Encapsulated GDNF-producing C2C12 cells for Parkinson's disease: a pre-clinical study in chronic MPTP-treated baboons

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Neurobiology of Disease 16 (2004) 428–439

Encapsulated GDNF-producing C2C12 cells for Parkinson’s disease:

a pre-clinical study in chronic MPTP-treated baboons

Haruhiko Kishima,a Thomas Poyot,a Jocelyne Bloch,b Julien Dauguet,c

Franc�oise Conde,a Frederic Dolle,c Franc�oise Hinnen,c William Pralong,b

Stephane Palfi,a,d Nicole Deglon,b Patrick Aebischer,b and Philippe Hantrayea,c,*

aResearch Associate Unit URA CEA CNRS 2210, Service Hospitalier Frederic Joliot, Orsay, FrancebSwiss Federal Institute of Technology Lausanne (EPFL), Institute of Neuroscience, Lausanne, Switzerlandc Isotopic Imaging, Biochemical and Pharmacological Unit (UI2BP), Department of Medical Research, Service Hospitalier Frederic Joliot, Orsay, FrancedNeurosurgery Service, CHU Henri Mondor Hospital, AP-HP, 94010 Creteil, France

Received 25 November 2003; revised 1 March 2004; accepted 22 March 2004

Available online 6 May 2004

Glial cell line-derived neurotrophic factor (GDNF), a potent neuro-

trophic factor with restorative effects in a variety of rodent and primate

models of Parkinson’s disease (PD), could be of therapeutic value to

PD. In this study, we show that intraventricular chronic infusion of low

doses of GDNF using encapsulated genetically engineered C2C12 cells

can exert: (1) transient recovery of motor deficits (hypokinesia); (2)

significant protection of intrinsic striatal dopaminergic function in the

immediate vicinity of the site of implantation of the capsule in the

caudate nucleus, and (3) significant—long-lasting—neurotrophic prop-

erties at the nigral level with an increase volume of the cell bodies.

These observations confirm the potent neurorestorative potential of

GDNF in PD and the safety/efficacy of the encapsulation technology as

a means to deliver in situ this neurotrophic cytokine even using an

intraventricular approach.

D 2004 Elsevier Inc. All rights reserved.

Keywords: Glial cell line-derived neurotrophic factor; Parkinson disease;

Neurotrophic factor; Macroencapsulation; 1-methyl-4-phenyl-1,2,3,6-tetra-

hydropyridine; Baboon

Introduction

Parkinson’s disease (PD) is a neurodegenerative pathology

characterized by a progressive dysfunction, followed by an actual

degeneration of midbrain dopaminergic neurons. The pathology is

clinically characterized by severe motor symptoms such as hypo-

kinesia, bradykinesia, rigidity, and resting tremor. The main therapy

for PD is L-3,4-dihydroxyphenylalanine (L-dopa) or dopamine

agonists. However, as the disease progresses, patients typically

0969-9961/$ - see front matter D 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.nbd.2004.03.012

* Corresponding author. URA CEA CNRS 2210, Service Hospitalier

Frederic Joliot, 4 Place du Gal Leclerc, 91401 Orsay, France. Fax: +33-1-

69-86-77-45.

E-mail address: [email protected] (P. Hantraye).

Available online on ScienceDirect (www.sciencedirect.com.)

become less responsive to L-dopa and develop motor side effects.

As a complement to such pharmacological treatments, surgical

therapies including deep brain stimulation (Kumar et al., 1998,

1999; Lanotte et al., 2002; Pollak et al., 2002) and transplantation

of fetal dopamine neurons (Freed et al., 2001; Lindvall et al., 1990;

Peschanski et al., 1994; Remy et al., 1995) have been developed.

But all these approaches remain essentially symptomatic therapies

and as such are unable to halt or even compensate for the continual

loss of dopamine neurons. In this context, an optimal therapeutic

strategy would aim at both preventing neuronal dysfunction and

neurodegeneration in the patient’s brain.

Glial cell line-derived neurotrophic factor (GDNF), a potent

neurotrophic factor with restorative effects in a variety of rodent

and primate models of PD exert neuroprotective and neurotrophic

properties on dopaminergic neurons, both in vitro and in vivo

(Beck et al., 1995; Bjorklund et al., 1997; Gash et al., 1996;

Kordower et al., 2000; Lin et al., 1993; Tomac et al., 1995). In a

recent phase 1 safety trial, GDNF was delivered directly into the

putamen of five parkinsonian patients (Gill et al., 2003). This

resulted in major improvement in motor performances as well as

significant improvement in the activities of daily living. Even

medication-induced dyskinesias were reduced by 64% and were

not observed off medication during chronic GDNF delivery.

However, chronic direct intraparenchymal or intraventricular de-

livery of substances is often associated with undesirable side

effects or complications such as necrotic lesions (Kordower et

al., 1999).

The present study was designed to investigate whether contin-

uous GDNF delivery through encapsulated GDNF-producing cells

implanted into the lateral ventricle, a structure close to the caudate

nucleus, would not only oppose but also reverse the motor deficits

and nigrostriatal dopaminergic degeneration associated with a

chronic neurotoxic treatment in a primate model of PD. Systemic

administration of the mitochondrial complex I inhibitor 1-methyl-

4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to nonhuman pri-

mates has been shown to mimic the typical pathological features

H. Kishima et al. / Neurobiology of Disease 16 (2004) 428–439 429

observed in PD including the classical triad of motor symptoms

(akinesia, rigidity, tremor) and a preferential degeneration of

substantia nigra pars compacta dopaminergic neurons resulting in

an uneven pattern of caudate–putamen dopaminergic deafferenta-

tion (Hantraye et al., 1993; Varastet et al., 1994). In the chronic

version of the lesion model (Hantraye et al., 1993; Varastet et al.,

1994; Wullner et al., 1994), MPTP-treated primates undergo a

gradual two-step neurodegenerative process evolving from striatal

dopaminergic dysfunction to actual neurodegeneration of midbrain

dopamine neurons. The sequence of events in this model are

characterized by a first stage of mild hypokinesia–bradykinesia

(15–20% decrease in spontaneous locomotor activity) but no

significant changes in striatal dopaminergic function, as assessed

by positron emission tomography (PET) (<5% decrease in striatal18F-DOPA uptake). This first stage is followed by a second phase

of more aggressive progression of motor deficits (up to 50%

decrease in spontaneous locomotor activity), severe loss in striatal18F-DOPA uptake (up to 80% decrease compared to normal

values), and actual nigral cell loss (Varastet et al., 1994; Wullner

et al., 1994).

To mimic more precisely the clinical situation in which

GDNF would be infused in early symptomatic patients, we used

the chronic lesion model and delivered the cytokine to the

MPTP-treated baboons immediately before reaching the second

stage of MPTP intoxication, that is, 8 weeks after initiation of

the MPTP treatment. To ensure continued delivery of GDNF

during the entire duration of the MPTP neurotoxic treatment (i.e.,

60 weeks), we used an ex vivo gene therapy approach based on

the intracerebral implantation of encapsulated, genetically mod-

ified, GDNF-producing cells (Sautter et al., 1998). This macro-

encapsulation technique has already demonstrated its feasibility

Fig. 1. Study design and experimental paradigm. Chronic MPTP treatment was ini

dose 0.5 mg/kg, final dose of 1.0 mg/kg) for 60 weeks. Repetitive 18F-DOPA PET

arrows) before MPTP, and every 6 weeks during the entire course of the neuroto

GDNF-producing C2C12 cells were implanted during three consecutive surgery se

and behavioral test sessions were regrouped into five different time points roug

placement (prelesion, 6–20, 21–40, 42–52, 54–60 weeks).

and efficacy in delivering trophic factors under various pre-

clinical and clinical situations (Aebischer et al., 1996; Bach-

oud-Levi et al., 2000; Deglon et al., 1996; Mittoux et al., 2000;

Sautter et al., 1998; Tseng and Aebischer, 2000; Zurn et al.,

2000).

We show here that chronic intraventricular infusion of low

doses of GDNF can exert: (1) a transient recovery of motor deficits

(hypokinesia); (2) a significant protection of striatal dopaminergic

function in the immediate vicinity of the capsule; and (3) a

significant neurotrophic effect in the caudate with an increased

number of tyrosine hydroxylase (TH)-immunoreactive (ir) cells

and in the substantia nigra with an increased cell volume.

These observations largely confirm the therapeutic potential of

the encapsulation technology for the chronic delivery in situ of

large molecules such as cytokines and the potent neurorestorative

(neurotrophic) potential of GDNF even when the cytokine is

delivered at very low concentrations and in the lateral ventricle

of parkinsonian baboons.

Materials and methods

Animals

Nine adolescent male Papio anubis baboons (weight 11–15 kg)

were used in this study. Six of these animals were submitted to a

chronic MPTP regimen of intoxication that induces a severe

degeneration of mesencephalic dopaminergic neurons (Varastet et

al., 1994). Three other animals, without MPTP treatment, were

used as controls. Eight, 25, and 47 weeks after initiation of the

MPTP treatment, three of the MPTP-treated baboons (GDNF #1–

tiated in six baboons that received weekly intramuscular injections (starting

imaging and behavioral tests were performed at various time points (vertical

xic treatment. Capsules containing either native or genetically engineered

ssions at 8, 25, and 47 weeks post-MPTP. For statistical comparisons, PET

hly corresponding to the different surgery sessions necessary for capsules

H. Kishima et al. / Neurobiology of Disease 16 (2004) 428–439430

3) were implanted with encapsulated GDNF-producing C2C12

cells as the experimental therapy, with the remaining three

MPTP-treated baboons (MPTP-only #1–3) receiving capsules

containing native C2C12 cells (Fig. 1). During the entire study,

weekly MPTP injections were performed for a total 60-week

duration to mimic an ongoing parkinson-like pathology, after

which animals were sacrificed for histological analysis. All nine

animals were housed individually in standard primate cages with

free access to food and water. All procedures were in strict

accordance with the recommendations of the European Economic

Community (86/609/EEC) and the French National Committee for

the care and use of laboratory animals (87/848).

MPTP intoxication

The six baboons treated with the dopaminergic neurotoxin

received weekly intramuscular injections of 0.5–1 mg/kg MPTP

for 60 weeks according to the regimen represented in Fig. 1. The

neurotoxic solution was freshly dissolved at a final concentration

of 1 mg/ml hydrochloride MPTP in 0.9% NaCl before each weekly

injection. As previously shown, such a chronic MPTP treatment

results in the progressive appearance of a parkinsonian syndrome

characterized by hypokinesia, bradykinesia, balance impairment,

and postural alterations (Hantraye et al., 1993), associated with

progressive loss of 18F-DOPA caudate-putamen uptake and severe

nigrostriatal degeneration (Varastet et al., 1994).

Motor testing

Behavioral assessments were performed before and during the

MPTP intoxication to detect any signs of acute intoxication and the

presence of spontaneous abnormal movements. In addition, an

absolute quantification of kinetic parameters was performed. To

this aim, the freely moving animals were placed in a plexiglass

video cage (100 � 100 � 180 cm) and video-recorded from the

front for 40 min. The total distance moved (TDM) (an index of

spontaneous locomotor activity) was measured during the 40-min

test period using a dedicated video-based movement tracking and

analyzing software (EthoVision, Noldus, Netherlands). These be-

havioral assessments were performed every 6 weeks during the

entire duration of the study (see Fig. 1).

C2C12 cell line and encapsulation

The pCI-neo expression vector (Promega, Madison, WI) was

modified as previously described (Rinsch et al., 1997). The

fragment containing the mutated dihydrofolate reductase gene

(DHFR) under the control of the SV40 promoter and the

hepatitis virus B 3V untranslated region (HBV-3V UTR) was

obtained from the RP3224D plasmid (Deglon et al., 1996)

digested with PvuII. The isolated fragment was subcloned into

the pCI-Neo vector digested with BamHI and blunted using the

T4 polymerase to fill in the protruding ends. The resulting vector

pCI-ND was digested with BglII and EcoRI to remove the

cytomegalovirus (CMV) promoter. The murine phosphoglycerate

kinase 1 promoter and a chimeric intron excised from the pCI-

PGK-GIP-R/IRES/GLP-1 vector (Moens et al., 1996), via BglII/

EcoRI digestion, were subcloned into the BglII/EcoRI site of the

pCI-ND plasmid, forming the pPI-DN vector. The plasmid

pcDNA3 containing the cDNA coding for the human GDNF

with a consensus Kozak sequence (a 636-bp fragment: positions

1–151 and 1–485 genbank accession numbers L19062 and

L19063) was digested with BamHI/HindIII and blunted. The

GDNF fragment was subcloned into the pPI-DN vector-digested

EcoRI and blunted to create the pPI-DN-hGDNF. Finally, the

Herpes Simplex Virus Thymidine kinase (HSV-tk) gene obtained

from the plasmid RP3224D digested with NotI was cloned into

the BglII site of the pPI-DN-hGDNF. The final plasmid named

pPI-DNT-KZ-hGDNF was amplified in a standard Escherichia

coli strain (DH10B) and purified by Cesium chloride gradient.

The integrity of the vector was verified by restriction analysis

and sequencing.

C2C12 mouse myoblasts derived from leg skeletal muscles of

an adult C3H mouse were obtained from American Type Culture

Collection (ATCC; CRL 1772, Rockville, MD). C2C12 cells

transfected by calcium phosphate precipitation (Mammalian trans-

fection kit, Stratagene) with the pPI-DNT-KZ-hGDNF plasmid

were selected with 0.8 mg/ml G418 for 2 weeks, and the integrated

plasmid was then amplified with increasing concentrations of

methotrexate (1–200 AM) over a 4-week time period. Stability

of transgene expression was achieved by alternating cell incubation

weekly in media containing either 0.8 mg/ml G418 or 200 AMmethotrexate for an additional 6 weeks. The C2C12-engineered

cells were then maintained in culture media supplemented with 0.8

mg/ml G418. Untransfected C2C12 cells and clones # 7 and #19

producing approximately 0.5 and 0.26 Ag hGDNF/106 cells/day

were used for encapsulation. The cells were harvested using

0.125% trypsin-EDTA and suspended at 25,000 cells/Al in DMEM,

10% FCS.

Matrices were obtained from a polyvinyl alcohol (PVA) sponge

(Rippey Corporation, El Dorado Hills, CA, USA) using a hollow

drill with an internal diameter corresponding to the inner dimen-

sions of the capsule. The PVA rods were washed in ultra pure water

and dried at room temperature. The matrices were inserted into 1.2

cm long, semipermeable, polyethersulfone (PES #5) hollow fibers

(OD: 720 Am; ID: 524 Am; molecular weight cutoff: 280 kDa)

(Akzo Nobel AG, Wupperthal, Germany). The fibers were steril-

ized with ethylene oxide and kept 10 days at room temperature to

eliminate traces of gas. Using a 50-Al syringe (Hamilton Bonaduz

AG, Bonaduz, Switzerland) fitted with an adaptor hub, 12 Al of cellsuspension was injected into the device (300,000 cells). The hubs

were removed and the extremity of the capsules sealed by photo-

polymerization of an acrylate-based glue at a wavelength of 460

nm (Luxtrak LCM 23, Ablestik, Electronic Materials & Adhe-

sives). The capsules were maintained in differentiating media for 3

days and DMEM, 10% FCS (5% CO2, 37jC) for 4 days and then

implanted into the lateral ventricle of the animals.

GDNF assay

Before implantation and following explantation (i.e., up to 3

months in situ), the amount of GDNF released by each capsule over

a 1-h incubation in 1 ml of DMEM, 10% FCS was measured with a

sandwich enzyme-linked immunosorbent assay (ELISA) GDNF kit

(R&D Systems, Minneapolis, MN). Standards and samples were

assayed in duplicate according to the procedure specified by the

manufacturer. The detection limit of the test was 31.2 pg/ml.

Surgery

Eight weeks after initiation of the MPTP treatment, animals

were anesthetized with an intramuscular injection of a mixture of

Fig. 2. Location of the implanted capsules. (A and B) Surgical targeting of

implanted capsules observed on sagittal and coronal T1-weighted MRI scans

obtained in the GDNF #3 baboon immediately post-surgery. The capsules

appear as black tracks running from the superior frontal gyrus through the

cingulate gyrus and corpus callosum, finally reaching the lateral ventricle at

the level of the caudate nuclei. L, left side of the brain. (C) Frontal cresyl

violet photomicrograph obtained post-mortem from the same GDNF #3

animal and corresponding to the white inset in B. All capsules were found

running through the corpus callosum and terminating their course in the

lateral ventricle just beside the caudate nucleus. Very limited damage could

be observed around the capsule despite the repeated implantation/

explantation procedures. CN, caudate nucleus; CC, corpus callosum; LV,

lateral ventricle. Calibration bar indicates 1 cm in A and B; 2 mm in C.


ketamine (Ketalar, Parke-Davis, France 15 mg/kg) and xylazine

(Rompun, Bayer, France, 1.5 mg/kg; Banknieder et al., 1978) and

orally intubated. Animals were randomly selected to be implanted

with either native C2C12 capsules or GDNF-producing C2C12

capsules. To this aim, animals were placed into the stereotaxic

frame (Kopf Instruments, CA, USA) and the capsules bilaterally

Table 1

GDNF releases of implanted capsules (ng/24 h)

Animals GDNF #1 GDNF #2

Surgical sessions Clones 1st #7 2nd #7 3rd #19 1st #19 2

Pre-implantation release 122 26 33 36 27 29 103 76 4

Post-explanation release nd nd nd nd 1.7 2.9 10 7 2

nd: not detectable.

implanted into the lateral ventricle using an adapted micromanip-

ulator (coordinates: anterior: 18 mm, lateral: 4.7 mm, ventral: 5

mm, according to the atlas of Riche et al., 1968) (Figs. 2A, B).

Three series of capsules were implanted in each animal at 8, 25,

and 47 weeks post-MPTP (Table 1). At 8, 25, and 47 weeks post-

MPTP, each animal received one capsule in each side. However, as

an attempt to increase the amount of GDNF delivered, baboon

GDNF #2 received 4 capsules (two in each ventricle) and baboon

GDNF #3 received three capsules (two capsules in the right

ventricle, one capsule in the left ventricle) during the last (47

weeks) implantation series (Fig. 1 and Table 1).

Magnetic resonance imaging

Correct placement of the capsules was verified post-operatively

using magnetic resonance imaging (MRI). To this aim, baboons

were anesthetized by intramuscular injection of a mixture of

ketamine (15 mg/kg) and xylazine (1.5 mg/kg) and positioned into

an MRI-compatible frame in the MR magnet aligned with laser

beams. MRI examinations were performed on a 1.5-T MR magnet

(Signa, General Electric) in the coronal plane using a receive-only

pseudo-Helmholtz probe. A T1-weighted inversion-recovery se-

quence in 3D mode and a 256 � 192 matrix over 124 slices of

1.5 mm in thickness was used to generate the MR images. The

same sets of MRI images were used for PET-MRI co-registration

(see below).

18F-DOPA positron emission tomography

6-[18F]fluoro-L-DOPA (18F-DOPA) was prepared as previously

described (Dolle et al., 1998; Namavari et al., 1992) by regio-

selective radiofluorodestannylation using [18F]fluorine ([18F]F2,

cyclotron-produced isotope, half-life: 110 min) and N-(formyl or

tert-butoxycarbonyl)-3,4-di(tert-butoxycarbonyloxy)-6-trimethyl-

stannyl-L-phenylalanine ethyl ester as the labeling precursor.

Radiochemical, chemical and enantiomerical purities of the radio-

ligand determined by HPLC were found to be higher than 98%,

95% and 99%, respectively.18F-DOPA PET measurements were acquired with an ECAT

Exact HR+ tomograph (Siemens CTI) operating in 3D acquisition

mode (63 planes, axial field of view 155 mm, isotropic resolution

4.5 mm FWHM; Adam et al., 1997). Ketamine-xylazine anaes-

thetized animals were positioned in the ECAT Exact HR+ tomo-

graph, their head held in a fixed position using the same stereotaxic

head holder as for MRI. A 15-min transmission scan was first

performed using 68Ge sources, to correct for g-ray attenuation. 18F-

DOPA (148 MBq on average, in 10 ml of 0.9% NaCl) was then

injected intravenously over 60 s The 18F-DOPA scanning protocol

included 9 frames of 10 min each, starting immediately after tracer

injection. These PET studies were performed once before MPTP

and every 6 weeks during the entire duration of the study (i.e. each

baboon received 11 PET scan examinations, see Fig. 1). Between

GDNF #3

nd #19 3rd #19 1st #19 2nd #19 3rd #19

5 55 83 68 61 67 88 83 43 59 89 82 95

0 nd 26 26 53 4 29 25 29 8 46 50 54

Fig. 3. Locomotor activity in MPTP-treated baboons: effect of GDNF.

Percent changes in locomotor activity in the MPTP-only group (black

circles) and MPTP-GDNF treated baboons (grey squares). Alterations in

locomotor activity in MPTP-only animals was characterized by a gradual

(linear) decrease in total distance moved (TDM), reaching 55% of control

value by weeks 42–52, with no recovery observed at the latest time-point

studied (54–60 weeks). In contrast, the initial decrease in TDM values

(hypoactivity) observed in the GDNF-treated group was ameliorated by

GDNF treatment between weeks 21–40 and weeks 54–60. Due to the

limited number of animals included, the only significant difference between

both groups was observed for the 42- to 52-week time-point. Data are

expressed as means F SE. *P < 0.05 (Mann–Whitney U test).


group comparisons were performed at various time-points, before

MPTP (control), and at 6–20, 21–40, 42–52 and 54–60 weeks

after initiation of the MPTP treatment. These time points were

selected to perform statistical comparisons matching the various

capsule implantation times, so that 6–20 weeks would correspond

to capsule 1, 21–40 weeks to capsule 2, 42–52 weeks to capsules

2–3, 54–60 weeks to capsule 3.

Image analysis was performed using two different methods: a

voxel-to-voxel comparison using a statistical parametric mapping

approach (SPM) (Friston et al., 1991) and quantitative measure-

ments using regions of interest (ROI).

For the SPM method (SPM99; Wellcome Department of

Cognitive Neurology, London UK, http://www.fil.ion.ucl.ac.uk/

spm/spm99.html), MRI and integrated 18F-DOPA PET images

(0–80 min) from three baboons were used to generate template

images. Images of each animal were realigned to the first volume

and normalized to the MRI and 18F-DOPA PET template (Friston

et al., 1995a) to account for variation in gyral anatomy and inter-

individual variability in structure–function relationships, and to

improve the signal-to-noise ratio. We also constructed a voxel-

based parametric image of 18F-DOPA influx constants (Ki, min�1)

from time frames 30–80 min after 18F-DOPA injection using a

dedicated in-house software and the multiple-time graphical anal-

ysis method (Patlak and Blasberg, 1985). This parametric image of

Ki was also normalized to the template using the same trans-

forming parameter of each baboon as that used for normalizing

integrate PET image (Gill et al., 2003; Ito et al., 1999). All six

baboons were included in the same statistical analysis, using voxel

to voxel comparison. Statistical parametric maps were then gener-

ated using an ANOVA model implemented through the General

Linear Model formulation of SPM (Friston et al., 1995b). We

analyzed the main effect of GDNF by comparing images obtained

in GDNF-treated with those obtained in MPTP-only animals with a

statistical threshold set at P < 0.001 for peak height, corrected for

spatial extend (>8 voxels per cluster). We tested for relative

increases and decreases in Ki by categorical comparisons of

MPTP-only vs. GDNF at the various time-points described above.

This generated statistical parametric mapping (t) maps for the 18F-

DOPA changes associated with each comparison. For between-

group comparisons, the statistical parametric mapping (t) maps

were subsequently transformed into statistical parametric mapping

(z) maps, and the level of significance of areas of activation was

assessed by the peak height of their foci with estimations based on

the theory of random Gaussian fields. Significance was accepted if

voxels survived an uncorrected threshold of P = 0.001.

For the ROI method, the time frames collected from 30 to 90

min were summed to create an integrated image used to define the

regions-of-interest (ROIs). The integrated PET images were then

co-registered to the individual magnetic resonance images. ROIs

were drawn over the paraventricular caudate regions and the entire

putamen in each hemisphere and an additional circular ROI was

drawn over the occipital region. Regional time–activity curves

were obtained for all ROIs. The 18F-DOPA uptake constant (Ki,

min�1) was measured in caudate and putamen using a multiple-

time graphical analysis with occipital activity as input function.

(Patlak and Blasberg, 1985).

Histological analysis

All animals were killed with a lethal dose of pentobarbital

(100 mg/kg iv, Sanofi, France). Immediately after death, brains

were removed and cut into coronal blocks (10–20 mm thickness).

Blocks from one hemisphere were immersed in isopentane at

�80jC and stored at �80jC. Blocks from the other hemisphere

were immersed for 5 days in PLP (paraformaldehyde 2%, sodium

m-periodate 0.20%, L-lysine monohydrochloride 1.4% in 0.1 M

phosphate buffer) at 4jC. They were then treated in a graded

series of sucrose-phosphate-buffered solutions (12%, 16%, and

18%). Coronal sections (40 Am thickness) were cut with a

freezing microtome (Microm, Francheville, France), kept in ana-

tomical series in a cryoprotective solution (glycerol and ethylene

glycol 30% in 0.1-M phosphate buffer), and stored at �20jC until

further processing. The presence of catecholaminergic neurons

was revealed by tyrosine hydroxylase (TH) immunocytochemis-

try. Free-floating sections were incubated for 30 min at room

temperature in phosphate-buffered saline (PBS; pH 7.4) contain-

ing 0.3% Triton-X 100 (TX-100) and 5% normal goat serum. The

sections were then incubated for 48 h at room temperature or 72

h at 4jC in PBS containing 0.3% TX-100, 3.5% normal goat

serum, 0.05% bovine serum albumin, and a rabbit anti-TH

antiserum (Institut Jacques Boy, Reims, France) diluted

1:10,000. The sections were then processed by the avidin-biotin

peroxidase method of Hsu et al. (1981) using Vectastain and VIP

kits (Vector laboratories, USA). Additional sections, also kept in

anatomical series were stained with cresyl violet for anatomical

location of brain structures.

Stereological evaluation of the number of TH-immunoreactive (ir)

neurons in the substantia nigra

Cell counts were performed on a computer-assisted image

analysis system consisting of an Olympus Provis microscope

equipped with a computer-controlled motorized stage, a Sony

http:\\www.fil.ion.ucl.ac.uk\spm\spm99.html


HAD Power 3CCD video camera, an Olympus Pentium II com-

puter (Olympus, Rungis, France), and C.A.S.T.-Grid, the Olympus

software for stereological cell counts (Olympus, Albertslund, Den-

mark). Stereologic analyses were performed using the optical

fractionator (West et al., 1991, 1996). Using a random starting

point, 1:10 series of sections was sampled throughout the whole

substantia nigra (yielding 20–25 sections per animal, depending

on the case). After outlining the boundaries of the substantia nigra

on the computer graphic display in each section separately, the

C.A.S.T.-grid software placed within each boundary a set of optical

dissector frames (100 � 100 Am) in a systematic, randomized

fashion, corresponding to a predetermined percentage of the

sampled area that was kept constant throughout the study (35%).

TH-ir neurons were then counted in each stack of optical dissectors

Fig. 4. 18F-DOPA positron emission tomography striatal changes. (A, B, and C) 18

normal (prelesioned) animal (A) and 55-week post-MPTP in MPTP-only #2 anima

decreased with MPTP treatment in both experimental groups compared to control

regional differences between MPTP-only and GDNF groups. (D and E) Statistical

the three GDNF-treated animals at 42–52 weeks using the SPM99 image analysis

the baboon’s brain (grey colors) fused with areas (color coded pixels) of significa

MPTP-only baboons. Z score threshold for significance was set to P < 0.05. Axial

function was increased by the intraventricular delivery of GDNF. (F and G) ROI an

putamen (G). Again, the only significant difference between both experimental gro

54–60 week). Data are expressed as mean F SE. *P < 0.05 (ANOVA, Fisher’s PL

C); 1 cm in D and E.

(each dissector was 1 Am in depth), according to stereological

principles. The thickness of these dissector stacks was kept

constant within each animal and was set at 8 Am in this study.

All analyses were performed using a �60 Plan-Neofluar objective.

The total number of TH-ir neurons in the substantia nigra was then

obtained using the formula developed by West et al. (1991): N =

AQ�. 1/asf. 1/ssf. 1/tsf, in which the total number of neurons (N) is

defined by the number of neurons counted in the dissectors located

within the defined subdivisions of the sampled sections AQ�

multiplied by the area, section, and thickness sampling fractions

(asf, ssf, and tsf, respectively).

The primate striatum contains intrinsic TH-ir neurons, the

numbers of which are augmented after dopamine depletion and

further increased by GDNF treatment (Palfi et al., 2002). To assess

F-DOPA PET scan images integrated from 0 to 80 min post-injection in one

l (B) or MPTP-GDNF #1 animal (C). 18F-DOPA striatal uptake dramatically

PET scan. On gross visual inspection, it was difficult to identify significant

analysis of 18F-DOPA PET scans between the three MPTP-only animals and

software. (D and E) panels represent coronal (D) and axial (E) MRI views of

nt difference (increases in 18F-DOPA uptake) between GDNF-treated and

and coronal views are shown to demonstrate that only caudate dopaminergic

alysis of 18F-DOPA PET scans in the periventricular caudate region (F) and

ups was observed in the caudate nucleus at the latter time-points (42–52 and

SD post hoc test). Calibration bars: 1 cm in A (same scale apply for B and


the possible trophic effect of GDNF delivery on these intrinsic

striatal neurons, TH-ir cell counts were performed at �1250

magnification within the caudate and putamen of the nine animals

(3 controls, 3 MPTP-only, 3 GDNF) from five different, anatom-

ically matched levels evenly distributed along the rostro-caudal

axis. TH-ir cell numbers were expressed as total number of TH-ir

cells observed per structure.

Statistical analysis

All values are expressed as means F SEM. Differences among

means were analyzed with an unpaired t test, factorial, or repeated

Fig. 5. Local neurotrophic effects of GDNF on Tyrosine hydroxylase (TH)-immun

C) Immunohistochemical staining of TH in the periventricular caudate nucleus of

(C). Whereas, a dense network of TH-positive fibers was noted in control animals,

MPTP. However, in GDNF-treated animals (C), a relative preservation of TH-po

compared to MPTP-only animals (B). (D and E) Intrinsic TH-positive cell numbers

(black bars) and GDNF-treated baboons (Grey bars). As previously described (P

numbers in both the caudate nucleus and the putamen of MPTP-only and GDNF-

was only observed in caudate nucleus in the GDNF-group as compared to MPTP-o

to affect periventricular regions (such as caudate nuclei) and not regions located a

0.01 (ANOVA, post hoc Fisher PLSD test). Calibration bar: 200 Am for (A, B, a

analysis of variance (ANOVA) followed by post hoc test, or

Mann–Whitney U test.

Results

Release of GDNF by the encapsulated cells

Table 1 summarizes all data related to the encapsulated GDNF-

producing cells and their implantation into the respective host

animals. Two different clones of GDNF-producing C2C12 cells

were used (clone #7, #19). Only the GDNF #1 animal received the

opositive fiber densities and intrinsic TH-positive cell numbers. (A, B, and

one control (A), one MPTP-only animal (B), and one GDNF-treated animal

a severe depletion could be observed in both experimental groups receiving

sitive fibers was noted in the periventricular region of the caudate nucleus

in caudate nucleus (D) and putamen (E) of control (open bars), MPTP-only

alfi et al., 2002), MPTP treatment significantly increased TH-positive cell

treated animals, as compared to controls, a further increase in cell numbers

nly. This indicates that GDNF delivery to the ventricular space was only able

t distance such as the putamen. Data are expressed as mean F SE. **P <

nd C).


two clones, whereas the remaining two received clone #19 capsules

only. At implantation, clone #7 capsules released an average of

(54.3 F 22.7 ng/24 h, mean F SE), whereas clone #19 capsules

were characterized by a higher release of GDNF (67.9 F 5.48 ng/

24 h, mean F SE). None of the clone #7 capsules had detectable

GDNF releases at explantation due to poor cell survival, whereas

clone #19 cells still demonstrated significant GDNF-release capac-

ity (23.0F 4.55 ng/24 h, meanF SE) as well as better preservation

within the capsule. As no sign of side effects were noted after the

first two surgeries and to further increase the amount of GDNF

released, the number of implanted capsules was increased to three

capsules in the GDNF #3 animal and four capsules in GDNF #2 on

Fig. 6. Neurotrophic effect of GDNF in mesencephalic dopaminergic nuclei. Low-

of TH-immunostained coronal sections at the level of the ventral tegmental area (V

D), and one GDNF-treated (E, F) baboon. SNpc and VTA TH-immunoreactive (

experimental groups compared to controls. Whereas stereological cell counts in

groups of parkinsonian animals compared to controls, a significant increase in ce

control and MPTP-only baboons. Calibration bars: 2 mm for (A, C, and E); 200 Am(ANOVA, post hoc Fisher PLSD test).

the third surgery. As a result, the total amount of GDNF delivered

in the GDNF #1 animal can be assumed to be smaller than for the

two others (GDNF #2 and GDNF #3). In addition, the highest

release of GDNF was obtained with the third set of capsules

implanted in GDNF #2 and GDNF #3 animals.

Capsules placement

Figs. 2A, B show the placement of the capsules within the two

lateral ventricles as observed in the post-operative MRI. The black

artifacts correspond to the capsule’s tracks running from the superior

frontal gyrus through the cingulate gyrus and corpus callosum and

magnification (A, C, E) and high-magnification (B, D, F) photomicrographs

TA) and substantia nigra (SNpc) of one control (A, B), one MPTP-only (C,

TH-ir) cell numbers dramatically decreased after MPTP treatment in both

SNpc-VTA regions evidenced a similar 65% TH-positive cell loss in both

ll volume could be detected in the GDNF-treated animals as compared to

for (B, D, and F). Data are expressed as meanF SE. *P < 0.05; **P < 0.01

H. Kishima et al. / Neurobiology of436

finally reaching the lateral ventricle at the level of the caudate nuclei

(Figs. 2A, B). Post-mortem histological evaluation of the tracks left

after the capsule removal (Fig. 2C) showed that most of the capsules

penetrated the corpus callosum just beside caudate nucleus and

exposed their tips into the lateral ventricles. Fig. 2C also demon-

strates the very limited trauma caused by the successive implanta-

tion and explantation of three different capsules over a 9-month

period. Over the six baboons implanted three times bilaterally,

hemorrhage was only observed on one occasion, restricted to the

very track of the explanted capsule. In all cases, there was no

noticeable inflammatory reaction along the surgical tracks, confirm-

ing the good tolerability of these implants by the primate brain.

Transient recovery in locomotor activity

Implantation of GDNF-producing capsules resulted in limited

behavioral changes over time. Fig. 3 depicts the time-course

changes in locomotor activity (total distance moved -TDM- over

40 min) observed over the entire duration of the study in MPTP-

only and GDNF-treated animals. Before MPTP, no significant

differences were observed between groups (MPTP-only group:

15,800 F 1400 cm, GDNF group: 12,600 F 660 cm, P > 0.4).

Under MPTP intoxication, a progressive decrease in TDM values

was observed in the MPTP-only group, reaching a maximal 50%

decrease by 45 weeks of neurotoxic treatment (black circles in Fig.

3). While a similar reduction of TDM was also observed in the

GDNF-treated group during the early phase of intoxication, a

transient increase of TDM values was observed between weeks

30 and 45, achieving statistically significant difference between the

two experimental groups at weeks 42–52 (grey squares in Fig. 3)

(Mann–Whitney U test P < 0.05). No significant differences

between groups were noted at any time point of the study on other

parkinsonian symptoms including balance impairment, postural

alteration, bradykinesia, tremor, and fur condition (data not

shown).

18F-DOPA PET examinations

The chronic MPTP intoxication induced a progressive reduc-

tion in 18F-DOPA uptake in caudate and putamen, which on visual

inspection, appeared quite similar in both experimental groups

(Figs. 4 A, B, C). Statistical parametric image analysis between

groups at the various times matching the behavioral evaluations

only demonstrated a significant difference between groups for the

42–52 and 54–60 week time points (Figs. 4D, E). These changes

consisted of an increase in 18F-DOPA uptake in the GDNF group

compared to the MPTP-only groups, restricted to the most para-

ventricular parts of the caudate nucleus, bilaterally. Notably, no

other differences were observed between groups in any other

dopaminergic region of the brain including the putamen, nucleus

accumbens or midbrain nuclei, at any time-point.

Having selected for ROI analysis the periventricular caudate

regions (caudate in Fig. 4F) and the entire putamen (Fig. 4G), we

further studied the progressive decrease in 18F-DOPA uptake

between the two groups of animals. Whereas GDNF significantly

attenuated the MPTP-induced loss of 18F-DOPA uptake in the

periventricular caudate nucleus (ANOVA, unpaired Student’s t test,

P < 0.05 GDNF vs. MPTP-only at 42–52 and 54–60 weeks), there

was no significant difference in the putamen (Fig. 4G) or midbrain

dopaminergic nuclei between both experimental groups (not

shown).

Post-mortem observations

Post-mortem analysis of striatal (caudate, putamen) TH-ir

showed that dopamine fibers were dramatically depleted in both

groups of MPTP-treated baboons when comparing to control

animals (Fig. 5A). In both MPTP-treated groups, TH-ir fiber

densities were greatly affected after 60 weeks of chronic MPTP

treatment, in both caudate nucleus (Figs. 5B, C) and putamen (not

shown). Densitometric measurements did not disclose significant

difference between the two experimental groups. However, as

observed previously (Palfi et al., 2002), intrinsic TH-ir striatal

neuron numbers were significantly increased by the MPTP treat-

ment in both experimental groups when compared to control

animals (Figs. 5D, E, P < 0.05 control vs. MPTP-only, P < 0.01

control vs. GDNF), with a further significant increase in cell

numbers observed in the caudate nucleus (Fig. 5D; P < 0.05

GDNF vs. MPTP-only), but not the putamen of GDNF-treated

animals compared to MPTP-only-treated animals (Fig. 5E).

As shown in Figs. 6A–F, the chronic MPTP treatment induced

a severe depletion of nigral TH-ir neurons and fibers in both

experimental groups. Low magnification photomicrographs dis-

play the preferential degeneration of TH-ir cells in the ventral and

most lateral part of the substantia nigra, similar to the pattern of cell

loss described previously in baboons with chronic MPTP intoxi-

cation (Hantraye et al., 1993). To assess whether GDNF-producing

encapsulated cells could have exerted neurotrophic/neuroprotective

effects on TH-ir cells at the somatic level, stereological cell counts

(Fig. 6G) and somatic volume assessments (Fig. 6H) were per-

formed in the substantia nigra-ventral tegmental area of all animals.

Cell counts showed that TH-ir cell loss accounted for more than

65% decrease in both experimental groups, which was highly

significant compared to control animals (P < 0.001, control vs.

MPTP-only and control vs. GDNF). However, no significant

difference in TH-ir cell numbers could be noted between MPTP-

only and GDNF-treated animal groups.

When assessing TH-ir cell volumes, a significant increase could

be demonstrated in GDNF-treated animals compared to both

control (P < 0.05) and MPTP-only animals (P < 0.01) (Fig. 6H).

Disease 16 (2004) 428–439

Discussion

The present study was undertaken to assess the neurotrophic

and neuroprotective potential of GDNF-producing encapsulated

cells in a pre-clinical setting before embarking into a clinical trial.

The experimental protocol was therefore purposely designed to

cope at best with the clinical situation that would be encountered in

the PD patients. To this aim, we first choose a chronic primate

model of nigrostriatal degeneration to mimic the progressive

nigrostriatal cell death occurring in PD. Second, we deliberately

decided to introduce the GDNF-producing cells 8 weeks after

initiation of the neurotoxic treatment to study not only the neuro-

protective, but also the neurorestorative potential of GDNF on

MPTP-induced behavioral and neurochemical deficits. Third, the

chronic MPTP treatment was maintained for 60 weeks to assess the

therapeutic potential of GDNF-producing cells at various stages of

nigrostriatal degeneration. Fourth, potential GDNF-induced alter-

ations in striatal and nigral dopamine metabolism were repeatedly

assessed using longitudinal 18F-DOPA PET, a standard follow-up

imaging technique in neuroprotective clinical trials. Finally, the

experimental design focused on using the same intraventricular


placement of the GDNF-producing encapsulated cells than that

previously demonstrated to be safe and efficacious in various pre-

clinical and clinical trials (Aebischer and Pralong, 2003; Bachoud-

Levi et al., 2000; Mittoux et al., 2000; Zurn et al., 2001).

With such an experimental design, we here show that despite

the low doses of trophic factor delivered, GDNF-producing en-

capsulated cells implanted into the lateral ventricle induced a

transient recovery in locomotor activity and focal although signif-

icant changes of striatal 18F-DOPA uptake in severely parkinsonian

baboons. Furthermore, post-mortem histological observations con-

firm that GDNF-producing cells implanted in the lateral ventricle

close to the caudate nuclei also induced detectable changes in

various dopaminergic markers not only locally (at the terminal

level), but also more distantly within the mesencephalic dopami-

nergic nuclei (i.e., at the somatic level of dopaminergic neurons).

Analysis of available data suggest that the transient changes in

locomotor activity and mild although persistent changes in 18F-

DOPA caudate uptake observed in these baboons could be attrib-

utable to neurotrophic changes in striatal and nigral dopaminergic

markers rather than to a true neuroprotection of the dopaminergic

cells. Altogether, the present pre-clinical study points to a potential

therapeutic efficacy of encapsulated GDNF as a neurorestorative

treatment for PD.

Post-mortem cell counts in mesencephalic dopaminergic nuclei

clearly indicate that intraventricular GDNF delivery was not

neuroprotective against chronic MPTP neurotoxicity. TH-ir cell

numbers in mesencephalic nuclei were depleted to a similar extent

in both experimental groups compared to controls. This finding

was rather unexpected as GDNF has clearly been shown to exert

neuroprotective effects against a variety of neurotoxins including

6-hydroxydopamine and MPTP itself (Bjorklund et al., 2000;

Georgievska et al., 2002; Shingo et al., 2002; Wang et al., 2002).

The lack of neuroprotection observed in the present study may be

attributable to various parameters such as the dose of GDNF

delivered, the site of administration for the trophic factor, or the

timing of cell implantation chosen. Regarding the first point, doses

of GDNF delivered in the present study were in the nanomolar

range which is in marked contrast with the higher doses of GDNF

shown effective in previous primate studies (Grondin et al., 2002)

as well as in patients (Gill et al., 2003). In the perspective of a

clinical application, it may be possible to increase the dose of

GDNF delivered by increasing the number of capsules implanted

in each patient. In line with this, it should be noted that in a very

similar preclinical study using Ciliary Neurotrophic Factor

(CNTF)-producing encapsulated cells as a potential treatment for

Huntington’s disease (Mittoux et al., 2000), full therapeutic effi-

cacy (both neurorestorative and neurotrophic) was observed fol-

lowing implantation of four capsules in the primate’s brain. The

therapeutic efficacy of encapsulated GDNF-producing cells may

also be improved by selecting another site of administration for

GDNF. In the present study, we chose to implant the cells in the

lateral ventricle as this site of delivery was judged to be safer than

the intraparenchymal route according to findings in previous

preclinical and clinical trials (Mittoux et al., 2000), and multiple

implantation of capsules into the same intraventricular/periventric-

ular space did not cause major deleterious effects nor inflammatory

responses. These observations are in marked contrast with a recent

study (Blanchet et al., 2003) in which all implants were surrounded

by an intense immune reaction with prominent ‘‘foreign body’’

inflammatory infiltrates. As postulated by the authors, membrane

breaches, the xenogeneic nature of the cells used, and the intra-

parenchymal site of implantation may have contributed to the

negative outcome of their study. However, in line with the post-

mortem data presented here, which did not show any sign of

rejection, it now seems reasonable to reconsider intraparenchymal

implantations as a more appropriate site for GDNF delivery. This

would bear more clinical significance, since GDNF delivery to the

sensorimotor striatum such as the post-commissural putamen is

likely of major importance for motor recovery, as shown by a

recent clinical trial (Gill et al., 2003). Future testing in nonhuman

primates of this form of chronic intracerebral delivery of GDNF

using similar encapsulated implants should concentrate on the

potential therapeutic efficacy of intra-putaminal implants.

Despite the lack of evidence for a neuroprotective action, GDNF-

producing cells implanted into the ventricular space were neverthe-

less able to trigger detectable neurotrophic effects on mesencephalic

dopaminergic neurons—not only locally (i.e., in the caudate nucle-

us), but also more distally, that is, at the level of the dopaminergic

(TH-positive) cell bodies. As a strong evidence for a local trophic

effect of GDNF within the infused area, one can point to the

significant increase in intrinsic TH-ir cell numbers specifically

observed in the caudate nucleus and not in the putamen of GDNF-

treated animals compared to MPTP-only animals. In contrast to

lower species, the primate striatum contains a population of TH-ir

neurons whose function currently remains elusive. These cells were

initially described in the nonhuman primate striatum by Dubach et

al. (1987) and have now been identified in the human striatum as

well (Porritt et al., 2000). Their numbers have been reported to

increase up to 3-fold in monkeys submitted to MPTP toxicity

(Betarbet et al., 1997). We have shown recently that GDNF can

further increase the number of these cells up to 7-fold in MPTP

primates additionally treated with the cytokine (Palfi et al., 2002). In

the present study, intraventricular delivery of GDNF resulted in a

significant increase in striatal TH-ir neurons that was restricted to

caudate nuclei compared with MPTP-only animals. This corre-

sponded to a more than 8-fold increase in the number of TH-ir cells

in comparison with normal cell numbers in the same region.

Interestingly, no such increase in TH-ir cell numbers was observed

in the putamen, a dopamine-depleted region similar to the caudate

nucleus, but located more distantly from the site of GDNF delivery.

This indicates that the GDNF released by the capsule was able to

reach the most lateral part of the caudate nucleus—a distance

corresponding to 1–3 mm from the ventricular space—but not the

putamen located 5–10 mm away from the ventricular space. These

observations may prove useful in optimizing the location of sites of

delivery in future clinical trials aiming at delivering GDNF in situ

and strongly support the use of intraparenchymal implantation sites

to restore dopamine function within the putamen.

Not only was intraventricular GDNF delivery capable of local

trophic effects, but also it significantly increased the somatic

volume of midbrain dopaminergic neurons. These neurons are in

the substantia nigra and ventral tegmental area, several centimeters

away from the site of administration. It is likely that GDNF acted

on these neurons with retrograde transport of the cytokine along

the axons of the remaining nigrostriatal neurons, as shown previ-

ously (Bjorklund et al., 2000). In line with this, it is interesting to

note that both in the chronic MPTP lesion model and in the human

pathology, the uneven degeneration of dopamine neurons also

results in a differential pattern of fiber depletion between caudate

nucleus and putamen. The relative preservation of dopamine

neurons projecting to the caudate nucleus, together with the

intraventricular site of GDNF delivery, may be two important


features that can explain the neurotrophic effects of GDNF

observed in the mesencephalic dopaminergic nuclei.

Acknowledgments

The authors thank Christophe Jouy and Frederic Sergent for the

support of the animals. The study was supported by grants from the

European Community (NeuroGet program QLRT KA3-1999-

00702).

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Encapsulated GDNF-producing C2C12 cells for Parkinson's disease: a pre-clinical study in chronic MPTP-treated baboons