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UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE – UFRN
CENTRO DE CIÊNCIAS DA SAÚDE
DEPARTAMENTO DE FARMÁCIA
PÓS-GRADUAÇÃO EM DESENVOLVIMENTO E INOVAÇÃO TECNOLÓGIA EM
MEDICAMENTOS - PPgDITM
ALDAIR JOSÉ SARMENTO SILVA
ALFA-TOCOFEROL PREVINE OS DÉFICITS COGNITIVOS, MOTORES E
NEURONAIS EM UM MODELO DE PARKINSON PROGRESSIVO EM RATOS
Tese apresentada à Universidade Federal do Rio Grande do Norte, para obtenção de título de doutor no curso de Desenvolvimento e Inovação Tecnológica em Medicamentos.
NATAL – RN, 2014
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UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE – UFRN
CENTRO DE CIÊNCIAS DA SAÚDE
DEPARTAMENTO DE FARMÁCIA
PÓS-GRADUAÇÃO EM DESENVOLVIMENTO E INOVAÇÃO TECNOLÓGIA
EM MEDICAMENTOS
ALDAIR JOSÉ SARMENTO SILVA
ALFA-TOCOFEROL PREVINE OS DÉFICITS COGNITIVOS, MOTORES E
NEURONAIS EM UM MODELO DE PARKINSON PROGRESSIVO EM
RATOS
Tese apresentada à Universidade Federal do Rio Grande do Norte, para obtenção de título de doutor no curso de Desenvolvimento e Inovação Tecnológica em Medicamentos.
ORIENTADORA: Profa. Dra. Regina Helena da Silva
NATAL – RN, 2014
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Este trabalho foi desenvolvido com auxílio da: (1) CAPES, através da
concessão de uma bolsa doutorado; (2) FAPERN, através da concessão de
auxílio deslocamento para cursar disciplinas em outros Estados e insumos e
equipamentos para pesquisa; (3) CNPq, através da concessão do suporte
financeiro e logística; do programa de pós-graduação em desenvolvimento e
inovação tecnológica em medicamentos – PPgDITM e do programa de pós-
graduação em Psicobiologia (Departamento de Fisiologia/UFRN), onde foram
desenvolvidas as atividades experimentais.
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Dedico este trabalho a minha família,
em especial, aos meus pais (Zé
Furtado e Maria José) e aos meus
avós (Chico Furtado e Mocinha), que
mesmo diante de tantas
dificuldades, nunca perderam as
esperanças.
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AGRADECIMENTOS
É diante da conclusão de uma escrita que sou remetido a defender uma
tese, tese esta que se coloca como possível e me traz certo sentido quando
remetida a estruturação familiar que me permitiu chegar aonde cheguei. A
chegada não significa o fim, e sim, a abertura de novos passos, novas
caminhadas e novas experiências. Sou grato àqueles que me concederam a
confiança, que me ensinaram a determinação, que impulsionaram a seguir com
a fé Divina.
A transmissão, que surgiu antes mesmo de uma sistematização formal de
conhecimentos, me fez acreditar em um investimento profissional que me
impulsionaria para a conquista de meus objetivos. Hoje agradeço em especial:
Aos meus avós paternos (Chico Furtado e Mocinha Furtado – in memorian)
que me ensinaram a seguir pelo caminho certo, lembro-me muito bem quando
vovô me transmitia confiança e serenidade diante das circunstâncias da vida.
Aos meus pais (Zé Furtado e Maria José), que acreditaram no saber. Minha
mãe com sua fiel e feliz transmissão sendo minha primeira e eterna professora,
me passando algo além de um ensinamento em sala de aula, devo a ela
também os ensinamentos de fé, me lembro com ternura dos agradecimentos a
Mãe Rainha quando passei no vestibular, dando assim o primeiro passo para
minha vida profissional. Meu pai, agricultor, com seu saber irredutível, que
sempre soube nos apoiar em cada momento, se doou a roça para assim
investir nos estudos dos seus oito filhos, me ensinou a calar e a falar somente
o necessário. Hoje me passa um filme de quando sai do sítio e fui estudar na
cidade, de início não quis ir, mas, meu pai me convenceu me presenteando
com uma bicicleta, cor azul e do ano. Lembro até hoje e a cada dia identifico
mais ainda o valor da frase que sempre me falaram: “a maior herança que
deixo para os meus filhos é o estudo, porque essa ninguém tira”, sou muito
grato pela significação desta transmissão.
Aos meus irmãos, por me estruturarem nesta união e colaborarem com minha
formação, a qual é enlaçada pelo amor, um afeto advindo de nossos pais que
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nos permitiu crescer na firmeza deste sentimento. Socorro Sarmento, formada
em Ciências e graduanda em Pedagogia, Tânia Sarmento Phd em Farmácia,
Eva Mônica, Dra. Em zootecnia, Gerlania Sarmento Dra. Em Farmácia,
Francisco Furtado, graduado em química, Maria José doutoranda em
medicamentos e Gorete Sarmento mestre em Psicologia. Gostaria de
agradecer aos meus tios, em especial Assis Furtado e Socorro Sarmento pela
cumplicidade e apadrinhamento.
Quando fui ao Maranhão em 2003, pude construir um novo enlace familiar,
conheci minha esposa Neysa Saiki, a quem tenho grande amor e enorme
gratidão por sua coragem, determinação e companheirismo, pois, quando
tomei a decisão de vir para o RN fazer doutorado, imediatamente arrumou as
malas e a mudança. Fruto dessa relação amorosa com Neysa, vinheram Airton
José, até então com quatro anos e Cássio Saiki, com três meses de vida, após
esses anos e a cada dia que eles crescem me deixam cada ver mais feliz e
orgulhoso, me contagiam com suas alegrias, brincadeiras e com muito amor.
Agradeço ainda, em especial, à Marinalva, que veio comigo do Maranhão a
qual considero parte da família, com sua paciência e dedicação no que se
propõe a fazer. Quero agradecer também a Nelson Saiki e Isabel Silva, pela
forma que me acolheram em sua família, com muita confiança, respeito e
incentivos, também a Nelysa, Gustavo e Rita. Como também aos meus
cunhados Gil, Celso, Maurício, Zey e Mirelly pelo apoio, aos primos, e aos
amigos que deixei em São Luís do Maranhão. Para falar dos companheiros do
LEME, prefiro iniciar agradecendo a uma pessoa que a considero intelectual e
que sabe lidar com as diversas situações para qual é solicitada, conheci
Regina via e-mail, e não poderia imaginar que estaria diante de uma pessoa
especial, obrigado Rê por sua confiança e dedicação, aprendi muito com você
e com Alessandra Mussi, à qual tenho muita admiração. Cheguei um pouco
tímido no laboratório, com uma lata de refrigerante Jesus que tinha trazido do
Maranhão para presentear as professoras e os novos colegas que foram muito
importantes durante todo o doutorado, com suas ajudas providencias e também
nas manutenções do biotério e laboratórios, são eles: Geison, Flávio, Alícia,
Fernando, Thieza, Anderson, André, Gênedi, Sophia, Priscila, Aline, Ezequiel,
Isabella, Diego, Clarissa, Ywlliane, as Jéssicas, João, Anatildes, Luiz Eduardo,
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Diana, Fernanda, Ivon, Luane, Cintia a professora Rovena, ao professor
Jeferson, e por fim ao pós-doc Ramon Hypolito Lima, que tenho muito a
agradecer por sua dedicação e capacidade de desenvolver pesquisa, a sua
contribuição foi muito importante nesta reta final do doutorado. Agradeço
também ao pessoal do laboratório da professora Miriam, a professora Elaine
Gavioli, a secretaria da PPgDITM, aos colegas de sala pelo companheirismo
durante as viagens para pagar disciplinas em outros estados. Agradeço
também à Faculdade Pitágoras – unidade São Luis/MA, em nome do Prof.
Hermínio, pelo apoio e compreenção. É com a sensação de dever cumprido,
atravessado por uma jornada árdua e gratificante, que chego ao final de uma
escrita e inicio de uma nova carreira, enlaçada pela ética, respeito,
determinação e amor à profissão.
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SUMÁRIO
ABREVIATURAS ...........................................................................................................................i
RESUMO.......................................................................................................................................iii
ABSTRACT.....................................................................................................................................iv
1. INTRODUÇÃO ....................................................................................................................... 1
1.1. TRATAMENTOS FARMACOLÓGICOS E MODELOS ANIMAIS PARA O
ESTUDO DA DOENÇA DE PARKINSON .......................................................................... 6
1.2. DOENÇA DE PARKINSON, ESTRESSE OXIDATIVO E AGENTES
ANTIOXIDANTES. ................................................................................................................. 9
2. JUSTIFICATIVA ................................................................................................................... 11
3. OBJETIVOS .......................................................................................................................... 12
3.1. OBJETIVO GERAL ........................................................................................................... 12
3.2. OBJETIVOS ESPECÍFICOS ........................................................................................... 12
3.2.1 Objetivos artigo 1 ........................................................................................................ 12
3.2.2 Objetivos artigo 2 ........................................................................................................ 12
3.2.3 Objetivos artigo 3 ........................................................................................................ 13
4. Artigo 1 ................................................................................................................................. 14
5. Abstract .................................................................................................................................. 15
6. Introduction ........................................................................................................................... 16
7. Material and Methods .......................................................................................................... 17
7.1. Animals ........................................................................................................................... 17
7.2. Drugs .............................................................................................................................. 17
7.3. Experimental design ..................................................................................................... 18
7.4. Statistical Analysis ........................................................................................................ 19
8. Results ................................................................................................................................... 19
8.1 Catalepsy ........................................................................................................................ 19
8.2. Novel object recognition .............................................................................................. 20
9. Discussion ............................................................................................................................. 22
10. Acknowledgments: ............................................................................................................. 27
11. References .......................................................................................................................... 28
12. Artigo 2 ............................................................................................................................... 34
13. Abstract ............................................................................................................................... 35
14. Introduction ......................................................................................................................... 36
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15. Material and Methods ........................................................................................................ 37
15.1. Animals ......................................................................................................................... 37
15.2. Drugs ............................................................................................................................ 38
15.3. Experimental design and general procedures ....................................................... 38
16. Behavioral tests .................................................................................................................. 39
16.1. Catalepsy test ............................................................................................................. 39
16.2. Oral movements ......................................................................................................... 39
16.3. Rotarod test ................................................................................................................. 40
16.4. Immunohistochemistry for tyrosine hydroxylase (TH) ........................................... 40
16.5. Enzymatic Analysis .................................................................................................... 42
16.6. Statistical Analysis ...................................................................................................... 42
17. Results ................................................................................................................................. 43
17.1. Catalepsy ..................................................................................................................... 43
17.2. Oral movements ......................................................................................................... 44
17.3. Rotarod test ................................................................................................................. 44
17.4. Immunohistochemistry for tyrosine hydroxylase (TH) ........................................... 45
17.5. Determination of CAT and SOD activities .............................................................. 48
18. Discussion ........................................................................................................................... 50
19. Acknowledgments: ............................................................................................................. 54
20. References .......................................................................................................................... 55
21. Artigo 3 ............................................................................................................................... 60
22. Abstract ............................................................................................................................... 61
23. Introduction ......................................................................................................................... 62
23.1 Animal models of PD ....................................................................................................... 63
23.2. Motor and non-motor behavioral impairment in the reserpine model ..................... 65
23.3. Pharmacological and predictive quality of the reserpine model .............................. 67
23.4. Molecular and neurochemical features of the reserpine model .............................. 68
24. Final considerations ........................................................................................................... 74
25. References .......................................................................................................................... 77
26. CONSIDERAÇÕES FINAIS ............................................................................................. 96
27. REFERÊNCIAS .................................................................................................................. 97
28. Anexos ............................................................................................................................... 105
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ABREVIATURAS
%EON - % Exploração do objeto novo
5-HT - Serotonina
6-OHDA - 6-hidroxidopamina
CEUA - Comissão de ética no uso de animais
COMT - Catecol-o-metiltransferase
CPu - Núcleo Caudado Putamen
DA - Dopamina
DAB - Diaminobenzidina
DOR - Densitometria óptica relativa
DP - Doença de Parkinson
DS - Dorsal striatum
DV - Dorsoventral
ED - Estriado dorsal
EPM - Erro Padrão da Média
GABA - Via gabaérgica
GD - Giro denteado
Glu - Via glutamatérgica
GP - Globo pálido
GPe - Globo pálido extermo
GPi - Globo pálido interno
HIP - Hipocampo
HPLC - High-Performance Liquid Chromatography
L-DOPA L-3,4-dihydroxyphenylalanine
MAO-B - Monoamino oxidase-B
MPP+ -1metilfenilpiridina
MPPP - 1-metil-4-fenilphenyl-4-propionpiperidina
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MPTP - 1-metil-4-fenil-1,2,3,6-tetraidropiridina
NA - Noradrenalina
NE - Noradrenergic
NST - Núcleo subtalâmico
PB - Tampão fosfato
PBS - Tampão fosfato salina
PD - Parkinson´s disease
PFA - Paraformaldeído
PFC - Pré-frontal córtex
REM - Rapid eye movement
RES – Grupo tratado com reserpina
RESt - Grupo Reserpine treated
RESw - Grupo Reserpine withdrawn
ROD - Relative optic density
RON – Reconhecimento do objeto novo
s.c. - Subcutânea
SNpc - Substância negra parte compacta
SNpr - Substância negra parte reticulada
TH - Tirosina hidroxilase
TOC – Grupo tratado com α-tocoferol
VEI - Grupo Controle
VMAT-2 - Vesicular monoamine transporter - 2
VTA - Ventral tegmental area
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RESUMO
A doença de Parkinson (DP) é um distúrbio neurodegenerativo progressivo que
afeta aproximadamente de 1-2% da população mundial, com maior prevalência
entre os homens. Os principais sintomas são motores, e incluem bradicinesia,
rigidez, instabilidade postural e tremor em repouso. Além disso, ocorrem
sintomas não motores, tais como distúrbios do sono, ansiedade, depressão, e
déficits cognitivos. Tais alterações clínicas são consequência da perda
irreversível de neurônios dopaminérgicos principalmente na substância negra
parte compacta. O tratamento mais eficaz para a DP é o uso da levodopa,
porém, esta medicação trata apenas os sintomas, apresentando limitações
após o uso prolongado. Sendo assim, consideram-se alternativas de
tratamento que pudessem conferir neuroproteção, retardando a progressão da
doença e/ou prevenindo o surgimento dos sintomas. Um exemplo seria o uso
de antioxidantes, dentre eles, o α-tocoferol. Os mecanismos, assim como a
natureza crônica da doença, podem ser mimetizados e estudados a partir do
uso de modelos animais. Dessa forma, o principal objetivo do nosso estudo foi
investigar os efeitos da administração do α-tocoferol sobre os danos motores,
cognitivos e neuronais em um modelo animal para doença de Parkinson.
Utilizamos a administração repetida de uma dose baixade reserpina,
concomitante com a aplicação de α-tocoferol. Nós observamos que o
tratamento repetido com reserpina provocou déficits cognitivos e motores de
forma progressiva, além de uma diminuição na marcação para a enzima
tirosina hidroxilase (envolvida na síntese de dopamina) na via nigroestriatal. No
entanto, esses déficits não foram apresentados pelo grupo de animais tratados
com α-tocoferol, evidenciando um provável efeito neuroprotetor provacado pelo
antioxidante. Podemos concluir que a aplicação de α-tocoferol foi capaz de
previnir as alterações causadas pela administração de reserpina. Ainda, o
nosso estudo sugere que a indução de danos motores e cognitivos
progressivos pela reserpina quando aplicada em baixas doses são adequados
para o estudo de possíveis intervenções neuroprotetornas para a DP.
Palavras-chave: Reserpina, Doença de Parkinson; α-tocoferol; Detrimento
motor, Prejuízo mnemônico.
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vi
ABSTRACT
Parkinson’s disease (PD) is a progressive neurodegenerative disorder that
affects 1-2% of world population, with a higher prevalence among men. The
main symptoms are of motor nature, and include bradikynesia, rigidity, postural
instability and tremor. In addition, non-motor symptoms may occur, such as
sleep disturbances, anxiety, depression, and cognitive deficits. The motor
alterations are a consequence of the irreversible loss of dopaminergic neurons
mainly in the substantia nigra pars compacta. The most effective current
treatment for PD is L-DOPA administration. However, this drug, despite
amegliorating symptoms, does not interfere with the neurodegeneration, and
thus has limitations at long term. Thus, alternative treaments that could act by
neuroprotective mechanisms have been considered, such as antioxidant
agents. The mechanisms related to the symptoms and progressive nature of PD
can be studied in animal models. In this sense, the aim of the present study was
to investigate the effects of the antioxidant α-tocopherol on the motor, cognitive
and neuronal deficits induced by repeated treatment with reserpine (a
progressive pharmacological model of parkinsonism). Rats submitted to the
reserpine protocol were concomitantly treated with α-tocopherol. The results
showed that the repeated treatment with reserpine, as expected, induced
progressive motor and cognitive decrements, as well as dimished tyrosine
hydroxylase immunostaining in the substantia nigra pars compacta and
striatum. These deficits were not present in the animals that were co-treated
with α-tocoferol, suggesting a possible neuroprotective effect induced by this
antioxidant agent. In conclusion, α-tocoferol was able to prevent the alterations
caused by repeated reserpine administration. In addition, our study suggest that
low-dose reserpine-induced progressive motor and cognitive deficits can be
useful in the study of possible neuroprotective strategies for PD.
Keywords: Reserpine, Parkinson’s disease, α-tocopherol, motor impairment,
short-term memory impairment.
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1. INTRODUÇÃO
A doença de Parkinson (DP) é uma doença neurodegenerativa
progressiva, a qual foi descrita pela primeira vez em 1817 por James Parkinson
como “paralisia agitante” (Teive,1998).
Atualmente, a DP é uma das doenças neurodegenerativas mais comuns
em indivíduos com idade avançada, surgindo em média aos 55 anos. Por volta
dos 70 anos de idade há um aumento na incidência da doença (Hald &
Lotharius, 2005; Chesselet, 2011). Nos EUA o risco de desenvolvimento da DP
entre mulheres é de 1,3%, e entre os homens 2%, enquanto que em indivíduos
com mais de 80 anos de idade a prevalência chega a 5% (De Lau & Breteler,
2006; Nussbaum & Ellis, 2003; Wood-Kaczmar et al., 2006). No Brasil essa
prevalência é de 3,3%, para a população acima de 65 anos (Barbosa et al.,
2006). Pode ser diagnosticada em qualquer idade, porém, apenas 3% dos
casos são reconhecidos em indivíduos com menos de 50 anos de idade (Van
Den Eeden, 2003). A doença se instala progressivamente, comprometendo
diretamente a qualidade de vida dos pacientes acometidos (Chesselet, 2011).
A etiologia da DP apresenta características multifatoriais e a doença está
relacionada com os fatores de risco ambientais, tais como, exposição a
herbicidas, inseticidas (Elbaz & Tranchant, 2007), metais pesados,
envenenamento por monóxido de carbono (Nicholson et al., 2002), exposição a
substâncias tóxicas tais como 6-hidroxidopamina (6-OHDA) e 1-metil-4-fenil-
1,2,3,6-tetrahidropiridina (MPTP), entre outros (Calne, 2007; Mayeux, 2003).
De 5% a 10% dos casos observam-se ligações entre a ocorrência da doença e
as mutações gênicas que podem causar diversas formas de parkinsionismo e
da DP, dentre estes genes, estão o Parkin. Pink1, DJ-1, para formas recessivas
de início precoce, e os genes da α-sinucleína, LRRK-2 e GBA para mutações
dominantes, que levam ao desenvolvimento da DP (Revesz, 2009; Przedborski,
2004; Hald & Lotharius, 2005). Outro fator seria o estresse oxidativo que está
envolvido na patogênese da DP, e é descrito como um desequilíbrio entre a
formação e a eliminação de espécies reativas de oxigênio (ERO) e de
nitrogênio (ERN; Barnham et al., 2004; Calabrese et al., 2007). O oxigênio
pode gerar ERO seja por absorção de energia ou transferência de elétrons
2
(Barreiros et al., 2006), aumentando as EROs e promovendo neurotoxicidade e
danos na membrana neuronal, que como consequência, pode provocar morte
celular (Dauer & Przedborski., 2003). O sistema nervoso apresenta uma alta
vulnerabilidade às espécies reativas de oxigênio, e várias evidências sugerem
que a formação de radicais livres e estresse oxidativo possa desempenhar um
papel importante na patogênese da DP (Russel et al., 1998; Halliwell &
Gutteridge, 1999). Quando a produção das espécies reativas de oxigênio -
ROS excede a capacidade do sistema antioxidante em eliminá-las, ocorrem os
danos oxidativos (Jenkins & Goldfard, 1993), e causa danos na membrana
neuronal, que pode ter como consequência apoptose celular (Farooqui &
Farooqui, 2011; Patel & Chu, 2011). Este evento está associado à presença de
agregados ou inclusões citoplasmáticas nucleares, os corpúsculos de Lewy
(Tgo et al., 2001; Dauer & Przedborski, 2003), tais agregados, são constituídos
principalmente pela proteína neural α-sinucleína (Koo et al., 2008) e parkina
(Singh & Dikshi, 2007). O acúmulo desses corpúsculos desencadeiam
excitoxicidade com consequente morte neuronal da via dopaminérgica
nigroestriatal (Corti et al., 2001; Przedborski, 2005; Swinner et al., 2011), dessa
forma, há uma redução dos níveis de dopamina no estriado, bem como em
outros núcleos da base (Gerlach & Riederer, 1996) e surgimento dos sintomas
clínicos da DP.
O diagnóstico clínico da DP atualmente é baseado na presença de sinais
e sintomas manifestados ou relatado pelos pacientes, principalmente: (1) os
sintomas motores, tais como, bradicinesia (dificuldade em iniciar movimento),
rigidez (aumento do tônus muscular), tremor em repouso e instabilidade
postural, os quais se iniciam quando ocorre uma perda de >50% de neurônios
da substância negra parte compacta (SNpc), com consequente redução de
>80% dos níveis de dopamina (DA) do estriado (Deumens et al., 2002; Fahn,
2003; Klockgether, 2004; Reeve, 2014); (2) sintomas não-motores, tais como
alterações cognitivas com perda progressiva da memória e/ou demência
(Schapira et al., 2006), e comprometimento na aprendizagem, ansiedade,
depressão (Brown et al., 2011; Higginson et al., 2005), distúrbios do sono
(Clarenbach, 2000) e na resposta à terapia com medicações tradicionalmente
utiladas na clínica médica (Nicholson et al., 2002; Klockgether, 2004; Jankovic,
2008).
3
Os distúrbios não motores da DP passaram a ser considerados com
maior importância nas últimas décadas, já que os prejuízos cognitivos afetam
significativamente a qualidade de vida das pessoas acometidas e influenciam
de forma negativa cuidadores e familiares (Korczyn, 2001; Nieoullon, 2002;
Zgaljardic et al., 2004). Ainda assim, muitos desses sintomas cognitivos não
são diagnosticados com antecedência ou não são tratados, muitas vezes
devido a poucas evidências científicas ou a abordagens profissionais mais
detalhadas (Slawek et al., 2005). Em alguns casos, os prejuízos cognitivos não
parecem estar relacionados aos sintomas motores, pois, em geral, se
manifestam antes de tais alterações (Fénelon, 1997; Shults, 2003). Além disso,
têm sido correlacionados a disfunções nas projeções das vias dopaminérgicas
envolvidas em funções de áreas fronto-corticais, tais como planejamento de
ações e a memória operacional (Pillon et al., 1997; 1997a; Cools et al., 2002),
podendo acometer a memória, atenção, linguagem, habilidades visuoespaciais,
visuoconstrutivas e funções executivas (Zgaljardic et al., 2004).
Sabe-se também que na DP os neurônios dopaminérgicos da área
tegmentar ventral (VTA) podem estar diminuídos, com consequente redução de
DA nas vias mesocortical e mesolímbica, estando relacionadas com as
alterações cognitivas e emocionais da doença (Dymecki et al., 1996; Vernier et
al., 2004).
Os neurônios dopaminérgicos originam-se de certos núcleos específicos
do cérebro, dentre eles: a SNpc, a VTA e os núcleos hipotalâmicos. As
projeções dos feixes de fibras desses núcleos formam as quatro principais vias
dopaminérgicas: a via nigroestriatal (corpos celulares da SNpc projetam-se
para os núcleos caudado e putamen); a via mesocortical (corpos celulares em
VTA projetam-se para o córtex pré-frontal); a via mesolimbica (corpos celulares
de VTA projetam-se para o núcleo accumbens, amígdala e hipocampo); e a via
tuberoinfundibular (corpos celulares no hipotálamo projetam-se para hipófise;
Machado, 2005).
Estas projeções dopaminérgicas atuam sobre dois tipos distintos de
receptores, os das classes D1 e D2. Os receptores da classe D1 são formadas
pelas famílias D1 e D5 e possuem propriedades excitatórias, enquanto os da
classe D2, são formados pelas famílias D2, D3 e D4, e possuem propriedades
inibitórias. As duas classes atuam em diversas funções, dentre elas o
4
planejamento, a regulação e a execução dos movimentos voluntários
automáticos (Guttman, 1992). A ação dopaminérgica pode promover no
estriado a ativação da via direta, através dos receptores D1 e a inibição da via
indireta, através da estimulação dos receptores do tipo D2, nos circuitos
reguladores do movimento nos gânglios da base.
A via direta tem início no córtex cerebral, especificamente em áreas
motoras primária, suplementar, pré-motora e somestésica, que se projetam
para o estriado, substância negra parte reticula (SNpr) e em seguida para a
porção interna do globo pálido, cujas aferências terminam nos núcleos ventral
anterior, lateral e centro mediano do tálamo e daí os impulsos nervosos são
direcionados para a área motora primária do córtex cerebral. A via direta
provoca a perda da inibição talâmica e maior excitação do córtex motor. Já a
via indireta, inibe o movimento reduzindo a atividade do globo pálido externo, o
que consequentemente causa desinibição do núcleo subtalâmico, ao qual
possui neurônios glutamatégicos, direcionados ao globo pálido interno,
ativando os núcleos de saída e inibindo a atividade do tálamo (Figura 1 -
Ponzoni & Garcia-Cairasco, 1995; Siegel, 2006; Albin et al., 1989; Smith et al.,
1998).
5
Figura 1: Representação esquemática do circuito dos gâglios basais. O esquema à esquerda (A) representa a transmissão normal e o da direita (B) representa o desequilíbrio na transmissão em pacientes com a Doença de Parkinson. Figura adaptada de Bravo et al., 2014. Abreviaturas: globo pálido interno – GPi, globo pálido externo – GPe, núcleo subtalâmico – NST, substância negra parte compacta – SNpc, substância negra parte reticulada – SNr, receptor de dopamina D1 e receptor de dopamina D2.
Com a morte das células dopaminérgicas ocorre uma diminuição da DA
liberada em estruturas que recebem projeções dopaminérgicas (Centonze et
al., 1999). A perda neuronal altera o planejamento motor, produzindo quadros
hipocinéticos devido à ação desregulada das vias direta e indireta. A morte dos
neurônios dopaminérgicos da SNpc, provoca diminuição da DA no estriado e
desinibição GABAégica dos neurônios da via indireta, gerando acentuada
hipoatividade do globo pálido externo (GPE), seguido por desinibição do núcleo
subtalâmico. Portanto, a bradicinesia e acinesia da DP, resultam do aumento
da inibição GABAérgica sobre os neurônios dos grupos ventral anterior, lateral
e centro-mediano do tálamo, por excesso informações excitatória advindas do
núcleo subtalâmico sobre o globo pálido interno (GPI) esubstância negra parte
reticulada (SNpr).
DP
Normal
6
1.1. TRATAMENTOS FARMACOLÓGICOS E MODELOS ANIMAIS PARA O
ESTUDO DA DOENÇA DE PARKINSON
Apesar dos avanços científicos para o tratamento da DP, as abordagens
terapêuticas e cirúrgicas apresentam pouca eficácia no controle dos sintomas
motores da doença a longo prazo (Lees et al., 2009). Desde a década de 1960,
os tratamentos mais utilizados para a DP são sintomáticos, visando restaurar
ou disponibilizar níveis de DA nas vias dopaminérgicas. O uso da Levodopa (L-
DOPA) é uma das primeiras estratégias de tratamento a ser utilizada para o
tratamento da DP, que melhor promove diminuição dos sintomas,
principalmente os relacionados à bradicinesia e rigidez. Apesar de atualmente
ser o método mais utilizado para aliviar os sintomas, apresenta após o uso
prolongado, efeitos colaterais como: discinesias, coreia, atetose, distonias;
alucinações (Lees et al., 2009; Stayte & Vissel, 2014) e oscilações motoras
decorrentes do aumento e diminuição dos níveis plasmáticos da DA (fenômeno
“On/Off”) (Massano, 2011). Estima-se que cerca de 90% dos indivíduos
enfrentam o aparecimento desses efeitos indesejáveis após 10 anos do início
do tratamento com o fármaco (Ahlskog et al., 2001).
O sucesso inicial dessas estratégias de tratamento baseou-se no
entendimento de que a DP seria um distúrbio relacionado com a deficiência de
DA. Em face da diminuição da eficácia a longo prazo e dos importantes efeitos
colaterais, ocorre a utilização de outras medicações que agem em conjunto
com a levodopa, como os agonistas dopaminérgicos (pramipexole, ropinirone,
bromocriptina, pergolide, lisuride, e outros) (Lees et al., 2009). Além dos
fármacos acima citados, outras medicações também são usadas no tratamento
da DP, como os inibidores da monoamina oxidase - MAO-B (selegilina e
rasagilina), a amantandina e os inibidores de catecol-O-metil-transferase –
COMT (entacapone) (Nicholson et al., 2002; Lees et al., 2009). Vale ressaltar
que a maioria dos sintomas não motores apresenta baixa resposta à terapia
dopaminérgica por também serem manifestações de acometimento em vias
noradrenégicas e serotoninérgicas (Chaudhuri et al., 2011).
Entretanto, apesar das descobertas científicas sobre a etiologia,
fisiopatologia e terapêutica da DP, ainda não existe um consenso sobre os
mecanismos da doença (Shimohama, 2003). Dessa forma, utilizamos modelos
7
animais para estudar os mecanismos patogênicos, os possíveis do tratamento
e os sintomas das patologias humanas, podendo inclusive sugerir novas
formas de abordagem terapêutica a serem testadas na clínica (Gerlach &
Riederer, 1996).
Sabe-se também que a DP é uma doença que tipicamente de humanos,
apresentando poucos relatos do aparecimento desta doença
neurodegenerativa em animais (Dauer & Przedborski, 2003). Dessa forma,
essas características presentes nas diferentes fases na DP só podem ser
observadas em animais através da administração de agentes neurotóxicos
(Lima et al., 2006). Infelizmente, não existe um modelo fidedigno, que
represente todos os sintomas da doença, principalmente no que diz respeito à
natureza progressiva do surgimento dos sintomas (Da Cunha et al., 2008). Nas
últimas décadas alguns modelos foram desenvolvidos e os mais estudados
utilizavam toxinas tais como 6-OHDA e MPTP (Gerlach & Riederer, 1996).
Porém, esses dois modelos têm demonstrado resultados com perdas
específicas e imediatas de células do sistema nervoso (SN), não apresentando
um processo neurodegenerativo progressivo (Meredith et al., 2008).
Outro modelo que pode ser utilizado para se estudar a DP em animais, é
a administração de reserpina. Este fármaco atua através da depleção de
monoaminas, causando distúrbios sobre a atividade motora e cognitiva dos
animais (Colpaert, 1987; Alves et al., 2000; Silva et al., 2002; Skalisy et al.,
2002). A reserpina é uma droga que evita o armazenamento de monoaminas
nas vesículas sinápticas, através do bloqueio dos transportadores da
membrana que captam as monoaminas para dentro da vesícula (Verheij &
Cools, 2007). Dessa forma, as vesículas sinápticas permanecem vazias e
consequentemente não há neurotransmissores para serem liberados na fenda
sináptica quando um potencial de ação atinge o botão sináptico (Rang et al.,
2004).
A depleção de dopamina estriatal tem demonstrado ser um bom modelo,
por promover sintomas típicos da DP, como a acinesia, a rigidez, tremores e
déficits cognitivos visuoespaciais (Colpaert, 1987; Johnston et al., 1999; Skalisy
et al., 2002; Fernandes et al., 2008; Aguiar et al., 2009). Contudo, é importante
ressaltar que o tratamento com reserpina, como um modelo de DP, apresenta
limitações, pois a administração da droga não provoca depleção de
8
neurotransmissores apenas na via nigroestriatal e nem age exclusivamente em
vias dopaminérgicas. Por outro lado, como comentado acima, essa pode ser
uma vantagem em relação aos modelos seletivos para vias dopaminérgicas,
uma vez que alguns sintomas da DP, especialmente os não-motores, vem
sendo relacionados a déficits em outras vias monoaminérgicas (Chaudhuri et
al., 2011).
Outro aspecto é o fato da administração de reserpina, a priori, não
promover uma degeneração neuronal progressiva. Nesse sentido, a
administração crônica de reserpina em baixas doses pode promover déficits
cognitivos e motores de forma progressiva (Fernandes et al., 2012; Santos et
al., 2013), podendo assim ser utilizado eficazmente como um modelo para DP
em animais. A grande maioria dos trabalhos envolvendo a DP, tanto em
humanos quanto em animais, procuram responder questões relacionadas aos
prejuízos motores. Além disso, como jámensionado, os trabalhos com modelos
animais, em sua maioria, verificam respostas comportamentais após
tratamento agudo, o que leva a prejuízos motores intensos e imediatos,
impedindo a avaliação de qualquer tipo de alteração comportamental além da
motora. Esse fato é relevante, porque além dos prejuízos motores, existem
outras manifestações sintomáticas em pacientes com a DP, como alterações
na cognição, no humor e no sistema sensorial. Dessa forma, o surgimento
gradual dos sintomas pode ser vantajoso para a detecção desses sinais não
motores.
Além disso, a reserpina pode promover uma elevação no estresse
oxidativo celular, possivelmente pelo aumento da metabolização da dopamina
acumulada no citoplasma pela enzima monoaminaoxidase (Abílio et al., 2002).
Esse efeito é encontrado também após a administração crônica de baixas
doses (Fernandes et al., 2012). Nesse sentido, sugere-se que a administração
repetida de uma dose baixa de reserpina pode ser um protocolo adequado para
estudar tentativas terapêuticas que interfiram com a progressão da doença.
De fato, diante do surgimento de efeitos colaterais graves e da eficácia
apenas sintomática das drogas citadas acima, inúmeras pesquisas tem
buscado estudar tratamentos que agissem não apenas sobre os sintomas, mas
também retardando o processo neurodegenerativo.
9
1.2. DOENÇA DE PARKINSON, ESTRESSE OXIDATIVO E AGENTES
ANTIOXIDANTES.
O estresse oxidativo é derivado de um desequilíbrio entre a formação e
a eliminação de espécies reativas de oxigênio – ERO, e de nitrogênio - ERN
(Barnham et al., 2004; Calabrese et al., 2007). O oxigênio pode gerar ERO por
absorção de energia ou transferência de elétrons (Barreiros et al., 2006),
promovendo neurotoxicidade e danos na membrana neuronal, que como
consequência, pode provocar morte celular (Dauer & Przedborski, 2003).
O sistema nervoso apresenta uma alta vulnerabilidade às EROs, e
evidências sugerem que a formação desses radicais livres possa desempenhar
um papel importante na patogênese da DP (Halliwell & Gutteridge, 1999;
Farooqui & Farooqui, 2011). Este evento também está associado à presença
de agregados ou inclusões citoplasmáticas nucleares, os corpúsculos de Lewy
(Dauer & Przedborski, 2003), os quais são constituídos principalmente pela
proteína neural α-sinucleína (Koo et al., 2008) e pela parkina (Singh & Dikshi,
2007). Estes agregados proteicos podem desencadear elevações na
excitoxicidade e, consequentemente, morte neuronal, principalmente na via
dopaminérgica nigroestriatal (Corti et al., 2001; Przedborski, 2005). O
desequilíbrio da via dopaminérgica leva a uma redução dos níveis de dopamina
no estriado, assim como em outros núcleos ou gânglios da base (Gerlach &
Riederer, 1996; Mizuno, 1999). Também há uma redução na atividade de
enzimas responsáveis pela síntese de dopamina, como a tirosina hidroxilase
(TH) e a DOPA-descarboxilase (Gerlach & Riederer, 1996).
A produção contínua de radicais livres durante os processos metabólicos
gera muitos mecanismos antioxidantes intracelulares para impedir a indução de
danos à célula. Os antioxidantes são agentes responsáveis pela inibição e
redução das lesões causadas pela produção de ERO nas células (Bianchi &
Antunes, 1999), atuando no retardo e/ou inibição da oxidação destes agentes
tóxicos (Halliwell & Gutteridge,1999).
Os agentes antioxidantes podem ser classificados em enzimáticos e não
enzimáticos. O sistema enzimático de defesa é constituído pelas enzimas
superóxido dismutase (SOD), catalase (CAT), glutationa peroxidase (GPx) e a
glutationa redutase (GR) (Grissa, 2007). A presença desses antioxidantes no
10
sistema celular é conhecida por prevenir danos oxidativos (Therond, 2000;
Schneider & Oliveira, 2004; Wu et al., 2005), constituindo a primeira defesa
endógena de neutralização das EROs. Através delas, as células tentam manter
baixas as quantidades do radical superóxido e de peróxidos de hidrogênio,
evitando a formação do radical OH (Halliwell & Gutteridge, 1999).
Os agentes antioxidantes não enzimáticos complementam ação
antioxidante protetora do sistema biológico sendo constituídos por substâncias
como a vitamina E (tocoferóis), vitamina C (ácido ascórbico) e β-caroteno
(carotenoides) (Grissa, 2007). Estas substâncias são os antioxidantes mais
estudados em animais e humanos por atuarem na redução de radicais livres,
principalmente do radical peroxil, e do oxigênio singlete (Filipe et al., 2001). A
vitamina E, o principal antioxidante lipossolúvel presente nas membranas
celulares, tem o α-tocoferol como seu componente mais importante e atua no
bloqueio da etapa de propagação da peroxidação lipídica dos ácidos graxos
poliinsaturados das membranas e lipoproteínas, ao doar um átomo de
hidrogênio aos radicais peroxil e alcoxil. A capacidade do α-tocoferol é
conferida pela regeneração do radical α-tocoferoxil por agentes redutores,
principalmente o ácido ascórbico, exercendo assim a sua atividade antioxidante
(Halliwell & Gutteridge, 1999).
A administração exógena da vitamina E tem sido considerada para o
tratamento de distúrbios degenerativos, em especial a DP, geralmente em
associação com a terapia convencional sintomatológica (De Araújo et al., 2011;
Magyar et al., 2004; Mayo et al., 2005; Weber & Ernst, 2006; Weinreb et al.,
2010; Hsieh et al., 2012; Marin & Aguiar, 2011; Salamone & Baskin, 1996).
Embora os estudos geralmente apontem para retardo na progressão dos
sintomas, fica difícil diferenciar os efeitos da terapia complementar daqueles da
terapia principal sintomatológica. Nesse sentido, em modelos animais, tem-se a
oportunidade de avaliar o efeito do tratamento antioxidante per se, em ratoss
não-tratados com L-DOPA ou outro agonista dopaminérgico. Assim, o efeito
neuroprotetor da vitamina E na DP foi proposto por estudos in vitro e em
modelos animais de parkinsonismo (Miklya et al., 2003; Butterfield et al., 2002;
Roghani & Behzadi, 2001; Azzi, 2007; Azzi et al., 2004; Ferri et al., 2006).
Porém, na maioria desses estudos em modelos animais, os efeitos desse
agente antioxidante não foi avaliado quanto à progressão das alterações
11
comportamentais inerentes à DP, focando apenas em grau de lesão neuronal
ou avaliações comportamentais não relacionadas aos sintomas da doença (por
exemplo, comportamento rotacional induzido por agonistas dopaminérgicos).
A partir disso, seria importante ampliar as pesquisas em animais,
buscando avaliar a progressão tanto dos déficits motores quanto dos cognitivos
associados ao aparecimento da doença.
Em resumo, as pesquisas para o tratamento da DP tendem a se focar
em agentes neuroprotetores que pudem atuar no processo neurodegenerativo
da patologia, retardando ou até mesmo impedindo esse processo. Nesse
sentido, os processos neurodegenerativos estariam associados ao estresse
oxidativo, levando à ideia de que várias doenças neurológicas podem ser
evitadas e/ou atenuadas pelo tratamento com agentes antioxidantes, como a
vitamina E (α-tocoferol) discutida neste estudo.
2. JUSTIFICATIVA
Uma grande variedade de intervenções tem sido estudada com o
objetivo de solucionar alguns aspectos no tratamento da DP. Ainda, pesquisas
que buscam entender o funcionamento dos mecanismos básicos que envolvem
esta doença neurodegenerativa tem atraído a atenção da comunidade científica
há algumas décadas. Os modelos animais que buscam mimetizar os sintomas
cognitivos e motores encontrados na DP estão entre as pesquisas que mais se
destacam no que diz respeito ao entendimento do fisiopatologia da doença.
Entretanto, os modelos animais mais estudados se baseiam na administração
aguda de fármacos que causam lesões em neurônios dopaminérgicos na via
nigroestriatal (o principal foco de neurodegeneração descrito na etiologia da
doença). Todavia, para tratamentos com proposta neuroprotetora, o uso de
modelos que induzem sintomas parkinsonianos crônicos ainda não foi
explorado.
Diante do exposto, a proposta desse trabalho é a utilização de um
modelo farmacológico progressivo com a administração crônica de uma dose
baixa de reserpina (um depletor de monoaminas) para estudar a ação de um
tratamento com potencial neuroprotetor.
12
3. OBJETIVOS
3.1. OBJETIVO GERAL
Analisar os efeitos da administração da vitamina E sobre os danos
motores, cognitivos e neuronais em um modelo animal de doença de Parkinson
induzido pela administração repetida de reserpina. Os objetivos serão
apresentados ao longo do texto subdivididos em três distintos artigos
científicos, com isso os objetivos específicos serão distribuídos da mesma
forma.
3.2. OBJETIVOS ESPECÍFICOS
3.2.1 Objetivos artigo 1
a) Avaliar as alterações motoras, a partir do teste diário de catalepsia,
para definir o período do aparecimento desses sintomas ao longo do
tratamento, e se a vitamina E é capaz de retardar ou impedir o aparecimento
desses prejuízos motores;
b) Avaliar as alterações cognitivas, a partir do teste de reconhecimento
de objetos no campo aberto, a cada 3 injeções, para verificar o período do
aparecimento desses sintomas, através de uma tarefa de memória de
reconhecimento e se a vitamina E é capaz de retardar ou impedir o
aparecimento desses prejuízos cognitivos.
3.2.2 Objetivos artigo 2
a) Avaliar as alterações motoras, a partir do teste do rotarod, a cada 3
injeções, para definir o período do aparecimento desses sintomas motores, e
se a vitamina E é capaz de retardar ou impedir o aparecimento desses
prejuízos motores;
b) Avaliar as alterações motoras, a partir da avaliação dos movimentos
orais – protrusão da língua, tremor de queixo e mastigação não direcionada a
nenhum objeto, a cada 3 injeções, para definir o período do aparecimento
desses sintomas, e a vitamina E é capaz de retardar ou impedir o aparecimento
desses prejuízos motores;
13
c) Avaliar parâmetros de estresse oxidativo pela quantificação da
atividade das enzimas antioxidantes (catalase e superóxido desmutase) no
estriado e hipocampo de ratos submetidos ao tratamento crônico com reserpina
com ou sem o tratamento com vitamina E;
d) Analisar os efeitos do tratamento repetido com reserpina com ou sem
o tratamento com a vitamina E sobre os níveis de tirosina hidroxilase (TH) em
diferentes regiões cerebrais através da imunohistoquímica.
3.2.3 Objetivos artigo 3
a) Revisar na literatura os mecanismos neuroquímicos, moleculares e
comportamentais da utilização de reserpina como modelo animal para a DP.
14
4. Artigo 1
(Aceito para publicação no periódico “Biochemistry & Pharmacology”)
ALPHA-TOCOPHEROL COUNTERACTS COGNITIVE AND MOTOR
DEFICITS INDUCED BY REPEATED TREATMENT WITH RESERPINE
Aldair José Sarmento-Silvaa, Ramón Hypolito Limaa, Alicia Cabrala, Ywlliane
Meurera Alessandra Mussi Ribeiroa,b, Regina Helena Silvaa,c*
aMemory Studies Laboratory, Physiology Department, Federal University of Rio
Grande do Norte, Natal, Brazil.
bDepartment of Biosciences, Federal University of São Paulo, Santos, Brazil.
cDepartment of Pharmacology, Federal University of São Paulo, São Paulo,
Brazil.
*Corresponding Author
Departamento de Farmacologia – UNIFESP
Rua Botucatu, 862, Edifício Leal Prado, 1º.andar
CEP 04023062 - São Paulo, SP, Brasil
Email: [email protected]
15
5. Abstract
Previous studies showed that chronic administration of the monoamine
depleting agent reserpine in low doses promotes progressive cognitive and
motor impairments in rats, and this protocol has been used as a
pharmacological progressive model of Parkinson's disease. These behavioral
alterations are accompanied by increased brain oxidative stress. We aimed to
verify the effects of the concomitant treatment with the antioxidant agent alpha-
tocopherol on the motor and cognitive deficits induced by chronic reserpine in
rats. Rats were repeatedly treated with 0.1 mg/kg reserpine with or without a
concomitant treatment with 40 mg/kg alpha-tocopherol. Across the treatment,
motor and cognitive performances were evaluated by the catalepsy and novel
object recognition tests, respectively. As expected, reserpine-treated rats
showed progressively increased duration of catalepsy together with short-term
memory deficits in the object recognition test. Importantly, these detrimental
outcomes due to reserpine treatment were prevented by concomitant daily
administration of the antioxidant agent alpha-tocopherol. The results show a
preventive role of alpha-tocopherol on behavioral alterations induced by
repeated reserpine treatment. This is relevant to the investigation of possible
neuroprotective interventions in Parkinson’s disease.
Keywords: Reserpine, Parkinson’s disease, α-tocopherol, motor impairment, short-term
memory impairment.
Abbreviations: NOR- Novel Object Recognition; PD – Parkinson´s Disease; RES –
reserpine; ROS – reactive oxygen species; TOC – alpha-tocopherol; VR – vehicle for
reserpine; VT – vehicle for alpha-tocopherol; PKC – protein kinase C
16
6. Introduction
Reserpine precludes the storage of monoamines through the blockage of
the synaptic vesicles transporters [1]. Consequently, synaptic vesicles are still
available but there is a reduction in the amount of dopamine in the synaptic
cleft. Because an important loss of dopaminergic neurons is the core feature of
Parkinson´s disease (PD) [2], reserpine administration to rodents is a valid
approach to study this disease in animal models [3-5]. The acute administration
of a high dose of reserpine (above 1.0 mg/kg) leads to severe motor impairment
[4]. In addition, acute injection of reserpine in lower doses causes memory
deficits in the absence of motor damage [6,7]. However, although both cognitive
and motor impairments are symptoms of PD, their emergence shortly after an
acute injection is not compatible with the gradual progression of symptoms
found in the clinical situation. More recently, studies have shown that the
chronic administration of reserpine in low doses can promote progressive
cognitive and motor impairments, along with decreased tyrosine hydroxylase
levels in the nigrostriatal pathway [8]. This protocol is suggested as a
progressive pharmacological model of PD [8,9].
Besides its classical mechanism of action (i.e. blockage of the vesicular
transport of monoamines), there is clear evidence that reserpine also causes an
increase in cellular oxidative stress, possibly potentiated by the rise in the levels
of dopamine in the cytoplasm, which undergoes oxidative metabolism [10]. In
this respect, the central nervous system is quite vulnerable to reactive oxygen
species (ROS), which play a very important function in the pathogenesis of
neurodegenerative disorders, including PD [11]. For example, there is evidence
that the inclusion of antioxidant agents in the pharmacological treatment of PD
has advantages over the treatment based only in dopamine replacement [11-
13]. In addition, the repeated treatment with reserpine that induces progressive
features compatible with PD also leads to increased brain oxidative stress [9].
However, it is unclear if a possible oxidative damage is responsible for the
behavioral deficits presented by animals repeatedly treated with reserpine.
Antioxidant agents mainly act as a reinforcement of endogenous
antioxidant defenses. An important antioxidant agent is vitamin E (alpha-
tocopherol; TOC), which plays an essential role in protecting the body against
17
the damaging effects of ROS. Specifically, TOC blocks the propagation step of
lipid peroxidation of polyunsaturated fatty acids in membranes and lipoproteins
[14], mainly by neutralizing the effects of peroxides and oxygen free radicals
[15].
The aim of this study was to evaluate the effects of the antioxidant agent
TOC on motor, cognitive and neuronal parameters in animals submitted to a
progressive pharmacological animal model of PD, i.e., the repeated treatment
with a low dose of reserpine.
7. Material and Methods
7.1. Animals
We used 75 five-month-old male Wistar rats (300-500g). The animals
were obtained from the Physiology Department at the Federal University of Rio
Grande do Norte, and were housed in groups of four, in plastic cages, under
controlled conditions of ventilation, temperature (23 ± 1°C), and light/dark cycle
(12h/12h, lights on 6:30 a.m.), with free access to water and food. The rats were
handled according to the Brazilian law for the use of animals in scientific
research (Law Number 11.794) and all the procedures described were
approved by the local ethical committee (CEUA/UFRN nº 051/2011).
7.2. Drugs
Reserpine (RES; Sigma Chemical Co., St. Louis, MO) was dissolved in
acetic acid and further diluted in distilled water at the concentration of 0.1
mg/mL, pH ≈ 6.5. We used this vehicle (glacial acetic acid diluted in water) as a
control for reserpine treatment (VR). RES and VR were given s.c. on alternate
days. The antioxidant alpha-tocopherol (TOC; Sigma Chemical Co., St. Louis,
MO) was diluted in distilled water with Tween-80 at the concentration of 40
mg/mL. We used the vehicle used to dilute TOC (VT) as a control for TOC
treatment. These solutions were injected i.p. daily. The volume of injection was
18
1mL/kg of body weight in all cases. We prepared all solutions every 48 hours
and kept them at 4ºC between administrations.
7.3. Experimental design
The rats were randomly assigned to the following groups: VR + VT
(n=18), RES + VT (n=19), RES + TOC (n=19) and VR + TOC (n=19). Drug
treatment lasted 30 days. Animals received 15 s.c. injections of RES (0.1
mg/kg) or VR every 48 hours, concomitantly to daily i.p. administration of TOC
(40 mg/kg) or VT.
Before the beginning of the experiments, all animals were submitted to a
daily 5-minute handling session for five consecutive days. Throughout the
treatment, all the animals were subjected to catalepsy tests (performed daily)
and part of the animals (n=35, 7-11 per group) went through the novel object
recognition (NOR) tasks (days 2, 12 and 18 of treatment). The experimental
design is shown in Figure 1. Both behavioral tests were performed as described
in our previous study [8] and were conducted before the injections of that day.
Thus, all behavioral evaluations were performed 48h after the last injection of
reserpine in order to avoid acute effects of the drug. NOR sessions were
recorded with a digital camera fixed above the arena and the behavior was
analyzed through a video-tracking software (Anymaze, Stoelting Co, Wood
Dale, Illinois, USA). Before each experimental procedure, the apparatuses were
cleaned with a 5% alcohol solution, and the experimental groups were
alternated across testing.
Figure 1. Schematic illustration of the experimental design.
19
7.4. Statistical Analysis
We analyzed the performances in catalepsy test (total time spent in
immobility until the animal removed both forepaws of the bar) by the two-way
ANOVA with repeated measures followed by Tukey’s multiple comparison post
hoc test. In the NOR task we conducted one-way ANOVA followed by
Bonferroni’s multiple comparison post hoc test in order to compare old versus
familiar object exploration. Analyses for the exploration ratio throughout test
sessions and among experimental groups were conducted through two-way
ANOVA followed by Tukey’s Post Hoc test.
8. Results
8.1 Catalepsy
Figure 2 shows that from day 15 onwards there was an increase in
catalepsy behavior of the group RES+VT compared to all other groups (RM
two-way ANOVA; days of treatment [F (29,2130) = 16.72, P < 0.0001], treatment [F
(3,2130) = 211.0, P < 0.0001] and days of treatment × treatment interaction effects
[F (87,2130) = 4.876, P < 0.0001]). This increase was not detected for the group
RES+TOC.
Figure 2. Repeated administration of reserpine increases catalepsy duration and this
effect is prevented by α-tocopherol. Animals were placed daily in a catalepsy bar and
20
the latency to step-down was registered. Arrows indicate reserpine (RES; 0.1 mg/kg) or
vehicle (VR) s.c. injections, while α-tocopherol (TOC; 40 mg/kg) or its vehicle (VT) were
administered through daily i.p. injections. Data are expressed as mean + SEM; (*) P <
0.05 for RES+VT vs RES+TOC; (#) P < 0.01 for RES+VT vs VR+VT; (***) P < 0.001
and (****) P < 0.0001 for RES + VT vs all experimental groups in Tukey’s multiple
comparison post hoc test after RM two-way ANOVA.
8.2. Novel object recognition
We found that all animals spent more time exploring the new object in the
second day of protocol (first test; Fig. 3A; one-way ANOVA [F (7,62) = 11.23; P <
0.0001]). Reserpine treatment impaired short-term memory after the 12th day of
protocol (second and third tests). Conversely, treatment with α-tocopherol was
able to prevent the short-term memory impairment (Fig. 3B; one-way ANOVA [F
(7,74) = 6.864; P < 0.0001] and Fig. 3C; one-way ANOVA [F (7,68) = 10.00; P <
0.0001). We also performed statistical analyses in order to evaluate the effect of
drug administration in objects exploration ratio throughout test sessions and
among experimental groups. We found that in the third test session animals’
receiving RES differs on exploration rate of new (Table 1; two-way ANOVA [F
(6,89) = 2.843; P < 0.05]) and old objects (Table 1; two-way ANOVA [F (6,89) =
2.843; P < 0.05]) when comparing to both VR+VT and RES+TOC. Yet, we
found that only RES+VT group presented alterations in object discrimination
across tests. More accurately, exploration of old and new objects increased and
decreased, respectively, comparing first and second tests (Table 1; two-way
ANOVA [F (3,89) = 2.760; P < 0.05]) and first and third tests (Table 1; two-way
ANOVA [F (3,89) = 2.649; P < 0.05]).
21
Figure 3. Animals were treated with reserpine (RES; 1.0 mg/kg) or vehicle (VR)
through s.c. injections, and α-tocopherol (TOC; 40 mg/kg) or its vehicle (VT) with daily
i.p. injections. Animals were tested on the following days of experiment: (A) 2nd, (B) 12th
and (C) 18th. In each day, training (with two identical objects, data not shown) and test
(with one familiar and one novel object) were performed with a one-hour interval in an
open field arena. Data are expressed as mean ± SEM. (*) P < 0.05; (**) P < 0.01; (***)
P < 0.001 and (****) P < 0.0001 when comparing old vs new object exploration ratio in
one-way ANOVA followed by Bonferroni’s multiple comparison post hoc test.
22
Table 1. Exploration rate in the NOR task throughout the test sessions. Data are
expressed as mean ± SEM. (*) P < 0.05 and (€) P < 0.01 when comparing RES+VT vs
RES+TOC and VR+VT vs RES+VT respectively. (¥) P < 0.05 and (#) P < 0.001 when
comparing the first vs second test and first vs third test respectively. All statistical
analyses were conducted through two-way ANOVA followed by Tukey’s Post Hoc test.
GROUPS
TESTS OBJECTS VR+VT RES+VT RES+TOC TOC
First Test
Post 1st
injection
OLD 36.94 ± 6.61 26.06 ± 5.01 34.11 ± 5.84 40.6 ± 4.91
NEW 63.06 ± 6.61 73.94 ± 5.01 65.89 ± 5.84 59.4 ± 4.91
Second Test
Post 6th
injection
OLD 34.69 ± 5.92 43.97 ± 5.31¥ 26.85 ± 5.87 39.17 ± 6.32
NEW 65.31 ± 5.92 56.03 ± 5.31¥ 73.15 ± 5.87 60.83 ± 6.32
Third Test
Post 9th
injection
OLD 27.71 ± 4.97 59.99 ± 6.95€ #
31.99 ± 6.43* 37.07 ± 3.25
NEW 72.28 ± 4.97 40.01 ± 6.95€ #
68.01 ± 6.43* 62.93 ± 3.25
9. Discussion
In this study, we investigated the effects of concomitant treatment with
TOC on catalepsy behavior and NOR task in rats submitted to a chronic
treatment with a low dosage of reserpine. We observed that the motor and
cognitive impairments induced by chronic treatment with reserpine were
prevented by treatment with TOC. These results can be seen in the evaluation
of catalepsy behavior performed 48 h after each reserpine injection (Figure 2)
as well as in the analysis of exploration time in the novel object recognition task
(Figure 3 and Table 1).
As previously observed in studies by our group [8,9], repeated treatment
with a low dose (0.1 mg/kg) of reserpine in rats induced the progressive
appearance of motor impairment. This impairment is marked by a gradual
23
increase in the duration of catalepsy behavior. Indeed, as one can see in Figure
2, reserpine-treated (RES+VT) animals start differing from control subjects after
7 reserpine s.c. injections. It is well documented that catalepsy in rodents
indicates akinesia and rigidity that are important symptoms of PD [16-18].
Importantly, we did not observe this impairment in the group that was
concomitantly treated with TOC. Indeed, the group RES+TOC (Figure 2)
presented catalepsy duration similar to control across the treatment.
Besides motor assessment, the protocol used in the present study
includes the cognitive evaluation. Cognitive deficits have been reported as
symptoms of PD, and can even appear before the motor deficits. In a previous
study, we have shown that the protocol of reserpine treatment used here
induces short-term memory deficits before the appearance of increased
catalepsy behavior and other motor signs [8]. The present study corroborates
those findings. We used the NOR task, which involves recognition memory and
executive functions, both functions that can be impaired in PD [19,20]. Our
results corroborated the previous study showing that animals treated with
reserpine failed to discriminate the objects in the test session (in the second
and third tests, see Figure 3). Further, similarly to that described for motor
evaluations, the deficit was prevented by TOC administration. Indeed, animals
treated with both reserpine and TOC presented increased novel object
exploration in all tests, similarly to control subjects. In addition, comparisons
among experimental groups showed that animals treated with RES had worse
object discrimination compared to both control and RES+TOC groups in the
third test. Finally, when performances across the three tests were analyzed,
only the group treated with reserpine alone presented discrimination deficits in
the second and third tests compared to the first test (Table 1). These additional
analyses reinforce the prevention of the reserpine-induced object recognition
impairment by co-treatment with TOC.
As mentioned, reserpine is a non-selective inhibitor of the vesicular
monoamine transporter [1]. Thus, one could raise the possibility that the
behavioral alterations induced by reserpine treatment are related exclusively to
the dopamine depletion caused by this blockage. In other words, the alterations
could be a consequence of an additive effect on dopaminergic function.
However, there is evidence that favors the hypothesis that the progressive
24
effect of the repeated treatment with reserpine is due to oxidative damage. First,
a previous study has shown that the classical acute treatment (with a dose 10
times higher than the one we used) did not cause a reduction in tyrosine
hydroxylase staining (an indicative of dopaminergic neuronal function), although
causing an important motor impairment [21]. Conversely, the protocol used here
(repeated treatment with a low dose) reduced tyrosine hydroxylase staining in
the substantia nigra and striatum, and part of the alterations induced by the
treatment were not recovered after 30 days of treatment withdrawal [8]. Second,
it has been shown that reserpine treatment increases brain oxidative stress and
this alteration is accompanied by behavioral deficits [10, 22, 23]. In addition, in a
previous study [9] the repeated treatment with a low dose of reserpine induced
an increase in striatal level of lipid peroxidation, which occurred concomitantly
to the motor impairment. These results lead us to question if co-treatment with
TOC would prevent the progressive motor and cognitive alterations induced by
the repeated treatment with a low dose of reserpine. As discussed above,
treatment with TOC was able to prevent these deficits. This preventive effect
might be explained by a neuroprotection mechanism, probably by a reduction
the in neurotoxic dopamine oxidation bioproducts [24].
Despite the well-known antioxidant properties of vitamin E, it is important
to mention that tocopherol and other antioxidant agents can have pro-oxidant
effects as well. Indeed, the ability of these compounds to accept and donate
electrons enables them to cause oxidative damage under certain conditions
[25]. However, this pro-oxidant action is mainly found in vitro, and under high
concentrations [26, 27]. Some in vivo studies have also shown pro-oxidant
effects of classical antioxidants, but they are variable depending on substance,
concentration, age of the subject and target molecules [25, 28-30]. Further, it
seems that their preferential action is antioxidant when an oxidant insult from
another source is present [31]. In the case of the present results, there was no
evidence of a pro-oxidant action regarding possible behavioral alterations.
Nevertheless, an antioxidant role of vitamin E in ameliorating
neurodegeneration in PD has been consistently proposed by in vitro and animal
studies [32-37]. On the other hand, despite strong evidence favoring an
antioxidant effect, the exact mechanism of action of vitamin E in Parkinson´s
disease is still under investigation [32]. There is evidence that vitamin E,
25
particularly alpha-tocopherol, can act through other mechanisms not related to
modulation of oxidative stress. For example, studies showed that alpha-
tocopherol regulates the expression of several genes [38, 39] and inhibits
protein kinase C (PKC) activity [40, 41]. The later could be related to the
neuroprotective action of this compound, because PKC activation has been
implicated in cell death signaling pathways related to PD [42]. This relationship
was found in studies with animal models of PD induced by the toxins 1-methyl-
4-phenylpyridinium [43] and paraquat [44]. If PKC activation is also relevant for
reserpine-induced parkinsonism it is still unknown.
Regardless of the specific mechanism related to the prevention of
behavioral alterations found in the present study, there is evidence that
increased oxidative stress underlies the physiopathology of neurodegenerative
diseases such as PD [45-48]. Further, clinical data suggest that neuroprotective
treatments based on increasing antioxidant defenses are able to delay the
progression of the pathology [49-56]. Thus, a neuroprotective intervention could
be a relevant line of investigation in animal models of this disease. However,
the usual acute pharmacological models includes severe motor impairment
upon a single injection of reserpine or specific neurotoxins [4, 57-61]. This
approach is not suitable for the investigation for testing neuroprotective
interventions because they usually present a preventive and/or a
neurodegeneration delaying profile. Further, most of the previous studies
investigating the effects of vitamin E treatments on PD models did not
investigate progressive behavioral deficits related to the clinical symptoms of
the disease [33, 35, 37, 62]. In this sense, the need for animal models of PD
more compatible with clinical outcomes when investigating neuroprotective
therapies has been pointed out [63]. Thus, the present findings reinforce the
idea that the protocol of progressive parkinsonism induction with reserpine is
suitable for investigating possible neuroprotective interventions in animal
models of PD.
In conclusion, concomitant treatment with alpha-tocopherol prevents
behavioral alterations induced by repeated reserpine. Although the antioxidant
action of vitamin E is probably related, the exact mechanism underlying this
preventive effect remains to be investigated. Finally, the progressive behavioral
motor and cognitive alterations induced by repeated reserpine treatment seems
26
an adequate protocol to investigate possible neuroprotective interventions for
PD.
27
10. Acknowledgments:
The authors would like to thank Antonio Carlos Queiroz de Aquino for
capable technical assistance. This study was supported by grants from
Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq,
Brazil), Fundação de Amparo a Pesquisa do Estado do Rio Grande do Norte
(FAPERN, Brazil), and Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior (CAPES, Brazil).
28
11. References
1. Henry JP, Sagne C, Botton D, Isambert MF, Gasnier B (1998). Molecular
pharmacology of the vesicular monoamine transporter. Adv Pharmacol 42: 236-
239.
2. Daeur W, Przedborski S (2003). Parkinson’s disease: mechanisms and models.
Neuron 39: 889-909.
3. Alves CSD, Andreatini R, da Cunha C, Tufik S, Vital MABF (2000).
Phosphatidylserine reserves reserpine-induced amnesia. Eur J Pharmacol 404:
161-167.
4. Colpaert FC (1987). Pharmacological characteristics of tremor, rigidity and
hypokinesia induced by reserpine in rat. Neuropharmacology 26: 1431-1440.
5. Skalisy LL, Beijamini V, Joca SL, Vital MABF, da Cunha C et al. (2002).
Evaluation of the face validity of reserpine administration as an animal modelo
of depression- Parkinson’s disease association. Prog Neuro-Psychopharmacol
Biol Psychiatry 26: 879-883.
6. Carvalho RC, Patti CC, Takatsu-Coleman AL, Kameda SR, Souza CF et al.
(2006). Effects of reserpine on the plus-maze discriminative avoidance task:
dissociation between memory and motor impairments. Brain Res 1122: 179-83.
7. Fernandes VS, Ribeiro AM, Melo TG, Godinho M, Barbosa FF et al. (2008).
Memory impairment induced by low doses of reserpinein rats: possible
relationship with emotional processing deficits in Parkinson´s disease. Prog
Neuro-Psychopharmacol Biol Psychiatry 32:1479–83.
8. Santos JR, Cunha JAS, Dierschnabel AL, Campêlo CLC, Leão AHFF et al.
(2013). A new progressive model of Parkinson´s disease: different temporal
courses of motor and cognitive deficits. Behav Brain Res 253, 68–77.
9. Fernandes VS, Santos JR, Leão AH, Medeiros AM, Melo TG et al. (2012).
Repeated treatment with a lowdose of reserpine as a progressive model of
Parkinson’s disease. Behav Brain Res 231:154–63.
10. Abílio VC, Araujo CCS, Bergamo M, Calvente PRV, D’Almeida V, et al. (2003).
Vitamin E attenuates reserpine-induced oral dyskinesia and striatal oxidized
glutathione/reduced glutathione ratio (GSSG/GSH) enhancement in rats. Prog
Neuro-Psychopharmacol Biological Psychiatry 27:109–14.
29
11. Ebadi M, Srinivasan SK, Baxi MD (1996). Oxidative stress and antioxidant
therapy in Parkinson’s disease. Prog Neurobiol 48: 1–19.
12. Bavarsad-Shahripour R, Harrigan MR, Alexandrov AV (2014). N-acetylcysteine
(NAC) in neurological disorders: mechanisms of action and therapeutic
opportunities. Brain Behav 4: 108–122.
13. Pérez HJ, Carrillo SC, García E, Ruiz-Mar G, Pérez-Tamayo R et al. (2014).
Neuroprotective effect of silymarin in a MPTP mouse model of Parkinson's
disease. Toxicology 319: 38-43.
14. Halliwell B, Gutteridge JM (2007). Free Radicals in Biology and Medicine.
Oxford University Press.
15. Jiang Q (2014). Natural forms of vitamin E: metabolism, antioxidant and anti-
inflammatory activities and the role in disease prevention and therapy. Free
Radic Biol Med 72: 76-90.
16. Sanberg PR, Bunsey MD, Giordano M, Norman AB (1988). The catalepsy test:
its ups and downs. Behav Neurosci 102: 748-59.
17. de Lau LM, Breteler MM (2006). Epidemiology of Parkinson's disease. Lancet
Neurol 5: 525-35.
18. Duty S, Jenner P (2011). Animal models of Parkinson's disease: a source of
novel treatments and clues to the cause of the disease. Br J Pharmacol 164:
1357-91.
19. Higginson CI, Wheelock VL, Carroll KE, Sigvardt KA (2005). Recognition
memory in Parkinson's disease with and without dementia: evidence
inconsistent with the retrieval deficit hypothesis. J Clin Exp Neuropsychol 27:
516-28.
20. Lewis SJ, Dove A, Robbins TW, Barker RA et al. (2003). Cognitive impairments
in early Parkinson's disease are accompanied by reductions in activity in
frontostriatal neural circuitry. J Neurosci 23: 6351-6.
21. Caudle WM, Colebrooke RE, Emson PC, Miller GW (2008). Altered vesicular
dopamine storage in Parkinson’s disease: a premature demise. Trends
Neurosci 31: 303-308.
22. Bergström T, Ersson C, Bergman J, Möller L (2012). Vitamins at physiological
levels cause oxidation to the DNA nucleoside deoxyguanosine and to DNA--
alone or in synergism with metals. Mutagenesis 27(4): 511-517.
30
23. Osiecki M, Ghanavi P, Atkinson K, Nielsen LK, Doran MR (2010). The ascorbic
acid paradox. Biochem Biophys Res Commun 400(4): 466-470.
24. Palozza P, Calviello G, Serini S, Maggiano N, Lanza P, Ranelletti FO, Bartoli
GM (2001). beta-carotene at high concentrations induces apoptosis by
enhancing oxy-radical production in human adenocarcinoma cells. Free Radic
Biol Med 30(9): 1000-1007.
25. de Oliveira BF, Veloso CA, Nogueira-Machado JA, Martins Chaves M (2012).
High doses of in vitro beta-carotene, alpha-tocopherol and ascorbic acid induce
oxidative stress and secretion of IL-6 in peripheral blood mononuclear cells from
healthy donors. Curr Aging Sci 5(2): 148-156.
26. Winterbone MS, Sampson MJ, Saha S, Hughes JC, Hughes DA (2007). Pro-
oxidant effect of alpha-tocopherol in patients with type 2 diabetes after an oral
glucose tolerance test--a randomised controlled trial. Cardiovasc Diabetol. Feb
22;6:8.
27. Nadeem N, Woodside JV, Kelly S, Allister R, Young IS, McEneny J (2012). The
two faces of α- and γ-tocopherols: an in vitro and ex vivo investigation into
VLDL, LDL and HDL oxidation. J Nutr Biochem 23(7): 845-851.
28. Reich EE, Montine KS, Gross MD, Roberts LJ 2nd, Swift LL, Morrow JD,
Montine TJ (2001). Interactions between apolipoprotein E gene and dietary
alpha-tocopherol influence cerebral oxidative damage in aged mice. J Neurosci
21(16): 5993-5999.
29. Vatassery GT (1992). Vitamin E. Neurochemistry and implications for
neurodegeneration in Parkinson's disease. Ann N Y Acad Sci 669: 97-109.
30. Casani S, Gómez-Pastor R, Matallana E, Paricio N (2013). Antioxidant
compound supplementation prevents oxidative damage in a Drosophila model
of Parkinson's disease. Free Radic Biol Med 61:151-160.
31. Fariss MW, Zhang JG (2003). Vitamin E therapy in Parkinson's disease.
Toxicology 189(1-2): 129-46.
32. Miklya I, Knoll B, Knoll J (2003). A pharmacological analysis elucidating why, in
contrast to (-)-deprenyl (selegiline), alpha-tocopherol was ineffective in the
DATATOP study. Life Sci 72(23): 2641-2648.
33. Butterfield DA, Castegna A, Drake J, Scapagnini G, Calabrese V (2002).
Vitamin E and neurodegenerative disorders associated with oxidative stress.
Nutr Neurosci 5(4): 229-239.
31
34. Roghani M, Behzadi G (2001). Neuroprotective effect of vitamin E on the early
model of Parkinson's disease in rat: behavioral and histochemical evidence.
Brain Res 892(1): 211-217.
35. Azzi A (2007). Molecular mechanism of alpha-tocopherol action. Free Radic
Biol Med 43(1): 16-21.
36. Azzi A, Gysin R, Kempná P, Munteanu A, Negis Y, Villacorta L, Visarius T,
Zingg JM (2004). Vitamin E mediates cell signaling and regulation of gene
expression. Ann N Y Acad Sci 1031:86-95.
37. Ferri P, Cecchini T, Ambrogini P, Betti M, Cuppini R, Del Grande P, Ciaroni S
(2006). alpha-Tocopherol affects neuronal plasticity in adult rat dentate gyrus:
the possible role of PKCdelta. J Neurobiol 66(8): 793-810.
38. Azzi A, Ricciarelli R, Zing JM (2002). Non-antioxidant molecular functions of
alpha-tocopherol (vitamin E). FEBS Letters 519: 8-10.
39. Kanthasamy A, Jin H, Mehrotra S, Mishra R, Kanthasamy A, Rana A (2010).
Novel cell death signaling pathways in neurotoxicity models of dopaminergic
degeneration: relevance to oxidative stress and neuroinflammation in
Parkinson's disease. Neurotoxicology 31(5): 555-561.
40. Chalimoniuk M, Stolecka A, Ziemińska E, Stepień A, Langfort J, Strosznajder
JB (2009). Involvement of multiple protein kinases in cPLA2 phosphorylation,
arachidonic acid release, and cell death in in vivo and in vitro models of 1-
methyl-4-phenylpyridinium-induced parkinsonism-the possible key role of PKG.
J Neurochem 110(1): 307-317.
41. Cristóvão AC, Barata J, Je G, Kim YS (2013). PKCδ mediates paraquat-
induced Nox1 expression in dopaminergic neurons. Biochem Biophys Res
Commun 437(3): 380-385.
42. Beal MF (2002). Oxidatively modified proteins in aging and disease. Free Radic
Biol Med 32: 797-803.
43. Beal MF (2003). Mitochondria, oxidative damage, and inflammation in
Parkinson's disease. Ann N Y Acad Sci 991:120-31.
44. Cadenas E, Davies KJ (2000). Mitochondrial free radical generation, oxidative
stress, and aging. Free Radic Biol Med 29: 222-30.
45. Younes-Mhenni S, Frih-Ayed M, Kerkeni A, Bost M, Chazot G (2007).
Peripheral blood markers of oxidative stress in Parkinson's disease. Eur Neurol
58: 78-83.
32
46. Abdel-Salam OM (2008). Drugs used to treat Parkinson's disease, present
status and future directions. CNS Neurol Disord Drug Targets 7: 321-42.
47. Beal MF (2009). Therapeutic approaches to mitochondrial dysfunction in
Parkinson's disease. Parkinsonism Relat Disord 15 Suppl 3: S189-94.
48. Chen JJ, Ly AV (2006). Rasagiline: A second-generation monoamine oxidase
type-B inhibitor for the treatment of Parkinson's disease. Am J Health Syst
Pharm 63: 915-28.
49. De Araújo DP, Lobato R de F, Cavalcanti JR, Sampaio LR, Araújo PV et al.
(2011). The contributions of antioxidant activity of lipoic acid in reducing
neurogenerative progression of Parkinson's disease: a review. Int J Neurosci
121: 51-7.
50. Magyar K, Pálfi M, Tábi T, Kalász H, Szende B et al. (2004). Pharmacological
aspects of (-)-deprenyl. Curr Med Chem 11: 2017-31.
51. Mayo JC, Sainz RM, Tan DX, Antolín I, Rodríguez C et al. (2005). Melatonin
and Parkinson's disease. Endocrine 27: 169-78.
52. Weber CA, Ernst ME (2006). Antioxidants, supplements, and Parkinson's
disease. Ann Pharmacother 40: 935-8.
53. Weinreb O, Amit T, Bar-Am O, Youdim MB (2010). Rasagiline: a novel anti-
parkinsonian monoamine oxidase-B inhibitor with neuroprotective activity. Prog
Neurobiol 92: 330-44.
54. Hsieh MH, Gu SL, Ho SC, Pawlak CR, Lin CL et al. (2012). Effects of MK-801
on recognition and neurodegeneration in an MPTP-induced Parkinson's rat
model. Behav Brain Res 229: 41-7.
55. Marin C, Aguilar E (2011). In vivo 6-OHDA-induced neurodegeneration and
nigral autophagic markers expression. Neurochem Int 58: 521-6.
56. Salamone JD, Baskin P. (1996). Vacuous jaw movements induced by acute
reserpine and low-dose apomorphine: possible model of parkinsonian tremor.
Pharmacol Biochem Behav 53: 179-83.
57. Salamone JD, Ishiwari K, Betz AJ, Farrar AM, Mingote SM et al. (2008).
Dopamine/adenosine interactions related to locomotion and tremor in animal
models: possible relevance to parkinsonism. Parkinsonism Relat Disord 14
Suppl 2: S130-4.
58. Tetrud JW, Langston JW (1989). MPTP-induced parkinsonism as a model for
Parkinson's disease. Acta Neurol Scand Suppl 126:35-40.
33
59. Itoh N, Masuo Y, Yoshida Y, Cynshi O, Jishage K, Niki E. (2006). gamma-
Tocopherol attenuates MPTP-induced dopamine loss more efficiently than
alpha-tocopherol in mouse brain. Neurosci Lett 403(1-2): 136-140.
60. Kamat CD, Gadal S, Mhatre M, Williamson KS, Pye QN, Hensley K (2008).
Antioxidants in central nervous system diseases: preclinical promise and
translational challenges. J Alzheimers Dis 15(3): 473-493.
34
12. Artigo 2
(Submetido à publicação no periódico “Progress in
Neuropsychopharmacology & Biological Psychiatry”)
EFFECTS OF ALPHA-TOCOPHEROL ON BEHAVIORAL AND NEURONAL
PARAMETERS IN A RAT MODEL OF PARKINSON'S DISEASE
Aldair José Sarmento-Silvaa, Ramón Hypolito Limaa, Ywlliane Meurera, André
Macedo Medeirosb, Lisandro Lungatoc, Rovena Clara Galvão Januário
Engelbertha,d , Jeferson de Souza Cavalcanted, Alessandra Mussi Ribeiroa,e,
Vania D´Almeidac, Regina Helena Silvaa,b*
a Memory Studies Laboratory, Department of Physiology, Federal University of
Rio Grande do Norte, Natal, Brazil.
b Department of Pharmacology, Federal University of São Paulo, São Paulo,
Brazil.
c Department of Psychobiology, Federal University of São Paulo, São Paulo,
Brazil
d Laboratory of Neurochemical Studies, Department of Physiology, Federal
University of Rio Grande do Norte, Natal, Brazil.
e Department of Biosciences, Federal University of São Paulo, Santos, Brazil.
*Corresponding author: Regina H. Silva
Departamento de Farmacologia – UNIFESP
Rua Botucatu, 862, Edifício Leal Prado, 1º.andar
CEP 04023062 - São Paulo, SP, Brasil
Email: [email protected]
35
13. Abstract
Parkinson's disease (PD) is a chronic and progressive syndrome that
reduces the levels of dopamine in the nigrostriatal dopaminergic pathway.
Previous studies showed that chronic administration of reserpine (RES, a
monoamine depleting agent) in low doses promotes progressive cognitive and
motor impairments in rats, along with increased brain oxidative stress. We have
recently verified that the object recognition impairment and increased catalepsy
behavior induced by repeated RES were prevented by alpha-tocopherol (TOC)
coadministration. In the present study, we verify if (1) the effects of concomitant
TOC are extensive to other motor deficits (oral movements and motor
coordination) and (2) if the prevention of behavioral alterations is accompanied
by modifications compatible with neuroprotection (tyrosine hydroxylase (TH)
immunostaining and activity of antioxidant enzymes). Rats were treated with 15
s.c. injections of RES (0.1 mg/kg) or its vehicle every 48h, concomitantly to daily
30 i.p. administrations of TOC (40 mg/kg) or its vehicle. As expected, rats
treated exclusively with reserpine presented increased catalepsy behavior,
increased oral movements, motor impairment in the rotarod and decreased TH
staining in the substantia nigra pars compacta, ventral tegmental area,
hippocampus and striatum. All these detrimental outcomes due to RES
treatment were prevented by concurrent daily administration of the antioxidant
TOC. However, we did not find any changes in the activity of catalase and
superoxide dismutase after treatment with RES and/or TOC. The data indicate
that the effect of coadministration with TOC is extensive to behavioral and
neuronal alterations induced by repeated RES, suggesting a neuroprotective
effect with relevant implications for the treatment of PD. The relationship
between the effects found here and a possible antioxidant mechanism of TOC
remains to be further investigated.
Keywords: Reserpine, Parkinson’s disease, α-tocopherol, motor impairment, short-term
memory impairment.
36
14. Introduction
Parkinson's disease (PD) is a chronic and progressive syndrome that
reduces the levels of dopamine (DA) in the nigrostriatal dopaminergic pathway,
as a result from the death of dopaminergic neurons mainly in the substantia
nigra pars compacta (SNpc - Dauer & Przedborski, 2003). Commonly
associated with aging, this pathology is clinically characterized by motor
symptoms such as tremor, bradykinesia, muscular rigidity and postural
instability, as well as cognitive impairments and depression (Fahn, 2006). In
fact, those typical motor symptoms may be accompanied by cognitive
impairments threatening the patient’s life quality (Korczyn, 2001; Nieoullon,
2002; Zgaljardic et al., 2004).
Studies with animal models of PD have been useful in understanding the
mechanisms leading to the clinical symptoms, as well as potential treatments.
One of the most common approaches is administration of reserpine to rodents
(Alves et al., 2000; Colpaert, 1987; Skalisy et al., 2002). Reserpine prevents the
storage of monoamines through the blockage of the synaptic vesicles
transporters (Henry et al., 1998). Consequently, synaptic vesicles are still
available but with a reduced amount of available dopamine in the synaptic cleft.
Moreover, the treatment with reserpine causes an increase in cellular oxidative
stress, possibly potentiated by the rise in the levels of dopamine in the
cytoplasm, which undergoes oxidative metabolism (Abílio et al., 2002).
The central nervous system (CNS) is quite vulnerable to reactive oxygen
species (ROS), which play a very important function in the pathogenesis of
neurodegenerative disorders, including PD (Ebadi et al., 1996). In this respect,
there is evidence that the inclusion of antioxidant agents in the pharmacological
treatment of PD has advantages over the treatment based only in dopamine
replacement (Bavarsad et al., 2014; Ebadi et al., 1996; Pérez et al., 2014).
Importantly, previous studies showed that chronic administration of reserpine in
low doses promotes progressive cognitive and motor impairments in rats, along
with neuronal alterations compatible with damage in the nigrostriatal pathway,
including increased brain oxidative stress (Fernandes et al., 2012; Santos et al.,
2013).
37
The antioxidant agent vitamin E (α-tocopherol) can be formed
metabolically or found in the environment and plays an essential role in
protecting the body against the damaging effects of ROS. Specifically, vitamin E
blocks the propagation step of lipid peroxidation of polyunsaturated fatty acids
in membranes and lipoproteins (Halliwell & Gutteridge, 2007) and is the major
lipid-soluble antioxidant present in cell membranes. We have recently
demonstrated that the object recognition impairment and increased catalepsy
behavior induced by repeated reserpine treatment were prevented by the
concomitant treatment with α-tocopherol (Sarmento-Silva et al., paper 2). The
aim of this study was to evaluate the effects of the antioxidant agent α-
tocopherol on motor and neuronal parameters in rats submitted to the repeated
treatment with a low dose of reserpine. Specifically, we verify if (1) the effects of
concomitant α-tocopherol are extensive to other motor deficits (oral movements
and motor coordination) and (2) if the prevention of behavioral alterations is
accompanied by modifications compatible with neuroprotection (tyrosine
hydroxylase (TH) immunostaining and activity of antioxidant enzymes).
15. Material and Methods
15.1. Animals
We used 4-5-month-old 96 male Wistar rats (300-500g). The animals
were obtained from the Physiology Department at the Federal University of Rio
Grande do Norte, and were housed in groups of four, in plastic cages, under
controlled conditions of ventilation, temperature (23 ± 1°C), and light/dark cycle
(12h/12h, lights on 6:30 a.m.), with free access to water and food. The rats were
handled according to the Brazilian law for the use of animals in scientific
research (Law Number 11.794) and all the procedures described were
approved by the local ethical committee (CEUA/UFRN nº 051/2011).
38
15.2. Drugs
Reserpine (RES - Sigma Chemical Co., St. Louis, MO) was dissolved in
acetic acid and further diluted in distilled water at the concentration of 0.1
mg/ml, pH ≈ 6.5. We used this vehicle (glacial acetic acid diluted in water) as a
control for reserpine treatment (VR). RES and VR were given s.c. on alternate
days. The antioxidant α-tocopherol (TOC - Sigma Chemical Co., St. Louis, MO)
was diluted in distilled water with Tween-80 at the concentration of 40 mg/ml.
We used the vehicle used to dilute tocopherol as a control for TOC treatment
(VT). TOC and VT were injected i.p. daily. The volume of injection was 1ml/kg
of body weight in all cases.
15.3. Experimental design and general procedures
The rats were randomly assigned to the following groups: (1) VR + VT,
(2) RES + VT, (3) RES + TOC and (4) VR + TOC. Drug treatment lasted for 30
days. Animals received 15 s.c. injections of RES (0.1 mg/kg) or VR every 48
hours, concomitantly to daily i.p. administration of TOC (40 mg/kg) or VT.
Before the beginning of the experiments, all animals were submitted to a
daily 5-minute handling session for five consecutive days. Throughout the
treatment, the animals were subjected to the following evaluations: catalepsy
test (performed daily); evaluation of oral movements (days 1, 6, 10, 24, 30);
locomotor activity in the rotarod (days 1, 17, 25 and 31). The enzymatic assay
and immunohistochemistry for TH were performed at the end of the treatment.
The experimental design is shown in Fig. 1. All behavioral tests were conducted
before the injections of that day. Before each experimental procedure, the
apparatuses were cleaned with a 5% alcohol solution, and the experimental
groups were alternated across testing.
39
Figure 1. Schematic illustration of the experimental design.
16. Behavioral tests
16.1. Catalepsy test
To evaluate the catalepsy behavior, the animal’s forepaws were daily
placed on a horizontal bar positioned at 9 cm above the bench surface (n=9
animals per group). The catalepsy behavior was measured considering the total
time spent in immobility until the animal removed both forepaws of the bar. The
animals were submitted to three consecutive exposures, up to 80 seconds for
each trial and the mean value was considered to statistical analyses.
16.2. Oral movements
The oral movements measured were: (1) the duration of oral tremor, in
seconds, (2) the number of chewing movements that were not directed to any
object (vacuous chewing movements), and (3) the number of tongue
protrusions. The animals were individually placed in a barred cage measuring
approximately 0.21 cm x 0.29 cm x 0.24 cm (L x W x H) with mirrors fixed under
and behind the cage to allow the observation of the animal´s snout by the
experimenter. This evaluation was performed on the 1st, 6th, 10th, 24th and 30th
treatment days (n=18-19 animals per group).
40
16.3. Rotarod test
Animals were exposed on a rotarod test (Insight®, Brazil) to detect a
potential impairment on coordination or motor performance (Dekundy et al.,
2006). The experiments were conducted in two phases: training and test. The
training was held for 5 days prior to treatment (n=8-10 animals per group). In
each training day, animals were trained to walk on a rotary cylinder in a
constant acceleration of 8 rpm for 3 successive trials with a 40-second interval
after each fall. We considered the average of the three trials in order to
establish a baseline value. A sensor at the platform detected the fall off and
automatically recorded the latency to fall (in seconds). A cut off latency of 240
seconds was considered (Monville et al., 2006). The test session was carried
out on the 6th, 14th, 22nd and 30th days of treatment (48h after the 3rd, 8th, 12th
and 15th injection of reserpine or vehicle).
16.4. Immunohistochemistry for tyrosine hydroxylase (TH)
Upon completion of the behavioral procedures and treatment, animals
(n=5-6 animals per group) were euthanized (sodium thiopental, 40 mg/kg, i.p.)
and perfused transcardially with 200 ml phosphate-buffered saline (PBS), pH
7.4, containing 500 IU heparin (Liquemin, Roche, Brazil), followed by 300 ml
4.0% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. We removed
animals’ brains through craniotomy and immersed in a fixative solution at 4°C
for 24 hours followed by cryoprotection for 72 hours in 30% sucrose in 0.1 M
PBS, pH 7.4. We serially sliced in 30 μm coronal sections using a cryostat
(Leica, USA). Slices were collected and allocated into five compartments
distributed in sequence with approximately 150 µm distance between sections.
Compartments were stored at 4°C in an ethylene glycol and phosphate buffer
based antifreeze solution for cryoprotection for further immunohistochemical
analysis.
Afterwards, sections were washed five times with phosphate buffered
saline (PBS, 0.1M, pH 7.4) for 5 minutes each followed by incubation in PBS
41
plus hydrogen peroxide (0.3%) for 20 minutes. The brain sections were first
incubated overnight with a monoclonal anti-TH primary antibody (1:10,000;
Chemicon, CA, USA) containing 2% BSA (Sigma, St Louis, MO, USA) and
diluted in 0.3% Triton X-100 and 0.1M PBS, pH 7.4. Afterwards, the sections
were incubated with the biotinylated secondary antibody goat anti-mouse
1:10000 (Vector Labs, USA) diluted in Triton X-100 0.4% for 2 hours. Then, the
sections were incubated with the avidin-biotin-perioxidase solution (ABC Elite
kit, Vector Labs, USA) for 2 hours. The reaction was developed by the addition
of diaminobenzidine (DAB; Sigma, St Louis, MO, USA) at 2.5%, diluted in 0.1 M
PBS, pH 7.4. Between each step the tissues were washed four times with 0.1 M
PB pH 7.4 (for 5 min each). Then, the sections were dried, dehydrated in a
graded alcohol series, cleared in xylene, and coverslipped with Entellan
(Merck). All the immunostainings were performed concomitantly, minimizing
possible differences in background between the animals. An adjacent series
was stained with tionine to serve as a reference series for cytoarchitetural
purposes. Sections were examined under brightfield illumination (Olympus
Microscope, BX-41), and captured using a CCD camera (Nikon, DXM-1200).
In order to estimate the number of dopaminergic neurons, we selected
four sections of each region evaluated (SNpc and VTA): one at the rostral level,
two at medium level and one at the caudal level, representative of the
rostrocaudal extension of each area of interest. We used the Paxinos and
Watson rat brain atlas (2007) to assess the exact location of the brain regions.
Additionally, we assessed TH+ levels through analysis of relative optical
densitometry (OD) in the striatum and hippocampal subregions (CA1, CA3 and
DG) using the Image J software (Version 1.46i, NIH). We choose four
representative sections of the rostrocaudal extension of each region. In each
section, we analyzed four fields evenly distributed throughout the areas of
interest. The medium pixels in the target area were subtracted from de medium
values of a control region (areas that should not have specific TH staining) of
the same tissue (cortex or corpus calosum). Finally, we normalized all values
considering the control group, in order to evaluate proportional alterations.
42
16.5. Enzymatic Analysis
Two days after the last session of behavioral tests, we euthanized the
animals by decapitation (n=5 animals per group). Subsequently, we removed
and washed the brain with ice-cold saline and rapidly dissected both
hippocampus and striatum followed by weighing and storage at −80 ºC for
further use on biochemical analysis. Before enzymatic assays, the tissues were
mechanically macerated in Hanks buffer salt solution, and the homogenate was
centrifuged in 0.1 M PO4K buffer (pH 7.0) at 1100 X g for 15 min at 4°C.
Afterwards, obtained supernatants were centrifuged at 18000 X g for 15 min at
4 °C.
We performed spectrophotometric assays for catalase (CAT) and
superoxide dismutase (SOD) activities following standard protocols (Beutler,
1975; Ewing & Janero, 1995). We measured CAT activity following hydrogen
peroxide disappearance at 240 nm. One unit of CAT corresponds to the amount
of the enzyme that hydrolyses 1lmol of hydrogen peroxide per minute, at 25ºC.
Then, we analyzed SOD activity from nitro blue tetrazolium (NBT) reduction by
superoxide anion produced via photoreduction of riboflavin at 560 nm.
According to protocol, we defined one unit of SOD as the amount that caused
50% inhibition of rate of NBT reduction. For mitochondrial SOD (MnSOD)
activity measurement, we add 15 mM potassium cyanide (KCN) in reaction
medium to inhibit the cytosolic SOD (CuZnSOD) activity. In order to normalize
antioxidant enzymes activities of brain structures, we measured the content of
total proteins in the respective homogenates using a Bio-Rad kit. Results were
expressed as U (units)/mg of protein.
16.6. Statistical Analysis
All data were tested for normality using through Kolmogorov–Smirnov
test and analyzed accordingly. We analyzed the performances in catalepsy, oral
movements, rotarod tests along treatment using two-way ANOVA with repeated
measures followed by Tukey’s multiple comparison post hoc test. We used an
ANOVA followed by the Sidak post hoc test to analyze data from TH+ neurons
43
counting and relative optical densitometry (OD). To analyze the enzymatic
assay experiments, we used Kruskal Wallis test followed by Mann–Whitney U-
test comparing all groups. Results were expressed as mean ± SEM (overall) or
median for enzymatic assay (minimum; maximum values) and P < 0.05 was
considered to reflect significant differences.
17. Results
17.1. Catalepsy
Figure 2 shows that from day 17 onwards there was an increase in
catalepsy behavior of the group RES+VT compared to all other groups (RM
two-way ANOVA; days of treatment [F (29,960) = 9.005, P < 0.0001], treatment [F
(3,960) = 93.82, P < 0.0001] and days of treatment × treatment interaction effects
[F (87,960) = 2.948, P < 0.0001]). This increase was not detected for the group
RES+TOC. Data confirm that chronic administration of reserpine promotes
progressive motor impairments, starting after the 8th reserpine injection. Yet,
this detrimental effect is prevented by α-tocopherol co-administration.
Figure 2. Repeated administration of reserpine increases catalepsy duration
and this effect is prevented by α-tocopherol. Arrows indicate reserpine (RES;
0.1 mg/kg) or vehicle (VR) s.c. injections, while α-tocopherol (TOC; 40 mg/kg)
or its vehicle (VT) were given daily i.p.. Data are expressed as mean + SEM; (*)
P < 0.05 for RES+VT vs RES+TOC; (**) P < 0.01 for RES+VT vs TOC+VR and
(****) P < 0.0001 for RES + VT vs all experimental groups in Tukey’s multiple
comparison post hoc test after RM two-way ANOVA.
44
17.2. Oral movements
We found an increase in the oral twitching after the 5th reserpine injection
and this impairment was attenuated by α-tocopherol treatment (Fig. 3A; two-
way ANOVA for days of treatment [F (5,384) = 14.58, P < 0.0001], treatment [F
(3,384) = 101.7, P < 0.0001], days of treatment × treatment interaction effects [F
(15,384) = 6.822, P < 0.0001]). Likewise, we found a gradual increase on vacuous
chewing movements after the 5th reserpine injection and this effect was
attenuated by α-tocopherol administration (Fig. 3B; two-way ANOVA for days of
treatment [F (5, 384) = 10.57, P < 0.0001], treatment [F (3,384) = 80.41, P < 0.0001],
days of treatment × treatment interaction effects [F (15,384) = 3.840, P < 0.0001]).
We also found that reserpine treatment increases the frequency of tongue
protrusions. However, the group RES+TOC presented significant improvement
only in the test performed on day 6 (Fig. 3C; Two-way ANOVA for days of
treatment [F (5, 384) = 1.052, P = 0.3867], treatment [F (3,384) = 28.25, P < 0.0001],
days of treatment × treatment interaction effects [F (15,384) = 0.6488, P =
0.8338]). Overall, data show that the increase in oral movements induced by
RES was prevented by concomitant TOC.
17.3. Rotarod test
We found that the reserpine administration impaired coordination and
balance. The effects due to treatment started after the 8th reserpine injection.
These motor deficits are characterized by a decrease in time spent in
behavioral apparatus and this effect is prevented by daily injections of α-
tocopherol (Fig. 3D; RM two-way ANOVA for days of treatment [F (4,165) = 6.259,
P = 0.0001], treatment [F (3,165) = 26.32, P < 0.0001], days of treatment ×
treatment interaction effects [F (12,165) = 5.862, P < 0.0001]).
45
Figure 3. α-tocopherol prevented increased oral movements and impaired
motor coordination in the rotarod test induced by repeated administration of
reserpine. The animals were treated with reserpine (RES; 1.0 mg/kg) or vehicle
(VR) s.c. injections, and α-tocopherol (TOC; 40 mg/kg) or its vehicle (VT) were
given daily i.p.. (A) Oral tremor; (B) Vacuous chewing movements; (C) Tongue
protrusions; (D) Rotarod test. Data are expressed as mean + SEM; (*) P < 0.05
for RES+VT vs VR + VT; (#) P < 0.05 for RES+VT vs RES+TOC in Tukey’s post
hoc test after RM two-way ANOVA.
17.4. Immunohistochemistry for tyrosine hydroxylase (TH)
Reserpine treatment promoted a significant reduction in the total number
of TH+ immunoreactive neurons in the SNpc (Fig. 4A and 6A; ANOVA followed
by Sidak’s post hoc test [F (3,19) = 19.58, P < 0.0001]) and in the VTA (Fig. 4B
and 6B; ANOVA followed by Sidak's post hoc test [F (3,19) = 4.480, P < 0.05]).
However, daily administration of α -tocopherol prevented this effect in both
SNpc and VTA.
46
We found that reserpine chronic administration produces a decrease of
OR in the striatum (Fig. 5A and 6C; ANOVA [F (3,19) = 4.480, P < 0.05] followed
by Sidak's post hoc test). Moreover, we found a reduction in the OR in the
dentate gyrus hippocampal subregion (Fig. 5D and 6D; ANOVA [F (3,19) = 3.916,
P < 0.05] followed by Sidak’s post hoc test). However, this effect was prevented
by α-tocopherol chronic treatment. Conversely, no changes were found in the
dorsal hippocampus CA1 subregion (Fig. 5A and 6D; ANOVA [F (3,19) = 0.7547,
P = 0.5332]) and CA3 (Fig. 5B and 6D; ANOVA [F (3,19) = 0.5947, P = 0.6261]).
Figure 4. Treatment with α-tocopherol reversed the neuronal damage caused
by repeated administration of reserpine. The animals were divided into following
groups: VR + VT (n = 6), RES + VT (n = 5), RES + TOC (n = 6), and VR + TOC
(n = 6). Data are expressed as mean ± SEM. (A) (****) P < 0.0001 to RES + VT
vs VR + VT and (####) P < 0.0001 to RES + VT vs RES + TOC; (B) (#) P < 0.05
and (**) P < 0.01 to RES + VT vs RES + TOC and RES + VT vs VR + VT,
respectively, in ANOVA followed by Sidak’s post hoc test.
47
Figure 5. Effects of repeated administration of reserpine on TH level in optical
density in different subregions of dorsal hippocampus and Striatum. (A)
Striatum, (B) CA1, (C) CA3 and (D) GD. The animals were divided into following
groups VR + VT (n = 6), RES + VT (n = 5), RES + TOC (n = 6), and VR + TOC
(n = 6). Data are expressed as mean ± SEM. (*) P < 0.05 comparing VR + VT
vs RES + VT and (#) P < 0.05 comparing RES + VT vs RES + TOC (ANOVA
followed by Sidak’s post hoc test).
48
Figure 6. Representative photomicrographs of brain coronal sections of (A)
Substantia nigra pars compacta (SNpc), (B) Ventral Tegmental Area (VTA), (C)
striatum (STR) and (D) hippocampal subregions (GD, CA3 and CA1). Animals
were repeatedly treated with vehicle (VR + VT), reserpine (RES + VT),
reserpine + tocopherol (RES + TOC) or tocopherol (VR + TOC). All the rats
were perfused on the 31st day of treatment. Scale bar in (A), (B) and (D): 200
µm; (C): 1000 µm.
17.5. Determination of CAT and SOD activities
Table 1 shows the levels of catalase (CAT) and superoxide dismutase
(SOD) in the hippocampus and striatum of all studied animals. Kruskal–Wallis
ANOVA detected no significant effects for CAT in the hippocampus [H(3) = 0.16;
P = 0.98] or striatum [H(3) = 2.10; P = 0.55] between groups. Also, Kruskal–
Wallis ANOVA revealed no significant effects for total [H(3) = 2.93; P = 0.40],
SNpc
VTA
STR
HIP
49
cytosolic [H(3) = 4.89; P = 0.17] or mitochondrial [H(3) = 1.08; P = 0.78] SOD in
hippocampus neither in total [H(3) = 4.16; P = 0.24], cytosolic [H(3) = 2.58; P =
0.45] or mitochondrial [H(3) = 1.46; P = 0.69] SOD in striatum between all
groups.
Table 1. Determination of oxidative stress parameters by antioxidant enzymes
activities in hippocampus and striatum of rats subjected to different treatments.
Median (minimum; maximum values) for catalase (CAT), superoxide dismutase
(SOD) and its fractions in U (units)/mg protein of animals treated with saline
(control), reserpine (RES), tocopherol (TOC) and tocopherol + reserpine
(TOC+RES).
Brain area Oxidative stress
enzymes Control RES TOC TOC+RES
Hippocampus
CAT 7.33 (3.83; 15.66) 21.00 (2.90; 26.65) 5.87 (4.19; 22.38) 4.34 (3.19; 25.68)
Total SOD 42.25 (7.69; 53.15) 19.22 (6,90; 55,03) 10.41 (8.54; 37.31) 9.92 (9.68; 31.93)
Mitochondrial MnSOD 8.39 (1.55; 14.38) 5.53 (1.53; 14.91) 3.08 (1.82; 10.37) 2.93 (2.07; 6.06)
Cytosolic CuZnSOD 33.86 (6.14; 38.77) 7.39 (6.66; 25.87) 7.25 (5.92; 26.94) 12.49 (11.53; 13.07)
Striatum
CAT 6.81 (3.44; 10.83) 5.31 (3.05; 7.69) 7.06 (4.43; 15.04) 7.22 (4.92; 8.99)
Total SOD 18.16 (14.44; 23.24) 17.63 (15.41; 20.38) 19.75 (18.43; 21.51) 19.17 (18.13; 21.97)
Mitochondrial MnSOD 7.54 (6.68; 9.92) 8.44 (5.75; 9.61) 7.35 (5.62; 12.24) 7.14 (5.64; 9.28)
Cytosolic CuZnSOD 10.58 (6.89; 13.32) 8.98 (6.96; 14.62) 11.76 (8.25; 14.12) 12.49 (11.53; 13.07)
50
18. Discussion
In this study, we show that different types of motor impairment induced
by chronic treatment with reserpine were prevented by α-tocopherol treatment,
as demonstrated by the evaluation of catalepsy behavior (Fig. 2), evaluation of
oral movements (Fig. 3A-C), and observation of balance and motor coordination
in the rotarod test (Fig. 3D). In addition, neuronal analyses showed that
reserpine treatment decreased number of TH+ cells in SNpc and VTA, and co-
treatment with α-tocopherol reversed the detrimental effect caused by reserpine
treatment on the levels of TH positive cells (Fig. 4 and Fig. 6). Likewise,
reserpine treatment diminished staining in the striatum (Fig. 5A and Fig. 6) and
dentate gyrus (Fig. 5D and Fig. 6) and α-tocopherol co-treatment reverted this
detrimental effect. However, we did not observe alterations in SOD and CAT
activity as a consequence of either reserpine or α-tocopherol treatments (Table
1).
Corroborating previous studies (Fernandes et al., 2012; Santos et al.,
2013) we showed that repeated treatment with a low dose (0.1 mg/kg) of
reserpine in rats induces short-term memory deficit followed by gradual
appearance of motor impairments, accompanied by neurochemical changes
compatible with PD pathology. Indeed, this protocol of reserpine-induced
parkinsonism is characterized by a gradual increase of catalepsy behavior, in
which reserpine-treated animals consistently start differing from control subjects
after 7 or 8 reserpine injections (present results and those previous studies). In
this respect, catalepsy in rodents reflects akinesia and rigidity that are important
clinical signs of PD (Sanberg et al., 1988; de Lau & Breteler, 2006; Duty &
Jenner, 2011). Nevertheless, we did not observe this increase in the group that
was concomitantly treated with RES and TOC, which presented catalepsy
duration similar to control until the end of the treatment. This result corroborates
a recent study (Sarmento-Silva, unpublished), indicating that the effects
observed are replicable. However, it is reasonable to speculate if the protective
effect of vitamin E would still be significant if the reserpine treatment lasted
longer. Regardless, we can suggest that TOC treatment promoted at least a
clear-cut delay in the catalepsy behavior progression. Nevertheless, the
catalepsy test evaluates only some aspects of the main motor symptoms of PD
51
such as bradykinesia and akinesia. Thus, in the present study, we evaluated
other aspects of motor function under the same treatment regimen.
Some studies have suggested the induction of oral movements as a
model of tremor-related symptoms of PD (Salamone & Baskin, 1996; Salamone
et al., 2008). Indeed, these abnormal oral movements can be induced by a
number of interventions that decreases dopaminergic function (Jicha &
Salamone, 1991; Salamone & Baskin, 1996; Andreassen et al., 2003;
Salamone et al., 2008), including the repeated treatment with a low dose of
reserpine (Fernandes et al., 2012). In the present study, the animals treated
with reserpine presented increased oral twitching, vacuous chewing movements
and number of tongue protrusions from the 6th day of treatment onwards.
Conversely, animals receiving TOC concomitant to reserpine treatment did not
show this increase throughout treatment, similarly to what happened to
catalepsy behavior. However, some cases, especially regarding tongue
protrusions, RES+VT animals did not present statistical difference when
compared to RES+TOC animals, which suggests that prevention of the
alteration was not complete. This concern notwithstanding, overall analysis of
oral movement parameters shows that co-treatment with α-tocopherol
diminished the motor impairments caused by reserpine.
The rotarod test is a useful tool to evaluate motor function, and studies
on animal models of PD have classically shown deficits in motor coordination
and balance using this apparatus (Rozas et al., 1998; Klivenyi & Vecsei, 2011;
Thornton & Vink, 2012; Didonet et al., 2014). This test is relevant because it
evaluates postural instability, which is a different kind of motor impairment than
those evaluated by catalepsy and oral movement tests. However, this
evaluation has not been previously performed regarding the progressive
reserpine-induced parkinsonism. In the present study, we show that this feature
of motor behavior is also progressively impaired by the repeated treatment with
a low dose of reserpine. Indeed, animals treated with this drug have shown
decreased latency to fall from the apparatus towards the end of the treatment.
Importantly, once again the treatment with TOC prevented this alteration,
keeping the animals from the group RES+TOC similar to controls.
An important observation in animal models of PD is the reduction of brain
TH levels, especially those induced by acute administration of neurotoxins
52
(Mogi et al., 1987; Jackson-Lewis et al., 1995; Bové et al., 2005; Fitzpatrick et
al., 2005; Li et al., 2005; Pérez et al., 2014). In a previous study, we have
shown that the protocol of repeated reserpine treatment induced a decrease in
the TH staining in several brain areas, including the dorsal striatum, as well as
in the number of TH+ neurons in the main dopaminergic nuclei (SNpc and VTA
– Santos et al., 2013). The present results corroborated those findings, and
added the prevention of those neuronal alterations by the co-administration of
TOC. In this respect, the decrease of TH immunostaining in the nigro-striatal
pathway and in the VTA caused by repeated reserpine correlated with the motor
and cognitive impairments induced by this treatment, respectively (Santos et al.,
2013). Thus, the prevention of TH deficiency in SNpc, striatum and VTA by
TOC could be related to the behavioral improvement observed in RES+TOC
treated animals here and in our recent previous study (Sarmento-Silva et al.,
unpublished). Alternatively, the object recognition deficit caused by reserpine
(Santos et al., 2013) and its reversion by TOC (Sarmento-Silva et al.,
unpublished) can both be related to the TH levels in the DG (see figure 5D)
because this hippocampal subregion has been implicated in some aspects of
this memory task (Barbosa et al. 2012).
In summary, both the progressive behavioral alterations and the
decrement in TH staining in the nigrostriatal pathway, VTA and DG were
prevented by a neuroprotective treatment, i.e., the antioxidant agent α-
tocopherol. As discussed previously, reserpine-induced dopamine depletion
could be implicated in the motor impairments, suggesting that reserpine
repeated treatment induces some level of neurotoxicity underlying behavioral
impairment, probably due to the increase in cytoplasmatic DA degradation.
Indeed, RES treatment increases oxidative stress in the striatum (Abílio et al.,
2003; 2004; Burger et al., 2003; Bilska et al., 2007) and the protocol of
reserpine treatment used here caused striatal lipid peroxidation in a previous
study (Fernandes et al., 2012). Nevertheless, studies regarding RES and
oxidative stress are not always consistent, and differs on dosage, days of
treatment, moment of animals’ sacrifice and molecular targets (Spina & Cohen,
1989; Abílio et al., 2002, 2003, 2004; Burger et al., 2003; Faria et al., 2005;
Bilska et al., 2007; Teixeira et al., 2008, 2009; Pereira et al., 2011; Fernandes
et al., 2012; Reckziegel et al., 2013). Yet, given the known results on RES
53
acute treatment and oxidative stress, and the prevention of reserpine-induced
behavioral and neuronal alterations by classical antioxidant agents (present
results; Sarmento-Silva et al., unpublished; Burger et al., 2003; Faria et al.,
2005), we investigated parameters related to antioxidant defenses in the brain,
such as CAT and SOD activities. However, our study did not show any changes
in CAT or SOD activities in either hippocampus or striatum due to RES or TOC
treatment (See table 1). We decided to collect samples 48 hours after the last
injections of RES or TOC in order to avoid conflict with the acute effect of both
drugs. Therefore, the lack of change on enzymatic activity found on this study
might be a response to the pharmacological time window. Another possibility is
that CAT and SOD have a rapid metabolism (Muzykantov, 2001; Baureder et
al., 2014) in order to preserve the survival of neuronal cells and therefore, we
could not observe any changes in enzymatic activity. In other words, possible
rapid changes in the enzymes’ activity following day-by-day injections were not
detected due to our euthanizing schedule. In this respect, the evaluation of CAT
and SOD activities at different time points throughout the treatment, as well as
the evaluation of other parameters indicative of oxidative stress and/or
endogenous antioxidant status could be helpful in clarifying the mechanism of
the protective action of TOC. These proposals are currently under investigation
in our laboratory.
In conclusion, the present findings reinforce the idea that the prolonged
treatment with TOC is an interesting preventive approach regarding behavioral
and neuronal alterations related to PD. Further, we suggest that the protocol of
repeated reserpine-induced parkinsonism is a suitable protocol for investigating
potential neuroprotective interventions.
54
19. Acknowledgments:
The authors would like to thank Antonio Carlos Queiroz de Aquino for
capable technical assistance. This study was supported by grants from
Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq,
Brazil), Fundação de Amparo à Pesquisa do Estado do Rio Grande do Norte
(FAPERN, Brazil), Pós-Graduação em Desenvolvimento e Inovação
Tecnológica em Medicamento (PPgDITM, Brazil) and Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil).
55
20. References
1. Abílio VC, Araujo CCS, Bergamo M, Calvente PRV, D’Almeida V, Ribeiro RA,
Frussa-Filho R. Vitamin E attenuates reserpine-induced oral dyskinesia and
striatal oxidized glutathione/reduced glutathione ratio (GSSG/GSH)
enhancement in rats. Prog Neuro-Psychopharmacol Biological Psychiatry 2003;
27:109–14.
2. Abílio VC, Silva RH, Carvalho RC, Grassl C, Calzavara MB, Registro S,
D'Almeida V, Ribeiro R de A, Frussa-Filho R. Important role of striatal catalase
in aging- and reserpine-induced oral dyskinesia. Neuropharmacology 2004;
47(2):263-72
3. Abílio VC, Vera Jr JAR, Ferreira LSM, Duarte CRM, Carvalho RC, Grass C,
Mar-tins CR, Torres-Leite D, Bignotto M, Tufik S, de Ribeiro RA, Frussa-Filho
R. Effects of melatonin on orofacial movements in rats. Psychopharmacology
2002; 161:340–7.
4. Alves CSD, Andreatini R, da Cunha C, Tufik S, Vital MABF. Phosphatidylserine
reserves reserpine-induced amnesia. Eur J Pharmacol 2000; 404, 161-167.
5. Andreassen OA, Ferrante RJ, Aamo TO, Beal MF, Jørgensen HA. Oral
dyskinesias and histopathological alterations in substantia nigra after long-term
haloperidol treatment of old rats. Neuroscience 2003; 122(3):717-25.
6. Barbosa FF, Pontes IM, Ribeiro S, Ribeiro AM, Silva RH. Differential roles of
the dorsal hippocampal regions inthe acquisition of spatial and temporal aspect
s ofepisodic-like memory. Behav Brain Res. 2012 Jun 15;232(1):269-77.
7. Baureder M, Barane E, Hederstedt L. In vitro assembly of catalase. J Biol
Chem. 2014 Oct 10;289(41):28411-20.
8. Bavarsad SR, Harrigan MR, and Alexandrov AV. N-acetylcysteine (NAC) in
neurological disorders: mechanisms of action and therapeutic opportunities.
Brain Behav 2014; 4(2): 108–122.
9. Beutler E. Disorders in glutathione metabolism. Life Sciences 1975; 16:1499-
1506.
10. Bilska A, Dubiel M, Sokołowska-Jezewicz M, Lorenc-Koci E, Włodek L. Alpha-
lipoic acid differently affects the reserpine-induced oxidative stress in the
striatum and prefrontal cortex of rat brain. Neuroscience 2007; 146(4):1758-71.
56
11. Bové J, Prou D, Perier C, Przedborski S. Toxin-induced models of Parkinson's
disease. NeuroRx 2005; 2(3):484-94.
12. Burger ME, Alves A, Callegari L, Athayde FR, Nogueira CW, Zeni G, Rocha JB.
Ebselen attenuates reserpine-induced orofacial dyskinesia and oxidative stress
in rat striatum. Prog Neuropsychopharmacol Biol Psychiatry 2003; 27(1):135-
40.
13. Colpaert FC. Pharmacological characteristics of tremor, rigidity and hypokinesia
induced by reserpine in rat. Neuropharmacology 1987; 26(9): 1431-1440.
14. Dauer W, Przedborski S. Parkinson’s disease: Mechanisms and models.
Neuron 2003; 39: 889-909.
15. de Lau LM, Breteler MM. Epidemiology of Parkinson's disease. Lancet Neurol
2006; 5(6):525-35.
16. Dekundy A, Pietraszek M, Schaefer D, Cenci MA, Danysz W. Effects of group I
metabotropic glutamate receptors blockade in experimental models of
Parkinson's disease. Brain Res Bull 2006; 69(3):318-26.
17. Didonet JJ, Cavalcante JC, Souza L de S, Costa MS, André E, Soares-Rachetti
V de P, Guerrini R, Calo' G, Gavioli EC. Neuropeptide S counteracts 6-OHDA-
induced motor deficits in mice. Behav Brain Res 2014; 266:29-36.
18. Duty S, Jenner P. Animal models of Parkinson's disease: a source of
noveltreatments and clues to the cause of the disease. Br J Pharmacol 2011;
164(4):1357-91.
19. Ebadi M, Srinivasan SK, Baxi MD. Oxidative stress and antioxidant therapy in
Parkinson’s disease. Prog. Neurobiol 1996; 48: 1–19.
20. Ewing JF & Janero DR. Microplate superoxide dismutase assay employing a
nonenzymatic superoxide generator. Anal Biochem 1995; 10;232(2):243-8.
21. Fahn S. Levodopa in the treatment of Parkinson's disease. J Neural Transm
Suppl 2006; 71:1–15.
22. Faria RR, Abílio VC, Grassl C, Chinen CC, Negrão LTR, Castro JPMV,
Fukushiro DF, Rodrigues MSD, Gomes PHZ, Registro S, Carvalho RC,
D'Almeida V, Silva RH, Ribeiro RA, Frussa-Filho R. Beneficial effects of vitamin
C and vitamin E on reserpine-induced oral dyskinesia in rats: Critical role of
striatal catalase activity, Neuropharmacology 2005; Volume 48(7):993-1001.
23. Fernandes VS, Santos JR, Leão AH, Medeiros AM, Melo TG, Izídio GS, Cabral
A,Ribeiro RA, Abílio VC, Ribeiro AM, Silva RH. Repeated treatment with a
57
lowdose of reserpine as a progressive model of Parkinson’s disease. Behav
Brain Res 2012; 231:154–63.
24. Fitzpatrick E, Ashkan K, Wallace BA, Benabid AL, Mitrofanis J.
Differentialsurvival patterns among midbrain dopaminergic cells of MPTP-
treated monkeys and 6OHDA-lesioned rats. Anat Embryol (Berl) 2005;
210(2):101-23.
25. Halliwell B, Gutteridge JM. Free Radicals in Biology and Medicine. New York:
Oxford University Press, 2007. 851P.
26. Henry JP, Sagne C, Botton D, Isambert MF, Gasnier B. Molecular
pharmacology of the vesicular monoamine transporter. Adv Pharmacol 1998;
42:236-239.
27. Jackson-Lewis V, Jakowec M, Burke RE, Przedborski S. Time course and
morphology of dopaminergic neuronal death caused by the neurotoxin 1-
methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Neurodegeneration 1995; 4(3):257-
69.
28. Jicha GA, Salamone JD. Vacuous jaw movements and feeding deficits in rats
with ventrolateral striatal dopamine depletion: possible relation to parkinsonian
symptoms. J Neurosci 1991; 11(12):3822-9.
29. Klivenyi P, Vecsei L. Pharmacological models of Parkinson's disease in rodents.
Methods Mol Biol 2011; 793:211-27.
30. Korczyn AD. Dementia in Parkinson’s disease. J Neurol 2001; 248 (suppl3):
III/1- III/4.
31. Li X, Yin J, Cheng CM, Sun JL, Li Z, Wu YL. Paraquat induces selective
dopaminergic nigrostriatal degeneration in aging C57BL/6 mice. Chin Med J
(Engl) 2005; 118(16):1357-61.
32. Mogi M, Harada M, Kojima K, Kiuchi K, Nagatsu I, Nagatsu T. Effects of
repeated systemic administration of 1-methyl-4-phenyl-1,2,3,6-
tetrahydropyridine (MPTP) on striatal tyrosine hydroxylase activity in vitro and
tyrosine hydroxylase content. Neurosci Lett 1987; 80(2):213-8.
33. Monville C, Torres EM, Dunnett SB. Comparison of incremental and
accelerating protocols of the rotarod test for the assessment of motor deficits in
the 6-OHDA model. J Neurosci Methods 2006; 158 (2): 219-23.
58
34. Muzykantov VR. Targeting of superoxide dismutase and catalase to vascular
endothelium. J Control Release. 2001 Mar 12;71(1):1-21.
35. Nieoullon A. Dopamine and the regulation of cognition and attention. Progress
in Neurobiology 2002; 67, 53-83.
36. Pereira RP, Fachinetto R, de Souza Prestes A, Wagner C, Sudati JH, Boligon
AA, Athayde ML, Morsch VM, Rocha JB. Valeriana officinalis ameliorates
vacuous chewing movements induced by reserpine in rats. J Neural Transm.
2011; 118(11):1547-57.
37. Pérez HJ, Carrillo SC, García E, Ruiz-Mar G, Pérez-Tamayo R, Chavarría A.
Neuroprotective effect of silymarin in a MPTP mouse model of Parkinson's
disease. Toxicology 2014; 319:38-43.
38. Reckziegel P, Peroza LR, Schaffer LF, Ferrari MC, de Freitas CM, Bürger ME,
Fachinetto R. Gallic acid decreases vacuous chewing movements induced by
reserpine in rats. Pharmacol Biochem Behav. 2013 Mar; 104:132-7.
39. Rozas G, López-Martín E, Guerra MJ, Labandeira-García JL. The overall rod
performance test in the MPTP-treated-mouse model of Parkinsonism. J
Neurosci Methods 1998; 83(2):165-75.
40. Salamone JD, Baskin P. Vacuous jaw movements induced by acute reserpine
and low-dose apomorphine: possible model of parkinsonian tremor. Pharmacol
Biochem Behav 1996; 53(1):179-83.
41. Salamone JD, Ishiwari K, Betz AJ, Farrar AM, Mingote SM, Font L, Hockemeyer
J,Müller CE, Correa M. Dopamine/adenosine interactions related to locomotion
andtremor in animal models: possible relevance to parkinsonism. Parkinsonism
Relat Disord 2008; 14 Suppl 2:S130-4.
42. Sanberg PR, Bunsey MD, Giordano M, Norman AB. The catalepsy test: its ups
and downs. Behav Neurosci 1988; 102(5):748-59.
43. Santos JR, Cunha JAS, Dierschnabel AL, Campêlo CLC, Leão AHFF, Silva AF,
Engelberth RCGJ, Izídio GS, Cavalcante JS, Abílio VC, Ribeiro A M, Silva RH.
A new progressive model of Parkinson´s disease: Different temporal courses of
motor and cognitive deficits. Behav Brain Res 2013; 253, 68–77.
44. Silva RH, Abílio VC, Torres-Leite D, Bergamo M, Chinen CC, Claro FT,
Carvalho R de C, Frussa-Filho R. Concomitant development of oral dyskinesia
59
and memory deficits in reserpine-treated male and female mice. Behav Brain
Res 2002; 132, 171-177.
45. Skalisy LL, Beijamini V, Joca SL, Vital MABF, da Cunha C, Andreatini R.
Evaluation of the face validity of reserpine administration as an animal modelo
of depression- Parkinson’s disease association. Prog Neuro-Psychopharmacol
Biol Psychiatry 2002; 26, 879-883.
46. Spina MB & Cohen G. Dopamine turnover and glutathione oxidation:
implications for Parkinson disease. Proc Natl Acad Sci U S A. 1989; 86(4):
1398–1400.
47. Teixeira AM, Trevizol F, Colpo G, Garcia SC, Charão M, Pereira RP, Fachinetto
R, Rocha JB, Bürger ME. Influence of chronic exercise on reserpine-induced
oxidative stress in rats: behavioral and antioxidant evaluations. Pharmacol
Biochem Behav. 2008; 88(4):465-72.
48. Teixeira AM, Reckziegel P, Müller L, Pereira RP, Roos DH, Rocha JB, Bürger
ME. Intense exercise potentiates oxidative stress in striatum of reserpine-
treated animals. Pharmacol Biochem Behav. 2009; 92(2):231-5.
49. Thornton E, Vink R. Treatment with a substance P receptor antagonist is
neuroprotective in the intrastriatal 6-hydroxydopamine model of early
Parkinson's disease. PLoS One 2012; 7(4):e34138.
50. Zgaljardic DJ, Foldi NS, Borod JC. Cognitive and behavioral dysfunction in
Parkinson’s disease: neurochemical and clinicopathogical contributions. J
Neural Transm 2004; 111, 1287-1301.
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21. Artigo 3
(Submetido à publicação no periódico “Brain Pathology”)
MOLECULAR, NEUROCHEMICAL AND BEHAVIORAL HALLMARKS OF
RESERPINE AS A MODEL FOR PARKINSON’S DISEASE: NEW
PERSPECTIVES TO A LONG-STANDING MODEL
Running head: Reserpine as a model of Parkinson’s disease
Anderson H.F.F. Leão1*, Aldair J. Sarmento-Silva1*, Alessandra M. Ribeiro1,2,
Regina H. Silva1,3#
1Memory Studies Laboratory, Department of Physiology, Universidade Federal
do Rio Grande do Norte, Natal, RN, Brazil.
2Department of Biosciences, Universidade Federal de São Paulo, Santos, SP,
Brazil
3Department of Pharmacology, Universidade Federal de São Paulo, São Paulo,
SP, Brazil
*These authors contributed equally to this work
#Corresponding Author
Departamento de Farmacologia – UNIFESP
Rua Botucatu, 862, Edifício Leal Prado, 1º.andar
CEP 04023062 - São Paulo, SP, Brasil
Email: [email protected]
61
22. Abstract
The administration of reserpine to rodents was one of the first models employed
to investigate the pathophysiology and screening for potential treatments of
Parkinson’s disease (PD). The reserpine model was critical to the
understanding of the role of monoamine system in the regulation of motor and
affective disorders, as well as the efficacy of current PD treatments, such as L-
DOPA and dopamine agonists. Nevertheless, with the introduction of toxin-
induced and genetic models of PD, reserpine became underused. The main
rationale to this drawback was the supposed absence of reserpine construct
validity with PD. Here, we highlight classical and recent experimental findings
that support the face, pharmacological and construct validity of reserpine PD
model, and reason against the current rationale for its underuse. We also aim to
shed a new perspective upon the model by discussing the main challenges and
potentials for the reserpine model of PD.
Keywords: Parkinson’s disease, reserpine, rodent, animal model, dopamine.
62
23. Introduction
Parkinson’s disease (PD) is the second most common neurodegenerative
disorder after Alzheimer’s disease. Its onset is rarely before the age of 50 years
and a sharp increase of the incidence is seen after the age of 60 years (18). PD
affects approximately 1-2% of the population over the age of 60 (58), with a
higher prevalence in men than in women (18,57). Most importantly, it is a
disorder with progressive onset and escalating deterioration of quality of life
(25). Therefore, PD is recognized as a social and economic burden to countries
with increasing life expectancy, and for this reason, the scientific interest in the
disorder is continuously emphasized (18).
PD diagnose is oriented by its cardinal motor symptoms, which include
bradykinesia, rigidity, resting tremor, and postural instability (100). However,
even though PD is considered a motor disorder, patients present equally
incapacitating non-motor symptoms. Further, those symptoms may appear
previously or concomitantly to motor symptoms (116), and include sleep
disorders (78,123,140), anxiety (141), depression (14,89), neuropathic pain and
nociceptive sensitization (24,66,178), impulsivity disorder (147,185,186),
dementia and executive function impairment (1,6,45,113), olfactory dysfunction
(6,55) and constipation (44,140).
The motor alterations are a consequence of dopaminergic neuronal loss in the
substantia nigra (SN) (84,100), which originates the main dopaminergic
projection to the motor regulating nucleus in the basal ganglia (48,110).
Nonetheless, loss of dopaminergic neurons in the ventral tegmental area (VTA)
– projecting to limbic areas and to pre-frontal cortex – is also reported in PD
(174,179). This loss results in emotional and cognitive deficits (141,152).
Furthermore, other neurotransmission disturbances are described, as revealed
by histopathological markers in serotonergic (93,176), noradrenergic
(25,193,195) and cholinergic (179,193) neurons.
Studies have also characterized the neurochemical alterations in PD at the
cellular and genetic levels. 5-10% of PD cases can be traced to familial
heritage, subsequently allowing the identification of some genes that underlie
63
rare familial forms of the disease (188). This approach highlighted genes
involved in main cellular pathways implicated in synaptic function (SNCA: α-
sinuclein), ubiquitin-proteasome protein degradation (Parkin and UCHL1),
respiratory chain (PINK1), protein phosphorylation (LRRK2), and oxidative
stress response (DJ-1) (54,150,184,188). Consequently, impairment of these
pathways leads to oxidative stress and defective protein folding, signalization
and degradation (43,96,104,167). Finally, the accumulation of defective protein
aggregates - mainly constituted by α-sinuclein, parkin and ubiquitin, known as
Lewy’s bodies (182) – is followed by cell death. Thus, the pathogenesis of PD
primarily relates to the generation of oxidative stress and accumulation of
defective proteins.
These genetic alterations are in accordance with epidemiological associations
to PD. These associations comprise exposure to environmental toxins that act
on the respiratory chain (37,132,177) – such as pesticides, heavy metals and
carbon monoxide – and neuroinflammation (80,182). Both events result in the
generation of toxic reactive oxygen (ROS) and nitrogen (RNS) species giving
rise to cell damage and eventually cell death. In brief, PD harbors the oxidative
imbalance as a common molecular pathway to cellular stress and
neurodegeneration. Thus, animal models of PD aim to reproduce the
aforementioned cellular and molecular damages (40,56,118), while clinical and
preclinical therapeutic strategies target different candidate steps of these
pathways to slow PD progression (30,83).
23.1 Animal models of PD
Current studies use genetic and neurotoxic approaches to reproduce PD
pathophysiological hallmarks in animal models. In genetic studies, some
strategies focus on the overexpression of normal or truncated autosomal
dominant genes – as SNCA (20,97,126,187) and LRRK2 (107,108) - and
knockout or knockdown of autosomal recessive genes, as Parkin, PINK1 or DJ-
1 (98,99,144,173). Nevertheless, none of these strategies recapitulates key
clinical and neuropathological features of PD, and they only account for 5-10%
of PD cases (188). As a result, the most frequently adopted strategy is to induce
64
oxidative imbalance and dopamine depletion by the administration of toxins or
drugs that act upon dopaminergic neurons (34,40,56,65,118,125,154,162,192).
MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) and 6-OHDA (6-
hydroxydopamine) are the most used toxins in animal models of PD due to their
rather selective actions upon dopaminergic neurons (8,17,56,118). Both enter
the dopaminergic neuron by the dopamine transporter (DAT) and inhibit the
complex I in the respiratory chain, causing reduction in ATP, oxidative damage,
protein aggregation, cell death and dopamine depletion (56,86,118,166). MPTP
is a highly lipophilic protoxin that readily crosses the blood-brain barrier when
peripherally administered (148). Once in the brain, MPTP is converted by glial
MAO-B into its intermediate 1-methyl-4-phenyl-2,3,dihydropyridinium (MPDP+),
which is rapidly oxidized into 1-methyl-4-phenylpyridinium (MPP+) and then
reabsorbed by the dopaminergic neuron through the DAT (41). A disadvantage
of this model is that rodents are more resilient to cell damage induced by MPTP
compared to primates. This results in the need for higher dosages and
increased variability in neurodegeneration within treated animals (39,56,155). In
addition, the handling of large doses of MPTP and biological waste products
represents a high risk of contamination to researchers (142).
6-OHDA, on the other hand, do not cross the blood-brain barrier, and is
required to be directly delivered to the brain (17,23,56,155). Contrastingly from
MPTP, 6-OHDA also enters noradrenergic neurons through DAT (26), and
usually this lack of specificity is resolved by the coadministration of the inhibitors
of noradrenaline (NA) and serotonin (5-HT) reuptake, nortriptiline or
desipramine (24,52,171). Although safer regarding contamination risk compared
to MPTP, bilateral administration of 6-OHDA results in extensive neuronal loss
and severe motor impairment incompatible with survival. Thus, animals are
required to be tube-fed due to aphagia and adipsia (51,180). In order to avoid
these issues, most studies perform the unilateral lesion with 6-OHDA, and
assess motor deficit by inducing unilateral rotating behavior with dopaminergic
agonists (156,171). Thus, unilateral administration of 6-OHDA lack face validity
with PD (51).
65
Alternatively, environmental toxins like rotenone, paraquat and maneb have
been employed to model PD in rodents (8). Of those, rotenone is the most used
due to its lipophilic structure, easiness to cross biological membranes, and
ability to inhibit complex I and generate ROS (15,85,157). However, despite its
close relationship to epidemiological expositions on PD, rotenone’s absence of
selective tissue action results in systemic and peripheral toxicity (68,139,145)
and highly variable dopaminergic lesions (19,39,157,194).
Finally, the administration of reserpine – an inhibitor of the vesicular transporter
of monoamines in the central nervous system (VMAT2) – was one of the
earliest animal models of PD. Reserpine is an alkaloid extracted from Rauwolfia
serpentine, and was first used as a potent antihypertensive drug due to its
capacity to deplete cellular monoamine content (70,115,138). The clinical use of
reserpine was then followed by the observation that patients chronically treated
with reserpine developed lethargy, depression and motor dyskinesia, implicating
the monoamine system in the pathophysiology of affective and motor disorders
(70,94). Readily after, reserpine was used in rodents to mimic parkinsonian
motor and non-motor impairments (16,35,36,47,63,151,160). Although
considered outdated in comparison to the aforementioned models, the
reserpine model recapitulates key features of PD symptomatology,
neurochemistry and pharmacology. For this reason, the model was practical to
elucidate the relevance of dopaminergic neurotransmission to motor control as
well as to screen for candidate drugs for treatment of PD. Under this reference,
this review will highlight a new perspective upon the model and reason against
the current rationale for the undervaluation of the reserpine-induced
parkinsonism model.
23.2. Motor and non-motor behavioral impairment in the reserpine model
The relationship between reserpine and PD was first established by Carlsson et
al. (1957) by the observation that the akinetic state induced by reserpine in
rodents was alleviated by L-DOPA (35,36). At doses varying from 1 to 10
mg/kg, the reserpine induces a wide range of motor impairments reminiscent of
PD, mainly akinesia, hypokinesia, catalepsy, limb rigidity and facial tremor
(16,47,151). These motor features are a consequence of the blockage of
66
VMAT2 (183), leading to total monoamine depletion - including dopamine,
noradrenaline (NA) and serotonin (5-HT).
Besides the typical motor impairment, reserpine is also able to produce aversive
(64,159) and recognition (154) memory deficits, anxiety-like behavior (22,103),
depressive and anhedonic-like behaviors (9,10,160) and nociceptive
sensitization (9,10,109,133). Moreover, the memory impairment and anxiety-like
behavior were described in a dose range (0.1-0.5 mg/kg) that did not produce
motor impairment (22,64,154,159), dissociating an important confounding factor
in behavioral analysis.
More recently, the repeated treatment with low doses of reserpine (0.1 mg/kg)
has been suggested as a progressive model of PD (65,154). Under this
treatment regimen, animals progressively developed motor impairment in the
open field, catalepsy bar, and oral movement tests after repeated injections of a
low dose (0.1 mg/kg) of reserpine. Deficits in these motor tests recapitulate
main motor symptoms of PD, such as hypokinesia and bradykinesia in the open
field and catalepsy bar test (i.e., slowness and difficulty to initiate movements),
and resting tremor in the oral movement test.
In the aforementioned study (154), the motor impairments were preceded by
cognitive impairment in the novel object recognition task and accompanied by
neuronal alterations compatible with the pathophysiology of PD – i.e. reduction
in tyrosine hydroxylase (TH) immunostaining (154) and increased lipid
peroxidation in the striatum (65). Furthermore, the object recognition index
positively correlated with VTA immunostaining for TH, suggesting that neuronal
pathways disruption other than nigrostriatal pathway may play an important role
in non-motor symptoms of PD. In addition, object recognition deficit occurred
after a 1h (154), but not 24h (65), interval between training and test sessions.
These results are in accordance with executive function and procedural memory
deficits in PD patients (105,147,149,186). In parallel, immobility in the forced
swim test correlated with pain indexes, indicating a comorbid relationship
between reserpine-induced non-motor symptomatology (9). Similarly, PD non-
motor impairments comprise anxiety (141), depression (14,89), and nociceptive
sensitization (27,66,178). Thus, non-motor findings induced by reserpine
67
resemble non-motor PD symptoms, reinforcing reserpine’s face validity as a PD
model.
23.3. Pharmacological and predictive quality of the reserpine model
The use of reserpine was critical to the first demonstration of the therapeutic
efficacy of L-DOPA (35,163). This effect was shortly after reproduced in
humans (50) and the reserpine model was established for screening of potential
symptomatic treatment efficacy of new drugs for PD. Indeed, besides L-DOPA,
the reserpine model predicted other current symptomatic anti-Parkinson
treatments: apomorphine (76), pramipexole (62,112), ropinirole (71), rotigotine
(181), pergolide (47,90), bromocriptine (90,91) and cabergoline (122). Likewise,
reserpine-induced motor impairment is also reverted by other clinically utilized
agents in association with L-DOPA, for example: muscarinic antagonists, such
as benztropine and trihexyphenidyl (76); MAO-B or COMT inhibitors such as
selegiline (47,161), rasagiline (67) and tolcapone (111); and amantadine
(47,49,76,92,161). Table 1 summarizes different types of motor impairment
induced by reserpine that are reverted by these drugs. In fact, reserpine is still
currently used to assess anti-parkinsonian efficacy of novel agents, such as D3
receptor agonists (73), inhibitors of glutamate release (95), group III
metabotropic glutamate receptor (mGlu) agonists or positive allosteric
modulators (13,29,131), group I muscarinic metabotropic receptor (mAchR)
antagonists or allosteric modulator (189), and mixed adenosine A2A/A1
antagonists (12,158).
Reserpine is also employed in the screening for antioxidant and anti-
inflammatory treatments to prevent motor impairments such as dyskinesia
(5,9,21,60,128,135,136). Current rationale on oral dyskinesia accounts for
oxidative stress on the pathophysiology of the disorder (2,4,125,169,170).
Accordingly, monoamine depletion in reserpine-treated rats is followed by
increase of ROS/NOS and cell damage (164). The metabolism of
catecholamine (CA) intrinsically results in ROS formation, which is increased as
a consequence of free CA in the cytoplasm of reserpine-treated rats (117,143).
Thus, oxidative stress and cell damage sums up to the monoamine depletion to
impair motor performance. For this reason, treatment with antioxidants is able to
68
revert reserpine-induced oxidative stress and oral dyskinesia (2,136). Finally,
treatment with 40 mg/kg vitamin E concomitant to the repeated treatment with
0.1 mg/kg reserpine protocol (65,154) prevented cognitive and motor
impairment, as well as the TH immunostaining reduction in rats (unpublished
data).
These neurochemical imbalances resemble PD features, as oxidative stress
and dopamine depletion are keystones of the disease pathophysiology (31,72).
Thus, reserpine pharmacological rationale comprises important qualities of PD
pathophysiology and constitutes a good model for screening for candidate
drugs to both symptomatic treatment and possible slowing of disease
progression. This advantage is reinforced by its low toxicity to researchers, low
cost, and reproducibility among laboratories, which points out the reserpine
model of PD as a suitable model for drug screening.
23.4. Molecular and neurochemical features of the reserpine model
Despite the robust face and pharmacological validity of the reserpine model, the
current literature does not recognize the reserpine as a useful model, arguing
the lack of construct validity (56). This drawback is due to the experimental
observations that (1) reserpine do not induce neurodegeneration and protein
aggregation (56,190); (2) motor performance, monoamine content, and TH
staining are partially restored after treatment interruption (133,154); and (3)
reserpine lacks specificity regarding dopaminergic neurotransmission
(9,10,109,129,133).
Nevertheless, the behavioral and neurochemical features of reserpine
administration are highly reproducible with little variance across studies.
Reserpine peripherally administered in the dose range of 1-10 mg/kg is known
to produce a robust (70-95%) depletion of monoamine content in several brain
areas (9,10,53,59,77,82,109,129,133,172; for a summary, see Table 2. This
monoamine depletion starts 30 min after reserpine injection and may endure up
to 14 days, finally returning to normal levels after 21 days of retrieval (82,133).
At first, the absence of specificity was taken as a disadvantage in the model to
accurately reflect PD neurochemistry. However, the realization that PD also
69
comprises 5-HT and NA neurotransmission (25,93,176,193,195) imbalances
argues in favor of the reserpine model as a satisfactory model to reproduce PD
neurochemistry disruptions.
Table 2. Monoamine content depletion induced by different reserpine treatment
regimens in rodents. STR: striatum; SN: substantia nigra; BLA: basolateral amygdala;
CTX: cortex; HPC: hippocampus; THA: thalamus; PFC: prefrontal cortex. Time
window refers to time last reserpine injection. #microdialysis studies.
DOSE STRUCTURE TIME WINDOW DA NA 5-HT REFERENCES
(50x) 0.01 mg/kg STR 24h 0% ~45% 0% 129
(50x) 0.1 mg/kg STR 24h ~90% ~90% ~65%
(50x) 1.0 mg/kg STR 24h ~95% ~90% ~90%
5.0 mg/kg
SN 2h ~85% N/A N/A 82
24h ~70%
STR 2h >95%
24h >95%
1.0 mg/kg STR 6h ~80% N/A ~50% unpublished
data 24h ~90% ~80%
96h ~75% ~80%
5.0 mg/kg STR 24h ~95% N/A N/A 59
5.0 mg/kg STR 24h ~70% N/A N/A 172
10.0 mg/kg STR 18h ~95% N/A N/A 77
STR# 18h >95%
1.0 mg/kg STR 24h ~55% N/A N/A 53
(3x) 1.0 mg/kg BLA 24h ~75% ~80% ~70% 109
(3x) 1.0 mg/kg CTX 48h ~75% ~60% ~70% 9
(3x) 1.0 mg/kg CTX 48h ~80% ~70% ~80% 10
HPC 48h ~70% ~60% ~85%
3.0 mg/kg THA# 24h ~75% >95% >95% 133
PFC# 24h ~90% >95% ~90%
70
Table 1. Predictive validity of reserpine PD model effectiveness for symptomatic treatment of different motor disturbances in PD. The table was constructed
and updated according to the table presented by Duty and Jenner (2011). The drug list was compiled from the Parkinson’s UK web site:
parkinsons.org.uk/content/dopamine-agonists. Accessed 06/10/2014.
TREATMENT RIGIDITY HYPOKINESIA CATALEPSY TREMOR ORAL DYSKINESIA REFERENCES L-DOPA ± Carbidopa + + + + - 47,76,91,122,161 DA agonistas
Bromocriptine + + + - - 90,91,122,161 Cabergoline + + + - - 122 Pergoline + + + + - 47,90,112 Pramipexole - + + - - 62,112 Ropinirole - - + - - 71 Apomorphine + + + - - 76,90,91
Glutamate antagonists
Amantadine + + - + - 47,76,161 Anticholinergics - - - - - -
Orphenadrine - - - - - - Procyclidine - - - - - - Trihexyphenidyl + - - - - 76 Benztropine + - - - - 76
COMT inhibitors
Entacapone - - - - - - Tolcapone - - - - - -
MAO-B inhibitors
Rasagiline - + - - - 67 Slegiline + + - - + 47,161
Anti-oxidative and Dietary therapy
Vitamin E - - - - + 2,60
Co-enzime Q10 - - - - - - Miscelous - - - - + 5,21,128,135,136
71
Moreover, this characteristic is especially important to the aforementioned non-
motor deficits of PD. For instance, NA and 5-HT transmissions are related to
cognitive and emotional function (119,160). Accordingly, reserpine treatment
results in monoamine depletion in areas involved in emotional processing - as
the amygdala (109) - and cognition - as the hippocampus, cortex (9,10) and
pre-frontal cortex (133). Further, repeated reserpine treatment reduces TH
staining in the hippocampus, pre-frontal cortex, dorsal striatum, VTA, SNpc, and
locus coeruleus (154). Another highly reproducible biochemical alteration in the
reserpine model is the induction of oxidative stress. Reserpine in the dose
range of 1-10 mg/kg is able to induce decreases in catalase, superoxide
dismutase, total content of reduced glutathione, and ATP as well as an increase
in glutathione peroxidase activity, oxidized glutathione, lipid peroxidation, nitric
oxide and iron (2–
4,9,10,21,32,33,59,60,65,109,120,128,136,137,146,153,159,164,169,170; for a
summary, see Table 3). Overall, there is an increase in oxidative damage.
Nevertheless, some studies report contradicting results. Those differences
seem to emerge from different dosage, treatment regimen, and brain area
studied. For example, repeated treatment with low doses of reserpine (0.1
mg/kg) produced cumulative effects upon lipid peroxidation in the striatum, but
not hippocampus, of rats (65). As well, catalase activity is generally reduced in
all brain areas - except for the striatum in which some studies found increased
activity (169,170) or no significant differences (4,60). This opposite outcome
may be due to a differential fine-tuning of catalase activity regulation in the
striatum, as catecholaminergic metabolism intrinsically leads to oxidative stress
(117,143). In fact, oxygen peroxide (H2O2) is one of the main products of
catecholamine metabolism by MAO-A (117,143), and naturally one may
speculate that catalase in catecholaminergic neurotransmission is differentially
modulated by increases in H2O2 in order to provide antioxidant protection.
Indeed, this is endorsed by the observation that catecholaminergic neurons are
relatively abundant in populations of catalase-positive microperoxisomes (114).
Thus, it seems that treatment duration and brain area studied defines the extent
of oxidative damage induced by reserpine.
72
The oxidative stress induced by reserpine is related to increased DA
metabolism as a result of reduction on the number of DA molecules in the
vesicle (134) and increased DA turnover (61,129,164). Accordingly, MAO-A
inhibitor reverts L-DOPA and reserpine induced increase in oxidized glutathione
(164,165). In addition, free DA and metabolites in the cytoplasm results in auto-
oxidation of DA and DOPAC to their corresponding reactive quinones - DA-Q
and DOPAC-Q, respectively – (11,117,143), which contributes do cell apoptosis
and synuclein dimerization (75).
The generation of highly reactive molecules results in early cell damage as
consistently evidenced by lipid peroxidation (Table 3) resulting in pro-
inflammatory signalization by TNF-α and IL1-β (9,10). Subsequently, the
increase in pro-inflammatory cytokines activates microglia initiating a vicious
circle of adhesion, inflammation and liberation of more cytokines. Activated
microglia upon dopaminergic neurons also results in increased nitric oxide (NO)
(9,10,21). Afterwards, NO - in the presence of superoxide (O2−) - produces
peroxynitrite (NO3−) (117,143), which is highly reactive and has been shown to
inactivate TH via S-thiolation on cysteine residues (7,88,101,102). In this
context, repeated treatment with a low dose of reserpine (0.1 mg/kg) resulted in
reduced TH immunostaining in several brain areas – i.e. hippocampus, pre-
frontal cortex, dorsal striatum, SNpc and VTA (154).
Ultimately, these events may terminate in the commitment with apoptotic
pathways. In other words, there is a reduction in anti-apoptotic molecules, as
Bcl-2 (59,109), and an increase in pro-apoptotic molecules, as capastase-3
(9,10,109). Nevertheless, whether such signalization leads to actual apoptosis
and cell death is not yet established. Another important feature in favor of the
construct validity of the reserpine model is the observation that VMAT2 deficient
mice, which express only 5% of functional VMAT2, presents age-associated
neurodegeneration in SNpc, locus coeruleus and dorsal raphe, followed by α-
synuclein accumulation and TH and tyramine transporter immunostaining
reduction (38,168). This VMAT2 deficient mice also presents L-DOPA
responsive motor impairment, two-fold increase in DA concentration in cytosol,
reduction in TH phosphorylation associated with catechol feedback, 95% of DA
depletion and increased DA turnover (46,124,168).
73
Table 3. Molecular changes related to oxidative stress induced by different reserpine treatment regimens in rodents.
STRUCTURE DOSE (MG/KG) TIME WINDOW CAT SOD GPX GST GSH GSSG GSSG/GSH LPO NO REFERENCES
Total brain
5.0 24 h ↓ ↓ ↑ 59
(3x) 1.0 3 h ↓ ↓ ↓ ↑ 136
(3x) 1.0 24 h ↓ ↓ ↓ ↑ 127
(3x) 1.0 24 h ↓ ↓ ↓ ↑ 128
(3x) 1.0 17 days ↓ ↓ ↓ ↑ 153
Cortex
(2x) 1.0 24 h ns 137
(3x) 1.0 24 h ns 32
(3x) 1.0 48 h ↓ ↓ ↓ ↑ ↑ 10
(3x) 1.0 48 h ↓ ↓ ↑ ↑ 9
(3x) 1.0 96 h ns 146
10 2 h ns 164
Striatum (10x) 0.1 24 h ↑ 3
(10x) 0.1 48 h ↑ 65
(2x) 0.5 24 h ns 60
(2x) 1.0 24 h ns ns 4
(2x) 1.0 24 h ↑ ↑ 169
(2x) 1.0 24 h ↑ 2
(2x) 1.0 24 h ↑ 33
(2x) 1.0 24 h ↑ ↓ 170
(2x) 1.0 24 h ns 137
(3x) 1.0 24 h ↑ 32
(3x) 1.0 96 h ns 146
5.0 90 min ↑ ns ↑ ↑ 21
10 2 h ↑ 164
Hippocampus (10x) 0.1 48 h ns 65
(2x) 1.0 24 h ns 137
(3x) 1.0 48 h ↓ ↓ ↓ ↑ ↑ 10
(3x) 1.0 48 h ↓ ↓ ↑ ↑ 9
5.0 90 min ns ns ↑ ↑ 21
Substantia nigra (2x) 1.0 24 h ns 137
Basolateral amygdala (3x) 1.0 24 h ↓ ↑ 109
CAT: catalase; SOD: superoxide dismutase; GPx: glutathione peroxidase; GST: glutathione-S-transferase; GSH: reduced glutathione; GSSG:
oxidized glutathione; LPO: lipid peroxide; NO: nitric oxide; ns: not significant. Time window refers to time after last reserpine injection.
74
Moreover, these alterations are accompanied by non-motor impairments, such
as deficit in olfactory discrimination, delayed gastric emptying, altered sleep
latency, anxiety-like behavior and age-dependent depressive behavior (168). In
short, all behavioral and neurochemical alterations in VMAT2 deficient mice
recapitulates the effects of reserpine treatment. Thus, neurodegeneration
seems plausible in long-term VMAT2 functional blockade by reserpine
treatment. For instance, treatment with 1 mg/kg of reserpine every other day for
6 week resulted in persistent neurochemical changes – DA depletion and D1
and D2 receptor up-regulation – up to 60 days after treatment interruption (130),
suggesting long-lasting or permanent neurochemical changes within a chronic
reserpine treatment. Accordingly, repeated treatment with 0.1 mg/kg of
reserpine every other day for 20 days resulted in reduction of TH
immunohistochemistry that was only partially reversed after 30 days of
treatment interruption (154).
As follows, acute or short-term DA depletion by reserpine treatment results in
up-regulation of D1, but not D2 (42,121,172). Nevertheless, long-term treatment
also leads to D2 up-regulation (130,175). These neurochemical modifications
also relate to dopaminergic denervation in untreated PD patients. Functional
imaging techniques report up-regulation of D2 receptor, whereas up-regulation
of D1 is not yet clearly defined (79,87).
In conclusion, reserpine treatment is able to induce (1) monoamine depletion,
(2) oxidative stress, (3) inflammation, (4) pro-apoptotic commitment, (4) tyrosine
hydroxylase immunostaining reduction and (5) dopamine receptors up-
regulation (for summary of neurochemical events after reserpine administration,
see Figure 1). Most of these reserpine-induced neurochemical alterations are
clearly reminiscent of PD pathophysiology and thus holds a satisfactory
resemblance to PD phenomenology.
24. Final considerations
The exposed prospect of reserpine-induced behavioral, pharmacological and
neurochemical findings restates the use of reserpine as a valuable and
promising model for PD study. Thus, the current underuse of reserpine to
75
understand PD pathophysiology should be reconsidered. As well, the use of
reserpine could be substantive to the relevance of VMAT2 functionality to PD in
humans. Indeed, polymorphisms in promoter regions that increases
transcription of VMAT2 are protective against PD (28,74), and reduction in
VMAT2 and its mRNA in nigrostriatal neurons have been reported in PD
patients (81,120). Further, VMAT2 is present in Lewy’s bodies in the substantia
nigra of PD patients (191), and ventral tegmental area dopaminergic neurons
that are spared in PD harbors higher levels of VMAT2 (120). Finally, increased
cytoplasmatic dopamine influences the conformational state of α-synuclein,
promoting stabilization of its pathogenic form (69,106). Thus, because
functional VMAT2 expression is protective against dopaminergic
neurodegeneration, its long-term blockage might represent an interesting
approach to model PD.
We believe that the scientific effort on reserpine PD model validation should
focus in answering whether neurodegeneration and cell death occurs in the
chronic treatment with reserpine, as well as the exploitation of the model to
investigate progressive neurochemical features of PD. We recently presented a
low dose reserpine-induced progressive model of PD that could be useful to
investigate such inquiry (65,154). Therefore, in view of the presented
experimental evidences, the reserpine-induced PD model in rodents reaches
face, pharmacological and phenomenological validity criteria, and closely
resembles major molecular pathways to PD progression.
76
Figure 1. Neurochemical and molecular events after reserpine treatment. (1)
Reserpine precludes dopamine (DA) storage. (2) Increased DA is metabolized in the
cytoplasm (3) generating reactive oxygen species (ROS) and (4) highly reactive
quinones (DA-Q and DOPAC-Q) (5) resulting in oxidative stress and (6) lipid
peroxidation.(7) Accumulation of ROS and reactive quinones leads to cell damage and
pro-inflammatory signalization. (8) Activation of microglia byTNF-α and IL-1β (9)
amplify pro-inflammatory signalization resulting in (10) nitric oxide (NO)increase and
peroxynitrite (NO3-) formation with free superoxide (O2
-). (11) NO3- inhibits TH activity
and (12) reinforces cell damage committing cell fate in pro-apoptotic signalization.At
the same time, (13) monoamine depletion in synaptic cleft results in (14) up-regulation
of D1 and D2 receptorson the post-synaptic and pre-synaptic membrane.ALDH:
aldehyde dehydrogenases; MAO: monoamine oxidase; AADC: aromatic L-amino acid
decarboxylase; TH: tyrosine hydroxylase.
77
25. References
1. Aarsland D, Andersen K, Larsen JP, Lolk A (2003) Prevalence and
characteristics of dementia in Parkinson disease. Arch Neurol 60(3):387-92.
2. Abílio V, Araujo CCS, Bergamo M, Calvente PRV, D’Almeida V, Ribeiro R de A,
Frussa-Filho R (2003) Vitamin E attenuates reserpine-induced oral dyskinesia
and striatal oxidized glutathione/reduced glutathione ratio (GSSG/GSH)
enhancement in rats. Prog Neuro-Psychopharmacology Biol Psychiatry
27(1):109-14.
3. Abílio V, Vera J, Ferreira L, Duarte C, Carvalho R, Grassl C, Martins
CR, Torres-Leite D, Bignotto M, Tufik S, Ribeiro RA, Frussa-Filho R (2002)
Effects of melatonin on orofacial movements in rats. Psychopharmacology
(Berl)161:340-7.
4. Abílio VC, Silva RH, Carvalho RC, Grassl C, Calzavara MB, Registro S,
D’Almeida V, Ribeiro R de A, Frussa-Filho R (2004) Important role of striatal
catalase in aging- and reserpine-induced oral dyskinesia. Neuropharmacology
47(2):263-72.
5. Al-Bloushi S, Safer A-M, Afzal M, Mousa SA. (2009) Green tea modulates
reserpine toxicity in animal models. J Toxicol Sci 34(1):77-87.
6. Anang JBM, Gagnon J-F, Bertrand J-A, Romenets SR, Latreille V, Panisset M,
Montplaisir J, Postuma RB (2014). Predictors of dementia in Parkinson disease:
A prospective cohort study. Neurology 83(14):1253-60.
7. Ara J, Przedborski S, Naini AB, Jackson-Lewis V, Trifiletti RR, Horwitz J,
Ischiropoulos H (1998) Inactivation of tyrosine hydroxylase by nitration following
exposure to peroxynitrite and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MPTP). Proc Natl Acad Sci USA 95(13):7659-63
8. Arif IA, Khan HA (2010) Environmental toxins and Parkinson’s disease: Putative
roles of impaired electron transport chain and oxidative stress. Toxicol Ind
Health 26(2):121-8.
9. Arora V, Chopra K (2013) Possible involvement of oxido-nitrosative stress
induced neuro-inflammatory cascade and monoaminergic pathway:
underpinning the correlation between nociceptive and depressive behaviour in a
rodent model. J Affect Disord 151(3):1041-52.
78
10. Arora V, Kuhad A, Tiwari V, Chopra K (2011) Curcumin ameliorates reserpine-
induced pain-depression dyad: behavioural, biochemical, neurochemical and
molecular evidences. Psychoneuroendocrinology 36(10):1570-81.
11. Asanuma M, Miyazaki I, Ogawa N (2003). Dopamine- or L-DOPA-induced
neurotoxicity: The role of dopamine quinone formation and tyrosinase in a
model of Parkinson’s disease. Neurotox Res 5(3):165-76.
12. Atack JR, Shook BC, Rassnick S, Jackson PF, Rhodes K, Drinkenburg WH,
Ahnaou A, Riele P, Langlois X, Hrupka B, de Haes P, Hendrickx H, Aerts N,
Hens K, Wellens A, Vermeire J, Megens AAHP (2014) JNJ-40255293, A novel
adenosine A2A/A1 antagonist with efficacy in preclinical models of Parkinson’s
disease. ACS Chem Neuroscience. 5(10):1005-19.
13. Austin PJ, Betts MJ, Broadstock M, O’Neill MJ, Mitchell SN, Duty S (2010)
Symptomatic and neuroprotective effects following activation of nigral group III
metabotropic glutamate receptors in rodent models of Parkinson’s disease. Br J
Pharmacol 160(7):1741-53.
14. Barone P (2011) Treatment of depressive symptoms in Parkinson’s disease.
Eur J Neurol 18(1):11-5.
15. Bashkatova V, Alam M, Vanin A, Schmidt WJ (2004) Chronic administration of
rotenone increases levels of nitric oxide and lipid peroxidation products in rat
brain. Exp Neurol 186(2):235-41.
16. Baskin P, Salamone J (1993) Vacuous jaw movements in rats induced by acute
reserpine administration: interactions with different doses of apomorphine.
Pharmacol Biochem Behav 46(4):793-7.
17. Beal MF (2001) Experimental models of Parkinson’s disease. Nat Rev Neurosci
2:325-32.
18. Benito-León J, Porta-Etessam J, Bermejo F (2006) Epidemiology of Parkinson
disease. Lancet Neurol 5:525-35.
19. Betarbet R, Sherer TB, Mackenzie G, Garcia-osuna M, Panov A V, Greenamyre
JT (2000) Chronic systemic pesticide exposure reproduces features of
Parkinson’s disease. Nat Neurosci 3(12):1301-6.
20. Bianco C Lo, Ridet J-L, Schneider BL, Déglon N, Aebischer P (2002) Alpha-
synucleinopathy and selective dopaminergic neuron loss in a rat lentiviral-based
model of Parkinson’s disease. Proc Natl Acad Sci USA 99(16):10813-8.
79
21. Bilska A, Dubiel M, Sokołowska-Jezewicz M, Lorenc-Kocib E, Włodek L (2007)
Alpha-lipoic acid differently affects the reserpine-induced oxidative stress in the
striatum and prefrontal cortex of rat brain. Neurosci 146(4):1758-71.
22. Bisong SA, Brown R, Osim EE (2010) Comparative effects of Rauwolfia
vomitoria and chlorpromazine on locomotor behaviour and anxiety in mice. J
Ethnopharmacol 132(1):334-9.
23. Blesa J, Phani S, Jackson-Lewis V, Przedborski S (2012) Classic and new
animal models of Parkinson’s disease. J Biomed Biotechnol 2012:1-10.
24. Bonito-Oliva A, Masini D, Fisone G (2014) A mouse model of non-motor
symptoms in Parkinson’s disease: focus on pharmacological interventions
targeting affective dysfunctions. Front Behav Neurosci 8:290.
25. Braak H, Del K, Rüb U, de Vos RAI, Jansen ENH, Braak E (2003) Staging of
brain pathology related to sporadic Parkinson’s disease. Neurobiology of Aging
24:197-211.
26. Breese GR, Traylor TD (1971) Depletion of brain noradrenaline and dopamine
by 6-hydroxydopamine. Br J Pharmacol 42(1):88-99.
27. Brefel-Courbon C, Ory-Magne F, Thalamas C, Payoux P, Rascol O (2013)
Nociceptive brain activation in patients with neuropathic pain related to
Parkinson’s disease. Parkinsonism Relat Disord 19(5):548-52.
28. Brighina L, Riva C, Bertola F, Saracchi E, Fermi S, Goldwurm S, Ferrarese, C
(2013) Analysis of vesicular monoamine transporter 2 polymorphisms in
Parkinson’s disease. Neurobiol Aging 34(6):1712.e9-13.
29. Broadstock M, Austin PJ, Betts MJ, Duty S (2012) Antiparkinsonian potential of
targeting group III metabotropic glutamate receptor subtypes in the rodent
substantia nigra pars reticulata. Br J Pharmacol 165(4b):1034-45.
30. Brundin P, Barker RA, Conn PJ, Dawson TM, Kieburtz K, Lees AJ,
Schwarzschild MA, Tanner CM, Isaacs T, Duffen J, Matthews H, Wyse, RKH
(2013) Linked clinical trials--the development of new clinical learning studies in
Parkinson’s disease using screening of multiple prospective new treatments. J
Parkinsons Dis 3(3):231-9.
31. Brundin P, Li J-Y, Holton JL, Lindvall O, Revesz T (2008) Research in motion:
the enigma of Parkinson’s disease pathology spread. Nat Rev Neurosci
9(10):741-5.
80
32. Burger M, Fachinetto R, Calegari L, Paixão MW, Braga AL, Rocha JBT. (2004)
Effects of age on reserpine-induced orofacial dyskinesia and possible protection
of diphenyl diselenide. Brain Res Bull 64(4):339-45.
33. Burger ME, Alves A, Callegari L, Athayde FR, Nogueira CW, Zeni G, Rocha
JBT (2003) Ebselen attenuates reserpine-induced orofacial dyskinesia and
oxidative stress in rat striatum. Prog Neuropsychopharmacol Biol Psychiatry
27(1):135-40.
34. Cannon JR, Tapias VM, Na HM, Honick AS, Drolet RE, Greenamyre JT (2009)
A highly reproducible rotenone model of Parkinson’s disease. Neurobiol Dis
34(2):279-90.
35. Carlsson A, Lindqvist M, Magnusson T (1957) 3,4-dihydroxyphenylalanine and
5-hydroxytryptophan as reserpine antagonists. Nature 180(4596):1200.
36. Carlsson M, Carlsson A (1989) Marked locomotor stimulation in monoamine-
depleted mice following treatment with atropine in combination with clonidine. J
Neural Transm Park Dis Dement Sect 1(4):317-22.
37. Caudle WM, Guillot TS, Lazo CR, Miller GW (2012) Industrial toxicants and
Parkinson’s disease. Neurotoxicology 33(2):178-88.
38. Caudle WM, Richardson JR, Wang MZ, Taylor TN, Guillot TS, McCormack AL,
Colebrooke RE, di Monte, DA, Emson PC, Miller GW (2007) Reduced vesicular
storage of dopamine causes progressive nigrostriatal neurodegeneration. J
Neurosci 27(30):8138-48.
39. Cenci MA, Whishaw IQ, Schallert T (2002) Animal models of neurological
deficits: how relevant is the rat? Nat Rev Neurosci 3(7):574-9.
40. Chesselet M-F, Richter F (2011) Modelling of Parkinson’s disease in mice.
Lancet Neurol 10(12):1108-18.
41. Chiba K, Trevor A, Castagnoli N (1984). Metabolism of the neurotoxic tertiary
amine, MPTP, by brain monoamine oxidase. Biochem Biophys Res Commun
120(2):574-8.
42. Chipkin RE, McQuade RD, Iorio LC (1987) D1 and D2 dopamine binding site
up-regulation and apomorphine-induced stereotypy. Pharmacol Biochem Behav
28(4):477-82.
43. Chuang RS, Gitler AD (2013) Parallel PARKing: Parkinson’s Genes Function in
Common Pathway. Neuron 77(3):377-9.
81
44. Clairembault T, Leclair-Visonneau L, Neunlist M, Derkinderen P (2014) Enteric
glial cells: New players in Parkinson’s disease? Mov Disord.
45. Coelho M, Marti MJ, Sampaio C, Ferreira JJ, Valldeoriola F, Rosa MM, Tolosa
E (2014) Dementia and severity of parkinsonism determines the handicap of
patients in late-stage Parkinson’s disease: the Barcelona-Lisbon cohort. Eur J
Neurol 15.
46. Colebrooke RE, Humby T, Lynch PJ, McGowan DP, Xia J, Emson PC (2006)
Age-related decline in striatal dopamine content and motor performance occurs
in the absence of nigral cell loss in a genetic mouse model of Parkinson’s
disease. Eur J Neurosci 24(9):2622-30.
47. Colpaert FC (1987) Pharmacological characteristics of tremor, rigidity and
hypokinesia induced by reserpine in rat. Neuropharmacology 26(9):1431-40.
48. Da Cunha C, Wietzikoski EC, Dombrowski P, Bortolanza M, Santos LM,
Boschen SL, Miyoshi E (2009) Learning processing in the basal ganglia: a
mosaic of broken mirrors. Behav Brain Res 199(1):157-70.
49. Danysz W, Gossel M, Zajaczkowski W (1994) Are NMDA antagonistic
properties relevant for antiparkinsonian-like activity in rats? Case of amantadine
and memantine. J Neural Transm 7:155-66.
50. Degkwitz R, Frowein R, Kulenkampff C, Mohs U (1960) On the effects of L-
dopa in man and their modification by reserpine, chlorpromazine, iproniazid and
vitamin B6. Klin Wochenschr 38:120-3.
51. Deumens R, Blokland A, Prickaerts J (2002) Modeling Parkinson’s disease in
rats: an evaluation of 6-OHDA lesions of the nigrostriatal pathway. Exp Neurol
175(2):303-17.
52. Didonet JJ, Cavalcante JC, Souza LDS, Costa MSMO, André E, Soares-
Rachetti VDP, Guerrini R, Calo G, Gavioli EC (2014) Neuropeptide S
counteracts 6-OHDA-induced motor deficits in mice. Behav Brain Res 266:29-
36.
53. Dluzen DE, Bhatt S, McDermott JL (2008) Differences in reserpine-induced
striatal dopamine output and content between female and male mice:
implications for sex differences in vesicular monoamine transporter 2 function.
Neuroscience 154(4):1488-96.
54. Dodson MW, Guo M (2007) Pink1, Parkin, DJ-1 and mitochondrial dysfunction
in Parkinson’s disease. Curr Opin Neurobiol 17(3):331-7.
82
55. Driver-Dunckley E, Adler CH, Hentz JG, Dugger BN, Shill HA, Caviness JN,
Sabbagh MN, Beach TG (2014) Olfactory dysfunction in incidental Lewy body
disease and Parkinson’s disease. Parkinsonism Relat Disord 20:1260-1262.
56. Duty S, Jenner P (2011) Animal models of Parkinson’s disease: a source of
novel treatments and clues to the cause of the disease. Br J Pharmacol
164:1357-91.
57. Eeden SKVD, Tanner CM, Bernstein AL, Fross RD, Leimpeter A, Bloch DA,
Nelson LM (2003) Incidence of Parkinson’s disease: Variation by age, gender,
and race/ethnicity. Am J Epidemiol 157(11):1015-22.
58. Elbaz A, Bower JH, Maraganore DM, McDonnell SK, Peterson BJ, Ahlskog JE,
Schaid DJ, Rocca WA (2002) Risk tables for parkinsonism and Parkinson’s
disease. J Clin Epidemiol 55(1):25-31.
59. El-Ghazaly MA, Sadik NAH, Rashed ER, Abd-El-Fattah AA (2013)
Neuroprotective effect of EGb761(R) and low-dose whole-body γ-irradiationin a
rat model of Parkinson’s disease. Toxicol Ind Health 21:1-17.
60. Faria RR, Abílio VC, Grassl C, Chinen CC, Negrão LTR, de Castro JPMV,
Fukushiro DF, Rodrigues MSD, Gomes PHZ, Registro S, de Carvalho RDC,
D'Almeida V, Silva RH, Ribeiro RA, Frussa-Filho R (2005) Beneficial effects of
vitamin C and vitamin E on reserpine-induced oral dyskinesia in rats: critical role
of striatal catalase activity. Neuropharmacology 48(7):993-1001.
61. Fekete MI, Szentendrei T, Herman JP, Kanyicska B (1980) Effects of reserpine
and antidepressants on dopamine and DOPAC (3,4-dihydroxyphenylacetic
acid) concentrations in the striatum, olfactory tubercle and median eminence of
rats. Eur J Pharmacol 64(4):231-8.
62. Ferger B, Buck K, Shimasaki M, Koros E, Voehringer P, Buerger E (2010)
Continuous dopaminergic stimulation by pramipexole is effective to treat early
morning akinesia in animal models of Parkinson’s disease: A pharmacokinetic-
pharmacodynamic study using in vivo microdialysis in rats. Synapse 64(7):533-
41.
63. Fernagut PO, Diguet E, Labattu B, Tison F (2002) A simple method to measure
stride length as an index of nigrostriatal dysfunction in mice. J Neurosci
Methods 113(2):123-30.
64. Fernandes VS, Ribeiro AM, Melo TG, Godinho M, Barbosa FF, Medeiros DS,
Munguba H, Silva RH (2008) Memory impairment induced by low doses of
83
reserpine in rats: possible relationship with emotional processing deficits in
Parkinson disease. Prog Neuropsychopharmacol Biol Psychiatry 32(6):1479-83.
65. Fernandes VS, Santos JR, Leão AHFF, Medeiros AM, Melo TG, Izídio GS,
Cabral A, Ribeiro RA, Abílio VC, Ribeiro AM, Silva RH (2012) Repeated
treatment with a low dose of reserpine as a progressive model of Parkinson’s
disease. Behav Brain Res 231(1):154-63.
66. Fil A, Cano-de-la-Cuerda R, Muñoz-Hellín E, Vela L, Ramiro-González M,
Fernández-de-Las-Peñas C (2013) Pain in Parkinson disease: a review of the
literature. Parkinsonism Relat Disord 19(3):285-94.
67. Finberg JPM, Youdim MBH (2002) Pharmacological properties of the anti-
Parkinson drug rasagiline; modification of endogenous brain amines, reserpine
reversal, serotonergic and dopaminergic behaviours. Neuropharmacology
43(7):1110-8.
68. Fleming SM, Zhu C, Fernagut PO, Mehta A, DiCarlo CD, Seaman RL,
Chesselet M (2004) Behavioral and immunohistochemical effects of chronic
intravenous and subcutaneous infusions of varying doses of rotenone. Exp
Neurol 187(2):418-29.
69. Follmer C, Roma L, Einsiedler CM, Lara A, Moncores M, Weissmu G, Lashuel
HA, Lansbury P, Neto VM, Silva JL, Foguel D (2007). Dopamine affects the
stability, hydration, and packing of protofibrils and fibrils of the wild type and
variants of α-synuclein. Biochemistry 46:472-82.
70. Freis ED (1954) Mental depression in hypertensive patients treated for long
periods with large doses of reserpine. N Engl J Med 251(25):1006-8.
71. Fukuzaki K, Kamenosono T, Nagata R (2000) Effects of ropinirole on various
parkinsonian models in mice, rats, and cynomolgus monkeys. Pharmacol
Biochem Behav 65(3):503-8.
72. Gao HM, Hong JS (2011) Gene-environment interactions: key to unraveling the
mystery of Parkinson’s disease. Prog Neurobiol 94(1):1-19.
73. Ghosh B, Antonio T, Reith M, Dutta A (2010) (4-(2-((5-Hydroxy-1, 2, 3, 4-
tetrahydronaphthalen-2-yl)(propyl) amino) ethyl) piperazin-1-yl) quinolin-8-ol
and its analogues as highly potent dopamine D2/D3 agonists. J Med Chem
53(5):2114-25.
84
74. Glatt CE, Wahner AD, White DJ, Ruiz-Linares A, Ritz B (2006) Gain-of-function
haplotypes in the vesicular monoamine transporter promoter are protective for
Parkinson disease in women. Hum Mol Genet 15(2):299-305.
75. Goldstein DS, Sullivan P, Cooney A, Jinsmaa Y, Sullivan R, Gross DJ, Holmes
C, Kopin IJ, Sharabi Y (2012) Vesicular uptake blockade generates the toxic
dopamine metabolite 3,4-dihydroxyphenylacetaldehyde in PC12 cells:
relevance to the pathogenesis of Parkinson’s disease. J Neurochem
123(6):932-43.
76. Goldstein J, Barnett A, Malick J (1975) The evaluation of anti-parkinson drugs
on reserpine-induced rigidity in rats. Eur J Pharmacol 33:183-8.
77. Gołembiowska K, Dziubina A (2012) The effect of adenosine A(2A) receptor
antagonists on hydroxyl radical, dopamine, and glutamate in the striatum of rats
with altered function of VMAT2. Neurotox Res 22(2):150-7.
78. Gómez-Esteban JC, Tijero B, Somme J, Ciordia R, Berganzo K, Rouco I,
Bustos JL, Valle MA, Lezcano E, Zarranz JJ (2011) Impact of psychiatric
symptoms and sleep disorders on the quality of life of patients with Parkinson’s
disease. J Neurol 258(3):494-9.
79. Guttman M (1992) Dopamine receptors in Parkinson’s disease. Neurol Clin
10(2):377-86.
80. Hald A, Lotharius J (2005) Oxidative stress and inflammation in Parkinson’s
disease: Is there a causal link? Experimental Neurology 193:279-90.
81. Harrington KA, Augood SJ, Kingsbury AE, Foster OJF, Emson PC (1996)
Dopamine transporter (DAT) and synaptic vesicle amine transporter (VMAT2)
gene expression in the substantia nigra of control and Parkinson’s disease.
Molecular Brain Research 36:157-62.
82. Heeringa MJ, Abercrombie ED (1995) Biochemistry of somatodendritic
dopamine release in substantia nigra: an in vivo comparison with striatal
dopamine release. J Neurochem 65(1):192-200.
83. Henchcliffe C, Severt WL (2011) Disease modification in Parkinson’s disease.
Drugs Aging 28(8):605-15.
84. Henderson JM, Stanic D, Tomas D, Patch J, Horne MK, Bourke D, Finkelstein
DI (2005) Postural changes after lesions of the substantia nigra pars reticulata
in hemiparkinsonian monkeys. Behav Brain Res 160(2):267-76.
85
85. Höglinger GU, Féger J, Prigent A, Michel PP, Parain K, Champy P, Ruberg M,
Oertel WH, Hirsch EC (2003) Chronic systemic complex I inhibition induces a
hypokinetic multisystem degeneration in rats. J Neurochem 84(3):491-502.
86. Hsieh Y, Mounsey RB, Teismann P (2011) MPP+-induced toxicity in the
presence of dopamine is mediated by COX-2 through oxidative stress. Naunyn-
Schmiedeberg's Arch Pharmacol 384:157-67.
87. Hurley MJ, Jenner P (2006) What has been learnt from study of dopamine
receptors in Parkinson’s disease? Pharmacol Ther 111(3):715-28.
88. Ischiropoulos H (2003) Biological selectivity and functional aspects of protein
tyrosine nitration. Biochem Biophys Res Commun 305(3):776-83.
89. Ishihara L, Brayne C (2006) A systematic review of depression and mental
illness preceding Parkinson’s disease. Acta Neurol Scand 113(4):211-20.
90. Johnels B (1982) Locomotor hypokinesia in the reserpine-treated rat: drug
effects from the corpus striatum and nucleus accumbens. Pharmacol Biochem
Behav 17(2):283-9.
91. Johnson A, Loew D, Vigouret J (1976) Stimulant properties of bromocriptine on
central dopamine receptors in comparison to apomorphine, (+)-amphetamine
and L-dopa. Br J Pharmacol 56:59-68.
92. Jurna I, Grossmann W, Nell T (1973) Depression by amantadine of tremor
induced by reserpine and oxotremorine in the rat. Naunyn-Schmiedeberg’s Arch
Pharmacol 152:141-52.
93. Kalaitzakis ME, Gentleman SM, Pearce RKB (2013) Disturbed sleep in
Parkinson’s disease: anatomical and pathological correlates. Neuropathol Appl
Neurobiol 39(6):644-53.
94. Kane JM (1982) Tardive Dyskinesia. Arch Gen Psychiatry American Medical
Association 39(4):473.
95. Kaur S, Starr M (1996) Motor effects of lamotrigine in naive and dopamine-
depleted mice. Eur J Pharmacol 304(1-3):1-6.
96. Kim SW, Ko HS, Dawson VL, Dawson TM (2005) Recent advances in our
understanding of Parkinson’s disease. Drug Discov Today Dis Mech 2(4):427-
33.
97. Kirik D, Annett LE, Burger C, Muzyczka N, Mandel RJ, Bjo A (2003)
Nigrostriatal alpha-synucleinopathy induced by viral vector-mediated
86
overexpression of human alpha-synuclein : A new primate model of Parkinson’s
disease. Proc Natl Acad Sci USA 100(5):2884-9.
98. Kitada T, Asakawa S, Hattori N, Matsumine H, Yamamura Y, Minoshima S,
Yokochi M, Mizuno Y, Shimizu N (1998) Mutations in the parkin gene cause
autosomal recessive juvenile parkinsonism. Nature 392(6676):605-8.
99. Kitada T, Pisani A, Porter DR, Yamaguchi H, Tscherter A, Martella G, Bonsi P,
Zhang C, Pothos EM, Shen J (2007) Impaired dopamine release and synaptic
plasticity in the striatum of PINK1-deficient mice. Proc Natl Acad Sci USA
104(27):11441-6.
100. Klockgether T (2004) Parkinson’s disease: clinical aspects. Cell Tissue Res
318(1):115-20.
101. Kuhn DM, Aretha CW, Geddes TJ (1999) Peroxynitrite inactivation of tyrosine
hydroxylase: mediation by sulfhydryl oxidation, not tyrosine nitration. J Neurosci
19(23):10289-94.
102. Kuhn DM, Sakowski SA, Sadidi M, Geddes TJ (2004) Nitrotyrosine as a marker
for peroxynitrite-induced neurotoxicity: the beginning or the end of the end of
dopamine neurons? J Neurochem 89(3):529-36.
103. Labuda CJ, Fuchs PN (2002) Catecholamine depletion by reserpine blocks the
anxiolytic actions of ethanol in the rat. Alcohol 26:55-9.
104. Lee FJS, Liu F (2008) Genetic factors involved in the pathogenesis of
Parkinson’s disease. Brain Res Rev 58(2):354-64.
105. Lees AJ, Smith E (1983) Cognitive deficits in the early stages of Parkinson’s
disease. Brain 106(2):257-70.
106. Li HT, Lin DH, Luo XY, Zhang F, Ji LN, Du HN, Song GQ, Hu J, Zhou JW
(2005) Inhibition of alpha-synuclein fibrillization by dopamine analogs via
reaction with the amino groups of alpha-synuclein. Implication for dopaminergic
neurodegeneration. FEBS J 272(14):3661-72.
107. Li X, Patel JC, Wang J, Avshalumov M V, Nicholson C, Buxbaum JD, Elder GA,
Rice ME, Yue Z (2010) Enhanced striatal dopamine transmission and motor
performance with LRRK2 overexpression in mice is eliminated by familial
Parkinson’s disease mutation G2019S. J Neurosci 30(5):1788-97.
108. Lin X, Parisiadou L, Gu X-L, Wang L, Shim H, Sun L, Xie C, Long C-X, Yang W-
J, Ding J, Chen ZZ, Gallant PE, Tao-Cheng J-H, Rudow G, Troncoso JC, Liu Z,
Li Z, Cai H (2009) Leucine-rich repeat kinase 2 regulates the progression of
87
neuropathology induced by Parkinson’s-disease-related mutant alpha-
synuclein. Neuron 64(6):807-27.
109. Liu S, Zhao R, Li X, Guo H, Tian Z (2014) Attenuation of reserpine-induced
pain/depression dyad by gentiopicroside through downregulation of GluN2B
receptors in the amygdala of mice. Neuromol Med 16:350-9.
110. Lotharius J, Brundin P (2002) Pathogenesis of Parkinson’s disease: dopamine,
vesicles and alpha-synuclein. Nat Rev Neurosci 3(12):932-42.
111. Maj J, Rog Z, Skuza G, Sowifiska H, Superata J (1990) Behavioural and
neurochemical effects of Ro 40-7592, a new COMT inhibitor with a potential
therapeutic activity in Parkinson’s disease. J Neural Transm 2:101-12.
112. Maj J, Rogóz Z, Skuza G, Kołodziejczyk K (1997) The behavioural effects of
pramipexole, a novel dopamine receptor agonist. Eur J Pharmacol 324(1):31-7.
113. Mata IF, Leverenz JB, Weintraub D, Trojanowski JQ, Hurtig HI, Van Deerlin VM,
Ritz B, Rausch R, Rhodes SL, Factor SA, Wood-Siverio C, Quinn JF, Chung
KA, Peterson AL, Espay AJ, Revilla FJ, Devoto J, Hu S-C, Cholerton BA, Wan
JY, Montine TJ, Edwards KL, Zabetian CP (2014) APOE, MAPT, and SNCA
Genes and Cognitive Performance in Parkinson Disease. JAMA Neurol
71(11):1405-12.
114. McKenna O, Arnold G, Holtzman E (1976) Microperoxisome distribution in the
central nervous system of the rat. Brain Res 117:181-94.
115. McQueen EG, Doyle AE, Smirk FH (1954) Mechanism of hypotensive action of
reserpine, an alkaloid of Rauwolfia serpentina. Nature 174(4439):1015.
116. Mehndiratta M, Garg RK, Pandey S (2011) Nonmotor symptom complex of
Parkinson’s disease--an under-recognized entity. J Assoc Physicians India
59:302-8.
117. Meiser J, Weindl D, Hiller K (2013) Complexity of dopamine metabolism. Cell
Commun Signal 11:34.
118. Meredith GE, Sonsalla PK, Chesselet MF (2008) Animal models of Parkinson’s
disease progression. Acta Neuropathol 115(4):385-98.
119. Millan MJ, Agid Y, Brüne M, Bullmore ET, Carter CS, Clayton NS, Connor R,
Davis S, Deakin B, DeRubeis RJ, Dubois B, Geyer MA, Goodwin GM, Gorwood
P, Jay TM, Joëls M, Mansuy IM, Meyer-Lindenberg A, Murphy D, Rolls E,
Saletu B, Spedding M, Sweeney J, Whittington M, Young LJ (2012) Cognitive
88
dysfunction in psychiatric disorders: characteristics, causes and the quest for
improved therapy. Nat Rev Drug Discov 11(2):141-68.
120. Miller GW, Erickson JD, Perez JT, Penland SN, Mash DC, Rye DB, Levey AI
(1999) Immunochemical analysis of vesicular monoamine transporter (VMAT2)
protein in Parkinson’s disease. Exp Neurol 148:138-48.
121. Missale C, Nisoli E, Liberini P, Rizzonelli P, Memo M, Buonamici M, Rossi A,
Spano P (1989) Repeated reserpine administration up-regulates the
transduction mechanisms of D1 receptors without changing the density of
[3H]SCH 23390 binding. Brain Res 483(1):117-22.
122. Miyagi M, Arai N, Taya F, Itoh F, Komatsu Y, Kojima M, Isaji M (1996) Effect of
cabergoline, a long-acting dopamine D2 agonist, on reserpine-treated rodents.
Biol Pharm Bull 11:1499-502.
123. Monderer R, Thorpy M (2009). Sleep disorders and daytime sleepiness in
Parkinson’s disease. Curr Neurol Neurosci Rep 9(2):173-80.
124. Mooslehner KA, Chan PM, Xu W, Liu L, Smadja C, Humby T, Allen ND,
Lawrence SW, Piers CE (2001) Mice with very low expression of the vesicular
monoamine transporter 2 gene survive into adulthood: potential mouse model
for parkinsonism. Molecular and cellular biology 21(16):5321-31.
125. Morin N, Jourdain VA, Di Paolo T (2013) Modeling dyskinesia in animal models
of Parkinson disease. Exp Neurol 256:105-16.
126. Mulcahy P, O’Doherty A, Paucard A, O’Brien T, Kirik D, Dowd E (2013) The
behavioural and neuropathological impact of intranigral AAV-α-synuclein is
exacerbated by systemic infusion of the Parkinson’s disease-associated
pesticide, rotenone, in rats. Behav Brain Res 243:6-15.
127. Nade VS, Shendye N V, Kawale LA, Patil NR, Khatri ML (2013) Protective
effect of nebivolol on reserpine-induced neurobehavioral and biochemical
alterations in rats. Neurochem Int 63(4):316-21.
128. Naidu PS, Singh A, Kulkarni SK (2006) Effect of Withania somnifera root extract
on reserpine-induced orofacial dyskinesia and cognitive dysfunction. Phytother
Res 20(2):140-6.
129. Neisewander JL, Castañeda E, Davis DA (1994) Dose-dependent differences in
the development of reserpine-induced oral dyskinesia in rats: support for a
model of tardive dyskinesia. Psychopharmacology (Berl) 116(1):79-84.
89
130. Neisewander JL, Lucki I, McGonigle P (1991) Neurochemical changes
associated with the persistence of spontaneous oral dyskinesia in rats following
chronic reserpine treatment. Brain Res 558(1):27-35.
131. Niswender C, Johnson K, Weaver C, Jones C, Xiang Z, Luo Q, Rodriguez AL,
Mario JE, Paulis T (2008) Discovery, characterization, and antiparkinsonian
effect of novel positive allosteric modulators of metabotropic glutamate receptor
4. Mol Pharmacol 74(5):1345-58.
132. Noyce AJ, Bestwick JP, Silveira-Moriyama L, Hawkes CH, Giovannoni G, Lees
AJ, Schrag A (2012) Meta-analysis of early nonmotor features and risk factors
for Parkinson disease. Ann Neurol 1-9.
133. Oe T, Tsukamoto M, Nagakura Y (2010) Reserpine causes biphasic nociceptive
sensitivity alteration in conjunction with brain biogenic amine tones in rats.
Neuroscience 169(4):1860-71.
134. Omiatek DM, Bressler AJ, Cans A-S, Andrews AM, Heien ML, Ewing AG.
(2013) The real catecholamine content of secretory vesicles in the CNS
revealed by electrochemical cytometry. Sci Rep 3:1447.
135. Patil R, Dhawale K, Gound H, Gadakh R (2012) Protective effect of leaves of
Murraya koenigii on reserpine-induced orofacial dyskinesia. Iran J Pharm Res
11(2):635-41.
136. Patil R, Kasture S (2012) Protective effect of Rubia cordifolia on reserpine-
induced orofacial dyskinesia. Nat Prod Res 26(22):2159-61.
137. Pereira R, Fachinetto R, Prestes AS, Wagner C, Sudati JH, Boligon
AA, Athayde ML, Morsch VM, Rocha JB (2011) Valeriana officinalis ameliorates
vacuous chewing movements induced by reserpine in rats. J Neural Transm
118:1547-57.
138. Peter D, Jimenez J, Liu Y, Kim J, Edwards RH (1994) The chromaffin granule
and synaptic vesicle amine transporters differ in substrate recognition and
sensitivity to inhibitors. J Biol Chem 269(10):7231-7.
139. Phinney AL, Andringa G, Bol JGJM, Wolters EC, van Muiswinkel FL, van Dam
A-MW, Drukarch B (2006) Enhanced sensitivity of dopaminergic neurons to
rotenone-induced toxicity with aging. Parkinsonism Relat Disord 12(4):228-38.
140. Plouvier AOA, Hameleers RJMG, van den Heuvel EAJ, Bor HH, Olde Hartman
TC, Bloem BR, van Weel C, Lagro-Janssen ALM (2014) Prodromal symptoms
90
and early detection of Parkinson’s disease in general practice: a nested case-
control study. Fam Pract 31(4):373-8.
141. Prediger RDS, Matheus FC, Schwarzbold ML, Lima MMS, Vital MABF (2012)
Anxiety in Parkinson’s disease: a critical review of experimental and clinical
studies. Neuropharmacology 62(1):115-24.
142. Przedborski S, Jackson-lewis V, Naini AB, Petzinger G, Miller R, Akram M
(2001) The parkinsonian toxin (MPTP): a technical review of its utility and
safety. Journal of neurochemistry 76:1265-74.
143. Qi Z, Miller GW, Voit EO (2008) Computational systems analysis of dopamine
metabolism. PLoS One 3(6):e2444.
144. Ramsey CP, Tsika E, Ischiropoulos H, Giasson BI (2010) DJ-1 deficient mice
demonstrate similar vulnerability to pathogenic Ala53Thr human alpha-syn
toxicity. Hum Mol Genet 19(8):1425-37.
145. Ravenstijn PGM, Merlini M, Hameetman M, Murray TK, Ward MA, Lewis H, Ball
G, Mottart C, Goyet CV, Lemarchand T, van Belle K, O'Neill MJ, Danhof M, de
Lange ECM (2008) The exploration of rotenone as a toxin for inducing
Parkinson’s disease in rats, for application in BBB transport and PK-PD
experiments. J Pharmacol Toxicol Methods 57(2):114-30.
146. Reckziegel P, Peroza LR, Schaffer LF, Ferrari MC, de Freitas CM, Bürger ME,
Fachinetto R (2013) Gallic acid decreases vacuous chewing movements
induced by reserpine in rats. Pharmacol Biochem Behav 104:132-7.
147. Redgrave P, Rodriguez M, Smith Y, Rodriguez-Oroz MC, Lehericy S, Bergman
H, Agid Y, DeLong MR, Obeso JA (2010) Goal-directed and habitual control in
the basal ganglia: implications for Parkinson’s disease. Nat Rev Neurosci
11(11):760-72.
148. Riachi NJ, Lamanna C, Harik I (1989) Entry of 1 -Methyl-4-Phenyl-1,2,3,6-
Tetrahydropyridine into rat brain. J Pharmacol Exp Ther 249(3):744-8.
149. Rodriguez-Oroz MC, Jahanshahi M, Krack P, Litvan I, Macias R, Bezard E,
Obeso JA (2009) Initial clinical manifestations of Parkinson’s disease: features
and pathophysiological mechanisms. Lancet Neurol 8(12):1128-39.
150. Sai Y, Zou Z, Peng K, Dong Z (2012) The Parkinson’s disease-related genes
act in mitochondrial homeostasis. Neurosci Biobehav Rev 36(9):2034-43.
91
151. Salamone J, Baskin P (1996) Vacuous jaw movements induced by acute
reserpine and low-dose apomorphine: possible model of parkinsonian tremor.
Pharmacol Biochem Behav 53(1):179-83.
152. Salgado-Pineda P, Delaveau P, Blin O, Nieoullon A (2005) Dopaminergic
contribution to the regulation of emotional perception. Clin Neuropharmacol
28(5):228-37.
153. Sanghavi CR, Barhate SA, Mahajan MS, Mohan M, Kasture SB (2011) Korean
ginseng extract attenuates reserpine-induced orofacial dyskinesia and improves
cognitive dysfunction in rats. Nat Prod Res 25(7):704-15.
154. Santos JR, Cunha JAS, Dierschnabel AL, Campêlo CLC, Leão AHFF, Silva AF,
Engelberth RCGJ, Izídio GS, Cavalcante JS, Abílio VC, Ribeiro AM, Silva RH
(2013) Cognitive, motor and tyrosine hydroxylase temporal impairment in a
model of parkinsonism induced by reserpine. Behav Brain Res 253:68-77.
155. Schober A (2004) Classic toxin-induced animal models of Parkinson’s disease:
6-OHDA and MPTP. Cell Tissue Res 318(1):215-24.
156. Schwarting RKW, Huston JP (1996) The unilateral 6-hydroxydopamine lesion
model in behavioral brain research: analysis of functional deficits, recovery and
treatments. Progress in Neurobiology 50:275-331.
157. Sherer TB, Kim JH, Betarbet R, Greenamyre JT (2003) Subcutaneous rotenone
exposure causes highly selective dopaminergic degeneration and alpha-
synuclein aggregation. Exp Neurol 179(1):9-16.
158. Shook BC, Rassnick S, Osborne MC, Davis S, Westover L, Boulet J, Hall D,
Rupert KC, Heintzelman GR, Hansen K, Chakravarty D, Bullington JL, Russell
R, Branum S, Wells KM, Damon S, Youells S, Li X, Beauchamp DA, Palmer D,
Reyes M, Demarest K, Tang Y, Rhodes K, Jackson PF (2010) In vivo
characterization of a dual adenosine A2A/A1 receptor antagonist in animal
models of Parkinson’s disease. J Med Chem 53(22):8104-15.
159. Silva RH, Abílio VC, Torres-Leite D, Bergamo M, Chinen CC, Claro FT,
Carvalho RC, Frussa-Filho R (2002) Concomitant development of oral
dyskinesia and memory deficits in reserpine-treated male and female mice.
Behav Brain Res 132(2):171-7.
160. Skalisz LL, Beijamini V, Joca SL, Vital MABF, Da Cunha C, Andreatini R (2002)
Evaluation of the face validity of reserpine administration as an animal model of
92
depression – Parkinson’s disease association. Prog Neuropsychopharmacol
Biol Psychiatry 26:879-83.
161. Skuza G, Rogoz Z, Quack G, Danysz W (1994) Memantine, amantadine, and L-
deprenyl potentiate the action of L-dopa in monoamine-depleted rats. J Neural
Transm 98:57-67.
162. Sonsalla PK, Zeevalk GD, German DC (2008) Chronic intraventricular
administration of 1-methyl-4-phenylpyridinium as a progressive model of
Parkinson’s disease. Parkinsonism Relat Disord 14(Suppl 2):S116-8.
163. Sourkes TL (1971) Actions of Levodopa and Dopamine in the Central Nervous
System. JAMA J Am Med Assoc 218(13):1909.
164. Spina MB, Cohen G (1989) Dopamine turnover and glutathione oxidation:
implications for Parkinson disease. Proc Natl Acad Sci USA 86(4):1398-400.
165. Spina N, Cohen GS, York B, York N, July NY (1988) Exposure of school
synaptosomes of oxidized glutathione1 to L-Dopa increases levels. J Pharmacol
Exp Ther 247(2):502-7.
166. Sriram K, Pai KS, Boyd MR, Ravindranath V (1997) Evidence for generation of
oxidative stress in brain by MPTP: in vitro and in vivo studies in mice. Brain Res
749(1):44-52.
167. Takahashi H, Wakabayashi K (2001) The cellular pathology of Parkinson’s
disease. Neuropathology 21:315-22.
168. Taylor TN, Caudle WM, Shepherd KR, Noorian A, Jackson CR, Iuvone PM,
Weinshenker D, Greene JG, Miller GW (2009) Nonmotor symptoms of
Parkinson’s disease revealed in an animal model with reduced monoamine
storage capacity. J Neurosci 29(25):8103-13.
169. Teixeira AM, Reckziegel P, Müller L, Pereira RP, Roos DH, Rocha JBT, Bürger
ME (2009) Intense exercise potentiates oxidative stress in striatum of reserpine-
treated animals. Pharmacol Biochem Behav 92(2):231-5.
170. Teixeira AM, Trevizol F, Colpo G, Garcia SC, Charão M, Pereira RP, Fachinetto
R, Rocha JBT, Bürger ME (2008) Influence of chronic exercise on reserpine-
induced oxidative stress in rats: behavioral and antioxidant evaluations.
Pharmacol Biochem Behav 88(4):465-72.
171. Thiele SL, Warre R, Nash JE (2012) Development of a unilaterally-lesioned 6-
OHDA mouse model of Parkinson’s disease. J Vis Exp (60):1-10.
93
172. Thomas KL, Rose S, Jenner P, Marsden CD (1992) Acute reserpine treatment
induces down regulation of D1 dopamine receptor associated adenylyl cyclase
activity in rat striatum. Biochem Pharmacol 44(1):83-91.
173. Tohru K, Youren T, Clement A, Gautier, Jie S (2010) Absence of nigral
degeneration in aged parkin/DJ-1/PINK1 triple knockout mice. J Neurochem
111(3):696-702.
174. Torack RM, Morris JC (1992) Tyrosine hydroxylase-like (TH) immunoreactivity
in Parkinson’s disease and Alzheimer's disease. J Neural Transm Park Dis
Dement Sect 4(2):165-71.
175. Traub M, Reches a, Wagner HR, Fahn S (1986) Reserpine-induced up-
regulation of dopamine D2 receptors in the rat striatum is enhanced by
denervation but not by chronic receptor blockade. Neurosci Lett 70(2):245-9.
176. Tredici K, Braak H (2012) Spinal cord lesions in sporadic Parkinson’s disease.
Acta Neuropathol 124(5):643-64.
177. Tsuboi Y (2012). Environmental-Genetic Interactions in the Pathogenesis of
Parkinson’s Disease. Experimental neurobiology 21(3):123-8.
178. Tykocki T, Kornakiewicz A, Mandat T, Nauman P (2013) Pain perception in
patients with Parkinson’s disease. J Clin Neurosci 20(5):663-6.
179. Uhl GR, Hedreen JC, Price DL (1985) Parkinson’s disease: loss of neurons
from the ventral tegmental area contralateral to therapeutic surgical lesions.
Neurology 35(8):1215-8.
180. Ungerstedt U (1971) Adipsia and Aphagia after 6-Hydroxydopamine induced
degeneration of the nigro-striatal dopamine system. Acta Physiol Scand
82(S367):95-122.
181. van der Weide J, de Vries J, Tepper PG, Horn AS (1986) Pharmacological
profiles of three new, potent and selective dopamine receptor agonists: N-0434,
N-0437 and N-0734. Eur J Pharmacol 125:273-82.
182. Venderova K, Park DS (2012) Programmed cell death in Parkinson’s disease.
Cold Spring Harb Perspect Med 2:a009365.
183. Vergo S, Johansen JL, Leist M, Lotharius J (2007) Vesicular monoamine
transporter 2 regulates the sensitivity of rat dopaminergic neurons to disturbed
cytosolic dopamine levels. Brain Res 1185:18-32.
94
184. Vives-Bauza C, de Vries RL a, Tocilescu MA, Przedborski S (2009) Is there a
pathogenic role for mitochondria in Parkinson’s disease? Parkinsonism Relat
Disord 15(Suppl 3):S241-4.
185. Voon V, Dalley JW (2011) Parkinson disease: Impulsive choice-Parkinson
disease and dopaminergic therapy. Nat Rev Neurol 7(10):541-2.
186. Voon V, Mehta AR, Hallett M (2011) Impulse control disorders in Parkinson’s
disease: recent advances. Curr Opin Neurol 24(4):324-30.
187. Wakamatsu M, Ishii A, Iwata S, Sakagami J, Ukai Y, Ono M, Kanbe D,
Muramatsu S, Kobayashi K, Iwatsubo T, Yoshimoto M (2008) Selective loss of
nigral dopamine neurons induced by overexpression of truncated human alpha-
synuclein in mice. Neurobiol Aging 29(4):574-85.
188. Wood-Kaczmar A, Gandhi S, Wood NW (2006) Understanding the molecular
causes of Parkinson’s disease. Trends Mol Med 12(11):521-8.
189. Xiang Z, Thompson AD, Jones CK, Lindsley CW, Conn PJ (2012) Roles of the
M1 muscarinic acetylcholine receptor subtype in the regulation of basal ganglia
function and implications for the treatment of Parkinson’s disease. J Pharmacol
Exp Ther 340(3):595-603.
190. Yadav S, Dixit A, Agrawal S, Singh A, Srivastava G, Singh AK, Srivastava G,
Singh AK, Srivastava PK, Prakash O, Singh MP (2012) Rodent models and
contemporary molecular techniques: Notable feats yet incomplete explanations
of Parkinson’s disease pathogenesis. Mol Neurobiol 46(2):495-512.
191. Yamamoto S, Fukae J, Mori H, Mizuno Y, Hattori N (2006) Positive
immunoreactivity for vesicular monoamine transporter 2 in Lewy bodies and
Lewy neurites in substantia nigra. Neurosci Lett 396(3):187-91.
192. Yazdani U, German DC, Liang C-L, Manzino L, Sonsalla PK, Zeevalk GD
(2006) Rat model of Parkinson’s disease: chronic central delivery of 1-methyl-4-
phenylpyridinium (MPP+). Exp Neurol 200(1):172-83.
193. Zarow C, Lyness SA, Mortimer JA, Chui HC (2003) Neuronal loss is greater in
the locus coeruleus than nucleus basalis and substantia nigra in Alzheimer and
Parkinson diseases. Arch Neurol 60(3):337-41.
194. Zhu C, Vourc’h P, Fernagut P-O, Fleming SM, Lacan S, Dicarlo CD, Seaman
RL, Chesselet MF (2004) Variable effects of chronic subcutaneous
administration of rotenone on striatal histology. J Comp Neurol 78(4):418-26.
95
195. Zweig RM, Cardillo JE, Cohen M, Giere S, Hedreen JC (1993) The locus
ceruleus and dementia in Parkinson’s disease. Neurology 43(5):986-91.
96
26. CONSIDERAÇÕES FINAIS
Este trabalho aborda uma perspectiva de tratamento da DP, através do
uso de uma molécula antioxidante, o alpha-tocoferol. Neste estudo, esta
substância minimizou os danos motores, cognitivos e neuronais
dopaminérgicos induzidos pela administração repetida de reserpina, um
modelo animal farmacológico e progressivo da DP. Especificamente, o
tratamento concomitante com esse antioxidante preveniu os déficits
promovidos pela reserpina na tarefa de reconhecimento de objetos (artigo 1),
que foi realizado com um intervalo de 1 h entre treino e teste, compatível com
os déficits de memória de curto prazo e funções executivas apresentados pelos
pacientes com DP. Além disso, o fármaco também foi eficaz em prevenir as
alterações induzidas pela reserpina nos testes motores da catalepsia (artigos 1
e 2), rotarod (artigo 2) e movimentos orais (artigo 2), sugerindo uma
abrangência da ação em vários tipos de sintomas motores da doença. Por fim,
mostramos que a imunomarcação específica para neurônios dopaminérgicos
(por meio da enzima TH) que se encontra reduzida após o tratamento com
reserpina é prevenida pela administração com o alpha-tocoferol (artigo 2).
Desta forma, os resultados sugerem uma ação neuroprotetora do alpha-
tocopherol, prevenindo as alterações comportamentais e neuronais induzidas
no modelo utilizado.
Além de reforçar a possibilidade de um tratamento concomitante com um
antioxidante promover um retardo na progressão dos sintomas parkinsonianos
(corroborando algumas evidências clínicas), nosso estudo propõe a
administração repetida de uma dose baixa de reserpina como um protocolo
adequado para o estudo de possíveis intervenções neuroprotetoras na DP.
Além disso, este estudo constitui-se em evidência da participação do sistema
oxidativo nos danos progressivos observados neste modelo animal, o que
contribui para sua validação como modelo desta doença. Esse aspecto de
nosso estudo foi abordado no artigo 3, onde revisamos a literatura pertinente
ao uso da reserpina como indutora de parkinsonismo, compilando evidências
que reforçam sua validação como bom modelo para o estudo da DP.
97
27. REFERÊNCIAS
1. Abílio, V. C. et al. Effects of melatonin on orofacial movements in rats.
Psychopharmacology, v. 161, n. 4, p. 340-7, jun. 2002.
2. Aguiar Jr., Aderbal S., Araújo, Andréa L., da-Cunha, Thaise R., Speck, A. E.,
Ignácio, Z. M., De-Mello, N. & Prediger, R. D. S. 2009. Physical exercise
improves motor and short-term social memory deficits in reserpinized rats. Brain
Research Bulletin, 79, 452–457.
3. Ahlskog, J.E., Muenter, M.D., 2001. Frequency of levodopa-related dyskinesias
and motor fluctuations as estimated from the cumulative literature. Mov. Disord.
16, 448–458.
4. Albin RL, Young AB, Penney JB (1989) The functional anatomy of basal ganglia
disorders. Trends Neurosci 12:366–376.
5. Alves, C.S., Andreatini, R., da Cunha, C., Tufik, S., Vital, M.A., 2000.
Phosphatidylserzine reverses reserpine-induced amnesia. Eur J Pharmacol.
404(1-2), 161-167.
6. Barbosa, M.T., Caramelli, P., Maia, D.P., Cuningham, M.C.Q., Guerra, H.L.,
Lima-Costa, M.F., Cardoso, F., 2006. Parkinsonims anda parkinson´s disease
in the elderly: a community-based survey in Brazil (The Bambuí Study).
Moviment Disorders. 21(6):800-808.
7. Barnham J.K., Masters A.B., Bush A. Neurodegenerative disease and oxidative
stress. Nat Rev Drug Discov, 3, 205-214, 2004.
8. Barreiros, A. L. B. S.; David, J. M.; David, J. P.; Quim. Nova 2006, 29,113.
9. Bianchi, M.L.P.; Antunes, L.M.G. Radicais livres e os principais antioxidantes
da dieta. Rev. Nutr. Campinas, v. 12, p. 123-130, 1999.
10. Bravo, S. A., Rangel-Barajas C., Garduño B. F., Pathophysiology of L-Dopa
Induced Dyskinesia-Changes in D1/D3 Receptors and Their Signaling Pathway.
Book edited by Abdul Qayyum.Published: March 26, 2014.
11. Brown, R.G., Landau, S., Hindle, J.V., Playfer, J., Samuel, M., Wilson, K.C.,
Depression and anxiety related subtypes in Parkinson's disease. J Neurol
Neurosurg Psychiatry. 2011 Jul;82(7):803-9.
12. Calabrese V. , Mancuso C., Calvani M., Rizzarelli E., Butterfield D. A., A. Stella.
Nitric oxide in the central nervous system: neuroprotection versus neurotoxicity.
Nat Rev Neurosci, 8, 766-775, 2007.
98
13. Calne B. D. Parkinson’s disease is not one disease. Parkinsonism Relat Disord,
7, 3-7, 2001.
14. Centonze, D., Gubellini, P., Picconi, B., Calabresi, P., Giacomini, P., Bernardi,
G., 1999. Unilateral dopamine denervation blocks corticostriatal LTP. J
Neurophysiol, 82(6):3575-79.
15. Chaudhuri, K.R.; Odin, P.; Antonini, A. Martinez-Martin, P. Parkinson’s Disease:
The non-motor issues. Parkinsonism Pelat Disord, v. 17, n. 10, p. 711-723,
2011.
16. Chesselet M-F, Richter F. Modelling of Parkinson’s disease in mice. Lancet
Neurol [Internet]. Elsevier Ltd; 2011 Dec [cited 2014 Sep 1];10(12):1108–18.
17. Clarenbach P. Parkinson's disease and sleep. J Neurol. 2000 Sep;247 Suppl
4:IV/20-3.
18. Colpaert, F C. Pharmacological characteristics of tremor, rigidity and
hypokinesia induced by reserpine in rat. Neuropharmacology, 1987; 26, No. 9,
pp. 1431-1440.
19. Cools R, Stefanova E, Barker RA, Robbins TW, Owen AM. Dopaminergic
modulation of high-level cognitition in Parkinson‘s disease: the role of the
prefrontal cortex revealed by PET. Brain 2002; 125: 584-594.
20. Corti, O., Lesage, S., Brice, A., What genetics tells us about the causes and
mechanisms of Parkinson’s disease. Physiol Rev. 91(4):1161-218, 2011.
21. Da Cunha, C., Angellucci, M.E.M., Canteras, N.S., Wonnacott, S., Takahashi,
R.N., 2002. The lesion of the rat substantia nigra pars compacta dopaminergic
neurons as a model for Parkinson’s disease memory disabilities. Cell Mol
Neurobiol. 22, 227-37.
22. Dauer, W., Przedborski, S., 2003. Parkinson’s Disease: Mechanisms and
Models. Neuron. 39, 889-909.
23. De Lau LM, Breteler MM. Epidemiology of Parkinson's disease. Lancet Neurol
2006; 5(6):525-35.
24. Deumens, R; Blokland, A; Prickarts, J (2002). Modeling Parkinson’s disease in
rats: an evaluation of 6-OHDA lesion of the nigrostriatal pathway. Experimental
Neurology 175, 303-317.
99
25. Dymecki, J., Lechowicz, W., Bertrand, E., Szpak, G.M., 1996. Changes in
dopaminergic neurons of the mesocorticolimbic system in Parkinson's disease.
Folia Neuropathol. 34(2):102-6.
26. Elbaz A., Tranchant C., Epidemiologic studies of environmental exposures in
Parkinson's disease. J Neurol Sci. 2007 Nov 15;262(1-2):37-44. Epub 2007 Jul
27.
27. Fahn, S., 2003. Description of Parkinson's disease as a clinical syndrome, Ann.
N. Y. Acad. Sci. 991, pp. 1–14.
28. Farooqui, T; Farooqui, AA. Lipid-mediated oxidative stress inflammation in the
pathogenesis of Parkinson’s disease. Parkinson’s Disease.
29. Fénelon, G. 1997. Parkinson disease. Medical treatment. Presse Med, 26,
1357-61.
30. Fernandes, V. S. et al. Memory impairment induced by low doses of reserpine
in rats: possible relationship with emotional processing deficits in Parkinson
disease. Progress in neuro-psychopharmacology & biological psychiatry, v. 32,
n. 6, p. 1479-83, 1 ago. 2008.
31. Fernandes, V. S. et al. Repeated treatment with a low dose of reserpine as a
progressive model of Parkinson ’ s disease. Behavioural Brain Research, v.
231, p. 154-163, 2012.
32. Filipe, P. et al. Flavonoids and urate antioxidant interplay in plasma oxidative
stresse. Mol. Cell. Biochem., v. 221, p. 79-87, 2001.
33. Gerlach M., Riederer P. Animal models of Parkinson’s disease: an empirical
comparison with the phenomenology of the disease in man. J Neural Transm,
103, 987-1041, 1996.
34. Grissa, O; Ategbo, J.; Yessoufou, A; Tabka, Z; Miled, A; Jerbi, M; Dramane, K;
Moutairou, K; Prost, J; Hichami, A. Antioxidant status and circulating lipids are
altered in human gestational and diabetes, 2007.
35. Guttman, M., 1992. Dopamine receptors in Parkinson´s disease. Neurol. Clin.
10(2): 377-86.
36. Hald A., Lotharius J. Oxidative stress and inflamation in Parkinson’s disease: Is
there a casual link? Experim Neurol, 184, 561-564, 2005.
37. Halliwell, B.; Gutteridge, J.M. Free Radicals in Biology and Medicine. New York:
Oxford University Press, 1999. 851P.
100
38. Higginson, D.S. King, D. Levine, V.L. Wheelock, N.O. Khamphay, K.A. Sigvardt
The relationship between executive function and verbal memory in Parkinson’s
disease Brain and Cognition, 52 (2003), pp. 343–352
39. Jankovic, J. Parkinson’s disease: Clinical Features and Diagnosis. J. Neurol
Neurosurg Psychiatry, v. 79, p. 368-376, 2008.
40. Johnston, R E; Schallert, T; Becker, J B B. Akinesia and postural abnormality
after unilateral dopamine depletion. Behavioural Brain Research, 1999; 104,
189-196.
41. Klockgether, Thomas. Parkinson’s disease: clinical aspects. Cell Tissue Res,
2004; 318: 115–120.
42. Koo, H-J., Lee, H-J., Im, H., 2008. Sequence determinants regulating fibrillation
of human _-synuclein. Biochem Biophys Res Commun. 368, 772-78.
43. Korczyn, A D. Dementia in Parkinson’s disease. J. Neurol, 2001; 248 (suppl3):
III/1- III/4.
44. Lees, A.J.; Hardy, J.; Revesz, T. Parkinson’s disease. Lancenet, v. 373, p.
2055-2066, 2009.
45. Lima M.M.S, Braga Reksidler A, Marques Zanata S, Bueno Machado H, Tufik
S, Vital M.A: Different parkinsonism models produce a time-dependent
induction of COX-2 in the substantia nigra of rats. Brain Res. 1101, 117-125;
2006.
46. Machado, Â.B.M., 2005. Neuroanatomia funcional. 2. ed. Rio de Janeiro:
Atheneu.
47. Massano, M. Doença de Parkinson – Atualização Clínica. Acta Med Port, v. 24
(S4), p. 827-834, 2011.
48. Mayeux R. Epidemiology of neurodegeneration. Annu. Rev. Neurosci., 26: 81–
104, (2003).
49. Meredith, G. E.; Totterdell, S.; Potashkin, J. A.; Surmeier D. J. Modeling PD
pathogenesis in mice: Advantages of a chronic MPTP protocol. Parkinsonism
and Related Disorders, 2008 (A); 14,112-115.
50. Mizuno Y. Genetics of Parkinson’s disease. Biomed Pharmacother, 53, 109-
116, 1999.
51. Nicholson, G.; Pereira, A.C.; Hall, G.M. Parkinson’s Disease and Anaesthesia.
Br J Anaesth, v. 89, n. 06, p. 904-16, 2002.
101
52. Nieoullon, A. Dopamine and the regulation of cognition and attention. Progress
in Neurobiology, 2002; 67, 53-83.
53. Nussbaum RL., Ellis CE., Alzheimer's disease and Parkinson's disease. N Engl
J Med. 2003 Apr 3;348(14):1356-64.
54. Olanow C. and Tatton W. (1999) Etiology and pathogenesis of Parkinson
disease. Annu. Rev. Neurosci. 22, 123–144.
55. Pearce, R.K.; Jackson, M.; Smith, L.; Jennifer, P.; Marsden, C.D. Chronic
Levodopa administration induces dyskinesias in the 1-methyl-4phenyl-1,2,3,6-
tetrhydropyridine-treated common marmoset (Callithrix Jacchus). Mov Disord, v.
10, p. 731-740, 1995.
56. Pillon, B., Deweer, B., Vidailhet, M., Bonnet, A.M., Hahn-Barma, V. & Dobois, B.
1997. Is impaired memory for spatial location in Parkinson‘s disease domain
specific or dependent on strategic processes. Neuropsychologia, 36:1, 1-9.
57. Pillon, B., Ertle, S., Deweer, B., Bonnet, A. M., Vidailhet, M. & Dubois, B. 1997a.
Memory for spatial location in parkinsonian patients. Neurochologia, 35:3, 221-
228.
58. Ponzoni, S., Garcia-Cairasco, N., 1995. Neurobiologia do Parkinsonismo:
substratos neurais e neuroquímicos dos gânglios basais. Arq Neuropsiquiatria.
53(3-B):706-710.
59. Przedborski, S. Pathogenesis of nigral cell death in Parkinson’s disease.
Parkinsonism Relat Disord, 11, 3-7, 2005.
60. Rang, H.P., Dale, M.M., Ritter, J.M. & Moore, P.K. 2004. Farmacologia. Rio de
Janeiro: Elsevier, 5 ed., 903p.
61. Reeve A., Simcox E., Turnbull D., Ageing and Parkinson's disease: why is
advancing age the biggest risk factor? Ageing Res Rev. 2014 Mar;14:19-30.
doi: 10.1016/j.arr.2014.01.004. Epub 2014 Feb 3.
62. Santos JR, Cunha JA, Dierschnabel AL, Campêlo CL, Leão AH, Silva AF,
Engelberth RC, Izídio GS, Cavalcante JS, Abílio VC, Ribeiro AM, Silva RH.
Cognitive, motor and tyrosine hydroxylase temporal impairment in a model of
parkinsonism induced by reserpine. Behav Brain Res. 2013 Sep 15;253:68-77
63. Schapira A.H.V., Bezard E., Brotchie J., et al. Novel pharmacological targets for
the treatment of Parkinson’s disease. Nat Rev Drug Discov, 5, 854-854, 2006.
102
64. Schneider, C.D.; Oliveira, A.R. Radicais livres de oxigenio e exercicio:
mecanismos de formacao e adaptacao ao treinamento fisico. Rev Bras Med
Esporte. v. 10, p. 308-313, 2004.
65. Shimohama S., Sawada H., Kitamura Y., Taniguchi T. Disease model:
Parkinson’s disease. Trends Mol Med, 9, 360-365, 2003.
66. Shults, C.W. 2003. Treatments of Parkinson Disease. Arch Neurol, 60:
1680:1684.
67. Siegel, G.J., 2006. Basic Neurochemistry. Molecular, Cellular and Medical
Aspects. 7 ed. Londres: ed. Elsevier.
68. Silva, R H; Abílio, V C; Torres-Leite, D; Bergamo, M; Chinen, C C; Claro, F T;
Cravalho, R de C; Frussa-Filho, R. Concomitant development of oral dyskinesia
and memory deficits in reserpine-treated male and female mice. Behavioural
Brain Research, 2002; 132, 171-177.
69. Singh S., Dikshit M. Apoptotic neural death in Parkinson’s disease:
Involvemente of nitric oxide. Brain Res Rev, 54, 233-250, 2007.
70. Skalisy, L L; Beijamini, V; Joca, S L; Vital, M A B F; da Cunha, C; 50.
Andreatini, R. Evaluation of the face validity of reserpine administration as an
animal modelo of depression- Parkinson’s disease association. Progress in
Neuro-Psychopharmacology & Biological Psychiatry, 2002; 26, 879-883.
71. Slawek, J.; Derejko, M.; Lass, P. Factors affecting the quality of life of patients
with idiopathic Parkinson’s Disease – a cross-sectional study in an out patient
clinic attendrees. Parkinsonism Relat Sidord, v. 11, n.7, p. 465-568, 2005.
72. Smith Y, Bevan MD, Shink E, Bolam JP (1998) Microcircuitry of the direct and
indirect pathways of the basal ganglia. Neuroscience 86:353–387.
73. Stayte S, Vissel B. Advances in non-dopaminergic treatments
for Parkinson's disease. Frontiers in Neurocience; 2014 May 22.
74. Teive, HAG (1998). O papel de Charcot na Doença de Parkinson. Arq. Neuro-
Psiquiatr. Vol.56 n.1 São Paulo.
75. Therond, P. et al. Biomarkers of oxidative stress: an analytical approach.
Clinical Nutrition and Metabolic Care. v. 3, p. 373-384, 2000.
76. Van Den Eeden SK, Tanner CM, Bernstein AL, Fross RD, Leimpeter A, et
al. 2003. Incidência da doença de Parkinson: Variação por idade, gênero e raça
/ etnia Am. J. Epidemiol.157:1015-22.
103
77. Verheij, M.M.M. & Cools, A.R. 2007.Differential contribution of storage pools to
the extracellular amount of accumbal dopamine in high and low responders to
novelty: effects of reserpine. Journal of Neurochemistry, 100, 810-821.
78. Vernier, P., Moret, F., Callier, S., Snapyan, M., Wersinger, C., Sidhu, A., 2004.
The degeneration of dopamine neurons in Parkinson's disease: insights from
embryology and evolution of the mesostriatocortical system. Ann N Y Acad Sci.
1035: 231-49.
79. Wood-Kaczmar A., Gandhi S., Wood NW., Understanding the molecular causes
of Parkinson's disease. Trends Mol Med. 2006, Nov;12(11):521-8.
Epub 2006 Oct 5.
80. Wu., T.; Hallet., M. The influence of normal human ageing on automatic
movements. J Physiol, v. 552, n. 2, p. 605-615, 2005.
81. Zgaljardic DJ, Foldi NS, Borod JC. Cognitive and behavioral dysfunction in
Parkinson's disease: neurochemical and clinicopathogical contributions. J.
Neural Transm 2004; 111: 1287–301.
82. De Araújo DP, Lobato R de F, Cavalcanti JR, Sampaio LR, Araújo PV et al.
(2011) The contributions of antioxidant activity of lipoic acid in reducing
neurogenerative progression of Parkinson's disease: a review. Int J Neurosci
121: 51-7. http://www.ncbi.nlm.nih.gov/pubmed/21126109
83. Magyar K, Pálfi M, Tábi T, Kalász H, Szende B et al. (2004) Pharmacological
aspects of (-)-deprenyl. Curr Med Chem 11: 2017-31.
http://www.ncbi.nlm.nih.gov/pubmed/15279565
84. Mayo JC, Sainz RM, Tan DX, Antolín I, Rodríguez C et al. (2005) Melatonin and
Parkinson's disease. Endocrine 27: 169-78.
http://www.ncbi.nlm.nih.gov/pubmed/16217130
85. Weber CA, Ernst ME (2006) Antioxidants, supplements, and Parkinson's
disease. Ann Pharmacother 40: 935-8.
http://www.ncbi.nlm.nih.gov/pubmed/16622156
86. Weinreb O, Amit T, Bar-Am O, Youdim MB (2010). Rasagiline: a novel anti-
parkinsonian monoamine oxidase-B inhibitor with neuroprotective activity. Prog
Neurobiol 92: 330-44. http://www.ncbi.nlm.nih.gov/pubmed/20600573
87. Hsieh MH, Gu SL, Ho SC, Pawlak CR, Lin CL et al. (2012) Effects of MK-801
on recognition and neurodegeneration in an MPTP-induced Parkinson's rat
104
model. Behav Brain Res 229: 41-7.
http://www.ncbi.nlm.nih.gov/pubmed/22227506
88. Marin C, Aguilar E (2011) In vivo 6-OHDA-induced neurodegeneration and
nigral autophagic markers expression. Neurochem Int 58: 521-6.
http://www.ncbi.nlm.nih.gov/pubmed/21237229
89. Salamone JD, Baskin P (1996) Vacuous jaw movements induced by acute reserpine and low-dose apomorphine: possible model of parkinsonian tremor. Pharmacol Biochem Behav 53: 179-83. http://www.ncbi.nlm.nih.gov/pubmed/8848448
90. Miklya I, Knoll B, Knoll J (2003) A pharmacological analysis elucidating why, in
contrast to (-)-deprenyl (selegiline), alpha-tocopherol was ineffective in the
DATATOP study. Life Sci 72(23): 2641-2648.
http://www.ncbi.nlm.nih.gov/pubmed/12672509
91. 33. Butterfield DA, Castegna A, Drake J, Scapagnini G, Calabrese V (2002)
Vitamin E and neurodegenerative disorders associated with oxidative stress.
Nutr Neurosci 5(4): 229-239. http://www.ncbi.nlm.nih.gov/pubmed/12168685
92. 34. Roghani M, Behzadi G (2001) Neuroprotective effect of vitamin E on the
early model of Parkinson's disease in rat: behavioral and histochemical
evidence. Brain Res 892(1): 211-217.
http://www.ncbi.nlm.nih.gov/pubmed/11172767
93. 35. Azzi A (2007) Molecular mechanism of alpha-tocopherol action. Free Radic
Biol Med 43(1): 16-21. http://www.ncbi.nlm.nih.gov/pubmed/17561089
94. 36. Azzi A, Gysin R, Kempná P, Munteanu A, Negis Y, Villacorta L, Visarius T,
Zingg JM (2004) Vitamin E mediates cell signaling and regulation of gene
expression. Ann N Y Acad Sci 1031:86-95.
http://www.ncbi.nlm.nih.gov/pubmed/15753136
95. 37. Ferri P, Cecchini T, Ambrogini P, Betti M, Cuppini R, Del Grande P, Ciaroni
S (2006) alpha-Tocopherol affects neuronal plasticity in adult rat dentate gyrus:
the possible role of PKCdelta. J Neurobiol 66(8): 793-810.
http://www.ncbi.nlm.nih.gov/pubmed/16673395
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28. Anexos
Carta de aceite artigo 1
Carta de submissão artigo 2
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Carta de submissão artigo 3