Administração repetida de baixas doses de …...VALÉRIA FERNANDES DE SOUZA Administração repetida de baixas doses de reserpina: um possível modelo para o estudo de déficits

VALÉRIA FERNANDES DE SOUZA

Administração repetida de baixas doses de reserpina:

um possível modelo para o estudo de déficits cognitivos e motores associados à Doença de

Parkinson

Tese apresentada à Universidade

Federal do Rio Grande do Norte,

para obtenção de título de doutor no

curso de pós-graduação em

Psicobiologia.

NATAL/RN

2011

II

VALÉRIA FERNANDES DE SOUZA

Administração repetida de baixas doses de reserpina: um possível modelo para o estudo de déficits cognitivos e

motores associados à Doença de Parkinson

Tese apresentada à

Universidade Federal do Rio Grande

do Norte, para obtenção de título de

doutor no curso de pós-graduação

em Psicobiologia.

ORIENTADORA: Profa. Dra. Regina Helena da Silva

NATAL/RN

III

Título: Administração repetida de baixas doses de reserpina: um possível modelo para o estudo de déficits cognitivos e motores associados à Doença de Parkinson

Autor: Valéria Fernandes de Souza

Data da defesa: 15/ 09/ 2011

Banca Examinadora:

___________________________________

Prof. Vanessa Costhek Abílio

Universidade Federal de São Paulo, UNIFESP

___________________________________

Prof. Ângela Maria Ribeiro

Universidade Federal de Minas Gerais, UFMG

___________________________________

Prof. Elaine Cristina Gavioli

Universidade Federal do Rio Grande do Norte, UFRN

___________________________________

Prof John Fontenele Araujo

Universidade Federal do Rio Grande do Norte, RN

___________________________________

Prof. Regina Helena da Silva

Universidade Federal do Rio Grande do Norte, RN

IV

“O homem não teceu a teia da vida, ele é dela apena um fio. O que ele fizer estará

fazendo para si mesmo. O que ele fizer para si mesmo estará fazendo para a Teia.”

Chefe Seattle

V

AGRADECIMENTOS

À Universidade Federal do Rio Grande do Norte pela oportunidade concedida.

A minha orientadora, Profa. Regina Helena da Silva, por acreditar e tornar

possível a realização desse sonho e por sua amizade.

Aos meus colegas do Laboratório de Estudo de Memória (LEME), pela

contribuição nos experimentos e amizade. Em especial, ao Ronaldo, Thieza, Anderson,

Alicia e Geison pela colaboração nos experimentos e nas análises dos vídeos.

A colaboração da Profa. Angela Maria Ribeiro (Laboratório de Neurociências e

Comportamento, LaNeC/UFMG) na coleta de meus dados, incentivo nas horas difíceis

e por sua amizade.

A minha família, em especial minha mãe, pelo seu apoio e amor incondicional

que sempre me incentivaram na busca da realização de meus sonhos. Meu pai, que

sempre cultivou na minha criação a busca pela curiosidade e estudos, obrigada. Ao

meu irmão. A Tula que sempre manteve sua fidelidade e amizade. Aos meus parentes

(Tias, Tios, Primos e Primas) pelo reconhecimento do meu esforço, pelo carinho e

apoio. Muito obrigada!!!

Aos meus amigos do LaNeC pela troca constante de conhecimento e alegria em

trabalhar dentro de um laboratório.

Aos meus amigos e amigas Potiguares que tornaram minha estada em Natal

agradável.

Às minhas amigas e amigos da minha terra natal (Belo Horizonte) que estão

comigo na caminhada da vida em vários momentos, sempre demonstrando apoio,

carinho.

VI

Gostaria muito de agradecer aos animais que deram suas vidas em prol do meu

trabalho de doutoramento e para a melhoria da ciência brasileira.

A todos os momentos difíceis que enfrentei para chegar a conclusão deste

trabalho pois eles me ensinaram a buscar soluções, ter força e determinação que

servirão como características essenciais para exercer minha profissão. Além disso, a

todos os momentos maravilhosos que me deram motivação, alegria e a certeza que

tudo passa.

Enfim, a todos que diretamente ou indiretamente me ajudaram a concluir esse

trabalho.

VII

SUMÁRIO

Página

1. Introdução..................................................................................................................

13

1.1. Apresentação........................................................................................................... 14

1.2. Introdução Geral...................................................................................................... 14

1.2.1. Doença de Parkinson.............................................................................................

14

1.2.2. Transtornos motores na Doença de Parkinson...................................................... 17

1.2.3. Déficits cognitivos na Doença de Parkinson.......................................................... 22

1.2.4. Sistemas de neurotransmissão na Doença de Parkinson...................................... 24

1.2.5. Estudo da doença de Parkinson em modelos animais.......................................... 27

1.2.6. Estresse oxidativo e doença de Parkinson............................................................ 35

1.3. Justificativa.............................................................................................................. 39

1.4. Objetivo geral.......................................................................................................... 41

1.4.1. Objetivos específicos.................................................................................. 41

2. Experimentos.............................................................................................................. 42

2.1. Experimento I................................................................................................. 43

2.2. Experimento II................................................................................................ 72

3. Discussão geral e conclusões.................................................................................. 104

4. Referências................................................................................................................. 113

5. Anexo.......................................................................................................................... 135

VIII

Resumo

A doença de Parkinson (DP) é um dos transtornos cerebrais neurodegenerativos

mais comuns e se caracteriza primariamente por uma progressiva degeneração dos

neurônios dopaminérgicos nigroestriatais. Os sintomas principais dessa doença são

aqueles de origem motora (bradicinesia, rigidez, tremor em repouso), porém alterações

na cognição, no humor e no sistema sensorial também podem ser observadas.

Modelos animais que tentam mimetizar características clínicas da DP vêm sendo

utilizados para compreender as alterações comportamentais e mecanismos neuronais

subjacentes ao distúrbio neurofisiológicos dessa doença Contudo, a maioria dos

modelos promove um comprometimento motor intenso e imediato, compatível com

estágios avançados da doença, invalidando estes estudos quanto à avaliação da

natureza progressiva da manifestação sintomatológica (motora ou cognitiva) da DP.

A administração de reserpina (um depletor de monoaminas) em roedores tem

sido considerada um modelo animal para o estudo da DP. Recentemente verificamos

que a reserpina (em doses menores que as usualmente empregadas para produzir os

sintomas motores) promove um déficit de memória em uma tarefa de discriminação

aversiva, sem alterar a atividade motora. A partir desse estudo sugeriu-se que a

administração desse fármaco em doses baixas pode ser útil para o estudo dos déficits

de memória encontrados na DP. Corroborando esse dado, em outro estudo, a

administração aguda subcutânea de reserpina, em doses que não afetam a função

motora, levou a alterações em memória que envolve contexto emocional enquanto as

sem conotação emocional não foram afetadas.

Os objetivos do presente trabalho foram estudar os déficits cognitivos e motores

associados à administração repetida de baixas doses de reserpina e desenvolver um

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possível modelo que mimetize uma neurodegeneração progressiva. Para isso, ratos

Wistar machos com idade de 5 meses foram submetidos a um tratamento repetido, em

dias alternados, com veículo ou diferentes doses de reserpina. Parâmetros cognitivos e

motores, bem como possíveis alterações na função neuronal, foram avaliados ao longo

do tratamento. Os principais resultados encontrados foram: a administração repetida de

0,1 mg/Kg de reserpina em ratos é capaz de induzir o aparecimento gradual de sinais

motores compatíveis com as características progressivas encontrados em pacientes

com DP; os sinais motores foram acompanhados por um aumento dos níveis de

estresse oxidativo no estriado; alterações nas concentrações de glutamato no estriato

nos grupos tratados com doses repetidas de 0,1 e 0,2 mg/Kg foram observadas cinco

dias após o final do tratamento; em animais tratados com doses repetidas de 0,1 mg/kg,

déficits cognitivos foram observados apenas após o surgimento dos sinais motores,

mas não em avaliações feitas anteriormente ao surgimento desses sinais; na dose de

0,2 mg/kg a avaliação cognitiva foi comprometida pela presença de déficits motores

intensos. Dessa forma, os dados obtidos indicam que o protocolo de tratamento com a

reserpina utilizado neste trabalho seja uma alternativa viável para os estudos do

processo progressivo de aparecimento de sinais parkinsonianos em ratos,

principalmente no que diz respeito aos sinais motores. Quanto aos sinais cognitivos,

sugere-se que mais estudos são necessários, possivelmente em outros modelos

comportamentais e/ou alterando-se o esquema de tratamento.

X

Abstract

Parkinson's disease (PD) is one of the most common neurodegenerative brain

disorders and is characterized primarily by a progressive degeneration of dopaminergic

neurons nigroestriatais. The main symptoms of this disease are motor alterations

(bradykinesia, rigidity, tremor at rest), which can be highly disabling in advanced stages

of the condition. However, there are symptomatic manifestations other than motor

impairment, such as changes in cognition, mood and sensory systems.

Animal models that attempt to mimic clinical features of PD have been used to

understand the behavioral and neural mechanisms underlying neurophysiological

disturbance of this disease. However, most models promote an intense and immediate

motor impairment, consistent with advanced stages of the disease, invalidating these

studies for the evaluation of its progressive nature.

The administration of reserpine (a monoamine depletor) in rodents has been

considered an animal model for studying PD. Recently we found that reserpine (in doses

lower than those usually employed to produce the motor symptoms) promotes a memory

deficit in an aversive discrimination task, without changing the motor activity. It was

suggested that the administration of this drug in low doses can be useful for the study of

memory deficits found in PD. Corroborating this data, in another study, acute

subcutaneous administration of reserpine, while preserving motor function, led to

changes in emotional context-related (but not neutral) memory tasks.

The goal of this research was to study the cognitive and motor deficits in rats

repeatedly treated with low doses of reserpine, as a possible model that simulates the

progressive nature of the PD. For this purpose, 5-month-old male Wistar rats were

submitted to a repeated treatment with vehicle or different doses of reserpine on

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alternate days. Cognitive and motor parameters and possible changes in neuronal

function were evaluated during treatment. The main findings were: repeated

administration of 0.1 mg / kg of reserpine in rats is able to induce the gradual

appearance of motor signs compatible with progressive features found in patients with

PD; an increase in striatal levels of oxidative stress and changes in the concentrations of

glutamate in the striatum were observed five days after the end of treatment; in animals

repeatedly-treated with 0. 1 mg/kg, cognitive deficits were observed only after the onset

of motor symptoms, but not prior to the onset of these symptoms; 0.2 mg / kg reserpine

repeated treatment has jeopardized the cognitive assessment due to the presence of

severe motor deficits. Thus, we suggest that the protocol of treatment with reserpine

used in this work is a viable alternative for studies of the progressive appearance of

parkinsonian signs in rats, especially concerning motor symptoms. As for the cognitive

symptoms, we suggest that more studies are needed, possibly using other behavioral

models, and / or changing the treatment regimen.

XII

Lista de abreviações

ALDH2: adeído desidrogenase mitocondrial

DOPAC: ácido 3-4-dihidroxifenilacetico

DOPAL: 3-4-dihidroxifenilacetaldeido

DP: Doença de Parkinson

DSM-IV: ―Diagnostic and Statistic Manual of Mental Disorders of the American

Psychiatry Association IV‖

4HNE: 4-hidroxi-2-nonenal

GSH: glutationa

O2-: superóxido

OH-: radical hidroxila

6-OHDA: 6-hidroxidopamina

MAO-B: Monoamina oxidase B

MAO: monoamina oxidase

MDA: malondialdeído

NMDA: N-metil-D-aspartato

NE: norepinefrina

NO-: oxido nítrico

MPP+: 1-metil-4-fenil-piridina

MPTP: 1-metil-4-fenil-1,2,3,6-tetrahidropiridina

ROS: “oxygen reactive species”

SN: Sistema nervoso

VMAT2: transportadores vesiculares de monoaminas 2

13

1.Introdução

14

1.1. Apresentação

Esta tese foi organizada em formato de artigos científicos. Dessa forma,

apresentamos uma introdução geral e, em seguida, os experimentos I e II, estes

expostos como manuscritos para submissão. Finalizamos com uma discussão geral

e conclusão, onde os resultados obtidos nos experimentos realizados foram

unificados.

1.2. Introdução Geral

1.2.1. Doença de Parkinson

A Doença de Parkinson (DP) foi descrita pela primeira vez por James

Parkinson em 1817 (2002). Atualmente, é um dos transtornos cerebrais

neurodegenerativos mais comuns, atingindo em torno de 1% da população com 65

anos ou mais (Bennett et al. 1996, Mayeux 2003).

Transtornos motores como tremores, bradicinesia (lentificação dos

movimentos), rigidez e anormalidades na postura ou na marcha são considerados as

características primárias da DP (Grossman 1999, Korczyn 2001, Nieoullon 2002).

Contudo, existem outras manifestações sintomáticas além das motoras, dentre as

quais podemos citar alterações na cognição, no humor e no sistema sensorial

(Higginson et al. 2001, Korczyn 2001, Richard et al. 2004, Zgaljardic et al. 2004,

Perbal et al. 2005, Shohamy et al. 2005, Koerts et al. 2007, Monchi et al. 2007,

15

Huang et al. 2007, Schmitt-Eliassen et al. 2007). Além disso, por ser uma doença de

evolução progressiva, dependendo do estágio da doença pode ser observado

disfunção autonômica, alterações de personalidade, distúrbios do sono, dificuldade

na fala e disfunção sexual (Mayeux 2003, Klochgether 2004).

Classicamente, a maioria dos pesquisadores tem utilizado a idade de 40 anos

para classificar os pacientes quanto às manifestações clínicas da DP (Quinn et al.

1987). Quinn et al. (1987) propuseram que casos da DP que se iniciassem entre a

idade de 21-40 anos deveriam ser denominados de portadores da ―DP de início

precoce‖. Além disso, resultados de estudos com portadores da ―DP de início

precoce‖ indicaram uma relação com fatores genéticos relacionados ao risco de

desenvolvimento da doença, especialmente se houver um histórico familiar positivo

(Quinn et al. 1987, Schrag & Schott 2006). A maioria destes pacientes também

apresenta idiopatia de corpos de Lewys (Schrag & Schott 2006). Os pacientes que

manifestam os sintomas da DP com 70 anos ou mais são classificados como de

inícios tardio (Jankovic et al. 1990).

Estudos têm indicado existir uma heterogeneidade clínica em pacientes com

DP que sugerem diferentes mecanismos bioquímicos e degenetarivos (Jankovic &

Kapadia 2001, Lewis et al. 2005, Reijnders et al. 2009). Relatos da literatura

mostram evidências que a progressão da degeneração não é linear (Jankovic &

Kapadia 2001, Nurmi et al. 2001). No início da doença, há uma progressão mais

rápida e em estágios mais avançados, uma taxa de deterioração desacelerada

(Jankovic & Kapadia 2001). Alguns pesquisadores propõem uma classificação para

estes pacientes sendo estes separados em quatro principais subtipos de evolução

da doença: (1) que se inicia na juventude; (2) de rápida progressão; (3) de tremor

não dominante associado a psicopatologias; (4) de tremor dominante. Os sintomas

16

relacionados a déficits cognitivos, depressão, apatia, alucinações podem ser

inclusos no subtipo tremor não dominante, associado a psicopatologias que também

acompanham distúrbios motores como hipocinesia (movimentos lentos ou

ausentes), rigidez, instabilidade postural e distúrbios da marcha. Essas

características fisiopatológicas subjacentes aos subtipos indicam possíveis

implicações neuropatológicas diferenciadas (Reijnders et al. 2009).

Estudos que enfocam deficits motores e cognitvos na DP apresentam

resultados consideravelmente variados (Aarsland et al. (2004, 2007), Borek et al.

2006, Reijnders et al. 2009). Alterações cognitivas que se manifestam antes das

disfunções motoras em pacientes com DP são descritos na literatura (Fenelon 1997,

Shults 2003). Por outro lado, é comum encontrar a associação de um declínio

funcional e rápido das funções motoras com a presença de prejuízos cognitivo que

caracteriza um quadro demencial em indivíduos com DP (Aarsland et al. (2004,

2007)). Geralmente, o início do desenvolvimento do quadro demencial pode oscilar

do diagnóstico até 10 anos ou mais após detectada a doença. Existem vários fatores

preditores do declínio cognitivo, dentre eles podemos destacar os sintomas motores

graves, a presença de alucinações, a presença de corpos de Lewys e os distúrbios

na fala (Aarsland et al. 2004, Burn et al. 2006). Apesar da heterogeneidade dos

pacientes com DP, é encontratado um consenso na literatura quanto à influência dos

déficits cognitivos na qualidade de vida destes pacientes (Aarsland et al. (2004,

2007), Borek et al. 2006).

Existe uma grande dificuldade em identificar os fatores de risco que podem

causar a DP embora exista um consenso de que seja uma doença multifatorial.

Contudo, alguns estudos destacaram fatores como a predisposição genética,

infecção viral (influenza A), traumas físicos e exposição a substâncias tóxicas tais

17

como 6-hidroxidopamina (6-OHDA) e 1-metil-4-fenil-1,2,3,6-tetrahidropiridina (MPTP)

(Calne 2007, Mayeux 2003). Adicionalmente, estudos sobre as taxas de mortalidade

e prevalência da DP mostram que a incidência é maior nos homens do que nas

mulheres. As razões para o aumento do risco de homens desenvolverem DP não

são conhecidas (Wooten et al. 2004).

1.2.2. Transtornos motores na Doença de Parkinson

Os primeiros sintomas manifestados durante o desenvolvimento da DP

podem ser tremor de repouso unilateral no braço ou na perna. Contudo, sintomas

como a bradicinesia (lentificação e escassez de movimentos), incapacidade de

realizar movimentos (acinesia), membros rígidos, andar e postura inclinados, podem

também estar presentes em fases iniciais da doença (Mayeux 2003). Outra

dificuldade motora frequentemente relatada por pacientes com DP é a incapacidade

de realizar movimentos suaves e coordenados com as mãos, que dificulta o

desempenho na escrita e o desenvolvimento de movimentos precisos (Van Gemmert

et al. 2001).

O sintoma de bradicinesia afeta todos os movimentos voluntários e

involuntários. Os movimentos automáticos e habituais, tais como a movimentação

dos braços durante a caminhada, o piscar dos olhos e a deglutição da saliva, são

fortemente reduzidos. Outras características da bradicinesia são: dificuldade em

iniciar movimentos voluntários, lentidão e passos pequenos ao andar. Os pacientes

com DP também apresentam uma menor mobilidade na expressão facial e uma fala

monotônica, que leva a déficits na habilidade de comunicação, apesar de poder

18

apresentar uma função intelectual preservada (Hallett & Khoshbin 1980, Klockgether

2004). A presença de bradicinesia nas extremidades superiores manifesta-se como

micrografia e, com o processo degenerativo, as pessoas afetadas desenvolvem

dificuldade na execução de movimentos finos, como o de abotoar roupas

(Klockgether 2004).

O tremor parkinsoniano normalmente manifesta-se durante o repouso e afeta

principalmente os membros superiores, podendo também afetar as pernas e, com

menos frequência a cabeça. Nos casos típicos de DP, o tremor de repouso possui

uma frequência de 4-7 Hz. Este sintoma não é necessariamente incapacitante, mas

muitos pacientes sofrem porque o tremor os estigmatiza como portadores da DP

(Klockgether 2004).

A rigidez muscular é definida como um aumento da resistência da

movimentação passiva em consequência da rigidez da articulação, que se manifesta

em toda a amplitude do movimento (Xia et al. 2009). Em pacientes nos quais a

rigidez é acompanhada pelo tremor de repouso, um tipo muito característico de

resistência pode ser observado e tem sido denominado como rigidez de roda

denteada. Relatos subjetivos dos pacientes com rigidez a descrevem como

sensações de rigidez e diminuição da capacidade de relaxar os músculos dos

membros (Klockgether 2004).

Na maioria dos casos de pacientes com DP, os sintomas de tremor, rigidez e

bradicinesia estão presentes. Entretanto, a extensão e a gravidade destes sintomas

apresentam variações (Louis et al. 1999, Bertram et al. 2005) . Um sintoma que

claramente está presente é a incapacidade de realizar movimentos suaves e

coordenados (Bertram et al. 2005).

19

O diagnóstico de DP utilizado na clínica geralmente inclui a presença de

bradicinesia e pelo menos uma das três características primárias que são: (1) rigidez

muscular dos membros; (2) tremor postural ou residual; (3) instabilidade postural ou

transtorno postural. Apesar de por vezes presentes, características como demência

ou disfunções autonômicas não contemplam os sintomas utilizados para o

diagnóstico (Mayeux 2003). Assim sendo, os sintomas motores têm sido ressaltados

como os mais importantes transtornos associados com a DP (Klockgether 2004).

Geralmente, os sintomas motores da DP são atribuídos à perda progressiva

dos neurônios dopaminérgicos da substância negra, que leva ao comprometimento

dos tratos extrapiramidais que controlam movimentos corporais complexos

(Grossman 1999, Korczyn 2001, Nieoullon 2002). Entretanto, os mecanismos

subjacentes da depleção de dopamina relacionados aos distúrbios motores não

estão completamente esclarecidos e continuam sob investigação. Alguns estudos

que correlacionam disfunções motoras, dopamina e DP têm sugerido que estes

fatores envolvem alterações do funcionamento dos circuitos cortico-estriatais

(Antonini et al. 1997, Costa et al. (2004, 2006)).

Os pesquisadores Glendinning & Enka (1994) relatam que os mecanismos

subjacentes a alterações da unidade motora na DP são ainda pouco entendidos.

Entretando, estes pesquisadores apontam uma diminuição na atividade muscular e

alteração da unidade motora devido: (1) a irregularidade e intermitência dos padrões

de descargas nas unidades motoras; (2) ao fato de que os músculos antagonistas

(os quais possuem ação anatômica oposta à dos músculos agonistas e usualmente

no movimento permanencem relaxados permitindo a maior facilidade do movimento)

são coativos. Uma possível hipótese para estas mudanças está em um desequilíbrio

entre os impulsos excitatórios e inibitórios para os neurônios motores.

20

Resultados de estudos post mortem em pacientes com DP suregem que

características parkinsonianas diferentes podem ter alterações diferenciadas nos

circuitos neuronais. Estes estudos mostram que pacientes com parkinsonismo do

tipo acinético-rígido possuem perdas mais significativas de células da porção

ventrolateral da substância negra e do locus coeruleus em comparação à pacientes

com parkinsonismo com tremor predominante (Paulus & Jellinger 1991, Jellinger

1999).

Geralmente, os sintomas motores da DP são atribuídos à perda progressiva

dos neurônios dopaminérgicos da substância negra, que levam ao comprometimento

dos tratos extrapiramidais que controlam movimentos corporais complexos

(Grossman 1999, Korczyn 2001, Nieoullon 2002). Entretanto, os mecanismos

subjacentes da depleção de dopamina relacionados distúrbios motores não estão

completamente esclarecidos e continuam em discussão. Alguns estudos que

correlacionam disfunções motoras, dopamina e DP têm sugerido que estes fatores

envolvem alterações do funcionamento dos circuitos cortico-estriatais (Antonini et al.

1997, Costa et al. (2004, 2006), Cilia et al. 2007). De fato, num estudo desenvolvido

por Cilia et al. 2007, no qual foram avaliadas características clínicas e imagem de

ressonância magnética do cérebro de um paciente com tremor palatal (caracterizado

clinicamente por contrações rítmicas e involuntárias dos músculos do palato mole) e

ataxia progressiva (falta de coordenação dos movimentos, podendo afetar a força

muscular e o equilíbrio de uma pessoa), foi encontrada redução dos transportadores

de dopamina no estriado direito. Este estudo também revelou uma degeneração

hipertrófica dos núcleos olivares e significante hipometabolismo nos núcleos rubros,

sugerindo que os sintomas de tremor palatal e ataxia progressiva podem estar

21

relacionados a danos nas vias dentato-rubro-olivar e a disfunções dopaminérgicas

nigro-estriatais.

Um estudo utilizando tomografia por emissão de pósitrons (TEP) com

marcadores fluoroso F18, fluorodopa (FDOPA) e raclopride (RACLO) utilizados para

estudar o metabolismo da glicose estriatal e de DOPA, e marcação de receptor D2

de dopamina mostraram em seus resultados sintomas motores e atrofia de múltiplos

sistemas dopaminérgicos. Este achado indica que a degeneração de sistemas

dopaminérgicos pré e pós-sinápticos estriatais é responsável pelas alterações

motoras em humanos (Antonini et al. 1997).

Outras evidências do envolvimento do sistema dopaminérgico com as

alterações motoras estão no efeito de medicamentos utilizados no tratamento de tais

sintomas, os quais aumentam a função dopaminérgica. Estudos têm demonstrado

que medicamentos pró-dopaminérgicos (exemplo: L-DOPA, selegelina, entre outros)

melhoram a rigidez de pacientes com DP (Benecke et al. 1987, Xia et al. 2009).

A gravidade do transtorno motor pode também estar associada ao declínio

cognitivo (Aarsland et al. (2004, 2005), Burn et al. 2006). Um estudo indicou uma

relação entre pacientes com instabilidade na marcha e dificuldade postural com o

risco de desenvolver um quadro demencial (Burn et al. 2006). Além disso, Louis et

al. (1999) também encontraram que sintomas como rigidez, bradicinesia, tremor e

instabilidade postural em pacientes com DP são preditores do desenvolvimento de

demência. Nesse sentido, podemos observar que as diversidades de sintomas

motores podem ser consequência de uma neurodegeneração e alterações

neuroquímicas que ainda não estão esclarecidas.

22

1.2.3. Déficits cognitivos na Doença de Parkinson

Como escrito no item anterior evidências surgerem que os sintomas motores

são devido à perda progressiva dos neurônios dopaminérgicos da substância negra

que promovem depleção dos níveis de dopamina estriatal (Johnston et al. 1999,

Lindner et al. 1999, Ridley et al. 2006). Contudo, os distúrbios motores podem estar

acompanhados também por prejuízos intelectuais que afetam significativamente a

qualidade de vida de uma pessoa acometida (Korczyn 2001, Nieoullon 2002,

Scherfler et al. 2004, Zgaljardic et al. 2004). Em alguns casos, esses prejuízos

cognitivos se manifestam antes das alterações motoras, e sugere-se que estejam

envolvidos com circuitos neuronais diferentes (Fenelon 1997, Shults 2003). Além

disso, as alterações cognitivas têm sido correlacionadas 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).

Estudos de neuroimagem em pacientes com Parkinson evidenciam uma base

neural específica para os danos cognitivos encontrados nesses casos clínicos

(Owen et al. 1998, Cools et al. 2002, Koerst et al. 2007). Conforme já mencionado,

esses déficits cognitivos na DP têm sido freqüentemente atribuídos a prejuízos em

projeções dopaminérgicas corticais (Cools et al. 2002). Contudo, outras evidências

sugerem que os déficits também podem estar relacionados a danos em regiões

subcorticais (Pillon et al. 1996, Pillon et al. 1997). Além disso, a própria via nigro-

estriatal (onde ocorre a degeneração característica da doença causadora dos

sintomas motores) pode estar relacionada a alguns tipos de funções cognitivas

23

(Perry et al. 2004, Albouy et al. 2008, Ferreira et al. 2008). Finalmente, deve-se

ressaltar que embora as disfunções executivas sejam as mais bem estudadas em

pacientes com DP, o DSM-IV (―Diagnostic and Statistic Manual of Mental Disorders

of the American Psychiatry Association IV‖) coloca déficits de memória como

característica básica da demência associada à DP.

O declínio cognitivo mais acelerado na DP tem sido relacionado a alguns

fatores preditivos como idade avançada, ocorrência de alucinações, presença de

sintomas motores graves (Aarsland et al. 2004).

A prevalência de demência na DP possui resultados variados em estudos

encontrados na literatura científica onde métodos de avaliações distintos são usados

na população estudada. Entretando, estima-se que a demência afete

aproximadamente 40% dos pacientes com DP e a incidência nestes pacientes é de

até seis vezes maior que em pessoas saudáveis. Existe uma forte discussão sobre

alterações em outras vias neuronais envolvidas na demência da DP, pois danos na

via nigroestriatal não são suficientes para explicar o desenvolvimento da demência.

Vários sintomas da DP são constantemente associados a fatores de risco do

desenvolvimento da demência, entre eles estão: a idade avançada; idade avançada

e início de sintomas motores; início precoce de confusões mentais relacionados à

levodopa ou psicose; presença de comprometimento axial e da fala; sintomas

motores graves, em especial bradicinesia; escores em teste de cognição baixos, em

especial na fluência verbal; e depressão (Murat 2003, Aarsland et al. (2004, 2007),

Borek et al. 2006). Assim sendo, a etiologia dos déficits cognitivos associados à DP

ainda não está muito bem esclarecida.

24

1.2.4. Sistemas de neurotransmissão na Doença de Parkinson

A execução correta dos movimentos depende do circuito dos núcleos da base

que processam sinais que chegam do córtex. Assim sendo, alguns pesquisadores

desenvolveram um modelo de funcionamento dos núcleos da base a fim de

compreender melhor os mecanismos envolvidos na execução dos movimentos na

condição normal e em transtornos como a DP (DeLong & Wichmann 2007, Blandini

et al. 2000). De acordo com o modelo do circuito dos núcleos da base, a entrada do

sinal do circuito seria na substância negra pars compacta que projeta vias

dopaminérgicas para o estriado. Os neurônios estriatais expressam receptores

dopaminérgicos do tipo D1 e D2 , os quais são distintos funcionalmente. O subgrupo

de neurônios estriatais que expressa receptores D1 projeta vias GABAérgica para a

substância negra pars reticulata e para globo pálido medial, denominada via direta.

O subgrupo de neurônios estriatais que expressa receptores D2 projeta vias

Gabaérgicas para o globo pálido lateral, denominada via indireta, que envia

projeções GABAérgicas para o núcleo subtalâmico. O núcleo subtalâmico, por sua

vez, envia eferências glutamatérgicas para o globo pálido medial e globo pálido

lateral. A substância negra pars reticulata e o globo pálido medial formam um núcleo

que envia projeções Gabaérgicas (inibitórias) que atingem o tálamo motor que

projeções glutamatérgicas para o córtex motor, fechando o circuito (Blandini et al.

2000) (Ver figura 1).

O processo de neurodegeneração dopaminérgica na substância negra que

ocorre na DP resulta em uma consequente diminuição da dopamina no estriado,

desencadeando alterações secundárias, as quais contribuem para os complexos

sintomas parkinsonianos subjacentes. Foi postulado que uma perda gradativa de

25

dopamina estriatal levaria ao aumento da atividade dos núcleos de saída dos

núcleos da base. Dessa forma, este aumento da atividade dos núcleos de saída

resultaria no aumento do controle inibitório sobre o tálamo motor e subseqüente

redução das saídas glutamatérgicas para o córtex motor (Blandini et al. 2000,

Ossowska et al. 2002) (Ver figura 1).

Figura 1: Representação esquemática da organização funcional do circuito dos Núcloes da base de acordo com o modelo clássico das vias diretas e indiretas (Adaptada de Blandini et al. 2000).

Estudos de afinidade realizados em tecido cerebral de pacientes com DP

mostraram um aumento da ligação em receptores NMDA (N-metil-D-aspartato, um

subtipo de receptor glutamatérgico) no estriado. Esses pacientes apresentavam

26

características neurológicas e psiquiátricas do quadro clínico da DP (Ulas et al.

1994). Os resultados ressaltam uma possível relação de alterações das vias

glutamatérgicas que fazem parte do circuito dos núcleos da base com os complexos

sintomas parkinsonianos (Lange et al. 1997, Ulas et al. 1994).

Outra possível relação do glutamato com a DP está na hipótese de esse

transmissor desencadear um processo gradual de eliminação de células resultante

da ativação de programas de apoptose. Uma via seria através da excitotoxidade por

estimulação excessiva do receptor NMDA pelo glutamato, que causaria morte

celular. Outra via seria através da capacidade do glutamato em induzir a formação

de espécies reativas de oxigênio (oxygen reactive species, ROS) que resultariam em

danos nas células neuronais (Tan et a. 1998, Blandini et al. 2000).

Entretanto, existem controvérsias na literatura quanto a este modelo de

funcionamento do circuito dos gânglios da base, que tem sido uma proposta para

explicar aspectos dos distúrbios motores associados a alterações anatômicas e

neuroquímicas. Um estudo realizado com pacientes com sintomas parkinsonianos

avaliou a concentração de neurotransmissores (GABA e Glutamato) em 18 regiões

das vias tálamo-cortical do circuito dos gânglios da base e compararou com tecidos

das mesmas regiões de indivíduos que morreram sem históricos de distúrbios

neurológicos ou psiquiátricos. Os resultados desta pesquisa mostram uma

diminuição da concentração de GABA apenas na região centromedial do tálamo nos

pacientes com sintomas parkinsonianos (Gerlach et al. 1996).

27

1.2.5. Estudo da doença de Parkinson em modelos animais

Modelos animais vêm sendo utilizados para estudar as alterações causadas

pela DP. Alguns modelos tentam mimetizar características clínicas da DP em

roedores, a fim de compreender melhor os mecanismos subjacentes ao distúrbio

neurofisiológicos dessa doença. Contudo, há muitas controvérsias quanto aos

modelos que expressariam a natureza progressiva da DP e dos estágios ―pré-clinico‖

e ―clínico‖.

Nas últimas décadas, alguns modelos famacológicos foram criados e os mais

estudados utilizam toxinas tais como 6-hidroxidopamina (6-OHDA) e 1-metil-4-fenil-

1,2,3,6-tetrahidropiridina (MPTP). Estas duas substâncias promovem lesões

específicas de células do sistema nervoso (SN), promovendo um comprometimento

motor intenso e imediato, sem estabelecer um processo gradativo

neurodegenerativo (Meredith et al. 2008).

A neurotoxina, 6-OHDA, tem uma estrutura similar à dopamina e à

norepinefrina (NE), o que proporciona uma alta afinidade pelos transportadores de

catecolaminas das membranas. Dessa forma, essa toxina é transportada para

dentro do neurônio, onde promove reações de oxidação e produção de paraquinona

e peróxido hidrogênio, ambos com alta toxicidade. A 6-OHDA não atravessa a

barreira hematoencefálica, por isso deve ser administrada diretamente no tecido

nervoso, onde causa lesões específicas nos neurônios liberadores de dopamina e

norepinefrina (Meredith et al. 2008).

A 6-OHDA induz a morte de células neuronais dentro das primeiras 12 horas

após a administração. A depleção de dopamina estabelece-se entre o segundo e o

terceiro dia após a administração. Em geral, esta neurotoxina normalmente é

28

administrada unilateralmente, que induz o aparecimento de um comportamento

esteriotipado de rotação contralateral a lesão (Marin et al. 2007, Blandini et al. 2008),

e o hemisfério contralateral é utilizado como controle. As injeções bilaterais da 6-

OHDA são evitadas devido à alta taxa de mortalidade dos animais submetidos a

este procedimento (Ferro et al. 2005, Blandini et al. 2008), pelo menos nas doses

usuais. Outro aspecto interessante é que os efeitos funcionais induzidos pela lesão

por 6-OHDA não dependem apenas do total de doses injetadas, mas também do

local ou sub-região em que a toxina provoca morte celular.

A MPTP é uma toxina que causa sintomas motores, semelhantes à DP, por

destruir especificamente neurônios dopaminérgicos do SN. Nas últimas décadas,

sua administração tem sido usada como um modelo mais eficaz para estudar

mudanças moleculares subjacentes as disfunções mitocondriais da DP. Após a

administração de MPTP, ocorre uma perda rápida de neurônios dopaminérgicos,

apresentando-se assim os transtornos motores característicos da doença de

Parkinson (Meredith et al. 2008a). A MPTP, uma vez no tecido nervoso, é oxidada

para 1-metil-4-fenil-2,3-dihidropiridinium (MPDP+) pela monoamina oxidase B (MAO-

B). A MPDP+ é, então, convertida em MPP+ (1-metil-4-fenil-piridina, uma molécula

altamente tóxica), que pode entrar nas células dopaminérgicas por ser um substrato

com alta afinidade pelos transportadores dopaminérgicos. Dentro das células

dopaminérgicas: (1) pode ser armazenado nas vesículas sendo transportado pelos

transportadores vesiculares de monoaminas 2 (VMAT2); (2) pode ser armazenado

dentro das mitocôndrias, através de um mecanismo dependente do potencial

transmembrana mitocondrial e, dentro da mitocôndria, agir bloqueando o

componente I de transporte de elétrons que induz o aumento de espécies reativas

de oxigênio (não mostrado na Figura 2) e diminuição da síntese de ATP e (3) pode

29

permanecer no citosol celular interagindo com as enzimas (Ver figura 2) (Dauer et al.

2003).

Recentemente, alguns estudos têm proposto modelos crônicos com o MPTP

em roedores, através da infusão crônica intra-cerebral. Contudo, os estudos crônicos

com MPTP têm encontrado algumas limitações, como uma alta mortalidade dos

animais, pelo nível de toxidade da substância, e uma alta variabilidade dos sinais

que caracterizam a DP. Apesar dessas limitações esse modelo tem evidenciado

algumas vantagens, como a presença de alterações mitocondriais e a possibilidade

de avaliar processos neuroprotetores em estágios do desenvolvimento dos sinais da

DP (Sonsalla et al. 2008, Meredith et al. 2008 a).

Figura 2: Representação esquemática das vias intracelulares do MPP+ dentro das células dopaminérgicas (Adaptada de Dauer et al. 2003).

Mitocôndria Vesícula sináptica

Enzimas MPP+ bloqueia a cadeia

de transporte de elétrons.

30

Estudos em animais que expressam 5% do transportados vesicular de

monoaminas 2 (VMAT 2) tem sido proposto como um novo modelo para o estudo da

DP (Taylor et al. 2009). Os animais deficientes de VMAT 2 apresentam aumento do

estresse oxidativo, perda progressiva dos terminais de dopamina assim como

acumulação de -sinucleína (Caudle et al. 2007). Além disso, disfunções

monoaminérgicas também são encontradas, os níveis de dopamina, de

norepinefrina e de serotonina são severamente diminuídos (Caudle et al. 2007,

Taylor et al. 2009). Alterações no sono, gastrointestinais, sintomas de ansiedade e

depressão foram observados em resultados de estudos com camundongos

deficientes de VMAT 2 (Taylor et al. 2009).

Outro modelo utilizado para se estudar a DP é a administração de reserpina

em roedores, baseado nos efeitos de agentes de depleção de monoaminas sobre a

atividade motora (Colpaert 1987, Kim et al. 1999, Alves et al. 2000, Silva et al. 2002,

Skalisy et al. 2002). Tanto o modelo farmacológico da administração de reserpina

dos animais deficientes de VMAT 2 quanto o modelo resultam na disfunção do

transportados vesicular de monoaminas. 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

(Liu et al. 1996, 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). 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 neurotransmissores apenas na via nigroestriatal e nem age

exclusivamente em vias dopaminérgicas. Outro aspecto é o fato da administração

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única de reserpina não promover uma degeneração neuronal progressiva. Por outro

lado, a reserpina pode promover um aumento no estresse oxidativo celular,

possivelmente pelo aumento da metabolização da dopamina acumulada no

citoplasma pela enzima monoaminoxidase (Abílio et al. 2002). Dessa forma, a

administração repetida de doses reduzidas dessa substância poderia ser um melhor

modelo para estudar uma doença neurodegenerativa progressiva.

A maioria dos modelos farmacológicos desenvolvidos utiliza avaliações

comportamentais que são realizadas para observação de sinais motores e cognitivos

semelhantes às características clínicas da DP. As avaliações dos sinais motores são

estudadas através de: avaliações da passada (Kirik et al.1998, Chang et al. 1999),

parâmetros motores no campo aberto (distância percorrida, frequência em levantar

as patas dianteiras, latência em iniciar o movimento, tempo de imobilidade,

velocidade) (Peixoto et al. 2005, Perry et al. 2005, Reksidler et al. 2008), tempo no

comportamento de catalepsia (Namba et al. 1981, Perry et al. 2005), distância

percorrida e velocidade em labirintos (Carvalho et al. 2006), entre outros. As

avaliações dos sinais cognitivos são realizadas através de: tarefas do labirinto

aquático de Morris (Da Cunha et al. 2002, Bellissimo et al. 2004, Perry et al. 2004),

teste do medo condicionado ao contexto (Fernandes et al. 2008), teste da esquiva

discriminativa em labirinto em cruz elevado (Carvalho et al. 2006), entre outros.

As lesões induzidas pela 6-OHDA em parte do corpo estriado dorso medial

provocam alterações no comportamento motor de uma forma geral. Entretanto,

lesão na via ventrolateral dos núcleos caudado-putamen provocam alteração no

início do movimento e na orientação sensório-motora (Cousins & Salamone 1996,

Kirik et al.1998). Além disso, um estudo demonstrou que uma depleção de dopamina

32

no estriado superior a 80% induz reduções significativas da capacidade dos ratos

em ajustar os passos enquanto que redução dopaminérgica estriatal inferior a 80%

não provoca déficits detectáveis (Chang et al. 1999).

Ratos com lesão na substância negra, induzida pela MPTP, 24 horas após a

administração aguda mostram redução no número de quadrantes percorridos e na

freqüência em levantar as patas dianteiras, parâmetros avaliados no campo aberto.

Contudo, este efeito não persiste ao longo do tempo, indicando um possível

mecanismo de compensação do circuito neuronal (Perry et al. 2005). Outro trabalho

com administração repetida de MPTP demonstrou que as perdas de neurônios

dopaminérgicos na substância negra compacta foram significativas somente após a

primeira aplicação. Porém, as alterações motoras (diminuição distância percorrida e

velocidade no campo aberto e aumento do tempo no comportamento de catalepsia)

permaneceram estáveis durante o tratamento com três aplicações. Uma provável

explicação para a alteração motora não regredir está na consequente diminuição

dopaminérgica nas vias estriatais, reafirmada pela diminuição expressiva da enzima

tirosina hidroxilase, da primeira aplicação ao último dia de análises (Reksidler et al.

2008).

Alguns trabalhos demonstraram que déficits de memória ocorrem ainda na

fase inicial da DP, quando sinais motores são pouco observados (Da Cunha et al.

(2001, 2002), Bellissimo et al. 2004, Perry et al. 2004, Fernandes et al. 2008). Neste

sentido, trabalhos nos quais foram utilizado o tratamento com MPTP como modelo

animal de DP, aplicado agudamente na substância negra pars compacta, os dados

mostraram déficits na aquisição da memória e nos processos de retenção no teste

de esquiva ativa (Da Cunha et al. (2001, 2002)), assim como prejuízos na memória

espacial de ratos na tarefa do labirinto aquático de Morris (Da Cunha et al. 2002,

33

Bellissimo et al. 2004, Perry et al. 2004). Contudo, os estudos de Bellissimo et al.

(2004) e Da Cunha et al. (2002) também demonstraram a possibilidade da

participação do comprometimento motor, a negligência sensorial e/ou prejuízo da

representação espacial contralateral neste prejuízo da tarefa do labirinto aquático.

Os resultados destes estudos ainda revelaram que os ratos com lesão pelo MPTP

apresentaram perda acentuada de células dopaminérgicas da substância negra

(parte compacta), assim como uma significativa depleção da dopamina no estriado

(Da Cunha et al. ( 2001, 2002), Bellissimo et al. 2004).

Um estudo comparativo dos modelos de Parkinson, utilizando MPTP (100 µg)

e 6-OHDA (6 µg), infundidos bilateralmente na região central da substância negra de

ratos adultos, detectou que ambas as neurotoxinas causavam perdas significativas

de células marcadas pela tirosina hidroxilase, assim como levaram a depleção de

dopamina no estriado. Entretanto, este estudo demonstrou que a 6-OHDA causa

perda de células mais intensa e ampla, além de levar o animal a ter uma perda de

peso mais intensa e mortalidade mais acentuada que a do MPTP (Ferro et al. 2005).

A administração de reserpina promove sinais parkinsonianos como acinesia,

rigidez, tremores e déficits cognitivos visuoespaciais (Colpaert 1987, Johnston et al.

1999, Lindner et al. 1999, Skalisz et al. 2002, Delfino et al. 2004, Peixoto et al. 2005,

Carvalho et al. 2006, Aguiar Jr et al. 2009). Além disso, a hipocinesia induzida pela

reserpina parece estar relacionada ao decréscimo de dopamina, e um estudo

demonstrou que a L-DOPA é capaz de reverter estes efeitos de catalepsia (Namba

et al. 1981). Além de induzir uma diminuição na atividade locomotora, a reserpina

também causa concomitantemente anedonia (uma menor resposta a recompensas)

e estes sintomas estão associados à DP (Skalisz et al. 2002). Dessa forma, a

administração de reserpina constitui um modelo farmacológico de DP capaz de

34

mimetizar não somente os aspectos motores, mas também outros sintomas

presentes no desenvolvimento da patologia referida.

Carvalho et al. (2006) demonstraram que os efeitos da reserpina induzem um

prejuízo no desempenho da memória na esquiva discriminativa em labirinto em cruz

elevado, uma tarefa que associa uma estimulação aversiva com um determinado

local do labirinto. É importante ressaltar que nesse estudo (Carvalho et al. 2006),

foram utilizadas doses de reserpina menores que as usuais (0,1 a 0,5 mg/kg) e que

o comprometimento cognitivo foi observado mesmo em doses que não afetaram a

função motora. O fato de doses pequenas de reserpina induzirem déficits cognitivos,

sem alterarem a atividade motora está de acordo com a observação de que déficits

cognitivos podem preceder os sinais motores tanto em pacientes com DP (Cooper et

al. 1991, Owen et al. 1992) quanto em modelos animais (Schneider & Pope-

Coleman 1995, Carvalho et al. 2006). De fato, evidências sugerem que pequenas

perturbações na transmissão dopaminérgica levariam a déficits cognitivos, enquanto

que um alto nível de alteração nessa neurotransmissão levaria a déficits motores, os

quais poderiam até sobrepor prejuízos cognitivos pré-existentes (Schneider & Pope-

Coleman 1995, Pillon et al. 1997, Owen et al. 1998).

Recentemente, em nosso laboratório, com base no estudo de Carvalho et al.

(2006) citado acima, investigamos os efeitos da reserpina (0,1 - 0,5 mg/Kg) no

desempenho de ratos no reconhecimento de objetos, na memória operacional

espacial (alternação espontânea) e na memória emocional (condicionamento

contextual da resposta de medo). Na tarefa de reconhecimento de objetos e de

alternação espontânea os animais não foram afetados pelo tratamento com

reserpina, ao contrário do condicionamento contextual da resposta de medo, que foi

prejudicado. Associados a estudos prévios, esses resultados sugerem que uma

35

depleção moderada de monoaminas pode preferencialmente induzir déficits em

tarefas que envolvem contextos emocionais (Fernandes et al. 2008). Tomados em

conjunto, os estudos até o momento realizados sugerem que o efeito amnésico da

reserpina em ratos pode ser uma abordagem comportamental para o estudo dos

sintomas cognitivos da DP podendo estar correlacionados com disfunções nas

projeções dopaminérgicas envolvidas no controle de funções de áreas fronto-

corticais e nigroestriatais. Tais estudos prévios, entretanto, foram realizados com a

administração sistêmica (subcutânea) aguda de reserpina, de modo que seria

interessante verificar os efeitos da reserpina em um tratamento prolongado, o que

poderia mimetizar com mais fidedignidade as etapas relacionadas com déficits

cognitivos que surgiriam ao longo do processo neurodegenerativo progressivo da

DP.

1.2.6. Estresse oxidativo e doença de Parkinson

O oxigênio (O2) é uma molécula essencial para a vida dos seres vivos,

contudo, é capaz de produzir espécies altamente reativas (radicais livres)

denominado ―espécies reativas de oxigênio‖ (reactive oxygen species – ROS), que

ocorrem durante a fosforilação oxidativa mitocondrial. Exemplos de radicais livres

derivados de reações com o O2 são o superóxido (O2-), o radical hidroxila (OH-) e o

oxido nítrico (NO-). Entretanto, existem defesas naturais do organismo para proteger

contra as ROS. Normalmente, as células mantêm um controle homeostático sobre o

estado oxidativo, equilibrando a produção de ROS e das defesas antioxidantes.

36

Quando o equilíbrio é afetado, favorecendo a produção de ROS, ocorre o estresse

oxidativo, o que resulta no acúmulo de moléculas oxidativas que alteram a atividade

normal da célula (Bains & Shaw 1997, Tsang & Chung 2009). Todos os

componentes celulares são vulneráveis à ação das ROS, mas a membrana é um

dos mais atingidos em decorrência da peroxidação lipídica, que gera alterações

estruturais e na permeabilidade iônica. Além disso, a peroxidação lipídica pode ser

catalizada por íons ferro (Ferreira & Matsubara 1997, Rauhala et al. 1996). Os

neurônios são particularmente susceptíveis ao estresse oxidativo, pois podem

apresentar altas taxas de atividade metabólicas oxidativas e baixos níveis de

enzimas antioxidantes, o que pode resultar na morte celular (Bains & Shaw 1997,

Tsang & Chung 2009).

O desequilíbrio entre eventos oxidativos e as defesas antioxidantes pode

gerar estresse oxidativo que por sua vez pode induzir a morte neuronal. Dessa

forma, este desequilíbrio pode aumentar a produção de ROS e reduzir agentes

antioxidantes, como as moléculas de glutationa (GSH) (Tsang & Chung 2009).

Nesse sentido, um dos fatores propostos como mecanismo de perdas de células

nigroestriatais na DP é o estresse oxidativo neuronal (Beal 2003). Contudo, o

estresse oxidativo também é proposto como causa do processo de envelhecimento

normal (Cadenas & Davies 2000, Beal 2002), assim como em doenças

neurodegenerativas relacionadas ao envelhecimento, como a doença de Alzheimer

entre outras (Beal (2000, 2002)).

Alguns prováveis indicativos da relação entre o estresse oxidativo e a DP têm

sido relatados, como redução dos níveis de glutationa (GSH, agente óxido-redutor)

no mesencéfalo, indicando um aumento dos níveis de radicais livres, aumento de

teor de ferro na substância negra, propiciando reações de peroxidação lipídica e

37

alterações no complexo I da cadeia respiratória mitocondrial (Bains & Shaw 1997,

Tsang & Chung 2009). Além disso, o metabolismo de dopamina é uma fonte de ROS

nos neurônios nigroestriatais (Tsang & Chung 2009). O processo oxidativo da

dopamina é catalizado pela ação da enzima monoamina-oxidase (MAO). A

metabolização da dopamina produz quinonas e semi-quinonas que podem atuar

como oxidantes, sustentando a hipótese da formação da ROS (Tsang & Chung

2009). Alguns pesquisadores acreditam que o aumento da reciclagem (―turnover‖) da

dopamina está associado a eventos oxidativos na célula, através do aumento da

produção de peróxido de hidrogênio. O peróxido de hidrogênio é formado durante a

degradação da dopamina pela enzima monoamina oxidase (MAO) ou pela oxidação

do anel catecol (Ver figura 3). Um produto desta reação é o H2O2 (Ver figura 3) que

pode interagir com metais de transição (por exemplo, o ferro) e formar radicais

hidroxilas que causam danos em proteínas, lipídeos e no DNA celular. Essa reação

pode ser bloqueada por antioxidantes como a glutationa (Rabinovic & Hastings

1998). Além do H2O2 também é produzido o 3,4-dihidroxifenilacetaldeido (DOPAL)

(Ver figura 3) que em seguida passa por uma oxidação mediada pela ALDH2

(adeído desidrogenase mitocondrial) produzindo o ácido 3-4-dihidroxifenilacetico

(DOPAC) (Jinsmaa et al. 2009, Marchitti et al. 2010). Em estudos recentes foi

evidenciada a existência de enzimas que podem compensar a oxidação do DOPAL

podendo inibir o ALDH2 (Marchitti et al. 2010). Portanto, a peroxidação lipídica e os

pordutos desta que são 4HNE (4-hidroxi-2-nonenal) e MDA (malondialdeído) podem

prejudicar o catabolismo de dopamina celular via inibição do ALDH2, produzindo

nível elevados de aldeídos (Jinsmaa et al. 2009).

38

Figura 3: Formação de peróxido de hidrogênio durante a degradação da dopamina por uma reação de oxidação catalisada pela enzima monoamina oxidase. (Adaptado de Spina & Cohen 1989)

O MPTP, depois de ser convertido em MPP+ pela MAO B no cérebro, induz a

formação de radicais livres, como o radical hidroxila (OH-). A elevação do nível do

radical hidroxila leva a peroxidação lipídica que, como já foi explicado, pode levar à

morte celular. Assim sendo, alguns trabalhos têm relacionado o modelo do MPTP

como um método interessante de estudar a hipótese do estresse oxidativo e os

possíveis mecanismos patofisiológicos da DP (Obata 2002).

Alguns autores sugerem que as lesões dopaminérgicas nigroestriatais

induzidas pela 6-OHDA ocorrem pela geração de peróxido de hidrogênio e radicais

hidroxilas (Heikkila & Cohen 1971, Riobó et al. 2002).

A reserpina parece exercer um efeito sobre estes eventos oxidativos citados

acima. Resultados de estudos demonstraram que a reserpina induz uma queda dos

níveis de glutationa do estriado (Abílio et al. 2003, Teixeira et al. 2008), aumento da

peroxidação lipídica e da atividade de catalase do estriado (Abílio et al. 2002, Nade

et al. 2009) e de glutationa oxidada no estriado e no córtex pré-frontal (Spina &

Cohen 1989).

Dessa forma, podemos concluir que existem evidências dos danos do

estresse oxidativo em cérebro de pacientes com DP (Beal 2002), e em modelos

farmacológicos da doença, tais como o MPTP (Obata, 2002), a 6-OHDA (Riobó et al.

2002) e a reserpina (Bilska et al. 2007, Spina & Cohen 1989). Contudo, a relação

Dopamina

O

2

H2

O

H2O

2 NH

3

3,4-dihidroxifenilacetaldeido

(DOPAL)

+ + +

MAO

+

39

entre o estresse oxidativo e fatores relacionados à degeneração progressiva,

alterações motoras e cognitivas não estão esclarecidos.

Em face do exposto nesta introdução, propomos o estudo da administração

repetida de baixas doses de reserpina como um modelo de DP que possa abranger

as características comportamentais e bioquímicas citadas acima, em semelhança

aos sintomas observados em humanos afetados por esta patologia.

1.3. Justificativa

A maioria dos modelos utilizados para estudar os déficits cognitivos e motores

da DP é baseada em efeitos de administrações agudas de substâncias como 6-

OHDA e MPTP. Contudo, esses dois modelos acarretaram em perdas específicas e

imediatas de células do sistema nervoso (SN) além de um acentuado número de

mortes de animais, não apresentando um processo neurodegenerativo progressivo

(Ferro et al. 2005, Meredith et al. 2008).

Uma alternativa proposta nesse trabalho seria a utilização de um modelo

farmacológico crônico, administrando-se repetidamente baixas doses de reserpina,

que possibilitasse o aparecimento progressivo de sinais semelhantes aos sintomas

encontrados na DP. Estudos prévios com administração aguda de reserpina têm

mostrado alterações motoras (Skalisz et al. 2002, Aguiar Jr et al. 2009), mas

também um comprometimento cognitivo que ocorreria independentemente do

declínio motor, como quadros clínicos encontrados em humanos com DP (Carvalho

et al. 2006, Fernandes et al. 2008). Além disso, nesses estudos anteriores com

40

animais, a administração aguda subcutânea de reserpina (em doses que não afetam

a função motora) levou a alterações de memória que envolve contexto emocional

enquanto as sem conotação emocional não foram afetadas. Outro fator importante a

ser considerado é que além da dopamina, há evidências da participação de outros

sistemas de neurotransmissão, como o GABA e o glutamato na gênese dos

sintomas parkinsonianos, tanto em modelos animais quanto em humanos (Bezard et

al, 1997; Bianchi et al, 2003; Bonsi et al, 2007; DeLong e Wichmann, 2007). Existe

ainda o fator da alteração de mecanismos de neuroproteção a eventos oxidativos

induzidos pela reserpina (Bilska et al. 2007, Spina & Cohen 1989), os quais

corroboram os danos do estresse oxidativo em cérebro de pacientes com DP (Beal

2002), e em modelos farmacológicos como o MPTP (Obata 2002) e a 6-OHDA

(Riobó et al. 2002). Contudo, a relação entre o estresse oxidativo, o envolvimento de

sistemas de neurotransmissão não dopaminérgicos, fatores relacionados à

degeneração progressiva e alterações motoras e cognitivas na DP ainda não está

completamente esclarecida.

Tomados em conjunto, dados prévios sugerem que o efeito induzido pela

reserpina sobe a memória e parâmetros motores de ratos pode ser uma abordagem

adequada para o estudo dos sintomas cognitivos e motores da DP. Entretanto, até o

momento, tais estudos foram realizados apenas com a administração aguda de

reserpina. Assim sendo, propomos o estudo da administração repetida de baixas

doses de reserpina como um modelo farmacológico de DP que possa abranger as

características citadas acima, assemelhando aos sintomas observados em humanos

afetados por esta patologia.

41

1.4. Objetivo geral

No presente trabalho, propomos desenvolver um possível modelo

farmacológico em ratos que mimetize uma neurodegeneração progressiva

semelhante às encontradas em pacientes com DP, através da administração

repetida de baixas doses de reserpina.

1.4.1. Objetivos específicos

Avaliamos os efeitos da administração repetida de reserpina (em doses que

causariam pouco ou nenhum comprometimento motor agudamente) sobre:

1. O desempenho de ratos em modelos comportamentais de memória ao longo

do tratamento;

2. A atividade motora ao longo do tratamento;

3. Os níveis de GABA e glutamato em regiões cerebrais possivelmente

relacionadas com o surgimento de déficits cognitivos ou sintomas motores ao

final do tratamento;

4. Os níveis de peroxidação lipídica como indicativo de dano causado por

processos oxidativos decorrentes da administração repetida de reserpina.

42

2.Experimentos

43

2.1. Experimento I: Artigo científico que será submetido ao periódico Psychology &

Neuroscience

BEHAVIORAL AND NEUROCHEMICAL EFFECTS OF REPEATED ADMINISTRATION OF LOW DOSES OF

RESERPINE: A PROGRESSIVE MODEL FOR THE STUDY OF PARKINSON’S DISEASE?

Valéria S. Fernandes1, Anderson H.F.F. Leão1, Angela Maria Ribeiro2, Alessandra M. Ribeiro1, Regina H. Silva1,*

1Memory Studies Laboratory, Physiology Department, Federal University of Rio Grande do Norte, Natal, Brazil 2Departamento de Bioquímica e Imunologia, Laboratório de Neurociência Comportamental e Molecular – LaNeC. Universidade Federal de Minas Gerais, Brazil

44

Abstract

Parkinson's Disease (PD) has been studied in models that attempt to mimic the

neurophysiologic and behavioral changes found in the development of this disease.

However, in general, these protocols induce an immediate severe motor impairment,

similar to advanced stages of PD. The administration of reserpine (a monoamine

depletor) in rodents has been considered a model for studying PD. In this study,

repeated treatment with 0.1 and 0.2 mg/kg (but not 0.05 mg/kg) reserpine have

induced progressive motor alterations in rats when compared with the vehicle-treated

group, as shown by the evaluation of the catalepsy behavior across the treatment.

Additionaly, animals repeatedly treated with 0.1 mg/kg reserpine showed

concomitant memory impairment when tested in the plus-maze discriminative

avoidance task. At the end of the treatment (5 days after the 15th injection) striatal

GABA and gluatamate levels were determined. While no changes were observed for

the GABAergic system, a decrease in glutamate striatal concentration was found in

0.1 and 0.2 mg/kg reserpine-treated animals. Thus, repeated treatment with low

doses of reserpine appears to be promising as a model of PD, since it induces

progressive motor alterations. By the end of the treatment, these motor symptoms

were accompanied by cognitive and neurochemical changes. However, more studies

are needed to verify if the memory deficits and neurochemical alterations would

present a progressive profile as well.

Keywords:

Reserpine, Parkinson‘s Disease, cognition, GABA, glutamate, animal model

45

1. Introduction

Parkinson's disease (PD) is a progressive neurodegenerative disease in which

the ability to perform voluntary movements is gradually lost. The clinical condition of

PD includes rigidity, tremor and bradykinesia (slowness of movement) (Klockgether,

2004). However, cognitive changes can also be observed in patients with PD

(Aarsland et al., 2004; Mahieux et al., 1998; Verbaan et al., 2007).

Animal models have been used to study the changes caused by PD. Some

studies try to mimic the clinical features of PD in rodents in order to better understand

the neurophysiological mechanisms underlying the disorder (Meredith et al. 2008).

However, the the effectiveness of models regarding the progressive nature of the

"preclinical" and "clinical" stages of PD is controversial (Deumens et al., 2002).

Indeed, in recent decades some pharmacological animal models of PD have been

developed, with the most studied toxins being 6-hydroxydopamine (6-OHDA) and 1-

methyl 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP) (Meredith et al., 2008; Schober,

2004). These two models have shown specific loss of cells related to PD in the

central nervous system (CNS), although not presenting a neurodegenerative

process, instating already an advanced stage of the disease upon administration

(Meredith et al., 2008).

The administration of reserpine (an irreversible blocker of monoamine

vesicular carrier) in rodents has been considered an animal model for the study of

PD (Colpaert, 1987; Kim et al., 1999; Alves et al., 2000; Silva et al., 2002; Skalisy et

al., 2002). Reserpine interferes with the storage of monoamines in intracellular

vesicles, causing monoamine depletion in nerve terminals and transient

hypolocomotion and muscular rigidity, depending on the dose (Colpaert, 1987).

46

Recently, lower doses of reserpine have also been found to promote a memory

deficit in an aversive discrimination task without any effects on motor activity,

suggesting that the administration of this drug in low doses can be useful to study

memory deficits found in PD (Carvalho et al., 2006). Similar results were found in a

different aversively motivated behavioral model, the contextual fear conditioning

(Fernandes et al., 2008). In summary, the data from the literature suggest that acute

reserpine is able to induce motor and cognitive alterations similar to those found in

PD patients, although in different dose ranges.

Although hypofunction of the dopaminergic nigrostriatal system is considered

to be the core of the physiopathology of PD, there is evidence that other

neurotransmitter systems are involved in the symptoms of the disease (Bezard et al.,

1997; Bianchi et al., 2003; Bonsi et al., 2007; DeLong & Wichmann, 2007). Studies

have suggested that the depletion of dopamine (DA) in the striatum consequently

leads to inhibition of the GABAergic striato-pallidal projections, as well as changes in

thalamo-nigral glutamatergic projections (Filloux & Townsend, 1993; DeLong &

Wichmann, 2007). Indeed, a study with rats treated with 6-OHDA showed that loss of

dopaminergic neurons in the forebrain induce increase on the GABA levels in

pallidum globe (Bianchi et al., 2003). Furthermore, studies with glutamatergic drugs

(in particular, group II mGluR agonists) show improvement of motor signs in mice

treated with MPTP (Bonsi et al., 2007). However, these studies in animals were

performed with acute pharmacological models, and have shown controversial results.

Further, studies with brain tissue from PD patients show altered levels of GABA in the

medial center thalamus (Gerlach et al., 1996). These studies indicate that the

relationship between dopaminergic, glutamatergic, GABAergic and behavioral

changes is somewhat complex, and still unclear.

47

Considering the importance of an animal model that simulates the progressive

nature of the disease, we evaluated the effects of a repeated treatment with low sub-

effective doses of reserpine on motor and cognitive behaviors. In addition, we also

addressed possible changes in GABAergic and glutamatergic systems as a

consequence of this treatment.

2. Methods

2.1. Subjects

Five-month old male Wistar rats (n= 29) were used. All animals were

maintained in groups of four or five per cage, under a 12 h light 12 h dark cycle and

at a constant temperature of 25 1 C, with food and water available ad libitum. The

rats were handled according to Brazilian law procedures for the use of animals in

scientific research (Law Number 11.794) and all procedures were approved by the

local research ethics committee (final opinion number 149/2008). All efforts were

made to minimize animal pain, suffering or discomfort, and to minimize the number of

rats used.

2.2. Drug treatment, general procedures and experimental design

Reserpine (methyl reserpate 3,4,5-trimetothoxycinnamic acid ester: Sigma

Chemical Co. St. Louis, MO) was dissolved in glacial acetic acid and diluted to the

correct concentration in distilled water. Vehicle consisted of the same amount of

48

acetic acid and water as in the reserpine solution. These solutions were injected

subcutaneously (s.c.).

Rats received 15 s.c. injections of vehicle (VEH; n=8), 0.05 mg/Kg (RES 0.05;

n=7), 0.1 mg/Kg (RES 0.1; n=7) or 0.2 mg/kg (RES 0.2; n=7) of reserpine, at a

volume of 1 ml/kg body weight, on alternate days. During treatment rats went through

the following procedures: (1) assessment of catalepsy behavior 24h after the 3rd, 6th,

9th, 12th and 15th injections; (2) plus-maze discriminative avoidance task 24h and 48 h

after the 10th injection; (3) assessment of orofacial movements 24 h after the 14th

injection; (4) Contextual fear conditioning 48 and 72 h after the 15th injection; (5)

evaluation of GABAergic and glutamatergic parameters in the striatum 5 days after

the 15th injection (Figure 1).

Figure 1: Experimental design

Every rat was submitted to 10 min of gentle handling once a day for five days

before the beginning of the experimental procedures. The analyses of catalepsy

49

behavior and orofacial movements were performed by direct observation (by

researchers blind to the treatment). All other behavioral sessions were recorded by a

camera placed above the apparatus and the behavioral parameters were registered

by an animal video-tracking software (Any maze Stoelting, USA).

All apparatus were washed with a water–alcohol (5%) solution before

behavioral testing to eliminate possible bias due to odors left by previous subjects.

2.3. Apparatus

2.3.1. Catalepsy Test:

The catalepsy was assessed placing the animal‘s front paws on a horizontal

bar positioned at 9 cm above the bench surface. The duration of catalepsy, which

was defined as an immobile posture, keeping the two front paws on the bar, was

measured within a maximum of 180 s.

2.3.2. Plus-maze discriminative avoidance task:

The apparatus employed was a modified elevated plus-maze made of wood

containing two enclosed arms (50 X 15 X 40 cm) opposite to two open arms (50 X 15

cm). A 100-watt lamp was placed over the middle of one of the enclosed arms

(aversive enclosed arm). In the training session, each rat was placed in the centre of

the apparatus and, over a period of 10 min, every time the animal entered the

enclosed arm containing the lamp, an aversive situation was produced until the

animal left the arm. The aversive stimuli were the 100-watt light and an 80 dB noise

50

applied through a speaker placed over the aversive enclosed arm. In the test

session, held 24h later, the rats were again placed in the apparatus for 10 min,

without receiving the aversive stimulation, with the lamp and the speaker still present

over the aversive arm, but turned off. Distance traveled in the apparatus (used for

motor activity evaluation) and time spent in each arm (aversive, non-aversive and

open arms) were registered. Percent time in aversive arm (time spent in aversive

enclosed arm/time spent in both enclosed arms) and percent time spent in open

arms (time spent in open arms/time spent in both open and enclosed arms)

considering the whole duration of behavioral sessions were used to evaluate memory

and anxiety, respectively (Silva et al., 2000). Percent time spent in the aversive

enclosed arm assessed minute by minute across the training and test sessions were

used to evaluate learning and extinction of the task, respectively (Ribeiro et al.,

2010).

2.3.3. Orofacial movements assessment:

Rats were placed individually in wired cages (29 cm × 24 cm × 21 cm) with

mirrors positioned under the floor and behind the back wall of the cage to allow

behavioral quantification when the animal faced away from the observer. The number

of tongue protrusions (projection of the tongue out of the oral cavity), vacuous

chewing movement frequency (mouth openings in the vertical plane not directed

toward physical material), and facial twitching (duration (in seconds) of twitching of

the facial musculature) were measured continuously for 15 min.

51

2.4. Biochemical analysis: Evaluation of GABA and glutamate levels:

After the animals were sacrificed by decapitation, the brains were quickly

removed from the cranial cavity, weighed and dissected according to the stereotactic

coordinates provided by Paxinos & Watson (Paxinos & Watson 1997). The sample of

striatum was then stored at -80 ° C to achieve the biochemical assays.

Samples of striatum were weighed and homogenized in 15 volumes of

methanol: water (85:15 v / v) in automatic homogenizing. Then the homogenate was

centrifuged at 4 ° C for 15 minutes at 7800x g (Sorvall RC-5B). The supernatant

obtained after centrifugation was collected and kept on ice until subjected to

derivatization.

Due to the absence of electroactive or fluorescent characteristics inherent in

the amino acid glutamate and GABA, several works have used the technique of pre-

column derivatization for the chromatographic separation and identification of these

compounds. One of the most widely used derivatising agents is o-phthalaldehyde

(OPA), which reacts with primary amines in the presence of thiol and generates

electroactive and fluorescent derivatives (Freitas et al. 2009). The derivatization

reaction was made by mixing 100 mL of sample, 20 mL of methanolic OPA (5 mg /

mL) prepared daily, 75 mL of borate buffer (pH 9.9) and 5 mL of 3-

mercaptopropiônico acid (MPA). The resulting solution was stirred and injected into

the chromatographic system after 1 minute at room temperature.

The chromatographic system used to determine of GABA and glutamate

consisted of a Shimadzu chromatograph (LC-10AD, Tokyo, Japan) with 200 mL

injector valve (Rheodyne 7725-I, California, USA) and fluorescence detector (FLD-

Shimadzu spectrofluorometric detector RF-551, Tokyo, Japan) coupled to a pump

52

LC-10. The wavelengths of excitation and emission used were 337 nm and 454 nm,

respectively. A reversed phase chromatographic column C18 (150 mm x 4.6 mm ID)

and a guard column (E. Merck RT 250-4, ER Darmsdt, Germany) were used in the

analysis. The isocratic mobile phase consisted of a 0.05 M solution of sodium

acetate, tetrahydrofuran and methanol (50:1:49 v / v), pH 4.0. The mean elution of

GABA and glutamate is 8.0 to 3.0 minutes, respectively, the concentrations in µg / g

of tissue were calculated using peak areas and their standard curves which was

provided by an integrator (R7Ae Shimadzu C-plus) coupled to the chromatographic

system (Freitas et al., 2009).

2.5. Statistics

All data were tested for homogeneity of variances (Levene's test) and

normality (Kolmogorov-Smirnov test) and parametric tests were performed for all

data. Data on the percentage of time spent in the aversive arm (measured every

minute, in training and test sessions) and catalepsy behavior across the treatment

(24h after the 3rd, 6th, 9th, 12th and 15th injections) were analyzed by analysis of

variance (ANOVA) with repeated measures. For catalepsy behavior analysis,

between-subject comparisons were held in each timepoint with one-way ANOVA with

sequential Bonferroni‘s post hoc. Other data were analyzed by one-way ANOVA

followed by Duncan's test for post hoc analysis. The results were expressed as

means ± SE. The level of significance in all tests was p <0.05.

53

3. Results

3.1- Effects of repeated administration of low doses of reserpine on the

catalepsy behavior:

Catelpsy behavior results are shown in figure 2. ANOVA with repeated

measures revealed time (quantity of injections) [F(5, 125)=22.36; p=0.000],

treatment (reserpine (0.05, 0.1 or 0.2 mg/Kg) or vehicle) [F(3, 25)=16.92; p=0.000]

and time X treatment interaction [F(5, 125)=7.54; p=0.000] effects. Analyzing the

data of each observation, we found no significant effects of treatment until the

observation held after the 6th injection, although treatment with 0,2 mg/kg reserpine

induced a marginally significant effect after six injections (p=0.068 compared to

vehicle). Significant effects of treatment were observed after the 9th, 12th and 15th

subcutaneous injections [F (3, 25) = 8.32, p = 0.001; F (3, 25) = 11.22, p = 0.000; F

(3, 25) = 8.28, p = 0.000, respectively]. The Bonferroni‘s post hoc analysis showed

that RES 0.1 and RES 0.2 groups presented increased immobility time in the bar

when compared to the VEH and RES 0.05 groups after the 9th, 12th and 15th

injections.

54

Fig. 2: Effects of repeated administration of low doses of reserpine (RES- 0.05, 0.1 or

0.2 mg/Kg) or vehicle (VEH) on catalepsy behavior before the beginning (basal) and

24h hours after the 3rd, 6th, 9th, 12th and 15th injections. Data are expressed as

mean±S.E.M. (s). ANOVA with repeated measures revealed time, treatment and time

x treatment interaction effects. *p<0.05 compared to VEH and RES 0.05 group

(ANOVA and Bonferroni‘s test).

3.2- Effects of repeated administration of low doses of reserpine on plus-maze

discriminative avoidance task

In the training session, we observed a significant effect of treatment in the

distance traveled in the apparatus (Figure 3 A; ANOVA, F (3, 25) = 15.98, p = 0.000).

The post hoc analysis (Duncan‘s test) revealed that RES 0.1 and 0.2 groups showed

decreased motor activity when compared to groups VEH and RES 0.05 in the

training session (24 h after administration of the 10th injection). In the test session, 48

0

20

40

60

80

100

0 3rd 6th 9th 12th 15th

Ca

tale

ps

y d

ura

tio

n (

s)

VEH RES 0.05 RES 0.1 RES 0.2

55

h after administration of the 10th injection, there was no significant difference between

groups in the distance traveled (Fig. 3 B; ANOVA, F (3, 25) = 0.51, p = 0.681).

Fig. 3: Effects of repeated administration of low doses of reserpine (RES - 0.05, 0.1

or 0.2 mg/Kg) or vehicle (VEH) on the plus-maze discriminative avoidance apparatus

during training (A) and test (B) sessions performed 24 and 48 h after the 10th

injection, respectively. * p <0.05 compared with vehicle and RES 0.05 group (ANOVA

and Duncan‘s test)

In the training session (24h after the 10th injection), we found no significant

differences in the percentage of time in the open arms (%TO) (Figure 4 A; ANOVA, F

(3, 25) = 0.39, p = 0.761). However, in the test (48h after the 10th injection), a

significant effect of treatment was found for %TO. The post hoc analysis (Duncan

test) revealed that RES 0.2 group showed a significant increase in the percentage of

time in open arms in the test session when compared with all other groups (Fig. 4 B,

ANOVA, F (3, 25) = 8.99, p = 0.000).

56


or 0.2 mg/Kg) or vehicle (VEH) on percent time in the open arms (%TO) of the plus-

maze discriminative avoidance apparatus during training (A) and test (B) sessions,

performed 24 and 48 h after the 10th injection, respectively. * p <0.05 compared with

all other groups (ANOVA and Duncan‘s test)

Regarding the percent time in the aversive arm, we found no significant effects

when the whole training session duration was considered for analysis (Figure 5 A,

ANOVA, F (3, 25) = 1.99, p = 0. 142). When the same analysis was applied to the

test session, we found a significant effect of treatment for percentage of time in the

aversive arm. The post hoc analysis (Duncan test) showed that RES 0.1 group

showed increased aversive arm exploration when compared with groups VEH and

RES 0.05 (Figure 5 B, ANOVA, F (3, 25) = 3.25, p = 0. 039).

In the training session, significant effects of time (minutes) [F (9, 225)= 8.41;

p=0.000] and the time X treatment interaction [F (9, 225)= 2.38; p=0.019] were found

when the percentage of time in the aversive enclosed arm (%TAV) was evaluated

across the session. No effect of the treatment (RES (0.05, 0.1 or 0.2 mg/Kg) or VEH)

[F (3, 25)= 2.75; p=0.064] was found (see Fig 5 C).

57

In the test session, a significant effect of the time X treatment interaction [F (9,

225)= 2.04; p=0.029] was found when the percentage of time in the aversive

enclosed arm (%TAV) was evaluated across the session. No effects of time (minutes)

[F (9,225)= 1.38; p=0.246] or treatment (RES (0.05, 0.1 or 0.2 mg/Kg) or VEH) [F

(3,25)= 0.96; p=0.429] were found (see Fig 5 D).

58


or 0.2 mg/Kg) or vehicle (VEH) on the percent time spent in the aversive arm (%TAV)

of the discriminative avoidance apparatus. Data are expressed as the mean±S.E.M.

for the whole training (A) and test (B) sessions and minute by minute during training

59

(C) and test (D) sessions, performed 24 and 48 h after the 10th injection, respectively.

ANOVA with repeated measures revealed time (minutes) effect, in training session,

and time x treatment interaction effects, in training and test sessions (C and D)* p

<0.05 compared with vehicle and RES 0.05 group (ANOVA and Duncan‘s test)

3.3- Effects of repeated administration of low doses of reserpine on orofacial

movements

The analysis of the orofacial movements, held 24 h after the 14th injection,

showed significant differences between groups for the number of vacuous chewing

movements (Figure 6 A, F (3, 25) = 60.46, p = 0.000), the duration of facial twitching

(Figure 6 B, F (3, 25) = 40.99, p = 0.000) and the number of tongue protrusions

(Figure 6 C, F (3, 25) = 13.34, p = 0.000). The post hoc analysis (Duncan‘s test)

showed that, in all orofacial movement measures, RES 0.1 and RES 0.2 groups

showed significantly increased values when compared with the VEH and RES 0.05

groups. In addition, RES 0.2 group presented increased duration of facial twitching

when compared to RES 0.1 animals.

60

61

Figure 6: Effects of repeated administration of low doses of reserpine (RES - 0.05,

0.1 or 0.2 mg/Kg) or vehicle (VEH) on orofacial movements 24 h after the 14th

subcutaneous injection. Data are expressed as the mean±S.E.M. of the number of

vacuous chewing movements (A), the duration of facial twitching (B) and the number

of tongue protrusions (C). * p <0.05 compared with vehicle group and RES 0.05

group, # p <0.05 compared with RES 0.1 group (ANOVA and Duncan‘s test).

3.4- Effects of repeated administration of low doses of reserpine on GABA and

glutamate striatal levels

Significant differences were observed between groups for concentrations (µg/g)

of glutamate in the striatum, 5 days after the administration of 15 subcutaneous

injections (Figure 7 A; ANOVA, F (3, 25) = 4.72, p = 0.010). The post hoc analysis

(Duncan test) showed that reserpine RES 0.1 and 0.2 groups showed decreased

glutamate striatal levels when compared with the VEH and RES 0.05 groups.

However, we found no significant difference between groups of concentrations in

µg/g of GABA was observed (Figure 7 B, ANOVA, F (3, 25) = 0.88, p = 0.466) in the

striatum, after the administration of 15 subcutaneous injections.

62

Figure 7: Effects of repeated administration of low doses of reserpine (RES - 0.05,

0.1 or 0.2 mg/Kg) or vehicle (VEH) on the concentration of glutamate or GABA in

striatum 5 days after the 15th injection. Data are expressed as the mean±S.E.M. of

the concentrations of glutamate (A) and GABA (B) present in striatum samples. *p

<0.05 compared with vehicle and RES 0.05 groups (ANOVA and Duncan‘s test).

63

4. Discussion

This study evaluated the effect of repeated treatment with low doses of

reserpine on motor and cognitive parameters. The results revealed that repeated

treatment with 0.05 mg/kg of reserpine did not change the performance of cognitive

and motor tasks or in the concentrations of glutamate and GABA in the striatum. On

the other hand, the results obtained by repeated treatment with higher doses of

reserpine (RES 0.1 and 0.2 mg / kg) showed changes in the performance of animals

on contextual fear conditioning and plus-maze discriminative avoidance behavior.

More importantly, repeated treatment with 0.1 and 0.2 mg/kg of reserpine showed a

progressive onset of motor alterations, as shown by increased on catalepsy behavior

from the 9th injection onwards (Fig. 2), decreased distance traveled in the

discriminative avoidance apparatus 24 h after the 10th injection (Fig. 3A) and

increase in orofacial movements 24 h after the 14th injection (Fig 6 A, B and C). We

also found a significant decrease of striatal glutamate in levels in both groups RES

0.1 and 0.2 five days after the 15th injection (Fig. 7A).

The motor symptoms are characteristic of the clinical presentation of PD and

are associated with the neurodegenerative process of the basal ganglia (Klockgether,

2004; Johnston et al., 1999; Lindner et al., 1999; Mayeux, 2003, Ridley et al., 2006).

Studies in rodents with drugs that change the dopaminergic system (haloperidol,

MPTP, 6-OHDA, reserpine) induces the appearance of signs of hypokinesia and

rigidity (catalepsy) similar to Parkinsonian symptoms (Shiozaki et al., 1999; Díaz et

al., 2001; Góngora-Alfaro et al., 2009; Corona et al., 2010). Additionaly, the presence

of hypokinesia can be addressed by the evaluation of locomotor activity after these

pharmacological treatments (Carvalho et al., 2006; Díaz et al., 2001; Capitelli et al.,

2008). As mentioned above, the present study revealed that rats exposed to a

64

repeated administration of 0.1 and 0.2 mg/kg reserpine showed both kinds of

alterations (increased catalepsy behavior - Fig. 2; decreased distance traveled in the

plus-maze discriminative avoidance apparatus- Fig. 3A). Previous studies have

demonstrated these effects of reserpine on catalepsy behavior and locomotor

activity. However, in these studies, one or two injections of considerably higher doses

(1.0 or 5.0 mg / kg) were administred (Shiozaki et al., 1999; Dutra et al., 2002) to

produce catalepsy and hipolocomotion. Therefore, the results found in the present

work suggest that the alterations observed during treatment could indicate

progressive features of those signs.

The evaluation of orofacial movements has been used in animal studies of

tardive dyskinesia, using reserpine to induce the appearance of oral dyskinesia

(Neisewander et al., 1991; Neisewander et al., 1994; Abílio et al., 2003; Abílio et al.,

2004). The tardive dyskinesia is characterized by severe motor symptoms where the

face, mouth and tongue are often involved (called orofacial dyskinesia) and can

manifest as a side effect of neuroleptics both in humans and in animals (Hansen et

al., 1997; Andreassen et al., 2000). On the other hand, some researchers apply the

evaluation of orofacial movements as a model of parkinsonism, especially of tremor-

related symptoms (Salamone et al., 1996; Salamone et al., 2008). In this study, we

observed that after administration of the 14th injection, groups RES 0.1 and 0.2 (but

not RES 0.05) showed an increase in orofacial movements (Fig.6 A, B and C).

Studies have shown that changes in the ventrolateral striatal dopamine system (Jicha

et al., 1991) or changes in striatal levels of oxidative stress (Abílio et al., 2003; Abílio

et al., 2004) may be related to the increase of orofacial movements. Both changes in

striatal dopaminergic system as changes in levels of oxidative stress in brain are

factors related to PD (Nieoullon et al., 2002; Beal, 2003). In addition, there is

65

evidence that orofacial movements alterations co-exist with other kinds of motor

changes, including parkinsonism (Harten et al., 1997). On the other hand, a study of

specific dopamine lesions in the ventrolateral striatum in rats showed oral motor

disorders in the absence of locomotor deficits (Jicha et al., 1991). Thus, different

changes in the dopaminergic system can cause various motor signs showing that it is

unclear which is the neurochemical mechanism underlying the simultaneous onset of

orofacial dyskinesia and other motor alterations.

Besides evaluating memory, we used the plus maze discriminative avoidance

task (performed after the 10th injection) to assess learning, memory, anxiety and

motor behavior, since it has been shown that these evaluations can be performed

concomitantly, and by different parameters, in this paradigm (Silva et al., 2002; Silva

et al., 2000). In the training session, we observed that RES 0.1 and 0.2 groups

showed a decrease in distance traveled in the maze (Fig. 3A). The possibility that

this motor impairment could have interfered with the acquisition of the task should be

considered, since the animals have to enter the aversive arm during training to learn

the assossiation with the aversive stimuli. However, the RES 0.1 animals did

explored the aversive enclosed arm in the first three minutes of the session,

gradually avoing this arm across the training, indicating they have learned the task

(as shown by the evaluation of behavior minute by minute throughout the training

session in Fig 5C). On the other hand, the animals from RES 0.2 group almost did

not enter the aversive arm during the training session. Thus, these results suggest

that the RES 0.2 group had an impairment in the acquisition of the task due to the

motor deficit.

The evaluation of the data obtained in the test session has shown two altered

parameters as a consequence of reserpine repeated treatment. First, animals treated

66

with 0.2 mg/kg reserpine presented increased percent time in the open arms (Fig

4B). This result would usually indicate changes in anxiety-like behavior. However, the

severe motor impairment presented by this group at this point of the treatment could

be the reason of this increase. Indeed, the observation of individual data of this

parameter (data not shown) revealed that most animals minimally advanced from the

central platform to one of the open arms and remained there. The other alteration

induced by reserpine treatment was the increase in percent time in the aversive

enclosed arm, specifically at 0.1 mg/kg (Fig. 5C). In this respect, we found that these

animals showed motor deficits during the acquisition of the task, which could

jeopardize an interpretation related to the cognitive aspect of animal behavior in the

test session. However, as mentioned before, the animals in this group did learn the

task, so the increased aversive arm exploration in the test session is probably

reflects a retrieval deficit. Nevertheless, it would be interesting to verify if reserpine

would induce cognitive impairments earlier during treatment, when the motor

impairment would be absent or mild.

One of the possible consequences of the neurodegeneration of the

nigrostriatal pathway in PD would be the modulation in the neural circuitry of the

basal ganglia, through alterations of the GABAergic and glutamatergic systems

(Blandini et al., 2000; Ossowska et al., 2002). Thus, we investigated the levels of

glutamate and GABA in the striatum region and found a significant decrease in the

concentration of glutamate groups RES 0.1 and 0.2. This result corroborates the

work of Day et al (2006) that showed that in a period of 5 days after initiation of

dopamine depletion, there were significant losses striatopallidal glutamatergic

synapses. Moreover, the loss of glutamatergic synapses after depletion of dopamine

has already been described in previous studies in animal models and in patients with

67

Parkinson's disease (McNeill et al., 1988; Ingham et al., 1998; Dunah et al., 2000).

On the other hand, no alterations were found in GABA striatal levels after treatment,

corroborating reports that show absence of alterations in the striatal GABAergic

system in humans with PD (Gerlach et al., 1996).

In summary, we observed motor alterations during repeated treatment with 0.1

and 0.2 mg/kg reserpine consistent with a progressive development of motor

impairment. The RES 0.1 group showed a concomitant cognitive impairment,

although the possible progressive nature of this alteration remains to be investigated.

Finally, motor changes (observed in tests of catalepsy) were accompanied by a

decreased level of glutamate (but not GABA) in the striatum, which is consistent with

neurochemical changes previously shown in animal models and in patients with PD

(see above). Thus, although further investigation is undoubtedly necessary, repeated

treatment with low doses of reserpine appears to be a promising animal model to

study the progressive motor changes of PD.

5. Acknowledgments

The authors would like to thank Ana Raquel Borges Pereira Caixeta, Fabio

Antonio Vigil and Patricia da Silva Oliveira (LaNeC/UFMG) for methodological

assistance and Dra. Vanessa C. Abílio (LiNC/UFSP) for helpful suggestions. This

research was supported by fellowships from Conselho Nacional de Desenvolvimento

Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal

de Nível Superior (CAPES).

68

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2.2. Experimento II: Artigo científico que será submetido ao periódico Progress in

Neuropsychopharmacology & Biological Psychiatry

Repeated treatment with reserpine as a possible progressive model of

Parkinson’s disease

Valéria S. Fernandes1, José R. Santos1, Thieza G. Melo1, Anderson H.F.F. Leão1,

André M. Medeiros1, Geison S. Izídio1, Alicia Cabral1, Rosana A. Ribeiro2, Vanessa

C. Abílio2,3, Alessandra M. Ribeiro1, Regina H. Silva1,*

1Memory Studies Laboratory, Physiology Department, Federal University of Rio

Grande do Norte, Natal, Brazil

2Department of Pharmacology, Universidade Federal de São Paulo, São Paulo,

Brazil.

3Laboratório Interdisciplinar de Neurociência Clínica (LiNC), Department of

Psychiatry, Universidade Federal de São Paulo, São Paulo, Brazil.

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ABSTRACT

Animal models are widely used to study alterations caused by the Parkinson‘s

Disease (PD). However, in general, pharmacological models do not express the

progressive nature of the disease, causing immediate severe motor impairment after

acute administration. Reserpine administration in rodents has been suggested as a

pharmacological model of PD based on the effects of this monoamine-depleting

agent on motor activity. We found that repeated administration with a low dose (0.1

mg/Kg) of reserpine in rats induces a gradual appearance of motor signs.

Furthermore, these motor signs were accompanied by increased levels of striatal lipid

peroxidation. However, treatment with reserpine was unable to induce cognitive

abnormalities and alterations in hippocampal lipid peroxidation. Thus, repeated

treatment with low alternated doses of reserpine progressively induced alterations in

motor function, indicating a possible application of this model in the study of the

progressive nature the motor signs in PD.

Keywords: reserpine, Parkinson‘s Disease, cognition, movement disorders,

oxidative stress, animal model

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1- Introduction

Parkinson‘s disease (PD) is a progressive neurodegenerative disorder,

characterized by bradykinesia, tremor, rigidity and postural abnormalities

(Klockgether, 2004). However, cognitive impairments can also be observed in PD

patients (Mahieux et a., 1998; Aarsland et al., 2004; Verbaan et al., 2007). The

pattern of cognitive disturbances associated with PD includes learning impairments

(Schmitt-Eliassen et al., 2007), deficits of executive functions such as planning or

working memory (Morris et al.,1988; Cools et al., 2002; Cox et al., 2002) and

attentional deficits (Bronnick et al., 2006).

Animal models are extensively used to study alterations caused by the PD

(Beal, 2001). However, in general, pharmacological models do not express the

progressive nature of the disease, causing immediate severe motor impairment with

a single administration (Da Cunha et al., 2002; Bellissimo et al., 2004; Henderson et

al., 2005).

The administration of reserpine to rodents has been suggested as a

pharmacological model of PD based on the effects of this monoamine-depleting

agent on motor activity. Reserpine interferes with the storage of monoamines in

intracellular vesicles, causing monoamine depletion in nerve terminals and transient

hypolocomotion and muscular rigidity, depending on the dose (Colpaert 1987;

Gerlach and Riederer, 1996). The range dose usually used to induce such motor

alterations in rodents is 1 to 5 mg/kg, and the severe motor impairment prevents

other kinds of behavioral evaluations, such as memory tests and other

cognitive/emotional assessments. However, previous results from our group

(Fernandes et al., 2008) have shown that a single administration of reserpine in low

doses (0.1-0.5 mg/kg) can induce deficits in emotional memory without causing motor

alterations. Carvalho et al. (2006) obtained similar results, in a different behavioral

model. These findings corroborate studies with PD patients showing deficits in

emotional processing while motor deficits were absent (Schneider et al., 2003;

Salgado-Pineda et al., 2005; Bowers et al., 2006). Therefore, the previous studies

suggest that depending on the dose, reserpine is able to induce changes in rodents

similar to the symptoms found in humans with PD.

The reserpine is an irreversible inhibitor of the vesicular monoamine

transporter 2 (VMAT2). The blockage of dopamine vesicular uptake results in the

75

accumulation of neurotoxic dopamine oxidation byproducts (Caudle et al., 2008). For

instance, dopamine (DA) reacts with molecular oxygen to form dopamine-quinones

which can deplete glutathione, generating reactive oxygen species (ROS) during this

process (Tsang and Chung, 2009). When the production of ROS exceeds the ability

of the antioxidant system to eliminate them, oxidative damage occurs (Cadenas and

Davies, 2000). These neuronal damages caused by oxidative stress can induce

alterations in both motor (Faria et al., 2005; Teixeira et al., 2009) and cognitive skills

(Chen et al., 2010).

The brain is particularly sensitive to oxidative damage when compared to other

organs or systems, mainly because it contains high levels of membrane lipids,

excitotoxic amino acids, low levels of antioxidant defenses and autoxidizable

neurotransmitters (Cadenas and Davies, 2000).Therefore, it is quite understandable

that many studies have been performed to investigate the role of oxidative injury in

neurodegenerative diseases including PD (Cadenas and Davies, 2000; Beal, 2003).

In fact, evidences of oxidative stress damage are found in both brain tissue from PD

patients (Beal, 2002) and in pharmacological models such as the 1-methyl 4-phenyl

1,2,3,6-tetrahydropyridine (MPTP) (Obata, 2002), 6-hydroxydopamine (6-OHDA)

(Riobó et al., 2002) and reserpine (Spina and Cohen, 1989; Bilska et al., 2007)

models. However, the relationship among oxidative stress, PD‘s progressive

degeneration, motor and cognitive deficits remains unclear.

Considering the importance of PD, the progressive nature of the disease and

the possible relationship with oxidative stress, we evaluate the repeated

administration of reserpine as a possible pharmacological model with progressive

effects similar to those in patients with PD. Therefore we submit Wistar rats to a

repeated treatment with a sub-effective dose of reserpine and evaluate motor

behavior and memory performance. Furthermore, we evaluated oxidative stress in

the striatum and hippocampus by measuring lipid peroxidation.

2- Materials and methods

2.1- Subjects

Five-month old male Wistar rats (n= 74) were used. All animals were

maintained in groups of four or five per cage, under a 12 h light 12 h dark cycle and

76

at a constant temperature of 25 1 C, with food and water available ad libitum. The

rats were handled according to Brazilian law procedures for the use of animals in

scientific research (Law Number 11.794) and all procedures were approved by the

local research ethics committee (final opinion number 149/2008). All efforts were

made to minimize animal pain, suffering or discomfort, and to minimize the number of

rats used.

2.2- Drug treatment, general procedures and experimental design

Reserpine (methyl reserpate 3,4,5-trimetothoxycinnamic acid ester: Sigma

Chemical Co. St. Louis, MO) was dissolved in glacial acetic acid and diluted to the

correct concentration in distilled water. Vehicle consisted of the same amount of

acetic acid and water as in the reserpine solution. These solutions were injected

subcutaneously (s.c.).

Rats received 7-10 subcutaneous injections of vehicle (VEH) or 0.1 mg / kg of

reserpine (RES), at a volume of 1 ml/kg body weight, on alternate days. During

treatment rats went through the following procedures: (1) catalepsy test two days

before the beginning, and every day throughout the treatment, i.e., 24 h and 48 h

after each injection (n=13 (VEH); n=12 (RES)); (2) quantification of open field

behavior 24 h after the 4th injection (n=17 per group); (3) assessment of orofacial

movements before starting the treatment, 24 h after the 5th and 10th injections and 48

h after the 10th injection (n=8 per group); (4) training and test sessions of novel object

recognition task, 24 h and 48 h after the 5th injection, respectively (n= 7 per group);

(5) training and test sessions of plus-maze discriminative avoidance task, 24h and 48

h after the 7th injection, respectively (n=23 per group); (6) body weighing 24 h before

1st, 3rd, 5th and 7th injections, (7) quantification of striatal and hippocampal lipid

peroxidation 48 h after the 7th (n=10 per group) and 10th injections (n = 12 per group)

(Figure 1).

77

Fig. 1. Outline: experimental design

Every rat was submitted to 10 min of gentle handling once a day for five days

before the beginning of the experimental procedures. The behavioral analysis of the

catalepsy test and orofacial movements assessment were performed manually by

direct observation. All other behavioral sessions were recorded by a camera placed

above the apparatus and the behavioral parameters were registered by an animal

video-tracking software (Anymaze, Stoelting, USA).

During behavioral sessions, all apparatuses were washed with a water–

alcohol (5%) solution before behavioral testing to eliminate possible bias due to

odors left by previous subjects.

During treatment rats went through the following procedures:

2.3- Behavioral testing

2.3.1- Catalepsy Test

The catalepsy was assessed placing the animal‘s front paws on a horizontal

bar positioned at 9 cm above the bench surface. The duration of catalepsy, which

78

was defined as an immobile posture, keeping the two front paws on the bar, was

measured with a maximum of 180 s. Three trials were carried out for each animal in

each observation day and the results were analyzed considering the mean value of

the three trials.

2.3.2- Open field

The apparatus, made of wood and painted in black, was a circular open-field

arena (84 cm in diameter) with 32 cm high walls. We quantified the distance traveled

(in meters), the frequency of rearing (partial or total rising onto hind limbs), immobility

duration (time of complete absence of paw movements), the latency to start

movement (time spent to move one or more paws from the initial position) and the

time in center (time spent in the center of the open field).

2.3.3- Orofacial movements

Rats were placed individually in wired cages (40 cm×40.5 cm×20 cm) with

mirrors positioned under the floor and behind the back wall of the cage to allow

behavioral quantification when the animal was faced away from the observer. The

number of tongue protrusions (projection of the tongue out of the oral cavity),

vacuous chewing movement frequency (mouth openings in the vertical plane not

directed toward physical material), and facial twitching (duration, in seconds, of

twitching of the facial musculature) were measured continuously for 15 min.

2.3.4- Novel object recognition task

The task was carried out in a circular open-field arena (84 cm in diameter) with

32 cm high walls, made of wood and painted in black. The objects used were a sugar

bowl and a plastic stem glass, which were alternately familiar or new to avoid the

effect of possible preference. In the training session, rats were exposed to the

experimental chamber and two copies of an object for 10 min. In the test session, 24

h after the training, one object was replaced for a new object and the rats were

allowed to explore them for ten min. The time to explore each object was measured.

79

Exploration behavior included touching with forepaws or nose, sniffing and biting

each object.

2.3.5- Plus-maze discriminative avoidance task

The apparatus employed was a modified elevated plus-maze made of wood

containing two enclosed arms (50 X 15 X 40 cm) opposite to two open arms (50 X 15

cm). A 100-watt lamp was placed over the middle of one of the enclosed arms

(aversive enclosed arm). In the training session, each rat was placed in the centre of

the apparatus and, over a period of 10 min, every time the animal entered the

enclosed arm containing the lamp, an aversive situation was produced until the

animal left the arm. The aversive stimuli were the 100-watt light and an 80 dB noise

applied through a speaker placed over the aversive enclosed arm. In the test

session, held 24h later, the rats were again placed in the apparatus for 10 min,

without receiving the aversive stimulation, with the lamp and the speaker still present

over the aversive arm, but turned off. Distance traveled in the apparatus (used for

motor activity evaluation) and time spent in each arm (aversive, non-aversive and

open arms) were registered. Percent time in aversive arm (time spent in aversive

enclosed arm/time spent in both enclosed arms) and percent time spent in open

arms (time spent in open arms/time spent in both open and enclosed arms)

considering the whole duration of behavioral sessions were used to evaluate memory

and anxiety, respectively (Silva & Frussa-Filho, 2000). Percent time spent in the

aversive enclosed arm assessed minute by minute across the training and test

sessions were used to evaluate learning and extinction of the task (Ribeiro et al.,

2010).

2.4- Tissue preparation and oxidative stress parameters

Rats were decapitated, 48 h after the 7th (n=10 per group) and 10th injections (n

= 12 per group); and their brains were removed and put on ice. The striatum and

hippocampus were dissected. Each tissue sample of the hippocampus and striatum

was homogenized in ice-cold 0.1 M phosphate buffer (1:50, w:v). A duplicate of each

sample was used to determine MDA by measurement of fluorescent product formed

from the reaction of this aldehyde with thiobarbituric acid, as described by Tanizawa

80

et al. (1981). The results are expressed as nmol MDA/g tissue, calculated by plotting

the obtained fluorescence (excitation at 315 nm, emission at 553 nm) against an

MDA concentration standard curve.

2.5. Statistical analysis

Catalepsy behavior across the treatment (before, 24 h and 48 hours after each

injection), the orofacial movements (before starting the treatment, 24 h after the 5th

and 10th injections and 48 h after the 10th injection), the percentage of time in

aversive arm across behavioral sessions of the discriminative avoidance task and the

body weight (24 h before 1st, 3rd, 5th and 7th injections), were compared using ANOVA

with repeated measures. When necessary, pair wise comparisons were held with

multiple t tests with sequential Bonferroni‘s correction. The independent samples t

test was used to analyze differences between groups RES and VEH in all

parameters of open field behavior and also distance traveled, percent time spent in

the aversive or in the open arms in the discriminative avoidance task. In the novel

object recognition task, comparisons within groups for percentage of time to explore

old x new objects were performed with paired-samples t test. Results are expressed

as mean±SE and the significant threshold considered was p< 0.05.

3- Results

3.1- Effects of repeated administration of reserpine on catalepsy behavior

ANOVA with repeated measures revealed time (days of treatment) [F(21,

483)=18.16; p=0.000], treatment (reserpine or vehicle) [F(1, 23)=12.19; p=0.002] and

time X treatment interaction effects [F(21, 483)=9.29; p=0.000]. Rats repeatedly

treated with reserpine showed progressive increases in the catalepsy behavior,

which were significantly different from VEH on days 16 (48 h after the 7th injection) (t=

3.44; p= 0.002), and from day 18 onwards: 48 h after the 8th injection (t=4.24;

p=0.000), 24 h after the 9th injection (t=5.52; p=0.000), 48 h after the 9th injection

(t=4.43; p=0.000), 24 h after the 10th injection (t=7.40; p=0.000) and 48 h after the

10th injection (t= 6.41; p=0.000) (See Fig 2).

81

Fig.2. Effects of repeated administration of reserpine (RES - 0.1 mg/Kg) or vehicle (VEH) on catalepsy behavior. Data are expressed as mean±S.E.M. (s). ANOVA with repeated measures revealed time, treatment and time x treatment interaction effects. *p<0.05 compared to VEH group (independent samples t test with Bonferroni‘s correction).

3.2- Effects of repeated administration of reserpine in an open field

No effects of repeated administration of reserpine were found in open field

behavior. The distance traveled (t= 0.74; p=0.466), the frequency of rearing (t=0.91;

p= 0.368), the immobility duration (t=0.24; p=0.816), the latency to start movement

(t=0.69; p=0.498) and the time spent in the center of the open field (t=0.53; p=0.597)

of the reserpine group were not different from those presented by the vehicle group

(Fig. 3 A, B, C, D and E, respectively).

82

Fig.3. Effects of repeated administration of reserpine (RES - 0.1 mg/Kg) or vehicle (VEH) on open field behavior evaluated 24 h after the 4th injection. Data are expressed as the mean±S.E.M. of distance traveled (A), rearing frequency (B), immobility duration (C), latency to start the movement (D) and time in center (E).

3.3- Effects of repeated administration of reserpine on orofacial movements

ANOVA with repeated measures revealed time (days of treatment) [F(3,

42)=16.35; p=0.000], treatment (reserpine or vehicle) [F(1, 14)=7.66; p=0.015] and

time X treatment interaction effects [F(3, 42)=7.58; p=0.001] for the number of

vacuous chewing movements. Significant increases due to reserpine treatment

compared to VEH were detected on days 21 (24 h after the 10th injection) (t=3.37; p=

0.005) and 22 (48 h after the 10th injection) (t=2.90; p= 0.012) (see Fig. 4 A).

Regarding duration of facial twitching, ANOVA with repeated measures

revealed a treatment (reserpine or vehicle) effect [F (1,14)= 7.52; p=0.016]. No effect

A

B

C

D

E

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of time (days or treatment) [F(3, 42)=0.83; p=0.473] or time X treatment interaction

[F(3, 42)=1.75; p=0.178] were found. Significant increases due to reserpine treatment

compared to VEH were detected on day 21 (24 h after the 10th injection) (t=3.73; p=

0.002) (see Fig 4 B).

For the number of tongue protrusions, ANOVA with repeated measures

revealed a treatment (reserpine or vehicle) effect [F (1,14)= 6.45; p=0.024]. No effect

of time (days of treatment) [F(3, 42)=2.57; p=0.091] or time X treatment interaction

[F(3, 42)=1.29; p=0.291] were found. Significant increases due to reserpine treatment

compared to VEH were detected on days 21 (24 h after the 10th injection) (t=2.98; p=

0.010) and 22 (48 h after the 10th injection) (t=2.92; p= 0.011) (see Fig 4 C).

84

Fig.4. Effects of repeated administration of reserpine (RES - 0.1 mg/Kg) or vehicle (VEH) on orofacial movements. Data are expressed as the mean±S.E.M. of the

85

number of vacuous chewing movements (A), the duration of facial twitching (B) and the number of tongue protrusions (C). ANOVA with repeated measures revealed treatment effects for all parameters, as well as time and time x treatment interaction effects for vacuous chewing. *p<0.05 compared to VEH group (independent samples t test with Bonferroni‘s correction).

3.4- Effects of repeated administration of reserpine on novel object recognition task

Both groups showed an increased percentage of novel object exploration

compared to the old object (t= 3.73; 3.36 and p= 0.010; 0.015 for VEH and RES,

respectively, Fig 5), indicating adequate performance in the task.

Fig.5. Effects of repeated administration of reserpine (RES - 0.1 mg/Kg) or vehicle (VEH) on novel object recognition task performed 48 h after the 5th injection. Data are expressed as the mean±S.E.M. *p<0.05 compared to percent of old object exploration (paired-samples t-test).

3.5- Effects of repeated administration of reserpine on plus-maze discriminative

avoidance task

Repeated treatment with reserpine decreased the distance traveled in the

maze 24 h after the 7th injection (t= 3.32 and p= 0.002, Fig 5A), in the training

session, and 48 h after the 7th injection (t= 2.39 and p= 0.021, Fig 5C), in the test

session, when compared to the vehicle group. However, as shown in Fig. 6 (B and

* *

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D), no effects of repeated administration of reserpine were found on the latency to

start the movement, in the training or test sessions.

Fig.6. Effects of repeated administration of reserpine (RES - 0.1 mg/Kg) or vehicle (VEH) on plus-maze discriminative avoidance training (A,B) and test (C,D) sessions performed 24 and 48 h after the 7th injection, respectively. Data are expressed as the mean±S.E.M. of the distance traveled (m) (A, C) and latency to start the movement (s) (B,D). *p<0.05 compared to VEH group (independent samples t test).

Regarding anxiety-like behavior, the percentage of time in the open arms

(%TO) in the training (t= 0.44 and p= 0.665) and in the test (t= 1.08 and p= 0. 286)

presented by the reserpine group were not different from those presented by the

vehicle group (Fig. 7 A, and B, respectively).

Fig.7. Effects of repeated administration of reserpine (RES - 0.1 mg/Kg) or vehicle (VEH) on percent time in the open arms (%TO) of the plus-maze discriminative

A B

87

avoidance apparatus during training (A) and test (B) sessions, performed 24 and 48 h after the 7th injection, respectively. Data are expressed as the mean±S.E.M.

No effects of repeated administration of reserpine were found on the percent

time in the aversive enclosed arm (%TAV), in the training (t= 1.77; p=0.084) and test

(t=0.63; p=0.533) (see Fig 8 A and B) when the whole sessions were considered for

analysis.

In the training session, a significant effect of time (minutes) [F (9,396)= 8.19;

p=0.000] was found when the percentage of time in the aversive enclosed arm

(%TAV) was evaluated across the session. No effect of the treatment (reserpine or

vehicle) [F (1,44)= 0.64; p=0.429] and time X treatment interaction [F (9,396)= 1.34;

p=0.251] were found (see Fig 8 C).

In the test session, significant effects of time (minutes) [F( 9,396)=2.58;

p=0.024] was found when the percentage of time in the aversive enclosed arm

(%TAV) was evaluated across the session. No effect of the treatment (reserpine or

vehicle) [F (1,44)= 1.25; p=0.27] and the time X treatment interaction [F(9,396)=1.03;

p=0.406] were found (see Fig 8 D).

88

Fig.8. Effects of repeated administration of reserpine (RES - 0.1 mg/Kg) or vehicle (VEH) on percent time in the aversive enclosed arm (%TAV) of the plus-maze discriminative avoidance apparatus during the whole sessions (A, B) or minute by minute across the sessions (C,D), for training (A, C) and test (B,D), performed 24 and 48 h after the 7th injection, respectively. Data are expressed as the mean±S.E.M. ANOVA with repeated measures revealed time (minutes) effects in C and D.

C

D

A B

89

3.6- Effects of repeated administration of reserpine in the body weight

Regarding the body weight, ANOVA with repeated measures revealed no effect

of time (number of injections) [F(4, 96)=0.73; p=0.536], treatment (reserpine or

vehicle) [F (1,24)= 0.82; p=0.374] or time X treatment interaction [F(4, 96)=0.66;

p=0.572] (see Fig 9).

Fig.9. Effects of repeated administration of reserpine (RES - 0.1 mg/Kg) or vehicle in the body weight across the treatment. Data are expressed as the mean±S.E.M.

3.7- Effects of repeated administration of reserpine on striatal and hippocampal lipid

peroxidation

Fig. 10 (A and B) shows striatal and hippocampal levels of lipid peroxidation,

respectively, 48 h after 7th and 10th injection of rats repeatedly treated with reserpine.

No effects of repeated administration of reserpine were found after the 7th injection in

striatum (t= 1.35, p = 0.195) or hippocampus (t= 1.15, p = 0.269), and after the 10th

injection in hippocampus (t= 2.09; p=0.055). However, the reserpine-treated rats

showed increased levels of lipid peroxidation in the striatum 48 h after the 10th

injection (t=3.00; p=0.011) (see Fig 10 A).

0

100

200

300

400

500

2 6 10 14 20

Bo

dy w

eig

ht

(g)

Days of treatment

VEHRES

90

Fig.10. Effects of repeated administration of reserpine (RES - 0.1 mg/Kg) or vehicle (VEH) on striatal (A) and hippocampal (B) levels of lipid peroxidation. Data are expressed as the mean±S.E.M. of MDA levels per gram of tissue.*p<0.05 compared to VEH group (independent samples t test).

91

4- Discussion

In this study, we investigated the effects of repeated administration with a low

dose of reserpine on motor and cognitive parameters. We observed that this

repeated treatment with reserpine induced a progressive motor impairment. In fact,

these results can be seen in the evaluation of catalepsy behavior performed before,

24 and 48 h after each injection (Figure 2). In addition, the motor parameters

evaluated in the open field were not altered in the RES group 24 h after 4th injection

(Figure 3) but hypolocomotion was detected when the distance traveled in the

discriminative avoidance maze was measured 24 and 48 h after the 7th injection

(Figures 6 A and C). However, none of the memory tests performed were affected by

the treatment with reserpine (Figures 5 and 8). Furthermore, the present study

demonstrates that repeated reserpine treatment can induce motor abnormalities and

concomitant increases in striatal levels of lipid peroxidation, an indicative of oxidative

stress-induced neuronal damages (Figure 10).

PD is a neurodegenerative disorder of the basal ganglia characterized for a

complex situation of behavioral disorders, including tremor, rigidity and bradkinesia

(Johnston et al., 1999; Lindner et al., 1999; Ridley et al., 2006). These motor

symptoms have been highlighted as those that characterize the clinical status of an

affected person, so they are considered the most important disorders associated with

PD (Mayeux, 2003; Klockgether, 2004). In rodents, dopamine hypofunction lead to

parkinsonian symptoms as the emergence of signs of akinesia and rigidity

(catalepsy). The evaluation of catalepsy has been used as an important parameter

for the detection of motor signs in animal models of PD (Chinen and Frussa-Filho,

1999; Diaz et al., 2001). These signs can be induced not only by drugs that block

dopamine receptors such as haloperidol (Shiozaki et al., 1999; Gongora-Alfaro et al.,

2009) but also by substances that are potential inhibitors of mitochondrial complex I

as MPTP (Shiozaki et al., 1999) and rotenone (Corona et al., 2010), or the neurotoxin

6-OHDA (Diaz et al., 2001). Additionaly, these effects are also present after

monoamine vesicle depletion induced by reserpine (Shiozaki et al., 1999).

Reserpine interferes with the storage of catecholamines by blocking the presynaptic

vesicular carriers, resulting in depletion of monoamines in nerve terminals (Caudle et

al., 2008) and induction of hypolocomotion and muscular rigidity (Colpaert 1987;

Gerlach and Riederer, 1996). This study revealed that rats exposed to repeated

92

administration of reserpine at 0.1 mg / kg showed a gradual increase of cataleptic

immobility time when compared with the control group (Figure 2). Previous studies

have demonstrated that a short treatment with high doses of reserpine (1.0 mg / kg

every other day for 4 days) (Dutra et al., 2002) or an acute injection of an even higher

dose (5 mg / kg) (Shiozaki et al., 1999) produce catalepsy as well hypolocomotion.

However, in the present study, it is unlikely that the increase in catalepsy behavior is

due to the acute effect of the previous administration since it is still present even 48 h

after the last injection from the 7th injection onwards. Thus, a progressive neuronal

effect of the repeated treatment leading to the motor impairment could be

hypothesized.

Data from catalepsy evaluation are corroborated by the fact that the repeated

treatment with a low-dose of reserpine was not able to impair motor parameters

evaluated after the 4th injection in the open field (distance traveled, rearing frequency,

immobility duration and latency to start movement) (Figure 3). Previous research has

demonstrated that acute administration of higher doses of reserpine induced

locomotor alterations (Faria et al., 2005; Peixoto et al., 2005; Tadaiesky et al., 2006).

In this respect, hypokinesia is an important feature of animal models of PD and is

often related to a significant loss of dopamine cells (Da Cunha et al., 2002; Delfino et

al., 2004, Ferro et al., 2005; Capitello et al., 2008).The present results suggest that

there was no acute effect of the dose used on motor behavior, and the continuation

of the repeated treatment was necessary to produce motor abnormalities that were

observed in the catalepsy test only after the 7th injection (Figure 2). In this respect,

another motor parameter used in this study was the distance traveled and the latency

to start the movement in the elevated plus-maze used in the discriminative avoidance

task, which was performed 24 and 48 h after the 7th injection (Figure 6).

Corroborating the data from the catalepsy evaluation, animals treated with reserpine

showed a significant decrease in distance traveled in the maze in both sessions.

However, no significant differences between groups were found in latency to start

movement. Studies with MPTP model evaluated the latency in initiating the first

movement and demonstrated that there was an increase 24 h after administration of

the toxin, however, this change disappeared after 7 days (Capitelli et al., 2008).

Thus, these differences in results found in motor discriminative avoidance test may

reflect a gradual period of motor alteration, as can be found in humans with PD (Burn

et al., 2006).

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Previous studies have suggested oral dyskinesia induced in rodents by

reserpine as a possible animal model of tardive dyskinesia (Neisewander et al.,

1991; Neisewander et al., 1994; Abilio et al., 2003 and 2004). The tardive dyskinesia

is a side effect of long-term treatment with neuroleptics characterized by severe

motor symptoms where the face, mouth and tongue are frequently involved (orofacial

dyskinesia) (Hansen et al., 1997; Andreassen and Jorgensen, 2000). On the other

hand, some authors advocate the induction of orofacial movements as a model of the

tremor-related symptoms found in patients with PD (Salamone and Baskin, 1996,

Salamone et al., 2008). These authors argue that the difference of tremor seen in

rodents is restricted to the observation of the jaw which does not occur in humans

where this symptom can be observed in the limbs (Salamone and Baskin, 1996).

These movement alterations in rodents can be induced by a series of conditions

related to the neurochemistry and pathophysiology of parkinsonism such as depletion

of dopamine levels caused by reserpine (Salamone and Baskin , 1996, Salamone et

al., 2008), dopamine antagonists such as haloperidol (Andreassen et al., 2003) and

neurotoxins such as 6-OHDA (Jicha and Salamone, 1991). Here we found that the

treatment with repeated administration with a low-dose of reserpine was able to

induce an increase in orofacial movements 24h after the 10th injection (Figure 4). It

should be noted that neither orofacial movements nor catalepsy behavior were

altered 24 h after the 5th injection (Figures 2 and 4). However, 24h after the 10th

injection, concomitant motor alterations were observed in the catalepsy test and the

oral dyskinesia evaluation. Indeed, previous research has shown that acute reserpine

administration (at higher doses) induced decreased locomotion and increased

duration of immobility concomitantly to the oral dyskinesia (Faria et al., 2005; Peixoto

et al., 2005). In addition, there are descriptions of cases of PD patients who have

concomitant usual motor symptoms (bradykinesia, disorders in walking, among

others) and impaired oromotor control (Robertson et al., 2001). On the other hand,

Sussman et al. (1997) showed that reserpine-induced oral dyskinesia persisted

despite repletion of dopamine in the caudate-putamen, suggesting that the persistent

neuropathological change underlying this behavior occurs in a neural pathway other

than the dopaminergic nigrostriatal pathway. Thus, the pathophysiological

characteristics of orofacial movements are still controversial. However, the data

presented here indicate a progressive increase in orofacial movements

simultaneously to catalepsy behavior. Additionaly, this result is corroborated by

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recent data from our laboratory showing the same pattern of concomitant

appearance of both kinds of symptoms across a repeated treatment with 6-OHDA

(unpublished results).

As mentioned before, besides motor symptoms, PD patients also have other

manifestations such as cognitive, mood and sensory system alterations (Higginson et

al., 2001; Korczyn, 2001; Zgaljardic et al., 2004; Koerts et al., 2007). We have

recently verified that single administrations of reserpine — at doses that do not

modify motor function — impair memory in the discriminative avoidance task (a

rodent model of aversive discrimination – Carvalho et al., 2006), while no effects of

the same acute doses were detect in the novel object recognition task (Fernandes et

al., 2008). Thus, in the present study we investigated the effects of the repeated

treatment with 0.1 mg/kg reserpine on the performance of rats in these two tasks.

Due to evidence that cognitive deficits can precede the appearance of the motor

symptoms in the progress of the disease, we attempt to evaluate cognitive deficits

before an expressive motor impairment was instated.

The novel object recognition task (performed after the 5th injection) is based on

the fact that rats recognize a previously presented object, and therefore would spend

more time exploring the new object presented in the test session (Figure 5). The

preference for exploring new objects was shown by both groups, indicating that the

repeated administration of reserpine did not affect this kind of memory. Similar results

were found with acute administration of reserpine (0.1, 0.25 or 0.5 mg / kg)

(Fernandes et al., 2008). In this respect, the lack of alteration in this task is in

accordance with some clinical studies showing intact recognition memory in PD

patients (Gabrieli, 1996; Postle et al., 1997).

Besides evaluating memory, we used the plus maze discriminative avoidance

task (performed after the 7th injection) to assess learning, anxiety and motor

behavior, since it has been shown that these evaluations can be performed

concomitantly, and by different parameters, in this paradigm (Silva and Frussa-Filho,

2000; Silva et al., 2002a). The results have shown that there were no significant

differences in the percentage of time in the aversive arm between the VEH and RES

groups, both in the training and test sessions, indicating that repeated treatment with

reserpine was not able to promote changes in learning or retrieval of the aversive

task (Figure 8). Moreover, by evaluating the distance traveled in the maze, we

observed that animals treated with reserpine showed motor deficits during the

95

acquisition and retrieval of the task (Figure 6), corroborating the increase in catalepsy

duration also shown at this point of the treatment (Figure 2). This motor activity

decrement, however, did not interfere with the analysis of the data related to the

cognitive aspect of the task, since rats‘ performances were evaluated by time spent

in the aversive enclosed arm. Indeed, previous studies conducted with this paradigm

have shown the viability of separate and reliable analysis and interpretation of the

two parameters (Silva & Frussa-Filho, 2000; Silva et al., 2002a,b; Carvalho et al.,

2006; Kameda et al., 2007;Niigaki et al., 2010). Furthermore, the evaluation of

behavior minute by minute throughout the training session indicated that even in the

presence of motor deficits, animals treated with reserpine learned the task, as shown

by a decrement of aversive arm exploration by the end of the session (Figure 8 C).

Similarly, the analysis of aversive arm exploration throughout the test session

indicated that animals treaded with reserpine or vehicle showed retrieval of the task

(low aversive arm exploration in the first minutes) followed by extinction of the task

(increase in exploration) (Figure 8 D).

In summary, in the present study, the repeated administration with a low-dose

of reserpine did not produce changes in the memory task involving an emotional

context, as opposed to what has been previously observed after single injections in

this same paradigm (Silva et al., 2002a; Carvalho et al., 2006) or in the contextual

fear conditioning task (Fernandes et al., 2008). In this respect, research has shown

that excessive or insufficient levels of dopamine may have a negative effect on

emotional memory (Cools et al., 2002; Halbig et al., 2008). Thus, the previous

studies performed with acute treatments (with doses from 0.1 to 1 mg/kg) could

reflect the effects of acute dopamine depletion on emotional memory. Although the

dose used in this study is within this range (0.1 mg / kg), it was given several times,

and it is possible that the decrease in the levels of dopamine depletion was

outweighed by up regulation of D1 and D2 receptors in the caudate-putamen

(Neisewander et al., 1991) or by other compensatory mechanisms of plasticity

(Castaneda et al., 1990; Bezard and Gross 1998; Berzard et al., 2001; Cropley et al.,

2006). Also, the repeated treatment, which was efficient in inducing progressive

motor impairment, did not induce cognitive impairments in any of the paradigms used

here. Thus, a dissociation between the cognitive deficits induced by reserpine

treatment (observed in previous studies) and a possible degenerative process

induced by the repeated treatment conducted here is suggested. It is important to

96

note, however, that cognitive impairments can be observed in rats injured with MPTP

(Da Cunha et al., 2002) and PD patients (Bowers et al., 2006; Halbig et al., 2008).

Thus, it would be interest to verify the effects of the repeated treatment used here in

other animal models of memory, or even in other aspects of cognitive function.

Another parameter evaluated in the plus-maze discriminative avoidance task

was the time of exploration of the open arms of the maze, indicative of anxiety-like

behavior (Silva & Frussa-Filho, 2000). Results showed that treatment with reserpine

did not induce alterations in anxiety-like behavior (Figure 7), corroborating the

previous studies that investigated the effects of reserpine in this task (Silvia et al.,

2002a; Carvalho et al., 2006).

Increased oxidative stress with cumulative free radical damage is present in

brain aging and neurodegenerative diseases such as PD (Cadenas and Davies,

2000; Beal, 2003). In this respect, treatment with reserpine can result in the

accumulation of neurotoxic dopamine oxidants that can induce the production of

ROS exceeding the ability of the antioxidant system to eliminate them, thus resulting

in oxidative damage (Cadenas and Davies, 2000; Caudle et al., 2008). Recent

studies showed that acute administration of high doses of reserpine increases lipid

peroxidation on striatum and antioxidant agents are able to reverse the behavioral

effects induced by reserpine (Abílio et al., 2003, 2004; Faria et al., 2005). Herein, the

repeated treatment with a low dose of reserpine has shown to induce an increased

striatal level of lipid peroxidation 48 h after the 10th injection (Figure 10A), when an

important motor impairment was also present (Figure 2). On the other hand,

hippocampal levels of lipid peroxidation were not modified by the treatment.

Interestingly, the absence of memory impairment may be related to lack of neuronal

damage caused by oxidative stress in the hippocampus (Figure 10B). These findings

suggest that the treatment used here may induce a progressive neuronal damage

similar to what is found in patients with PD, at least considering motor aspects of the

pathology.

Although quantitative assessment was not conducted, there were no important

peripheral autonomic changes in reserpine-treated animals throughout the treatment.

Additionally, no change was found in body weight of rats during the repeated

treatment of reserpine (Figure 9), and all animals survived to the treatment. In this

respect, Ferro et al. (2005) found a significant change in weight in pharmacological

models of MPTP and 6-OHDA when compared to control groups, with 20% death of

97

treated animals. In light of these findings, we suggest that reserpine may be a more

favorable drug to the development of a pharmacological progressive model, which

requires repeated treatment over time, compared to MPTP or 6-OHDA models. On

the other hand, it is important to mention that reserpine, as a pharmacological model

of PD, is considered to be unspecific, because this drug acts on the depletion of all

monoamines. However, there is evidence in the literature that the physiopathology of

PD itself is not exclusively related to dopamine, since other neurotransmitter systems

have shown to be involved in the PD symptoms such as the serotonergic and the

GABAergic systems, among others (Ossowska et al., 2002; Borah et a., 2007).

Reserpine is an irreversible inhibitor of the vesicular monoamine transporter

(VMAT). As mentioned above the action of reserpine prevents the storage of

monoamines in synaptic vesicles (Caudle et al., 2008). There are studies suggesting

that animals express 5% of VMAT as a promising model for the study of PD. The

VMAT deficient animals have increased oxidative stress, progressive loss of

dopamine terminals and accumulation of -synuclein (Caudle et al. 2007). In addition,

monoaminergic dysfunctions are also found levels of dopamine, norepinephrine and

serotonin are severely diminished (Caudle et al. 2007, Taylor et al. 2009). Sleep

disturbances, gastrointestinal symptoms of anxiety and depression were observed in

results from studies with mice deficient for VMAT (Taylor et al. 2009). As can be seen

the VMAT2-deficient animals exhibit similar alterations in the animals treated with

reserpine. Data from this study show increased oxidative stress and data from other

studies show alteration of dopamine levels in rats treated with reserpine (Sussman et

al., 1997) as well as can be seen in animals deficient for VMAT. In addition, results of

studies using Western blot analysis showed reductions markers for VMAT2

immunoreactivity in putamen, caudate and nucleus accumbens of PD brain

compared to control cases (Miller et al., 1999). These findings show that alterations

in VMAT can be one of the factors related to the development of PD.

In conclusion, we found that repeated administration with a low dose of

reserpine in rats induces a gradual appearance of motor signs compatible with the

progressive nature of PD (Klockgether, 2004). These motor signs are accompanied

by increased levels of oxidative stress in the striatum that support studies showing an

increase in free radicals as a possible factor in PD (Cadenas and Davies, 2000; Beal,

2003). Nevertheless, the treatment protocol applied was not able to induce cognitive

deficits, at least in the behavioral models used, which was corroborated by the

98

absence of oxidative damage in the hippocampus. More studies are required to verify

the progressive possible changes of the dopaminergic and other neurotransmitter

systems in the PD model proposed here. Further investigation with other behavioral

models could also clarify if the cognitive deficits related to PD can be observed in this

new progressive pharmacological model.

Acknowledgements

The authors would like to thank Claudenice Moreira dos Santos and Josué

Cândido Macedo for capable technical assistance. This research was supported by

fellowships from Conselho Nacional de Desenvolvimento Científico e Tecnológico

(CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES),

Fundação de Apoio à Pesquisa do Estado do Rio Grande do Norte (FAPERN), and

Pró-reitoria de Pesquisa da Universidade Federal do Rio Grande do Norte

(PROPESQ/UFRN).

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3.Discussão geral

e conclusão

105

3. Discussão geral e conclusão

Neste trabalho, investigamos os efeitos da administração repetida com doses

baixas de reserpina em parâmetros motores e cognitivos. Podemos observar nos

resultados tanto do experimento I quanto no experimento II que o tratamento

repetido com reserpina (0,1 e 0,2 mg/Kg) é capaz de induzir alterações motoras. No

experimento I, observamos alteração no comportamento de catalepsia avaliado vinte

e quatro horas após a nona, a décima segunda e a decima quinta injeções para os

grupos RES 0,1 e 0,2. No experimento II, pode ser observado na avaliação do

comportamento de catalepsia, realizada antes, 24 e 48 horas após cada injeção, que

a alteração motora aparenta ser progressiva. Na avaliação de outros parâmetros

motores, como os movimentos orofaciais, também podemos observar uma alteração

vinte e quatro horas após a décima quarta injeção, no experimento I, e vinte e quatro

e quarenta e oito horas após a décima injeção, no experimento II. Nos parâmetros

cognitivos observamos no experimento I, as alterações encontradas foram

acompanhadas por déficits motores nos grupos RES 0,1 e 0,2. Entretanto, no

experimento II, foram encontrados déficits motores sem alterações cognitivas no

grupo RES 0,1 vinte e quatro e quarenta e oito horas após a sétima injeção. Os

fatores bioquímicos analisados revelaram um aumento dos níveis de estresse

oxidativo no estriado dos animais tratados com RES 0,1, quarenta e oito horas após

a décima injeção (experimento II) e, uma diminuição da concetração de glutamato

estrital dos animais dos grupos RES 0,1 e 0,2, cinco dias após a décima quinta

injeção (experimento I).

No experimento I, observamos que os ratos tratados com doses repetidas de

reserpina (0,1 e 0,2 mg/Kg) apresentaram alteração motora durante o periodo de

106

tratamento. Estes dados podem ser observados na avaliação do comportamento de

catalepsia (24 horas após a nona, a décima segunda e a décima quinta injeções), na

distância percorrida no labirinto em cruz da esquiva discriminativa (na sessão de

treino, 24 horas após a décima injeção), nas tentativas de escapar no teste de medo

condicionado ao contexto (48 e 72 horas após a décima quinta injeção). Contudo, os

animais do grupo RES 0,05 não apresentaram alterações motoras durante o

tratamento. Os resultados dos grupos RES 0,1 e 0,2 indicaram que o tratamento

proposto pode apresentar estágios com ausência ou presença de alterações

motoras. Entretanto, somente com estes dados não era possível determinar se as

alterações motoras eram decorrentes de uma modificação progressiva permanente

nas vias motoras ou de um efeito imediato de uma determinada injeção de

reserpina. Dessa forma, no experimento II, escolhemos a dose de 0,1 mg/ Kg de

reserpina pois esta foi capaz de induzir estágios nas alterações motoras (ausência e

presença de alterações motoras) e avaliamos o efeito da reserpina sobre a

motricidade (por meio da avaliação da catalepsia, um parâmetro importante para a

detecção de sinais motores em modelos animais de PD) (Chinen & Frussa-Filho

1999, Diaz et al. 2001) vinte e quatro horas e quarenta e oito horas após as

administrações, durante todo o tratamento. Assim, no experimento II, os resultados

demonstraram que a administração repetida de 0,1 mg/kg de reserpina induziu um

aumento gradual do tempo de imobilidade cataléptica, quando comparado com o

grupo controle. Estes dados indicam uma possível semelhança ao processo

neurodegenerativo encontrados em humanos com DP onde as alterações gradativas

dos circuitos dos núcleos da base provocam um aparecimento progressivo dos

sintomas parkinsonianos (distúrbios comportamentais nos quais a rigidez, o tremor e

a bradicinesia são caracteríticos) (Johnston et al. 1999, Lindner et al. 1999,

107

Deumens et al. 2002, Ridley et al. 2006, Meredith et al. 2008a). Além disso,

evidências sugerem que o comportamento de catalepsia em animais está

diretamente relacionado à alterações no sistema dopaminérgico (Chinen et al. 1999,

Gongora-Alfaro et al. 2009). Um estudo em ratos com administração intraventricular

de 6-OHDA demonstrou um efeito dose dependente no tempo de permanência da

posição caléptica (Díaz et al. 2001). Dessa forma, os dados apresentados no

experimento II parecem corroborar estudos que mostram que a reserpina induz

alterações motoras (Kannari et al. 2000, Farley et al. 2006). Outro dado importante é

que no experimento II encontramos um aumento dos níveis de peroxidação lipídica

no estriado concomitante ao aumento do comportamento cataléptico sugerindo uma

relação entre os fatores.

Outro parâmetro motor avaliado nos experimento I e II foi os movimentos

orofaciais. Esta avaliação foi realizada através de três parâmetros que são: o

número de protrusões de língua; o número de movimentos de mastigação que não

fosse direcionado a nenhum objeto (vacuous chewing movements); e a duração de

tremores de queixo (em segundos). O comportamento de mastigação é um

movimento repetitivo da mandíbula (de freqüência de 03-06 Hz) que pode ser

induzido por alterações dopaminérgicas (Jicha & Salamone 1999, Díaz et al. 2001) e

tem sido proposto por alguns pesquisadores como um modelo de tremor

parkinsoniano (Salamone & Baskin 1996, Dutra et al. 2002). No experimento I,

observamos que os animais dos grupos RES 0,1 e 0,2 apresentaram um aumento

em todos os parâmetros orofacias avaliados, vinte e quatro horas após a décima

quarta injeção. Aparentemente, este resultado foi concomitante com as alterações

motoras encontradas na avaliação do comportameto cataléptico. Entretanto, não foi

possível confirmar esta hipótese já que não havíamos feito a mensuração da

108

catalepsia neste período do tratamento. Dessa forma, no experimento II, avaliamos

os movimentos orofaciais vinte e quatro horas após a quinta e a décima injeção, e

quarenta e oito horas após a décima injeção. Os resultados obtidos mostraram que

os animais do grupo RES 0,1 apresentaram alterações nos movimentos orofaciais

24 e 48 horas após a décima injeção, concomitantemente às alterações motoras das

análises do comportamento de catalepsia. Estudos anteriores com administração

aguda de reserpina corroboram com estes resultados encontrados (Faria et al. 2005,

Peixoto et al. 2005). Além disso, Sussman et al. (1997) demonstraram que a

discinesia oral induzida pela reserpina persistiu por 84 dias e produziu uma

dimunição de 74% da dopamina da via caudado-putâmen três dias após a injeção

mas esta diminuição não foi observada 84 dias após a injeção. Dessa forma, as

mudanças neurobiológicas subjacentes ao comportamento em debate ainda não

estão bem compreendidas.

Um parâmetro cognitivo avaliado no experimento I foi o modelo da esquiva

discriminativa em labirinto em cruz elevado. Este modelo permite a avaliação

concomitantemente, por parâmetros diferentes, da memória, da ansiedade e do

comportamento motor (Silva & Frussa-Filho 2000, Silva et al 2002a). Os resultados

mostraram que o grupo RES 0,05 não apresentou alteração nos parâmentros

avaliados. Entretando, o grupo RES 0,1 apresentou alteração na porcentagem de

tempo de permanência no braço aversivo concomitantemente às alterações

motoras. As alterações motoras encontradas no grupo RES 0,1 não prejudicaram a

aquisição da tarefa, no treino, prejuízo que foi constatado no grupo RES 0,2. Dessa

forma, no experimento II, decidimos utilizar a dose de 0,1mg/Kg de reserpina em um

período do tratamento onde não houvesse (ou fossem mínimas) alterações motoras

na avaliação de catalepsia. Assim sendo, a esquiva discriminativa foi realizada vinte

109

e quatro e quarenta e oito horas após a sétima injeção. A administração repetida de

0,1mg/Kg de reserpina não induziu alteração na memória, mas houve alteração na

distância percorrida no labirinto. Estes resultados são contrários a outros estudos

nos quais os autores utilizaram um tratamento agudo de reserpina no mesmo

modelo comportamental (Silvia et al. 2002a, Carvalho et al. 2006). A este respeito

existem evidências na literatura mostrando que níveis excessivos ou insuficientes de

dopamina podem alterar a memória emocional (Cools et al. 2002, Halbing et al.

2008). Além disso, estudos indicam que as vias dopaminérgicas têm propriedades

intrínsecas capazes de ativar mecanismos compensatórios que são diferenciados

dependendo nos níveis de alteração (Castaneda et al. 1990, Bezard & Gross 1998,

Berzard et al. 2001, Mawlawi et al. 2001, Cropley et al. 2006). Assim sendo, a dose e

o protocolo de tratamento utilizado no experimento II provavelmente pode ter

induzido um decréscimo dos níveis de dopamina que foram contrabalanceados por

um mecanismo compensatório de plasticidade nos sistemas neuronais relacionados

à memória. Portanto, o tratamento repetido com reserpina, foi eficiente na indução

de déficits motores, mas não em alterações cognitivas, pelo menos nos paradigmas

utilizados neste trabalho. Entretanto, deficiências cognitivas foram observadas em

ratos lesados com MPTP nos testes de esquiva ativa e labirinto aquático (Da Cunha

et al. 2002) e em pacientes com DP (Bowers et al. 2006, Halbig et al. 2008). Dessa

forma, seria interessente verificar os efeitos do tratamento repetido de reserpina 0,1

mg/kg em outros modelos de memória ou mesmo em outras funções cognitivas.

No desenvolvimento da DP ocorrem mortes de neurônios dopaminérgicos,

entretanto, existe um período que precede o aparecimento dos primeiros sinais

clínicos onde o sistema mantem certo nível de funcionamento (Bezard & Gross

1998). Tudo indica que as vias dopaminérgicas possuem propriedades intrínsecas

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capazes de acionar mecanismos compensatórios (Castaneda et al. 1990, Bezard &

Gross 1998, Berzard et al. 2001, Mawlawi et al. 2001, Cropley et al. 2006). Estes

mecanismos compensatórios parecem ser diferenciados dependendo do nível de

perda de dopamina (Bezard & Gross 1998). Estes mecanismos podem ser através

da regulação dos receptores de dopamina assim como super sensibilização deste ou

aumento de liberação de dopamina em terminais remanescentes como demonstrado

em animais lesados com 6-OHDA (Castaneda et al. 1990). Entretanto, existem

contradições quanto à relação entre perdas de células dopaminérgicas e sintomas

clínicos (Bezard & Gross, 1998). Este fato sugere que os sintomas clínicos poderiam

estar vinculados não somente com as alterações dopaminérgicas, mas também com

a atuação de outros sistemas não-dopaminérgicos (Bezard & Gross 1998, Blandini

et al. 2000). Assim sendo, no experimento I, foi observada diminuição na

concentração do nível de glutamato estriatal dos grupos RES 0,1 e 0,2 cinco dias

após a décima quinta injeção. Este resultado corrobora o estudo de Day et al. (2006)

que mostraram que ocorre perda de sinapses glutamatérgicas no estriato-pallidal

após a depleção dopaminérgica. Seria interessante ter realizado análises

bioquímicas dos níveis de dopamina no estriado dos ratos do experimento I.

Contudo, por problemas de ordem técnica, não foi possível realizar estas análises.

Em vista destes fatores, e de evidências que indicam uma relação de estresse

oxidativo cerebral com a fisiopatologia da doença de Parkinson (Cadenas & Davies

2000, Beal 2003), resolvemos avaliar os danos oxidativos no estriado e no

hipocampo causado pela reserpina no experimento II. Assim sendo, no experimento

II, foi observado um aumento nos níveis de estresse oxidativo do estriato, 24 horas

após a décima injeção, o que mostra que possíveis danos celulares podem estar

ocorrendo nesta área. De uma forma geral, uma hipótese que podemos levantar dos

111

dados encontrados nos dois experimentos seria: a redução da atividade

dopaminérgica nigroestriatal induzida por danos oxidativos alteraria a atividade do

globo pallidum e da substância nigra reticutala, resultando na diminuição da

atividade talâmina e cortical, que finalmente, levaria a redução da atividade

glutamatérgica corticoestriatal. Roberts et al. (1982) sugerem que um possível

mecanismo compensatório para a redução da atividade glutamatérgica

corticoestriatal que seria o aumento de receptores NMDA levando a uma

supersensibilidade que compensaria a regulação da liberação de dopamina.

A reserpina é uma droga que evita o armazenamento de monoaminas nas

vesículas sinápticas através da inibição da ação dos transportadores da membrana

que captam as monoaminas para dentro da vesícula (Liu et al. 1996, Verheij & Cools

2007). Dessa forma, as vesículas sinápticas permanecem vazias e

conseqüentemente não há neurotransmissores para serem liberados na fenda

sináptica quando um potencial de ação atinge o botão sináptico (Rang et al. 2004).

Então, a atuação da reserpina não se restringe apenas nas vias dopaminérgicas,

atuando também nas vias noradrenérgicas e serotoninérgicas. Este fator poderia ser

uma limitação da utilização desta droga como um modelo farmacológico de

Parkinson se não houvesse relatos na literatura demonstrando que existem também

alterações em outras vias monoaminérgicas na DP (Devos et al. 2010, Fox et al.

2009).

Podemos concluir através dos resultados obtidos nos experimentos I e II que:

1. A administração repetida de 0,1 mg/Kg de reserpina em ratos é capaz de

induzir o aparecimento gradual de sinais motores compatíveis com as

características progressivas encontrados em pacientes com DP (Klockgether

2004);

112

2. Os sinais motores induzidos através da administração repetida de 0,1 mg/Kg

de reserpina em ratos foram acompanhados por um aumento dos níveis de

estresse oxidativo no estriado que está de acordo com trabalhos que

sustentam a hipótese do aumento de radicais livres estarem relacionados à

DP (Cadenas & Davies 2000, Beal 2003, Abílio et al. 2004, Faria et al. 2005,

Teixeira et al. 2009);

3. Foram encontradas alterações nas concentrações de glutamato no estriato

nos grupos tratados com doses repetidas de 0,1 e 0,2 mg/Kg, cinco dias após

a décima quinta injeção, corroborando os estudos prévios;

4. O protocolo de tratamento aplicado não foi capaz de induzir déficits

cognitivos sem alteração motora, dados corroborados pela ausência de

alteração dos níveis de estresse oxidativo no hipocampo.

Dessa forma, são necessários mais estudos para a compreensão das

mudanças nos sistemas de neurotransmissão durante o processo de

aparecimento dos sinais parkinsonianos no modelo proposto.

113

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5. Anexo

Documents

Administração repetida de baixas doses de …...VALÉRIA FERNANDES DE SOUZA Administração repetida de baixas doses de reserpina: um possível modelo para o estudo de déficits