| The vitreous body is a highly hydrated (>98%water) extracellular matrix that fills the eye. The structure of the vitreous gel derives from interactions between collagens (II, EX and XI), hyaluronic acid and proteoglycans (eg. Chondromodulin-1, versican, opticin). In humans and many other primates the vitreous body undergoes irreversible age-related liquefaction (also termed vitreous degeneration or synchesis), which often leads to detachment of the vitreous from the inner surface of the retina (posterior vitreous detachment; PVD). PVD usually occurs without incident, but stress on the retina resulting from vitreous traction may cause severe vision-threatening retinal diseases, including retina tear, rhegmatogenous retinal detachment, macular edema, macular hole, macular traction syndrome, epiretinal membrane formation, even facilitating the development of choroidal neovascularization (CNV) and exudative AMD. Besides, degraded vitreous is also a risk factor for nuclear cataract and primary open angle glaucoma (POAG).However, till now there is not many researches for this vitreous area. Part of the reasons can be ascribed to the difficulties of describing this special material, and the introduction of pars-plana vitrectomy (PPV) for most kinds of vitreo-retinal diseases just simply remove the vitreous. It needs to be mentioned that PPV is also a risk factor for nuclear cataract and POAG development. Researches have found the reason is after removing the vitreous, so removes its antioxidant system as well. Like in aqueous humor,68%of the antioxidants come from ascorbic acid (AsA), so we presume the major antioxidant in vitreous is also AsA. After removing the whole vitreous, excessive oxygen can easily diffuse into the cavity of vitreous space from retina vessels, keep diffusing to the anterior part of eyes, causing oxidative stress and damage to the eye tissues all around.To address of this problem, we have several issues need to understand:1. We need to know what happens to the vitreous components and its structure when people get older and suffer vitreous liquefaction;2. What is the mechanism for vitreous liquefaction;3. Is there a possible method to prevent vitreous liquefaction aiming to the mechanism of vitreous liquefaction;4.Except to the side effects of artificial vitreous, could the efforts trying to keep the original vitreous or renew its structure really help lower these post-operation complications.So, we used bovine and human eyes as the main source for vitreous and designed the following experiments:1. Trying to exam the different strucutre of Cloquet’s channel, and compare anti-oxidant system difference between bovine and human vitreous;2. Trying to modeling vitreous liquefaction in bovine eyes, and test the efficiency of one type of chondroitin sulphate proteoglycan mimics in restoring the structure of bovine vitreous;3. Using proteomics method to identify the vitreous structure protein changes and related protein modifications during people aging;4. Using quick freeze deep-etch electron microscope to exam the original structure of human vitreous and bovine vitreous being treated with different proteases, and immuno-gold labeling to identify specific vitreous components.The results found:1. In bovine vitreous there exists an apparent Cloquet’s channel, being made of the residual vitreous membrane. We did not find any multi-or single channel structure in young human vitreous; the multiple-channel like structure in the previous report may actually be caused by vitreous liquefaction. 2. In aqueous humor and vitreous, there is an opposite trend of AsA distribution in human and bovine vitreous. We tested the concentrations of AsA in different location of the eyes, and correlated with the oxygen concentration, which may provide a way to explain the working mechanism of anti-oxidants system in the eyes;3. We used trypsin and collagenase to build a bovine vitreous liquefaction model, and then applied with a type of chondroitin sulphate proteoglycan mimics, which could either bind to type Ⅱ collagen or hyaluronan or both. With the tests including rheology, percentage of vitreous liquefaction calculation, western blot and quick freeze deep-etch electron microscope exam, we found these CS-proteoglycan mimics have the ability of preserving or restoring the physical properties and ultra-structure of vitreous tending to liquefy;4. We primarily set up a customized proteomics method for screening the vitreous structure proteins, and got first preliminary data from a human donor vitreous. Through Western blot, we found that as aging, one of the major structure proteins in vitreous-type Ⅱ collagen-underwent decreased presence in human vitreous.5. We used quick-freeze deep etch electron microscope to first reveal the original vitreous structure in human with different ages, and in bovine vitreous after treating with several different enzymes. Combined with immune-gold labeling, we identified several structure proteins in vitreous including type II collagen, type IX collagen, OPTC et al, and made the possible quantification measure.In general, by detecting morphology, using proteomics and chemical analysis, we tried to discover and discuss the special structure and function of the vitreous. With bioengineer methods we applied new insight into prevention of vitreous liquefaction and further vitreous degeneration. Hopefully this method could be broadly used, and advancing the prevention and therapy of vitreous degeneration related eye diseases. |
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