doi:10.1093/brain/awh644 Brain (2005), 128, 2961–2976

Olfactory ensheathing glial co-grafts improvefunctional recovery in rats with 6-OHDA lesions

Saga Johansson, I-Hui Lee, Lars Olson and Christian Spenger

Department of Neuroscience, Karolinska Institutet, S-171 77 Stockholm, Sweden

Correspondence to: Saga Johansson, Department of Neuroscience, Karolinska Institutet, S-171 77 Stockholm, SwedenE-mail: saga.johansson@neuro.ki.se

Olfactory ensheathing cells (OEC) transplanted to the site of a spinal cord injury can promote axonal sparing/regeneration and functional recovery. The purpose of this study was to investigate if OEC enhance the effects ofgrafted dopamine-neuron-rich ventral mesencephalic tissue (VM) in a rodent model of Parkinson’s disease. Weco-grafted VM with either OEC or astrocytes derived from the same olfactory bulbs as the OEC to rats with aunilateral 6-hydroxydopamine lesion of the nigrostriatal system. Co-grafting fetal VM with OEC, but not withastrocytes enhanced dopamine cell survival, striatal reinnervation and functional recovery of amphetamine-and apomorphine-induced rotational behaviour compared with grafting embryonic VM alone. Grafting OEC orastrocytes alone had no effects. Intriguingly, only in the presence of OEC co-grafts, did dopamine neuronsextend strikingly long neurites that reached peripheral striatal compartments. Comparable results wereobserved in a co-culture system where OEC promoted dopamine cell survival and neurite elongation througha mechanism involving both releasable factors and direct contact. Cell type analysis of fetal VM grafts suggestedthat dopamine neurons of the substantia nigra rather than of the ventral tegmental area were increased in thepresence of OEC co-grafts. We conclude that the addition of OEC enhances efficacy of grafted immaturedopamine neurons in a rat Parkinson’s disease model.

Keywords: transplantation; dopamine neurons; olfactory ensheathing cells; astrocytes; Parkinson’s disease

Abbreviations: GFAP = glial fibrillary acidic protein; OEC = olfactory ensheathing cells; SN = substantia nigra;VM = ventral mesencephalic tissue

Received March 1, 2005. Revised August 16, 2005. Accepted August 30, 2005. Advance Access publication October 26, 2005

IntroductionParkinson’s disease is a progressive neurodegenerative

disorder. A cardinal feature is degeneration of dopamine

neurons projecting from substantia nigra (SN) to striatum.

To date, there is no cure and the causes are largely unknown,

although there is increasing evidence for causative genetic

factors in familial forms and genetic susceptibility factors

in idiopathic forms. Grafting fetal ventral mesencephalic tis-

sue (VM) to the adult dopamine-depleted striatum can partly

restore dopamine innervation and counteract functional

deficits in animal models of the disorder (Bjorklund and

Stenevi, 1979; Perlow et al., 1979; Dunnett et al., 1997), and

in some patients with severe Parkinson’s disease (Lindvall

et al., 1988; Bjorklund et al., 2003). Dopamine cell trans-

plantation protocols have been developed and refined in

several laboratories but remain experimental (Lindvall and

Hagell, 2000). The success of transplantations as a clinical

approach is hampered by limited availability of embryonic

tissue, limited graft survival, restricted dopaminergic

reinnervation of striatum, suboptimal functional effects

and, sometimes, negative side-effects (Freed et al., 2001).

Low cell survival can be partially counteracted by

neuroprotective (Brundin et al., 2000; Nakao et al., 1994)

or neurotrophic agents (Hurelbrink and Barker, 2001;

Fernandez-Espejo, 2004) but even when cell survival is

improved, reinnervation appears to remain insufficient

(Barker et al., 1996; Stromberg and Bickford, 1996). It has

been suggested that glial cells play key roles in survival and

function of the grafted neurons. Immature mesencephalic

glial cells that accompany immature grafted dopamine

neurons in most experimental grafting protocols, as well as

adult host striatal glial cells, may influence survival and fibre

outgrowth from grafted neuroblasts (Schwab and Thoenen,

1985; Gates et al., 1993; Moon et al., 2001; Bradbury et al.,

2002; Morgenstern et al., 2002). Olfactory ensheathing cells

(OEC) are unique in supporting continuous olfactory nerve

fibre growth from the olfactory mucosa into the CNS

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throughout adulthood (Graziadei and Monti Graziadei,

1980; Barber, 1982; Barber and Lindsay, 1982; Doucette

et al., 1983; Doucette, 1984). Several studies have suggested

a therapeutic potential of OEC grafted to the injured spinal

cord (Li et al., 1997; Ramon-Cueto et al., 1998, 2000; Lu et al.,

2001). Denis-Donini and Estenoz (1988) have also observed

extraordinary neurite outgrowth from nigral dopamine

neurons while co-cultured with OEC.

To determine if OEC improves effects of embryonic

dopamine neurons grafted to the adult dopamine-depleted

striatum, we implanted single grafts of fetal VM, OEC or

astrocytes derived from the same source as the OEC, and

compared the outcome of these three types of single grafts

to double grafts containing VM + OEC or VM + astrocytes.

We show that OEC, but not astrocytes, increase survival

of grafted dopamine neurons, enhance dopaminergic rein-

nervation of host striatum and promote functional recovery

compared with grafting embryonic VM alone. Grafts consist-

ing of OEC or astrocytes alone have no effects. Using an

in vitro system we further demonstrate that OEC are able

to promote dopamine cell survival and neurite elongation

through releasable factors.

Materials and methods6-Hydroxydopamine (6-OHDA) lesionsAdult female Sprague–Dawley rats (Scanbur, Sollentuna, Sweden)

were kept on a 12:12-h day:night cycle with free access to food pellets

and water. Experiments were approved by the Animal Research

Ethics Committee of Stockholm.

Unilateral lesions were performed by stereotaxic injection of

6-OHDA (Sigma, Sweden: 2 mg/ml in 0.9% NaCl containing

0.2 mg/ml ascorbic acid). The rats (5-week-old, 150 g body wt)

were anesthetized with halothane and positioned in a stereotaxic

frame. Injections into the medial forebrain bundle of the nigrostriatal

pathway (coordinates 4.4 mm posterior and 1.2 mm lateral to

bregma and 7.8 mm below the dural surface) were performed

using a micropump connected to a 10 ml Hamilton syringe and

at a rate of 1 ml/min with a total volume of 4 ml, totalling 8 mg

of 6-OHDA/animal. The syringe was withdrawn 2 min after the

injection was completed. Animals received subcutaneous injections

of buprenorphine (Temgesic, 0.3 mg/kg) every twelfth hour during

the first 2 days after surgery to reduce pain.

Rotational behaviourAmphetamine- and apomorphine-induced rotational behaviour

was used to confirm completeness of the lesion and to determine

effects of engraftment protocols. Rats were placed in plastic roto-

meter bowls and connected to a computerized system registering

the number of turns. After 10 min, when spontaneous rotational

behaviour had ceased, amphetamine (2 mg/kg intraperitoneally,

diluted in 0.9% NaCl) or apomorphine (0.05 mg/kg subcuta-

neously in the flank region, diluted in 0.9% NaCl) was injected.

Rotational behaviour was followed for another 70 (apomorphine)

or 90 (amphetamine) min. Rotational tests were carried out 2 and

4 weeks post-lesion. Amphetamine, which releases dopamine, was

used to detect unilateral losses of dopamine fibres in the form of

ipsilateral rotations. Since it is difficult to determine completeness

of a striatal dopamine denervation by amphetamine (Hefti et al.,

1980a, b; Heikkila et al., 1981; Casas et al., 1988; Carman et al., 1991;

Hudson et al., 1993; Moore et al., 2001) we also used the D2 receptor

agonist apomorphine, which causes contralateral rotations as a way

to determine completeness of the 6-OHDA lesions in living animals.

To ensure selection of well denervated animals, care was taken to

select only animals with at least 7.0 turns/min after amphetamine

injection and that responded to apomorphine with totally >450 turns

and displayed the characteristic two-peak rotation curve known to be

seen only in completely or almost completely denervated animals

(Herrera-Marschitz and Ungerstedt, 1984). Behavioural recovery

was assessed by amphetamine-induced rotations 2, 3 and 7 weeks

after grafting, and by apomorphine-induced rotations 8 weeks after

grafting.

OEC and astrocyte culturesPrimary OEC and astrocyte cultures were prepared from the

olfactory bulb of adult female Sprague–Dawley rats (5-week-old,

150 g body wt, Scanbur, Sollentuna, Sweden) based on a previously

described method (Nash et al., 2001). Isolation of the different types

of glia was accomplished by the different rates of cell attachment.

Briefly, animals were anaesthetized with isoflurane and decapitated.

The olfactory bulbs were removed and transferred into Hank’s bal-

anced salt solution (HBSS, Sigma). After removal of the meninges

and vessels, the medioventral superficial aspects of the bulbs (mainly

olfactory nerve and glomerular layer) were collected, minced, and

incubated with 0.1% trypsin at 37�C for 10 min. The trypsination was

stopped by addition of culture medium: Dulbecco’s modified Eagle’s

medium (DMEM)/Ham’s/F-12 (1:1 mixture, Sigma, St Louis, MO)

supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine

and penicillin (100 U/ml, Gibco), streptomycin (100 mg/ml, Gibco),

amphotericin B (1.75 mg/ml, Gibco). Following two washes in the

medium, cells were resuspended, triturated by fire-polished glass

Pasteur pipettes, and plated on uncoated Petri dishes. Many fibro-

blasts attach to the Petri dishes during this first 18-h incubation

period (37�C, 5% CO2). The supernatants, enriched for astrocytes

and OEC, were transferred to an uncoated culture flask and incub-

ated for another 36 h to allow for the attachment of astrocytes. OEC

do not attach to uncoated surfaces for 96–120 h, therefore remain in

the supernatant, which was then transferred onto poly-D-lysine-

coated (PDL, 0.1 mg/ml, Sigma) culture flasks. OEC and astrocytes

were allowed to grow to confluency in 10–14 days on PDL-coated

and uncoated flasks, respectively. On the day of transplantation, the

cells were trypsinized with 0.25% trypsin for 3 min at 37�C. The

detached cells were transferred to DMEM containing FBS and cent-

rifuged at 2800 r.p.m. for 8 min. After washing, the cell pellet was

resuspended in 1.5 ml DMEM and the cells were counted. Finally the

cells were centrifuged at 6000 r.p.m. for 7.5 min before the volume

was adjusted to the appropriate cell concentration for transplanta-

tion. More flasks of astrocyte cultures had to be harvested per graft

because less number of cells survived on the uncoated surface.

Analysis of OEC and astrocyte culturesTo determine purity of OEC and astrocytes in the primary cultures,

additional cell cultures were plated onto 2-well glass chamber slides

(PDL-coated and uncoated, respectively; LabTek, Naperville IL) and

processed for immunohistochemistry. Following a week in culture,

cells were fixed for 20 min with 4% paraformaldehyde in 0.1 M

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phosphate buffered saline (PBS) containing 0.4% picric acid,

washed several times in PBS, and preincubated in blocking solution

containing 10% donkey serum (Sigma) and 0.3% Triton X-100 in

PBS for 1 h at room temperature. Triple staining was performed

using primary antibodies raised against the low affinity neurotrophin

receptor p75 (p75, mouse, 1:300, Abcam), glial fibrillary acidic

protein (GFAP; chicken, 1:50, Abcam), and fibronectin (rabbit,

1:100, Chemicon) diluted in blocking solution. Following an over-

night incubation at 4�C, cells were washed three times in PBS and

incubated with Cy3/Cy2/Cy5 conjugated secondary antibodies

(1:300/1:50/1:50, Jackson ImmunoResearch Lab.) diluted in blocking

solution for 1 h at room temperature. Slides were coverslipped with

mounting medium containing DAPI (Vectashield/4,6-diamidino-

2-phenylindole, Vector, Burlingame, CA) before microscopy. The

purity of the cell cultures was determined by counting the immuno-

reactive cell types attached to the slides (Fig. 1). Cells with p75

immunoreactivity were designated OEC, these cells can also be dif-

fuse GFAP or weak fibronectin immunoreactive, as has been reported

previously (Ramon-Cueto and Nieto-Sampedro, 1992; Nash et al.,

2001); cells labelled strongly by fibronectin-immunohistochem-

istry alone were designated fibroblasts/meningeal cells; GFAP+ but

p75-negative cells were designated astrocytes. Five sample areas per

well and six wells totally at 20· magnification were used to calculate

the average percentage of each cell type. Using this protocol, out of

the cell types that could be observed, we obtained 43% astrocytes,

56% fibroblasts and 1% OEC in the astrocyte culture and 44%

OEC, 51% fibroblasts and 5% astrocytes in the OEC culture.

Thus fibroblasts/meningeal cells are abundant and present in the

same relative amounts in both types of cultures, while the two

types of cell preparations are relatively pure with respect to con-

taining either OEC or astrocytes. While fibroblasts/meningeal

cells together with OEC have been reported beneficial in spinal

cord injury (Li et al., 1998; Lakatos et al., 2003b), these cells do

not appear critical for the different results obtained in the present

in vivo experiments.

TransplantationsAnimals were anaesthetized with halothane. A stereotaxic frame was

used for implantation. VM from fetuses was dissected in DMEM

from embryonic day 14.5. The tissue from 20 fetuses was pooled

and trypsinated in 100 ml 0.5% trypsin for 5 min in 37�C. After

addition of 1.4 ml DMEM with 10% FBS the tissue was centrifuged at

1000 r.p.m. for 5 min. The cell pellet was washed with DMEM,

centrifuged at 2000 r.p.m. for 2 min, resuspended in 500 ml

DMEM and mechanically dissociated by trituration (10–20 strokes)

using a Pasteur pipette, and finally a 23G needle. Thereafter the cells

were counted in a Burker chamber and the cell suspension was

centrifuged for 7.5 min at 6000 r.p.m.. The cell pellet was resuspen-

ded in DMEM. For co-transplantation, the cell suspension from

the cultured OEC or astrocytes was mixed with the embryonic

VM suspension.

In one experiment, as illustrated in Table 1, animals received

either 100 000 embryonic cells together with 200 000 OEC (OEC/VM

co-grafts) or with 200 000 astrocytes (astrocyte/VM co-grafts),

or 200 000 OEC alone (OEC grafts) or 200 000 astrocytes alone or

100 000 embryonic cells alone (VM grafts). In a second experiment,

the number of VM cells was increased from 100 000 to 250 000,

and the animals therefore received 250 000 embryonic cells plus

200 000 OEC (OEC/VM co-grafts), or only 250 000 embryonic cells

(VM grafts) or only 200 000 OEC (OEC grafts).

The grafts were placed in the lower lateral part of the upper medial

quadrant of striatum (coordinates 0.5 mm anterior and 2.0 mm

lateral to bregma, and 5.0 mm below the dural surface). Injections

were performed at a rate of 1 ml/min. The syringe was slowly

Fig. 1 Examples of cell types found in OEC cultures from theadult olfactory bulb. Confocal images of triple-labelled cellcultures (A) and (E). As expected, many cells display p75immunoreactivity located in a Golgi-like perinuclear pattern andas puncta in the cell membrane (B) and (F). Other cells havestrong fibrillar GFAP immunoreactivity typical of astrocytes (C).OEC can be weakly GFAP + (C) or fibronectin + (D). Cellscharacterized by fibronectin immunoreactivity, presumably fibro-blasts/meningeal cells (H) are also found. Circle = OEC, asterisk =astrocyte, square = fibroblast/meningeal cell. Scale bar = 50 mm.

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withdrawn 5 min after the injection was completed to minimize

back-flow. Non-grafted controls received an injection of DMEM

at the same coordinates used for grafting. The animals received

subcutaneous injections of buprenorphine every twelfth hour during

the first 2 days after surgery to reduce pain, and they were sacrificed

8.5 weeks after transplantation. From the day prior to transplanta-

tion, the rats received a daily injection of cyclosporin A (10 mg/kg

body wt, Sandimmun, diluted in Ringer’s solution with an addition

of 2 mg/kg doxycycline) to avoid graft rejection.

In vitro experimentsOEC cultures were seeded onto PDL-coated 3.24 cm2 cover slips

placed in 6-well culture plates or onto PDL-coated membrane inserts

suspended 0.8 mm above the well’s bottom (4.2 cm2 with pore size

1.0 mm, Falcon). After 7 days in culture the OEC had reached 70%

confluency and a cell suspension of fetal VM was added to the cover

slips. The mesencephalic cell suspension was prepared as described

above for transplantation, 75 000 VM cells were added to each

cover slip. The cultures were divided into three groups: Group 1,

VM cells that were in direct contact with OEC (n = 10); Group 2,

VM cells on PDL-coated cover slips beneath a membrane with an

OEC monolayer (n = 10) and Group 3, VM cells on PDL-coated

cover slips with no OEC in the culture (n = 11). The culture

medium was switched from DMEM with 10% FBS, to a culture

medium consisting of two-thirds DMEM and one-third HBSS

supplemented with 10% FBS, 1.5% glucose and 1% HEPES when

VM cells were added to the cultures. The medium was changed

every third day. After 8 days in culture, cover slips were fixed in

2% paraformaldehyde for 30 min, rinsed in PBS and processed for

immunohistochemistry.

ImmunohistochemistryAnimals were intraperitonally injected with an overdose of pento-

barbital and intracardially infused with 100 ml Ca2+-free Tyrode’s

solution containing 0.1 ml heparin followed by 250 ml 4% para-

formaldehyde in PBS containing 0.4% picric acid. After perfusion,

brains were post-fixed for 1 h and rinsed in cold 10% sucrose in PBS

for at least 24 h. The tissue was rapidly frozen with gaseous CO2 and

30 or 14 mm cryostat sections generated. After thawing, slides were

rinsed in PBS before processing for immunohistochemistry.

Single, double or triple labelling was performed using primary

antibodies raised against tyrosine hydroxylase (TH, mouse, Diasorin,

1 : 5000 or rabbit, 1 : 300, Pelfreeze) to label dopamine neurons, p75

to label OEC (mouse, 1 : 500, Abcam) or GFAP (rabbit, 1 : 100, Sigma,

or goat, 1 : 50, Santa Cruz) to label astrocytes. To identify different

sub-populations of TH+ neurons in the grafts, sections were double

labelled with antibodies raised against TH and the G-protein-gated

inwardly rectifying K+ channel subunit (Girk2, rabbit, 1 : 80,

Alomone Labs, Israel) or calbindin (CB, mouse, 1 : 500, Sigma).

All antibodies were diluted in 0.3 or 0.6% Triton X-100 in PBS

for 14 or 30 mm sections, respectively. Incubation with primary

antibodies was performed for 48 h at 4�C, and double and triple

labelling was performed in a sequence such that one type of antibody

was applied at a time. Between incubations, sections was rinsed thrice

for 10 min in PBS. Secondary fluorescent antibodies, either conjug-

ated with Cy3 (1 : 400, Jackson ImmunoResearch Lab.), ALEXA454

(1 : 500, Molecular Probes, USA), or Cy5 (1 : 50, Jackson Immun-

oResearch Lab.) were applied for 1 h at room temperature. Finally,

sections were rinsed in PBS, mounted in 90% glycerol in PBS and

coverslipped. The 30 mm thick sections were processed for stereology

and histology by pretreatment with 2 M HCl for 30 min in 37�C and

rinsing in PBS before incubation. Sections processed for stereology

were single labelled and were, after incubation with primary anti-

bodies, incubated with biotinylated anti-mouse secondary antibodies

(1 : 200 in 0.6% Triton X-100 in PBS, Vector, USA) at room

temperature for 60 min, followed by incubation with an avidin–

biotinylated-peroxidase complex (Vectastain ABC kit, Vector,

USA, diluted 1 : 100 in 0.6% Triton X-100 in PBS) for 2 h at

room temperature. The substrate of 3,30-diaminobenzidine (DAB;

0.7 mg/ml, Sigma) was used as a chromogen in the presence of

0.01% H2O2. Finally, sections were washed in PBS and dehydrated

in alcohol and xylene before mounting with Entellan� (Merck).

Cultures were double or triple labelled with antibodies

against TH, GFAP and p75. The staining protocol used was the

same as for 14 mm slides except that the antibodies were diluted

in 0.6% Triton X-100.

Image analysisHistological evaluation of co-localization of the different cell types in

intrastriatal grafts and cultures were performed using multi-tract

scanning with confocal microscopy (Zeiss LSM 510 Meta).

Quantification of different sub-populations ofdopamine neuronsThe majority of dopamine neurons in the ventral tegmental area

(VTA) are small rounded cells that co-express TH and CB, but

not Girk2. The dopamine neurons in SN on the other hand are

typically larger cells with an elongated morphology, and the majority

of the nigral TH+ neurons express Girk2, but not CB (Mendez

et al., 2005; Thompson et al., 2005). Counting of TH+/Girk2+ and

Table 1 Experimental groups

Groupsa Grafted cells Numberof graftedanimalsVM cells OEC Astrocytes

Experiment 1a 100 000 15b* 200 000 15c 100 000 200 000 18d 200 000 5e 100 000 200 000 8f** 5

Experiment 2a 250 000 10b* 200 000 10c 250 000 200 000 12d** 5

aWell dopamine-denervated rats, as determined by

amphetamine- and apomorphine-induced rotational behaviour,were randomly selected to six groups in Experiment 1.Similarly, such animals were randomly selected to four groupsin Experiment 2.*Animals grafted with OEC from the two experimentswere pooled for behavioural and histologicalanalyses. The pooled group thus consists of 25 animals.**Sham-operated controls were pooled for behavioural andhistological analyses. The pooled sham group thus consistsof 10 animals.

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TH+/CB+ cells was performed on digital images (AxioVison image

software) of sections at the level of + 0.2 mm with respect to bregma.

Sections from five randomly selected animals from each sub-group

from the first experiment (Group 1a–f, Table 1) were analysed. The

cell diameter was also measured on all counted neurons, and the

position within the graft (central versus peripheral) was also noted.

StereologyNumbers of TH+ neurons in intrastriatal grafts, lengths of TH-

immunoreactive fibres in striatum and numbers of TH+ neurons

within the lesioned SN were determined using computerized

image analysis (Nikon microscope and Stereologer�, SPA inc.).

The software utilizes a stereological three-level fraction-based sam-

pling design based on the fractionator sampling method (Gundersen,

1986). Every fifth section throughout the transplants [section sam-

pling fraction (ssf) = 1/5] and every tenth section (ssf = 1/10) of the

�1.8 mm long rostro–caudal distribution of the SN (between 4.5 and

6.30 mm posterior to bregma) were systematically sampled using the

‘optical fractionator’ after randomly selecting the first section within

the first interval. A rectangular counting frame, ‘the dissector’, with

known area was superimposed on the field of view by the computer

software, and then the numbers of cell nuclei that come into focus

within the height of the optical dissector was counted. Counting

frames were systematically distributed with known x and y steps

throughout the marked region from a random starting point. The

area of the counting frame relative to the area associated with the x

and y steps gives the second fraction [area sampling fraction (asf)].

The height of the optical dissector relative to the thickness of the

section results in the third fraction [thickness (t)/height (h)]. The

total number of neurons is given by Ntotal =P

Q� (1/ssf) (1/asf)

(t/h), where Q� is the number of neurons counted in the dissectors.

Optical fractionator estimates are free of assumptions about cellular

shape and size and are unaffected by tissue shrinkage.

The area of the intrastriatal graft and the SN was manually

outlined using a 10· lens. The area of SN was defined based on

neuro-anatomical landmarks, and the position of TH+ neurons

on the contralateral intact side. Cell counts were performed with

a ·60 lens (numerical aperture = 1.4). For cell counts of the intra-

striatal graft, eight animals from each sub-group from the first and

second experiment were randomly chosen for stereology with excep-

tion of the VM/astrocytes co-grafted and astrocytes grafted group

in which intrastriatal cell counts were performed on all eight and

five rats, respectively. The measured sections were randomly selected

and coded. Cell counts in SN were performed on sections from five

randomly selected animals from each treatment sub-group from

the first experiment (Group 1a–f, Table 1).

The total length of TH-immunoreactive fibres innervating

defined volumes of striatum was measured by superimposing a vir-

tual 3D-probe in the shape of a sphere with a diameter of 10 mm in

the rectangular dissector (Mouton et al., 2002; Hofstetter et al., 2005,

for further illustration of the stereology method, see supplementary

Fig. 1). Fibres that traverse the virtual sphere surface at any angle

were counted at the level of +0.2 mm with respect to bregma

(Paxinos and Watson). Again, the counting frames, with the virtual

spheres, were distributed with known x and y steps throughout

the marked region. The total length of fibres/volume is given by

Ltotal = 2 ·P

Q� · (v/a) · (1/ssf) · (1/asf) · (t/h), where (v/a) is

the volume of the dissector relative to the surface area of the probe

and Q� is the number of fibres transversing the probe surface.

The length of TH+ fibres was counted in two volumes; the entire

caudate-putamen (CPu) on the lesioned side, excluding the area of

the graft, to estimate the total amount of fibres in striatum, and the

area of CPu most lateral to the ventricle to calculate the amount of

fibres furthest away from the graft (illustrated in Fig. 2G). The area of

the entire CPu was defined by the borders to the lateral ventricle,

corpus callosum, external capsule and the ventral pallidum. The area

of the CPu most distal to the graft was determined by the border

between CPu and the external capsule and the swelling of the olfact-

ory tubercle (Fig. 2G). This corresponds approximately to the area

of CPu between 3.8 and 4.8 mm lateral to bregma. The areas were

outlined using a ·4 lens and fibre length was measured with a

·100 lens (numerical aperture = 1.4).

In vitro cell counts and morphometricanalysisQuantification of TH-immunoreactive neuron numbers in cell cul-

ture was performed on digital images (AxioVision image software)

on 10 cover slips from each group. The cover slip was divided into an

81 square grid; each individual square had an area of 4 mm2. To

obtain cell counts the numbers of neurons in 18 squares were coun-

ted in a randomized fashion and the total number of neurons on

the cover slip was calculated. For quantative morphometry the

following parameters were investigated for 30 randomly chosen

individual TH+ neurons in each group: number of primary neurites,

total number of branch points, and length of the longest neurite.

Statistical analysisStatistical analysis of rotational data, cell counts, fibre length and

morphometric results were performed on mean differences in the

treatment groups using one-way analysis of variance (ANOVA)

followed by Tukey–Kramer post hoc analysis.

ResultsAmphetamine- and apomorphine-inducedrotational behaviourSignificant reduction of amphetamine- and apomorphine-

induced rotations was seen in all groups that received fetal

VM grafts, either alone or in combination with OEC or astro-

cytes. Reductions were seen at all time points investigated. In

the first experiment, when rats where grafted with 100 000

cells derived from fetal VM, OEC/VM co-grafted rats showed

a 78% reduction in amphetamine-induced rotations while

animals given single VM grafts showed a 55% reduction in

amphetamine-induced rotation (P < 0.01) at 7 weeks post-

grafting (Fig. 2A and B). Generally, rats with OEC/VM co-

grafts demonstrated a significantly larger decrease in both

amphetamine and apomorphine-induced rotations compared

with animals with astrocyte/VM co-grafts at all times. No

significant reduction of rotational behaviour compared

with the situation prior to transplantation was observed in

animals that received a single astrocyte graft or no graft at all.

However, animals that received a single OEC graft showed

a modest, but significant reduction (�8%; P < 0.05, Fig. 2A)

of amphetamine-induced rotational behaviour 7 weeks

after transplantation, but no reduction in the number of

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Fig. 2 Effect of co-grafting VM with OEC or astrocytes on drug-induced rotational behaviour, survival of dopamine neurons and length ofTH-positive fibres in host striatum. Grafting VM cells caused significant reduction of amphetamine- (A) and (C) and apomorphine-induced(B) and (D) rotations (expressed in percentage of pre-grafting values) in all animals. When OEC were mixed with 100 000 VM cells,there was a significant further decrease of both amphetamine- and apomorphine-induced rotations at all time points compared to animalsgiven only 100 000 VM cells or the same number of VM cells mixed with astrocytes, or grafted with OEC or AC alone (A) and (B). WhenOEC were mixed with 250 000 VM cells there was a significantly larger reduction of amphetamine-induced rotations compared to ratsgiven 250 000 VM cells alone 2 weeks after transplantation (C, D). No such effect of OEC mixed with 250 000 VM cells on amphetamine-induced rotations were seen at later time points; apomorphine-induced rotations were not influenced at any time point (C) and (D). Thehost SN on the denervated side contained a few percent of the original population of TH+ dopamine neurons. There were no significantdifferences between the different groups in numbers of surviving TH+ neurons (expressed in percentage of intact contralateral side) in thelesioned SN (E). The number of surviving grafted TH+ neurons was significantly higher in animals in which OEC and 100 000 VM cellswere grafted together, compared to groups co-grafted with astrocytes or given 100 000 VM cells alone (F). Increasing the number ofgrafted VM cells to 250 000 doubled the number of surviving grafted TH+ neurons, but there was no significant difference in cell survivalbetween the group grafted with 250 000 VM cells alone and the group co-grafted with OEC and 250 000 VM cells (F). The amount of TH+

fibres was quantified in the entire CPu on the lesioned side (grey and blue area) and in the distal lateral part of CPu (blue area) in (G).Both the total amount of TH+ fibres in the striatal volume (H) and the amount of TH+ fibres in the lateral part of the host CPu (I) wassignificantly increased in the group co-grafted with OEC and 100 000 VM cells compared to animals with single VM grafts or co-graftedwith astrocytes and 100 000 VM cells. Similarly, co-grafting OEC with 250 000 VM cells significantly increased both the total (H) andlateral (I) amount of TH+ fibres in striatum compared to grafting 250 000 VM cells alone. CPu = caudate-putamen; ec = external capsule;LV = lateral ventricle; Tu = olfactory tubercle; AC = astrocytes. Error bars 6 SEM, *P < 0.05, **P < 0.01, ***P < 0.005.

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apomorphine-induced rotations 8 weeks after transplantation

was apparent (Fig. 2B).

When we increased the number of grafted embryonic cells

2.5-fold in the second experiment, a significantly improved

reduction of amphetamine-induced rotational behaviour

caused by the addition of OEC was seen at 2 weeks after

transplantation compared with animals with VM grafts

alone (37 and 20% decrease in number of rotations, respect-

ively; P < 0.05, Fig. 2C). However, the difference between

OEC/VM co-grafted and VM-grafted animals diminished

and was no longer significant at 3 weeks (57 and 52% reduc-

tion in rotations, respectively) and 7 weeks (88 and 83%

reduction in rotations, respectively) post-grafting due to a

ceiling effect. Similarly, co-grafting of OEC with 100 000

or 250 000 VM cells gave a very similar maximal

reduction in amphetamine-induced (79 and 88%, respect-

ively) or apomorphine-induced (76 and 79%, respectively,

Fig. 2A and C, and B and D, respectively) rotation. However,

a significant difference was observed between animals that

received single VM grafts of either 100 000 or 250 000 cells

(reduction of amphetamine-induced rotation at 7 weeks 55%

compared with 83%, P < 0.01; reduction of apomorphine-

induced rotation at 8 weeks 44% compared with 76%,

P < 0.05).

Graft analysis—general observationsHistological analysis 8 weeks post-grafting revealed viable

grafts in striatum in all grafted brains. Immunohistochemistry

demonstrated surviving TH+ neurons, GFAP+ astrocytes and

p75+ OEC as expected in the respective grafts (Figs. 3 and 5A

and B). TH+ neurons with different sizes were observed within

all grafts with VM cells. TH+ neurons with large elongated cell

bodies were mainly located at the periphery of the grafts and

were observed in single VM grafts and co-grafts with OEC or

in co-grafts with astrocytes whereas TH+ neurons with smaller

rounded cell bodies were mainly located in the centre of the

grafts and were observed in single VM grafts and co-grafts

with OEC or astrocytes. In OEC co-grafts, small TH+ neurons

were also located in the periphery of the graft.

A dense TH+ fibre network was present within the VM

grafts and also extended to variable distances into host

striatum (Fig. 3A–C, E–G, I and J). In animals with OEC

co-grafts dopamine neurons gave rise to an extensive TH+

fibre network reaching as far as the lateralmost periphery of

the striatum (Fig. 3C). This peripheral fibre network though

was less dense than that close to the graft site. Only a few,

scattered TH+ fibres could be observed in peripheral regions

of striatum in animals with single VM grafts and co-grafts

with astrocytes (Fig. 3G). As expected, groups grafted with

250 000 VM cells had a more dense TH+ fibre outgrowth

compared with groups grafted with 100 000 VM cells. Also,

no TH+ cells were observed in animals grafted with astrocytes

alone, OEC alone (Fig. 3H) or in non-grafted, sham-injected

controls. Neither were there any TH+ fibres observed within

the respective graft areas. However, a very small number of

TH-immunoreactive fibres, presumably residual fibres spared

from the lesion, were occasionally seen randomly scattered

in striatum in all groups. These fibres reflect the very low

number (1–6%) of remaining dopamine neurons found

in SN.

Olfactory ensheathing cells were identified by p75

immunohistochemistry. Co-grafted OEC were present both

in the centre and the periphery of the graft (Figs. 3D and 5A).

No significant migration of OEC into host striatum was

observed in either single grafts (Fig. 3L) or co-grafts (Fig.

3D and 5A). However, it cannot be excluded that some

OEC, either inside or outside the graft area, had become

p75-negative and thus were not detected. No p75+ cells

were found in host striatum in animals grafted with VM

alone or in non-grafted controls. A few p75+ cells were occa-

sionally found at the graft site in some animals with a single

astrocyte graft or astrocyte/VM co-graft.

Single grafts and co-grafts with astrocytes were labelled

with GFAP antibodies. Due to the general and widespread

presence of astrocytes in all brain tissues, the origin of

GFAP+ cells is not easily determinable. However, single astro-

cyte grafts (Fig. 3K) were more intensely GFAP+ than areas

surrounding the injection tracts in sham-operated controls.

Single OEC and VM grafts and co-grafts of OEC with VM

were associated with less GFAP+ immunoreactivity than

astrocyte/VM co-grafts.

Girk2 and CB immunoreactivity ofgrafted dopamine neuronsTo further characterize TH+ neurons in the grafts we used

Girk2 and CB immunoreactivity. Analysis of the number of

Girk2+ and CB+ cells in the grafts showed that co-grafting

VM with OEC significantly increased the percentage of

TH+ neurons that express Girk2 (VM/OEC co-grafts: 69.3%;

68 6 3 TH+/Girk2+ cells of 98 6 3 TH+ cells; P < 0.01;

VM/astrocyte co-grafts: 48.1%; TH+/Girk2+ cells 37 6 2

TH+/Girk2+ cells of 74 6 2 TH+ cells; single VM grafts:

49.6%; 39 6 2 TH+/Girk2+ cells of 78 6 2 TH+ cells). The

percentage of TH+/CB+ neurons did not significantly differ

within different groups (an average of 27 6 2% of TH+ cells

were also CB+ in all groups). The vast majority of TH+/Girk2+

neurons were large cells with a mean diameter of �18 mm.

These cells were located at the periphery of the graft

(Fig. 4A–C). In OEC/VM co-grafts, however, there were

also smaller TH+/Girk2+ cells with a diameter between 13

and 15 mm. Most of the TH+/CB+ neurons were small roun-

ded cells with an average diameter of �13 mm and located

in the centre of the grafts (Fig. 4D–F).

Co-localization of OEC and TH+

neurons in co-graftsSections triple labelled for TH, GFAP and p75 were studied

by confocal microscopy to evaluate possible co-localization

and cell–cell contacts among the grafted cells. OEC were

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found in close contact with TH+ cell bodies and fibres within

the graft (Fig. 5A and B). More TH-immunoreactive fibres

growing into the host brain were observed to emerge from

OEC rich areas. Interestingly, there was no detectable migra-

tion of p75+ OEC outside the graft area, and therefore no

contact between TH-positive fibres innervating striatum and

OEC were observed. However, some OEC may lose their p75

immunoreactivity after grafting and we can therefore not

exclude the possibility of p75� OEC migrating outside of

the graft site.

Fig. 3 Histology of intrastriatal grafts. All pictures show TH-immunoreactivity except (D) and (L) (p75) and (I) (GFAP). The boxed areasin (A), (E) and (I) are shown in higher magnification in (B), (F) and (J), respectively. Grafts with VM cells had surviving TH+ neurons andTH+ fibres reinnervating the host striatum (A)–(C), (E)–(G), (I) and (J). Animals co-grafted with OEC and 100 000 VM cells (A)–(C) had amore extensive TH+ reinnervation compared to animals grafted with 100 000 VM cells alone (E)–(G) or co-grafted with astrocytesand 100 000 VM cells (I) and (J). There was a striking difference between the amount of TH+ fibres in the peripheral part of striatum inanimals co-grafted with OEC (C) and animals with single VM grafts (G). No TH+ fibres were observed at the transplantation site in animalsgrafted with OEC alone (H). Immunohistochemical labelling of p75 demonstrated surviving OEC in animals grafted with OEC alone (L),shows the area corresponding to panel (H) as well as in OEC/VM co-grafts (D). No p75+ cells were found in non-grafted controls oranimals grafted with VM alone, and on rare occasions a few p75+ cells were present in animals grafted with astrocytes alone or co-graftedwith astrocytes and VM (data not shown). Animals grafted with astrocytes alone had an intense GFAP+ labelling of the graft area (K).The illustration in (M) shows the location of the graft versus pictures of the peripheral striatum (shown in C and G). AC = astrocytes; LV =lateral ventricle; asterisk = border between cortex and striatum. Scale bars: (A), 300 mm; (E) and (I), 300 mm; (B)–(D), (F)–(H) and (J)–(L),300 mm.

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Number of TH+ neurons in hostsubstantia nigraThe number of dopamine nerve cells in SN, as defined by TH

immunoreactivity, was determined by stereology. The values

obtained on the lesioned side were expressed as a percentage

of neurons found on the intact contralateral side. Consistent

with the rotational responses to apomorphine, few TH+

neurons were observed within SN on the lesioned side, ran-

ging between 1 and 6% of the number of TH-immunoreactive

neurons found on the intact side for individual animals

(156–963 dopamine neurons on the lesioned side, an average

of 15 644 dopamine neurons on the intact side). There was

no significant difference in TH+ cell numbers between the

different treatment groups or the denervated non-grafted

Fig. 4 Sub-populations of dopamine neurons in VM grafts. To identify sub-populations of VM dopamine cells, sections were double labelledfor TH and Girk2 or CB, which preferentially label SN or VTA dopamine neurons, respectively. Adjacent sections of an OEC/VM co-graftare shown in (A) and (D). The boxed areas in (A) and (D) are shown at higher magnification in (B) and (C) and (E) and (F), respectively.TH+/Girk2+ dopamine neurons cells were located primarily in the peripheral part of the graft area (B) and (C) whereas TH+/CB+ neuronswere mainly located at the core of the graft (E) and (F). No or few TH�/Girk2+ cells were observed in the grafts. A population of TH�/CB+

cells were also observed in the grafts (E) and (F). Scale bars: (A, D) 400 mm, (B), (C), (E), (F) 200 mm.

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controls (Fig. 2E). This clearly demonstrates that the lesions

in all animals were complete or close to complete. This is in

agreement with the classic observation by Ungerstedt (1968)

that the majority of the dopamine neurons in SN have died

at the time point used for transplantation.

Survival of grafted dopamine neuronsA significantly higher number of TH+ neurons were found

within OEC/VM co-grafts (611 6 30; mean 6 SEM), com-

pared with VM single grafts (438 6 19; mean 6 SEM; P <

0.001) and astrocyte/VM co-grafts (mean 438 6 15; P < 0.001)

Fig. 5 Co-localization of dopamine neurons and OEC in vivo, and morphology and quantification of cell survival and neurite growth in vitro.Panels (A) and (B) show a section from an OEC/VM co-graft, triple-labelled for TH, GFAP and p75. The boxed area in (A) is enlarged in(B). TH+ fibres were observed within the graft and growing into host striatum [arrow in (A)]. Close contact between TH+ cell bodiesand fibres and p75+ OEC were observed within the graft (B). TH+ neurons in control cultures (C) and (D) had a more branchedmorphology compared to neurons cultured in OEC conditioned medium (E) and (F) or in direct contact with OEC (G) and (H). Dopamineneurons cultured in direct contact with the OEC monolayer had a more elongated bipolar morphology (G) and were often seengrowing aligned to p75+ OEC (H). Culturing fetal VM cells in direct contact with OEC or in OEC-conditioned medium increased survival ofTH+ neurons significantly compared to control cultures (I). Both direct contact with OEC and conditioned medium also enhanced thelength of the longest neurite significantly compared to control (J). TH+ neurons cultured in direct contact with OEC had significantlyfewer primary neurites and branching points compared to neurons in control and conditioned medium cultures (K) and (L).OEC-conditioned medium appeared to be intermediate between direct contact and control with respect to cell survival, neuritelength and branching points (I)–(L). Scale bars: (A) 100 mm, (B) 20 mm, (C), (E) and (G) 50 mm, (D), (F) and (H) 50 mm. Errorbars 6 SEM, *P < 0.05, **P < 0.01, ***P < 0.005.

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in animals that received grafts with 100 000 VM cells (Fig. 2F).

Approximately 5% of the cells prepared from the dissected

VM are dopamine neurons prior to transplantation (Fawcett

et al., 1995). The number of neurons found in the grafts

corresponds to a survival rate of 12.2% in OEC/VM

co-grafts and 8.8% in animals grafted with VM alone or

with astrocyte/VM co-grafts.

There was no significant difference in the number of

surviving grafted TH+ neurons between animals with

OEC/VM co-grafts (1306 6 146; mean 6 SEM) and VM

single grafts (1202 6 144; mean 6 SEM) when 250 000

VM cells were grafted (Fig. 2F). The survival rate of the

TH+ neurons in the groups that received 250 000 VM cells

was 10.5% (OEC/VM) and 9.6% (VM). However, grafts with

250 000 VM cells generated more surviving TH+ neurons

than grafts with 100 000 VM cells (1306 compared with

611 for the OEC/VM groups, and 1202 compared with 438

for the VM groups, P < 0.001).

Amount of TH+ fibres innervating thelesioned striatumVM grafts generated a significant higher total amount of

TH-immunoreactive fibres in CPu in all animals compared

with non-grafted controls (Fig. 2H). Co-grafting with OEC

increased the total fibre amount by 181% (865 6 73 mm;

mean 6 SEM) compared with animals with single VM grafts

(479 6 89 mm; mean 6 SEM), and by 168% compared with

astrocyte co-grafts (515 6 83 mm; mean 6 SEM, P < 0.005) in

animals receiving 100 000 VM cells. In animals that received

250 000 VM cells, co-grafting with OEC significantly

increased the total fibre amount by 153% (1422 6 159 mm;

mean 6 SEM) compared with animals grafted with single VM

grafts (928 6 64 mm; mean 6 SEM, Fig. 2H). Increasing the

number of grafted neurons by 2.5-fold increased the total

amount of fibres by 164 and 194% in the OEC/VM co-grafted

group and the VM grafted group, respectively. A total amount

of 71 6 16 mm TH+ fibres/striatum was observed in animals

grafted with OEC alone; however, this was not significant

compared with rats with astrocyte grafts (13 6 3 mm;

mean 6 SEM) or non-grafted controls (10 6 3 mm;

mean 6 SEM).

No, or almost no TH+ fibres were located in the lateralmost

area of striatum in animals without a VM graft (Fig. 2I). In

animals with single VM grafts lateral striatal regions remained

essentially non-innervated by the graft. However, when

OEC were added to the VM cells, a significant amount of

TH+ nerve fibres reached the lateral aspects of striatum,

(length of TH-immunoreactive fibres in the lateral striatal

area; 74 6 11 and 181 6 64 mm mean 6 SEM; in animals

co-grafted with OEC and 100 000 or 250 000 VM cells,

respectively). Co-grafting with OEC strikingly increased the

number of fibres found in the lateralmost peripheral

portion of striatum 10–14-fold (100 000 VM cells and

250 000 VM cells, respectively) compared with single VM

grafts (Fig. 2I).

Effect of OEC on dopamine neurons in vitroAnalysis of TH+ cell numbers showed that both direct contact

with OEC (816 6 132 cells; mean 6 SEM, Fig. 5I) and OEC

conditioned medium (579 6 31 cells; mean 6 SEM) signi-

ficantly increased the number of TH+ neurons in culture by

255 (P < 0.001) and 182% (P < 0.05), respectively, compared

with control VM cultures (318 6 27 cells; mean 6 SEM).

The number of primary neurites, total number of branch

points and length of longest neurite for TH+ neurons revealed

that direct contact with OEC increased neurite length and

decreased branching (Fig. 5J–L). There was a significant

increase in length of neurite extension from neurons cultured

in direct contact with OEC monolayers, 9476 43 mm com-

pared with 624 6 34 mm (mean 6 SEM, P < 0.005) in control

cultures (Fig. 5J). However, neurons in control cultures had

significantly more branch points, 7.4 6 0.54 branch points

compared with 4.4 6 0.25 (mean 6 SEM, P < 0.001) in direct

contact co-cultures (Fig. 5K–L). TH+ nerve cells grown

in OEC conditioned medium appeared to be intermediate

between the control and direct cell contact conditions,

with a neurite length of 801 6 61 mm (mean 6 SEM) and

6.0 6 0.49 (mean 6 SEM) branch points. Furthermore,

TH-immunoreactive neurons growing in direct contact with

OEC tended to display a bipolar morphology and appeared

to be growing aligned with p75+ OEC (Fig. 5G–H). The

differences in neuronal morphology in the different cultures

are illustrated in Fig. 5C–H.

DiscussionGrafted embryonic dopamine neurons can partly reinnervate

the dopamine-denervated striatum and counteract some

symptoms in experimental animals and patients with

Parkinson’s disease. A key problem with this approach is

yield in terms of numbers of surviving grafted dopamine

neurons and amount of axonal arborization. In this study

the possible beneficial effect of adding olfactory ensheathing

cells to the grafted dopamine neurons was investigated. We

find that the addition of OEC leads to significantly better

functional effects of grafted VM cells, in terms of both

amphetamine- and apomorphine-induced rotational behavi-

our in the rat 6-OHDA model, than seen with VM cells alone.

We further show that this improvement is paralleled by an

increased number of surviving grafted dopamine neurons,

preferentially of the Girk2+ rather than the CB+ type, and

that there is an increased amount of new dopamine nerve

fibres in the denervated host striatum when OEC are present.

This effect is specific to the OEC, since it is not seen when

instead an astrocyte-rich, OEC-poor cell suspension, derived

from the same starting material as the OEC-rich preparation,

is mixed with the VM cells. We rule out any major direct effect

of OEC, since such cells alone have minor or no functional

or structural effects. A striking and unique effect of adding

OEC is that the grafted dopamine neurons continue to extend

axonal branches to lateral regions of host striatum, regions

that remain permanently denervated when VM cells alone are

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grafted. Finally, our in vitro experiments show that the effects

of OEC on embryonic dopamine neurons on survival and

neurite elongation are exerted by mechanisms that are not

exclusively cell–cell contact dependent.

OEC/VM co-grafted rats have a 1.5 times higher total nerve

fibre length in striatum than VM grafted rats. The nerve fibre

length in the lateralmost portion of striatum was remarkably

increased by 10–14-fold (100 000 VM cells and 250 000 VM

cells, respectively) in OEC/VM co-grafted rats compared with

VM grafted rats. Equally remarkably, these fibres were present

at distances from the grafted neurons that were almost not at

all reached by axons from TH neurons in single VM grafts.

Comparable results were observed in the co-culture system;

OEC promoted dopamine cell survival and neurite

elongation. The extended innervation of the striatum is a

long sought-for effect, which has not been reported before

in rodent allograft models. Human embryonic dopamine

neurons grafted to rodent hosts can show more extended

innervation patterns which might be related to the extended

period of development in humans as compared with

rodents (Stromberg et al., 1986, 1989; Brundin et al.,

1988b; Wictorin et al., 1992).

The number of grafted neurons is known to be closely

correlated to the resulting decrease of amphetamine- and

apomorphine-induced behaviour (Brundin et al., 1985,

1988a; Rioux et al., 1991; Nakao et al., 1994). When we

increased the number of grafted VM cells from 100 000 to

250 000, we observed an enhanced reduction of drug-induced

rotations in animals that received VM grafts alone. Except for

observations 2 weeks after grafting, such an enhanced effect of

adding OEC on rotational behaviour was not as evident when

we increased the number of grafted dopamine neurons. The

reduction in drug-induced rotational behaviour (Brundin

et al., 1988b) has been shown to follow a saturation curve,

where after a certain degree of dopaminergic reinnervation

(Bjorklund et al., 1980) or neuronal survival (Brundin et al.,

1988a), further increases have no additional effect. Pre-

sumably, the high number of neurons grafted in the second

experiment was sufficient to almost completely reverse the

lesion-induced rotational behaviour already in the group

that received single VM grafts, thus masking any possible

additional effects of OEC co-grafts. Although the survival-

promoting effect of co-grafting with OEC is not evident

when the number of grafted VM cells increases, the ability

to support wide-spread neurite growth was not diminished.

Thus the OEC-induced effects on dopamine cell survival and

neurite extension are separate events.

There is a wide range in numbers of grafted VM cells,

numbers of surviving neurons and effects of grafted neurons

on rotational behaviour reported in the literature. Differences

can largely be explained by differences in protocols used for

cell suspension preparation and transplantation. The survival

rate in our study and the subsequent decrease in rotational

behaviour are in agreement with several other studies

(Costantini and Snyder-Keller, 1997; Sortwell et al., 1998;

Wilby et al., 1999; Tornqvist et al., 2000; Ostenfeld et al.,

2002). Increased numbers of surviving TH+ cells do not auto-

matically result in better reversal of rotational behaviour

(Rosenblad et al., 1999; Wilby et al., 1999; Ostenfeld

et al., 2002); the amount and location of graft-derived

TH+ intrastriatal innervation is also an important deter-

minant of the functional outcome. It should be noted that

while drug-induced rotational behaviour provides a sensit-

ive indicator of graft survival, it only provides limited

information about the function of the graft. Other studies

have reported a complete reversal of drug-induced rotational

behaviour, yet limited effects on complex motoric tasks

such as front paw reaching and stepping (Georgievska et al.,

2004). Further studies are needed to evaluate the effects of

co-grafting VM with OEC on more complex motor tasks.

Interestingly, the presence of OEC led to an increase in the

Girk2+ sub-population of TH+ neurons, while there was no

increase of the CB+ sub-population. Other studies have

demonstrated that the majority of the dopamine neurons

in SN express Girk2, whereas the majority of dopamine

cells in VTA express CB (Mendez et al., 2005; Thompson

et al., 2005). Thompson et al. (2005) further demonstrated

that Girk2+ dopamine neurons become mainly located in the

periphery of a graft and project preferentially within striatum,

while CB+ neurons are found located in the centre of the

VM grafts and project to frontal cortex. Our observations

of the location of Girk2+ and CB+ TH neurons in the grafts

are in agreement with these studies.

The mechanisms underlying the growth-promoting ability

of OEC have yet to be clarified. OEC induce little astrocytic

response and chondroitin sulphate proteoglycan expression

in vitro and in vivo following transplantation into adult

CNS, providing a more permissive substrate for nerve out-

growth (Lakatos et al., 2000, 2003). OEC also express

membrane surface molecules, neurite-promoting molecules

(e.g. amyloid precursor protein), soluble neuregulins and

neurotrophic factors that may contribute to the observed

effects in the co-grafting situation (Ramon-Cueto and

Avila, 1998; Chuah and West, 2002; Moreno-Flores et al.,

2002; Lipson et al., 2003; Chung et al., 2004). While co-grafted

OEC stimulate TH+ neurites to grow extensively beyond the

grafted site, the OEC themselves are primarily confined to

the site of engraftment. These are in agreement with others

and our previous findings that OEC grafts, identified by p75

immunoreactivity or by lentiviral-transferred green fluores-

cent protein, exhibit limited migration from a severe lesion

site and can hardly extend across an astrocytic barrier

(Ruitenberg et al., 2002; Lee et al., 2004). We suggest that

OEC grafts in the lesioned striatum are primarily confined

close to the injection site, although the possibility that some

OEC could have lost their p75 immunoreactivity and

migrated outside the graft can not be completely excluded.

This phenomenon is somewhat akin to the original observa-

tion of Li et al. (1998) that the effect of OEC grafted into

the focally lesioned spinal cord was to form a ‘patch’ across

which the cut central axons regenerated. We suggest that

OEC act on fetal dopamine neurons by providing a cell–cell

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contact-mediated, and/or paracrine stimulation of dopamine

neurons, and/or stimulate neurites to grow longer distances or

to grow for a longer-than-normal time to reach the striatal

periphery. In our co-culture experiment, dopamine neurons

cultured in direct contact with an OEC monolayer were less

branched and had a more fusiform morphology, suggesting

that OEC may retard dopamine neuroblast maturation, thus

prolonging the period of active nerve fibre growth.

The present results are partly seemingly at variance with a

recent study by Agrawal et al. (2004) in which OEC and VM

cells were grafted together or separately to rats with unilateral

6-OHDA denervations. Agrawal et al. (2004) reported not

only that OEC/VM co-grafted rats have better functional

recovery than rats with single VM grafts, but also surprisingly

that grafting OEC alone is superior to VM alone in terms of

striatal dopamine fibre density and that this effect was cor-

related to an increased number of surviving dopamine neur-

ons within the lesioned SN. The extent of the lesion is critically

important in determining the outcome of grafting strategies

using the 6-OHDA model (Blanchard et al., 1995, 1996;

Hansen et al., 1995; Kirik et al., 1998; Finkelstein et al.,

2000; Song and Haber, 2000; Stanic et al., 2003). The most

parsimonious explanation of the differences between the

present study and that of Agrawal et al. (2004) is that they

used rats that were partially denervated, thus allowing for

effects of OEC on remaining dopamine fibres in striatum,

and possibly trophic stimulation of these neurons reflected

also in increased cell counts in SN. Thus our study demon-

strates direct effects of OEC on grafted neurons only, while

Agrawal et al. noted what appear to be effects of OEC both on

grafted neurons and on remaining host dopamine neurons.

Fetal VM has also been co-grafted with a wide variety of

other tissues or cells, including peripheral nerve/Schwann

cells (van Horne et al., 1991; Collier et al., 1994), immature

astrocytes from several regions of CNS (Krobert et al., 1997;

Pierret et al., 1998), striatal tissue (Costantini et al., 1994;

Costantini and Snyder-Keller, 1997; Emgard-Mattson et al.,

1997; Sortwell et al., 1998), carotid body (Shukla et al., 2004),

Sertoli cells (Borlongan et al., 1997; Sanberg et al., 1997) or

fetal kidney (Granholm et al., 1998; Chiang et al., 2001).

Improvements were obtained in all these cases. The common

denominator behind the increase in survival and/or fibre

density and function in the different studies may be the pro-

duction of neurotrophic factors. One neurotrophic factor

that has been demonstrated to have a strong effect on dopam-

ine cell survival and morphological maturation both in vitro

and in vivo is GDNF (Tomac et al., 1995; Widenfalk et al.,

1997; Rosenblad et al., 1996; Granholm et al., 1997, Sautter

et al., 1998; Espejo et al., 2000; Tornqvist et al., 2000; Widmer

et al., 2000; Chaturvedi et al., 2003; Schaller et al., 2005). The

increase in survival rate by co-grafting VM with OEC in our

study is similar to the increase seen after treatment of VM

grafts with GDNF (Wilby et al., 1999; Tornqvist et al., 2000;

Ostenfeld et al., 2002). The GDNF treatment can also increase

TH+ intrastriatal innervation (Rosenblad et al., 1996;

Granholm et al., 1997; Tornqvist et al., 2000) although that

has not always been the case (Yurek, 1998). There is, however,

no previous study where VM grafts have been treated with

neurotrophic factors that has demonstrated a similar remark-

able increase in long-distance intrastriatal nerve fibre innerva-

tion as seen in the present study.

We observed no effect of co-grafting VM with astrocytes

from the adult olfactory nerve. Astrocytes differ in survival

and growth-promoting capacity between different regions

of the brain and between different states of maturation

(Autillo-Touati et al., 1988; Denis-Donini and Estenoz,

1988; Garcia-Abreu et al., 1995; Le Roux and Reh, 1995;

Krobert et al., 1997). In previous in vitro studies we have

observed the existence of at least two different types of

immature astrocytes within the fetal VM tissue, one type

associated with elongation of neurites and another type

stimulating neurite branching and target innervation

(Johansson and Stromberg, 2002, 2003). Since the VM grafts

already contained both these types of astrocytes, it appears

a priori unlikely that the addition of a third population of

astrocytes would have additional effects.

We conclude that the addition of OEC can markedly

enhance the effects of fetal dopamine neuron grafts in the

completely dopamine-denervated rat striatum. Of particular

significance is the fact that neurites from OEC-stimulated

grafted dopamine neurons are able to reach a much larger

host striatal territory than single grafted dopamine neurons.

Supplementary materialThe Supplementary material cited in this article is available at

Brain online.

AcknowledgementsThis work was supported by the Swedish Research Council,

AFA, NIDA, and Yen Tjing Ling Medical Foundation in

Taiwan.

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