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. 2011 May 17;52(6):3309-20.
doi: 10.1167/iovs.10-6873.

Use of an adult rat retinal explant model for screening of potential retinal ganglion cell neuroprotective therapies

Natalie D Bull  1 , Thomas V Johnson, Guncha Welsapar, Nicholas W DeKorver, Stanislav I Tomarev, Keith R Martin
Affiliations

Use of an adult rat retinal explant model for screening of potential retinal ganglion cell neuroprotective therapies

Natalie D Bull et al. Invest Ophthalmol Vis Sci. .

Abstract

PURPOSE. To validate an established adult organotypic retinal explant culture system for use as an efficient medium-throughput screening tool to investigate novel retinal ganglion cell (RGC) neuroprotective therapies. METHODS. Optimal culture conditions for detecting RGC neuroprotection in rat retinal explants were identified. Retinal explants were treated with various recognized, or purported, neuroprotective agents and cultured for either 4 or 7 days ex vivo. The number of cells surviving in the RGC layer (RGCL) was quantified using histologic and immunohistochemical techniques, and statistical analyses were applied to detect neuroprotective effects. RESULTS. The ability to replicate previously reported in vivo RGC neuroprotection in retinal explants was verified by demonstrating that caspase inhibition, brain-derived neurotrophic factor treatment, and stem cell transplantation all reduced RGCL cell loss in this model. Further screening of potential neuroprotective pharmacologic agents demonstrated that betaxolol, losartan, tafluprost, and simvastatin all alleviated RGCL cell loss in retinal explants, supporting previous reports. However, treatment with brimonidine did not protect RGCL neurons from death in retinal explant cultures. Explants cultured for 4 days ex vivo proved most sensitive for detecting neuroprotection. CONCLUSIONS. The current adult rat retinal explant culture model offers advantages over other models for screening potential neuroprotective drugs, including maintenance of neurons in situ, control of environmental conditions, and dissociation from other factors such as intraocular pressure. Verification that neuroprotection by previously identified RGC-protective therapies could be replicated in adult retinal explant cultures suggests that this model could be used for efficient medium-throughput screening of novel neuroprotective therapies for retinal neurodegenerative disease.

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Figures

Figure 1.
Figure 1.
RGCL changes in retinal explant cultures over time ex vivo. Three approaches were used to assess RGCL changes in transverse sections of retinal explants. Immunohistochemistry was used to label RGCs for either Islet-1+ (A) or NeuN+ (B) expression, and the number of positive cells (arrows, positive labeling) in the ganglion cell (GC) layer was counted. The number of DAPI+ nuclei (C) and calbindin+ (D) cells in the GC layer were also quantified (arrows, positive labeling). Examples of negative control goat anti-mouse (E) and goat anti-rabbit (F) secondary antibody labeling are also shown (note: these images were acquired using a laser intensity 3 to 4 times higher than that used in (A–D) because no background fluorescence was visible under normal excitation intensity). Representative images shown are all from explants cultured for 4DEV; an identical pattern of staining was observed at all time points. Quantification over time (G; n = 4 explants per time point) revealed a fairly steady loss of RGC marker expression in cultures up to approximately 7 days ex vivo, after which the rate of loss appeared to plateau. Furthermore, loss of calbindin+ neurons (mostly displaced amacrine cells) from the RGCL was comparatively flat over time ex vivo. All further comparative experiments were quantified at 4DEV and 7DEV. Values are mean ± SEM. INL, inner nuclear layer. Scale bar, 50 μm (A–F).
Figure 2.
Figure 2.
Effect of reduced oxygen tension on neuronal survival and explant morphology. RGC survival in explants cultured in 3% O2 (37°C) was compared with that in control cultures (37°C, air) at 4DEV (A; n = 4 explants/group) and 7DEV (B; n = 4 explants/group). Explant culture in reduced oxygen was found to significantly impair RGC survival. Given retinal explants are normally kept at 35°C, RGC survival at 37°C/air was also compared with that in explants cultured in normal conditions at 35°C/air. An increase in culture temperature did not greatly impact explant health. Data from freshly isolated explants (0DEV) are included for comparison. Values are mean ± SEM. *P < 0.05, **P < 0.01 using one-way ANOVA with Bonferroni post hoc comparison. Immunohistochemical labeling of βIII tubulin in whole-mounted retinal explants at 7DEV (C–E; n = 4 explants/group) demonstrated explant culture in reduced oxygen (E) severely impaired RGC survival and axonal health compared with control cultures (D). Specifically, RGC density was greatly reduced, RGC axons appeared disrupted, and axonal swellings were common. Explants cultured at 37°C (D) showed no obvious difference in RGC morphology compared with those kept in normal conditions at 35°C (C). In contrast to negative RGC effects, DAPI staining of 7DEV explant sections indicated that reduced oxygen culture conditions (I) alleviated retinal thinning over time ex vivo compared with control retinas (H). No difference in retinal thickness was observed in cultures at 37°C (H) compared with those in normal conditions at 35°C (G). An example of a DAPI-stained transverse section of freshly isolated explant at 0DEV (F) is included for comparison. Morphometric analysis of DAPI-stained explant sections revealed that in 3% O2, explants were significantly thicker, compared with controls, at both 4DEV and 7DEV (J; n = 4 explants/group). The ONL was also significantly thicker in explants cultured in 3% O2, compared with controls, at both 4DEV and 7DEV (K; n = 4 explants/group). Culture of explants in 3% O2 did not significantly affect the thickness of other retinal layers compared with controls (data not shown). Data from freshly isolated explants (0DEV) are included for comparison. Values are mean ± SEM. *P < 0.05, **P < 0.01 for groups indicated. #P < 0.05, ##P < 0.01 compared with 0DEV data. Statistical analysis one-way ANOVA with Bonferroni post hoc comparison. GC, ganglion cell layer; IP, inner plexiform layer; IN, inner nuclear layer; ON, outer nuclear layer; OP, outer plexiform layer. Scale bar, 50 μm (C–I).
Figure 3.
Figure 3.
Inhibition of apoptosis or delivery of neurotrophic support enhances RGC survival in retinal explants. Culture of explants with a pan-caspase inhibitor significantly reduced RGC death at both 4DEV (A; n = 6 explants/group) and 7DEV (B; n = 6 explants/group), compared with controls (vehicle). In addition, culture of explants with BDNF plus forskolin (F) was found to improve RGC survival, as assessed by Islet-1 immunohistochemistry, at 7DEV (D; n = 8 explants/group) but did not affect RGC survival when assessed at 4DEV (C; n = 8 explants/group). Values are mean ± SEM. **P < 0.01 using two-tailed Student's t-test.
Figure 4.
Figure 4.
Coculture of MSCs with retinal explants strongly enhanced RGC survival in explants. GFP+-MSCs were cocultured on the inner retinal surface of explants for 4DEV (A; n = 4 vehicle, n = 3 MSCs) and 7DEV (B; n = 4 explants/group). At both time points, GFP+-MSC coculture significantly enhanced RGC survival, as assessed immunohistochemically and histologically (MSC nuclei were excluded from these counts by their colocalization with GFP). (A, B) Values are mean ± SEM. *P < 0.05, **P < 0.01 using two-tailed Student's t-test. Robust, significant MSC-mediated RGC neuroprotection was also observed in explants in which RGCs were injured prior to culture by 7 days' OHT (OHT; C), 14 days' OHT (D), and 7 days after ONC (E), compared with injured retinas treated with vehicle alone (OHT/ONC+MSCs vs. OHT/ONC+Vehicle). In addition, neither 7 days' OHT nor 14 days' OHT potentiated RGC death ex vivo (OHT+Vehicle vs. Naive+Vehicle; C, D). In comparison, ONC 7 days before explantation tended to enhance RGC loss where a significant difference in survival was detected using NeuN immunohistochemical analysis (Naive+ Vehicle vs. ONC+Vehicle; E). (C–E) All explants were cultured for 7DEV. All values are mean ± SEM. *P < 0.05, **P < 0.01 using one-way ANOVA with Bonferroni post hoc comparison.
Figure 5.
Figure 5.
Representative immunohistochemical images of retinal explants cocultured with GFP+ MSCs. Retinal explants were cultured, for 4DEV or 7DEV, untreated (control), or cocultured with GFP+ MSCs (green) on the inner retinal surface. Transverse explant sections were immunohistochemically labeled for Islet-1 (A, red), NeuN (B, red), βIII-tubulin (β3T; C, red), or glial fibrillary acidic protein (GFAP; D, red). Nuclei were counterstained with DAPI (blue). Cocultured MSCs did not migrate into the retinal tissue, but were associated with increased Islet-1+ and NeuN+ cell density within the RGCL at both 4DEV and 7DEV (quantification presented in Figs. 4A, 4B). In addition, MSC coculture resulted in a thicker retinal explant overall, a thicker inner plexiform layer (IPL, white vertical lines), and a well-preserved nerve fiber layer (NFL, arrows) at both time points (C). GFAP immunoreactivity (D, red) in astrocytes within the NFL and Müller cells throughout the retina was present in all explants but appeared upregulated, particularly in Müller cells (arrowheads), with MSC coculture at 7DEV. INL, inner nuclear layer; IPL, inner plexiform layer; NFL, nerve fiber layer; B3T, βIII-tubulin. Scale bar, 100 μm.
Figure 6.
Figure 6.
Screening RGC neuroprotection by pharmacologic agents using retinal explant cultures. Retinal explants were cultured for either 4DEV or 7DEV and were treated with a variety of drugs. Explants were continuously exposed to the drugs during culture. Betaxolol (A; n = 6/group), losartan (E; n = 6/group), tafluprost (G; n = 6/group), and simvastatin (I; n = 6/group) were all found to reduce RGC loss in 4DEV explants. In contrast, brimonidine (C, D; n = 6/group, C; n = 5 vehicle, n = 6 brimonidine, D) did not affect RGC loss in retinal explants. Pharmacologic neuroprotection did not prevent RGC death, as no neuroprotective effects by betaxolol (B; n = 6/group), losartan (F; n = 6/group), tafluprost (H; n = 6/group), or simvastatin (J; n = 6/group) were detected at 7DEV, but instead delayed neurodegeneration thereby providing a temporal window in which to observe direct effects on RGC survival. All values are mean ± SEM. *P < 0.05, **P < 0.01, two-tailed Student's t-test.
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