Herein we show for the first time that repetitive preconditioning with sublethal hypoxia before disease onset can prevent the subsequent neurodegeneration characterizing glaucoma. The uniquely sustained adaptive response to RHP robustly abrogates the ongoing apoptotic death of RGC soma as well as axonal injury/loss. This novel demonstration of “glaucoma tolerance” implies the existence of intrinsic, cytoprotective regulatory systems in the CNS, the protracted activation of which can prevent or slow both the somatic and axonal degeneration associated with this disease. In turn, these findings advocate for an expansion of the traditional definitions of preconditioning and tolerance in the CNS to incorporate this novel form of induced neuroplasticity, and suggest the exciting possibility of achieving similarly protracted periods of protection against chronic cellular injury in other tissues.
In glaucoma, RGC axons die by mechanisms distinct from those governing the demise of the soma (11
). Thus, we measured RGC viability at both somatic and axonal levels in the present study, not to elucidate primary and secondary injury events per se, but to demonstrate the potential pan-cellular, multifactorial protective effects of RHP. With respect to RGC soma, RHP robustly attenuated the 20–30% progressive loss of RGCs we documented by brn3 immunolabeling during the initial 3–10 wks of intraocular hypertension. The brn3-positive cell bodies we quantified comprise a specific, but relatively large, subpopulation of RGCs that express this RGC-specific gene product (8
); displaced amacrine cells are not identified with this immunolabel. Although the relative susceptibility to glaucomatous injury of RGCs carrying the brn3 gene compared with other RGC subpopulations is unclear, the magnitude of RGC soma loss in our model was similar to that independently measured in other inducible mouse glaucoma models (21
). RHP-induced soma protection was also confirmed by immunolabeling for the nonphosphorylated neurofilament heavy-chain marker SMI32. The nuclear condensation of the label, truncation of positively stained dendrites and somatic shrinking we witnessed in non-preconditioned glaucomatous retinas, described previously for the DBA/2J model (11
), defines an RGC soma phenotype that was not observed in RHP-treated mice. Finally, our finding of hyperphosphorylated SMI34-positive RGC soma, axons and dendrites only in the glaucomatous retinas of nonpreconditioned mice is consonant with similar soma labeling in mouse RGCs disconnected from their distal axons in the DBA/2J model (19
) and in rat RGCs after optic nerve crush (10
); the shifting of neurofilament phosphorylation from axon to soma is common to many neurological disorders and may be a harbinger for the eventual demise of the cell (13
). Taken together, our findings support the concept that innate responses can be induced in RGCs by preconditioning that promote the survival of the soma in the face of neurodegeneration-inducing glaucoma.
Histological, biochemical, molecular and genetic evidence, in animal models (14
), monkeys (28
) and humans (29
), collectively support the contention that RGC soma loss in glaucoma occurs by apoptosis. Our documentation of expected changes in several apoptotic endpoints in the current study are consonant with previous findings in these different models with respect to the altered expression of cleaved caspase-9 (30
), cleaved caspase-3 (21
) and bcl-2/bax (21
). That changes in these aforementioned apoptotic endpoints were largely abrogated in RHP-treated mice indicates that RHP clearly established a robust antiapoptotic phenotype for RGC soma. In fact, at least for bcl-2 and bax, we confirmed that RHP appears to “prime the pump” for such an effect, given the changes we observed after RHP in mice without subsequent IOP elevation. Whether RHP promotes such a phenotype secondary to transcriptional and/or posttranslational regulation of these and other proteins is not yet known. Overall, our results indicate that, mechanistically, when RHP precedes the period of intraocular hypertension, the somatic expression of several hallmark pro- and antiapoptotic proteins is altered in a sustained fashion in the retina such that the apoptotic demise of RGC soma is dramatically reduced.
With respect to RGC axons, our findings demonstrate that RHP treatment robustly protected against glaucomatous axonal loss, both distally in the postlaminar optic nerve and more proximally in the retinal nerve fiber layer. This result is a critical finding, given the accumulating evidence indicating that, in glaucoma and other neurodegenerative diseases, axonal injury is a fundamental, and perhaps primary, event that ultimately leads to the apoptotic death of the soma (11
). Although the extent and pattern of IOP elevation varies between models, and temporal correlations are not exact, the 32% axonal loss in the postlaminar optic nerve we quantified after 3 wks of IOP elevation was similar to the magnitude of loss (20–50%) observed after 2–4 wks in other inducible mouse glaucoma models (24
). We also quantified robust preservation of proximal RGC axon integrity within the retina in RHP-treated mice. Because of their role in regulating and, to a minor extent, participating in axonal transport secondary to microtubule contacts, changes in phosphorylation status of the different neurofilament subunits can serve as surrogate markers for axonal injury and degeneration (34
). Because SMI32 recognizes a nonphosphorylated epitope on the neurofilament heavy subunit that comprises the axonal cytoskeleton in healthy RGCs (11
), expression changes are thought to reflect axonal dysfunction or degeneration and not a reduction in neurofilament transport; however, whether reductions in SMI32 labeling truly reflect axonal loss remains to be demonstrated conclusively. The greater axonal loss in superior regions of the postlaminar optic nerve that we observed at 10 wks is consistent with the regional difference reported for adult mice 12 wks after IOP elevation by limbal laser photocoagulation (36
). Similar changes also occur in progressive fashion in the DBA/2J model with advancing disease (11
Collectively, our quantitative and qualitative assessments of axonal integrity, at two locations and at two distinct time points, make it clear that the preservation of RGC axons was promoted by prior RHP treatment despite the protracted period of intraocular hypertension that causes significant axonal loss in untreated mice. Although this protection may, in part, be secondary to RHP-induced changes in astrocytes, oligodendrocytes, non-RGC neurons and/or other cells, the endogenous, intra-axonal protective mechanisms that are uniquely activated by RHP to prevent or slow this axonopathy will be important to elucidate not only for this disease, but for brain and spinal cord injury and other white matter neuropathies. In glaucoma, this step may involve reductions in the extent of distal and proximal axonal transport deficits; inhibition of calcium influx, modulations in amyloid precursor protein (APP), caspase-6, nicotinamide mononucleotide adenylyltransferase (Nmnat), Jun NH2
-terminal kinase (JNK) or other putative mediators of the Wallerian degeneration-resistant phenotype; and perhaps even synaptic and other physiological adaptations at the level of the superior colliculus (17
). The lack of axonal protection in DBA/2J mice deficient in bax (14
) would suggest the axon survival- promoting effects of RHP are bax-independent and largely distinct from the antiapoptotic-based protection that we showed RHP affords to RGC soma.
Many studies support the contention that astrocytes (particularly those in the optic nerve head) contribute importantly to glaucoma pathology (37
). Moreover, glial cell activation, as evidenced by the prototypical upregulation of GFAP, is thought to be a hallmark of CNS injury. However, this prevailing wisdom is being countered by the notion that, in glaucoma, reactive astrogliosis and other functional/morphological responses of astrocytes may actually be protective for nearby axons and soma depending on the spatio-temporal context of the response to elevated IOP (38
). The astrocyte hypertrophy and enhancement in GFAP immunostaining intensity that we observed in the postlaminar optic nerve are well-established, relatively early responses in other inducible (27
) and genetic (11
) glaucoma models and may be a progressive response to fill spaces vacated by degenerating axons (11
). That these glial changes did not occur in mice with prior RHP may reflect an astrocyte-specific adaptive response that contributes to the preservation of neighboring axons, or it may simply be that this secondary response to axonal loss was never initiated because RGC axonal dysfunction/loss was minimized by preconditioning.
Somatic and axonal protection of RGCs by RHP was demonstrated herein using an inducible mouse model of primary open-angle glaucoma in which a sustained, moderate elevation in IOP was achieved by weekly ligation of patent episcleral veins. Another group performed episcleral vein ligation in mice a single time and also obtained similar levels of RGC loss after a couple of weeks (23
); however, as also observed in rats (25
), elevations in IOP were not as consistently maintained in these single ligation models. Laser photocoagulation of limbal and episcleral veins (21
) and injection of microbeads (24
) represent other ways of inducing transient IOP elevations in mice. Whereas reviews of the pros and cons of these rodent models will likely continue (16
), we would predict that the robust RGC survival-promoting effects of RHP that we documented in our model will ultimately be demonstrable in other inducible models of glaucoma and perhaps other neurodegenerative diseases, given that preconditioning-induced ischemic tolerance appears to reflect fundamental, evolutionarily conserved, adaptive mechanisms on the part of all the cells in the body (41