Noninvasive measurements of pyridine nucleotide fluorescence from the cornea.

The autofluorescence of reduced pyridine nucleotides (NADH and NADPH) and oxidized flavoproteins within the rabbit cornea were noninvasively measured as a function of depth. This was accomplished by combining a corneal specular microscope with a time-shared spectrofluorometer. When either 8 mM sodium pentobarbital or sodium sulfide, known inhibitors of mitochondrial respiration were applied to cornea, the autofluorescence at 440 nm (excited at 366 nm) increased and that at 540 nm (excited at 460 nm) decreased. No autofluorescence was measurable following destruction of the cellular membranes by freezing and leaching of the cellular constituents. The 440 nm autofluorescence is from reduced pyridine nucleotides, whereas the 540 nm autofluorescence is from the oxidized flavoproteins. The time course of the pyridine nucleotide autofluorescence after the application of the pentobarbital to either the endothelial or epithelial bathing solutions made it possible to measure the diffusion properties of this drug through the cornea. The method used is useful studying the diffusion and effects of metabolically active drugs upon the cornea.

Noninvasive measurements of pyridine nucleotide and flavoprotein in the lens.

Abnormalities in glucose metabolism are thought to be among the main causes of cataract formation. The authors have made noninvasive biochemical measurements of the lens that provide information concerning glucose metabolism in the lens epithelium. The autofluorescence of reduced pyridine nucleotides (PN) and oxidized flavoproteins (Fp) within the rabbit lens were noninvasively measured as a function of depth using redox fluorometry. The peak of the autofluorescence at 440 nm (excited at 360 nm) and 540 nm (excited at 460 nm) were determined at the lens epithelium. When 8 mM sodium pentobarbital, a known inhibitor of mitochondrial respiration, was applied to the lens, the autofluorescence peak at 440 nm increased and that at 540 nm decreased. The 440 nm autofluorescence is thought to be from reduced pyridine nucleotides, whereas the 540 nm autofluorescence is from the oxidized flavoprotein. Blocking lens respiration with pentobarbital caused an increase in the PN/Fp ratio by a factor of 3 within 3.5 hr after pentobarbital application.

Correlation of redox fluorometry and analytical measurements of pyridine nucleotide.

Pyridine nucleotide levels in the corneal epithelium were measured using redox fluorometry, a noninvasive method for monitoring the metabolic status of corneal tissue, and a sensitive bioluminescent assay and an improved extraction procedure that allows the simultaneous extraction and measurement of both NADH and NAD. The same corneas were measured using each of the two methods to enable comparison of the results. The NADH/(NADH + NAD) fraction in the normal epithelium measured by the bioluminescent assay was 0.14 +/- 0.06. Incubation of corneas in 1 mM potassium cyanide (KCN) to mimic the anoxic state increased the NADH/(NADH + NAD) fraction significantly to 0.24 +/- 0.03 (P less than 0.001). The autofluorescence from reduced pyridine nucleotide measured by redox fluorometry also increased with KCN from 2840 +/- 605 to 5147 +/- 738 (P less than 0.0001). A plot of the fluorescence and analytical data for each cornea showed a positive correlation between the two methods, with a correlation coefficient (r value) of 0.80. The correlation was improved but was not dependent on the high values of the KCN treated corneas; an r value of 0.73 was obtained for the non-KCN treated corneas alone. Additional measurements of the temperature dependence of the fluorescence intensity of an NADH solution and the cornea gave a decrease in intensity of 17% from 25 degrees C to 35 degrees C for the NADH solution and 11% (P = 0.0004) for the reduced pyridine nucleotide fluorescence in the cornea over the same temperature range.

Metabolic and morphologic changes in the corneal endothelium. The effects of potassium cyanide, iodoacetamide, and ouabain.

The metabolic pathways of glycolysis and mitochondrial respiration in the corneal endothelial cell are the primary sources of the adenosine triphosphate (ATP) necessary to maintain endothelial structure and pump fluid to maintain the corneal stroma in its normally dehydrated and transparent state. The correlation between endothelial metabolism and morphology in rabbits was studied for 7 days after the application of three different agents: (1) iodoacetamide, used to inhibit ATP synthesis from both glycolysis and respiration; (2) potassium cyanide (KCN), used to inhibit ATP synthesis from respiration only; and (3) ouabain, used to inhibit fluid pumping but not ATP synthesis. After application of each of these three drugs to the corneal endothelium, changes in endothelial morphology were measured. The greatest change resulted from the use of iodoacetamide. Specular microscopic examination of the endothelium after the application of iodoacetamide showed progressive degradation of the integrity of the cellular structure; after 6 hr, there were no discernible cell borders. In those corneas treated with either KCN or ouabain, only minor changes in the endothelium were seen during the full 7 days of the investigation. Computer-assisted morphometric analysis showed an increase in the coefficient of variation of both cell area and perimeter in all cases. This increase was greater in the corneas treated with ouabain than those treated with either iodoacetamide or KCN. Redox fluorometry showed that the metabolic ratio (autofluorescence of reduced pyridine nucleotides divided by that of oxidized flavoproteins) decreased significantly in the iodoacetamide-treated corneas, increased significantly in the KCN group, and showed no significant change in the corneas in the ouabain group, all compared with a control group. The results showed that (1) when ATP produced by both glycolysis and respiration was inhibited by 0.1 mmol/l iodoacetamide, the endothelial cells could not survive, but (2) when ATP synthesis produced by respiration alone was inhibited by 1.0 mmol/l KCN, the cells could survive for at least 1 wk on the ATP produced by anaerobic glycolysis. Furthermore, the polymegathism seen after application of ouabain, a drug that is not believed to affect ATP synthesis but inhibits the endothelial pump function, is greater than that seen as a result of reduced pump function caused by inhibited respiration produced by 1.0 mmol/l KCN. Combining specular microscopy, computer-assisted morphometric analysis, redox fluorometry, and corneal pachymetry allowed correlations between corneal endothelial metabolism, pump function, and morphology to be studied.

Effects of EGF and indomethacin on rabbit corneal endothelial wound closure in excised corneas.

Corneal endothelial cells of rabbit corneas stored in M-K medium at 37 degrees C were wounded by touching them lightly with a micropipet under video specular microscope observation. Three groups were studied: control, with EGF, and with EGF + indomethacin. The wound closure process (initial wound area about 8500 microns 2) was observed and recorded with time-lapse videography for 6 hr. The recorded video images were digitized and computer assisted morphometric analysis was performed. (1) Addition of either EGF (10 ng/ml) + indomethacin (1 microM), or EGF (10 ng/ml) alone to the M-K medium statistically significantly shortened the wound closure time as compared with the control group. (2) Both EGF + indomethacin and EGF alone resulted in a greater average percent relative change of the shape factor, more than three times greater with EGF + indomethacin and more than two times greater with EGF alone, than in the control group 150 min after wounding. (3) The maximum cell shape change occurred at about 150 min after wounding in the groups EGF + indomethacin and EGF alone, and at about 200 min in the control group. After this time in all three groups the cells began to approach a normal shape. (4) The cells near the wound boundary moved faster in the EGF + indomethacin and the EGF groups as compared with the control group. These results suggest that EGF and indomethacin may be of therapeutic value in promoting closure of traumatized human corneal endothelium.

Thermal cycling effects on the stored rabbit cornea.

Specular microscopy has proven itself a useful tool for evaluation of donor corneas. In many eye banks, corneas are stored at 4 degree C and warmed to room temperature for specular microscopic evaluation. On occasion it would be desirable to put the cornea back into storage provided there were no detrimental effects of the recooling and subsequent rewarming. The effects of repeated cooling and rewarming (thermal cycling) on the corneal endothelium were determined using rabbit corneas stored in M-K medium at 4 degree C. Some corneas were left in the refrigerator for a 7-day storage interval while others were removed daily, warmed to room temperature, evaluated morphologically and biochemically with the specular microscope and the redox fluorometer, respectively, and then placed back in the refrigerator. At the end of the 7-day storage period there were no statistically significant differences in either the biochemical signals or the specular appearance between thermally cycled corneas and corneas that were continuously stored at 4 degrees C for the same period of time. Repeated warming, specular microscopic observation, and cooling of the cornea appear not to be detrimental to the corneal endothelium.

Automated morphometric analysis of corneal endothelial cells.

This paper describes a feasibility study for computer image analysis of corneal endothelial specular micrographs. The analysis involved image enhancement, boundary detection of individual cells, and subsequent calculation of cell area, cell area distribution, cell perimeter, cell orientation, and cell diameter. Cell areas produced by automated analysis agreed well with manually measured cell areas from the same photomicrographs. The computer methods required little human guidance or correction, produced accurate results, and could be made to run fairly quickly with modest computing resources.

Corneal endothelial changes following minor trauma.

The healing processes that occur when corneal endothelial cells are subjected to only mild trauma are not known. To study these processes we have developed a system that enables only a few endothelial cells to be traumatized or destroyed under continuous specular microscopic observation in vitro. Experiments in which a small group of cells were traumatized by gentle wounding with a microglass tip produced an immediate and distinct dark area having the same size as the tip. Using this method we have produced and observed two types of wound by controlling the force of wounding. The first type of wound, produced by a gentle touch, recovered within 1 hr. The second type of wound, produced by moderate touch, took about 24 hr to recover completely from the trauma. In the second type of wound, we observed migration, elongation, coalescence and mitosis during the healing process. Histological examination revealed that in the first type of wound, the cells remained intact with no apparent damage seen by vital staining and light microscopy. For the second type of wound, the cells were completely missing although there was no apparent damage to Descemet's membrane.

In vivo photomicrography of the crystalline lens.

We describe a technique and apparatus for observing and photographing the crystalline lens in vivo and in the enucleated eye. Magnifications of x25 to x100 are easily obtainable on the film plane. The method is suitable for diagnostic observation and clinical research on cataract formation and prevention in humans, as well as for animal experimentation studies.

Specular microscopic appearance of damaged and dead endothelial cells in corneas following short-term storage.

Specular microscopic evaluation of rabbit, pig, and cat corneas was performed after storage in a moist chamber in McCarey-Kaufman (M-K) medium or in tissue culture medium 199. Irrespective of the storage method, cooling at 4 degrees C resulted in a deterioration of the specular microscopic image. Most of the changes proved to be reversible if the tissue was quickly rewarmed to 35 degrees C and maintained at that temperature for 20 minutes. One change, dark areas larger than a single cell, was not reversible and increased in prevalence with increased storage time. Trypan blue staining revealed that the dark areas seen with the specular microscope contained damaged endothelial cells. Deep corneal striae tended to increase as the storage interval increased, and considerable cell damage occurred in the endothelium covering these folds. Storage in M-K medium was the most effective of the three methods in preventing deep corneal folds and endothelial damage.

Clinical specular microscopy. I. Optical principles.

The clinical specular microscope yields a corneal endothelial image. Depending on the slit width of the illumination source, a typical endothelial photomicrograph contains three or four distinct zones. The appearance of the boundary between the endothelial cell pattern and the adjacent dark zone, called the dark boundary, reflects the configuration of the endothelial cell-aqueous humor interface and provides important information about the posterior corneal surface.

Clinical specular microscopy. II. Qualitative evaluation of corneal endothelial photomicrographs.

The clinical specular microscope shows the morphological appearance of the endothelium in normal and abnormal corneas. This instrument resolves the endothelial mosaic of the normal cornea into a quasiregular pattern of contiguous cells having well-defined cell boundaries. Cell size varies over a wide range in a number of disorders, and endothelial cells may assume shapes that are substantially different from their usual hexagonal appearance. Cell boundaries are dark and most commonly appear as a straight, narrow line. However, other types of cell boundaries, collectively referred to as doubled boundaries, have been encountered. Cell boundaries normally intersect in a manner that results in three angles of intersection, each approximately 60 degrees, but variations from this pattern are seen. A number of noncellular structures also can be seen in the endothelial zone.

In vivo photomicrography of the corneal endothelium.

A technique and apparatus for observing and photographing the corneal endothelium in vivo at a magnification of approximately times 200 is described. The method is suitable for animal experimentation and for diagnostic observation and clinical research in humans.

Specular microscopy of corneal epithelium.

Specular microscopy was used to study the morphologic appearance of normal and regenerating rabbit corneal epithelium before and after corneal abrasion or heptanol application. Specular photomicrographs of the denuded area and healing epithelium were taken for two to three weeks as the epithelium regenerated, as well as prior to trauma. During the healing process the cells appeared much larger than normal and had an abnormal morphologic appearance. In addition, specular photomicrographs of the corneal epithelium in topically anesthetized human subjects were obtained. These results indicate that this method is suitable for diagnostic observation and clinical research in human subjects, as well animal experimentation.

Coalescence of endothelial cells in the traumatized cornea. I. Experimental observations in cryopreserved tissue.

After thawing, cryopreserved rabbit corneas were stained with trypan blue and cellular damage was evaluated with the specular microscope using indirect illumination. Thereafter, the cornea was maintained at 35 degrees C, and specular microscopy, performed from the endothelial side, was used to observe the same area of endothelium for several hours. Dead endothelial cells interfered with specular reflection and appeared as dark areas devoid of cellular definition. Viable cells, presumably damaged by freeze-thaw injury, began to effect repair within one hour after thawing and coalesced to form a single larger cell. Our photographic evidence suggests that coalescence of injured corneal endothelial cells represents an important step in the early repair process, resulting in a reduction of the total number of cells but enabling damaged cells to survive.

Coalescence of endothelial cells in the traumatized cornea. II. Clinical observations.

In the rabbit, cells coalesce to repair a damaged corneal endothelial layer. Clinical specular microscopy showed that this phenomenon also occurs in human beings. The resulting endothelial cells are large, irregularly shaped, and multinucleated. They are quite different in their specular microscopic appearance from corneal endothelial cells seemingly undergoing mitosis, which was observed in a successful penetrating keratoplasty and which represents another apparent mode of repair. Additional evidence for coalescence of endothelial cells is derived from evaluation of histograms of cell size v age that have been published previously.

Endothelial mosaic in Fuchs' dystrophy. A qualitative evaluation with the specular microscope.

The progressive morphological changes of cornea guttata in Fuchs' endothelial dystrophy have been characterized by clinical specular photomicroscopy. Five specific stages in the development of excrescences can be discerned using this in vivo technique. Several stages can be observed in the same cornea at a given time, although in most cases the majority of guttate changes seemed to have progressed to the same stage of development. All five stages of cornea guttata discerned in this study can occur in a cornea clinically free of edema. Two types of cornea guttata can be identified in vivo. One has a smooth, regular posterior surface, whereas the posterior contour of the second is irregular.

The human corneal endothelium in keratoconus: A specular microscopic study.

The corneal endothelium in 12 cases of keratoconus was examined with the clinical specular microscope. There appeared to be an increase in cellular pleomorphism with many cells considerably smaller than normal distributed throughout the endothelial cell population. There were also many large, elongated cells whose long axis showed a definite tendency to assume a similar directional orientation. The long axis of these cells seemed oriented toward the apex of the cone, and the cells appeared to have been stretched by the ectatic process. Many endothelial cells contained dark intracellular structures. Their significance is unknown. The single cornea in this series with a history of acute hydrops contained a localized area in which the endothelial cells were seven to ten times larger than normal. This suggests that rupture of the endothelium and Descemet's membrane, responsible for the acute edematous process, occurs at this site, and that the adjacent cells enlarged to fill the defect.

Epithelialization of the anterior chamber: clinical investigation with the specular microscope.

Four patients, each of whom had had an uncomplicated cataract extraction, were examined because of an apparent epithelialization of the anterior chamber. In each instance, the diagnosis was later verified histopathologically. The involved eye was photographed with the clinical specular microscope and the endothelial photomicrographs were analyzed. It was noted that considerable endothelial cell loss had occurred, as evidence by the larger size of the remaining cells. Endothelial cells were present but they were grossly abnormal well below the demarcation line visible with the slit-lamp biomicroscope. These in vivo observations support the thesis that damage to the corneal endothelium is a necessary factor for epithelial invasion of the anterior chamber.

Corneal edema secondary to vitreocorneal contact.

Aphakic eyes, some with edematous corneas, and others with corneas free of edema, but all with formed vitreous humor in contact with the corneal endothelium, were studied. Removal of vitreous humor from the anterior chamber by closed vitrectomy resulted in substantial improvement in the state of corneal hydration and, in some cases, in the elimination of clinically notable corneal edema. Specular microscopy showed endothelial abnormalities in the edematous corneas that were not present in corneas with vitreous contact that remained free of edema. However, those endothelial changes seen in edematous corneas before vitrectomy seemed to persist after vitrectomy and corneal deturgescence. Thus, elimination of vitreous contact may result in clinical reversal of corneal edema despite a prolonged period of vitreous contact and in the face of seemingly irreversible endothelial changes.