Abstract
Abstract: :
Purpose: Abnormal zinc metabolism has been described in retinal disorders such as age–related macular degeneration (AMD). One of the hallmarks of AMD is the appearance of drusen both peripherally and in the macula. Previous studies looking at the zinc content of drusen were inconclusive. Our present study concentrated on demonstrating the presence of readily available or chelatable zinc in drusen in human donor eyes. Methods: Frozen donor eyes from the Montana Eye Bank were used and were photographed before dissection. Using a trephine, 6 mm diameter regions of the peripheral and central retinae were dissected. Following defrosting, the neuronal retina and the retinal pigment epithelial cells were removed to expose the underlying Bruch’s membrane with or without drusen. Paired samples from individual eyes were treated in two ways. One of the samples was immersed in phosphate buffer saline (PBS) that contained 100 µM of the zinc specific chelator TPEN and the second sample was in PBS only overnight before 10 µM Zinpyr1 (a zinc specific fluorescence probe) in PBS was applied to both samples for 5 min. Excess Zinpyr1 was removed by rinsing the samples in fresh PBS and the changes in fluorescence was viewed by a NIKON fluorescence microscope (excitation: 460–500 nm; emission: 530–560 nm). Results: The samples that were treated with PBS only there was a robust increase in fluorescence in the presence of Zinpyr1, but in the TPEN treated samples the increase in fluorescence was not detectable, signifying the presence of chelatable zinc. There was an overall increase in baseline fluorescence that appeared to be associated with elastin, a known zinc binding protein, in Bruch’s membrane and vascular walls. Long incubation with Zinpyr1 resulted in punctate staining within drusen that appears to be located within caveoli in some drusen. Conclusions: Zinc is present in drusen and may play a role in the pathogenesis of AMD although its relevance to the disease process is yet to be determined.
Keywords: age-related macular degeneration • drusen • microscopy: light/fluorescence/immunohistochemistry