Taras Ardan

Effect of Two Different UVA Doses on the Rabbit Cornea and Lens

The aim of the present paper was to examine the irradiation effect of two doses of UVA rays (365 nm) on the rabbit cornea and lens. Corneas of anesthetized adult albino rabbits were irradiated with UVA rays for 5 days (daily dose 1.01 J cm−2 in one group of rabbits and daily dose 2.02 J cm−2 in the second group of animals). The third day after the last irradiation, the rabbits were killed, and their eyes were employed for spectrophotometrical, biochemical and immunohistochemical investigations. Normal eyes served as controls. Absorption spectra of the whole corneal centers were recorded over the UV–VIS (visible) spectral range. Levels of antioxidant and prooxidant enzymes, nitric oxide synthases and nitric oxide (indirectly measured as nitrate concentration) were investigated in the cornea. Malondialdehyde, a byproduct of lipid peroxidation, was examined in the cornea and lens. The results show that the staining for endothelial nitric oxide synthase was more pronounced in corneas irradiated with the higher UVA dose. Otherwise, UVA rays at either dose did not significantly change corneal light absorption properties and did not cause statistically significant metabolic changes in the cornea or lens. In conclusion, UVA rays at the employed doses did not evoke harmful effects in the cornea or lens.

The Effect of Actinoquinol with Hyaluronic Acid in Eye Drops on the Optical Properties and Oxidative Damage of the Rabbit Cornea Irradiated with UVB Rays

Irradiation of the cornea with UVB rays leads to its oxidative damage, swelling and increased light absorption. We investigated changes in the corneal optics (evaluated by changes of corneal hydration and light absorption) and microscopical disturbances of corneas irradiated with UVB rays as influenced by eye drops containing actinoquinol with hyaluronic acid. Rabbit corneas were irradiated with a daily dose of 0.5 or 1.01 J cm−2 of UVB rays (312 nm) for 4 days. During irradiation, the eye drops were applied on the right eye and buffered saline (or hyaluronic acid) on the left eye. On day 5 the rabbits were sacrificed and the corneas examined spectrophotometrically for light absorption. The corneal thickness (hydration) was measured using a pachymeter. Corneas of some other rabbits were examined immunohistochemically. After buffered saline treatment UVB rays evoked changes in the corneal optics and induced oxidative damage of the corneas. After actinoquinol‐hyaluronic acid application, these changes were diminished. Hyaluronic acid alone was less effective. In conclusion, actinoquinol‐hyaluronic acid eye drops decreased changes in corneal optics and suppressed oxidative damage in the UVB‐irradiated cornea. However, the effective corneal protection by these eye drops was limited to the lower UVB dose.

Immunohistochemical expression of matrix metalloproteinases in the rabbit corneal epithelium upon UVA and UVB irradiation.

Matrix metalloproteinases (MMPs) are proteolytic enzymes involved in tissue remodeling and wound healing. These enzymes degrade and also synthesize components of the extracellular matrix. Overexpression of MMPs results in excessive extracellular matrix degradation and tissue destruction. In the cornea, destructive processes may lead to scarring and loss of vision. In this study MMPs (types 1, 2, 7, 8, 9 and 14) were examined immunohistochemically in the normal rabbit corneal epithelium and in epithelium irradiated in vivo with similar doses of UVB or UVA radiation (UVB rays 312 nm, UVA rays 365 nm, daily dose 1.01 J/cm(2) for four days). Results show that MMPs studied revealed low expression in the normal corneal epithelium, whereas after repeated UVB irradiation the expression of MMPs was significantly increased in the corneal epithelium, in ascending order: MMP-2, MMP-9, MMP-1, and MMP-7 with MMP-8. In contrast, compared to normal corneas, repeated UVA radiation did not significantly change the expression of MMPs in the irradiated corneal epithelium. MMP-14 was expressed at very low levels in all studied corneas, whereas no significant changes were detected upon UV exposure. In conclusion, UV radiation of shorter wavelength (UVB) induced an increase in expression of all MMPs except MMP-14. It is suggested that overexpression of MMPs in the corneal epithelium contributes to the damaging effect of UVB radiation to the cornea.

A frame-supported ultrathin electrospun polymer membrane for transplantation of retinal pigment epithelial cells

We report on the design and fabrication of a frame-supported nanofibrous membrane for the transplantation of retinal pigment epithelial (RPE) cells, which is a promising therapeutic option for the treatment of degenerative retinal disorders. The membranous cell carrier prepared from 640 nm-thick poly(DL-lactide) fibres uniquely combines high porosity, large pore size and low thickness, to maximize the nutrient supply to the transplanted cells in the subretinal space and thus to enhance the therapeutic effect of the transplantation. The carrier was prepared by electrospinning, which made it easy to embed a 95 μm-thick circular supporting frame 2 mm in diameter. Implantations into enucleated porcine eyes showed that the frame enabled the ultrathin membrane to be handled without irreversible folding, and allowed the membrane to regain its flat shape when inserted into the subretinal space. We further demonstrated that the minimum membrane thickness compatible with the surgical procedure and instrumentation employed here was as low as 4 μm. Primary porcine RPE cells cultivated on the membranes formed a confluent monolayer, expressed RPE-specific differentiation markers and showed transepithelial resistance close to that of the native RPE. Most importantly, the majority of the RPE cells transplanted into the subretinal space remained viable. The ultrathin, highly porous, and surgically convenient cell carrier presented here has the potential to improve the integration and the functionality of transplanted RPE cells.

Hydration and transparency of the rabbit cornea irradiated with UVB-doses of 0.25 J/cm(2) and 0.5 J/cm(2) compared with equivalent UVB radiation exposure reaching the human cornea from sunlight.

Purpose: Exposure of the cornea to UV radiation from sunlight evokes intraocular inflammation, photokeratitis. Photokeratitis is caused by UVB radiation. It is accompanied by changes of corneal hydration and light absorption. The aim of this study was to examine the effect of two UVB doses on corneal optics in rabbits and to compare these UVB doses with the equivalent exposure of UVB radiation reaching the human cornea from sunlight. Materials and Methods: Rabbit corneas were irradiated with a daily UVB dose of 0.25 J/cm2 or 0.5 J/cm2 for 4 days. One day after finishing the irradiations the rabbits were sacrificed and corneal light absorption measured using our spectrophotometrical method. Corneal hydration was examined using an ultrasonic Pachymeter every experimental day before the irradiation procedure and the last day before sacrificing the animals. Results: Changes in corneal optics appeared after the repeated exposure of the cornea to a UVB dose of 0.25 J/ cm2 and massively increased after the repeated exposure of the cornea to a UVB dose of 0.5 J/cm2. The first significant changes in corneal hydration appeared after a single exposure of the cornea to a UVB dose of 0.25 J/cm2. Conclusions: Changes in corneal hydration appeared after the exposure of the rabbit cornea to a single UVB dose equivalent to 2.6 hours of solar UVB radiation reaching the human cornea, as measured by UVB sensors embedded in the eyes of mannequin heads facing the sun on a beach at noon in July. Repeated exposure of the rabbit cornea to the same UVB dose evoked profound changes in corneal optics. Although comparison of experimental and outdoor conditions are only approximate, the results in rabbits point to the danger for the human eye from UVB radiation when short stays in sunlight are repeated for several consecutive days without UV protection.

The influence of various toxic effects on the cornea and changes in corneal light transmission

PurposeNormal corneal hydration is necessary for the maintenance of corneal transparency. Damage of the corneal epithelium or endothelium by various external influences disturbs the mechanism by which the cornea maintains normal hydration and transparency. The cornea swells, and the corneal thickness increases, resulting in increased scatter and the development of corneal opacity. The transmission of light across the cornea is changed. The purpose of this study is to investigate spectrophotometrically the corneal light transmission under the influence of the various factors affecting the cornea.MethodsWe developed a spectrophotometric method to measure the light transmission across the cornea under the influence of various factors affecting the cornea, such as treatment with 0.9% NaCl, saline, or phosphate buffered saline (PBS), solutions employed as placebo eye drops (negative controls) in experimental studies, agents toxic to the cornea, such as diluted acids or alkalis. The method distinguishes between changes in corneal light transmission caused by altered corneal thickness (the level of hydration) and changes resulting from other corneal disturbances which in turn affect corneal light transmission.ResultsThe results obtained show that the corneal light transmission is decreased following the application of toxic substances on the corneal surface. This decrease is highly dependent on the severity of the corneal injury evoked by individual noxes, and the resulting changes in corneal hydration and transparency.ConclusionsThe influence of various influences applied to the cornea, manifested as changes in corneal light transmission, can be measured using our spectrophotometric method with a high degree of sensitivity.

Reduced Levels of Tissue Inhibitors of Metalloproteinases in UVB-Irradiated Corneal Epithelium

Tissue inhibitors of metalloproteinases (TIMPs) are the major endogenous regulators of metalloproteinase activity in tissues. TIMPs are able to inhibit activity of all known matrix metalloproteinases (MMPs) and thus participate in controlling extracellular matrix synthesis and degradation. We showed previously elevated expressions of MMPs in the rabbit corneal epithelium upon UVB exposure and suggested that these enzymes might be involved in corneal destruction caused by excessive proteolysis. The aim of this study was to investigate TIMPs in the corneal epithelium after UV irradiation using immunohistochemical and biochemical methods. We found that as compared to control rabbit corneas where relatively high levels of TIMPs were present in the epithelium, repeated irradiation of the cornea with UVB rays (not with UVA rays of similar doses) significantly decreased TIMPs in corneal epithelial cells. The results of this study point to the suggestion that the decrease in TIMPs in the corneal epithelium after UVB irradiation contributes to increased proteolytic activity of MMPs in UVB‐irradiated corneal epithelium found previously.

Gradual Phenotype Development in Huntington Disease Transgenic Minipig Model at 24 Months of Age

Background: Huntington disease (HD) is an incurable neurodegenerative disease caused by the expansion of a polyglutamine sequence in a gene encoding the huntingtin (Htt) protein, which is expressed in almost all cells of the body. In addition to small animal models, new therapeutic approaches (including gene therapy) require large animal models as their large brains are a more realistic model for translational research. Objective: In this study, we describe phenotype development in transgenic minipigs (TgHD) expressing the N-terminal part of mutated human Htt at the age of 24 months. Methods: TgHD and wild-type littermates were compared. Western blot analysis and subcellular fractionation of different tissues was used to determine the fragmentation of Htt. Immunohistochemistry and optical analysis of coronal sections measuring aggregates, Htt expression, neuroinflammation, and myelination was applied. Furthermore, the expression of Golgi protein acyl-CoA binding domain containing 3 (ACBD3) was analyzed. Results: We found age-correlated Htt fragmentation in the brain. Among various tissues studied, the testes displayed the highest fragmentation, with Htt fragments detectable even in cell nuclei. Also, Golgi protein ACBD3 was upregulated in testes, which is in agreement with previously reported testicular degeneration in TgHD minipigs. Nevertheless, the TgHD-specific mutated Htt fragments were also present in the cytoplasm of striatum and cortex cells. Moreover, microglial cells were activated and myelination was slightly decreased, suggesting the development of a premanifest stage of neurodegeneration in TgHD minipigs. Conclusions: The gradual development of a neurodegenerative phenotype, accompanied with testicular degeneration, is observed in 24- month-old TgHD minipigs.

C9 Different forms of huntingtin in various tissues of transgenic minipig model increase with age

Background The progression of Huntington’s disease (HD) in human patients is predominantly linked with formation of aggregates, oligomers, and fragments of huntingtin (Htt). Aggregates have been related to cell death, but on the contrary, smaller soluble forms of mHtt and huntingtin oligomers were described to be toxic to the cells and to be the key factors of cellular dysfunction. Therefore we focus on the detection of these forms of Htt in our unique large animal model; transgenic minipig (TgHD) expressing mutant N-terminal (548 aa, 124 CAG/CAA) part of human huntingtin. Aims To follow disease development in transgenic minipigs by detecting different forms of Htt and comparing them with wild-type (WT) siblings. Methods We perform immunohistochemical and biochemical methods (Western blots, filter retardation assay, velocity sedimentation, etc.) on different tissues at various ages with main focus on brain in order to detect different forms of Htt. Results We observed that in cortex of TgHD minipigs, fragmentation increases with age. In addition, we have detected that fragmentation is tissue-specific. For example from all the tissues tested, we are able to see fragments mainly in the cortex, cerebellum, lung and testes of TgHD minipigs, and significantly less in other tissues. Using velocity sedimentation we have identified unphosphorylated mHtt in higher structures in TgHD minipigs. Conclusion We provide information about HD phenotype development in transgenic minipig model, to be able to test new therapeutic approaches. Acknowledgement This study was supported by CHDI Foundation (A-5378) and by National Sustainability Programme, project number LO1609 (Czech Ministry of Education, Youth and Sports). The research leading to these results has received funding from the Norwegian Financial Mechanism 2009–2014 and the Ministry of Education, Youth and Sports under Project Contract no. MSMT-28477/2014 (project ID 7F14308).