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CK Vorwerk, R Banaskiewicz, AU Bayer, C Knop, W Behrens-Baumann; Noninvasive Measurement of RGC Survival by In Vivo Microscopy . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4341.
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Purpose: The loss of retinal ganglion cell (RGC) neurons is a well-recognized feature of glaucoma. The present pilot study was focused on the potential of a new fluorescence stereomicroscope (Leica MZ FLIII) as a noninvasive in vivo method for imaging of RGCs in rat eyes. This will allow us to monitor progression of RGC loss over a period of weeks or months in rat models of RGC loss. Methods: The methodology of in vivo fluorescence stereomicroscopy can be carried out easily with the commercially available equipment. For all in vivo experiments adult hooded rats were used within a weight range of 250-300g. Prior retrograde fluorescent labeling of retinal ganglion cells was carried out by injection the fluorescent tracer Fluorogold® into the colliculus superior. After 4-5 days, to allow the tracer to be transported to the cell soma of the retinal ganglion cells, direct imaging of RGC was carried out. The head of the animal was fixed in a three-point device connected to a ball joint for easy positioning. For imaging RGCs we used the new fluorescence stereomicroscope MZ FLIII (Leica, Germany) with appropriate filters for fluorogold. A video camera system (MV-CAM XC003, Sony, Japan) with a digital imaging computer software (Image-Pro Plus Version 4.1, Media Cybernetics, USA) connected to the microscope was used to store the images. Results: The quality of the images allows a quantification of RGC in the stored pictures. For comparison of the same area of the retina, images can be obtained and stored multiple times at various time points. Conclusion: The availability of noninvasive in vivo imaging of retinal ganglion cells and subsequent quantification in the rat retina by fluorescence stereomicroscopy opens new avenues for the research of potential neuroprotective drugs. Ongoing research using this technique will test the usefulness for visualizing dynamic changes, such as retinal ganglion cell loss or change in cell size sequentially in the same rat eye following optic nerve crush, NMDA administration, or elevation of intraocular pressure (various glaucoma models).
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