V. Bantseev

Mitochondrial “Movement” and Lens Optics following Oxidative Stress from UV‐B Irradiation

Abstract:  Mitochondria provide energy generated by oxidative phosphorylation and at the same time play a central role in apoptosis and aging. As a byproduct of respiration, the electron transport chain is known to be the major intracellular site for the generation of reactive oxygen species (ROS). Exposure to solar and occupational ultraviolet (UV) radiation, and thus production of ROS and subsequent cell death, has been implicated in a large spectrum of skin and ocular pathologies, including cataract. Retinal pigment epithelial cell apoptosis generates photoreceptor dysfunction and ultimately visual impairment. The purpose of this article was to characterize in vitro changes following oxidative stress with UV‐B radiation in (a) ocular lens optics and cellular function in terms of mitochondrial dynamics of bovine lens epithelium and superficial cortical fiber cells and (b) human retinal pigment epithelial (ARPE‐19) cells. Cultured bovine lenses and confluent cultures of ARPE‐19 cells were irradiated with broadband UV‐B radiation at energy levels of 0.5 and 1.0 J/cm2. Lens optical function (spherical aberration) was monitored daily up to 14 days using an automated laser scanning system that was developed at the University of Waterloo. This system consists of a single collimated scanning helium–neon laser source that projects a thin (0.05 mm) laser beam onto a plain mirror mounted at 45° on a carriage assembly. This mirror reflects the laser beam directly up through the scanner table surface and through the lens under examination. A digital camera captures the actual position and slope of the laser beam at each step. When all steps have been made, the captured data for each step position is used to calculate the back vertex distance for each position and the difference in that measurement between beams. To investigate mitochondrial movement, the mitochondria‐specific fluorescent dye Rhodamine 123 was used. Time series were acquired with a Zeiss 510 (configuration Meta 18) confocal laser scanning microscope equipped with an inverted Axiovert 200 M microscope and 40‐× water‐immersion C‐Apochromat objective (NA 1.2). The optical analysis showed energy level‐dependent increases in back vertex distance variability (loss of sharp focus) from 0.39 ± 0.04 mm (control, n= 11) to 1.63 ± 0.33 mm (1.0 J/cm2, n= 10) and 0.63 ± 0.13 mm (0.5 J/cm2, n= 9). Confocal laser scanning microscopy analysis of both bovine lenses and ARPE‐19 cells showed that following treatment at 0.5 J/cm2 the mitochondria stopped moving immediately whereas at 1.0 J/cm2 not only did the mitochondria stop moving, but fragmentation and swelling was seen. Untreated control tissue exhibited up to 15 μm/min of movement of the mitochondria. This could represent normal morphological change, presumably allowing energy transmission across the cell from regions of low to regions of high ATP demand. Lack of mitochondrial movement, fragmentation, and swelling of mitochondria may represent early morphological changes following oxidative stress that may lead to activation of caspase‐mediated apoptotic pathways.

The lens of the eye as a focusing device and its response to stress

The continued peripheral growth of the lens, resulting in the concentration of older tissue toward the center, has the important optical consequence of producing a lens of variable refractive index. An approach consisting of the projection of fine laser beams through excised lenses in physiological solution has been used for in vitro study of lens optical quality. By varying the separation of the incident beams and/or the wavelength characteristics of the laser used, lens refractive properties and relative transparency may be examined. In the review provided, these optical properties are correlated to lens suture anatomy, lens mitochondrial morphology and function and the function of lens heat shock proteins. In addition, lens spherical aberration is evaluated as a function of accommodation. This work can be highlighted as follows: Mammalian lens suture morphology has a direct impact on lens optical function and, while suture structure of mammalian and avian lenses are very different, they both show an age-related deterioration in morphology and focusing ability. The distribution and appearance of mitochondria of the lens epithelium and superficial fiber cells are similar in all vertebrates. Lens mitochondrial integrity is correlated to lens focusing ability, suggesting a correlation between lens optical properties and lens metabolic function. The induction of cold cataract measured optically in cultured mammalian lenses is enhanced by thermal (heat) shock and this effect is prevented by inhibiting heat shock protein production. Finally, lens accommodative function can be studied by measuring lens refractive change using a physiological model involving an intact accommodative apparatus.

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.

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.

The Effects of Tween 20 on in vitro Bovine Lenses

ABSTRACT The optical properties of the cultured bovine lens were analyzed after exposure to various concentrations of Tween 20, a nonionic surfactant, to find a nonirritating concentration for commercial products. Bovine lenses were extracted and placed into a culture chamber for 24 hours at 37°C with 4–5% CO2. The lenses were placed into three treatment (1%, n = 10; 10%, n = 9; and 100%, n = 10 Tween 20) and one control group (n = 7) for 15 minutes. For 8 days following treatment, the lens optics were analyzed periodically for back vertex distance (focal length) and back vertex distance variability (sharpness of focus) using a laser-scanning device. For both the control and the 1% Tween 20 condition, no significant change was seen from the beginning of the experiment (p > 0.05). The 10% Tween 20 solution induced significant loss of sharp focus (0.62 ± 0.1 mm SEM) 4 hours after exposure, increasing to BVD = 1.69 ± 0.3 mm SEM by the end of experimentation (p < 0.05). At full strength (100%), Tween 20 began to cause damage after 4 hours (BVD = 0.50 ± 0.06 mm SEM), and this change increased to BVD = 4.46 ± 0.59 mm SEM after 8 days following treatment (p < 0.05). Therefore, a dose-dependant increase in back vertex distance (BVD) variability was detected. This research suggests that using 1% Tween 20 in commercial solutions should not produce ocular irritation, whereas concentrations above 10% will cause significant irritation. As well, the bovine lens assay, paired with the automated lens scanner, provided a sensitive approach to measure mild ocular irritation.