Developmental Brain Research, 17 (1985) 105-116 105 Elsevier

BRD 50131

PC12 Neurite Regeneration and Long-Term Maintenance in the Absence of Exogenous Nerve Growth Factor in Response to Contact with Schwann Cells

MICHAEL COCHRAN and MARK M. BLACK

Department of Anatomy, Temple University School of Medicine, 3400 North Broad Street, Philadelphia, PA 19140 (U.S.A.)

(Accepted July 5th, 1984)

Key words: PC12 cells - - Schwann cells - - neurite regeneration - - neurite-promoting factors - - cell surface - - nerve growth factor

We have investigated mouse and rat ganglionic Schwann cells as possible sources of neurite outgrowth-promoting factors by co-cul- turing Schwann cells with nerve growth factor (NGF)-responsive PC12 pheochromocytoma cells primed by pretreatment with NGF. NGF-primed PC12 cells are capable of neurite regeneration when provided with an appropriate neurite promoting factor such as NGF. When primed PC12 cells were co-cultured with Schwann cells in the absence of exogenous NGF, PC12 cells that directly con- tacted Schwann cells became enlarged and flattened, attaining a neuron-like morphology within one day. When contact with Schwann cells was established, PC12 cells regenerated neurites by the first day of co-culture and these were maintained throughout the experi- ments (7 weeks). Most PC12 cells cultured in the same collagen-coated dishes with Schwann cells, but not directly in contact with them, failed to regenerate neurites. Instead, they began to proliferate, forming cell clusters. Neurite regeneration by PC12 cells in contact with Schwann cells was not blocked by antibody to NGF. These results demonstrate the presence of a neurite-promoting activ- ity localized to the vicinity of the Schwann cell surface which is capable of eliciting regeneration and long-term maintenance of PCI2 neurites in the absence of exogenous NGF. This activity does not appear to be due to NGF.

INTRODUCTION

Peripheral non-neuronal cells can provide t rophic

support for long-term neuronal survival, and the mi-

croenvironment provided by these cells is impor tant for the success of axonal regenera t ion 6,t0,33,39,43.

When per iphera l sensory or sympathet ic neurons are

cultured in isolation from non-neuronal cells, their

long-term survival is dependen t upon an exogenous

supply of the t rophic agent, nerve growth factor

(NGF)tO, 39,41. Per ipheral non-neuronal cells der ived

from dorsal root ganglia ( D R G s ) can provide an

NGF-l ike t rophic activity that supports neuron at-

tachment and long-term survival in the absence of an

exogenous source of N G F , and this suppor t is

blocked by ant ibodies to NGFt0, 4°-42. These results

have led to the proposa l that the t rophic suppor t to

per ipheral neurons in vivo is normal ly provided by

their associated non-neuronal cells, and that exoge-

nous sources of neuronot rophic factors are required

for survival only when neurons are exper imenta l ly

deprived of these associated cells, as occurs in tissue culture38,39.

In this repor t , we describe the use of the PC12 line

of nerve growth factor (NGF)- respons ive pheochro-

mocytoma cells co-cul tured with Schwann cells de-

rived from mouse and rat dorsal root ganglia (DRGs)

to investigate the capacity of Schwann cells to sup-

port neuri te regenerat ion. The use of PC12 cells per-

mits the assay of neur i te -promot ing activities in pe-

r ipheral non-neuronal cells, independent of their ca-

pacity to support neuronal survival. This is because

PC12 cells do not require an exogenous source of

N G F or o ther neuronot rophic factors for survival or

proliferat ion. However , when provided with N G F at

physiological levels, PC12 cells begin to acquire char-

acteristics of di f ferent ia ted sympathet ic neurons 24,

including the cessation of cell division, product ion of

long, branching neuri tes, acquisit ion of neuron-l ike

electrical proper t ies , and the product ion of synaptic

Correspondence: Michael Cochran, Department of Anatomy, Temple University School of Medicine, 3400 North Broad Street, Phil- adelphia, PA 19140, U.S.A.

0165-3806/85/$03.30 © 1985 Elsevier Science Publishers B.V.

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vesicles 18,22,25,26,36. The long-term maintenance of

PC12 neurites requires a trophic influence that can be provided by NGF, and removal of this influence results in degeneration of neurites 26. Finally, when PC12 ceils, previously treated with NGF, are mecha- nically divested of their neurites and then replated onto collagen substrates, they regenerate neurites in the presence of NGF, but not in its absencell,Z3. 24.

Results from the present study provide evidence that contact with Schwann cells can promote PC12 cell neurite regeneration and long-term maintenance in the absence of exogenous NGF. Moreover, the Schwann cell-associated neurite regeneration-pro- moting activity is not blocked by antibody to NGF.

MATERIALS AND METHODS

Preparation of Schwann cell beds Explants consisting of dorsal root ganglia from

neonatal mice or rats were transferred into Aclar plastic mini-dishes29 coated with reconstituted rat tail tendon collagen. To eliminate fibroblasts, cultures were cycled with antimitotic agents on alternating feeding days, by methods modified from Wood 46. DRG explants were initially grown in an antimitotic medium A containing 87% (v/v) Eagles MEM, 10% (v/v) fetal calf serum, 2% (v/v) chick embryo extract (EE), 0.015 M KC1, 1.4 mM glutamine, 0.6% glu- cose, 50 ng/ml 2.5 S nerve growth factor (NGF), 0.01

mM fluorodeoxyuridine (FuDR), 0.02 mM cytosine arabinoside (Ara C), and 0.01 mM uridine. This anti- mitotic medium was alternated every feeding cycle with medium B containing 70% Eagles MEM, 20% fetal calf serum, 10% EE, 1.4 mM L-glutamine, and 50 ng/ml NGF. Cultures were fed every 2-3 days, al- ternating between medium A and B during the first 6 feedings. At this time, DRG explant cultures having radial neurite outgrowths which were populated by non-neuronal cells were selected, and the ganglionic explants were excised with razor blade fragments. These were transplanted into fleshly prepared col- lagen-coated dishes, in B medium. Most of these transplanted ganglia produced neurite outgrowths that were subsequently populated by Schwann cells and which were also devoid of fibroblasts. By 3-4 weeks after transplantation, the neurite outgrowth regions contained large populations of Schwann cells. Cultures that were flee of fibroblasts (identi-

lied as flattened cells situated on the collagen sub- strate between neurite fascicles) were selected, and the D R G explants in these cultures were excised and discarded. The resulting pure Schwann cell cultures were then maintained in NGF-flee RPMI medium containing 10% horse serum and 5% fetal calf serum, without NGF, for 1-2 weeks to effect a washout of exogenous NGF and to allow time for neurite degen- eration. RPMI medium was selected for its suitability for long-term PC12 cell culture. The resulting cul-

tures contained only dense islands of Schwann cells sit- uated in central regions of the dishes. These central islands occupied areas 5-10 mm in diameter in the centers of each dish, or approximately 4-15% of the total surface area of each culture. These cultures were used directly in PC12 addition experiments.

Preparation of PC12 cells PC12 pheochromocytoma cells were grown on col-

lagen-coated culture dishes in RPMI medium con- taining 1% horse serum and 50 ng/ml 2.5 S NGF for 21 days. Nerve growth factor was prepared by the method described by Mobley et al. 30. These cells were then removed from the dishes by treatment with 0.2% trypsin in calcium- and magnesium-free PBS for 30 min at 36 °C. This was followed by 3 washes by centrifugation and resuspension in RPMI medium containing 10% horse serum and 5% fetal calf serum,

without NGF. Prior to addition of these cells to Schwann cell cul-

tures, each PC12 cell suspension was divided into two

portions. To one of these, 2.5 S NGF was added to 50 ng/ml final concentration, and these cells were used in control cultures. The other portion of PC12 cells remained in NGF-free medium. Schwann cell cul- tures were also divided into two groups; one group remained in NGF-free medium, and the other was

shifted into medium containing 50 ng/ml NGF. PC12 cells in NGF-flee RPMI medium were then aliquoted onto Schwann cell cultures in the same NGF-flee me- dium. As controls, PC12 cells in NGF-containing RPMI medium were plated onto Schwann cell cul- tures in the same NGF-containing medium. Addi- tional control cultures consisting of isolated NGF- primed PC12 cells (not recombined with Schwann cells) were prepared by replating these cells onto fresh collagen-coated dishes in RPMI medium, either in the presence or absence of 50 ng/ml NGF, at

the same cell densities.

Light microscopic quantitation Cultures were monitored on feeding days for the

presence of PC12 neurites by either phase contrast or Hoffman interference contrast microscopy. PC12 cells, occurring either as single cells or clusters, were

counted and scored as having neurites if they had one or more processes that extended at least 2 × the cell body diameter. Clusters of PC12 cells were counted as single cells with or without neurites, regardless of the numbers of neurites extending from them. The arrangement of neurons on beds of Schwann cells sometimes resulted in ambiguous images in which slender cellular processes could not be identified as originating from either PC12 or underlying Schwann

cells. In these cases, PC12 cells or clusters were scored as cells without neurites. This scoring method inevitably results in an underestimate of the total number of PC12 cells bearing neurites because it dis- regards the proportion of PC12 cells or clusters that produced neurites which were indistinguishable from background cell processes, or which fasciculated along Schwann cells.

The problem of identifying neurites on Schwann

cell beds was partially overcome by scoring cultures for neurite-bearing cells after fixation for electron microscopy and plastic embedding. Osmium post-fix- ation tended to increase the contrast between neu- rites and underlying Schwann cell processes when fixed cultures were viewed by phase microscopy (see

Fig. 1A, C, D).

Electron microscopy The presence of neurites in Schwann cell bed re-

gions of PC12-Schwann cell co-cultures was con- firmed by electron microscopy. For ultrastructural analysis, cultures were fixed at approximately one week intervals up to 7 weeks after cell combination. Cultures were rinsed in Hank's BSS and then fixed with 2% glutaraldehyde in 0.1 M arsenate buffer con- taining 0.1% sucrose at room temperature for 1 h. These were rinsed in the same buffer with sucrose, and post-fixed for 1 h (5 °C) in 0.1 M arsenate buffer containing 1% osmium tetroxide and 1.5% potassi- um ferrocyanide. Cultures were dehydrated in a graded ethanol series, and embedded in low viscosity plastic. The Aclar mini-dishes were removed and the

107

cultures were then scored for the percentage of PC12

cells containing neurites. Subsequently, silver-gold thin sections were taken randomly throughout the Schwann cell bed, stained in uranyl acetate and lead citrate, and examined in a Philips 300 electron micro- scope operating at 60 kV.

Treatment with antibodies to mouse NGF Additional cultures were treated with rabbit anti-

serum to purified mouse 2.5 S NGF (BRL Laborato- ries). In an initial trial, we determined that a 1:100 di- lution of the antiserum was effective in completely blocking the neurite regenerative response of isola- ted PC12 cells to 10 ng/ml of 2.5 S NGF. Antiserum was applied to Schwann cell cultures either 3 days prior to plating PC12 cells, or at the same time that cells were added. Experimental co-cultures were treated with a 1:100 dilution of the antiserum in the absence of NGF. Control co-cultures were treated with 10 ng/ml of 2.5 S NGF and antiserum. NGF-pri- med PC12 cells were divested of their neurites and washed as described above prior to replating onto the Schwann cell beds. Additional control cultures in- cluded PC12 cells grown in the absence of Schwann cells, either: (1) without NGF or antiserum; (2) with 10 ng/rnl 2.5 S NGF and without antiserum; (3) with 10 ng/ml NGF and antiserum; (4) with 500 ng/ml NGF and antiserum; and (5) with 10 ng/ml NGF and a 1:100 dilution of non-immune rabbit serum. These cultures were maintained for 5 days. Cell counts in Schwann cell bed regions and in off-bed regions of the cultures were made daily.

RESULTS

PC12-Schwann cell co-cultures Purified Schwann cells occupied a central 5-10

mm diameter region in each culture dish. In the ab- sence of neurons, these cells are mitotically quies- cent 47 and do not migrate into peripheral regions of the culture dishes. At the time of PC12 cell addition, these cultures therefore contained a central Schwann cell bed region, surrounded by a cell-free collagen substratum, or off-bed region. The Schwann cell den- sity in the central beds was variable, although the densities were generally greater in the central region of the bed than at the periphery. When PC12 cells were added to these cultures, some of the PC12 cells

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Fig. 1. PC12 cells co-cultured with mouse Schwann cells. When cultured for 4 days without exogenous NGF (A), PC12 cells flattened, enlarged, and produced abundan t neurites (arrowheads) when cells were situated over the Schwann cell beds. Al though visibility of neurites was limited by the background of Schwann cell processes, PC12 cells which extended neuri tes over open spaces between Schwann cells, and cells with neurites oriented at right angles to the Schwann cell processes, were more easily seen. Most PC12 cells sit-

109

settled on and became attached to the Schwann cell bed, while others became attached to surrounding

off-bed regions of collagen. This permitted compari- son of the responses of PC12 cells that had estab-

lished contact with Schwann cells with the responses of PC12 cells not directly in contact with the Schwann

cell beds, but which were continuously exposed to

medium conditioned by Schwann cells in the same dish.

Primed PC12 cells replated in the absence o f Schwann

cells

Before discussing the behavior of PC12 cells in the presence of Schwann cells, we will describe control

PC12 cell cultures grown without Schwann cells in

the presence or absence of NGF. PC12 cells treated

with NGF for 21 days were divested of their neurites and replated onto collagen-coated culture dishes.

When these cells were plated in NGF-containing me-

dium, they rapidly regenerated neurites (Tables I and II, Fig. 1), as previously described by Greene 22.

In contrast, when such cells were plated into NGF-

free medium, they failed to produce neurites (Tables I and II). In the absence of NGF, the PC12 cells

formed small colonies that increased in size with time, presumably due to cell proliferation. These re-

suits confirm that PC12 cells primed by pretreatment

with NGF have the ability to regenerate neurites, but that they only express this ability when provided with

an appropriate neurite-promoting agent such as NGF 22. In the experiments described below, primed

PC12 cells are plated onto cultures containing a cen-

trally located Schwann cell bed to assay Schwann

cells for neurite regeneration-promoting activity.

PC12 cell spreading on Schwann cells

In PC12-Schwann cell co-cultures, PC12 cells that

attached to either mouse or rat Schwann cell beds be-

gan to flatten within several hours after establishing

contact with the beds, both in the presence or ab-

sence of exogenous NGF. By the first day of co-cul-

ture, these cells had enlarged to achieve a neuron-

like appearance in the phase microscope, with a large

pale nucleus, prominent nucleolus, and phase-dark

cytoplasm typically associated with cell spreading

(Figs. 1A, 3A). After 4 days of co-culture in the ab- sence of exogenous NGF, 74% of all PC12 cells con-

tacting Schwann cells had attained a flattened mor- phology. PC12 cells that were attached to the sur- rounding off-bed regions of collagen, however, re-

mained small, spherical and phase-bright when exog-

enous N G F was not provided (Fig. 1B). Most (86%)

of these cells retained these features and were not flattened on the collagen substratum by the fourth day of co-culture. With time in culture, clusters of

PC12 cells became prominent in the surrounding off- bed regions of collagen, indicative of PC12 cell prolif-

eration in these areas. Some PC12 cell clustering also

occurred in the Schwann cell bed regions, but clusters

on the Schwann cell beds were less prominent and re-

mained smaller than in off-bed regions, raising the

possibility that contact with the Schwann cell bed lim-

TABLE I

PC12 neurite regeneration and maintenance -- PC12 + mouse Schwann cell co-cultures

Percentages of PC12 cells exhibiting neurites on and off Schwann cell beds as a function of time in culture. Figures rep- resent percentages of PC12 cells bearing neurites, with the numbers of cells counted indicated in parentheses. Control NGF-treated cultures were given 50 ng/ml 2.5 S NGF.

-NGF medium +2.5 S NGF medium

On SC bed Off SC Bed On SC bed Off SC bed

Days after cell combination 1 26.3 (407) 0.8 (382) 29.1 (367) 67.2 (350) 4 22.4 (397) 3.6 (350) 27.4 (364) 74.6 (355) 8 58.7 (386) 6.4 (360) 64.3 (352) 97.5 (363)

12 64.7 (364) 7.2 (356) 65.2 (367) 98.0 (345) 19 66.3 (371) 9.4 (287) 76.8 (353) 98.8 (247) 24 67.9 (343) 8.7 (324) 69,1 (322) 97.8 (288) 29 65.4 (302) 7.4 (306) 70.3 (326) 98.6 (274) 34 69.5 (324) 8.8 (311) 74.8 (318) 97.0 (304)

Isolated PC12 cells (controls) 4 - 0.9 (298) - 84.9 (307)

12 - 0.0 (312) - 99.7 (344) 19 - 0.0 (301) - 97.8 (312)

uated on the collagenous substrate peripheral to the Schwann cell bed regions (B) were devoid of neurites at 4 days. After 19 days of co-culture without exogenous NGF (C), PC12 cells situated on Schwann cells also exhibited abundant neurites (arrowheads). In these cultures, PC12 cells in surrounding off-bed regions of the collagen substrate (D) continued to proliferate, producing small colonies of cells with few neurites. Control isolated PC12 cells grown in the presence of 50 ng/ml NGF produced abundant neurites at both 4 days (E) and 19 days (F). All panels are phase photomicrographs. A, C, and D were photographed after osmium fixation for electron mi- croscopy to enhance contrast between neurites and background Schwann cell processes. Bars: 30/~m.

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TABLE II

Percentage of PC12 cells with neurites in cultures treated with antiserum to 2.5 S NGF

Percentages of PC12 cells exhibiting neurites on and off Schwann cell beds in the presence or absence of rabbit antiserum to mouse 2.5 S nerve growth factor (NGF). Anti-NGF was applied at a concentration sufficient to block PC12 neurite regeneration in response to 10 ng/ml 2.5 S NGF, and to block about 50% of neurite regeneration in response to 500 ng/ml NGF. Anti-NGF pretreated Schwann cell cultures were treated for 3 days with antiserum prior to plating PC12 cells, and antiserum treatment was continued during co-culture. Anti-NGF was unable to block PC12 neurite regeneration and maintenance in response to contact with Schwann cells.

+Anti-NGF +Anti-NGFpretreatment -Anti-NGF

PC12 + Schwann cell co-cultures without NGF On Schwann cell bed

Day 1 2 3 4

Off Schwann cell bed Day 1

2 3 4

Controls without Schwann cells PC12 cells - NGF - Anti-NGF

Day 1 2 3 4

PC12 cells + 10 ng/ml 2.5 S NGF Day 1 9.3 (376)

2 3.7 (375) 3 3.0 (367) 4 1.5 (326)

PC12 cells + 500 ng/ml 2.5 S NGF Day 1

2 29.6 (395) 3 49.4 (354) 4 51.2 (383)

PC12 cells + 10 ng/ml 2.5 S NGF + non-immune rabbit serum

Day 1 2 3 4

56.0 (697) 65.7 (402) 60.7 (632) 58.1 (689) 66.9 (375) 61.2 (668) 59.5 (706) 69.5 (357) 68.0 (679) 63.8 (639) 66.3 (674) 73.5 (321)

7.1 (693) 4.2 (456) 10.9 (763) 2.6 (693) 2.6 (430 2.9 (653) 0.6 (653) 2.5 (448) 2.0 (711) 0.2 (569) 2.2 (730) 0.5 (563)

8.3 (349) 4.5 (374) 0.9 (318) 0.4 (241)

86.9 (396) 94.1 (392) 92.8 (305) 94.8 (344)

79.0 (403) 88.2 (372) 88.9 (423)

its PC12 cell proliferation. Under the culture condi-

tions used in the present experiments, the limiting ef-

fect of Schwann cell contact upon PC12 cell prolifer-

ation was not transmitted through the medium to oth-

er PC12 cells that did not contact the Schwann cell

beds.

Neurite regeneration and maintenance in the presence

o f Schwann cells

PC12 cells that were co-cultured with mouse

Schwann cells in the absence of exogenous NGF be-

gan to regenerate neurites by the first day after cell

combination, and the proport ion of neuri te-bearing

cells remained high for up to 49 days. The neurite-

bearing cells, however, were largely restricted to the

central Schwann cell bed areas of the cultures when

exogenous N G F was not provided (see Tables I and

II). Control PC12-Schwann cell co-cultures main-

tained in the presence of exogenous NGF, however,

developed neurites both on and off the Schwann cell

beds (see Table I). Within one day after replating,

many (26-60%) of the PC12 cells situated over the'

Schwann cell beds had neurites, but few (1-10%) of

the cells in the same dishes which were situated off

the Schwann cell beds had neurites.

In the absence of exogenous NGF, the apparent

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Fig. 2. Electron micrographs of PC12 neurites si tuated over mouse Schwann cell beds after 19 (A), 34 (B), and 49 (C) days of co-cul- ture in the absence of NGF. Long- term maintenance of PC12 neurites without exogenous NGF was evident only when the PC12 cells or their neurites established contact with Schwann cells. The Schwann cells and their processes are identifiable by their darker, fibrillar cytoplasm in contrast to the lighter-staining PC12 neurites, which have axon-like distributions of microtubules. Note the absence of Schwann cell basal lamina. Bars: 0 .5#m.

112

Fig. 3. PC12-Schwann cell co-cultures grown in the absence of NGF and treated with antiserum to mouse 2.5 S NGF. Cultures were treated with a concentration of antiserum sufficient to inactivate 10 ng/ml 2.5 S NGF. When PC12 cells were plated onto Schwann cells in the absence of NGF, abundant neurites (arrowheads) were apparent on the Schwann cell bed within 24 h (A). When PC12 cells were situated off the Schwann cell beds, neurites were not produced (B). Bars: 30/~m.

proport ions of neur i te-bear ing PC12 cells increased

with time in Schwann cell bed regions. By 8 days of

co-culture, most (58%) of the PC12 cells that con-

tacted Schwann cell beds had neurites, but the num-

bers of neur i te-bear ing PC12 cells lying off the

Schwann cell beds remained less than 10% through-

out the measurements . The numbers of neuri te-bear-

ing PC12 cells on the Schwann cell beds reached

69-75% in both NGF-dep r ived and in control NGF-

containing cultures. This p robably represents an un-

derest imate of the actual numbers of neuri te bearing-

PC12 cells on the Schwann cell beds, because of the

difficulty in distinguishing neuri tes that may have fas-

ciculated along Schwann cells. Note , for example ,

that while PC12-Schwann cell co-cultures t reated

with N G F would be expected to contain equivalent

proport ions of neur i te-bear ing PC12 cells both on

and off the Schwann cell beds, the numbers of neu-

r i te-bearing cells on Schwann cell beds in these NGF-

t rea ted co-cultures were somewhat less than in off-

bed regions, and were similar to those observed in

the NGF-depr ived cultures.

The numbers of PC12 cells having neuri tes at early

times after co-culture with Schwann cells were more

variable than after longer periods of co-culture (com-

pare Tables I and II). While the reason for this varia-

bility is not known, we suggest that the visibility of

neurites on Schwann cell beds increases with t ime in

vitro as the neuri tes fasciculate and continue to grow

in length and diameter . The visibility of small, newly

regenera ted neurites on the Schwann cell beds may

be expected to depend upon the density and distribu-

tion of Schwann cells; a high density of Schwann cells

would result in a lower visibility of neuri tes and hence

lower apparent percentages of neur i te-bear ing cells.

PC12 cells si tuated immedia te ly off of the Schwann

cell beds (50-100 # m from the edge) general ly did

not have neuri tes at any time during the exper iments ,

while nearby PC12 cells s i tuated on the Schwann cell

bed frequently had neurites. Occasionally, however ,

PC12 cells s i tuated immedia te ly off the Schwann cell

bed had neurites. In such cases, the neuri tes con-

tacted the Schwann cell bed. The occasional PC12

cells with neuri tes immedia te ly off the Schwann cell

bed could be explained by stabil ization of initial neu-

rite sprouts which contacted the Schwann cell bed.

Because the PC12 cell line was originally derived

from a rat pheochromocytoma, we also carried out

experiments in which PC12 cells were combined with

rat DRG-de r ived Schwann cells. The results of

these exper iments were similar to those in which

mouse Schwann cells were used. Cell f lat tening and

neurite regenerat ion commenced within one day af-

ter cell combinat ion in the rat Schwann cell bed re-

gions. The numbers of neurite bearing cells both on

and off the Schwann cell beds were also similar to those obtained using mouse Schwann cells, indicating that both mouse and rat Schwann cells can support PC12 neurite regeneration.

Electron microscopy Electron micrographs of PC12-Schwann cell co-

cultures maintained in NGF-free medium confirm the presence of abundant fascicles of PC12 neurites distributed along the surfaces of Schwann cells at each time point examined up to 7 weeks (Fig. 2). PC12 neurites were identified by their shape, lightly staining cytoplasm, and distribution of microtubules, in contrast to the denser, fibrillar cytoplasm of adja- cent Schwann cells. These processes often occupied shallow indentations of the Schwann cell surface reminiscent of early axon-Schwann cell interactions prior to ensheathment. PC12 neurites were largely unensheathed by Schwann cell processes at any of the examined times, and the Schwann cell basal lami- na was absent over most of the cell surfaces. The fail- ure of Schwann cell ensheathment of PC12 neurites, and the failure of PC12 support of Schwann cell basal lamina assembly, has been described in a preliminary report 12 and will be fully described in another paper.

Neurite regeneration in the presence of anti-NGF Rabbit antiserum against purified mouse nerve

growth factor was applied to co-cultures of PC12 cells and mouse Schwann cells to determine whether the neurite regenerative effect was due to the presence of NGF. Antiserum sufficient to inactivate 10 ng/ml of 2.5 S NGF was applied to Schwann cell cultures both immediately prior to addition of primed PC12 cells and 3 days prior to plating PC12 cells. In control PC12 cultures we determined that 10 ng/ml of 2.5 S NGF was sufficient to elicit neurite regeneration in primed PC12 cells, confirming the previous report of Greene 22. The concentration of antiserum used in

these experiments blocked neurite regeneration by isolated PC12 cells when grown in the presence of 10 ng/ml 2.5 S NGF (see Table II). Furthermore, when 2.5 S NGF was supplied at 500 ng/ml in the presence of antibody, only about 50% of the PC12 cells were able to regenerate neurites after 4 days of treatment.

In the presence of the indicated amount of anti- body to NGF, PC12 cells situated over mouse

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Schwann cell beds flattened and produced neurites in

a manner that was indistinguishable from co-cultures established without antibody (Table II, Fig. 3A). Neurites were apparent within 24 h after plating PC12 cells, and the percentage of cells with neurites on the Schwann cell beds remained at high levels for the duration of the antiserum treatment (Table II). When Schwann cell beds were pretreated with anti- serum (1:100) for 3 days prior to addition of PC12 cells, and then continuously incubated in the antise- rum during PC12-Schwann cell co-culture, a similar positive neurite regenerative response resulted (Table II). PC12 cells situated off the Schwann cell beds in the same antiserum-treated cultures failed to produce neurites either in the presence or absence of' NGF. When PC12 cells were grown in 10 ng/ml NGF with a 1:100 dilution of non-immune rabbit serum, neurites were produced at similar levels to those in cultures that did not receive the non-immune serum. These results demonstrate that the regenerative re- sponse of PC12 cells in contact with Schwann cell beds is not blocked by anti-NGF, and suggest that this response results from a neurite-promoting activ- ity other than NGF that is found near the Schwann cell surface.

DISCUSSION

PC12 cells cultured in isolation require an exoge-

nous source of NGF for neurite initiation and mainte- nance 26. Furthermore, primed PC12 cells, previously

grown in the presence of NGF, also require exoge- nous NGF for the regeneration of neurites when these cells are replated onto collagenous sub- strates11. 23. The results presented here demonstrate that PC12 neurite regeneration and long-term main- tenance can be effected in the absence of exogenous NGF when PC12 cells are co-cultured with mouse or rat Schwann cells. Under the present NGF-free cul- ture conditions, PC12 cells only regenerated neurites when they contacted a Schwann cell bed, and failed to produce neurites when situated on a collagen sub- stratum immediately adjacent to Schwann cells. These observations raise the possibility that the PC12 neurite-promoting activity reported here is restricted to Schwann cell surfaces and is not a diffusable, medi- um-conditioning factor. These results could also be explained by the secretion of a soluble neurite-pro-

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moting factor that is only found in sufficient concen-

tration near the Schwann cell surface to elicit the

PC12 regenerative response. However, in the pres- ent study we have observed many PC12 cells situated

on collagen in close proximity to the edge of Schwann

cell beds, and these cells rarely regenerated neurites.

While these results do not distinguish between these possibilities, they nevertheless demonstrate that con-

tact or close apposition with a Schwann cell bed can

elicit PC12 neurite regeneration in the absence of ex- ogenous NGF.

Furthermore, the Schwann cell-mediated neurite regenerative response is not blocked by antiserum to mouse 2.5 S nerve growth factor, suggesting that the

neurite promoting activity is antigenically different

from NGF. A possible candidate for the neurite-pro- moting activity is the extracellular matrix (ECM)

produced by Schwann cells. Schwann cell basal lami- na 27 and some of the molecular constituents of basal lamina 45 have previously been implicated in the proc-

ess of axonal regeneration in peripheral nerves.

Moreover, PC12 cells have been shown to initiate neurites in NGF-free medium in response to growth

on a basement membrane-like extracellular matrix (ECM) 21,a'4. However, neurite maintenance under

these conditions is transient (5-10 days21 or 3-5

days44), suggesting that the ECM provides a suitable

stimulus for initiation of PC12 neurites but not their

long-term maintenance. Schwann cells in the experi-

ments reported here could be providing PC12 cells with a similar ECM-associated neurite-promoting

factor; however, the long-term neurite survival (at least 7 weeks) that we have observed suggests that an additional influence that is not provided by isolated

ECM components may be present. Furthermore, in preliminary experiments we have observed PC12 cell

neurite regeneration to occur on replated beds of

Schwann cells that had previously been dissociated by collagenase and trypsin treatment. Trypsinization of Schwann cells has previously been shown to result in the complete removal of the Schwann cell basal lamina and, in isolation, Schwann cells do not reas- semble basal lamina9. The regeneration of PC12 neu- rites on beds of trypsinized Schwann cells therefore suggests that the Schwann cell-associated PC12 neu- rite-promoting activity does not require the presence of an intact basal lamina. Electron micrographs of Schwann cells co-cultured with PC12 cells also dem-

onstrate that basal lamina is neither assembled nor

maintained after long periods of Schwann cell con- tact with PC12 cell neurites. This observation further

suggests that a morphologically identifiable basal

lamina may not be required for Schwann cell-me-

diated PC12 neurite regeneration in the absence of

NGF, and raises the possibility that the Schwann cell- derived neurite promoting activity is associated with

the Schwann cell plasmalemma.

Neurite-promoting factors (NPFs) have been found in a variety of tissue extracts and in condi- tioned media derived from several sources 3,7,13-16, 19,20,2s,31 35.40 42. Some of these NPFs are active only

when bound to non-collagen substrates such as poly- ornithinel-3,13 15,2s. Perhaps Schwann cell surfaces in

vivo function similarly to poly-ornithine substrates in

vitro by binding NPFs. A neurite regeneration-pro- moting factor situated at Schwann cell surfaces could

be important during the repair of injury to peripheral nerves. In cultures of peripheral ganglionic cells, neurites tend to extend along parallel arrays of

Schwann cell processes 37. In vivo, Schwann cells re-

spond to axotomy by proliferating to fill the original

endoneurial compartment of the distal nerve seg- ment. The surfaces of such Schwann cells, initially

devoid of basal lamina, may provide the substrates upon which regenerating axons extend. A neurite-

promoting factor, specifically localized to the

Schwann cell surface, could therefore facilitate the growth of regenerating neurites along the axis of

Schwann cells and ensure the correct orientation of axons during repair.

The ability of Schwann cells to facilitate the regen- eration of PC12 cell neurites may be indicative of a

more general property of Schwann cells. In this re- spect, several lines of evidence already suggest that

the microenvironment of peripheral nerves is impor-

tant to the success of axonal regeneration for PNS n e u r o n s 15,27,33,34,43. Moreover, axons of CNS neu-

rons, which largely fail to regenerate in the CNS, are capable of extensive regeneration when they extend into implanted segments of peripheral nerves con- taining Schwann cells 4.s,jT.

The present study demonstrates that PC12 cells can be used to identify neurite-promoting activities independently of neuronal survival factors. Because PC12 cells do not require exogenous trophic factors such as NGF for survival, as do peripheral sympa-

115

thet ic and sensory neu rons in isolat ion, the ex is tence

of n e u r i t e - p r o m o t i n g factors can be assayed di rec t ly

by using these ceils. F u r t h e r w o r k is cur ren t ly in pro-

gress to charac te r ize the Schwann ce l l -assoc ia ted

n e u r i t e - p r o m o t i n g activity.

ACKNOWLEDGEMENTS

We wish to thank Dr . L loyd A . G r e e n e for p rov id-

ing A C 1 2 cells and Mr. T h o m a s K r u p a for his excel-

lent assistance with e l ec t ron microscopy . This re-

search was suppor t ed by grants f rom the Muscu la r

Dys t rophy Assoc ia t ion ( M . C . ) , by Na t iona l Insti-

tutes of H e a l t h G r a n t NS17681 ( M . M . B . ) and by a

Resea rch C a r e e r D e v e l o p m e n t A w a r d NS00698

( M . M . B . ) .

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