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Neuroprotective effect of pioglitazone in the 6-hydroxydopamine model of Parkinson’s disease
S652
P.5.c. Dementia and neurological disorders − Neurological disorders (basic)
P.5.c.012 Neuroprotective effect of pioglitazone in the 6-hydroxydopamine model of Parkinson’s disease M.M.F. Machado1 , T.B. Bassani1 , E.L.R. Moura1 , V. C´oppolaSegovia2 , S. Zanata2 , M. Vital1 ° 1 Universidade Federal do Paran´a Setor de Ciˆencias Biol´ogicas, Pharmacology, Curitiba, Brazil; 2 Universidade Federal do Paran´a Setor de Ciˆencias Biol´ogicas, Basic Pathology, Curitiba, Brazil Background: Depression is one of the most common non-motor symptoms observed in Parkinson’s disease (PD) patients. Although the primary cause of PD remains unknown some factors are related to the progression of neuronal death such as neuroinflammation [1]. NF-úB is a nuclear transcription factor which is involved with inflammation process in neurons of the substantia nigra pars compacts (SNpc) and microglial cells in which the persistent NF-úB activation promotes the increase of inflammatory response and neurodegeneration [2]. Pioglitazone (pio) is an insulin sensitizing drug used in the treatment of type 2 diabetes, characterized pharmacologically as agonist of peroxisome proliferator-activated receptor gamma (PPARg). When the PPARg is activated it is able to inhibit the transcriptional initiation of NF-úB causing repression of genes involved in the inflammatory cascade [3]. Objective: The aim of the study was to assess the potential neuroprotective and behavioral effects of pioglitazone in the 6-OHDA animal model of Parkinson’s disease. Methods: Male wistar rats were divided into 4 groups: sham + vehicle, sham + pio, 6OHDA + vehicle and 6OHDA + pio. Bilateral infusions of 6-OHDA (6mg dissolved in CSF; 1ml per site injection) in the SNpc was performed through stereotaxic surgery, according to the following coordinates: antero-posterior (AP): −5.0 mm from the bregma; medio-lateral (ML): ±2.1 mm from the midline; dorso-ventral (DV): 8.0 mm from the skull [4]. The rats were treated for 5 days with pioglitazone 30 mg/kg (pio) or vehicle orally. These animals were submitted to the open field test (OFT) which was held on 1, 7, 14, 21 days after the surgery. The forced swimming test (FST) was held on 21st and 22nd days. After that the rats were euthanized with removal of SN for later analysis total IúB, nuclear p65 and dosage of tyrosine hydroxylase (TH) by western blot. Statistical significance was performed using one-way ANOVA and two-way ANOVA with repeated measures, Tukey’s test and Bonferroni when was appropriate. Results: In the OFT (n = 8−10/group), the 6-OHDA group presented hypolocomotion in the 1st and 7th days after toxin infusion when compared to the sham group (P = 0.0013). However, the animals of the 6-OHDA+pio group present hypolocomotion only in the 1st day (P = 0.0075). Moreover, in the 7, 14 and 21 days the rats of the 6-OHDA+pio group showed a significant increase in locomotion in comparison with the 6-OHDA+veh (P < 0.05). In FST (n = 12−15/group) the rats of 6-OHDA group compared with the sham group exhibited an increase of immobility time but without significance (P > 0.05) and the swimming time was decreased but also had no significant difference (P > 0.05). No difference in climbing time was observed between the groups (P > 0.05). The TH levels in 6-OHDA group were decreased when compared with the sham group however, not significantly (P > 0.05). There was not difference between the groups in relation to the levels of NFúB and IúB (P > 0.05). Conclusions: This study shows that pioglitazone in 6-OHDA model of PD promote a neuroprotective effect due to its ability
to restore the hypolocomotion of injured animals, however it does not present any effect on NfúB pathway. References [1] Hirsch, E., Vyas, S., Hunot, S., 2012. Neuroinflammation in Parkinson’s disease. Parkinsonism and Related Disorders, 18S1:210–212. [2] Pranski, E., Sanford, C.D.V., Dalai, N., Orr, A.L., Karmali, D., Cooper, D.S., Gearing, M., Lah, J.J., Levely, A.I., Betarbet, R., 2013. NF-kB activity is inversely correlated to RNF11 expression in Parkinson’s disease. Neurosci Lett 28(547):16−20. [3] Sauer, S., 2015. Ligands for the nuclear peroxisome proliferatoractivated receptor gamma. Trends in Pharmacological Sciences, 36(10):688–704. [4] Paxinos, G., Watson, C., 2005. The rat brain in stereotaxic coordinates: the new coronal set. 5th ed. San Diego: Academic Press.
P.5.c.013 Inhibition of pro-apoptotic signalling by fingilimod and pramipexole in Parkinson’s disease experimental model J. Motyl1 , P.L. Wencel2 ° , J.B. Strosznajder1 1 Mossakowski Medical Research Centre Polish Academy of Sciences, Department of Cellular Signalling, Warsaw, Poland; 2 Mossakowski Medical Research Centre Polish Academy of Sciences, Department of Neurosurgery, Warsaw, Poland Background: The etiologic basis of Parkinson’s disease (PD), the second most common age-related neurodegenerative disorder in the world is still poorly understood and its therapy is not successful. Sphingosine kinase (Sphk1) and its product, bioactive sphingolipid − sphingosine-1-phoshate (S1P) can play key roles in controlling cell fate, which makes them attractive targets in the development of neurodegenerative disorders therapy. Our last data demonstrated significant inhibition of Sphk1 expression/ activity in PD in vitro model, induced by neurotoxin 1-methyl4-phenylpyridinium (MPP+). At the same time we indicated the neuroprotective effect of S1P and its analog phospho-fingolimod (FTY720-P) in PD cellular model. Moreover, presented previously preliminary results showed alteration of Sphk1 in PD animal model. The aim of current research was to investigate the effect of S1P receptors modulator − fingolimod (FTY720) and dopamine D2/D3 receptors agonist − pramipexole (PPX) on molecular changes in two parts of mouse brain (striatum and midbrain) in PD model, induced by neurotoxin 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP). Methods: Adult males of C57BL/6 mice were used in the experiments. Pharmacological PD model was induced by intraperitoneally (i.p.) administration of neurotoxin MPTP (given three times with 2 hours intervals in total dose of 40 mg/kg body weight). Control mice were inoculated with sterile saline. Some mice had received i.p. injection of FTY720 (1 mg/kg body weight) or PPX (1 mg/kg body weight) 1 hour after the last MPTP injection and in sequence during 10 days. Then in following four days several behavioral tests (open field, rota-rod) were performed, and finally mice were decapitated on 15th day. Whole striatum and midbrain (including the substantia nigra) were used for biochemical analysis. The spectrofluorometrical, immunochemical (Western blot) and Real Time PCR methods were applied. For statistical comparison we have used: Student’s t-test or one-way ANOVA followed by Newman–Keuls post-hoc test. The values of p < 0.05 was considered statistically significant. Results: Our data demonstrated and confirmed significant lower midbrain activity of Sphk1 in MPTP-administrated mice (80% of
P.5.c. Dementia and neurological disorders − Neurological disorders (basic)
P.5.c.012 Neuroprotective effect of pioglitazone in the 6-hydroxydopamine model of Parkinson’s disease M.M.F. Machado1 , T.B. Bassani1 , E.L.R. Moura1 , V. C´oppolaSegovia2 , S. Zanata2 , M. Vital1 ° 1 Universidade Federal do Paran´a Setor de Ciˆencias Biol´ogicas, Pharmacology, Curitiba, Brazil; 2 Universidade Federal do Paran´a Setor de Ciˆencias Biol´ogicas, Basic Pathology, Curitiba, Brazil Background: Depression is one of the most common non-motor symptoms observed in Parkinson’s disease (PD) patients. Although the primary cause of PD remains unknown some factors are related to the progression of neuronal death such as neuroinflammation [1]. NF-úB is a nuclear transcription factor which is involved with inflammation process in neurons of the substantia nigra pars compacts (SNpc) and microglial cells in which the persistent NF-úB activation promotes the increase of inflammatory response and neurodegeneration [2]. Pioglitazone (pio) is an insulin sensitizing drug used in the treatment of type 2 diabetes, characterized pharmacologically as agonist of peroxisome proliferator-activated receptor gamma (PPARg). When the PPARg is activated it is able to inhibit the transcriptional initiation of NF-úB causing repression of genes involved in the inflammatory cascade [3]. Objective: The aim of the study was to assess the potential neuroprotective and behavioral effects of pioglitazone in the 6-OHDA animal model of Parkinson’s disease. Methods: Male wistar rats were divided into 4 groups: sham + vehicle, sham + pio, 6OHDA + vehicle and 6OHDA + pio. Bilateral infusions of 6-OHDA (6mg dissolved in CSF; 1ml per site injection) in the SNpc was performed through stereotaxic surgery, according to the following coordinates: antero-posterior (AP): −5.0 mm from the bregma; medio-lateral (ML): ±2.1 mm from the midline; dorso-ventral (DV): 8.0 mm from the skull [4]. The rats were treated for 5 days with pioglitazone 30 mg/kg (pio) or vehicle orally. These animals were submitted to the open field test (OFT) which was held on 1, 7, 14, 21 days after the surgery. The forced swimming test (FST) was held on 21st and 22nd days. After that the rats were euthanized with removal of SN for later analysis total IúB, nuclear p65 and dosage of tyrosine hydroxylase (TH) by western blot. Statistical significance was performed using one-way ANOVA and two-way ANOVA with repeated measures, Tukey’s test and Bonferroni when was appropriate. Results: In the OFT (n = 8−10/group), the 6-OHDA group presented hypolocomotion in the 1st and 7th days after toxin infusion when compared to the sham group (P = 0.0013). However, the animals of the 6-OHDA+pio group present hypolocomotion only in the 1st day (P = 0.0075). Moreover, in the 7, 14 and 21 days the rats of the 6-OHDA+pio group showed a significant increase in locomotion in comparison with the 6-OHDA+veh (P < 0.05). In FST (n = 12−15/group) the rats of 6-OHDA group compared with the sham group exhibited an increase of immobility time but without significance (P > 0.05) and the swimming time was decreased but also had no significant difference (P > 0.05). No difference in climbing time was observed between the groups (P > 0.05). The TH levels in 6-OHDA group were decreased when compared with the sham group however, not significantly (P > 0.05). There was not difference between the groups in relation to the levels of NFúB and IúB (P > 0.05). Conclusions: This study shows that pioglitazone in 6-OHDA model of PD promote a neuroprotective effect due to its ability
to restore the hypolocomotion of injured animals, however it does not present any effect on NfúB pathway. References [1] Hirsch, E., Vyas, S., Hunot, S., 2012. Neuroinflammation in Parkinson’s disease. Parkinsonism and Related Disorders, 18S1:210–212. [2] Pranski, E., Sanford, C.D.V., Dalai, N., Orr, A.L., Karmali, D., Cooper, D.S., Gearing, M., Lah, J.J., Levely, A.I., Betarbet, R., 2013. NF-kB activity is inversely correlated to RNF11 expression in Parkinson’s disease. Neurosci Lett 28(547):16−20. [3] Sauer, S., 2015. Ligands for the nuclear peroxisome proliferatoractivated receptor gamma. Trends in Pharmacological Sciences, 36(10):688–704. [4] Paxinos, G., Watson, C., 2005. The rat brain in stereotaxic coordinates: the new coronal set. 5th ed. San Diego: Academic Press.
P.5.c.013 Inhibition of pro-apoptotic signalling by fingilimod and pramipexole in Parkinson’s disease experimental model J. Motyl1 , P.L. Wencel2 ° , J.B. Strosznajder1 1 Mossakowski Medical Research Centre Polish Academy of Sciences, Department of Cellular Signalling, Warsaw, Poland; 2 Mossakowski Medical Research Centre Polish Academy of Sciences, Department of Neurosurgery, Warsaw, Poland Background: The etiologic basis of Parkinson’s disease (PD), the second most common age-related neurodegenerative disorder in the world is still poorly understood and its therapy is not successful. Sphingosine kinase (Sphk1) and its product, bioactive sphingolipid − sphingosine-1-phoshate (S1P) can play key roles in controlling cell fate, which makes them attractive targets in the development of neurodegenerative disorders therapy. Our last data demonstrated significant inhibition of Sphk1 expression/ activity in PD in vitro model, induced by neurotoxin 1-methyl4-phenylpyridinium (MPP+). At the same time we indicated the neuroprotective effect of S1P and its analog phospho-fingolimod (FTY720-P) in PD cellular model. Moreover, presented previously preliminary results showed alteration of Sphk1 in PD animal model. The aim of current research was to investigate the effect of S1P receptors modulator − fingolimod (FTY720) and dopamine D2/D3 receptors agonist − pramipexole (PPX) on molecular changes in two parts of mouse brain (striatum and midbrain) in PD model, induced by neurotoxin 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP). Methods: Adult males of C57BL/6 mice were used in the experiments. Pharmacological PD model was induced by intraperitoneally (i.p.) administration of neurotoxin MPTP (given three times with 2 hours intervals in total dose of 40 mg/kg body weight). Control mice were inoculated with sterile saline. Some mice had received i.p. injection of FTY720 (1 mg/kg body weight) or PPX (1 mg/kg body weight) 1 hour after the last MPTP injection and in sequence during 10 days. Then in following four days several behavioral tests (open field, rota-rod) were performed, and finally mice were decapitated on 15th day. Whole striatum and midbrain (including the substantia nigra) were used for biochemical analysis. The spectrofluorometrical, immunochemical (Western blot) and Real Time PCR methods were applied. For statistical comparison we have used: Student’s t-test or one-way ANOVA followed by Newman–Keuls post-hoc test. The values of p < 0.05 was considered statistically significant. Results: Our data demonstrated and confirmed significant lower midbrain activity of Sphk1 in MPTP-administrated mice (80% of