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Medium requirements for neuritic outgrowth from goldfish retinal explants and the trophic effect of taurine
Int. J. Devl Neuroscience 20 (2002) 607–617
Medium requirements for neuritic outgrowth from goldfish retinal explants and the trophic effect of taurine S. Cubillos, F. Fazzino, L. Lima∗ Laboratorio de Neuroqu´ımica, Centro de Biof´ısica y Bioqu´ımica, Instituto Venezolano de Investigaciones Cient´ıficas, Apdo. 21827, Caracas 1020-A, Venezuela Received 12 June 2002; received in revised form 7 October 2002; accepted 9 October 2002
Abstract The use of culture media of known composition are necessary for studying the role of trophic molecules. Since most of the in vitro research on regeneration of the optic nerve has been performed in the presence of fetal calf serum, the aim of this study was to obtain a medium in which the neuritic outgrowth from post-crush goldfish retinal explants could take place without adding fetal calf serum. After the lesion of the optic nerve (10 days), the retina of goldfish was dissected and explants were cultured for 5 and 10 days in the absence or in the presence of fetal calf serum, at which time the neuritic outgrowth was determined. Various concentrations and combinations of glucose, albumin, calcium, HEPES and taurine were used. The highest neuritic outgrowth was observed in the presence of fetal calf serum, in which condition the amino acid taurine increased length and density of neurites. Media supplemented with albumin, calcium or HEPES did not modify the outgrowth of neurites from the explants. However, glucose favored the neuritic outgrowth in a bell-shaped manner, although fibers were thinner than those observed in the presence of fetal calf serum. Taurine did not stimulate outgrowth of neurites from explants growing in a medium with optimal concentrations of glucose, indicating that elements of the fetal calf serum are determinant for the trophic effect of taurine. The present results contribute to further studies, such as those related to the effect of taurine and of trophic factors derived from the optic tectum, which would be performed in the presence of a medium free of fetal calf serum. © 2002 ISDN. Published by Elsevier Science Ltd. All rights reserved. Keywords: Culture medium; Optic nerve; Regeneration
1. Introduction Outgrowth of neurites from goldfish retinal explants occurs after lesion of the optic nerve (Lima et al., 1988). Conditions of the culture include the addition of fetal calf serum (Landreth and Agranoff, 1979), since in the absence of it neuritic outgrowth is dramatically restricted, even in the presence of trophic molecules, such as taurine (Lima et al., 1988). There have been several approaches to solve the important issue of studying the influence of stimulating agents per se without the concomitant effect of several substances present in the serum (Caffe et al., 2001; Espinosa de los Monteros et al., 1997; Sholl-Franco and Araujo, 1997; Winkler et al., 2000). A number of reports are related to the trophic effect of taurine, those touch aspects of concentration-dependency, time-related modifications, variations in the transport of ∗ Corresponding author. Tel.: +58-212-504-1213; fax: +58-212-504-1295. E-mail address: [email protected] (L. Lima).
taurine, among others (Lima, 1999). However, there is no consistent evaluation of the possible neuritic outgrowth obtained in the absence of fetal calf serum for studying the requirements of specific molecules during the regeneration in vitro. These facts are specifically important in our studies, because it was previously reported that the medium from optic tectum cultures and the co-culture of optic tectum and retina result in differential patterns of neuritic outgrowth (Cubillos et al., 2000), indicating the presence of determinant elements in those conditions, although previous studies were performed in the presence of fetal calf serum (Cubillos et al., 2000; Lima et al., 1988). The aims of the present report are to evaluate the neuritic outgrowth from goldfish retinal explants after the lesion of the optic nerve in the absence of fetal calf serum, and to study the possible effect of taurine by itself as a trophic agent in the retina. According to previous reports (Fischer, 1982; Ishida and Cheng, 1991; Lansel and Niemeyer, 1997; Lima et al., 1988; Lleu and Rebel, 1990; Matus et al., 1997; Winkler et al., 2000), we selected specific molecules to reach the objectives.
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2. Experimental procedures 2.1. Animals Goldfish (Carassius auratus), 5–6 cm in length were used after a lesion of the optic nerve. The lesion was performed by crushing the nerve around 1 mm behind the eye with fine forceps 10 days prior to dissection of the retina. After
this period of time they were adapted to dark for 30 min while anesthetized in 0.05% tricaine (Sigma) for obtaining the retina. 2.2. Retinal explants and evaluation of neuritic outgrowth Squares of 500 m (5–6 per retina) were prepared by sectioning the retina with a Mcllwain tissue chopper and
Fig. 1. Length of neurites from goldfish retinal explants at 5 or 10 days in culture. (A) In the absence or in the presence of seven increasing concentrations of albumin, 0, 0.01, 0.05, 0.1, 0.5, 1, 5 and 10%, 5 days F(7,388) = 3.98, P < 0.001; 10 days F(7,327) = 20.56, P < 0.001. (B) In the absence or in the presence of six concentrations of fetal calf serum without albumin, 0, 0.1, 0.5, 1, 2, 5 and 10%, 5 days F(6,339) = 126.53, P < 0.001; 10 days F(6,339) = 132.13, P < 0.001; with 0.1% albumin, 5 days F(6,298) = 115.97, P < 0.001; 10 days F(6,318) = 77.12, P < 0.001. (∗) P < 0.05 with respect to 5 days by Student’s t-test.
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placed 10–14 on poly-l-lysine (Sigma) pre-coated tissue flasks (25 mm2 ). Fetal calf serum (FCS) was used in some cultures in a concentration of 10%, and was omitted in those in which the following conditions were tested: calcium, glucose or albumin at different concentrations. Taurine, 4 mM (Lima et al., 1988), was added to some explants. The nutrient medium was Leibovitz, L-15, 3 ml per dish (Sigma) with 0.1 mg/ml of gentamicin and 20 mM or variable concentrations of (N-[2-hydroethyl]piperazine-N -[2-ethanesulfonic acid]) (HEPES). Five and ten days after plating, the density
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of neurites was evaluated using a predetermined scale (Landreth and Agranoff, 1979; Lima et al., 1988) and length (five neurites per explant) was determined by using the program SigmaScanPro (Jandel). 2.3. Statistical analysis Two ways analysis of variance was performed and pairs of samples, such as 5 versus 10 days in culture were compared by Student’s t-test. Significance corresponds to P < 0.05.
Fig. 2. Density of neurites from goldfish retinal explants at 5 and 10 days in culture, as described in Fig. 1. (A) 5 days F(7,72) = 1.11, P = 0.364; 10 days F(7,62) = 2.97, P < 0.01. (B) Without albumin, 5 days F(6,63) = 21.24, P < 0.001; 10 days F(6,63) = 10.75, P < 0.001. With albumin, 5 days F(6,56) = 14.00, P < 0.001; 10 days F(5,58) = 4.47, P < 0.001. (∗) P < 0.05 with respect to 5 days by Student’s t-test.
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Results are the mean ± standard error of the mean (S.E.M.), which is shown in the figures, except for length of neurites if it was too low (5–8% of the mean). 3. Results 3.1. Effect of fetal calf serum and albumin on neuritic outgrowth In the absence of serum, there was a small basal neuritic outgrowth that was not modified by the addition of albumin at 5 days in culture, except with 0.05 and 0.1% albumin, in which case there was a small decrease in length of neurites (Fig. 1A). A significant increase in the length of neurites after 10 days in culture was observed as compared with 5 days (Fig. 1A). Neurite length increased in the presence of FCS (0.1–10%) in a dose-dependent manner (Fig. 1B). The addition of albumin to retinal explants growing in the presence of variable concentrations of FCS did not modify the dose-dependency observed with FCS alone, but there was a decrease in the length of neurites as compared with the explants growing in the presence of FCS, but in the absence of albumin (Fig. 1B). Concerning the density of neurites (Fig. 2A and B), no significant changes were observed between 5 and 10 days in culture, and the density of fibers in the presence of FCS was not different from that with FCS and albumin. 3.2. Effect of fetal calf serum and glucose on neuritic outgrowth The length of neurites was significantly increased by the addition of glucose to the medium in the absence of FCS (Fig. 3A), this effect was evident at 0.5 mM glucose and was significantly smaller with 25 mM as compared to 0.5 mM, although it was still higher than control or lower concentrations of glucose. The presence of 10% FCS, and increasing concentrations of glucose in the medium of outgrowing explants did not further elevate the length of neurites already increased by FCS alone, as compared to those in the absence of FCS. Density of neurites was modified in a similar manner (Fig. 3B). The differences of magnitude in the effect obtained with glucose alone or with glucose and FCS was more evident at lower concentrations of glucose (Fig. 4). 3.3. Effect of calcium, albumin and glucose on neuritic outgrowth The addition of calcium at several concentrations did not increase the length of neurites from retinal explants, rather it reduced outgrowth of neurites at high concentrations (Fig. 5A). The combination of albumin and calcium produced similar results as calcium alone. In both cases, there was a significant increase in the length of neurites between 5 and 10 days in culture. Density of neurites was decreased
Fig. 3. Effect of glucose and fetal calf serum on neuritic outgrowth from goldfish retinal explants at 5 days in culture. (A) Length of neurites in the absence and in the presence of increasing concentrations of glucose 0, 0.01, 0.05, 0.5, 1, 2, 5, 25 mM without fetal calf serum F(7,224) = 17.50, P < 0.001; with 10% fetal calf serum F(7,549) = 5.68 P < 0.001. (B) Density of neurites, without fetal calf serum F(7,45) = 3.37, P < 0.01; with 10% fetal calf serum F(7,101) = 23.55, P < 0.001.
in the presence of high concentration of calcium, and no further modification was observed during the time in culture, except for a significant increase at 10 days as compared to 5 days in the presence of 12 mM calcium (Fig. 5B). The elevation of neurite length or density by 0.5 mM glucose was not altered by the simultaneous addition of increasing concentrations of calcium (Fig. 6A and B). The combination of albumin and glucose produced an increase in length and density of neurites as compared to albumin alone (Fig. 7A and B), but was not different from glucose alone.
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Fig. 4. Neuritic outgrowth from goldfish retinal explants at 5 days in culture. (A) In the presence of basic medium, absence of glucose or fetal calf serum. (B) In the presence of 0.5 mM glucose. (C) In the presence of 10% fetal calf serum. (D) In the presence of 0.5 mM glucose and 4 mM taurine. The line correspond to 500 m.
3.4. Effect of HEPES and fetal calf serum on neuritic outgrowth The outgrowth of neurites was significantly and slightly increased with augmenting concentrations of HEPES in the medium without FCS, but this effect was not observed in the presence of FCS, in which case there was a decrease in the length of neurites and no further elevation between 5 and 10 days in culture (Fig. 8A). Similar pattern of neuritic outgrowth was obtained for density of neurites in the presence of HEPES without FCS, but not with FCS (Fig. 8B). 3.5. Effect of taurine on neuritic outgrowth from explants in the presence of variable medium composition The presence of taurine in the medium without FCS produced a significant increase in the length of neurites in a concentration of 10 mM and in the density at various concentrations (Fig. 9A and B). In the presence of glucose there was a higher neuritic outgrowth (length and density), and the addition of taurine increased it according to the concentration added (Fig. 9A and B). In the presence of glucose and FCS there was an increase of length and density
of neurites produced by taurine, following a bell-shaped concentration-dependency. In addition, the presence of sucrose decreased the effect of 10 mM taurine, but did not have any effect by itself in concentrations up to 10 mM (data not shown). Taurine, 4 mM, did not modify outgrowth of neurites from explants growing in the presence of variable concentrations of calcium, neither the combination of FCS and calcium was different respecting FCS alone (Fig. 10A and B). The addition of taurine to explants cultured in the absence or in the presence of HEPES produced a significant increase with 20 mM HEPES and 4 mM taurine at 5 days in culture. Increased length occurred between 5 and 10 days in culture with taurine in the medium. HEPES alone reduced the length of neurites at 10 days in culture as compared to controls (Fig. 11A). Density of neurites was unaffected in the presence of HEPES or HEPES and taurine (Fig. 11B).
4. Discussion We previously demonstrated that the neuritic outgrowth from goldfish retinal explants was stimulated by the amino acid taurine (Lima et al., 1988), this effect was not observed
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Fig. 5. Effect of calcium and albumin on neuritic outgrowth from goldfish retinal explants at 5 and 10 days in culture. (A) Length of neurites in the absence and in the presence of 3, 6 and 12 mM calcium without albumin, 5 days F(3,161) = 10.39 P < 0.001; 10 days F(3,137) = 2.53, P = 0.059; with 0.1% albumin, 5 days F(3,161) = 8.12, P < 0.001; 10 days F(3,104) = 8.80, P < 0.001. (B) Density of neurites without albumin, 5 days F(3,31) = 4.72, P < 0.01; 10 days F(3,26) = 10.10, P < 0.001; with 0.1% albumin, 5 days F(3,30) = 3.27, P < 0.05; 10 days F(3,22) = 0.74, P = 0.538. (∗) P < 0.05 with respect to 5 days by Student’s t-test.
in isolated retinal ganglion cells in culture (Matus et al., 1997), since only the number of outgrowing cells was elevated by taurine, indicating that the integrity of the tissue was playing a determinant role on the effect of taurine as a trophic agent. This might be related to the interaction
Fig. 6. Effect of calcium and glucose on neuritic outgrowth from goldfish retinal explants at 5 and 10 days in culture. (A) Length of neurites in the absence and in the presence of 0, 3 and 6 mM calcium without glucose, 5 days F(2,129) = 0.58, P = 0.560; 10 days F(2,125) = 6.19, P < 0.01; with 0.5 mM glucose, 5 days F(2,147) = 1.26, P = 0.287, P < 0.001; 10 days F(2,147) = 7.92, P < 0.001. (B) Density of neurites without glucose, 5 days F(2,27) = 0.18, P = 0.838; 10 days F(2,27) = 4.14, P < 0.05; with 0.5 mM glucose, 5 days F(2,27) = 1.68, P = 0.205; 10 days F(2,27) = 0.74, P = 0.489.
between glial Müller cells, which in the presence of glucose have a low oxygen consumption (Winkler et al., 2000), and might spare oxygen for retinal neurons and then maintain the capability of extending neurites. Moreover, the present results support that glucose is sufficient to preserve the outgrowth of neurites from retinal explants, however, taurine
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Fig. 7. Effect of albumin and glucose on neuritic outgrowth from goldfish retinal explants at 5 and 10 days in culture. (A) Length of neurites in the absence and in the presence of 0, 0.1 and 1% albumin without glucose, 5 days F(2,112) = 3.27, P < 0.05; 10 days F(2,92) = 0.70, P = 0.05; with 0.5 mM glucose 5 days F(2,136) = 3.56, P < 0.05; 10 days F(2,147) = 1.29, P = 0.277. (B) Density of neurites without glucose, 5 days F(2,21) = 1.07, P = 0.360; 10 days F(2,17) = 1.88, P = 0.182; with 0.5 mM glucose 5 days F(2,25) = 0.56, P = 0.578; 10 days F(2,27) = 0.77, P = 0.473.
was not effective unless the elements of FCS were present. Differences in glucose metabolism between glia and neurons have been reported, which correspond to a physiological interaction between these cells (Lansel and Niemeyer, 1997). For instance, the electrical function of cat retina is known to be preserved by glucose in vitro (Onoe and Niemeyer,
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Fig. 8. Effect of HEPES and fetal calf serum on neuritic outgrowth from goldfish retinal explants at 5 and 10 days in culture. (A) Length of neurites in the absence and in the presence of 0, 10 and 20 mM without fetal calf serum 5 days F(2,145) = 9.26, P < 0.001; 10 days F(2,145) = 14.91, P < 0.001; with 10% fetal calf serum 5 days F(2,147) = 6.90, P < 0.01; 10 days F(2,147) = 37.30, P < 0.001. (B) Density of neurites without fetal calf serum 5 days F(2,27) = 0.12, P = 0.887; 10 days F(2,27) = 1.71, P = 0.200; with 10% fetal calf serum, 5 days F(2,27) = 3.23, P = 0.055; 10 days F(2,27) = 3.42, P < 0.05. (∗) P < 0.05 with respect to 5 days by Student’s t-test.
1992). It is interesting to show that concentrations of glucose lower than those considered as normal, around 5 mM (Lansel and Niemeyer, 1997), were sufficient to elevate the neuritic outgrowth, although the stimulatory effect was lower with 25 mM. This observation might be related to the regulation of the osmolarity, however, sucrose up to 10 mM did not have any effect by itself, but when added at lower
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Fig. 9. Effect of taurine, glucose, fetal calf serum and sucrose on neuritic outgrowth from goldfish retinal explants at 5 and 10 days in culture. (A) Length of neurites in the absence and in the presence of taurine 0, 0.2, 2, 4 and 10 mM, 5 days F(4,143) = 4.37, P < 0.01; with 0.5 mM glucose, 5 days F(2,414) = 3.10, P < 0.05; with 10% fetal calf serum F(4,298) = 13.11, P < 0.001; with 0.5 mM sucrose F(4,172) = 3.79, P < 0.01. (B) Density of neurites in the absence and in the presence of taurine 0, 0.2, 2, 4 and 10 mM, 5 days F(4,28) = 1.17, P = 0.346; with 0.5 mM glucose F(4,79) = 1.38, P = 0.249; with 10% fetal calf serum F(4,58) = 0.64, P = 0.638; with 0.5 mM sucrose F(4,33) = 3.74, P < 0.05.
concentrations with taurine, a small increase in neuritic outgrowth was observed, except with 10 mM taurine (Fig. 8). The components of a reported chemically defined medium when added in different combinations to oligodendrocytes in culture result in variable degrees of maturation (Espinosa de los Monteros et al., 1997), contributing to the understanding of the requirements for myelination potential of central nervous system. Although the evidence is in favor of the
importance of FCS for outgrowth neuritic an elevated rate and magnitude, goldfish retinal explants outgrow in the absence of FCS in a modest manner. It has been recently demonstrated that neonatal mouse retinal explants can be cultured and remain viable for more than 4 weeks in a serum-free medium (Caffe et al., 2001), in addition, it was previously shown that cerebellar cells of the mouse survive in hormonally defined medium, which includes
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Fig. 10. Effect of calcium, taurine and fetal calf serum on neuritic outgrowth from goldfish retinal explants at 5 and 10 days in culture. (A) Length of neurites in the absence and in the presence of 0, 3 and 6 mM calcium without taurine or fetal calf serum, 5 days F(2,93) = 0.68, P = 0.512; 10 days F(2,83) = 4.02, P < 0.05; with 4 mM taurine, 5 days F(2,112) = 1.59, P = 0.213; 10 days F(2,70) = 6.09, P < 0.01, with 10% fetal calf serum, 5 days F(1,153) = 1.07, P = 0.302; 10 days F(1,103) = 13.43, P < 0.001. (B) Density of neurites without taurine or fetal calf serum, 5 days F(2,20) = 0.97, P = 0.396; 10 days, F(2,16) = 0.94, P = 0.413; with 4 mM taurine, 5 days F(2,26) = 0.56, P = 0.575; 10 days F(2,14) = 0.01, P = 0.991, with 10% fetal calf serum, 5 days F(1,29) = 0.02, P = 0.876; 10 days F(1,19) = 0.25, P = 0.620. (∗) P < 0.05,with respect to 5 days by Student’s t-test.
albumin, insulin, transferrin, selenium, thyroxine and a protease inhibitor (Fischer, 1982). Moreover, one of a group of molecules associated with survival of cells are cytokines from activated lymphocytes (Sholl-Franco and Araujo, 1997). The characterization of minimal conditions
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Fig. 11. Effect of HEPES and taurine on neuritic outgrowth from goldfish retinal explants at 5 and 10 days in culture. (A) Length of neurites in the absence and in the presence of 0, 10 and 20 mM without taurine, 5 days F(2,147) = 1.26, P = 0.286; 10 days F(2,147) = 6.14, P < 0.01; with 4 mM taurine, 5 days F(2,147) = 9.47, P < 0.001; 10 days F(2,147) = 12.40, P < 0.001. (B) Density of neurites without taurine, 5 days F(2,27) = 0.22, P = 0.805; 10 days F(2,27) = 2.32, P = 0.117; with 4 mM taurine, 5 days F(2,27) = 4.57, P < 0.05; 10 days F(2,27) = 0.94, P = 0.403. (∗) P < 0.05 with respect to 5 days by Student’s t-test.
for preserving the capacity of culturing nervous tissue is essential in the study of individual components or unidentified factors of different sources, such as constituents of the optic tectum, as has been our experience in the goldfish retinal model (Cubillos et al., 2000).
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There are a number of reports showing the successful results concerning neuritic outgrowth in the presence of calcium (Ishida and Cheng, 1991). Chelating of extracellular or intracellular calcium produces a decrease of outgrowth of neurites from goldfish retinal explants (Lima et al., 1993), also calcium channel antagonists partially inhibit neurite outgrowth from adult rat olfactory cells (Sonigra et al., 1999). Despite these facts, the supplementation with calcium only slightly increased neuritic outgrowth up to 3 mM, and produced inhibition at 6 and 12 mM. The addition of albumin to the medium did not produce any effect on outgrowth of neurites, although it has been reported that transferrin and albumin improve differentiation of neural tissue of the rat used for in vivo transplantation (Belovari et al., 2001), also albumin seems to participate in visual-cycle transport (Adler and Edwards, 2000). It is also known that the interaction of soluble and attached factors influences neurite outgrowth (Michler et al., 1989). For instance, goldfish retinal explants cultured on a number of substrates present a differential elongation of fibers and variable response to taurine (Lima et al., 1989). As it is well supported, the optic tectum (Mizrachi et al., 1986) and the optic nerve (Caday et al., 1989) produce trophic substances which have positive influence on retinal regeneration. We have shown that the optic tectum is not only a growth-stimulatory factor producer, but it is also involved in the secretion of inhibitory molecules (Cubillos et al., 2000). In addition, FCS-mediated inhibition of neurite elongation has been noticed in ciliary ganglion neurons in culture (Davis et al., 1984). For studying factors derived from the optic tectum, a serum-free medium is required. Taurine is known to possess a trophic influence on the central nervous system. For instance, taurine promotes proliferation and differentiation of human fetal neuronal cells (Chen et al., 1998), also it is related to migration of cells (Maar et al., 1995) increases differentiation of retinal cells (Altshuler et al., 1993), and stimulates neuritic outgrowth from retinal explants and ganglion cells (Lima et al., 1989; Matus et al., 1997). However, at least concerning goldfish retinal explants, this effect needs the presence of FCS, and only a small stimulatory effect is observed in the absence of FCS in the medium, as previously shown (Lima et al., 1988). For this and all above exposed reasons, the definition of a free-FCS medium is crucial for studying specific elements or target-derived preparations, which will contribute to the understanding of the mechanisms of action of taurine as a trophic agent in the retina. It is relevant to quote the fact that HEPES, used in the cultures to stabilize pH, produces an inhibition of taurine uptake (Lleu and Rebel, 1990), by this way it could modify the effect of taurine in some manner, although taurine seems to exert the stimulation of elongation inside the cell (Lima et al., 1993). However, the present results show a very small increase on neuritic outgrowth by HEPES alone, and a decrease of elongation if FCS is present in the medium. Independent of the relation of HEPES with
taurine transport, the magnitude of the effects are not of relevance for described taurine effects. In our experience, HEPES could be avoided from the medium without an important modification of reported data on the trophic effect of taurine. According to the results of the present study, the serumfree medium for evaluating the effect of the tectum on retinal outgrowth of neurites would be composed by an optimal concentration of glucose added to the basal medium.
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Matus, P., Cubillos, S., Lima, L., 1997. Differential effect of taurine and serotonin on outgrowth from explants or isolated cells of the retina. Int. J. Devl. Neuroscience 15, 785–793. Michler, A., Tytko, H., Wolff, J.R., 1989. Interaction of soluble and substratum-bound factors influences neurite elongation. J. Hirnforsch 30, 197–202. Mizrachi, Y., Rubinsteinn, M., Kimhi, Y., Schwartz, M., 1986. A neurotrophic factor derived from goldfish brain: characterization and purification. J. Neurochem. 46, 1675-1682. Onoe, S., Niemeyer, G., 1992. Changing glucose concentration affects rod-mediated response in the perfused cat eye. Nippon Ganka Gakkai Zasshi 96, 634–640. Sholl-Franco, A., Araujo, E.G., 1997. Conditioned medium from activated spleen cells supports the survival of rat retinal cells in vitro. Braz. J. Med. Biol. Res. 30, 1299–1303. Sonigra, R.J., Brighton, P.C., Jacoby, J., Hall, S., Wigley, C.B., 1999. Adult rat olfactory nerve ensheathing cells are effective promoters of adult central nervous system neurite outgrowth in coculture. Glia 25, 256–269. Winkler, B.S., Arnold, M.J., Brassell, M.A., Puro, D.G., 2000. Energy metabolism in human retinal Müller cells. Invest. Ophthalmol. Vis. Sci. 41, 3183–3190.
Medium requirements for neuritic outgrowth from goldfish retinal explants and the trophic effect of taurine S. Cubillos, F. Fazzino, L. Lima∗ Laboratorio de Neuroqu´ımica, Centro de Biof´ısica y Bioqu´ımica, Instituto Venezolano de Investigaciones Cient´ıficas, Apdo. 21827, Caracas 1020-A, Venezuela Received 12 June 2002; received in revised form 7 October 2002; accepted 9 October 2002
Abstract The use of culture media of known composition are necessary for studying the role of trophic molecules. Since most of the in vitro research on regeneration of the optic nerve has been performed in the presence of fetal calf serum, the aim of this study was to obtain a medium in which the neuritic outgrowth from post-crush goldfish retinal explants could take place without adding fetal calf serum. After the lesion of the optic nerve (10 days), the retina of goldfish was dissected and explants were cultured for 5 and 10 days in the absence or in the presence of fetal calf serum, at which time the neuritic outgrowth was determined. Various concentrations and combinations of glucose, albumin, calcium, HEPES and taurine were used. The highest neuritic outgrowth was observed in the presence of fetal calf serum, in which condition the amino acid taurine increased length and density of neurites. Media supplemented with albumin, calcium or HEPES did not modify the outgrowth of neurites from the explants. However, glucose favored the neuritic outgrowth in a bell-shaped manner, although fibers were thinner than those observed in the presence of fetal calf serum. Taurine did not stimulate outgrowth of neurites from explants growing in a medium with optimal concentrations of glucose, indicating that elements of the fetal calf serum are determinant for the trophic effect of taurine. The present results contribute to further studies, such as those related to the effect of taurine and of trophic factors derived from the optic tectum, which would be performed in the presence of a medium free of fetal calf serum. © 2002 ISDN. Published by Elsevier Science Ltd. All rights reserved. Keywords: Culture medium; Optic nerve; Regeneration
1. Introduction Outgrowth of neurites from goldfish retinal explants occurs after lesion of the optic nerve (Lima et al., 1988). Conditions of the culture include the addition of fetal calf serum (Landreth and Agranoff, 1979), since in the absence of it neuritic outgrowth is dramatically restricted, even in the presence of trophic molecules, such as taurine (Lima et al., 1988). There have been several approaches to solve the important issue of studying the influence of stimulating agents per se without the concomitant effect of several substances present in the serum (Caffe et al., 2001; Espinosa de los Monteros et al., 1997; Sholl-Franco and Araujo, 1997; Winkler et al., 2000). A number of reports are related to the trophic effect of taurine, those touch aspects of concentration-dependency, time-related modifications, variations in the transport of ∗ Corresponding author. Tel.: +58-212-504-1213; fax: +58-212-504-1295. E-mail address: [email protected] (L. Lima).
taurine, among others (Lima, 1999). However, there is no consistent evaluation of the possible neuritic outgrowth obtained in the absence of fetal calf serum for studying the requirements of specific molecules during the regeneration in vitro. These facts are specifically important in our studies, because it was previously reported that the medium from optic tectum cultures and the co-culture of optic tectum and retina result in differential patterns of neuritic outgrowth (Cubillos et al., 2000), indicating the presence of determinant elements in those conditions, although previous studies were performed in the presence of fetal calf serum (Cubillos et al., 2000; Lima et al., 1988). The aims of the present report are to evaluate the neuritic outgrowth from goldfish retinal explants after the lesion of the optic nerve in the absence of fetal calf serum, and to study the possible effect of taurine by itself as a trophic agent in the retina. According to previous reports (Fischer, 1982; Ishida and Cheng, 1991; Lansel and Niemeyer, 1997; Lima et al., 1988; Lleu and Rebel, 1990; Matus et al., 1997; Winkler et al., 2000), we selected specific molecules to reach the objectives.
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2. Experimental procedures 2.1. Animals Goldfish (Carassius auratus), 5–6 cm in length were used after a lesion of the optic nerve. The lesion was performed by crushing the nerve around 1 mm behind the eye with fine forceps 10 days prior to dissection of the retina. After
this period of time they were adapted to dark for 30 min while anesthetized in 0.05% tricaine (Sigma) for obtaining the retina. 2.2. Retinal explants and evaluation of neuritic outgrowth Squares of 500 m (5–6 per retina) were prepared by sectioning the retina with a Mcllwain tissue chopper and
Fig. 1. Length of neurites from goldfish retinal explants at 5 or 10 days in culture. (A) In the absence or in the presence of seven increasing concentrations of albumin, 0, 0.01, 0.05, 0.1, 0.5, 1, 5 and 10%, 5 days F(7,388) = 3.98, P < 0.001; 10 days F(7,327) = 20.56, P < 0.001. (B) In the absence or in the presence of six concentrations of fetal calf serum without albumin, 0, 0.1, 0.5, 1, 2, 5 and 10%, 5 days F(6,339) = 126.53, P < 0.001; 10 days F(6,339) = 132.13, P < 0.001; with 0.1% albumin, 5 days F(6,298) = 115.97, P < 0.001; 10 days F(6,318) = 77.12, P < 0.001. (∗) P < 0.05 with respect to 5 days by Student’s t-test.
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placed 10–14 on poly-l-lysine (Sigma) pre-coated tissue flasks (25 mm2 ). Fetal calf serum (FCS) was used in some cultures in a concentration of 10%, and was omitted in those in which the following conditions were tested: calcium, glucose or albumin at different concentrations. Taurine, 4 mM (Lima et al., 1988), was added to some explants. The nutrient medium was Leibovitz, L-15, 3 ml per dish (Sigma) with 0.1 mg/ml of gentamicin and 20 mM or variable concentrations of (N-[2-hydroethyl]piperazine-N -[2-ethanesulfonic acid]) (HEPES). Five and ten days after plating, the density
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of neurites was evaluated using a predetermined scale (Landreth and Agranoff, 1979; Lima et al., 1988) and length (five neurites per explant) was determined by using the program SigmaScanPro (Jandel). 2.3. Statistical analysis Two ways analysis of variance was performed and pairs of samples, such as 5 versus 10 days in culture were compared by Student’s t-test. Significance corresponds to P < 0.05.
Fig. 2. Density of neurites from goldfish retinal explants at 5 and 10 days in culture, as described in Fig. 1. (A) 5 days F(7,72) = 1.11, P = 0.364; 10 days F(7,62) = 2.97, P < 0.01. (B) Without albumin, 5 days F(6,63) = 21.24, P < 0.001; 10 days F(6,63) = 10.75, P < 0.001. With albumin, 5 days F(6,56) = 14.00, P < 0.001; 10 days F(5,58) = 4.47, P < 0.001. (∗) P < 0.05 with respect to 5 days by Student’s t-test.
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Results are the mean ± standard error of the mean (S.E.M.), which is shown in the figures, except for length of neurites if it was too low (5–8% of the mean). 3. Results 3.1. Effect of fetal calf serum and albumin on neuritic outgrowth In the absence of serum, there was a small basal neuritic outgrowth that was not modified by the addition of albumin at 5 days in culture, except with 0.05 and 0.1% albumin, in which case there was a small decrease in length of neurites (Fig. 1A). A significant increase in the length of neurites after 10 days in culture was observed as compared with 5 days (Fig. 1A). Neurite length increased in the presence of FCS (0.1–10%) in a dose-dependent manner (Fig. 1B). The addition of albumin to retinal explants growing in the presence of variable concentrations of FCS did not modify the dose-dependency observed with FCS alone, but there was a decrease in the length of neurites as compared with the explants growing in the presence of FCS, but in the absence of albumin (Fig. 1B). Concerning the density of neurites (Fig. 2A and B), no significant changes were observed between 5 and 10 days in culture, and the density of fibers in the presence of FCS was not different from that with FCS and albumin. 3.2. Effect of fetal calf serum and glucose on neuritic outgrowth The length of neurites was significantly increased by the addition of glucose to the medium in the absence of FCS (Fig. 3A), this effect was evident at 0.5 mM glucose and was significantly smaller with 25 mM as compared to 0.5 mM, although it was still higher than control or lower concentrations of glucose. The presence of 10% FCS, and increasing concentrations of glucose in the medium of outgrowing explants did not further elevate the length of neurites already increased by FCS alone, as compared to those in the absence of FCS. Density of neurites was modified in a similar manner (Fig. 3B). The differences of magnitude in the effect obtained with glucose alone or with glucose and FCS was more evident at lower concentrations of glucose (Fig. 4). 3.3. Effect of calcium, albumin and glucose on neuritic outgrowth The addition of calcium at several concentrations did not increase the length of neurites from retinal explants, rather it reduced outgrowth of neurites at high concentrations (Fig. 5A). The combination of albumin and calcium produced similar results as calcium alone. In both cases, there was a significant increase in the length of neurites between 5 and 10 days in culture. Density of neurites was decreased
Fig. 3. Effect of glucose and fetal calf serum on neuritic outgrowth from goldfish retinal explants at 5 days in culture. (A) Length of neurites in the absence and in the presence of increasing concentrations of glucose 0, 0.01, 0.05, 0.5, 1, 2, 5, 25 mM without fetal calf serum F(7,224) = 17.50, P < 0.001; with 10% fetal calf serum F(7,549) = 5.68 P < 0.001. (B) Density of neurites, without fetal calf serum F(7,45) = 3.37, P < 0.01; with 10% fetal calf serum F(7,101) = 23.55, P < 0.001.
in the presence of high concentration of calcium, and no further modification was observed during the time in culture, except for a significant increase at 10 days as compared to 5 days in the presence of 12 mM calcium (Fig. 5B). The elevation of neurite length or density by 0.5 mM glucose was not altered by the simultaneous addition of increasing concentrations of calcium (Fig. 6A and B). The combination of albumin and glucose produced an increase in length and density of neurites as compared to albumin alone (Fig. 7A and B), but was not different from glucose alone.
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Fig. 4. Neuritic outgrowth from goldfish retinal explants at 5 days in culture. (A) In the presence of basic medium, absence of glucose or fetal calf serum. (B) In the presence of 0.5 mM glucose. (C) In the presence of 10% fetal calf serum. (D) In the presence of 0.5 mM glucose and 4 mM taurine. The line correspond to 500 m.
3.4. Effect of HEPES and fetal calf serum on neuritic outgrowth The outgrowth of neurites was significantly and slightly increased with augmenting concentrations of HEPES in the medium without FCS, but this effect was not observed in the presence of FCS, in which case there was a decrease in the length of neurites and no further elevation between 5 and 10 days in culture (Fig. 8A). Similar pattern of neuritic outgrowth was obtained for density of neurites in the presence of HEPES without FCS, but not with FCS (Fig. 8B). 3.5. Effect of taurine on neuritic outgrowth from explants in the presence of variable medium composition The presence of taurine in the medium without FCS produced a significant increase in the length of neurites in a concentration of 10 mM and in the density at various concentrations (Fig. 9A and B). In the presence of glucose there was a higher neuritic outgrowth (length and density), and the addition of taurine increased it according to the concentration added (Fig. 9A and B). In the presence of glucose and FCS there was an increase of length and density
of neurites produced by taurine, following a bell-shaped concentration-dependency. In addition, the presence of sucrose decreased the effect of 10 mM taurine, but did not have any effect by itself in concentrations up to 10 mM (data not shown). Taurine, 4 mM, did not modify outgrowth of neurites from explants growing in the presence of variable concentrations of calcium, neither the combination of FCS and calcium was different respecting FCS alone (Fig. 10A and B). The addition of taurine to explants cultured in the absence or in the presence of HEPES produced a significant increase with 20 mM HEPES and 4 mM taurine at 5 days in culture. Increased length occurred between 5 and 10 days in culture with taurine in the medium. HEPES alone reduced the length of neurites at 10 days in culture as compared to controls (Fig. 11A). Density of neurites was unaffected in the presence of HEPES or HEPES and taurine (Fig. 11B).
4. Discussion We previously demonstrated that the neuritic outgrowth from goldfish retinal explants was stimulated by the amino acid taurine (Lima et al., 1988), this effect was not observed
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Fig. 5. Effect of calcium and albumin on neuritic outgrowth from goldfish retinal explants at 5 and 10 days in culture. (A) Length of neurites in the absence and in the presence of 3, 6 and 12 mM calcium without albumin, 5 days F(3,161) = 10.39 P < 0.001; 10 days F(3,137) = 2.53, P = 0.059; with 0.1% albumin, 5 days F(3,161) = 8.12, P < 0.001; 10 days F(3,104) = 8.80, P < 0.001. (B) Density of neurites without albumin, 5 days F(3,31) = 4.72, P < 0.01; 10 days F(3,26) = 10.10, P < 0.001; with 0.1% albumin, 5 days F(3,30) = 3.27, P < 0.05; 10 days F(3,22) = 0.74, P = 0.538. (∗) P < 0.05 with respect to 5 days by Student’s t-test.
in isolated retinal ganglion cells in culture (Matus et al., 1997), since only the number of outgrowing cells was elevated by taurine, indicating that the integrity of the tissue was playing a determinant role on the effect of taurine as a trophic agent. This might be related to the interaction
Fig. 6. Effect of calcium and glucose on neuritic outgrowth from goldfish retinal explants at 5 and 10 days in culture. (A) Length of neurites in the absence and in the presence of 0, 3 and 6 mM calcium without glucose, 5 days F(2,129) = 0.58, P = 0.560; 10 days F(2,125) = 6.19, P < 0.01; with 0.5 mM glucose, 5 days F(2,147) = 1.26, P = 0.287, P < 0.001; 10 days F(2,147) = 7.92, P < 0.001. (B) Density of neurites without glucose, 5 days F(2,27) = 0.18, P = 0.838; 10 days F(2,27) = 4.14, P < 0.05; with 0.5 mM glucose, 5 days F(2,27) = 1.68, P = 0.205; 10 days F(2,27) = 0.74, P = 0.489.
between glial Müller cells, which in the presence of glucose have a low oxygen consumption (Winkler et al., 2000), and might spare oxygen for retinal neurons and then maintain the capability of extending neurites. Moreover, the present results support that glucose is sufficient to preserve the outgrowth of neurites from retinal explants, however, taurine
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Fig. 7. Effect of albumin and glucose on neuritic outgrowth from goldfish retinal explants at 5 and 10 days in culture. (A) Length of neurites in the absence and in the presence of 0, 0.1 and 1% albumin without glucose, 5 days F(2,112) = 3.27, P < 0.05; 10 days F(2,92) = 0.70, P = 0.05; with 0.5 mM glucose 5 days F(2,136) = 3.56, P < 0.05; 10 days F(2,147) = 1.29, P = 0.277. (B) Density of neurites without glucose, 5 days F(2,21) = 1.07, P = 0.360; 10 days F(2,17) = 1.88, P = 0.182; with 0.5 mM glucose 5 days F(2,25) = 0.56, P = 0.578; 10 days F(2,27) = 0.77, P = 0.473.
was not effective unless the elements of FCS were present. Differences in glucose metabolism between glia and neurons have been reported, which correspond to a physiological interaction between these cells (Lansel and Niemeyer, 1997). For instance, the electrical function of cat retina is known to be preserved by glucose in vitro (Onoe and Niemeyer,
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Fig. 8. Effect of HEPES and fetal calf serum on neuritic outgrowth from goldfish retinal explants at 5 and 10 days in culture. (A) Length of neurites in the absence and in the presence of 0, 10 and 20 mM without fetal calf serum 5 days F(2,145) = 9.26, P < 0.001; 10 days F(2,145) = 14.91, P < 0.001; with 10% fetal calf serum 5 days F(2,147) = 6.90, P < 0.01; 10 days F(2,147) = 37.30, P < 0.001. (B) Density of neurites without fetal calf serum 5 days F(2,27) = 0.12, P = 0.887; 10 days F(2,27) = 1.71, P = 0.200; with 10% fetal calf serum, 5 days F(2,27) = 3.23, P = 0.055; 10 days F(2,27) = 3.42, P < 0.05. (∗) P < 0.05 with respect to 5 days by Student’s t-test.
1992). It is interesting to show that concentrations of glucose lower than those considered as normal, around 5 mM (Lansel and Niemeyer, 1997), were sufficient to elevate the neuritic outgrowth, although the stimulatory effect was lower with 25 mM. This observation might be related to the regulation of the osmolarity, however, sucrose up to 10 mM did not have any effect by itself, but when added at lower
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Fig. 9. Effect of taurine, glucose, fetal calf serum and sucrose on neuritic outgrowth from goldfish retinal explants at 5 and 10 days in culture. (A) Length of neurites in the absence and in the presence of taurine 0, 0.2, 2, 4 and 10 mM, 5 days F(4,143) = 4.37, P < 0.01; with 0.5 mM glucose, 5 days F(2,414) = 3.10, P < 0.05; with 10% fetal calf serum F(4,298) = 13.11, P < 0.001; with 0.5 mM sucrose F(4,172) = 3.79, P < 0.01. (B) Density of neurites in the absence and in the presence of taurine 0, 0.2, 2, 4 and 10 mM, 5 days F(4,28) = 1.17, P = 0.346; with 0.5 mM glucose F(4,79) = 1.38, P = 0.249; with 10% fetal calf serum F(4,58) = 0.64, P = 0.638; with 0.5 mM sucrose F(4,33) = 3.74, P < 0.05.
concentrations with taurine, a small increase in neuritic outgrowth was observed, except with 10 mM taurine (Fig. 8). The components of a reported chemically defined medium when added in different combinations to oligodendrocytes in culture result in variable degrees of maturation (Espinosa de los Monteros et al., 1997), contributing to the understanding of the requirements for myelination potential of central nervous system. Although the evidence is in favor of the
importance of FCS for outgrowth neuritic an elevated rate and magnitude, goldfish retinal explants outgrow in the absence of FCS in a modest manner. It has been recently demonstrated that neonatal mouse retinal explants can be cultured and remain viable for more than 4 weeks in a serum-free medium (Caffe et al., 2001), in addition, it was previously shown that cerebellar cells of the mouse survive in hormonally defined medium, which includes
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Fig. 10. Effect of calcium, taurine and fetal calf serum on neuritic outgrowth from goldfish retinal explants at 5 and 10 days in culture. (A) Length of neurites in the absence and in the presence of 0, 3 and 6 mM calcium without taurine or fetal calf serum, 5 days F(2,93) = 0.68, P = 0.512; 10 days F(2,83) = 4.02, P < 0.05; with 4 mM taurine, 5 days F(2,112) = 1.59, P = 0.213; 10 days F(2,70) = 6.09, P < 0.01, with 10% fetal calf serum, 5 days F(1,153) = 1.07, P = 0.302; 10 days F(1,103) = 13.43, P < 0.001. (B) Density of neurites without taurine or fetal calf serum, 5 days F(2,20) = 0.97, P = 0.396; 10 days, F(2,16) = 0.94, P = 0.413; with 4 mM taurine, 5 days F(2,26) = 0.56, P = 0.575; 10 days F(2,14) = 0.01, P = 0.991, with 10% fetal calf serum, 5 days F(1,29) = 0.02, P = 0.876; 10 days F(1,19) = 0.25, P = 0.620. (∗) P < 0.05,with respect to 5 days by Student’s t-test.
albumin, insulin, transferrin, selenium, thyroxine and a protease inhibitor (Fischer, 1982). Moreover, one of a group of molecules associated with survival of cells are cytokines from activated lymphocytes (Sholl-Franco and Araujo, 1997). The characterization of minimal conditions
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Fig. 11. Effect of HEPES and taurine on neuritic outgrowth from goldfish retinal explants at 5 and 10 days in culture. (A) Length of neurites in the absence and in the presence of 0, 10 and 20 mM without taurine, 5 days F(2,147) = 1.26, P = 0.286; 10 days F(2,147) = 6.14, P < 0.01; with 4 mM taurine, 5 days F(2,147) = 9.47, P < 0.001; 10 days F(2,147) = 12.40, P < 0.001. (B) Density of neurites without taurine, 5 days F(2,27) = 0.22, P = 0.805; 10 days F(2,27) = 2.32, P = 0.117; with 4 mM taurine, 5 days F(2,27) = 4.57, P < 0.05; 10 days F(2,27) = 0.94, P = 0.403. (∗) P < 0.05 with respect to 5 days by Student’s t-test.
for preserving the capacity of culturing nervous tissue is essential in the study of individual components or unidentified factors of different sources, such as constituents of the optic tectum, as has been our experience in the goldfish retinal model (Cubillos et al., 2000).
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There are a number of reports showing the successful results concerning neuritic outgrowth in the presence of calcium (Ishida and Cheng, 1991). Chelating of extracellular or intracellular calcium produces a decrease of outgrowth of neurites from goldfish retinal explants (Lima et al., 1993), also calcium channel antagonists partially inhibit neurite outgrowth from adult rat olfactory cells (Sonigra et al., 1999). Despite these facts, the supplementation with calcium only slightly increased neuritic outgrowth up to 3 mM, and produced inhibition at 6 and 12 mM. The addition of albumin to the medium did not produce any effect on outgrowth of neurites, although it has been reported that transferrin and albumin improve differentiation of neural tissue of the rat used for in vivo transplantation (Belovari et al., 2001), also albumin seems to participate in visual-cycle transport (Adler and Edwards, 2000). It is also known that the interaction of soluble and attached factors influences neurite outgrowth (Michler et al., 1989). For instance, goldfish retinal explants cultured on a number of substrates present a differential elongation of fibers and variable response to taurine (Lima et al., 1989). As it is well supported, the optic tectum (Mizrachi et al., 1986) and the optic nerve (Caday et al., 1989) produce trophic substances which have positive influence on retinal regeneration. We have shown that the optic tectum is not only a growth-stimulatory factor producer, but it is also involved in the secretion of inhibitory molecules (Cubillos et al., 2000). In addition, FCS-mediated inhibition of neurite elongation has been noticed in ciliary ganglion neurons in culture (Davis et al., 1984). For studying factors derived from the optic tectum, a serum-free medium is required. Taurine is known to possess a trophic influence on the central nervous system. For instance, taurine promotes proliferation and differentiation of human fetal neuronal cells (Chen et al., 1998), also it is related to migration of cells (Maar et al., 1995) increases differentiation of retinal cells (Altshuler et al., 1993), and stimulates neuritic outgrowth from retinal explants and ganglion cells (Lima et al., 1989; Matus et al., 1997). However, at least concerning goldfish retinal explants, this effect needs the presence of FCS, and only a small stimulatory effect is observed in the absence of FCS in the medium, as previously shown (Lima et al., 1988). For this and all above exposed reasons, the definition of a free-FCS medium is crucial for studying specific elements or target-derived preparations, which will contribute to the understanding of the mechanisms of action of taurine as a trophic agent in the retina. It is relevant to quote the fact that HEPES, used in the cultures to stabilize pH, produces an inhibition of taurine uptake (Lleu and Rebel, 1990), by this way it could modify the effect of taurine in some manner, although taurine seems to exert the stimulation of elongation inside the cell (Lima et al., 1993). However, the present results show a very small increase on neuritic outgrowth by HEPES alone, and a decrease of elongation if FCS is present in the medium. Independent of the relation of HEPES with
taurine transport, the magnitude of the effects are not of relevance for described taurine effects. In our experience, HEPES could be avoided from the medium without an important modification of reported data on the trophic effect of taurine. According to the results of the present study, the serumfree medium for evaluating the effect of the tectum on retinal outgrowth of neurites would be composed by an optimal concentration of glucose added to the basal medium.
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