorophotometry , Longitudinal Studies , Ouabain/metabolism , Sodium-Potassium-Exchanging ATPase/metabolismABSTRACT
Maintenance of high autopsy rates is associated with specific benefits, especially for clinical practice and for clinical and epidemiologic research. We have compiled and evaluated (on the basis of related costs and benefits) a comprehensive list of recommendations to resurrect the autopsy and reestablish it as a central contributor to medical practice, teaching, and research.