Cografts of adrenal medulla with C6 glioma cells in rats with 6-hydroxydopamine-induced lesions

Neuroscience Vol. 34, No. 3, pp. 687697, 1990 Printed in Great Britain

03~-4322/90 $3.00 i- 0.00 Pergamon Press plc

0 1990 IBRO

COGRAFTS OF ADRENAL MEDULLA WITH C6 GLIOMA CELLS IN RATS WITH

6-HYDROXYDOPAMINE-INDUCED LESIONS

G. BING,*~$ M. F. D. NOTTER,* J. T. HANSEN,* C. KELLOGG,$ J. H. KORDOWER* and D. M. GASHED

*Department of Neurobiology and Anatomy, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, U.S.A.

TThe Beijing Institute for Neuroscience, Beijing, The People’s Republic of China §Department of Psychology, University of Rochester, Rochester, NY 14642, U.S.A.

Abstract-Amitotic [‘Hlthymidine-labeled C6 glioma cells, which are known to produce neurotrophic factor(s), were grafted alone and with adrenal chromaffin cells in an attempt to improve chroma%n cell survival and phenotypic differentiation. Long-Evans rats with unilateral 6-hydroxydopamine-indu~d lesions of the nigrostriatal pathway were divided into four groups: (1) those receiving adrenal medullary cells co-transplanted with C6 glioma cells; (2) those receiving adrenal medullary graft alone; (3) those receiving C6 glioma grafts alone; and (4) those serving as a vehicle control group. All rats were killed one month after transplantation. Immunohistochemical, neurochemical, and autoradiographic methods were used to identify and characterize the grafted cells. Tyrosine hydroxylase-immunoreactive cells were found in all animals that received grafts of the adrenal medulla alone or of adrenal medulla co-transplanted with C6 glioma cells. The cograft recipients had more tyrosine hydroxyla~-immunoreactive cells than the hosts receiving just adrenal chromaffin cells (P < 0.05). Additionally, more grafted chromaffin cells formed processes in the former group. All three tissue recipient groups (adrenal medullary, C6 glioma cell, and cografted animals) had a significant reduction (P < 0.05) in ipsilateral rotations after amphetamine (0.5 mg/kgi.p.) injections as compared to the control vehicle recipient group. Moreover, the reduction in rotation was more marked in the cografted hosts than in the other two implanted groups (P < 0.05).

Significantly higher dopamine levels were found in the transplant sites of both cograft and adrenal medullary graft recipients than in sham grafted control animals.

Implants of adrenal medullary and embryonic substantia nigral tissues placed into the striatum of rats with unilateral lesions of the nigrostriatal pathway survive and ameliorate the drug-induced motor deficits resulting from the lesion.6*7*‘1,29 These promis- ing results have led to recent clinical trials using adrenal medulla autografts for the treatment of Parkinsonism.2.‘s*i9 Parkinson’s disease is character- ized by the extensive loss of dopamine neurons in the substantia nigra and dopaminergic denervation of the nigra’s principal target, the striatum.5~‘3.20 Although improvement has been achieved by grafting adrenal medulla tissue into the striatum in laboratory animals, fundamental questions about the mechanisms of action remain.

Recent experimental studies have demonstrated that survival of adrenal medullary cells transplanted

$To whom correspondence should be addressed. Abbreviations: CMF, calcium, magnesium-free; DA, dopa-

mine; DOPAC, 3,~ihydroxyphenyla~tic acid; EC, electrochemical detection; EDTA, ethyIen~iaminetetra- acetate; HPLC, high-performance liquid chromatog- - . raphy; MPTP, 1-methyl-4-phenyl-l&,6-tetrahydro- uvridine; NGF, nerve growth factor: 6-OHDA. 6-hv- droxydopamine; TH-IRT tyrosine hydroxylase-immunb- reactive.

into the parenchyma ofrodents is rather low, with only 5% or less of implanted adrenal medullary cells surviving in the st~atum.4~*~‘1~27~33 Most of the surviving cells retained an endocrine phenotype or possessed only short processes; synaptic connections between the grafted chromaffin cells and the host brain have not been observed except with adrenal grafts from newborn rats. Bohn et ~2. reported that although adrenal transplants in I-methyl-4-phenyl- 1,2,5,6-tetrahydropyridine (MPTP)-treated mice had a poor survival, the transplant site was filled with tyrosine hydroxylase-immunoreactive (TH-IR) fibers that appeared to be of host origin.* If the improvement of symptoms of Parkinson’s disease is due to the release of dopamine into the host brain by grafted adrenal medullary cells, then a cograft of the adrenal medulla with neurotrophic factor-producing cells might improve the survival of the adrenal medullary cells. On the other hand, if recovery is the result of host neuronal regeneration and sprouting induced by the grafted tissue, then implantation of the appropriate neurotrophic factor-producing cells alone into the striatum should reverse the behavioural abnormality.

One cell line that produces neurotrophic factors is the C6 glioma cell line. The C6 glioma cells were

687

688 G. BING er al

derived from an N-nitrosomethylurea-induced rat glial tumor.4,2’ C6 glioma cells can release high concentrations of nerve growth factor (NGF)-like sub- stances into the culture medium.3~‘8~23~24~32~39 Culture

medium conditioned by C6 ghoma cells can support survival and fiber outgrowth of peripheral sensory neurons’.” and PC12 pheochromocytoma cells.’ More recently, Unsicker et a1.35.37 reported that

adrenal medullary cells respond to C6 glioma-conditioned medium by forming neurites, and increasing the catecholamine content of the cultures. Moreover, coculture of adrenal medulla cells from different species with C6 ghoma cells induced a greater neuri- togenic effect on chromaffin cells than treatment with NGF alone.‘”

The present study was designed to improve the survival of adrenal medullary grafts in the denervated host striatum by transplanting them with C6 ghoma cells. Immunohistochemical, neurochemical and autoradiographic methods were used to identify and characterize the grafted cells. Functional recovery in the graft recipients was assessed by measuring reductions in amphetamine-induced rotational behavior. The results were compared with animals receiving either adrenal medullary or C6 glioma cells alone.

EXPERIMENTAL PROCEDURES

Animals

The graft recipients were male LonggEvans specific pathogen-free rats (Charles River Breeding Laboratories, Portage, ME) with an initial weight of 200-225 g. All animals were maintained on a 12-h light/dark cycle with food and water provided ad libitum. Rats were housed individually in plastic cages covered with Envio-gard R bonnets.

Surgical procedures

Unilateral nigrostriatal lesions were produced by stereotaxic injections of 6-hydroxydopamine (6-OHDA) into the anterior substantia nigra according to the atlas of Paxinos and Watsor? (coordinates: -4.8 mm anterior-posterior, 1.8 mm lateral, and 8.1 mm dorsoventral with toothbar setting at 3.3 mm below the interaural line). All lesions were made on the non-dominant side, relative to amphetamine- induced rotations in intact animals.)’ The neurotoxin 6- OHDA (8 bg) was dissolved in 4 ~1 of isotonic saline containing 0.2 mg/ml of ascorbic acid and injected through a Hamilton syringe (10 ~1) at a rate of 1 nl/min. The injection solution was prepared immediately before adminis- tration and kept on ice. Before implantation, animals were injected with amphetamine (5 mg/kg, i.p.) at least twice at one-week intervals and tested with a modified Ungerstedt rotometerj4 before implantation. Because a 95% depletion in dopamine levels is necessary to elicit significant rotational behavior, only those animals that showed more than seven turns per minute over a 60-min period were selected for grafting. There is about a 99% reduction of dopamine levels in the striatum of the animals chosen by this criterion (see Results).

Grqjiing procedures

Transplants were performed 14 days following the lesion. Host animals were anesthetized with Chloropent (3 ml/kgi.p.) and placed in a Kopf stereotaxic apparatus. Donor cells, suspended in culture medium, were drawn up

into a Hamilton syringe and injected into four sites in the dorsal striatum at a concentration of 10,OOOcells/~l. The coordinates for the first two sites were: I .O mm anterior to bregma, 2.0 mm lateral, and 4.0 mm (site 1) and 5.5 mm (site 2) below the dura. For the second two sites, the coordinates were: 1 mm rostra1 to bregma, 3.3 mm lateral, and 5.0 mm (site 3) and 6.5 mm (site 4) below the dura. The injections were made at the rate of 1 pl/min, and each injection site received 2~1 of the cell suspension. Control animals received an infusion of the culture medium at the same coordinates. Ten, 24 and 30 days following implantation, both experimental and control rats were tested for amphetamine (5 mg/kg)-induced rotational behavior. They were killed 32 days post-transplantation.

Donor tissue

Adrenal medullae were dissected from young (100-125 g) male Long-Evans rats. Fragments of adrenal medulla were rinsed in calcium, magnesium-free (CMF) buffer, and then trypsin (0.3%) (Sigma) and collagenase (Worthington) (0.3%) were added to the CMF buffer containing 0.02% EDTA. The tissue was placed in a 37°C water bath,“agitated frequently for 30mit-1, and triturated through a Pasteur pipette until single cells were obtained (see Fig. 1 for schematic illustration of grafting procedure). Fetal calf serum, at a final volume of 10%. was added to quench the trypsin activity and 100 ,ug/ml deoxyribonuclease (DNAase) was added to prevent clumping. Cells were pelleted in a centrifuge, rinsed in CMF and counted with a hemocytometer. The cell number was adjusted to 10,000 cells/PI for transplantation.

The C6 glioma cell line was obtained from the American Type Culture Collection and monolayer cultures of C6 cells were grown in Eagle’s Minimal Essential Medium with 10% fetal calf serum, 50 ng/ml gentamicin, and 2.5 pg/ml fungi- zone. Cells were subcultured with 0.06% trypsin and 0.02% EDTA and kern at 37°C in humid 95% air and 5% CO,. For transplaniation, C6 cells were labeled with 1 pCi/ml [)H]thymidine (Amersham, specific activity of 40 Ci/mmol) for 36 h and then rendered amitotic by treatment with 0.5 pg/ml mitomycin C followed by 10e5M bromo- deoxyuridine 6 h later. After 24 h, amitotic C6 cells were trypsinized to remove them from their substrate and treated with 10% fetal calf serum to quench enzyme activity. Cells were then counted with a hemocytometer and examined for viability by Tryptan Blue dye exclusion. Glioma cells were pelleted by centrifugation in an IEC centrifuge at 1000 rpm and cell number adjusted to 10,000 cells/n1 in CMF bufferZS for transplantation.

Histology

Animals were anesthetized with Chloropent (0.5 ml/100 g body weight) and killed by intracardiac perfusion with 4% paraformaldehyde and 0.025% glutaraldehyde in phosphate-buffered saline (pH 7.4). The brain was removed and 25pm frozen coronal sections were cut on a sliding knife microtome. The sections were collected in a cryoprotective solution,‘* and alternate sections processed for the immunohistochemical localization of TH-IR cells, or stained with Cresyl Violet for demonstrating Nissl substance. The visual- ization of TH-containing cells was carried out via the avidin-biotin procedure. I4 After extensive washing of the tissue, the sections were incubated in 3% normal goat serum for 30 min. The tissue was incubated overnight at 4°C in the primary antiserum (Eugene Tech.) at a dilution of 1: 1000. On the second day after washing, the sections were incubated in a biotinylated secondary goat antirabbit antiserum (Vector 1: 200) for 60 min. Subsequently, sections were rinsed twice in 50 mM Tris-buffered saline (pH 7.4) and incubated with the avidin-biotin complex (Vector 1: 200) for 60 min. The peroxidase reaction was developed using 0.05% 3.3-diamino-benzidine and 0.01% hydrogen peroxide. The reaction was terminated with washes in Tris-buffered saline.

Cografts of chromaffin cells with C6 cells

COGRAFT PREPARATlON

Rat Adrenal Medulla CB Glioma Cells

CEplTRlFUGATlON +

ANTI-TUMOR DRUG rFiim?dEKf

CELL COLLECTION

689

I RESUSPEND AND MIX

FOR GRAFT

Fig. 1. A schematic of the cografting procedures. See Experimental Procedures for detail.

Sections were then mounted, dehydrated and coverslipped with Permount. Every sixth section was collected for TH immunocytochemistry, and cell counts were made manually on these sections. Cell number was corrected for section thickness by the method of A~rcrombie.’ Selected sections of rat brain containing cografts were processed for autoradiography-immunocytochemistry. They were first stained for TH, then dipped in NTB-2 emulsion (Eastman Kodak Company, Rochester, NY) and, after a two-week exposure, were developed according to previously described proto- cols.‘2

sigh-~rform~~ce liquid chromutograph~~lectrochemical detection analysis of catechoiamines and metabolites

Levels of catecholamines and their metabolites in striata containing adrenal medullary and C6 glioma cells, and in intact and lesioned striata were measured using high-performance liquid chromatography (HPLC) and

electrochemical detection (EC). To measure the levels of catecholamines and metabolites in the grafted striatum of intact and 6-OHDA-denervated rats, the animals were killed by decapitation. Brains were immediateiy removed and l-mm-thick coronal sections along the injection needle tract cut under a dissecting microscope. A piece of tissue 2 x 3 mm containing the needle tract was cut from the section as site 1, and a similar section lateral to this was cut as site 2. The tissue was weighed on an analytical balance (average weight was 10 mg) and placed in 0.1 M perchloric acid (lO&150~1). The tissue was then sonicated using a cup-horn attachment on a Heat Systems sonicator until a homogeneous solution was obtained (tubes were kept in an ice water bath during sonication). The homogenates were centrifuged (at 4°C) in a table top centrifuge (approximately 16OOg), the supematant was removed and 20 ~1 were injected onto the HPLC column (Alltech, C-18 reverse phase, 5 cl). The mobile phase was a potassium phosphate

690 G. BING et al.

buffer (0.07 M, pH 4.0) containing EDTA (0.5 mM), acetonitrile (8%), methanol (10%) and sodium octyl sulfate (100 mg/l). The medium was pumped over the column at a flow rate of 1 ml/min. The working electrode for EC detection was a glassy carbon (Bioanalytic Systems) set at 0.65 V with a sensitivity of 2nAjV (at these settings the limit of detection is 20-22 pg). The amount of material, expressed as nmol/g, was based upon comparison of the peak heights of the sample to the peak heights of known standards. Peak heights were measured using a recording integrator (Hewlett Packard). Specific samples were injected at different dilutions to allow for accurate measurement of all compounds.

Host rats were anesthetized with sodium pentobarbital (40 mg/kgi.p.) and killed by intracardiac perfusion of a solution containing 3% glutaraldehyde, 1% paraformaldehyde in 0.1 M cacodylate buffer at room temperature. The fixative was preceded by a saline flush. Following a 20-min perfusion, the brain was removed and the appropriate area around the transplant site removed in a series of coronal sections approximately 2mm in thickness. Sectioning the brain in this manner preserved the orientation, which was critical to localizing the transplanted cells. The sections were immersed in fixative for 2 h, washed in buffer, and post-fixed in osmium tetroxide and potassium ferrocyanide. Then the sections were stained en bloc in many1 acetate, dehydrated in acetone, and embedded Aat in Spurr’s low viscosity resin. One-micrometer sections stained with Totuidine Blue were used to find appropriate areas for thin sectioning. Thin sections, cut on an ultramicrotome with a diamond knife, were on grid stained with lead citrate and viewed in the electron microscope at 60 kV.

The behavioral data were anaiysed by a two-way analysis of variance with repeated measures on one factor, followed by sample main effects tests. Tests of significance for sample main effects with more than two levels were followed by a Newman-Keuls test at the P < 0.05 level of significance.

RESULTS

General

All of the animals appeared healthy at the time of killing. On histological examination, macrophages were found along the needle tract and in the implantation site of all recipients. These yellow- colored macrophages were easily distinguished from the grafted chromaffin cells, which exhibited an inten- sive dark brown immunohistochemical staining reaction.

TH immunohistochemistry revealed that the unilateral intranigral 6-OHDA injection had destroyed almost all TH-IR-containing cell bodies in the substantia nigra on the lesioned side. Moreover, the TH-IR in the striatum ipsilateral to the lesion was reduced to virtually background levels. All of the rats grafted with adrenal medulla and C6 glioma cells or adrenal medullary cells alone had surviving catecholamine-containing cells in the host striatum. How- ever, there were more TH-IR cells and more intense

Table 1. The numbers of adrenal medullary and C6 glioma cells in the host striatum after one month implantation

Groups

Cograft

(nA;7)

(“G5) (n = 5)

Number of AM cells Number of C6 cells

399.6 f 126.8* 3062 f 892.2’

I I 1.6 + 36.7 N/A

N/A 1483.6 k 762.8

Values are mean + S.E.M. *Significant (P < 0.05) difference from the controls (Student’s t-test). N/A, not applicable.

TH immunohistochemical reactivity in the striatum of cografted animals than in the striatum of those grafted with adrenal medullary cells alone (Table 1). Grafted cells were found adjacent to the injection site and often in close proximity to blood vessels (Fig. 2).

One month after grafting, most adrenal medullary cells grafted alone displayed rounded morphological profiles with either no processes or only very short processes (less than 10 pm). Most adrenal medullary cells grafted with C6 glioma cells had an elongated cell body with longer observable processes (10-50 pm). In general, adrenal medullary cells in cografts (Fig. 3A) were more neuronal in appearance than adrenal medullary cells grafted alone (Fig. 3B). In the control animals that received injections of culture media only, no TH-IR cells and few TH-IR fibers were detected in the striatum ipsilateral to the nigral lesion.

The average number of cells surviving for one month in all groups is shown in Table 1. In general, there were more TH-positive adrenal chromaffin cells in the striatum cografted with C6 glioma cells than in that grafted with only adrenal medullary cells (P < 0.05). Migration of the grafted cells from the injection site is a general phenomenon; however, the injected C6 glioma cells migrated farther from the needle tract than the adrenal cells. The longest distances of migration measured were 200 pm in adrenal medullary cell grafts and 800pm in C6 glioma grafts. Both chromaffin and C6 glioma cells had the tendency to cluster around host blood vessels (Fig. 4).

The glioma cells were distinguished by autoradiographic granules concentrated in the nuclei, and seemed to integrate well with the host striatum, since there was less evidence of glial scarring and fewer macrophages in the injection site than there were in the host striatum receiving adrenal medullary cells.

Rotational behavior

The results of rotational behavioral testing are summarized in Figs 5 and 6. Control animals injected with culture media showed a slightly increased number of rotations ipsilateral to the lesioned side following amphetamine injection over time after implantation. Rats receiving either C6 glioma or adrenal medullary cell implants showed a significant

n

Cografts of chromaffin cells with C6 cells

Gg. 2. ned Ull

‘Y a ut

A< 3renal medullary cells cografted with C6 glioma cells in a denervated striatum. (A) 1 lacy cells (black arrowhead) were stained for TH-IR, and C6 cells (open arrowhead) were ora diographic methods. (B) A dark-field view of A more clearly demonstrates the distritu

al Jtoradiographically-labeled C6 glioma cells (open arrowheads). Magnification: x 400.

4 drenal abeled tion of

692 G. BINC et al.

Fig. 3. A higher power photomicrograph reveals: (A) adrenal medullary cells cografted with C6 glioma cells. Note the cell processes (black arrowhead) of the grafted medullary cells and the autoradiographic granules (open arrowhead) over grafted C6 ghoma cells. (B) Adrenal grafts alone in the host striatum.

Note the round cell bodies and clustered appearance of the adrenal grafts. Magnification: x 800.

Fig. 4. A double-labeled section with TH immunocytochemistry and autoradiography, counterstained with Cresyl Vio- let to show the relationship of TH-IR adrenal medullary cells, autoradiographically labeled C6 glioma cells and blood vessels. Note the adrenal medullary cells (black arrowhead) and C6 glioma cells (open arrowhead) between

two blood vessels. Ma~ification: x400.

reduction in amphetamine-induced rotations compared to control animals [F(4, 12) = 90.15, P < 0.05)]. Rats with surviving grafts also showed a small contralateral rotation, an indication. of dopamine release in the striatum in the 30-min period after amphetamine injection (see Fig. 6). The maximal decrease in ipsilateral rotations by both adrenal medullary and C6 glioma cells grafts, seen 10 days post-implantation, was approximately 45%. The reductions in both groups were less pronounced at 24 and 30 days following implantation, although still si~ificantly less than control. In contrast, the cografted animals showed a continuous decline in their ipsilateral rotational behavior. There was about a 52% reduction at 30 days post-implantation, which was not only significantly lower than control animals but also lower than the animals grafted with adrenal medullary cells alone (P < 0.05). The animals cografted with adrenal medullary and C6 glioma cells also showed a continued increase with time after implantation in contralateral rotations after the amphetamine injection. At 30 days, the amount of contralateral rotation was about six times greater in cografted animals as compared to the control group.

Cografts of chromaffin cells with C6 cells 693

tt

prelesion post-lesion day 10 day 24 day 30 days post-transplantation

Fig. 5. Amphetamine (5 mg/kg)-induced ipsilateral rotation to the lesioned side over a 60-min testing period in rats with 6-OHDA lesions of the substantia nigra, grafts with C6 glioma (c6), adrenal medullary cell transplants (am), cografts (am + c6) and in significant (P < 0.05 level) difference compared to the control group and x indicates a significant (P < 0.05) difference compared to the adrenal medullary cell

graft group (two-way ANOVA with post-hoe Newman-Keuls test).

High-performance liquid chromatography

The summary of results of the biochemical deter- minations of catecholamines, 3,4-dihydroxyphenyl- acetic acid (DOPAC), and dopamine (DA) turnover rate (DOPAC/DA) is shown in Table 2. In the 6-OHDA-lesioned control animals the DA content in the denervated striatum was less than 1% of that in the intact striatum at 30 days post-transplantation. The intrastriatal adrenal medullary grafts increased the levels of all three catecholamines (Table 2) in the lesioned host striatum 30 days after transplantation. The C6 glioma grafts also increased DA and norepinephrine content in the host striatum though there were no catecholamines in the grafted cells. Brains of animals receiving cografts of adrenal medullary and C6 glioma cells showed increased levels of dopamine in the lesioned striatum which were significantly

• 16

, , .

i::z Contmlutlxel RolatlomB Anlr Amplw~mlne InJi~lon

i l l l ~ B e ~

poet-k~on day 10 day 24 J

day

Fig. 6. Contralateral rotation to the lesioned side in the first 30 min of tests. Bar represents mean _+ S.E.M. The asterisk indicates a significant (P < 0.05 level) difference compared to the controls, and x indicates a significant difference (P < 0.05) compared to the adrenal medullary cell graft group (two-way ANOVA with post-hoc Newman-Keuls

test).

higher than levels seen in recipients of adrenal medullary and C6 glioma cell grafts alone (P < 0.05). The catecholamine turnover rate expressed by the ratio of DOPAC/DA was remarkably higher in the denervated striatum than in the intact one. Intra- striatal grafts in all three groups significantly lowered the catecholamine turnover rate in the lesioned striatum toward the intact level.

Electron microscopy

After one month, transplanted chromaffin cells in the host striatum still possessed a distinct basal lamina and other features typical of chromaffin cells. The implanted chromaflin cells contained numerous dense-core vesicles in their cytoplasm. In our prep- arations, synaptic contacts between chromaflin cells and host were not observed (Fig. 7A). C6 glioma cells in the host striatum appeared healthy, and many of them were closely associated with capillaries and seemed well integrated with the host. The interstitial spaces in the grafts were filled with fragments of the host neuropil, and nerve terminals. Mitotic figures were not found in the present study (Fig. 7B).

DISCUSSION

These experiments demonstrate that cografts of adrenal medullary cells with C6 glioma cells are more effective in reducing the abnormal rotational behavior induced by amphetamine in rats with 6-OHDA- induced lesions of the nigrostriatal DA system than grafts of either adrenal medullary or C6 glioma cells alone. The levels of catecholamines and their metabolites measured in the lesioned striata are consistent with this improved rotational behavior; that is, a much higher level of dopamine is seen in the striata

694 G. BING et al.

Group

Table 2. Catecholamine and DOPAC concentrations in striatum

Dopamine DOPAC n nmol/g wet weight DOPAC/DA NE EPi

Lesioned controls Intact side Lesioned side

AM grafts Site 1 Site 2

C6 grafts Site 1 Site 2

AM+C6 Site 1 Site 2

6 93.3 + 15 20.1 * 4.0 0.22 + 0.01 6 0.62 + 0.25 0.84 + 0.1 1.35 + 0.6

6 2.5 k 0.6* 0.98 k 0.4 0.39 & 0.2* 4 1.3kO.3 0.7 & 0.2 0.51 * 0.1

6 2.0 & 0.5 4 1.2kO.4

6 4.5 _+ 0.8*t 1.6 k 0.7 0.36kO.l” 4 0.7 * 0.1 0.6 f 0.2 0.76 + 0.14

1.2kO.3 0.59 + 0.18* 0.5&0.1 0.42 k 0.3

N.D. N.D.

2.99 + 2.4 N.D.

0.52 i 0.2 0.27 Ifr 0. i

2.11 + 0.4 1.19io.5

N.D. N.D.

2.30 k 1.1 N.D.

N.D. N.D.

1.87 + 0.26 0.19 + 0.06

Values are mean & S.E.M. Dopamine, DOPAC, norepinephrine (NE) and epinephrine (EPi) concentrations were obtained with HPLC followed by EC. Site 1 contained the grafts and site 2 was adjacent to the injection site. *Significant difference (P < 0.05) compared to the 6-OHDA-lesioned control; tsignificant difference (P < 0.05) compared to the striatum grafted with C6 glioma cells alone (two-way ANOVA with post-hoc Newman-Keuls test). N.D., not detected.

of the cografted rats than in grafts of adrenal medullary or C6 glioma cells alone.

Cografting adrenal chromaffin cells with C6 glioma cells induced adrenal chromaffin cells to change from an endocrine phenotype towards a neuronal phenotype. Adrenal grafts alone, examined one month after transplantation, consisted mostly of clustered chromaffin cells with very short processes. Adrenal chromaffin cells in a cograft were more neuronal-like and had elongated processes, with a large nucleus-to-cytoplasmic ratio. Altered chromaffin cell morphology in the cografts is probably due to the absence of glucocorticoids and to the

presence of NGF and other trophic factors in the environment of the cograft.26,4’

Cografting substantially improved the survival of the adrenal chromaffin cells in the denervated striatum. Equally interesting, the numbers of surviving C6 glioma cells were also increased by cografting with adrenal cells. This result indicates that both adrenal medullary and C6 glioma cells may produce reciprocal trophic effects that promote their mutual survival.

The present neurochemical data strongly support our findings that cografting was more effective at improving the rotational behavior than any single cell

Fig. 7. (A) Electron micrograph of implanted dissociated chromaffin cells. The cells appear robust, contain numerous dense-core vesicles, and possess a distinct basal lamina. Processes of supporting cells and fibrous astrocytes separate groups of chromaffin cells. Magnification: x8000. (B) Electron micrograph of implanted C6 glioma cells (arrowhead). These cells appear healthy. The interstitial spaces are filled with fragments of the host neuropil, including myelin figures and several isolated nerve terminals as well as a flocculent material. A capillary appears in the lower right corner of the micrograph. Magnification:

x 2250.

Cografts of chromaffin cells with C6 cells 695

type implant. The striatal dopamine levels taken from using NGF infusion. Firstly, although some NGF is the cografted injection site were not only higher than contained in C6 glioma cell~,‘~ other trophic factors in the lesioned control but were also significantly released by C6 glioma cells might be quite different higher than in the adrenal chromaffin cell implant from NGF.3s*37 The conditioned medium from C6 (P < 0.05). Several factors may contribute to this glioma cells is consistently more potent than NGF result: (1) better survival of adrenal chromaffin cells with respect to supporting adrenal chromaffin cell in the cograft group; (2) NGF-like factor(s) released survival, eliciting fiber outgrowth and inducing cate- by C6 glioma cells stimulating adrenal chromaffin cholamine content of cultures3’ Secondly, the time cells to produce more dopamine; and (3) the host interval between lesion and transplantation may be response to the trophic factor(s) released by both important. In the present study, 14 days elapsed adrenal medullary and C6 glioma cells. There was a between lesion and transplantation while in the other 99% depletion of dopamine in the rat striatum studies there was a wait of one or two months before unilateral to the lesioned substantia nigra in animals implementation. Substantia nigra neurons that have reaching behavioral criteria. The striatal tissue taken not been completely lesioned may have regenerated in from a site adjacent to the injection site but without response to the trophic substance from the implant. adrenal chromaffin cells, also had a higher dopamine Thirdly, C6 glioma cells migrated quite far from the content than did the lesioned control striatum. This injection site, and were more evenly distributed in the result suggests that dopamine released by chromaffin host striatum, dispersing neurotrophic factors more cells may diffuse into limited regions of the brain consistently as compared with a single dialysis fiber parenchyma. infusion of the NGF.33

It has been proposed that functional restoration facilitated by fetal brain transplantation after frontal cortex lesions may be due to a trophic mechanism.17 This concept is strongly supported by a recent study showing that transplants of purified astrocytes can promote behavioral recovery after frontal cortex ablation.16 The neurotrophic factor(s) released by grafted tissue are still largely unknown. It has been shown that intraventricular infusions of NGF can promote behavioral recovery after damage to septo- hippocampal connections4’ and improve spatial memory in the aged rat.” However, NGF application in nigrostriatal lesions has not been shown to correct rotational behavior.30,33 Stromberg et al., reported that NGF significantly increased survival of adrenal chromaIKn cells in the host striatum and the grafts treated with NGF were also more effective in counteracting asymmetric rotational behavior.33 Recently, Pezzoli et al., reported that conjunction of NGF infusion with either fat tissue or adrenal medullary tissue were equally effective in reducing apomo~hine-induced rotation in 6-OHDA-lesioned rats3” However, in both Stromberg et d’s and Pozzoli et d’s studies. rats injected with NGF alone did not demonstrate significant improvement of apomorphine-induced rotations.30*33 In contrast, rats in our study did show significant improvement in rotational behavior with just the implantation of C6 glioma cells.

The finding that striata implanted with C6 glioma cells had detectable norepinephrine levels very strongly suggests that the trophic factor secreted by C6 glioma cells may induce neuronal sprouting from noradrenergic systems in the brain or from the sympathetic system. The other possible source of the noradrenaline found in the C6 glioma implanted striatum may be derived from the proliferation of blood vessels stimulated by angiogenic factors released by C6 glioma cells.

CONCLUSION

Our results with cografts of C6 and adrenal medulla suggest that at least two mechanisms are working in these implants to promote functional recovery. The grafts possess neurotrophic activities which stimulate catecholaminergic innervation of the denervated striatum. In addition, increased numbers of surviving adrenal medullary cell provide increased quantities of dopamine and other catecholamines to compensate for dopamine deficiencies in the lesioned striatum. Therefore, cografting adrenal medulla~ cells with neurotrophic factor-producing cells seems to have potential utility as an approach for improving the efficacy of adrenal grafts.

There are several possible factors that could contribute to the differences in our studies and those

Acknowledgements-The authors wish to thank Dr Gloria Pleger for developing HPLC-EC procedures and Barbara Ferbel and Vickie Meyers for excellent technical assistance. This work was supported by USPHS grants NS25778 and 57RR05403.

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(Accqred 5 June 1989)

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Cografts of adrenal medulla with C6 glioma cells in rats with 6-hydroxydopamine-induced lesions