Implantation of PC12 cells into the corpus striatum of rats with lesions of the dopaminergic nigrostriatal neurons

Brain Research, 348 (1985) 283-288

283

BRE 11160

Implantation of PC12 Cells into the Corpus Striatum of Rats with Lesions of the Dopaminergic Nigrostriatal Neurons F. HEFTI 1,*, J. HARTIKKA 1and M. SCHLUMPF2 ISandoz Ltd., PreclinicalResearch, 4002 Basle and 2Institute of Pharmacology, University of Zurich, 8006 Zurich 'Switzerland)

(Accepted February 12th, 1985) Key words: circling behavior-- 6-hydroxydopamine - - nigrostriatal lesion - - PC12 cell - - striatum - - transplantation - - grafting

PC12 cells, which have the capacity to synthesize and release catecholamines, were implanted into the corpus striatum of adult rats with lesions of the dopaminergic nigrostriatal pathway. PC12 cells survived for 1-2 weeks in the host striatum and degenerated thereafter. The presence of these cells was associated with an attenuation of the apomorphine-induced circling behavior in rats with unilateral nigrostriatal lesions. These findings indicate that cells from a cell line are able, at least temporarily, to survive in the rat brain and to compensate functional deficits caused by lesions of specific neuronal systems. Cell lines might therefore be used for intracerebral grafting instead of neurons derived from fetal brains, if their survival can be prolonged and their proliferation in the host brain prevented.

INTRODUCTION Grafts of e m b r y o n a l brain tissue are able to compensate functional deficits induced by lesions of specific neuronal systems in the rat brain2, 4.8-13.2o-22. In particular, tissue pieces or dissociated cells from the substantia nigra of fetal rats i m p l a n t e d into the corpus striatum of adult animals with lesions of the dopaminergic nigrostriatal pathway reduce the behavioral deficits associated with these lesions4, s-13. These studies are likely to have great implications for the treatment of h u m a n Parkinson's disease, since they suggest the possibility that behavioral alterations caused by the d e g e n e r a t i o n of d o p a m i n e r g i c neurons occurring in this disease might be corrected by implants of fetal tissue from the substantia nigra. Successful application of the grafting technique to humans, however, is h i n d e r e d by substantial technical and ethical p r o b l e m s that arise, if the t r a n s p l a n t e d tissue has to be of human origin. Such p r o b l e m s could be avoided with the use of cell lines grown in culture and available any time for implantation. Cells to be used for implantation into the h u m a n brain would

have to satisfy several criteria. (1) They should grow in culture but stop proliferating once implanted into the brain. This could possibly be achieved by producing temperature-sensitive mutants or cells that differentiate when s u r r o u n d e d by brain tissue. (2) The implanted cells must have the capacity to synthesize and store dopamine. (3) The cells must release d o p a m i n e after implantation into the striatum. This requires that they either tonically release d o p a m i n e or that they express receptors for other transmitter substances released from neurons of the host brain which stimulate d o p a m i n e release from PC12 cells, D o p a mine must be released in sufficient quantities to stimulate d o p a m i n e receptors of the host brain. To study the feasibility of the a p p r o a c h with cell lines, we decided to implant PC12 cells into the corpus striatum of rats with lesions of the dopaminergic nigrostriatal system. PC12 cells were chosen because of their ability to synthesize and store large amounts of d o p a m i n e and n o r e p i n e p h r i n e and to release these transmitter substances when depolarized 14.15. Furthermore, PC12 cells can be induced to stop proliferating and to acquire a neuron-like m o r p h o l o g y when

Correspondence: F. Hefti. Present address: Department of Neurology, University of Miami Medical School, P.O. Box 016960, Miami, FL 33101, U.S.A.

0006-899385/$(13.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

284 exposed to nerve growth factor (NGF)14. MATERIALS AND METHODS Unilateral nigrostriatal lesions were produced in male rats (Wistar, 200 g) by injecting 8/~g of 6-hydroxydopamine into the right anteromedial substantia nigra 17. Lesioned animals were injected twice weekly with apomorphine (0.25 mg/kg, s.c.) and placed in a cylinder (30 cm diameter, fiat bottom) to test for rotational behavior. The number of apomorphine-induced contralateral rotations (full body turns) was counted in the time interval between 15 and 20 min after the injection of apomorphine. Animals exhibiting a constant frequency of rotations during at least 5 test sessions were taken for the injection of PC12 cells. For intrastriatal injections of PC12 cells, animals were anesthetized with pentobarbital and placed in a stereotaxic apparatus. PCI2 cells suspended in 5 10 pl of growth medium (cf. below) were taken up into a Hamilton syringe attached to the stereotaxic apparatus. For single injections, the syringe was inserted into the brain at the anteroposterior level of the bregma and 2.5 mm lateral to the midline. The tip was lowered to a position at 4.5 mm below the surface of the dura. This site of injection corresponded to position A7500, lateral 2.5, and dorsal +0.5 according to the atlas of K6nig and KlippeU 9. For double injections, the tip of the syringe was lowered into the striatum at the same lateral and dorsoventral positions, but at 0.5 mm anterior and posterior to the bregma (corresponding to levels A8000 and A700019, respectively). The cell suspension was injected over a period of 10 min, and the syringe was slowly retrieved 2 min after termination of the injection. Control animals received equal volumes of medium injected at two striatal sites. Rats injected with cells or control medium were tested twice weekly for apomorphine-induced rotational behavior as before performing the injection. PC12 cells were grown in polystyrene culture dishes (35 mm diameter, Falcon) in Dulbecco's modified Eagles medium containing 10% horse serum and 5% fetal calf serum. For intracerebral injections, the cells were washed several times in medium and suspended in a minimal volume of medium. At various times after injection of cells, rats were

taken for histochemical analysis. They were decapitated and their brains were rapidly frozen on dry ice. Sections of 30/~m were cut on a freezing microtome and were taken for the visualization of catecholamines using the histofluorescence method of De la Torre 7 or for Nissl staining using cresyl violet. Chemicals used were of analytical grade and purchased from Merck (Darmstadt, F.R.G.) or Sigma (St. Louis, MO). Culture media and sera were obtained from Gibco Europe. RESULTS Histochemical visualization of catecholamines in sections revealed that the intranigral 6-hydroxydopamine injections had destroyed virtually all catecholamine-containing cell bodies in the substantia nigra. Accordingly, the catecholamine-specific fluorescence was reduced to background level in the striatum ipsilateral to the lesion. Lesioned animals injected intrastriatally with medium did not differ from those with 6-hydroxydopamine lesions alone. PC12 cells injected intrastriatally were easily discerned by their high content of catecholamines. All animals killed within the first week after injection contained a large number of highly fluorescent PC12 cells (Fig. 1A, B). They usually formed an agglomerate at the site of injection. The boundary between the agglomerate and the striatal tissue was always distinct, indicating that individual PC12 cells had not pen, etrated into striatal tissue. The injected cells retained the round morphological appearance they had exhibited in culture, and there was no difference in morphology between cells in the center of the agglomerate and those near the host brain tissue, indicating that the contact with striatal tissue did not induce morphological changes of injected PC12 cells. PCt2 cells did not survive permanently after intrastriatal injections. In animals analyzed 1-2 weeks after the operation, the agglomerate of injected material still was clearly visible. However, as judged by Nissl staining, the central part of the agglomerate was composed of amorphous material probably derived from degenerated PC12 cells. Intact PC12 cells, exhibiting strong catecholamine-specific fluorescence after staining, were only seen in areas near the striatal tissue (Fig. IC). In animals killed later than 2 weeks after the injections, no catecholamine-

285

Fig. 1. PCI2 cells injected into the corpus striatum of rats with nigrostriatal lesions. Catecholamine-containing PC12 cells were visualized using a glyoxylicacid histofluorescence method. A, B: agglomeration of PC12 cells in the corpus striatum 5 days after injection. C: PC12 cells 10 days after intrastriatal injections. Fluorescent, catecholamine-containing cells are exclusively localized near the striatal tissue. They surround degenerated material remaining at the site of injection. D: agglomeration of PCI2 cells in the lateral ventricle observed at 10 days after intracerebral injections. Bars represent 0.1 mm. containing PC12 cells but only degenerated material was present at the injection site. In some animals fluorescent PC12 were found in the ventricles (Fig. 1D). Similarly to PC12 cells localized intrastriatally, these cells degenerated within 1-2 weeks after injection. lntrastriatal injection of PC12 cells at two intrastriatal sites attenuated the a p o m o r p h i n e - i n d u c e d ro-

tational behavior during the 2 weeks after operation (Fig. 2). In contrast, the frequency of rotation was not affected by injections of medium alone. To quantify the effect of PC12 cells on a p o m o r p h i n e - i n d u c e d circling behavior, we averaged the frequency of rotation measured during 2 weeks after injection (corresponding to 5 test sessions) and compared it with the

286 ROTATIONS PER 5 M I N

PC12 cells growing in culture are induced to acquire a neuron-like morphology,, when nerve growth factor (NGF) is included in the culture medium!L We test-

100

ed whether PC I2 cells exhibiting a neuron>like mor-

-5o "J"~

MEDIUM

1

0

-o

1

2

WEEKS

t. . . .

2

phology survive better in rat striata, and ~Ae lhetef(~e injected PC12 cells grown in presence <~1 N(}F or added N G F to the medium during the injection. Neither of those treatments improved the survival of

P C "~2

'1' 2

~-~

"

,,,,

1

,

0

1

2

PC12 cells after intrastriatal injections (data not shown}.

WEEKS

DISCUSSION Fig. 2. Attenuation of apomorphine-induced circling behavior by intrastriatal injections of PC12 cells into rats with unilateral nigrostriatal lesions. Animals received unilateral nigrostriatal injections of 6-hydroxydopamine and their apomorphine-induced rotations were measured twice weekly, The frequency of rotations was assessed in the time interval from 15 to 20 min after injection of apomorphine (0.25 mg/kg s.c.). After establishment of a stable baseline, animals were injected with PC12 cells into the lesioned striatum at two injection sites. Controls received equal volumes of vehicle. The figure shows frequencies of rotations of individual animals. The average frequencies are given in Table I. Animals not tested during the entire period of 2 weeks after injection of PC12 cells were taken for histochemical analysis.

average frequency during the 2 weeks before the in-

The results of the present study show that PC12 cells injected into the striatum of rats with nigrostriatal lesions survive during a period of 1-2 weeks in the host brain. During that time, they appear to be able to compensate for functional deficits associated with the lesions. The mechanism by which PC12 cells produce this functional compensation has not been elucidated. When injected intrastriatally, they might tonically release catecholamines without being stimulated by host striatal neurons. Eventually, release of catecholamines might be induced by striatal transmitter substances acting through receptors located on the PC12 cells. These cells have been shown to ex-

cells at one intrastriatal site marginally reduced the average frequency by 9%. Injections of PC12 cells at

press rnuscarinic and nicotinic cholinergic receptors as well as receptors for enkephalins j.i~',>. Stimulation of the nicotinic receptors elevates the influx of

two sites resulted in a significant reduction of the average frequency by 27%. Control injections of medium failed to significantly affect the average rate of

sodium and the release of catecholamincs ~,>. However, it cannot be excluded that part of the functional compensation observed in the present study was me-

circling.

diated by catecholamines liberated from degenerat-

jection (Table I). Intrastriatal injections of PC12

TABLE t Apomorphine-induced rotations o f rats with unilateral nigrostriatal lesions ~{[ier intrastriatal rejections ~1' I'( 72 cells

Animals with unilateral nigrostriatal lesions were tested twice weekly before and after the injection of PC I2 cells into tile s~riatum. For experimental details cf. Fig. 2 and Methods. The values given represent mean frequencies of rotation averaged from 5 measurements before and after the injections of PCI2 cells. Values are means ± S.E.M., numbers in parentheses represent the number of animals tested. Rotations per 5 rain

Untreated controls (6) Vehicle injected at two sites (8) PC12 cells injected at one site (7) PC12 cells injected at two sites (11)

Befi)re injection

After injection

53.6 -+ 4.1 65.1 +_9.2 55.9 ± 4.6 61.9 ± 7,4

58.2 ± 9.5 58.3 _+9.7 48.4 _+8.3 43.4_+ 9.9

' ~ ( hange

+4.5 7_ ~() -5.6 + 7.8 --9. I z~ 7.2 27.2 ± ~).3'

* Significantlydifferent from mean value established before injection of PCI2 cells, P < 0.05 (analysis of variance).

287 ing PC12 cells.

causes. The fact that PC12 cells located near striatal

The time of survival of PC12 cells injected into rat

tissue survived best in the present study suggests that

striata appears to be very short when c o m p a r e d with

brain tissue exerts a beneficial influence on them.

that of grafted dopaminergic neurons obtained from

H o w e v e r , striatal tissue failed to affect the PC12 cells

fetal rat brains. Dissociated dopaminergic neurons

in a morphologically recognizable way. We found no

from fetal substantia nigra survive for several months

indication that PC12 cells were induced to migrate

in striata of rats with nigrostriatal lesions and contin-

away from the injection site and to integrate them-

uously increase their network of processes during

selves into the brain tissue or to acquire a neuron-like

that time 3-5,6. Correspondingly, behavioral changes

morphology as they do in presence of N G F . Further

observed after p e r f o r m i n g these grafts had a slow on-

studies clearly are needed to i m p r o v e the survival of

set and lasted for the entire observation time 9. In the

grafted PC12 cells, or other cell lines showing better

present study, the onset of the behavioral changes

survival have to be found.

was rapid, and their duration was limited to 1 - 2 weeks. Nevertheless, since PC12 cells d e g e n e r a t e

ACKNOWLEDGEMENTS

within a few hours when exposed to unfavorable conditions in culture, a survival time of 1 - 2 weeks in vivo

We thank Dr. B. G/ihwiler for stimulating dis-

can be considered as significant. T h e reason why in-

cussions and his contribution to the initiation of this

jected PC12 cells d e g e n e r a t e in the host brain is not

study. We are grateful to Dr. U. Otten for providing

known. Lack of essential nutrients or growth factors

us with PC12 cells, and we thank Miss Ch. Messer for

or an immunological response of the host are possible

excellent technical assistance.

REFERENCES

and Iversen, S.D., Intracerebral grafting of neuronal cell suspensions. IV. Behavioral recovery in rats with unilateral 6-OHDA lesions following implantation of nigral cell suspensions in different forebrain sites, Acta Physiol. Scand., Suppl. 522 (1983) 29-37. 10 Dunnett, S.B., Bj6rklund, A., Schmidt, H.R., Stenevi, U. and Iversen, S.D., Intracerebral grafting of neuronal cell suspensions. V. Behavioral recovery in rats with bilateral 6-OHDA lesions following implantation of nigral cell suspensions, Acta Physiol. Scand., Suppl. 522 (1983) 39-47. 11 Dunnen, S.B., Bunch, S.T., Gage, F.H. and Bj6rklund, A., Dopamine-rich transplants in rats with 6-OHDA lesions of the ventral tegmental area. I. Effects on spontaneous and drug-induced locomotor activity. Behav. Brain Res,, 13 (1984) 71-82. 12 Fray, P.J., Dunnett, S.B., Iversen, S.D., Bj6rklund, A. and Stenevi, U., Nigral transplants reinnervating the dopamine-depleted neostriatum can sustain intracranial selfstimulation, Science. 219 (1983) 416-419. 13 Freed, W.J., Ko, G.N.. Niehoff, D.L., Kuhar, M.J., Hoffer, B.J., Olson, L., Cannon-Spoor, H.E., Morihisa, J.M. and Wyatt, R.J., Normalization of spiroperidol binding in the denervated rat striatum by homologous grafts of substantia nigra, Science, 222 (1983) 937 939. 14 Greene, L.A. and Tischler, A.S., Establishment of noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor, Proc. Natl. Acad. Sci. U.S.A.. 73 (1976) 2424-2428. 15 Greene, L.A. and Rein, G., Release, storage and uptake of catecholamines by a clonal cell line of nerve growth factor responsive pheochromocytoma cells. Brain Research, 129 (1977) 247-263. 16 Greene, L.A. and Rein, G., Release of pH]norepinephfine from a clonal line of pheochromocytoma cells (PC12) by nicotinic cholinergic stimulation, Brain Research, 138

1 Amy, C.M. and Bennett, E.L., Increased sodium ion conductance through nicotinic acetylcholine receptor channels in PC12 cells exposed to nerve growth factors, J. Neurosci., 3 (19831 1547-1553. 2 Arendash, G.W. and Gorski, R.A., Enhancement of sexual behavior in female rats by neonatal transplantation of brain tissue from males, Science, 217 (1982) 1276-1278. 3 Bj6rklund, A. and Stenevi, U., Reconstruction of the nigrostriatal dopamine pathway by intracerebral nigral transplants, Brain Research, 177 (1979) 555-560. 4 Bj6rkhmd, A., Schmidt, R.H. and Stenevi, U., Functional reinnervation of the neostriatum in the adult rat by use of intraparenchymal grafting of dissociated cell suspensions from the substantia nigra, Cell. Tiss. Res., 212 (1980) 39-45. 5 Bj6rkhmd, A., Stenevi, U., Schmidt, R.H., Dunnett, S.B. and Gage, F.H., lntracerebral grafting of neuronal cell suspensions. I. Introduction and general methods for preparation, Acta Physiol. Scand., Suppl. 522 (1983) 1-7. 6 Bj6rkiund, A., Stenevi. U., Schmidt, H.R., Dunnen, S.B. and Gage, F.H., Intracerebral grafting of neuronal cell suspensions. I1. Survival and growth of nigral cell suspensions implanted in different brain sites, Acta Physiol. Scan&, Suppl. 522 (1983) 9 18. 7 De la Torre, J.C., An improved approach to histofluorescencc using the SPG method for tissue monoamines, J. Neurosci. Meth., 3 (1980) 1-5. 8 Dunnett, S.B., Bj6rkluud, A., Stenevi, U. and lversen, S,D., Behavioral recovery following transplantation of substantia nigra in rats subjected to 6-OHDA lesions of the nigrostriatal pathway. II. Bilateral lesions, Brain Research, 229 (1981) 457-47/). 9 Dunnett, S.B., Bj6rklund, A., Schmidt, H.R., Stenevi. U.

2S8 (1977) 521-528. 17 Hefti, F., Melamed, E. and Wurtman, R.J.. Partial lesions of the dopaminergic nigrostriatal system in rat brain: bio~ chemical characterization, Brain Research, i 95 (1980) 123 137, I8 Jumblatt, J.E. and Tischler. A.S., Regulation of muscarinic ligand binding sites by nerve growth factor in PCI2 pheochromocytoma cells, Nature (London), 297 (1982) 152-154. 19 KOnig, J.F.R. and Klippel, R.A., The Rat Brain. A Stereotaxic Atlas of the Forebrain and Lower Parts ~f the Brain Stem, Williams and Wilkins, Baltimore, MD, 1963. 2(1 Krieger, D T , and Pertow, M.J., Gibson. M.J,, Davies,

I . F . , Z i m m e r m a n n , E.A.. Form. M and(iimril{}n, ti.M , Brain grafts reverse hypogonadism c}f gonad(;'[ropin releasing hormone deficiency, \;amre (L(md,~;; 298 (I9~2} 468-471. 21 Labbe, R., Firl. A., Mufson. E,J. and Stcili, i).(i., la,c~al brain transplants: reduction of cognitive dclicii~ in rats with frontal cortex lesions, Science, 221 (1983) 47(!-~72~ 22 Low, W.C., Lewis, P.R., Bunch, S.T., I)unrmtt. S.B., Tho.man, S.R., [versen, S.D.. Bj6rklund, A. and Stenevi, l .. Function recovery following neural transplantation of cm~ bryonic septal nuclei in adult rats with septohippocampal lesions, Nature (London), 300 (I 982) 260-2(~2