- Email: [email protected]
Extract from brain stimulates neurite outgrowth from fetal rat retinal explants
Developmental Brain Research, 6 (1983) 77-83 Elsevier Biomedical Press
77
Extract from Brain Stimulates Neurite Outgrowth from Fetal Rat Retinal Explants JAMES E. TURNER*, YVES-ALAIN BARDE**, MARTIN E. SCHWAB and HANS THOENEN Department of Neurochemistry, Max-Planck-lnstitute for Psychiatry, 8033 Martinsried (F. R. G.) (Accepted April 22nd, 1982) Key words: neurotrophic factors - - nerve growth factor - - retinal explants
Explants from rat fetal retina were placed in culture and assayed for fiber outgrowth. In contrast to results obtained with lower vertebrates, nerve growth factor (NGF) does not seem to play a role in this system: NGF is not able to stimulate fiber outgrowth and antibodies to NGF do not block the spontaneously occurring tibet outgrowth. However, an extract prepared from pig brain is able to stimulate fiber outgrowth in a dose-dependent manner. It is suggested that such an extract can be used as a source of putative neurotrophic factors exhibiting in the mammalian central nervous system (CNS) an action similar to that of NGF in the peripheral nervous system (PNS) of mammals and in the CNS of lower vertebrates like fishes and amphibia. INTRODUCTION Recent studies in lower vertebrates have shown that regeneration o f severed optic nerves (evaluated by morphological criteria) can be enhanced by injection of a well-characterized protein, N G F za-'6 Conversely, injection o f antiserum to N G F delays the process o f regeneration 8,26. Interestingly, these in vivo effects have their parallel in vitro under welldefined conditions. Explants from goldfish retina can be placed in culture and spontaneously send out processes l°. This p h e n o m e n o n can be simulated by the addition o f low levels o f N G F . Conversely, the spontaneously occurring fiber outgrowth can be inhibited by the addition o f antibodies to N G F z6. This is therefore a useful model to study nerve regeneration in the C N S o f lower vertebrates. In higher vertebrates such as mammals, a role for N G F in normal development or after lesion has been convincingly demonstrated only in the P N S : injection o f N G F prevents the normally occurring cell death and stimulates neurite outgrowth, for example in the superior cervical ganglion o f the rat.
On the other hand, injection o f antibodies to N G F destroys the neurons which depend on N G F for survival during certain stages of development. In the rat, for example, extensive neuronal death in spinal ganglia can be induced by exposure o f antibodies to N G F before birth. The same is truc for sympathetic neurons o f the paravertebral chain, until a few days after birth (for reviews see refs, 12, 22). In this study, retinal explants from fetal rats were tested for fiber outgrowth, as has been done previously with goldfish retina explants. In the system described here, it appears that N G F and its antiserum have no effect but that an extract prepared from pig brain markedly enhances fiber outgrowth. MATERIALS AND METHODS Dissection and culture of explants Rat fetuses (gestation days 18-20) were removed from ether-anesthetized pregnant females (Wistar strain) and placed in sterile phosphate-buffered saline (PBS). Eyes were dissected, placed in PBS, opened and retinas were removed using watchmakers for-
* Present address: Department of Anatomy, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, N.C. 27103, USA. ** To whom correspondence should be addressed. 0165-3806/83/(R)0(O0000/$03.00(© 1983 Elsevier Biomedical Press
78 ceps under a dissecting microscope. At this stage of development, retinas can be cleanly stripped 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 were collected in PBS and minced into small pieces (approximately 400 600/~m squares) using forceps. Culture dishes were coated with polyornithine (Sigma, Type IB) as a substrate and equilibrated with culture medium for 2 h in 5',!,~iCO~,/95 ~',~air at 37 :C in an incubator. The culture medium consisted of L-15 (GIBCO) supplemented with 5',',~i (v/v) rat serum, additional amino acids and vitamins l~;, penicillin and streptomycin, and methylcellulose (Dow). Just prior to cxplantation, the excess medium was aspirated from the dishes. The explants were then placed on the dish with a Pasteur pipette. One drop of medium was placed over thc explants which were separated and positioned (20-30 explants/dish) by means of glass manipulators. Explants were allowed to equilibrate in a minimum amount of medium (2-3 drops/dish) overnight which allowed them to attach, after which time 1 ml of medium was added. The medium was exchanged every 4 days. Culturcs were pulsed with 10/~M cytosine arabinoside (Sigma) once a week for 4 days to prevent non-neuronal cell outgrowth. Treatment was begun on day 4 ot" culture. In order to determine if BE binds to the polyornithine substrate to exert its effects, BE (50 #g/ml) containing medium was incubated in coated dishes for 2-4 h before explantation. After 3 washes cxplants were cultured in control medium.
Evaluation of neurite outgrowth Neurite outgrowth was measured in the same manner as previously established for goldfish retinal explants l0 by determining a neurite growth index ( N G I ) score which takes into consideration two aspects of neurite outgrowth, tiber density and fiber length. The NGI was detined as the product of the neurite density score, based on a scale of 0-4, and the length score. The length score was measured by an ocular micrometer (one unit equals 10 ~m using a 10 × objective and a 12.5 x ocular) where a length of 200/~m was assigned a score of 1,400/~m a score of 2 etc. The length score for each explant was an
average of 4 measurements, one from each side of the explant. The neurite density score was determined from a standard atlas of representative explants demonstrating various degrecs of neuritc outgrowth. Triplicate culttues were established for each assay point with a total of 30 explants analyzed pcr point. For consistcncy, for each point (control and experimental) the 30 explants showing the largest tibet outgrowth wcre selected. NGI scorcs were analyzed statistically by the Student's /-test. In order to ensure that the outgrowth from explants was of neuronal origin, fibers were stained by the Holmes silvcr method 1'~ and observed by light microscopy.
Preparation of pig brain extract (BE) Whole pig brains wcre homogenized in 0.15 M (NH4),)SO4 and the pH brought to 4.5 with 1 N HCI. After centrifugation for 40 min at 20,O00g, the pH of the supernatant was brought to 6.5 with 1 N NaOH and solid (NH4)..,SO4 was added to a saturation of 70','41. After ccntrifugation for 40 rain at 20,000 g, the resulting pellet was dialyzed against 0. I M sodium phosphate buffer, pH 6.0, and applied to a CM-cellulose column equilibrated with the same buffer. Thc material cluted with a step of 0.5 M NaCI added to the phosphate buffer (after a first elution with 0.15 M NaCI) was dialyzed against watcr and lyophilized. The protein content of this fraction was determined by the method of Lowry et al. la using bovine serum albumin as a standard. In comparison to the starting homogenate, this preparation had a hundred times higher specific activity whcn tested for survival activity on 10-day-old chick embryo sensory neurons, as described pleviously (rcf. 2, and Barde, Edgar and Thoenen, submitted for publication).
Preparation of NGF and NGF-antiserum The 2.5S N G F used in this experiment was prepared according to the method of Bocchini and Angeletti 5 as modified by Suda et al.ek In this preparation one biological unit (BU) of 2.5S N G F was equivalent to 5 ng/ml as determined by the bioassay method of Fenton 7. One biological unit of N G F was neutralized by a 1:10,000 dilution of the NGF-As. In these experiments, all sera were heat inactivated at 56 ')C fbr 30 min.
79
Fig. I. Phase-contrast micrographs of 20-day-old fetal rat retinal explants 5 days after culture. A: control explant demonstrating outgrowth of mostly short, broad processes (P). B: experimental explants treated with BE (50/~g/ml). Note the extensive fiber network which appears denser and longer than those of controls. Magnification : 128 x .
~0 RESIJI.TS Under the present culture conditions there was a sparse, but distinct spontaneous outgrowth o f fibers from the rat retinal explants (Fig. 1A). With the addition o f BE to the medium (50 ,ag/tnl), the neurites became much denser with a considerable increase in length (Fig. I B). Whcn these cxplants were stained by the Holmes silver method, the fibers demonsttated a positive reaction confirming their neuritic nature (Fig. 2). Cytosine arabinoside pulses were used to prevent the outgrowth o f non-neuronal cells along ncuritcs (Fig. 1). Cytosine arabinosidc had no eff;ects on the extent of fiber outgrowth. in order for BE to elicit its effects it must bc present in the medium: BE will not bind to the polyornithine substrate, since no stimulatory effects were noted when explants were placed in control medium aftcr the substrate had been prctreated with BE.
When BE was added in increasing conccntrations to thc culture medium, there was a dose-dependent increase in the density and length o f neuritc outgrowth from the explants between l and 100/tg/ml, leveling off between I00 and 500 tlg/ml (Fig. 3). The BE-mediated increase in NG1 was approximately 5fold at 100/~g/ml when compared to control values. BE administration (50/~g/ml) to retinal explants caused a continued, statistically significant, elcvation of N G I s over controls for at least 2 weeks in culture (Fig. 4). Control explants ceased ncurite outgrowth after day 11, and subsequently began to show signs of degeneration. Exposure of the explants to N G F or N G F - A s had no effect on neurite density or length compared to controls (Fig. 5). For unknown reasons, the N G I values were slightly lower in the prescncc o f N G F than in its absence, although the difference is not statistically significant. In addition, N G F did not augment the BE-mediated response when added
Fig. 2. I,ight micrograph of 18-day-old fetal rat retinal explant treated with BE (50 !~giml~, 7 days after culture, stained by the I-Iolmes silver method for nerve fibers. Note the positively stained processes which correspond to those seen in Fig. I. Magnification : 176 ,.:.
8-
(30)
6-
/ -9
5
_=4 ¢_o
=~3
....L-
:z:
~3-
,, P'~ 0 050 w,, P.~ 0001
l-
o
i
16
I
BE CONCENIRAIION(ug/ml)
BE
Fig. 3. Dose-response study demonstrating a 5-fold increase
in NGI values elicited by BE over a concentration range of 1-100/~g/ml after 5 days in culture. Vertical bars represent S.E.M. The number in parentheses indicates total explants analyzed per point.
BE.As 8E.NGF
NGF
As
Ss
Fig. 5. NGI values were determined after 5 days in culture. Thirty explants were analyzed per group and the vertical bars represent S.E.M. BE was used at a concentration of 50 ttg/ml, NGF at 50 ng/ml, NGF-As at a 1:1000 dilution and SS (nonimmune sheep serum) at 1:1000. C is control (no addition to the medium).
20-
-
T
IB
together, nor did the c o m b i n a t i o n of BE ÷ N G F As significantly reduce the N G I s compared to BE alone (Fig. 5).
115
DISCUSSION 14
These results show that retinal explants from rat fetuses display a s p o n t a n e o u s fiber outgrowth that can be m a i n t a i n e d for up to 1 1 days in culture. The fibers could be stained positively with the Holmes silver method, indicating that they are of n e u r o n a l origin. The use of cytosine arabinoside prevented the outgrowth of n o n - n e u r o n a l cells. In contrast to goldfish retinal explants, the fiber outgrowth from rat explants is not influenced by N G F or its antiserum. Whereas in the goldfish
12-
10
8
6-
4-
2-
/*
:
"7" B15°'g'm*
0 1
2
3
t,
5
6 7 8 9 DAYS IN CbLTURE
10
'I
12
13
14
Fig. 4. Effects of BE (50 tzg/ml) on the NGI values during the first two weeks in culture. Note that BE can stimulate and sustain a significant elevation in neurite outgrowth during this time period. Growth from control explants ceases after 11 days in culture. Vertical bars represent S.E.M. Numbers in parentheses indicate total explants analyzed per group.
~2 system it is possible to markedly enhance the spontaneous fiber outgrowth by the addition of N G F and to reduce it by' NGF-As, neither effect could be observed here with rat explants. "l'hesc results are in line with what is known so far on the effect of N G F in the CNS of various species: in the goldlish and the newt, regeneration of crushed optic nerves is enhanced by injection of N G F and delayed by that of NGF-As. In mammals there is so far no detinitive evidence that N G F is able to influence fiber outgrowth in the CNS. Although there is a report by BjYrklund et al. 4 that N G F can influence the regeneration of central eatecholamine-containing tibers into pieces of iris implanted into the midbrain, others have demonstrated that N G F does not play any role in development, regeneration and regulation of central catecholamine neurons <'~.lT.ls. This lack ofeffect is in contrast to what is observed in the PNS of mammals where it has been firmly established that N G F plays a key role in the development of some sympathetic and sensory neurons. In view of the negative results obtained with NGF, it is of interest that fiber outgrowth can be enhanced by the use of extracts of CNS tissue. These experiments were prompted by the observation that medium conditioned by glioma cells a or astrocytes la as wcll as extracts from the brain" have a distinct 'neurotrophic' effect : they all are able to support the survival of cultured peripheral embryonic neurons which would otherwise die. It remains to be demonstrated that the molecule which induces fiber outgrowth from rat retinal explants is the same as that supporting the survival of peripheral embryonic neurons. This demonstration must await the purification of the survival factor(s). However, it is interesting to note that preliminary results obtained with a fraction from pig brain (obtained after further fractionation on hydroxylapatite) having a specific activity a hundred times higher on the survival of chick embryonic sensory neurons than that used in this study, turned out to be also a hundred times more active in the retinal explant system described here,
Due to the nature of the system used in this study, we do not know ~.hether or not the effcct of BE b, a direct one: the expkmts contain a variety of cell type:< including Mi.iller cells which have begun to differentiate at this stage of development~,'% making it possible that the enhanced fiber outgro~th seen ill the presence of BE is mediated via nonneuronal cells. Furthermore, the present study doc:~ not indicate which types of neurons cxhibit fiber outgrowth. Ho~vever, ganglionic cells are likely' to hc responsible since a study using a horseradish pcroxidase tracer indicated that the primary nemitc outgrowth fl'om mouse fetal retina is of ganglionic cell originl:L A degeneration of fibers and ganglion cells during the second week of culture, a phenomenon which could be prevented by co-culture ~.ith optic tectum (the normal target tissue of ganglion cells) was also noted in that study. Our control cxplants showed a similar trend in that neurite outgro~th stopped after I I day.s and there ~as some degene,ation thereafter. This was not seen whcn BE ~as added to the culture. We conclude from the data presented here that a trophic factor(s) present in the mammalian central nervous system can stimulate ncurite outgrowth from mammalian fetal retina. Some of the neurons of the fetal retina may therefore require one or more specific molecules in order to survive and/or regenerate neurites during normal development of after lesion. Pig brain extracts prepared as described here could be a source of such molecules which might play a role in the CNS of mammals similar to that played b y N G F in the PNS of mammals as well as in the CNS of lower vertebrates. A(_KNOWLEDG EM ENTS This work was made possible in part by an NIH Research Career Development Award, NS 00338. awarded to J.E.T., who was a visiting professor in the Department of Neurochemistry.
83 REFERENCES 1 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. 2 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. 3 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. 4 Bj6rklund, A., Bjerre, B. and Stenevi, V., Has nerve growth factor a role in the regeneration of central and peripheral catecholamine neurons? In K. Fuxe, L. Olson and Y. Zotterman (Eds.), Dynamics of Degeneration and Growth in Neurons, Pergamon Press, New York, 1974, pp. 389~,09. 5 Bocchini, V. and Angeletti, P. U., The nerve growth factor: purification as a 30,000 molecular weight protein, Proc. nat. Acad. Sci. U.S.A., 64 (1969) 787-794. 6 Coyle, J. T., Jacobowitz, D., Klein, D. and Axelrod, J., Dopaminergic neurons in explants of substantia nigra in culture, J. NeurobioL, 4 (1973) 461-470. 7 Fenton, E. L., Tissue culture assay of nerve growth factor and of the specific antiserum, Exp. Cell Res., 59 (1979) 83-92. 8 Glaze, K. A. and Turner, J. E., Regenerative repair in the severed optic nerve of the newt (Triturus virideseens); Effect of nerve growth factor antiserum, Exp. Neurol., 58 (1978) 500-510. 9 Konkel, R. T., Mailman, R. B., Bendeich, E. G., Garrison, A. M., Mueller, R. A. and Breese, G. R., Evaluation of the effects of nerve growth factor and anti-nerve growth factor on the development of central catecholcontaining neurons, Brain Res., 144 (1978) 277-285. 10 Landreth, G. E. and Agranoff, B. W., Explant culture of adult goldfish retina. A model for the study of CNS regeneration, Brain Res., 161 (1979) 39-53. 11 Letourneau, P . C . , Possible roles for cell-to-cell substratum adhesion in neuronal n,orphogenesis, Develop. Biol., 44 (1975) 77-91. 12 Levi-Montalcini, R. and Angeletti, P. U., Nerve growth factor, Physiol. Rev., 48 (1968) 534-569. 13 Lindsay, R. M., Adult rat brain astrocytes support survival of both NGF-dependent and NGF-independent neurons, Nature (Lond.), 282 (1979) 80-82. 14 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. bioL Chem., 193 (1951) 265-275.
15 Luna, L. G., Methods for nerve cells and fibers: Holmes method for nerve cells and fibers. In L. G. Luna (Ed.),
Manual of the Histologic Staining Methods oJ the Armed Forces Institute of Pathology, McGraw-Hill, New York, 1968, pp. 196-198. 16 Mains, R. E. and Patterson, P. A., Primary cultures of dissociated sympathetic neurons. 1. Establishment of long-term growth in culture and studies of differentiated properties, J. Cell Biol., 59 (1973) 329-345. 17 Olson, L., Ebendal, T. and Seiger, ,~., N G F and antiN G F : evidence against effects on fiber growth in locus coeruleus from cultures of perinatal CNS tissues, Develop. Neurosci., 2 (1979) 160-176. 18 Schwab, M. E., Otten, U., Agid, Y. and Thoenen, H., Nerve growth factor (NGF) in the rat CNS: absence of specific retrograde axonal transport and tyrosine hydroxylase induction in locus coeruleus and substantia nigra, Brain Res., 168 (1979) 473--483. 19 Smalheiser, N. R., Crain, S. M. and Bornstein, M. M., Development of ganglion ceils and their axons in organized cultures of fetal mouse retinal explants, Brain Res., 204 (1981) 159-178. 20 Stoeckel, K., Gagnon, C., Guroff, G. and Thoenen, H., Purification of nerve growth factor antibodies by affinity chromatography, J. Neurochem., 26 (1976) 1207-1211. 21 Suda, K., Barde, Y. A. and Thoenen, H., Nerve growth factor in mouse and rat serum: correlation between bioassay and radioimmunoassay determinations, Proc. nat. Acad. Sci. U.S.A., 75 (1978) 4042-4046. 22 Thoenen, H. and Barde, Y. A., Physiology of nerve growth factor, Physiol. Rev., 60 (1980) 1284-1335. 23 Turner, J. E. and Glaze, K. A., Regenerative repair in the severed optic nerve of the newt (Triturus viridescens): effects of nerve growth factor, Exp. Neurol., 57 (1977) 687-697. 24 Turner, J. E. and Delaney, R. K., Retinal ganglion cell response to axotomy and nerve growth factor in the regenerating visual system of the newt (Notophthalamus viridescens), Brain Res., 171 (1979) 197-212. 25 Turner, J. E., Delaney, R. K. and Johnson, J. E., Retinal ganglion cell response to nerve growth factor in the regenerating and intact visual system of the goldfish (Carassius attratus), Brain Res., 197 (1980) 319-330. 26 Turner, J. E., Delaney, R. K. and Johnson, J. E., Retinal ganglion cell response to axotomy and nerve growth factor antiserum treatment in the regenerating visual system of the goldfish (Carussius auratus): an in vivo and in vitro analysis, Brain Res. 204 (1981) 283-294. 27 Weidman, T. A. and Kuwabara, R., Postnatal development of the rat telina, Arch. Ophthal., 79 (1968) 470-484.
77
Extract from Brain Stimulates Neurite Outgrowth from Fetal Rat Retinal Explants JAMES E. TURNER*, YVES-ALAIN BARDE**, MARTIN E. SCHWAB and HANS THOENEN Department of Neurochemistry, Max-Planck-lnstitute for Psychiatry, 8033 Martinsried (F. R. G.) (Accepted April 22nd, 1982) Key words: neurotrophic factors - - nerve growth factor - - retinal explants
Explants from rat fetal retina were placed in culture and assayed for fiber outgrowth. In contrast to results obtained with lower vertebrates, nerve growth factor (NGF) does not seem to play a role in this system: NGF is not able to stimulate fiber outgrowth and antibodies to NGF do not block the spontaneously occurring tibet outgrowth. However, an extract prepared from pig brain is able to stimulate fiber outgrowth in a dose-dependent manner. It is suggested that such an extract can be used as a source of putative neurotrophic factors exhibiting in the mammalian central nervous system (CNS) an action similar to that of NGF in the peripheral nervous system (PNS) of mammals and in the CNS of lower vertebrates like fishes and amphibia. INTRODUCTION Recent studies in lower vertebrates have shown that regeneration o f severed optic nerves (evaluated by morphological criteria) can be enhanced by injection of a well-characterized protein, N G F za-'6 Conversely, injection o f antiserum to N G F delays the process o f regeneration 8,26. Interestingly, these in vivo effects have their parallel in vitro under welldefined conditions. Explants from goldfish retina can be placed in culture and spontaneously send out processes l°. This p h e n o m e n o n can be simulated by the addition o f low levels o f N G F . Conversely, the spontaneously occurring fiber outgrowth can be inhibited by the addition o f antibodies to N G F z6. This is therefore a useful model to study nerve regeneration in the C N S o f lower vertebrates. In higher vertebrates such as mammals, a role for N G F in normal development or after lesion has been convincingly demonstrated only in the P N S : injection o f N G F prevents the normally occurring cell death and stimulates neurite outgrowth, for example in the superior cervical ganglion o f the rat.
On the other hand, injection o f antibodies to N G F destroys the neurons which depend on N G F for survival during certain stages of development. In the rat, for example, extensive neuronal death in spinal ganglia can be induced by exposure o f antibodies to N G F before birth. The same is truc for sympathetic neurons o f the paravertebral chain, until a few days after birth (for reviews see refs, 12, 22). In this study, retinal explants from fetal rats were tested for fiber outgrowth, as has been done previously with goldfish retina explants. In the system described here, it appears that N G F and its antiserum have no effect but that an extract prepared from pig brain markedly enhances fiber outgrowth. MATERIALS AND METHODS Dissection and culture of explants Rat fetuses (gestation days 18-20) were removed from ether-anesthetized pregnant females (Wistar strain) and placed in sterile phosphate-buffered saline (PBS). Eyes were dissected, placed in PBS, opened and retinas were removed using watchmakers for-
* Present address: Department of Anatomy, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, N.C. 27103, USA. ** To whom correspondence should be addressed. 0165-3806/83/(R)0(O0000/$03.00(© 1983 Elsevier Biomedical Press
78 ceps under a dissecting microscope. At this stage of development, retinas can be cleanly stripped 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 were collected in PBS and minced into small pieces (approximately 400 600/~m squares) using forceps. Culture dishes were coated with polyornithine (Sigma, Type IB) as a substrate and equilibrated with culture medium for 2 h in 5',!,~iCO~,/95 ~',~air at 37 :C in an incubator. The culture medium consisted of L-15 (GIBCO) supplemented with 5',',~i (v/v) rat serum, additional amino acids and vitamins l~;, penicillin and streptomycin, and methylcellulose (Dow). Just prior to cxplantation, the excess medium was aspirated from the dishes. The explants were then placed on the dish with a Pasteur pipette. One drop of medium was placed over thc explants which were separated and positioned (20-30 explants/dish) by means of glass manipulators. Explants were allowed to equilibrate in a minimum amount of medium (2-3 drops/dish) overnight which allowed them to attach, after which time 1 ml of medium was added. The medium was exchanged every 4 days. Culturcs were pulsed with 10/~M cytosine arabinoside (Sigma) once a week for 4 days to prevent non-neuronal cell outgrowth. Treatment was begun on day 4 ot" culture. In order to determine if BE binds to the polyornithine substrate to exert its effects, BE (50 #g/ml) containing medium was incubated in coated dishes for 2-4 h before explantation. After 3 washes cxplants were cultured in control medium.
Evaluation of neurite outgrowth Neurite outgrowth was measured in the same manner as previously established for goldfish retinal explants l0 by determining a neurite growth index ( N G I ) score which takes into consideration two aspects of neurite outgrowth, tiber density and fiber length. The NGI was detined as the product of the neurite density score, based on a scale of 0-4, and the length score. The length score was measured by an ocular micrometer (one unit equals 10 ~m using a 10 × objective and a 12.5 x ocular) where a length of 200/~m was assigned a score of 1,400/~m a score of 2 etc. The length score for each explant was an
average of 4 measurements, one from each side of the explant. The neurite density score was determined from a standard atlas of representative explants demonstrating various degrecs of neuritc outgrowth. Triplicate culttues were established for each assay point with a total of 30 explants analyzed pcr point. For consistcncy, for each point (control and experimental) the 30 explants showing the largest tibet outgrowth wcre selected. NGI scorcs were analyzed statistically by the Student's /-test. In order to ensure that the outgrowth from explants was of neuronal origin, fibers were stained by the Holmes silvcr method 1'~ and observed by light microscopy.
Preparation of pig brain extract (BE) Whole pig brains wcre homogenized in 0.15 M (NH4),)SO4 and the pH brought to 4.5 with 1 N HCI. After centrifugation for 40 min at 20,O00g, the pH of the supernatant was brought to 6.5 with 1 N NaOH and solid (NH4)..,SO4 was added to a saturation of 70','41. After ccntrifugation for 40 rain at 20,000 g, the resulting pellet was dialyzed against 0. I M sodium phosphate buffer, pH 6.0, and applied to a CM-cellulose column equilibrated with the same buffer. Thc material cluted with a step of 0.5 M NaCI added to the phosphate buffer (after a first elution with 0.15 M NaCI) was dialyzed against watcr and lyophilized. The protein content of this fraction was determined by the method of Lowry et al. la using bovine serum albumin as a standard. In comparison to the starting homogenate, this preparation had a hundred times higher specific activity whcn tested for survival activity on 10-day-old chick embryo sensory neurons, as described pleviously (rcf. 2, and Barde, Edgar and Thoenen, submitted for publication).
Preparation of NGF and NGF-antiserum The 2.5S N G F used in this experiment was prepared according to the method of Bocchini and Angeletti 5 as modified by Suda et al.ek In this preparation one biological unit (BU) of 2.5S N G F was equivalent to 5 ng/ml as determined by the bioassay method of Fenton 7. One biological unit of N G F was neutralized by a 1:10,000 dilution of the NGF-As. In these experiments, all sera were heat inactivated at 56 ')C fbr 30 min.
79
Fig. I. Phase-contrast micrographs of 20-day-old fetal rat retinal explants 5 days after culture. A: control explant demonstrating outgrowth of mostly short, broad processes (P). B: experimental explants treated with BE (50/~g/ml). Note the extensive fiber network which appears denser and longer than those of controls. Magnification : 128 x .
~0 RESIJI.TS Under the present culture conditions there was a sparse, but distinct spontaneous outgrowth o f fibers from the rat retinal explants (Fig. 1A). With the addition o f BE to the medium (50 ,ag/tnl), the neurites became much denser with a considerable increase in length (Fig. I B). Whcn these cxplants were stained by the Holmes silver method, the fibers demonsttated a positive reaction confirming their neuritic nature (Fig. 2). Cytosine arabinoside pulses were used to prevent the outgrowth o f non-neuronal cells along ncuritcs (Fig. 1). Cytosine arabinosidc had no eff;ects on the extent of fiber outgrowth. in order for BE to elicit its effects it must bc present in the medium: BE will not bind to the polyornithine substrate, since no stimulatory effects were noted when explants were placed in control medium aftcr the substrate had been prctreated with BE.
When BE was added in increasing conccntrations to thc culture medium, there was a dose-dependent increase in the density and length o f neuritc outgrowth from the explants between l and 100/tg/ml, leveling off between I00 and 500 tlg/ml (Fig. 3). The BE-mediated increase in NG1 was approximately 5fold at 100/~g/ml when compared to control values. BE administration (50/~g/ml) to retinal explants caused a continued, statistically significant, elcvation of N G I s over controls for at least 2 weeks in culture (Fig. 4). Control explants ceased ncurite outgrowth after day 11, and subsequently began to show signs of degeneration. Exposure of the explants to N G F or N G F - A s had no effect on neurite density or length compared to controls (Fig. 5). For unknown reasons, the N G I values were slightly lower in the prescncc o f N G F than in its absence, although the difference is not statistically significant. In addition, N G F did not augment the BE-mediated response when added
Fig. 2. I,ight micrograph of 18-day-old fetal rat retinal explant treated with BE (50 !~giml~, 7 days after culture, stained by the I-Iolmes silver method for nerve fibers. Note the positively stained processes which correspond to those seen in Fig. I. Magnification : 176 ,.:.
8-
(30)
6-
/ -9
5
_=4 ¢_o
=~3
....L-
:z:
~3-
,, P'~ 0 050 w,, P.~ 0001
l-
o
i
16
I
BE CONCENIRAIION(ug/ml)
BE
Fig. 3. Dose-response study demonstrating a 5-fold increase
in NGI values elicited by BE over a concentration range of 1-100/~g/ml after 5 days in culture. Vertical bars represent S.E.M. The number in parentheses indicates total explants analyzed per point.
BE.As 8E.NGF
NGF
As
Ss
Fig. 5. NGI values were determined after 5 days in culture. Thirty explants were analyzed per group and the vertical bars represent S.E.M. BE was used at a concentration of 50 ttg/ml, NGF at 50 ng/ml, NGF-As at a 1:1000 dilution and SS (nonimmune sheep serum) at 1:1000. C is control (no addition to the medium).
20-
-
T
IB
together, nor did the c o m b i n a t i o n of BE ÷ N G F As significantly reduce the N G I s compared to BE alone (Fig. 5).
115
DISCUSSION 14
These results show that retinal explants from rat fetuses display a s p o n t a n e o u s fiber outgrowth that can be m a i n t a i n e d for up to 1 1 days in culture. The fibers could be stained positively with the Holmes silver method, indicating that they are of n e u r o n a l origin. The use of cytosine arabinoside prevented the outgrowth of n o n - n e u r o n a l cells. In contrast to goldfish retinal explants, the fiber outgrowth from rat explants is not influenced by N G F or its antiserum. Whereas in the goldfish
12-
10
8
6-
4-
2-
/*
:
"7" B15°'g'm*
0 1
2
3
t,
5
6 7 8 9 DAYS IN CbLTURE
10
'I
12
13
14
Fig. 4. Effects of BE (50 tzg/ml) on the NGI values during the first two weeks in culture. Note that BE can stimulate and sustain a significant elevation in neurite outgrowth during this time period. Growth from control explants ceases after 11 days in culture. Vertical bars represent S.E.M. Numbers in parentheses indicate total explants analyzed per group.
~2 system it is possible to markedly enhance the spontaneous fiber outgrowth by the addition of N G F and to reduce it by' NGF-As, neither effect could be observed here with rat explants. "l'hesc results are in line with what is known so far on the effect of N G F in the CNS of various species: in the goldlish and the newt, regeneration of crushed optic nerves is enhanced by injection of N G F and delayed by that of NGF-As. In mammals there is so far no detinitive evidence that N G F is able to influence fiber outgrowth in the CNS. Although there is a report by BjYrklund et al. 4 that N G F can influence the regeneration of central eatecholamine-containing tibers into pieces of iris implanted into the midbrain, others have demonstrated that N G F does not play any role in development, regeneration and regulation of central catecholamine neurons <'~.lT.ls. This lack ofeffect is in contrast to what is observed in the PNS of mammals where it has been firmly established that N G F plays a key role in the development of some sympathetic and sensory neurons. In view of the negative results obtained with NGF, it is of interest that fiber outgrowth can be enhanced by the use of extracts of CNS tissue. These experiments were prompted by the observation that medium conditioned by glioma cells a or astrocytes la as wcll as extracts from the brain" have a distinct 'neurotrophic' effect : they all are able to support the survival of cultured peripheral embryonic neurons which would otherwise die. It remains to be demonstrated that the molecule which induces fiber outgrowth from rat retinal explants is the same as that supporting the survival of peripheral embryonic neurons. This demonstration must await the purification of the survival factor(s). However, it is interesting to note that preliminary results obtained with a fraction from pig brain (obtained after further fractionation on hydroxylapatite) having a specific activity a hundred times higher on the survival of chick embryonic sensory neurons than that used in this study, turned out to be also a hundred times more active in the retinal explant system described here,
Due to the nature of the system used in this study, we do not know ~.hether or not the effcct of BE b, a direct one: the expkmts contain a variety of cell type:< including Mi.iller cells which have begun to differentiate at this stage of development~,'% making it possible that the enhanced fiber outgro~th seen ill the presence of BE is mediated via nonneuronal cells. Furthermore, the present study doc:~ not indicate which types of neurons cxhibit fiber outgrowth. Ho~vever, ganglionic cells are likely' to hc responsible since a study using a horseradish pcroxidase tracer indicated that the primary nemitc outgrowth fl'om mouse fetal retina is of ganglionic cell originl:L A degeneration of fibers and ganglion cells during the second week of culture, a phenomenon which could be prevented by co-culture ~.ith optic tectum (the normal target tissue of ganglion cells) was also noted in that study. Our control cxplants showed a similar trend in that neurite outgro~th stopped after I I day.s and there ~as some degene,ation thereafter. This was not seen whcn BE ~as added to the culture. We conclude from the data presented here that a trophic factor(s) present in the mammalian central nervous system can stimulate ncurite outgrowth from mammalian fetal retina. Some of the neurons of the fetal retina may therefore require one or more specific molecules in order to survive and/or regenerate neurites during normal development of after lesion. Pig brain extracts prepared as described here could be a source of such molecules which might play a role in the CNS of mammals similar to that played b y N G F in the PNS of mammals as well as in the CNS of lower vertebrates. A(_KNOWLEDG EM ENTS This work was made possible in part by an NIH Research Career Development Award, NS 00338. awarded to J.E.T., who was a visiting professor in the Department of Neurochemistry.
83 REFERENCES 1 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. 2 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. 3 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. 4 Bj6rklund, A., Bjerre, B. and Stenevi, V., Has nerve growth factor a role in the regeneration of central and peripheral catecholamine neurons? In K. Fuxe, L. Olson and Y. Zotterman (Eds.), Dynamics of Degeneration and Growth in Neurons, Pergamon Press, New York, 1974, pp. 389~,09. 5 Bocchini, V. and Angeletti, P. U., The nerve growth factor: purification as a 30,000 molecular weight protein, Proc. nat. Acad. Sci. U.S.A., 64 (1969) 787-794. 6 Coyle, J. T., Jacobowitz, D., Klein, D. and Axelrod, J., Dopaminergic neurons in explants of substantia nigra in culture, J. NeurobioL, 4 (1973) 461-470. 7 Fenton, E. L., Tissue culture assay of nerve growth factor and of the specific antiserum, Exp. Cell Res., 59 (1979) 83-92. 8 Glaze, K. A. and Turner, J. E., Regenerative repair in the severed optic nerve of the newt (Triturus virideseens); Effect of nerve growth factor antiserum, Exp. Neurol., 58 (1978) 500-510. 9 Konkel, R. T., Mailman, R. B., Bendeich, E. G., Garrison, A. M., Mueller, R. A. and Breese, G. R., Evaluation of the effects of nerve growth factor and anti-nerve growth factor on the development of central catecholcontaining neurons, Brain Res., 144 (1978) 277-285. 10 Landreth, G. E. and Agranoff, B. W., Explant culture of adult goldfish retina. A model for the study of CNS regeneration, Brain Res., 161 (1979) 39-53. 11 Letourneau, P . C . , Possible roles for cell-to-cell substratum adhesion in neuronal n,orphogenesis, Develop. Biol., 44 (1975) 77-91. 12 Levi-Montalcini, R. and Angeletti, P. U., Nerve growth factor, Physiol. Rev., 48 (1968) 534-569. 13 Lindsay, R. M., Adult rat brain astrocytes support survival of both NGF-dependent and NGF-independent neurons, Nature (Lond.), 282 (1979) 80-82. 14 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. bioL Chem., 193 (1951) 265-275.
15 Luna, L. G., Methods for nerve cells and fibers: Holmes method for nerve cells and fibers. In L. G. Luna (Ed.),
Manual of the Histologic Staining Methods oJ the Armed Forces Institute of Pathology, McGraw-Hill, New York, 1968, pp. 196-198. 16 Mains, R. E. and Patterson, P. A., Primary cultures of dissociated sympathetic neurons. 1. Establishment of long-term growth in culture and studies of differentiated properties, J. Cell Biol., 59 (1973) 329-345. 17 Olson, L., Ebendal, T. and Seiger, ,~., N G F and antiN G F : evidence against effects on fiber growth in locus coeruleus from cultures of perinatal CNS tissues, Develop. Neurosci., 2 (1979) 160-176. 18 Schwab, M. E., Otten, U., Agid, Y. and Thoenen, H., Nerve growth factor (NGF) in the rat CNS: absence of specific retrograde axonal transport and tyrosine hydroxylase induction in locus coeruleus and substantia nigra, Brain Res., 168 (1979) 473--483. 19 Smalheiser, N. R., Crain, S. M. and Bornstein, M. M., Development of ganglion ceils and their axons in organized cultures of fetal mouse retinal explants, Brain Res., 204 (1981) 159-178. 20 Stoeckel, K., Gagnon, C., Guroff, G. and Thoenen, H., Purification of nerve growth factor antibodies by affinity chromatography, J. Neurochem., 26 (1976) 1207-1211. 21 Suda, K., Barde, Y. A. and Thoenen, H., Nerve growth factor in mouse and rat serum: correlation between bioassay and radioimmunoassay determinations, Proc. nat. Acad. Sci. U.S.A., 75 (1978) 4042-4046. 22 Thoenen, H. and Barde, Y. A., Physiology of nerve growth factor, Physiol. Rev., 60 (1980) 1284-1335. 23 Turner, J. E. and Glaze, K. A., Regenerative repair in the severed optic nerve of the newt (Triturus viridescens): effects of nerve growth factor, Exp. Neurol., 57 (1977) 687-697. 24 Turner, J. E. and Delaney, R. K., Retinal ganglion cell response to axotomy and nerve growth factor in the regenerating visual system of the newt (Notophthalamus viridescens), Brain Res., 171 (1979) 197-212. 25 Turner, J. E., Delaney, R. K. and Johnson, J. E., Retinal ganglion cell response to nerve growth factor in the regenerating and intact visual system of the goldfish (Carassius attratus), Brain Res., 197 (1980) 319-330. 26 Turner, J. E., Delaney, R. K. and Johnson, J. E., Retinal ganglion cell response to axotomy and nerve growth factor antiserum treatment in the regenerating visual system of the goldfish (Carussius auratus): an in vivo and in vitro analysis, Brain Res. 204 (1981) 283-294. 27 Weidman, T. A. and Kuwabara, R., Postnatal development of the rat telina, Arch. Ophthal., 79 (1968) 470-484.