Michael J. Doughty

Long-term effects of a single dose of ultraviolet-B on albino rabbit cornea--I. in vivo analyses.

Abstract Both eyes of female albino rabbits (1.9 kg) were exposed to a single dose of UV‐B (300 ± 9 nm; 0.125 J/cm2 total dose) between 13.30 and 15.00 h. The average irradiance was 209 ± 4 μW/cm2 delivered over 612 ± 13 s. At various time periods thereafter (every 12 h for 3 days, 6, 7, 14, 28, 42, 56, 112, 224 and 336 days post‐irradiation), the animals were subjected to a full slit lamp examination to evaluate the status of the cornea and the anterior segment along with optical or ultrasonic pachometry of central corneal thickness. The results were compared with studies on age‐matched rabbits over the same time period. In response to the UV‐B irradiation, the corneas showed a modest edema (20% increase in central corneal thickness) that peaked at 48 h. Nearly normal central cornea] thickness returned in 6 days and followed by a secondary very slight swelling (< 5%) that resolved by 14 days. The edema was accompanied by keratitis over the same period. Thereafter, both control and UV‐B irradiated corneas progressively increased in thickness with age. Biomicroscopy also revealed the appearance of granular opacities in the corneal epithelium that peaked at 72–96 h and resolved over 28 days. In addition, very small microdot opacities of the corneal epithelium were present in the UV‐B irradiated corneas that reached maximum at 72 h but persisted to some degree throughout the evaluation period. Biomicroscopy also revealed a progressive disruption of the homogeneous nature of the corneal stroma by the appearance of large ‘bread crumb’‐like opacities that started at 72 h and was still present at the end of the evaluation period. These results suggest that long‐term evaluation of the cornea is important after acute UV‐B exposure and indicate that acute exposure to UV‐R can produce corneal changes resembling those reported following chronic exposure to UV‐R‐rich environments.

Evaluation of the effects of saline versus bicarbonate‐containing mixed salts solutions on rabbit corneal epithelium in vitro

Rabbit corneas were incubated, over 4 h in vitro, with the corneal epithelial surface exposed to various solutions to assess their utility as incubation solutions for physiological, pharmaceutical or toxicological studies. The corneal endothelium was perfused with a 35 mM bicarbonate-mixed salts solution equilibrated at 36 degrees C. Corneal thickness, corneal hydration or epithelial cell appearance (as assessed by scanning electron microscopy) were found to be similar to in vivo if a 35 mM bicarbonate, mixed salts solution (equilibrated with 5% CO2-air) was used for the epithelium. Some swelling (14 microns h-1), increased hydration and minor cell exfoliation were seen if this 35 mM bicarbonate solution was equilibrated with 5% CO2-95% O2 (hyperoxia). Solutions with only 5 mM bicarbonate (0.5% CO2-air) produced rapid swelling, large increases in hydration and marked cellular damage. Slightly hypertonic (310 mOsm kg-1) solutions containing 5 mM bicarbonate caused some swelling at 15 microns h-1, small increases in hydration and some cell damage but the swelling and cellular damage were further reduced by making the solution slightly more hypertonic (325 mOsm kg-1) by addition of NaCl and KCl. Saline (NaCl 0.9% or 0.97%) or phosphate-buffered saline (PBS) (300 mOsm kg-1) produced swelling at 21-28 microns h-1, 30% increases in hydration and almost total destruction of the superficial cell layers. These studies confirm in vivo experiments that saline (and also buffered saline solutions) are rather toxic to the corneal epithelium and thus should not be used as epithelial incubation solutions. Even when using mixed salts solutions and even with bicarbonate present, small differences in composition can have marked effects on corneal thickness, hydration or cell appearance. Hyperoxic solutions appear to be mildly cytotoxic compared with normoxic solutions.

Optical effects of UV-A and UV-B radiation on the cultured bovine lens

The effects of repeated exposures to UV-A (335 nm) and UV-B (305 nm) radiation on the crystalline lens were studied by treating cultured bovine lenses daily or weekly. The effects of irradiation on lens optical quality were monitored using an automated scanning laser system that records both relative transmittance and focal length across the lens. Relatively low radiant exposures of UV-B were used (0.06, 0.03, 0.01 J/cm2) compared to UV-A (1.44 J/cm2). In total, 38 treated lenses and 32 controls were cultured for times ranging from 400-1000 hours. Results indicate that this range of UV-B exposure may represent the threshold for in vitro UV-B induced opacification. Lenses treated weekly with 0.06 J/cm2 UV-B showed a significant decrease in transmittance compared to controls 69 hours after the first treatment and an increase in focal length variability. The ability of the lens to repair itself, as found in a previous single dose study, was absent after repeated doses. Lenses exposed daily to 0.03 and 0.01 J/cm2 UV-B showed no significant change in transmittance or focal length variability compared to controls. Daily exposure to 1.44 J/cm2 UV-A resulted in no significant change in transmittance or focal length variability compared to controls.

Analyses of areas and shapes of cells on the corneal surface of the albino rabbit by scanning electron microscopy.

The surface of 8 different corneal quadrants was assessed by scanning electron microscopy. Cells were identified by their electron reflex (light, medium, dark) and their areas, longest and shortest dimensions measured using a manual digitizer tablet/computer-interfaced system. Analyses of over 8200 cells show that while light cells are predominantly small (uncorrected mean area of 39.7 microns2), there can be light cells ten times larger than this. Medium reflex cells show a very wide range of area (uncorrected values ranging from 16 to 1257 microns2) while dark reflex cells show an even wider range (24 to 2218 microns2). Analysis of the longest to shortest dimensions (LS ratio) reveals light cells to be more elongate than medium cells while dark cells tend to be rounder.

Topographical differences in cell area at the surface of the corneal epithelium of the pigmented rabbit

Corneas from female pigmented rabbits were prepared for scanning electron microscopy at 15.00 h. The superior-temporal and inferior-nasal quadrants were evaluated at 500X at-stage magnification, normal to the corneal surface, at central, paracentral and peripheral sites. Non-parametric statistical analyses of the histograms of cell surface areas at each site from seven corneas revealed significant differences between the three sites but not between superior and inferior quadrants.

Microscopical evaluation of the crystalline lens of the squid (Loligo opalescens) during embryonic development

The similarity between the cephalopod lens and the teleost (vertebrate) lens can be considered an optical example of convergent evolution. However, the embryology and ultrastructure of the cephalopod lens appear to be different from that of vertebrates, and perhaps unique to the animal kingdom. Using light and scanning electron microscopy, the morphogenesis of the squid (Loligo opalescens) lens is characterized. Results indicate that the posterior lens primordium appears first during development and is derived from cellular processes which extend from a middle group, group 2, of lentigenic (ectodermal) cells. The processes extend from the basal aspect of the lentigenic cells, project down into the optic vesicle during early stages of development, and fuse to form the posterior lens primordium. During later stages, the processes extend from surrounding lentigenic cells and are applied to the stalk of the lens, where they form bud-shaped protrusions. Once applied to the lens, the processes form lens elements that later fuse into plate-like elements evident in later-staged embryo and adult lenses. The anterior lens primordium is derived from an anterior group, group 1, of lentigenic cells, during later stages of development. Lentigenic processes extend from these lentigenic cells and are laid down in a circumferential fashion to form the anterior lens cap. As in the posterior lens, evidence indicates that the anterior lens elements fuse to form plate-like elements. The ultrastructure and morphogenesis of the cephalopod lens is discussed and contrasted with other strategies of lens development.

Large sample study of the effects of phenylephrine 2.5% eyedrops on the amplitude of accommodation in man

A single drop of phenylephrine 2.5% (MYDFRIN TM) was instilled into the eyes of 160 volunteers (aged 20 to 30 years) and their amplitude of accommodation assessed under standard illumination using a push‐up technique without an artificial pupil‐ The amplitude was assessed every 5 or 10 min over a period of 90 min. Within 30 min, nearly 75% of the subjects showed a measurable change in amplitude. Overall, the amplitude declined slowly with time to realize an average 11 % reduction from an average starting amplitude of 10.7 D. A large range of responses from a 30% increase to a 60% decrease in amplitude was. however, observed. Subjects with large amplitudes of accommodation were more likely to show significant decreases while some subjects with smaller amplitudes showed an apparent increase in accommodation (r2= 0.176). The phenomenon was observed in both blue‐ and brown‐eyed subjects and in non‐contact lens wearers and soft contact lens wearers (r2 values of 0.144 to 0.265).

Impact of glutaraldehyde versus glutaraldehyde-formaldehyde fixative on cell organization in fish corneal epithelium.

The purpose of this study was to examine the impact of a low osmolality glutaraldehyde fixative and a high osmolality glutaraldehyde-formaldehyde fixative on the structural organization of a tissue that could be exposed to low and high osmolality environments. The corneas of freshwater trout were prepared for transmission and scanning electron microscopy using either a fixative of 2% glutaraldehyde in 60 mM cacodylate buffer (pH 7.8, 260 mOsm/l) or a fixative prepared by adding 2.5% glutaraldehyde to a solution of 1% formaldehyde and buffering the solution with 0.1 M cacodylate (pH 7.6, 850 mOsm/l; Karnovsky-type fixative). The corneal epithelial cell layer thickness was greater after glutaraldehyde compared to glutaraldehyde-formaldehyde fixation (67 vs 55 microm), as was the thickness of the superficial cells (5.1 vs 3.4 microm) and basal cells (43 vs 38 microm). The intermediate (wing) cells of the epithelium were, however, less thick after glutaraldehyde fixation (15 vs 18 microm). The width of the squamous, intermediate and basal cells was greater following glutaraldehyde fixation with the effect being greatest in the superficial layers and insignificant at the level of the basal cells. The results show that chemical fixatives with extremes of osmolality cannot only produce different cell sizes in a tissue but also determine the overall organization of the cells in a positional-dependent fashion.

Protection from acute exposure to ultraviolet radiation by ultraviolet-absorbing RGP contact lenses*

One eye in each of 12 female pigmented rabbits was irradiated with UVB while wearing an EqualensR I rigid gas‐permeable contact lens. The other eye served as a control. The irradiance level was 625 μ W cm ‐2 (285‐315 nm total band pass) with exposure times up to 2364s. Eyes evaluated 24 h and 48 h after exposure by biomicroscopy and pachometry, and later by scanning electron microscopy (SEM). The contact lens was found to protect the central cornea from radiant exposures up to 60 times threshold. The exposed areas of cornea showed characteristic ultraviolet damage with an arc of demarcation indicating the position of the lens. With time the area formed a ridge of damaged cells (confirmed by SEM). At radiant exposure over 0.75 J cm ‐2 some oedema was seen in the ‘protected’ region, apparently by extension rather than as a direct effect. These findings suggest that in the human being exposed to ultraviolet radiation this ultraviolet‐obsorbing RPG lens would permit nasal and temporal photoketatitis with sparing of the central cornea and provide some protection of more posterior ocular structures.

LONG-TERM EFFECTS OF A SINGLE DOSE OF ULTRAVIOLET-B ON ALBINO RABBIT CORNEA–II. DETURGESCENCE and FLUID PUMP ASSESSED in vitro

Abstract— Both eyes of female albino rabbits (1.9 kg;9–10 wk old) were exposed to a single dose of UV‐B (300 ± 9 nm; 0.125 J/cm2 total dose) between 13.30 and 15.00 h. At various time periods thereafter (every 12 h for 3 days, 6, 7, 14, 28, 42, 56, 112, 224 and 336 days post‐irradiation), animals were sacrificed, samples of aqueous humor taken for analysis and stroma‐endothelium preparations obtained from the corneas. Following such threshold irradiation, small increases in aqueous humor tonicity and protein levels were observed. The preparations were mounted in a specular microscope assembly (for measuring the rate and magnitude of corneal deturgesccnce) or between two half chambers (for measuring fluid pump) and equilibrated for 2 h with a CO2‐equilibrated glucose‐adenosine‐glutathione‐supplemented Ringer solution at 37°C and a hydrostatic pressure of 20 cm H2O. After equilibration the stromal thickness showed large variation with large reductions in both the rate and amplitude of deturgescence function observed by 36 h. Large reductions in fluid pump activity were also observed by 36 h. The magnitude of the effects on fluid pump were somewhat greater than the effects on deturgescence. Both functions recovered to pre‐irradiation levels by 112 days post‐irradiation.