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Neurobiology of Disease 16 (2004) 516–526

Olfactory ensheathing cell transplantation restores functional deficits

in rat model of Parkinson’s disease: a cotransplantation approach

with fetal ventral mesencephalic cells

A.K. Agrawal,a,* S. Shukla,a R.K. Chaturvedi,a K. Seth,a N. Srivastava,b

A. Ahmad,a and P.K. Setha

aDevelopmental Toxicology Division, Industrial Toxicology Research Centre, M.G. Marg, Lucknow 226 001, IndiabSchool of Studies in Biochemistry, Jiwaji University, Gwalior, India

Received 19 August 2003; revised 22 April 2004; accepted 27 April 2004

Different strategies have been worked out to promote survival of

transplanted fetal ventral mesencephalic cells (VMCs) using trophic

and nontrophic support. Olfactory ensheathing cells (OECs) express

high level of growth factors including NGF, bFGF, GDNF, and NT3,

which are known to play important role in functional restoration or

neurodegeneration. In the present investigation, an attempt has been

made to study functional restoration in 6-hydroxydopamine (6-

OHDA)-lesioned rat model of Parkinson’s disease (PD) following

cotransplantation of VMC and OECs (cultured from olfactory bulb,

OB) in striatal region. The functional restoration was assessed using

neurobehavioral, neurochemical, and immunohistochemical approach.

At 12 weeks, post-transplantation, a significant recovery ( P < 0.001) in

D-amphetamine induced circling behavior (73%), and spontaneous

locomotor activity (SLA, 81%) was evident in cotransplanted animals

when compared with 6-OHDA-lesioned animals. A significant restora-

tion ( P < 0.001) in [3H]-spiperone binding (77%), dopamine (DA)

(82%) and 3,4-dihydroxy phenyl acetic acid (DOPAC) level (75%) was

observed in animals cotransplanted with OECs and VMC in

comparison to lesioned animals. A significantly high expression and

quantification of tyrosine hydroxylase (TH)-positive cells in cotrans-

planted animals further confirmed the supportive role of OECs in

viability of transplanted dopaminergic cells, which in turn may be

helping in functional restoration. This was further substantiated by our

observation of enhanced TH immunoreactivity and differentiation in

VMC cocultured with OECs under in vitro conditions as compared to

VMC alone cultures. The results suggest that cotransplantation of

OECs and VMC may be a better approach for functional restoration in

6-OHDA-induced rat model of Parkinson’s disease.

D 2004 Elsevier Inc. All rights reserved.

Keywords: D-Amphetamine; 6-OHDA; Tyrosine hydroxylase; OECs;

VMC; Parkinson’s disease

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

doi:10.1016/j.nbd.2004.04.014

* Corresponding author. Developmental Toxicology Division, Indus-

trial Toxicology Research Centre, PO Box 80, M.G. Marg, Lucknow 226

001, India. Fax: +91-522-2228227.

E-mail address: aka33@rediffmail.com (A.K. Agrawal).

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

Introduction

Parkinson’s disease (PD) is a chronic and progressive neu-

rodegenerative disorder characterized by selective degeneration

of mesencephalic dopaminergic neurons in substantia nigra pars

compacta (SNpc), with a subsequent loss of dopamine (DA) at

axon terminals innervating in the striatum. Dysfunction of

nigrostriatal pathway results in typical motor syndromes (Lang

and Lozano, 1998a,b). Since PD is due to striatal DA depletion,

a therapeutic approach of L-dihydroxyphenylalanine (L-DOPA)

treatment remains the most effective treatment for PD. However,

its neurotoxic and other long-term side effects limit the efficacy

of the treatment (Jenner and Brin, 1998).

In last decades, transplantation of fetal dopaminergic tissue

into the denervated striatum has been considered as an alterna-

tive approach to replenish striatal DA level and reform the

nigrostriatal pathway (Bjorklund, 1992; Nikkhah et al., 1995).

However, the clinical applicability of this therapy is limited by

numerous factors such as the controversial ethical or legal issues

raised in the use of human fetal tissue, the difficulty in obtaining

sufficient viable fetuses necessary for the surgical procedure, and

the low survival of transplanted cells (Kordower et al., 1995).

To improve fetal cell survival in transplant, several attempts

have been made using other DA-producing tissue (Lopez-Loz-

ano et al., 1999), trophic factor support (Johansson et al., 1995;

Mayer et al., 1993; Rosenblad et al., 1996; Sanberg et al., 1997;

Sinclair et al., 1996; Takayama et al., 1995; Yurek et al., 1996),

antioxidants (Dugan et al., 2001; Nakao et al., 1994), and anti-

apoptotic agents (Mytilineou et al., 1997; Schierle et al., 1999).

Olfactory ensheathing cells (OECs) derived from olfactory

bulbs (OBs) are continuously dividing cells and can grow and

multiply in culture (Ramon-Cueto and Nieto-Sampedro, 1991).

These cells have a highly malleable phenotype as a result of

coexpressing phenotypic features of both astrocytes and

Schwann cells (Doucette, 1990; Ramon-Cueto and Nieto-Sampe-

dro, 1992). OECs have also been shown to express nerve

growth factor (NGF), brain-derived neurotrophic factor (BDNF),

glial cell line-derived neurotrophic factor (GDNF), ciliary neuro-

trophic factor (CNTF), and their receptors (Woodhall et al.,

A.K. Agrawal et al. / Neurobiology of Disease 16 (2004) 516–526 517

2001). In recent studies, OECs have been shown to promote

growth and remyelination of damaged axons (Doucette, 1990;

Ramon-Cueto and Avila, 1998). Li et al. (1997) has demon-

strated that cultured OECs transplanted in electrolytic lesioned

corticospinal tract exhibited elongated growth of damaged axons

and improves neurobehavioral deficits. Recently, functional re-

covery of paraplegic rats and motor axon regeneration of spinal

cord by OECs transplantation has been demonstrated (Ramon-

Cueto et al., 2000).

The present study was aimed to evaluate efficacy of cotrans-

plantation approach of cultured OECs and fetal ventral mesen-

cephalic cell (VMC) in 6-hydroxydopamine (6-OHDA)-lesioned

rats. Functional restoration was assessed using neurobehavioral,

neurochemical, and immunohistochemical studies.

Materials and methods

Chemicals

6-OHDA-HBr, D-amphetamine, haloperidol, trypsin, DNase,

3-3V diaminobenzidine (DAB) with metal enhancer, primary

monoclonal antibodies–anti-tyrosine hydroxylase (TH) and anti-

p75NTR, secondary antibodies biotinylated peroxidase linked,

goat anti-mouse IgG-TRITC conjugate, goat anti-rabbit IgG-FITC

conjugate, and poly-L-lysine (PLL) were purchased from Sigma

Co. (USA). DMEM: F12 medium, fetal bovine serum (FBS),

Hank’s balanced salt solution (HBSS), antibiotic–antimycotic,

and sodium bicarbonate were obtained from Gibco BRL

(USA), while sodium pentobarbital was procured from Merck

(Germany). [3H]-spiperone (specific activity, 15.7 Ci/mmol) was

obtained from Amersham (UK). All the other chemicals used in

the study were of AR grade, available locally. The culture ware

used in the study was of Nunc brand (Denmark).

Animals

Adult Wistar rats of either sex (200–250 g) obtained from

Industrial Toxicology Research Centre breeding colony were used

in the study. Experimental protocol has been approved by ethical

committee of our Center. Animals were housed two/cage under a

12:12 h light–dark cycle and permitted food and water ad

libitum.

6-OHDA lesions

Rats were anesthetized with sodium pentobarbital (40 mg/kg in

0.9% saline, ip), fixed in stereotaxic apparatus (Stoelting Co.

USA), and given two stereotaxic injections of 6-OHDA (4 Ag/Al in0.2% L-ascorbate saline) unilaterally into the right ascending

nigrostriatal dopaminergic pathway using a 10 Al Hamilton sy-

ringe at following coordinates (in millimeters with respect to

bregma): (I) 3 Al at AP �4.4, L 1.2, V 7.8, (ii) 2 Al at AP

�4.0, L 0.8,V 8.0 (Paxinos and Watson, 1998). The injection rate

was 1 Al/min using auto-injector device attached with stereotaxic

apparatus, and the cannula was left in place for 5 min and slowly

retracted thereafter. Control rats were subjected to the same

protocol except that a 6-OHDA-free solution (0.9% NaCl and

0.2% ascorbic acid, ip) was injected. After 4 weeks of lesioning,

rats were challenged with D-amphetamine (5 mg/kg) to check

rotational scores for 30 min. Animals exhibiting at least 8 full

turns/min, ipsilateral to the lesioned side, were chosen further for

transplantation studies (Hashitani et al., 1998). Lesioned rats were

divided, equally, into four groups and were transplanted as per the

experimental protocol.

Fetal VMC preparation

DA-rich cell suspension was prepared from fetal ventral

mesencephalic (VM) tissue according to the modified method

of Nikkhah et al. (1994). In brief, VM tissue from 13- to 14-day-

old rat fetuses was dissected and kept in DMEM: F12 medium.

Single cell suspension was prepared by incubating the tissue in

0.1% trypsin/DMEM: F12 at 37jC for 20 min followed by

rinsing four times with 0.05% DNase/DMEM: F12. The me-

chanical dissociation of the tissue pieces was performed in

requisite volume of 0.05% DNase/DMEM: F12 by repeated

trituration followed by centrifugation at 600 rpm for 5 min,

and the pellet was resuspended in a final volume of 0.05%

DNase/DMEM: F12 to achieve approximately 125,000 cells/Al.The viability of cells was established before transplantation by

Trypan blue dye exclusion test and was found to be 95%.

Approximately 250,000 cells/2 Al DMEM: F12 media were

transplanted in striatal region of lesioned rats following the

coordinates AP �0.3, L 3.5,V 4.5.

OEC culture

OECs obtained from the glomerular layers of adult olfactory

bulb were cultured following the method of Ramon-Cueto and

Nieto-Sampedro (1992). In brief, adult Wistar rats (200 g) of

either sex, obtained from ITRC breeding colony, were sacrificed

by decapitation, and the olfactory bulbs (OBs) were dissected in

HBSS in sterile condition. After careful removal of the pia from

OB, the olfactory nerve fiber and glomerular layers (ONGL)

were dissected out. Tissue was washed twice with HBSS (Ca2+,

Mg2+ free), diced in small fragments, and incubated with 0.1%

trypsin at 37jC for 15 min. Trypsinization was stopped by

adding DMEM: F12 medium supplemented with 10% fetal

bovine serum. The cell suspension was centrifuged at 800 rpm

for 3 min, resuspended in DMEM: 12 medium with 10% serum,

and this step was repeated twice. Cell dissociation was achieved

by passing the tissue 15–20 times through a fire polished

siliconized Pasteur pipette. Viable cells, in the form of single

cell suspension, were then plated in culture flasks precoated with

PLL at a density of 6.5�106 per flask and maintained in

DMEM: F12 supplemented with 2 mM glutamine, 10,000

U/ml penicillin, 10,000 Ag/ml streptomycin, and 25 Ag/ml

amphoterecin B. Cultures were maintained in 5% CO2 atmo-

sphere at 37jC and medium was changed after every 2 days.

For OEC alone or cotransplantation, after 14 days of culture, the

cells were trypsinized and centrifuged for 10 min at 1000 � g

after being neutralized with DMEM: F12 supplemented with

10% FBS. Cells were resuspended in DMEM: F12 at a density

of 1,25,000 cells/Al and used for transplantation study.

Coculture of VMC and OEC

Tissue for preparation of embryonic mesencephalic cultures was

obtained from embryonic day 15 rats. Single cell suspension was

made by repeated pipetting with fire polished pipette as described

earlier (Nikkhah et al., 1994). Cells were plated on PLL-coated

A.K. Agrawal et al. / Neurobiology of Disease 16 (2004) 516–526518

coverslips in DMEM: F12 medium or conditioned DMEM: F12

medium of OEC cultures. A set of coverslips containing 3-day-old

VMC culture maintained in conditioned mediumwas then removed,

washed gently with medium, and were placed in a culture dish above

an established (14-day-old) culture of OECs. Cells were allowed to

grow in coculture environment. After 11 days, the coverslips with

cultured VMC grown with or without OEC were removed and

processed for TH positivity (immunocytochemically) and morpho-

logical assessment.

Transplantation procedure

Lesioned rats were divided into four groups and cells were

transplanted in striatal region 5 weeks post-lesioning following

the coordinates AP �0.3, L 3.5,V 4.5. For cotransplantation, a

homogenous suspension of cultured OECs and VMC was made

by mixing equal number of each cell type (Roy et al., 1999).

DMEM: F12 (2 Al) was injected in control animals using same

coordinates, which served as sham-operated control.

Group I: Received 250,000 cells of VMCGroup II: Received 250,000 cells of OECs

Group III: Received 250,000 cells each of VMC and OECs

Group IV: Lesioned group

After transplantation, rats were housed two per cage. Twelve

weeks post-transplantation, rats were subjected to neurobehavioral,

neurochemical, and immunohistochemical evaluation.

Neurobehavioral studies

D-Amphetamine-induced circling behavior

Five rats from each group (sham, lesioned, all transplanted

groups) were assessed for circling behavior after injecting 5 mg/kg

D-amphetamine intraperitoneally following the method of Wenning

et al. (1996). Rotational behavior was recorded after 30 min of

injection and assessed for a period of 30 min and expressed as

rotations/30 min.

Spontaneous locomotor activity

Spontaneous locomotor activity (SLA, as distance traveled) was

monitored in a computerized Optovarimex (Columbus Instruments,

Ohio, USA) system following the method of Ali et al. (1990). Rats

were individually placed in the test apparatus, acclimatized for a

period of 5 min, and their activity scores were recorded for 10 min

sessions. Interruptions in the photo beams positioned in parallel,

inside the chamber, resulted in an activity count, which was

recorded for data analysis.

Neurochemical studies

DA-D2 receptor binding assay

DA-D2 receptor binding assay was carried out in striatal

region of sham, lesioned, and all transplanted groups. Striatal

synaptic membrane preparation was made following the method

of Agrawal et al. (1981). In brief, crude synaptic membrane

fraction was obtained by homogenizing the tissue in 19 volume

of prechilled 0.32 M sucrose followed by centrifugation at

50,000 � g for 10 min. The resultant pellet was rehomogenized

in 5 mM Tris–HCl, pH 7.4, in same volume and recentrifuged

for 10 min at 4jC. The pellet was finally suspended in 40 mM

Tris–HCl, pH 7.4, and stored at �20jC until assay. The binding

incubation was carried out, in triplicate, at 37jC for 15 min

using 100 Al (250–300 Ag protein) synaptic membrane fraction

with 1 nM [3H]-spiperone as specific ligand for DA-D2 receptor

in 40 mM Tris–HCl buffer, pH 7.4. To determine nonspecific

binding, parallel assay, in triplicate, using high concentration

(1 AM) of unlabelled haloperidol (DA-receptor antagonist) was

done. After 15 min of incubation at 37jC, the reaction was

terminated by cooling in ice and the reaction mixture was

filtered through glass microfiber filter (GF/C) under vacuum

using Brandel cell Harvester (USA). The filter papers were

washed twice with same buffer, dried, and radioactivity counted

in LKB Rack h Liquid Scintillation counter (Packard Instru-

ment, Germany) having an efficiency of 50% for tritium.

Specific binding was calculated by subtracting nonspecific

binding from total binding obtained in absence of haloperidol.

The results were expressed in terms of pmol of ligand bound/g

protein. Protein was estimated by the method of Lowry et al.

(1951). Scatchard (1949) analysis was performed using varying

concentrations (0.1–10 nM) of [3H]-Spiperone. Affinity (Kd)

and the maximum number of binding sites (Bmax) were

calculated using linear regression analysis.

DA and DOPAC levels

The striatal tissue level of dopamine (DA) and its metabolite

3,4-dihydroxy phenyl acetic acid (DOPAC) were measured in

the experimental animals following the method of Kim et al.

(1987) using HPLC (Merck) with the help of electrochemical

detector (Merck, L-3500 A). The results are expressed in terms

of pg DA and DOPAC/g wet weight of tissue.

Immunohistochemical or immunocytochemical studies

Rats from each group were perfused with 150 ml of 0.1 M,

pH 7.2, phosphate-buffered saline (PBS) followed by 250 ml of

ice-cold 4% paraformaldehyde in PBS for fixation of tissues.

Brains were removed and postfixed in the same fixative over-

night, followed by cryopreservation in 10%, 20%, and 30% (W/

V) sucrose in PBS. Serial coronal sections were cut on a freezing

microtome (Slee Mainz Co., Germany) at 20-Am thickness. For

immunocytochemistry, the cultured cells were fixed in 4% para-

formaldehyde in PBS. These tissue sections and cell cultures were

then incubated for 48 h in respective primary antibodies (anti-TH

antibody, anti-p75NTR, 1:500). After removing the primary

antibody, sections or cultures were washed three times with

PBS and incubated in secondary antibody biotinylated peroxidase

linked or conjugated to FITC/TRITC (1: 200) for 2 h at room

temperature followed by three washes with PBS. Color was

developed for peroxidase-linked antibody with 3-3V-diaminoben-

zidine as chromogen. Sections were transferred onto gelatinized

glass slides, dehydrated, cleared, mounted in DPX, cover slipped,

and then visualized under microscope. The fluorescent labeled

sections were mounted in semipermanent mount (glycerol + PBS)

and were visualized under fluorescent microscope (Leica, Ger-

many) using appropriate filters.

Image analysis

The density of TH-immunoreactive (IR) fibers in striatum

region and total number of TH–IR neurons in the substantia

nigra pars compacta (SNpc) was determined using a computer-

Table 1

Circling behavior and spontaneous locomotor activity in sham, 6-OHDA

lesioned, OEC transplanted, VMC transplanted, and VMC + OEC

cotransplanted rats 12 weeks post-transplantation

Treatment Amphetamine

(5 mg/kg, ip)-induced

rotations

(number of

rotations/30 min)

Spontaneous

locomotor activity

(distance traveled

in cm/10 min)

Sham 20 F 4.13 1598 F 37.36

Lesioned 241 F 35.20***a 1139 F 54.73***a

OEC transplanted 175 F 13.33*b 1257 F 14.91*b

VMC transplanted 149 F 19.48**b 1334 F 13.72**b

Cotransplanted 79 F 11.79***b,**c 1513 F 45.98***b, **c

For circling behavior, rats were challenged with 5 mg/kg b.wt, ip, D-

amphetamine, and ipsilateral rotations were counted for 30 min. In SLA,

locomotor activity was observed for 10 min. Values represent mean F SE

of five rats. a = vs. sham, b = vs. lesion, c = vs. VMC transplant. df = (4,

24), f value = 18.71 for amphetamine-induced rotations and 25.26 for SLA.

*One-way ANOVA P < 0.05.

**One-way ANOVA P < 0.01.

***One-way ANOVA P < 0.001.

A.K. Agrawal et al. / Neurobiology of Disease 16 (2004) 516–526 519

ized image analysis system (Leica Qwin 500 image analysis

software) as described by Shingo et al. (2002). The unbiased

stereological method was applied, where a person unknown to

the experimental design carried out the image analysis. Com-

puterized analysis enabled the percent area of a selected field

that was occupied by TH–IR fibers to be assessed. This area

was expressed as Am2 per total field view (300 � 300 Am,

90,000 Am2). The density of TH–IR fibers was measured in the

striatum of the transplanted or lesioned and intact contralateral

side at the level of bregma +1.0, +0.6, +0.2 (Paxinos and

Watson, 1998). The total number of TH–IR neurons in the

SNpc was counted on both sides at three levels: �5.2, �5.5,

and �5.7 mm with respect to the bregma (Paxinos and Watson,

1998), as previously reported by Kearns and Gash (1995).

Analyzed values obtained in the experimental side were

expressed as a percentage of those present on the intact

contralateral side. The data obtained were then averaged for

statistical analysis.

Statistical analysis

Mean significant difference in the treatment groups was

determined using one-way analysis of variance (ANOVA).

Before this, homogeneity of variance between the transplanted

groups was ascertained. Further, the individual treatment be-

tween the two groups was assessed by comparison of least

significant differences taking t values for error df at the 5%

level of significance. Values of P < 0.05 were considered to be

statistically significant. For cell counting and fibers density,

intergroup comparisons were performed with ANOVA.

Results

General observation

No significant change in the body weight was observed

between animals of lesioned and transplanted group when com-

pared to sham, and all the animals have been included in the study.

Neurobehavioral studies

D-Amphetamine-induced circling behavior and SLA

To understand the extent of neurodegeneration caused by 6-

OHDA and the efficacy of transplantation in ameliorating behav-

ioral deficits, we have studied neurobehavioral changes. Agonist-

induced stereotypy was monitored by measuring unilateral circling

behavior, whereas SLAwas quantified as distance traveled. Results

of behavioral study are summarized in Table 1. A significant

increase (P < 0.001) in circling behavior was observed in 6-

OHDA-lesioned group when compared with sham-operated animals

12 weeks post-transplantation. Animals transplanted with OECs or

VMC exhibited attenuation in circling behavior by 30% (P < 0.05)

or 42% (P < 0.01), respectively. However, rats cotransplanted with

OECs and VMC exhibited a restoration of 73% (P < 0.001) in

comparison to lesioned rats (df = 4,24 and f value = 18.71).

A significant decrease (P < 0.001) in SLA was observed in 6-

OHDA-lesioned group when compared with sham, which was

restored by 26% (P < 0.05) and 43% (P < 0.01) in OECs and

VMC transplanted groups, respectively, after 12 weeks post-

transplantation. However, cotransplanted groups exhibited attenu-

ation in motor activity to an extent of 81% (P < 0.001) when

compared with lesioned rats (df = 4,24 and f value = 25.26).

Neurochemical studies

Dopaminergic degeneration following 6-OHDA lesioning

results in neurochemical changes such as a decrease in DA content

in addition to the reduced immunoreactivity against the marker

enzyme TH. Further, to correlate neurobehavioral changes and their

restoration with neurochemical alterations, [3H]-spiperone binding

reflecting specifically the effect on DA-D2 receptors and estimation

of level of DA and its metabolite DOPAC was done in the striatum

region.

DA-D2 receptor binding

Results of DA-D2 receptor binding are summarized in Table 2.

The results revealed significant increase (P < 0.001) in DA-D2

receptor binding in 6-OHDA-lesioned rats as compared to sham.

VMC transplantation, alone, was found to attenuate dopamine

receptor binding to an extent of 45% (P < 0.01) when compared

with lesioned group. Restoration was more pronounced in animals

receiving cotransplantation of both VMC and OEC (77%, P <

0.001). OEC transplantation, alone, also showed marked restora-

tion (33%, P < 0.05) in receptor binding (df = 4,24 and f value =

12.74).

DA and DOPAC levels

The results are summarized in Fig. 1. A significant decrease in

DA and DOPAC level was observed in striatal region of 6-OHDA-

lesioned rats (P < 0.001) compared to sham, indicating significant

loss in DA neurons in lesioned animals. VMC transplantation

restored the DA and DOPAC levels by 31% and 40% (P < 0.01),

respectively, whereas OEC transplantation showed restoration in

these levels by 21% and 29% (P < 0.05) when compared with

lesioned group. Cotransplanted rats exhibited more pronounced

and significant restoration in these levels (82% and 75%, P <

0.001) in comparison to lesioned rats indicating functional viability

of DA neurons post-transplantation (df = 4,24, f value = 39.44 for

dopamine level and 18.47 for DOPAC level).

Table 2

[3H] Spiperone DA-D2 receptor binding in striatal synaptic membranes of sham, 6-OHDA lesioned, VMC transplanted, OEC transplanted, and VMC + OEC

cotransplanted rats 12 weeks post-transplantation

Treatment groups analysis [3H]-spiperone binding Scatchard

(pmol bound/g protein) Kd (nM) Bmax (pmol bound/g protein)

Sham 557 F 34.57 0.78 852 F 57.19

6-OHDA lesioned 880 F 50.44***a 0.47 1299 F 68.23**a

VMC transplanted 735 F 27.02**b 0.66 956 F 65.20**b

OEC transplanted 775 F 38.07*b 0.70 1100 F 61.11*b

VMC + OEC transplanted 630 F 17.36***b, **c 0.77 817 F 45.12***b

Values represent mean F SE of five rats. Kd and Bmax values are mean from three separate sets of Scatchard analysis. a = vs. sham, b = vs. lesion, c = vs.

VMC transplant. df = (4, 24), f value = 12.74.

*One-way ANOVA P < 0.05.

**One-way ANOVA P < 0.01.

A.K. Agrawal et al. / Neurobiology of Disease 16 (2004) 516–526520

Immunohistochemistry

The functional viability of dopaminergic neurons at the trans-

planted site was further assessed by mapping the rate-limiting

enzyme, TH, for DA-biosynthesis using monoclonal antibody

against TH. Animals with intrastriatal grafts had healthy grafts

with numerous cell bodies and fibers within the graft and fiber

outgrowth into the surrounding host striatum. Viable intrastriatal

grafts were present and demonstrable in all test groups. Numerous

TH-immunoreactive cell bodies were seen mainly in the periphery

of the grafts. Dense TH-immunoreactivity was present within the

graft and extended to variable distances into the host striatum (Fig.

2). In 6-OHDA-lesioned rats, expression of TH was significantly

less (Fig. 2b) as compared to sham group (Fig. 2a). VMC and OEC

***One-way ANOVA P < 0.001.

Fig. 1. Dopamine and its metabolite (DOPAC) levels in striatal regions of sham, 6

cotransplanted rats 12 weeks post-transplantation. Values represent mean F SE of

sham, b = vs. lesion, c = vs. VMC transplant. df = (4, 24), f value = 39.44 for d

transplantation, alone, indicated a significant improvement in TH

expression (Figs. 2c and d), when compared to lesioned animals. In

Fig. 2c, TH-positive cell bodies derived from transplanted VMC

are visible. In Fig. 2d, OEC transplantation, alone, can be seen to

enhance the TH-positive dendritic fibers of residual dopaminergic

neurons of host striatum originating from substantia nigra. A

significantly higher expression of TH was observed in cotrans-

planted rats (Figs. 2e and f), as compared to OEC and VMC

transplanted group where TH-positive cell bodies, neurite exten-

sion, and fiber innervation are visible. The higher TH expression in

the cotransplanted animals further supports the enhanced function-

ally viable transplanted fetal VMC.

To quantify TH expression, image analysis was performed in

TH-positive sections. The results have been expressed in terms of

-OHDA lesioned, OEC transplanted, VMC transplanted, and VMC + OEC

five rats. ***P < 0.001, **P < 0.01, *P < 0.05 One-way ANOVA a = vs.

opamine level and 18.47 for DOPAC level.

Fig. 2. Photomicrographs of striatal sections illustrating TH (DAB)-immunoreactive cells 12 weeks post-transplantation. Lesioned rats (b) have shown

diminished TH positivity as compared to sham (a). VMC alone (c) and OEC alone graft (d) have shown considerable TH-immunopositive cells in comparison

to lesioned rats (b). Higher magnification (f) of cotransplanted rat striatum (e) has shown TH-positive cell bodies, neurites extension, and fibers innervation.

The intensity was more in the grafted area and the area adjacent to it. Maximum TH immunoreactivity was observed in cografted animals where some

migratory cells could also be visualized. Arrowheads indicate immunoreactivity for TH. Scale bar = (a–b) 300 Am, (c–e) 150 Am, and (f) 50 Am.

A.K. Agrawal et al. / Neurobiology of Disease 16 (2004) 516–526 521

% TH–IR fibers density in striatum and TH–IR neurons counts

in SNpc. The results are summarized in Fig. 3. It is evident from

the results that number of TH-positive cells as well as the density

of TH-positive fibers is significantly (P < 0.001) high in cotrans-

planted animals as compared to VMC/OEC alone transplanted

animals. It can be attributed to the neurotrophic support provided

by OEC, on one hand enhancing the survival of transplanted VMC

while simultaneously protecting the residual striatal dopaminergic

fibers, originating from host substantia nigra. The number of TH–

IR neurons in grafted rats in the ipsilateral side was enumerated as

a percentage of the TH–IR neurons in the intact contralateral side.

Twelve weeks post-transplantation, the number of TH–IR neurons

in the cotransplanted animals (70 F 7.9%, P < 0.001) was

significantly greater than those receiving VMC (26 F 5.2%, P <

0.05) or OEC alone (50 F 7.1%).

Graft analysis

Immunohistochemical analysis of brains of rats receiving VMC

graft or VMC and OEC cografts, 12 weeks post-grafting, was

performed to confirm the presence of functional transplanted cells.

TH (TRITC) immunohistochemistry demonstrated surviving do-

paminergic neurons (Figs. 4a and c), while p75NTR (FITC)

positivity (Fig. 4b) confirmed the presence of viable OECs. Fig.

4d shows TH (TRITC) positivity in OEC alone graft in striatal

region, where the TH-positive fibers are visible, which have

probably regenerated from endogenous residual dopaminergic

neurons. However, no TH-positive cell bodies could be seen in

Fig. 4d as transplanted OEC does not exhibit any TH positivity.

In vitro studies

In vitro coculture study provided additional evidence of OEC

efficacy in enhancing growth and functionality of VMC cells. A

qualitative analysis showed that TH-positive neurons cocultured

with OEC (Fig. 5a) had more pronounced neurite outgrowth than

VMC alone culture (Fig. 5b).

Discussion

The pathological changes in PD are relatively isolated and

involve specific degeneration of the dopaminergic neurons. Thus,

several research groups have employed different strategies to

ascertain the feasibility of replacing dying cells with an alterna-

tive vital population, including adrenal medullary cells (Freed et

al., 1990), myoblasts (Patridge and Davies, 1995), monocytes

(Ling and Wong, 1993), progenitor cells (Lundberg et al.,

1996a,b), and genetically engineered cells (Ono et al., 1997) that

produce DA. Transplantation of cells from different origins offers

an exciting alternative to the limitation of the existing pharma-

cological approaches (Lindvall and Bjorklund, 1989; Nikkhah et

al., 1994).

Though the major pathological changes in PD involve degene-

ration of dopaminergic cells of substantia nigra, the possible

contribution of altered glial function in manifestation of PD has

also been suggested (Chao et al., 1996a; Hirsch et al., 1998).

Recently, morphological evidences of reactive gliosis or glial

dysfunction following MPTP/6-OHDA injections have been pre-

sented (Chen et al., 2002; Kevin et al., 1999). An indirect support

of glial involvement was also provided by the studies of Sullivan et

al. (1998) where apart from other neurotrophic factors, GDNF was

shown to be very effective in protecting dying DA neurons.

In the present investigation, an attempt was made to explore the

feasibility, for the first time, of cotransplantation approach, using

fetal VMC as source of DA neurons along with cultured OECs, a

specialized glial cell with dual characteristics of CNS and PNS

(astrocytes and Schwann cell), to understand the possible role of

Fig. 3. Effect of OEC, VMC, and VMC +OEC transplantation on the (a) density of TH–IR fibers in striatum and (b) TH–IR neurons count in SNpc in 6-OHDA-

lesioned animals (ratio lesioned– intact side, transplanted site– intact side). Animals cotransplanted with VMC and OEC showed significant increase in TH–IR

fibers density and TH–IR neurons count as compared to VMC or OEC alone transplanted groups. One-way ANOVA ***P <0.001, **P <0.01, *P <0.05. a = vs.

lesioned, b = vs. VMC transplanted.

A.K. Agrawal et al. / Neurobiology of Disease 16 (2004) 516–526522

OEC for glial support in long-term functional viability and better

survival of fetal VMC in 6-OHDA-lesioned rat model of PD.

We observed a marked increase in D-amphetamine-induced

rotational behavior and a decrease in SLA activity in 6-OHDA-

lesioned rats. These observations are in agreement with the

previous reports, which conclude that symptoms of altered motor

functioning possibly result from reduced striatal, DA levels or

specific DA cell death (Zigmond et al., 1984). 6-OHDA-lesioned

Fig. 4. Photomicrographs showing functionally viable VMC and OEC cells in cograft/VMC alone graft following 12 weeks post-transplantation. TH (TRITC)

immunopositive VMC cells in cograft (a) appeared to be more differentiated then VMC cells of VMC alone graft (c). Presence of OEC (b) was represented by

intense immunolabeling for p75NTR (FITC). d shows TH (TRITC) positivity in OEC alone graft, where the TH-positive fibers derived from residual

endogenous dopaminergic neurons are visible. Arrowhead indicates immunoreactive cells. Scale bar = 20 Am.

A.K. Agrawal et al. / Neurobiology of Disease 16 (2004) 516–526 523

animals when grafted with VMC showed a significant improve-

ment in motor deficit, which could be possibly due to upregulation

of DA by VMC grafts. It is relevant to note that fetal VMC is rich

in dopaminergic neurons, and in recent years, has emerged as a

potential or ready source of DA for neuronal replacement strategies

(Nikkhah et al., 1995). However, a major constraint is its low

survival following grafting. The low survival of VMC could be due

to initial surgical trauma and conditions prevailing at the time of

surgery such as altered microenvironment of lesioned host brain,

leading to their gradual degeneration (Kordower et al., 1998). To

overcome this limitation, protective strategies have been proposed

such as supplementation of growth factors, MAO inhibitors, and

antioxidants. However, biophysical and practical considerations

present obstacle for the desired and continuous delivery of neuro-

trophic factors to CNS neurons. For continuous supplementation of

neurotrophic factors cultured, neuroprecursor cells and polymer-

encapsulated cells genetically modified to secrete growth factors

Fig. 5. Photomicrographs showing TH (DAB) immunoreactive staining for DA

increased number and length of fibers in the neurons of cocultured group as com

have been recently tested (Ostenfeld et al., 2002; Shingo et al.,

2002). Lack of such cells to provide multiple trophic factor

support, essential for long-term restoration, remains a major

limitation (Aszmann et al., 2002). OECs are source of multiple

growth factors and it has been reported that combination of more

than one growth factor, such as GDNF and BDNF together, is more

effective in survival of DA neurons (Erickson et al., 2001; Sautter

et al., 1998a,b). NGF, the other growth factor secreted by OECs,

has also been reported to enhance the number of DA neurons in

lesioned nigral region (Melchior et al., 2003).

Ostenfeld et al. (2002) have shown that cultured neurospheres

increase fiber outgrowth of primary embryonic dopamine neurons

in coculture, and when these neurospheres were cografted with

VMC, they significantly increased the initial survival of dopamine

graft in 6-OHDA-lesioned rats. However, no long-term behavioral

recovery could be obtained at 6-week post-lesioning and immu-

nohistopathological studies confirmed gradual death of grafted

neurons in VMC cultured alone (a) or cocultured with OEC (b). Note the

pared to VMC alone culture. Scale bar = 20 Am.

A.K. Agrawal et al. / Neurobiology of Disease 16 (2004) 516–526524

neurons. Contrary to these observations, our study clearly dem-

onstrated persistent behavioral recovery at 12 weeks post-grafting;

maximum in OEC + VMC cografted animals, probably indicating

an increased number of functional neurons. It could be hypoth-

esized that OECs, being rich in growth factors, render long-term

or multiple trophic support to VMC. Not only this, an attenuation

of behavioral deficits by OEC transplantation, per se, in this study

further implicates its trophic support to host DA neurons resulting

in improved functionality and regrowth. It has been shown that

glial cells actively communicate with neurons, where even

neuronal dopaminergic sprouting was evident in response (Ald-

skogius and Kozlova, 1998; Kim et al., 1994; Kimelberg, 1995).

An indirect support to such an explanation was provided by

studies of Connor et al. (2001), who stated that injection of

GDNF, before 6-OHDA lesioning, imparts protection and func-

tional recovery in animals. Our results exhibiting increased level

of DA and DOPAC in VMC + OEC cografted rats, compared to

VMC or OEC alone transplanted animals, is consistent with the

above findings where also the restoration was more marked in

cografted animals.

TH immunopositivity is downregulated following 6-OHDA

lesioning in animals, due to loss of DA cells and their decreased

functionality. The most striking support of significant recovery in

OEC and VMC cotransplanted group is evident by increased TH-

immunopositive fibers density in striatum in the order of OEC +

VMC > VMC > OEC transplanted animals. The increase in

number of TH–IR neurons in SNpc of transplanted animals further

suggests the existence of functional graft, and it can be speculated

that the retrograde axonal transport of neurotrophic factors such as

GDNF and BDNF secreted by OEC and other dopaminotrophic

factors secreted by the embryonic VMC or residual dopamine

neurons might have lent contributory effect to this recovery (Ehlers

et al., 1995; Kearns and Gash, 1995; Mufson et al., 1999; Tomac et

al., 1995). Further, the neurotrophins secreted by OEC could

possibly have helped in the recovery by reaching to the target area

(SNpc) through retrograde vesicular transport by binding to trkA/

ret receptor complex (Ehlers et al., 1995). Transplanted VMC

contributes to enhance TH positivity themselves directly, while

OEC, being glial in nature, does not show any TH positivity.

Further, a support by OEC can be explained by the possibility that

OEC, being rich in trophic factors (Woodhall et al., 2001),

provided trophic support to transplanted VMC on one hand,

whereas increasing resprouting and elongation of residual DA cells

of host striatum originating from substantia nigra. This was

consistent with the reports showing that growth factor support

through genetically modified cells enhances the survival of dopa-

minergic neurons or cells (Chaturvedi et al., 2003; Sautter et al.,

1998a,b; Wang et al., 2003). Such a combined and coordinated

action might have helped in cell-to-cell communication and neu-

ronal path finding (Oland and Tolbert, 2002). Substantial evidence

to this was provided by our in vitro studies on coculture of VMC

and OEC where an enhanced survival and neurite outgrowth was

observed in TH-positive neurons. These observations are consis-

tent with the reports of trophic support provided by the cotrans-

plantation or coculturing of fetal kidney cells or Sertoli cells with

VMC leading to increased survival and functioning of VMC

(Chiang et al., 2001; Granholm et al., 1998; Sanberg et al.,

1997). It was further strengthened by the studies of Thompson et

al. (2000), where formation of glial bridges by OECs at the

transplanted site within 48 h post-transplantation was reported.

Further, an increased transcription of GDNF following transplan-

tation of SVG and SVG-TH a glial cell line has also been reported

(Yadid et al., 1999).

An increase in DA-D2 receptor binding even in the conditions

of cell loss following 6-OHDA lesioning has been reported to

represent a classical phenomenon of denervated supersensitivity.

Such an increase was considered to be a response offered by

residual striatal dopaminergic neurons or post-synaptic cells to

mitigate the initial disturbances in striatal loops. The decreased

DA-receptor binding in rats receiving VMC + OEC and VMC

graft, 12 weeks post-grafting, compared to 6-OHDA-lesioned

animals, further suggests the increased number of functional viable

neurons due to VMC survival. However, relatively significant

recovery in cotransplanted group confirms the increased survival

of transplanted cells. This was further supported by the significant

attenuation in binding as observed also in OEC alone group.

Cotransplantation of VMC and OEC in 6-OHDA-lesioned rats

suggests better approach toward functional restoration over VMC

or OEC transplantation.

Conclusion

The results of OECs cotransplantation in 6-OHDA-lesioned

animals suggest a significant functional restoration in cotrans-

planted group as compared to individual VMC or OEC trans-

planted group, although the use of OECs in cotransplantation may

help in long-term functional restoration in clinical cases of PD.

This work should stimulate additional preclinical and clinical

studied directed at characterizing the transplanted OECs at a

cellular or molecular level overtime. Also, it is necessary to rule

out possible harmful outcomes caused by introducing apparent

‘‘trophic factories’’ into the striatum.

Acknowledgments

We are thankful to Dr. M.M. Ali for helping in neurobehavioral

studies and Mr. N. Mathur for statistical analysis. This work was

supported by grants from the Indian Council of Medical Research

(ICMR) New Delhi. S. Shukla and R.K. Chaturvedi is recipient of

senior research fellowship (SRF) from CSIR, New Delhi, India. K.

Seth is a recipient of Woman Scientist award from DST, New

Delhi. Technical assistance of Mr. S.K. Shukla and Mr. Kailash

Chandra is acknowledged. ITRC MS communication No-178.

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