次声作用对血－视网膜屏障功能影响的离子机制研究

Infrasound refers to very low frequency sound, falling below what is usually considered to be the range of human hearing 20-20000Hz. Several studies has indicated that infrasound could affect the pituitary adrenocortical system, cardiovascular system, respiratory system, digestive organ and others of animals or human beings. Claim that infrasound adversely causes pilots visual deficit and substantially reduced visual acuity has not been clearly demonstrated in any experimental study. The purpose of this study is to evaluate the possible adverse effects of infrasound on the visual system from the electrophysiology points of view. Therefore, we try to explore the possible effect of infrasound on the rat's retinal function from visual electrophysiological, morphological points of view and the molecular mechanisms indicated by expression of VEGF and Occludin as well as it's signal transduction pathway by measuring changes of [Ca2+]i and ionic channel.Part one: The influence effects of the visual system exposing to infrasound.To determine the influence of the visual system exposing to infrasound, the visual function were evaluated from the electrophysiological points of view. The results showed that infrasound do harm to the visual system. There was a statistically significant reduction in the amplitude of the electrophysiological parameter with increasing stimulus time while the latency time showed the prolonged trend, which tells that infrasound causes extensive damage to all layers of the retina.Part two: The study of the injury of rat's retina under subjection to infrasound: Morphological changes Morphologically, taking lanthanum nitrate (La) as blood vessel tracer, combined the albumin immunohistochemical staining to localize the breakdown of blood-retinal barrier (BRB) we found that there were no obvious abnormity in the morphology of the eyeball before and after the exposure, but the result of electron microscope showed that during the exposure procedure, the damage which infrasound caused to the BRB stepped up.Part three: Effects of infrasound on VEGF, Occludin expression in tissues of the rat's retina.To further discover the molecular mechanism of BRB breakdown under infrasound, VEGF and Occludin expressions were evaluated by immunohistochemical staining technique as well as Western-blot. The results showed that VEGF was generally absent in the majority of retinas, but can be upregulated by infrasound exposure, which may contribute to the breakdown of the blood-retinal barrier. Meanwhile, by western-blot, semi-quantitatively, the Occludin of retina showed decrease after the exposure. We conclude that retina is in the status of lack of blood and oxygen under infrasound exposure, which inducing the high expression of VEGF in the inner retina. VEGF, possibly through lowering the expression of Occludin or making it in functional disorder, causes the functional disorder of the tight junction of endothelium of BRB, which results the damage of the barrier function.Part four: Effects of infrasound on the current of the Ca2+ activated K+ channel of the cultured retinal microvascular endothelial cells. In order to make initiatory discussion about the signal transduction responsible for the breakdown of BRB under infrasound exposure, we used patch-clamp whole cell technique and laser scanning confocal microscopy (LSCM) to discover the change of cultured bovine retinal endothelial cell(BREC) ion channel and the cytoplasmic free calcium concentration ([Ca2+]i). Being lack of voltage-gated calcium channel (VDCC), the magnitude of the calcium influx into endothelial cells is modulated by the membrane potential which is determined by all kinds of ion channel, the most important one of those is big conductance calcium-activated potassium channel (BKCa). Infrasound exposure caused the increase of BKCa channel activity, and the hypolarization of the membrane potential, which provide the driving force for calcium entering into the endothelium, and thus a series of physical-chemical changes taken place. Using patch clamp

采用免疫沉淀检测技术、免疫荧光技术、膜片钳技术等，从不同角度观察次声作用对动物血－视网膜屏障功能及对培养的视网膜微血管内皮细胞电生理、细胞内钙离子浓度等影响，探讨不同参数的次声作用对眼损伤的特点和对动物血－视网膜屏障功能影响的离子机制，初步阐明次声作用对眼生物医学效应的机理，为次声的卫生防护提供实验依据。