Expression of glucose transporters in the prelaminar region of the optic-nerve head of the pig as determined by immunolabeling and tissue culture.

BACKGROUND: To develop the use of cultured tissue of the prelaminar optic nerve of the pig to explore possible alterations of the astrocyte-axon metabolic pathways in glaucoma, we map the distribution of the glucose transporters GLUT1 and GLUT3 in fresh and cultured tissue. METHODS: We monitor cell survival in cultures of the prelaminar optic-nerve tissue, measuring necrosis and apoptosis markers biochemically as well as morphologically, and establish the presence of the glucose transporters GLUT1 and GLUT3. We map the distribution of these transporters with immunolabeling in histological sections of the optic nerve using confocal and electronic transmission microscopy. RESULTS: We find that the main death type in prelaminar culture is apoptosis. Caspase 7 staining reveals an increment in apoptosis from day 1 to day 4 and a reduction from day 4 to day 8. Western blotting for GLUT1 shows stability with increased culture time. CLSM micrographs locate GLUT1 in the columnar astrocytes and in the area of axonal bundles. Anti-GLUT3 predominantly labels axonal bundles. TEM immunolabeling with colloidal gold displays a very specific distribution of GLUT-1 in the membranes of vascular endothelial cells and in periaxonal astrocyte expansions. The GLUT-3 isoform is observed with TEM only in axons in the axonal bundles. CONCLUSIONS: Tissue culture is suitable for apoptosis-induction experiments. The results suggest that glucose is transported to the axonal cleft intracytoplasmically and delivered to the cleft by GLUT1 transporters. As monocarboxylate transporters have been reported in the prelaminar region of the optic-nerve head, this area is likely to use both lactate and glucose as energy sources.

Fenestrations and preferential flow routes in the prelaminar optic nerve through wet scanning electron microscope and perfusion of tracers.

PURPOSE: We study the vitreous interface of the optic disc to delimit the passages for the flow of fluids through the prelaminar tissue of porcine eyes. METHODS: Wet scanning electron microscope (SEM), conventional SEM and transmission electron microscope (TEM) were used to explore the surface of the optic nerve of the pig. The vitreous cavity was perfused with a fluorescent marker and colloidal gold at controlled pressure. Samples of perfused optic nerve head were cryosectioned and observed with the confocal laser microscope (lectin) or resin embedded and observed under TEM (gold). RESULTS: Fenestrations were present under the SEM in all three regions of the vitreous interface. SEM results were confirmed at the TEM level and under the wet-SEM. Perfusion experiments traced the flow of a fluorescent molecule delineating routes of preferential flow with origin in the fenestrations. Colloidal gold marked the site of entrance in the prelaminar tissue identifying major fenestrations in the basal membrane. CONCLUSIONS: Interchange of fluid between the optic nerve and the vitreous cavity in the pig is facilitated by fenestrations of varied sizes in the basal membrane and preferential flow routes through the prelaminar tissue. Preferential flow routes exist in the extracellular spaces of Elschnig and Kuhn' astrocytes and give a sharply distinct image when compared with flow through zones in which astrocytes envelope axons. Escape routes may be instrumental in preventing oedemas of the optic nerve head, but they could also serve as entrance doors for fluids from the vitreous and aqueous and play a pathogenic role in ageing and glaucoma.