Before addressing the usefulness of the explant model for screening RGC neuroprotective therapies, it was necessary to define RGC degeneration over time ex vivo. We predicted that to detect RGC neuroprotection optimally, active cell loss must be ongoing rather than have reached a maximum plateau. Three parameters were used to assess RGCL cell survival in explant sections, as a proxy for RGC survival. Quantification of the number of cells in the RGCL of explant sections over 2 weeks ex vivo demonstrated that the rate of cell loss was most rapid from 0DEV up to approximately 5DEV to 7DEV, after which the rate of loss slowed. To determine the stage at which cultures were reliably sensitive for detecting neuroprotection, we compared two time points in subsequent experiments. By 4DEV, sufficient cell death had occurred to permit the detection of neuroprotection and cell death was steadily ongoing (injured cells were available to protect). By 7DEV, the rate of RGCL cell loss had slowed (fewer injured cells to protect) however a large number of cells had died, thereby providing a larger potential difference to detect preservation. We also observed that the rate of cell death was somewhat user and/or laboratory dependent, with slightly different survival rates detected by individual experimenters. Thus, it is advisable for end-users to characterize the time course of RGC death in this system before use. It is also important to note that RGC survival can vary between experiments conducted at different times, even within the same laboratory, probably because of the slight variability in culture conditions or tissue viability after dissection. Thus, it is critical that experimental and control explants be cultured in parallel (with explants from each group distributed evenly across culture plates) to ensure identical culture conditions for meaningful comparison of results. Interestingly, RGC injury before explant culture had little effect on subsequent RGC survival ex vivo. It could be expected that injury before explantation would initiate cell death pathways in vivo and, thus, accelerate RGC loss in explant cultures. In fact, no effect of previous OHT on explant RGC survival was found, and only a mild trend to increased cell death was noted in explants after previous ONC injury.
It should be noted that the histologic assessments used in this model are unlikely to represent a pure measure of RGC survival. Choice of a selective RGC marker to assess survival after injury requires careful consideration because available RGC markers are imperfect. Specifically regarding the assessments used in this study, quantification of RGCL nuclei by DAPI overestimates RGC survival because of the inclusion of glial, vascular, and immune cells. It was for this reason that more specific immunohistochemical markers were also assessed. Immunohistochemical methods, rather than retrograde RGC labeling, were chosen because the latter technique would have necessitated intracranial surgery in vivo before explantation. An ideal antigen for RGC quantification would be RGC specific, with nuclear expression (for accurate quantification) unaffected by cellular injury. Both Islet-1 and NeuN are nuclear antigens expressed by RGCs, although both markers are also expressed by some displaced amacrine cells.21,22
However, we examined displaced amacrine cell loss in retinal explants over time ex vivo and found it to be modest (D). Thus, we concluded that the loss of immunohistochemically positive displaced amacrine cells would not significantly influence assessment of RGC degeneration. In addition to imperfect specificity, neuronal protein markers may be downregulated after neuronal injury but before cell death,23–26
which could underestimate the numbers of surviving cells. Despite these caveats, all three cell labels used here have been used previously to quantify RGCs in situ.27–31
Although other antigens are more specifically expressed by RGCs (e.g., βIII-tubulin, Thy1, γ-secretase), their additional labeling of RGC processes produces a staining pattern too disorganized for accurate quantification. In addition, immunohistochemistry for Brn3, another RGC-specific marker, was tested but failed to produce a clear and consistent signal in explants, possibly due to gene downregulation after explantation. Overall, however, it is important to note that the same assumptions applied to quantification in all explants, including parallel controls. Therefore, quantification caveats should be comparable across groups and controlled for with respect to individual treatments.
Tissue/cell culture experiments are normally conducted at atmospheric oxygen tension (i.e., ~21% O2
), despite most cells experiencing lower oxygen concentrations in vivo. Interestingly, maintenance of cultures in reduced oxygen can enhance neuronal survival in vitro, presumably through reduced oxygen toxicity.32–34
Thus, we examined whether the maintenance of retinal explants in reduced oxygen improved explant longevity and enhanced neuronal survival. Analysis of RGC survival and morphology clearly demonstrated that low oxygen tension exacerbated RGC degeneration in explant cultures. In contrast, the survival of photoreceptors, as assessed by ONL morphometric analysis, appeared potentiated in these cultures. This could, however, be partially attributed to tissue relaxation or swelling in culture conditions. This may indicate a difference between RGC and photoreceptor sensitivity to oxygen toxicity, and/or their respective oxygen demands. These differential features may reflect the conditions to which each cell type is typically exposed in vivo, where oxygen tension is lower in the outer retina compared with that of the vascularized inner retina.35
The purpose of the initial treatment experiments was to verify that RGC neuroprotection previously demonstrated in vivo could be replicated in vitro using the retinal explant model. The first compounds tested were a pan-caspase inhibitor and BDNF. BDNF has been well established as an important prosurvival factor in RGCs, and dysregulation of BDNF signaling has been strongly implicated in RGC degenerative diseases such as glaucoma.36,37
It has also been demonstrated that apoptosis plays a key role in glaucomatous RGC death and that the blockade of apoptosis can enhance RGC survival after injury.38,39
In addition, we recently demonstrated that the intravitreal transplantation of MSCs strongly protects RGCs in vivo10
and, therefore, examined whether this effect could also be replicated ex vivo. Application of either the caspase inhibitor or BDNF enhanced RGC survival in adult retinal explant cultures, thereby replicating results previously observed in other systems. Intriguingly, the neuroprotective effects of both caspase inhibition and BDNF in retinal explants were modest. There could be several reasons the protective effects observed were perhaps less than expected. Prosurvival signaling by BDNF in RGCs appears to exhibit variability depending on which cellular compartment (soma or axon) initiates the signaling cascade, with retrograde BDNF apparently more potent than endogenous retinal signaling.40
Thus somatic application in explants may be less effective. Moreover, the efficacy of BDNF neuroprotection is notoriously short-lived, perhaps because of changes in receptor expression after chronic ligand exposure or in disease states.23,37,41
A larger reduction in RGC death by caspase inhibition might also have been expected. It is unclear why only a moderate, though strongly significant, neuroprotective effect was observed after caspase inhibition in retinal explants. It is possible that the inhibitor dose chosen was inadequate for robust signaling blockade in explants, or perhaps the axotomy injury was sufficient to overcome blockade of cell death pathways. In contrast, coculture of MSCs with retinal explants was found to confer strong RGC protection, comparative to that observed previously in vivo, which lasted for a relatively long time (for at least 7DEV). Almost all other pharmacologic neuroprotection was detected only at 4DEV. Indeed, MSC coculture was by far the most effective neuroprotective treatment tested in this study. Furthermore, injury to RGCs before explantation for culture did not impact the strong MSC-mediated RGC neuroprotection in vitro. These results, taken with the BDNF and caspase inhibitor observations, suggest that organotypic adult retinal explants can provide a system in which to model in vivo retinal neuroprotection. Given that retinal cells are maintained in situ and within an environment more similar to that in vivo, compared with other in vitro systems, it is possible that the retinal explant model will provide a useful tool for screening novel neuroprotectants. However, the accuracy of its predictive ability remains to be established.
Based on these results we used the adult retinal explant system to investigate RGC neuroprotection by a number of pharmacologic compounds, some previously reported to be efficacious alongside more novel compounds. The main purpose of these experiments was to demonstrate the potential of the adult retinal explant system rather than to explore the therapeutic potential of any single agent in great detail. One advantage of an organotypic model over in vivo preparations is its ability to isolate direct retinal effects from systemic effects. For example, it appears that several ophthalmic drugs routinely used to control IOP in glaucoma may also protect RGCs by acting on the retina directly. Here, use of the explant system would permit the study of direct RGC protective effects independently of effects on IOP, which may indirectly protect RGCs. In the present study, we examined the direct retinal neuroprotective effects of several clinical ocular hypotensive drugs on RGC survival in retinal explants. All had been previously reported to protect RGCs from death using in vivo glaucoma models, results from which are potentially complicated by IOP effects. An additional advantage of the explant model over dissociated retinal cultures may be sensitivity to neuroprotective drugs that work through indirect mechanisms involving other retinal cell types (e.g., glia).
Betaxolol is a selective β1-adrenoceptor antagonist reported to alleviate RGC death both in vitro and in vivo.42–44
There is some experimental evidence that betaxolol treatment may elicit better visual field outcomes in glaucoma patients, compared with other IOP lowering β-blockers (e.g., timolol), despite lower reductions in IOP.43
The interpretation of these human studies remain controversial because the possibility remains that the apparent neuroprotective effect of betaxolol could actually occur through an effect on the IOP profile not captured by the study design. Nevertheless, such studies have been used to support the idea that betaxolol may modulate RGC survival by both IOP-dependent and IOP-independent mechanisms. Betaxolol has been postulated to confer neuroprotection by blocking sodium and calcium channel activity.43,44
In the present study, betaxolol was found to reduce RGC loss robustly in retinal explants. Clearly, this is a direct cellular effect given that explants are isolated from IOP, and it supports the conclusion that betaxolol not only lowers IOP but could potentially have a direct neuroprotective effect if delivered to RGCs at an appropriate concentration.
In contrast, no direct neuroprotective effect was found after treatment of the explants with the α2-adrenoceptor agonist brimonidine. This drug has also been reported to protect RGCs from death in vitro and in vivo,45–48
possibly by modulating Ca2+
influx or intracellular signaling pathways.45,46,49
However, a lack of effect on Ca2+
-channel activity has also been reported,16
and neuroprotective efficacy has not been confirmed in an evidence-based review of relevant clinical trials.50
Lack of neuroprotection by brimonidine in retinal explants would appear consistent with these later data.
Losartan is a selective, competitive angiotensin-II receptor (type 1) antagonist reported to lower IOP in glaucoma patients,51
and drugs from this class (in particular candesartan) can reduce retinal degeneration in rat models of diabetic retinopathy,52
This neuroprotective effect may be mediated by modulating retinal production of reactive oxygen species.53
In retinal explant cultures, we detected a modest losartan-mediated protective effect on RGCs, suggesting direct neuroprotection independent of IOP reduction. This effect was not as strong as that observed after betaxolol application.
Tafluprost is a preservative-free synthetic prostaglandin-F2alpha
receptor analog. Prostanoid agonists have been found to protect RGCs from injury in vivo,55–57
perhaps by suppressing apoptosis through the inhibition of caspase-3 activation.58
In support of previously published work, application of tafluprost to retinal explants enhanced RGC survival significantly, suggesting a direct neuroprotective ability concomitant with ocular hypotensive effects.
Simvastatin is an HMG-CoA reductase inhibitor belonging to the statin class of drugs that were traditionally developed as hypolipidemic drugs to control hypercholesterolemia in cardiovascular disease. These drugs have no ocular hypotensive effect and are not used for glaucoma therapy. Since their original clinical application several off-target effects have been discovered, including a potential neuroprotective capacity.59,60
Indeed, in vivo statin treatment can protect RGCs after axotomy and ischemia/reperfusion.61–63
The neuroprotective mechanism of statins appears to be multimodal (see Ref. 59
for a review). However, importantly for RGC neuroprotection, they are reported to modulate Bcl-2, BAX, BDNF, and heat shock protein expression, and also activation of Akt, Wnt, and ERK signaling pathways,59,62–66
which are all known mediators of RGC survival. In support of previous studies, simvastatin treatment of adult retinal explant cultures offered significant RGC neuroprotection, suggesting the ability of this drug to alleviate RGC death in glaucoma/OHT should be further investigated.
Of all pharmacologic agents tested, none prevented RGC death completely but instead only delayed degeneration. This is perhaps unsurprising given that RGCs are axotomized upon explantation. This observation suggests there is a temporal window in which to detect neuroprotective explant effects. In the present study this time point was approximately 4DEV. However, variation in explant survival was noted between laboratories and, to a lesser degree, between experiments. Therefore, the system requires optimization by end-users for the detection of neuroprotection. Furthermore, some variability in RGC marker ability to reveal differences in survival was observed, with NeuN appearing more consistent and sensitive in the present study. This finding, in conjunction with other considerations discussed, highlights the need to choose the RGC marker/assessment used with care. We also demonstrated that no previous RGC injury is needed to generate an ex vivo system in which to detect RGC protection. Replication of previously established in vivo neuroprotective treatments, such as caspase inhibition and MSC transplantation, suggests that the organotypic adult retinal explant model is a useful medium-throughput system in which to screen novel neuroprotective therapies. Treatments identified in this way could then be tested in preclinical models to verify efficacy in vivo. For example, the identification of robust RGC protection by MSC and simvastatin treatment in the present study suggests these novel therapies should be investigated further as potential adjunctive glaucoma therapies.