efeitos neuroquímicos da administração de olanzapina e/ou

UNIVERSIDADE DO EXTREMO SUL CATARINENSE - UNESC
UNIDADE ACADÊMICA DE CIÊNCIAS DA SAÚDE - UNASAU
PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE - PPGCS
EFEITOS NEUROQUÍMICOS DA ADMINISTRAÇÃO DE
OLANZAPINA E/OU FLUOXETINA EM RATOS WISTAR:
IMPLICAÇÕES NO ENTENDIMENTO DA TERAPÊUTICA DA
DEPRESSÃO BIPOLAR
FABIANO ROSA AGOSTINHO
CRICIÚMA, MARÇO DE 2011
FABIANO ROSA AGOSTINHO
EFEITOS NEUROQUÍMICOS DA ADMINISTRAÇÃO DE
OLANZAPINA E/OU FLUOXETINA EM RATOS WISTAR:
IMPLICAÇÕES NO ENTENDIMENTO DA TERAPÊUTICA DA
DEPRESSÃO BIPOLAR
Tese apresentada ao Programa de Pós-Graduação
em Ciências da Saúde da Universidade do Extremo
Sul Catarinense, para obtenção do título de Doutor
em Ciências da Saúde.
Orientador: Prof. Dr. João Quevedo
CRICIÚMA, MARÇO DE 2011
Ao meu Pai, meu maior agradecimento.
AGRADECIMENTOS
À Deus sobre todas as coisas, que por intermédio de seu Filho, me acalmava
e abria meus olhos para a dura, frágil e ao mesmo tempo maravilhosa realidade da vida;
Aos meus pais Walter Rodrigues Agostinho e Maria Odete Rosa Rodrigues,
e meu tio Antonio Adalberto Buozi Rosa que mesmo distantes me ampararam e se
esforçaram muito para que eu seguisse bem e que com certeza não descansarão;
Ao professor Dr. João Quevedo, amigo antes de tudo, o qual tive a sorte de
poder ser orientado desde a primeira fase do curso de medicina. Portador de um dos
conhecimentos mais impressionantes, resultado de uma “energia pragmática” que
transforma tudo em produtividade e que, cuja presença, contagia até mesmo as mentes
mais inertes;
Gustavo Feier, verdadeiro amigo e prontamente Doutor, seu mérito não cabe
somente nas nossas incansáveis horas de laboratório e graduação em medicina. Sua
disponibilidade é incomum, tanto para a ciência quanto para a amizade;
Minha colega Doutora Gislaine Zilli Réus que, diante de minhas maiores
dificuldades, sacrificava momentos preciosos de seu tumultuado tempo de estudos para
que esse trabalho fosse levado a termo;
Á Força Área Brasileira que mesmo antes de me incorporar já havia me
apoiado proporcionando condições a realizar o doutorado;
Dois amigos que, nesta época da minha vida, foram meus “dois” braços
direitos na amizade verdadeira, 2 Ten Brandão e 1 Ten Gustavo, colaborando
respectivamente na fabricação artesanal da cerveja perfeita e na maior inspiração
musical da nossa banda “Dr. Yathros”.
Aos todos os pesquisadores do Laboratório de Neurociências por terem
proporcionado um ambiente de colaboração no mais virtuoso sentido da palavra;
Por último, mas poderia ser primeiro, ao meu verdadeiro amor, Daiane
Breda, minha companheira, pelo apoio, carinho, dedicação, amor e por dar sentido a
isso tudo.
“Temos que acreditar em alguma coisa, e
sobretudo
temos
de
ter
um
sentimento
de
responsabilidade coletiva, segundo o qual cada um
de nos será responsável sobre todos os outros.”
(José Saramago)
∆ 16 de novembro de 1922
† 18 de junho de 2010
RESUMO
O transtorno do humor bipolar (THB) é um transtorno com importante prejuízo
na qualidade de vida. Atualmente, a depressão bipolar (DB) representa a fase de maior
dificuldade de tratamento. O metabolismo energético cerebral e fatores neurotróficos estão
associados ao THB. Recentemente, uma combinação fixa de um antipsicótico atípico
olanzapina (OLZ) e um inibidor seletivo de serotonina fluoxetina (FLX) (SymbiaxTM) foi
aprovada pela FDA para o tratamento da DB. O presente estudo avalia o efeito agudo e
crônico da monoterapia e da combinação de OLZ e FLX no metabolismo energético e
expressão de neurotrofinas. Para isso, foram estudadas as atividades da enzima creatina
quinase (CK), citrato sintase (CS), cadeia respiratória mitocondrial e a expressão de BDNF,
NGF e NT-3 em córtex prefrontal, hipocampo, cerebelo, estriado e córtex cerebral em
cérebro de ratos. O tratamento agudo, ratos Wistar machos receberam uma aplicação de
OLZ (3 e 6 mg/kg) e/ou FLX (12,5 e 25 mg/kg) e para o tratamento crônico por 28 dias. Os
resultados mostram a atividade da CK, no tratamento agudo com OLZ (3 e 6 mg/kg), estava
inibida no cerebelo e prefrontal. Tanto a combinação como a monoterapia com OLZ (3 ou 6
mg/kg) com FLX (12,5 ou 25 mg/kg) inibiu a atividade da CK no prefrontal, cerebelo,
hipocampo estriado e córtex. No tratamento crônico 2 horas após a ultima administraçao,
observa-se inibida tanto na monoterapia com FLX (12,5 ou 25 mg/kg) quanto na
combinação com OLZ (3 ou 6 mg/kg). Neste mesmo protocolo, o tratamento com OLZ (3
ou 6 mg/kg) não alterou a atividade da CK. Porem, no tratamento crônico 24h após a ultima
administração, não observaram-se alterações na atividade da CK. A CS, no tratamento
agudo, a administração de OLZ (6 mg/kg) e OLZ/FLX (3/25 mg/kg) aumentou a atividade
da CS no prefrontal. No hipocampo OLZ (3 mg/kg) e OLZ/FLX (3/25 mg/kg) aumentou a
atividade da CS. No estriado a OLZ (3 e 6 mg/kg), FLX (25 mg/kg) e a combinação
OLZ/FLX (3/25 mg/kg) também aumentou a atividade da CS. No tratamento crônico não
foram observados alterações. Na cadeia respiratória, no tratamento agudo e crônico com
OLZ e FLX, encontrou-se aumentada a atividade dos complexos na monoterapia e na
combinação, porem na combinação observa-se maiores alterações. A expressão das
neurotrofinas BDNF e NGF não esta alterada pela combinação OLZ/FLX. Porem a
expressão de NT-3 esta aumentada na administração OLX/FLZ 6/25 mg/kg no prefrontal.
Finalmente, este estudo apóia a hipótese do metabolismo energético e as expressões de
neurotrofinas podem estar envolvidas no tratamento combinado OLZ/FLX no tratamento do
THB.
Palavras Chave: combinação olanzapina/fluoxetina, creatina quinase, citrato sintase,
cadeia respiratória mitocondrial, NGF, BDNF, NT-3, transtorno do humor bipolar.
ABSTRACT
Bipolar disorder (BD) is a prevalent condition in adults determining a
significant impairment in quality of life . Bipolar depression represents a difficult-to-treat
and disabling form of depression. It has been hypothesized that BD is associated with
mitochondrial dysfunction and neurotrophic factors. Recently, a fixed combination of the
antipsychotic drug olanzapine (OLZ) and the antidepressant fluoxetine (FLX) (SymbiaxTM)
has been introduced for the treatment of BD. This study evaluates the effects of OLZ alone,
FLX alone, or a combination of both; in energetic metabolism and neurotrophines
expression. For this, we studied the activity of the enzyme creatine kinase (CK), citrate
synthase (CS), and mitochondrial respiratory chain, expression of BDNF, NGF and NT-3 in
the prefrontal cortex, hippocampus, cerebellum, striatum and cerebral cortex in the rat
brain. The acute treatment, male Wistar rats received an application OLZ (3 and 6 mg/kg)
and / or FLX (12.5 and 25 mg/kg) and chronic treatment for 28 days. The results show CK
activity in the acute treatment OLZ (3 and 6 mg/kg) was inhibited in the cerebellum and
prefrontal. Both the combination and monotherapy OLZ (3 or 6 mg/kg) and FLX (12.5 or
25 mg/kg) inhibited CK activity in the prefrontal cortex, cerebellum, hippocampus, striatum
and cortex. In the chronic treatment 2 hours after the last administration, there was inhibited
both in monotherapy FLX (12.5 or 25 mg/kg) and in combination with OLZ (3 or 6 mg/kg).
In this same protocol, treatment with OLZ (3 or 6 mg/kg) did not alter CK activity. In the
present study, the activity of CS, in the acute treatment, the administration of OLZ (6
mg/kg) and OLZ/FLX (3/25 mg/kg) increased activity in prefrontal CS. OLZ hippocampus
(3 mg/kg) and OLZ/FLX (3/25 mg/kg) increased the activity of CS. In the striatum OLZ (3
and 6 mg/kg), FLX (25 mg/kg) and combination OLZ/FLX (3/25 mg/kg) also increased the
activity of CS. In the chronic treatment were not observed changes. Respiratory chain in
acute and chronic treatment with FLX and OLZ, we found increased activity of complexes
in monotherapy and in combination, but the combination was observed major changes. The
expression of neurotrophins BDNF and NGF is not altered by the combination OLZ/FLX.
But the expression of NT-3 increased in this administration OLX/FLZ 6/25 mg/kg in the
prefrontal. Finally, this study supports the hypothesis of energy metabolism and the
expression of neurotrophins may be involved in the combined treatment OLZ/FLX in the
treatment of bipolar disorder.
Keywords:
combination
olanzapine/fluoxetine,
creatine
kinase,
mitochondrial respiratory chain, NGF, BDNF, NT-3, bipolar disorder.
citrate
synthase,
LISTA DE ILUSTRAÇÕES
Figura 1. Representação esquemática da cadeia respiratória mitocondrial. ______________________ 21
Figura 2. Representação esquemática da atividade da enzima creatina quinase. __________________ 22
Figura 3. Respresentaçao esquemática da atividade da enzima citrato sintase. ____________________ 23
Figura 4. Representação esquemática ligação das neurotrofinas com os receptores tirosina quinase. _ 24
LISTA DE ABREVIATURAS E SIGLAS
5-HTTR – Gene Transportador de Serotonina
ACTH – Hormônio de Liberação de Adrenocorticotrofinas
ADP – Adenosina Difosfato
AMPc – Monofosfato de Adenosina Cíclico
APA – American Psychiatry Association
ATP – Adenosina Trifosfato
BDNF – Fator Neurotrófico Derivado do Cérebro (brain-derived neurotrophic factor)
CID-10 – Código Internacional de Doenças
CK – Creatina Quinase
COMT – Catecol – O – Metil Transferase
CRH – Hormônio de Liberação de Corticotrofinas
CS – Citrato Sintase
DNA – Ácido Desoxirribonucléico
DSM-IV-TR – Manual Diagnóstico e Estatístico dos Transtornos Mentais
ECA – Epidemiologic Captation Area (Área de Captação Epidemiológica)
ECT – Eletroconvulsoterapia
FDA – Administração de Alimentos e Medicamentos (Food and Drug Administration)
FLX – Fluoxetina
fMRI – Espectroscopia por Ressonância Magnética Funcional
HPA – Eixo Hipotálamo Pituitária Adrenal
ISRS – Inibirdor Seletivo da Recaptaçao de Serotonina
Li – Lítio
MAO A – Mono Amino Oxidase A
MRS – Espectroscopia por Ressonância Magnética
NADH – Nicotinamida Adenina Dinucleotídeo
NGF – Fator de Crescimento Neurotrófico (Nerve Grow Factor)
NIMH – National Institute Mental Health (Instituto Nacional de Saúde Mental)
NT – 4 – Neurotrofina 4
NT-3 – Neurotrofina 3
OLZ – Olanzapina
PET – Tomografia por Emissão de Pósitrons
Pi – Fósforo Inorgânico
SNC – Sistema Nervoso Central
SOE – Sem Outra Especificação
TDHA –Transtorno do Déficit de Atenção e Hiperatividade
TEHB – Transtorno do Espectro do Humor Bipolar
THB – Transtorno do Humor Bipolar
THB I – Transtorno do Humor Bipolar tipo I (maníaco depressivo)
THB II – Transtorno do Humor Bipolar tipo II (hipomaníaco depressivo)
TOC – Transtorno Obsessivo Compulsivo
TrK – Receptor Tirosina Quinase
VPZ – Valproato
YMRS – Escala Específica para Avaliação de Mania -Young Mania Rating Scale.
SUMÁRIO
PARTE I ____________________________________________________________ 13
1 INTRODUÇÃO ____________________________________________________ 14
1.1 DEFINIÇÃO __________________________________________________________ 14
1.2 EPIDEMIOLOGIA ____________________________________________________ 14
1.3 ETIOLOGIA __________________________________________________________ 17
1.4 FISIOPATOLOGIA ____________________________________________________ 18
1.4.1
Áreas Cerebrais e THB _____________________________________________ 18
1.4.2
Bases Biológicas e THB ____________________________________________ 19
1.5 DIAGNÓSTICO _______________________________________________________ 25
1.6 TRATAMENTO _______________________________________________________ 26
2 JUSTIFICATIVA __________________________________________________ 29
3 OBJETIVOS_______________________________________________________ 30
3.1 OBJETIVO GERAL ___________________________________________________ 30
3.2 OBJETIVOS ESPECIFICOS ____________________________________________ 30
PARTE II ___________________________________________________________ 31
4 ARTIGOS _________________________________________________________ 32
4.1 ARTIGO I ____________________________________________________________ 32
4.2 ARTIGO II ___________________________________________________________ 48
4.3 ARTIGO III __________________________________________________________ 65
4.4 ARTIGO IV ___________________________________________________________ 89
PARTE III _________________________________________________________ 110
5 DISCUSSÃO______________________________________________________ 111
REFERÊNCIAS ____________________________________________________ 117
13
PARTE I
14
1
INTRODUÇÃO
1.1
Definição
O Transtorno do Humor Bipolar (THB) é uma forma de transtorno de
humor caracterizado pela variação extrema do humor entre uma fase maníaca ou
hipomaníaca, (hiperatividade, euforia, inconsequência, risco a segurança financeira,
problemas de relacionamentos, irritabilidade, pensamento acelerado, idéias grandiosas,
grande imaginação), uma fase de depressão (humor depressivo, idéias de ruína, inibição,
lentidão para conceber e realizar idéias, ansiedade ou tristeza) (Shansis & Cordioli,
2005) e uma apresentação mista (sintomas maníacos e depressivos associados)
(Kapczinsky & Quevedo, 2009).
Esta definição está baseada no Diagnostic and Statistical Manual of Mental
Disorders – Text Revision (DSM IV-TR, 2000) publicado pela American Psychiatry
Association (APA, sigla do Inglês) e classificado pelo International Statistical
Classification of Diseases and Related Health Problems (CID-10, sigla do Inglês)
(World Health Organization, 1992).
O DSM IV-TR faz uma distinção entre as duas formas maiores do THB:
Tipo I (mania e depressão) e Tipo II (hipomania e depressão), além de definir uma
forma mais leve que é a ciclotimia (flutuações entre depressão subsindrômica e
hipomania) e conceituar outras formas que não se encaixariam nos três subgrupos
anteriores: THB SOE (sem outra especificação), dentre os quais estariam as
apresentações do chamado “espectro bipolar” (DSM IV-TR, 2002; Cordioli et al.,
2010).
1.2
Epidemiologia
O THB é um transtorno crônico, recorrente e, muitas vezes, com remissão
incompleta, potencialmente letal e que afetam um grande número de pessoas (Goodwin
15
& Jamison, 2003). Cerca de 30% dos pacientes fazem tentativas de suicídio e cerca de
20% acabam por completar este intento (Moreno & Andrade, 2005). Estes transtornos
estão associados a grandes custos pessoais e sociais e pelo menos uma proporção de
casos é refratária ao tratamento convencional (Schatzberg et al., 1998).
A prevalência é similar entre os sexos tanto para o THB I (Weissman et al.,
1991; Kessler et al., 1997; Szádóczky et al., 1998; Tem Have et al., 2002; Schaffer et
al., 2006) quanto para o THB II (Moreno & Andrade, 2005; Merikangas et al., 2007),
embora exista claramente uma predominância para o sexo feminino (Moreno & Dias,
2002).
Vários estudos epidemiológicos foram desenvolvidos em diferentes países a
partir da década de 80 (Jenkins & Meltzer, 1995; Mason & Wilkonson, 1996; Mithcell
et al., 2004) . Dois destes estudos são os de maior relevância – Epidemiologic
Catchment Area (ECA, sigla do inglês) (Posternak & Zimmerman, 2002) e National
Comorbidity Survey (NCS, sigla do inglês) (Kessler et al., 1994).
O Estudo ECA–NIMH (Área de Captação Epidemiológica do Instituto
Nacional de Saúde Mental, sigla do Inglês) nos Estados Unidos encontrou prevalência
durante a vida para o THB I de 0,8%, e, para o THB II 0,5% (Robbins & Regier, 1991;
Weissman et al., 1996). Freeman e colaboradores (2002) demonstraram que pacientes
com transtorno bipolar apresentam prevalência de 21% para transtorno do pânico e
transtorno obsessivo compulsivo (TOC), comparados a 0,8% e 2,6% para a população
em geral. No estudo do NCS, a prevalência de transtornos ansiosos em geral no THB foi
de 92,9% contra 24,9% da população.
Observa-se, entretanto, que trata-se de um transtorno com grande
probabilidade de coexistência com outras comorbidades características de outros
transtornos psiquiátricos, tais como, transtorno de déficit de atenção e hiperatividade
(TDHA), depressão e transtorno obsessivo compulsivo (TOC) (Katzow et al., 2003;
Angst et al., 2007; Mcintyre et al., 2008) o que dificulta o diagnóstico.
Em media 30% dos pacientes com THB não recebem tratamento, não foram
diagnosticados ou receberam tratamento equivocado (Faravelli et al., 2009). Quando o
conceito de THB é ampliado para o chamado Transtorno do “Espectro” do Humor
Bipolar (TEHB), a prevalência aumenta para até 6% (Goodwin & Jamison, 2003; Judd
et al., 2003). Em clínicas psiquiátricas, em torno de 50% dos pacientes apresentam
THB (Schatzberg et al., 2003).
16
O Estudo Multicêntrico Brasileiro de Morbidade Psiquiátrica (AlmeidaFilho et al., 1997) obteve estimativas de prevalência de transtornos mentais em três
grandes centros urbanos brasileiros: Brasília, São Paulo e Porto Alegre. Neste estudo foi
encontrada a prevalência de 0,3 - 1,1% para THB (Moreno & Andrade, 2005; Lima et
al., 2005).
Após o primeiro episódio, há um risco de aproximadamente 90% de o
paciente ter outro episódio em algum momento de sua vida. Dentre os pacientes que se
apresentam com um episódio depressivo, há uma chance de 5 a 15% de que estes sejam
efetivamente
bipolares
e
não
unipolares
(European
College
of
Neuropsychopharmacology, 2001). Estes transtornos iniciam-se antes dos 30 anos de
idade, mas podem começar em qualquer idade (inclusive na infância) o que
possivelmente o início precoce aumenta o risco de piores prognósticos em geral, e
particularmente de ciclagem rápida, ideação suicida e comorbidades com transtornos
relacionados a substâncias (Bauer & Pfennig, 2005; Leboyer et al., 2005). Em um
estudo comunitário transversal, onde mesmo com a dificuldade na estimativa da idade
de início (pelo viés da memória de início), situa a idade de início entre o fim da
adolescência e o começo da idade adulta (Leboyer et al., 2005). Na realidade, hoje em
dia já se sabe que, muitos anos antes do surgimento de um primeiro episódio maior (seja
de mania, depressão ou misto), o portador de THB já apresentava desde muito
precocemente na sua vida sintomas ditos subsindrômicos. Inclusive, cada vez mais, têm
sido valorizadas as apresentações mistas que se iniciariam muito precocemente na
infância ou adolescência e que merecem uma abordagem psicofarmacológica
diferenciada (Chengappa et al., 2003; Leboyer et al., 2005).
O THB ocorre com igual frequência em homens e mulheres. Salienta-se,
ainda, que o curso da doença varia de um paciente para outro e pode ocorrer, inclusive,
uma grande variação de um episódio para outro em um mesmo paciente (Hirschfeld et
al., 2002b); ainda que, geralmente, a forma de apresentação no mesmo paciente tende a
ser mais ou menos semelhante nos vários episódios ao longo de sua vida (Schaffer et al.,
2006). Indivíduos com transtorno bipolar têm uma maior probabilidade de dependerem
de recursos públicos e de estarem desempregados (Ten Have et al., 2002; Kessler et al.,
2007).
Dados genéticos indicam fortemente que um fator significativo no
desenvolvimento dos transtornos é a genética, sendo esta mais fortemente associada ao
transtorno bipolar e esquizofrenia (Bass et al., 2009; Hamshere et al., 2009) do que para
17
o transtorno depressivo (Levinson et al., 2006). Ainda que não avaliada em estudos de
comunidade, a história familiar deve ser incluída como importante fator de risco. A
relação entre THB e história familiar está bem estabelecida e o risco é consistentemente
aumentado em parentes de primeiro grau quando comparados com controles normais ou
com depressão maior (Lima et al., 2005).
O estado civil parece estar implicado no THB, sendo que a história de
divórcio, independente do estado civil atual, tem sido associado ao transtorno (Kessler
et al., 1997; Merikangas et al., 2007).
Variações étnicas significativas não são encontradas nos grandes estudos
populacionais (Lima et al., 2005).
1.3 Etiologia
A base causal para os transtornos do humor é desconhecida, mas os fatores
causais podem ser divididos em genéticos, biológicos e psicossociais. Essa divisão é
didática, em razão da probabilidade de os três campos interagirem entre si (Frey et al.,
2004). Os THB são vistos atualmente como transtornos predominantemente
neurobiológicos com expressão psicológica nos quais há em geral, porém não
necessariamente, a presença de múltiplos eventos estressores psicossociais inespecíficos
agindo como desencadeadores ou mantenedores de episódios (Frey et al., 2004).
O modelo de interação gene e ambiente, adotado por Caspi e Moffitt (2006)
assume que eventos (estressores) ambientais podem desencadear trastornos mentais em
indivíduos geneticamente suscetíveis. O termo alostase se refere ao esforço extra que o
organismo emprega na adaptação dos sistemas fisiológicos às diversas situações de
estresse - “estabilidade através da mudança”. Este “esforço adaptativo” atua por meio
de vias neuronais, neurohormonais, metabólicas, imunológicas e de controle do
metabolismo energético. A constante ativação deste mecanismo de resgate geraria um
desgaste cumulativo, que é chamado de carga alostática. Assim, Kapczinsky e
colaboradores (2008) mostram que existe uma complexa interação entre as alterações
genéticas e ambientais que podem induzir mudanças já documentadas no eixo
18
hipotálamo-hipofise-adrenal (HPA – sigla do inglês), no sistema imunológico, o
aumento do estresse oxidativo e alterações em neurotrofinas (O’Connor et al., 2000).
Finalmente, as bases biológicas do THB mostram existir um quadro
complexo de interação entre os múltiplos genes que causam suscetibilidade, bem como
a relação destes com os fatores ambientais e suas consequências para o organismo
(Fernández et al., 2006). Este conceito corresponde ao modelo da “diátese do estresse”
(Eberhart & Hammen, 2010; Schnittker et al., 2010).
1.4
Fisiopatologia
1.4.1 Áreas Cerebrais e THB
Como acima discutido, apesar de THB ser um transtorno psiquiátrico
importante e comum, a base neuropatológica específica é desconhecida (Chen et al,
2011). Entretanto, nos últimos 15 anos os estudos post mortem e o aprimoramento de
técnicas de neuroimagem - principalmente a de ressonância magnética (MRI, sigla do
inglês), a tomografia por emissão de pósitrons (PET, sigla do inglês), e mais
recentemente a espectroscopia por ressonância magnética (MRS, sigla do inglês) e
espectroscopia por ressonância magnética funcional (fMRI, sigla do inglês) tem
produzido vários estudos a fim de clarear os substratos neurais do THB (Drevets WC,
2001; Strakowski et al., 2005; Chen et al., 2011).
Os principais sintomas de THB que são instabilidade afetiva, anormalidades
neurovegetativas, impulsividade e psicose sugerem que o sistema límbico anterior age
controlando esses comportamentos (Strakowski et al., 2005). Essa disfunção envolve as
principais estruturas do sistema límbico, como a amídala que modula um sistema
interativo córtex pré-frontal-estriado-hipotálamo que controlam os comportamentos
sócio-emocionais (Strakowski et al., 2005; Cummings JL, 1993).
Em relação à amídala, um estudo de neuroimagem mostrou uma diminuição
significativa dessa área em pacientes bipolar (Pearlson et al., 1997; Drevets et al., 2001).
No córtex pré-frontal, dois estudos também demonstraram diminuição em pacientes
com THB (Strakowski et al., 2000; 2002). Estudos de espectroscopia têm demonstrado
19
anormalidades na membrana e no metabolismo de segundos mensageiros no estriado e
no córtex pré-frontal de pacientes bipolares (Strakowski et al., 2000; Cummings et al.,
1993).
Adicionalmente, alterações e diminuição no hipocampo também têm sido
descrito no THB (Rosokija et al., 2000; Bertolino et al., 2003). Chen e colaboradores
(2006), em um estudo de neuroimagem funcional demonstraram uma ativação anormal
no hipocampo durante testes de emoção, atenção e memória. Concordando com achados
neurofisiológicos sobre a deficiência cognitiva nos episódios agudos do humor e uma
significante diminuição da memória declarativa durante a remissão (Beardem et al.,
2006a; 2006b).
Esses estudos juntos sugerem fortemente que THB está associado
principalmente a alterações na amídala, córtex pré-frontal, estriado e hipocampo,
resultando em prejuízo cognitivo e desregulação do humor em pacientes bipolares.
1.4.2 Bases Biológicas e THB
As bases biológicas do THB estão relacionados com a genética, as vias
neurohormonais, de transcrição de sinal, neurotrofinas, metabolismo energético, entre
outros (Frey et al., 2004).
Embora o THB apresente um elevado padrão de herdabilidade, a busca por
genes de suscetibilidade tem demonstrado que a maioria das pesquisas envolvendo
estudos de genes únicos apresenta resultados negativos. Dados na literatura sugerem,
entretando, que alguns genes funcionais apresentam modesta, mas significativa
associação com aumento de suscetibilidade para THB. Estão associados com a
suscetibilidade o gene transportador de serotonina (5HTTR), o gene da enzima
degradadora de catecolaminas, catecol-O-metiltransferase (COMT), o gene da enzima
que degrada as monoaminas, monoamine oxidase A (MAOA), e o gene do fator
neurotrófico derivado do cérebro (BDNF) (Frey et al., 2004).
O eixo HPA integra respostas a um estressor e sua ativação resulta na
liberação de cortisol ou corticosterona do córtex da glândula adrenal. A ativação do eixo
HPA, juntamente com o aumento da atividade catecolaminérgica e a integração com
outros sistemas neuroedócrinos regula a função vascular e a captação de energia,
20
facilitando as respostas comportamentais adequadas e servindo para manter a
homeostase (Meaney et al., 1993). Este processo ocorre com a produção e secreção do
hormônio liberador de corticotrofina (CRH, sigla do Inglês) e arginina-vasopressina
pelo hipotálamo, esses hormônios ativarão a hipófise anterior promovendo assim a
liberação do hormônio adrenocorticotrófico (ACTH, sigla do Inglês), o qual promoverá
a secreção de glicocorticóides pelo córtex da adrenal (Handa et al.,1994; Francis et al.,
1996; Herman & Cullinan, 1997). Os glicocorticóides por sua vez regulam diversas
funções que visam a manutenção dos processos metabólicos básicos, assim como a
regulação do organismo em resposta ao estresse (Levine et al., 2001). Estudos prévios
reportaram que o estresse pode causar dano e atrofia de neurônios em certas estruturas
cerebrais, mais notavelmente no hipocampo (Magariños & Mcewen, 1995; Sapolsky et
al., 1996; Duman RS, 2004). Entre as funções hipocampais estão incluídas controle do
aprendizado e da memória e regulação do eixo HPA, as quais estão envolvidas na
depressão e THB (Meaney et al., 1993; Frey et al, 2004; Machado-Vieira et al, 2005).
Além disso, o hipocampo tem conexões com o córtex pré-frontal e a amigdala, regiões
que estão diretamente envolvidas na emoção e na cognição, podendo estar associados a
outros principais sintomas da depressão e do THB (Belmaker RH, 2004; Duman &
Monteggia, 2006).
A alteração do metabolismo energético mitocondrial com consequente
prejuízo na produção energética da célula é uma hipótese atrativa para explicar a
fisiopatologia do THB (Stork & Renshaw, 2005). Um estado energético celular anormal
pode levar a perda da função e da plasticidade neuronal, e consequentemente, a
alterações cognitivas e comportamentais característicos da depressão e THB (Manji &
Lenox, 2000; Dzeja & Terzic, 2003).
O cérebro desenvolve intensa atividade metabólica e para isso ele utiliza
energia na forma de trifosfato de adenosina (ATP, sigla do inglês) (Ames A, 2000),
parte dessa energia é formada na cadeia de transporte de elétrons. A cadeia de transporte
de elétrons é composta por cinco complexos enzimáticos (complexos I, II, III, IV e V) e
dois componentes que não fazem parte dos complexos, a coenzima Q, que transporta
elétrons do complexo I e II ao complexo III, e o citocromo c, que transporta elétrons do
complexo III ao complexo IV. A energia é utilizada ao permitir-se o fluxo de prótons a
favor do gradiente de concentração através da enzima ATP sintase ou complexo V
(Törnroth-Horsefield & Neutze, 2008). Os elétrons presentes nas coenzimas NADH e
FADH2 são transferidos para o complexo I, do complexo I para coenzima Q, depois
21
para o complexo III, citocromo c, complexo IV e finalmente para o oxigênio (Wallace
DC, 1999). A sequência da reação, coordenada pela presença do íon magnésio, é utilizar
um ADP em presença de um fósforo inorgânico (Pi) sendo catalizada pelo complexo V
em ATP (Törnroth-Horsefield & Neutze, 2008).
Figura 1. Representação esquemática da cadeia respiratória mitocondrial.
As mitocôndrias das células neurais e gliais são particularmente importantes
para as funções cerebrais, pois fornecem a energia requerida para a atividade da sinapse
e também estão ligadas às funções metabólicas (Ly & Verstreken, 2006; Ledoux et al.,
2007). Alguns autores têm demonstrado uma relação entre o THB e o metabolismo
energético (Konradi et al., 2004; Kato & Kato, 2000; Calabrese et al., 2001).
Em estudo post mortem avaliando genes nucleares em hipocampo de
pacientes bipolares, foi encontrada diminuição na expressão de inúmeros genes
diretamente relacionados à atividade mitocondrial (Konradi et al., 2004). Esse estudo
corrobora dados anteriores e a hipótese de Kato, da universidade de Tóquio, que sugere
depleção mitocondrial e alteração no metabolismo energético de pacientes bipolares
(Kato & Kato, 2000). Entretanto, os resultados que reforçam essa hipótese são ainda
pouco conclusivos, justificando a necessidade de mais estudos sobre o metabolismo
energético no THB.
De fato, a mitocôndria esta envolvida nas comunicações intercelulares (Ly
et al., 2006) e sua disfunção esta ligada ao THB (Konradi et al., 2004). Efeitos de
antipsicóticos, inclusive a olanzapina (OLZ), mostraram inibir o metabolismo
energético em cérebro de ratos (Streck et al., 2007). Por outro lado Bosch e
22
colaboradores (2005) mostram que os efeitos da combinação de OLZ com Fluoxetina
(FLX) sustenta o nível extracelular de monoaminas no córtex prefrontal de ratos.
Além da cadeia respiratória mitocondrial, a creatina quinase (CK, sigla do
inglês), uma enzima com papel central no metabolismo energético, recentemente vem
sendo estudada em transtornos psiquiátricos (Andres et al., 2008). No cérebro a CK é
importante em funções, tais como o tamponamento energético (regenerando ATP) e a
transferência do ATP dos sítios produtivos para os de consumo (Wyss et al., 1992;
Kaldis et al., 1996; Wyss & Kaddurah-Daouk, 2000).
Figura 2. Representação esquemática da atividade da enzima creatina quinase.
Recentemente foi mostrada uma diminuição na atividade da enzima CK em
cérebro de ratos submetidos ao um modelo animal de mania (Streck et al., 2008). Em
humanos com transtorno do humor bipolar também foram encontrados níveis anormais
da enzima CK (Meltzer HI, 2000). Além disso, Segal e colaboradores (2007) avaliaram
os níveis séricos da enzima CK em uma amostra que continha indivíduos com
transtorno depressivo maior em episódio depressivo psicótico e não psicótico.
Adicionalmente a amostra incluiu sujeitos com transtorno esquizoafetivo e transtorno
bipolar que se apresentavam em episódios depressivos. Os resultados apontaram para
um aumento nos níveis de CK na depressão maior não-psicótica, comparado aos outros
grupos. Recentemente, Assis e colaboradores (2007) mostram que a OLZ (10 mg/kg),
em um modelo agudo, aumenta a atividade da CK no estriado de ratos. Entretanto
baixas doses (2,5 e 5 mg/kg) inibe a atividade da enzima no cerebelo e prefrontal.
Alguns inibidores seletivos da recaptação de serotonina (ISRS, sigla do inglês) mostam
alterações na atividade da enzima CK (Assis et al., 2009).
A enzima citrato sintase (CS) inicia o primeiro passo do ciclo de Krebs
(Marco, et al., 1974). Esta localizada na matriz mitocondrial onde cataliza a
condensação de oxaloacetato e o grupo acetil da acetil coenzima-A (acetil CoA) mais
H2O gerando citrato e CoA. Esta enzima é inibida por altas concentrações de ATP,
23
acetil CoA e NADH quando o suprimento de energia celular esta alta. Esta regulação
assegura que o ciclo de Krebs não oxide o piruvato e acetil CoA em excesso quando a
concentração de ATP esta alta (Shepherd & Garland, 1969).
Figura 3. Respresentaçao esquemática da atividade da enzima citrato sintase.
Estudos demonstraram que ratos submetidos a um modelo animal de mania
tiveram uma redução na atividade das enzimas CS em tecido cerebral (Correa et al.,
2007) sugerindo que esta enzima esta envolvida em distúrbios do humor.
Antidepressivos e estabilizadores do humor e a OLZ foram associados a modularem a
atividade da enzima CS em cérebro de ratos (Hroudova et al., 2010). Scaini e
colaboradores (2010) encontraram aumentada a atividade da CS na administração
crônica com os ISRS no prefrontal, estriado, córtex e hipocampo de ratos.
As neurotrofinas são fatores intermediários que regulam diferenciação e
sobrevivência de neurônios e modulam a plasticidade e a transmissão sináptica. Além
disso, agora se sabe também que a inibição da morte celular (apoptose) é outro
componente
importante
da
sua ação.
Estes
fatores podem ser secretados
constitutivamente em um curto intervalo ou ainda dependendo da atividade neuronal.
As neurotrofinas ligam-se e ativam uma família específica de receptor de tirosina
quinase (TrK) promovendo uma regulação complexa no SNC. Devido a esses fatores,
essenciais para funcionamento e sobrevivência neuronal supõem-se que a viabilidade
neuronal pode ser afetada pela redução persistente dessas neurotrofinas no SNC (Du et
al., 2003). Um peptídeo chamado de fator de crescimento neuronal (NGF, da sigla em inglês) foi o
primeiro fator trófico a ser identificado. O NGF é produzido pelos alvos dos axônios na divisão
sináptica do sistema nervoso (Caggiula et al., 2005). Esse peptídeo, produzido e liberado pelos
tecidos-alvos, é absorvido pelos axônios sinápticos e transportado retrogradamente, promovendo a
sobrevivência neuronal; entretanto, mesmo com a liberação de NGF pelos tecidos-alvos pode
ocorrer morte celular se o transporte axoplasmático for interrompido (Schramm et al., 2005). As
expansões neuronais em desenvolvimento projetam-se em áreas com concentrações elevadas de
24
NGF, que se liga a receptores específicos para sofrer passagem endocitótica, uma vez no citoplasma,
o NGF é carreado por transporte axonal retrógado rápido para o soma, onde promove ações tróficas
(Caggiula, et al., 2005). O NGF é um dos membros da família das proteínas tróficas coletivamente
chamadas de neurotrofinas. Os membros dessa família incluem a neurotrofina-3 (NT-3), a
neurotrofina-4 (NT-4) e o fator neurotrófico derivado do cérebro (BDNF, sigla do inglês).
São requeridos para a diferenciação e a sobrevivência de subpopulações neuronais
específicas, tanto no sistema nervoso periférico, como no SNC (Kalb et al., 2005). O BDNF
promove a expansão dos axônios dos neurônios da dopamina e da acetilcolina. Níveis
mais altos de atividade neuronal estimulam a liberação de BDNF. Em alguns estudos,
foram demonstrados que os ratos que não podem produzir o BDNF morrem em poucas
semanas e os animais que estão vivendo em ambientes de alto estresse produzem níveis
mais baixos desse fator (Frey et al., 2006 a;b;c; Obata & Noguchi, 2006).
Figura 4. Representação esquemática ligação das neurotrofinas com os receptores tirosina quinase.
Recentes trabalhos demonstram que uma diminuição de plasticidade e
resiliências celulares podem estar envolvidas no THB, e que antidepressivos e
estabilizadores do humor exercem efeitos em vias de sinalização que regulam a
plasticidade celular (Payne et al., 2003; Coyle & Duman, 2003; Duman RS, 2004;
Duman & Monteggia, 2006). Vários estudos demonstram uma redução no volume de
algumas regiões do cérebro acompanhada pela atrofia e perda celular em pacientes com
25
THB. Assim como estudos estruturais de imagem que demonstram um volume de massa
cinzenta reduzida no córtex pré-frontal, estriado e ventrículo em transtornos do humor
(Drevets WC, 2001; Goodwin, et al., 2003; Manji & Duman, 2001). Além disso,
estudos neuropatológicos posmortem têm mostrado redução no volume cortical e no
tamanho das células gliais no córtex pré-frontal e na amígdala (Drevets et al., 2001;
Goodwin et al., 2003; Manji & Duman, 2001). Permanece ainda a duvida se esses
danos promovem anormalidades em episódios do humor ou se essas sequelas são
produzidas por episódios recorrentes do humor (Shaltiel et al., 2007). Em estudos
recentes do nosso laboratório foi demonstrado um aumento de BDNF (Frey et al.,
2006a), de NGF (Frey et al., 2006c) e de NT-3 (Walz et al., 2007) em tecido cerebral de
ratos Wistar submetidos ao modelo animal de mania que foram parcialmente revertidos
quando tratados com Li e VPA. Estes resultados reforçam a idéia de que os efeitos
terapêuticos dos estabilizadores do humor podem estar associados com o aumento de
fatores neurotróficos, implicando a possível participação dos mesmos no THB.
Além disso, foram encontrados níveis reduzidos de BDNF em pacientes
com recaída de esquizofrenia crônica tratados com antipsicoticos típicos ou atípicos
(Rizos, et al., 2010). Também recentemente, um estudo demonstrou um aumento na
transcição do gene para BDNF com olanzapina e que esta associada a vias de
sinalizaçao celular. Assim, sugere que a OLZ pode exercer um efeito de proteção para
células neuronais (Lee et al., 2010).
1.5
Diagnóstico
O diagnóstico do THB consiste da identificação de três possíveis fases
(maníaca, hipomaníaca ou mista) acompanhado ou não de episódio depressivo. Um
episódio de humor deprimido consiste na diminuição do interesse ou prazer, perda ou
ganho de peso significativo, insônia ou hipersonia, agitação ou retardo psicomotor,
sentimento de inutilidade ou culpa excessiva, capacidade diminuída de pensar ou
concentrar-se, pensamentos de morte recorrentes (DSM IV, 2004; CID-10, 2002). Uma
das fases depressivas com maior dificuldade de tratamento ocorre no THB (Tohen et al.,
2010).
26
O episódio maníaco consiste em um quadro psicótico grave caracterizado
por grande agitação, loquacidade, euforia, insônia, perda do senso crítico,
grandiosidade, prodigalidade, exaltação da sexualidade, e heteroagressividade (DSM
IV-TR, 2004; CID-10, 2002).
O episódio hipomaníaco consiste na alteração de humor semelhante à
mania, porém com menor intensidade e que, segundo o DSM IV, pelo menos três destes
sintomas deverão ser encontrados: auto-estima em alta ou grandiosidade sem delírios,
pouca necessidade de sono, compulsão para falar demais; fuga de idéias e pouca
concentração; prática de mais atividades dirigidas a objetivos; agitação psicomotora;
excesso de atividades prazerosas com alto potencial para conseqüências danosas ou
ainda episódio misto (DSM IV-TR, 2004; CID-10, 2002).
Episódios mistos representam uma mistura simultânea de manifestações
depressivas e maníacas ou hipomaníaca. Uma minoria de pacientes experimenta apenas
um episódio maníaco e a maioria apresenta episódios que oscilam entre os dois pólos
(DSM IV, 2004; CID-10, 2002).
1.6
Tratamento
Desde a década de 60, até o início da década de 80, o Li, considerado o
primeiro estabilizador do humor, teve um papel predominante no tratamento agudo e
profilático do THB (Schou et al.,1954; Maggs R, 1963; Goodwin et al., 1969, Stokes et
al.,1971).
Desde
então,
a
Carbamazepina
e
após,
gradualmente,
o
ácido
valpróico/valproato foram sendo usados (Okuma et al., 1979; 1981; 1990; Post et al.,
1983; Pope et al., 1991; Freeman et al., 1992; Bowden et al., 1994). Estes dois
anticonvulsivantes passaram a ser utilizados em função da percepção de que nem todos
os pacientes respondiam ao Li (a resposta ao Li, em geral, situa-se em torno de 50 a
70%, a depender do tipo de apresentação do episódio e de outros fatores) e também
passaram a ser consideradas como alternativas aos efeitos colaterais causados por esse
estabilizador (o que acarretava menor aderência ao tratamento ao Li).
Durante a década de 90 novas drogas anticonvulsivantes foram sendo
testadas no tratamento do THB como a Gabapentina, a Lamotrigina e o Topiramato,
27
bem como um análogo da Carbamazepina, a Oxcarbamazepina (Frye et al., 2000; Pande
et al., 2000; Calabrese et al., 1999; Hummel, et al., 2002). Além disso, nesta mesma
época, foram comprovadas funções neuroprotetoras do Li (Chuang et al., 2002).
Ainda, nesta última década foram sendo demonstradas propriedades
estabilizadoras de alguns antipsicóticos atípicos como a Risperidona, a Ziprazidona, a
Quetiapina e, em especial, a OLZ (Yatham et al., 2004; Keck et al., 2003; Tohen, et al.,
2003; Macritchie et al., 2003).
Cabe ressaltar que as antigas e já comprovadas propriedades da
eletroconvulsoterapia (ECT) permanecem sendo, ao longo de todas estas décadas,
medidas eficazes e de ação rápida em determinadas apresentações bipolares (Medda et
al., 2010).
Desde então, a definição do que é uma droga estabilizadora do humor tem
sido motivo de debate na literatura especializada em função de uma variedade cada vez
maior de drogas utilizadas no tratamento do THB (Licht et al., 2000; Ghaemi et al.,
2001; Ghaemi SN, 2001; Perlis et al., 2002).
Os antipsicóticos têm sido amplamente utilizados no tratamento do THB
especialmente em combinação com o Li (Vieta et al., 2004). A clorpromazina e o
haloperidol são os antipsicóticos típicos mais bem estudados no tratamento no THB. A
literatura sugere que o haloperidol tem propriedades contra a mania e um início de ação
mais rápido em comparação com os antipsicóticos atípicos (Yildiz et al., 2011; Chan et
al., 2010). Efeitos colaterais dos típicos como sintomas extrapiramidais e discinesia
tardia e a as taxas maiores de indução da depressão (Yildiz et al., 2011) levou o FDA
(Food and Drug Administration, sigla do Inglês) ate 2008 a aprovar antipsicóticos
atípicos para o tratamento da mania bipolar (Maloney & Sikich, 2010).
A OLZ foi estudada em comparação com outros antipsicóticos típicos e
atípicos, estabilizadores do humor e anticonvulsivantes no tratamento do THB. (Vieta et
al., 2004; 2010; Tollefson et al., 1998; Tohen et al., 1999; 2000; 2002; 2003a;b; 2005).
A superioridade da OLZ foi demonstrada a partir das taxas de remissão
significativamente maiores nos pacientes bipolares que não apresentavam sintomas
psicóticos com menores taxas para indução de depressão quando comparados com
outros típicos e pontuação total da escala YMRS menores. Foram encontrados também
menores pontuações na escala YMRS quando comparada com outros estabilizadores
como o divalproato (Tohen et al., 2002) e o Li (Tohen et al., 2005). Portanto, a OLZ
28
tem se mostrado efetiva também para a depressão severa (Shelton et al., 2001) e THB
(Tohen et al., 1999; 2000; 2002; 2003; 2005).
Recentes estudos mostram um sinergismo entre o efeito a OLZ e a FLX
(Kodama et al., 2004; Maragnoli et al., 2004; Seager et al., 2004). A combinação destes
fármacos tem se mostrado eficaz no tratamento do THB (Rothschild et al., 2004; Shi et
al., 2004). O mecanismo neural preciso, responsável por esta eficácia ainda não está
claramente entendido (Seager et al., 2005). Sabe-se que a combinação de inibidores de
recaptação seletiva de serotonina (SSRI, sigla do inglês) como a FLX, com
antipsicóticos atípicos como a OLZ melhoram significantemente o tempo e a
intensidade da resposta no tratamento em pacientes depressivos resistentes quando
comparados com outros tratamentos sem combinações. A coadministração de OLZ e
FLX têm mostrado efeitos sinérgicos ligados à transmissão noradrenérgica e
dopaminérgica no córtex pré-frontal (Bosch, et al., 2005; Vieta, et al., 2010).
Mais recentemente o FDA aprovou o uso da combinação fixa de OLZ e
FLX (SymbyaxTM – Lilly) para o tratamento de episódios depressivos associados á
depressão bipolar (Shelton RC, 2006; Tohen et al., 2010, Bobo & Shelton 2010; Vieta
et al., 2010).
29
2
JUSTIFICATIVA
Tratar um paciente potencialmente bipolar possui problemas especiais pelo
risco de virada maníaca, alta propensão a recorrências inclusive depressivas, o risco
aumentado de suicídio e a pobre resposta aos antidepressivos (Rihmer & Kiss, 2002;
Katzow et al., 2003; Hawton et al., 2005; McIntyre et al., 2008). Estudos indicam que
pacientes que se encontram na fase depressiva do THB permanecem mais tempo nesta
fase quando comparada com a fase maníaca e, consequentemente levando mais tempo
para se recuperarem, mesmo quando em tratamento com estabilizadores do humor
(Angst et al., 2007). Porém, o uso de antidepressivos durante essa fase pode
potencialmente levar ao suicídio (Ghaemi et al., 2001; Faravelli et al., 2009). Dois
estudos estão de acordo. Os estudos conduzido por Shelton e colaboradores (2001) e
Matthews e colaboradores (2002) mostraram que a combinação de OLZ com FLX
obteve resultados significativamente maiores e melhora mais rápida do que a
monoterapia em pacientes com depressão resistente ao tratamento.
A qualidade de vida dos portadores de THB depende, portanto, em grau
importante, do uso de medicações. Sabe-se que com um tratamento adequado as
consequências do THB podem ser bastante controladas e reduzidas. Os efeitos positivos
de um tratamento efetivo são de que muitos pacientes conseguem passar um tempo
maior em condições de levarem suas vidas de forma mais adequada. Para outros, o
tratamento pode, pelo menos, reduzir as consequências negativas do transtorno.
O papel da combinaçao da OLZ/FLX mostra-se eficiente tanto para rápida
resposta para episódios depressivos, taxas de remissão significativamente maiores
associado a baixas possibilidades de virada maníaca em pacientes bipolares (Shelton et
al., 2006). Porém, existe a necessidade de mais estudos para um maior entendimento
deste sinergismo encontrado com a combinação da OLZ/FLX no tratamento do THB.
30
3
3.1
OBJETIVOS
Objetivo Geral
Avaliar as alterações neuroquímicas induzidas pela administração aguda e
crônica de OLZ e/ou FLX em ratos Wistar adultos.
3.2
Objetivos Específicos
Avaliar os efeitos da combinaçao de OLZ e FLX na atividade da enzima CK
em cérebro de ratos Wistar adultos,
Avaliar os efeitos da combinaçao de OLZ e FLX na atividade da enzima CS
no cérebro de ratos Wistar adultos,
Avaliar os efeitos da combinaçao de OLZ e FLX no metabolismo energético
de ratos Wistar adultos,
Avaliar os efeitos da combinação de OLZ e FLX na expressão de
neurotrofinas (BDNF, NGF e NT-3) em cérebro de ratos Wistar adultos,
31
PARTE II
32
4
ARTIGOS
4.1
Artigo I
Artigo publicado na revista Brain Research Bulletin, 2009.
33
EFFECTS OF OLANZAPINE, FLUOXETINE AND
OLANZAPINE/FLUOXETINE ON CREATINE KINASE ACTIVITY IN RAT
BRAIN
Fabiano R. Agostinho, Giselli Scaini, Patricia M. Santos,
Gabriela K. Ferreira, Isabela C. Jeremias, Gislaine T. Rezin, Alexandra I. Zugno, João
Quevedo, Emilio L. Streck
1
Laboratório de Fisiopatologia Experimental, Programa de Pós-graduação em Ciências
da Saúde, Universidade do Extremo Sul Catarinense, 88806-000 Criciúma, SC, Brazil;
2
Laboratório de Neurociências, Programa de Pós-graduação em Ciências da Saúde,
Universidade do Extremo Sul Catarinense, 88806-000 Criciúma, SC, Brazil.
Correspondence: Prof. Emilio L. Streck,
Laboratório de Fisiopatologia Experimental,
Universidade do Extremo Sul Catarinense, 88806-000, Criciúma, SC, Brazil.
Fax: +55 48 3341 2644.
E-mail: emiliostreck@gmail.com
34
Abstract
Bipolar disorder is a devastating major mental illness associated with higher
rates of suicide and work loss. Recently, a fixed combination of the atypical
antipsychotic olanzapine and the serotonin selective reuptake inhibitor (SSRI)
fluoxetine (SimbiaxTM) has been approved in the US for the treatment of bipolar I
depression. It is well described that inhibition of creatine kinase (CK) activity has been
implicated in the pathogenesis of a number of diseases like bipolar I depression,
especially in the brain. In this work, we evaluated the effect of acute and chronic
administration of fluoxetine, olanzapine and the association of fluoxetine/olanzapine on
CK activity in the brain of rats. For acute treatment, adult male Wistar rats received one
single injection of olanzapine (3 or 6 mg/kg) and/or fluoxetine (12.5 or 25 mg/kg). For
chronic treatment, adult male Wistar rats received daily injections of olanzapine (3 or 6
mg/kg) and/or fluoxetine (12.5 or 25 mg/kg) for 28 days. In the present study we
observed that acute administration of OLZ inhibited CK activity in cerebellum and
prefrontal cortex. The acute administration of FLX inhibited creatine kinase in
cerebellum, prefrontal cortex, hippocampus, striatum and cerebral cortex. In the chronic
treatment, when the animals were killed 2 hours after the last injection a decrease in
creatine kinase activity after FLX administration, alone or in combination with OLZ, in
cerebellum, prefrontal cortex, hippocampus, striatum and cerebral cortex of rats
occurred. However, when the animals were killed 24 hours after the last injection, we
found no alterations in the enzyme. Although it is difficult to extrapolate our findings to
the human condition, the inhibition of creatine kinase activity by these drugs may be
associated to the occurrence of side effects.
Keywords: creatine kinase, olanzapine, fluoxetine.
35
INTRODUCTION
Bipolar disorder is a devastating major mental illness associated with higher
rates of suicide and work loss [1,2], and characterized by manic, major depressive,
and/or mixed episodes. The clinical hallmark in the diagnosis of bipolar disorder is the
presence of manic symptoms [1,3]. On the other hand, the depressive phase of bipolar
disorder has been especially difficult to treat. Most controlled clinical research on the
treatment of bipolar disorder has focused on the manic phase, with relatively little
research on treatment of the depressive phase [4].
A fixed combination of antipsychotic and antidepressants drugs was widely
used in medicine and, at one time, was common in psychiatry. A generation ago,
combinations of antidepressants with either antipsychotics (e.g., amitriptyline and
perphenazine [Etrafon™, Triavil™]) or benzodiazepines (e.g., amitriptyline and
chlordiazepoxide [Limbitrol™]) were widely used by both psychiatrists and other
medical practitioners [5]. Recently, a fixed combination of the antipsychotic drug
olanzapine (OLZ) and the antidepressant fluoxetine (FLX) (Symbyax™) has been
introduced for the treatment of bipolar depression [5]. In a controlled study of Shelton et
al. [6], subjects with treatment-resistant depression received OLZ alone, FLX alone, or a
combination of both; the combination was associated with significantly greater and
faster improvement than was either drug alone.
While there is clear clinical benefit from this combination, the precise
neural mechanisms responsible for its efficacy are not clearly understood. Therefore, it
is important to investigate the mechanisms of action of this combination in order to not
only better understand the etiology of the clinical syndromes, but also to eventually
facilitate the development of improved drugs to treat them [7].
Creatine kinase (CK, EC 2.7.3.2) is a crucial enzyme for high energyconsuming tissues like brain, skeletal muscle and heart. This enzyme works as a
buffering system of cellular ATP levels, playing a central role in energy metabolism.
CK catalyses the reversible transfer of the phosphoryl group from phosphocreatine to
ADP, regenerating ATP [8-10]. It is also known that a decrease in CK activity is
associated with neurodegenerative pathways that result in neuronal death in brain
ischemia [11], neurodegenerative diseases [12,13], bipolar disorder [14] and other
pathological states [15,16].
36
Recently, Assis et al. [17] found that OLZ (at 10.0 mg/kg) increased the
activity of CK in striatum in rats; this drug did not affect the enzyme activity in
hippocampus and cerebral cortex. However, the results showed that lower doses of OLZ
(2.5 and 5.0 mg/kg) inhibited the enzyme activity, only in cerebellum and prefrontal
cortex. The inhibitory effect of OLZ on CK activity in cerebellum and prefrontal cortex
suggest that this drug may impair energy metabolism in these brain areas [17].
Considering that the effects of OLZ and FLX on brain energy metabolism
are still poorly known, we evaluated the effects of these drugs (alone or in combination)
on CK activity in rat brain.
MATERIALS AND METHODS
Animals
Adult male Wistar rats (300 g) were obtained from the Central Animal
House of University of Extremo Sul Catarinense (UNESC), Criciúma, SC, Brazil. The
animals were caged in groups of five with free access to food and water and were
maintained on a 12-hr light/dark cycle (lights on 7 a.m.), at a temperature of 22±1ºC.
All experimental procedures were carried out in accordance with the National Institutes
of Health Guide for the Care and Use of Laboratory Animals and the Brazilian Society
for Neuroscience and Behavior (SBNeC) recommendations for animal care, with the
approval of UNESC Ethics Committee.
Drugs
OLZ (Zyprexa™) and FLX (Prozac™) were provided from Eli Lilly do
Brasil Ltda, São Paulo, Brazil.
37
Administration of antipsychotics
Animals received daily intraperitoneal injections of OLZ (3 or 6 mg/kg),
FLX (12 or 25 mg/kg) or combination of both drugs for 28 days in two protocols of
chronic model, and, in the acute model for one day. All the drugs were dissolved in
Tween 1% solution (vehicle). Control animals received vehicle (1.0 mL/kg). In the
acute protocol, after the single injection, the animals were killed 2 hours after by
decapitation, and the hippocampus, striatum, cerebellum, cortex and prefrontal cortex
were immediately removed. Similarly, in the chronic protocols, the animals were killed
2 and 24 hours after the last injection, and the same areas are removed. After that, the
activity of CK was measured.
Tissue and homogenate preparation
Hippocampus, striatum, cerebellum, cortex and prefrontal cortex were
homogenized (1:10, w/v) in SETH (sucrose, EDTA, tris, heparin) buffer, pH 7.4 (250
mM sucrose, 2 mM ethylenediaminetetraacetic acid, 10 mM Trizma base, 50 IU/ml
heparin). The homogenates were centrifuged at 800 × g for 10 min. and the supernatants
kept at −70ºC until used for CK activity determination. The maximal period between
homo-genate preparation and enzyme analysis was always less than 5 days. Protein
content was determined by the method described by Lowry et al. [18] using bovine
serum albumin as standard.
Creatine kinase activity
CK activity was measured in brain homogenates pre-treated with 0.625 mM
lauryl maltoside. The reaction mixture consisted of 60 mM Tris-HCl, pH 7.5, containing
7 mM phosphocreatine, 9 mM MgSO 4 and approximately 0.4–1.2 µ g protein in a final
volume of 100µl. After 15 min. of preincubation at 37ºC, the reaction was started by the
38
addition of 0.3 µmol of ADP plus 0.08µmol of reduced glutathione. The reaction was
stopped after 10 min. by the addition of 1 µmol of hydroxymercuribenzoic acid. The
creatine formed was estimated according to the colorimetric method of Hughes [19].
The colour was developed by the addition of 100 µL 2% α-naphthol and 100 µL 0.05%
diacetyl in a final volume of 1 mL and read spectrophotometrically after 20 minutes at
540 nm. Results were expressed as units/min.mg protein.
Statistical analysis
Data were analyzed by one-way of variance (ANOVA) followed by Tukey
test when F was significant and are expressed as mean ± standard deviation. All
analyses were performed using the Statistical Package for the Social Science (SPSS)
software version 16.0.
RESULTS
In the present study, we evaluated the acute (Figure 1) and chronic (Figures
2 and 3) effects of OLZ and FLX on creatine kinase activity in hippocampus, striatum,
cerebellum, cortex and prefrontal cortex of rats. In the acute protocol (Figure 1), we
verified that OLZ inhibited CK activity only in cerebellum and prefrontal cortex. On the
other hand, hippocampus, striatum and cerebral cortex were not affected. Moreover,
FLX (alone or in combination with OLZ) decreased the activity of creatine kinase in
cerebellum, prefrontal cortex, hippocampus, striatum and cerebral cortex.
In the chronic treatment, when the animals were killed 2 hours after the last
injection (Figure 2), we observed a decrease in creatine kinase activity after FLX
administration, alone or in combination with OLZ, in cerebellum, prefrontal cortex,
hippocampus, striatum and cerebral cortex of rats. OLZ administered alone did not alter
creatine kinase activity. Finally, in the chronic treatment (Figure 3), when the animals
were killed 24 hours after the last injection, we found no alterations in creatine kinase
activity caused by OLZ and FLX.
39
DISCUSSION
The creatine/phosphocreatine/creatine kinase system is important for normal
energy homeostasis by exerting several integrated functions, such as temporary energy
buffering, metabolic capacity, energy transfer and metabolic control [20,21]. The brain
of adult rats, like other tissues with high and variable rates of ATP metabolism, presents
high phosphocreatine concentration and creatine kinase activity. It is well described that
inhibition of creatine kinase activity has been implicated in the pathogenesis of a
number of diseases, especially in the brain [20,21] including depression, bipolar
disorder, and schizophrenia.
The addition of an atypical antipsychotic drug to an antidepressant drug is
observed in clinical practice as a strategy to treat bipolar depression [22, 23] as well as
treatment-resistant depression [20, 21, 24, 25], and psychotic depression [26]. Rapid
remission has been described in relation to OLZ and risperidone augmentation. In a
controlled study [27], subjects with treatment-resistant depression received OLZ alone,
FLX alone, or a combination of both; the combination was associated with significantly
greater and faster improvement than was either drug alone. In this work, we performed
the first study examining the effects of an atypical antipsychotic and a standard
antidepressant association on creatine kinase activity.
Some side effects of antipsychotics limit their long-term use and are
probably associated with oxidative stress and/or energy impairment [4]. However, the
atypical antipsychotic OLZ is associated with less occurrence of side effects than
conventional drugs [17,28].
It is well known that creatine kinase is sensitive to free radicals [29]. We
have already shown that clozapine administration caused oxidative injury in rat brain.
However, we did not find the same effect for OLZ [30]. We also observed that chronic
administration of clozapine inhibited creatine kinase activity in cerebellum and
prefrontal cortex of rats [17]. In the present study we observed that acute administration
of OLZ inhibited CK activity in cerebellum and prefrontal cortex. The acute
administration of FLX inhibited creatine kinase in cerebellum, prefrontal cortex,
hippocampus, striatum and cerebral cortex.
In the chronic treatment, we observed interesting results; when the animals
were killed 2 hours after the last injection a decrease in creatine kinase activity after
40
FLX administration, alone or in combination with OLZ, in cerebellum, prefrontal
cortex, hippocampus, striatum and cerebral cortex of rats occurred. However, when the
animals were killed 24 hours after the last injection, we found no alterations in the
enzyme. With these findings, we suggest that FLX did not present a chronic effect on
creatine kinase activity.
Brain and other high energy tissues are more susceptible to reduction of
energy metabolism. In this context, neuropsychiatry disorders, such as schizophrenia,
depression and bipolar disorder, have been related to dysfunction in brain metabolism
[31-33]. Metabolism impairment includes mitochondrial dysfunction [34,35], increase
in reactive oxygen species production and expression of biochemical markers of cellular
degeneration [36-38].
The inhibitory effect of OLZ and FLX on creatine kinase activity in the
brain of rats leads us to speculate whether these drugs impair energy metabolism.
Although it is difficult to extrapolate our findings to the human condition, the inhibition
of creatine kinase activity by these drugs may be associated to the occurrence of side
effects. Further studies are important to evaluate whether other enzymes involved in
brain metabolism are also affected by these drugs.
ACKNOWLEDGEMENTS
This research was supported by grants from Eli Lilly, Programa de Pósgraduação em Ciências da Saúde – Universidade do Extremo Sul Catarinense (UNESC)
and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).
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46
LEGENDS AND FIGURES
Figure 1. Effect of acute administration of olanzapine (OLZ) and fluoxetine (FLX) on creatine kinase
activity in the brain of rats. hours after the administration of the drugs. Values are expressed as mean ±
S.D. for five independent experiments.
Different from control (saline group), *p<0.05 (ANOVA) followed by Tukey test.
Figure 2. Effect of chronic administration of olanzapine (OLZ) and fluoxetine (FLX) on creatine kinase
activity in the brain of rats. The animals were killed 2 hours after the last administration of the drugs.
Values are expressed as mean ± S.D. for five independent experiments.
Different from control (saline group), *p<0.05 (ANOVA) followed by Tukey test.
47
Figure 3. Effect of chronic administration of olanzapine (OLZ) and fluoxetine (FLX) on creatine kinase
activity in the brain of rats. The animals were killed 24 hours after the last administration of the drugs.
Values are expressed as mean ± S.D. for five independent experiments.
48
4.2
ARTIGO II
Artigo publicado na revista Neuroscience Letters, 2011.
49
TREATMENT WITH OLANZAPINE, FLUOXETINE AND
OLANZAPINE/FLUOXETINE ALTERS CITRATE SYNTHASE ACTIVITY IN
RAT BRAIN
Fabiano R. Agostinhoa, Gislaine Z. Réusa, Roberto B. Stringaria, Karine F. Ribeiroa,
Ana K. Ferraroa, Joana Benedetb, Natália Rochib, Giselli Scainib, Emílio L. Streckb, and
João Quevedoa*.
a
Laboratório de Neurociências and Instituto Nacional de Ciência e Tecnologia
Translacional em Medicina (INCT-TM), Programa de Pós-Graduação em Ciências da
Saúde, Unidade Acadêmica de Ciências da Saúde, Universidade do Extremo Sul
Catarinense, 88806-000, Criciúma, SC, Brazil;
b
Laboratório de Fisiopatologia Experimental and Instituto Nacional de Ciência e
Tecnologia Translacional em Medicina (INCT-TM), Programa de Pós-Graduação em
Ciências da Saúde, Unidade Acadêmica de Ciências da Saúde, Universidade do
Extremo Sul Catarinense, 88806-000 Criciúma, SC, Brazil.
*Corresponding author:
Prof. João Quevedo, MD, PhD
Laboratório de Neurociências,
Programa de Pós-Graduação em Ciências da Saúde,
Unidade Acadêmica de Ciências da Saúde,
Universidade do Extremo Sul Catarinense,
88806-000 Criciúma, SC, Brazil
Fax: +55 48 3443 4817.
E-mail: quevedo@unesc.net
50
Abstract
A growing body of evidence has indicated that energy metabolism
impairment may be involved in pathophysiology of some neuropsychiatric disorders. In
this study, we evaluated the effect of acute and chronic administration of fluoxetine,
olanzapine and the combination of fluoxetine/olanzapine on citrate synthase activity in
brain of rats. For acute treatment, Wistar rats received one single injection of olanzapine
(3 or 6 mg/kg) and/or fluoxetine (12.5 or 25 mg/kg). For chronic treatment, rats
received daily injections of olanzapine (3 or 6 mg/kg) and/or fluoxetine (12.5 or 25
mg/kg) for 28 days. In the present study we observed that acute administration of
olanzapine inhibited citrate synthase activity in cerebellum and prefrontal cortex. The
acute administration of olanzapine increased citrate synthase activity in prefrontal
cortex, hippocampus and striatum and fluoxetine increased citrate synthase activity in
striatum. Olanzapine 3 mg/kg and fluoxetine 12.5 mg/kg in combination increased
citrate synthase activity in prefrontal cortex, hippocampus and striatum. In the chronic
treatment we did not observed any effect on citrate synthase activity. Our results
showed that olanzapine and fluoxetine increased citrate synthase activity after acute, but
not chronic treatment.
Keywords: Citrate Synthase. Olanzapine. Fluoxetine. Mood disorders
51
INTRODUCTION
Mood disorders are among the most prevalent forms of mental illness.
Severe forms of depression affect 2%–5% of the U.S. population, and up to 20% suffer
from milder forms of the illness. Another roughly 1%–2% are afflicted by bipolar
disorder (also known as manic-depressive illness) or its less severe variants [32] and
were associated with higher rates of suicide and work loss [3, 18, 40].
Among persons with major depression, 75–85% have recurrent episodes
[29]; 10–30% recover incompletely and have persistent, residual depressive symptoms
[21]. Several combinations of effective treatments have been used in the search for
higher response rates or more rapid responses than monotherapy to diminish treatmentresistant depression [7, 26, 36]. One strategy is to combine of the antipsychotic drug
olanzapine plus antidepressant drugs [31]. Accordingly, olanzapine plus fluoxetine is an
approved therapy for treating human depression [6]. In preclinical studies in male rats,
olanzapine [8] or fluoxetine [41], alone or combined [24], produce antidepressant-like
effects. In addition, a fixed combination of olanzapine and fluoxetine has been
introduced for the treatment of bipolar depression [3].
Evidence from the literature strongly indicates that metabolism
impairment may be involved in pathophysiology of some neuropsychiatric disorders,
such as bipolar disorder and major depression [5, 16, 17, 20, 33, 38, 42]. In fact, our
group recently demonstrated that rats submitted to the chronic mild stress increased
oxidative stress in submitochondrial particles into the brain [20]. In addition studies
reported that rats submitted to the animal model of mania were reduced the creatine
kinase and citrate synthase activity in brain [5, 38], suggesting that creatine kinase and
citrate synthase are involved in mood disorder. Very recently, we also demonstrated that
acute and chronic treatments with olanzapine and fluoxetine (alone or in combination)
alter creatine kinase activity in rat brain [1]. In addition, abnormalities in respiratory
complexes activity and energy production may lead to cellular degeneration [4].
Citrate synthase (EC 4.1.3.7) is localized within cells in the mitochondrial
matrix and catalyzes the condensation of oxaloacetate and the acetyl group of acetyl
coenzyme-A (acetyl CoA), the first step of Krebs cycle. In this step, oxaloacetate reacts
with acetyl CoA and H2O to yield citrate and CoA. This enzyme is inhibited by high
amounts of ATP, acetyl CoA and NADH, when the cell energy supplied is high. This
52
regulation ensures that Krebs cycle will not oxidise an excess of pyruvate and acetyl
CoA when ATP concentrations in cell are high [37]. In addition, citrate synthase has
been used as a quantitative enzyme marker for the presence of intact mitochondria [23].
Therefore, considering that citrate synthase plays an important role in brain energy
metabolism and that mitochondrial dysfunction is probably involved in the
pathophysiology of bipolar disorder and major depression, the objective of this study
was to investigate the effects of olanzapine and fluoxetine (alone or in combination) on
citrate synthase activity in rat brain.
METHODS AND MATERIALS
2.1. Animals
Male Adult Wistar rats (60 days old) were obtained from UNESC
(Universidade do Extremo Sul Catarinense, Criciúma, SC, Brazil) breeding colony.
They were housed five per cage with food and water available ad libitum and were
maintained on a 12-h light/dark cycle (lights on at 7:00 AM). All experimental
procedures involving animals were performed in accordance with the NIH Guide for the
Care and Use of Laboratory Animals and the Brazilian Society for Neuroscience and
Behavior (SBNeC) recommendations for animal care and with approval by local Ethics
Committee under protocol number 510/2006.
2.2. Drugs and treatments
Olanzapine (Zyprexa™) and Fluoxetine (Prozac™) were provided from Eli
Lilly do Brasil Ltda, São Paulo, Brazil. Animals received daily intraperitoneal injections
of olanzapine (3 or 6 mg/kg), fluoxetine (12.5 or 25 mg/kg) or combination of both
drugs for 28 days in two protocols of chronic model, and in the acute model for one day.
The doses and protocols were executed following study described by Agostinho et al.
53
[1]. All the drugs were dissolved in Tween 1% solution (vehicle). Control animals
received vehicle (1.0 mL/kg). In the acute protocol, after the single injection, the
animals were killed 2 hours after by decapitation, and the prefrontal cortex,
hippocampus and striatum were immediately removed. Similarly, in the chronic
protocols, the animals were killed 2 and 24 hours after the last injection, and the same
areas are removed [1]. After that, the activity of citrate synthase was measured.
2.3. Citrate synthase activity
Prefrontal cortex, hippocampus and striatum were homogenized (1:10, w/v)
in SETH buffer (0.25 M sucrose, 1 mM EDTA, 10 mM Tris–HCl, pH 7.4). The
homogenates were centrifuged at 800 ×g for 10 min and the supernatants were kept at
−80°C until it will be used for enzyme activity determination. Protein content was
determined by the method described by Lowry et al [19] using bovine serum albumin as
standard. Citrate synthase activity was assayed according to the method described by
Shepherd and Garland [37]. The reaction mixture contained 100 mM Tris, pH 8.0, 100
mM acetyl CoA, 100 mM 5,5′-di-thiobis-(2- nitrobenzoic acid), 0.1% triton X-100, and
2–4 µg supernatant protein and was initiated with 100 µM oxaloacetate and monitored
at 412 nm for 3 min at 25°C (the final volume of reaction mixture was 0.3 mL).
2.4. Statistical analysis
Data were analyzed by one-way of variance (ANOVA) followed by Tukey
test when F was significant and presented as mean ± S.E.M. All analysis were
performed using the Statistical Package for the Social Science (SPSS) software version
16.0.
54
RESULTS
As depicted in Fig. 1A, the acute administration of olanzapine in dose of 6
mg/kg and olanzapine 3 mg/kg in combination with fluoxetine 25 mg/kg increased
citrate synthase activity in prefrontal cortex of rats (ANOVA, F (8-27) = 18,08; p < 0.05;
Fig.1A). Acute administration of olanzapine in dose of 3 mg/kg alone and in
combination with fluoxetine 25 mg/kg increased citrate synthase activity in
hippocampus of rats (ANOVA, F
(8-27)
= 15,51; p < 0.05; Fig.1A). Moreover, the
activity of citrate synthase increased in striatum of rats after acute administration of
olanzapine in doses of 3 and 6 mg/kg, fluoxetine in dose of 25 mg/kg. In addition
olanzapine in dose of 3 mg/kg in combination with fluoxetine, 25 mg/kg also increased
citrate synthase activity in striatum of rats compared to saline group (ANOVA, F (8-26) =
12,69; p < 0.05; Fig.1A). Acute treatment with fluoxetine in dose of 12.5 mg/kg did not
alter citrate synthase activity when administrated alone or in combination with
olanzapine (p > 0.05).
The intraperitoneal treatment with olanzapine and flouxetine alone or in
combination in all doses did not alter the activity of citrate synthase in prefrontal cortex,
hippocampus and striatum in both, chronic treatments (p > 0.05; Fig.1B and C).
DISCUSSION
In the present study we evaluated the effects of the antipsychotic olanzapine
and antidepressant fluoxetine (alone or in combination) on citrate synthase activity in rat
brain. We showed that acute treatment with olanzapine and fluoxetine (alone) in some
doses increased the citrate synthase activity in rat brain. Interestingly acute treatment
with olanzapine 3 mg/kg and fluoxetine 12.5 mg/kg in combination increased citrate
synthase activity in prefrontal cortex, hippocampus and striatum (Fig. 1A). Chronic
treatment with olanzapine and fluoxetine alone or in combination did not alter the
citrate synthase activity when the rats were killed 2 or 24 hours (Fig. 1B and C) after
last administration.
55
Our results indicate that the effects of the olanzapine and fluoxetine might
involve mitochondrial function, but this effect is treatment regime-related in some rat
brain areas. In fact, we recently showed that acute and chronic treatments with
olanzapine and fluoxetine (alone or in combination) alter creatine kinase activity in
some rat brain areas, such as, hippocampus and prefrontal cortex [1].
The hippocampus is one of several limbic structures that has been
implicated in mood disorders. In addition, the hippocampus has connections with the
prefrontal cortex, region that is more directly involved in emotion and cognition and
thereby contributes to other major symptoms of mood disorders [9, 13, 14]. The
striatum is a dopaminergic area involved to memory [11], mood disorders [22].
Additionally, striatum also has relation to the start, stop and direction of motor
movement [12].
The addition of an atypical antipsychotic drug to an antidepressant drug is
observed in clinical practice as a strategy to treat bipolar depression [15, 28] as well as,
treatment-resistant depression [10, 27, 30] and psychotic depression [35]. In a
controlled study [25], subjects with treatment-resistant depression received olanzapine
and fluoxetine alone, or a combination of both; the combination was associated with
significantly greater and faster improvement than was either drug alone. Tamayo and
colleagues [39] studied the effectiveness of olanzapine/fluoxetine combination
treatment in Puerto Rican patients with bipolar depressive episode and showed
improvements resulting from 7 weeks of acute olanzapine/fluoxetine combination
treatment at a starting once-daily dose of 12/25 mg (mean modal dose was 10.8/27.8
and were maintained in responses for additional 12 weeks with olanzapine and
fluoxetine in some doses, in addition fluoxetine was more effective than olanzapine at
10 mg monotherapy. One other study in patients with mixed depression in bipolar I
disorder showed that olanzapine/fluoxetine combination treatment 6/25 mg (mean
modal dose was 7.4/39.3 mg/d) demonstrated that olanzapine/fluoxetine was not
significantly different from that of olanzapine alone, but showed a trend in favor of a
possible superiority of olanzapine/fluoxetine combination [2].
It is well known that citrate synthase plays an important role in brain energy
metabolism [37]. Our study showed that acute treatment with fluoxetine and olanzapine
alters citrate synthase activity, suggesting that fluoxetine and olanzapine should be
involved in mitochondrial function changes. In fact, treatment with fluoxetine and
olanzapine alters the creatine kinase activity in rat brain [1] and mitochondrial
56
respiratory chain (I, II, III and IV) (unpublished data yet), and both creatine kinase and
mitochondrial respiratory chain play a central role in energy metabolism. Moreover,
mitochondrial disease results from a malfunction in biochemical cascade and the
damage to the mitochondrial electron transport chain has been suggested to be an
important factor in the pathogenesis of a range of neuropsychiatric disorders, such as,
bipolar disorder, depression and schizophrenia [33].
In this work, we showed increased citrate synthase activity after acute
treatment with olanzapine and fluoxetine in combination in prefrontal cortex,
hippocampus and striatum. However, chronic treatment did not alter citrate synthase
activity. The reason for this discrepancy this study is unclear but could be related to
desensitization to the effects of repeated olanzapine and fluoxetine or still for adaptation
mechanism. In this manner, the effects of fluoxetine and olanzapine in this study might
be due to the defense mechanism. Scaini and colleagues [34] demonstrated that chronic
administration of antidepressant paroxetine increased citrate synthase and succinate
dehydrogenase activities in prefrontal cortex, hippocampus, striatum and cerebral cortex
of rats; in contrast chronic administration of antidepressants nortriptiline and
venlafaxine did not alter citrate synthase activity in rat brain. We suggest that effects
under citrate synthase activity may be dependent action mechanism of different
antidepressants.
In conclusion we suggested an involvement of citrate synthase activity in
mood disorders, as well as, olanzapine and fluoxetine effects, but these effects were
regime-related in some rat brain areas.
ACKNOWLEDGEMENTS
This study was supported in part by grants from ‘Conselho Nacional de
Desenvolvimento Científico e Tecnológico’ (CNPq-Brazil – JQ and ELS), FAPESC
(JQ), and from the Instituto Cérebro e Mente (JQ) and UNESC (JQ and ELS). JQ and
ELS are recipients of CNPq (Brazil) Productivity fellowships. GZR is holder of a
FAPESC/CAPES studentship.
57
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62
FIGURES AND LEGENDS
FIGURES
Figure 1
A
Acute Treatment
Citrate synthase activity
[nmol TNB/min x mg protein]
6
5
*
*
Saline + Saline
OLZ 3 + Saline
*
4
** * *
*
3
OLZ 6 + Saline
FLX 12,5 + Saline
FLX 25 + Saline
OLZ 3 + FLX 12,5
2
OLZ 3 + FLX 25
OLZ 6 + FLX 12,5
1
OLZ 6 + FLX 25
0
Prefrontal cortex
Hippocampus
Striatum
B
Chronic treatment 2h
Citrate synthase activity
[nmol TNB/min x mg protein]
2,5
Saline + Saline
OLZ 3 + Saline
2
OLZ 6 + Saline
FLX 12,5 + Saline
1,5
FLX 25 + Saline
1
OLZ 3 + FLX 12,5
OLZ 3 + FLX 25
0,5
OLZ 6 + FLX 12,5
OLZ 6 + FLX 25
0
Prefrontal cortex
Hippocampus
Striatum
63
C
Chronic treatment 24h
Citrate synthase activity
[nmol TNB/min x mg protein]
3
Saline + Saline
2,5
OLZ 3 + Saline
OLZ 6 + Saline
2
FLX 12,5 + Saline
FLX 25 + Saline
1,5
OLZ 3 + FLX 12,5
1
OLZ 3 + FLX 25
OLZ 6 + FLX 12,5
0,5
OLZ 6 + FLX 25
0
Prefrontal cortex
Hippocampus
Striatum
64
LEGEND OF FIGURE
Figure 1 - Effects of the acute (A), chronic 2h (animals were killed 2 hour after the last
injection; B) and chronic 24h (animals were killed 24 hour after the last injection; C) administration of
fluoxetine, olanzapine or fluoxetine/olanzapine (i.p.) on the citrate synthase activity in prefrontal cortex,
hippocampus and striatum of rats. Data was analyzed by one-way analysis of variance followed by Tukey
test when F was significant. Values are expressed as nmol TNB formed per minute per mg proteint (mean
± S.E.M.) of 5 rats. * p <0.05 vs. saline according to ANOVA followed by Tukey post-hoc test.
65
4.3
ARTIGO III
Artigo submetido na revista Acta Neuropsychiatrica, 2011.
OLANZAPINE PLUS FLUOXETINE TREATMENT ALTERS
MITOCHONDRIAL RESPIRATORY CHAIN ACTIVITY IN THE RAT BRAIN.
Fabiano R. Agostinho, Gislaine Z. Réus, Roberto B. Stringari, Karine F. Ribeiro,
Gabriela K. Ferreira, Isabela C. Jeremias, Giselli Scaini, Gislaine T. Rezin, Emílio
Streck and João Quevedo.
66
OLANZAPINE PLUS FLUOXETINE TREATMENT ALTERS
MITOCHONDRIAL RESPIRATORY CHAIN ACTIVITY IN THE RAT BRAIN
Fabiano R. Agostinhoa, Gislaine Z. Réusa, Roberto B. Stringaria, Karine F.
Ribeiroa, Gabriela K. Ferreirab, Isabela C. Jeremiasb, Giselli Scainib, Gislaine T. Rezinb,
Emílio Streckb, and João Quevedoa*.
a
Laboratório de Neurociências and Instituto Nacional de Ciência e Tecnologia
Translacional em Medicina (INCT-TM), Programa de Pós-Graduação em Ciências da
Saúde, Unidade Acadêmica de Ciências da Saúde, Universidade do Extremo Sul
Catarinense, 88806-000, Criciúma, SC, Brazil;
b
Laboratório de Fisiopatologia Experimental and Instituto Nacional de Ciência e
Tecnologia Translacional em Medicina (INCT-TM), Programa de Pós-Graduação em
Ciências da Saúde, Unidade Acadêmica de Ciências da Saúde, Universidade do
Extremo Sul Catarinense, 88806-000 Criciúma, SC, Brazil.
Running title: Olanzapine plus fluoxetine and energy metabolism
*Corresponding author:
Prof. João Quevedo, MD, PhD
Laboratório de Neurociências,
Programa de Pós-Graduação em Ciências da Saúde,
Unidade Acadêmica de Ciências da Saúde,
Universidade do Extremo Sul Catarinense,
88806-000 Criciúma, SC, Brazil
Fax: +55 48 3443 4817.
E-mail: quevedo@unesc.net
67
Abstract
Evidence is emerging for a role for dysfunctional mitochondria in
pathophysiology and treatment of mood disorders. In this study, we evaluated the
effects of acute and chronic administration of fluoxetine, olanzapine and the
combination of fluoxetine/olanzapine on mitochondrial respiratory chain activity in the
rat brain. For acute treatment, Wistar rats received one single injection of olanzapine (3
or 6 mg/kg) and/or fluoxetine (12 or 25 mg/kg) and for chronic treatment, rats received
daily injections of olanzapine (3 or 6 mg/kg) and/or fluoxetine (12 or 25 mg/kg) for 28
days and we evaluated mitochondrial respiratory chain complexes I, II, II-III and IV
activity in prefrontal cortex, hippocampus and striatum. Our results showed that both
acute and chronic treatments with fluoxetine and olanzapine alone or in combination
altered respiratory chain complexes activity in the rat brain, but in combination we
observed larger alterations. Finally, these findings further support the hypothesis that
metabolism energy could be involved in the treatment with antipsychotic and
antidepressant in combination to mood disorders.
Keywords: Olanzapine; Fluoxetine; Mitochondrial respiratory chain; Bipolar Disorder;
Bipolar Depression.
68
INTRODUCTION
Mood disorders are among the most prevalent forms of mental illness.
Severe forms of depression affect 2%–5% of the U.S. population, and up to 20% suffer
from milder forms of the illness. Another roughly 1%–2% are afflicted by Bipolar
Disorder (BD) or its less severe variants [1, 2] and were associated with higher rates of
suicide and work loss [3-5].
Tissues with high energy demands, such as the brain, contain a large
number of mitochondria, and are therefore more susceptible to reduction of the aerobic
metabolism [6] Mitochondrial disease results from a malfunction in biochemical
cascade and the damage to the mitochondrial electron transport chain has been
suggested to be an important factor in the pathogenesis of a range of neuropsychiatries
disorders, such as bipolar disorder, depression and schizophrenia [7, 8]. Several studies
have demonstrated that abnormalities in energy metabolism lead to cellular
degeneration [9]. This effect may occur because when the mitochondrial dysfunction is
severe it can lead to cell death by apoptosis or necrosis [10, 11]. In fact, mitochondria
are involved in essential processes, such as apoptosis and calcium homeostasis [12-14],
which are involved in cell death.
Mitochondria are intracellular organelles which play a crucial role in ATP
production [9]. Most cell energy is obtained through oxidative phosphorylation, a
process requiring the action of various respiratory enzyme complexes located in a
special structure of the inner mitochondrial membrane, the mitochondrial respiratory
chain [15]. In most organisms, the mitochondrial respiratory chain is composed of four
complexes, where the electron transport couples with translocation of protons from the
mitochondrial matrix to the intermembrane space. The generated proton gradient is used
by ATP synthase to catalyze the formation of ATP by the phosphorylation of adenosine
diphosphate (ADP) [7,16].
A fixed combination of antipsychotic and antidepressant drugs was widely
used in medicine and, at one time, was common in psychiatry. A generation ago,
combinations of antidepressants with either antipsychotics (e.g., amitriptyline and
perphenazine [EtrafonTM, TriavilTM]) or benzodiazepines (e.g., amitriptyline and
chlordiazepoxide [LimbitrolTM]) were widely used by both psychiatrists and other
medical practitioners [17]. Recently, a fixed combination of the antipsychotic drug
69
olanzapine (OLZ) and the antidepressant fluoxetine (FLX) (SymbyaxTM) has been
introduced for the treatment of BD [3,18]. In a controlled study of Shelton and
colleagues [19], subjects with treatment-resistant depression received OLZ alone, FLX
alone, or a combination of both; the combination was associated with significantly
greater and faster improvement than was either drug alone. While there is a clear
clinical benefit from this combination, the precise neural mechanisms responsible for its
efficacy are not clearly understood. Therefore, it is important to investigate the
mechanisms of action of this combination in order to not only better understand the
etiology of the clinical syndromes, but also to eventually facilitate the development of
improved drugs to treat them [4, 20].
Considering the effects of OLZ, FLX and these combinations on brain
energy metabolism is still unknown we evaluated the effects of these drugs on
mitochondrial respiratory chain in the rat prefrontal cortex, hippocampus and striatum.
Is important to note that we chose the prefrontal cortex, hippocampus and striatum in
the current study, because these brain areas are implicated in mood disorders [21-22].
MATERIAL AND METHODS
Animals
Male adult Wistar rats (60 days old) were obtained from UNESC
(Universidade do Extremo Sul Catarinense, Criciúma, SC, Brazil) breeding colony.
They were housed five per cage with food and water available ad libitum and were
maintained on a 12-h light/dark cycle (lights on at 7:00 a.m.). All experimental
procedures involving animals were performed in accordance with the NIH Guide for the
Care and Use of Laboratory Animals and the Brazilian Society for Neuroscience and
Behavior (SBNeC) recommendations for animal care and with approval by local Ethics
Committee under protocol number 510/2006.
70
Drugs and treatments
Olanzapine (Zyprexa™) and Fluoxetine (Prozac™) were provided from Eli
Lilly do Brasil Ltda, São Paulo, Brazil. Animals received daily intraperitoneal injections
of OLZ (3 or 6 mg/kg), FLX (12 or 25 mg/kg) or combination of both drugs for 28 days
in two protocols of the chronic model (A and B), and in the acute model for one day.
Protocols and doses of drugs were performed in accordance with previous studies [2324]. All the drugs were dissolved in saline (NaCl 0.9%) solution (vehicle). Control
animals received saline (NaCl 0.9%) (1.0 ml/kg). In the acute protocol, after the single
injection, the animals were killed 2 hours later by decapitation, and the prefrontal
cortex, hippocampus and striatum were immediately removed. Similarly, in the chronic
protocols, the animals were killed 2 (A) and 24 (B) hours after the last injection, and the
same areas are removed. The analysis were performed after different times of
decapitation (2 and 24 hours) after the last injection to be sure that the effects of the
studied parameters were due to a chronic effect [23-24]. After that, the activity of
mitochondrial respiratory chain was measured (n = 5 each).
Tissue and homogenate preparation
Hippocampus, striatum, and prefrontal cortex were homogenized (1:10,
w/v) in SETH (sucrose, EDTA, tris, heparin) buffer, pH 7.4 (250 mM sucrose, 2 mM
ethylenediaminetetraacetic acid, 10 mM Trizma base, 50 IU/mL heparin). The
homogenates were centrifuged at 800×g for 10 min and the supernatants kept at −70oC
until used for mitochondrial respiratory chain activity determination. The maximal
period between homogenate preparation and enzyme analysis was always less than 5
days. Protein content was determined by the method described by Lowry and colleagues
[25] using bovine serum albumin as standard.
71
Respiratory chain enzyme activities
NADH dehydrogenase (complex I) was evaluated by the method described
by Cassina and Radi [26] by the rate of NADH-dependent ferricyanide reduction at 420
nm.
The
activities
of
succinate-2,6-dichloroindophenol
(DCIP)-oxidoreductase
(complex II) and succinate: cytochrome c oxidoreductase (complex II–III) were
determined by the method described by Fischer and colleagues [27]. Complex II activity
was measured by following the decrease in absorbance due to the reduction of 2,6-DCIP
at 600 nm. Complex II-III activity was measured by cytochrome c reduction from
succinate at 550 nm. The activity of cytochrome c oxidase (complex IV) was assayed
according to the method described by Rustin and colleagues [28], measured by
following the decrease in absorbance due to the oxidation of previously reduced
cytochrome c at 550 nm. The activities of the mitochondrial respiratory chain
complexes were calculated as nmol/min mg protein.
Statistical analysis
All data is presented as mean ± S.E.M (Standard Error of the Mean).
Differences among experimental groups in the assessment of mitochondrial respiratory
chain activity were determined by one-way ANOVA, followed by Tukey post-hoc test
when ANOVA was significant; P < than 0.05 were considered to be statistical
significant.
RESULTS
As depicted in Fig. 1A, complex I activity increased in the prefrontal cortex
of rats treated acutely with OLZ 6 mg/kg and OLZ 3 mg/kg plus FLX 12 mg/kg (Fig.
1A; F = 35.63; p < 0.05); in the hippocampus complex I activity increased with FLX 25
mg/kg (Fig. 1A; F = 180.27; p < 0.05); in the striatum complex I activity increased with
72
OLZ 6 mg/kg (Fig. 1A; F = 3.69; p < 0.05). The complex II activity increased in
prefrontal cortex (Fig. 1B; F = 2.28; p < 0.05) and hippocampus (Fig. 1B; F = 36.43; p
< 0.05) after acute treatment with OLZ 6 mg/kg alone. The complex II-III activity
increased in the prefrontal cortex (Fig. 1C; F = 17.03; p < 0.05) with OLZ 3 mg/kg plus
FLX 25 mg/kg and OLZ 6 mg/kg plus FLX 25 mg/kg, in the hippocampus (Fig. 1C; F =
6.92; p < 0.05) with OLZ 3 mg/kg plus FLX 25 mg/kg and OLZ 6 mg/kg plus FLX 25
mg/kg and striatum (Fig. 1C; F = 15.71; p < 0.05) with OLZ 3 mg/kg plus FLX 12 or 25
mg/kg and OLZ 6 mg/kg plus FLX 25 mg/kg. After acute treatment the complex IV
activity increased in the prefrontal cortex (Fig. 1D; F = 5.7; p < 0.05) with OLZ 3
mg/kg plus FLX 25 mg/kg and OLZ 6 mg/kg plus FLX 12 or 25 mg/kg and striatum
(Fig. 1D; F = 8.68; p < 0.05) with OLZ 3 mg/kg plus FLX 12 or 25 mg/kg. In the
chronic treatment, when the animals were killed 2 h after the last injection (Fig. 2), there
was an increase in complex I activity in the striatum after OLZ 6 mg/kg and FLX 25
mg/kg in combination (Fig. 2A; F = 4.02; p < 0.05). The complex II activity decreased
in the striatum after chronic treatment with OLZ 6 mg/kg plus FLX 12 mg/kg (Fig. 2B;
F = 1.87; p < 0.05), and they were not altered in the prefrontal cortex (Fig. 2B; F = 0.92;
p > 0.05) and hippocampus (Fig. 2B; F = 1.93; p > 0.05). The complex II-III activity
increased in the striatum with OLZ 3 mg/kg alone compared to control group (Fig. 2C;
F = 7.81; p < 0.05), and they were not altered in prefrontal cortex (Fig. 2C; F = 1.87; p
> 0.05) and hippocampus (Fig. 2C; F = 1.91; p > 0.05). The complex IV activity did not
alter in the prefrontal cortex (Fig. 2D; F = 0.36; p > 0.05) and striatum (Fig. 2D; F =
3.52; p < 0.05) compared to control group; in contrast in the hippocampus the complex
IV activity increased after treatment with OLZ 6 mg/kg and FLX 25 mg/kg in
combination compared to control group (Fig. 2D; F = 2.4; p < 0.05).
In the chronic treatment, when the animals were killed 24 h after the last
injection (Fig. 3) we showed that complex I activity decreased in the prefrontal cortex
with FLX 12 mg/kg alone, compared to control group (Fig. 3A; F = 3.9; p < 0.05);
however the complex I activity did not alter in hippocampus (Fig. 3A; F = 2.21; p >
0.05) and striatum (Fig. 3A; F = 1.15; p > 0.05). In the complex II activity (Fig. 2B) did
not alter in the prefrontal cortex (F = 3.45), hippocampus (F = 4.83) and striatum (F =
1.39), compared to control group. Treatment with FLX 25 mg/kg alone decreased
complex II-III activity in the striatum, compared to control group (Fig. 3C; F = 4.99; p <
0.05). In the hippocampus (Fig. 3C; F = 3.42; p > 0.05) and prefrontal cortex (Fig. 3C; F
= 2.18; p > 0.05) did not observe alteration in the complex II-III activity. The Fig. 3D
73
shows that complex IV activity did not alter in the prefrontal cortex (F = 2.13). In the
striatum the complex IV activity increased after treatment with OLZ 6 mg/kg and FLX
12 mg/kg in combination, compared to control group (F = 8.18; p < 0.05). In contrast in
the hippocampus the complex IV activity decreased after treatment with OLZ 3 and 6
mg/kg alone, as with FLX 12 and 25 mg/kg alone. In addition, OLZ 3 mg/kg plus FLX
12 or 25 mg/kg also decreased the complex IV activity, compared to control group (F =
6.87; p < 0.05).
DISCUSSION
In the present study we evaluated the effects of the antipsychotic OLZ
and of the antidepressant FLX (alone or in combination) on mitochondrial respiratory
chain activity in the rat brain. We showed that both acute and chronic treatments with
FLX and OLZ alone or in combination altered respiratory chain complex activity in the
rat brain, but in combination we observed larger alterations. We demonstrated that these
alterations were related to treatment regime, complex, brain area and drug
concentration.
Recent studies from our group showed that acute administration of FLX
inhibited creatine kinase in the rat brain. This study also showed that chronic treatment,
when the animals were killed 2 h after the last injection, showed a decrease in the
creatine kinase activity after FLX administration, alone or in combination with OLZ. In
contrast when the animals were killed 24 h after the last injection, we did not observe
alterations in the enzyme [23]. In addition, acute, but not chronic treatment with FLX
and OLZ alone or in combination increased citrate synthase activity in the rat brain [24].
The creatine kinase works as a buffering system of cellular ATP levels and the citrate
synthase has been used as a quantitative enzyme marker for the presence of intact
mitochondria [29] both enzymes play an important role in brain energy metabolism. In
fact several studies have been appointed to mitochondrial abnormalities in a number of
disorders, including depression, bipolar disorder and schizophrenia [7, 30-31].
Studies have identified that some brain regions from bipolar disorder
patients presented a decrease energy metabolism and abnormalities in mitochondrial
DNA [32-33]. Moreover, reductions of mitochondrial respiratory chain were found in
74
patients with depression, schizophrenia and bipolar disorder [34-35]. Additionally,
animal models evaluating the molecular pharmacology of mood stabilizing drugs have
implicated mitochondrial energy metabolism as a target for these drugs [36-37].
Dror and colleagues [38] showed alteration in complex I activity and in
levels of mRNA and protein of the 24 and 51-kDa iron-sulfur flavoprotein subunits of
the complex from platelets of schizophrenia patients, suggesting that these alterations
may result in abnormal neural transmission, synaptic plasticity and connectivity, leading
to abnormal behavioral symptoms in schizophrenia. Moreover, another study has shown
abnormalities in energy metabolism in the basal ganglia of chronic schizophrenics [39].
In addition, Iwamoto and colleagues [32] showed mitochondrial dysfunction in
postmortem brains of schizophrenic patients, however, this dysfunction was due to the
patients’ medication, especially antipsychotics. Additionally, a study showed that OLZ,
clozapine and haloperidol inhibited succinate dehydorgenase (SDH, an important
enzyme of Krebs cycle and part of the mitochondrial respiratory chain as an electron
transferring protein); however aripiprazole antipsychotic increased the enzyme in the rat
brain [40]. Several studies have showed that antipsychotic drugs inhibited the
respiratory electron transport chain [41-44]. In the present study we showed that OLZ
alone or in combination with FLX inhibited the complex IV activity in the hippocampus
when the animals were killed 24 h after the last injection, and OLZ in combination with
FLX inhibited the complex II activity in the striatum when the animals were killed 2 h
after the last injection; however, in most cases, OLZ alone or in combination acted to
increase the complex respiratory chain in the rat brain.
The effects of OLZ and FLX found in this study could be also related to
oxidative stress. In fact, mitochondria can produce an excess of reactive oxygen species
(ROS), which will cause oxidative damage to cellular constituents, such as membrane
lipids and proteins [45]. In addition, mtDNA mutations in elevated production of ROS
in turn proved to increase the number of mtDNA mutations [46]. Several studies have
generally suggested a compromised oxidative stress in psychiatric disorders, such as
bipolar disorder, depression and schizophrenia [47-49]. Additionally, chronic exposure
to antipsychotics, haloperidol and clozapine, but not OLZ, caused changes in the
activities of antioxidant enzymes and oxidative damage in the rat brain [50-51].
Researchers have reported that some side effects of antipsychotics are associated to
oxidative stress [52-53] and metabolism impairment [54]. Recently, a study from our
group showed that OLZ and FLX treatment inhibited creatine kinase activity [23],
75
suggesting that inhibition of enzyme may be associated to the occurrence of some side
effects of OLZ and FLX. However, OLZ exerted antioxidant effects though modulating
ROS levels, superoxide dismutase activity and Bax expression to provide protective
effects against N-methyl-4-phenylpyridinium (MPP+) induced oxidative stress in PC12
cells [55]. FLX also have shown an antioxidant effects [56-58].
Reductions in mRNA and protein of complex I subunits NADH
dehydrogenase ubiquinone flavoprotein (NDUFV1), NADH-ubiquinone oxidoreductase
flavoprotein gene (NDUFV2) and NADH dehydrogenase (ubiquinone) Fe-S protein
1(NDUFS1) have been demonstrated in the cerebellum postmortem from patients with
depression [30]. Many animal models of mania and depression have revealed alterations
in metabolism energy. Studies from our group demonstrated reduced creatine kinase
and citrate synthase activity in brain of rats submitted to the animal model of mania
[36,59] Moreover, in another study from our group, it was demonstrated that
antidepressants imipramine [60] and paroxetine [61] increased creatine kinase activity
in the rat brain, suggesting that modulation of energy metabolism by antidepressants
could be an important mechanism of action of these drugs. Nevertheless, our group also
showed that mitochondrial respiratory chain complexes I, II-III and IV were inhibited
after chronic mild stress in the cerebral cortex and cerebellum [62]. Madrigal and
colleagues [16] also reported that complexes I–III and II–III of mitochondrial
respiratory chain were inhibited in rat brains after chronic stress (immobilization for six
hours during 21 days). Hroudova and Fisar [63] demonstrated that several
antidepressant drugs inhibited complex I and IV of the mitochondrial respiratory chain,
suggesting that in pathophysiology of mood disorders and therapeutic effects of
antidepressant could have changes in energetic metabolism of cells determined by
mitochondria.
In clinical practice, atypical antipsychotic drugs in combination with
antidepressant drugs have been used as a strategy to treat [64] treatment-resistant
depression [1,65-66] and psychotic depression [67]. In an elegant controlled study,
Matthews and colleagues [68] showed that subjects with treatment-resistant depression
that received OLZ and FLX alone, or a combination of both; the combination was
associated with significantly greater and faster improvement than was either drug alone.
In the present study we also demonstrated greater effects of OLZ and FLX in
combination
under
metabolism
energy
parameters,
antidepressant FLX
and
antipsychotic OLZ alone or in combination increased or decreased mitochondrial
76
respiratory chain, dependently of treatment regime, enzymatic complex, brain area and
drug concentration. The reason for this different alteration in this study is unclear but
could be related to desensitization to the effects of repeated olanzapine and fluoxetine
administration, or to the adaptation mechanism of mitochondria. The differences of
OLZ and FLX found in the present findings could be related to brain distribution of the
drugs, or differences in toxicity of its metabolites.
In conclusion, taking together the present findings and evidence from the
literature, we hypothesize that FLX and OLZ in combination could be involved in
mitochondrial function, which is altered in several mood disorders. However, it remains
to be seen if effects of the combination of drugs on the mitochondrial respiratory chain
are related to the therapeutic or to side effects of pharmacotherapy.
ACKNOWLEDGEMENTS
This study was supported in part by grants from ‘Conselho Nacional de
Desenvolvimento Científico e Tecnológico’ (CNPq-Brazil – JQ, ELS), from the
Instituto Cérebro e Mente (JQ) and UNESC (JQ and ELS). JQ and ELS are recipients of
CNPq (Brazil) Productivity fellowships. GZR is holder of a FAPESC/CAPES
studentship.
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FIGURES
Figure 1:
A
ACUTE
Complex I activity
[nmol/min x mg protein]
20000
*
18000
16000
Sal+Sal
Sal+OLZ 3
14000
Sal+OLZ 6
*
12000
10000
8000
6000
Sal+FLX 12
*
Sal+FLX 25
*
OLZ 3+FLX 12
OLZ 3+FLX 25
4000
2000
OLZ 6+FLX 12
OLZ 6+FLX 25
Prefrontal cortex
Hippocampus
Striatum
B
Complex II activity
[nmol/min x mg protein]
ACUTE
200
180
160
140
120
100
80
60
40
20
*
Sal+Sal
Sal+OLZ 3
Sal+OLZ 6
Sal+FLX 12
Sal+FLX 25
OLZ 3+FLX 12
OLZ 3+FLX 25
*
OLZ 6+FLX 12
OLZ 6+FLX 25
Prefrontal cortex
Hippocampus
Striatum
83
C
Complex II-III activity
[nmol/min x mg protein]
ACUTE
10,0
9,0
8,0
7,0
6,0
5,0
4,0
3,0
2,0
1,0
,0
*
*
*
*
*
Sal+OLZ 3
Sal+OLZ 6
*
*
Sal+Sal
Sal+FLX 12
Sal+FLX 25
OLZ 3+FLX 12
OLZ 3+FLX 25
OLZ 6+FLX 12
OLZ 6+FLX 25
Prefrontal cortex
Hippocampus
Striatum
D
ACUTE
Complex IV activity
[nmol/min x mg protein
900
*
800
**
700
600
300
Sal+OLZ 3
Sal+OLZ 6
Sal+FLX 12
500
400
Sal+Sal
Sal+FLX 25
* *
*
OLZ 3+FLX 12
OLZ 3+FLX 25
200
OLZ 6+FLX 12
100
OLZ 6+FLX 25
Prefrontal cortex
Hippocampus
Striatum
84
Figure 2:
A
CHRONIC 2 h
Complex I activity
[nmol/min x mg protein]
2500
Sal+Sal
*
2000
Sal+OLZ 3
Sal+OLZ 6
1500
Sal+FLX 12
Sal+FLX 25
1000
OLZ 3+FLX 12
OLZ 3+FLX 25
500
OLZ 6+FLX 12
OLZ 6+FLX 25
Prefrontal cortex
Hippocampus
Striatum
B
CHRONIC 2 h
Complex II activity
[nmol/min x mg protein]
9
Sal+Sal
8
Sal+OLZ 3
7
Sal+OLZ 6
6
Sal+FLX 12
5
Sal+FLX 25
4
OLZ 3+FLX 12
3
*
2
1
OLZ 3+FLX 25
OLZ 6+FLX 12
OLZ 6+FLX 25
Prefrontal cortex
Hippocampus
Striatum
85
C
CHRONIC 2 h
Complex II-III activity
[nmol/min x mg protein]
9
*
8
Sal+Sal
7
Sal+OLZ 3
6
Sal+OLZ 6
5
Sal+FLX 12
4
Sal+FLX 25
3
OLZ 3+FLX 12
2
OLZ 3+FLX 25
1
OLZ 6+FLX 12
OLZ 6+FLX 25
Prefrontal cortex
Hippocampus
Striatum
D
CHRONIC 2 h
Complex IV activity
[nmol/min x mg protein]
250
Sal+Sal
*
200
Sal+OLZ 3
Sal+OLZ 6
150
Sal+FLX 12
Sal+FLX 25
100
OLZ 3+FLX 12
50
OLZ 3+FLX 25
OLZ 6+FLX 12
OLZ 6+FLX 25
Prefrontal cortex
Hippocampus
Striatum
86
Figure 3:
A
CHRONIC 24 h
Complex I activity
[nmol/min x mg protein]
25000
Sal+Sal
20000
Sal+OLZ 3
Sal+OLZ 6
15000
Sal+FLX 12
Sal+FLX 25
10000
OLZ 3+FLX 12
OLZ 3+FLX 25
5000
OLZ 6+FLX 12
*
Prefrontal cortex
OLZ 6+FLX 25
Hippocampus
Striatum
B
CHRONIC 24h
Complex II activity
[nmol/min x mg protein]
20
Sal+Sal
18
16
Sal+OLZ 3
Sal+OLZ 6
14
12
10
Sal+FLX 12
Sal+FLX 25
8
6
OLZ 3+FLX 12
4
2
OLZ 6+FLX 12
OLZ 3+FLX 25
OLZ 6+FLX 25
Prefrontal cortex
Hippocampus
Striatum
87
C
CHRONIC 24 h
Complex II-III activity
[nmol/min x mg protein]
14
Sal+Sal
12
Sal+OLZ 3
10
Sal+OLZ 6
8
Sal+FLX 12
6
Sal+FLX 25
4
OLZ 3+FLX 12
*
OLZ 3+FLX 25
2
OLZ 6+FLX 12
OLZ 6+FLX 25
Prefrontal cortex
Hippocampus
Striatum
D
CHRONIC 24 h
Complex IV activity
[nmol/min x mg protein]
3000
*
2500
Sal+Sal
Sal+OLZ 3
Sal+OLZ 6
2000
Sal+FLX 12
1500
Sal+FLX 25
1000
OLZ 3+FLX 12
OLZ 3+FLX 25
*
** * * *
500
OLZ 6+FLX 12
OLZ 6+FLX 25
Prefrontal cortex
Hippocampus
Striatum
88
LEGENDS AND FIGURES
Figure 1: Effects of the acute administration of OLZ and FLX on the complex I (A); II (B);
II-III (C) and IV (D) activities in the rats prefrontal cortex, hippocampus and striatum. Bars represent
means±S.E.M.* p <0.05 vs. saline according to ANOVA followed by Tukey post-hoc test.
Figure 2: Effects of the chronic administration of OLZ and FLX on the complex I (A); II
(B); II-III (C) and IV (D) activities in the rats prefrontal cortex, hippocampus and striatum. The animals
were killed 2 h the last administration of the drugs. Bars represent means±S.E.M.* p <0.05 vs. saline
according to ANOVA followed by Tukey post-hoc test.
Figure 3: Effects of the chronic administration of OLZ and FLX on the complex I (A); II
(B); II-III (C) and IV (D) activities in the rats prefrontal cortex, hippocampus and striatum. The animals
were killed 24 h the last administration of the drugs. Bars represent means±S.E.M.* p <0.05 vs. saline
according to ANOVA followed by Tukey post-hoc test.
89
4.4
ARTIGO IV
Artigo publicado na revista Neuroscience Letters 2011.
90
OLANZAPINE PLUS FLUOXETINE TREATMENT INCREASES NT-3
PROTEIN LEVELS IN THE RAT PREFRONTAL CORTEX
Fabiano R. Agostinhoa, Gislaine Z. Réusa, Roberto B. Stringaria, Karine F. Ribeiroa,
Bianca Pfaffensellerb, Laura Stertzb, Bruna S. Panizzuttib, Flávio Kapczinskib and João
Quevedoa*.
a
Laboratório de Neurociências and Instituto Nacional de Ciência e Tecnologia
Translacional em Medicina (INCT-TM), Programa de Pós-Graduação em Ciências da
Saúde, Unidade Acadêmica de Ciências da Saúde, Universidade do Extremo Sul
Catarinense, 88806-000, Criciúma, SC, Brazil;
b
Laboratório de Psiquiatria Molecular and Instituto Nacional de Ciência e Tecnologia
Translacional em Medicina (INCT-TM), Centro de Pesquisas, Hospital de Clínicas de
Porto Alegre, 90035-003 Porto Alegre, RS, Brazil.
*Corresponding author:
Prof. João Quevedo, MD, PhD
Laboratório de Neurociências
Programa de Pós-Graduação em Ciências da Saúde
Unidade Acadêmica de Ciências da Saúde
Universidade do Extremo Sul Catarinense
88806-000 Criciúma, SC, Brazil
Fax: +55 48 3431-2736.
E-mail: quevedo@unesc.net
91
Abstract
Evidence is emerging for a role for neurotrophins in the treatment of mood
disorders. In this study, we evaluated the effects of chronic administration of fluoxetine,
olanzapine and the combination of fluoxetine/olanzapine on the brain-derivedneurotrophic factor (BDNF), nerve growth factor (NGF), and neurotrophin-3 (NT-3) in
the rat brain. Wistar rats received daily injections of olanzapine (3 or 6 mg/kg) and/or
fluoxetine (12.5 or 25 mg/kg) for 28 days, and we evaluated for BDNF, NGF and NT-3
protein levels in the prefrontal cortex, hippocampus and amygdala. Our results showed
that treatment with fluoxetine and olanzapine alone or in combination did not alter
BDNF or NGF protein levels, but NT-3 protein levels were increased by olanzapine 6
mg/kg/fluoxetine 25 mg/kg combination. Finally, these findings further support the
hypothesis that NT-3 could be involved in the effect of treatment with antipsychotic and
antidepressant combination in mood disorders.
Keywords: Brain-derived-neurotrophic factor; Nerve growth factor; Neurotrophin-3;
Fluoxetine; Olanzapine; Mood disorder.
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INTRODUCTION
Bipolar disorder (BD) is a prevalent condition in adults determining a
significant impairment in quality of life [27]. Bipolar depression represents a difficultto-treat and disabling form of depression. Studies indicate that patients with BD spend
more time in and take longer to recover from the depressive phase than the manic phase
[33, 47].
Olanzapine (OLZ) has demonstrated efficacy in the treatment of acute
bipolar mania [59, 61-63, 65] stabilizing effects [11, 60, 62, 65-66] and has been found
to improve depressive symptoms in patients with schizophrenia [58, 64]. In a controlled
study of Shelton et al. [53] subjects with treatment-resistant depression received OLZ
alone, fluoxetine (FLX) alone, or a combination of both. The combination was
associated with significantly greater and faster improvement than was either drug alone.
In a critical review, Fountoulakis et al. [26] show that several practice guidelines
disagree on how best to initiate treatment of bipolar depression.
Neurotrophic factors, as such, brain derived neurotrophic factor (BDNF),
nerve growth factor (NGF), and neurotrophin-3 (NT-3) are critical regulators of the
formation, survival, differentiation, and outgrowth of select peripheral and central
neurons throughout adulthood [32, 51] and plasticity of neural networks [28, 32, 51].
Thus, based on the literature findings, the present study was aimed to
investigate physiological effects of the combination of OLZ and FLX on the BDNF,
NT-3 and NGF protein levels in the prefrontal cortex, hippocampus and amygdala.
MATERIALS AND METHODS
Animals
Male adult Wistar rats (60 days old) were obtained from UNESC
(Universidade do Extremo Sul Catarinense, Criciúma, SC, Brazil) breeding colony.
They were housed five per cage with food and water available ad libitum and were
93
maintained on a 12-h light/dark cycle (lights on at 7:00 a.m.). All experimental
procedures involving animals were performed in accordance with the NIH Guide for the
Care and Use of Laboratory Animals and the Brazilian Society for Neuroscience and
Behavior (SBNeC) recommendations for animal care and with approval by local Ethics
Committee under protocol number 510/2006.
Drugs and treatments
Olanzapine (Zyprexa™) and Fluoxetine (Prozac™) were provided from Eli
Lilly do Brasil Ltda, São Paulo, Brazil. Animals received daily intraperitoneal injections
of OLZ (3 or 6 mg/kg), FLX (12.5 or 25 mg/kg) or combination of both drugs for 28
days. All the drugs were dissolved in Tween 1% solution (vehicle). Control animals
received vehicle (1.0 ml/kg). After the chronic treatment the animals were killed by
decapitation 24 hours after the last injection and prefrontal cortex, hippocampus and
amygdala were immediately removed and stored at −70°C for biochemical analysis.
Neurotrophins measurement
BDNF, NT-3 and NGF levels in prefrontal cortex, hippocampus and
amygdala were measured by sandwich-ELISA, according to the manufacturer
instructions (Chemicon, USA for BDNF and Millipore, USA & Canada for NT-3 and
NGF). Briefly, rat prefrontal cortex, hippocampus and amygdala were homogenized in
phosphate buffer solution (PBS) with protease inhibitor cocktail (Sigma). Microtiter
plates (96-well flat-bottom) were coated for 24 h with the samples diluted 1:2 in sample
diluent and standard curve ranged from 7.8 to 500 pg/ml of BNDF, NT-3 or NGF. The
plates were then washed four times with sample diluent and a monoclonal anti-BNDF,
anti-NT-3 or anti-NGF rabbit antibody (diluted 1:1000 in sample diluent) was added to
each well and incubated for 3 h at room temperature. After washing, a peroxidase
conjugated anti-rabbit antibody (diluted 1:1000) was added to each well and incubated
at room temperature for 1 h. After addition of streptavidin-enzyme, substrate and stop
94
solution, the amount of each neurotrophin was determined by absorbance in 450 nm.
The standard curve demonstrates a direct relationship between Optical Density (OD)
and the concentration. Total protein was measured by Lowry's method using bovine
serum albumin as a standard, as previously described by Lowry et al. [39].
Statistical analysis
All data are presented as mean ± S.E.M. Differences among experimental
groups in the assessment of BDNF, NGF and NT-3 protein levels were determined by
one-way ANOVA, followed by Tukey post-hoc test when ANOVA was significant; P <
0.05 were considered to be statistical significant.
RESULTS
As depicted in Figure 1A, the NT-3 protein levels did not alter in the
hippocampus (Figure 1A; F
(8-42)
= 0.51; p = 0.83) and amygdala (Figure 1A; F
(8-43)
=
0.45; p = 0.88) after treatment with olanzapine or fluoxetine alone or in combination.
However treatment with olanzapine at the dose of 6 mg/kg in combination with
fluoxetine at the dose of 25 mg/kg we found an increase in NT-3 protein levels in the
prefrontal cortex (Figure 1A; F
(8-37)
= 2.4; p = 0.03); but olanzapine or fluoxetine
treatment alone did alter NT-3 protein levels in the prefrontal cortex.
The BDNF and NGF protein levels are illustrated in the Figure 1B and 1C,
respectively. After treatment with olanzapine or fluoxetine alone or in combination,
both BDNF and NGF protein levels did not alter in the prefrontal cortex (Figure 1B; F
(8-46)
= 1.11; p = 0.37; Figure 1C; F (8-24) = 0.65; p = 0.72), hippocampus (Figure 1B; F (8-
51)
= 0.98; p = 0.45; Figure 1C; F (8-18) = 1.48; p = 0.23) and amygdala (Figure 1B; F (8-
47)
= 0.57; p = 0.79; Figure 1C; F (8-18) = 0.74; p = 0.64).
95
DISCUSSION
In the present study we evaluated the effects of the antipsychotic OLZ and
of the antidepressant FLX (alone or in combination) on the NT-3, BDNF and NGF
protein levels in the rat brain. We showed that chronic treatment with FLX and OLZ in
combination increased the NT-3 protein levels in the prefrontal cortex. Treatment with
FLX and OLZ alone or in combination did not alter BDNF or NGF protein levels in the
rat brain.
The hippocampus regulates hypothalamic-pituitary-adrenal (HPA) axis, and
has connections with amygdala and prefrontal cortex. In addition, brain imaging studies
have indicated volumes of the hippocampus, prefrontal cortex or amygdala in patients
with major depression or bipolar disorder [14, 20].
Several studies have highlighted the role of neurotrophins in the
pathophysiology and treatment of psychiatric disorders. The BDNF, NT-3 and NGF
promote survival and cellular plasticity [4, 38]. Reductions of BDNF have been found
in humans, as well as in animal model [22, 34], on the other hand, antidepressant
treatment produces antidepressant effect [55]. In addition, patients with schizophrenia
and bipolar disorder showed reduced serum BDNF levels in relation to healthy
volunteers [17, 49, 50]. Moreover, antipsychotic, as well as mood stabilizers treatment
seems to alter levels of BDNF [9, 29, 49, 50].
In the present findings we showed that treatment with FLX and OLZ alone
or in combination did not alter BDNF protein levels in the prefrontal cortex,
hippocampus or amygdala. Our investigation did not confirm a previous study showed
by other authors, who reported alteration in BDNF after treatment with FLX or OLZ.
Lee et al. [35] showed that OLZ treatment (10–100 µM) increased basal BDNF gene
promoter activity in a dose dependent manner and increased protein levels at high dose.
In addition, OLZ increased BDNF levels in the cortex and hippocampus [18] and frontal
cortex [10]. In addition, FLX administration increased BDNF mRNA levels in the
nucleus accumbens and hippocampus [42-43]. We cannot explain the reason for this
different result, but similar discrepancies have been observed by the other author. In
fact, plasma BDNF levels did not alter after 8 weeks of treatment with OLZ [31, 67].
Additionally, FLX did not alter BDNF mRNA isoforms in the rat hippocampus [5, 19].
FLX (10 mg/kg) increased BDNF protein levels in the frontal cortex, but not in the
96
hippocampus, amygdala, olfactory, and brain stem [10]. Other study showed still that
FLX treatment decrease BDNF mRNA expression in the rat hippocampus [41].
Moreover, OLZ decreased BDNF in the hippocampus and frontal cortex [7]. In
addition, OLZ treatment did not alleviate the decreases in RNA expression of BDNF
and NGF produced by neonatal quinpirole treatment [15]. Furthermore, both BDNF and
NGF seem to be activity-dependent [36]. In the present data, we did not alteration in
BDNF or NGF protein levels in the rat brain, suggesting that the effects exerted by FLX
or OLZ on the BDNF and NGF levels may have been manner-dependent.
NGF is very important to neuronal survival, neurite outgrowth and synapse
formation [4] and has shown an association between NGF and mood disorders or
psychiatric diseases. In fact, plasma NGF concentrations were decreased in BD patients
when compared to that seen with controls and BD individuals in mania had lower NGF
levels than euthymic patients or controls [12]. Moreover, NGF has been found
decreased in plasma, liquor or postmortem brains of schizophrenic patients [3, 13, 23].
In addition, the antipsychotics, haloperidol, chlorpromazine, risperidone and OLZ
reduced BDNF and NGF protein levels in the striatum and hippocampus [48], in this
study the authors showed that second-generation antipsychotics compared to firstgeneration antipsychotics induce less deleterious on neurotrophic factor in the brain. In
contrast, Parikn et al. [46], demonstrated that OLZ, but not antipsychotic risperidone
increased levels of NGF levels in the hippocampus. Additionally, administration of
OLZ for 29 days water at the doses of 3 and 15 mg/kg increased BDNF in the
hippocampus and occipital cortex [7]. Discrepancies between studies may be related to
route of drug administration.
Little is known about antidepressants and NGF. In the present we did not
showed alteration on the NGF protein levels after treatment with FLX alone or in
combination with OLZ. The antidepressants, amitriptyline and paroxetine also did not
alter NGF serum concentrations from depressed patients [30]. However, very recently
was demonstrated that intranasal NGF had significant antidepressant effects on animal
models of depression [54]. On the other hand, NGF was reduced in brain regions of the
Flinders Sensitive Line rat, a genetic animal model of depression [2000]. In addition, an
escitalopram-dependent NGF reduction in stressed rats was detectable in the cortex, but
not in the frontal cortex, cerebellum and serum [52]. Moreover, the antidepressant
fluvoxamine potentiated the NGF-induced neurite outgrowth [44] and desipramine or
fluoxetine for 48 h elevated the NGF mRNA expression [37] in PC12 cells.
97
NT-3 is a neurotrophin that plays key roles in neuronal survival,
differentiation, connectivity and plasticity [32, 51]. Moreover, human studies have
demonstrated participation of NT-3 in the pathophysiology of stress, major depression
and BD [25, 34, 56]. In the present data we interestingly showed that administration of
FLX and OLZ in combination increased NT-3 protein levels in the prefrontal cortex.
Lesions of the prefrontal cortex are associated with development of depression or
aggression [8].
Serum NT-3 levels in drug-free and medicated patients with BD during
manic and depressive episodes were increased when compared with controls [25]. In a
study conducted by Otsuki et al. [45] was showed reduced expression levels of NT-3
mRNAs, but not in BDNF, NGF and neurotrophin-4 mRNAs in peripheral white blood
cells in patients with major depressive disorder in a current depressive state, but not in a
remissive state, suggesting that the changes in the NT-3 mRNAs might be statedependent and associated with the pathophysiology of major depression. Our findings
showed that FLX plus OLZ increased NT-3 protein levels, but not BDNF and NGF,
suggesting that treatment with these drugs may be important to current depressive state.
The gene expression for NT-3 was not affected by single or repeated administration of
antidepressants drugs, including FLX [16]. Our study also showed that FLX alone did
not alter NT-3 protein levels, but in combination with OLZ increased NT-3 protein
levels. A study conducted by Matthews et al. [40] showed that the combination of OLZ
and FLX significantly greater and faster improvement than was either drug alone in
subjects with treatment-resistant depression.
In addition, studies have shown an association between neurotrophins and
glucocorticoids. It was shown an Increase of NGF and NT-3 mRNA in the hippocampus
by glucocorticoids, probably as a compensatory response to stress-induced damage [56].
Additionally a study showed that glucocorticoids exert biphasic effects on neuronal
mitochondrial dynamics, with low levels potentiating and chronic high levels
attenuating various aspects of mitochondrial function [21]. Studies from our group
recently demonstrated that FLX and OLZ alone or in combination altered the creatine
kinase and citrate synthase activities [1-2], which are involved with energy metabolism.
In conclusion, the present findings suggest that NT-3 may be involved in the
therapeutic action of olanzapine/fluoxetine combination in mood disorders.
98
ACKNOWLEDGEMENTS
This study was supported in part by grants from ‘Conselho Nacional de
Desenvolvimento Científico e Tecnológico’ (CNPq-Brazil – JQ and FK), from the
Instituto Cérebro e Mente (JQ) and UNESC (JQ). JQ and FK are recipients of CNPq
(Brazil) Productivity Fellowships. GZR is holder of a FAPESC studentship.
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107
FIGURES
Figure 1:
A
,50
*
,45
Sal+Sal
NT3 pg/µg
,40
Sal+OLZ 3
,35
Sal+OLZ 6
,30
Sal+FLX 12.5
,25
Sal+FLX 25
,20
OLZ 3+FLX 12.5
,15
OLZ 3+FLX 25
OLZ 6+FLX 12.5
,10
OLZ 6+FLX 25
,05
,00
Prefrontal cortex
Hippocampus
Amygdala
B
,25
Sal+Sal
,20
BDNF pg/µg
Sal+OLZ 3
Sal+OLZ 6
,15
Sal+FLX 12.5
Sal+FLX 25
OLZ 3+FLX 12.5
,10
OLZ 3+FLX 25
OLZ 6+FLX 12
,05
OLZ 6+FLX 25
,00
Prefrontal cortex
Hippocampus
Amygdala
108
C
1,0
0,9
Sal+Sal
NGF pg/µg
0,8
Sal+OLZ 3
0,7
Sal+OLZ 6
0,6
Sal+FLX 12.5
0,5
Sal+FLX 25
0,4
OLZ 3+FLX 12.5
0,3
OLZ 3+FLX 25
0,2
OLZ 6+FLX 12
OLZ 6+FLX 25
0,1
Prefrontal cortex
Hippocampus
Amygdala
109
LEGEND OF FIGURES
Figures 1A, 1B e 1C: Effects of the chronic administration of OLZ and FLX on the NT-3 (A), BDNF (B)
and NGF (C) protein levels in the rat prefrontal cortex, hippocampus and amygdala. Bars represent means
± S.E.M.* p <0.05 vs. saline according to ANOVA followed by Tukey post-hoc test.
110
PARTE III
111
5
DISCUSSÃO
A combinação de um antipsicótico atípico a um antidepressivo é observado
na prática clínica como uma estratégia para a depressão resistente ao tratamento (Fava
et al., 2000; Hirschfeld et al., 2002a; Benazzi et al., 2009), depressão psicótica
(Schatzberg et al., 2003; Dodd et al., 2005) e também para o tratamento da depressão
bipolar (Müller-Oerlinghausen et al., 2002; Tohen et al., 2004; Shelton et al., 2006). A
combinação de OLZ/FLX encontra-se associada a uma rápida e significativa melhora do
quadro clínico quando comparado a monoterapia em pacientes resistentes ao tratamento
para depressão (Shelton et al., 2001; Bobo & Shelton, 2010). Sabe-se que a fase mais
difícil de tratamento de um paciente bipolar é a depressiva (Calabrese et al., 1999).
Além disso, esta combinação está associada a eficácia, segurança e tolerabilidade no
tratamento da depressão resistente (Bobo et al., 2010).
Em virtude dos resultados encontrados com a combinaçao foi recentemente
aprovado pelo FDA, nos Estados Unidos (EUA), a combinação de OLZ/FLX para a
depressao bipolar (Shelton et al., 2006) industrializada pela Eli Lilly e comercializada
pelo nome de Symbyax TM.
Entende-se que, por um lado o benefício clínico da combinação OLZ/FLX
para o tratamento da depressão bipolar parece ser evidente (Tohen et al., 2004; Shelton
et al., 2006) porém, os mecanismos neuronais responsáveis por esta eficácia não são
totalmente conhecidos (Ghaemi et al., 1999; Tamayo et al., 2009; Tohen et al., 2010).
Evidências na literatura indicam que o cérebro possui um intenso
metabolismo energético e que pode estar envolvido na fisiopatologia de muitos
transtornos psiquiátricos como a depressão maior e o THB (Kato & Kato, 2000;
Konradi et al., 2004; Corrêa et al., 2007; Lucca et al., 2009; Rezin et al., 2009; Streck et
al., 2008).
Efeitos colaterais de antipsicóticos e antidepressivos, que são limitantes do
tratamento crônico e sua adesão, foram associados com estresse oxidativo e/ou a
diminuição de energia (Hamman et al., 1995; Gross et al, 1996; Szasz et al., 2007;
Martins et al., 2008).
112
Cérebro de ratos adultos, como outros tecidos com metabolismo que
necessitem de altas e variadas quantidades de ATP, apresentam altas concentrações de
fosfocreatina e atividade da enzima CK (Wallimann et al., 1992). A CK catalisa uma
transferência reversiva do grupo fosforil (P) da fosfocreatina para a ADP regenerando
ATP (Dzeja et al., 1996). Uma diminuição da atividade da enzima CK esta associado a
vias neurodegenerativas que resultam e morte neuronal por isquemia, (Tomimoto et al.,
1993), doenças neurodegenerativas (Akzenov et al., 2000; David et al., 1998), THB
(Streck et al., 2008) e outras patologias (Gross et al., 1996; Hamman et al., 1995). A
disfunção desta enzima é encontrada também na depressão resistente, esquizofrenia e no
THB (Kato & Kato, 2000; Prabakan et al., 2004; Andresa et al., 2008). Alguns
antidepressivos seletivos demonstram aumentar a atividade da CK em cerebros de ratos
(Santos et al., 2009)
Evidencias demonstram que antioxidantes, como a ketamina, podem
reverter a inibição da cadeia respiratoria em cerebro de ratos induzidos por um modelo
de estresse crônico moderado (Rezin et al., 2009). De fato, outros estudos recentes com
o mesmo modelo, demonstram que existe um aumento do estresse oxidativo em regiões
submitocondriais do cérebro. Descontroles nas atividades dos complexos respiratórios
mitocondriais e produção de energia podem levar inclusive a degeneração celular
(Rustin et al., 1998). Além disso, outras evidências demonstram que antipsicóticos
alteram parâmetros oxidativos em cérebros de ratos (Agostinho et al., 2007; Martins et
al., 2008).
Em um modelo animal de mania, a atividade das enzimas CK e CS esta
inibida (Streck et al., 2008; Corrêa et al., 2007). Recentemente, Assis e colaboradores
(2009) encontraram que a OLZ 10mg/kg aumenta a atividade de CK no estriado de ratos
sem afetar a atividade da enzima no hipocampo e no córtex. Entretanto, com baixas
doses da OLZ (2,5 e 5mg/kg) a atividade da CK é inibida no cerebelo e córtex
prefrontal. Antidepressivos, como a imipramina, podem inibir a atividade da CK em
cérebro de ratos, provavelmente envolvendo o metabolismo energético (Assis et al.,
2009).
O presente estudo mostrou que após a administraçao aguda de OLZ, a
atividade da CK foi inibida no cerebelo e no córtex prefrontal. Após administração
aguda de FLX a atividade da CK foi inibida nas mesmas áreas e também no hipocampo,
estriado e no córtex cerebral. No tratamento crônico, 2 horas após a última injeção, a
atividade da CK estava diminuída após FLX monoterapia e em combinação com a OLZ
113
no cerebelo, córtex prefrontal, hipocampo, estriado e córtex cerebral. Entretanto, no
tratamento crônico, 24 horas após a última injeção da monoterapia OLZ ou FLX ou em
combinação, não se observaram alterações na atividade da enzima. Estes dados sugerem
que a FLX não apresenta um efeito crônico na atividade da CK e podem estar
associados ao período após a última administração.
Em conjunto estes dados sugerem que o efeito inibitório da OLZ e FLX na
atividade da CK em cérebro de ratos pode levar a especular se estas drogas influenciam
no metabolismo energético. Entretanto é difícil correlacionar estes resultados com a
sintomatologia clínica. Porém, a inibição da atividade da CK pode estar associada ao
aparecimento de alguns efeitos colaterais destes medicamentos (Fava et al., 2000).
A CS esta localizada na matriz mitocondrial e tem sido utilizada como
enzima marcadora quantitativa da presença de dano mitocondrial (Marco et al., 1974).
O presente estudo mostrou que o tratamento agudo com OLZ 6mg/kg em monoterapia e
a combinação OLZ/FLX 3/25 mg/kg aumenta a atividade da CS no córtex prefrontal e
hipocampo. O tratamento agudo com OLZ nas doses de 3 e 6 mg/kg e FLX 25mg/kg em
monoterapia e a combinação OLZ/FLX 3/25mg/kg aumentou a atividade da CS no
estriado. No tratamento crônico tanto a monoterapia quanto a combinação de FLX e
OLZ não alteram a atividade da enzima após 2 e 24 horas da última administração.
Este estudo indica que os efeitos da OLZ e FLX estão envolvidos em
alterações na função mitocondrial. A razão destas alterações não estão esclarecidas,
porém podem estar relacionadas com dessensibilização para os efeitos reparativos da
OLZ e FLX, por mecanismo de adaptação ou mecanismo de defesa e associado ao
sinegismo do mecanismo de ação da combinação.
Os tecidos com alta demanda de energia como o cérebro, contém um grande
número de mitocôndrias sendo, entretanto, mais suscetível a redução do metabolismo
aeróbico. (Boekema et al., 2007).
A mitocôndria é uma organela que promove um papel crucial na produção
de ATP (Calabrese et al., 2001). A maior parte da energia celular é obtida através da
fosforilação oxidativa, um processo que requer a ação de complexos enzimáticos
respiratórios, localizados na membrana mitocondrial interna (Horn & Barrientos, 2008).
A cadeia respiratória é denominada pelo conjunto dos complexos sendo composta por
cinco complexos que transportam pares de elétrons pela translocação de prótons da
matriz mitocondrial para o espaço intermembrana. O gradiente de prótons gerado é
114
utilizado pela ATP sintase para formar ATP pela fosforilação de adenosina difosfato
(ADP) (Madrigal et al., 2001; Fattal et al., 2006).
Distúrbios no funcionamento fisiológico da mitocôndria resultante de um
malfuncionamento da cascata bioquímica e dano na cadeia trasportadora de elétrons tem
sido sugerido com sendo um importante fator na patogênese de uma serie de distúrbios
neuropsiquiátricos como o THB, depressão e esquizofrenia (Fattal et al., 2006; Prakaran
et al., 2004). Diversos estudos tem demostrado que anormalidades no metabolismo
energético pode levar a degeneraçao celular (Calabrese et al., 2001) além de estar
envolvida em processos essenciais, como apoptose (Gur et al., 1987; Gigante et al.,
2010).
Outros estudos tem identificado regiões cerebrais especificas em pacientes
bipolares uma diminuição no metabolismo energético e anormalidades no DNA
mitocondrial (Iwamato et al., 2005; Kato et al., 2000; 2005). Modelos animais que
avaliam a farmacologia molecular dos estabilizadores do humor tem implicado que o
metabolismo energético mitocondrial como um alvo para estas drogas (Wang et al.,
2004; Correa et al., 2007).
O presente estudo mostra que o tratamento agudo e crônico com FLX e
OLZ em monoterapia ou combinação alteram a atividade dos complexos da cadeia
respiratória em cérebro de ratos. Porém na combinação pode ser observado uma grande
alteração relacionada com a concentração das drogas, a área cerebral e o protocolo
empregado.
No tratamento agudo a atividade do complexo I apresenta-se aumentada no
córtex prefrontal com OLZ 6mg/kg e a combinação OLZ/FLX 3/12,5 mg/kg, no
hipocampo com FLX 25mg/kg e no estriado com OLZ 6mg/kg. A atividade do
complexo II encontra-se aumentada no córtex prefrontal, hipocampo e estriado com
OLZ 6 mg/kg em monoterapia. A atividade do complexo II-III também está aumentada
no prefrontal, hipocampo, estriado na terapia combinada de OLZ 3 e 6 mg/kg com FLX
12,5 e 25 mg/kg. A atividade do complexo IV também apresenta-se aumentada no
prefrontal estriado na terapia combinada de OLZ 3 e 6 mg/kg com FLX 12,5 e 25
mg/kg.
No tratamento crônico, 2 horas após a ultima administração, houve aumento
na atividade do complexo I com OLZ/FLX 6/25 mg/kg no estriado. A atividade do
complexo II esta também está aumentada no estriado na dose combinada de OLZ/FLX
6/12,5 mg/kg. A atividade do complexo III encontra-se aumentada no estriado na
115
monoterapia com OLZ 3mg/kg. A atividade do complexo IV apresenta-se aumentada no
hipocampo na combinação OLZ/FLX 6/25 mg/kg. Estes resultados mostram o estriado
e o hipocampo com aumento na atividade da cadeia respiratória 2 horas após a última
administração
com
o
tratamento
crônico
em
diferentes
concentrações
dos
medicamentos.
No tratamento crônico 24 horas após a última administração a atividade do
complexo I encontra-se aumentada no prefrontal na monoterapia com FLX 12,5 mg/kg.
Não houve alterações na atividade do complexo II. A atividade do complexo III
encontra-se aumentada no estriado no tratamento com FLX 12,5 mg/kg. A atividade do
complexo IV esta aumentada com a combinação de OLZ/FLX 6/12,5 mg/kg. Em
contraste há uma inibição da atividade do complexo IV no hipocampo após monoterapia
com OLZ 3 e 6 mg/kg, FLX 12,5 e 25 mg/kg e a combinação de OLZ 3 mg/kg com
FLX 12,5 e 25 mg/kg. Em conjunto, estes resultados indicam que a atividade dos
complexos da cadeia respiratória encontra-se aumentada. Este efeito pode ser
considerado positivo quando comparado com estudos que indentificaram uma
diminuição no metabolismo energético em cérebros de pacientes com o THB (Iwamoto
et al., 2005; Kato & Kato, 2000; Kato et al., 2005). Levando em consideração que o
metabolismo energético mitocondrial esta sendo considerado um alvo para novas
estratégias de tratamento (Correa et al., 2007; Wang et al., 2004) a ação da combinação
OLZ/FLX na cadeia respiratória pode ter uma contribuição para a resposta rápida e
intensa observada nos pacientes com THB.
As neurotrofinas BDNF, NGF, NT-3 promovem a sobrevivência neuronal e
a plasticidade neuronal (Lindsey et al., 1994; Altar & Distefano, 1998). Além disso,
Smith e colaboradores (1995) demonstram o envolvimento do estresse e o aumento nos
níveis de glicocorticóides afetam a expressão de BDNF e NT-3. As reduções nos níveis
de BDNF sérico são observados em pacientes com esquizofrenia e THB (Cunha et al.,
2006; Rizos et al., 2010), sendo que relatos na literatura mostram que antidepressivos,
antipsicóticos e estabilizadores do humor alteram os níveis de BDNF (Gonzáles-Pinto et
al., 2010; Bai et al., 2003; Rizos et al., 2010; Shirayama et al., 2002). Assim, diversos
estudos têm relacionado o papel das neurotrofinas na fisiopatologia e tratamento de
transtornos psiquiátricos como depressão, esquizofrenia e o THB (Lindsey et al., 1994;
Duman & Monteggia, 2006; Karege et al., 2002; Cunha et al., 2006; Rizos et al., 2010).
A concentração sérica da NGF encontra-se diminuída em pacientes com
THB. Pacientes com este transtorno em episódio maníaco apresentam níveis diminuídos
116
de NGF quando comparados com controles e pacientes THB eutímicos (Barbosa et al.,
2010). Alem disso, evidencias demonstram um efeito sinérgico da combinação da
OLZ/FLX na modulação do NGF (Maragnoli et al., 2006).
O NT-3 é uma neurotrofina que tem um papel chave na sobre a
sobrevivência, diferenciação, conexão e plasticidade neuronal (Huang & Reichardt,
2001; Schinder & Poo, 2000). Estudos em humanos têm descrito a participaçao do NT-3
na fisiopatologia do estresse, depressão maior e THB (Fernandez et al., 2010; Smith et
al., 1996).
No presente estudo a avaliação dos efeitos da OLZ, FLX e a sua
combinação nos níveis de NT-3, BDNF e NGF no cérebro de ratos mostrou que o
tratamento crônico com a combinação FLX/OLZ aumenta os níveis de NT-3 no córtex
prefrontal. Lesões em neurônios do córtex prefrontal estão associados ao
desenvolvimento da depressão (Arango et al., 1995). Este resultado sugere que o
tratamento com a combinação OLZ/FLX pode ser importante no tratamento para THB
no estado atual depressivo. Em conjunto, estes dados sugerem que o NT-3 pode estar
envolvido na ação terapêutica da combinação de OLZ/FLX no THB.
No presente estudo foi concluído que a administração da combinação da
OLZ/FLX aumenta a expressão de NT-3 no córtex préfrontal de ratos, sugerindo que
esta neurotrofina pode estar envolvida nos efeitos terapêuticos da combinação de
OLZ/FLX, observados em pacientes bipolares. Além disso, foi observado que a
combinação de OLZ/FLX alterou enzimas do metabolismo energético, sendo
necessários maiores estudos sobre o metabolismo energético para avaliar os efeitos
dessa combinação em pacientes com THB.
117
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