J.G. Sivak

Impairment of Eye Lens Cell Physiology and Optics by Broadband Ultraviolet A–Ultraviolet B Radiation¶

The phototoxicity of ultraviolet A (UVA) alone and UVA plus ultraviolet B (UVB) combined on cultured porcine lenses was investigated by analyzing cellular function as measured with a fluorescence bioassay approach and optical integrity, in terms of sharpness of the lens focus as measured with a scanning laser system. The bioassay consisted of carboxyfluorescein diacetate‐acetoxymethyl ester and alamarBlue fluorescent dyes. Aseptically dissected porcine lenses were maintained in modified medium 199 without phenol red supplemented with 1% penicillin–streptomycin and 4% porcine serum. At 1 week of preincubation, baseline measurements were obtained. Then the lenses were treated with single exposures of different UVA and UVB energy levels. The lenses treated with 86 J/cm2 UVA alone showed a significant (P < 0.05) decrease in cellular and optical integrity at 48 h after exposure, whereas those treated with 43 J/cm2 UVA alone did not show significant phototoxic effect. Lenses treated with 15.63 J/cm2 UVA plus 0.019 J/cm2 UVB combined showed significant adverse effects beginning from 48 h after exposure. Also, there was no recovery. These findings show that a high UVA dose alone and relatively low UVA in combination with low UVB radiant exposure can impair lens cellular and optical functions, respectively.

Optical function and mitochondrial metabolic properties in damage and recovery of bovine lens after in vitro carbonyl cyanide m-chlorophenylhydrazone treatment

In order to elucidate the correlation between lens optical function and metabolic function, in vitro bovine lens optical quality and mitochondrial integrity was measured following treatment with carbonyl cyanide m-chlorophenylhydrazone (the mitochondrial depolarizing agent, CCCP). The results indicate that in vitro exposure to CCCP resulted in concentration and time-dependent loss of sharp focus. The concentrations tested included 65.0, 32.5, 16.25 and 8.125 microm CCCP. Lenses treated with two lower concentrations show recovery from damage at the 24-h scan point. In lenses treated with 65 microM CCCP, mitochondria in lens epithelial and superficial cortical fibre cells appeared short and swollen. The results of this study indicate that lens optical function and mitochondrial integrity are closely correlated.

Long-term Lens Organ Culture System with a Method for Monitoring Lens Optical Quality¶

Abstract The Scan Tox™ System is a method for monitoring lens optical quality (focus or lack of focus) in culture conditions, which mimic conditions inside the eye. The ocular lens is an ideal organ for long-term culture experiments because it has no direct blood supply and no connection to the nervous system. The Scan Tox™ System makes it possible to keep lenses for long-term studies of up to a few weeks. The use of cultured lenses, mainly bovine, replaces the need for testing the effects of potentially damaging agents on live animals. This optical monitoring apparatus uses a computer-operated scanning laser beam, a video-camera system and a video frame analyzer to record the focal length and transmittance of the cultured lens. The scanner is designed to measure the focal length at points across the diameter of the lens. The lens container permits the lens to be exposed to a vertical laser beam from below. The laser source projects its light onto a plain mirror, which is mounted at 45° on a carriage assembly. The mirror reflects the laser beam directly up through the test lens. The mirror carriage is connected to a positioning motor, which moves the laser beam across the lens. The camera sees the cross section of the beams and, by examining the image at each position of the mirror, Scan Tox™ software is able to measure the quality of the lens by calculating the back vertex distance for each beam position. The cultured lenses continue to maintain their original refractive function. When foreign substances are introduced to a cultured lens, the Scan Tox™ System measures the resulting optical response. This provides a very sensitive means to follow early damage to the eye lens. Because the lens is maintained in an intact state in solutions that are similar to those inside the eye, the lens retains its normal recuperative powers. So in addition to measuring early damage, this system allows measurement of recovery from damage.

Dose-response of the cultured bovine lens to butyl, methyl and propyl parabens.

Pre‐screening of cosmetic ingredients is vital for consumer safety. Previous in vivo techniques, such as the Draize test, have proved to be unreliable in predicting ocular irritancy and therefore there is a need for alternate testing methodologies. One such test is the scanning laser in vitro assay system which quantifies irritancy based on the focusing ability of the cultured bovine lens. In combination with confocal microscopy, a more thorough documentation of ocular irritancy can be achieved. This study investigates the response of cultured bovine lenses over time to butyl, methyl and propyl parabens, which are common antimicrobial agents found in cosmetic and ophthalmic products. The focusing ability of the lens was measured with an automated laser scanner over a period of 96 h. At 120 h post‐treatment, the lenses were analysed by using a confocal laser scanning microscope to determine the characteristics of nuclei, and the morphology and distribution of mitochondria within the lenses. Irritancy to the three parabens was investigated at both an optical and cellular level. Each of the parabens was tested at 0.002% and 0.2%, where the 0.2% butyl paraben was found to be the most irritating.