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/ . Embryo!, exp. Morph. Vol. 31,1, pp. 151-167, 1974
Printed in Great Britain
Effects of nerve growth factor
from the venom of Vipera russelli on dispersed
sensory ganglion cells from the embryonic chick
By D. V. BANTHORPE, F. L. PEARCE AND C. A. VERNON 1
From the The Christopher Ingold Laboratories, The Department of
Chemistry, University College London
SUMMARY
A quantitative study has been made of the effects of nerve growth factor (NGF), isolated
from the venom of Vipera russelli, on the growth of dispersed cells from the sensory ganglia
of the embryonic chick. The main effects were to increase the viability of the sensory neurons
in culture and to promote the regeneration of nerve fibres. In the presence of NGF a higher
proportion of neurons produced fibres and the fibres were longer and slightly more branched
than in control cultures. The number of fibres produced per neuron was unaltered. Supporting
cells were unaffected by the presence of NGF. No stimulation of mitotic activity among
either type of cell was observed. It is concluded that, under the stated conditions, NGF does
not promote the differentiation of immature cells but acts, both in a maintenance and a
stimulatory capacity, only on those cells recognizable as neurons.
INTRODUCTION
Nerve growth factor (NGF) is the name given to a family of proteins that
specifically stimulate the growth of fibres from embryonic sympathetic and
sensory neurons in vitro. The biochemical and biological properties of NGF
have been extensively reviewed (Levi-Montalcini & Angeletti, 1968; Zaimis,
1972). Complete purification has been achieved using mouse salivary gland and
certain snake venoms as sources (Angeletti, 1970; Bocchini & Angeletti, 1969;
Pearce et al. 1912a, b; Varon, Nomura & Shooter, 1967). Although the various
forms of NGF differ in chemical (Pearce et al. 1912a, b) and immunological
properties (Zanini, Angeletti & Levi-Montalcini, 1968) their biological effects
in vitro are apparently indistinguishable.
In vivo NGF is most active in embryos and neonates. It produces characteristic hypertrophic and hyperplastic changes in the sympathetic ganglia when
administered to chick embryos, newborn mice, rats and kittens (Levi-Montalcini
& Booker, 1960 a; Edwards et al. 1966). The catecholamine content of the tissues
innervated by the sympathetic ganglia increases almost in proportion to the
increase in ganglion size (Edwards et al. 1966). The administration of an anti1
Author's address: The Department of Chemistry, University College London, 20 Gordon
Street, London WC1H0AJ, U.K.
152
D. V. BANTHORPE, F. L. PEARCE AND C. A. VERNON
serum prepared using NGF from mouse salivary gland results, in neonates, in
extensive destruction of the neurons of the sympathetic ganglia (Levi-Montalcini
& Booker, 1960 b). The procedure has become known as immuno-sympathectomy
(for a review see Zaimis, 1967). Neurons of the sensory ganglia are also said to
be responsive to NGF but only during a limited period of their embryonic
development (Levi-Montalcini & Angeletti, 1968). These experiments have led
to the view that NGF may play an important part in the development and,
perhaps, maintenance of the peripheral nervous system. However, in spite of
intensive research the physiological role of NGF, if any, remains obscure.
The effects of NGF in culture on explants of sensory and sympathetic ganglia
from birds and mammals have been extensively studied. It has been claimed that
oxidative and synthetic processes are stimulated in the ganglion cells and that an
increased production or RNA, lipid and protein occurs (Angeletti, LeviMontalcini & Calissano, 1968). Such results are, however, to be expected in
view of the considerable growth effects produced by the factor. It is also said
that NGF promotes a premature maturation of sympathetic and sensory
neurons, the latter being not only larger than controls but showing a massive
increase in neurofibrillar material, an increase in endoplasmic reticulum and a
dilation of the Golgi complex (Levi-Montalcini, Caramia, Luse & Angeletti,
1968).
Some experiments have been done with cell cultures made by dissociation of
sensory or sympathetic ganglia. It is claimed (Levi-Montalcini & Angeletti, 1963)
that the neurons do not survive in such cultures unless NGF is added to the
medium but no quantitative data have been given and, in any case, the methods
used for dispersion probably resulted in considerable cellular damage (see
Banks et al. 1970). However, in a sense cell cultures provide the simplest model
for the study of NGF and, since we have developed methods of dispersion which
we believe produce less damage than those previously used, we have carried out
a quantitative investigation of the effects of NGF on cells isolated from sensory
ganglia of chick embryos. A preliminary account of some of the results has
already been published (Vernon et al. 1969).
MATERIALS AND METHODS
Nerve growth factor. This was purified from the venom of Viper a russelli as
previously described (Pearce et al. 1912 a, b). The material used in each experiment was assayed using whole sensory ganglia from 8-day chick embryos in
hanging-drop culture on collagen (Lamont, 1968). Only samples which showed
a standard response (3-4 on the accepted arbitrary scale (Cohen, 1958)) at a
concentration in the culture medium of ca. 10~8 g ml" 1 were used. Unless otherwise stated this concentration of NGF (denned as 1 B.U. ml" 1 (Levi-Montalcini
& Angeletti, 1968)) was used in all the experiments.
Mitotic inhibitors. Colchicine was obtained from Ciba Laboratories ('Col-
Effects of NGF on dispersed sensory cells
153
cemid'). The compound was homogeneous to thin-layer chromatography on
silica gel G (Merk Ltd; 100 fivaplate) with chloroform-diethylamine (9:1, v/v as
eluent) and iodoplatinate as spray reagent. Ouabain was obtained from the
Sigma Chemical Co. Ltd., London. It was homogeneous to chromatography as
specified above using methylene dichloride-methanol-formamide (80:19:1, v/v)
as eluent and antimony chloride in chloroform as spray reagent.
Preparation of ceil suspensions. Sensory ganglia were dissected from the
lumbosacral and thoracic regions of 8-day chick embryos (White Leghorn) and
dissociated with pronase as previously described (Banks et ai. 1970). Typically,
the dissociation of ca. 75 ganglia gave about 1 ml of suspension containing
approximately 5xlO 5 cells ml- 1 . Incubation conditions were as previously
described and the cultures obtained were similar to those previously illustrated
(Banks et al. 1970): specific variations in technique for individual experiments
in the present work are given below.
Identification of cells. Neurons in culture were identified by their characteristic
appearance and by their strong affinity for methylene blue and silver stains
(Costero & Pomerat, 1951; Holmes, 1943). The remaining cells were classified
as supporting cells: most had the characteristic morphology of fibroblasts or
spindle cells. Neurons were conveniently counted under phase-contrast whereas
the total cell count in a given preparation was more easily made using dark-field
microscopy. Analyses of significance were made using standard methods
(Stanley, 1963).
Measurement of cell viability
Method 1. This method requires the dissection of only a small number of
ganglia at any given time and leads to a matched pair of cultures one of which
contains NGF whereas the other does not. Its disadvantage is that in a series of
matched pairs the cell density is not constant.
Dispersed cells from about 15 ganglia were resuspended in medium 199
{ca. 200 ju,\). The suspension was divided equally into two parts by pipetting
drops into a pair of glass rings (6 mm diameter; 4 mm high; ca. 100 JLL\ suspension per ring) mounted on collagen-coated coverslips. Cockerel serum (ca. 50 /A)
and either buffered saline or a solution of NGF in buffered saline (ca. 50 ju\)
were then added and the cultures incubated for appropriate periods of time.
Some cultures were incubated in a serum-free medium consisting of medium 199
(3 parts) and buffered saline (1 part) with or without NGF. After incubation the
cultures were stained with methylene blue, fixed in ammonium molybdate and
mounted in Canada balsam (Banks et al. 1970). Counts of neurons and of total
cells were made by examining between 10 and 40 randomly selected fields
(400 x final magnification) under phase contrast and dark field respectively so
as to give a total count of approximately 1000 cells of all types for each culture.
The areas examined were distributed throughout the culture to obtain representative sampling and individual fields were selected blind to avoid subjective
bias.
154
D. V. BANTHORPE, F. L. PEARCE AND C. A. VERNON
Method 2. This method requires a large number of ganglia (ca. 350) and gives a
set of cultures (28) with the same cell density. The dispersed cells were resuspended
in a solution (3 ml) composed of medium 199 and buffered saline (2:1). The
suspension was gently agitated mechanically and a sample was withdrawn and
examined using a modified Fuchs-Rosenthal haemocytometer. The cell density
was adjusted to be in the range 2-3 x 105 cells ml"1, i.e. about half the cell density
considered optimal for the survival of neurons. The suspension was then dispensed from a wide-bore micropipette, in aliquots of 100 /d, into glass rings
mounted on collagen-coated coverslips contained in sterile Petri dishes. Agitation
of the suspension was continued throughout the procedure and cell counts were
made at the half-way and end stages to ensure that the cell density had remained
constant. Serum, both with and without NGF was then added to the rings so
that each Petri dish contained a control and a culture containing NGF. The
final composition of the culture medium was medium 199, 2 parts, serum or
serum containing added NGF, 1 part, and buffered saline, 1 part. The 28 cultures,
of which half were controls, were numbered at random and incubated for periods
of 4, 10, 24, 34, 48 and 72 h. After incubation the cultures were stained with
methylene blue and prepared for microscopic examination. Counts of neurons
and of supporting cells were made on sixty randomly selected fields (400 x final
magnification). The combined areas of these fields represented ca. 20% of the
total culture area.
Measurement ofmitotic coefficients
Suspensions were cultured in liquid medium on collagen as described above
for varying periods of time. Since mitotic activity is influenced by cell density
(Wilmer, 1933) the cultures were arranged so that the final plated cell densities
were in the range 200-1000 cells mm2. After incubation, the cultures were fixed
in formal saline (24 h), dehydrated in 70 % ethanol (12 h) and then stained with
Mallory's haematoxylin. They were then washed in running tap water (2 h),
dehydrated in the usual way and mounted in Canada balsam. Cell counts were
then made and between 500 and 2000 cells were examined to determine the
percentage of cells exhibiting mitotic figures (i.e. the mitotic coefficient). It was
found that cells showing such figures were difficult to classify since entry into
mitosis was accompanied by loss of normal shape and streaming of the cytoplasm. However, the nuclei of the neurons were found to be more basophilic
than those of the supporting cells and treatment of the cultures with haematoxylin for 20 sec resulted in a satisfactory staining of the neurons whilst leaving
the supporting cells virtually unstained. Consistently, treatment with haematoxylin for 10 min resulted in a satisfactory staining of the supporting cells but
stained the neurons so intensely as to obscure all nuclear detail.
The mitotic activity of cells in the periphery of sensory ganglia was also
examined by the method described above. These cells were predominantly supporting cells although neurons were occasionally seen to migrate from the body
of the explant (Lamont & Vernon, 1967).
Effects of NGF on dispersed sensory cells
155
Measurements on the regeneration of nerve fibres
1. On collagen supports. The procedure was as described under method 2
above except that the cells were plated at a density of approximately ten times
less. It was found possible to measure only the maximum lengths of the fibres
produced by individual cells and to determine the number of bipolar and of
unipolar cells using this method of culture. Other quantitative work was not
possible because the cells actively migrated and formed clusters. Complex networks of fibres were observed to form between the clusters.
2. In clotted media. The production of nerve fibres in clotted media was
most conveniently studied using ganglia from rather older embryos (9-10 days)
than used in other experiments. The cells obtained in this way responded more
favourably to the restrictions imposed by the semi-solid medium. Suspensions
of cells were collected in a solution of thrombin (Calbiochem, 1 mg ml"1) in
medium 199 and were diluted to give a final plated cell density approximately
one-fiftieth that normally used (see above). The cell suspension (ca. 25 fi\) was
then clotted on to sterile glass coverslips either (a) by addition of a solution
containing equal parts buffered saline, with and without NGF, and cockerel
plasma (ca. 25 /A) (plasma clots); or (b) by addition of a solution containing
equal parts cockerel serum, with and without NGF, and a 1 % solution of bovine
fibrinogen (Calbiochem) in buffered saline (ca. 25 fi\) (fibrin clots); or (c) by
addition of a solution containing equal parts of medium 199 and 1 % fibrinogen
in buffered saline with and without NGF (ca. 25 fi\) (serum-free fibrin clots).
To prevent lysis of the clot under these conditions it was necessary to add soyabean trypsin inhibitor (Sigma Chemical Co., London, 1 mgml"1) to the original
thrombin solution.
Each method led to matched pairs of cultures differing only in the presence
or absence of added NGF. Plasma clots sometimes gave irreproducible results
and the cockerel plasma occasionally showed high NGF activity in conventional
assay. Fibrin clots showed negligible NGF activity.
The cultures were incubated for 24 h, fixed, stained with methylene blue and
cell counts made on each preparation. The percentage of nerve cells which
regenerated fibres, the average and maximum lengths of the fibres and the
number of fibres produced by individual cells were recorded. The degree of
branching of the fibres was assessed on an arbitrary scale between 0 and 5 where
unity was taken to represent a single bifurcation and a score of 5 corresponded
to about 10 branch points.
RESULTS
Measurement of cell viability
Method 1. Six matched pairs of cultures were incubated for each of the periods
4,24,48 and 72 h, the number of neurons that survived in each culture are given
as a percentage of the total number of cells in Table 1. For any given time period
156
D. Y. BANTHORPE, F. L. PEARCE AND C. A. VERNON
Table 1. Effect of NGF on percentage of neurons grown
in cultures containing serum
Culture number
(0 Incubation period 4 h
N*
ct
P value
33-4 ±1-2
34-7 ±1-2
29-2 ±1-1
26-5 ±1-6
N.S.
N.S.
00
N
c
P value
N
C
P value
N
c
P value
26-4 ±1-8
31-9±2-2
31-3±l-8
31-6±2-6
28-8 ±1-8
29-3 ±1-4
27-3 ±1-6
28-6 ±1-4
N.S.
N.S.
N.S.
N.S.
25-7 ±2-0
23-4 ±2-2
N.S.f
25-9 ±2-4
240 ±1-6
N.S.f
8-7 ±0-6
4-4 ±0-3
< 001§
7-4 ±0-7
4-3 ±0-4
< 001§
141+0-8
12-3 ±0-6
15-2±l-3
13-2±10
N.S.
N.S.
Incubation period 24 h
29-3 ±2-6
27-8 ±2-2
19-6 ±2-2
18-9 ±1-7
< 0-01J
< 0-01J
Incubation period 48 h
14-6±1-1
17-0 ±1-0
101 ±0-8
101 ± 1 0
< 001
< 001
10-4 ±0-7
7-1 ±1-1
< 002
9-2 ±0-6
4-6 + 0-4
< 001
15-9 + 10
9-3 ± 1 0
< 001
(iii)
14-8 + 0-6
7-7 ±0-6
< 001
16-7 ±0-9
10-4±0-9
< 001
(iv) Incubation period 72 h
15-2 ±0-9
13-8 ±0-6
12-3 ±0-9
9-6±l-l
9-6±l-4
9-6 ±0-8
< 001
< 001
N.S.
* Nis percentage of neurons in NGF treated culture.
f C is percentage of neurons in control culture.
All counts were made on 20 randomly selectedfieldswith the exception of:
(t) 10 fields and (§) 40 fields. All values are means ±S.E.M.; N.S. = not significant.
the percentages vary markedly from culture to culture. This arises partly because
of the effect of cell density on the viability of dispersed neurons. However, for
each matched pair the percentage of neurons surviving in the culture containing
NGF (N) may be directly compared with that shown by the control (C). At
4 h there are no differences which are statistically significant. With the longer
time periods for each matched pair N > C; the differences being statistically
significant at the level P < 0-05 in 13 out of 18 pairs.
A method of combining all the data obtained at any given period of time is
to define either of the two parameters, p and ^ as
P =
N
C'
N-C
The parameters are clearly related and represent alternative methods of presenting the same data. For completeness, both methods will be used in the
present work. The values of the two quantities may be calculated for each of
the six matched pairs in turn and the mean values and the standard errors of
Effects of NGF on dispersed sensory cells
157
Table 2. Comparison of effects of NGF in cultures containing serum
Time (h)
p
4
24
48
72
0-97
1-43
1-74
1-37
<j>
±003
±014
±008
±008
-0-38 ±0-40
2-72 ±0-66
4-20 ± 0-27
2-55 ±0-46
p and $5 are defined in the text. Values are means ± S.E.M. based on six observations for each
time period.
Table 3. Effect of NGF on percentage of neurons in cultures
grown in a serum-free medium
Culture number
1
2
N*
21-4±l-8
c*
20-9 ± 3 1
3
(0 Incubation period 4 h
18-3 ±2-7
18-2± 1-9
180±2-3
15-8±2-3
14-5 ±1-7
18-9±2-6
P value
N.S.
N.S.
TV
C
P value
25-9 ±1-7
12-1 ±1-4
< 001
25-4+1-8
100±l-3
< 001
N
15-8±l-4
0-2 ±0-2
(iii) Incubation period 72 h
16 1 ± 16
0-2 ±0-2
< 0011
N.S.
5
6
27-2 ±1-9
27-5 ±2-6
26-0 ±2-3
27-2 ±1-9
N.S.
N.S.
4
N.S.
00 Incubation period 24 h
c
P value
< ooit
47-1 ±2-8
3-7 ±0-9
< OOlf
17-6±l-5
7-3 ±1-2
< 001 f
8-6 ±0-9
3-7 ±0-5
< 0-Olf
27-2 ± 1 - 7
9 1 ±1-2
< ooit
* N and C as defined in Table 1. All counts made on 20 randomly selected fields with the
exception of (t) 30 fields. All values are means ± S.E.M.
Table 4. Comparison of effects of NGF in serum-free cultures
Time (h)
p
4
24
106 ± 0 0 5
2-48 + 014
<fi
0-68 ±0-35
5-91 ±0-22
p and ^ are defined in the text. Values are means ± S.E.M. based on six observations at 4 h
and five observations at 24 h. The latter does not include the data obtained from culture 3,
Table 3.
the mean values can then be obtained for each set. When NGF has no effect on
the viability of the neurons the values of p and ^ will not differ significantly
from unity and zero respectively. As shown in Table 2 this is the case at 4 h
incubation. The values increase at 24 h (4-24 h, P < 0-01, P < 0-01, respectively) show a further slight increase at 48 h (24-48 h, P < 0-1, P ca. 0-05) and
then decrease (48-72 h, P < 0-01, P < 0-02).
158
D. V. BANTHORPE, F. L. PEARCE AND C. A. VERNON
16
£ 14
8. 12
13 10
-5Q
18
36
Time (h)
54
72
he
Fig. 1. The effect of NGF on the viability of dispersed sensory nerve cells.
O—O, Cultures treated with NGF; O - - O , control cultures.
30 —
o 29 _
eel
c
28 27 _
un
co
26 —
o 25 _
o.
C.
24
3
23
22
21
—
-o
—
/ •
_
18
36
Time (h)
54
72
Fig. 2. The effect of NGF on the viability of dispersed sensory supporting cells.
O—O, Cultures treated with NGF; O - - O , control cultures.
A similar set of experiments using fewer cultures were carried out in a serumfree medium. The results are given in Tables 3 and 4. They show essentially the
same pattern as that detailed above except that the effect of the presence of
NGF, is at the longer incubation periods, much more pronounced. The values
of p and 0 again approximate to unity and zero respectively at 4 h of incubation,
but increase significantly after 24 h in culture (4-24 h, P < 0-01, P < 0-01).
A comparison of the values of p and 0 for cultures grown in the presence and
absence of serum (Tables 2, 4) shows that after 4 h the respective values do not
differ significantly, but that this difference becomes highly significant after
24 h (P < 0-01, P < 0-01).
Method 2. The behaviour of dispersed ganglion cells with increasing time was
studied by examination of a number of equivalent cultures. The average numbers
of neurons and supporting cells which survived per field are shown graphically
in Figs. 1 and 2. Points and symbols represent mean values and standard errors
Effects of NGF on dispersed sensory cells
159
of the means, typically for counts made on 60 fields for each of two duplicate
cultures. The number of neurons surviving in culture fell markedly as the time of
incubation increased (Fig. 1). The decrease was most marked between the fourth
and twenty-fourth hours of incubation but a much higher proportion of neurons
survived in those cultures containing NGF. The behaviour of supporting cells,
on the other hand, was independent of the presence of NGF (Fig. 2).
Measurement of mitotic coefficients
1. In dispersed cell cultures. Six pairs of control cultures and cultures treated
with NGF were incubated in a medium containing 25 % (v/v) of cockerel serum
for each of the periods 4, 24, 48 and 72 h. The mitotic coefficients (± S.E.M.) for
the supporting cells in the treated cultures were found to be 0-1 ±0-1, 0-4 ±0-1,
0-3 ±0-1 and 0-4 ±0-1 respectively, compared with 0-2 ±0-1, 0-4 ±0-1, 0-3 ±0-1
and 0-4 ±0-1 respectively, in the control cultures. A total of 15000 neurons were
examined and 17 possible mitoses were observed of which 9 were in cultures
treated with NGF.
Similar experiments were carried out with cultures grown in serum-free media.
After 4 h incubation the supporting cells showed mitotic coefficients of 0-1 ±0-1
both in the treated and control cultures. In the 18 cultures incubated for longer
periods of time only 14 mitotic figures were observed of which 11 were in cultures
treated with NGF. Approximately 10000 neurons were examined in these
cultures but no mitotic figures were seen.
Concentrations of colchicine greater than 2 x 10~12 g ml" 1 were found to be
highly toxic to the cultures of dispersed cells and few cells survived. However,
at concentrations between 2 x 10~13 g ml" 1 and 2 x 10~15 g rah1 appreciable
numbers of neurons survived and produced fibres in cultures containing NGF.
A limited number of supporting cells also survived. No mitotic figures were
seen in these experiments.
The addition of ouabain at a concentration of 10~6 M led to a marked vacuolization of the supporting cells. This effect has been previously reported by
Stefanelli, Palladini & Ieradi (1965). Large numbers of neurons survived in these
cultures only in the presence of NGF. At concentrations between 10~7 and
10~ 8 M, less widespread vacuolization of the supporting cells occurred but
appreciably more neurons again survived and regenerated fibres in those cultures
treated with NGF than in the controls. As before, no mitotic figures were
detected in these experiments.
2. In outgrowths from explants of sensory ganglia. Mitotic coefficients (± S.E.M.)
for cells which had migrated from six explants of sensory ganglia cultured for
24 h in a medium containing 2 5 % serum were 2-04 ±0-44 (with NGF) and
1-60 + 0-47 (controls). In cultures for 48 h, 14 treated ganglia and 16 controls
gave mitotic coefficients of 1-84 ±0-08 and 1-74 + 0-11, respectively. In comparison, the corresponding values for fibroblasts in the periphery of explants of
chick heart (six cultures) after 48 h were 1-59 ±0-33 and 1-68 ±0-20, respect-
160
D. V. BANTHORPE, F. L. PEARCE AND C. A. YERNON
ively. None of the differences between treated and control cultures are statistically significant.
In the absence of serum the outgrowth of cells from explants of sensory ganglia
was considerably reduced and very low mitotic coefficients were recorded
(< 0-1). However, the characteristic outgrowth of nerve fibres was unaffected by
the absence of serum providing that NGF was present.
The effects of a large excess of NGF were investigated using four cultures,
each with two explants of sensory ganglia, grown for 48 h in a medium containing 1000 B.U. ml" 1 (10~5 g ml"1) of NGF. Parallel cultures were grown in a
medium containing the normal amount of NGF (1 B.U. ml"1) and in control
medium. The mitotic coefficients in the peripheral supporting cells were found
to be l-04±0-08, 1-73 ±010 and 1-51 ±0-10 respectively. Again there is no
significant difference between the control cultures and those containing the
normal amount of NGF. However, the reduction in mitotic activity at high
concentrations of NGF is significant (P < 0-01) and may reflect a physical
inhibition of cell division by the very dense outgrowth of nerve fibres which
occurs under these conditions.
Concentrations of colchicine in the range 2x 10~12 g m l - 1 - 2 x l O ^ g m l " 1
prevented mitotic activity in the periphery of explants of sensory ganglia and
led to an accumulation of arrested metaphases and prophases corresponding to
formal mitotic coefficients in the range 0-5-5. Those cultures treated with NGF
did not contain a greater number of arrested mitotic figures than the controls.
The outgrowth of nerve fibres produced by the addition of NGF was unaffected
under these conditions.
Similarly concentrations of ouabain in the range 10~7 to 10~8 M inhibited cell
division in the periphery of the explants without affecting their characteristic
response to NGF.
Measurements on the regeneration of nerve fibres
1. On collagen supports. Most of the neurons obtained by dispersion of sensory
ganglia were initially spheroid or ellipsoid in shape and devoid of processes.
Some, particularly from older embryos, possessed axon stumps. Preliminary
studies on the growth of fibres from these cells were made on cultures in a liquid
medium on collagen. A total of 597 neurons were examined in cultures treated
with NFG compared with 100 neurons in control cultures. The difference
reflects the greater viability of neurons in the presence of NGF, particularly at
low cell densities. The maximum lengths of nerve fibres in isolated cells in the
treated and control cultures were 1030 and 430 jam respectively. The maximum
numbers of fibres regenerated from individual cells were 5 and 4, and in cultures
treated with NGF 43-3 ±1-8% of the cells were unipolar compared with
68-9 ±4-4% in control cultures {P < 0-01).
2. In clotted media. Matched pairs of cultures were maintained in plasma
clots, serum-free fibrin clots and in fibrin clots supplemented with serum: a total
161
Effects of NGF on dispersed sensory cells
Table 5. Regeneration of nerve fibres in clotted media
Control cultures
Treated cultures
•>
Medium
247
Plasma clots
291
Fibrin clots +
serum
61
Fibrin clotsserum
191
Fibrin clots.
Excess (treated)
+ optimal (control)
cones, of NGF.
All cultures +
serum
°/
P value
«i
/o
"2
02
1916
1947
12-9 ±0-8
14-9 ±0-8
145
145
2278
2408
6-4 ±0-5
60 ±0-5
< 001
< 001
358
170±2-0
19
188
10-1 ±2-2
< 005
725
26-3 ±1-6
156
1035
15 1 ± 1 1
< 001
/o
The table records the number of neurons that regenerated fibres (nu n2) expressed as a
percentage (%) of the total number of neurons (a^a^). The percentages are expressed as
means ± S.E.M. significance levels refer to a comparison of the two percentages in each case.
Experimental details are given in the text.
Table 6. Polarity of neurons in clotted media
Treated cultures
Control cultures
A
Medium
"i
'h
/o
V
P value
/o
Plasma clots
230
Fibrin clot +
283
serum
Fibrin clot —
61
serum
Fibrin clots. Ex188
cess (treated) +
optimal (control)
cones, of NGF.
All cultures + serum
< 005
< 001
247
291
931 ± 1-6
97-2 ± 1 0
126
131
145
145
86-9 + 2-8
90-3 ±2-5
61
100
19
19
100
N.S.
191
98-4 ±0-9
151
156
96-8 ±1-4
N.S.
The table records the number of neurons that were unipolar (b^ b2) expressed as a percentage (%) of the total population that produced fibres (A71? «2). The percentages are means ±
S.E.M., significance levels refer to a comparison of the two percentages in each case. Experimental details are given in the text.
of ten pairs were prepared for each experiment. An additional 30 cultures were
grown in fibrin clots supplemented with serum, half containing the normal
concentration of NGF (1 B.U. ml"1) and the other half a very high concentration
of NGF (1000 B.U. ml"1). The effects of NGF on the regeneration of fibres, the
length and extent of branching and on the polarity of the cells are recorded
in Tables 5-8.
II
EMB
31
162
D. V. BANTHORPE, F. L. PEARCE AND C. A. VERNON
Table 7. Degree of branching of nerve fibres in clotted media
Treated cultures
/.
Degree
'h
Medium
Plasma clots
70
Fibrin clots + serum 42
Fibrin clots —serum 9
39
Fibrin clots.
Excess (treated)+
optimal (control)
cones, of NGF.
All cultures + serum
247
291
61
191
0-62 ±007
0-31 ±004
0-26 ±009
0-40 ±006
Control cultures
c2
'h
Degree
P value
63
6
0
39
145
145
19
156
0-63 ±007
009 ± 004
< 001
0
0-43 ±007
N.S.
N.S.
N.S.
The table records the degree of branching (scored on an arbitrary scale of 0-5, where 1
indicates a single bifurcation) measured for the number of neurons that branched {cx, c2) in
the total population that produced fibres (nu n2). The values are means ±S.E.M., significance
levels refer to a comparison of the degree of branching in each case. Experimental details are
given in the text.
Table 8. Length of nerve fibres in clotted media
Treated cultures
Medium
>h
Lx (/on)
Plasma clots
Fibrin clots + serum
Fibrin clots —serum
Fibrin clots.
Excess (treated) +
optimal (control)
cones, of NGF.
All cultures + serum
247
291
61
191
99±4
97 ±3
98 ±7
69 ±3
Control cultures
c
145
145
19
156
L2 (/tm)
P value
68±4
45 ±3
78 ±15
54±2
< 001
< 001
N.S.
< 001
The table records the mean lengths (Lx, L2) ± S.E.M. of the nerve fibres produced by the
number of neurons examined (nu n2). Significance levels refer to a comparison of the mean
fibre lengths in each case. Experimental details are given in the text.
DISCUSSION
Using an improved method (Banks et al. 1970) we were able to prepare
suspensions of high cell density by dissociation of embryonic chick sensory
ganglia. The cells were then readily cultured on collagen in a liquid medium
containing serum. After 4 h of culture neurons could be distinguished from
supporting cells by their affinity for methylene blue, by silver staining and,
partly, by morphological appearance. After longer periods of culture distinction
between the two cell types could easily be made by inspection; the supporting
cells taking up the typical appearance of fibroblast, spindle and other cells.
Estimations of the percentage of the cells which were neurons could, therefore,
be made with reasonable precision especially for the longer periods of culture.
The results using the method of matched pairs (method 1) were complicated
Effects ofNGF on dispersed sensory cells
163
because the viability of neurons depends upon, among other things, the total
cell density. Nevertheless, the effect of NGF is quite clear: from the data in
Tables 1 and 2 it can be seen that for periods of culture greater than 4 h a higher
proportion of neurons survived in the presence of NGF than in its absence.
These results must be interpreted in terms of an increased viability of the nerve
cells in the presence of NGF, since we have shown that the factor does not
stimulate mitotic activity among these cells and also does not affect either the
mitotic activity or the viability of the supporting cells. The increased viability
of the nerve cells was most evident during the period between 4 and 48 h of
incubation. In the period between 48 and 72 h some deterioration apparently
occurred in the experimental cultures, although the latter still contained a considerably higher proportion of nerve cells than the controls. If this deterioration
was real, it may reflect a loss of NGF activity in the medium during the longer
period of incubation. It is possible that the survival of neurons requires the
continuous presence of NGF and that the substance slowly disappears under
culture conditions. Support for this hypothesis is provided by the finding that
the medium removed from cultures grown for 72 h contained, on conventional
assay, less NGF that the original medium. The effect of NGF on cell viability
is even more pronounced in a serum-free medium (Tables 3, 4). In such a
medium, after 72 h of culture in the presence of NGF, a considerable proportion of neurons were still present. In the absence of NGF, however, virtually no
neurons remained and the cultures consisted almost entirely of supporting cells.
The effect of NGF on the viability of neurons was more clearly demonstrated
using a series of equivalent cultures all of the same initial cell density (method 2).
It was found (Fig. 2) that there was some slight proliferation of the supporting
cells during the earlier period of culture but that the number of these cells
thereafter remained sensibly constant and was unaffected by the presence of
NGF. On the other hand, the number of neurons over the same time period
dropped by almost a factor often in the absence of NGF (Fig. 1). In the presence
of NGF, however, although some decrease occurred in the period between
10 and 34 h, the overall decrease was much smaller, i.e. less than twofold. There
is no doubt, therefore, that NGF markedly increases the viability of the neurons
in these particular culture conditions.
The origin of the small decrease in the number of neurons even in the presence
of NGF is of interest. It is possible that the cell population is not homogeneous
and that some neurons are unresponsive to NGF. Alternatively it may be that
some cells are damaged in the course of the dispersion and subsequently cease
to be viable.
It must be emphasized that the experiments summarized in Figs. 1 and 2 were
deliberately carried out at rather low cell density. At higher cell density the
effect of NGF, although still real, is less marked. The reason for this is not
known. It may be that the cell suspension contains some cells capable of secreting
NGF but we have been unable to obtain any experimental support for this view.
164
D. V. BANTHORPE, F. L. PEARCE AND C. A. VERNON
An alternative explanation for the apparent ability of NGF to maintain the
neurons in culture is that the death of these cells is partially balanced by
differentiation to recognizable neurons from a less differentiated pool. This
hypothesis must be rejected, however, since the numbers of supporting cells
remaining in culture were unaffected by the presence of NGF. Thus, the effect
cannot be accounted for by the removal and conversion of undifferentiated cells
and must be explained directly in terms of an increased neuron viability. The
experiment then demonstrates that the cells which respond to NGF are those
which have already passed the earlier stages of differentiation and have acquired
the characteristic appearance and staining properties of neurons.
The independent behaviour of the supporting cells was confirmed in studies
on mitotic activity. It was found that the mitotic coefficients observed for these
cells were very low (0-1-0-4) and were unaffected by the addition of NGF. These
low values may be a consequence of the dispersion procedure: fibroblasts
obtained by dispersal of embryonic chick heart showed mitotic coefficients in
the same range.
Supporting cells which had migrated out of explants of sensory ganglia
grown in hanging-drop culture showed much higher mitotic coefficients (~ 2).
Concentrations of NGF of 1 B.U. ml" 1 produced the characteristic outgrowth of
fibres but was without effect on the mitotic activity of the supporting cells.
Large concentrations of NGF (~ 1000 B.U. ml"1) actually depressed the value
of the mitotic coefficient for the supporting cells. In the absence of serum mitotic
activity proceeded at a very low level but the effect of NGF in promoting fibre
outgrowth was unchanged. Appropriate concentrations of colchicine and of
ouabain effectively arrested mitotis but left the response of the neurons unaltered. In all the experiments described the mitotic activity of recognizable
neurons was, predictably, very small.
We conclude, therefore, that the response to NGF observed in our experiments
is unrelated to mitotic activity and involves only those cells which have already
differentiated into neurons. This is consistent with the well established fact that
mitosis in sensory ganglia cells of chick embryos has practically ceased by the
ninth day of development, i.e. approximately at the stage the explants were taken
(Levi-Montalcini, 1966). We do not wish to claim that NGF never affects
mitotic activity but only that under the conditions described by us its effects
are unrelated to mitosis. It has been claimed that NGF released from sarcoma
grafts or isolated from mouse submaxillary glands and certain snake venoms
can stimulate mitotic activity in receptive sensory cells in vivo (Levi-Montalcini,
1958, 1966). This may be a reflexion of some distinct biological activity which
is not revealed by our relatively short-term experiments in vitro. On the other
hand, sarcomas may contain agents which stimulate mitosis, as has been reported for other tumour cells (Argyris & Argyris, 1962), and the NGF samples
used were, by current standards, relatively impure (Angeletti, 1970). However,
the hypertrophic and hyperplastic effects of NGF on sympathetic ganglia in
Effects of NGF on dispersed sensory cells
165
chick embryos and neonate mammals have been well documented (LeviMontalcini & Booker, 1960«; Edwards et ah 1966) and are in keeping with the
fact that mitotic activity persists in the sympathetic system for a much longer
period of normal development (Levi-Montalcini, 1966).
In addition to its role in maintaining cell viability NGF also had the effect of
promoting the growth of fibres from dispersed neurons. Experiments using
clotted media showed that, in the presence of NGF, a larger number of cells
distinguishable as neurons produced fibres than in control media (Table 5). The
biggest effect was found in fibrin clots containing an excess amount of NGF.
However, even under these conditions, only about 26 % of the neurons grew
fibres. Culture in liquid medium on collagen proved to be an unsuitable method
for the quantitative investigation of fibre growth because of the migration and
aggregation of the neurons. Nevertheless, under these conditions practically all
of the neurons produced fibres. The difference between the results obtained by the
two methods of culture probably arises from the very much lower cell densities
used in the clotted media.
Measurements of the length of nerve fibres in clotted media showed that the
presence of NGF also resulted in longer fibres. In three out of four of the experiments summarized in Table 8 the mean values of the fibre lengths are significantly greater in treated than in control cultures. The less significant results
obtained in the absence of serum probably reflect the limited data obtained
from cultures in which relatively fewer cells survived. The average length of the
fibres in treated cultures was ca. 100 jum; much less than the average value
associated with the outgrowth from whole ganglia cultured under the same conditions (ca. 400 /im). The maximum fibre lengths observed for treated and
control cultures were 500 and 300 jam respectively.
Most of the neurons in clotted media were unipolar and although NGF
appeared to increase this tendency in plasma clots and in fibrin clots containing
serum the effect is marginal (Table 6). The degree of branching of the fibres was
also found not to be highly dependent on the presence of NGF (Table 7). In
liquid medium on collagen supports, however, the neurons were observed to
produce highly branched fibres: this may be general phenomenon when the
supporting medium lacks substructure (Weiss, 1934).
Our observation of an increased response in dispersed cell cultures as the
NGF concentration was raised (Tables 5,8) contrasts with the stunted appearance
of whole ganglia treated with comparably large doses of the factor (LeviMontalcini, 1966). This appearance was originally attributed to a toxic effect,
but is now believed (Levi-Montalcini & Angeletti, 1968) to reflect an inhibition
of normal fibre outgrowth by the overcrowded population of nerve fibres. The
present results are consistent with the latter explanation.
A number of problems remain unresolved in the present work. In particular,
the possible relationship between the production of nerve fibres and the consequent viability of the cells is not clear. The primary function of NGF may be
166
D. V. BANTHORPE, F. L. PEARCE AND C. A. VERNON
to initiate the growth of fibres and. cells not stimulated in this way may cease to
be viable through a reduced metabolism. The effect of NGF on the rate of
growth of individual fibres is also ill defined. The nerve fibres produced by those
cells maintained in the presence of NGF for 24 h are longer than the controls,
but whether the factor actually increases the rate of growth of fibres or merely
initiates their production after a shorter period of incubation is uncertain. It is
also unknown whether NGF triggers the de novo formation of fibres from
neuroblasts previously lacking them or causes the regeneration of previously
existing fibres lost during the excision of the ganglia and. the subsequent dissociation of the tissue.
The extension of any conclusions from the present work to the possible role
of NGF in vivo must be made with care, since it is possible that the dispersion
procedure selects an atypical sample of ganglion cells which respond in an
abnormal way to a foreign environment. However, such an extension is supported by the observation of a similar overall response to the factor under both
conditions. Endogeneous NGF could then have an effect in the intact organism
similar to that in tissue culture and thus act to trigger and maintain receptive
neurons during the period in which nerve fibres are growing towards their end
organs. The factor could also have a precise function in directing the growth of
fibres toward their final targets of innervation. In this context, the recent
findings of Charlwood, Lamont & Banks (1972) are of considerable interest.
These workers have shown that the fibres from sensory ganglia incubated in
tissue culture will grow toward a suitable source of NGF such as a capillary
containing a solution of the active material. This experiment appears to provide
an example of genuine chemotaxis and may provide further information of the
role of NGF in the development of the peripheral nervous system.
The authors wish to thank the Whitehall Foundation of New York for generous financial
assistance. The award of University of London Postgraduate Studentships to F. L.P. are
gratefully acknowledged. Thanks are due to Drs D. M. Lamont and K. A. Charlwood for
helpful discussion and to Miss Janet Cook and Miss Janet Drew for skilled technical
assistance.
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