Phenotypic map of porcine retinal ganglion cells

Abstract

Purpose: Porcine retina is an excellent model for studying diverse retinal processes and diseases. The morphologies of porcine retinal ganglion cells (RGCs) have, however, not yet been described comprehensively. The aim of the present study was to créate a classification of the RGCs using the 1, 1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) tracing method.

Methods: About 170 RGCs were retrogradely labeled by injecting DiI into the optic nerve of postmortem eyes and statistically analyzed by two different clustering methods: Ward's algorithm and the K-means clustering. Major axis length of the soma, soma area size, and dendritic field area size were selected as main parameters for cluster classification.

Results: RGC distribution in clusters was achieved according to their morphological parameters. It was feasible to combine both statistical methods, thereby obtaining a robust clustering distribution. Morphological analysis resulted in a classification of RGCs in three groups according to the soma size and dendritic field: A (large somas and large dendritic fields), B (medium to large somas and medium to large dendritic fields), C (medium to small somas and medium to small dendritic fields). Within groups, fine clustering defined several subgroups according to dendritic arborization and level of stratification. Additionally, cells stratifying in two different levels of the inner plexiform layer were observed within the clusters.

Conclusions: This comprehensive study of RGC morphologies in the porcine retina provides fundamental knowledge about RGC cell types and provides a basis for functional studies toward selective RGC cell degeneration in retinal disorders.

Figure 1

Distribution of retinal ganglion cells in porcine retina with respect to their soma area sizes and dendritic field sizes. The retinal ganglion cells (RGCs) from nine retinas were measured for three different parameters: soma major axis length, soma area size and dendritic field area size. Cells were grouped into three different groups according to (A) soma area: <100 μm², 100–200 μm², >200 μm² as well as (C) dendritic field area (<10,000 μm², 10,000–20,000 μm², >20,000 μm². Major axis length and the soma area sizes of RGCs correlate linearly (B), whereas soma area 1 sizes do not correlate linearly with dendritic field area sizes (D).

Figure 2

Ward’s dendrogram for retinal ganglion cells. The Ward’s method showed that retinal ganglion cells (RGCs) could be differentiated into three clusters (A, B, C). The obtained dendrogram for cluster analysis is represented here. The relative similarity of cells (x-axis) was shown in the linkage distance (y-axis) for all RGCs analyzed (n=170). The cluster origin for the cluster obtained in this work was designated with the corresponding letter. A continuous 3 pt-weight line divides the three main clusters in the study.

Figure 3

K-means analysis of the diverse porcine retinal ganglion cell population. A: The Silhouette plot was elaborated according the best clustering distribution, taking into account the given parameters of soma major axis length, soma area size, and dendritic field area size for all retinal ganglion cells. The number of individuals in each cluster is defined and the mean value of the silhouette plot (0.49) is also indicate (where n is the sampling size and represent the total Number of analyzed cells; a, b, and c represent the three obtained clusters; the cluster a contains 96 cells; the cluster b 60 and the cluster c 14). B: The representation of the principal analysis components was carried out. The representative clustering plot for all retinal ganglion cells showed their distribution into three major clusters (+: cells into cluster a; Δ: cells into cluster b; ¢′: cells into cluster c).

Figure 4

Representative pictures from the A cluster. A: The A1 subcluster contains cells with the largest somas and dendritic fields from the total population. B: The cells contained in the A2 subcluster display large somas and large dendritic fields. Scale bars are 50 µm. Arrows indicate the cell belonging to the specific subcluster. C: Schematic representation of retinal ganglion cells showing the possible branching and levels of stratification for the subclusters. INL is the inner nuclear layer; GCL is the ganglion cell layer.

Figure 5

Representative pictures of the B cluster cells. Retinal ganglion cells (RGCs) representing the subclusters are shown. A: B1 subcluster contains cells with large soma areas and medium to large dendritic fields. B: B2 subcluster contains cells with medium soma areas and medium dendritic fields. C: B3 subcluster contains cells with medium soma areas and small dendritic fields. The scale bar is 50 µm. Arrows indicate the cell belonging to the specific subcluster. D: This is a schematic representation of RGCs showing the possible branching and levels of stratification for the subclusters. INL is the inner nuclear layer; GCL is the ganglion cell layer.

Figure 6

Cluster C cells containing medium to small soma areas and medium to small dendritic fields. A: C1 subcluster contains cells with medium to small soma size and large to medium dendritic fields. B: C2 subcluster contains cells with medium soma sizes and small dendritic fields. C: C3 subcluster contains cells with small soma size and medium dendritic fields. D: C4 subcluster contains the smallest retinal ganglion cells (RGCs) with small soma size and small dendritic fields. E: This is a schematic representation of RGCs showing the possible branching and levels of stratification for the subclusters. INL is the inner nuclear layer; GCL is the ganglion cell layer.

Figure 7

Schematic diagrams of the representative models of retinal ganglion cells for the different subclusters noted above. The differences between the dendritic fields along the different subclusters can be observed. The scale bar is 100 µm.