Konstantin Galichanin

Evolution of damage in the lens after in vivo close to threshold exposure to UV-B radiation: Cytomorphological study of apoptosis

The purpose of the present study was to investigate cataractogenesis and recovery of lens damage after in vivo close to threshold ultraviolet (UV)-B radiation around 300 nm. Eighty six-week-old albino Sprague-Dawley rats were familiarized to a rat restrainer five days prior to exposure. Groups of non-anesthetized rats were exposed unilaterally to 8 kJ/m(2) UVR-300 nm. The animals were sacrificed at 1, 7, 48 and 336h following exposure. The lenses were extracted for imaging of dark-field lens macro anatomy and measurement of intensity of forward lens light scattering to quantify lens opacities. Three exposed lenses and one non-exposed lens from each time interval were examined with light and transmission electron microscopy (TEM). Macro anatomy and lens light scattering revealed that all contralateral non-exposed lenses were clear. The degree of lens opacity (difference in lens light scattering between exposed and non-exposed lenses) increased during the 336h, reaching a plateau towards the end of the observation period. Light microscopy and TEM demonstrated that apoptotic features appeared in the epithelium already 1h after UVR exposure, and small vacuoles were seen in the outer cortex. Epithelial damage occurs during the first 48h after exposure and is followed by regenerative repair at 336h post-exposure. Apoptotic epithelial cells were phagocytized by adjacent epithelial cells. Cortical fiber cells exhibited increasing damage throughout the observation period without any clear repair after 336h. In conclusion, acute UVR-induced cataract is partly a reversible. Lens epithelium is a primary target for UVR exposure. Damage to cortical fiber cells remained irreversible.

Evolution of light scattering and redox balance in the rat lens after in vivo exposure to close‐to‐threshold dose ultraviolet radiation

Acta Ophthalmol. 2010: 88: 779–785

Protective effect of the thioltransferase gene on in vivo UVR-300 nm-induced cataract.

PURPOSE To determine the protection factor (PF) for glutaredoxin-1 (Grx1) with regard to UVR-induced cataract by comparison of in vivo ultraviolet radiation (UVR) lens toxicity between double knockout Grx1⁻/⁻ and Grx1⁺/⁺ mice. METHODS Twenty Grx1⁺/⁺ mice and 20 Grx1⁻/⁻ mice were unilaterally exposed in vivo to UVR for 15 minutes. Groups of four animals each received 0.0, 2.1, 2.9, 3.6, and 4.1 kJ/m(2) UVR-300 nm. At 48 hours after UVR exposure, light-scattering in the exposed and contralateral nonexposed lenses was measured quantitatively. Macroscopic lens changes were documented with dark-field illumination photography. RESULTS UVR-300 nm induced subcapsular and cortical cataract in Grx1⁻/⁻ and Grx1⁺/⁺ mice. In both Grx1⁻/⁻ and Grx1⁺/⁺, the light-scattering intensified with increased in vivo exposure doses of UVR-300 nm. The intensity of forward light-scattering was higher in the lenses of Grx1⁻/⁻ mice than in the lenses of Grx1⁺/⁺ mice. The threshold dose for in vivo UVR-300 nm-induced cataract, expressed as MTD(2.3:16), was 3.8 in the Grx1⁺/⁺ group and 3.0 in the Grx1⁻/⁻ group, resulting in a PF of 1.3. CONCLUSIONS The PF is an objective relative measure of protective properties. The Grx1 gene is associated with an in vivo PF of 1.3. This result signifies that the presence of the gene allows a 1.3 times longer in vivo exposure to UVR, at equivalent irradiance, than the absence of the gene before early-onset, UVR-induced cataract occurs. This finding indicates the important role of the Grx1 gene in the oxidation defense system of the lens.

Cataract after repeated daily in vivo exposure to ultraviolet radiation.

AbstractEpidemiological data indicate a correlation between lifelong exposure to ultraviolet radiation and cortical cataract. However, there is no quantitative experimental data on the effect of daily repeated in vivo exposures of the eye to UVR. Therefore, this experiment was designed to verify whether the dose additivity for UVR exposures holds through periods of time up to 30 d. Eighty rats were conditioned to a rat restrainer 5 d prior to exposure. All animals were divided into four exposure period groups of 1, 3, 10, and 30 d of exposure to UVR. Each exposure period group of 20 animals was randomly divided into five cumulated UVR dose subgroups. Eighteen-wk-old non-anesthetized albino Sprague-Dawley rats were exposed daily to UVR-300 nm for 15 min. One week after the last exposure, animals were sacrificed. The lenses were extracted for macroscopic imaging of dark-field anatomy, and degree of cataract was quantified by measurement of the intensity of forward lens light scattering. Maximum tolerable dose (MTD2.3:16), a statistically defined standard for sensitivity for the threshold for UVR cataract, was estimated for each exposure period. Exposed lenses developed cataract with varying appearance on the anterior surface. Single low doses of UVR accumulated to cause cataract during periods up to 30 d. MTD2.3:16 for 1, 3, 10, and 30 d of repeated exposures was estimated to 4.70, 4.74, 4.80, and 6.00 kJ m−2, respectively. In conclusion, the lens sensitivity to UVR-B for 18-wk-old Sprague-Dawley rats decreases with the increasing number of days being exposed.

Kinetics of GADD45α, TP53 and CASP3 gene expression in the rat lens in vivo in response to exposure to double threshold dose of UV-B radiation.

The purpose of the present study was to investigate the evolution of expression of mRNA message for the genes for the genome stress sensor GADD45α, the apoptosis initiator TP53 and the apoptosis executor CASP3 in the rat lens in vivo in response to exposure to UVR around 300 nm. Forty six week old female albino Sprague-Dawley rats were unilaterally exposed to double threshold dose for cataract induction, 8 kJ/m(2) (8.9 W/m(2) for 15 min), of UVR (λ(max) = 300 nm). The animals were sacrificed at 1, 5, 24 and 120 h following exposure to UVR-B. For each of the GADD45α, TP53 and CASP3 genes, respectively, mRNA expression in the lenses was measured by quantitative RT-PCR. It was found that expression of mRNA for GADD45α transiently increases between 5 and 24 h after exposure. TP53 is slightly downregulated in exposed lenses at 1 and 5 h after exposure and thereafter the mRNA expression increases with a constant rate of 9.4\ 10(-3) rel. units/h to a 1.8 fold increase at 120 h after exposure. Expression of mRNA for CASP3 is downregulated at 1, 5 and 24 h after in vivo exposure and then increases with a constant rate of 4.7 10(-3) rel. units/h, upto a 1.3 fold upregulation at 120 h. Double threshold dose of UVR, for short delay onset of cataract, in vivo causes a transient upregulation of the stress sensor GADD45α, a concurrent downregulation of TP53 and CASP3, followed by a constant upregulation of TP53 that precedes a constant upregulation of CASP3.

A new universal rat restrainer for ophthalmic research

Acta Ophthalmol. 2011: 89: e67–e71

Does infrared or ultraviolet light damage the lens

In daylight, the human eye is exposed to long wavelength ultraviolet radiation (UVR), visible radiation and short wavelength infrared radiation (IRR). Almost all the UVR and a fraction of the IRR waveband, respectively, left over after attenuation in the cornea, is absorbed in the lens. The time delay between exposure and onset of biological response in the lens varies from immediate-to-short-to-late. After exposure to sunlight or artificial sources, generating irradiances of the same order of magnitude or slightly higher, biological damage may occur photochemically or thermally. Epidemiological studies suggest a dose-dependent association between short wavelength UVR and cortical cataract. Experimental data infer that repeated daily in vivo exposures to short wavelength UVR generate photochemically induced damage in the lens, and that short delay onset cataract after UVR exposure is photochemically induced. Epidemiology suggests that daily high-intensity short wavelength IRR exposure of workers, is associated with a higher prevalence of age-related cataract. It cannot be excluded that this effect is owing to a thermally induced higher denaturation rate. Recent experimental data rule out a photochemical effect of 1090 nm in the lens but other wavelengths in the near IRR should be investigated.

Characterization of molecular mechanisms of in vivo UVR induced cataract.

Cataract is the leading cause of blindness in the world (1). The World Health Organization defines cataract as a clouding of the lens of the eye which impedes the transfer of light. Cataract is a multi-factorial disease associated with diabetes, smoking, ultraviolet radiation (UVR), alcohol, ionizing radiation, steroids and hypertension. There is strong experimental (2-4) and epidemiological evidence (5,6) that UVR causes cataract. We developed an animal model for UVR B induced cataract in both anesthetized (7) and non-anesthetized animals (8). The only cure for cataract is surgery but this treatment is not accessible to all. It has been estimated that a delay of onset of cataract for 10 years could reduce the need for cataract surgery by 50% (9). To delay the incidence of cataract, it is needed to understand the mechanisms of cataract formation and find effective prevention strategies. Among the mechanisms for cataract development, apoptosis plays a crucial role in initiation of cataract in humans and animals (10). Our focus has recently been apoptosis in the lens as the mechanism for cataract development (8,11,12). It is anticipated that a better understanding of the effect of UVR on the apoptosis pathway will provide possibilities for discovery of new pharmaceuticals to prevent cataract. In this article, we describe how cataract can be experimentally induced by in vivo exposure to UVR-B. Further RT-PCR and immunohistochemistry are presented as tools to study molecular mechanisms of UVR-B induced cataract.