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Promotion of neurite outgrowth and cell survival in dissociated fetal rat retinal cultures by a fraction derived from a brain extract
DevelopmentalBrain Research, 18 (1985) 265-274
265
Elsevier BRD 50167
Promotion of Neurite Outgrowth and Cell Survival in Dissociated Fetal Rat Retinal Cultures by a Fraction Derived From a Brain Extract JAMES E. TURNER
Departmentof Anatomy, Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, NC27103 (U.S.A.) (Accepted September llth, 1984)
Key words: CNS neurotrophic factor - - dissociated retinal cells - - neurite outgrowth - - cell survival
We have reported previously that a fraction (BE) derived from a pig brain extract stimulated neurite outgrowth and cell survival from fetal rat retinal explants. The BE effects were dose dependent and could not be altered by NGF or its antiserum. In the present study we have observed that under similar culture conditions BE was also capable of stimulating neurite outgrowth and cell survival from fetal rat dissociated retinal cells. More specifically, the neurite-promoting activity of BE was found to be dose dependent over a concentration range of 0-50 pg/ml with a half-maximal response between 5 and 10 mg/ml. The ability of BE to stimulate neurite outgrowth was also age-related. There was a progressive decrease in the BE-mediated response between fetal day 17 and the second neonatal day. Viable process bearing cells could also be maintained in culture for at least two weeks in the presence of BE (25 pg/ml). In contrast, after 1 day in culture control cells began to rapidly degenerate and by days 3-5 no process-bearing cells were observed. The BE was found to exert its action primarily through a soluble factor(s) in the culture medium. However, we also report evidence for a substrate bound component of the BE which may aid in the attachment and/or neurite outgrowth phenomena.
INTRODUCTION It is becoming increasingly clear that the growth of neuronal processes in the peripheral nervous system (PNS) is controlled by a n u m b e r of extrinsic factors. Neurotrophic factors are believed to play a very important role in the development, m a t u r a t i o n and/or maintenance of a n u m b e r of sympathetic 14, sensory 6 and parasympathetic 2s neurons. In addition, in vitro studies have recently pointed out the importance of substrate binding neurite-promoting factors (NPFs), which must be present u n d e r certain culture conditions in order for neurotrophic factors to exert their effects on peripheral neuronsl-3,12,25. On the other hand, very little is k n o w n about the nature of these p h e n o m e n a in the central nervous system (CNS). However, a recent report has described the purification to homogeneity of a new neurotrophic factor (PF) from the m a m m a l i a n brain. The purification of this factor was stimulated by previous results which had indicated that glioma-condi-
tioned m e d i u m 8 and a rat brain extract 6 Could support the survival of embryonic sensory n e u r o n s cultured in the absence of NGF. The biological activity of the newly isolated protein is similar to that previously reported for both glioma-conditioned medium and the brain extract. The new growth factor is functionally and immunologically distinct from N G F , and it is a basic protein with a molecular weight of 12,300 and a PI i> 10.17. Results also suggest that this protein from the m a m m a l i a n brain may be produced by glial cells6, 8. The outcome of these observations has been the discovery that a CM-cellulose column fraction (BE), which is derived from an intermediate step in the purification of PF from a pig brain extract, stimulated neurite outgrowth from fetal rat retinal explants23. More specifically, we found that BE stimulation was specific, dose d e p e n d e n t and age-related23,z4. In addition, B E was found to increase the maturation and cell survival of n e u r o n s in the fetal retinal explants 24. We have formed an hypothesis from these obser-
Correspondence: J. E. Turner, Department of Anatomy, Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, NC 27103, U.S.A. 0165-3806/85/$03.30 (~) 1985 Elsevier Science Publishers B.V. (Biomedical Division)
266 vations that there are neurotrophic factors in the mammalian CNS which are capable of supporting and/ or stimulating neurite outgrowth in a manner similar to that found in the PNS. The present study further confirms our hypothesis by demonstrating similar BE-mediated phenomena in a newly established dissociated fetal rat retinal culture system.
glucose and 2.0 mM L-glutamine; N I supplements ~ (Sigma): insulin (5ktg/ml), transferrin (5ktg/ml), progesterone (2 x 10-8 M), selenium (3 x 10-8 M) and putrescine (1 x 10-3 M); and (3) antibiotics; penicillin (10,000 ktg/ml) and streptomycin (10 mg/ml).
Preparation of the culture substrate MATERIALS AND METHODS
Culture of dissociated cells Fetuses from 17 to 21 day pregnant anesthesized albino rats were surgically removed and placed in sterile phosphate-buffered saline (PBS). Eyes from the fetuses and 2-day neonates were dissected, placed in PBS and retinas removed by watchmakers forceps (no. 4), using a surgical microscope with a cool fiber optic system. At this stage of development, retinas can be stripped cleanly away from surrounding tissue and the developing lens. It is also possible to remove the vascular net which begins to form on the vitreal surface of the retina and around the lens. Dissected retinas are collected in 5 ml of Ca 2÷,Mg 2÷free PBS (CMF) to which was added 60HI of 1% trypsin (Worthington) and 60 #1 of 0.1% DNase I (Sigma). After incubation at 37 °C for 15 min, digestion was stopped by the addition of 200 H1 of soybean trypsin inhibitor (Gibco, 2 mg/ml). The retinas were centrifuged at 1000 g for 5 min, resuspended in CMF and dissociated by 25-30 strokes through a siliconcoated Pasteur pipette. After hemocytometer counts, cells were diluted with CMF to a density of 2 × 106 cells/ml, aliquoted to various tubes, centrifuged at 100 g for 5 min, resuspended in control or BE-containing medium and except for one experiment seeded at 1 ml onto fibronectin- (50 Hg/ml in PBS, pH 6.5, Biomedical Technologies) coated dishes (35 mm, 6-well, Costar) which had been previously equilibrated with 1 ml of the appropriate medium (i.e. total volume equals 2 ml). The Trypan blue dye exclusion test indicated that 90% of the cells plated survived and attached after 2 h in culture. Part of the medium (0.5-0.8 ml) was exchanged every 3 - 4 days. The culture medium was the same as that described in a previous report 24. More specifically, the media consisted of: (1) Eagle's basal medium (Gibco) supplemented with 26.4 mM NaHCO3, 33.3 mM
In most experiments the dishes were first coated with 1 ml of fibronectin 4 (50 ktg/ml) for 1 h at room temperature washed once with 1 ml of sterile distilled water and equilibrated in the tissue culture incubator with 1 ml of control or BE-containing medium at 37 °C with 5% CO 2 for 1-2 h. Control dishes were incubated in medium with no BE present. In one set of experiments dealing with substrate bound phenomena dishes were prepared as previously described except fibronectin was omitted in one group leaving the plastic substrate to be conditioned by BE. Control cells were cultured on the plastic substrate in the absence of BE. In another set of experiments dealing with substrate-bound phenomena dish surfaces were first equilibrated with fibronectin followed by BE- (25 ~g/ml) containing medium as previously described. However, equilibration was allowed to continue overnight for 12 h in BE-containing medium at which time: (1) 2 x 106 cells ml were seeded in 1 ml of BE- (25 Hg/ml) containing medium as described previously; and (2) another set of dishes was first washed 3 times with control medium to remove the unbound BE before cells (2 x 106 ml) were seeded in control medium onto the BE-modified substrate. Control cells were cultured on fibronectin in the absence of BE.
Scanning electron microscopy Cultures were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer for 1 h. After 3 changes in buffer, cultures were dehydrated in an alcohol series and dried by the critical point method. Small pieces of the dish surface were mounted on aluminum studs, coated with gold-palladium (60:40) and photographed with a 501 Phillips SEM.
Tetanus toxin binding studies The staining of BE (25 Hg/ml)-exposed, 17-day fetal rat dissociated retinal cells (2 x 106 cells/dish), treated after 5 days in culture with tetanus toxin was
267
Fig. 1. Phase-contrast photomicrographs demonstrating BE (25 #g/ml)-mediated stimulation of fiber outgrowth and cell survival from 17-day fetal rat dissociated retinal cell cultures. A: appearance of fiber outgrowth from BE-treated cells after 4 days in culture. Note the numerous contacts made between cells. Also note non-responding necrotic cells among responding neurons (arrows). B: control cells after 4 days in culture demonstrating extensive cell necrosis (dark cells) and no fiber outgrowth. C: cell aggregation and process formation in BE-treated group after 7 days in culture. D: control cells after 7 days in culture demonstrating no aggregation and no process formation. Note phase-bright areas are extruded nuclei and not intact cells. E: higher magnification of a retinal neuron-like cell body (arrowhead) with an extensive process resulting after 7 days of BE treatment. F: between 7 and 14 days in culture a few aggregating cells with long processes appear attached to a flattened non-neuronal cell. Mag.: A-B and E-F, 300× ; C-D 150×.
268 performed using an indirect immunofluorescence method19. Four-well 35 mm culture dishes (Greiner and Sohne) with an optional volume of 80 ~1 were used for these studies. Culture dishes were washed twice with phosphate-buffered saline containing 0.1% BSA (PBS/A), incubated for 20 min with tetanus toxin (25 #g/ml) in PBS/A and washed 3 times in PBS/A. Cultures were next fixed for 20 min in 6% formaldehyde in Ca2+,Mg2÷-free phosphate-buffered saline (CMF), washed 3 times in CMF/A and incubated 20 min with human anti-tetanus toxin (1:50 dilution), washed 3 times with CMF/A, incubated for 20 min with FITC-conjugated goat anti-human-aglobulin (1:100 dilution), washed 3 times in CMF/A and mounted in CMF/glycerin (1:1). All procedures were carried out at room temperature. The tetanus toxin was a gift from Dr. B. Bizzin, Institute Pasteur, Paris; human anti-tetanus toxin was a gift from Dr. Johannsen, Behring-Werke, Marbury; and FITCconjugated goat anti-human-a-globulin was purchased from Behring-Werke; Marburg.
Preparation of brain extract (BE) The pig brain extract fraction (BE) was a gift from Dr. Yves Barde and was prepared according to the procedure reported by Barde et al.7. In comparison to the starting homogenate, the BE had a hundred times higher specific activity when tested for survival activity on 20-day-old chick embryo sensory neurons, as described previously 7. The BE preparation is equivalent to step III of the purification procedure for PF 7.
Evaluation of neurite outgrowth Cultures were analyzed by phase-contrast microscopy and quantitations performed by determining the number of process bearing cells/dish. Processbearing cells were only scored if their length was at least twice the cell body diameter. The number of cells/dish was estimated by a strip counting method 26. The data were analyzed for significance of difference by the Student's t-test. All observations were made from duplicate or triplicate sister cultures. RESULTS The majority of fetal rat dissociated retinal cells will not extend neurites in a defined medium with fi-
bronectin as a substrate. Those few retinal cells that show initial signs of neurite outgrowth under control conditions are withdrawn by 48 h, and by 4 days most cells have become necrotic and begin to float off the substrate (Figs. 1B, D and 2B). However, the presence of BE (25/~g/ml) in the medium causes a dramatic enhancement of cell attachment and neurite outgrowth, which reaches a peak by culture day 4 (Fig. 1A). The normally rounded cells become more phase-dark and extend 2 - 4 branching processes which interdigitate to form fascicles (Figs. 1A and 2A). After 1 week in culture many of the BE-treated retinal cells have a tendency to form small processbearing aggregates (Fig. 1C). However, many individual cells with long processes can still be observed after one week in culture (Fig. 1E). The presence of flattened, dividing non-neuronal cells are seldom observed in these cultures; however, when they are seen they usually serve as a substrate on which several process bearing neurons are aggregated (Fig. 1E). Tetanus toxin binding revealed that greater than 95% of the viable BE-treated process-bearing cells are positively labeled (Fig. 3). Although BE treatment illicits a dramatic response with respect to retinal cell survival and neurite outgrowth, the total number of responding cells is equivalent to only 20-40% of the total cells plated. More specifically, of the 2 x 106 cells plated per dish only 4-8 x 105 cells respond to BE treatment depending on the embryonic age. Therefore, even in BE treated dishes there is normally a great amount of cell necrosis (Fig. 1A and E). The BE-mediated response drops off dramatically with decreased plating density to where there are virtually no surviving cells left to respond after several days at either 1 x 106 or 5 x 105 cells/ml, respectively. The BE-mediated neurite outgrowth response is a dose-dependent phenomenon over a concentration range of 0-50/xg/ml, with a half-maximal response at approximately 5/xg/ml (Fig. 4). Viable process-bearing cells can be maintained in culture in the presence of BE (25/xg/ml) for at least 2 weeks (Fig. 5). The BE-mediated response becomes maximal between days 2 and 4 at which time control cells have withdrawn their processes, become necrotic and have begun to lift up off the substrate (Fig. 1B and D). Retinal cells responding to BE do so in an age-dependent fashion. More specifically, there are dramatic step-
269
Fig. 2. Scanning electron micrographs of 17-day fetal rat dissociated retinal cells after 6 days in culture. A: cultures treated wi*h BE (25/~g/ml) demonstrating the profuse mat of interdigitated and fasciculated processes from the rounded retinal neuronal cell bodies. B: control cultures showing the dramatic loss of viable cells and the complete absence of processes. Mag. 2000x.
270
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Fig. 3. Phase-contrast and companion fluorescent photomicrographs of control and tetanus toxin-treated 17-day fetal rat dissociated retinal cells exposed to BE (25/~g/ml) for 6 days in culture. A: phase-contrast photomicrograph of tetanus toxin-treated cells. B: fluorescent photomicrograph of A demonstrating that all viable, process-bearing cells are positively labeled. C: phase contrast photomicrograph of control cells without tetanus toxin treatment. D: fluorescent photomicrograph of C showing lack of specific labeling in control dishes. Mag. 222 x.
wise decreases in the number of BE-responding cells between 17 fetal day, 21 fetal day and 2 neonatal day retinas, respectively (Fig. 6). Between 17 and 21 fetal days there was a 2-fold reduction in numbers of process-bearing cells, compared to a 4-fold reduction between 21 fetal and 2 neonatal days in response to BE treatment. If fibronectin-coated culture dishes were treated with BE (25/~g/ml)-containing medium for 12 h, washed free of the soluble BE and cells plated in control medium onto the modified substrate to which B E was bound, the number of process-bearing cells was significantly reduced by 5-fold from 8 x 105 to 1.5 × 105 responding neurons (Fig. 7). However, the reduced number of responding cells was still significantly higher than cells plated under the absolute control conditions of no substrate or medium expo-
sure to BE (Fig. 7). In turn, experiments conducted in the presence or absence of fibronectin as an initial substrate revealed that in those dishes incubated only with BE (25/.tg/ml) for 3 h and washed free of BE approximately 1.5 x 106 cells responded compared to 6 × 106 process-bearing cells cultured under normal experimental conditions (Fig. 8). Similarly, the number of reduced process-bearing cells was significantly higher than control cells plated on only a plastic substrate in the absence of B E (Fig. 8). In both of these experiments (Figs. 7 and 8) BE-containing medium was equilibrated on fibronectin-coated dishes for 3-12 h prior to cell plating which was not the case during the earlier experiments (Figs. 4-6). When these figures are compared, it is quite evident that increased prior plating time with BE significantly enhances the number of process-bearing cells.
271
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I 5 10 25 50 I 0 0 BE CONCENTRATION (,ug/ml) Fig. 4. A dose-response curve demonstrating the number of 17-day fetal rat dissociated retinal process-beating cells induced by BE treatment°after 4 days in culture. Vertical lines represent S.E.M. All points are significantly different from control values (P < 0.001) and 1, 2.5, 5 and 10/~g/ml concentration points are significantly different (P < 0.001). However, the 3 highest concentration points are not significantly different from each other (P > 0.05). Concentration points represent an average measurement from two sister cultures.
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Fig. 6. Graph depicting the age-dependent BE (25/~g/ml)-mediated dissociated retinal cell response in 17 fetal day (17F), 21 fetal day (21F) and 2 day neonatal (2DN) cultures. All group values are significantly different from one another (P < 0.001). Vertical bars represent the S.E.M. Each group value represents an average measurement from two sister cultures.
DISCUSSION In the present study we have reported that u n d e r defined tissue culture conditions an extract prepared
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BE Treatment (25~9/ml) o--.o Untreated Controls
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Fig. 5. Graph demonstrating the stimulation and long-term survival of 17-day fetal dissociated retinal process-bearing cells in the presence of BE treatment. Note that after 3 days in culture
there are no surviving process-bearing cells in control dishes. All group values from the same time points are significantlydifferent from one another (P < 0.001). Vertical bars represent the S.E.M. Each group value represents an average measurement from two sister cultures.
from pig brain (BE) was capable of stimulating neurite outgrowth and cell survival from fetal rat dissociated retinal cells. This neurotrophic response was quite dramatic since after 1 day in culture control cells began to rapidly degenerate and by 3 - 5 days no process-bearing cells were observed. Subsequently, by 4 days most control cells had begun to float off the substrate. This observation is in agreement with our previous reports in which BE was found to stimulate neurite outgrowth and cell survival in fetal rat retinal explants23,24. More specifically, we found that long radial fiber outgrowth could be stimulated from explants in excess of 1 m m where controls exhibited no growth. In addition, B E appeared to accelerate retinal ganglion-like cell m a t u r a t i o n and to significantly reduce cell death in explants. It is also important to note that avian brain extracts have also been shown to stimulate neurite outgrowth from 6-day chick embryonic retina u. After 1 week in culture many of the BE-treated retinal cells displayed a tendency to form small process-bearing aggregates. The reason for this p h e n o m -
272 6O F
enon cannot be explained readily, except to say that BE may ensure a greater n u m b e r of healthy and/or surviving cells, as well as affecting the cell adhesive or binding (recognition) qualities through a memb r a n e - b o u n d molecule like C A M 21. Further evidence
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for this possibility was reported earlier. Specifically, the BE-mediated retinal response dropped off pre-
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cipitously with decreasing plating density. Virtually no surviving cells were left to respond after several days at 1 x 106 cells/ml, a density which would greatly diminish the chances for cell-to-cell contacts. The BE-mediated dissociated cell neurite outgrowth response was found to be a dose-dependent p h e n o m e n o n over a concentration range of 0-50/~g/ml with a half-maximal response at approximately 5/~g/ml. These results are complementary to our earlier explant work in which we reported a dose-
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Fig. 8. Graph demonstrating that to a limited extent a component of BE can bind to the plastic dish surface in the absence of a fibronectin substrate (BE-F) to exert a stimulatory action on dissociated 17-day fetal rat retinal cells. In the BE-F experiment, BE (25 ktg/ml) was equilibrated onto the plastic dish surface for 3 h, washed 3 times and cells plated in control medium without BE. Controls consisted of BE (25 Izg/ml) substrate equilibration with cells plated in BE (25 ~g/ml) containing medium on fibronectin substrate (BE+F) and conditions using no fibronectin or BE in the medium (C-F). All group values are significantly different from one another (P < 0.001). Vertical bars represent the S.E.M. Each group value represents an average measurement from two sister cultures taken after 4 days in culture.
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Fig. 7. Graph demonstrating stimulatory actions of BE (25 Izg/ml) present as either a soluble molecule in the medium (BE-S), bound only to the substrate and not present in the medium (BE-B) or no BE present at all (C). BE was equilibrated onto the fibronectin (50/~g/ml) substrate for 12 h before exchange with either fresh BE-containing medium (BE-S) or 3 washes with regular medium (BE-B). Note that BE can still exert some stimulatory activity while bound (BE-B) to the substrate. All group values are significantly different from one another (P < 0.001). Vertical bars represent the S.E.M. Each group value represents an average measurement from two sister cultures taken after 4 days in culture. Dissociated retinal cells are from 17-day fetuses.
dependent p h e n o m e n o n over a very similar concentration range23,24. In a similar fashion, we have observed in this and a previous study 24 that retinal cells respond to BE in an age-dependent fashion. More specifically, there were dramatic stepwise decreases in the n u m b e r of BE-responding cells between 17 fetal day, 21 fetal day and 2 day neonatal retinas, respectively. Similar age-related trends have been reported for the response of chick sensory ganglia to both N G F , glioma-conditioned m e d i u m and rat brain extracts 6. In addition, T h o m p s o n and Rapoport reported a significant age-related decrease in the percentage and average length of neurites formed in 24 h cultures of dissociated chick retina between 6 and 16 days of embryonic age. W h e t h e r the age-dependent decrease in B E responsiveness is due to diminished cell viability and/or receptor site modifica-
273 tion has not been determined. However, it has been demonstrated that there is a direct correlation between reduced responsiveness to NGF and loss of receptor activity in dorsal root neurons 20. The retinal cell type(s) responding to BE in both the dissociated and explant systems are not known at the present time. However, at this stage of development (i.e. 17-21-day fetuses) the rat retina is relatively undifferentiated since all ten layers are not distinguishable until postnatal day 215,27. At birth the rat retina contains two distinguishable layers: (1) the inner cell layer consisting of a row of well differentiated ganglion cells; and (2) an outer layer formed by an abundance of undifferentiated neuroepithelial cells 27. Even as early as embryonic day 15, differentiating ganglion cells were observed to possess axons which extended into the optic stalkS. Therefore, it seems plausible from this evidence that the retinal ganglion cells are the most likely neurons to extend their axons onto the culture substrate and be affected in this manner by BE in our system. Our explant studies would tend to strengthen further this argument since BE treatment was shown to stimulate maturation and survival of ganglion-like cells 24. However, this does not rule out the possibility that over a 1-2-week culture period other retinal cell types could differentiate in vitro 16 and be stimulated by BE treatment. This may be especially evident since 20-40% of the plated cells survive in the presence of BE which most likely exceeds the number of cultured ganglion cells. Alternatively, BE may shift neuroepithelial differentiation towards ganglion cells. We also have no direct evidence as to whether BE acts on neurons, the substrate or indirectly through glial cell elements, since at least, Mfiller cells have begun to differentiate at this time5,Z7. However, from our present tetanus toxin binding results, we concluded that the retinal culture consisted primarily of a highly enriched (t>95%) neuronal population. This was not surprising since our defined media conditions have been reported not to support non-neuronal cell survival 10. In further support of this conclusion, tetanus toxin binding has been shown to be specific for cultured rat retinal neurons with no non-specific binding to the non-neuronal (i.e. glial and connective tissue) components of the culture 9. Therefore, we would speculate that a direct action of BE on retinal neurons is most likely, since in a similar situation BE
and the purified factor were required in the medium for the survival and neurite outgrowth of embryonic chick sensory neurons, when seeded at low densities in the absence of glial elements6. The BE was found to exert its action primarily through a soluble factor(s). We know from previous studies that BE is different from N G F and is not involved in this phenomenon23. However, we have also reported in the present study evidence for a substrate-bound component of the BE which may aid in the attachment and/or neurite outgrowth phenomena. More specifically, a component of the BE could not be washed entirely from a fibronectin or plastic substrate and thus allowed some cell survival and neurite outgrowth to occur, although at a significantly reduced amount compared to conditions where BE was present in the medium. The necessity for multiple components to ensure cultured neuronal cell survival and/or neurite outgrowth (i.e. soluble and substrate-bound factors) is further substantiated by studies in which concurrent addition of ciliary neurotrophic factor (CNF) to the medium of 8-day-old dissociated chick embryo ciliary ganglionic neurons was shown to enhance neurite outgrowth only in the presence of PNPF 1-3,12,13,25. In a number of cases PNPFs do not, by themselves, support the survival or neurite outgrowth but require the presence of neurotrophic factors1,17,TM. However, there is some evidence that for PC 12 cells substrate factors can support neurite outgrowth to some extent in the absence of NGF 15. Further studies need to be conducted to conclusively demonstrate that different components within BE are synergistic and that substrate-bound BE is similar to PNPFs. In conclusion, we have reported for the first time that a dissociated cell population from the mammalian CNS responds in a typical neurotrophic manner to an extract prepared from pig brain. This evidence speaks very strongly for our hypothesis that there are trophic factors in the mammalian CNS capable of maintaining neurons and stimulating neurite outgrowth during development. In addition, we conclude that the BE offers a rich source of molecules which may be capable of exerting neurotrophic actions on certain developing and regenerating CNS neurons.
274 ACKNOWLEDGEMENTS
to thank Drs. Y v e s B a r d e and H a n s T h o e n e n for their g e n e r o u s supply of brain extract.
This w o r k was s u p p o r t e d by a grant f r o m the National E y e Institute ( E Y 04377). T h e a u t h o r wishes
REFERENCES 1 Adler, R. and Varon, S., Cholinergic neurotrophic factors: V. Segregation of survival and neurite-promoting activities in heart conditioned medium, Brain Res., 188 (1980) 437-448. 2 Adler, R. and Varon, S., Neurotrophic guidance by polyornithine-attached materials of ganglionic orgin, Develop. Biol., 81 (1981) 1-11. 3 Adler, R. and Varon, S., Neuritic guidance by noneuronal cells of ganglionic origin, Develop. Biol., 86 (1981) 69-81. 4 Akers, R. M., Mosher, D. F. and Lilien, J. E., Promotion of retinal neurite outgrowth by substratum-bound fibronectin, Develop. Biol., 86 (1981) 179-188. 5 Armstrong, C. and Monie, I. W., Congenital eye defects in rats following maternal folic acid deficiency during pregnancy, J. Embryol. exp. Morph., 16 (1966)531-543. 6 Barde, Y. A., Edgar, D. and Thoenen, H., Sensory neurons in culture: changing requirements for survival factors during embryonic development, Proc. nat. Acad. Sci. U.S.A., 77 (1980) 1199-1203. 7 Barde, Y. A., Edgar, D. and Thoenen, H., Purification of a new neurotrophic factor from mammalian brain, EMBO J., 1 (1982) 549-553. 8 Barde, Y. A., Lindsay, R. M., Monard, D. and Thoenen, H., New factor released by cultured glioma cells supporting survival and growth of sensory neurons, Nature (Lond.), 274 (1978) 818. 9 Beale, R., Nicholas, D., Neuhoff, V. and Osborne, N. N., The binding of tetanus toxin to retinal cells, Brain Res., 248 (1982) 141-149. 10 Bottenstein, J. E., Skaper, S. K., Varon, S. S. and Sato, G. H., Selective survival of neurons from chick embryo sensory ganglionic dissociates serum-free supplemented medium, Exp. Cell Res., 125 (1980) 183-190. 11 Carri, N. G. and Ebendal, T., Organotypic cultures of neural retina: neurite outgrowth stimulated by brain extracts, Develop. Brain Res., 6 (1983) 219-229. 12 Collins, F., Axon initiation by ciliary neurons in culture, Develop. Biol., 65 (1978) 50-57. 13 Collins, F., Induction of neurite outgrowth by a conditioned medium factor bound to culture substratum, Proc. nat. Acad. Sci. U.S.A., 75 (1978) 5210-5213. 14 Edgar, D., Barde, Y. A. and Thoenen, H., Subpopulations of cultured chick sympathetic neurons differ in their requirements for survival factors, Nature (Lond.), 289 (1981) 294-295.
15 Fujii, D. R., Massoglia, S. L., Savion, N. and Gospodarowicz, D., Neurite outgrowth and protein synthesis by PC 12 cells as a function of substratum and nerve growth factor, J. Neurosci., 2 (1982) 1157-1194. 16 LaVail, M. and Hild, W., Histotypic organization of the rat retina in vitro, Z, Zellforsch., 114 (1971) 557-579. 17 Manthorpe, M. S., Skaper, S., Adler, R., Landa, K. and Varon, S., Cholinergic neurotrophic factors: IV. Fractionation properties of an extract from selected chick embryonic eye tissues, J. Neurochem., 34 (1980) 69-75. 18 Manthorpe, M. S., Varon, S. and Adler, R., Neurite-promoting factor in conditioned medium from RN 22 schwannoma cultures: bioassay, fractionation and properties, J. Neurochem., 37 (1981) 759-767. 19 Mirsky, R., Wendon, L. M. B., Black, P., Stolkin, C. and Bray, D., Tetanus toxin: a cell surface marker for neurons in culture, Brain Res., 148 (1978) 251-259. 20 Rohrer, H. and Barde, Y. A., Presence and disappearance of nerve growth factor receptors on sensory neurons in culture, Develop. Biol., 89 (1982) 309-315. 21 Ruthishauser, V., Thiery, J. P., Brackenbury, R. and Edelman, G. M., Adhesion among neural cells of the chick embryo. III. Relationship of the surface molecule CAM to cell adhesion and the development of histotypic patterns, J. CellBiol., 79 (1978) 371-381. 22 Thompson, J. M. and Rapoport, S. I., Developmental decrease in neurite extension in cultured chick embryo retina and spinal cord neurons, Develop. Biol., 84 (1981) 244-246. 23 Turner, J. E., Barde, Y. A., Schwab, M. E. and Thoenen, H., Extract from brain stimulates neurite outgrowth from fetal rat retinal explants, Develop. Brain Res., 6 (1983) 77-83. 24 Turner, J. E., Barde, Y. A. and Thoenen, H., A new neurotrophic factor isolated from mammalian brain: in vitro studies on fetal rat retina, Soc. Neurosci. Abstr., (1983) 838. 25 Varon, S., Manthorpe, M. and Adler, R., Cholinergic neurotrophic factors: I. Survival, neurite outgrowth and choline acetyltransferase activity in monolayer cultures from chick embryo ciliary ganglia, Brain Res., 173 (1979) 29-45. 26 Varon, S. and Raiborn, C., Dissociation fraction and culture of chick embryo sympathetic ganglionic cells, J. Neurocytol., 1 (1972) 211-221. 27 Weidman, T. A. and Kuwabara, R., Postnatal development of the rat retina, Arch. Ophthalmol., 79 (1968) 470-484.
265
Elsevier BRD 50167
Promotion of Neurite Outgrowth and Cell Survival in Dissociated Fetal Rat Retinal Cultures by a Fraction Derived From a Brain Extract JAMES E. TURNER
Departmentof Anatomy, Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, NC27103 (U.S.A.) (Accepted September llth, 1984)
Key words: CNS neurotrophic factor - - dissociated retinal cells - - neurite outgrowth - - cell survival
We have reported previously that a fraction (BE) derived from a pig brain extract stimulated neurite outgrowth and cell survival from fetal rat retinal explants. The BE effects were dose dependent and could not be altered by NGF or its antiserum. In the present study we have observed that under similar culture conditions BE was also capable of stimulating neurite outgrowth and cell survival from fetal rat dissociated retinal cells. More specifically, the neurite-promoting activity of BE was found to be dose dependent over a concentration range of 0-50 pg/ml with a half-maximal response between 5 and 10 mg/ml. The ability of BE to stimulate neurite outgrowth was also age-related. There was a progressive decrease in the BE-mediated response between fetal day 17 and the second neonatal day. Viable process bearing cells could also be maintained in culture for at least two weeks in the presence of BE (25 pg/ml). In contrast, after 1 day in culture control cells began to rapidly degenerate and by days 3-5 no process-bearing cells were observed. The BE was found to exert its action primarily through a soluble factor(s) in the culture medium. However, we also report evidence for a substrate bound component of the BE which may aid in the attachment and/or neurite outgrowth phenomena.
INTRODUCTION It is becoming increasingly clear that the growth of neuronal processes in the peripheral nervous system (PNS) is controlled by a n u m b e r of extrinsic factors. Neurotrophic factors are believed to play a very important role in the development, m a t u r a t i o n and/or maintenance of a n u m b e r of sympathetic 14, sensory 6 and parasympathetic 2s neurons. In addition, in vitro studies have recently pointed out the importance of substrate binding neurite-promoting factors (NPFs), which must be present u n d e r certain culture conditions in order for neurotrophic factors to exert their effects on peripheral neuronsl-3,12,25. On the other hand, very little is k n o w n about the nature of these p h e n o m e n a in the central nervous system (CNS). However, a recent report has described the purification to homogeneity of a new neurotrophic factor (PF) from the m a m m a l i a n brain. The purification of this factor was stimulated by previous results which had indicated that glioma-condi-
tioned m e d i u m 8 and a rat brain extract 6 Could support the survival of embryonic sensory n e u r o n s cultured in the absence of NGF. The biological activity of the newly isolated protein is similar to that previously reported for both glioma-conditioned medium and the brain extract. The new growth factor is functionally and immunologically distinct from N G F , and it is a basic protein with a molecular weight of 12,300 and a PI i> 10.17. Results also suggest that this protein from the m a m m a l i a n brain may be produced by glial cells6, 8. The outcome of these observations has been the discovery that a CM-cellulose column fraction (BE), which is derived from an intermediate step in the purification of PF from a pig brain extract, stimulated neurite outgrowth from fetal rat retinal explants23. More specifically, we found that BE stimulation was specific, dose d e p e n d e n t and age-related23,z4. In addition, B E was found to increase the maturation and cell survival of n e u r o n s in the fetal retinal explants 24. We have formed an hypothesis from these obser-
Correspondence: J. E. Turner, Department of Anatomy, Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, NC 27103, U.S.A. 0165-3806/85/$03.30 (~) 1985 Elsevier Science Publishers B.V. (Biomedical Division)
266 vations that there are neurotrophic factors in the mammalian CNS which are capable of supporting and/ or stimulating neurite outgrowth in a manner similar to that found in the PNS. The present study further confirms our hypothesis by demonstrating similar BE-mediated phenomena in a newly established dissociated fetal rat retinal culture system.
glucose and 2.0 mM L-glutamine; N I supplements ~ (Sigma): insulin (5ktg/ml), transferrin (5ktg/ml), progesterone (2 x 10-8 M), selenium (3 x 10-8 M) and putrescine (1 x 10-3 M); and (3) antibiotics; penicillin (10,000 ktg/ml) and streptomycin (10 mg/ml).
Preparation of the culture substrate MATERIALS AND METHODS
Culture of dissociated cells Fetuses from 17 to 21 day pregnant anesthesized albino rats were surgically removed and placed in sterile phosphate-buffered saline (PBS). Eyes from the fetuses and 2-day neonates were dissected, placed in PBS and retinas removed by watchmakers forceps (no. 4), using a surgical microscope with a cool fiber optic system. At this stage of development, retinas can be stripped cleanly away from surrounding tissue and the developing lens. It is also possible to remove the vascular net which begins to form on the vitreal surface of the retina and around the lens. Dissected retinas are collected in 5 ml of Ca 2÷,Mg 2÷free PBS (CMF) to which was added 60HI of 1% trypsin (Worthington) and 60 #1 of 0.1% DNase I (Sigma). After incubation at 37 °C for 15 min, digestion was stopped by the addition of 200 H1 of soybean trypsin inhibitor (Gibco, 2 mg/ml). The retinas were centrifuged at 1000 g for 5 min, resuspended in CMF and dissociated by 25-30 strokes through a siliconcoated Pasteur pipette. After hemocytometer counts, cells were diluted with CMF to a density of 2 × 106 cells/ml, aliquoted to various tubes, centrifuged at 100 g for 5 min, resuspended in control or BE-containing medium and except for one experiment seeded at 1 ml onto fibronectin- (50 Hg/ml in PBS, pH 6.5, Biomedical Technologies) coated dishes (35 mm, 6-well, Costar) which had been previously equilibrated with 1 ml of the appropriate medium (i.e. total volume equals 2 ml). The Trypan blue dye exclusion test indicated that 90% of the cells plated survived and attached after 2 h in culture. Part of the medium (0.5-0.8 ml) was exchanged every 3 - 4 days. The culture medium was the same as that described in a previous report 24. More specifically, the media consisted of: (1) Eagle's basal medium (Gibco) supplemented with 26.4 mM NaHCO3, 33.3 mM
In most experiments the dishes were first coated with 1 ml of fibronectin 4 (50 ktg/ml) for 1 h at room temperature washed once with 1 ml of sterile distilled water and equilibrated in the tissue culture incubator with 1 ml of control or BE-containing medium at 37 °C with 5% CO 2 for 1-2 h. Control dishes were incubated in medium with no BE present. In one set of experiments dealing with substrate bound phenomena dishes were prepared as previously described except fibronectin was omitted in one group leaving the plastic substrate to be conditioned by BE. Control cells were cultured on the plastic substrate in the absence of BE. In another set of experiments dealing with substrate-bound phenomena dish surfaces were first equilibrated with fibronectin followed by BE- (25 ~g/ml) containing medium as previously described. However, equilibration was allowed to continue overnight for 12 h in BE-containing medium at which time: (1) 2 x 106 cells ml were seeded in 1 ml of BE- (25 Hg/ml) containing medium as described previously; and (2) another set of dishes was first washed 3 times with control medium to remove the unbound BE before cells (2 x 106 ml) were seeded in control medium onto the BE-modified substrate. Control cells were cultured on fibronectin in the absence of BE.
Scanning electron microscopy Cultures were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer for 1 h. After 3 changes in buffer, cultures were dehydrated in an alcohol series and dried by the critical point method. Small pieces of the dish surface were mounted on aluminum studs, coated with gold-palladium (60:40) and photographed with a 501 Phillips SEM.
Tetanus toxin binding studies The staining of BE (25 Hg/ml)-exposed, 17-day fetal rat dissociated retinal cells (2 x 106 cells/dish), treated after 5 days in culture with tetanus toxin was
267
Fig. 1. Phase-contrast photomicrographs demonstrating BE (25 #g/ml)-mediated stimulation of fiber outgrowth and cell survival from 17-day fetal rat dissociated retinal cell cultures. A: appearance of fiber outgrowth from BE-treated cells after 4 days in culture. Note the numerous contacts made between cells. Also note non-responding necrotic cells among responding neurons (arrows). B: control cells after 4 days in culture demonstrating extensive cell necrosis (dark cells) and no fiber outgrowth. C: cell aggregation and process formation in BE-treated group after 7 days in culture. D: control cells after 7 days in culture demonstrating no aggregation and no process formation. Note phase-bright areas are extruded nuclei and not intact cells. E: higher magnification of a retinal neuron-like cell body (arrowhead) with an extensive process resulting after 7 days of BE treatment. F: between 7 and 14 days in culture a few aggregating cells with long processes appear attached to a flattened non-neuronal cell. Mag.: A-B and E-F, 300× ; C-D 150×.
268 performed using an indirect immunofluorescence method19. Four-well 35 mm culture dishes (Greiner and Sohne) with an optional volume of 80 ~1 were used for these studies. Culture dishes were washed twice with phosphate-buffered saline containing 0.1% BSA (PBS/A), incubated for 20 min with tetanus toxin (25 #g/ml) in PBS/A and washed 3 times in PBS/A. Cultures were next fixed for 20 min in 6% formaldehyde in Ca2+,Mg2÷-free phosphate-buffered saline (CMF), washed 3 times in CMF/A and incubated 20 min with human anti-tetanus toxin (1:50 dilution), washed 3 times with CMF/A, incubated for 20 min with FITC-conjugated goat anti-human-aglobulin (1:100 dilution), washed 3 times in CMF/A and mounted in CMF/glycerin (1:1). All procedures were carried out at room temperature. The tetanus toxin was a gift from Dr. B. Bizzin, Institute Pasteur, Paris; human anti-tetanus toxin was a gift from Dr. Johannsen, Behring-Werke, Marbury; and FITCconjugated goat anti-human-a-globulin was purchased from Behring-Werke; Marburg.
Preparation of brain extract (BE) The pig brain extract fraction (BE) was a gift from Dr. Yves Barde and was prepared according to the procedure reported by Barde et al.7. In comparison to the starting homogenate, the BE had a hundred times higher specific activity when tested for survival activity on 20-day-old chick embryo sensory neurons, as described previously 7. The BE preparation is equivalent to step III of the purification procedure for PF 7.
Evaluation of neurite outgrowth Cultures were analyzed by phase-contrast microscopy and quantitations performed by determining the number of process bearing cells/dish. Processbearing cells were only scored if their length was at least twice the cell body diameter. The number of cells/dish was estimated by a strip counting method 26. The data were analyzed for significance of difference by the Student's t-test. All observations were made from duplicate or triplicate sister cultures. RESULTS The majority of fetal rat dissociated retinal cells will not extend neurites in a defined medium with fi-
bronectin as a substrate. Those few retinal cells that show initial signs of neurite outgrowth under control conditions are withdrawn by 48 h, and by 4 days most cells have become necrotic and begin to float off the substrate (Figs. 1B, D and 2B). However, the presence of BE (25/~g/ml) in the medium causes a dramatic enhancement of cell attachment and neurite outgrowth, which reaches a peak by culture day 4 (Fig. 1A). The normally rounded cells become more phase-dark and extend 2 - 4 branching processes which interdigitate to form fascicles (Figs. 1A and 2A). After 1 week in culture many of the BE-treated retinal cells have a tendency to form small processbearing aggregates (Fig. 1C). However, many individual cells with long processes can still be observed after one week in culture (Fig. 1E). The presence of flattened, dividing non-neuronal cells are seldom observed in these cultures; however, when they are seen they usually serve as a substrate on which several process bearing neurons are aggregated (Fig. 1E). Tetanus toxin binding revealed that greater than 95% of the viable BE-treated process-bearing cells are positively labeled (Fig. 3). Although BE treatment illicits a dramatic response with respect to retinal cell survival and neurite outgrowth, the total number of responding cells is equivalent to only 20-40% of the total cells plated. More specifically, of the 2 x 106 cells plated per dish only 4-8 x 105 cells respond to BE treatment depending on the embryonic age. Therefore, even in BE treated dishes there is normally a great amount of cell necrosis (Fig. 1A and E). The BE-mediated response drops off dramatically with decreased plating density to where there are virtually no surviving cells left to respond after several days at either 1 x 106 or 5 x 105 cells/ml, respectively. The BE-mediated neurite outgrowth response is a dose-dependent phenomenon over a concentration range of 0-50/xg/ml, with a half-maximal response at approximately 5/xg/ml (Fig. 4). Viable process-bearing cells can be maintained in culture in the presence of BE (25/xg/ml) for at least 2 weeks (Fig. 5). The BE-mediated response becomes maximal between days 2 and 4 at which time control cells have withdrawn their processes, become necrotic and have begun to lift up off the substrate (Fig. 1B and D). Retinal cells responding to BE do so in an age-dependent fashion. More specifically, there are dramatic step-
269
Fig. 2. Scanning electron micrographs of 17-day fetal rat dissociated retinal cells after 6 days in culture. A: cultures treated wi*h BE (25/~g/ml) demonstrating the profuse mat of interdigitated and fasciculated processes from the rounded retinal neuronal cell bodies. B: control cultures showing the dramatic loss of viable cells and the complete absence of processes. Mag. 2000x.
270
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Fig. 3. Phase-contrast and companion fluorescent photomicrographs of control and tetanus toxin-treated 17-day fetal rat dissociated retinal cells exposed to BE (25/~g/ml) for 6 days in culture. A: phase-contrast photomicrograph of tetanus toxin-treated cells. B: fluorescent photomicrograph of A demonstrating that all viable, process-bearing cells are positively labeled. C: phase contrast photomicrograph of control cells without tetanus toxin treatment. D: fluorescent photomicrograph of C showing lack of specific labeling in control dishes. Mag. 222 x.
wise decreases in the number of BE-responding cells between 17 fetal day, 21 fetal day and 2 neonatal day retinas, respectively (Fig. 6). Between 17 and 21 fetal days there was a 2-fold reduction in numbers of process-bearing cells, compared to a 4-fold reduction between 21 fetal and 2 neonatal days in response to BE treatment. If fibronectin-coated culture dishes were treated with BE (25/~g/ml)-containing medium for 12 h, washed free of the soluble BE and cells plated in control medium onto the modified substrate to which B E was bound, the number of process-bearing cells was significantly reduced by 5-fold from 8 x 105 to 1.5 × 105 responding neurons (Fig. 7). However, the reduced number of responding cells was still significantly higher than cells plated under the absolute control conditions of no substrate or medium expo-
sure to BE (Fig. 7). In turn, experiments conducted in the presence or absence of fibronectin as an initial substrate revealed that in those dishes incubated only with BE (25/.tg/ml) for 3 h and washed free of BE approximately 1.5 x 106 cells responded compared to 6 × 106 process-bearing cells cultured under normal experimental conditions (Fig. 8). Similarly, the number of reduced process-bearing cells was significantly higher than control cells plated on only a plastic substrate in the absence of B E (Fig. 8). In both of these experiments (Figs. 7 and 8) BE-containing medium was equilibrated on fibronectin-coated dishes for 3-12 h prior to cell plating which was not the case during the earlier experiments (Figs. 4-6). When these figures are compared, it is quite evident that increased prior plating time with BE significantly enhances the number of process-bearing cells.
271
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I 5 10 25 50 I 0 0 BE CONCENTRATION (,ug/ml) Fig. 4. A dose-response curve demonstrating the number of 17-day fetal rat dissociated retinal process-beating cells induced by BE treatment°after 4 days in culture. Vertical lines represent S.E.M. All points are significantly different from control values (P < 0.001) and 1, 2.5, 5 and 10/~g/ml concentration points are significantly different (P < 0.001). However, the 3 highest concentration points are not significantly different from each other (P > 0.05). Concentration points represent an average measurement from two sister cultures.
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Fig. 6. Graph depicting the age-dependent BE (25/~g/ml)-mediated dissociated retinal cell response in 17 fetal day (17F), 21 fetal day (21F) and 2 day neonatal (2DN) cultures. All group values are significantly different from one another (P < 0.001). Vertical bars represent the S.E.M. Each group value represents an average measurement from two sister cultures.
DISCUSSION In the present study we have reported that u n d e r defined tissue culture conditions an extract prepared
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Fig. 5. Graph demonstrating the stimulation and long-term survival of 17-day fetal dissociated retinal process-bearing cells in the presence of BE treatment. Note that after 3 days in culture
there are no surviving process-bearing cells in control dishes. All group values from the same time points are significantlydifferent from one another (P < 0.001). Vertical bars represent the S.E.M. Each group value represents an average measurement from two sister cultures.
from pig brain (BE) was capable of stimulating neurite outgrowth and cell survival from fetal rat dissociated retinal cells. This neurotrophic response was quite dramatic since after 1 day in culture control cells began to rapidly degenerate and by 3 - 5 days no process-bearing cells were observed. Subsequently, by 4 days most control cells had begun to float off the substrate. This observation is in agreement with our previous reports in which BE was found to stimulate neurite outgrowth and cell survival in fetal rat retinal explants23,24. More specifically, we found that long radial fiber outgrowth could be stimulated from explants in excess of 1 m m where controls exhibited no growth. In addition, B E appeared to accelerate retinal ganglion-like cell m a t u r a t i o n and to significantly reduce cell death in explants. It is also important to note that avian brain extracts have also been shown to stimulate neurite outgrowth from 6-day chick embryonic retina u. After 1 week in culture many of the BE-treated retinal cells displayed a tendency to form small process-bearing aggregates. The reason for this p h e n o m -
272 6O F
enon cannot be explained readily, except to say that BE may ensure a greater n u m b e r of healthy and/or surviving cells, as well as affecting the cell adhesive or binding (recognition) qualities through a memb r a n e - b o u n d molecule like C A M 21. Further evidence
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for this possibility was reported earlier. Specifically, the BE-mediated retinal response dropped off pre-
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cipitously with decreasing plating density. Virtually no surviving cells were left to respond after several days at 1 x 106 cells/ml, a density which would greatly diminish the chances for cell-to-cell contacts. The BE-mediated dissociated cell neurite outgrowth response was found to be a dose-dependent p h e n o m e n o n over a concentration range of 0-50/~g/ml with a half-maximal response at approximately 5/~g/ml. These results are complementary to our earlier explant work in which we reported a dose-
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Fig. 8. Graph demonstrating that to a limited extent a component of BE can bind to the plastic dish surface in the absence of a fibronectin substrate (BE-F) to exert a stimulatory action on dissociated 17-day fetal rat retinal cells. In the BE-F experiment, BE (25 ktg/ml) was equilibrated onto the plastic dish surface for 3 h, washed 3 times and cells plated in control medium without BE. Controls consisted of BE (25 Izg/ml) substrate equilibration with cells plated in BE (25 ~g/ml) containing medium on fibronectin substrate (BE+F) and conditions using no fibronectin or BE in the medium (C-F). All group values are significantly different from one another (P < 0.001). Vertical bars represent the S.E.M. Each group value represents an average measurement from two sister cultures taken after 4 days in culture.
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Fig. 7. Graph demonstrating stimulatory actions of BE (25 Izg/ml) present as either a soluble molecule in the medium (BE-S), bound only to the substrate and not present in the medium (BE-B) or no BE present at all (C). BE was equilibrated onto the fibronectin (50/~g/ml) substrate for 12 h before exchange with either fresh BE-containing medium (BE-S) or 3 washes with regular medium (BE-B). Note that BE can still exert some stimulatory activity while bound (BE-B) to the substrate. All group values are significantly different from one another (P < 0.001). Vertical bars represent the S.E.M. Each group value represents an average measurement from two sister cultures taken after 4 days in culture. Dissociated retinal cells are from 17-day fetuses.
dependent p h e n o m e n o n over a very similar concentration range23,24. In a similar fashion, we have observed in this and a previous study 24 that retinal cells respond to BE in an age-dependent fashion. More specifically, there were dramatic stepwise decreases in the n u m b e r of BE-responding cells between 17 fetal day, 21 fetal day and 2 day neonatal retinas, respectively. Similar age-related trends have been reported for the response of chick sensory ganglia to both N G F , glioma-conditioned m e d i u m and rat brain extracts 6. In addition, T h o m p s o n and Rapoport reported a significant age-related decrease in the percentage and average length of neurites formed in 24 h cultures of dissociated chick retina between 6 and 16 days of embryonic age. W h e t h e r the age-dependent decrease in B E responsiveness is due to diminished cell viability and/or receptor site modifica-
273 tion has not been determined. However, it has been demonstrated that there is a direct correlation between reduced responsiveness to NGF and loss of receptor activity in dorsal root neurons 20. The retinal cell type(s) responding to BE in both the dissociated and explant systems are not known at the present time. However, at this stage of development (i.e. 17-21-day fetuses) the rat retina is relatively undifferentiated since all ten layers are not distinguishable until postnatal day 215,27. At birth the rat retina contains two distinguishable layers: (1) the inner cell layer consisting of a row of well differentiated ganglion cells; and (2) an outer layer formed by an abundance of undifferentiated neuroepithelial cells 27. Even as early as embryonic day 15, differentiating ganglion cells were observed to possess axons which extended into the optic stalkS. Therefore, it seems plausible from this evidence that the retinal ganglion cells are the most likely neurons to extend their axons onto the culture substrate and be affected in this manner by BE in our system. Our explant studies would tend to strengthen further this argument since BE treatment was shown to stimulate maturation and survival of ganglion-like cells 24. However, this does not rule out the possibility that over a 1-2-week culture period other retinal cell types could differentiate in vitro 16 and be stimulated by BE treatment. This may be especially evident since 20-40% of the plated cells survive in the presence of BE which most likely exceeds the number of cultured ganglion cells. Alternatively, BE may shift neuroepithelial differentiation towards ganglion cells. We also have no direct evidence as to whether BE acts on neurons, the substrate or indirectly through glial cell elements, since at least, Mfiller cells have begun to differentiate at this time5,Z7. However, from our present tetanus toxin binding results, we concluded that the retinal culture consisted primarily of a highly enriched (t>95%) neuronal population. This was not surprising since our defined media conditions have been reported not to support non-neuronal cell survival 10. In further support of this conclusion, tetanus toxin binding has been shown to be specific for cultured rat retinal neurons with no non-specific binding to the non-neuronal (i.e. glial and connective tissue) components of the culture 9. Therefore, we would speculate that a direct action of BE on retinal neurons is most likely, since in a similar situation BE
and the purified factor were required in the medium for the survival and neurite outgrowth of embryonic chick sensory neurons, when seeded at low densities in the absence of glial elements6. The BE was found to exert its action primarily through a soluble factor(s). We know from previous studies that BE is different from N G F and is not involved in this phenomenon23. However, we have also reported in the present study evidence for a substrate-bound component of the BE which may aid in the attachment and/or neurite outgrowth phenomena. More specifically, a component of the BE could not be washed entirely from a fibronectin or plastic substrate and thus allowed some cell survival and neurite outgrowth to occur, although at a significantly reduced amount compared to conditions where BE was present in the medium. The necessity for multiple components to ensure cultured neuronal cell survival and/or neurite outgrowth (i.e. soluble and substrate-bound factors) is further substantiated by studies in which concurrent addition of ciliary neurotrophic factor (CNF) to the medium of 8-day-old dissociated chick embryo ciliary ganglionic neurons was shown to enhance neurite outgrowth only in the presence of PNPF 1-3,12,13,25. In a number of cases PNPFs do not, by themselves, support the survival or neurite outgrowth but require the presence of neurotrophic factors1,17,TM. However, there is some evidence that for PC 12 cells substrate factors can support neurite outgrowth to some extent in the absence of NGF 15. Further studies need to be conducted to conclusively demonstrate that different components within BE are synergistic and that substrate-bound BE is similar to PNPFs. In conclusion, we have reported for the first time that a dissociated cell population from the mammalian CNS responds in a typical neurotrophic manner to an extract prepared from pig brain. This evidence speaks very strongly for our hypothesis that there are trophic factors in the mammalian CNS capable of maintaining neurons and stimulating neurite outgrowth during development. In addition, we conclude that the BE offers a rich source of molecules which may be capable of exerting neurotrophic actions on certain developing and regenerating CNS neurons.
274 ACKNOWLEDGEMENTS
to thank Drs. Y v e s B a r d e and H a n s T h o e n e n for their g e n e r o u s supply of brain extract.
This w o r k was s u p p o r t e d by a grant f r o m the National E y e Institute ( E Y 04377). T h e a u t h o r wishes
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