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Peptide growth factors but not ganglioside protect against excitotoxicity in rat retinal neurons in vitro
Brain Research 767 Ž1997. 279–288
Research report
Peptide growth factors but not ganglioside protect against excitotoxicity in rat retinal neurons in vitro Valerie ´ Heidinger ) , David Hicks, Jose´ Sahel, Henri Dreyfus Laboratoire de Physiopathologie retinienne, INSERM CJF 92 r 02, Medicale A, Centre Hospitalier et UniÕersitaire de Strasbourg, BP 426, 67091 ´ ´ Strasbourg Cedex, France Accepted 30 April 1997
Abstract Glutamate is the major excitatory neurotransmitter in the retina, but excessive stimulation of its receptors leads to widespread neuronal stress and death. Both growth factors and gangliosides display important influences on responses to neuronal injury and degeneration. In this study, we have investigated the potential protective effects of two well characterized growth factors, epidermal and basic fibroblast growth factor ŽEGF and bFGF respectively., and the monosialoganglioside GM1, on cultured rat retinal neurons submitted to toxic levels of excitatory amino acids. Application of 1 mM glutamic acid reduced global neuronal viability by 80% when compared to control untreated cultures, whereas treatment with the glutamic acid agonist kainic acid Ž1 mM. led to specific, large decreases Ž75% reduction. in amacrine cell numbers. 24 h pretreatment with either EGF or bFGF Ž500 pM each. prevented the majority of excitatory amino acid-induced neuronal death, whereas similar treatment with 10y5 M GM1 did not block neuronal degeneration. These findings demonstrate that EGF and bFGF act as neuroprotective agents against retinal excitotoxicity in vitro, whereas ganglioside GM1 is not effective in this particular paradigm. q 1997 Elsevier Science B.V. Keywords: Excitotoxicity; Growth factor; Ganglioside; Retinal neuron; Excitatory amino acid
1. Introduction Glutamate is the major excitatory neurotransmitter in the brain and mediates fast synaptic transmission in the vertebrate central nervous system ŽCNS. w27,67x. It can also act as a toxic substance to neurons when present in excess and lead to either rapid- or delayed-type neurotoxicity w14,47,59x. Glutamate has also long been known to exert neurotoxic actions on different populations of neurons in the inner retina w42x. Epidermal growth factor ŽEGF. and basic fibroblast growth factor ŽbFGF. are polypeptides with pleiotropic actions and a widespread tissue distribution w6,10,12,26,29x. They are both prototypic members of different trophic factor families and are capable of influencing the survival and neurite outgrowth of neurons. For example, both bFGF and EGF have been shown to act as neurite elongation and maintenance factors for cultured cortical neurons w50,51x.
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0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 6 0 5 - 7
Besides its potent neurotrophic activity, evidence has accumulated indicating that bFGF also acts as a protective agent in vivo and in vitro. It has been shown that bFGF treatment improved survival following lesions of various neuronal pathways w5,56x. In addition, bFGF was effective in preventing hippocampal neuronal damage following hypoglycemia w13x and Kirshner et al. w39x demonstrated that systemic administration of bFGF could protect against neuronal death from excitotoxicity and hypoxia. In vitro, EGF also exhibited protective effects against glutamate neurotoxicity in cerebellar neurons w1x. Such observations have led to various proposals to use neurotrophic growth factors for the treatment of neurodegenerative disorders w33x. Gangliosides are sialylated glycosphingolipids abundant in neuronal membranes within the CNS w41,63x. They have been implicated in cell–cell recognition, transmembrane signaling and adhesion w30,41,53x and they also modulate cell growth by regulating proliferative and maturational processes controlled by polypeptide growth factors w53x. Monosialoganglioside GM1 has been reported to reduce excitotoxic CNS injury. In vitro studies indicated that
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GM1 was capable of reducing EAA-related neurotoxicity in different types of neuronal cell cultures w23x and GM1 anti-excitotoxic activity has also been described in vivo after exogenous injection w38,40,62x. Basic FGF is involved in development, differentiation and survival of the neural retina w35,64x. Basic FGF was reported to delay the degeneration of Royal College of Surgeons rat retinal photoreceptors w21x. EGF is endogenous to the retina since mRNA for this factor is detected in bovine and rat retinal extracts w4,22x. Ganglioside presence and distribution within the retina have been well studied w16,18,19x but as for peptide growth factors precise knowledge of their physiological functions is lacking. In vitro and in vivo studies suggest that gangliosides exhibit neuroprotective effects against retinal excitotoxicity and ischemia w20,69x. In this study, we investigated the potential neuroprotective actions of EGF, bFGF and GM1 on EAA toxicity. We confirmed the neurotoxic effect of glutamic acid and its agonist kainic acid on distinct retinal cell populations in vitro, and demonstrate significant prevention of excitotoxic death by either growth factor under the experimental conditions used, whereas at the concentration tested GM1 did not significantly modify neuronal survival.
mediumrF12, DMEMrF12. and were then chopped into small fragments, rinsed with phosphate buffered saline ŽPBS. and digested with 0.05% trypsin in buffer: NaCl Ž8 grl., KCl Ž0.4 grl., NaHCO 3 Ž0.58 grl., glucose Ž1 grl., EDTA-Na 2 Ž0.2 grl. for 25 min at 378C. A monodisperse cell suspension was prepared by trituration through pasteur pipettes Žnormal and flame-constricted.. The resulting cell suspension was seeded onto 12 mm coverslips wcoated serially with poly-D-lysine Ž2 m grcm2 . for 30 min and laminin Ž1 m grcm2 . for 1 h and then washed with PBSx in 24 well plates. Seeding was done at 2.10 5 cellsrcm2 in DMEMrF12 supplemented with 10% fetal calf serum ŽFCS. and penicillin-streptomycin Ž10 IUrl.. Retinal cultures were incubated at 378C in a humidified atmosphere of 5% carbon dioxide-95% air for 3 days before drug treatment. After 24 h, DMEMrF12 10% FCS was substituted with chemically defined medium: DMEMrF12 plus insulin Ž5 m grml., transferrin Ž5 m grml., sodium selenite Ž5 ngrml., putrescine Ž16.1 m grml., progesterone Ž0.63 m grml., taurine Ž375 m grml., cytidine 5X-diphosphatidylethanolamine Ž1.28 m grml., cytidine 5X-diphosphatidylcholine Ž2.56 m grml., hydrocortisone Ž0.2 m grml., triiodotyrosine Ž0.02 m grml. and sodium pyruvate Ž110 m grml. w3x. Some wells were maintained throughout the experiments in DMEMrF12 10% FCS.
2. Materials and methods
2.3. Experimental treatment
2.1. Materials
The conditions of treatment were chosen based on preliminary experiments in which culture age and EAA concentrations were investigated. Cell cultures were treated according to the following: Ž1. control wells were left untreated, in either 10% FCS DMEMrF12 or CDM, receiving addition of 50 m l vehicle buffer only; Ž2. wells receiving growth factors wEGF 500 pM Ž2.5 m l. or bFGF 500 pM Ž5 m l.x or ganglioside GM1 Ž10y5 M. Ž5 m lrwell. were treated 3 days after cell seeding; Ž3. wells receiving EAA Žglutamic acid 1 mM or kainic acid 1 mM, 50 m l. were treated 4 days after seeding. Thus in those wells receiving both growth factorsrganglioside and EAA, growth factorrganglioside treatment occurred 24 h prior to EAA addition. All wells were rinsed twice in PBS after a total of 5 days in vitro and fixed in 4% paraformaldehyde in PBS for 20 min.
All tissue culture supplies Žmedia, sera, dishes. were obtained from GIBCO-BRL Life Technologies, CergyPontoise, France. Additional reagents for defined media, tissue culture substrates, EAA, fluorescent antibodies, fluorescent ExtrAvidin, anti-vimentin monoclonal antibody Žclone V9. and ganglioside GM1 were all purchased from Sigma-Aldrich, Saint Quentin Fallavier, France. EGF was bought from Chemicon International Inc., Temecula, CA, USA and bFGF from Pharma Biotechnologie, Hannover, Germany. HPC-1 antibody was the generous gift of Dr. C.J. Barnstable ŽYale University, CT, USA.; NSE polyclonal antibody was obtained from Polysciences Ltd., Eppelheim Germany. Biotinylated antibodies were from Amersham, Les Ulis, France. Rho-4D2 anti-opsin antibody was obtained from hybridomas derived from mice immunized against rat retinal proteins w34x. All other chemicals and reagents were of analytical grade. 2.2. Retinal neuronal cultures Handling of animals conformed to the ARVO Resolution on the Use of Animals in Research. Retinal neuronal cultures were prepared using slight modifications of previously described methods w36x. One postnatal day ŽPN1. Wistar rats were anesthetized by dry ice inhalation, decapitated and enucleated. Retinas were isolated from ocular tissue in culture medium ŽDulbecco’s modified Eagles
2.4. Immunocytochemistry Coverslips were incubated in PBS containing 0.1% bovine serum albumin ŽBSA., 0.1% Tween 20 ŽPBS buffer. for 5 min. Different primary antibodies were used: HPC-1 Žanti-syntaxin. monoclonal antibody which labels specifically amacrine cells in developing and adult retina w7x; Rho-4D2 monoclonal antibody has been previously characterized and binds to the amino terminal of rod photoreceptor opsin w34x; anti-rat neuron specific enolase ŽNSE. polyclonal anti-
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body, which is specific for the rat form of NSE and recognizes all differentiated retinal neurons w36x; vimentin monoclonal antibody, clone V9 which binds to intermediate filaments and recognises Muller glial cells ¨ in the retina w17x. For the two latter antibodies, cells were permeabilized 5 min with 0.1% Triton X-100 in PBS. Cultures were incubated with primary antibodies Ž10 m grml. for 1 h, washed several times with PBS buffer and then exposed for 1 h to biotinylated goat anti-mouse IgG Žfor monoclonal primary antibodies. Ž10 m grml. or biotinylated goat anti-rabbit IgG Žfor polyclonal primary antibody. Ž10 m grml.. After several washes with PBS buffer, cultures were exposed for 1 h to extravidin linked to tetrarhodamine isothiocyanate ŽTRITC. Ž10 m grml.. For double immunostaining, suitable combinations of monoclonal and polyclonal antibodies were used, one being detected by a secondary fluorescent antibody Žgoat anti-rabbit IgG-fluoresceine isothiocyanate ŽFITC. for polyclonals or rabbit anti-mouse IgGFITC for monoclonals.. Coverslips were finally washed thoroughly, mounted and viewed under a Nikon Optiphot 2 photomicroscope equipped with Nomarski differential interference optics and epifluorescence illumination.
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2.5. Assessment of cellular injury Neuronal cell injury was assessed by counting of viable cells Ždefined as those exhibiting continuous plasma membranes with no signs of vacuolation, and well-developed neuritic expansions, following immunolabelling; cells showing perikaryal swelling, nuclear pyknosis or fragmentation, or localized neurite swellings were not included in the counts. after different growth factor and EAA treatments compared to control cultures. A minimum of 50 optical fields, randomly chosen per coverslip, were counted Žunder a 40 = objective.. Cell number was expressed by mean neuronal number per optical field. Each experiment was repeated separately more than 3 times with triplicate treatments per experiment. Statistical analyses were done using the Peritz parametric test w32x. 3. Results 3.1. Culture morphology After 5 days in vitro, control Žuntreated. retinal cultures maintained in DMEMrF12 supplemented with 10% FCS
Fig. 1. Retinal neuronal cultures after 5 days in defined medium in vitro. ŽSame field in A, B and C.. ŽA. Nomarski image of typical field showing numerous rounded neurons and extensive fibre outgrowth. Rare glial cells are also observed. ŽB. Amacrine cells immunostained with anti-syntaxin antibody Žshort arrow indicates labelled cell in panels A and B.. ŽC. Photoreceptors labelled with anti-opsin antibody Žlong arrow indicates labelled cell in panels A and C.. ŽD. NSE antiserum labels all neurons. ŽE. Muller glial cells in untreated cultures immunostained with anti-vimentin antibody. ŽF. ¨ Cultures treated 48 h with EGF Ž500 pM. and immunostained with anti-vimentin antibody. Same results were obtained after bFGF Ž500 pM. pretreatment. Bar s 10 m m.
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exhibited two morphological cell types: rounded neuronlike cells and flattened glial cells ŽFig. 1A.. Immunostaining with different cell type-specific antibodies allowed identification of distinct subpopulations of neurons present in our culture system. Anti-syntaxin antibody labelled relatively large Žsoma diameter 10 m m. cells, exhibiting a
Fig. 3. Neuronal loss following glutamic acid exposure and protection afforded by EGF or bFGF pretreatment. For each experimental group Žno growth factor, EGF or bFGF, with or without serum., numbers of NSE immunoreactive cells in control cultures were expressed as 100%. The percentage of surviving neurons was calculated with respect to control values. For untreated cultures in CDM, 100% represented an average of 60"5 cells per field. There were no significant differences in cell numbers between control Žeither in CDM or 10% FCS. and cells treated only with growth factors Žeither EGF or bFGF.. ) ) ) P - 0.001, significance values for glutamic acid treated cells in comparison to their control untreated cells; 888 P - 0.001 and 8 P - 0.05, significance values for EGF or bFGF pretreated cells exposed to glutamic acid in comparison to their controls Žpretreated with growth factors but without glutamic acid exposure..
highly developed neuritic network ŽFig. 1B.. This amacrine cell population represented approximately 15% of all neuronal cells in our culture model. Anti-opsin antibody uniquely labelled cells with a small rounded cell soma and one or several short neurites ŽFig. 1C.. These rod photoreceptors represented 20% of the total neurons. When labelled with anti-NSE antibody, all large multipolar and smaller unipolar neurons were positively stained ŽFig. 1D.. Muller glial cells were labelled with anti-vimentin anti¨ body ŽFig. 1E.. When EGF or bFGF were added to the culture medium, glia increased rapidly and reached confluency after two days of growth factor exposure ŽFig. 1F., whereas neuronal cell numbers did not change. To reduce glial cell development, we maintained cultures in CDM after 1 day in vitro. In CDM, neuronal cells did not present any differences in number or morphology compared to cultures maintained in serum supplemented medium. 3.2. PreÕention of glutamic acid-induced toxicity on neuronal cells by EGF and bFGF pretreatment Fig. 2. Effects of bFGF on glutamic acid-induced neurotoxicity. Cultures were immunostained with anti-NSE antibody. ŽA. Control untreated cultures. ŽB. Cultures pretreated for 24 h with 500 pM bFGF only. ŽC. Cells treated with 1 mM glutamic acid for 24 h. ŽD. Cells pretreated with 500 pM bFGF followed by 1 mM glutamic acid exposure. Notice the well preserved neuronal morphology. The same results were obtained after EGF Ž500 pM. treatment prior to glutamic acid exposure. Bar s10 m m.
The toxic effects of glutamic acid application to retinal neuronal cultures, and the potential protective influences of growth factors against these insults, were tested in retinal cultures maintained in CDM. As described above, there were no significant differences in neuronal cell numbers
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between growth factor Žeither EGF or bFGF. pretreated and untreated cultures ŽFig. 2A and Fig. 2B.. Assessment of glutamic acid-induced toxicity was performed after labelling with anti-NSE antibody. NSE immunoreactive cells in control cultures exhibited intact, strongly stained cell bodies ŽFig. 2A.. After 1 mM glutamic acid exposure,
Fig. 5. Amacrine cell loss following kainic acid exposure and protection afforded by EGF or bFGF pretreatment. For each experimental group Žno growth factor, EGF or bFGF, with or without serum., numbers of amacrine cells in control cultures were expressed as 100%. The percentage of surviving neurons was calculated with respect to control values. For untreated cultures in CDM, 100% represented an average of 10"1 amacrine cells per field. There were no significant differences in cell numbers between controls Žeither in CDM or 10% FCS. and cells treated only with growth factors Žeither EGF or bFGF.. ) ) ) P - 0.001, significance values for kainic acid treated cells in comparison to their control untreated cells; 888 P - 0.001 and 88 P - 0.01, significance values for EGF pretreated cells exposed to kainic acid in comparison to their controls Žpretreated with EGF but without kainic acid exposure..
NSE-immunoreactive cell numbers decreased dramatically, and residual cell soma labelling was less intense and processes were no longer visible ŽFig. 2C.. EGF or bFGF treatment during 24 h prior to glutamic acid exposure prevented cell alterations as shown in Fig. 2D, with cell labelling similar to control cultures Žwell defined rounded cells and processes.. Counting of immunolabelled cells showed that 80% of neuronal cells disappeared after 1 mM glutamic acid treatment ŽFig. 3.. Double immunolabelling
Fig. 4. Effects of EGF on kainic acid-induced amacrine cells death. ŽA. Control untreated cultures. ŽB. Cultures pretreated for 24 h with 500 pM EGF only. ŽC. Cultures treated with 1 mM kainic acid for 24 h. ŽD. Cultures pretreated with 500 pM EGF followed by 1 mM kainic acid exposure for 24 h. Bar s10 m m.
Fig. 6. Pretreatment with 10y5 M GM1 does not prevent glutamic acid-induced neuronal death in vitro. ) ) ) P - 0.001, significance values above the black column refer to comparisons between glutamic acid treated cell with or without GM1 pretreatment and their respective controls untreated with glutamic acid.
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3.3. PreÕention of kainic acid toxicity on amacrine cells by EGF and bFGF pretreatment
Fig. 7. Pretreatment with 10y5 M GM1 does not prevent kainic acid-induced amacrine cell death in vitro. ) ) ) P - 0.001, significance values above the black column refer to comparisons between kainic acid treated cell with or without GM1 pretreatment and their respective controls untreated with kainic acid.
using anti-NSE and anti-syntaxin antibodies showed that approximately 20% of the glutamic acid-sensitive population was composed of amacrine cells Ždata not shown.. The majority of glutamic acid-sensitive neurons were not labelled with any cell-type specific markers, but were of small size Ž; 5 m m cell body diameter. with a bipolar morphology. Prior treatment with EGF led to maintenance of the majority of neurons, with only 25% neuronal loss. The number of surviving neurons was equally elevated in bFGF-pretreated cells ŽFig. 3.. Similar values were obtained when these experiments were performed using cultures maintained in 10% FCS supplemented medium ŽFig. 3..
Syntaxin immunostaining revealed that control amacrine cells exhibited well-defined cell bodies possessing highly developed and ramified processes ŽFig. 4A.. When cultures were pretreated by EGF or bFGF alone, amacrine cell numbers or morphology did not change ŽFig. 4B.. Kainic acid-treated cultures exhibited a dramatic loss of amacrine cells and disappearance of their neuritic network ŽFig. 4C.. When neuronal cultures were pretreated with EGF or bFGF, significant preservation of amacrine cells and process outgrowth was observed ŽFig. 4D.. Examination of brightfield images showed no obvious differences in the numbers of total neurons between untreated and 1 mM kainic acid treated cells. Immunostaining for amacrine cells showed that 1 mM kainic acid reduced their numbers by more than 75% compared to control untreated cells ŽFig. 5.. EGF treatment 24 h prior to EAA exposure inhibited kainic acid-induced amacrine cell loss by 50% Žcultured in CDM., whereas similar treatment with bFGF completely prevented the neuronal loss due to kainic acid. When similar studies were performed in 10% FCS supplemented cultures, no significant differences compared to experiments done in CDM medium were observed ŽFig. 5.. 3.4. Monosialoganglioside GM1 is not neuroprotectiÕe against excitatory amino acid toxicity Neuronal cultures were treated with glutamic acid Ž1 mM. or kainic acid Ž1 mM. as described above, and after
Fig. 8. EAA effects on photoreceptor cells immunostained with anti-opsin antibody. ŽA. Control untreated cultures. ŽB. Cells treated for 24 h with 1 mM glutamic acid. ŽC. Cells treated for 24 h with 1 mM kainic acid. Bar s 10 m m.
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Fig. 9. EAA are not excitotoxic for photoreceptor cells in vitro. ) ) ) P 0.001, significance values for all neuronal cell numbers treated with 1 mM glutamic acid compared to control untreated cells.
immunostaining cell numbers were counted. Pretreatment with 10y5 M GM1 alone for 24 h did not have any influence on the global retinal neuronal population or amacrine cell numbers. When GM1 pretreatment was performed 24 h before glutamic acid exposure, no protectiverrestorative effect was observed on neuronal cells ŽFig. 6.. Neither did GM1 pretreatment prevent kainic acid-induced amacrine cell death ŽFig. 7.. We also tested GM1 protection against lower EAA concentrations Ž200 m M glutamic acid and 100 m M kainic acid., also without effect Ždata not shown.. 3.5. Neither glutamic nor kainic acid exhibit any toxicity for photoreceptor cells EAA Ž1 mM glutamic acid or kainic acid. treatment did not lead to photoreceptor cell death. Photoreceptor numbers did not decrease when cultures were treated with glutamic or kainic acid ŽFig. 8.. In control, untreated cultures, photoreceptors represented 20% of the neuronal population, and after either glutamic or kainic acid exposure their numbers did not change significantly ŽFig. 9..
4. Discussion In this study, we have demonstrated that pretreatment with EGF or bFGF greatly reduces retinal neuronal death induced by excessive levels of glutamic or kainic acid. We confirmed the neurotoxic effect of elevated extracellular glutamic acid levels on retinal neurons, as originally described by Lucas and Newhouse w42x in whole retina and for which the term «excitotoxicity» was given by Olney et al. w55x. We used different cell type-specific antibodies to determine which subpopulations of neurons were altered by EAA exposure. Use of anti-syntaxin antibody to label specifically amacrine cells showed that this population was sensitive to both EAA. It accounted for the entire cell loss observed with kainic acid and 20% of that observed with
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glutamic acid. Photoreceptors labelled with anti-opsin antibody were not affected by either treatment, in agreement with previous studies w2,20,57x. The very low numbers of ganglion and horizontal cells Ž- 1% total neurons. in our cultures made it impossible to quantify EAA effects on these cells. However previous studies have demonstrated the susceptibility of ganglion cells to both glutamic acid w11x and kainic acid w66x treatment, which is to be expected given their glutamatergic input w31,37,52x. Hence, the bulk of the glutamic acid-induced neuronal loss is probably accounted for by bipolar cells as they represent one of the most abundant cell types in the rat retina and participate in glutamatergic pathways w8x. However, anti-PKC antibodies which label rod bipolar cells in adult retina w54x stain only lightly cells in these immature cultures, and so their identity remains unconfirmed. EAA treatments were all performed with a concentration of 1 mM EAA because preliminary experiments indicated that maximum cell death occurred at this concentration. The selection of EGF and bFGF was based on their protective properties against EAA-induced neuronal cell death in brain neurons w1,25,28x, and their presence in retina w4,22,64x. Moreover, previous studies on retina also suggest that growth factors could be used as good tools against numerous types of injury w21,65,70x. We used a concentration of 500 pM of growth factors because previous in vitro studies indicated that EGF or bFGF neuritogenic or survival properties were obtained under these conditions w36x. In our experiments growth factors were added prior to excitatory amino acid treatment, as previous reports have indicated that growth factor addition simultaneously or subsequently to excitotoxic treatment in vitro is ineffective w1,43x. Such observations indicate that growth factor neuroprotective action is through induction of de novo synthesis of compounds such as antioxidant enzymes or calcium binding proteins w13,45,46x. There were no differences between EGF and bFGF protection against glutamic acid-induced neuronal toxicity, whereas a difference was observed between EGF and bFGF-induced prevention of amacrine cell death: bFGF was more effective then EGF as all amacrine cells were preserved. This might indicate that downstream events after growth factor binding may differ for the two factors with respect to protection against excitotoxicity. Higher doses of EGF might induce better preservation of amacrine cell numbers. Mechanisms of neurotrophic factor protection against EAA-induced injury remain unclear and previous studies have evoked different hypotheses. Their involvement in the regulation of calcium concentration and their interaction with free radicals andror nitric oxide implicated in excitotoxic mechanisms have been described in CNS w13,15,43,45,71x. In our system, it was possible that growth factor protective effects were mediated by Muller glial ¨ cells as even in defined media some glial cells Ž- 10% total cells. were present. We used a chemically defined medium to reduce glial proliferation but EGF and bFGF
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have been defined as potent mitogens for Muller ¨ glial cells w44,58x and pretreatment with these growth factors increased glial numbers. As glial cells can take up glutamic acid from the extracellular medium w9,60x, an increase in Muller ¨ cell numbers could be responsible for the protective phenomena observed. However glial involvement in the observed protection is less likely for two reasons. Firstly, we observed protection against kainic acid-induced amacrine cell death by growth factors, whereas glial cell transporters are unable to take up kainic acid. Secondly, both glutamic and kainic acid-induced neuronal cell death were observed in mixed glial-neuronal cultures Žculture medium supplemented with serum. demonstrating the inefficiency of Muller glial cells to prevent excitotoxicity by ¨ themselves. On the other hand, we cannot rule out that the growth factors may have upregulated levels of other trophic molecules in the glial monolayers. One neuroprotective strategy has made use of monosialoganglioside GM1 since in vitro studies have indicated that GM1 was able to reduce EAA-induced neurotoxicity in cortical and cerebellar neurons w23x or retinal neurons w20x. Protective effects of GM1 in experimental models of cerebral or retinal ischemia have been described w38,40,69x. Mohand-Said et al. w49x also described protection of rat retina after post-ischemic intravitreal GM1 injection. The ability of gangliosides to protect neurons could be related to their effects on calcium fluxes w24x as EAA-induced neuronal damage is due to massive intracellular calcium influx w14x. The results reported in our study demonstrated, in contrast to previous in vitro studies w20x that GM1 was not able to protect neurons against excitotoxic injury. The GM1 concentration used in the present study was 20 times lower Ž10y5 M., approaching physiological concentrations more closely. At these lower concentrations Facci et al. did not observe increased survival either, so it is possible that high doses of GM1 may improve neuron survival in our model as well. Pretreatment with GM1 was done 24 h before EAA exposure to allow ganglioside incorporation into the cultured cells as previous studies on retinal glial cells w48x demonstrated a 15% increase in ganglioside concentration after ganglioside pretreatment under the same experimental conditions and at the same concentration used here. GM1 also failed to protect neurons at lower concentrations of glutamic acid Ž200 m M. or kainic acid Ž100 m M., these concentrations corresponding to the minimal doses inducing significant neuronal or amacrine cells death. Additional differences between the studies reported here and those of Facci et al. w20x concern the model systems, in their case embryonic chick retina, and in our case postnatal rat retina, which led to different proportions of cell types. An alternative explanation for the lack of ganglioside protection in our model is that although ischemic neuronal death is mediated by EAA other mechanisms are also possible, as for instance free radical production w68x or generation of lipid mediators w61x. Thus GM1 protection could conceivably occur at
these stages of ischemic cell death rather than affecting the excitotoxic pathway. In conclusion, the major findings of this study are: Ži. glutamic acid is excitotoxic for several retinal neuronal types, whereas kainic acid-induced toxicity is specific for amacrine cells in vitro; Žii. significant prevention of EAAinduced neuronal cell loss by the growth factors EGF or bFGF; Žiii. inability of ganglioside GM1 to prevent EAAinduced neuronal toxicity. Although the mechanisms of protection of growth factors remain undefined, our results suggest that growth factors could be considered interesting candidates for potential clinical use in neurodegenerative disorders as they can modulate responses to glutamic acid and promote cell survival.
Acknowledgements The authors would like to thank Dr. C.J. Barnstable for the generous gift of HPC-1 antibody; Valerie ´ Forster for technical assistance; and the Ministere ` de l’Enseignement Superieur et de la Recherche, ADRET-Alsace, IPSEN ´ Pharmaceuticals and MGENrINSERM for financial aid.
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Research report
Peptide growth factors but not ganglioside protect against excitotoxicity in rat retinal neurons in vitro Valerie ´ Heidinger ) , David Hicks, Jose´ Sahel, Henri Dreyfus Laboratoire de Physiopathologie retinienne, INSERM CJF 92 r 02, Medicale A, Centre Hospitalier et UniÕersitaire de Strasbourg, BP 426, 67091 ´ ´ Strasbourg Cedex, France Accepted 30 April 1997
Abstract Glutamate is the major excitatory neurotransmitter in the retina, but excessive stimulation of its receptors leads to widespread neuronal stress and death. Both growth factors and gangliosides display important influences on responses to neuronal injury and degeneration. In this study, we have investigated the potential protective effects of two well characterized growth factors, epidermal and basic fibroblast growth factor ŽEGF and bFGF respectively., and the monosialoganglioside GM1, on cultured rat retinal neurons submitted to toxic levels of excitatory amino acids. Application of 1 mM glutamic acid reduced global neuronal viability by 80% when compared to control untreated cultures, whereas treatment with the glutamic acid agonist kainic acid Ž1 mM. led to specific, large decreases Ž75% reduction. in amacrine cell numbers. 24 h pretreatment with either EGF or bFGF Ž500 pM each. prevented the majority of excitatory amino acid-induced neuronal death, whereas similar treatment with 10y5 M GM1 did not block neuronal degeneration. These findings demonstrate that EGF and bFGF act as neuroprotective agents against retinal excitotoxicity in vitro, whereas ganglioside GM1 is not effective in this particular paradigm. q 1997 Elsevier Science B.V. Keywords: Excitotoxicity; Growth factor; Ganglioside; Retinal neuron; Excitatory amino acid
1. Introduction Glutamate is the major excitatory neurotransmitter in the brain and mediates fast synaptic transmission in the vertebrate central nervous system ŽCNS. w27,67x. It can also act as a toxic substance to neurons when present in excess and lead to either rapid- or delayed-type neurotoxicity w14,47,59x. Glutamate has also long been known to exert neurotoxic actions on different populations of neurons in the inner retina w42x. Epidermal growth factor ŽEGF. and basic fibroblast growth factor ŽbFGF. are polypeptides with pleiotropic actions and a widespread tissue distribution w6,10,12,26,29x. They are both prototypic members of different trophic factor families and are capable of influencing the survival and neurite outgrowth of neurons. For example, both bFGF and EGF have been shown to act as neurite elongation and maintenance factors for cultured cortical neurons w50,51x.
) Corresponding author. Fax: q33 Ž3. 8824-3314; E-mail: [email protected]
0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 6 0 5 - 7
Besides its potent neurotrophic activity, evidence has accumulated indicating that bFGF also acts as a protective agent in vivo and in vitro. It has been shown that bFGF treatment improved survival following lesions of various neuronal pathways w5,56x. In addition, bFGF was effective in preventing hippocampal neuronal damage following hypoglycemia w13x and Kirshner et al. w39x demonstrated that systemic administration of bFGF could protect against neuronal death from excitotoxicity and hypoxia. In vitro, EGF also exhibited protective effects against glutamate neurotoxicity in cerebellar neurons w1x. Such observations have led to various proposals to use neurotrophic growth factors for the treatment of neurodegenerative disorders w33x. Gangliosides are sialylated glycosphingolipids abundant in neuronal membranes within the CNS w41,63x. They have been implicated in cell–cell recognition, transmembrane signaling and adhesion w30,41,53x and they also modulate cell growth by regulating proliferative and maturational processes controlled by polypeptide growth factors w53x. Monosialoganglioside GM1 has been reported to reduce excitotoxic CNS injury. In vitro studies indicated that
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GM1 was capable of reducing EAA-related neurotoxicity in different types of neuronal cell cultures w23x and GM1 anti-excitotoxic activity has also been described in vivo after exogenous injection w38,40,62x. Basic FGF is involved in development, differentiation and survival of the neural retina w35,64x. Basic FGF was reported to delay the degeneration of Royal College of Surgeons rat retinal photoreceptors w21x. EGF is endogenous to the retina since mRNA for this factor is detected in bovine and rat retinal extracts w4,22x. Ganglioside presence and distribution within the retina have been well studied w16,18,19x but as for peptide growth factors precise knowledge of their physiological functions is lacking. In vitro and in vivo studies suggest that gangliosides exhibit neuroprotective effects against retinal excitotoxicity and ischemia w20,69x. In this study, we investigated the potential neuroprotective actions of EGF, bFGF and GM1 on EAA toxicity. We confirmed the neurotoxic effect of glutamic acid and its agonist kainic acid on distinct retinal cell populations in vitro, and demonstrate significant prevention of excitotoxic death by either growth factor under the experimental conditions used, whereas at the concentration tested GM1 did not significantly modify neuronal survival.
mediumrF12, DMEMrF12. and were then chopped into small fragments, rinsed with phosphate buffered saline ŽPBS. and digested with 0.05% trypsin in buffer: NaCl Ž8 grl., KCl Ž0.4 grl., NaHCO 3 Ž0.58 grl., glucose Ž1 grl., EDTA-Na 2 Ž0.2 grl. for 25 min at 378C. A monodisperse cell suspension was prepared by trituration through pasteur pipettes Žnormal and flame-constricted.. The resulting cell suspension was seeded onto 12 mm coverslips wcoated serially with poly-D-lysine Ž2 m grcm2 . for 30 min and laminin Ž1 m grcm2 . for 1 h and then washed with PBSx in 24 well plates. Seeding was done at 2.10 5 cellsrcm2 in DMEMrF12 supplemented with 10% fetal calf serum ŽFCS. and penicillin-streptomycin Ž10 IUrl.. Retinal cultures were incubated at 378C in a humidified atmosphere of 5% carbon dioxide-95% air for 3 days before drug treatment. After 24 h, DMEMrF12 10% FCS was substituted with chemically defined medium: DMEMrF12 plus insulin Ž5 m grml., transferrin Ž5 m grml., sodium selenite Ž5 ngrml., putrescine Ž16.1 m grml., progesterone Ž0.63 m grml., taurine Ž375 m grml., cytidine 5X-diphosphatidylethanolamine Ž1.28 m grml., cytidine 5X-diphosphatidylcholine Ž2.56 m grml., hydrocortisone Ž0.2 m grml., triiodotyrosine Ž0.02 m grml. and sodium pyruvate Ž110 m grml. w3x. Some wells were maintained throughout the experiments in DMEMrF12 10% FCS.
2. Materials and methods
2.3. Experimental treatment
2.1. Materials
The conditions of treatment were chosen based on preliminary experiments in which culture age and EAA concentrations were investigated. Cell cultures were treated according to the following: Ž1. control wells were left untreated, in either 10% FCS DMEMrF12 or CDM, receiving addition of 50 m l vehicle buffer only; Ž2. wells receiving growth factors wEGF 500 pM Ž2.5 m l. or bFGF 500 pM Ž5 m l.x or ganglioside GM1 Ž10y5 M. Ž5 m lrwell. were treated 3 days after cell seeding; Ž3. wells receiving EAA Žglutamic acid 1 mM or kainic acid 1 mM, 50 m l. were treated 4 days after seeding. Thus in those wells receiving both growth factorsrganglioside and EAA, growth factorrganglioside treatment occurred 24 h prior to EAA addition. All wells were rinsed twice in PBS after a total of 5 days in vitro and fixed in 4% paraformaldehyde in PBS for 20 min.
All tissue culture supplies Žmedia, sera, dishes. were obtained from GIBCO-BRL Life Technologies, CergyPontoise, France. Additional reagents for defined media, tissue culture substrates, EAA, fluorescent antibodies, fluorescent ExtrAvidin, anti-vimentin monoclonal antibody Žclone V9. and ganglioside GM1 were all purchased from Sigma-Aldrich, Saint Quentin Fallavier, France. EGF was bought from Chemicon International Inc., Temecula, CA, USA and bFGF from Pharma Biotechnologie, Hannover, Germany. HPC-1 antibody was the generous gift of Dr. C.J. Barnstable ŽYale University, CT, USA.; NSE polyclonal antibody was obtained from Polysciences Ltd., Eppelheim Germany. Biotinylated antibodies were from Amersham, Les Ulis, France. Rho-4D2 anti-opsin antibody was obtained from hybridomas derived from mice immunized against rat retinal proteins w34x. All other chemicals and reagents were of analytical grade. 2.2. Retinal neuronal cultures Handling of animals conformed to the ARVO Resolution on the Use of Animals in Research. Retinal neuronal cultures were prepared using slight modifications of previously described methods w36x. One postnatal day ŽPN1. Wistar rats were anesthetized by dry ice inhalation, decapitated and enucleated. Retinas were isolated from ocular tissue in culture medium ŽDulbecco’s modified Eagles
2.4. Immunocytochemistry Coverslips were incubated in PBS containing 0.1% bovine serum albumin ŽBSA., 0.1% Tween 20 ŽPBS buffer. for 5 min. Different primary antibodies were used: HPC-1 Žanti-syntaxin. monoclonal antibody which labels specifically amacrine cells in developing and adult retina w7x; Rho-4D2 monoclonal antibody has been previously characterized and binds to the amino terminal of rod photoreceptor opsin w34x; anti-rat neuron specific enolase ŽNSE. polyclonal anti-
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body, which is specific for the rat form of NSE and recognizes all differentiated retinal neurons w36x; vimentin monoclonal antibody, clone V9 which binds to intermediate filaments and recognises Muller glial cells ¨ in the retina w17x. For the two latter antibodies, cells were permeabilized 5 min with 0.1% Triton X-100 in PBS. Cultures were incubated with primary antibodies Ž10 m grml. for 1 h, washed several times with PBS buffer and then exposed for 1 h to biotinylated goat anti-mouse IgG Žfor monoclonal primary antibodies. Ž10 m grml. or biotinylated goat anti-rabbit IgG Žfor polyclonal primary antibody. Ž10 m grml.. After several washes with PBS buffer, cultures were exposed for 1 h to extravidin linked to tetrarhodamine isothiocyanate ŽTRITC. Ž10 m grml.. For double immunostaining, suitable combinations of monoclonal and polyclonal antibodies were used, one being detected by a secondary fluorescent antibody Žgoat anti-rabbit IgG-fluoresceine isothiocyanate ŽFITC. for polyclonals or rabbit anti-mouse IgGFITC for monoclonals.. Coverslips were finally washed thoroughly, mounted and viewed under a Nikon Optiphot 2 photomicroscope equipped with Nomarski differential interference optics and epifluorescence illumination.
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2.5. Assessment of cellular injury Neuronal cell injury was assessed by counting of viable cells Ždefined as those exhibiting continuous plasma membranes with no signs of vacuolation, and well-developed neuritic expansions, following immunolabelling; cells showing perikaryal swelling, nuclear pyknosis or fragmentation, or localized neurite swellings were not included in the counts. after different growth factor and EAA treatments compared to control cultures. A minimum of 50 optical fields, randomly chosen per coverslip, were counted Žunder a 40 = objective.. Cell number was expressed by mean neuronal number per optical field. Each experiment was repeated separately more than 3 times with triplicate treatments per experiment. Statistical analyses were done using the Peritz parametric test w32x. 3. Results 3.1. Culture morphology After 5 days in vitro, control Žuntreated. retinal cultures maintained in DMEMrF12 supplemented with 10% FCS
Fig. 1. Retinal neuronal cultures after 5 days in defined medium in vitro. ŽSame field in A, B and C.. ŽA. Nomarski image of typical field showing numerous rounded neurons and extensive fibre outgrowth. Rare glial cells are also observed. ŽB. Amacrine cells immunostained with anti-syntaxin antibody Žshort arrow indicates labelled cell in panels A and B.. ŽC. Photoreceptors labelled with anti-opsin antibody Žlong arrow indicates labelled cell in panels A and C.. ŽD. NSE antiserum labels all neurons. ŽE. Muller glial cells in untreated cultures immunostained with anti-vimentin antibody. ŽF. ¨ Cultures treated 48 h with EGF Ž500 pM. and immunostained with anti-vimentin antibody. Same results were obtained after bFGF Ž500 pM. pretreatment. Bar s 10 m m.
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exhibited two morphological cell types: rounded neuronlike cells and flattened glial cells ŽFig. 1A.. Immunostaining with different cell type-specific antibodies allowed identification of distinct subpopulations of neurons present in our culture system. Anti-syntaxin antibody labelled relatively large Žsoma diameter 10 m m. cells, exhibiting a
Fig. 3. Neuronal loss following glutamic acid exposure and protection afforded by EGF or bFGF pretreatment. For each experimental group Žno growth factor, EGF or bFGF, with or without serum., numbers of NSE immunoreactive cells in control cultures were expressed as 100%. The percentage of surviving neurons was calculated with respect to control values. For untreated cultures in CDM, 100% represented an average of 60"5 cells per field. There were no significant differences in cell numbers between control Žeither in CDM or 10% FCS. and cells treated only with growth factors Žeither EGF or bFGF.. ) ) ) P - 0.001, significance values for glutamic acid treated cells in comparison to their control untreated cells; 888 P - 0.001 and 8 P - 0.05, significance values for EGF or bFGF pretreated cells exposed to glutamic acid in comparison to their controls Žpretreated with growth factors but without glutamic acid exposure..
highly developed neuritic network ŽFig. 1B.. This amacrine cell population represented approximately 15% of all neuronal cells in our culture model. Anti-opsin antibody uniquely labelled cells with a small rounded cell soma and one or several short neurites ŽFig. 1C.. These rod photoreceptors represented 20% of the total neurons. When labelled with anti-NSE antibody, all large multipolar and smaller unipolar neurons were positively stained ŽFig. 1D.. Muller glial cells were labelled with anti-vimentin anti¨ body ŽFig. 1E.. When EGF or bFGF were added to the culture medium, glia increased rapidly and reached confluency after two days of growth factor exposure ŽFig. 1F., whereas neuronal cell numbers did not change. To reduce glial cell development, we maintained cultures in CDM after 1 day in vitro. In CDM, neuronal cells did not present any differences in number or morphology compared to cultures maintained in serum supplemented medium. 3.2. PreÕention of glutamic acid-induced toxicity on neuronal cells by EGF and bFGF pretreatment Fig. 2. Effects of bFGF on glutamic acid-induced neurotoxicity. Cultures were immunostained with anti-NSE antibody. ŽA. Control untreated cultures. ŽB. Cultures pretreated for 24 h with 500 pM bFGF only. ŽC. Cells treated with 1 mM glutamic acid for 24 h. ŽD. Cells pretreated with 500 pM bFGF followed by 1 mM glutamic acid exposure. Notice the well preserved neuronal morphology. The same results were obtained after EGF Ž500 pM. treatment prior to glutamic acid exposure. Bar s10 m m.
The toxic effects of glutamic acid application to retinal neuronal cultures, and the potential protective influences of growth factors against these insults, were tested in retinal cultures maintained in CDM. As described above, there were no significant differences in neuronal cell numbers
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between growth factor Žeither EGF or bFGF. pretreated and untreated cultures ŽFig. 2A and Fig. 2B.. Assessment of glutamic acid-induced toxicity was performed after labelling with anti-NSE antibody. NSE immunoreactive cells in control cultures exhibited intact, strongly stained cell bodies ŽFig. 2A.. After 1 mM glutamic acid exposure,
Fig. 5. Amacrine cell loss following kainic acid exposure and protection afforded by EGF or bFGF pretreatment. For each experimental group Žno growth factor, EGF or bFGF, with or without serum., numbers of amacrine cells in control cultures were expressed as 100%. The percentage of surviving neurons was calculated with respect to control values. For untreated cultures in CDM, 100% represented an average of 10"1 amacrine cells per field. There were no significant differences in cell numbers between controls Žeither in CDM or 10% FCS. and cells treated only with growth factors Žeither EGF or bFGF.. ) ) ) P - 0.001, significance values for kainic acid treated cells in comparison to their control untreated cells; 888 P - 0.001 and 88 P - 0.01, significance values for EGF pretreated cells exposed to kainic acid in comparison to their controls Žpretreated with EGF but without kainic acid exposure..
NSE-immunoreactive cell numbers decreased dramatically, and residual cell soma labelling was less intense and processes were no longer visible ŽFig. 2C.. EGF or bFGF treatment during 24 h prior to glutamic acid exposure prevented cell alterations as shown in Fig. 2D, with cell labelling similar to control cultures Žwell defined rounded cells and processes.. Counting of immunolabelled cells showed that 80% of neuronal cells disappeared after 1 mM glutamic acid treatment ŽFig. 3.. Double immunolabelling
Fig. 4. Effects of EGF on kainic acid-induced amacrine cells death. ŽA. Control untreated cultures. ŽB. Cultures pretreated for 24 h with 500 pM EGF only. ŽC. Cultures treated with 1 mM kainic acid for 24 h. ŽD. Cultures pretreated with 500 pM EGF followed by 1 mM kainic acid exposure for 24 h. Bar s10 m m.
Fig. 6. Pretreatment with 10y5 M GM1 does not prevent glutamic acid-induced neuronal death in vitro. ) ) ) P - 0.001, significance values above the black column refer to comparisons between glutamic acid treated cell with or without GM1 pretreatment and their respective controls untreated with glutamic acid.
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3.3. PreÕention of kainic acid toxicity on amacrine cells by EGF and bFGF pretreatment
Fig. 7. Pretreatment with 10y5 M GM1 does not prevent kainic acid-induced amacrine cell death in vitro. ) ) ) P - 0.001, significance values above the black column refer to comparisons between kainic acid treated cell with or without GM1 pretreatment and their respective controls untreated with kainic acid.
using anti-NSE and anti-syntaxin antibodies showed that approximately 20% of the glutamic acid-sensitive population was composed of amacrine cells Ždata not shown.. The majority of glutamic acid-sensitive neurons were not labelled with any cell-type specific markers, but were of small size Ž; 5 m m cell body diameter. with a bipolar morphology. Prior treatment with EGF led to maintenance of the majority of neurons, with only 25% neuronal loss. The number of surviving neurons was equally elevated in bFGF-pretreated cells ŽFig. 3.. Similar values were obtained when these experiments were performed using cultures maintained in 10% FCS supplemented medium ŽFig. 3..
Syntaxin immunostaining revealed that control amacrine cells exhibited well-defined cell bodies possessing highly developed and ramified processes ŽFig. 4A.. When cultures were pretreated by EGF or bFGF alone, amacrine cell numbers or morphology did not change ŽFig. 4B.. Kainic acid-treated cultures exhibited a dramatic loss of amacrine cells and disappearance of their neuritic network ŽFig. 4C.. When neuronal cultures were pretreated with EGF or bFGF, significant preservation of amacrine cells and process outgrowth was observed ŽFig. 4D.. Examination of brightfield images showed no obvious differences in the numbers of total neurons between untreated and 1 mM kainic acid treated cells. Immunostaining for amacrine cells showed that 1 mM kainic acid reduced their numbers by more than 75% compared to control untreated cells ŽFig. 5.. EGF treatment 24 h prior to EAA exposure inhibited kainic acid-induced amacrine cell loss by 50% Žcultured in CDM., whereas similar treatment with bFGF completely prevented the neuronal loss due to kainic acid. When similar studies were performed in 10% FCS supplemented cultures, no significant differences compared to experiments done in CDM medium were observed ŽFig. 5.. 3.4. Monosialoganglioside GM1 is not neuroprotectiÕe against excitatory amino acid toxicity Neuronal cultures were treated with glutamic acid Ž1 mM. or kainic acid Ž1 mM. as described above, and after
Fig. 8. EAA effects on photoreceptor cells immunostained with anti-opsin antibody. ŽA. Control untreated cultures. ŽB. Cells treated for 24 h with 1 mM glutamic acid. ŽC. Cells treated for 24 h with 1 mM kainic acid. Bar s 10 m m.
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Fig. 9. EAA are not excitotoxic for photoreceptor cells in vitro. ) ) ) P 0.001, significance values for all neuronal cell numbers treated with 1 mM glutamic acid compared to control untreated cells.
immunostaining cell numbers were counted. Pretreatment with 10y5 M GM1 alone for 24 h did not have any influence on the global retinal neuronal population or amacrine cell numbers. When GM1 pretreatment was performed 24 h before glutamic acid exposure, no protectiverrestorative effect was observed on neuronal cells ŽFig. 6.. Neither did GM1 pretreatment prevent kainic acid-induced amacrine cell death ŽFig. 7.. We also tested GM1 protection against lower EAA concentrations Ž200 m M glutamic acid and 100 m M kainic acid., also without effect Ždata not shown.. 3.5. Neither glutamic nor kainic acid exhibit any toxicity for photoreceptor cells EAA Ž1 mM glutamic acid or kainic acid. treatment did not lead to photoreceptor cell death. Photoreceptor numbers did not decrease when cultures were treated with glutamic or kainic acid ŽFig. 8.. In control, untreated cultures, photoreceptors represented 20% of the neuronal population, and after either glutamic or kainic acid exposure their numbers did not change significantly ŽFig. 9..
4. Discussion In this study, we have demonstrated that pretreatment with EGF or bFGF greatly reduces retinal neuronal death induced by excessive levels of glutamic or kainic acid. We confirmed the neurotoxic effect of elevated extracellular glutamic acid levels on retinal neurons, as originally described by Lucas and Newhouse w42x in whole retina and for which the term «excitotoxicity» was given by Olney et al. w55x. We used different cell type-specific antibodies to determine which subpopulations of neurons were altered by EAA exposure. Use of anti-syntaxin antibody to label specifically amacrine cells showed that this population was sensitive to both EAA. It accounted for the entire cell loss observed with kainic acid and 20% of that observed with
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glutamic acid. Photoreceptors labelled with anti-opsin antibody were not affected by either treatment, in agreement with previous studies w2,20,57x. The very low numbers of ganglion and horizontal cells Ž- 1% total neurons. in our cultures made it impossible to quantify EAA effects on these cells. However previous studies have demonstrated the susceptibility of ganglion cells to both glutamic acid w11x and kainic acid w66x treatment, which is to be expected given their glutamatergic input w31,37,52x. Hence, the bulk of the glutamic acid-induced neuronal loss is probably accounted for by bipolar cells as they represent one of the most abundant cell types in the rat retina and participate in glutamatergic pathways w8x. However, anti-PKC antibodies which label rod bipolar cells in adult retina w54x stain only lightly cells in these immature cultures, and so their identity remains unconfirmed. EAA treatments were all performed with a concentration of 1 mM EAA because preliminary experiments indicated that maximum cell death occurred at this concentration. The selection of EGF and bFGF was based on their protective properties against EAA-induced neuronal cell death in brain neurons w1,25,28x, and their presence in retina w4,22,64x. Moreover, previous studies on retina also suggest that growth factors could be used as good tools against numerous types of injury w21,65,70x. We used a concentration of 500 pM of growth factors because previous in vitro studies indicated that EGF or bFGF neuritogenic or survival properties were obtained under these conditions w36x. In our experiments growth factors were added prior to excitatory amino acid treatment, as previous reports have indicated that growth factor addition simultaneously or subsequently to excitotoxic treatment in vitro is ineffective w1,43x. Such observations indicate that growth factor neuroprotective action is through induction of de novo synthesis of compounds such as antioxidant enzymes or calcium binding proteins w13,45,46x. There were no differences between EGF and bFGF protection against glutamic acid-induced neuronal toxicity, whereas a difference was observed between EGF and bFGF-induced prevention of amacrine cell death: bFGF was more effective then EGF as all amacrine cells were preserved. This might indicate that downstream events after growth factor binding may differ for the two factors with respect to protection against excitotoxicity. Higher doses of EGF might induce better preservation of amacrine cell numbers. Mechanisms of neurotrophic factor protection against EAA-induced injury remain unclear and previous studies have evoked different hypotheses. Their involvement in the regulation of calcium concentration and their interaction with free radicals andror nitric oxide implicated in excitotoxic mechanisms have been described in CNS w13,15,43,45,71x. In our system, it was possible that growth factor protective effects were mediated by Muller glial ¨ cells as even in defined media some glial cells Ž- 10% total cells. were present. We used a chemically defined medium to reduce glial proliferation but EGF and bFGF
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have been defined as potent mitogens for Muller ¨ glial cells w44,58x and pretreatment with these growth factors increased glial numbers. As glial cells can take up glutamic acid from the extracellular medium w9,60x, an increase in Muller ¨ cell numbers could be responsible for the protective phenomena observed. However glial involvement in the observed protection is less likely for two reasons. Firstly, we observed protection against kainic acid-induced amacrine cell death by growth factors, whereas glial cell transporters are unable to take up kainic acid. Secondly, both glutamic and kainic acid-induced neuronal cell death were observed in mixed glial-neuronal cultures Žculture medium supplemented with serum. demonstrating the inefficiency of Muller glial cells to prevent excitotoxicity by ¨ themselves. On the other hand, we cannot rule out that the growth factors may have upregulated levels of other trophic molecules in the glial monolayers. One neuroprotective strategy has made use of monosialoganglioside GM1 since in vitro studies have indicated that GM1 was able to reduce EAA-induced neurotoxicity in cortical and cerebellar neurons w23x or retinal neurons w20x. Protective effects of GM1 in experimental models of cerebral or retinal ischemia have been described w38,40,69x. Mohand-Said et al. w49x also described protection of rat retina after post-ischemic intravitreal GM1 injection. The ability of gangliosides to protect neurons could be related to their effects on calcium fluxes w24x as EAA-induced neuronal damage is due to massive intracellular calcium influx w14x. The results reported in our study demonstrated, in contrast to previous in vitro studies w20x that GM1 was not able to protect neurons against excitotoxic injury. The GM1 concentration used in the present study was 20 times lower Ž10y5 M., approaching physiological concentrations more closely. At these lower concentrations Facci et al. did not observe increased survival either, so it is possible that high doses of GM1 may improve neuron survival in our model as well. Pretreatment with GM1 was done 24 h before EAA exposure to allow ganglioside incorporation into the cultured cells as previous studies on retinal glial cells w48x demonstrated a 15% increase in ganglioside concentration after ganglioside pretreatment under the same experimental conditions and at the same concentration used here. GM1 also failed to protect neurons at lower concentrations of glutamic acid Ž200 m M. or kainic acid Ž100 m M., these concentrations corresponding to the minimal doses inducing significant neuronal or amacrine cells death. Additional differences between the studies reported here and those of Facci et al. w20x concern the model systems, in their case embryonic chick retina, and in our case postnatal rat retina, which led to different proportions of cell types. An alternative explanation for the lack of ganglioside protection in our model is that although ischemic neuronal death is mediated by EAA other mechanisms are also possible, as for instance free radical production w68x or generation of lipid mediators w61x. Thus GM1 protection could conceivably occur at
these stages of ischemic cell death rather than affecting the excitotoxic pathway. In conclusion, the major findings of this study are: Ži. glutamic acid is excitotoxic for several retinal neuronal types, whereas kainic acid-induced toxicity is specific for amacrine cells in vitro; Žii. significant prevention of EAAinduced neuronal cell loss by the growth factors EGF or bFGF; Žiii. inability of ganglioside GM1 to prevent EAAinduced neuronal toxicity. Although the mechanisms of protection of growth factors remain undefined, our results suggest that growth factors could be considered interesting candidates for potential clinical use in neurodegenerative disorders as they can modulate responses to glutamic acid and promote cell survival.
Acknowledgements The authors would like to thank Dr. C.J. Barnstable for the generous gift of HPC-1 antibody; Valerie ´ Forster for technical assistance; and the Ministere ` de l’Enseignement Superieur et de la Recherche, ADRET-Alsace, IPSEN ´ Pharmaceuticals and MGENrINSERM for financial aid.
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