A novel system for the classification of diseased retinal ganglion cells

Abstract

Retinal ganglion cell (RGC) dendritic atrophy is an early feature of many forms of retinal degeneration, providing a challenge to RGC classifi cation. The characterization of these changes is complicated by the possibility that selective labeling of any particular class can confound the estimation of dendritic remodeling. To address this issue we have developed a novel, robust, and quantitative RGC classifi cation based on proximal dendritic features which are resistant to early degeneration. RGCs were labeled through the ballistic delivery of DiO and DiI coated tungsten particles to whole retinal explants of 20 adult Brown Norway rats. RGCs were grouped according to the Sun classifi cation system. A comprehensive set of primary and secondary dendrite features were quantifi ed and a new classifi cation model derived using principal component (PCA) and discriminant analyses, to estimate the likelihood that a cell belonged to any given class. One-hundred and thirty one imaged RGCs were analyzed; according to the Sun classifi cation, 24% ( n = 31) were RGC A , 29% ( n = 38) RGC B , 32% ( n = 42) RGC C , and 15% ( n = 20) RGC D . PCA gave a 3 component solution, separating RGCs based on descriptors of soma size and primary dendrite thickness, proximal dendritic fi eld size and dendritic tree asymmetry. The new variables correctly classifi ed 73.3% ( n = 74) of RGCs from a training sample and 63.3% ( n = 19) from a hold out sample indicating an effective model. Soma and proximal dendritic tree morphological features provide a useful surrogate measurement for the classifi cation of RGCs in disease. While a defi nitive classifi cation is not possible in every case, the technique provides a useful safeguard against sample bias where the normal criteria for cell classifi cation may not be reliable.

Retinal ganglion cell dendrite pathology and synapse loss

“…Furthermore, the magnitude of some neuroprotective strategies differs substantially among different RGC classes (Lindsey et al, 2015). Although new and potentially useful methods to classify and analyze RGCs in disease are emerging (Sümbül et al, 2014;Tribble et al, 2014), at present there is no agreement on a universal classification system that would allow reliable comparison of data sets from different studies. Regardless of methodological issues, it is clear that the structural and functional diversity of RGCs needs careful consideration when studying pathological changes in dendrites as this variability may confound data interpretation.…”

Shared and Differential Retinal Responses against Optic Nerve Injury and Ocular Hypertension

“…Morphological criteria such as soma and dendritic arborisation size and levels of stratification within the inner plexiform layer, as well as electrophysiological responses to light stimuli and target region of the brain, may render over 30 different types of RGCs in the healthy rodent retina (Sun et al, 2002a,b; Coombs et al, 2006; for review see: Sanes and Masland, 2015). Many of these attributes change after injury and thus cannot be used to identify damaged RGCs (for review see Tribble et al, 2014). Molecular markers for RGCs are scarce, most do not label the entire population of RGCs and are downregulated in response to retinal injury (Chidlow et al, 2005; Lönngren et al, 2006; Agudo et al, 2008), thus rendering their use unreliable to identify RGCs.…”

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

“…Attempts have been made to reclassify RGC subgroups based on specific characteristics of the dendritic tree that are relatively stable during early stages of cell death with a success rate of between 60% and 75%, in a group of healthy, uninjured RGCs. 26 The present system provides a platform that could be used for subclassification based on morphology at the time of explantation prior to degenerative changes to the dendritic tree. While this limits experimental protocols to RGC insults that are introduced ex vivo, it eliminates uncertainty about the baseline morphology of the cells being studied.…”

“…Further, classification of RGC subtype can be hampered by lack of information about the dendritic field at baseline, prior to injury. 26 …”