Towards a dynamic approach of experimental parkinsonism

Roy.

NeumPsychopharmacd.

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Psych&d Copyri&t PrInted

1998. Vol. 22, pp. 0 1998 Ehwkr in the USA.

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TOWARDS A DYNAMIC APPROACH

OF EXPERIMENTAL

PARKINSONISM

ERWAN BEZARD, THOMAS BORAUD, CHRISTELLE IMBERT, SANDRA DOVER0 BERNARD BIOULAC and CHRISTIAN E. GROSS

Basal Gang, Labomtoire de Neurophysiologie, CNRS UMR 5543, Universite de Bordeaux II, Bordeaux, France.

(Final

form,September

1998)

Abstract

Bezard, Erwan, Thomas Boraud, Christelle Imbert, Sandra Dovero, Bernard Bioulac, and Christian of experimental parkinsonism. Prog. NeuroE. Gross. Towards a dynamic 1&roach Psychopharmacol. & Biol. Psychiat. 1 8 9 22. pp. 1317-1329. 0 1998 Elsevkr Science IIK.

1. In order to mimic the slow and continuous evolution of Parkinson’s disease, a chronic model of parkinsonism in monkeys and mice has been developed. 2. Seven monkeys were daily treated with low-dose of MPfP administered intravenously until their clinical scores reached a value of 30-35% of the maximal score. MPTP treatment was stopped at D15.5 i 1. 3. The evolution of clinical signs resembles Parkinson’s disease and monkeys showed rigidity, akinesia and resting tremor which is not alleviated by L-Dopa treatment. 4.90 mice were daily treated with low-dose of MPTP during 20 days. Every two days, 8 individuals processed for tyrosine-hydroxylase their mesencephalon euthanized and were immunohistochemistry. The kinetics of nigml degeneration evolves with an exponential decay. 5. This chronic model of MPfP treatment so could be of great interest for study of the dynamic physiopathological changes which occur in Parkinson’s disease.

Kevwords

: monkey, mouse, MPTP, slow evolution of disease.

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine Abbreviations : levodopa (L-Dopa), Parkinson’s disease (PD), tyrosine hydroxylase immunoreactivity (TH-IR)

(MPfP),

Introduction

The motor symptoms dopaminergic Hassler,

characteristic

of Parkinson’s disease (PD) are linked to a degeneration

neurons in the substantia

1938).

The

identification

nigm pars compacta of

a

specific

1317

neural

(Ehringer toxin,

and Homykiewicz,

of

1960;

I-methyl4phenyl-1,2,3,6-

1318

E. Bezard et aL

tetrahydropyridine

(MPTP), that induces parkinsonism

in human (Davis et al., 1979) and nonhuman

primates (Bums et al., 1983; Langston et al., 1984) has fed to the development

of valuable animal

models for this disease. The MPTP monkey model has been reported to reproduce virtually all the classic behavioural, cognitive, biochemical, al., 1986; Benazzouz

and histologic changes that occur in PD (Bankiewicz

et

et al., 1992; Bums et al., 1983; Chiueh et al., 1984; Doudet et al., 1985;

Langston et al., 1983; Langston et al., 1984; Schneider and Kovelowski,

1990; Schultz et al., 1985).

Some disparities have, however, been suggested between the MPTP models and human PD. Acute MPfP

administration

does not consistently

reproduce

all the clinical features of the disease,

for

example the typical resting tremor. None of the available animals models involve the progressive and continuous

nigral neurodegeneration

the onset and progression

of idiopathic

(McGeer et al., 1988) with an exponential

that is observed in human PD. It has been suggested that PD represents

a novel ongoing

degenerative

process

decay (Feamley and Lees, 1991). The preclinical

phase

was thought until recently to last at least twenty years (Agid, 1991; Clough, 1991; Hoehn and Yahr, 1967; Keller,

1992; Koller et al., 1991; Vingerhoets

et al., 1994) but Feamley

and Lees have

shortened this to 4.7 years (Fearnley and Lees, 1991) and Morrish et al. to 3.1 years (Morrish et al., 1996). While the lenght of the period preceeding to debate,

the duration

progression

of symptoms

disease

and

the

presymptomatic

of the symptomatic can vary enormously

symptomatic

period

the first apperance of clinical signs remains open period

has been well documented.

The rate of

(Hoehn and Yahr, 1967) but PD is not a lethal

is always

very

long.

Even

with

a relatively

short

period, PD is a long term evolutive disease and the numerous animal models at our

disposal only offer stable models of nigml lesion. Although these models are useful for the study of basal ganglia physiopathology, of neuroprotective

they are perhaps not the best adapted for assessment

of the efficacy

products and/or changes which occur during the process of nigral degeneration.

In order to dispose of such a model, the authors devised a dynamic model both for the monkey and the mouse, a daily low-dose MPTP protocol which would allow us to study the gradual progression of the disease. This report provides the evolution of motor symptoms in the monkey and the kinetics of the nigral degeneration

in the mouse.

Methods

Animals

Seven cynomolgus

monkeys (Macaca fascicularis)

weighing 3-4 kg were used in this study. The

animals were housed in individual primate cages and their care supervised in the healthcare

and maintenance

Webester male mice (Iffa-Credo, in a temperature-controlled

of non-human

primates.

by veterinarians

Ninety (90) eight-week

Saint Germain s/l’Arbresle, France) weighing 3436g

skilled

old Swisswere housed

room under a 12 h light/dark cycle with free access to food and water.

The laboratory operates under the guidelines laid down by the National Institute of Health and is reeognised

by the French Ministry of the Environment.

Surgical procedures

were performed

under

A dynamic approach general anaesthesia

(ketamine-hydrochloride

of Parktnson’s

1319

disease

40 mg/kg i.m., Panphanna,

France, and xylazine

5

mg/kg i.m., Sigma, St-Louis, USA).

Behavioural Assessment

of Monkevs

Animals’ behaviour was evaluated on a parkinsonian

monkey rating scale (Benazzouz

et al, 1995;

Bezard et al., 1997a; Bezard et al., 1997b) using videotape recordings of monkeys in their cages as well as clinical neurological

evaluation.

evaluated

animals’

levels of motor performance,

coaxing them to effect various tasks by offering appetizing

fruits. A

simultaneous

independent

During each session

and blind assessment

recording. The three observers

two examiners

was made by a third examiner

achieved a coefficient

of concordance

(Kendall’s) greater than 0.95

in their rating of all behavioum before actually beginning actual data collection. rating were discussed regularly to eliminate observer idiosyncrasy We assessed the following symptoms:

watching a video

Any differences

in

(Taylor et al., 1994).

tremor (O-3) variation in the general level of activity (O-3)

body posture (flexion of spine; O-3), vocalization

(O-2), freezing (O-2) rigidity of each arm (O-3 for

each upper limb), and arm movements

(reaching for food with each arm; O-3 for each upper limb).

Minimal score was 0 and maximum

disability

score was 25. A score 2 1.5 corresponds

to full

parkinsonism,

similarly to the stage IV of the scale of Hoen and Yahr (Hoehn and Yahr, 1%7).

Exnerimental

Protocols

Monkevs

: Assessment

was canied out twice daily, before the MPTP injection at 9 a.m. and the

second at 6 p.m. The authors first carried out a five-day assessment

of monkeys’ normal behaviour

before beginning the MPTP protocol (from D-7 to D-3). Animals were treated daily (MPTP hydrochloride,

Sigma, St-Louis, USA; 0.2 mg/kg i.v.) until

they reached a score over eight on the rating scale. Clinical symptoms then continued to develop for several days after the treatment was stopped since this time interval has been found to be sufficient for degeneration

of the tyrosine hydroxylase

immunoreactive

(TH-IR) neurones (Jackson-Lewis

al., 1995) (from DO to D21). Then animals reached a state of stable parkinsonism, maximum score over a period of seven consecutive Levodopa

(L-Dopa) testing was subsequently

days (from D22 to D29).

performed

syndrome. The dose of Mcdopar (levodopa/carbidopa, the six monkeys was 15 mg/kg and determined test

dose

of 20 mglkg,

recommended

corresponding

et

attested by a

to check the parkinsonian

nature of the

4: 1 ratio; Roche, France) required to release

between D30 and D39. The authors adopted an oral

to approximately

1.5 times

the releasing

dose

as

by the CAPIT Committee for human L-Dopa testing (Langston et al., 1992). The test

was carried out twice daily (9 a.m. and 6 p.m.) for five days (from 042 to D47). Animals were assessed just before administration water) and 45 min after.

of L-dopa (20 mg/kg in 15 ml of water) or placebo (15 ml of

1320

E. Bezard et al. &

: Ten were used as controls and received daily injections of saline for twenty days. The other

eighty were treated with daily (9:00 am) injections

of MPTP hydrochloride

(4 mg/kg, i.p.; Sigma,

St. Quentin Fallavier, France) in saline for twenty days. Every two days, eight mice were withdrawn and kept without further injections

for another

interval has been found to be sufficient midbrain

(Jackson-Lewis

et al,

seven days, before being sacrificed.

for degeneration

of the TH-IR neurones

1995). Control mice were also sacrificed

This time

in the mouse

7 days after the last

injection. eleven groups of mice : ten groups which had

We thus disposed of, at the end of the experiment,

received daily MPTP treatment for periods ranging from two to twenty days, and one control group.

Immunohistochemistrv

Since Jackson-Lewis hydroxylase

et al. (1995) have described

(TH)-immunoreactivity

a robust correlation

(IR) and the number of surviving

Nissl stained slices) from 5 days after the last MPTP injection, TH-IR of mesencephalon

in order to quantify the importance

between

the tyrosine

neurons (quantification

the authors have investigated

of the dopaminergic

of the

neuronal death

both in monkey and in mouse.

: They were sacrified at the end of experiment

Monkevs

(after D49). After ketamine anaethesia,

they were perfused with saline followed by 4% paraformaldehyde

in a phosphate buffer (pH 7.4) as

fixative. Brains were removed and postfixed for 12 h at 4°C in the same fixative. Tissue slices were then rinsed for 24 h at 4°C in 20% sucrose in Tris Buffered Saline (pH 7.4), frozen in isopentane cooled on dry ice, and cut into 30 pm frontal sections with a cryostat. They were then processed Briefly,

for visualisation

slices were incubated

of TH-IR as previously desribed (Bezard et al., 1997d).

first, in serum containing

TH-antibody

(anti-TH)

s/Seine, France) and second in goat anti-mouse IgG serum (Biosys, Compitgne, then incubated in avidin-peroxydase

complex ( Elite ABC, Biosys, Compitgne,

followed by treatment with 3,3 diaminobenzidine Fallavier, Fiance) diluted in PBS and H202. &I& : They were anaesthetised perfused intracardially

with Pentobarbital

tettahydrochloride

(Biorad,

Ivry

France). They were France). This was

(DAB, Sigma, St. Quentin

(Sanoli, Libourne, France) (30 mgkg, i.p.) and

with saline followed by 4% paraformaldehyde.

Brains were removed, post-

fixed in the same fixative for two days at 4”C, immersed in 20% sucrose in saline, frozen by slow immersion sections

in isopentane

cooled on dry ice, then stored at -80°C before sectioning.

(40 pm) encompassing

the entire mesencephalon

were collected

Cryostat cut

free floating

for each

mouse. Sections were processed for visualisation Briefly, serum containing TH-antibody

of TH-IR as previously described (Bezard et al., 1997~).

(anti-TH) (Jacques Boy, Reims, France)

and sections incubated first, overnight at 4°C and second 2 h at room temperature IgG serum

(Biosys,

Compicgne,

peroxidase-anti-peroxidase

France)

diluted

was

diluted 1:2000

in goat anti-rabbit

1:200. They were then incubated

in rabbit

complex (PAP, Dako, Tmppes, France) diluted 1500 for 2 h. This was

A dynamic approach of Parkinson’s followed

by treatment

with 3,3’diaminobenzidine

disease

tetrahydrcchlotide

132 1 (DAB, Sigma, St. Quentin

Fallavier, France) diluted in TBS and H202. TH-immunoreactivity

cell counts were performed,

as previously

using an imaging system (CCD camera, Hamamatsu) equipped

connected

described

(Bezard et al., 1997~)

up to a Macintosh II Cx computer

with an image program (image 1.31n, NIMH). The final magnification

The evolutions of the nigraI degeneration significantly

different.

was 100 times.

in the rostral, median and caudal parts of the SNc are not

Three sections corresponding

to a representative

median plane of the SNc

were so retained for each mouse. This plane is exactly in the middle of the rostro-caudal SNc (Nelson et al., 19%). For quantification, using the mouse-driven TH-immunoreactive hemisphere

the SNc of each hemisphere

axis of the

was first delimited, then,

“location” function of the software, marks were placed on each cell body of

neurones. The number of neurones per SNc was calculated three times for each

by one examiner

blind with regard to the experimental

condition,

as previously

described (Bezard et al, 1!497c).

Data Analvsis

Student’s t test was used to compare results of behaviouml assessments post-hoc analyses (Student-Newman-Keuls of immunohistochemistry

multiple comparisons

of monkeys. ANOVA with

test) was used to compare results

of mice’ mesencephalons.

Results

Monkev Model

Evolution

of Clinical Siens : Persistent

motor abnormalities

(score on clinical rating scale > 2)

appeared 11 days after MPTP treatment was started and increased gradually up to full parkinsonism (Fig. 1). Monkeys rigidity example

became

increasingly

of the limbs and decreased when reaching

bradykinetic

vocalization.

adopting

a flexed posture,

Their movements

for fruit, and there were occasional

episodes

became

with increased

less accurate,

of freezing.

for

Both postural

tremor and some resting tremor were observed. The point at which the score on the clinical rating scale reached 28 (and when MPTP treatment was consequently

stopped), was comparable for all six monkeys (15.5 days f 1.1) (Fig. 1). The total

number of injections received was therefore 15 f 1 (cumulative dose = 3.0 f 0.2 mglkg). Full parkinsonism

(beginning of stable state) was first obtained on the 22nd day of treatment (Fig.

I). The control monkey’s behaviour remained normal throughout the whole experiment

(score = 0).

1322

E. Bezard et al.

1

25

5

0

15

10

20

25

Days Fig. 1. Evolution of daily clinical scores (clinical score * standard deviation).

L-Doua Testing : No significant

difference

(~9.5)

was found between the MPfP

situation (from

D22 to D29, 18.5 f 2.1) and the MPfP+placebo

situation (from D42 to D47, 18.3 + 2.4), attesting

the failure to recover during all the experiments.

L-Dopa reversed parkinsonian

six MPWtreated

regarding tremor, flexed posture, akinesia, rigidity, eating and vocalization. the consistent between (p&001),

decrease in scores on the parkinsonian

the MPTP+placebo

situation (18.3 f 2.4) and the MPTP+L-Dopa

immunoreactive

: Macroscopic

and

microscopic

cell body density was dramatically with the control

monkey.

were noted

These were reflected in

rating scale. There was a significant difference

with scores improving 51% after the administration

Immunohistochemistry in comparison

clinical signs in all

monkeys and had no effect on the control monkey. Improvements

situation

(9.0 rt 1.8)

of L-Dopa. examination

showed

that

reduced in the SNc of MPTP-treated

Quantitative

assessment

of TH-immunoreactive

TH-

monkeys cells

revealed a loss of 90% (p < 0.0001).

Mice Model

The MPTP-treated

mice presented

no parkinsonian

motor abnormalities

reported in literature (Gerlach and Riederer, 1996; Heikkila et al., 1989).

as has been generally

1323

A dynamic approach of Parkinson’s disease No significant

differences

were found in any instance between the number of TH-IR neurones on

the left and right sides of the SNc (p > 0.5). The average determined

number

of TH-IR

neurones

was

for each mouse (i.e. average of the 6 SNc values for each animal). Mean and S.D.was

then calculated

from these average values for each group (n=lO mice for Control group and n=8

mice for the MPTP-treated The time course mesencephalic

groups).

of changes

in the percentage

of TH-IR

neurones

for each representative

plane, in relation to the cumulative dose of MPTP, is shown in Fig. 2. The ANOVA

analysis showed that there were significant differences the second injection of MPlP,

between the eleven groups (p