The role of pramipexole in a severe Parkinson s disease model in mice

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1 Therapeutic Advances in Neurological Disorders Original Research The role of pramipexole in a severe Parkinson s disease model in mice Seham Gad ElHak, Abdel Aziz Ghanem, Hasan Abdelghaffar, Sahar ElDakroury, Dina ElTantawy, Sara ElDosouky and Mohamed Salama Ther Adv Neurol Disord (2010) 3(6) DOI: / ! The Author(s), Reprints and permissions: journalspermissions.nav Abstract: Background: Pramipexole is one of a new generation of dopamine agonists. Recently there have been questions regarding its neuroprotective effects. These effects have been tested against various insults, which have yielded conflicting results. Methods: In this study, we introduced a combination of 1-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine (MPTP)/paraquat to induce a severe Parkinson s disease model. The mice, after receiving the combination of toxins, were evaluated using mortality rates and immunohistochemistry for degenerating tyrosine hydroxylase-positive neurons. Results and conclusions: Pramipexole was tested for its capacity to offer protection against neurotoxic the effects of MPTP/paraquat in this model; however, the results showed no improvement with pramipexole therapy. Keywords: MPTP, paraquat, neuroprotection, Parkinson s disease, pramipexole, mouse Introduction Parkinson s disease (PD) is the second most prevalent neurodegenerative disorder. Despite the long history of research regarding this disease, no satisfactory treatment has been found [Spencer et al. 2005]. One of the most valuable methods of studying PD is through animal models. Many neurotoxic models have been designed to study Parkinsonism in animals [Emborg, 2004]. These models, however, have failed to recapitulate all of the pathological findings in PD [Lane and Dunnett, 2008]. To improve accuracy, fusion of models has been suggested. This combinatorial method seems more relevant to the real pathogenesis of the disease, which makes it more capable of simulating the true pathological findings of PD [Dawson et al. 2002]. One popular toxin combination is the 1-methyl- 4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)/ paraquat regimen. MPTP is a well-known neurotoxin and one of the classical chemicals used in PD modeling. Paraquat, on the other hand, is a pesticide that is used as a PD-inducing toxin in animals [Shepherd et al. 2006]. Combining these two drugs increases the severity of neuronal degeneration in a fashion similar to that induced by local 6-OH dopamine injection. This makes the MPTP/paraquat combination a reasonable substitute that can resolve many of the problems of 6-OH dopamine regarding mode of administration, unilateral lesions and lack of the progressive course of the model [Richter et al. 2008]. Pramipexole is a second-generation dopamine agonist with a better profile than the older generations [Hedlund and Perlmann, 2009]. New research suggests its capacity for neuroprotective effects [Winner et al. 2009]. Many pathways have been suggested for pramipexole neuroprotection, e.g. stimulation of dopaminergic neuron regeneration [Riaz and Bradford, 2005], antioxidant effects, inhibition of oligomerization of human a-synuclein and increased Bcl-2 immunoreactivity in dopaminergic neurons [Inden et al. 2009]. Since there is no perfect PD model, a neuroprotectant should be tested using multiple models [Anderson et al. 2006]. We think that evaluation Correspondence to: Mohamed Salama, MD Toxicology Department and Medical Experimental Research Center (MERC), ElGomhourya Street, toxicsalama@hotmail.com Seham Gad ElHak, MD Abdel Aziz Ghanem, MD Toxicology Department, Hasan Abdelghaffar, MD Medical Experimental Research Center (MERC) and Clinical Pathology Department, Mansoura University, Mansoura, Egypt Sahar ElDakroury, MD Toxicology Department, Dina ElTantawy, PhD Pathology Department, Faculty of Medicine, Sara ElDosouky, MSc Medical Experimental Research Center (MERC), 333

2 Therapeutic Advances in Neurological Disorders 3 (6) of pramipexole in a severe progressive PD model using the MPTP/paraquat combination would improve our judgment of the drug s neuroprotectant capacity. Materials and methods MPTP/paraquat mouse model and drug administration: Thirty (8-month-old) male BALB/c mice (20 25 g) were purchased from Vacsera animal house (Cairo, Egypt). All animal experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the protocols were approved by the Ethical Committee for Research at Mansoura University. The animals were divided into three s (10 mice each):. The control received only saline via intraperitoneal route, twice per week.. The MPTP/paraquat received a combination of MPTP (Sigma-Aldrich, 30mg/kg i.p.) þ paraquat (Sigma-Aldrich, 15 mg/kg i.p.) twice per week.. The MPTP/paraquat þ pramipexole received a combination of MPTP (30mg/kg i.p.) þ paraquat (15 mg/kg i.p.) twice per week. Pramipexole was given orally at 1 mg/kg/day. In vivo assessment of the model (mortality rate) An endpoint of this experiment was the death of intoxicated animals. Each animal received both toxins twice weekly until either severe incapacitation and death or completion of eight consecutive doses. The severity of PD manifestations was measured according to the mortality rate in each. Immunohistochemistry After death (or completion of eight consecutive doses), treated mice were perfused through the aorta with 50 ml of 10 mm phosphate-buffered saline (PBS), followed by 150 ml of a cold fixative consisting of 4% paraformaldehyde, 0.35% glutaraldehyde, and 0.2% picric acid in 100 mm phosphate buffer (PB), under deep anesthesia with pentobarbital (100 mg/kg, i.p.). After perfusion, the brain was quickly removed and postfixed for 2 days with paraformaldehyde in 100 mm PB and then transferred to 15% sucrose solution in 100 mm PB containing 0.1% sodium azide at 4 C. Brains were cut into 30-mm-thick sections using a cryostat and collected in 100 mm PBS containing 0.3% Triton X-100 (PBS-T). After several washes, the sections were stored until use in a free-floating state at 4 C for immunohistochemical analysis. Brain slices were incubated with primary mouse monoclonal anti-tyrosine hydroxylase (TH) antibody (diluted 1 : 1000 Sigma) overnight at 4 C. After several washes, sections were incubated with biotinylated (goat antimouse) secondary antibody (1 : 500), for 1 h at room temperature. The sections were then incubated with ABC solution 1 : 200 for 1 h at room temperature. All of the sections were washed several times with PBS-T between each incubation, and labeling was then revealed by 3,3 0 -diaminobenzidine (DAB). Stereological analysis of dopamine neurons in the ventral midbrain TH-immunopositive neurons in the substantia nigra pars compacta (SNpc) were estimated using stereological counts of cell numbers, on a Stereo-investigator system and optical density measurements on a Leica Q-win system). Six (30-mm-thick) sections, from the anterior to the posterior midbrain, were used for counting in each case. The total number of TH-immunopositive neurons was estimated using the optical fractionator method. Statistical methods All data were given as the mean±standard error of the mean (SEM). Two s of data were analyzed by Student s t-test. Three s of data were analyzed by analysis of variance (ANOVA) with a Tukey post hoc test. For all tests, p < 0.05 was deemed significant. Results Effect of MPTP/paraquat on nigrostriatal dopamine neurons in mice As shown in Figures 1 and 2 the toxin combination severely reduced the number of TH-immunopositive neurons in the SNpc in combination with the control. Stereological analysis of nigral TH-immunopositive neurons showed that MPTP/paraquat caused severe loss of dopamine neurons (Table 1)

SG ElHak, AA Ghanem et al. Table 1. Stereological cell counts in the substantia nigra. (A) Control (B) MPTP/ paraquat (C) MPTP/paraquat þ pramipexole 18,700±120 4340±30* 4770±20* *p < 0.

Medium magnification (20x) images in dorsolateral region of substantia nigra show no evidence of improvement. Table 2. Mortalities in the s. Figure 2.

Medium magnification (20x) images in dorsolateral region of substantia nigra show severe cell loss (some areas showing complete absences of TH immunoreactive cells) in this particularly vulnerable

3 SG ElHak, AA Ghanem et al. Table 1. Stereological cell counts in the substantia nigra. (A) Control (B) MPTP/ paraquat (C) MPTP/paraquat þ pramipexole 18,700± ±30* 4770±20* *p < 0.05 compared with the control. Figure 1. Tyrosine hydroxylase immunohistochemistry in the control. Figure 3. Tyrosine hydroxylase immunohistochemistry in the MPTP/paraquat þ pramipexole. Medium magnification (20x) images in dorsolateral region of substantia nigra show no evidence of improvement. Table 2. Mortalities in the s. Figure 2. Tyrosine hydroxylase (TH) immunohistochemistry in the MPTP/paraquat. Medium magnification (20x) images in dorsolateral region of substantia nigra show severe cell loss (some areas showing complete absences of TH immunoreactive cells) in this particularly vulnerable area. (A) Control (B) MPTP/paraquat 0 10* 10* *p < 0.05 compared with the control. (C) MPTP/paraquat þ pramipexole Effect of MPTP/paraquat on mortality rate The toxin combination produced a 100% mortality rate. Effects of pramipexole on the induced neurodegeneration Upon investigation of whether treatment with pramipexole (oral 1 mg/kg/day) protects dopamine neurons from damage caused by the chronic MPTP/paraquat administration, we found no improvement regarding TH-immunopositive neurons number in the SNpc (Figure 3). Effects of pramipexole on mortality rates Pramipexole treatment did not show protection against mortality as the same mortality rate (100%) was observed in the pramipexole-treated (Table 2). Discussion Although animal models represent the first step in examining candidate anti- Parkinsonian drugs, 335

4 Therapeutic Advances in Neurological Disorders 3 (6) the results usually show different effects when applied clinically [Shimohama et al. 2003]. This limitation has led some authors to ask for a rethink of animal model strategy [Emborg, 2004]. Others have asked for more matching of the animal model to the intended objective, i.e. some will be suitable to study mechanisms while others are better suited for investigational drug testing [Lane and Dunnett, 2008]. Another suggestion has been to test candidate drugs among various models to get what is called broad spectrum neuroprotectants [Anderson et al. 2006]. The value of this multiple testing approach would apply in the case of pramipexole. Previous work has shown its potential as a neuroprotectant in PD models, e.g. MPTP and rotenone [Inden et al. 2009; Hall et al. 1996]. However, in our study pramipexole revealed no neuroprotective effect. This finding is in accordance with the work of Kemmerer and colleagues and Jeon and colleagues with 6-OH dopamine lesioned rats [Jeon et al. 2007; Kemmerer et al. 2003]. Our work represents a severe grade of PD, simulating the end stage of the disease. The use of death or severe debilitation of the tested animals can be used to assess the severity of neuronal degeneration [Cannon et al. 2009]. Furthermore, mortality rate can be used to assess the capacity of certain drug to offer neuroprotection. The variable effects of pramipexole among different models led Anderson and colleagues to assume that different toxin protocols will give different results [Anderson et al. 2006]. On our side, we think that different grades of severity would be the causative factor for variable pramipexole effects. What supports our opinion is that pramipexole failed in both 6-OH dopamine lesions and in our study using a combination of high doses of MPTP/paraquat, where in these models severe degeneration of dopaminergic neurons is expected. In contrast, pramipexole succeeded in cases of rotenone or MPTP models where there is mild neurodegeneration. These findings would suggest that severe neurodegeneration can overwhelm the protective capacity of pramipexole and diminish their effects on neurogenesis [Winner et al. 2009; He et al. 2008; Riaz and Bradford, 2005]. Conclusion Pramipexole failed to exert neuroprotection in a severe model of MPTP/paraquat-induced Parkinson s disease in mice. This failure underscores the importance of testing candidate drugs against multiple models with different degeneration severity in order to obtain a so-called wide spectrum neuroprotectant. Funding This work was supported by the Medical Experimental Research Center (MERC) of Mansoura University. Conflict of interest statement None declared. References Anderson, D.W., Bradbury, K.A. and Schneider, J.S. (2006) Neuroprotection in Parkinson models varies with toxin administration protocol. Eur J Neurosci 24: Cannon, J.R., Tapias, V., Mee, H., Honick, A., Drolet, R. and Greenamyre, J.T. (2009) A highly reproducible rotenone model of Parkinson s disease. Neurobiol Dis 34: Dawson, T.M., Mandir, A.S. and Lee, M.K. (2002) Animal models of PD: pieces of the same puzzle? Neuron 35: Emborg, M.E. (2004) Evaluation of animal models of Parkinson s disease for neuroprotective strategies. J Neurosci Meth 139: Hall, E.D., Andrus, P.K., Oostveen, J.A., Althaus, J.S. and Vonvoigtlander, P.F. (1996) Neuroprotective effects of the dopamine D2/3 agonist pramipexole against postischemic or methamphetamine-induced degeneration of nigrostriatal neurons. Brain Res 742: He, X.J., Yamauchi, H., Uetsuka, K. and Nakayama, H. (2008) Neurotoxicity of MPTP to migrating neuroblasts: Studies in acute and subacute mouse models of Parkinson s disease. NeuroToxicology 29: Hedlund, E. and Perlmann, T. (2009) Neuronal cell replacement in Parkinson s disease. J Intern Med 266: Inden, M., Kitamura, Y., Tamaki, A., Yanagida, T., Shibaike, T., Yamamoto, A. et al. (2009) Neuroprotective effect of the antiparkinsonian drug pramipexole against nigrostriatal dopaminergic degeneration in rotenone-treated mice. Neurochem Int 55: Jeon, M.Y., Lee, W.Y., Kang, H.Y. and Chung, E.J. (2007) The effects of L-3,4- dihydroxyphenylalanine and dopamine agonists on dopamine neurons in the 336

5 SG ElHak, AA Ghanem et al. progressive hemiparkinsonian rat models. Neurol Res 29: Kemmerer, E.S., Desmond, T.J., Albin, R.L., Kilbourn, M.R. and Frey, K.A. (2003) Treatment effects on nigrostriatal projection integrity in partial 6-OHDA lesions: comparison of L-DOPA and pramipexole. Exp Neurol 183: 1 3. Lane, E. and Dunnett, S. (2008) Animal models of Parkinson s disease and L-dopa induced dyskinesia: How close are we to the clinic? Psychopharmacology 199: Riaz, S.S. and Bradford, H.F. (2005) Factors involved in the determination of the neurotransmitter phenotype of developing neurons of the CNS: Applications in cell replacement treatment for Parkinson s disease. Progress Neurobiol 76: Richter, F., Hamann, M. and Richter, A. (2008) Moderate degeneration of nigral neurons after repeated but not after single intrastriatal injections of low doses of 6-hydroxydopamine in mice. Brain Res 1188: Shepherd, K., Lee, E., Schmued, L., Jiao, Y., Ali, S., Oriaku, E. et al. (2006) The potentiating effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on paraquat-induced neurochemical and behavioral changes in mice. Pharmacol Biochem Behav 83: Shimohama, S., Sawada, H., Kitamura, Y. and Taniguch, T. (2003) Disease model: Parkinson s disease. TRENDS Mol Med 9: Spencer, P.S., Palmer, V.S. and Ludolph, A.C. (2005) On the decline and etiology of high-incidence motor system disease in West Papua (Southwest New Guinea). Mov Disord 20(Suppl. 12): S199 S126. Winner, B., Desplats, P., Hagl, C., Klucken, J., Aigner, R., Ploetz, S. et al. (2009) Dopamine receptor activation promotes adult neurogenesis in an acute Parkinson model. Exp Neurol 219: Visit SAGE journals online

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