Dose-dependent Changes After Proton and Photon Irradiation in a Zebrafish Model.

BACKGROUND/AIM: The importance of hadron therapy in the cancer management is growing. We aimed to refine the biological effect detection using a vertebrate model. MATERIALS AND METHODS: Embryos at 24 and 72 h postfertilization were irradiated at the entrance plateau and the mid spread-out Bragg peak of a 150 MeV proton beam and with reference photons. Radiation-induced DNA double-strand breaks (DSB) and histopathological changes of the eye, muscles and brain were evaluated; deterioration of specific organs (eye, yolk sac, body) was measured. RESULTS: More and longer-lasting DSBs occurred in eye and muscle cells due to proton versus photon beams, albeit in different numbers. Edema, necrosis and tissue disorganization, (especially in the eye) were observed. Dose-dependent morphological deteriorations were detected at ≥10 Gy dose levels, with relative biological effectiveness between 0.99±0.07 (length) and 1.12±0.19 (eye). CONCLUSION: Quantitative assessment of radiation induced changes in zebrafish embryos proved to be beneficial for the radiobiological characterization of proton beams.

Light exposure during embryonic and yolk-sac alevin development of Chinook salmon Oncorhynchus tshawytscha does not alter the spectral phenotype of photoreceptors.

Colour vision is mediated by the expression of different visual pigments in photoreceptors of the vertebrate retina. Each visual pigment is a complex of a protein (opsin) and a vitamin A chromophore; alterations to either component affects visual pigment absorbance and, potentially, the visual capabilities of an animal. Many species of fish undergo changes in opsin expression during retinal development. In the case of salmonid fishes the single cone photoreceptors undergo a switch in opsin expression from SWS1 (ultraviolet sensitive) to SWS2 (blue-light sensitive) starting at the yolk-sac alevin stage, around the time when they first experience light. Whether light may initiate this event or produce a plastic response in the various photoreceptors is unknown. In this study, Chinook salmon Oncorhynchus tshawytscha were exposed to light from the embryonic (5 days prior to hatching) into the yolk sac alevin (25 days post hatching) stage and the spectral phenotype of photoreceptors assessed with respect to that of unexposed controls by in situ hybridization with opsin riboprobes. Light exposure did not change the spectral phenotype of photoreceptors, their overall morphology or spatial arrangement. These results concur with those from a variety of fish species and suggest that plasticity in photoreceptor spectral phenotype via changes in opsin expression may not be a widespread occurrence among teleosts.

High-precision, non-invasive anti-microvascular approach via concurrent ultrasound and laser irradiation.

Antivascular therapy represents a proven strategy to treat angiogenesis. By applying synchronized ultrasound bursts and nanosecond laser irradiation, we developed a novel, selective, non-invasive, localized antivascular method, termed photo-mediated ultrasound therapy (PUT). PUT takes advantage of the high native optical contrast among biological tissues and can treat microvessels without causing collateral damage to the surrounding tissue. In a chicken yolk sac membrane model, under the same ultrasound parameters (1 MHz at 0.45 MPa and 10 Hz with 10% duty cycle), PUT with 4 mJ/cm2 and 6 mJ/cm2 laser fluence induced 51% (p = 0.001) and 37% (p = 0.018) vessel diameter reductions respectively. With 8 mJ/cm2 laser fluence, PUT would yield vessel disruption (90%, p < 0.01). Selectivity of PUT was demonstrated by utilizing laser wavelengths at 578 nm or 650 nm, where PUT selectively shrank veins or occluded arteries. In a rabbit ear model, PUT induced a 68.5% reduction in blood perfusion after 7 days (p < 0.001) without damaging the surrounding cells. In vitro experiments in human blood suggested that cavitation may play a role in PUT. In conclusion, PUT holds significant promise as a novel non-invasive antivascular method with the capability to precisely target blood vessels.

Multifractal dimension and lacunarity of yolk sac vasculature after exposure to magnetic field.

Several studies have reported about the effects of magnetic fields (MFs) on vascular tissue. Extremely low frequency magnetic fields (ELF-MFs) can promote either inhibition or stimulation of vasculogenesis and angiogenesis, depending upon the intensity and time of exposure to the MF. To investigate the possible effects of ELF-MF on vascular processes, it is necessary to employ methods that allow parameterization of the vascular network. Vascular network is a structure with fractal geometry; therefore, fractal methods have been used to evaluate its morphometric complexity. Here, we used the lacunarity parameter (complementary method of fractal analysis) and multifractal analyses to investigate angiogenesis and vasculogenesis in the embryonic yolk sac membrane (YSM) of Japanese quails (Coturnix japonica) with and without exposure to an external MF of 1 mT and 60 Hz. Lacunarity results showed that the vascular density was lower for the group exposed to the magnetic field for 9 h/day. In addition, multifractal analysis showed reduced vascularization in the experimental groups (6 h/day and 9 h/day of exposure to MF). Furthermore, multifractal analysis showed difference between the groups exposed for 12 and 24 h/day. Using multifractal methods (generalized dimensions and singularity spectrum), it was possible to characterize the vascular network of the quail embryo YSM as a multifractal object, therefore proving this method to be a more appropriate application than the traditional monofractal methods.

Fractal analysis of extra-embryonic vascularization in Japanese quail embryos exposed to extremely low frequency magnetic fields.

Magnetic fields (MF) can alter the dynamic behavior of vascular tissue and may have a stimulatory or inhibitory effect on blood vessel growth. Fractal geometry has been used in several studies as a tool to describe the development of blood vascular networks. Due to its self-similarity, irregularity, fractional dimension, and dependence on the scale of vessel dimensions, vascular networks can be taken as fractal objects. In this work, we calculated the fractal dimension by the methods of box counting (D(bc)) and information dimension (D(inf)) to evaluate the development of blood vessels of the yolk sac membrane (YSM) from quail embryos exposed to MF with a magnetic flux density of 1 mT and a frequency of 60 Hz. The obtained results showed that when the MF was applied to embryos aged between 48 and 72 h, in sessions of 2 h (6 h/day) and 3 h (9 h/day) with exposure intervals between 6 and 5 h, respectively, blood vascular formation was inhibited. Exposure sessions shorter than 2 h or longer than 3 h had no observable change on the vascular process. In contrast, the magnetic field had no observable change on the YSM vascular network for embryos aged between 72 and 96 h, irrespective of the exposure time. In conclusion, these results show a "window effect" regarding exposure time.

The photoprotective effect of ascorbic acid, acetylsalicylic acid, and indomethacin evaluated by the photo hen's egg test.

The aim of the present study was to evaluate the supposed photoprotective effects of ascorbic acid, acetylsalicylic acid, and indomethacin by the photo hen's egg test, a recently developed new model for phototoxicity. Therefore, in three independent experimental settings the blood vessel system of the embryo's yolk sac of 24 incubated hens' eggs (2 test groups) were exposed to 60 mJ/cm2 ultraviolet B (UVB) to induce severe phototoxic damage. Before UVB irradiation, one of these test groups was exposed additionally to one of the test substances and the other one to 0.9% sodium solution alone. To exclude plain toxic reactions, two additional test groups were exposed only to 0.9% sodium chloride solution or to one of the test substances alone. Over a test observation period of 24 h, the embryo lethality as well as the morphological changes of the yolk sac blood vessel system were observed. Ascorbic acid led to a significant and remarkable reduction of the UVB-induced damage. Acetylsalicylic acid also showed a significant but lower photoprotective capacity. In contrast, indomethacin showed no photoprotective effects in the photo hen's egg test.

Photo hen's egg test: a model for phototoxicity.

The aim of this investigation was to establish a new model for phototoxicity which is more advanced than the widely used cultures of yeasts, bacteria or cells of various origin, and at the same time to avoid animal testing. We studied the extraembryonal vasculature of the incubated hen's egg. This model was originally introduced by toxicologists as an alternative to the rabbit's eye irritation test (Draize test). In the photo hen's egg test, substances are applied to the embryo's yolk-sac blood vessel system at a non-toxic concentration and are irradiated with 5 J/cm2 ultraviolet A (UVA) (320-400 nm). Promethazine, haematoporphyrin, ciprofloxacin and 8-methoxypsoralen were tested in this system. Death of the embryo, membrane discoloration and haemorrhage are parameters for phototoxic damage, which were recorded during an observation period of 24 h. These well-known phototoxic substances induced pronounced damage of the yolk-sac membrane and blood vessels which was not found in the controls (test substance alone, UVA alone or untreated) using a 2 x 2 factorial test design. The photo hen's egg test serves as a valid screening model for substances supposed to be photosensitizers owing to a phototoxic mechanism.

Effect of gamma-irradiation on the uptake and digestion of 125I-labeled bovine serum albumin by rat visceral yolk sac cultured in vitro.

The effect of gamma-irradiation on a major nutritional function of the yolk sac (the uptake and digestion of macromolecular materials) was studied in rat visceral yolk sacs cultured in vitro being used 125I-labeled bovine serum albumin (RISA) as the tracer protein. The uptake of RISA (per g wet weight) by rat yolk sacs irradiated with doses of 5-80 Gy was essentially the same as that in the unirradiated control yolk sacs. There were no significant differences in yolk sac uptake of RISA with respect to the radiation doses or to culture period up to 18 hours after irradiation. External gamma-irradiation with 10-80 Gy does also had no effect on the extracellular release of 125I from yolk sacs which had been taken it up as RISA. The ratios of the activity in ultrafiltrates of the medium to the total activity in the medium were slightly higher at doses of 40 and 80 Gy.