Abstract
Purpose: :
To explore the presence and distribution of the large extracellular matrix glycoprotein, Tenascin-X (TNX; 220/500 kDa), in the rat retina and surrounding ocular tissue.
Methods: :
TNX protein expression was examined using immunohistochemistry (Santa Cruz rabbit polyclonal 1:200; Molecular probes goat anti-rabbit Alexa 594 1:100) on 12um thick frozen sections from eyes fixed in 4% buffered formaldehyde, and by western blotting. Immunohistochemistry was also performed on eyes where the retinal ganglion cells (RGCs) were retrogradely labeled by injecting 4% Fluorogold (FG) into the superior colliculus. Retinal sections were examined by epifluorescence and confocal microscopy. Changes in TNX expression were also examined by western blotting of retinal lysates from eyes that had received an optic nerve transection 2 weeks earlier. Retinas from control and axotomized rats were homogenized, lysed, and protein levels determined by Bradford assays. Gels, optimized for high molecular weight proteins, were loaded with equivalent protein from control and axotomized retinas, and retinal ganglion cell (RGC-5), Müller Cell (rMC-1), and fetal lens (FHL 124) cell line lysates for comparison. Following electrophoresis, protein was transferred to nitrocellulose membranes, probed with the same antibody as used for immunohistochemistry, and detected using ECL detection reagents (Amersham).
Results: :
TNX immunoreactivity (IR) was localized to retinal cells bordering the inner nuclear layer. RGC layer cells were most intensely stained, while cells in the amacrine cell layer of the INL were weakly labeled. Both FG- and non-FG-labeled cells in the RGC layer were TNX-IR. Astrocytes at the optic nerve (ON) head were also TNX-IR. TNX-IR was localized to the cytoplasm of retinal and ON cells. Western analysis of retinas revealed a major band between 220-250 kDa, as expected for TNX. No bands were detected in lysates from RGC-5, rMC-1, or FHL cells. Densitometric analysis revealed a 50% increase in TNX following optic nerve transection.
Conclusions: :
TNX has been previously localized to connective tissues where it plays a role in cell-matrix, cell-cell adhesion, and cell migration. In the present study we have localized TNX to the cytoplasm of RGCs, amacrine cells and optic nerve astrocytes. Western analysis revealed the presence of protein in retina with the expected molecular weight of TNX, but not other cells. In contrast to an expected decrease in TNX protein following the loss of 90% of RGCs after axotomy, TNX expression went up. Although the significance of an intracellular localization of TNX in retinal and ON cells is unclear, the finding that TNX expression increases after axotomy suggests a role for TNX in injury.
Keywords: ganglion cells • neuroprotection • astrocytes: optic nerve head