ciliary neurotrophic factor stimulates neurite outgrowth from spinal cord neurons

THE JOURNAL OF COMPARATIVE NEUROLOGY 364:68-77 (1996)

Ciliary Neurotrophic Factor Stimulates Neurite Outgrowth From

Spinal Cord Neurons

NELSON M. OYESIKU AND DONALD J. WIGSTON Departments of Neurosurgery (N.M.O.) and Physiology (D.J.W.) and Neuroscience Graduate

Program (N.M.O., D.J.W.), Emory University School of Medicine, Atlanta, Georgia 30322

ABSTRACT Ciliary neurotrophic factor (CNTF) has been shown to promote the survival of motoneu-

rons, but its effects on axonal outgrowth have not been examined in detail. Since nerve growth factor (NGF) promotes the outgrowth of neurites within the same populations of neurons that depend on NGF for survival, we investigated whether CNTF would stimulate neurite outgrowth from motoneurons in addition to enhancing their survival. We found that CNTF is a powerful promoter of neurite outgrowth from cultured chick embryo ventral spinal cord neurons. An effect of CNTF on neurite outgrowth was detectable within 7 hours, and at a concentration of 10 ngiml, CNTF enhanced neurite length by about 3- to 4-fold within 48 hours. The neurite growth-promoting effect of CNTF does not appear to be a consequence of its survival-promoting effect. To determine whether the effect of CNTF on spinal cord neurons was specific for motoneurons, we analyzed cell survival and neurite outgrowth for motoneurons labeled with diI, as well as for neurons taken from the dorsal half of the spinal cord, which lacks motoneurons. We found that the effect of CNTF was about the same for motoneurons as it was for neurons from the dorsal spinal cord. The responsiveness of a variety of spinal cord neurons to CNTF may broaden the appeal of CNTF as a candidate for the treatment of spinal cord injury or disease. c 1996 Wiley-Liss, Inc.

Indexing terms: CNTF, motoneuron, axonogenesis, diI labeling, neuronal cell culture

Neurotrophic factors (NTFs) are of great interest because of their role in the regulation of neural development and regeneration. NTFs identified so far fall into 3 main classes: the neurotrophins (e.g., nerve growth factor, NGF), the cytokines (e.g., ciliary neurotrophic factor, CNTF), and the fibroblast growth factors (e.g., bFGF; Patterson, 1992; Korsching, 1993; Baird, 1994). Most NTFs have been identified or evaluated based on their ability to promote the survival of neurons. For instance, NGF promotes the survival of sympathetic and dorsal root ganglion neurons in culture (Levi-Montalcini and Angeletti, 1963; Hamburger et al., 1981). In addition, NGF can rescue dorsal root ganglion neurons from developmental cell death in vivo (Hamburger et al., 1981). On the other hand, some NTFs seem to function by regulating neuronal phenotype (Rao and Landis, 1990). It is likely that many NTFs regulate both cell survival and phenotype. Indeed, NGF promotes the survival of, and neurite outgrowth from, neurons of both dorsal root ganglia and sympathetic ganglia (Levi- Montalcini and Hamburger, 1951, 1953; Thoenen et al., 1971; Menesini-Chen et al., 1978). It is thus likely that other NTFs promote neurite outgrowth from the neurons whose survival they support.

A particularly valuable neurotrophic factor would be one that enhances the growth of processes of neurons in the mammalian spinal cord, because of the high frequency and severe consequences of spinal cord injury in humans. If such a factor were identified, it might be possible to improve the recovery of function after spinal cord injury by stimulating axonal growth. Several neurotrophic factors have been identified which affect spinal cord neurons. The most extensively studied of these is ciliary neurotrophic factor (CNTF), which was originally isolated based on its ability to promote the survival of motoneurons from the chick ciliary ganglion (Manthorpe et al., 1982; Barbin et al., 1984). Although CNTF has been known for several years to promote the survival of motoneurons both in culture and in vivo (Arakawa et al., 1990; Sendtner et al., 1990, 1991; Oppenheim et al., 1991), not much is known about the effect of CNTF on neurite outgrowth from motoneurons, or from other classes of spinal neurons. Evidence suggesting that CNTF has a neurite growth-promoting effect on ciliary

Accepted 6, July 1995 Address reprint requests to Dr. Donald J. Wigston, Dept. of Physiology,

Emory University School of Medicine, Atlanta, GA 30322.

O 1996 WILEY-LISS, INC.

CNTF PROMOTES NEURITE OUTGROWTH 69

ganglion neurons (Varon et al., 1979; Unsicker and Wie- gandt, 1988) encouraged us to study the effect of CNTF on neurite outgrowth from spinal cord neurons. We found that human recombinant CNTF is a strong promoter of neurite outgrowth from chick embryo spinal neurons grown in culture. Furthermore, although previous experiments inves- tigating the effects of CNTF on spinal neurons have emphasized the effects of CNTF on motoneurons, we found that the influence of CNTF on spinal cord neurons is not restricted to that class of spinal neurons, but extends to neurons of the dorsal spinal cord. Effects of CNTF on these neurons have not been reported before. Some of our results have been reported previously in abstract form (Oyesiku and Wigston, 1992).

MATERIALS AND METHODS Cell culture

Culture dishes were prepared by attaching a cleaned glass coverslip to the bottom of a 35-mm plastic tissue culture dish in which a 16-mm hole had been drilled. The coverslips were then coated with poly-DL-ornithine (0.5 mgiml in borate buffer), rinsed with phosphate-buffered saline (PBS), and incubated for 5-6 hours with laminin (10 pgiml in serum-free culture medium). The excess laminin was removed just before plating cells.

The lumbar spinal cords of chicken embryos (HiLine, Mansfield, GA) on the sixth day of incubation (E6) were removed and cut into short segments. Spinal cord segments from 5 to 8 embryos were divided into dorsal and ventral hemisegments, and the dorsal segments were discarded, except when we wished to examine the growth of dorsal spinal cord neurons. Spinal cord segments were treated with 0.03% trypsin in Hanks’ BSS for 20 minutes at 37”C, washed in Hanks’ without trypsin, and individual cells dissociated by trituration. The cell suspension was then filtered through nylon mesh (50 pm pore size; Spectra) to remove cell aggregates and centrifuged to recover the cells. The cells were then resuspended in a culture medium consisting of Leibovitz’s (L-15) medium supplemented with glucose (23 mM), sodium bicarbonate (24 mM), horse serum (10%; GIBCO), penicillin (100 Uiml), streptomycin (100 Kgiml). The recovered cells were counted in a hemocy- tometer, and tested for viability using propidium iodide to label the nuclei of nonviable cells.

The dissociated cells were normally plated at a density of about 5,000 cellsicm2 onto the laminin-coated glass coverslips. In some experiments, however, the initial plating density was systematically varied so that we could compare the extent of neurite outgrowth from neurons grown at different densities. After plating, cells were allowed to settle for 1 hour, then medium with or without CNTF (recombinant human CNTF, a g f t from Synergen) was added. In some experiments we also used bFGF (50 ngiml; R&D Systems) and BDNF (50 ngiml; Regeneron). Mitotic inhibitors (15 pg/ml 5-fluoro-2’-deoxyuridine and 35 pgiml 2‘-deoxyuridine) were also added at the time of plating to prevent the proliferation of non-neuronal cells. Neurons were cultured at 36°C in an atmosphere containing 5% C02.

DiI labeling To distinguish motoneurons from other types of spinal

cord neurons, in some experiments motoneurons were labeled with the fluorescent lipophilic dye diI (1,l’-dioctadecyl- 3,3,3’ 3-tetramethyl indocarbocyanine perchlorate; Molecu-

lar Probes, Eugene, OR). Lumbar spinal motoneurons were labeled retrogradely by injecting about 0.1 pl of diI dis- solved in ethanol from a glass micropipet into the muscle masses of left and right hindlimb buds of chicken embryos maintained in vitro (Honig and Hume, 1986). To obtain satisfactory numbers of dorsal neurons in culture it was necessary to use spinal cords from E8 embryos, instead of the usual E6, because the development of the dorsal spinal cord lags behind the ventral cord (Hamburger, 1948). Twelve to 16 hours later the lumbar spinal cords were removed and cells prepared for culture as described above. The spinal cords were divided into dorsal and ventral segments prior to dissociation, and dorsal and ventral neurons were cultured separately. Although a few motoneurons may not have been labeled by diI using this method, it is likely that the majority of unlabeled neurons in ventral spinal cord cultures were not motoneurons.

Data collection and analysis All cell counts and neurite length measurements were

made blind. Cell densitg. Although it was not a primary aim of these

experiments to analyze the influence of CNTF on cell suruiual, cell counts were made so that any influence of cell density on neurite outgrowth could be detected. Also, since the effect of CNTF on cell survival has been well docu- mented, we were able to use cell survival to verify the effectiveness of our sample of CNTF. Furthermore, by monitoring survival, our study would provide information regarding the effect of CNTF on the survival of dorsal spinal cord neurons. CNTF has not previously been tested with this class of neuron.

Neurons were counted in 10 randomly chosen microscope fields of a known area in each of 3 dishes per condition using a 20 x or 40x objective and phase-contrast optics. Counts of adherent cells were carried out 3 hours after plating, and counts of process-bearing cells were carried out on cells fixed at 48 hours. Cell survival was estimated by expressing the density of process-bearing cells at 48 hours as a percentage of the density of adherent cells at 3 hours. These criteria reduce the possibility that counts would be biased by the inclusion of non-neuronal cells, which do not elabo- rate extensive processes. However, they might result in a failure to count some neurons at 48 hours if those cells failed to extend processes. To further guard against this, in many experiments we also counted cells labeled with the C-fragment of tetanus toxin, which selectively binds to neurons (Mirski et a]., 1978). Cells binding tetanus toxin were visualized with an antibody to tetanus toxin followed by a fluorescent secondary antibody (fluorescein-anti-IgG; Boeringer-Manheim). This confirmed that the cells we were counting were neurons, and that there were very few non-neuronal cells present.

Neurite length. After 48 hours in culture, 10 randomly chosen neurons in each dish were examined using a 4 0 ~ objective on a Zeiss Axiovert 35 microscope, and either a Newvicon (Dage) or CCD (Optronics) camera. The length of individual neurites 10 pm or more in length was measured directly from live images using an image analysis system (Image-1). To help avoid problems distinguishing true neurites from other kinds of processes such as filopodia or lamellopodia, we made our measurements using phase contrast microscopy which does not resolve fine filipodia well. To simplify our earliest experiments, we initially measured only the length of each neurite that emerged

70 N.M. OTESIKU AND D.J. WIGSTON

from the cell body (“major neurite”), from its base to the end of its longest branch, and ignored side branches. In all subsequent experiments, however, from which we con- structed Figures 2 ,3 ,5 and 6, we measured the total length for each neurite emerging from the cell body, including all side branches. Since it was not possible to distinguish axons from dendrites in these cultures, all processes were consid- ered equivalent and referred to as neurites. These data were used to calculate the mean number of neurites per cell, the mean neurite length, and the mean total neuritic length per cell.

Except where noted, all tissue culture reagents were obtained from GIBCO (Grand Island, NY) and chemicals from Sigma (St. Louis, MO) or Fisher Scientific (Atlanta, GA).

RESULTS Ventral spinal cord neurons show enhanced

neurite outgrowth in CNTF In initial experiments we investigated the effect of CNTF

at 10 ngiml on neurons from the ventral half of the lumbar spinal cord. This population of neurons is enriched for motoneurons, which have been shown to be responsive to CNTF (Arakawa et al., 1990). Not only were more neurons present in CNTF-treated cultures, but they also appeared to have much longer processes (Fig. 1). To verify this impression, we measured the length of each major neurite, ignoring side branches (see Materials and Methods), on neurons cultured for 48 hours in the presence or absence of CNTF. We found that CNTF at 10 ngiml caused a 2.5-fold increase in the length of the major neurites on each neuron: the mean length in 61 CNTF-treated neurons was 365 % 16 pm (mean * standard error of the mean), whereas the mean in 67 nontreated neurons was 152 * 10 pm. Our results indicate therefore that CNTF stimulates neurite outgrowth from cultured chick spinal neurons, in addition to its well known effect on neuronal survival.

In additional experiments we characterized the effect of CNTF on neurite outgrowth from ventral spinal cord neurons by measuring the total length of all neurites, including all side branches, on each neuron examined. We also tested a range of concentrations of CNTF to establish a dose-response curve for the stimulation of neurite growth. Furthermore, we also examined the dose-response relationship for the effect of CNTF on neuronal survival. We chose a range of CNTF concentrations between 0.001 ngiml and 100 ngiml, over which CNTF has previously been shown to promote neuronal survival (Arakawa et al., 1990). The dose-response curve for the effect of CNTF on neurite length is shown in Figure 2. In CNTF-treated cultures there was a greater than 3-fold enhancement of neurite growth: the mean neurite length was 94 t 17 pm in 119 nontreated neurons, compared with 293 t 18 pm in 127 neurons treated with CNTF at 10 ngiml. At this concentration the effect of CNTF on neurite length seemed to saturate (see Fig. 2).

We also measured the total neurite length for each neuron, which depends on both the mean neurite length and the number of neurites per cell. CNTF at 10 or 100 ngiml produced about a 4-fold increase in the mean total neurite length per neuron (252 2 18 km versus 1,012 & 78 pm) compared with the approximately 3-fold increase in mean neurite length (Fig. 3). A somewhat surprising find- ing was that the effect of CNTF on mean neurite length and

Fig. 1. Effect of ciliary neurotrophic factor (CNTF) on chick embryn spinal cord neurons. Neurons were dissociated from E8 chick embryo ventral spinal cords and cultured without (A) and with (B) CNTF (10 ngiml). Cells were fixed after 48 hours in culture and photographed. The neurons grown in CNTF (B) show more extensive neurite outgrowth than those grown in control medium (A). Scale bar, 50 pm.

total neurite length per cell spanned a very wide range of concentrations, up to 4 orders of magnitude (Figs. 2,3).

CNTF does not promote the initiation of major neurites

To see if CNTF increased the number of neurites, we analyzed the total number of major neurites emerging from the soma of each cell for the same neurons used to construct Figure 2. The number of neurites per cell in the presence of CNTF at 0.001 ngiml was 2.4 t 0.1, whereas in 100 ngiml CNTF it was 3.5 * 0.3 (P < .025). Moreover, there was a trend towards a greater number of neurites in increasing concentrations of CNTF (Table 1). From this it appears that CNTF might increase the number of neurites, in addition to their length. However, we found that the mean number of neurites in control medium (2.9 * 0.4) was not significantly different from the values obtained at 100 ngiml (3.5 2 0.3; P < .3). Furthermore, in subsequent experiments carried out to examine the effect of cell density on neurite length, we found no evidence for a significant effect of CNTF on neurite number. For 466 neurons grown in the


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Fig. 2. Effect of CNTF on the length of individual neurites. Neurons from E6 chick embryo ventral spinal cords were cultured in the absence of CNTF (control), or in varying concentrations of CNTF for 48 hours. Neurons were then fixed and individual neurites were measured with video image analysis software. Each point represents the mean neurite length 2 the standard error of the mean for three different experiments in which we analyzed at least 10 neurons in each of 3 separate culture dishes for each condition. Thus, each point represents data from about 100 or more neurons. The standard error of the data a t ICNTFl = 0.1 ngiml was too small to display. The effect of CNTF on individual neurite length seemed to saturate between 1 and 10 ng!ml; a t these concentrations mean neurite length was increased about 3-fold compared with control.

absence of CNTF the mean number of major neurites was 2.55 2 0.05, whereas for 461 neurons grown for 48 hours in the presence of CNTF at 10 ngiml, the mean number of major neurites was not significantly different at 2.67 t 0.06 ( P < .1). While this suggests that CNTF does not promote the initiation of new neurites, it is still possible that CNTF might stimulate the branching of existing neurites.

The effect of CNTF on neurite length is not restricted to motoneurons

To investigate whether the effect of CNTF on spinal cord neurons is limited to motoneurons, we selectively labeled motoneurons with diI and cultured them for 48 hours (see Materials and Methods). The distribution of diI within the spinal cord is indicated in the fluorescence micrograph in Figure 4A. DiI was found primarily in the ventral horn of the spinal cord, but, as expected, also in neurons of dorsal root ganglia and their central axons within the dorsal horn. We found that CNTF enhanced the growth of neurites from diI-labeled motoneurons by about 4.5-fold: the total neurite length per neuron was increased from 260 ? 18 pm in control medium to 1158 t 51 pm in CNTF (Fig. 5). The relative magnitude of this increase was similar to that observed with unlabeled ventral neurons in other experiments (see Fig. 3). To examine the specificity of action of CNTF we compared the response of diI-labeled motoneu-

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individual neurons. Total neurite length per cell increased by 4-fold in the highest concentrations of CNTF tested, reflecting an increase in both the length of individual neurites (Fig. 2) and a trend towards an increased number of neurites per cell (Table 1).

TABLE 1. Effect of CNTF on Ventral Spinal Cord Neurons

ICNTFJ (ng/ml) Cell survival (% Neurites per cell2

Control 8.6 z 0.3 2.9 I 0.4 10 10.3 ? 1.6 2.4 2 0.1 10 '' 1 3 8 ~ 2 0 2 4 2 0 1 10 ' 15.3 i 2.5 2.9 ? 0.2 1 16.9 i 1.9 3.3 2 0.1

10 32.8 2 2.2 3.3 ? 0.1 1 0 2 20.0 i 2.0 3.5 z 0.3

'Cells surviving a t 4 X hours expressed as a percentage of the number of cells present a t 3 hours (mean z standard error of the mean 1. 'Mean number of neurites per cell.

rons to CNTF with that of neurons we could be certain were not motoneurons. Since some of the unlabeled neurons in our cultures of ventral spinal cord might be motoneurons that did not take up diI, we examined neurons cultured from the dorsal half of the lumbar spinal cord, in which there are no motoneurons. The results are shown in Figure 5. The total neurite length per cell of untreated dorsal neurons was less, on average, than that of untreated diI-labeled neurons (156 * 11 pm versus 260 ? 18 pm; P < .001). This may be related somehow to the fact that these two classes of neurons develop at different times in vivo, and were therefore obtained from embryos of different ages (see Materials and Methods). Furthermore, the spinal cords from diI-injected animals were maintained overnight before dissociation and plating, to allow for transfer of diI from the hindlimb buds to the cell bodies of the motoneurons, whereas the dorsal neurons were plated immediately. How- ever, in spite of the differences in absolute neurite length

72 N.M. OYESIKU AND D.J. WIGSTON

Fig. 4. Selective labeling of chick embryo motoneurons with diI. Hindlimb buds of E6 embryos were injected with diI (for this illustra- tion only the right side) to retrogradely label motoneurons. A is a photomicrograph of a cross-section of a spinal cord from such an embryo taken under simultaneous illumination with both fluorescence excitation light and polarized transmitted light. Fluorescently labeled motoneurons appear in the lateral motor column (lmc) on the right side, in addition to a few labeled neurons in the right dorsal root

ganglion (drg) and their projections into the dorsal horn of the spinal cord (dorsal funiculus, do. Scale bar = 100 pm. B is a phase contrast photomicrograph of a field of neurons dissociated from the ventral spinal cord of an injected embryo, fixed and photographed 24 hours aftcr placing the neurons in culture. C is a fluorescence micrograph of the same field as in B. Not all cells visible in B are labeled with diI (arrow in B). The diI-labeled cells are most likely motoneurons. Scale bar in C, 20 pm and applies also to B.

between these two samples of neurons, the relative increase in neurite length produced by CNTF treatment was about the same (approximately 4-fold) in cultures of dorsal neurons as for diI-labeled motoneurons from the ventral spinal cord (586 i 30 pm versus 156 i 11 pm; see Fig. 5).

treated cultures was higher than in control cultures. It has been noted that some central nervous system (CNS) neurons, for example, rat hippocampal neurons, tend to extend neurites more vigorously when grown at higher cell densities (Banker and Cowan, 1977; Fletcher et al., 1994). Therefore, the effect of CNTF on neurite outgrowth might be an indirect effect of the elevated cell density in CNTF- treated cultures. To investigate this, we examined the

CNTF promotes the survival of dorsal spinal cord neurons

We also examined whether CNTF would promote the survival of neurons from the dorsal spinal cord, by counting the number of neurons remaining after 48 hours in culture. From our counts we estimated that 61 & 12% of the neurons present at 3 hours after plating survived for 48 hours in the presence of CNTF at 10 ngiml, compared with only 37 * 5% for neurons cultured without CNTF. As far as we are aware, this is the first report of an effect of CNTF on survival of neurons from the dorsal spinal cord. While this result supports our conclusion that CNTF influences a variety of different neuronal types within the spinal cord, our results do not exclude the possibility that certain types of spinal cord neurons might not respond to CNTF, or that some might respond more than others.

relationship between cell density and neurite length by plating E6 ventral spinal cord neurons at different densities and culturing them in the presence or absence of CNTF. This was complicated by the fact that cell density is continuously changing in these cultures owing to the gradual death of neurons. Therefore we counted the number of neurons in randomly chosen microscope fields at 24 hours after plating, the median time-point of our culture period, and used these counts to estimate the density of surviving cells in each dish at 24 hours. As shown in Figure 6, we found no evidence for an influence of cell density on neurite length over a 30-fold range of density, far beyond that encountered in cultures plated at our standard density ( - 5,000 cells/cm2) with or without CNTF. Furthermore, across the range of densities tested, neurons cultured in

The effect of CNTF on neurite outgrowth is independent Of its effect On density

CNTF at 10 ng/ml showed about the same 4-fold increase in neurite length compared with control neurons as when dated at our standard densitv of 5.000 cells/cm2. We

One consequence of the increased survival of neurons in CNTF was that the final density of neurons in CNTF-

therefore conclude that there is"no detectable effect of cell densityonneuritelength.

CNTF PROMOTES NEURITE OUTGROWTH

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Fig. 5. Effect of CNTF on different types of spinal cord neurons. The average total neurite length per cell is compared for three different populations of neurons, in the presence of CNTF at 10 ngiml (CNTF) and in its absence icontrol). Ventral: neurons of the ventral spinal cord; motor: diI-labeled motoneurons from the ventral spinal cord; dorsal: neurons from the dorsal spinal cord. Although the mean total neurite length per neuron was less for dorsal spinal cord neurons than for ventral neurons or diI-labeled motoneurons, CNTF produced the same relative increase in neurite length for each of these three classes of neuron.

Individual neurites extend more in the presence of CNTF

Although the effect of CNTF on neurite outgrowth was not secondary to the elevated cell density which results from increased cell survival in CNTF, it is conceivable that decreased cell viability in the absence of CNTF compro- mised neuronal growth and contributed to the differences in neurite lengths which we observed in the presence and absence of CNTF. Cell survival over the first 24 hours in culture was 34.1 i_ 3.1% in CNTF (10 ngiml) versus 17.2 ? 1.6% in control medium, compared with 32.8 ? 2.2 and 8.3 f 0.6% a t 48 hours. Thus, about half the control neurons present at 24 hours died between 24 hours and 48 hours, whereas there was not significant further cell loss in CNTF-treated cultures over this time. To reduce the likeli- hood that declining viability in the second 24 hours of culture might contribute to our results, we studied the growth of identified neurites over a period of 19-21 hours, beginning at 7-9 hours after plating. Many of the cells we observed initially died between the time of the first observa- tion and the second one about 20 hours later. We measured the change in length of neurites on those cells that survived. Data from 60 control cells in 18 different culture dishes and 57 CNTF-treated cells in 17 culture dishes showed that the average neurite lengths increased from 40 5 3 pm to 91 t 11 pm in control medium and from 64 ? 4 pm to 162 _t 14 pm in 10 ng/ml CNTF. Furthermore, the mean change in length of individual neurites was 54 _t 10 pm in control versus 105 % 12 pm in CNTF. Thus, there is a significant effect of CNTF on neurite length within the first 24 hours in culture, which was detectable as early as 7

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ventral spinal cord neurons was varied to achieve a range of cell densities of 617-17,421 neuronsicm2 at 24 hours after plating. At 48 hours, cells were fixed and their neurite lengths measured. Each symbol represents the mean neurite length obtained from a single culture dish, usually from 10 neurons (range 8-14). There was no detectable influence of density on mean neurite length in either control cultures (466 neurons examined) or cultures treated with 10 ng/ml CNTF (461 neurons). The coefficient of determination, r2, was 0.013 for control neurons and 0.034 for CNTF-treated neurons. Neurite length was about 4-fold longer in CNTF-treated than control cultures.

hours after plating. The distribution of changes in neurite length for neurons in control medium and CNTF-containing medium are shown in Figure 7. Twenty-one percent of control neurites did not grow at all or retracted partially or completely. A similar number (23%) of CNTF-treated neurites behaved similarly. If neurite growth is arrested or retarded in the absence of CNTF, we would expect to observe a larger proportion of very short or retracting neurites in the absence of CNTF. However, this is not evident in Figure 7. Instead, our data indicate no increase in the number of stunted neurites in control cultures. On the contrary, there was an increase in the number of very long processes in the presence of CNTF. For example, 66 of 230 CNTF-treated neurites grew 100 pm or more over this time, compared with only 28 of 201 control neurites. Fourteen CNTF-treated neurites exceeded 500 km in length, whereas only 5 control neurites grew 500 pm or more. These data are consistent with a neurite-growth stimulating action of CNTF.

Other agents that promote survival do not promote neurite outgrowth

If the effect of CNTF on neurite growth was secondary to its effect on cell survival, other factors which promote cell survival should also have an effect on neurite outgrowth. To investigate this, in a separate set of 4 experiments we grew neurons for 48 hours in the presence or absence of CNTF


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Fig. 7. Changes in length of individual neurites within the first 24 hours in culture. Images of identified control and CNTF-treated cells were collected at 7-9 hours after plating and again 19-21 hours later. From the two images of each of 201 control neurites and 230 CNTF- treated neurites, the change in length over the 19-21 hour growth period was measured. The frequency distribution of these changes is shown here. Negative values on the x-axis indicate a net reduction in length by either partial retraction or total withdrawal of the neurite; zero values are included in the 0-100 bin.

(10 ngiml), bFGF (50 ng/ml) or BDNF (50 ngiml), all of which have been shown to promote survival of spinal cord neurons (Arakawa et al., 1990; Martinou et al., 1992; Oppenheim et al., 1992; Sendtner et al., 1992; Yan and Snider, 1992; Koliatsos et al., 1993). We examined between 160 and 193 neurons for each condition (total 721). As shown in Table 2, all three agents promoted cell survival. However, there appeared to be no correlation between the magnitude of the survival promoting effect and any increase in total neurite length per cell. For example, CNTF

TABLE 2. Comparison of the Effect of CNTF, BDNF and bFGF on Survival and Neurite Length

% Control % Control neurite Agent survival l length per cell

CNTF (10 ngimi) 139 ? 15 197 2 30 BDNF (50 ng/ml) 132 2 18 154 r 15 bFGF (50 ngiml) 159 2 28 114 2 18

‘From a separate set of experiments from that presented in Table 1. Data are expressed as a percentage of values in control medium. Means 2 standard errors of the mean are presented.

increased survival by 39% versus control, on average, and total neurite length per cell by 97%, while bFGF increased survival by 59%, but increased total neurite length per cell by only 14%. Thus, even though CNTF, BDNF and bFGF all increased neuronal survival to a roughly similar degree, only CNTF and BDNF significantly affected neurite length. Furthermore, the effect of BDNF on neurite growth was not as dramatic as that of CNTF. Neurite length does not therefore appear to be correlated with neuronal survival.

DISCUSSION We found that CNTF is a potent promoter of neurite

outgrowth from cultured chick embryo spinal cord neurons. At its most effective concentrations, CNTF induced a 3- to 4-fold increase in the length of individual neurites elaborated within a 48-hour period compared with untreated neurons. CNTF did not have a significant effect on the number of major neurites emerging from each neuron. However, since we did not analyze the number of minor branches per neurite separately, though they are included in our measurements of total neurite length, it is still possible that part of the effect of CNTF on neurite length might be attributed to an ability to promote the formation of side branches.

Previous studies of the effects of CNTF on spinal cord neurons have examined only motoneurons (Arakawa et al., 1990; Sendtner et al., 1990; Nurcombe et al., 1991; Oppen- heim et al., 1991). However, we found that the effect of CNTF on neurite length was not limited to motoneurons, but was also evident with other neurons in the ventral spinal cord and neurons of the dorsal spinal cord. This is consistent with reports indicating that CNTF has a survival- promoting effect on a relatively wide range of neuronal types in tissue culture, such as chick ciliary ganglion neurons, dorsal root ganglion neurons, sympathetic ganglion neurons and hippocampal neurons (Adler and Varon, 1982; Barbin et al., 1984; Manthorpe et al., 1986; Pettmann et al., 1988; Eckenstein et al., 1990; Ip et al., 19911, (but see also Oppenheim et al., 1991; Kessler et al., 1994). Although we cannot say that all chick embryo spinal neurons are sensitive to CNTF, our results show that CNTF-responsive neurons are distributed fairly widely within the spinal cord.

It is conceivable that the effects of CNTF on neurite outgrowth are a consequence of its effect on cell survival. For instance, some neurons tend to extend neurites more vigorously when grown at higher densities (Banker and Cowan, 1977; Fletcher et al., 19941, suggesting that the effect of CNTF on neurite outgrowth might be a consequence of the elevated cell density in CNTF-treated cultures resulting from increased cell survival. In our experiments we found that over a wide range of cell densities extending far beyond that found in our cultures of either control or CNTF-treated neurons there was no discernible correlation between neuronal density and neurite length


(Fig. 6). Therefore the effect of CNTF on neurite length is not secondary to the elevated cell density in CNTF-treated cultures. Nevertheless, our experiments do not directly address whether the survival-promoting and neurite- promoting effects of CNTF are strictly independent. Indeed the range of concentrations over which CNTF affected cell survival and neurite length was similar, consistent with both effects being mediated by a common receptor. Of particular interest, therefore, is the possibility that the apparent effect of CNTF on neurite outgrowth is not a direct effect, but is a consequence of the retarded or arrested growth of neurons in the CNTF-deprived cultures. This is unlikely for several reasons. First, the distribution of changes in the length of individual, identified neurites measured on live neurons at two separate times about 20 hours apart shows that neurons cultured in the absence of added CNTF are not strikingly abnormal. These neurons extend processes, and show no signs of an increased frequency of neurite retraction or growth arrest. The main difference between control and CNTF-treated neurons is that there tends to be a greater number of neurites which extend long distances in the presence of CNTF. Second, even as early as 7 hours after plating, neurites were on average larger in CNTF-treated cultures, which is difficult to attribute to poor control cell viability. Third, if CNTF- treated neurons had longer neurites than control neurons simply because of a reduced viability of neurons in the absence of CNTF, we would expect that other agents which promote neuronal survival would also cause an apparent increase in neurite outgrowth. Our findings show that this is not the case: bFGF and BDNF, agents known previously to promote motoneuron survival, both increased neuronal survival to about the same level as did CNTF. However, bFGF failed to show any significant effect on neurite outgrowth. BDNF showed an effect on neurite outgrowth, but less than that of CNTF. Thus, the stimulation of neurite growth by CNTF is not a necessary consequence of its effect on cell survival. Finally, it is worthy of note that exogenous CNTF promotes axonal sprouting from the synaptic terminals of motor axons in adult muscle (Gurney et al., 19921, demonstrating directly that CNTF can promote neurite outgrowth independently of its effect on cell survival.

Although CNTF has been found to have a survival- promoting effect on a variety of neuronal types, and we have shown that it also has a neurite growth-promoting effect, the physiological role of CNTF is not clear. Several aspects of the biology of CNTF do not seem to fit with the standard neurotrophic factor hypothesis (Davies, 1988; Korsching, 1993). For instance, CNTF is present in substan- tial quantities in vivo (Manthorpe et al., 19861, much more so than other neurotrophic factors such as NGF. This suggests that its supply may not be limiting and therefore that it is unlikely that neurons compete for its uptake. Competition for trophic factors is thought to be the mechanism by which neurotrophic factors regulate neuronal survival. Furthermore, CNTF mRNA is still barely detectable by the time normal motoneuron death is over (Stockli et al., 19891, making it unlikely that CNTF regulates the survival of motoneurons during early development. In addition, disruption of the CNTF gene in transgenic mice has not revealed any striking developmental abnormalities, except for slight neuromuscular dysfunction in adults (Masu et al., 1993). The main source of CNTF, at least for motoneurons, does not appear to be their synaptic target cells. On the contrary, the largest quantities of CNTF

mRNA are found within the myelinating Schwann cells of peripheral nerves, and CNTF mRNA is notably absent in skeletal muscles (Williams et al., 1984; Stockli et al., 1989; Dobrea et al., 1992; Friedman et al., 1992; Rende et al., 1992). Surprisingly, CNTF lacks the leader sequence char- acteristic of secreted proteins and therefore it is likely to be a cytosolic rather than a secreted protein (Lin et al., 1989; Stockli et al., 1989). However, the possibility remains that CNTF might be exported from the cell by a mechanism that does not require this sequence, such as by a specific transporter. Finally, the mRNA for the CNTF receptor molecule CNTFRa seems to be widely distributed through- out the nervous system, arguing that CNTF probably acts very widely (Davis et al., 1991). Thus, the CNTF gene is expressed late in development, and the distribution of CNTF protein and its receptor appears to be inconsistent with a role for CNTF as a specific neuronal survival factor.

What then might the normal role of CNTF be? One suggestion has been that CNTF acts as a trophic factor only under pathological conditions (Sendtner et al., 1990). Ac- cording to this idea, CNTF released by cell damage would then help rescue dying neurons and, as suggested by our work, promote process outgrowth from those that survive. In support of this, it has been shown that levels of CNTF and its mRNA increase in the vicinity of a lesion in neonatal and adult brains (Nieto-Sampedro et al., 1983; Ip et al., 1993), and in adult spinal cord (Oyesiku and Wigston, 1995). Furthermore, pieces of degenerating nerve release factors that promote axonal outgrowth (Richardson and Ebendal, 1982; KufRer, 1989). However, levels of CNTF and its mRNA in peripheral nerves fall after axotomy and are not replenished until after axonal regeneration (Fried- man et al., 1992; Mata et al., 1993). Nevertheless, retrograde transport of CNTF by motoneurons is enhanced after axotomy (Curtis et al., 1993), and the decline in CNTF levels in peripheral nerves after axotomy may be a consequence of the release of CNTF by Schwann cells.

Although our results show that CNTF has the ability to promote process outgrowth from spinal cord neurons it is not known whether CNTF will stimulate axonal regeneru- tion within the adult spinal cord. Our experiments used embryonic neurons, and it is not clear how our results relate to adult neurons. Furthermore, since we were unable to distinguish between axons and dendrites in our cultures, we do not know if CNTF is capable of stimulating the growth of axons or dendrites. Finally, a major reason that axonal regeneration in the CNS is typically very poor is that the growth of axons in the adult CNS is inhibited by proteins associated with CNS myelin (Schwab and Caroni, 1988; Schwab, 1990) and reactive astrocytes of the glial scar (McKeon et al., 1991). Neutralizing these inhibitors of neurite outgrowth has been reported to improve axonal regeneration in vivo (Schnell and Schwab, 1990; Schnell et al., 1994). I t remains to be determined whether stimulation of axonal growth in vivo by CNTF would be enough to overcome this growth inhibition. Nevertheless, the neurotrophic factor NT-3 has been found to enhance sprouting of axons in the rat corticospinal tract after a spinal cord lesion (Schnell et al., 1994). Moreover, exogenous CNTF has been shown to promote sprouting from the synaptic terminals of motor axons in adult muscle, demonstrating that agents like CNTF can have effects on axonal growth in adults (Gurney et al., 1992). I t will be important to determine whether CNTF can stimulate axonal regeneration in an appropriate model of spinal cord injury.


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ACKNOWLEDGMENTS We thank Anne Shirley for assistance, Jay Krutulis and

Scott Counts for carrying out some of the neurite length measurements, and Drs. Randy Blakely, Arthur English, Harish Joshi, Marla Luskin and Melody Siegler for advice. This research was supported by grants from the American Paralysis Association and the Paralyzed Veterans of America, Spinal Cord Research Foundation.

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ciliary neurotrophic factor stimulates neurite outgrowth from spinal cord neurons

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