probenecid treatment

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Neuroscience Vol. 106, No. 3, pp. 589^601, 2001 ß 2001 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522 / 01 $20.00+0.00

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MOUSE MODEL OF PARKINSONISM: A COMPARISON BETWEEN SUBACUTE MPTP AND CHRONIC MPTP/PROBENECID TREATMENT E. PETROSKE,a G. E. MEREDITH,b S. CALLEN,b S. TOTTERDELLc and Y.-S. LAUa * a

b

Division of Pharmacology, School of Pharmacy, University of Missouri-Kansas City, 2411 Holmes Street, M3-111, Kansas City, MO 64108, USA

Department of Basic Medical Science, School of Medicine, University of Missouri-Kansas City, 2411 Holmes Street, Kansas City, MO 64108, USA c

Department of Pharmacology, University of Oxford, Oxford OX1 3QT, UK

Abstractö1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is widely used to induce an animal model of Parkinsonism. The conventional mouse model, which usually involves acute or subacute injections of MPTP, results in a signi¢cant but reversible loss of dopaminergic functions. We have developed an alternative mouse model, in which co-administration of MPTP with probenecid results in the chronic loss of striatal dopamine for at least 6 months after cessation of treatment. In the present study, we compare the neurochemical, morphological and behavioral changes that occur in this alternative, chronic model with those in the conventional, subacute model. In the chronic model, we demonstrate an almost 80% loss of striatal dopamine and dopamine uptake 6 months after withdrawal from treatment. The neurochemical signs match unbiased stereological measures that demonstrate gradual loss of substantia nigra neurons. Rotarod performance further substantiates these ¢ndings by showing a progressive decline in motor performance. Based on the comparisons made in this study in mice, the chronic MPTP/probenecid model shows considerable improvements over the conventional, subacute MPTP model. The sustained alterations in the nigrostriatal pathway resemble the cardinal signs of human Parkinson's disease and suggest that this chronic mouse model is potentially useful to study the pathophysiology and mechanisms of Parkinsonism. It should also prove useful for the development of neuroprotection strategies. ß 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: dopamine, rotarod, stereology, striatum, substantia nigra, tyrosine hydroxylase.

model would allow further testing of mechanisms underlying the disease process and might lead to the discovery of novel, neuroprotective measures for slowing or arresting the progressive deterioration of the motor performance in PD patients. The induction of human Parkinsonism by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has produced new hypotheses related to the etiology and pathogenesis of PD. MPTP neurotoxicity in humans is clinically indistinguishable from idiopathic PD (Davis et al., 1979; Langston et al., 1983) and the toxic e¡ect in humans can be reproduced in primates with few di¡erences (Gerlach and Riederer, 1996; Mizuno et al., 1998). Nevertheless, while MPTP toxicity in humans is irreversible (Ballard et al., 1985; Burns et al., 1985), in non-human primates, the symptoms spontaneously reverse after about a month (Eidelberg et al., 1986). This is signi¢cant since conducting exploratory, preclinical studies in primates is not considered practical. In order to establish a rodent model that closely resembles PD, it is crucial that it exhibits as many of the phenotypic features of the disease as possible. These should include (1) at least 70^80% persistent depletion of striatal DA; (2) pronounced reduction of striatal sites for DA uptake; (3) signi¢cant (50^60%) loss of substantia nigral cells; (4) marked de¢ciencies in the ani-

Parkinson's disease (PD) is a progressive, age-associated, neurodegenerative disease characterized by bradykinesia, resting tremor, rigidity and gait disturbance. Although its etiology is still unknown, it is clear that PD is the consequence of substantial loss of nigral neurons and depletion of the transmitter, dopamine (DA). However, the clinical symptoms do not fully develop until there is a loss of 70^80% striatal nerve terminals and of 50^60% substantia nigra pars compacta cells (Bernheimer et al., 1973; Agid, 1991). Current pharmacological therapy with L-DOPA is limited to treating the symptomatology but does not alter the course of the underlying disease. Therefore, it would be extremely valuable to develop a long-term animal model that mimics the symptoms exhibited by PD patients. Availability of such an animal

*Corresponding author. Tel.: +1-816-235-1798; fax: +1-816-2351776. E-mail address: [email protected] (Y.-S. Lau). Abbreviations : AUC, area under the curve; DA, dopamine ; DMSO, dimethyl sulfoxide; EDTA, ethylenediaminetetra-acetate; MPP‡ , 1-methyl-4-phenylpyridinium; MPTP, 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine; PB, phosphate bu¡er; PBS, phosphate-bu¡ered saline; PD, Parkinson's disease; r.p.m., revolutions per minute ; TH, tyrosine hydroxylase ; TH-ir, tyrosine hydroxylase immunoreactive; VTA, ventral tegmental area. 589

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mal's motor performance; (5) appearance of inclusion bodies in nigral neurons. Induction of Parkinsonism by MPTP in rodents has generated a wealth of neurochemical, pharmacological and anatomical ¢ndings. Rats are generally resistant to MPTP neurotoxicity (Heikkila et al., 1984; Lau and Fung, 1986; Zuddas et al., 1994) but mice, such as the C57BL strain, are susceptible (Heikkila et al., 1984; Lau and Fung, 1986; Hamre et al., 1999). However, in order to deplete striatal DA in mice, large doses of MPTP and frequent injections are required (Sonsalla and Heikkila, 1986). Attempts to improve the mouse model include the use of more potent neurotoxic analogs of MPTP (Fuller et al., 1987; Youngster et al., 1987; Ikeda et al., 1992) and the use of older mice (Jarvis and Wagner, 1985; Ricaurte et al., 1987; Saitoh et al., 1987; Ali et al., 1993). Although the magnitude of striatal DA depletion can be increased under some circumstances, progressive and persistent DA loss over a long period has yet to be demonstrated. It is well established that MPTP in rodents, following its peripheral administration, is rapidly excreted through the kidney (Lau et al., 1988). After reaching the CNS, this toxin and its active metabolite, 1-methyl-4-phenylpyridinium (MPP‡ ), are quickly cleared from the brain (Johannessen et al., 1985). Hence approaches have been adopted to enhance MPTP neurotoxicity by using agents that inhibit the central clearance of MPP‡ and/or the renal excretion of MPTP. When MPTP is co-administered with diethyldithiocarbamate (Corsini et al., 1985; Irwin et al., 1987) or acetaldehyde (Zuddas et al., 1989), which tend to reduce the clearance of MPP‡ from the brain, MPTP neurotoxicity in mice is enhanced or prolonged. We have used probenecid as an adjuvant, and discovered that it potentiates neurotoxicity by reducing the clearance of MPTP and its metabolites from kidney and brain. The e¡ects on striatal DA depletion are chronic and do not reverse for 6 months after treatment (Lau et al., 1990). In the present study, we further investigate this chronic, mouse, MPTP/probenecid model and compare it with the more conventional, subacute MPTP treatment. The latter model is considered to be subacute, because the duration of treatment combined with the survival period is longer than acute but less than a chronic paradigm (Dirckx, 1997). The results have already been published in abstract form (Petroske et al., 2000).

EXPERIMENTAL PROCEDURES

Animals Eight- to 10-week-old, male, C57BL/6 mice (Charles River Laboratories, Wilmington, MA, USA), weighing 22^25 g at the beginning of the study, were housed two to ¢ve animals per cage with food pellets and water available ad libitum. The room was maintained at constant temperature and humidity on a 12-h light^dark cycle. A minimum number of mice required to produce reliable scienti¢c data was used in this study. All animal

treatments including anesthesia were carried out strictly according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23, revised 1978) and were approved by the University of Missouri-Kansas City Institutional Animal Care and Use Committee. Subacute MPTP treatment In the subacute study, mice were treated with a conventional MPTP paradigm. These mice received MPTP hydrochloride (25 mg/kg in saline, s.c.) once a day for 5 consecutive days. The control animals were injected with saline under the same regimen. Animals were killed 3 or 30 days after the last treatment. Chronic MPTP/probenecid treatment In the chronic study, mice received a total of 10 doses of MPTP hydrochloride (25 mg/kg in saline, s.c.) in combination with an adjuvant, probenecid (250 mg/kg in dimethyl sulfoxide, DMSO, i.p.). Mice were treated similarly with probenecid or MPTP alone as controls. The 10 doses were administered on a 5-week schedule, such that injections were given with an interval of 3.5 days between consecutive doses. Animals were killed 1, 3 or 24 weeks (6 months) after the last treatment. MPTP hydrochloride and probenecid were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Probenecid was used to inhibit the rapid clearance and excretion of MPTP from the brain and kidney following each injection. Neither probenecid nor DMSO at the concentrations used in this study signi¢cantly produced any e¡ect on the striatal DA contents (Lau et al., 1990). Dopamine content analysis Striata from each animal were isolated, weighed and suspended in 0.2 ml of 0.2 N perchloric acid. Each sample was sonicated and centrifuged at 11 000Ug for 15 min at 4³C. The supernatant was ¢ltered through a nylon syringe ¢lter (Gelman ACRO LC3A, 0.45 Wm). An aliquot of the ¢ltrate was injected into a high performance liquid chromatography system (Waters, Milford, MA, USA) equipped with a C18 reverse phase, 3 W LUNA column (100 mmU2.0 mm, Phenomenex, Torrance, CA, USA). The sample was eluted by a mobile phase made of 25 mM NaH2 PO4 , 50 mM Na-citrate, 0.03 mM EDTA, 10 mM diethylamine HCl and 2.2 mM sodium octyl sulfate (pH 3.2), 30 ml/l methanol and 22 ml/l dimethylacetamide at a £ow rate of 0.4 ml/min. DA peak was determined by electrochemical detection at a potential of 0.6 V. The DA content in the sample was calculated by extrapolating the peak area from a standard curve (range 1^200 pg of DA) constructed under the same conditions during each run by the Maxima Workstation (Waters). Dopamine uptake assay DA uptake studies were carried out in a mouse synaptosomal preparation as previously described (Lau et al., 1991). In brief, mouse striata were isolated and homogenized in nine volumes of Tris^HCl (50 mM), sucrose (0.32 M) bu¡er, pH 7.5. The homogenate was centrifuged at 1000Ug for 20 min at 4³C. The resultant supernatant was further centrifuged at 27 000Ug. The pellet was resuspended in the same amount of Tris^sucrose bu¡er and used as the crude striatal synaptosome preparation. The uptake assay medium contained Tris^HCl (50 mM), pH 7.5, sucrose (0.32 M), KCl (5 mM), NaCl (120 mM) (assay bu¡er), pargyline (0.01 mM), and striatal synaptosomes (100^150 Wg protein). [3 H]DA (0.05 WM, speci¢c activity 14 Ci/mmol, Amersham Life Science, Arlington Heights, IL, USA) was added to the assay tube, which was incubated at 37³C for 6 min. The reaction was terminated by an injection of ice-cold assay bu¡er (3 ml) into the sample tube and the content was immediately (within 3 s) ¢ltered through a bu¡er-saturated GF/B ¢lter (Whatman,

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Clifton, NJ, USA) under vacuum. The ¢lter was washed with an additional 6 ml of bu¡er, dried and counted for radioactivity by the liquid scintillation method. The non-speci¢c [3 H]DA uptake was determined in separate tubes containing a DA uptake inhibitor, mazindol (1035 M). The speci¢c uptake of [3 H]DA by the intact DA terminals was calculated by subtracting the non-speci¢c uptake from total uptake and expressed as pmol/mg of synaptosomal protein. Animal perfusion Mice used for immunohistochemistry or stereological estimates were anesthetized with pentobarbital (130 mg/kg; Sigma), transcardially perfused with 20^40 ml of 2.5% sucrose in 0.1 M phosphate-bu¡ered saline (PBS), followed by 70 ml of 3% paraformaldehyde and 2.5% sucrose in 0.1 M phosphate bu¡er (PB). Their brains were removed, placed in the perfusion ¢xative for 2 h, and then allowed to sink in 30% sucrose in 0.1 M PB overnight at 4³C. The brains were blocked in the coronal plane and cut into 50-Wm sections using a Vibratome (TPI, St. Louis, MO, USA). Every third section from the midbrain was stained for Nissl and adjacent sections were immunoreacted with mouse antibodies directed against tyrosine hydroxylase (TH). Tyrosine hydroxylase immunohistochemistry All immunoreactions were carried out with reagents from the mouse-on-mouse (M-O-M) kit (Vector Laboratories, Burlingame, CA, USA). Sections were ¢rst blocked with normal serum from the M-O-M kit for 1 h and then incubated in mouse anti-TH sera (DiaSorin, Stillwater, MN, USA), 1:2000 in 0.01 M PBS with 0.1% Triton X-100, for 48^72 h at 4³C. This was followed by a 45-min incubation at room temperature in biotinylated horse anti-mouse IgG (provided in the M-O-M kit), and subsequently in avidin^biotin^peroxidase complex (ABC Elite kit, Vector Laboratories) for 45 min. The bound peroxidase was revealed with 0.05% 3,3P-diaminobenzidine tetrahydrochloride with 0.01% hydrogen peroxide in Tris^HCl bu¡er, pH 7.6. The reaction was allowed to proceed for 5^10 min. Sections were mounted on subbed slides, dehydrated, and coverslips applied. TH-immunoreactive (TH-ir) cell bodies were counterstained with Cresyl Violet and counted. Only those cells which clearly fell within the con¢nes of the substantia nigra (see below) in three coronal sections, 50 Wm thick and separated from each other by 300 Wm, were counted. The sections (bregma 33.1, 33.4, 33.7 mm) were selected because the fullest extent of the substantia nigra pars compacta is found at these mid-rostrocaudal levels. Slides were coded, neurons were photographed at 200U magni¢cation using a Polaroid DMC camera mounted on a Nikon 400E microscope and interfaced with a Macintosh computer. Photographs were taken at two focal depths. An observer who was kept blind to the treatment counted TH-ir neurons according to a procedure established by others (Kupsch et al., 1995). A neuron was counted if a nucleus was visible and one or more clearly de¢ned processes tapered gradually from the cell body (Meredith et al., 1999). The goal of this study was to compare changes in TH-ir cells between experimental groups and controls. Thus, the mean number of TH-ir neurons found in each experimental group is expressed as percentage of those in time-matched control mice. Unbiased estimates of neuronal loss In order to establish whether neurons are lost, unbiased, design-based stereology was used (Sterio, 1984; Gundersen, 1986; West et al., 1991). The total number of neurons was estimated for the substantia nigra (in its entirety) for four to eight mice in each group (Table 1). The reference volume of the substantia nigra (pars compacta and pars reticulata) was calculated from low-power images using a point count array according to Cavalieri principles

591

(Gundersen and Jensen, 1987). The thickness of each cut section was 50 Wm. The cross-sectional area of these sections was measured and the reference volume (Vr ) for the entire nigra was estimated with the following equation: aX Pi Vr ˆ T p iˆ1 where T is the thickness of a section, a/p is the area of each point and Pi is the number of points that landed within the substantia nigra on the ith section. Anatomical boundaries were delineated with the aid of a mouse atlas (Franklin and Paxinos, 1996). In brief, the medial border was de¢ned by a vertical line passing through the medial tip of the cerebral peduncle, thereby excluding the neurons in the ventral tegmental area (VTA). The ventral border followed the dorsal boundary of the cerebral peduncle, thereby including neurons in the pars compacta, pars reticulata and pars lateralis. The dorsal border passed above pars compacta and below the ventral margin of the medial lemniscus. The entire rostro-caudal extent of the dopaminergic cells in the substantia nigra is approximately 1200 Wm. We collected sections, cut coronally on a Vibratome, from a point just caudal to the subthalamic nucleus at bregma 32.54 and continued caudally until the retrorubral (A8 ) ¢eld, bregma 34.04. After a random start, every third, 50-Wm-thick section (approximately eight sections total) through this midbrain region was collected. Dedicated software (StereoInvestigator, version 4.10, MicroBrightField, Brattleboro, VT, USA) was linked to drive a motorized stage in three axes (x, y, z) on a Leica DIALUX microscope. The slides were coded and the total number of neurons was estimated using the optical fractionator (Gundersen, 1986; West et al., 1991). A systematic random sample of neurons was achieved by positioning a sampling grid over the entire substantia nigra on each section. This grid was divided into counting frames (25U25 Wm) that were equidistant from each other and their spacing set so that only 100^200 neurons were sampled within each brain (West et al., 1991). Movement between coordinates was e¡ected by a computer-controlled stepping motor attached to the x and y axes of the stage. The optimal dimensions of each counting frame and the grid size were established following a restricted pilot study. The sampling fraction was delimited at low power and neurons had to have a distinct nucleus to be counted. Cells were sampled with a 100U oil immersion objective with high numerical aperture (1.4) and through a de¢ned depth with a guard zone of 2 Wm. Tissue shrinkage was estimated for all cases and did not di¡er signi¢cantly between animals or groups. Optical disector counting rules (Gundersen, 1986) were used to count the total Nissl-positive neurons (N) in each disector volume, which was determined by multiplying the area of the counting frame by the disector height: X t 1 1 Nˆ Q3 h asf ssf where 4Q3 is the total number of particles counted, t is the mean section thickness, h is the height of the optical disector, asf is the area sampling fraction, and ssf is the section sampling fraction. Rotarod performance Motor activity in mice was measured 3 or 30 days after subacute MPTP treatment and 1 or 3 weeks or 6 months after chronic MPTP/probenecid injections with an automated revolving rod (3.0 cm) apparatus (Rotamex, Columbus Instruments, Columbus, OH, USA). Time (in seconds) spent on the rod at each level of revolving speed (in revolutions per minute, r.p.m.) indicated motor performance. Mice were tested on the revolving rod sequentially at eight incremental speeds (8, 10, 12, 14, 16, 18, 20, 24 r.p.m.) for up to 150 s per speed. A 5-min rest period was provided between successive speeds to alleviate animal stress and fatigue. The maximum time (up to 150 s) that the animal was able to stay on the revolving rod was plotted against each corresponding speed of rotation. The cumulative motor

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activity score for each mouse was calculated and expressed as total area under the curve (AUC) (Rozas et al., 1998). One week before the ¢rst behavioral test and data collection, all mice were handled and trained for at least 1 h with positive reinforcement (peanut butter crackers) on three separate days until they could perform the entire procedure without undue stress. Mice that were tested at 1- or 2-week intervals for up to 6 months did not require retraining. The experimenter who performed the motor activity tests was blind to the treatment each animal had received. Statistical analysis Statistical comparisons of values between control and treated groups in neurochemical and behavioral experiments were carried out using the unpaired Student's t-test. When more than two groups of means were compared, a one-way analysis of variance was employed. For stereological comparisons where the distribution of samples was not normal due to small sample size, the Mann^Whitney U-test was applied. In all cases, a P value of less than 0.05 was considered to be signi¢cant. All data are presented as mean þ S.E.M.

RESULTS

Striatal dopamine levels The striatal DA contents among respective control groups in subacute and chronic studies did not di¡er signi¢cantly and thus, data arising from animals in those groups were pooled. As shown in Fig. 1A, 3 days after the subacute MPTP treatment, the loss of striatal DA was highly signi¢cant (76% loss). Thirty days after the treatment, the loss of striatal DA remained signi¢-

cant but to a lesser extent (53% loss) when compared to controls, suggesting that a partial recovery had occurred. Striatal DA levels in chronic MPTP/probenecidtreated mice were strikingly di¡erent for each survival period. As shown in Fig. 1B, 1 and 3 weeks after chronic treatment, striatal DA was robustly depleted (95^98% loss). Six months following treatment, striatal DA remained low (76% loss), even though levels had recovered by some 20% when compared to groups examined 1 or 3 weeks after chronic treatment. We also compared DA levels in mice 6 months after chronic treatment with MPTP/probenecid to those treated chronically with MPTP alone. We found that striatal DA levels in the latter recovered by 80% when compared to those in the former. Striatal dopamine uptake Three days following the subacute treatment of MPTP, there was a signi¢cant decrease of DA uptake (54% lower); however, 30 days after treatment, DA uptake function recovered fully (Fig. 2A). In contrast, 1, 3 weeks or 6 months after the chronic MPTP/probenecid treatment, striatal DA uptake was persistently depressed; in the 6-month post-treatment group, there was a 72% loss of functional DA uptake in the striatal terminals (Fig. 2B). In mice which were chronically treated with MPTP alone, DA uptake recovered fully 6 months after treatment (Fig. 2B). The DA uptake values between control groups at di¡erent time points did not di¡er signi¢cantly and thus, they are presented combined.

Fig. 1. Striatal DA levels in subacute MPTP and chronic MPTP/probenecid (MPTP/P) mice. The number of animals used is indicated in parentheses. (A) Mice were treated with a single daily injection of saline or MPTP (25 mg/kg, s.c.) for 5 days. Striatal DA was assayed 3 or 30 days after the last injection. ‡ Signi¢cantly lower than the saline control group (P 6 0.0001). ‡‡ Signi¢cantly lower than the saline control group (P 6 0.0002) and higher than the 3-day subacute MPTP-treated group (P 6 0.002). (B) Mice were treated with 10 injections of probenecid (250 mg/kg, i.p.), MPTP (25 mg/kg, s.c.) or combined MPTP/probenecid for 5 weeks. Striatal DA was assayed 1 or 3 weeks, or 6 months after the last treatment. *Signi¢cantly lower than the probenecid control group (P 6 0.0001). **Signi¢cantly lower than the probenecid control group (P 6 0.0001) and higher than either 1-week or 3-week MPTP/P-treated mice (P 6 0.002). ***Signi¢cantly lower than the probenecid control group (P 6 0.04) and signi¢cantly higher than the 6-month MPTP/P-treated group (P 6 0.0001).

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Tyrosine hydroxylase immunoreactivity The pattern and extent of TH-ir neurons in the substantia nigra of saline- and probenecid-treated controls were similar (Figs. 3A and 4A). The TH-positive neurons were distributed throughout both the dorsal and ventral tiers of the pars compacta and scattered in small islands of the pars reticulata. Cells in the dorsal tier had long TH-positive processes oriented along the axis of the pars compacta, while those in the ventral tier had dendrites that penetrated the pars reticulata (Figs. 3A and 4A). Three days after subacute MPTP treatment, TH-ir processes were greatly reduced in the pars compacta, pars reticulata, pars lateralis and VTA (compare Fig. 3C with A); 30 days following treatment, these immunoreactive processes were again present (compare Fig. 3E with C). Although it appears that TH-ir cells in the pars compacta were lost (Fig. 3), when counted 3 or 30 days after subacute treatment, 95% of TH-ir cells remained (Fig. 5). When animals were treated chronically with MPTP/ probenecid, TH-ir processes also appeared to be greatly reduced throughout the substantia nigra, and to a lesser extent in the VTA, at 1 and 3 weeks post-treatment (Fig. 4C, E). However, following 6 months survival, both the pattern and extent of TH-positive ¢bers partially recovered (compare Fig. 4G with C and E). In speci¢c regions, we detected loss of dopaminergic neurons in the pars lateralis at 1 week post-MPTP/probenecid treatment (Fig. 4C), and in the middle of the pars compacta at 3 weeks post-treatment (Fig. 4E). The loss of TH-ir cells throughout the pars compacta and VTA was still

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observed at 6 months after chronic administration (compare Fig. 4G with A), although it was partially recovered (compare Fig. 4G with C and E). When counted 1 or 3 weeks after chronic treatment, cells expressing TH were signi¢cantly reduced to 52% and 62% of probenecid controls, respectively (Fig. 5). Six months after treatment, the number of TH-ir cells was 79% of matched controls (Fig. 5). In the striatum (caudate^putamen) and nucleus accumbens, the pattern and distribution of TH-ir ¢bers did not di¡er between controls (Figs. 3B and 4B). Three days after subacute MPTP administration, we observed a decrease in ¢ber immunoreactivity in the central and medial parts of the caudate^putamen as well as in the nucleus accumbens (Fig. 3D). Nevertheless, 30 days after treatment, the pattern and intensity of TH-positive ¢bers resembled those seen in controls (compare Fig. 3F with B). In contrast, following chronic MPTP/probenecid treatment, there was a progressive loss of TH-ir ¢bers in both the caudate^putamen and nucleus accumbens. Fibers were ¢rst reduced in medial and central, dorsal striatum and the core of nucleus accumbens at 1 week post-treatment (Fig. 4D), and later in dorsolateral caudate^putamen and lateral shell of nucleus accumbens 3 weeks after treatment (Fig. 4F). Six months following treatment, TH immunoreactivity returned to a level similar to controls (compare Fig. 4H with B). Interestingly, as reported in MPTP-treated primates (Betarbet et al., 1997), we also noticed the appearance of small neurons immunoreactive for TH throughout the caudate^putamen following subacute and chronic treatments (pictures not shown). These cells were numerous and embedded among residual TH-ir ¢bers.

Fig. 2. Striatal DA uptake in subacute MPTP and chronic MPTP/probenecid (MPTP/P) mice. The number of animals used is indicated in parentheses. (A) Mice were treated with a single daily injection of saline or MPTP (25 mg/kg, s.c.) for 5 days. [3 H]DA uptake in striatal synaptosomes was assayed 3 or 30 days after the last injection. ‡ Signi¢cantly lower than the saline control group (P 6 0.0005) and lower than the 30-day subacute MPTP-treated group (P 6 0.0001). (B) Mice were treated with 10 injections of probenecid (250 mg/kg, i.p.), MPTP (25 mg/kg, s.c.) or combined MPTP/probenecid for 5 weeks. [3 H]DA uptake in the striatal synaptosomes was assayed 1 or 3 weeks, or 6 months after the last treatment. *Signi¢cantly lower than the probenecid control group (P 6 0.001). **Not di¡erent from the probenecid control group (P s 0.6).

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Fig. 3. Representative photomicrographs illustrating TH immunoreactivity in the midbrain (left) and striatum (right) of subacute saline- or MPTP-treated mice. Mice were treated with a single daily injection of saline or MPTP (25 mg/kg, s.c.) for 5 days. TH immunohistochemistry was performed 3 or 30 days after the last injection. Sections were counterstained with Cresyl Violet. The micrographs illustrate (A) TH-ir cells and dendrites in the substantia nigra (SN) and VTA of a salinetreated control mouse. Note the dendritic process (arrows) of TH-ir neurons. (B) TH-ir ¢bers in the caudate^putamen (CP) and nucleus accumbens (NAc) of the control mouse. Corresponding sections are from mice (C, D) 3 days and (E, F) 30 days following subacute MPTP treatment. Compare the reduction in TH-ir processes at 3 days (C, arrow) with the recovery at 30 days (E, arrow). Scale bar = 250 Wm (in A, also applicable to C and E); 500 Wm (in B, also applicable to D and F).

Neuronal loss In order to estimate changes in the number of nigral neurons and in the volume of the substantia nigra as a whole, we implemented design-based stereological quantitation directly on Nissl-stained sections of the midbrain. We found no change in the volume of the substantia nigra in either group of subacutely treated mice or in the 1- or 3-week post-chronic groups

(Table 1). In the 6-month post-chronic mice, the volume of the substantia nigra was also unchanged when compared to that of matched controls. In the subacute group, there was a marginal decrease in the median total number of neurons 3 days after MPTP treatment (P = 0.045, Table 1). However, by 30 days post-treatment, the loss of cells was not signi¢cant (P = 0.068, Table 1). When we examined the chronic groups, we found signi¢cantly fewer neurons (320% and

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Fig. 4 (Caption overleaf).

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Fig. 5. TH-ir cell counts in subacute MPTP and chronic MPTP/probenecid (MPTP/P) mice. The number of animals used is indicated in parentheses. The mean number of TH-ir neurons was determined for each experimental group and expressed as percent of those in matched control mice. (A) Mice were treated with a single daily injection of saline or MPTP (25 mg/kg, s.c.) for 5 days. The percent of TH-ir cells 3 or 30 days after the last injection was not signi¢cantly di¡erent from the matched control group. (B) Mice were treated with 10 injections of probenecid (250 mg/kg, i.p.) or combined MPTP/probenecid for 5 weeks. The TH-ir cells were counted 1 or 3 weeks, or 6 months after the last treatment. *Signi¢cantly lower than the matched probenecid control group (P 6 0.03).

339%, respectively) in the entire substantia nigra 1 and 3 weeks after MPTP/probenecid treatment, when compared to controls (Table 1). Following 6 months survival, the substantia nigra of chronically treated mice still had signi¢cantly fewer cells when compared to matched probenecid-treated controls (Table 1). We also detected a signi¢cant neuronal loss in the 6-month probenecid con-

trol mice when compared to either saline, or 1- or 3-week post-chronic probenecid controls. Rotarod motor performance The animal performance on the rotarod within the control and treated groups was highly variable, but usu-

Table 1. Unbiased estimates of the reference volume (Vr ), and the total number of neurons (N) in the substantia nigra (unilateral) of mice treated subacutely with MPTP or chronically with MPTP/probenecid (MPTP/P) and their matched controls Treatment Subacute model Saline 3 days post-MPTP 30 days post-MPTP Chronic model 1-, 3-week probenecid 1-week post-MPTP/P 3-week post-MPTP/P 6-month probenecid 6-month post-MPTP/P

n

Mean Vr þ S.E.M. (mm3 )

Median N (U103 )

Mean N þ S.E.M. (U103 )

P value

CE group

6 7 5

0.191 þ 0.014 0.177 þ 0.019 0.184 þ 0.017

23.39 15.98 18.16

23.47 þ 2.0 18.03 þ 1.9 17.77 þ 0.9

a

0.067 0.084 0.055

5 7 8 4 4

0.193 þ 0.014 0.177 þ 0.006 0.171 þ 0.024 0.205 þ 0.017 0.209 þ 0.012

24.42 19.52 14.99 14.86 11.93

25.98 þ 2.0 18.51 þ 1.1 14.86 þ 1.4 15.77 þ 1.2 11.83 þ 0.26

a

0.045 0.068 (NS) 0.005 0.003

b

0.021

0.042 0.046 0.060 0.108 0.135

Comparisons between treated groups and matched controls were performed by the Mann^Whitney U-test. P values are indicated. NS, not signi¢cantly di¡erent. CE, coe¤cient of error for estimates of N for all groups. a The neuronal numbers for subacute saline, 1- or 3-week post-chronic probenecid-treated mice are not statistically di¡erent (P s 0.05). b Signi¢cantly lower than either subacute saline, 1- or 3-week post-chronic probenecid-treated controls (P 6 0.02).

Fig. 4. Representative photomicrographs illustrating TH immunoreactivity in the midbrain (left) and striatum (right) of chronic probenecid- or MPTP/probenecid (MPTP/P)-treated mice. Mice were treated with 10 injections of probenecid (250 mg/kg, i.p.) or combined MPTP (25 mg/kg, s.c.) with probenecid (250 mg/kg, i.p.) for 5 weeks. Sections were counterstained with Cresyl Violet. The micrographs illustrate (A) TH-ir cells and dendrites in the substantia nigra (SN) and VTA of a probenecid-treated control mouse. Note the TH-ir processes (arrows). (B) TH-ir ¢bers in the caudate^putamen (CP), core and shell regions of the nucleus accumbens (NAc) of the control mouse. Corresponding sections from mice (C, D) 1 week, (E, F) 3 weeks, and (G, H) 6 months after chronic MPTP/P treatment. Compare the substantial reduction in TH-ir processes and neurons at 1 or 3 weeks (C, E, arrow) with partial recovery at 6 months (G, arrow). Scale bar = 250 Wm (in A, also applicable to C, E and G); 500 Wm (in B, also applicable to D, F and H).

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597

Fig. 6. Rotarod performance of subacute MPTP and chronic MPTP/probenecid (MPTP/P) mice. The number of animals used is indicated in parentheses. Details about pre-training the animals and data collection are described in Experimental Procedures. (A) In mice treated with a single daily injection of saline or MPTP (25 mg/kg, s.c.) for 5 days, the rotarod performance scores (AUC) were collected 3 days or 30 days after the last injection and compared with time-matched saline controls. No signi¢cant motor de¢cits were detected. (B) In mice treated with 10 injections of probenecid (250 mg/kg, i.p.), or combined MPTP/probenecid for 5 weeks, the rotarod performance scores were collected 1 or 3 weeks, or 6 months after the last treatment. *Signi¢cantly lower than the matched probenecid control group (P 6 0.03).

ally consistent for any individual animal from week to week. The performance score for each set of experimentally treated animals was compared with their matched control group. A large number of animals were used in this study to minimize the e¡ects of intra-group variability. Overall scores in control groups declined somewhat as the survival period prolonged, possibly related to increased body weight and age of the animal. No di¡erence in rotarod performance was found between subacute saline- and MPTP-treated mice, 3 or 30 days after treatment (Fig. 6A). In chronic MPTP/probenecid-treated mice, no di¡erences in performance were detected between the control and 1-week post-treated group. However, a signi¢cant decline in rotarod performance was ¢rst seen at 3 weeks and persisted until 6 months after the chronic MPTP/probenecid treatment when compared to matched controls (Fig. 6B). The difference in AUC scores was due mainly to behaviorally impaired mice spending progressively less time on the rotating rod at increasing speeds of rotation. At speeds resembling a normal walking pace (8^14 r.p.m.), all groups of mice could initially remain on the rod for the full 150-s test period. At higher speeds (16^24 r.p.m.), the mice were required to run to remain on the rod. Motor de¢cits ¢rst appeared as a decrease in the amount of time spent on the rod at these higher speeds. Behavioral scores declined steadily over the course of 6-month observation period (data not shown). By 2 months post-treatment, most chronic MPTP/probenecid-treated mice had di¤culty remaining on the rod for more than 60 s at speeds above 14 r.p.m. Four months after treatment ended, most chronic MPTP/probenecid-

treated mice had di¤culty remaining on the rod for more than 60 s at speeds above 10 r.p.m. By 6 months posttreatment, most chronic MPTP/probenecid mice were unable to remain on the rod for more than 60 s at any speed.

DISCUSSION

In order to identify the pathophysiological mechanisms of PD and to develop new strategies for neuroprotection, it is imperative to establish a long-term animal model that closely resembles human Parkinsonism. This report presents a close correlation between neurochemical, morphological and behavioral manifestations validating a new model of MPTP Parkinsonism in a single study. We provide evidence that, for 6 months after chronic MPTP/probenecid treatment, C57BL mice exhibit a marked depletion of DA, which is associated with a signi¢cant loss of functional DA uptake and substantia nigral cells as well as a de¢cit in rotarod motor performance. We found that nigrostriatal dysfunction develops progressively in this chronic model and the di¡erence is signi¢cantly beyond the natural process of aging. Furthermore, in a parallel study, we also detected an accumulation of K-synuclein and ubiquitin proteins in the substantia nigra of the chronic MPTP/ probenecid but not in the subacute MPTP mice (Petroske et al., 2000). These proteins are anatomical markers of Lewy bodies in human PD cases (Polymeropoulos et al., 1997; Spillantini et al., 1997; Trojanowski and Lee, 1998).

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E. Petroske et al.

Mouse models of Parkinsonism Since the discovery that MPTP selectively destroys nigrostriatal DA neurons in humans, a wide range of animal models for Parkinsonism has been studied. While the non-human primate model closely resembles human PD, use of this species may not be ethically or ¢nancially justi¢able for exploring neurological and pathological mechanisms of this disease. The development of a comparable model in small animals is desirable but much debate has centered on the validity of mouse MPTP models compared to human Parkinsonism. In mice MPTP is usually administered either by an acute or a subacute regimen. Most acute MPTP studies follow the model that had been examined by Sonsalla and Heikkila (1986), who injected mice four times with MPTP at 20 mg/kg at 1- or 2-h intervals within a day. Subacute treatment generally follows the model originally developed by Heikkila et al. (1984), in which mice are typically injected with a single daily dose of MPTP at 30 mg/ kg for 5^10 days. Most published results are based on studies conducted within a few days to a month after MPTP treatment is terminated. We did not include the acute model in the present study, because with rapid and frequent injections of MPTP within 1 day, all mice died after the third injection due to peripheral toxicity (data not shown). Since most studies on the neurotoxic e¡ects of MPTP are generated from either cell culture or acute/ subacute mouse models, the postulated mechanisms (see recent review by Zhang et al., 2000) may not be entirely relevant to the chronic, neurodegenerative e¡ects seen in human PD. In the acute and subacute models, MPTP produces decrements in the striatal level of DA and its metabolites along with a reduction in striatal synaptosomal DA uptake (Heikkila et al., 1984; Hallman et al., 1985; Ricaurte et al., 1986). However, when survival times are extended, the neurotoxic e¡ects of MPTP in mice are reversible (Hallman et al., 1985; Ricaurte et al., 1986). Loss of TH-ir cells in the substantia nigra has been shown in some studies (Heikkila et al., 1984; Seniuk et al., 1990; Bezard et al., 1997) but not in others (Hallman et al., 1985; Ricaurte et al., 1986). Morphologically, subacute (Tatton and Kish, 1997) but not acute (Jackson-Lewis et al., 1995) MPTP induces apoptosis in dopaminergic neurons. Despite evidence for DA reductions, animals treated acutely or subacutely with MPTP do not exhibit motor abnormalities (Heikkila et al., 1989; Gerlach and Riederer, 1996). The data presented in the current study are consistent with the above cited ¢ndings indicating that subacute MPTP treatment only produces a transient neurotoxic insult to the nigrostriatal neurons, which reverses spontaneously and has no longterm neurodegenerative consequences. Thus, the conventional methods for rapidly inducing neurotoxicity by MPTP may not be relevant to the slow, progressive nature of human PD nor mimic the long and latent, neurodegenerative syndromes associated with chronic exposure to environmental or industrial chemicals (Gorrell et al., 1996; Hanna et al., 1999).

To the best of our knowledge, this is the ¢rst chronic MPTP mouse model, which has shown the hallmarks of PD for a survival period of 6 months. In the earlier study we showed that a relatively low dose of MPTP (15 mg/ kg) produced about 60% DA depletion 6 months after MPTP/probenecid treatment (Lau et al., 1990). In the present study, an increased dose of MPTP (25 mg/kg) appears to be the maximally tolerated dose when given together with probenecid. All the treated animals survived under this regimen. It is also noteworthy that while all animals treated subacutely with ¢ve daily injections of MPTP at 25 mg/kg had survived, a similar dose combined with probenecid resulted in the death of all animals (data not shown). Our observations on animal survival further support the notion that probenecid potentiates the toxic e¡ects of MPTP. MPTP neurotoxicity and neuronal degeneration In the subacute MPTP model, we detected an initial loss of TH immunoreactivity mainly in the dendritic processes and striatal ¢bers, but not in the substantia nigra cells. This suggests that these processes are highly vulnerable to the subacute, neurotoxic insult. MPTP may trigger the release of endogenous excitatory amino acids acutely and cause oxidative stress to the neuronal processes as demonstrated in brain slices (Bywood and Johnson, 2000) and in fetal rat mesencephalic cell culture (Sanchez-Ramos et al., 1988). Nevertheless, the early loss of dendritic processes and striatal ¢bers is not permanent, as shown in the present study (Fig. 3) and by others (Mitsumoto et al., 1998; Song and Haber, 2000). Either they have recovered or new ones have sprouted long after MPTP is eliminated systemically. In the present study, two methods were used to measure neuronal degeneration and they seemed to have produced di¡erent trends over time. Historically, studies that describe MPTP- or 6-hydroxydopamine-induced cell loss refer to decreases in the number or density of TH-ir neurons in the substantia nigra (Gerlach and Riederer, 1996). Most investigators count such neurons in selected nigral sections (Varastet et al., 1994; Bezard et al., 1997; Mitsumoto et al., 1998), but some have used unbiased stereology to estimate their numbers (Chadi et al., 1993; Liberatore et al., 1999; Walters et al., 1999). Regardless of the method employed, these studies have yielded little consensus. Part of the problem underlying these inconsistent results lies with the highly variable dose and injection regimen for MPTP (see above). Nevertheless, fundamental to all these e¡orts is the premise that a reduction in the number of TH-ir neurons means that dopaminergic cells have been lost. In the subacute MPTP mice and early time points of the chronic MPTP/probenecid model, the reduction in DA content and uptake in the striatum are far greater than the loss of TH-ir cells and total substantia nigra neurons. This observation has also been described in the acute (Ara et al., 1998) and subacute (Seniuk et al., 1990) mouse models, as well as in human PD (Hornykiewicz and Kish, 1987; Pakkenberg et al.,

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A chronic mouse MPTP/probenecid model of Parkinsonism

1991). It is postulated that the decrease in striatal DA caused by MPTP coincides with the enzymatic inactivation of TH through a nitration process, without a¡ecting the actual level of TH protein and cell number (Ara et al., 1998). In the present study, we used stereology to investigate neuronal loss across the entire substantia nigra instead of only investigating the pars compacta, as in other studies (Liberatore et al., 1999; Walters et al., 1999). This is because we believe that a change in the number of TH-positive cells detected by immunohistochemistry may not re£ect the actual loss of neuronal cells. Furthermore, there appears to be some sprouting of TH-ir processes in the substantia nigra of the 6-month MPTP/probenecid mice, and as such gives the impression of neuronal recovery. Moreover, it is quite di¤cult to establish precisely the boundary of pars compacta in Nissl-stained sections for stereological study, and there are dopaminergic neurons also present in islands of pars reticulata (Gerfen, 1992). Oorschot (1996) has reported 33 500 neurons in the rat substantia nigra (unilateral), approximately 22% of which is con¢ned to the pars compacta. We calculated from this study that the total number of neurons in the normal, mouse substantia nigra is 26% less than that reported for the rat (Oorschot, 1996). This means that unilateral pars compacta of the mouse contains approximately 5500 neurons, which is comparable to the numbers published by other investigators based on bilateral pars compacta in the mouse (Chadi et al., 1993; Liberatore et al., 1999; Walters et al., 1999). Since MPTP selectively destroys the nigrostriatal dopaminergic neurons (Schneider et al., 1987), we think that the neurons that are lost in the chronic MPTP/probenecid mice are largely dopaminergic. If dopaminergic neurons comprise approximately 25^30% of the total cells in the mouse substantia nigra (Chadi et al., 1993; Oorschot, 1996), then at 1 week after MPTP/probenecid treatment, we estimate that there would be 58% fewer dopaminergic cells. Three weeks and 6 months after chronic MPTP/ probenecid treatment, the loss of dopaminergic neurons would be even greater. In humans, it is estimated that the loss of nigrostriatal neurons is linear at a rate of 4^7% per decade in normal brains but this is accelerated in PD brains (McGeer et al., 1989; Scherman et al., 1989). In the 10^12-monthold, mature, adult, probenecid control mice, we detected a loss of 38% of the substantia nigra neurons due to aging alone (Table 1), which is similar to the ¢ndings for C57BL/6 mice reported by Tatton et al. (1991). Therefore, permanent cell loss observed here at 3 weeks and further at 6 months after MPTP/probenecid treatment resembles a toxic insult added onto the normal course of an age-related, linear decrease in substantia nigra dopaminergic cells. MPTP neurotoxicity and motor de¢cit Investigations that have reported long-term reductions in the numbers of TH-ir cells following MPTP rarely

599

provide a motor correlate. Indeed, recovery of motor performance appears to be almost universal following conventional MPTP treatment (Gerlach and Riederer, 1996). The use of acetaldehyde as an adjuvant with chronic MPTP treatment has been shown to produce motor de¢cits and reduced striatal TH-ir ¢bers lasting up to 100 days post-treatment (Rozas et al., 1998). The rotarod test, developed for mice by Rozas et al. (1998), is well suited to distinguish persistent motor de¢cits from temporary inactivity that results from peripheral toxicity and that may interfere with infrared activity cage monitoring. Recovery of striatal and nigral TH immunoreactivity observed here 6 months after chronic MPTP/probenecid treatment clearly does not match the motor de¢cits. Such increases in TH may re£ect sprouting of residual ¢bers (Song and Haber, 2000) or the de novo appearance of TH-ir neurons in dopamine-depleted striata (Du et al., 1995; Betarbet et al., 1997; Meredith et al., 1999), but more likely represent a compensatory mechanism for chronically reduced dopamine levels as suggested from post-mortem studies of PD brains (Grima et al., 1987). In the conventional MPTP model, our data show that a rapid, subacute insult to the nigrostriatal pathway with MPTP produces a marginal decrease of neuronal numbers without a concomitant reduction in TH-ir cells. Nevertheless, it does not produce long-lasting motor deficits, presumably because cell loss does not surpass the threshold for behavioral impairment (Kirik et al., 1998). In our chronic model, motor de¢cits persist, and can be matched to reduced levels and uptake of DA. Moreover, these de¢cits are the result of massive cell death. We ¢nd that the onset of the motor disability follows rather than coincides with the cell loss, and alterations in TH immunoreactivity are apparently unrelated.

CONCLUSION

We have shown that the chronic treatment of mice with MPTP/probenecid is accompanied by sustained nigrostriatal degeneration and motor decline resembling human PD. Based on the comparisons made in this study, this chronic model is an improvement over the conventional acute and subacute models. The chronic MPTP/probenecid model has a potential for explorations of disease progression, mechanisms of neurodegeneration, and neuroprotection.

AcknowledgementsöThe authors thank Dr. P.L. Gabbott for providing the StereoInvestigator apparatus and Charlie Callison, Katy Schafbuch and Shari Buzolich for their technical contributions to the work. This research was supported in part by grants from the University of Missouri Research Board, the Health Future Foundation, Inc., the National Parkinson Foundation, Inc. (to Y.-S.L.), and the National Institute of Neurological Disorders and Stroke (R01 NS41799 to G.E.M., Y.-S.L. and S.T.).

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