In the current study, we have employed a rat model of optic neuropathy induced by chronic elevation of the IOP together with a combination of histology, immunohistochemistry, Western blotting and real-time RT-PCR to address the spatial and temporal nature of RGC pathology. As identified by Morrison et al. [33
], the advantage of such a model compared with spontaneous models of chronic ocular hypertension is that the timing of the IOP increase following the surgical intervention is known. This engenders greater confidence in conclusions drawn about the chronology of pathological events. The data presented here provide robust support for the hypothesis that the ONH is the pivotal, and likely the primary, site of RGC injury following moderate elevation of IOP, with resulting anterograde degeneration of axons and retrograde injury and death of somas.
Anterograde fast axonal transport conveys newly synthesized molecules away from the cell body. Obstruction of this process rapidly compromises the integrity of the distal axon. In glaucoma, the lamina cribrosa of the ONH has long been considered a likely site of axonal transport failure. This hypothesis was formed after pioneering work performed in monkeys, which demonstrated that radioactive leucine accumulated within axons at the ONH after moderate elevation of IOP [1
]. Similar results have been found in pigs [2
]. However, current glaucoma research is mainly performed in rodents, and rodents lack a true lamina cribrosa. Rats possess a rudimentary structure, whilst mice have no connective tissue [15
]. As such, it is important to ascertain whether the ONH is an important site of axonal transport failure in rodents. We achieved this aim by immunolabelling for proteins (APP, synpatophysin and BDNF) that are routinely synthesised by RGCs and conveyed along the ON by fast axonal transport [6
]. Because the molecules analysed are of different molecular weights and have distinct physiological roles, this approach provides biologically meaningful information about transport viability during chronic ocular hypertension. Our results showed accumulation of all three proteins within axons at the ONH, but not distal to this location in the myelinated ON or OT, results confirmed by the use of the neural tracer CTB. The time course correlated well with the early monkey studies, with detectable accumulation by 8 h and widespread dysfunction from 24 h. By 14 days, however, the mean IOP had decreased markedly and disruption was measurably lower. The reduced accumulation of APP at this time point can be accounted for in two ways: (1) in axons that were not irreversibly damaged, the lower IOP allows normal transport of APP to resume; (2) axons that were irreversibly damaged by high IOP have now degenerated. Quigley and Addicks [38
] noted that a return to normal IOP within 1 week restored transport in some axons in monkeys.
Previous studies in rats have found results compatible with the hypothesis that chronically elevated IOP disrupts active retrograde axonal transport to the retina at the level of the ONH [28
], findings consistent with this study. In contrast, Crish et al. [12
] showed that axonal transport dysfunction in both spontaneous and induced rodent models of IOP elevation appeared first at the superior colliculus and progressed distal proximal, with ONH deficits occurring much later. The disparity between these studies may relate to the models used. In the micro-bead model used by Crish et al., the IOP elevations were maximally 10 mmHg and maintained for long periods, whilst the laser model used here and by others produces typical IOP rises of 25 mmHg for shorter periods. It is possible that modest, prolonged increases in IOP gradually compromise axonal transport efficiency, which is first manifest at the distal synapses, whilst greater increases in IOP physically constrict axons at the ONH.
We found axonal cytoskeletal abnormalities, including neurofilament beading and swellings, in the ONH at 24 h after induction of raised IOP. This suggests that axonal transport disruption is mechanical, and not simply functional, in a subset of axons at very early time points. Nevertheless, in other axons, it is likely that active axonal transport dysfunction significantly preceded physical damage, an argument supported by the results of Salinas-Navarro et al. [42
], who counted fewer RGCs in retinas back-labelled by a tracer that undergoes active transport than in retinas back-labelled by a passively diffusing tracer. In contrast to the ONH, no axonal cytoskeletal abnormalities were present in the entire myelinated ON and OT until 3 days, with progressively greater damage at 7 and 14 days. The results support the findings of others that IOP elevations of the magnitude recorded in this study elicit an early insult at the lamina of the ONH with Wallerian-like degeneration of axons distal to the site of injury [16
]. Regarding axonal cytoskeletal degeneration, a previous study in monkeys showed accumulation of npNFH in the ON following raised IOP [22
]. Using immunohistochemistry, we found a similar, robust increase in npNFH labelling in degenerating axons; however, Western blotting of ON samples revealed no increase in the npNFH 200-kDa band, rather the appearance of a continuum of low-molecular weight bands. These bands almost certainly represent breakdown products and may account for the increased immunoreactivity in tissue sections. npNFH is more labile and sixfold more susceptible than pNFH to degradation by calpain [34
] and our data indicate that it degenerates more rapidly than pNFH.
The strikingly early nature of pathological changes at the ONH prompted the question as to whether RGC somas are irreversibly injured at this same time. Our data indicate not. Down-regulation of RGC-specific mRNAs, which are sensitive early indicators of RGC viability [10
], occurred subsequent to axonal changes at the ONH and markedly later than in retinas subjected to NMDA-induced somatic excitotoxicity. It can be argued that the elevated IOP placed a considerable physiological stress on a proportion of RGC somas as evidenced by their upregulation of the molecular chaperone Hsp27; yet, this response also occurred in rats with normal IOPs that underwent ON crush and may simply have been caused by damage to the axonal compartment.
The long-term objective of glaucomatous pharmacotherapy is not merely neuroprotection of surviving RGCs, but regeneration of injured/disconnected axons. Within the CNS, endogenous regenerative attempts are always unsuccessful. In the visual system, RGC axons display only transient, local sprouting, proximal to the lesion site after ON crush [3
], and interestingly, even this limited response occurs only when the injury is within 3 mm of the eye, not if it is administered to the distal ON [13
]. Unlike the catastrophic injury caused by traumatic axonopathies, such as ON crush, RGCs are lost gradually during chronic ocular hypertension and only a proportion of the population will die. It follows that the inhibitory environment for regeneration may be less pronounced and regeneration strategies more effective. Surprisingly, no data are available on the endogenous regenerative response of RGCs during experimental glaucoma. Delineating such information is of utmost importance. We have shown in the current study that RGC axonal injury is first evident at the ONH and that the somas remain viable for a number of days; thus, we hypothesised that endogenous RGC regeneration should proceed at least to the ONH. To examine putative axonal regeneration, we employed Gap43, the quintessential marker of axon growth, but one which can be expressed in non-regenerative situations [25
], hence the caveat “putative”. Upregulation of Gap43 protein in the retina was first detectable by 3d after IOP elevation. By 14 days, numerous Gap43-positive axons were observed in the pre-laminar ONH, some extending to the transition region of the ONH. For comparative purposes, we analysed Gap43 in rats subjected to NMDA-induced excitotoxicity and ON crush. After NMDA treatment, no Gap43 expression was detected, a result consistent with early RGC somal death. After ON crush, substantial Gap43 immunoreactivity was observed, which extended to the crush site. The overall results provide further evidence that the ONH is the principal site of axonal injury in this rat glaucoma model and that chronically raised IOP induces a crush-like insult at this location.