The method used to label RGCs in formalin-fixed retinas is relatively simple and is based on the ability of DiI to diffuse intramembranously and label the cell bodies and processes of ganglion cells, which can be visualized with conventional fluorescence microscopy.
6 7 8 31 The method’s applicability in the diseased retina indicates that the integrity of dendrites remains intact. However, pruned dendritic branches cannot be seen using this method, because they have been disconnected from the main dendritic tree or because they have degenerated and the debris was cleared away. Regardless of the final fate of these branches, their disappearance is probably pathognomonic in glaucoma, and most likely indicates that the RGCs not only atrophy retrogradely because of compression at the optic nerve head, but may also be directly influenced by the increased IOP. Morphologic changes in RGCs have been reported in induced glaucoma in rodents and primates.
25 It has been recognized that acute IOP elevation obstructs orthograde and retrograde axonal transport in experimental monkey models.
29 30 31 This blockage may involve the trophic supply to the RGCs, as shown by the inhibition of brain-derived neurotrophic factor transport after an acute increase in IOP.
12 The morphologic correlate of diseased axons in the glaucomatous retinas in the present study may be altered axoplasmic transport, although dynamic studies have yet to demonstrate this. The sensitivity of axonal transport to various retinal diseases was also demonstrated in the Royal College of Surgeons rat.
32 The irregular, swollen beads along the axons may represent aggregates of transportation vesicles, although blockage of axonal transport has been assumed to be involved in initial stages of RGC loss. By injecting lucifer yellow intracellularly, Weber et al.
25 reported on the absence of axonal beads, which had been described by Kolb et al.,
33 based on Golgi impregnation, and thereby confirmed earlier
18 observations of such beading. This difference may be of a methodological nature, because lucifer yellow diffuses into the axonal lumen, whereas the Golgi method stains argyrophilic compartments of the axon, including the beads. Therefore, the present data, which were obtained with a dye that is known to stain all parts of the axons, clearly shows a beading of normal axons and increased beading of glaucomatous axons, thus suggesting that this is an effect of advanced glaucoma’s effects on axonal transport.