These data are consistent with previous studies using FG labeling and Brn3A (POU4F1) antibodies to label RGCs in rat12
and together suggest that most rat RGCs project primarily or by collaterals to the superior colliculus. Brn3 antibodies and Sncg mRNA probes have a significant advantage over retrograde tracers such as FG, which can grossly underestimate RGC number in optic nerve injuries such as glaucoma because of their dependence on intact axon structure between the brain and the eye. Alternatively, prelabeling RGCs before degeneration is prone to overestimate RGC numbers as the tracers are taken up by microglia that phagocytose dead RGCs.1,13
A potential limitation of the use of both Brn3a and Sncg mRNA for identifying RGCs is that some RGCs may decrease their gene expression below detection limits. However, pNF+ RGCs and other nearby RGCs cannot be labeled by FG but still can be identified as RGCs based on Sncg mRNA expression. Thus, Sncg mRNA can identify RGCs even after they are severely injured.
The purpose of these studies was to determine whether three features of RGC degeneration observed in DBA/2J mice1
were model specific or, rather, represented general features of glaucomatous degeneration. First, the degeneration was sectorial. Second, in and near sectors with RGC degeneration, many RGCs maintained RGC gene expression but could not be labeled retrogradely. Third, many of the RGCs that could not be retrogradely labeled had phosphorylated neurofilaments in their soma, a feature observed in other neurodegenerative disorders, such as the neurofibrillary tangles of Alzheimer's disease.14
Translimbal laser IOP elevation in the rat produces a topographic pattern of degeneration similar to that reported in DBA/2J mice1,15–18
; the retinas of lasered eyes have pie-shaped sectors of RGC loss that span from the optic disc to the periphery of the retina. Sectorial degeneration patterns were observed in all lasered retinas except those with near complete loss of RGCs. This sectorial pattern is most likely explained by a focal axonal insult within the optic nerve head. In the fovea-containing human and monkey retina, the patterns of sectorial RGC loss take on arcuate shapes and are concentrated at the upper and lower poles of the nerve head in an hourglass pattern.19
Given the regular radial pattern of RGC axon paths in the rodent retina, the sectorial loss seen here and in DBA/2J mice is likely the phylogenetic equivalent of the pattern of RGC loss in human glaucoma. Because rodents have a glial lamina but lack collagen plates,20
something other than collagen plates must be able to mediate localized IOP-dependent damage to axons within the optic nerve head.
Compared with Sncg mRNA, which is present in RGC somas before and after glaucomatous injury, pNF marks RGC somas only after injury. Normally, phosphorylated neurofilaments are found in RGC axons starting approximately 200 to 1000 μm from the somas,21
consistent with their putative role in maintaining axonal caliber.22,23
However, specifically after axonal trauma, pNF appears in somas and dendrites of damaged RGCs.6–8
The present study confirms previous findings in DBA/2J mice that RGCs with somatic pNF are found preferentially in the degenerating sectors of damaged retina. Comparable increases in pNF+ RGCs and sectorial loss of RGCs have recently been reported in a similar ocular hypertension model in rats.24
As in DBA/2J, these pNF+ RGCs represent a significant proportion of the Sncg+ cells that cannot be retrogradely labeled. Whether they are a stage or a subset of degenerating RGCs is unknown. The reason most of these RGCs cannot be retrogradely labeled is likely that the portions of their axons within the optic nerve have already degenerated given that the loss of axons measured in optic nerve cross-sections 10 days after lasering is already extensive. Further, pNF+ RGCs are associated with dramatic reactive plasticity at the optic nerve head and even within the retina. Such extensive reactive plasticity is typically only seen when neurons are physically disconnected from their targets. Similar reactive plasticity has been recently reported after laser-induced increases of IOP in CD1 mice.25
Unfortunately, the current methods to count RGCs in the retina and axons in the optic nerve are too crude to compare numbers of axons and RGCs within individual eyes. However, on a population basis, most of the axon loss has already occurred by 10 days, whereas the loss of RGCs continues between 10 and 29 days. All these data add to a growing body of literature1,25–27
demonstrating that axons, or at least the extraocular portion of axons, degenerate before RGC somas in glaucoma.
There are two informative differences between the RGC degenerations observed in DBA/2J mice and the rat translimbal laser model. First, in DBA/2J mice, there are hundreds of pNF+ RGCs at the peak of degeneration, whereas in the translimbal laser photocoagulation rat model, pNF+ RGCs number in the thousands. This suggests that after the more acute IOP elevation, RGC degeneration is either more synchronous or faster, or both. The second difference is that after translimbal laser photocoagulation, the pNF+ RGCs tend to be either largely of the weak variety (at 10 days) or of the strong variety (at 29 days), whereas in the DBA/2J mice, there is a more even distribution of both at any one time. This difference is likely attributable to the primary IOP insult occurring more synchronously in the current model, whereas in DBA/2J the insult occurs over a more protracted period. The current results also support the view that weak and strong pNF+ RGCs represent distinct stages in the degeneration of the same cells, as previously suggested.1
Given that there are more pNF+ RGCs at 10 days than at 29 days, weak pNF+ RGCs must have at least two fates: to die quickly or to become strong pNF+ RGCs. Strong pNF+ RGCs have increased expression of Sncg mRNA and are associated with reactive plasticity; therefore, it may be that these cells find sufficient trophic support within the retina to delay their eventual demise. Another important implication of the current studies is that, because the insult that gives rise to sectorial degeneration after short IOP increases produced by translimbal laser photocoagulation is linked to increased IOP, that which gives rise to sectorial degeneration in DBA/2J mice is also likely linked to increased IOP rather than to an unrelated inflammatory process. Finally, if pNF+ RGCs are as common in human glaucomatous retinas as they are in animal models, it may be possible to develop clinical imaging techniques to exploit their large fold-increase in number to diagnose glaucoma or to monitor its progression.