neuroprotective effects of pramipexole in young and aged mptp-treated mice

Brain Research 905 (2001) 44–53www.elsevier.com/ locate /bres

Research report

Neuroprotective effects of pramipexole in young and agedMPTP-treated mice

*David W. Anderson, T. Neavin, J.A. Smith, J.S. SchneiderDepartment of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, 1020 Locust Street, JAH 521, Philadelphia, PA 19107, USA

Accepted 27 March 2001

Abstract

This study examined the effect of pramipexole (PPX), a selective dopamine (DA) D /D agonist, on 1-methyl-4-phenyl-1,2,3,6-3 2

tetrahydropyridine (MPTP)-induced damage to the nigrostriatal dopamine system in young (8-week-old) and aged (12-month-old) mice.Co-administration of PPX and MPTP to young or aged mice, followed by 2 or 14 days of additional PPX treatment, significantlyattenuated MPTP-induced striatal DA loss. Pramipexole treatment also significantly attenuated the loss of tyrosine hydroxylaseimmunoreactive neurons (TH-IR) within the substantia nigra pars compacta (SNc) in both young and aged animals. Effects of PPXadministration on dopaminergic cell survival were confirmed in Nissl-stained sections and by quantitation of retrogradely labeledFluorogold-positive SNc neurons. Protective effects of PPX on striatal DA levels and SNc DA neuron survival were similar in young andaged animals, although the magnitude of these effects was significantly less in aged animals. These findings support the early initiation ofPPX therapy in Parkinson’s disease patients. 2001 Elsevier Science B.V. All rights reserved.

Theme: Disorders of the nervous system

Topic: Degenerative disease: Parkinson’s

Keywords: PPX; MPTP; Parkinson’s disease; Dopamine; Cell survival; Neuroprotection; Neurorestoration

1. Introduction electron transport chain, impaired ATP synthesis, increasedgeneration of free radicals, lipid peroxidation [11,21,36]

Striatal dopamine (DA) depletion consequent to degene- and ultimately cell death.ration of substantia nigra pars compacta (SNc) neurons While current PD therapies are designed primarily tois associated with both idiopathic Parkinson’s disease improve symptoms, an additional goal of new PD therapies(PD) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine needs to be neuroprotection. Dopaminergic agonists (e.g.(MPTP)-induced Parkinsonism. While the mechanisms that bromocriptine and pergolide), used as symptomatic therapytrigger neuronal degeneration in the SNc in idiopathic PD for PD, have been suggested to have neuroprotectiveare still unknown, the mechanisms contributing to DA properties in addition to their symptomatic effectsneuron degeneration following MPTP exposure are better [9,10,23,25,27,32,42]. Although most therapeutic effects ofdefined [16,35]. MPTP is converted to the DA agonists are thought to be associated with postsynaptic

1methylpyridinium ion (MPP ) by monoamine oxidase-B receptor stimulation [25], recent evidence suggests that DAprimarily within glia, and actively taken up by dopa- agonist effects may also be mediated at least in partminergic (DAergic) neurons. Although additional factors through pre-synaptic stimulation of DA autoreceptors andhave been hypothesized to play a role in the death of SNc intracellular sites within presynaptic DAergic neurons [27].

1neurons in PD [7,11,12,34,35,37], accumulation of MPP Potential neuroprotective effects of DA agonists on pre-in mitochondria leads to inhibition of complex 1 of the synaptic neurons may involve altered control of DA

metabolism, resulting in reduced oxidative stress [15,41].Pramipexole (PPX) h(2)-2-amino-4,5,6,7-tetrahydro-6-*Corresponding author. Tel.: 11-215-503-0370; fax: 11-215-923-

propylamino-bensthiazol dihydrochloridej is a high affini-3808.E-mail address: [email protected] (J.S. Schneider). ty, synthetic, non-ergoline DA agonist which preferentially

0006-8993/01/$ – see front matter 2001 Elsevier Science B.V. All rights reserved.PI I : S0006-8993( 01 )02466-0

D.W. Anderson et al. / Brain Research 905 (2001) 44 –53 45

binds to the D subfamily of DA receptors (D , D , and Twelve animals (12 months old) received MPTP at a2 2 3

D ) [19]. Recent studies have identified a sevenfold greater dose of 10 mg/kg b.i.d. at 4-h intervals for five consecu-4

affinity of PPX for binding to D receptors, both pre- and tive days. The dose of MPTP was reduced to produce a3

post-synaptically, when compared to its affinity for either lesion equivalent to that produced in young animalsD or D receptors [2,19]. Similarly a greater affinity for without causing excessive mortality [31]. Six of the 122 4

binding to presynaptic D autoreceptors as opposed to animals received PPX 30 min prior to each MPTP in-2

post-synaptic D receptors has been identified. The clinical jection and once daily for an additional 14 days after2

relevance of this binding specificity is not clear. The major cessation of MPTP. The remaining six animals that re-concentration of D receptors is within the mesolimbic and ceived co-administration of MPTP and PPX received3

mesocortical DAergic pathways (primarily associated with saline injections for 14 days after MPTP administration.mood regulation), but D receptors have also been iden- Six additional animals received only PPX and four animals3

tified within the Islands of Calleja and the dorsal striatum received only saline for 19 days (5 days b.i.d. followed by[29]. Pramipexole has been shown to reduce the levels of 14 days q.d.).extracellular DA in a dose-dependent manner [19] andappears to possess antioxidant properties [4,5,10,32,43]. 2.2. Tissue collection and DA analysisPramipexole can also protect against postischemic ormetamphetamine-induced degeneration as well as MPTP, Brains were removed fresh and the striatum was dissec-6-hydroxydopamine and levodopa-induced toxicity and ted out over ice, snap frozen in powdered dry ice andresultant nigral cell death [4,10,32,40,43]. stored at 2808C until analyzed. Striatal tissue was ana-

In view of the putative neuroprotective effects of PPX lyzed for DA, dihydroxyphenylacetic acid (DOPAC) andreported elsewhere, the present study was designed to homovanilic acid (HVA) by high performance liquidassess the neuroprotective potential of PPX on striatal DA chromatography (HPLC) with electrochemical detectionlevels and DA cell survival in the substantia nigra pars (BAS, Inc., Lafayette, IN). Briefly, tissue was sonicated incompacta (SNc) in young and aged MPTP-treated mice. 0.4 N perchloric acid, and then centrifuged at 15 000

rev. /min at 48C for 5 min. The supernatant was transferredinto fresh tubes and combined with internal standard

2. Material and methods (isoproterinol) prior to filtration. All samples were thenstored on ice until analyzed. Fifty microliters of sample

2.1. MPTP/PPX administration were injected into a 100 ml injection loop attached to aRheodyne injector. Mobile phase (0.2 M NaH PO , 2.52 4

All animals used in this study were cared for according mM sodium octane sulphonate, 0.1 mM EDTA, 10%to institutional and NIH animal care and use guidelines. methanol, at pH 3.5) was pumped through a 10 cm, 0.3Ninety young (8-week-old) and 22 aged (12-month-old) mm ODS cartridge column (Bioanalytical Systems, Inc.,male C57Bl /6J mice (Taconic Animal Research, German- West Lafayette, IN). Peak heights were compared againsttown, NY) were used for this study. known external standard concentrations and isoproterinol

Fifty-four 8-week-old animals received MPTP–HCl internal standards using EZChrom Chromatography Data(Research Biochemical Inc., Natick, MA), dissolved in System software (Scientific Software, Inc., San Ramon,physiological saline and administered subcutaneously at a CA). Means and S.E.M.s were calculated for DA andconcentration of 20 mg/kg b.i.d. at 4-h intervals for five DOPAC levels followed by one-way analysis of varianceconsecutive days. Thirty-six of these 54 animals also and post-hoc group comparisons (Bonferoni t-test).received intraperitoneal (i.p.) injections of PPX (1.0 mg/kg; Pharmacia & Upjohn, Kalamazoo, MI) dissolved in 2.3. Tyrosine hydroxylase immunoreactivity /Nisslphysiological saline and administered 30 min prior to each stainingMPTP injection. The remaining 18 MPTP-treated animalsreceived saline injections 30 min prior to each MPTP The caudal extent of the brain (including the SN) wasinjection. After cessation of MPTP treatment the 36 submersion-fixed in 4% paraformaldehyde for 1 week andanimals that received co-administration of MPTP and PPX transferred to a 30% sucrose solution. Serial coronalreceived an additional 2 (N56) or 14 (N512) days of PPX sections through the full rostro-caudal extent of the sub-or 2 (N56) or 14 (N512) days of saline before being stantia nigra were cut frozen at a thickness of 30 mm andeuthanized. The 18 animals that received co-administration collected into cold 0.1 M phosphate buffered saline (PBS).of MPTP and saline for 5 days received either an addition- Tyrosine hydroxylase (TH) immunohistochemistry wasal 14 (N56) days of PPX or 2 (N56) or 14 (N56) days of performed on free-floating sections. After a peroxide /saline administration. Six animals received only PPX and methanol quench of endogenous peroxidase activity, sec-eight animals received only saline for 19 days (5 days tions were incubated in 1% bovine serum albumin (BSA)b.i.d. followed by 14 days q.d.). All treatment groups are in PBS for 45 min at room temperature. The sections werelisted in Table 1. incubated overnight at 48C with rabbit anti-TH (Pel-Freeze

46 D.W. Anderson et al. / Brain Research 905 (2001) 44 –53

Inc., Rogers, AR) at a 1:500 dilution in BSA. After rinsing defined inclusion and exclusion limits within the area ofwith 0.1 M PBS, sections were incubated in biotinylated interest was analyzed for the presence of clearly definablegoat anti-rabbit antibody (Vector Labs, Burlinghame, CA) neurons (the presence of a nucleus or nucleolus) and thein 1% BSA for 2 h, rinsed and incubated in Streptavidin number of neurons per dissector was counted. The optical(Vector Labs, Burlinghame, CA) for 1 h at room tempera- dissector was then moved randomly by the computer toture. After several PBS rinses, sections were reacted with another area within the SNc, and another cell count was0.5% diaminobenzidine-tetrahydrochloride and 0.025% generated. This process was repeated for TH-IR, Nissl andH O , rinsed, mounted in sequential order (rostral–caudal) FG-labeled sections. Estimation of total neuron number2 2

and cover-slipped. An alternate set of sections not used for (based upon actual cell counts, tissue thickness (30 mm),immunohistochemistry was stained with cresyl violet as total area of designated region and the total number ofdescribed previously [30]. sections (n58) analyzed per animal) and group means and

variances were produced. Statistical analysis of group2.4. Fluorogold labeling of SNc neurons differences was assessed by analysis of variance (ANOVA)

with pairwise comparisons performed using post-hoc t-Mice (n518) were anesthetized with ketamine /xylazine tests and the Bonferroni correction.

(25 mg/ml:1.67 mg/ml i.p.) and placed in a stereotaxicframe. Body temperature was maintained at 378C using ahomeothermic heating pad. Burr holes were drilled in the 3. Resultsskull overlying the striatum (B: 0.5, L: 1.8, D: 2.8)according to the atlas of Franklin and Paxinos [8]. Approx- 3.1. MPTP-induced DA depletionimately 2 ml of Fluorogold (FG) (2% solution in 0.9%saline), was pressure ejected (Neurophore Medical Sys- In mice that received MPTP and no PPX, significanttems, NY) unilaterally into the striatum using a glass reductions in striatal DA levels were observed. In youngmicropipette (50 mm tip diameter), which was left in place mice, striatal DA levels were depleted by 8661% andfor 5 min after injection and then withdrawn. Sterile bone 8362% at 2 days (Table 1, Group 6) and 14 days (Tablewax was placed in the skull defect and the scalp was 1, Group 2), respectively, after cessation of MPTP ad-sutured. Seven days following the FG injection animals ministration. In aged mice, striatal DA levels were de-were assigned to one of the following treatment groups: 5 creased by 8762% at 14 days (Table 2, Group 12) afterdays of MPTP (20 mg/kg b.i.d.) followed by 14 days MPTP. MPTP also significantly decreased DOPAC levelssaline; 5 days MPTP/PPX (1.0 mg/kg PPX 30 min prior and increased DOPAC/DA ratios (Tables 1 and 2).to each MPTP injection) followed by 14 days PPX; saline Co-administration of MPTP and PPX in young animalsonly control group. At the end of the study, the animals significantly attenuated the MPTP-induced striatal DAwere anesthetized with sodium pentobarbitol (25 mg/kg depletion (t56.24; P,0.001: Table 1, Group 4). Animalsi.p.) and perfused transcardially with cold PBS followed by that received PPX prior to each MPTP injection, plus an4% paraformaldehyde. Brains were removed, post fixed for additional 2 or 14 days of PPX treatment (Table 1, Groups48 h, then placed in cryopreservative (30% sucrose). Serial 8 and 5, respectively) had significantly higher striatal DAsections (30 mM) were cut through the rostro-caudal extent levels than animals that received MPTP/PPX co-adminis-of both the striatum and SNc, mounted, cover-slipped and tration and 2 or 14 days of subsequent saline treatmentanalyzed for injection site / size and location. Only animals (t53.18; P,0.05, and t53.08; P,0.05: Table 1, Groups 7with comparable striatal injections were used for unbiased and 4, respectively). Extending PPX treatment for 14 daysstereological assessment of FG-labeled SNc neurons. did not result in any greater attenuation of DA loss than

that observed in animals receiving only 2 additional days2.5. Cell counting using unbiased stereological of PPX treatment (t50.49; P.0.05) (Table 1).quantitation Animals that received MPTP/PPX co-administration and

no additional PPX treatment still had higher striatal DACoded slides were analyzed blindly for total number of levels at 2 (t52.70; P,0.05: Table 1, Group 7) and 14

Nissl-stained neurons and total number of TH-IR neurons days (t53.69; P,0.01: Table 1, Group 4), than animalsin the SNc using a three-dimensional, fractionator based that did not receive any PPX (Table 1, Group 3). MPTP-unbiased stereology software program, StereologerE (SPA treated animals given PPX for 14 days beginning only afterInc., MD) on a Mac 9600 computer attached to an completion of MPTP administration also had significantlyOlympus BX-90 microscope fitted with a motorized stage. greater striatal DA levels than animals treated with MPTPA similar analysis was performed on FG-labeled cells. At and administered saline for 14 days (t55.37; P,0.001:low magnification (43), the SNc was outlined and a Table 1, Group 2). However, post-MPTP treatment withrandom sampling grid was created within the designated PPX was not as effective in sparing striatal DA levels asarea at high magnification (1003 oil immersion). Using an co-administration of MPTP/PPX (t52.59; P,0.05) (Fig.optical dissector probe, the randomly assigned area with 1).


Table 1Effects of MPTP and pramipexole (PPX) on dopamine (DA) and dihydroxyphenylacetic acid (DOPAC) levels in the striatum of young mice

Treatment (Group number) N DA DOPAC DOPAC/DA

Normal (1) 8 10.5660.70 1.2160.28 0.1360.02a,b,c i m,n,oMPTP/Saline — 14 days saline (2) 6 1.4760.15 0.4960.04 0.3460.02d,e jMPTP/Saline — 14 days PPX (3) 6 2.7360.18 0.6760.04 0.2560.02f k pMPTP/PPX — 14 days saline (4) 12 3.9460.43 0.4360.05 0.1160.02

MPTP/PPX — 14 days PPX (5) 12 5.6160.43 1.4660.05 0.2660.01g qMPTP/Saline — 2 days saline (6) 6 1.7060.19 0.2960.04 0.2360.01h l rMPTP/PPX — 2 days saline (7) 6 3.0260.45 0.3960.04 0.1060.01

MPTP/PPX — 2 days PPX (8) 6 5.2660.49 1.1960.09 0.2260.02Saline /PPX — 14 days PPX (9) 3 10.3760.81 1.3760.16 0.1460.02Saline /PPX — 2 days PPX (10) 3 11.6760.88 1.6360.10 0.1460.01

Units for DA and DOPAC, mg/g wet tissue; N, number of animals. Numbers represent mean6S.E.M. Comparative analysis between groups: (a) P,0.001versus Group 3, (b) P,0.01 versus Group 4, (c) P,0.001 versus Group 5, (d) P,0.001 versus Group 5, (e) P,0.05 versus Group 4, (f) P,0.05 versusGroup 5, (g) P,0.05 versus Group 7, (h) P,0.05 versus Group 8, (i) P,0.01 versus Group 5, (j) P,0.01 versus Group 5, (k) P,0.01 versus Group 5, (l)P,0.01 versus Group 8, (m) P,0.05 versus Group 3, (n) P,0.01 versus Group 4, (o) P,0.05 versus Group 5, (p) P,0.01 versus Group 5, (q) P,0.01versus Group 7, (r) P,0.01 versus Group 8.

Table 2Effects of MPTP and pramipexole (PPX) on dopamine (DA) and dihydroxyphenylacetic acid (DOPAC) levels in the striatum of aged mice

Treatment (Group number) N DA DOPAC DOPAC/DA

Normal (11) 4 12.4861.22 1.3760.31 0.1160.02a b cMPTP/Saline — 14 days saline (12) 6 1.6660.19 0.3660.05 0.2260.02

PPX/Saline — 14 days PPX (13) 6 11.3060.70 1.9460.46 0.1460.03MPTP/PPX — 14 days PPX (14) 6 3.5660.52 0.4660.19 0.1460.06

Units for DA and DOPAC, mg/g wet tissue; N, number of animals; numbers represent mean6S.E.M. Comparative analysis between groups: (a) P,0.05versus Group 14, (b) P,0.05 versus Group 11, (c) P,0.01 versus Group 11.

Fig. 1. Dopamine (DA) sparing effect of pramipexole (PPX) in young (A) and aged (B) mice. (A) In young animals, MPTP administration followed by 14days of saline administration caused a significant (86%) loss of striatal DA. When PPX was co-administered with MPTP and followed by either 2 or 14days of additional PPX administration, there was a significant sparing of striatal DA. Pramipexole administered for 14 days only after the cessation ofMPTP injection was less effective in sparing in striatal DA levels. (B) In aged animals, MPTP administration followed by 14 days of saline treatmentresulted in an 87% depletion of DA. Pramipexole co-administration with MPTP and followed by PPX for an additional 14 days resulted in only a 73%depletion of striatal DA levels. *P,0.05 versus (MPTP/PPX)114 days PPX, **P,0.05 versus (MPTP/PPX)114 days saline, ***P,0.05 versus(MPTP/PPX)12 days PPX, ****P,0.05 versus (MPTP/PPX)114 days PPX.


MPTP administration caused a significant increase in P,0.05: Table 3, Group 2). No significant change instriatal DOPAC/DA ratios in animals euthanized 14 days TH-IR neuron number was detected at 2 days followingafter the last MPTP injection (Table 1, Group 2). MPTP/ MPTP administration (Fig. 2A). The number of TH-IRPPX co-administration followed by 14 days of saline SNc neurons was also decreased by 3763% (t536.75;injections (Table 1, Group 4) normalized DOPAC/DA P,0.01: Table 3, Group 12) 14 days post-MPTP in agedratios whereas MPTP/PPX co-administration followed by mice (Fig. 2B). In both young and aged mice, the loss of14 days of PPX did not (Table 1, Group 5). Pramipexole TH-IR neurons mirrored the loss of Nissl-stained neuronstreatment alone for 7 or 19 days did not significantly alter in the SNc (Table 3). The number of FG-labeled SNcthe levels of DA, DOPAC or the DOPAC/DA ratio in neurons was also significantly reduced at 14 days afternormal animals (Table 1). MPTP exposure (t552.91; P,0.01) (Figs. 4 and 5).

Aged mice co-administered PPX and MPTP followed by Young animals co-administered MPTP/PPX followed bycontinued PPX administration for 14 days (Table 2, Group 14 additional days of PPX treatment had significantly more14) had significantly higher striatal DA levels compared to TH-IR SNc neurons (t520.46; P,0.01: Table 3, Group 5)MPTP-saline treated animals (t52.16; P,0.05: Table 2, and more FG-labeled SNc neurons (t58.54; P,0.05) (Fig.Group 12). The degree to which PPX attenuated MPTP- 4) than animals that received MPTP only followed by 14induced striatal DA loss in aged mice (approximately a days of saline (Figs. 3–5). No attenuation of TH-IR SNctwofold sparing of striatal DA levels), was significantly cell loss was found with co-administration of MPTP/PPXless than that observed in similarly treated young animals followed by saline for 14 days, or when PPX was onlymice (approximately a fourfold sparing of striatal DA administered following cessation of MPTP treatment.levels) (t53.5; P,0.01) (Table 2). Pramipexole treatment alone for 7 or 19 days did not result

in statistically significant increases in the number of TH-IR3.2. Pramipexole effects on MPTP-induced cell loss in or Nissl-stained neurons in the SNc (Table 3). Further-the SNc more, analysis of TH-IR cell soma size showed that

following MPTP administration the remaining TH-IR cellsAdministration of MPTP to young mice produced a had reduced cell area /volume when compared to normal

3465% (Fig. 2A) reduction in the number of SNc TH-IR animals (t516.50; P,0.01: Table 4). The size of TH-IRneurons assessed 14 days after MPTP exposure (t516.08; cells in animals co-administered MPTP/PPX followed by

Fig. 2. Tyrosine hydroxylase immunoreactive (TH-IR) cell counts in pramipexole (PPX) treated young (A) and aged (B) mice. (A) In young animals,MPTP administration followed by 14 days of saline resulted in a significant decrease in the number of TH-IR cells in the SNc. When PPX wasco-administered with MPTP and followed by 14 days of additional PPX treatment, the MPTP-induced loss of TH-IR cells in the SNc was completelyblocked. Pramipexole co-administration with MPTP but without any additional PPX administration did not have a significant effect on the TH-IR cellsurvival, nor did PPX administration only after cessation of MPTP administration. There was a slight decrease in TH-IR cell counts 2 days after the lastMPTP injection, which was also blocked by PPX administration. (B) In aged animals, MPTP administration caused a significant loss of TH-IR cells in theSNc which, as in young animals, was significantly attenuated by PPX administration. *P,0.01 versus MPTP only: 14 days saline, **P,0.01 versusMPTP/PPX: 14 days saline.


Table 3Effects of MPTP and pramipexole (PPX) on tyrosine hydroxylase immunoreactive and Nissl-stained neurons within the substantia nigra pars compacta ofyoung and aged mice

Treatment (Group number) Age n TH cells Nissl cells

Normal (1) 8 weeks 5 45466232 49456168a,b g,hMPTP/Saline — 14 days saline (2) 8 weeks 5 29846235 36096188cMPTP/Saline — 14 days PPX (3) 8 weeks 5 36286267 44376642d i,jMPTP/PPX — 14 days saline (4) 8 weeks 5 34036254 34346325

MPTP/PPX — 14 days PPX (5) 8 weeks 5 50526374 46476298MPTP/Saline — 2 days saline (6) 8 weeks 5 42376294 41116301MPTP/PPX — 2 days saline (7) 8 weeks 5 48566182 47476359MPTP/PPX — 2 days PPX (8) 8 weeks 5 48016269 51536297Saline /PPX — 14 days PPX (9) 8 weeks 3 61406354 56686174Saline /PPX — 2 days PPX (10) 8 weeks 3 51196324 51896100

Normal (11) 12 months 4 61686347 61146238e kMPTP/Saline — 14 days saline (12) 12 months 6 38936181 36116202

PPX/Saline — 14 days PPX (13) 12 months 5 60016198 60766174f lMPTP/PPX — 14 days PPX (14) 12 months 5 50896283 55846363

Comparative analysis between groups: (a) P,0.01 versus Group 1, (b) P,0.01 versus Group 5, (c) P,0.05 versus Group 5, (d) P,0.01 versus Group 5,(e) P,0.01 versus Group 11, (f) P,0.05 versus Group 12, (g) P,0.01 versus Group 1, (h) P,0.05 versus Group5, (i) P,0.01 versus Group 1, (j)P,0.05 versus, Group 5, (k) P,0.01 versus Group 11, (l) P,0.01 versus Group 12.

Fig. 3. Photomicrographs of TH-IR cells in the SNc of normal (A, B), MPTP-treated (C, D), and MPTP/PPX-treated (E, F) mice. Mice receiving MPTPfollowed by 14 days of saline had significant loss of SNc TH-IR cells (C, D), compared to untreated animals (A, B). In comparison, animalsco-administered MPTP/PPX and 14 days of additional PPX had a significant sparing of TH-IR cells compared to lesioned control animals. Note theapparent increase in cell size in the PPX-treated animal (bar5100 mM).


4. Discussion

Previous studies have suggested that the DA receptoragonist, PPX, may be neuroprotective against differenttypes of damage [10,13,32], including that caused byMPTP [13,41,43]. It has also been suggested that PPX mayreduce reactive oxygen species formed in DAergic terminalfields through DA auto-oxidation [24], as well as inhibitthe formation of mitochondrial permeability transitionpores after MPTP administration [5].

In the current study, co-administration of PPX andMPTP to young or aged mice, followed by 2 or 14 days ofadditional PPX treatment, significantly attenuated MPTP-induced striatal DA loss as well as loss of TH-IR andNissl-stained SNc neurons. The neuroprotective effect ofPPX on SNc neurons was further confirmed in studiesshowing enhanced survival of FG-labeled SNc cells inMPTP-treated animals that received PPX treatment. Thesedata suggest that PPX administration protected SNc DAer-gic neurons from dying rather than merely protecting theDAergic phenotype of these cells.

In young MPTP-treated animals co-treated with MPTPand PPX followed by a further 14 days of PPX, TH-IRSNc neuron number was not significantly different from

Fig. 4. Counts of Fluorogold (FG)-labeled SNc dopaminergic cells in normal (Table 3). Yet, striatal DA levels in these animalsMPTP and pramipexole (PPX) treated young mice. MPTP administration

were only 53% of normal. Tyrosine hydroxylase inactiva-followed by 14 days of saline resulted in a significant loss of FG-labeledtion occurs in SNc neurons through nitration followingnigrostriatal projection neurons. In comparison, PPX co-administeredMPTP administration [1]. Although nitration of a singlewith MPTP and followed by an additional 14 days of PPX administration

caused a significant sparing of FG-labeled neurons. Insert shows a tyrosine residue can inactivate TH activity in vitro and in^representative size and location of the striatal FG injection. P,0.05 vivo [1], it does not alter TH-IR, therefore a reduction in

versus normal; **P,0.01 versus MPTP only; ***P,0.01 versus normal.striatal DA levels can be observed without reduction inTH-IR. Thus, PPX may protect more fully against MPTP-induced SNc DAergic cell death but only partially protectagainst MPTP-induced TH-inactivation and loss of striatal14 additional days of PPX treatment was significantlyDA levels.increased compared to animals that received only MPTP

The attenuated neuroprotective effects of PPX in agedand to normal untreated animals (P.0.01, Table 4).(12-month-old) animals are likely due to an inherentTwelve-month-old animals co-administered MPTP/PPXdecrease in responsiveness to whatever neuroprotectivefollowed by 14 days of continued PPX administration alsosignals are modulated or stimulated by PPX treatment.had less loss of TH-IR and Nissl-stained SNc neuronsAttenuated responses to neuroprotective signals in aged(t511.41; P,0.05, t529.85; P,0.01, respectively) com-SNc neurons have also been observed in other studies [31].pared to lesioned control animals. The protective effect ofThe aged mice used in this study were only 12 months oldPPX on TH-IR/Nissl-stained SNc neurons in aged animalsor ‘middle-aged’ for a mouse. The neuroprotective effectswas of a lesser magnitude than that observed in youngof PPX may be even further attenuated in older animals. Aanimals (Table 3).reduced neuroprotective effect of PPX with increasing agehas potentially important clinical implications. These datasuggest that early intervention with PPX in younger PDpatients may be preferable to achieve the full advantage ofTable 4this drug’s putative neuroprotective properties.Effects of MPTP and pramipexole (PPX) on tyrosine hydroxylase

immunoreactive neuron size and area Animals treated with PPX at the time of MPTP adminis-tration had significantly less striatal DA loss than animalsTreatment (Group number) Cell area Cell volumetreated with PPX for 14 days beginning only after the

Normal (1) 6.3260.21 19.5860.59a,b d,e conclusion of MPTP administration. However, the degreeMPTP/Saline — 14 days saline (2) 5.4360.14 17.0760.33c f of TH-IR cell loss in both PPX administration conditionsMPTP/PPX — 14 days PPX (5) 7.9460.25 22.9060.35

was the same. This suggests that the presence of PPX atComparative analysis between groups: (a) P,0.01 versus Group 1, (b)the time that MPTP-induced damage is occurring isP,0.01 versus Group 5, (c) P,0.01 versus Group 1, (d) P,0.01 versus

Group 1, (e) P,0.01 versus Group 5, (f) P,0.01 versus Group 1. important for at least partially protecting DA synthetic


Fig. 5. Photomicrographs of FG-labeling in the SNc of: normal (A, B), MPTP-treated (C, D) and MPTP/PPX (E, F) mice. Mice receiving MPTP followedby 14 days of saline had significant loss of SNc FG-labeled cells (C, D), compared to untreated animals (A, B). In comparison, animals co-administeredMPTP/PPX and 14 days of additional PPX had a significant sparing of FG-labeled cells (bar5250 mM).

capacity. Furthermore, PPX can not only protect SNc remain unclear. Pramipexole-induced autoreceptor stimula-DAergic neurons from the early damaging effects of MPTP tion can lead to inhibition of DA synthesis [18], reducebut can also attenuate the slow degenerative process that extracellular levels of DA [3], and metabolites within theoccurs during the 2-week post MPTP period when SNc DA striatum, and decrease the levels of DA auto-oxidativeneurons continue to die [39]. products (i.e. formation of highly reactive and potentially

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