Effects of Müller Glia on Cell Survival and Neuritogenesis in Adult Porcine Retina In Vitro

Mónica García; Valerie Forster; David Hicks; Elena Vecino

Abstract

purpose. To examine the effects of glia-derived and brain-derived neurotrophic factors on survival and morphology of cultured retinal ganglion cells (RGCs) from adult porcine retina.

methods. Adult porcine retinas were dissociated and cultured in different conditions: (1) on laminin- and poly-d-lysine–coated coverslips in chemically defined medium (CDM); (2) on laminin- and poly-d-lysine–coated coverslips in CDM supplemented with brain-derived neurotrophic factor (BDNF); (3) in confluent monolayer cultures of retinal Müller glia (RMG) in CDM; (4) in 1-day cultures of RMG in CDM; (5) in fixed RMG cultures in CDM; and (6) in laminin-poly-d-lysine substrate in conditioned medium obtained from RMG. RGCs were classified on the basis of the size, number of neurites, and length of the neurites, and the survival of the RGCs was assayed after each treatment.

results. Confluent RMG substrates and RMG-conditioned medium significantly increased the survival of cultured porcine RGCs. Moreover, these two conditions increased the size of the RGCs and enhanced growth and elongation of the neurite. Addition of BDNF to the culture medium or use of 1-day cultured RMG as a substrate did not modify survival but increased the size, neurite number, and neurite length in the RCGs.

conclusions. These findings demonstrate that factor(s) secreted by RMG exert beneficial effects on survival of adult RGCs and neurite regeneration in vitro and may constitute effective agent(s) for neuroprotection of RGC.

RGCs are especially affected by several diseases, including retinal ischemia,¹ ² diabetes,³ ⁴ and glaucoma.⁵ ⁶ ⁷ In glaucoma, RGCs die by apoptosis, as has been demonstrated in different species.⁸ ⁹ The precise pathogenic mechanisms leading to apoptotic cell death are unknown. Moreover, death of RGCs during glaucoma seems to be selective and does not affect large, medium, and small cells equally. A rapid and severe loss of medium-sized RGCs has been observed in studies of optic nerve damage in the cat¹⁰ and in the avian retina in experimental glaucoma.¹¹ Death of RGCs of all sizes has been reported in hypertensive eyes in rats,⁸ ¹² but large RGCs seem to be more resistant to axotomy in adult rats.¹³ However, in human glaucoma,¹⁴ ¹⁵ in experimental glaucoma in monkeys,⁵ ⁶ ¹⁶ ¹⁷ and in adult porcine RGCs cultured with high doses of glutamate,¹⁸ large RGCs appear to be the most susceptible to death.

Neurotrophic factors, such as BDNF, have been implicated in survival and growth of RGCs after axotomy in rats¹³ ¹⁹ ²⁰ and cats¹⁰ and in moderately chronic hypertensive eyes in rats.²¹ It has also been demonstrated that BDNF supports the survival of RGCs and influences neurite outgrowth in vitro.²² ²³ ²⁴ ²⁵ ²⁶

The major glial type in the mammalian retina is the retinal Müller glial cells (RMG), extensions of which surround RGC bodies and their dendrites. Many functions have been postulated for these cells, including structural and nutritional roles, and removal of ions and neurotransmitters from the extracellular space.²⁷ RMG can protect against the excitotoxic effects of glutamate and increase the survival of RGCs in culture.²⁸ ²⁹ ³⁰ It has been demonstrated than RMG can reduce glutamate levels in the culture medium³¹ and that neuroprotection by RMG depends on maturation of glutamine synthetase expression and neuron–glia signaling after glutamate treatment.²⁹ RMG are also known to synthesize certain neurotrophic factors, such as fibroblast growth factor³² and ciliary neurotrophic factor.³³

The purpose of our study was to investigate the responses of adult porcine RGCs to different culture conditions involving RMG or BDNF. We analyzed survival, cell size distribution, neurite branching capacity, and RGC neurite length in vitro. The data show that RMG can enhance all these parameters, mostly through the release of soluble factor(s).

Materials and Methods

Tissue Collection and Cell Culture

All animal experimentation adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Adult porcine eyes were obtained from a local abattoir and transported to the laboratory in cold CO₂-independent Dulbecco’s modified Eagle’s medium (DMEM/−CO₂; Gibco-Life Technologies, Cergy-Pontoise, France). Eyes were dissected within 1 to 2 hours after enucleation. Retinal cell cultures were prepared according to a method previously reported,¹⁸ ³⁴ ³⁵ with the following minor modifications.

In our study we used only an approximately 1-cm² circular area located in the central superior region immediately nasal to the optic nerve head between the principal blood vessels in the retina. This region was chosen because of the relatively constant size and density of the RGCs. The sample was chopped into approximately 1- to 2-mm² fragments, washed in Ringer’s solution, and incubated in 0.5 mL of 0.2% activated papain (Worthington, Lakewood, NJ) in the same buffer for 20 minutes in a water bath at 37°C. The pieces were gently dissociated by repeated trituration, and isolated cells were seeded in DMEM/Ham’s-F12 (Gibco), supplemented with 5% fetal calf serum (FCS; Gibco) and penicillin-streptomycin (10 IU).

After determination of cell number and viability by examination of trypan blue–treated aliquots on a hemocytometer, cells were seeded at 5 × 10⁵ cells/cm² onto sterile 12-mm glass coverslips precoated sequentially with poly-d-lysine (2 μg/cm² for 1 hour; Sigma-Aldrich, Lyon, France) and laminin (1 μg/cm² overnight; Sigma-Aldrich). Cells were maintained in a humidified incubator at 37°C with an atmosphere of 5% CO₂-95% O₂.

In Vitro Treatments

Porcine retinal ganglion cells were cultured in several different conditions.

Control.

RGCs were incubated directly on the laminin-poly-lysine substrate for 24 hours in DMEM-5% FCS, before they were rinsed twice in serum-free DMEM and then maintained in chemically defined medium (CDM) for 5 days before fixation.

Brain-Derived Neurotrophic Factor.

RGCs were cultured on laminin-poly-lysine–coated coverslips for 24 hours and then transferred to CDM containing 10 ng/mL BDNF (R&D Systems, Abingdon, UK). RGCs were maintained in these conditions for 5 days before fixation.

Confluent RMG.

RGCs were cultured on fully confluent RMG (CoRMG) monolayers. These monolayers had been prepared previously from porcine retinas, according to the method of Guidry.³⁶ RMG retrieved from the density gradient (Percoll; Pharmacia Upjohn; Uppsala, Sweden) were resuspended in DMEM-10% FCS. Cells were counted and seeded at 6 × 10⁵ cells/cm², onto sterile, 1-cm² glass coverslips pretreated with laminin and poly-d-lysine. After 6 days in vitro, RMG were confluent and could be used as the substrate for RGC cultures.

Subconfluent RMG.

RMG were maintained in a humidified incubator at 37°C with an atmosphere of 5% CO₂-95% O₂ for 1 day and then used in the subconfluent condition (ScRMG) as substrates. RGC seeding was performed as for CoRMG.

Fixed RMG.

Confluent Müller cells were fixed (FxRMG) for 10 minutes with 4% paraformaldehyde in PBS, rinsed six times with DMEM, and then used as substrates for RGC cultures.

Conditioned Medium.

Purified RMG were grown to confluence (6 days) and then maintained in the presence of CDM for 24 hours. The conditioned medium (CM) was then collected, filtered, and stored at –20°C until ready for use. The medium was diluted 1:1 (vol/vol) with fresh CDM and added to RGC cultures after 24 hours in DMEM-5% FCS.

Immunocytochemistry

After 6 days in vitro, cells were washed in PBS (pH 7.3) and fixed with 4% paraformaldehyde in PBS for 15 minutes at room temperature. Cells were rinsed in PBS, permeabilized with 0.1% Triton X-100 for 5 minutes, and incubated in PBS containing 0.1% BSA, 0.1% Tween 20, and 0.1% NaN₃ (buffer A) for 15 minutes. Samples were incubated for 2 hours with anti-neurofilament (NF) 68-kDa (NF68) subunit monoclonal or anti-NF 200-kDa (NF200) subunit polyclonal antibody³⁷ ³⁸ (both diluted in buffer A at a final concentration of 10 μg/mL; Sigma-Aldrich), washed with PBS five times, and exposed for 1 hour to goat anti-mouse IgG/Texas red (10 μg/mL, for monoclonal primary antibody) or goat anti-rabbit IgG/Bodipy FL (10 μg/mL, for polyclonal primary antibody) both from Molecular Probes (Eugene, OR). Nuclei of cells were stained with 4,6-diaminodiphenyl-2-phenylindole (DAPI; 10 μg/mL; SigmaAldrich), incubated together with a fluorescent secondary antibody. Preparations were washed with PBS, mounted, and observed under an epifluorescence microscope (Axioskop 2; Zeiss, Jena, Germany). Immunocytochemical control experiments consisted of omission of the primary antibody, omission of the second antibody, and the use of a corresponding nonimmune serum.

Quantification of Cell Survival

RGCs labeled with anti-neurofilament antibodies were manually counted in cultures prepared from retinas of 10 separate adult porcine eyes. RGCs of each retina were cultured in the six experimental conditions (n = 10 for each condition). After analysis of the total number of cells in each condition, we analyzed the survival in response to the different culture conditions by calculating the percentage of survival compared with CDM control cultures, accepting survival in the CDM condition to be 100%. Statistical analysis was performed by computer (SPSS software; SPSS Sciences, Chicago, IL), using the χ² method to compare frequencies.

Analysis of Cellular Morphology

Half of the total surface was analyzed for each coverslip. One hundred eighty-two fields, each microscopic field corresponding to 246 μm², were photographed with a digital camera (Coolsnap; RS Photometrics, Tucson, AZ). In the present study, 19,870 RGCs were photographed and measured. RGCs were examined for four parameters: soma size, neurite number, combined length of all neurites, and length of the single longest neurite. Measurements were performed directly on the digital images by image analysis on computer (Scion Image; Scion, Frederick, MD, and Spot; Diagnostic Instruments Inc., Sterling Heights, MI).

To classify the RGCs, the area and diameter of the cell soma were measured, and the relationship between both parameters allowed us to categorize them into three groups: small RCGs, with cell bodies less than 14 μm in diameter; medium RGCs, with cell bodies between 15 and 20 μm in diameter; and large RGCs, with cell bodies with a diameter more than 21 μm. Both mean area and RGC distribution on the basis of RGC soma size were analyzed for each culture condition. Statistical analysis of the results was performed by using the general linear model of two factors followed by the Tamhane test. Percentage of increase in soma area in each condition of the CDM control (set at 100%) was calculated in each experiment, and then the mean percentage of increase was analyzed. Differences in percentage increase in the mean area among different conditions were analyzed by using Student’s t-test for unpaired data.

RGCs were also classified into three groups on the basis of neurite number: RGCs without neurites (0 neurites), RCGs with a small number of neurites (one to three neurites), and RCGs with four or more neurites (more than three neurites). We calculated the mean number of neurites per cell as well as the percentage of RGCs with neurites in each culture condition. Statistical analysis was performed by using the general linear model of two factors followed by the Tamhane test.

Measurements of total neurite length and length of the longest neurite were also performed for each different culture condition. Statistical analysis of the results was performed by using the general linear model of two factors followed by the Tamhane test. Percentage of increase of total length and mean length of the longest neurite were calculated for each condition of the CDM control (set at 100%). Comparison of the percentage of increase in the mean length among different conditions was analyzed by using the Student’s t-test for unpaired data.

The minimum level of significant difference was defined as P < 0.05. All data are presented as the mean ± SEM.

Results

Effect of Culture Conditions on RGC Survival

Analysis of NF68 and NF200 immunostaining was performed separately to analyze possible variations in NF distribution. A total of 39,476 cells were counted, and half of these were digitally recorded and measured. To simplify the display of results, and because data obtained from NF68 and NF200 immunostained cultures were not significantly different, the tables show combined data from both NF68 and NF200 labeling, whereas figures depict data corresponding to NF68 immunolabeling only.

We first assessed the effect of different treatments on the survival of RGCs. Results obtained for NF68-immunolabeled RGCs are shown in Figure 1 and are expressed as percentages of CDM control (100%). Whereas addition of BDNF did not significantly modify RGC survival (104.6% ± 4.2%), when RGCs were cultured on laminin-poly-lysine substrates in the presence of CM, we observed a statistically significant increase in survival (116.7% ± 4.8% and 120.9% ± 4.8% in NF68- and NF200-immunolabeled cells, respectively; P < 0.01, Table 1 ). With CoRMG substrates, the percentage of surviving RGCs also increased significantly (P < 0.05), 110.4% ± 3.4% for NF68- and 113.2% ± 3.3% for NF200-immunopositive RGCs. RGC survival was significantly reduced when cells were grown on FxRMG (59.7% ± 2.2%, P < 0.01), and no differences in RGC survival were observed when they were cocultured with ScRMG (102.1% ± 4.9%).

Effects of Culture Conditions on RGC Size Distribution

Small RGCs showed moderately intense NF immunolabeling within the somata and neurites, exhibiting discontinuous NF staining in neurites. Medium RGCs were more intensely stained and showed more highly branched neurites. Large RGCs with oval or circular shape possessed a displaced nucleus, and NF immunolabeling was very intense in the soma as well as along the numerous neurites (most cells had more than three large branches growing from the soma).

Figure 2A shows the mean soma area of RGCs cultured in the different conditions. The mean soma area of control cells grown on laminin-poly-lysine–coated coverslips with CDM was 150.3 ± 2.1 μm². Adding BDNF to the culture medium led to an increase of 19.6% (P < 0.05; 179.6 ± 3.1 μm²; Table 2 ). Culture of RGCs on CoRMG monolayers or in CM caused a significant increase compared with CDM (36.5% and 24.9%, respectively, P < 0.01 for both conditions). Although there were no significant differences in RGC sizes between CoRMG and CM (Table 2) , cells grown on CoRMG were significantly larger than those grown with BDNF (P < 0.05). RGCs cocultured with ScRMG showed an increase in RGC soma size (17.2%; P < 0.05), whereas FxRMG substrates did not affect RGC mean area compared with the control (increase of 6.5% ± 5.3%).

RGCs had been classified on the basis of their size, and we investigated whether the different conditions used in the present study would differentially affect small, medium, or large RGCs. Figure 2B shows how the different conditions affected the distribution of RGCs of different sizes. Results are shown in absolute values, although percentages of cells belonging to each group were also calculated to perform statistical analyses. When RGCs were cultured in control CDM the size distribution was 24.3% ± 9.9% small, 67.9% ± 8.5% medium, and 7.8% ± 2.8% large RGCs. The percentage of small RGCs was reduced when BDNF was added to the culture medium (10.2% ± 5.3%), and this decrease was even more evident in the CoRMG and CM treatments (5.9% ± 1.4% and 6.8% ± 1.9%, respectively). Statistical analyses showed significant differences in both conditions compared with the control (P < 0.01). Neither the ScRMG nor FxRMG conditions modified the number of small RGCs (22.2% ± 8.4% and 19.9% ± 10.1%, respectively) compared with the control.

No significant changes in the percentage of medium RGCs were observed in any of the conditions used in the present study. However, the percentage of large RGCs increased in all experimental groups (Fig. 2B) : whereas the percentage in control cultures was 7.8% ± 2.8%, percentages of large RGCs with the BDNF, CoRMG, FxRMG, ScRMG, and CM treatments increased to 30.7% ± 9.9%, 44.8% ± 3.7%, 16.2% ± 3.7%, 27.6% ± 10.5%, and 27.4% ± 2.5%, respectively. Except for the percentage of large RGCs with the FxRMG treatment (P = 0.37), all treatments were significantly different from CDM (P < 0.01).

Effects of Culture Conditions on Neurite Growth

To determine whether culture conditions regulate the neurite number and length in RGCs, we analyzed these parameters from large numbers of sampled cells in the different conditions, determining the percentage of RGCs with neurites, the mean total length of neurites, and the mean length of individual longest neurites per cell. The percentage of cells with neurites is shown for each treatment in Figure 3 and Table 3 . Of the RGCs cultured on laminin-poly-lysine substrates in CDM, 59.6% ± 8.8% had neurites, with the mean number of neurites per cell being 1.69 ± 0.08. The number did not vary with cells grown on FxRMG (61.5% ± 7.5%), but was significantly higher in all other experimental conditions: BDNF (78.8% ± 7.4%), ScRMG (70.2% ± 9.9%; P < 0.05 for both conditions), CoRMG (99.5% ± 0.4%), and CM (93.6% ± 2.1%; P < 0.01 for both conditions).

Results of neurite length measurements are shown in Figure 4 . For cells cultured in CDM, the mean total length of neurites was 180 ± 7.8 μm. Addition of BDNF caused increased elongation of neurites (320.9 ± 16 μm, an increase of 78.6%, P < 0.01; Table 4 ). RGCs cultured on CoRMG had a mean total neurite length of 496.1 ± 13.7 μm, whereas, in CM, the length was 350.9 ± 12.8 μm (both P < 0.01). The only treatment that led to a decrease in mean total length of neurites was FxRMG (144.8 ± 6.5 μm).

Analysis of the mean length of the single longest neurite per cell within different conditions is shown in Figure 5 and Table 5 . In CDM this length was 99.1 ± 4.2 μm, whereas addition of BDNF increased the mean length to 170.9 ± 8.9 μm (P < 0.01). CoRMG and CM elicited even stronger effects, leading to increases of 204.6% (P < 0.01) and 89.6% (P < 0.01; Table 5 ) respectively. Finally, no differences were detected in the FxRMG group (97.8 ± 4.23 μm).

Representative images of RGCs from the different experimental culture conditions are shown in Figure 6 .

Discussion

In the present study we analyzed the survival and morphologic characteristics of adult porcine RGCs in response to different culture conditions. Identification of RGCs was based on their expression of NF immunoreactivity, according to our previously published criteria.¹⁸ It has been reported that expression of NF and morphology of retinal cells varies with growth on different substrates,³⁹ and differences in the regional distribution of NFs have been demonstrated in RGCs from mouse retina.⁴⁰ Our data concur with those in previous work showing large RGCs to be strongly NF immunoreactive across many mammalian species, whereas smaller RGCs exhibit relatively lower NF immunoreactivity.³⁷ ³⁸

Retinal Ganglion Cell Size

RGCs have been classified into groups according to cell size in cats¹⁰ and rats.¹³ ⁴¹ ⁴² ⁴³ It is also possible to identify several different types of RGCs in the primate retina based on soma size and dendritic field architecture.⁴⁴ ⁴⁵ ⁴⁶ ⁴⁷ In our study we classified cultured porcine RGCs into three groups and observed that distribution by cell size in control conditions is similar to that described in cultures from feline retina.¹⁰ It has been described that RGCs of different sizes are differentially susceptible to degeneration,¹⁴ ¹⁵ ¹⁶ ¹⁷ and differential cell type susceptibility to glutamate toxicity¹⁸ and hypoxia⁴⁸ has been demonstrated. In our study, the increase in mean area observed when RGCs were cultured on confluent RMG or with CM correlated with a significant reduction of small RGCs and an increase in large RGCs in both conditions. It is possible that a factor released by confluent RMG improves the survival of large but not of small RGCs. Another hypothesis could be a generalized trophic effect on soma size. The existence of plasticity in adult retina has been postulated, because increases in RGC soma size have been observed after experimental glaucoma in rats.⁴⁹ Other studies have shown a decrease in mean soma area of RGCs during glaucoma, and, although it has been suggested that this is due to preferential disappearance of large RGCs,⁵ ⁵⁰ the shrinkage of cells during glaucoma has also been suggested.⁵¹ ⁵²

Effect of BDNF on Survival and Morphology of RGCs

The exact physiological role of BDNF in the survival of RGCs is still not fully clear. Exogenous BDNF transiently increases the survival of RGCs after axotomy or optic nerve injury.¹³ ²⁰ ²¹ ⁵³ It has been demonstrated that BDNF retrogradely transported from the target plays a role in the maintenance of RGCs,⁵⁴ but other studies suggest that a substantial fraction of the BDNF found in retina is derived from local sources, suggesting that BDNF could act as an autocrine trophic factor supporting RGC survival.⁵⁵ ⁵⁶ Moreover, it has been demonstrated that BDNF-immunopositive RGCs and the percentage of RGCs expressing TrkB are increased after axotomy, suggesting that intrinsic rescue mechanisms may contribute to short-term survival.⁵⁷ Finally, BDNF-null mice⁵⁸ and TrkB-null mice⁵⁹ do not exhibit developmental losses in RGCs. In our study, administration of BDNF did not significantly modify the survival of cultured RGCs. Possible explanations are that endogenous BDNF levels produced by porcine RGCs in culture⁶⁰ are sufficient to support RGC survival or that the effect of BDNF on survival in vitro is lower than in vivo, due to the restricted number of factors.⁴¹ It has also been proposed that BDNF may limit its own neuroprotective effect through downregulation of its receptor TrkB after excessive BDNF application.⁶¹ ⁶² Finally, Meyer-Franke et al.⁶³ have demonstrated that purified postnatal rat RGCs require elevation of intracellular cAMP levels to render them fully sensitive to treatment with neurotrophic factor, and similar mechanisms may operate in the model used in the current study.

Although BDNF did not influence survival, it stimulated changes in soma size and neurite elongation. BDNF increased the mean soma area of RGCs in culture, which correlates with a higher number of large RGCs in this condition (Fig. 6B) . Such differential responses may reflect differences in the number and type of TrkB receptors, because it has been demonstrated that the number of TrkB-truncated receptors varies depending on the soma size of the RGCs.⁶⁴ In agreement with these data, BDNF has been shown to enlarge significantly the mean soma profile of human dopaminergic neurons,⁶⁵ and to exert different effects on survival of small and large RGCs in rat retina cultures,²⁵ with large RGCs exhibiting higher affinity to BDNF than medium RGCs.¹⁰

In the visual system, neurotrophins influence neurite outgrowth in vitro²⁴ and in vivo.²⁶ ⁶⁶ It has been demonstrated that exogenous BDNF regulates RGC dendritic and axonal arborization in Xenopus in vivo⁶⁷ and enhances axon growth in individual RGCs of rats,⁶⁸ possibly through the TrkB receptor.⁶⁹ In our study, BDNF increased both the length and number of neurites in adult porcine RGCs.

Effect of RMG on Survival and Morphology of RGCs

Glial cells maintain normal functioning of the nervous system, both by controlling the extracellular environment and by supplying metabolites and growth factors. It has been shown that RMG can protect against the excitotoxic effects of glutamate in the whole retina³⁰ and that RGCs cultured on RMG show significantly better survival rates under conditions of hypoxia²⁸ and exposure to glutamate.²⁸ ²⁹ ³¹ In our results, survival of RGCs was significantly higher when these cells were cultured with confluent RMG. This effect seems not to be mediated by substrate alone, because an increase was also observed in RMG-conditioned medium. Meyer-Franke et al.⁶³ observed that the long-term survival of postnatal RGCs is maintained in part by trophic factors produced by glial cells, and it has been observed that glial cell line–derived neurotrophic factor significantly attenuates degeneration of RGCs after transection of the optic nerve in adult rats.⁷⁰ The effects of RMG on RGC survival vary depending on their confluence,⁴¹ and in agreement with these findings, we did not observe improvement in survival when RGCs were cultured on subconfluent RMG.

In contrast, it has been demonstrated that glial cells support regeneration of RGCs,⁷¹ ⁷² and, in addition, neurite length and number of RGCs depend on culture substratum.⁷³ In our study, RMG enhanced these parameters compared with control substrates, and because fixed RMG did not affect outgrowth of neurites in RGCs, we suggest that some factor(s) released from RMG is responsible for enhancing outgrowth of neurites in cultured RGCs (Fig. 6) . This factor could act by associating with the substrate to promote growth of neurites, or it may act directly to stimulate transcription of cytoskeletal proteins.⁷⁴ It has been shown that NGF acts directly on the neuronal soma to increase cytoskeletal protein synthesis.⁷⁵ ⁷⁶ RMG also synthesize and secrete lipoproteins that may improve outgrowth of RGC neurites.⁷⁷ The effect of confluent RMG on outgrowth of RGC neurites is stronger than that observed with CM alone, suggesting possible synergistic effects of substrate and diffusible factors.

In conclusion, in our study confluent RMG exerted beneficial effects on cultured RGCs from adult porcine retina, leading to improved survival, increased mean soma size, and increased outgrowth of neurites in RGCs. The beneficial effect of RMG is not mediated principally by increased adhesion of RGCs, because CM produced similar responses in RGC cultures. We suggest that a currently unidentified factor(s) produced and released into the medium from RMG is involved in these effects. Characterization and identification of these molecules is of potential interest in devising therapeutic strategies for protecting RGCs against degeneration in diseases such as glaucoma.

Supported by University País Vasco Grant EB006/99, Government Vasco Grant PI-1998-81, Ministry of Education and Science Grant PM97-0047 and European Community Pro Age Ret QLK6-2000-00569.

Submitted for publication March 7, 2002; revised May 28, 2002; accepted June 17, 2002.

Commercial relationships policy: N.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Elena Vecino, Universidad del País Vasco, Facultad Medicina, Departmento Biología, 48940 Leioa, Vizcaya, Spain; [email protected].

Figure 1.

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Effects of different experimental conditions on the survival of cultured RGCs labeled with anti-NF68 antibody. Results are expressed as the mean ± SEM (n = 10 different retina) and are percentages of cells surviving in each condition, compared with CDM control (set at 100%). *P < 0.05; **P < 0.01, significantly different compared with control; †significantly different compared with CoRMG (P < 0.01); ‡significantly different compared with CM (P < 0.01); §significantly different compared with ScRMG (P < 0.01); and ∥significantly different compared with BDNF (P < 0.01).

Table 1.

View Table

Percentage of RGCs Surviving

Table 1.

Percentage of RGCs Surviving

Group	Immunolabel
Group		NF68	NF200
CDM	100	100
BDNF	104.6 ± 4.2^{, ‡}	104.2 ± 3.3^{, †} ^{, ‡} ^{, §}
CoRMG	110.4 ± 3.4^*	113.2 ± 3.3^{, **}
ScRMG	102.1 ± 4.9^{, ‡}	102.1 ± 3.9^{, †} ^{, ‡}
FxRMG	59.7 ± 2.2^{, **} ^{, †} ^{, ‡} ^{, §} ^{, ∥}	59.6 ± 3.9^{, **} ^{, †} ^{, ‡} ^{, §} ^{, ∥}
CM	116.7 ± 4.8^{, **}	120.9 ± 4.8^{, **} ^{, †}

Percentage of RGCs surviving determined by immunolabeling with NF68 and NF200 cultured in different conditions, normalized to CDM (taken as 100%). Results are the mean of surviving RGCs ± SEM (n = 10).

* P < 0.05;

** P < 0.01, significantly different compared with control.

† Significantly different compared with CoRMG (P < 0.01 for all conditions except for CM, which is P < 0.05).

‡ Significantly different compared with CM (P < 0.01).

§ Significantly different compared with ScRMG (P < 0.01 for FxRMG, P < 0.05 for BDNF).

∥ Significantly different compared with BDNF (P < 0.01).

Figure 2.

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Effect of different experimental conditions on the survival of different sized RGCs in culture. (A) Mean area ± SEM of the measured RGCs was calculated for each treatment. **P < 0.01, significantly different compared with control; †significantly different compared with CoRMG (P < 0.01 for all conditions except for CM, which is P < 0.05); ‡significantly different compared with CM (P < 0.01); §significantly different compared with ScRMG (P < 0.01); and ∥significantly different compared with BDNF (P < 0.01). (B) Number of large, medium, and small RGCs immunopositive to NF68 and NF200 in the different culture conditions. Results are shown as the total number of RGCs (n = 10).

Table 2.

View Table

Percentage of Increase in Mean Soma Area

Table 2.

Percentage of Increase in Mean Soma Area

Group	Immunolabel
Group		NF68	NF200
CDM	100	100
BDNF	119.6 ± 8.2^* ^{, †}	115.4 ± 9.1^* ^{, †}
CoRMG	136.5 ± 8.4^{, **}	149.3 ± 10.4^{, **}
ScRMG	117.2 ± 7.5^* ^{, †}	109.6 ± 3.6^* ^{, †}
FxRMG	106.5 ± 5.3^{, †} ^{, ‡} ^{, §} ^{, ∥}	95.6 ± 11.3^{, †} ^{, §}
CM	124.9 ± 9.9^{, **}	127.9 ± 10.6^{, **}

Percentage increase in mean soma area calculated for both NF68 and NF200 immunolabeling. Results are shown as the mean soma area ± SEM (n = 10).

* P < 0.05 and

** P < 0.01, significantly different compared with CDM.

† Significantly different compared with CoRMG (P < 0.05 for all conditions except FxRMG, which is P < 0.01).

‡ Significantly different compared with CM (P < 0.05).

§ Significantly different compared with ScRMG (P < 0.05).

∥ Significantly different compared with BDNF (P < 0.05).

Figure 3.

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Effect of different experimental conditions on the number of neurites in cultured RGCs. Results are the mean percentage of RGCs with neurites ± SEM (n = 10). *P < 0.05; **P < 0.01, significantly different compared with control; †significantly different compared with CoRMG (P < 0.01 for all conditions except for CM, which is P < 0.05); and ‡significantly different compared with CM (P < 0.01).

Table 3.

View Table

Percentage of RGCs Expressing Neurites

Table 3.

Percentage of RGCs Expressing Neurites

Group	Immunolabel
Group		NF68	NF200
CDM	59.6 ± 8.8	33.4 ± 4.8
BDNF	78.8 ± 7.4^* ^{, †} ^{, ‡}	44.3 ± 3.3^{, †} ^{, ‡}
CoRMG	99.5 ± 0.4^{, **}	97.0 ± 0.9^{, **}
ScRMG	70.2 ± 9.9^* ^{, †} ^{, ‡}	56.9 ± 9.3^* ^{, †} ^{, ‡}
FxRMG	61.5 ± 7.5^{, †} ^{, ‡}	26.6 ± 5.9^{, †} ^{, ‡}
CM	93.6 ± 2.0^{, **} ^{, †}	88.1 ± 4.1^{, **} ^{, †}

Percentage of NF68- and NF200-immunolabeled RGCs expressing neurites in different experimental conditions. Results are the mean ± SEM (n = 10).

* P < 0.05 and

** P < 0.01, significantly different from control.

† Significantly different from CoRMG (P < 0.01 for all conditions except for CM, which is P < 0.05).

‡ Significantly different compared with CM (P < 0.01).

Figure 4.

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Effect of different experimental treatments on the length of neurites in cultured RGCs. Results are expressed as mean neurite length ± SEM (n = 10). **P < 0.01, significantly different compared with control; †significantly different compared with CoRMG (P < 0.01); ‡significantly different compared with CM (P < 0.01); §significantly different compared with ScRMG (P < 0.01); and ∥significantly different compared with BDNF (P < 0.01).

Table 4.

View Table

Percentage Increase of Mean Total Neurite Length

Table 4.

Percentage Increase of Mean Total Neurite Length

Group	Immunolabel
Group		NF68	NF200
CDM	100	100
BDNF	178.6 ± 48.1^* ^{, †}	214.6 ± 27.2^* ^{, †}
CoRMG	275.8 ± 37.5^{, **}	326.7 ± 43.5^{, **}
ScRMG	133 ± 31.7^* ^{, †} ^{, ‡}	168.5 ± 32.3^* ^{, †} ^{, ‡}
FxRMG	80.5 ± 12.4^{, **} ^{, †} ^{, ‡}	109.9 ± 26.5^{, †} ^{, ‡}
CM	195.1 ± 36.6^{, **} ^{, †}	233.7 ± 44.1^{, **} ^{, †}

Percentage increase of mean total neurite length of NF68- and NF200-immunolabeled RGCs cultured in different conditions. Results are the mean total neurite length ± SEM (n = 10).

* P < 0.05 and

** P < 0.01, significantly different compared with control.

† Significantly different compared with CoRMG (P < 0.01).

‡ Significantly different compared with CM (P < 0.01 for FxRMG, P < 0.05 for ScRMG).

Figure 5.

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Effect of different experimental conditions on length of the longest individual neurite in cultured RGCs. Results are the mean length of the longest single neurite per cell ± SEM (n = 10) **P < 0.01, significantly different compared with control; †significantly different compared with CoRMG (P < 0.01); ‡significantly different compared with CM (P < 0.01); §significantly different compared with ScRMG (P < 0.01); and ∥significantly different compared with BDNF (P < 0.01).

Table 5.

View Table

Percentage Increase in Mean Length of the Longest Neurite

Table 5.

Percentage Increase in Mean Length of the Longest Neurite

Group	Immunolabel
Group		NF68	NF200
CDM	100	100
BDNF	172.54 ± 41.03^* ^{, †}	181.27 ± 27.2^* ^{, †}
CoRMG	304.59 ± 54.5^{, **}	361.3 ± 52.4^{, **}
ScRMG	134.59 ± 14.4^* ^{, †} ^{, ‡}	148.47 ± 12.3^* ^{, †} ^{, ‡}
FxRMG	98.69 ± 16.1^{, †} ^{, ‡}	117.5 ± 15.6^{, †} ^{, ‡}
CM	189.56 ± 45.9^{, **} ^{, †}	211.02 ± 36.3^{, **} ^{, †}

Percentage increase in mean length of the longest neurite of NF68- and NF200-immunolabeled RGCs cultured in different conditions.

* P < 0.05 and

** P < 0.01, significantly different compared with control.

† Significantly different compared with CoRMG (P < 0.01).

‡ Significantly different compared with CM (P < 0.01).

Figure 6.

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Representative images of RGCs from the different experimental culture conditions. RGCs grown (A) in control CDM conditions, (B) on laminin-poly-lysine substrates supplemented with BDNF, (C) on a confluent RMG monolayer, (D) on subconfluent RMG, (E) in fixed RMG cultures, and (F) in cultures supplemented with RMG-conditioned medium. Note the different extension and disposition of the neurites: larger (C, F) and growing parallel to the longest axis of the RMG (C). Photographs were obtained with a triple filter: NF200 (green), NF68 (red), and nuclei (DAPI; blue). Bar, 50 μm.

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