Neurotoxicidade induzida pelo cloreto de manganês: modelo experimental de manganismo em ratos Wistar

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Bioquímica, Florianópolis, 2016.O Manganês (Mn) é um metal essencial extremamente importante para os sistemas biológicos. Contudo, a exposição excessiva ao Mn causa toxidade a diversos órgãos/sistemas, incluindo o sistema nervosos central (SNC). O excesso de Mn pode levar à síndrome conhecida como manganismo, uma condição neuropatológica em que alguns sintomas se assemelham a aqueles encontrados na doença de Parkinson (DP) idiopática. Apesar das similaridades entre ambas as condições, há eventos específicos relacionados à intoxicação por Mn, dentre os quais destaca-se perda neuronal e gliose no globo pálido (GP) e estriado. O objetivo deste estudo foi investigar os possíveis efeitos neurotóxicos da exposição ao MnC12 em um modelo experimental em ratos Wistar de 3 meses de idade, o qual baseou-se na administração de 4 injeções intraperitoneais de uma solução de MnC12 (dose de 25 mg/kg), sendo administrada uma injeção por dia nos dias 1, 3, 5 e 7. Além disso, objetivou-se avaliar a potencial reversibilidade desta toxicidade através da realização de testes comportamentais, bioquímicos e imunohistoquímicos 24 h imediatamente após a última exposição ao MnC12 (dia 8), assim como depois de um período de latência de 30 dias. Vinte e quatro h após a última exposição ao Mn, observou-se uma significativa redução no número de cruzamentos e levantadas no teste do campo aberto e um significativo aumento no número de resvaladas no teste do beam walking nos ratos tratados como MnC12 quando comparados com animais do grupo controle. Não houve diferença significativa entre os grupos nos níveis estriatais de espécies reativas ao ácido tiobarbitúrico (TBARS), tióis não-proteicos (NPSH), na atividade das enzimas antioxidantes gluationa redutase (GR), glutationa peroxidase (GPx) e superóxido dismutase (SOD), nem na atividade dos complexos I e II da cadeia respiratória mitocondrial. A imunorreatividade para a enzima tirosina hidroxilase (marcadora de neurônios catecolaminérgicos, TH) diminuiu significativamente no estriado dos animais expostos ao Mn 24 h apóis o tratamento, tendo sido normalizado após à latência de 30 dias. Os níveis da proteína ácida fibrilar glial (GFAP) foram significativamente aumentados apenas no globo pálido (GP) dos animais expostos ao Mn somente aos 30 dias após o tratamento. Não houve diferença na imunorreatividade para a enzima glutamato descarboxilase (mercadora de neurônios GABAérgicos, GAD 65). A reversão dos danos motores e da imunomarcação da TH revelam que há um potencial reversibilidade que se segue após o término da exposição ao MnC12. Conclui-se que o desenho experimental deste trabalho se mostrou um bom modelo para se estudar os mecanismos que levam ao manganismo, após uma intoxicação aguda por MnC12. Ainda, a gliose reativa (aumento da reatividade da GFAP) observada no GP aos 30 dias após a exposição sugere a existência de um processo reativo/inflamatório tardio, o qual poderia ser responsável por eventos neurodegenerativos tardios decorrentes da exposição ao Mn.Abstract : Manganese (Mn) is an essential metal extremely important for biological systems. However, the exposure to excessive Mn causes toxicity to various organ/systems, including the central nervous system (CNS). Importantly, excessive exposure to Mn can lead to the syndrome known as manganism, a neuropathological condition whose symptoms are similar to those found in idiopathic Parkinson's disease (PD). Despite the similarities between both conditions, there are specific events linked only to Mn intoxication such as neuronal loss and gliosis in the globus pallidus (GP) and striatum. The aim of this study was to investigate the potential neurotoxic effects of exposure to MnCl2 in an experimental model with adult (3 months) rats, based on the administration of 4 intraperitoneal injections of MnCl2 (dose of 25 mg/kg), being administered at days 1, 3, 5 e 7. In addition, we aimed to evaluate the potential reversibility of such toxicity by using behavioral, biochemical and immunohistochemical tests 24 hours immediately after the last exposure to MnCl2 (at day 8), as well as after a 30 days latency period . Twenty-four h after the last Mn injection, there was a significant reduction in the number of crossings and rearings in the open field test, as well as a significant increase in footslips in the beam walking test on rats treated with MnCl2 when compared to the control group. There were no significant differences in the striatal levels of thiobarbituric acid-reactive substances (TBARS), nonprotein thiols (NPSH), in the activity of antioxidant enzymes glutathione reductase (GR), glutathione peroxidase (GPx) and superoxide dismutase (SOD), as well as in the activities of complex I and II of the mitochondrial respiratory chain. Tyrosine hydroxilase (a marker of catecholaminergic neurons, TH) immunoreactivity decreased in the striatum of Mn exposed rats at 24 h after treatment, but it returned to control levels after the latency period. The glial fibrillary acidic protein (GFAP) levels increased only in GP at 30 days after Mn exposure. There was no difference in the immunoreactivity for glutamate decarboxylase (a marker of GABAergic neurons, GAD 65). The absence of the motor impairment and changes in TH immunostaining at 30 days after Mn exposure revealed the reversibility of symptoms triggered by MnCl2 exposure. We conclude that the experimental design of this study represents a useful strategy to study the mechanisms that lead to manganism, especially after an acute exposure to MnCl2. Moreover, the observed reactive gliosis (increased GFAP reactivity) in the GP at 30 days after Mn exposure suggests the occurrence of delayed reactive/inflammatory processes, which could be responsible for neurodegenerative events following Mn exposure

Probucol Increases Striatal Glutathione Peroxidase Activity and Protects against 3-Nitropropionic Acid-Induced Pro-Oxidative Damage in Rats

<div><p>Huntington’s disease (HD) is an autosomal dominantly inherited neurodegenerative disease characterized by symptoms attributable to the death of striatal and cortical neurons. The molecular mechanisms mediating neuronal death in HD involve oxidative stress and mitochondrial dysfunction. Administration of 3-nitropropionic acid (3-NP), an irreversible inhibitor of the mitochondrial enzyme succinate dehydrogenase, in rodents has been proposed as a useful experimental model of HD. This study evaluated the effects of probucol, a lipid-lowering agent with anti-inflammatory and antioxidant properties, on the biochemical parameters related to oxidative stress, as well as on the behavioral parameters related to motor function in an <i>in vivo</i> HD model based on 3-NP intoxication in rats. Animals were treated with 3.5 mg/kg of probucol in drinking water daily for 2 months and, subsequently, received 3-NP (25 mg/kg i.p.) once a day for 6 days. At the end of the treatments, 3-NP-treated animals showed a significant decrease in body weight, which corresponded with impairment on motor ability, inhibition of mitochondrial complex II activity and oxidative stress in the striatum. Probucol, which did not rescue complex II inhibition, protected against behavioral and striatal biochemical changes induced by 3-NP, attenuating 3-NP-induced motor impairments and striatal oxidative stress. Importantly, probucol was able to increase activity of glutathione peroxidase (GPx), an enzyme important in mediating the detoxification of peroxides in the central nervous system. The major finding of this study was that probucol protected against 3-NP-induced behavioral and striatal biochemical changes without affecting 3-NP-induced mitochondrial complex II inhibition, indicating that long-term probucol treatment resulted in an increased resistance against neurotoxic events (i.e., increased oxidative damage) secondary to mitochondrial dysfunction. These data appeared to be of great relevance when extrapolated to human neurodegenerative processes involving mitochondrial dysfunction and indicates that GPx is an important molecular target involved in the beneficial effects of probucol.</p> </div

Probucol reduces 3-NP-induced lipid peroxidation in rats.

<p>Treatments were conducted as previously mentioned (see Methods Section). Striatal thiobarbituric acid reactive substance (TBARS) levels are expressed as nmol of MDA/mg protein. The data are presented as the mean ± S.E.M. (n = 6 rats/group). *** p< 0.001 compared with the control group and # # p < 0.01 and # # # p < 0.001 compared with the 3-NP group using two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test.</p

3-NP treatment inhibits complex II activity.

<p>Treatments were conducted as previously mentioned (see Methods Section). Complex II activity in striatum is expressed as nmol.min⁻¹.mg protein⁻¹ and presented as the mean ± S.E.M. (n = 6 rats/group). ** p < 0.01 and *** p < 0.001 compared with the control group using two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test.</p

Probucol attenuates motor impairment induced by 3-NP in rats.

<p>Treatments were conducted as previously mentioned (see Methods Section). Locomotor (A) and exploratory (B) activities in the open field test as well as the latency for the first fall (C) and the number of falls in the rotarod (D) were evaluated 24 h after the last 3-NP administration. These results are expressed as the total number of crossings (A), total number of rearings (B), the latency for the first fall(s) (C) and the total number of falls. The data are presented as the mean ± S.E.M. (n =10 rats/group). *p < 0.05 and *** p < 0.001 compared with the control group and # p < 0.05, # # p < 0.01 and # # # p < 0.001 compared with the 3-NP group using two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test.</p

Figure 7

<p><b>Effects of 3-NP and/or probucol on iNOS, GFAP</b> and <b>caspase 3/cleaved caspase 3 expression.</b></p

Probucol attenuates the increase in superoxide dismutase (SOD) and catalase activities in the rat striatum.

<p>Treatments were conducted as previously mentioned (see Methods Section). SOD activity (A) is expressed as SOD units/mg of protein. Catalase activity (B) is expressed as µmol of H₂O₂/min/mg protein. The data are presented as the mean ± S.E.M. (n = 6 rats/group). * p < 0.05 and ** p < 0.01 compared with the control group, and # p < 0.05 and # # p < 0.01 compared with the 3-NP group using two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test.</p

Probucol prevents 3-NP-induced decreases in body weight.

<p>The animals were pretreated with probucol (3.5 mg/kg/day) or vehicle (1% of DMSO) in drinking water daily for 2 months and administered intraperitoneally with 3-NP (25 mg/kg) or vehicle, once a day for 6 consecutive days. The body weight values are expressed as the percentage of change in body weight after 3-NP or vehicle administration and presented as the mean ± S.E.M. (n = 10 rats/group). ***p < 0.001 compared with the control group and ## p < 0.01 and ### p < 0.001 compared with the 3-NP group using two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test.</p

Effects of 3-NP and/or probucol on striatal GSH levels, glutathione peroxidase and glutathione reductase activities.

<p>Treatments were conducted as previously mentioned (see Methods Section). The GSH levels (A) are expressed as µmol GSH·mg protein^-1. GR activity (B) and GPx activity (C) are expressed as the nmol of NADPH oxidized/min/mg protein. The data are presented as the mean ± S.E.M. (n= 6 rats/group). †† p < 0.01 and † † † p < 0.001 main effect of probucol, ** p < 0.01 and *** p < 0.001 compared with the control group and # p < 0.05 compared with the 3-NP group using two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test.</p