Time-Lapse Retinal Ganglion Cell Dendritic Field Degeneration Imaged in Organotypic Retinal Explant Culture

The irreversible death of retinal ganglion cells (RGCs) is the central common end-point of all optic neuropathies, glaucoma being the most common. The cellular and molecular cascades implicated in glaucomatous RGC loss are extensive,1 and elucidating the mechanisms by which RGCs die in glaucoma is requisite to the development of targeted treatment strategies aimed to prevent vision loss by increasing neuronal survival.2–6 Moreover, reparative strategies aimed at improving vision through endogenous regeneration or cell transplantation must take into account the survival and functional status of residual host retinal architecture including the axons and dendrites of RGCs.7,8 Compartmentalization of the RGC death process within cellular domains is being increasingly appreciated, though a complete understanding of the sequence of events leading from RGC injury to vision loss remains elusive. It is generally thought that the site of primary RGC injury in glaucoma is axonal and is associated with biomechanical deformation of the lamina cribrosa.9–11 Eventually, this leads to anterograde and retrograde axonal degeneration, cellular apoptosis, and dendritic field loss with demise of the associated afferent and efferent neural synapses. Evidence suggests that these related processes hold some degree of molecular pathway independence and can be experimentally isolated, at least temporarily.12–14 Such evidence is strongest for processes occurring in the axon and cell body. Dendritic and afferent synapse pruning has been documented in animal models of glaucoma15 and imaging suggests that dendritic arbors shrink following insult, though the precise timing, patterns, and mechanisms of RGC dendrite degeneration in glaucoma are incompletely understood. Evaluation of the RGC dendritic tree requires advanced imaging and analysis techniques capable of discriminating fine details of complex submicrometer fiber arrays. This poses several significant challenges, particularly in the rodent model systems most commonly employed to study glaucoma. In vivo imaging requires the presence of an RGC-specific label that actively traffics to or passively localizes within dendrites. While confocal laser scanning ophthalmoscopy techniques with and without adaptive optics have generated an impressive ability to visualize RGC cellular structure in vivo over a period of days to months,16–19 present technology has difficulty achieving the resolution necessary to discern fine detail. In vivo experimentation also poses difficulties related to local delivery of experimental compounds and precludes simultaneous electrophysiological recording. Ex vivo imaging techniques, which generally achieve higher resolution than in vivo imaging, have been developed and applied to healthy tissue, retinal tissue with prior RGC injury induced in vivo, and short term ex vivo physiological stress. However, so far these techniques have been limited to very short experiments (on the order of hours)20 or single time-point analyses.21–25 The significant degree of heterogeneity in the size, shape, and patterns of RGC dendritic fields therefore necessitate that a large of number of cells be analyzed in order to discriminate population-level mean differences in dendritic field characteristics between experimental groups imaged at single time points following injury. Further, classification of RGC subtype can be hampered by lack of information about the dendritic field at baseline, prior to injury.26 In order to address these limitations, we used organotypic culture of retinal tissue from mice genetically engineered to express an RGC-specific fluorescent tracer to facilitate dendritic field imaging ex vivo at high resolution using confocal microscopy. The pathogenesis of RGC injury in this model is primarily one of mechanical axonal trauma, and therefore more comparable to in vivo optic nerve crush or transection injury models than ocular hypertension induction, however the greater experimental tissue access afforded by tissue culture permits study at higher resolution than is possible in other systems. We employed previously described tissue culture techniques to maintain living explants for a period of up to 7 days, allowing time-lapse imaging of dendritic fields of the same RGC over time, which permitted within-cell paired statistical comparisons of dendritic field structure. A semiautomated system was used to trace dendritic trees prior to analysis, and a careful determination of intra- and interoperator variability in data acquisition was carried out. Analyses characterized potential dendritic field attenuation that was induced by the attendant axotomy involved in producing organotypic culture and demonstrated that these degenerative changes are ameliorated, in part, by treatment with neurotrophins.