Automated assessment of keratocyte density in clinical confocal microscopy of the corneal stroma.

Cell density in the corneal stroma is typically determined by counting the number of bright objects, presumably keratocyte nuclei, in images from clinical confocal microscopy. We present a program that identifies bright objects and counts those that most likely represent cells. Selection variables were determined from 125 normal corneas with cell densities that had been assessed manually. The program was tested on 17 corneas of patients before and at several intervals to 5 years after laser in situ keratomileusis (LASIK) surgery. In these corneas, which showed a decrease in cell density after surgery, the program identified cells as well as human observers did.

Analysis of postoperative glare and intraocular lens design.

PURPOSE: To assess the potential for reflected glare images from commonly used intraocular lens (IOL) materials and designs. SETTING: Mayo Clinic, Rochester, Minnesota, USA. METHODS: The interaction of reflected light rays from 3 commonly used IOLs (Bausch & Lomb LI61U and P359UV; Alcon AcrySof(R) MA60BM) with different optic designs (equi-biconvex: 10.0 and 15.0 mm anterior radius of curvature; unequal biconvex: 32.0 mm anterior radius of curvature) and optic materials (silicone, poly[methyl methacrylate], and acrylic) were examined in an eye model using the Zemax optical design program. The potential of each IOL model to produce subjective glare was determined from the size of the defocused reflected glare image at the retina. RESULTS: The unequal biconvex design concentrated reflected light on a retinal area that was 60-fold smaller than that of the equi-biconvex design. Increasing the refractive index of the IOL material from 1.43 (silicone) to 1.55 (acrylic) increased the amount of reflected light 5-fold. Compared to an equi-biconvex design composed of a lower refractive index material, the unequal biconvex design with a higher refractive index material increased the relative intensity of reflected light at the retina 300-fold, and for eyes with low corneal power the intensity increased 3500-fold. Similarly, for external glare apparent to an outside observer, the intensity of reflected light increased 400-fold and for low corneal power it increased 6000-fold. CONCLUSION: An unequal biconvex IOL design (32.0 mm anterior radius of curvature) composed of a higher refractive index material increased the potential for postoperative glare and external reflections.

Laser pointers and the human eye: a clinicopathologic study.

We report the absence of photic retinal injury after exposing the retina to light from class 3A laser pointers for durations of up to 15 minutes. Three patients with uveal melanomas were scheduled to have an enucleation. Each agreed to have his or her retina exposed to laser light from a class 3A laser pointer prior to enucleation. Continuous exposure was directed to the fovea for 1 minute, to the retina 5 degrees below fixation for 5 minutes, and to the retina 5 degrees above fixation for 15 minutes. Ophthalmoscopic evaluation of the cornea, lens, and retina and fluorescein angiographic studies of the retina were conducted before, 24 hours after, and 11 days after laser exposure in the first case; before and 86 hours after exposure in the second case; and before, 96 hours after, and 15 days after exposure in the third case. Other than transient afterimages that lasted only a few minutes, we were unable to document any functional, ophthalmoscopic, fluorescein angiographic, or histologic evidence of damage to any structures of the eyes. Transmission electron microscopic studies of retinal sites targeted by the laser pointers in the second and third cases revealed ultrastructural abnormalities in the outer retina and the pigment epithelium that were similar to abnormalities seen in the retina approximately 8 mm away from the targeted sites. The risk to the human eye from transient exposure to light from commercially available class 3A laser pointers having powers of 1, 2, and 5 mW seems negligible.

Ascorbic acid content of human corneal epithelium.

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.

Latrunculins' effects on intraocular pressure, aqueous humor flow, and corneal endothelium.

PURPOSE: To determine the effects of latrunculin (LAT)-A or -B on intraocular pressure (IOP), aqueous humor flow (AHF), anterior chamber (AC) protein concentration ([protein]AC), corneal endothelial permeability and morphology, and corneal thickness in living cynomolgus monkeys. METHODS: Topical LAT-A or LAT-B was administered to one eye, and vehicle to the other. IOP was measured by Goldmann tonometry, AHF and corneal endothelium transfer coefficient (ka) by fluorophotometry, [protein]Ac by Lowry assay, corneal endothelial cell morphology by specular microphotography, and corneal thickness by ultrasound pachymetry. RESULTS: LAT-A began to lower IOP at 6 hours and maximally reduced IOP by 4.6 mm Hg at 9 hours. LAT-B lowered IOP within 1 hour and maximally reduced IOP by 3.1 mm Hg at 6 hours. LAT-A increased AHF by 87% for 3 hours and increased ka by 94% over 6 hours; LAT-B increased ka by 39% over 6 hours without affecting AHF. LAT-A increased IV fluorescein entry into the cornea approximately 10 fold, but did not affect IV fluorescein entry into the AC. LAT-A increased [protein]AC by 25% at 2 hours but not 5.5 hours. LAT-B variably and insignificantly increased [protein]AC: at 1 hour but not at 6.5 hours. LAT-A induced extensive corneal endothelial pseudoguttata within 1 hour, with normal cell counts by 7 days. LAT-B increased central corneal thickness maximally by 47 microm at 3.5 hours. CONCLUSIONS: LAT-A and -B significantly reduced IOP and were consistent in their facility-increasing effect, indicating that pharmacologic disorganization of the actin cytoskeleton in the trabecular meshwork by latrunculins may be a useful antiglaucoma strategy. However, effects on corneal endothelium or ciliary epithelium are a potential safety issue.

Comparison of recording systems and analysis methods in specular microscopy.

PURPOSE: To compare corneal endothelial cell images from contact and automated noncontact specular microscopes and to compare endothelial image analysis by the Konan Robo Center Method and the Bio Optics Bambi Corners Method. METHODS: Twenty-six normal corneas of 13 subjects and 41 penetrating keratoplasties (PKs) of 38 patients were photographed with a Keeler-Konan contact specular microscope and a Konan Noncon Robo automated noncontact specular microscope. (i) After measuring and calibrating the magnification of each instrument, we digitized the cellular apices and analyzed the images from both instruments by using the Corners Method modified to accept x and y calibrations. (ii) Using the internal calibration marks of the Konan Noncon Robo specular microscope for calibration of magnification (as required for the Center Method), we evaluated identical cells on images from this microscope by both the Center Method and the Corners Method. (iii) We evaluated the reproducibility of both methods by repeating measurements on the same image. RESULTS: (i) When the images were properly calibrated for magnification by using an external scale, endothelial cell density (ECD) of normal corneas was 2,703 +/- 354 (mean +/- SD) cells/mm2 by contact and 2,685 +/- 357 cells/mm2 by noncontact techniques (p = 0.51). ECD of PK corneas was 1,767 +/- 773 cells/mm2 by contact and 1,807 +/- 775 cells/mm2 by noncontact techniques (p = 0.31). (ii) When images from the Konan Noncon Robo specular microscope were calibrated for magnification on the internal marks, the measured ECD from the same noncontact photographs was 6% less (p < 0.001). ECD was then 2,519 +/- 294 cells/mm2 (means +/- SD) by the Center Method and 2,523 +/- 305 cells/mm2 by the Corners Method (p = 0.55) in normal corneas and 1,715 +/- 748 cells/mm2 by the Center Method and 1,731 +/- 763 cells/mm2 by the Corners Method (p = 0.04) in PK corneas. (iii) The coefficient of variation of repeated measurements on the same normal image was 0.0025 for the Centers Method and 0.0099 for the Corners Method. CONCLUSIONS: (i) Images from the automated noncontact specular microscope may be used interchangeably with those from the contact specular microscope to measure ECD, but only if both are properly calibrated by measuring an external scale. (ii) As a method of analysis, the Center Method is equivalent to the Corners Method in normal corneas, but the proprietary internal calibration of the Center Method, which is required for its use, yields ECDs approximately 6% less than when an external scale is used for distance calibration. (iii) Cell density measurements by both the Center Method and the Corners Method were reproducible within 1%.

A new video pachometer.

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.

Comparison of effects of topical ibopamine and epinephrine on the circadian rhythm of intraocular pressure of the rabbit eye as measured by telemetry.

The purpose of this study was to measure the effect of topical ibopamine and epinephrine on intraocular pressure in undisturbed rabbits. Six pigmented rabbits were outfitted with a telemetric transducer system connected via catheter to the vitreous cavity of one eye for continuous monitoring of intraocular pressure. After entrainment to a 12 hour light, 12 hour dark cycle and after stabilization of the circadian rhythm of intraocular pressure, the animals were tested with 2% ibopamine and 2% epinephrine. Each drug was instilled during the light phase and during the dark phase of the circadian cycle. When administered during the light phase, both drugs caused a transient pressure rise followed by prolonged hypotension. When administered during the dark phase, neither drug caused a pressure rise but both drugs caused prolonged hypotension. It was concluded that ibopamine and epinephrine cause identical intraocular pressure changes in the normal rabbit eye. The effect was dependent on the timing of administration during the circadian cycle. Since ibopamine is a pro-drug of deoxyepinephrine (N-methyl dopamine, epinine), its effects are assumed to be due to this metabolite, a metabolite that is structurally similar to epinephrine.

Estimation of corneal endothelial pump function in long-term contact lens wearers.

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.

Automated quantification of keratocyte density by using confocal microscopy in vivo.

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.

Keratocyte density in vivo after photorefractive keratectomy in humans.

PURPOSE: To determine changes in keratocyte density in central human corneas in vivo after photorefractive keratectomy (PRK). METHODS: Fifteen patients (25 eyes) received excimer PRK (VISX Star) with epithelial removal by laser-scrape (43 microns ablation followed by manual scrape) to correct myopia between -1.5 D and -7.25 D. Corneas were examined by using confocal microscopy in vivo before PRK and at 1 day, 5 days, 1 month, and 3 months after PRK. A custom automated image-processing algorithm identified bright objects (keratocytes) against a dark background and estimated keratocyte density by using the number and size of the objects. Cell density was quantified in anteroposterior stromal regions after PRK and compared to cell density in corresponding pre-PRK regions. RESULTS: One day after PRK, keratocyte density increased 9% in the anterior third of the stroma (pi = .003), was unchanged in the middle third of the stroma (pi = .481), and decreased 6% in the posterior third of the stroma (pi = .038). Keratocyte density remained elevated in the anterior stroma to 3 months after PRK; at this time, there was a 13% increase in keratocyte density throughout the full-thickness stroma (pi < .001). CONCLUSIONS: Keratocyte density was increased in the anterior stroma immediately after PRK in humans. Three months later, keratocyte density was increased in all anteroposterior stromal regions, suggesting that PRK affects keratocytes throughout the entire central cornea.

Books and Software: Getting serious about ICPMS.

A review of Inductively Coupled Plasma Mass Spectrometry.

Measuring oxygen tension in the anterior chamber of rabbits.

PURPOSE: Measuring the concentration of oxygen in the aqueous humor without penetrating the eye would provide a new dimension in understanding aqueous humor and corneal dynamics. In this study a preinvasive method was developed for determining the cameral oxygen concentration in anesthetized rabbits by measuring the excited-state lifetime of a phosphorescent dye. METHODS: A scanning ocular fluorometer was designed to excite phosphorescence with a brief flash of light and to measure the decay of luminescence for as long as 1000 microsec after excitation. The measurement window was scanned through the depth of the anterior chamber or fixed at the mid-anterior chamber. A depot of the phosphorescent dye Pd-uroporphyrin was injected into the vitreous of eight pigmented rabbits, and within a few days the dye was measurable in the anterior chamber. The excited-state lifetime of this dye is inversely correlated to oxygen concentration and was calibrated by measuring the lifetime of dye in cuvettes equilibrated with oxygen-nitrogen mixtures. Oxygen tensions were determined from lifetimes measured in the open eye, under a polymethylmethacrylate (PMMA) contact lens, under two oxygen-permeable contact lenses, and immediately after lid closure. RESULTS: Oxygen tension in the mid-anterior chamber before placing a PMMA contact lens was 23 +/- 3 mm Hg (mean +/- SD; n = 6). After 20 minutes of PMMA lens wear, oxygen tension decreased to 4 +/- 2 mm Hg. When the focal diamond was scanned through the anterior chamber, oxygen tension was 24 +/- 5 mm Hg near the corneal endothelium and decreased to 17 +/- 8 mm Hg near the crystalline lens. Under the PMMA contact lens this gradient reversed: Oxygen tensions near the endothelium and lens were 3 +/- 2 mm Hg and 6 +/- 2 mm Hg, respectively. Lid closure for 10 minutes or longer decreased the mid-anterior chamber oxygen tension from 21 +/- 2 mm Hg (n = 19 measurements from seven animals) to 10 +/- 3 mm Hg (n = 15 measurements from five animals). CONCLUSIONS: Measuring excited-state lifetime of phosphorescent dyes in the anterior chamber provides a useful method for determining oxygen concentration in vivo, without penetrating the eye. Cameral oxygen tension under PMMA contact lenses are significantly lower than in the uncovered eye. The profile of oxygen tension through the anterior chamber suggests that oxygen is supplied transcorneally to the aqueous humor.

Effect of dorzolamide on corneal endothelial function in normal human eyes.

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.

Response of the pupil to tropicamide is not a reliable test for Alzheimer disease.

OBJECTIVE: To confirm the putative hypersensitivity of the pupil to a weak mydriatic in persons with Alzheimer dementia. DESIGN: Twenty patients with Alzheimer dementia and 20 control subjects were examined. Automated binocular infrared pupillography was performed in the dark after instillation of 0.01% tropicamide or placebo. Ocular penetration of eye drops was assessed simultaneously using 2% fluorescein sodium as a tracer. SETTING: Rochester, Minn. SUBJECTS: Twenty patients and 20 cognitively normal control subjects from the Alzheimer's Disease Patient Registry of the Mayo Clinic, Rochester, Minn. MAIN OUTCOME MEASURE: Percent change in the diameter of the pupil following topical ocular instillation of a diluted concentration of the mydriatic drug tropicamide and penetration of topically applied fluorescein into the aqueous humor. RESULTS: No statistically significant difference was found between patients with Alzheimer disease and control subjects in either the mydriatic response of the pupil or in the rate of penetration of topically applied fluorescein. CONCLUSION: No evidence of pupillary hypersensitivity to an anticholinergic mydriatic drug was found in patients with Alzheimer disease or any evidence that this putative hypersensitivity could be used as an early, simple diagnostic test for Alzheimer disease.

Comparison of dorzolamide and acetazolamide as suppressors of aqueous humor flow in humans.

OBJECTIVE: To compare the efficacy of topical 2% dorzolamide hydrochloride (Trusopt) as a suppressor of aqueous humor flow in the human eye with the efficacy of systemically administered acetazolamide (Diamox). DESIGN: A randomized, double-masked, placebo-controlled study of 40 human subjects in 2 academic centers. The effect of dorzolamide on aqueous humor flow was compared with that of acetazolamide as measured by the rate of clearance of topically applied fluorescein. RESULTS: Acetazolamide reduced aqueous flow from 3.18 +/- 0.70 (mean +/- SD) to 2.23 +/- 0.48 microL per minute, a reduction of 30% (P < .001), and dorzolamide reduced the flow to 2.65 +/- 0.64 microL per minute, a reduction of 17% (P < .001). The difference between the effect of acetazolamine and dorzolamide was significant (P < .001). When acetazolamide is added to dorzolamide, the aqueous flow was reduced further to 2.21 +/- 0.47 microL per minute, an additional reduction of 16% (P < .001). When dorzolamide was added to acetazolamide, no additional reduction was observed (P = .73). Similar effects were observed for intraocular pressure. Acetazolamide reduced pressure from 12.5 +/- 2.2 (mean +/- SD) to 10.1 +/- 2.2 mm Hg, a decrease of 19% (P < .001) and dorzolamide reduced it to 10.8 +/- 2.1 mm Hg, or a decrease of 13% (P < .001). The greater effect of acetazolamide than dorzolamide was significant (P = .03). CONCLUSIONS: For reasons that are not known, the topically applied carbonic anhydrase inhibitor 2% dorzolamide hydrochloride is not as effective as systemically administered acetazolamide. Clinicians who prescribe dorzolamide should expect less of an ocular hypotensive effect than that experienced from systemically administered acetazolamide.

Continuous measurement of intraocular pressure in rabbits by telemetry.

PURPOSE: Intraocular pressure (IOP) is dynamic and can be influenced by the use of tonometers. The authors developed a technique to implant a telemetric pressure transducer to measure IOP continuously in unrestrained rabbits. METHODS: A commercial telemetric pressure transducer was implanted subcutaneously on the dorsal neck, between the scapulae, of pigmented rabbits. A fluid-filled catheter that conducts pressure to the transducer was routed to the orbit subcutaneously and implanted in the anterior chamber through a limbal opening. The transducer measured pressure and broadcast this information by amplitude modulation radio to a receiver in the animal's cage. Data were recorded at a rate of 50 or 100 samples per second for 60 seconds to study transient changes in IOP to tonometry, or for 15 seconds every 2.5 minutes to study circadian behavior of IOP. RESULTS: Intraocular pressure was recorded from seven rabbits for 180 to 370 days. Within 3 to 8 days of implant surgery, IOP began to follow a circadian rhythm: IOP decreased after room lights were turned on at 00:00 CT and increased after they were turned off at 12:00 CT. (The term CT refers to circadian time and begins at 00:00 CT when lights are turned on.) The maximum difference in IOP between light and dark phases ranged from 6.4 mm Hg to 16.6 mm Hg. When the lighting cycle was advanced by 6 hours, the time of nocturnal rise in IOP also advanced, but it did so gradually over 10 to 14 days. CONCLUSIONS: The implanted pressure transducer provides a convenient, preinvasive method to measure and study IOP in unrestrained experimental animals. This technique will be used to study circadian variations in IOP, the effects of environmental stimuli, and the oculohypotensive effects of therapeutic agents.

The effects of sleep on circulating catecholamines and aqueous flow in human subjects.

We measured the rate of aqueous flow and analysed its relation to the time of day, the state of wakefulness and the urinary excretion of catecholamines. Two groups of subjects were studied. One group comprised 20 normal subjects who were studied over two 22-hr periods. During one period, the subjects were permitted to sleep during their customary hours of sleep; during the other, they were not permitted to sleep, but remained active for all 22 hr. The other group comprised ten subjects with obstructive sleep apnea who were studied over a 22-hr period and slept during their customary hours of sleep but without the aid of any respiratory device. Aqueous flow was measured with fluorophotometry. Motion of the wrist was monitored by a seismograph (wrist Actigraph) and served as a surrogate of activity and wakefulness. Urinary catecholamine excretion was measured during different periods of the wake/sleep cycle. Both groups exhibited the normal nocturnal suppression of flow (59% lower compared to morning in the normal group; 56% lower compared to morning in the apneic group). During sleep deprivation, the rate of flow at night in normal subjects was 30% lower than during the morning (P < 0.001) and 60% higher than during sleep (P < 0.001). Lid closure during sleep deprivation had no effect on the results. Aqueous flow correlated with a 'catecholamine index', derived from the combined excretion of epinephrine and norepinephrine. Flow also correlated with an 'activity index', and 'sleep efficiency', indices derived from motion of the wrist. We conclude that the day-night difference of aqueous humor flow as measured by clearance of fluorescein from the human eye is driven partly by a factor that has a circadian rhythm and partly by a factor that depends on the activity of the subject. We hypothesize that these factors are the catecholamines, epinephrine and norepinephrine.

Measurement of transmission of ultraviolet and visible light in the living rabbit cornea.

Corneal transmittance in pigmented rabbits was measured at ultraviolet and visible wavelengths from the ratio of fluorescence of dyes in the anterior chamber to fluorescence of the same dyes in a quartz cuvette. Aqueous humor was drained through a limbal incision and the anterior chamber was reformed with a mixture of a viscoelastic material and a fluorophore (fluorescein, O-methyl pyranine, or sulforhodamine B). Excitation spectra, emission spectra, or both were measured in the anterior chamber with a new scanning ocular fluorophotometer, at wavelengths between 250 nm and 700 nm. Fluorescence spectra were also measured from the same fluorophore in a quartz cuvette. External transmittance of the cornea was calculated at each wavelength from the ratio of fluorescence in the anterior chamber to fluorescence in the cuvette. Transmittance was 93% +/- 1.4% (mean +/- SD, n = 8 rabbits) at 500 nm and was 89% to 93% between 370 nm and 500 nm. Transmittance decreased to 82% +/- 5.7% at 350 nm, to 50% +/- 5.9% at 310 nm, and to less than 2% at 290 nm. Between 370 nm and 500 nm, the wavelength range most frequently used in fluorophotometry, average transmittance varied by less than 5%. These results suggest that fluorescence measured at two or more wavelengths within this spectrum needs little if any correction for differential attenuation by the cornea. At wavelengths shorter than 370nm, measurements should be corrected. This technique provides a simple and minimally invasive means of studying visible and ultraviolet transparency of the cornea in the living eye.

A comparison of two methods of measuring aqueous flow in humans: fluorophotometry and flare measurement.

The technique of Anjou and Krakau for measuring flare and Krakau's method for estimating aqueous humor flow in humans was compared to the fluorophotometric procedure of Jones and Maurice. The precision of flare was measured in 20 normal human volunteers, and the precision of flow was measured with fluorophotometry in 24 volunteers. In addition, the circadian rhythms of both flow and flare were measured simultaneously in a separate group of 20 subjects. This simultaneous measurement allowed us to calculate the entry rate of scattering substances into the aqueous humor as a function of time of day. This entry rate is critical for measuring flow by the method of Krakau. The mean coefficient of variation (+/- SD) of repeated measurements of flare at the same time of day was 23.2% +/- 10.3%. The mean coefficient of variation of the measurement of aqueous flow by fluorophotometry was 15.9% +/- 8.2%, significantly better than the measurement of flare (p = 0.01). The daytime entry rate of scattering substances into the aqueous humor varied from 1.39 +/- 0.86 microgram/min (calculated as albumin) to 1.05 +/- 0.58 microgram/min. The rates in daytime hours were not significantly different from each other. At night during sleep, the entry rate was 0.67 +/- 0.48 microgram/min, significantly slower (p between 0.002 and < 0.001). These data indicate that the flare technique cannot be used alone to calculate the circadian rhythm of flow and that direct measurement of flow by fluorescein clearance is likely to be the most repeatable and accurate of the two.