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

Abstract

Purpose: To develop an ex vivo organotypic retinal explant culture system suitable for multiple time-point imaging of retinal ganglion cell (RGC) dendritic arbors over a period of 1 week, and capable of detecting dendrite neuroprotection conferred by experimental treatments.

Methods: Thy1-YFP mouse retinas were explanted and maintained in organotypic culture. Retinal ganglion cell dendritic arbors were imaged repeatedly using confocal laser scanning microscopy. Maximal projection z-stacks were traced by two masked investigators and dendritic fields were analyzed for characteristics including branch number, size, and complexity. One group of explants was treated with brain derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) added to the culture media. Changes in individual dendritic fields over time were detected using pair-wise comparison testing.

Results: Retinal ganglion cells in mouse retinal explant culture began to degenerate after 3 days with 52.4% surviving at 7 days. Dendritic field parameters showed minimal change over 8 hours in culture. Intra- and interobserver measurements of dendrite characteristics were strongly correlated (Spearman rank correlations consistently > 0.80). Statistically significant (P < 0.001) dendritic tree degeneration was detected following 7 days in culture including: 40% to 50% decreases in number of branch segments, number of junctions, number of terminal branches, and total branch length. Scholl analyses similarly demonstrated a significant decrease in dendritic field complexity. Treatment of explants with BDNF+CNTF significantly attenuated dendritic field degeneration.

Conclusions: Retinal explant culture of Thy1-YFP tissue provides a useful model for time-lapse imaging of RGC dendritic field degeneration over a course of several days, and is capable of detecting neuroprotective amelioration of dendritic pruning within individual RGCs.

Figure 1

Characterization of RGC loss in mouse retinal explants. Wild-type CD1 mouse retinal explants (N = 5 explants per time point) were cultured for 0, 1, 3, 7, 10, or 14 days prior to fixation and immunofluorescent labeling of all cells (DAPI⁺, [A, C, E]) or RGCs (β-III-Tubulin⁺, [B, D, F]). Representative micrographs from nonoverlapping fields are shown in (A–F); scale bars: 100 μm. Quantification of DAPI⁺ and β-III-Tubulin⁺ cell density is presented in G. Non-RGCs represent the difference between DAPI⁺ and β-III-Tubulin⁺ cells. Percent β-III-Tubulin⁺ cells in the RGC layer is presented in (H). Error bars represent SD. ***P ≤ 0.001 by Dunnett's post hoc test compared with day 0 time point.

Figure 2

Characterization of RGC dendritic fields at 0 and 8 hours of culture. Thy1-YFP retinal explants were cultured for 7 days. Z-stack confocal images of live YFP⁺ RGCs (N = 16 RGCs) were obtained on day 0 and day 7. Two investigators masked to RGC identity traced the dendritic field of each RGC. Representative micrographs of z-stack maximum projections (A, B) and investigator skeleton tracings (C–F) are shown; scale bars: 100 μm; arrow points to the axon, which was excluded from the tracing. Dendritic field parameters (G–L), changes in field parameters over 8 hours (M), and variance of change in field parameters (N) are shown. Scholl histograms (O) depict the number of intersections between dendritic fields and Sholl rings of increasing eccentricity; inset graph quantifies the AUC of the Scholl histogram. Sholl parameters are shown (P–S). Error bars in (G–L) and (O–S) represent SD; error bars in (M) represent standard error of the mean. P > 0.05 by Wilcoxon Sum Rank tests for all comparisons.

Figure 3

Retinal ganglion cell dendritic field changes over 7 days in culture. Thy1-YFP retinal explants were cultured for 7 days. Z-stack confocal images of live YFP⁺ RGCs (N = 75 RGCs) were obtained on day 0 and day 7. Two investigators masked to RGC identity traced the dendritic field of each RGC. Representative micrographs of z-stack maximum projections showing RGCs with minimal (A, B), moderate (C, D), and severe (E, F) levels of dendritic field degeneration over 7 days are shown; scale bar represents 100 μm; arrow points to the axon, which was excluded from the tracing. Dendritic field parameters (G–L) and changes in field parameters over 8 hours (M) are shown. Scholl histograms (N) depict the number of intersections between dendritic fields and Sholl rings of increasing eccentricity; inset graph quantifies the AUC of the Scholl histogram. The average change in Sholl histogram over 7 days (O) and Sholl parameters at both time points are shown (P–S). Error bars in (G–L, N, P–S) represent SD; error bars in (M, O) represent SEM. **P ≤ 0.01 and ***P ≤ 0.001 by paired t-test comparing individual RGCs at both time points. *P ≤ 0.05 by paired t-test comparing change in field parameter between investigator tracings.

Figure 4

Comparison of dendritic fields in RGCs living versus dead after 7 days in culture. Thy1-YFP retinal explants were cultured for 7 days. Z-stack confocal images of live YFP⁺ RGCs (N = 130 RGCs) were obtained on day 0 only and cells were divided according to whether they were still alive and visible by microscopy at 7 days (N = 75 RGCs) or were no longer detectable at 7 days (N = 55 RGCs). Dendritic field parameters (A–F) for RGCs imaged at day 0 are shown. Scholl histograms (G) depict the number of intersections between dendritic fields and Sholl rings of increasing eccentricity; inset graph quantifies the AUC of the Scholl histogram. Sholl parameters for day 0 images are shown (H–K). Error bars represent SD. P > 0.05 by unpaired t-test for all comparisons.

Figure 5

Amelioration of RGC dendritic field changes over 7 days by BDNF+CNTF. Thy1-YFP retinal explants were cultured for 7 days with forskolin (20 μg/mL)+BDNF (50 ng/mL)+CNTF (50 ng/mL; N = 74 RGCs) or with forskolin only as a negative control (N = 48 RGCs). Z-stack confocal images of live YFP⁺ RGCs were obtained on day 0 and day 7. Dendritic field parameters (A–F) and changes in field parameters over 8 hours (G) are shown. Scholl histograms (H–I) depict the number of intersections between dendritic fields and Sholl rings of increasing eccentricity; inset graph quantifies the AUC of the Scholl histogram. Sholl parameters at both time points, (K–N), and change in Sholl parameters over 7 days (J, O) are shown. Error bars in (A–F, H–I, K–N) represent SD; error bars in (G, J, O) represent SEM. (A–F, K–N, H, I): **P ≤ 0.01 and ***P ≤ 0.001 by paired t-test comparing individual RGCs at both time points. (G, J, O): *P ≤ 0.05, **P ≤ 0.01 by unpaired t-test comparing change in field parameter between treatment groups.