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Effects of Different Schedules of MPTP Administration on Dopaminergic Neurodegeneration in Mice
EXPERIMENTAL NEUROLOGY ARTICLE NO.
148, 288–292 (1997)
EN976648
Effects of Different Schedules of MPTP Administration on Dopaminergic Neurodegeneration in Mice Erwan Bezard,1 Sandra Dovero, Bernard Bioulac, and Christian Gross Basal Gang, Laboratoire de Neurophysiologie, CNRS UMR 5543, Universite´ de Bordeaux II, 146 rue Leo Saignat, 33076 Bordeaux Cedex, France Received April 9, 1997; accepted July 15, 1997
Although a valuable 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) animal model of human Parkinson’s disease has been developed, our knowledge of the course of nigral degeneration remains fragmentary. Experimental factors which could possibly influence the destructive process must be taken into account. To evaluate the impact of experimental design, we compared the effects of different schedules of injection of the same cumulative dose of MPTP, in mice, by measuring tyrosine hydroxylase immunoreactivity in the substantia nigra pars compacta. Massive injection of the total dose over 1 day (4 injections of 20 mg/kg) destroyed more dopaminergic neurons than did the longterm daily injections of a lower dose of MPTP (20 injections of 4 mg/kg). This suggests that different schedules of administration of MPTP might induce different mechanisms of neuronal death. These mechanisms need to be better understood if chronic models of intoxication that replicate the evolution of human Parkinson’s disease more precisely are to be developed. r 1997 Academic Press
INTRODUCTION
The principal pathological characteristic of Parkinson’s disease (PD) is the progressive death of pigmented neurons in the substantia nigra pars compacta (SNc) (12), since identified as nigrostriatal dopamine neurons (8). In order to better understand both the physiological and the physiopathological mechanisms which underly normal and parkinsonian motor behaviors, it has been necessary to develop animal models of PD (9, 10). The identification of a specific neural toxin, 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP), that induces parkinsonism in human and nonhuman primates (5) has led to the development of a valuable animal model for this disease. The systemic administration of
1 To whom correspondence should be addressed. Fax: (33) 5 56 90 14 21. E-mail: [email protected].
0014-4886/97 $25.00 Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.
MPTP to nonhuman primates has been shown to cause the appearance of bradykinesia, rigidity, tremor, and postural abnormalities (1, 19, 25). Other models have also been developed, such as the mouse model (10, 11, 13, 18). Although the MPTP model is now widely used, the mechanisms responsible for the destruction of dopaminergic neurons have not yet been completely elucidated. Several factors can influence the dopaminergic degenerative process, such as genus-dependent variations in sensitivity to MPTP, individual susceptibility, and age (10, 13). It is known that different protocols of administration of the same dose of MPTP (mode of injection, sc or ip, number and frequency of injections) can produce varying degrees of dopamine loss (14, 21, 28). It is, however, difficult to draw general conclusions from these results, insofar as they were obtained by different research teams using different protocols under different experimental conditions. It is therefore important to investigate the impact of experimental design on dopaminergic neuronal death. The present study on the mouse provides an illustration of the variations that can be observed in the number of tyrosyne hydroxylase-immunoreactive (THIR) neurons in the SNc when different schedules of injection of the same cumulative dose of MPTP are used. MATERIAL AND METHODS
Animals Eighty 8-week-old OF1 male mice (Iffa-Credo, Saint Germain s/l’Arbresle, France) weighing 34–36 g were housed in a temperature-controlled room under a 12-h light/dark cycle with free access to food and water. Their care was supervised by veterinarians skilled in the health care and maintenance of mice. Animals were sacrificed under pentobarbital (Sanofi, Libourne, France) anesthesia. Our laboratory operates under the guidelines laid down by the National Institutes of
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Health and is recognized by the French Ministry of the Environment. Experimental Protocol Series I. Eight mice were used as controls (Fig. 1) and received four saline injections at 2-h intervals. These were sacrificed 7 days later. Series II. Thirty-two mice were treated with daily (9:00 AM) injections of MPTP hydrochloride (4 mg/kg, ip; Sigma, St. Quentin Fallavier, France) in saline for 20 days (from D0 to D20). Every 5 days, 8 mice were withdrawn and kept without further injections for another 7 days, before being sacrificed. This time interval has been found to be sufficient for degeneration of the TH-IR neurons in the mouse midbrain (16). We thus produced, at the end of the experiment, four subgroups which had received daily MPTP treatment for periods ranging from 5 (20 mg/kg) to 20 days (80 mg/kg) (Fig. 1). Series III. Thirty-two mice were treated with up to four injections of MPTP (20 mg/kg, ip) performed at D1, D7, D14, and D20. Every week, 8 mice were withdrawn and kept without further injections for an additional week, before being sacrificed. We again produced four groups which had received weekly MPTP treatment: the first at D1 (20 mg/kg); the second at D1 and D7 (40 mg/kg); the third at D1, D7, and D14 (60 mg/kg); and the fourth at D1, D7, D14, and D20 (80 mg/kg) (Fig. 1). Series IV. Eight mice received four injections of MPTP (20 mg/kg, ip) at 2-h intervals. We thus produced a single group which had received a cumulative dose of 80 mg/kg in 1 day (Fig. 1). These animals were likewise sacrificed 7 days later. Tissue Processing Mice were anesthetised with pentobarbital (30 mg/ kg, ip) and perfused intracardially with 25 ml of normal
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saline followed by 75 ml of 4% paraformaldehyde in saline. Brains were removed, postfixed in the same fixative for 2 days at 4°C, immersed in 20% sucrose in saline, frozen by slow immersion in isopentane cooled on dry ice, and then stored at 280°C before sectioning. Cryostat-cut sections (30 µm) encompassing the entire mesencephalon were collected free floating for each mouse. TH Immunohistochemistry Sections were processed for visualization of tyrosine hydroxylase immunoreactivity. Serum containing TH antibody (anti-TH) (Jacques Boy, Reims, France) was diluted 1:2000 in TBS with 0.15% Triton X-100 and 0.25% bovine serum albumin and sections were incubated overnight at 4°C. After triple rinsing in TBS, these were incubated for 2 h at room temperature in goat anti-rabbit IgG serum (Biosys, Compie`gne, France) diluted 1:200 in TBS with 0.25% bovine serum albumin. They were then rinsed again in TBS and incubated in rabbit peroxidase–anti-peroxidase complex (Dako, Trappes, France) diluted 1:500 for 2 h. After more thorough rinsing, followed by treatment with 3,38-diaminobenzidine tetrahydrochloride (Sigma) diluted in TBS and H2O2, slices were then rinsed 3 3 1 min in TBS, mounted on gelatin-coated slides, dried, dehydrated in gradated concentrations of ethanol, cleared in xylene, and coverslipped in Neoantelan (Polylabo, Strasbourg, France). Quantitative Analysis of Immunostained Neurons TH-immunoreactive cell counts were performed, as previously described (2), using an imaging system (CCD camera, Hamamatsu) connected up to a Macintosh II Cx computer equipped with an image program (image 1.31n, NIMH). The final magnification was 1003.
FIG. 1. Experimental protocol. White square, saline injection; black squares, MPTP injection. The size of the square represents the volume of the dose injected. The mice of Series I are the control group and have received saline injections. Mice of Series II received a daily MPTP injection (4 mg/kg), and those of Series III received a weekly MPTP injection (20 mg/kg for a period up to 20 days). Mice of Series IV received the maximum total dose (80 mg/kg) in 1 day.
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Three sections corresponding to a representative median plane of the SNc were retained for each mouse. This plane is exactly in the middle of the rostrocaudal axis of the SNc (22). For quantification, the SNc of each hemisphere was first delimited and then, using the mouse-driven ‘‘location’’ function of the software, marks were placed on each TH-immunoreactive cell body. The number of neurons per SNc was calculated three times for each hemisphere by one examiner blind with regard to the experimental condition, as previously described (2). ANOVA with post hoc analyses (Student–Newman– Keuls multiple comparisons test) was used to compare results. RESULTS
The MPTP-treated mice presented parkinsonian motor abnormalities as generally reported in literature (10, 14). No significant differences were found in any instance between the number of TH-IR neurons on the left and right sides of the SNc (P . 0.5). The average number of TH-IR neurons was determined for each mouse (i.e., average of the six SNc values for each animal). Means and SD were then calculated from these average values for each group of eight mice. Whatever the dose or schedule of administration of MPTP, we observed a significant reduction (P , 0.001) in the mean number of TH-IR neurones for all the treated Series (II, III, IV) compared to controls (200 6 4 TH-IR neurons). For both Series II and Series III, there was a significant difference in the number of TH-IR neurons between any one dose of MPTP and the previous dose given (P , 0.001, except between 40 mg/kg group and 60 mg/kg group of Series III; P , 0.01). As far as the schedule of administration was concerned, significant differences were also observed between Series II and III in the number of TH-IR neurons for 20 and 40 mg/kg (P , 0.001) and for 60 mg/kg (P , 0.01) (Fig. 2). For the highest cumulative dose (80 mg/kg), a significant difference was observed between Series II (54 6 2 TH-IR neurons) and Series III (49 6 4 TH-IR neurons) (P , 0.01), Series II and Series IV (43 6 3 TH-IR neurons) (P , 0.001), and Series III and Series IV (P , 0.001) (Fig. 2). Figure 3 shows samples of mesencephalic slices from controls (Series I; Fig. 3A), Series II (Fig. 3B), Series III (Fig. 3C), and Series IV (Fig. 3D). DISCUSSION
Our present results show that the pattern of MPTP injection has a significant impact on the number of TH-IR neurons of the SNc in the mouse. It has already been shown that the slow progression of neuronal death
FIG. 2. Mean number (6SD) of TH-IR neurons of controls (Series I); of mice treated with 20, 40, 60, and 80 mg/kg of Series II and III; and of mice treated with 80 mg/kg of Series IV. n 5 8 in all groups. *Comparison between each Series and controls (P , 0.001). **Comparison between Series III or IV and Series II (minimal significant P , 0.01). ***Comparison between Series III and Series IV (P , 0.001).
induced by the daily administration of low doses of MPTP closely resembles the evolution observed in human Parkinson’s disease (2, 3). Our present study shows that gradual intoxication (Series II) causes less severe lesions than acute intoxication (Series IV). A progression of damage between Series II and Series III, and Series III and Series IV, can be seen. Since the pattern of administration has an impact on the extent of nigral degeneration, it is clear that the action of MPTP varies according to the schedule and the concentration of injection. There is still an important debate concerning the precise mechanism of cell death induced by MPTP. Neurotoxic effects have been shown to be due to its oxidation product, 1-methyl-4phenylpyridinium (MPP1), which is actively transported into the presynaptic dopaminergic nerve terminals through the plasma membrane dopamine transporter (4, 15, 17, 24). Once inside the cell, MPP1 is taken up into the mitochondria where it impairs respiration (6, 7). This may lead to the formation of superoxide radicals (23) which in turn raise Ca21 concentrations in the cytosol to cytotoxic levels, finally leading to cell death (26).
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FIG. 3. (A) SNc of a control mouse (197 TH-IR neurons); (B) SNc of an 80 mg/kg treated mouse of Series II (57 TH-IR neurons); (C) SNc of an 80 mg/kg treated mouse of Series III (47 TH-IR neurons); (D) SNc of an 80 mg/kg treated mouse of Series IV (44 TH-IR neurons).
It is possible that the schedule of MPTP administration determines the selective activation of a particular mechanism of neuronal death. If involvement of the plasma membrane dopamine transporter is critical for MPP1 toxicity, vesicular uptake, when MPP1 is in the cytosol of the dopaminergic neuron, may be of particular importance. The susceptibility of dopaminergic neurones to MPTP may be linked to their low vesicular amine transporter activity (20), compared with other aminergic populations. This dopaminergic neuronal characteristic can explain why the chronic administration of low doses of MPTP is less toxic than the acute injection of large doses because synaptic vesicles may be more efficient in sequestering small amounts of MPP1, keeping it away from the mitochondria, compared to larger doses of the toxin. However, this is probably not the only explanation for such observations.
The present study makes two points: (i) the same cumulative dose of MPTP injected in different quantities over the same total period (Series II and III) produces different degrees of nigral degeneration; and (ii) a massive brief intoxication causes more damage than does the same dose injected at a low dosage over a longer period (Series II and III compared to Series IV). These data have been corroborated by a previous study on striatal dopamine concentration in different strains of mice following different MPTP dosing schedules (27), although the study in question only investigated variations over a period of 2 days. It is clear, for any given cumulative dose, that the schedule of administration of MPTP has an impact on the extent of nigral degeneration as demonstrated by the number of dopaminergic neurons destroyed. Since different degrees of toxicity exist according to the way in which MPTP treatment is given, future studies on
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the physiopathology of the basal ganglia using this model should take into account the schedule, duration, and concentration of MPTP administration. The development of animal models replicating more accurately the progressive and continual neuronal death observed in human parkinsonism is essential for our understanding of the pathological process of nigral degeneration. If different patterns of injection lead to different mechanisms of neurotoxicity, the neurotoxic action of MPTP on dopaminergic neurons should be investigated in more chronic protocols of administration in order to develop a more realistic model to improve our understanding of the neurodegeneration of human Parkinson’s disease.
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ACKNOWLEDGMENTS
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We thank Miss Imbert for technical assistance. This study was supported in part by the CNRS and the IFR of Neuroscience (INSERM No. 8; CNRS No. 13) and in part by MESR Grant 95523629.
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Benazzouz, A., C. Gross, and B. Bioulac. 1992. MPTP-induced hemiparkinsonism in monkeys: Behavioral, mechanographic, electromyographic and immunohistochemical studies. Exp. Brain Res. 90: 116–120. Bezard, E., S. Dovero, B. Bioulac, and C. Gross. 1997. Kinetics of nigral degeneration in a chronic model of MPTP-treated mice. Neurosci. Lett., in press. Bezard, E., C. Imbert, X. Deloire, B. Bioulac, and C. Gross. 1997. A chronic MPTP model reproducing the slow evolution of Parkinson’s disease: Evolution of motor symptoms in monkey. Brain Res. [in press] Buck, K. J., and S. G. Amara. 1994. Chimeric dopamine– norepinephrine transporters delineate structural domains influencing selectivity for catecholamines and 1-methyl-4-phenylpyridinium. Proc. Natl. Acad. Sci. USA 91: 12584–12588. Burns, R. S., C. C. Chiueh, S. P. Markey, M. H. Ebert, D. J. Jacobowitz, and I. J. Kopin. 1983. A primate model of parkinsonism: selective destruction of dopaminergic neurons in the pars compacta of the substantia nigra by N-methyl-4-phenyl-1,2,3,6tetrahydropyridine. Proc. Natl. Acad. Sci. USA 80: 4546–4550. DiMonte, D., S. A. Jewell, G. Ekstrom, M. S. Sandy, and M. T. Smith. 1986. MPTP and MPP1 cause rapid ATP depletion in isolated hepatocytes. Biochem. Biophys. Res. Commun. 137: 310–315. DiMonte, D., M. S. Sandy, and M. T. Smith. 1987. Increase efflux rather than oxidation is the mechanism of glutathione depletion by MPTP. Biochem. Biophys. Res. Commun. 148: 153–160. Ehringer, H., and O. Hornykiewicz. 1960. Verteilung von Noradrenalin und Dopamin (3-Hydroxytyramin) im Gehirn des Menschen und ihr Verhalten bei Erkrankungen des extrapyramidalen Systems. Klin. Wochenschr. 38: 1236–1239. Gash, D. M. 1989. Direct comparison of CNS directed therapies are needed. Neurobiol. Aging 10: 645–646. Gerlach, M., and P. Riederer. 1996. Animal models of Parkin
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son’s disease: An empirical comparison with the phenomenology of the disease in man. J. Neural Transm. 103: 987–1041. Gerlach, M., P. Riederer, H. Przuntek, and M. B. H. Youdim. 1991. MPTP mechanisms of neurotoxicity and their implications for Parkinson’s disease. Eur. J. Pharmacol. 208: 273–286. Hassler, R. 1938. Zur Pathologie der Paralysis agitans und des postenzephalitischen Parkinsonismus. J. Psychol. Neurol. 48: 387–476. Heikkila, R. E., and P. Sonsalla. 1991. The MPTP-treated mouse as a model of parkinsonism: How good is it? Neurochem. Int. 20: 299–303. Heikkila, R. E., B. A. Steber, L. Manzino, and P. K. Sonsalla. 1989. Some features of the nigrostriatal neurotoxin MPTP in the mouse. Mol. Chem. Neuropathol. 10: 171–183. Irwin, I., and J. W. Langston. 1985. The selective accumulation of MPP1 in the substantia nigra: A key to neurotoxicity? Life Sci. 36: 207–212. Jackson-Lewis, V., M. Jakowec, R. E. Burke, and S. Przedborski. 1995. Time course and morphology of dopaminergic neuronal death caused by the neurotoxin 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine. Neurodegeneration 4: 257–269. Javitch, J., and S. H. Snyder. 1985. Uptake of MPP1 by dopamine neurons explains selectivity of parkinsonism-inducing neurotoxin MPTP. Eur. J. Pharmacol. 106: 455–456. Kopin, I. J., and S. P. Markey. 1988. MPTP toxicity: Implication for research in Parkinson’s disease. Annu. Rev. Neurosci. 11: 81–96. Langston, J. W., L. S. Forno, C. S. Rebert, and I. Irwin. 1984. Selective nigral toxicity after systemic administration of 1methyl-4-phenyl-1-1,2,3,6,tetrahydropyridine (MPTP) in the squirrel monkey. Brain Res. 292: 390–394. Liu, Y., A. Roghani, and R. H. Edwards. 1992. Gene transfer of a reserpine-sensitive mechanism of resistance to N-methyl-4phenylpyridinum. Proc. Natl. Acad. Sci. USA 89: 9074–9078. Marien, M., M. Briley, and F. Colpaert. 1993. Noradrenaline depletion exacerbates MPTP-induced striatal dopamine loss in mice. Eur. J. Pharmacol. 236: 487–489. Nelson, E. L., C. L. Liang, C. M. Sinton, and D. C. German. 1996. Midbrain dopaminergic neurons in the mouse: Computerassisted mapping. J. Comp. Neurol. 369: 361–371. Rossetti, Z. L., A. Sotgiu, D. E. Sharp, M. Hadjiconstantinu, and N. H. Neff. 1988. MPTP and free radicals in vitro. Biochem. Pharmacol. 37: 4573–4574. Santiago, M., A. Machado, and J. Cano. 1996. Nigral and striatal comparative study of the neurotoxic action of 1-methyl4-phenylpyridinium ion: Involvement of dopamine uptake system. J. Neurochem. 66: 1182–1190. Schultz, W., A. Studer, G. Jonsson, E. Sundstru¨m, and I. Mefford. 1985. Deficits in behavioral initiation and execution processes in monkeys with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced parkinsonism. Neurosci. Lett. 59: 225–232. Shapira, A. H. V. 1994. Evidence for mitochondrial dysfunction in Parkinson’s disease—A critical appraisal. Mov. Disord. 9: 125–138. Sonsalla, P. K., and R. E. Heikkila. 1986. The influence of dose and dosing interval on MPTP-induced dopaminergic neurotoxicity in mice. Eur. J. Pharmacol. 129: 339–345. Sundstro¨m, E., A. Fredriksson, and T. Archer. 1990. Chronic neurochemical and behavioral changes in MPTP-lesionned C57BL/6 mice: A model for Parkinson’s disease. Brain Res. 528: 181–188.
148, 288–292 (1997)
EN976648
Effects of Different Schedules of MPTP Administration on Dopaminergic Neurodegeneration in Mice Erwan Bezard,1 Sandra Dovero, Bernard Bioulac, and Christian Gross Basal Gang, Laboratoire de Neurophysiologie, CNRS UMR 5543, Universite´ de Bordeaux II, 146 rue Leo Saignat, 33076 Bordeaux Cedex, France Received April 9, 1997; accepted July 15, 1997
Although a valuable 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) animal model of human Parkinson’s disease has been developed, our knowledge of the course of nigral degeneration remains fragmentary. Experimental factors which could possibly influence the destructive process must be taken into account. To evaluate the impact of experimental design, we compared the effects of different schedules of injection of the same cumulative dose of MPTP, in mice, by measuring tyrosine hydroxylase immunoreactivity in the substantia nigra pars compacta. Massive injection of the total dose over 1 day (4 injections of 20 mg/kg) destroyed more dopaminergic neurons than did the longterm daily injections of a lower dose of MPTP (20 injections of 4 mg/kg). This suggests that different schedules of administration of MPTP might induce different mechanisms of neuronal death. These mechanisms need to be better understood if chronic models of intoxication that replicate the evolution of human Parkinson’s disease more precisely are to be developed. r 1997 Academic Press
INTRODUCTION
The principal pathological characteristic of Parkinson’s disease (PD) is the progressive death of pigmented neurons in the substantia nigra pars compacta (SNc) (12), since identified as nigrostriatal dopamine neurons (8). In order to better understand both the physiological and the physiopathological mechanisms which underly normal and parkinsonian motor behaviors, it has been necessary to develop animal models of PD (9, 10). The identification of a specific neural toxin, 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP), that induces parkinsonism in human and nonhuman primates (5) has led to the development of a valuable animal model for this disease. The systemic administration of
1 To whom correspondence should be addressed. Fax: (33) 5 56 90 14 21. E-mail: [email protected].
0014-4886/97 $25.00 Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.
MPTP to nonhuman primates has been shown to cause the appearance of bradykinesia, rigidity, tremor, and postural abnormalities (1, 19, 25). Other models have also been developed, such as the mouse model (10, 11, 13, 18). Although the MPTP model is now widely used, the mechanisms responsible for the destruction of dopaminergic neurons have not yet been completely elucidated. Several factors can influence the dopaminergic degenerative process, such as genus-dependent variations in sensitivity to MPTP, individual susceptibility, and age (10, 13). It is known that different protocols of administration of the same dose of MPTP (mode of injection, sc or ip, number and frequency of injections) can produce varying degrees of dopamine loss (14, 21, 28). It is, however, difficult to draw general conclusions from these results, insofar as they were obtained by different research teams using different protocols under different experimental conditions. It is therefore important to investigate the impact of experimental design on dopaminergic neuronal death. The present study on the mouse provides an illustration of the variations that can be observed in the number of tyrosyne hydroxylase-immunoreactive (THIR) neurons in the SNc when different schedules of injection of the same cumulative dose of MPTP are used. MATERIAL AND METHODS
Animals Eighty 8-week-old OF1 male mice (Iffa-Credo, Saint Germain s/l’Arbresle, France) weighing 34–36 g were housed in a temperature-controlled room under a 12-h light/dark cycle with free access to food and water. Their care was supervised by veterinarians skilled in the health care and maintenance of mice. Animals were sacrificed under pentobarbital (Sanofi, Libourne, France) anesthesia. Our laboratory operates under the guidelines laid down by the National Institutes of
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Health and is recognized by the French Ministry of the Environment. Experimental Protocol Series I. Eight mice were used as controls (Fig. 1) and received four saline injections at 2-h intervals. These were sacrificed 7 days later. Series II. Thirty-two mice were treated with daily (9:00 AM) injections of MPTP hydrochloride (4 mg/kg, ip; Sigma, St. Quentin Fallavier, France) in saline for 20 days (from D0 to D20). Every 5 days, 8 mice were withdrawn and kept without further injections for another 7 days, before being sacrificed. This time interval has been found to be sufficient for degeneration of the TH-IR neurons in the mouse midbrain (16). We thus produced, at the end of the experiment, four subgroups which had received daily MPTP treatment for periods ranging from 5 (20 mg/kg) to 20 days (80 mg/kg) (Fig. 1). Series III. Thirty-two mice were treated with up to four injections of MPTP (20 mg/kg, ip) performed at D1, D7, D14, and D20. Every week, 8 mice were withdrawn and kept without further injections for an additional week, before being sacrificed. We again produced four groups which had received weekly MPTP treatment: the first at D1 (20 mg/kg); the second at D1 and D7 (40 mg/kg); the third at D1, D7, and D14 (60 mg/kg); and the fourth at D1, D7, D14, and D20 (80 mg/kg) (Fig. 1). Series IV. Eight mice received four injections of MPTP (20 mg/kg, ip) at 2-h intervals. We thus produced a single group which had received a cumulative dose of 80 mg/kg in 1 day (Fig. 1). These animals were likewise sacrificed 7 days later. Tissue Processing Mice were anesthetised with pentobarbital (30 mg/ kg, ip) and perfused intracardially with 25 ml of normal
289
saline followed by 75 ml of 4% paraformaldehyde in saline. Brains were removed, postfixed in the same fixative for 2 days at 4°C, immersed in 20% sucrose in saline, frozen by slow immersion in isopentane cooled on dry ice, and then stored at 280°C before sectioning. Cryostat-cut sections (30 µm) encompassing the entire mesencephalon were collected free floating for each mouse. TH Immunohistochemistry Sections were processed for visualization of tyrosine hydroxylase immunoreactivity. Serum containing TH antibody (anti-TH) (Jacques Boy, Reims, France) was diluted 1:2000 in TBS with 0.15% Triton X-100 and 0.25% bovine serum albumin and sections were incubated overnight at 4°C. After triple rinsing in TBS, these were incubated for 2 h at room temperature in goat anti-rabbit IgG serum (Biosys, Compie`gne, France) diluted 1:200 in TBS with 0.25% bovine serum albumin. They were then rinsed again in TBS and incubated in rabbit peroxidase–anti-peroxidase complex (Dako, Trappes, France) diluted 1:500 for 2 h. After more thorough rinsing, followed by treatment with 3,38-diaminobenzidine tetrahydrochloride (Sigma) diluted in TBS and H2O2, slices were then rinsed 3 3 1 min in TBS, mounted on gelatin-coated slides, dried, dehydrated in gradated concentrations of ethanol, cleared in xylene, and coverslipped in Neoantelan (Polylabo, Strasbourg, France). Quantitative Analysis of Immunostained Neurons TH-immunoreactive cell counts were performed, as previously described (2), using an imaging system (CCD camera, Hamamatsu) connected up to a Macintosh II Cx computer equipped with an image program (image 1.31n, NIMH). The final magnification was 1003.
FIG. 1. Experimental protocol. White square, saline injection; black squares, MPTP injection. The size of the square represents the volume of the dose injected. The mice of Series I are the control group and have received saline injections. Mice of Series II received a daily MPTP injection (4 mg/kg), and those of Series III received a weekly MPTP injection (20 mg/kg for a period up to 20 days). Mice of Series IV received the maximum total dose (80 mg/kg) in 1 day.
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Three sections corresponding to a representative median plane of the SNc were retained for each mouse. This plane is exactly in the middle of the rostrocaudal axis of the SNc (22). For quantification, the SNc of each hemisphere was first delimited and then, using the mouse-driven ‘‘location’’ function of the software, marks were placed on each TH-immunoreactive cell body. The number of neurons per SNc was calculated three times for each hemisphere by one examiner blind with regard to the experimental condition, as previously described (2). ANOVA with post hoc analyses (Student–Newman– Keuls multiple comparisons test) was used to compare results. RESULTS
The MPTP-treated mice presented parkinsonian motor abnormalities as generally reported in literature (10, 14). No significant differences were found in any instance between the number of TH-IR neurons on the left and right sides of the SNc (P . 0.5). The average number of TH-IR neurons was determined for each mouse (i.e., average of the six SNc values for each animal). Means and SD were then calculated from these average values for each group of eight mice. Whatever the dose or schedule of administration of MPTP, we observed a significant reduction (P , 0.001) in the mean number of TH-IR neurones for all the treated Series (II, III, IV) compared to controls (200 6 4 TH-IR neurons). For both Series II and Series III, there was a significant difference in the number of TH-IR neurons between any one dose of MPTP and the previous dose given (P , 0.001, except between 40 mg/kg group and 60 mg/kg group of Series III; P , 0.01). As far as the schedule of administration was concerned, significant differences were also observed between Series II and III in the number of TH-IR neurons for 20 and 40 mg/kg (P , 0.001) and for 60 mg/kg (P , 0.01) (Fig. 2). For the highest cumulative dose (80 mg/kg), a significant difference was observed between Series II (54 6 2 TH-IR neurons) and Series III (49 6 4 TH-IR neurons) (P , 0.01), Series II and Series IV (43 6 3 TH-IR neurons) (P , 0.001), and Series III and Series IV (P , 0.001) (Fig. 2). Figure 3 shows samples of mesencephalic slices from controls (Series I; Fig. 3A), Series II (Fig. 3B), Series III (Fig. 3C), and Series IV (Fig. 3D). DISCUSSION
Our present results show that the pattern of MPTP injection has a significant impact on the number of TH-IR neurons of the SNc in the mouse. It has already been shown that the slow progression of neuronal death
FIG. 2. Mean number (6SD) of TH-IR neurons of controls (Series I); of mice treated with 20, 40, 60, and 80 mg/kg of Series II and III; and of mice treated with 80 mg/kg of Series IV. n 5 8 in all groups. *Comparison between each Series and controls (P , 0.001). **Comparison between Series III or IV and Series II (minimal significant P , 0.01). ***Comparison between Series III and Series IV (P , 0.001).
induced by the daily administration of low doses of MPTP closely resembles the evolution observed in human Parkinson’s disease (2, 3). Our present study shows that gradual intoxication (Series II) causes less severe lesions than acute intoxication (Series IV). A progression of damage between Series II and Series III, and Series III and Series IV, can be seen. Since the pattern of administration has an impact on the extent of nigral degeneration, it is clear that the action of MPTP varies according to the schedule and the concentration of injection. There is still an important debate concerning the precise mechanism of cell death induced by MPTP. Neurotoxic effects have been shown to be due to its oxidation product, 1-methyl-4phenylpyridinium (MPP1), which is actively transported into the presynaptic dopaminergic nerve terminals through the plasma membrane dopamine transporter (4, 15, 17, 24). Once inside the cell, MPP1 is taken up into the mitochondria where it impairs respiration (6, 7). This may lead to the formation of superoxide radicals (23) which in turn raise Ca21 concentrations in the cytosol to cytotoxic levels, finally leading to cell death (26).
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FIG. 3. (A) SNc of a control mouse (197 TH-IR neurons); (B) SNc of an 80 mg/kg treated mouse of Series II (57 TH-IR neurons); (C) SNc of an 80 mg/kg treated mouse of Series III (47 TH-IR neurons); (D) SNc of an 80 mg/kg treated mouse of Series IV (44 TH-IR neurons).
It is possible that the schedule of MPTP administration determines the selective activation of a particular mechanism of neuronal death. If involvement of the plasma membrane dopamine transporter is critical for MPP1 toxicity, vesicular uptake, when MPP1 is in the cytosol of the dopaminergic neuron, may be of particular importance. The susceptibility of dopaminergic neurones to MPTP may be linked to their low vesicular amine transporter activity (20), compared with other aminergic populations. This dopaminergic neuronal characteristic can explain why the chronic administration of low doses of MPTP is less toxic than the acute injection of large doses because synaptic vesicles may be more efficient in sequestering small amounts of MPP1, keeping it away from the mitochondria, compared to larger doses of the toxin. However, this is probably not the only explanation for such observations.
The present study makes two points: (i) the same cumulative dose of MPTP injected in different quantities over the same total period (Series II and III) produces different degrees of nigral degeneration; and (ii) a massive brief intoxication causes more damage than does the same dose injected at a low dosage over a longer period (Series II and III compared to Series IV). These data have been corroborated by a previous study on striatal dopamine concentration in different strains of mice following different MPTP dosing schedules (27), although the study in question only investigated variations over a period of 2 days. It is clear, for any given cumulative dose, that the schedule of administration of MPTP has an impact on the extent of nigral degeneration as demonstrated by the number of dopaminergic neurons destroyed. Since different degrees of toxicity exist according to the way in which MPTP treatment is given, future studies on
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the physiopathology of the basal ganglia using this model should take into account the schedule, duration, and concentration of MPTP administration. The development of animal models replicating more accurately the progressive and continual neuronal death observed in human parkinsonism is essential for our understanding of the pathological process of nigral degeneration. If different patterns of injection lead to different mechanisms of neurotoxicity, the neurotoxic action of MPTP on dopaminergic neurons should be investigated in more chronic protocols of administration in order to develop a more realistic model to improve our understanding of the neurodegeneration of human Parkinson’s disease.
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ACKNOWLEDGMENTS
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We thank Miss Imbert for technical assistance. This study was supported in part by the CNRS and the IFR of Neuroscience (INSERM No. 8; CNRS No. 13) and in part by MESR Grant 95523629.
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