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L. C. Berglin, L. Bergman, D. Berezovsky, E. Kim, B. Myles, C. Anderson, H. Grossniklaus, H. Edelhauser; Tracing of Intravitreally Injected Labeled Drugs and Nanoparticles in Human Vitreous and Retina Using a Liquid Nitrogen Snap-Freeze Thaw Technique. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3487.
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To dissect and mount the human vitreous body in situ ex vivo in balanced salt solution plus (BSS+) to visualize anatomy, monitor physiologic aging, and drug delivery to the posterior segment of the eye.
Human, pig and rabbit whole globe donor tissue was sutured onto a Lucite frame according to previous descriptions (Sebag J & Balazs EA). The whole globe was snap frozen in isopentane 2-Methylbutane + liquid nitrogen. While still frozen, the sclera, choroid, RPE, and retina were removed en bloc from an intact frozen vitreous body. The frozen Lucite mounted specimen was vertically immersed in a Lucite box filled with BSS+. The vitreous specimen thawed in the BSS+ and was photo documented immediately using darkfield technique. Globes were injected through the pars plana with 0.05 ml riboflavin, fluorescein, triamcinolone, doxorubicin and bevacizumab nanoparticles prior to freezing to visualize a vitreous drug diffusion within the vitreous .
Vitreous dissection of human, pig and rabbit globes was easily achieved. Physiologic aging in human vitreous was noted with increasing vitreous detachment and collapse of the vitreous body in older specimens. The compromised integrity of the posterior vitreous surface in the posterior pole and at the optic disc was noted by the vitreous prolapse and leakage of fluorescein from the vitreous body targeting the macular area in vitreous and retinal freeze mounts, evidenced by the presence of macular staining. The vitreous cisternae previously described by Worst were visualized by the early fluorescein distribution in the intact vitreous.
Human, pig and rabbit whole globes are well suited for Lucite mounting and snap freezing. The vitreous body can easily be dissected and visualized in situ ex vivo. Monitoring of physiologic aging and disease processes as well as drug delivery to the posterior segment of the eye is easily visualized using this technique and provides important information about drug diffusion following an intravitreal injection.
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