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UNIVERSIDADE FEDERAL DO CEARÁ
PRÓ-REITORIA DE PESQUISA E PÓS-GRADUAÇÃO
PROGRAMA DE PÓS-GRADUAÇÃO EM BIOTECNOLOGIA DA REDE
NORDESTE DE BIOTECNOLOGIA
RAQUELL DE CASTRO CHAVES
EFEITO ANTIDEPRESSIVO DA RIPARINA IV SOBRE PADRÕES
COMPORTAMENTAIS E NEUROQUÍMICOS DE CAMUNDONGOS EXPOSTOS
AO MODELO DE ESTRESSE CRÔNICO INDUZIDO PELA ADMINISTRAÇÃO DE
CORTICOSTERONA
FORTALEZA
2019
RAQUELL DE CASTRO CHAVES
EFEITO ANTIDEPRESSIVO DA RIPARINA IV SOBRE PADRÕES
COMPORTAMENTAIS E NEUROQUÍMICOS DE CAMUNDONGOS EXPOSTOS AO
MODELO DE ESTRESSE CRÔNICO INDUZIDO PELA ADMINISTRAÇÃO DE
CORTICOSTERONA
Tese apresentada ao Programa de Pós-Graduação em Biotecnologia da Rede Nordeste em Biotecnologia – RENORBIO, da Universidade Federal do Ceará, como requisito parcial à obtenção do título de Doutor em Biotecnologia. Área de concentração: Biotecnologia em Saúde. Orientadora: Profa. Dra. Francisca Cléa Florenço de Sousa. Coorientadora: Profa. Dra. Alyne Mara Rodrigues de Carvalho
FORTALEZA
2019
Dados Internacionais de Catalogação na Publicação Universidade Federal do Ceará
Biblioteca UniversitáriaGerada automaticamente pelo módulo Catalog, mediante os dados fornecidos pelo(a) autor(a)
C439e Chaves, Raquell de Castro. Efeito antidepressivo da Riparina IV sobre padrões comportamentais e neuroquímicos de camundongosexpostos ao modelo de estresse crônico induzido pela administração de corticosterona / Raquell de CastroChaves. – 2019. 130 f. : il. color.
Tese (doutorado) – Universidade Federal do Ceará, Pró-Reitoria de Pesquisa e Pós-Graduação, Programade Pós-Graduação em Biotecnologia (Rede Nordeste de Biotecnologia), Fortaleza, 2019. Orientação: Profa. Dra. Francisca Cléa Florenço de Sousa. Coorientação: Profa. Dra. Alyne Mara Rodrigues de Carvalho.
1. Depressão. 2. Corticosterona. 3. Citocinas. 4. Estresse oxidativo . 5. Riparina. I. Título. CDD 660.6
RAQUELL DE CASTRO CHAVES
EFEITO ANTIDEPRESSIVO DA RIPARINA IV SOBRE PADRÕES
COMPORTAMENTAIS E NEUROQUÍMICOS DE CAMUNDONGOS EXPOSTOS AO
MODELO DE ESTRESSE CRÔNICO INDUZIDO PELA ADMINISTRAÇÃO DE
CORTICOSTERONA
Tese apresentada ao Programa de Pós-Graduação em Biotecnologia da Rede Nordeste em Biotecnologia – RENORBIO, da Universidade Federal do Ceará, como requisito parcial à obtenção do título de Doutor em Biotecnologia. Área de concentração: Biotecnologia em Saúde.
Aprovada em: 28 / 01 / 2019.
BANCA EXAMINADORA
________________________________________ Profa. Dra. Francisca Cléa Florenço de Sousa (Orientador)
Universidade Federal do Ceará (UFC)
________________________________________
Profa. Dra. Alyne Mara Rodrigues de Carvalho (Coorientador) Universidade Federal do Ceará (UFC)
________________________________________
Profa. Dra. Marta Maria de França Fonteles Universidade Federal do Ceará (UFC)
________________________________________
Profa. Dra. Mirna Marques Bezerra Universidade Federal do Ceará (UFC)
________________________________________
Profa. Dra. Kelly Rose Tavares Neves Universidade Federal do Ceará (UFC)
________________________________________
Prof. Dr. José Eduardo Honório Júnior Faculdade UniChristus
AGRADECIMENTOS
Nenhuma batalha é vencida sozinha. O sucesso destes anos de trabalho é de todas
as pessoas que estiveram ao meu lado estimulando para que hoje se tornasse uma conquista.
A Deus, por sempre me guiar segundo a tua vontade, me iluminar nos momentos
mais sombrios e me dar tranquilidade para seguir em frente com os meus objetivos e não
desanimar com as dificuldades.
Aos meus pais, Isabel, Paulo, Fernando e Ethel, por sempre, e principalmente nas
dificuldades, me mostrarem que eu não estou sozinha. Agradeço por não medirem esforços para
que meus sonhos se tornem realidade.
Aos meus irmãos, por compreenderem minhas ausências e me ajudarem quando
surgia um pedido de “me ajuda a corrigir aqui?” (Caio).
Ao meu esposo, Fellipe, que me acompanhou durante toda essa trajetória, por todo
o apoio para seguir em frente, dia após dia. Agradeço toda a parceria, força, abdicações e
paciência com as minhas angústias. Obrigada por ser plateia, mesmo sem entender nada do
assunto. Sem você, eu não estaria aqui.
Às minhas maravilhosas Cobras, presente que a faculdade me deu, Vív, Auri, Bela,
Naty, Mile e Pri, por me estimularem a buscar sempre mais. Uma amizade que vai além do
acadêmico e se torna essencialmente parte de quem eu sou hoje.
À minha orientadora, Profa. Cléa, por me inspirar e acreditar em mim durante todo
esse tempo e, com seu imenso coração, se tornar minha amiga e mãezona. Agradeço por
compreender minhas dificuldades, angústias, inseguranças e sempre acreditar no meu potencial.
À minha amiga e coorientadora, Alyne Mara, por ser extremamente disponível para
me ajudar todas as vezes que eu precisei (que não foram poucas!).
À maravilhosa banca de professores, Marta Fonteles, Mirna Bezerra, Kelly Neves,
Renata Alves e Eduardo Honório, por disponibilizarem seu precioso tempo para agregarem a
este trabalho.
Aos colegas do Lab Neuro, em especial a Iris, Victor, Daniel, Tiago, Iardja,
Marianas, Dana, Lara, Gabriel, Ricardo, Samily, Otoni, por permitirem que esse trabalho se
concretizasse. Agradeço por todos os fins de semana e feriados perdidos em tratamento de
animais. Em especial à Auriana por ter dividido comigo todas as angústias desse tratamento
crônico, com a sua companhia ficou mais fácil continuar. Aos outros que não puderam
participar dos experimentos, agradeço a troca de experiências nos seminários e a amizade do
dia a dia.
Agradeço aos encontros nos cafés, com as “riparianas”, reuniões regadas a muitas
trocas de informações que engrandeceram este trabalho.
À minha nova família, pelo acolhimento, em especial a minha sogra, Anna Sophia,
por todo apoio de sempre, principalmente me socorrer nas impressões. Agradeço aos meus
cunhados, Marcelo e Henrique, pelas dúvidas e revisão do inglês.
Às técnicas do laboratório de Neurofarmacologia, Lena e Vilani, que sempre
fizeram o seu melhor para manter o laboratório funcionando, sempre disponível para ajudar.
Aos meus colegas e amigos, Emiliano, Joyce, Malena, Carlos Renato e Leo, por
todo o apoio nessa difícil jornada.
Aos meus alunos, por me mostrarem o amor pela docência e a vontade de mudar o
mundo. Com vocês eu aprendi mais que ensinei!
Aos professores José Barbosa Filho e Stanley Gutierrez, pela síntese e
disponibilização da riparina IV sempre que foi necessário.
À Renorbio, através do Prof. Ivanildo, que nessa reta final se mostrou extremamente
acessível e empenhado na resolução de todos os problemas, e ao Adil, que sempre se
disponibilizou para auxiliar em todos os “perrengues”.
À Unidade Multiusuário do Núcleo de Pesquisa e Desenvolvimento de
Medicamentos – NPDM, UFC, em nome da Giovanna Barbosa, pelo apoio técnico na
utilização do Citômetro de fluxo.
À FUNCAP, CAPES e CNPq pelo apoio financeiro e viabilização deste projeto.
“If you focus on what you left behind, you will
never see what lies ahead.” – Ratatouille.
RESUMO
Os transtornos mentais têm etiologia multifatorial e o estresse apresenta-se como um dos fatores
causais. Na depressão, sugere-se que a alta concentração de cortisol contribui diretamente para
a patologia desta doença. Com base nisso, o presente estudo tem como objetivo avaliar o
potencial efeito antidepressivo da Riparina IV (Rip IV) em camundongos submetidos ao
modelo de estresse crônico por administração repetida de corticosterona. Camundongos Swiss
fêmeas foram divididos em quatro grupos: controle (Controle), corticosterona (Cort), Riparina
IV (Cort + Rip IV) e fluvoxamina (Cort + Flu). Três grupos receberam corticosterona (20
mg/kg, por via subcutânea) durante vinte e um dias, enquanto o grupo controle recebeu apenas
veículo salina. Após o décimo quarto dia, foram administrados aos grupos as drogas testes:
Riparina IV (50 mg/kg), fluvoxamina (50 mg/kg) ou água destilada, por gavagem, uma hora
após as injeções subcutâneas. No final do esquema de dosagem, foram realizados testes
neurocomportamentais, como o teste de nado forçado (FST), suspensão da cauda (TST), campo
aberto (OFT), labirinto em cruz elevado (EPM), preferência pela solução de sacarose ( SPT),
labirinto em Y (YMT), esquiva passiva (SDIT), interação social (SIT) e o teste de inibição de
pré-pulso (PPI). Os testes comportamentais foram acompanhados por avaliação de
neuroinflamação, através da avaliação dos parâmetros do estresse oxidativo (níveis de
malondialdeído, de nitrito/nitrato e de glutationa reduzida, atividade da superóxido dismutase
e da catalase) e perfil de citocinas (TNF-a, IFN-g, IL-2, IL-4, IL-6 e IL-10) e neuroplasticidade
(níveis de fator neurotrófico derivado do cérebro - BDNF) por meio de análises bioquímicas no
córtex pré-frontal, no estriado e no hipocampo. Os testes comportamentais revelaram o
desenvolvimento do comportamento ansioso/ depressivo e déficit cognitivo em camundongos
do grupo Cort em comparação ao controle. O tratamento com a Cort também induziu ao estresse
oxidativo e neuroinflamação, levando à diminuição do BDNF e morte celular neuronal. O
tratamento com a Rip IV, de forma semelhante ao antidepressivo Flu, mostrou um efeito
antidepressivo com melhora da função cognitiva, revelando o seu efeito neuroprotetor sobre o
estresse oxidativo, a neurogênese e perfil de citocinas pro-inflamatórias e anti-inflamatórias.
Este efeito antioxidante e anti-inflamatório observado coloca a Riparina IV como um possível
medicamento no tratamento antidepressivo de pacientes não-responsivos relacionados a
sintomas graves e cognitivos.
Palavras-chave: Depressão. Corticosterona. Citocinas. estresse oxidativo. Riparina.
ABSTRACT
Mental disorders have a multifactorial etiology and stress presents as one of the causal factors.
In depression, it is suggested that high cortisol concentration contributes directly to the
pathology of this disease. Based on these findings, the study aimed to investigate the potential
antidepressant effect of Riparin IV (Rip IV) in mice submitted to chronic stress model by
repeated corticosterone administration. Female Swiss mice were divided into four groups:
control (Control), corticosterone (Cort), Riparin IV (Cort + Rip IV) and fluvoxamine (Cort +
Flu). Three groups were administrated with corticosterone (20 mg/kg, subcutaneous) during the
21-day study, while the control group received only saline vehicle. After the 14th day, the
groups were administrated the following tested drugs: Riparin IV (50 mg/kg), fluvoxamine (50
mg/kg) or distilled water vehicle, by gavage, one hour after the subcutaneous injections. At the
end of dosing schedule, neurobehavioral tests were conducted such as the forced swimming test
(FST), the tail suspension test (TST), the open field test (OFT), the elevated plus maze (EPM),
the sucrose preference test (SPT), the Y-maze test (YMT), the step-down inhibitory avoidance
test (SDIT), the social interaction test (SIT), and the prepulse inhibition test (PPI). Behavioral
tests were followed by neuroinflammation, through oxidative stress (malondialdehyde,
nitrite/nitrate and reduced glutathione levels, and superoxide dismutase and catalase activities)
and cytokine content (TNF-a, IFN-g, IL-2, IL-4, IL-6 e IL-10), and neuroplasticity (brain-
derived neurotrophic factor – BDNF - levels) evaluation through biochemical analysis in the
prefrontal cortex, the striatum and the hippocampus. Behavioral tests revealed the development
of anxiety/depressive-like behavior with an cognitive deficit in Cort mice as compared to the
control. Cort treatment also induced to oxidative stress and neuroinflammation, leading to a
decrease of brain-derived neurotrophic factor (BDNF) and neuronal cell death. Rip IV
treatment, in a similar manner to the antidepressant Flu, showed an antidepressant-like effect
improving cognitive function, reveling its neuroprotective effect regarding oxidative stress,
neurogenesis and pro-inflammatory and anti-inflammatory cytokine profile. This antioxidant
and anti-inflammatory effect observed indicates Riparin IV as a possible drug in the
antidepressant treatment of non-responsive patients related to severe and cognitive symptoms.
Keywords: Depression. Corticosterone. Cytokines. Oxidative stress. Riparin.
LISTA DE ABREVIATURAS E SIGLAS
5-HT 5-hidroxitriptamina ou serotonina
5-HT1A Receptor de 5-hidroxitriptamina 1A
5-HT2 Receptor de 5-hidroxitriptamina 2
5-HTTLPR Polimorfismo no transportador de serotonina
ACTH Hormônio adrenocorticotrópico
AMPA α-amino-3-hidroxi-5-metil-4- isoxazolepropionato
BDNF Fator Neurotrófico Derivado do Cérebro
CA3 Corno de Amon 3
CAT Catalase
CNS Sistema nervoso central
Cort Corticosterona
CORT Corticosterona
CRF Fator de liberação da corticotropina
DSM Manual de Diagnóstico e Estatístico de Transtornos Mentais
DTNB ácido 5,5'-ditio-bis-(2-nitrobenzóico) ou Reagente de Ellman
EPM Labirinto em cruz elevado
EROS Espécies reativas de oxigênio
Flu Fluvoxamina
FST Teste do nado forçado
GABA Ácido gama-aminobutírico
GR Receptor glicocorticoide
GSH Glutationa reduzida
GSHPx Glutationa peroxidase
GSR Glutationa redutase
GSSG Glutationa dissulfeto
H2O2 Peróxido de hidrogênio
HC Hipocampo
HHA Hipotálamo-hipófise-adrenal
HPA Eixo hipotálamo-pituitária-adrenal
IDO Indoleamina-2,3-dioxigenase
IFN-a Interferon alfa
IL-1b Interleucina-1 beta
IL-10 Interleucina-10
IL-2 Interleucina-2
IL-4 Interleucina-4
IL-6 Interleucina-6
INF-g Interferon gama
ISRN Inibidor seletivo da receptação de noradrenalina
ISRS Inibidor seletivo da recaptação de serotonina,
LTM Memória de longo prazo
LTP Long Term Potentiation
MAO Monoamino oxidase
MDA Malondialdeido
MDD Distúrbio Depressivo Maior
MR Receptor mineralocorticoide
NBT Nitroazul de tetrazólio
NMDA N- metil-D-aspartato
NMDAR Receptor de N- metil-D-aspartato
NO Óxido nítrico
NPV Núcleo paraventricular
O• Radical superóxido
OFT Teste do campo aberto
OG Oral Gavagem
OH• Radical hidroxila
OMS Organização Mundial de Saúde
ONOO Peroxinitrito
PFC Córtex pré-frontal
PPI Inibição pré-pulso
Rip IV Riparina IV
RNAm RNA Mensageiro
SC Subcutâneo
SDIT Esquiva passiva step-down
SDL Latência de descida
SEM Desvio padrão da média
SIT Interação social
SNRI Inibidor seletivo de recaptação de serotonina e noradrenalina
SOD Superóxido dismutase
SPT Testes da preferência pela solução de sacarose
SSRI Inibidor seletivo de recaptação de serotonina
ST Corpo estriado
STM Memória de curto prazo
TBARS Substâncias reativas ao ácido tiobarbitúrico
TNF-a Fator de necrose tumoral alfa
TNFR-1 Receptor do fator de necrose tumoral tipo 1
TrkB Tirosina-quinase relacionado a tropomiosina do tipo B
TST Teste da suspensão da cauda
vitamina C Ácido ascórbico
vitamina E a-tocoferol
WHO World Health Organization
YMT Labirinto em Y
SUMÁRIO
1 INTRODUÇÃO 12
2 REVISÃO DE LITERATURA 16
2.1 Estresse e depressão 16
2.1.1 Envolvimento do eixo hipotalâmico-hipofisário-adrenal (HHA) 16
2.1.2 Anormalidades funcionais e estruturais do cérebro 21
2.1.3 Processos cognitivos, memória e aprendizado 24
2.1.4 Plasticidade e sobrevivência neuronal 26
2.1.5 Envolvimento do sistema imunológico e o estresse oxidativo 27
2.1.6 Modelo da administração de corticosterona 33
2.2 Abordagens terapêuticas na depressão 36
2.3 Riparina IV 38
3 CAPÍTULOS 39
3.1 Capítulo I 40
3.2 Capítulo II 65
4 CONSIDERAÇÕES FINAIS 108
REFERÊNCIAS 109
ANEXO A – SUBMISSÃO DE ARTIGO CIENTÍFICO A REVISTA 130
12
1 INTRODUÇÃO
Os distúrbios do humor relacionados ao estresse atingem aproximadamente 17% da
população mundial resultando em enorme sofrimento pessoal, sobrecarga econômica e social
(KESSLER et al., 2009). Um desses distúrbios é a depressão, uma doença de curso crônico e
recorrente, cuja neurobiologia ainda não foi completamente identificada, mas acredita-se que é
resultante de anormalidades celulares e moleculares que interagem com fatores genéticos e
ambientais (KRISHNAN; NESTLER, 2008).
De acordo com a Organização Mundial de Saúde (OMS), cerca de 322 milhões de
pessoas vivem com depressão, compreendendo 4,4% da população mundial (2015). A
prevalência desse distúrbio é alta e aumenta de acordo com o crescimento da população
mundial, onde estimou-se um crescimento de 18,4% no número de pessoas com depressão de
2005 a 2015. A prevalência varia de acordo com o sexo e idade, acometendo o sexo feminino
com maior frequência que no masculino (quase o dobro) e os idosos (BARUA et al., 2011;
WORLD HEALTH ORGANIZATION, 2017).
No Brasil, poucos estudos são encontrados sobre a prevalência de distúrbios depressivos
em diferentes regiões, entretanto, a OMS estima que afeta cerca de 5,8% da população
brasileira. Estudos mostram que pessoas depressivas apresentam uma pobre qualidade de vida,
maior susceptibilidade a outras doenças como cardiopatias e diabetes, alto risco de
comportamento abusivo e suicídio, o que leva a uma alta utilização dos serviços de saúde
(KRISHNAN; NESTLER, 2008; SILVA et al., 2014; WORLD HEALTH ORGANIZATION,
2017).
De acordo com o Manual de Diagnóstico e Estatístico de Transtornos Mentais, 5ª edição
(DSM-V), apesar dos principais sintomas da depressão incluírem humor deprimido e anedonia
(falta de interesse em atos prazerosos), a doença é caracterizada por um complexo agrupamento
de sintomas clínicos que podem incluir agitação e/ou retardo psicomotor, diminuição de
energia, alteração do peso e do apetite, nervosismo, irritabilidade, distúrbios do sono e
deficiências cognitivas incluindo o impedimento da habilidade de pensamento, concentração e
tomada de decisões (AMERICAN PSYCHIATRIC ASSOCIATION, 2014). Além disso, os
13
indivíduos apresentam um aumento de doenças físicas, diminuição da interação social e uma
alta taxa de mortalidade (KESSLER; BROMET, 2013).
Atualmente os antidepressivos disponíveis, apesar de largamente prescritos para
depressão e doenças relacionadas ao humor e ansiedade, apresentam significantes limitações
incluindo um intervalo de tempo longo para início da resposta terapêutica (semanas a meses) e
baixos índices de resposta (apenas um terço respondem ao primeiro medicamento e até dois
terços respondem a vários fármacos) (TRIVEDI et al., 2006). Isto é particularmente
problemático para uma doença associada a altos índices de suicídio.
Os antidepressivos típicos agudamente bloqueiam a recaptação ou a metabolização das
monoaminas (serotonina e noradrenalina), sendo os inibidores seletivos da recaptação de
serotonina a classe de medicamentos mais amplamente prescritos para a depressão e distúrbios
relacionados. Este mecanismo de ação agudo dá suporte a hipótese monoaminérgica, mas o
longo intervalo de tempo para o início da resposta do tratamento indica um início lento nas
adaptações de sinalização e regulação de genes-alvo, que por sua vez, resultam na regulação de
múltiplos processos fisiológicos, incluindo neuroplasticidade, neuroproteção e neurogênese no
cérebro adulto, o que leva a demora das ações terapêuticas dos antidepressivos (KRISHNAN;
NESTLER, 2008; DUMAN et al., 1997).
Estudos recentes indicam que uma diminuição da plasticidade sináptica (neurogênese,
ramificação axonal, dendritogênese e sinaptogênese) em áreas específicas do SNC, em
particular o hipocampo, pode ser um fator importante na fisiopatologia do comprometimento
cognitivo de pacientes depressivos. A anormal plasticidade neuronal pode estar relacionada
com alterações nos níveis de fatores neurotróficos, como o fator neurotrófico derivado do
cérebro (BDNF), que desempenha um papel central na plasticidade. Como o BDNF é reprimido
pelo estresse, a regulação epigenética do gene BDNF pode desempenhar um papel importante
na depressão (LEAL; BRAMHAM; DUARTE, 2017; MCEWEN et al., 2015).
Fatores ambientais estressantes provocam a ativação do eixo hipotalâmico-pituitário-
adrenal e faz com que o cérebro seja exposto aos corticosteroides, afetando as funções
neurocomportamentais com uma forte regulação de diminuição da neurogênese, sendo então,
um grande fator de risco para a depressão (YANG et al., 2015). O tratamento antidepressivo
pode aumentar os níveis de BDNF, estimular a neurogênese e reverter os efeitos inibitórios do
estresse. Entretanto esse aumento não é evidenciado com todos os fármacos antidepressivos e
14
mesmo naqueles em que essa elevação é observada, a melhora só é evidente apenas após três a
quatro semanas de administração, que é o tempo necessário para a maturação de novos
neurônios, (FLECK et al., 2009; GOLD, 2015).
Portanto, esforços significantes têm sido direcionados para a caracterização da
neurobiologia da depressão com a promessa de identificar novos alvos terapêuticos. Estudos
sugerem novos possíveis alvos para a farmacoterapia da depressão como fatores neurotróficos,
seus receptores e cascatas afins de sinalização intracelular; agentes que podem neutralizar os
efeitos do estresse sobre a neurogênese no hipocampo (incluindo antagonistas de
corticosteroides, citocinas inflamatórias e seus receptores) e agentes que facilitam a ativação da
expressão do gene e aumentam a transcrição de neurotrofinas no cérebro (AL-HARBI, 2012;
GUPTA; RADHAKRISHNAN; KURHE, 2015; YOUNG; BRUNO; POMARA, 2014).
Diferentes abordagens científicas (modelos animais, estudos neuroendócrinos, post-
mortem, psicofarmacológicos, genéticos e de neuroimagem) têm sido empregadas para
investigar a depressão. Contudo, como essa doença apresenta-se sem uma fisiopatologia ou
etiologia completamente conhecidas, modelos animais tornam-se cada vez mais válidos para
seu estudo. Para este fim, inúmeros modelos atualmente estão relacionando a etiologia da
depressão com a cronicidade de eventos estressantes que levam a alterações neurobiológicas e
falhas na transmissão cerebral desencadeando o processo depressivo (MÉNARD; HODES;
RUSSO, 2016).
Mesmo com alguns importantes avanços no campo dos fármacos antidepressivas,
decorrentes da descoberta de vários antidepressivos atípicos, há necessidade do
desenvolvimento de novos fármacos que possam apresentar melhor eficácia, diminuição da
latência do efeito terapêutico, diminuição das recaídas e comprometimento cognitivo,
principalmente na população idosa, além de redução dos efeitos colaterais indesejáveis.
Nesse contexto, o presente trabalho buscou investigar de forma mais detalhada o
potencial efeito antidepressivo da riparina IV em um modelo de estresse crônico que induz o
desenvolvimento da sintomatologia da depressão. Deste modo, será verificado se a riparina IV
é capaz de reverter sintomas como anedonia, desamparo apreendido e comprometimento
cognitivo, além de observar se esta é capaz de normalizar a expressão de fatores neurotróficos,
como o BDNF, assim como diminuir o processo inflamatório neuronal visando fornecer
subsídios para a ampliação do arsenal terapêutico para o tratamento da depressão. Além disso,
15
a investigação de alterações comportamentais e neuroquímicas no modelo de estresse pela
administração de corticosterona poderá contribuir para o entendimento dos aspectos
fisiopatológicos desta doença.
16
2 REVISÃO DE LITERATURA 2.1 Estresse e depressão
2.1.1 Envolvimento do eixo hipotalâmico-hipofisário-adrenal (HHA)
Distúrbios depressivos podem ocorrer de forma idiopática, entretanto, estudos mostram
que vários fatores de risco podem desencadear sintomas depressivos incluindo fatores
genéticos, como polimorfismos no receptor de 5-hidroxitriptamina (5-HTTLPR) e BDNF
(Val66Met) (KIYOHARA; YOSHIMASU, 2009), e fatores ambientais (alguns tipos de câncer,
anormalidades endócrinas, luto e eventos estressantes) (HAMMEN, 2005; SOUTHWICK;
VYTHILINGAM; CHARNEY, 2005; WAGER-SMITH; MARKOU, 2011).
Em termos de depressão, estudos com gêmeos indicam a importância no
desencadeamento dos sintomas (HENN; VOLLMAYR; SARTORIUS, 2004), sendo exposição
ao estresse um dos mais importantes (CHARNEY; MANJI, 2004; DEAN; KESHAVAN, 2017;
GOLD, 2015; WILLNER; SCHEEL-KRÜGER; BELZUNG, 2013). De fato, até 85% dos
pacientes experienciam significantes eventos estressantes antes do início dos sintomas
depressivos (HAMMEN, 2005).
A conexão entre o estresse e a depressão pode ser relacionada com observações da
hiperatividade do eixo hipotálamo-hipófise-adrenal (HHA), níveis altos de cortisol e
interrupção da ritmicidade do cortisol.
As respostas fisiológicas e neurobiológicas normais estão bem caracterizadas. A
exposição a um fator estressante agudo, isto é, qualquer estímulo que altere o funcionamento
normal, desencadeia uma série de eventos fisiológicos e comportamentais com o objetivo de
reestabelecer a homeostase. Dessa forma, ocorre ativação do eixo HHA resultando em uma
cascata de eventos endócrinos que incluem liberação e transporte de dois importantes
neuropeptídios (fator de liberação da corticotrofina - CRF e vasopressina) de neurônios do
núcleo paraventricular do hipotálamo (NPV) para a pituitária anterior (ou hipófise anterior),
onde estes hormônios estimulam a liberação do hormônio adrenocorticotrópico (ACTH) para a
circulação sistêmica. O ACTH atua, então, sobre o córtex glândula adrenal, onde estimula a
produção e liberação de glicocorticoides (cortisol no ser humano e corticosterona em roedores)
para a circulação sistêmica. Uma vez liberados, estes hormônios agem nos tecidos corporais,
17
para limitar as funções não essenciais e mobilizar energia para lidar com o fator estressante,
ssim como também atuam a nível cerebral, onde exercem influência inibitória central, ou seja,
inibem as atividades do eixo HHA (feedback negativo) (DE KLOET; JOËLS; HOLSBOER,
2005; DUMBELL; MATVEEVA; OSTER, 2016; OGŁODEK et al., 2014; STEPHENS;
WAND, 2012; TSIGOS; CHROUSOS, 2002) ( Figura 1).
Figura 1 - Representação esquemática da ativação do eixo HHA em resposta a um
agente estressor.
Fonte: Adaptado de Maclaughlin et al. (2011) Legenda: O fator de liberação da corticotrofina (CRF) e vasopressina sintetizados pelo núcleo paraventricular e liberados para o sistema portal hipofisário estimulam a síntese e secreção do hormônio adrenocorticotrópico (ACTH) pela hipófise anterior. O ACTH desencadeia a liberação de glicocorticoides (cortisol ou corticosterona) pelo córtex da adrenal. Os glicocorticoides regulam a liberação de CRH e ACTH através de mecanismos de feedback. Os glicocorticoides, então, exercem ações disseminadas no corpo conforme necessário para restaurar e manter a homeostase fisiológica. Setas sólidas: regulação positiva; linhas pontilhadas: feedback negativo.
18
Em resposta ao estressor, os glicocorticoides normalmente alcançam um pico de
concentração sistêmica depois de 15-30 minutos e retornam a concentrações basais após 60-90
minutos. Dessa forma, a ação desses hormônios ao estresse agudo pode ser permissiva,
estimulatória ou supressiva com potencial para responder em magnitude adequada, limitando o
impacto da resposta ao estressor para prevenir hiperativação e dano (DE KLOET, 2014).
O cortisol é o mais importante hormônio liberado durante a resposta ao estresse e age
em vários órgãos e áreas cerebrais através de dois tipos de receptores homólogos: receptores
mineralocorticoides (MR) e receptores glicocorticoides (GR), (BANUELOS; LU, 2016; BAO;
MEYNEN; SWAAB, 2008; DUMBELL; MATVEEVA; OSTER, 2016; GOMEZ-SANCHEZ;
GOMEZ-SANCHEZ, 2014) que apresentam distribuição específica e seletiva em regiões
cerebrais como na glândula pituitária, núcleo paraventricular e sistema límbico (STEPHENS;
WAND, 2012).
O processo de retroalimentação negativa do eixo HHA parece ser fortemente
dependente da integridade do hipocampo. O hipocampo expressa tanto receptores de
mineralocorticoides quanto de glicocorticoides, que são os principais sítios de ação dos
glicocorticoides. Os MR apresentam uma alta afinidade pelo cortisol (até 10 vezes maior que
pelo GR) e, portanto, são ativados mesmo quando os níveis deste hormônio estão baixos. Em
contrapartida, os GR apresentam uma baixa afinidade e são ativados somente quando a
concentração basal de cortisol está relativamente elevada, o que ocorre durante os picos
circadianos e em situações de estresse moderado a intenso (BELLAVANCE; RIVEST, 2014;
NIKKHESLAT; PARIANTE; ZUNSZAIN, 2018; ZUNSZAIN et al., 2011).
Os receptores de glicocorticoides (GR) estão intimamente envolvidos no processo de
retroalimentação negativa do eixo HHA. Quando estes receptores estão disponíveis em nível
alto, a inibição por retroalimentação do NPV é aumentada e a atividade no eixo HHA é
fortemente controlada. Contudo, quando estão em nível baixo, a inibição por retroalimentação
é ineficiente e o estímulo que provoca a resposta no eixo HHA permite um aumento, maior que
o normal, nos níveis de cortisol (DE KLOET; JOËLS; HOLSBOER, 2005; DUMBELL;
MATVEEVA; OSTER, 2016). O hipocampo é particularmente suscetível aos efeitos danosos
do estresse prolongado, evidenciado pela diminuição da ramificação dendrítica, diminuição da
neurogênese e diminuição da expressão de RNAm do receptor de glicocorticoides no
hipocampo (SCHOENFELD; CAMERON, 2015). As implicações funcionais deste dano não
estão claras, mas presume-se que ele reduz o controle de retroalimentação exercida pelo
19
hipocampo sobre o eixo HHA, causando mais aumento nos níveis de cortisol e, assim,
danificando ainda mais o hipocampo (WILLNER; SCHEEL-KRÜGER; BELZUNG, 2013)
(Figura 2). Esse dano hipocampal corrobora com a hipótese da cascata de glicocorticoides como
sendo um dos mais importantes mecanismos patogênicos nas doenças degenerativas e
associadas a desregulação do eixo HHA, como a depressão, outros distúrbios afetivos e o
Alzheimer.
Figura 2 – Comprometimento da retroalimentação negativa exercida pelo hipocampo no
estresse crônico.
Fonte: Autoria própria.
Legenda: O estresse crônico leva a altos níveis sustentados de glicocorticoides, que com o tempo podem levar a
danos celulares no hipocampo, onde a aprendizagem e a memória de novas informações são transferidas para a
memória de longo prazo. Esse dano, consequentemente, pode interferir no ciclo de feedback que diz ao cérebro
quando "desligar" a resposta ao estresse, alimentando ainda mais o ciclo.
Fundamentalmente, a ativação do eixo HHA em resposta a um fator de estresse agudo
é essencial para a sobrevivência, sendo sua intenção primária preparar o organismo para
combate-lo através da resposta de luta ou fuga, e então restaurar a homeostase corporal
20
(SANDI; HALLER, 2015). Contudo, quando há exposição cumulativa a estes estressores, os
níveis de glicocorticoides permanecem aumentados, resultando em aumento do catabolismo,
peptídios de estresse e citocinas inflamatórias (STEPHENS; WAND, 2012). Portanto, a
ativação prolongada do eixo HHA, pode apresentar um sério risco à saúde, podendo levar a
imunosupressão, inibição do crescimento, distúrbios do sono, ansiedade, comprometimento da
memória, diminuição do comportamento sexual e disforia crônica (BAO; MEYNEN; SWAAB,
2008; SOUTHWICK; VYTHILINGAM; CHARNEY, 2005; SWAAB; BAO; LUCASSEN,
2005). Dessa forma, o mesmo hormônio do estresse que é vital para a sobrevivência do
organismo durante o estresse agudo pode também predispor o organismo à doença se o período
de estresse for prolongado.
Diversos estudos revelam uma estreita conexão entre a excessiva ativação do eixo HHA
e a depressão. Por exemplo, cerca de metade dos pacientes deprimidos apresentam
hipercortisolemia e ritmicidade do cortisol interrompida (DUMBELL; MATVEEVA; OSTER,
2016; SOUTHWICK; VYTHILINGAM; CHARNEY, 2005) que pode ser revertida pelo
tratamento com antidepressivos (DU; PANG, 2015; HINKELMANN et al., 2012; WILLNER;
SCHEEL-KRÜGER; BELZUNG, 2013). Além disso, existem evidências do aumento dos
níveis de CRF no fluido cerebroespinhal, do aumento do cortisol livre na urina e da diminuição
da supressão do cortisol plasmático após a administração de dexametasona em pacientes
deprimidos (BAO; MEYNEN; SWAAB, 2008; ZUNSZAIN et al., 2011). Em pessoas
saudáveis, a administração de dexametasona suprime o ACTH e a liberação de cortisol pela
ligação ao GR por retroalimentação negativa. Em pacientes deprimidos, se a supressão do
ACTH pela dexametasona está diminuída, a normalização ocorre durante um tratamento eficaz
com antidepressivos (KIM et al., 2016). Estudos mostraram que em pacientes com a síndrome
de Cushing, desordem marcada cronicamente pelos altos índices de cortisol no plasma,
frequentemente apresentam altos índices de depressão (CHATTARJI et al., 2015; SWAAB;
BAO; LUCASSEN, 2005; ZUNSZAIN et al., 2011) criando um forte argumento para a
influência da desregulação do sistema de estresse e o desenvolvimento do estado depressivo.
Portanto, se várias classes de drogas antidepressivas são capazes de agir em vias
neuroendócrinas para regularem a secreção de cortisol (SOUTHWICK; VYTHILINGAM;
CHARNEY, 2005), então novas terapias antidepressivas que inibam a secreção de cortisol
podem ser promissoras em ensaios clínicos (SCHÜLE et al., 2009).
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2.1.2 Anormalidades funcionais e estruturais do cérebro
Circuitos neuronais podem ser remodelados pela experiência e eventos estressantes
apresentam um efeito relevante na funcionalidade da árvore dendrítica, espinhas dendríticas e
número de sinapses em várias regiões cerebrais (MCEWEN; MORRISON, 2013).
Os distúrbios depressivos são marcados por profundas alterações na estrutura, função e
responsividade cerebral (GODSIL et al., 2013) e, consequentemente, pacientes deprimidos
apresentam uma incapacidade em se adaptar ao ambiente e podem estar mais vulneráveis a
desafios ou experiências estressantes. Geralmente, os padrões de mudanças metabólicas durante
os episódios de depressão maior sugerem que determinadas estruturas que apresentam um papel
fundamental nas respostas de estresse (hipocampo) e áreas que modulam ou inibem a expressão
emocional também estão ativadas (córtex pré-frontal subgenual), enquanto que outras áreas de
processamento sensorial e atenção estão desativadas (córtex pré-frontal dorsolateral). A
ativação patológica de determinadas áreas cerebrais é acompanhada de anormalidades
estruturais. Dessa forma, análises de neuroimagem e post-mortem de pacientes com depressão
revelam mudanças estruturais na região límbica e frontal, incluindo o hipocampo, amígdala e
córtex pré-frontal (CHATTARJI et al., 2015; FUNAHASHI, 2017; KRISHNAN; NESTLER,
2008; MCEWEN; MORRISON, 2013).
O hipocampo é a região mais extensivamente estudada no contexto da depressão e os
resultados encontrados sugerem que reduções no volume hipocampal estão associadas com o
distúrbio depressivo. De modo interessante, pequenos volumes hipocampais têm sido mais
comumente encontrados em pacientes que apresentaram diversos episódios de depressão
quando comparados com aqueles em remissão ou que estavam em seu primeiro episódio (BAO;
MEYNEN; SWAAB, 2008; CHATTARJI et al., 2015; GODSIL et al., 2013; MALBERG et
al., 2000; ZUNSZAIN et al., 2011). Isto sugere que a redução do volume hipocampal está
relacionado com a severidade da doença (LIU et al., 2017). De modo semelhante, existem
relatos consistentes de que há redução no volume do córtex pré-frontal em pacientes com
depressão, especificamente no córtex pré-frontal dorsolateral, orbitofrontal e subgenual
(ARNSTEN, 2009; CERQUEIRA et al., 2005; CHARNEY; MANJI, 2004). Na amígdala,
contudo, as mudanças volumétricas parecem ser dinâmicas durante todo o curso da doença,
com um aumento inicial seguido de uma diminuição do volume com o progresso da depressão
(LORENZETTI et al., 2009). Essas regiões são parte do circuito límbico-córtico-talâmico que
22
apresenta um papel integral no processo cognitivo e emocional (CHARNEY; MANJI, 2004).
O fato de todas essas regiões, em algum grau, funcionarem patologicamente na depressão dá
suporte a um modelo neural de depressão no qual as disfunções em determinadas áreas que
modulam ou inibem o comportamento emocional podem resultar em manifestações emocionais,
motivacionais, cognitivas e comportamentais da depressão.
O estresse tem sido implicado em algumas mudanças volumétricas no cérebro de
pacientes deprimidos. Mais especificamente, tem sido reportado que a desregulação do eixo
HHA e as mudanças subsequentes na secreção de glicocorticoides pode resultar em ambas
remodelação reversível e morte celular irreversível em regiões límbicas e frontais, que pode
resultar em mudanças volumétricas e subsequente funcionamento patológico visto em pacientes
com depressão (CHATTARJI et al., 2015; MILLER; HEN, 2015). De modo relevante, o
hipocampo, o córtex pré-frontal e a amígdala expressam receptores de mineralocorticoides e
glucocorticoides, tornando-se alvos da ação do cortisol e, portanto, particularmente susceptíveis
à atrofia ou hipertrofia neuronal induzida pelo estresse (LIU et al., 2017).
Estudos anatômicos indicam que as terminações límbicas que incidem sobre o NPV do
hipotálamo e neurônios GABAérgicos hipotalâmicos podem ser excitatórias no hipocampo e
córtex pré-frontal, e assim aumentar o tônus GABAérgico, ou inibitório da amígdala, e assim
reduzir o tônus GABAérgico (CHATTARJI et al., 2015). Dessa forma, o hipocampo e o córtex
pré-frontal inibem a atividade do eixo HHA enquanto que a amígdala aumenta esta atividade
(GOLD, 2015; SANDI; HALLER, 2015; WILLNER; SCHEEL-KRÜGER; BELZUNG, 2013).
Como os pacientes com depressão apresentam diminuição do volume do hipocampo e
do córtex pré-frontal e um aumento no volume da amígdala, isto pode indicar que o estresse
prolongado interfere com a habilidade do hipocampo e do córtex pré-frontal em inibir a
atividade no eixo HHA enquanto o aumento da atividade sobre o eixo HHA é facilitado pelo
aumento da amígdala. Para corroborar com essas afirmações, alguns estudos informam que a
elevação prolongada de glicocorticoides induz atrofia dendrítica, e em alguns casos, morte
neuronal no hipocampo e córtex pré-frontal e hipertrofia dendrítica na amígdala (LIU et al.,
2017; SOUSA; CERQUEIRA; ALMEIDA, 2008) (Figura 3).
Evidências comportamentais demonstram como a exposição a condições de estresse
afeta a aprendizagem e a memória dependentes do hipocampo ou da amígdala. Em roedores, o
estresse crônico facilita o medo, o comportamento ansioso e prejudica a memória espacial.
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Embora o estresse repetido produza atrofia dendrítica na região do corno de Amon 3 (CA3) e
prejudique o aprendizado dependente do hipocampo, a região basolateral da amígdala mostrou-
se essencial para a facilitação da aprendizagem aversiva induzida pelo estresse (CHATTARJI
et al., 2015).
Figura 3 - Áreas cerebrais implicadas em transtornos psiquiátricos relacionados ao estresse.
Fonte: Adaptado de Chattarji et al. (2015).
Legenda: A amígdala, o córtex pré-frontal (CPF) e o hipocampo sofrem alterações estruturais e funcionais em
condições de estresse prolongado e, por sua vez, regulam diferencialmente a resposta ao estresse por meio da
atividade do eixo HHA (tanto positiva quanto negativamente).
Portanto, pode-se afirmar que o comprometimento da integração da informação
hipocampal, amigdalar e/ou pré-cortical está relacionada com a disfunção do eixo HHA assim
como prejuízos no humor e na cognição.
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2.1.3 Processos cognitivos, memória e aprendizado
Estudos sobre os efeitos crônicos de níveis elevados de glicocorticoides elucidaram a
relação entre hipercortisolemia, depressão e memória (JOELS; SARABDJITSINGH; KARST,
2012; WILLNER; SCHEEL-KRÜGER; BELZUNG, 2013). Três importantes áreas do sistema
límbico mostram alterações com o aumento de glicocorticoides circulantes: o córtex pré-frontal,
o hipocampo e a amígdala. O córtex pré-frontal está envolvido na função executiva (memória
de trabalho e comportamento assertivo), o hipocampo está envolvido no aprendizado e memória
(espacial e declarativa) e a amígdala no processamento da memória emocional (GODSIL et al.,
2013).
O córtex pré-frontal participa de uma série de funções cognitivas, como pensar,
racionalizar, planejar e tomar decisões (MILLER, 2000). Estudos realizados em humanos e
animais observaram redução dendrítica no córtex pré-frontal em condições de estresse crônico
(CERQUEIRA et al., 2005; GOLD, 2015; MCEWEN; MORRISON, 2013). Como o córtex
pré-frontal está implicado no processamento cognitivo, redução da atividade nessa área levaria
a um mau julgamento, planejamento e comprometimento de decisões (DEAN; KESHAVAN,
2017; SEO et al., 2017). Pacientes depressivos mostram diminuição da memória de trabalho,
comprometendo o processamento cognitivo, refletindo parcialmente na redução no
funcionamento do córtex pré-frontal dorsolateral (FUNAHASHI, 2017). Esses achados são
mais comuns do que em condições normais de estresse.
O hipocampo é intensamente afetado em condições de estresse prolongado, havendo
comprometimento da performance cognitiva devido a alterações cumulativas na função e
morfologia hipocampal (BAO; MEYNEN; SWAAB, 2008; CHATTARJI et al., 2015; LIU et
al., 2017; ORTIZ et al., 2018; SHEN et al., 2016). A consolidação da memória é um processo
no qual um traço de memória de curto prazo é transferido em uma de longo prazo estável.
Entretanto, nem todas as informações são igualmente armazenadas a longo prazo. Sabe-se que
experiências emocionalmente excitantes são bem lembradas, mesmo depois de décadas. A
consolidação bem-sucedida da memória depende da síntese de novas proteínas e de mudanças
a longo prazo na plasticidade sináptica (QUERVAIN et al., 2009).
Altos níveis de glicocorticoides podem reduzir a capacidade de aprendizado e memória,
ao prejudicar a Long-Term Potentiation (LTP; alterações celulares responsáveis pela
25
manutenção da excitação nas sinapses que resultam na consolidação da memória) (JOELS;
SARABDJITSINGH; KARST, 2012; MAHEU et al., 2004), a plasticidade sináptica, e ainda
promover a atrofia da árvore dendrítica (CHATTARJI et al., 2015; JOELS;
SARABDJITSINGH; KARST, 2012; LIU et al., 2017; MCEWEN; MORRISON, 2013; ORTIZ
et al., 2018). Estudos revelam que animais cronicamente estressados exibiram uma redução na
plasticidade e o LTP em neurônios hipocampais mediados por receptores glicocorticoides (GR)
(ALFAREZ et al., 2002), levando a um comprometimento adaptacional e de aprendizado.
Em condições normais, os receptores de glicocorticoides participam da consolidação de
memória no sistema corticolímbico, promovendo alterações comportamentais para preparar o
organismo para situações futuras (QUERVAIN et al., 2009). Os altos níveis circulantes dos
glicocorticoides alteram o balanço entre os receptores MR:GR, o que pode causar efeitos
opostos na função cognitiva (DE KLOET, 2014; DE KLOET; DERIJK; MEIJER, 2007;
NIKKHESLAT; PARIANTE; ZUNSZAIN, 2018). Enquanto a ativação do MR aumenta em
processos relacionados à memória, o estresse associado à ativação do GR pode comprometer
esta função. Vários estudos realizados em humanos e animais indicaram que os glicocorticoides
prejudicam a recuperação da memória espacial ou contextual em ratos e a memória declarativa
(principalmente episódica) em humanos (COLUCCIA et al., 2008; KUHLMANN;
KIRSCHBAUM; WOLF, 2005; RASHIDY-POUR et al., 2004; ZUNSZAIN et al., 2011).
Dai et al. (2018) encontraram hipoatividade na translocação para o núcleo de GR (
mecanismo pelo qual há regulação da transcrição gênica, pela ligação com a região promotora
dos genes responsivos aos glicocorticoides, passando a facilitar ou reprimir a transcrição
gênica) no hipocampo de animais depressivos submetidos a condições crônicas de estresse.
Essas alterações no balanço podem levar a desregulação na adaptação comportamental e
neuroendócrina, comprometendo o feedback negativo do eixo HHA, sendo um fator de risco na
precipitação da depressão.
Diminuições volumétricas observadas no hipocampo e em outras regiões cerebrais em
pacientes deprimidos dão suporte a conhecida hipótese neurotrófica da depressão, a qual
envolve decréscimos em fatores neurotróficos que são fatores de crescimento expressos no
neurodesenvolvimento que também regulam a plasticidade no cérebro adulto (KRISHNAN;
NESTLER, 2008).
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2.1.4 Plasticidade e sobrevivência neuronal
A habilidade do cérebro de se adaptar e modificar em resposta a experiências ou
situações ambientais depende da plasticidade das conexões sinápticas. Esse processo exibe
várias propriedades fisiológicas que substanciam seu papel como um correlato celular para
múltiplos processos cognitivos, incluindo aprendizado e memória (WOO; LU, 2009).
O fator neurotrófico derivado do cérebro (brain-derived neurotrophic factor - BDNF) é
considerado um importante mediador de eficácia sináptica, plasticidade neuronal,
conectividade, sobrevivência e maturação celular, neurogênese e funções cognitivas (LEAL;
COMPRIDO; DUARTE, 2014; SOUTHWICK; VYTHILINGAM; CHARNEY, 2005). É
produzido principalmente pela glia e pelos núcleos neuronais e tem grande expressão no
hipocampo, neocórtex, amígdala e cerebelo (ARANGO-LIEVANO et al., 2015; SHIMIZU et
al., 2003). Diversos estudos em humanos e animais sugerem que o BDNF está implicado na
fisiopatologia de diversas desordens neurodegenerativas e psiquiátricas como, por exemplo, a
depressão (IHARA et al., 2016; LOPES et al., 2018; VASCONCELOS et al., 2015;
WOLKOWITZ et al., 2011; WOO; LU, 2009).
A expressão de fatores neurotróficos, principalmente o BDNF, no córtex pré-frontal,
hipocampo e outras regiões cerebrais está diminuída em condições de estresse agudo e crônico
(LEAL; BRAMHAM; DUARTE, 2017; WILLNER; SCHEEL-KRÜGER; BELZUNG, 2013),
contribuindo diretamente com a redução do volume dessas áreas, sendo essa diminuição
revertida pelo tratamento com antidepressivos (GOLD, 2015; IHARA et al., 2016; LOPES et
al., 2018; MALBERG et al., 2000; ORTIZ et al., 2018; SHEN et al., 2016; SHIMIZU et al.,
2003; VASCONCELOS et al., 2015; WOLKOWITZ et al., 2011; ZUNSZAIN et al., 2011).
Por outro lado, estudos sugerem que a expressão aumentada de BDNF em regiões específicas,
como no núcleo meso accumbens e amígdala, resulta em efeitos pró-depressivos (CHATTARJI
et al., 2015; KRISHNAN; NESTLER, 2008).
Em relação ao seu papel central na plasticidade sináptica, vários estudos avaliaram como
o BDNF regula a aquisição (aprendizado) e retenção (memória) da informação. Há uma forte
relação observada entre o BDNF e a memória dependente do hipocampo, que inclui memória
declarativa ou episódica e memória espacial. Durante um contexto de aprendizado, a expressão
27
de BDNF é rápida e seletivamente supraregulada no hipocampo (LEAL; BRAMHAM;
DUARTE, 2017; WOO; LU, 2009).
O BDNF desempenha um papel fundamental na LTP hipocampal e na aprendizagem.
Esta neurotrofina mostrou regular a indução e manutenção de uma LTP estável; induzir
alterações na liberação de neurotransmissores; modular os receptores glutamatérgicos pós-
sinápticos – NMDA e AMPA; regular a síntese proteica; ativar a transcrição e modular a
plasticidade estrutural nas espinhas dendríticas (LEAL; COMPRIDO; DUARTE, 2014).
Estudos conduzidos por Ortiz e colaboradores (2018) investigaram o envolvimento de
BDNF e seu receptor tirosina-quinase relacionado a tropomiosina do tipo B (tropomyosin-
related receptor tyrosine kinase B - TrkB) na região CA3 do hipocampo de ratos estressados
por 21 dias. Concluiu-se que a presença de BDNF e TrkB é um importante fator para o processo
de recuperação após exposição a situações de estresse crônico, com aumento da complexidade
dendrítica e melhora no déficit de memória espacial.
A estreita relação entre os glicocorticoides e o BDNF na resposta adaptativa ao estresse
foi explorada pelo estudo de Arango-Lievano et al. (2015) que demonstrou que os receptores
de glicocorticoides eram alvos da sinalização mediada por BDNF e TrkB. Foi observado que o
BDNF induz a fosforilação de GR e sugere que ações coordenadas entre BDNF e
glicocorticoides são essenciais para respostas de neuroplasticidade ao estresse. Dessa forma,
estes resultados corroboram com a hipótese que alguns dos efeitos crônicos dos glicocorticoides
podem resultar da diminuição dos níveis e desregulação na sinalização do BDNF.
2.1.5 Envolvimento do sistema imunológico e o estresse oxidativo
Como a depressão é um transtorno complexo, é provável que alterações em vários
sistemas, que interagem em conjunto, fundamentem a patogênese da doença. Há evidências de
que processos inflamatórios mediados por citocinas desempenham um papel importante no
desenvolvimento de distúrbios de humor.
Estudos relatam que cerca de 30-70% dos pacientes tratados com interferon alfa (IFN-
α) apresentam depressão como efeito adverso nos três primeiros meses (BONACCORSO et al.,
28
2002; CHIU et al., 2017; PINTO; ANDRADE, 2016). Além disso, foram observadas altas
concentrações de citocinas em pacientes depressivos e a administração destas em animais pode
induzir a comportamentos semelhantes a depressão (BEAUREPAIRE, 2002; KIM et al., 2016;
KRISHNAN; NESTLER, 2008; LOTRICH, 2015), corroborando com a hipótese inflamatória
em algumas desordens depressivas.
A ativação do eixo HHA estimula a liberação de noradrenalina na circulação sistêmica,
que por sua vez, estimula a produção de interleucina-6 (IL-6) e ambas estimulam a uma resposta
de fase aguda. Esta resposta gera a produção de várias proteínas que desempenham efeitos pró-
inflamatórios e pró-trombóticos relevantes para a resposta ao estresse (BORTOLATO et al.,
2015; GOLD, 2015; SCHIEPERS; WICHERS; MAES, 2005). Apesar dos glicocorticoides
apresentarem vários efeitos anti-inflamatórios, altos níveis de cortisol geram alterações que
resultam em estímulos pró-inflamatórios como aumento da gordura visceral, resistência a
insulina ou hiperinsulinemia e estímulo simpático (IZAOLA et al., 2015).
Altas concentrações de glicocorticoides também promovem a liberação de citocinas
pró-inflamatórias pelos macrófagos e células da micróglia, que contribuem para a
dessensibilização de receptores GR e estímulo direto do eixo HHA (MAES, 2011; ZUNSZAIN
et al., 2011). Das citocinas inflamatórias, o fator de necrose tumoral alfa (TNF-a), interleucina-
1 beta (IL-1b) e interleucina-6 (IL-6) são as que apresentam o maior efeito modulatório no eixo
associado a resposta imune, podendo estimular do eixo HHA sozinhas, ou em sinergia
(BEAUREPAIRE, 2002; DUMBELL; MATVEEVA; OSTER, 2016; MALEK et al., 2015;
RIVEST, 2010; SCHIEPERS; WICHERS; MAES, 2005; SINGHAL et al., 2014). Há
evidências que sugerem que a IL-6, a principal citocina endócrina, desempenha o papel
principal na estimulação imune do eixo, especialmente na inflamação crônica (MIURA et al.,
2008; TSIGOS; CHROUSOS, 2002) (Figura 4).
Figura 4 - Diagrama esquemático da interação de células imunes com o eixo HHA
através de citocinas inflamatórias.
29
Fonte: Adaptado de Glaser e Kiecolt-Glaser (2005) Legenda: HHA: eixo hipotálamo-hipófise-adrenal; CRF: fator de liberação da corticotrofina; ACTH: hormônio adrenocorticotrópico.; TNF-a: fator de necrose tumoral alfa; IL-1b: interleucina-1 beta; IL-6: interleucina-6; APC: célula apresentadora de antígeno NK, células natural killer.
Além de seus múltiplos efeitos periféricos, as citocinas têm efeitos pleiotrópicos no
sistema nervoso central. Elas não só influenciam a inflamação, mas também exercem papéis
fundamentais na função dos neurotransmissores, regulação neuroendócrina, neuroplasticidade
e suporte neurotrófico (BORTOLATO et al., 2015; GOLD, 2015; LOTRICH; ALBUSAYSI;
30
FERRELL, 2013; NIKKHESLAT; PARIANTE; ZUNSZAIN, 2018; YOUNG; BRUNO;
POMARA, 2014).
As citocinas inflamatórias podem influenciar duas vias neurofarmacológicas
importantes: monoaminérgicas (serotonina, dopamina e noradrenalina) e glutamatérgicas.
Ambos os sistemas são frequentemente envolvidos na etiologia de distúrbios depressivos. A
presença de citocinas pode levar a diminuição de serotonina (5-HT) disponível através da
diminuição de triptofano para a síntese, aumento da liberação e metabolismo, além de
influenciar a expressão de receptores 5-HT1A e 5-HT2 (BORTOLATO et al., 2015;
SCHIEPERS; WICHERS; MAES, 2005). De forma similar, IL-6, IL-2 e TNF-α diminuem os
níveis de dopamina e noradrenalina ao diminuir a síntese, alteram a recaptação na fenda
sináptica e reduzem o conteúdo vesicular de dopamina (LOTRICH, 2015; LOTRICH;
ALBUSAYSI; FERRELL, 2013; MILLER; MALETIC; RAISON, 2009). Em relação ao
glutamato, a citocinas inflamatórias promovem liberação, diminuem a sua receptação ou atuam
como agonista NMDAR (SCHWARCZ et al., 2012), predispondo à excitotoxicidade.
Estudos mostram que além das citocinas inflamatórias, o estresse oxidativo afeta
negativamente a neuroplasticidade e neurogênese. A superprodução de espécies reativas de
oxigênio (EROS), como o radical superóxido (O•), hidroxila (OH•) e peróxido de hidrogênio
(H2O2) e espécies reativas de nitrogênio, como óxido nítrico (NO•) e peroxinitrito (ONOO),
pode induzir a clivagem do DNA nuclear (promovendo a apoptose), favorecer a peroxidação
lipídica (BARBOSA et al., 2008), reduzir a função de receptores catecolaminérgicos e
serotoninérgicos, além de aumentar a atividade da monoamino oxidase (MAO), enzima
responsável pela degradação de monoaminas (WILLNER; SCHEEL-KRÜGER; BELZUNG,
2013).
A estimulação desses receptores glutamatérgicos, aumenta o influxo de cálcio nos
neurônios, contribuindo para a geração de ROS e radicais livres, estimulando ainda a
peroxidação lipídica da membrana, e comprometendo, assim, a sua fluidez e permeabilidade,
com consequente dano neuronal (CARVALHO et al., 2017).
A inflamação e a fosforilação oxidativa mitocondrial geram espécies reativas e radicais
livres. Quando a produção desses radicais excede a capacidade sequestrante do sistema
antioxidante, ocorre oxidação extensa de proteínas e peroxidação lipídica, causando dano
oxidativo, degeneração celular e até declínio funcional. Esse desequilíbrio é conhecido como
31
estresse oxidativo, que está cada vez mais estabelecido no desfechos e progressão de uma ampla
gama de patologias, como diabetes, doenças coronarianas e depressão (BOUAYED;
RAMMAL; SOULIMANI, 2009; HALLIWELL, 2007; JÖRGENS; AROLT, 2018;
NIKKHESLAT et al., 2015; PALTA et al., 2014).
O sistema nervoso central é uma das áreas mais vulneráveis ao estresse oxidativo. O
cérebro consome grandes quantidades de oxigênio, carece de compostos antioxidantes e
apresenta altas concentrações de ácidos graxos poli-insaturados e íons metálicos (KIM et al.,
2016). Dessa forma, o estresse oxidativo torna-se particularmente perigoso para o
funcionamento normal do cérebro.
O sistema de enzimas antioxidantes e os antioxidantes de baixo peso molecular
representam o mecanismos de proteção que operam no cérebro para enfrentar ameaças
representadas pelas espécies reativas de oxigênio e nitrogênio. O sistema enzimático
antioxidante inclui a superóxido dismutase (SOD), glutationa redutase (GSR), glutationa
peroxidase (GSHPx) e catalase (CAT). As enzimas SOD, facilitam a dismutação espontânea
dos radicais superóxido para gerar H2O2, que é posteriormente removido pelas enzimas CAT e
glutationa peroxidase (Figura 5).
32
Figura 5 – Mecanismos antioxidantes de defesa contra o dano oxidativo.
Fonte: Adaptado de Valle, Oliver e Roca (2010)
Legenda: SOD: superóxido dismutase; GSH: Glutationa reduzida; GSSG: glutationa dissulfeto;
glutationa redutase (GSR), glutationa peroxidase (GSHPx)
Os antioxidantes de baixo peso molecular incluem glutationa, ácido úrico, ácido
ascórbico (vitamina C), a-tocoferol (vitamina E) e melatonina, que oferecem funções
neutralizantes, causando quelação de metais de transição. A glutationa, que ocorre na forma
reduzida (GSH) e também na forma oxidada (glutationa dissulfeto - GSSG), é o antioxidante
endógeno não enzimático mais importante e pode ser regenerada pela glutationa redutase com
o consumo de NADPH (HUBER; ALMEIDA; DE FÁTIMA, 2008; OZCAN et al., 2004;
SALIM, 2017), mantendo níveis ótimos de GSH reduzida. A glutationa, em particular,
desempenha um papel importante no processo de oxiredução e afeta o cérebro por desintoxicar
diretamente xenobióticos e EROS (KIM et al., 2016; KOGA et al., 2011; SALIM, 2017).
Na presença de estresse oxidativo, a constituição rica em lipídios do cérebro favorece a
peroxidação lipídica, com produção de moléculas tóxicas como malondialdeido (MDA), que
resulta em diminuição da fluidez da membrana e danos em proteínas de membrana, inativando
receptores, enzimas e canais iônicos (BOUAYED; RAMMAL; SOULIMANI, 2009; KIM et
al., 2016; WIGNER et al., 2018). Como resultado, o estresse oxidativo pode alterar a
neurotransmissão, a função neuronal e a atividade cerebral geral.
33
Dessa forma, os níveis de ansiedade, depressão e comprometimento cognitivo
correlacionam-se positivamente com os níveis de citocinas circulantes, um achado que
confirma mais uma vez o envolvimento de citocinas na mediação das respostas emocionais e
cognitivas as condições de estresse crônico. Desafios imunes são capazes provocar o estresse
oxidativo, aumentar os níveis de outras citocinas inflamatórias (como TNF- α, IL-6, IL-1b),
diminuir a expressão de BDNF no hipocampo de animais (BORTOLATO et al., 2015) e em
humanos (LOTRICH; ALBUSAYSI; FERRELL, 2013), estimular o eixo HHA (KIM et al.,
2016) alterar os níveis de neurotransmissores (LOTRICH, 2015), além de inibir a long-term
potentiation (ZUNSZAIN et al., 2011), com comprometimento da aprendizagem e memória,
fatores frequentemente afetados em distúrbios depressivos.
2.1.6 Modelo da administração de corticosterona
Diante de tudo isso, o modelo da administração de corticosterona foi desenvolvido com
o intuito de determinar a influência do estresse no desenvolvimento da depressão, sendo
amplamente utilizado (KIM; HAN, 2006).
Neste modelo, a corticosterona (Cort) pode ser administrada por um período de semanas
a meses por diversas vias como a injeção subcutânea, a implantação de pellets, a infusão de
bombas osmóticas ou através da administração passiva na bebida permitindo um controle mais
rigoroso sobre os níveis hormonais (MÜLLER et al., 2009). Uma vantagem desse modelo é que
ele permite examinar a influência direta de glicocorticoides no desenvolvimento da
sintomatologia da depressão.
Apesar de ser impossível examinar todos os sintomas da depressão manifestados em
pacientes através de modelos animais, uma ampla gama de medidas comportamentais tem sido
usadas com o intuito de “medir” a depressão em roedores como a perda de peso, o impedimento
da memória, o distúrbio do sono, a exploração no campo aberto, anedonia e os comportamentos
de desamparo, sendo os dois últimos os mais frequentemente utilizados (YIN; GUVEN;
DIETIS, 2016).
A anedonia (falta de interesse em atitudes agradáveis) é tipicamente inferida pela
medida da ingestão de solução de sacarose comparada com a ingestão de água. Uma vez que
34
camundongos normais preferem a solução de sacarose, então, uma diminuição da ingestão é
indicativa de depressão que pode ser revertida pelo tratamento com antidepressivos
(WILLNER, 2005).
O desamparo aprendido pode ser examinado em camundongos de várias formas, sendo
o mais utilizado o teste do nado forçado. Neste teste, o aumento do comportamento passivo,
como a imobilidade, e a diminuição do comportamento ativo, como o nado e a escalada, são
indicativos de comportamentos depressivos (NESTLER et al., 2002). Este teste é considerado
válido porque todas as formas de tratamento que são eficazes em humanos, incluindo os
antidepressivos típicos e atípicos e a terapia por eletrochoque são eficazes em diminuir a
imobilidade neste teste (CRYAN; VALENTINO; LUCKI, 2005; PORSOLT; BERTIN;
JALFRE, 1977).
Uma extensa gama de trabalhos indicam que a administração prolongada de
corticosterona acarreta em mudanças consistentes e confiáveis em uma variedade de
comportamentos em roedores que podem ser considerados sintomas depressivos como o
desamparo aprendido, verificado pelo aumento do tempo de imobilidade nos testes do nado
forçado e suspensão da cauda (CRYAN; VALENTINO; LUCKI, 2005; GUPTA et al., 2012;
IIJIMA et al., 2010; LOPES et al., 2018; OLIVEIRA, 2017; VASCONCELOS et al., 2015) e a
anedonia, representada pela diminuição do consumo de solução de sacarose (DAVID et al.,
2009; GUPTA et al., 2012; LOPES et al., 2018; VASCONCELOS et al., 2015), diminuição da
resposta para o reforço alimentar (GOURLEY; WU; TAYLOR, 2008), inibição do
comportamento sexual (GORZALKA; HANSON; HONG, 2001) e diminuição do grooming
(DAVID et al., 2009; VASCONCELOS et al., 2015).
Além disso, estudos evidenciam de que a administração repetida de Cort produz
comportamentos ansiosos em diversas tarefas incluindo o campo aberto (DAVID et al., 2009;
SKÓRZEWSKA et al., 2006; VASCONCELOS et al., 2015), o labirinto em cruz elevado
(SKÓRZEWSKA et al., 2006) e o modelo do claro/escuro (MURRAY; SMITH; HUTSON,
2008). Estes achados são extremamente relevantes uma vez que a manifestação de sintomas
depressivos são frequentemente associados a desordens ansiosas em pacientes com depressão
maior (MILLER; HEN, 2015; WORLD HEALTH ORGANIZATION, 2017), o que torna este
modelo altamente plausível e revela sua validade de face. Outras mudanças indicativas de
depressão que ocorrem com a administração de Cort incluem a diminuição do ganho de peso
(ZHAO et al., 2008) e desregulação da função do eixo HHA (ZHU et al., 2014).
35
Os estudos mostram ainda que a exposição ao estresse prolongado induz considerável
grau de plasticidade estrutural no cérebro adulto (LIU et al., 2017; MCEWEN; MORRISON,
2013). É importante ressaltar que essas mudanças neurobiológicas formam a base da
sintomatologia da depressão e que têm sido fielmente reproduzidas com o modelo de
administração de Cort. Por exemplo, alguns trabalhos mostraram que este modelo tem induzido
remodelação dendrítica no hipocampo (JACOBSEN; MØRK, 2006; SOUTHWICK;
VYTHILINGAM; CHARNEY, 2005), amígdala (MITRA; SAPOLSKY, 2008) e córtex pré-
frontal (GOURLEY; WU; TAYLOR, 2008; JACOBSEN; MØRK, 2006) semelhante ao que
tem sido documentado no cérebro post-mortem de pacientes com depressão (KONARSKI et
al., 2008). Além disso, é capaz de induzir extensa atrofia dendrítica no hipocampo
(BRUMMELTE; GALEA, 2010; SOUSA et al., 2000) e em doses altas e/ou prolongadas pode
causar morte celular (SOUSA; MADEIRA; PAULA-BARBOSA, 1998). De modo semelhante,
verifica-se atrofia do córtex pré-frontal (ZOLADZ et al., 2008) e redução na proliferação de
glia e células endoteliais (COTTER, 2002).
De maneira importante, muitas formas de estresse crônico, incluindo a deste modelo,
têm um profundo efeito na neurogênese hipocampal, causando rápidas e consistentes reduções
na proliferação e sobrevivência de neurônios recém-formados no cérebro adulto (DAVID et al.,
2009; MURRAY; SMITH; HUTSON, 2008). Tais alterações também são encontradas no
homem e podem ser revertidas por diversos tipos de tratamento para a depressão, como os
antidepressivos típicos e atípicos, eletrochoque e atividade física (DAVID et al., 2009;
DWIVEDI; RIZAVI; PANDEY, 2006). Esses achados são particularmente importantes como
uma das hipóteses atuais mais convincentes a respeito da etiologia da depressão de que o
estresse crônico provoca plasticidade patológica dentro do hipocampo levando a diminuição da
neurogênese e, eventualmente, aos sintomas depressivos (GODSIL et al., 2013).
A interação entre o estresse crônico e as mudanças celulares e moleculares que
influenciam no desenvolvimento da depressão tem se tornado cada vez mais clara (PARIANTE
et al., 2004). Por exemplo, tanto a administração de Cort quanto a depressão em humanos estão
associadas com reduções de fatores de transcrição os quais induzem genes efetores que
contribuem para a estabilização da plasticidade sináptica (EGELAND; ZUNSZAIN;
PARIANTE, 2015). Dentre estes está o fator neurotrófico derivado do cérebro que tem um
papel crucial na estabilização de neurônios durante o desenvolvimento (FU et al., 2016) e é
importante para a sobrevivência e função de neurônios maduros. Sendo assim, alguns trabalhos
36
mostram que a exposição aos glicocorticoides leva ao impedimento da sinalização mediada por
BDNF em regiões límbicas e frontais (FU et al., 2016; GOURLEY; WU; TAYLOR, 2008;
JACOBSEN; MØRK, 2006; SOUSA et al., 2015) semelhantes as observadas em pacientes com
depressão (CASTRÉN; RANTAMÄKI, 2010a; CASTRÉN; VÕIKAR; RANTAMÄKI, 2007).
Dessa forma, a validade do modelo de administração corticosterona para simular a
sintomatologia da depressão está bem estabelecida, visto que induz mudanças
comportamentais, morfológicas e celulares convincentes e reprodutíveis para o estudo dessa
patologia.
2.2 Abordagens terapêuticas na depressão
Todos os fármacos utilizados atualmente para o tratamento da depressão estão
relacionados de algum modo com o aumento da transmissão monoaminérgica em regiões
cerebrais. Os diferentes antidepressivos, inibidores seletivos da recaptação de serotonina
(ISRS), inibidores seletivos da receptação de noradrenalina (ISRN), inibidores da MAO,
tricíclicos e atípicos têm eficácia semelhante para a maioria dos pacientes deprimidos, variando
em relação ao seu perfil de efeitos colaterais e potencial de interação com outros medicamentos
(RACAGNI; POPOLI, 2008).
A eletroconvulsoterapia (ECT) é o tratamento antidepressivo agudo disponível mais
eficaz. No entanto, ele não é utilizado como tratamento inicial para depressão em função de
seus efeitos colaterais, necessidade de anestesia geral e estigma social (WILKINS; OSTROFF;
TAMPI, 2008).
A resposta ao tratamento com antidepressivos ocorre entre 2 a 4 semanas após o início
do uso, embora alguns pacientes respondam apenas em 6 semanas. As estratégias utilizadas
quando um paciente não responde ao tratamento com medicamento antidepressivo consiste em
aumento de dose, potencialização com lítio ou triiodotironina (T3), associação ou troca de
antidepressivos, eletroconvulsoterapia e associação com psicoterapia. Existem evidências
limitadas sobre qual estratégia seria a melhor alternativa quando não há resposta a um
tratamento inicial proposto (FLECK et al., 2003).
37
O planejamento de um tratamento com antidepressivos envolve a fase aguda, de
continuação e de manutenção, cada uma com objetivos específicos. O tratamento da fase aguda
inclui os 2 a 3 primeiros meses de tratamento e tem como objetivo a diminuição dos sintomas
depressivos (resposta) ou idealmente sua remissão completa (remissão). A continuação do
tratamento corresponde aos 4 a 6 meses que seguem ao tratamento da fase aguda e tem como
objetivo manter a melhora obtida evitando recaídas dentro de um mesmo episódio depressivo.
Ao final dessa fase, se o paciente permanece com a melhora obtida após o tratamento da fase
aguda, é considerado recuperado do episódio índice. Já a fase de manutenção tem por objetivo
evitar que novos episódios ocorram (recorrência) e é, em geral, por longo prazo. A terapia de
manutenção, portanto, é recomendada para aqueles pacientes com probabilidade de recorrência
(FLECK et al., 2009).
De acordo com a Associação Médica Brasileira, um terço dos pacientes com episódio
depressivo com remissão inicial recai no primeiro ano. Os índices de recaída são estimados em
20% a 24% nos primeiros 2 meses, 28% a 44% aos 4 meses, 27% a 50% aos 6 meses e 37% a
54% aos 12 meses (FLECK et al., 2009).
O Manual Diagnóstico e Estatístico de Transtornos Mentais, DSM-V, identifica o
comprometimento cognitivo (como prejuízo na concentração e indecisão) como critério de
diagnóstico de episódios depressivos (AMERICAN PSYCHIATRIC ASSOCIATION, 2014).
Há evidências que os sintomas cognitivos podem ser revertidos com a utilização de
antidepressivos, sendo alguns mais efetivos que outros (HERRERA-GUZMÁN et al., 2008).
Sendo assim, o entendimento da etiologia da depressão e a busca de novos fármacos
eficazes no tratamento desta doença que diminua o tempo de latência para o início do efeito
terapêutico, diminua os índices de recaída e melhore os sintomas de cognição é de suma
importância para conseguir controlar esta doença heterogênea, crônica e incapacitante.
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2.3 Riparina IV
A riparina IV, ou (O-Metil)-N-(3,4,5-trimetoxibenzoil)-tiramina, é uma molécula
sintética obtida pela primeira vez por Barbosa-filho, da Silva e Bhattacharyya em 1990.
Recebeu esse nome devido a semelhança estrutural com as riparinas I, II e III, alcamidas
naturais isoladas da Aniba riparia (BARBOSA-FILHO et al., 1987). Apresenta em sua estrutura
uma molécula de tiramina, uma monoamina derivada do aminoácido tirosina, e um derivado do
ácido benzoico, o trimetil-éter do ácido gálico, conforme figura 6.
Figura 6 - Estrutura química da riparina IV.
Fonte: Dias (2012).
Estudos anteriores com a riparina IV mostraram atividade anti-inflamatória e
antinociceptiva em testes de contorções abdominais induzidas por ácido acético; placa quente;
teste da formalina (DIAS, 2012; NASCIMENTO et al., 2016); nocicepção mecânica induzida
pela carragenina, capsaicina, mentol e glutamato (DIAS, 2012). Também foi observado um
efeito antimicrobiano contra cepas de Staphylococcus aureus e Escherichia coli (CATÃO et
al., 2005).
Devido a atividade comprovada das riparinas I, II e III nos modelos comportamentais
de depressão (DE SOUSA et al., 2014; LOPES et al., 2018; MELO et al., 2013, 2006;
OLIVEIRA, 2017; SOUSA et al., 2005, 2004; TEIXEIRA et al., 2013; VASCONCELOS et al.,
2015) e ansiedade (MELO et al., 2006; OLIVEIRA, 2012; SOUSA et al., 2005, 2007, 2004),
sendo a riparina IV um análogo estrutural dessas substâncias, torna-se relevante a investigação
do seu potencial farmacológico em animais submetidos a modelos de estresse crônico que
melhor representam as alterações comportamentais e neuroquímicas da depressão.
39
3 CAPÍTULOS
Os resultados foram divididos em artigos, como segue abaixo:
• ARTIGO 1: onde constam os resultados referentes aos efeitos da riparina IV no
comportamento ansioso (campo aberto e labirinto em cruz elevado), desamparo
aprendido (nado forçado, suspensão da cauda) e sintomas anedônicos ( preferência pela
solução de sacarose) em camundongos submetidos ao modelo de estresse induzido pela
administração de corticosterona. Foram avaliadas também os efeitos na
neuroplasticidade hipocampal através da dosagem dos níveis de BDNF. Artigo
submetido a revista Pharmacology, Biochemistry and Behavior.
• ARTIGO 2: onde constam os resultados referentes aos efeitos da riparina IV no
comportamentos de avaliação cognitiva e memória (labirinto em Y, esquiva passiva,
interação social e inibição pré-pulso), além de avaliar o efeito neuroprotetor nos
parâmetros de estresse oxidativo (nitrito, glutationa reduzida, lipoperoxidação e
atividade da superóxido dismutase e catalase) e níveis das citocinas (IL-2, IL-4, IL-6,
IL-10, IFN-γ, TNF-a) em camundongos estressados.
40
3.1 Capítulo I REVERSAL EFFECT OF RIPARIN IV IN DEPRESSION AND ANXIETY CAUSED
BY CORTICOSTERONE CHRONIC ADMINISTRATION IN MICE
Raquell de Castro Chavesa*; Auriana Serra Vasconcelos1; Natália Ferreira Oliveiraa; Iris
Cristina Maia Oliveiraa; Victor Celso Cavalcanti Capibaribea; Daniel Moreira Alves da Silvaa;
Iardja Stéfane Lopesa; José Tiago Valentima; Alyne Mara Rodrigues de Carvalhoa; Danielle
Silveira Macêdoa; Silvânia Maria Mendes Vasconcelosa; Stanley Juan Chaves Gutierrezb; José
Maria Barbosa Filhoc; Francisca Cléa Florenço de Sousaa
a Drug Research and Development Center, Department of Physiology and Pharmacology,
School of Medicine, Federal University of Ceará, Fortaleza, Ceará, Brazil. b Department of Biochemistry and Pharmacology, Faculty of Pharmacy, Federal University of
Piauí, Teresina, Piauí, Brazil. c Laboratory of Pharmaceutics Technology, Federal University of Paraiba, João Pessoa-Paraiba,
Brazil.
ABSTRACT
Mental disorders have a multifactorial etiology and stress presents as one of the causal factors.
In depression, it is suggested that high cortisol concentration contributes directly to the
pathology of this disease. Based on that, the study aims to evaluate the potential antidepressant
effect of riparin IV (Rip IV) in mice submitted to chronic stress model by repeated
corticosterone administration. Female Swiss mice were selected into four groups: control
(Control), stressed (Cort), riparin IV (Cort + Rip IV) and fluvoxamine (Cort + Flu). Three
groups were administrated subcutaneously (SC) with corticosterone (20 mg/kg) during twenty-
one days, while the control group received only vehicle. After the fourteenth day, groups were
administrated tested drugs: riparin IV (50 mg/kg), fluvoxamine (50 mg/kg) or distilled water
vehicle, by gavage, one hour after subcutaneous injections. After the final treatment, animals
were exposed to behavioral models such as forced swimming test (FST), tail suspension test
41
(TST), open field test (OFT), elevated plus maze (EPM) and sucrose preference test (SPT).
Hippocampus was also removed for the determination of BDNF levels. Corticosterone
treatment alters all parameters in behavior tests. Riparin IV and fluvoxamine exhibit
antidepressant effect in FST, TST and SPT. In EPM and OFT, treatment shown anxiolytic effect
without alter locomotor activity. Corticosterone administration decreased BDNF levels and
riparin IV could reestablish them. These findings suggest that riparin IV improves the
depressive and anxious symptoms after chronic stress and could be a new alternative treatment
for patients with depression.
Keywords: riparin; corticosterone administration; chronic stress; depression.
Highlights
• Corticosterone treatment can induce chronic stress and depressive symptoms in mice.
• Riparin IV shows antidepressant and anxiolytic effect after chronic stress.
• Riparin IV was able to normalize BDNF levels in mice hippocampus.
INTRODUCTION
Depression is a chronic and complex disorder with an enormous impact on society and
is associated with functional impairment and high morbidity and mortality. The prevalence of
major depression is high and is still increasing. Data confirmed that women are more vulnerable
than men and that it happens more frequently in young people and in the elderly (CAPRIOTTI,
2006; SILVA et al., 2014). According to The World Health Organization (WHO) by 2020
depression is estimated to be the second leading global burden of illness.
Depressive symptoms include depressed mood, irritability, lack of concentration,
psychomotor retardation or agitation, anhedonia (reduced ability to experience pleasure from
natural rewards), and abnormalities in appetite and sleep (ANISMAN; MATHESON, 2005).
Anxiety disorders have substantial co-morbidity with depression and could add, as regular
symptoms, nervous dread of the future, hypervigilance, increased heart rate and blood pressure
to regular symptoms (GREGUS et al., 2005; GRILLO, 2016; MILLER; HEN, 2015).
42
Most depression occurs idiopathically, but some risk factors could trigger depressive
symptoms, such as some types of cancers, endocrine abnormalities, side effects of drugs,
stressful life events, among many others (WAGER-SMITH; MARKOU, 2011). Stress is
presented as one of the causal factors of many mental disorders (ANISMAN; MATHESON,
2005; WANG et al., 2008) and it is suggested that hypersecretion of cortisol contributes directly
to the pathology of anxiety and depression (ROHLEDER; WOLF; WOLF, 2010;
SKÓRZEWSKA et al., 2006).
During depression, disturbs in the limbic system may result in alterations in the HPA
axis where the hippocampus appears to be involved in negative feedback control of the
glucocorticoid levels (KRISHNAN; NESTLER, 2008; STERNER; KALYNCHUK, 2010;
WARNER-SCHMIDT; DUMAN, 2006).
This inhibition appears to be dependent on the integrity of the hippocampus. Studies
suggest that depressed patients show hippocampal volumetric reductions (LORENZETTI et al.,
2009) and neurogenesis in rodent hippocampus is reduced by stress and increased by various
types of antidepressant treatments (MILLER; HEN, 2015; SAHAY; HEN, 2007; WARNER-
SCHMIDT; DUMAN, 2006). The neurotrophic alterations observed in the hippocampus of
depressed patients may be attributed in part to the reductions of the brain-derived neurotrophic
factor (BDNF) (AUTRY; MONTEGGIA, 2012).
Many strategies can take advantage of the molecular diversity of natural products in the
designing of combinatorial synthesis collections. Structural modifications of the skeleton of an
existing bioactive natural product are intended to promote improvements in their inherent
biological activity or pharmacological properties at a reasonable cost. This can be achieved by
means of semi-synthetic modifications of the molecule or by synthetic methods (HAUSTEDT
et al., 2006; KOEHN; CARTER, 2005).
Riparin IV is a synthetic alkamide drug analogue to Aniba riparia’s natural compounds
(Figure 1). The synthesis involves the condensation between acyl chlorides and O-
methyltyramine in a very high yield (BARBOSA-FILHO; DA SILVA; BHATTACHARYYA,
1990). Studies conducted by Dias (2012) and Nascimento et al. (2016) evidenced that riparin
IV has an antinociceptive and anti-inflammatory activity in the model of nociception induced
by acetic acid, formalin, and carrageenan.
43
Figure 1. Chemical structures of riparin IV. Source: Adapted from Barbosa-Filho et al. (1990)
Due to the similarity with the tyramine chemical structure, a sympathomimetic amine,
and central effects of riparins I, II and III, it becomes relevant to investigate the pharmacological
potential of riparin IV in anxiety and depression models. Based on these findings, the goal of
this study is to evaluate the potential antidepressant effect of riparin IV in mice submitted to a
chronic stress model through repeated corticosterone injections.
MATERIALS AND METHODS
Animals
Female Swiss mice (22–25 g) were used in this study. The animals were maintained on
a 12/12 h light/dark cycle, with access to water and food ad libitum, randomly distributed into
specified experimental groups. All experiments were performed at 23 ± 2 °C room temperature
and were carried out between 12:00 and 16:00 h, with each animal used only once. Experiments
were performed in accordance with the current laws and the National Institute of Health Guide
for the Care and Use of Laboratory Animals and under the consent and surveillance of the
Ethics Committee from the Department of Physiology and Pharmacology of Federal University
of Ceará (Protocol number 112/2014).
44
Drugs
Corticosterone (Sigma®, St Louis, MO, USA) was dissolved in a 0.9% saline solution
containing 0.1% polysorbate (Tween®) 80 (VETEC™, USA) and 0.1% dimethyl sulfoxide
(DMSO) (VETEC™, USA) and it was administered in a dose of 20 mg/kg, subcutaneously
(SC).
The riparin IV, a total of three batches, were provided by Laboratory Chemistry of
Bioactive Natural and Synthetic Products, Federal University of Piaui, Teresina, PI, Brazil.
Riparin IV was emulsified with 2% Tween® 80 and administered intragastric (oral gavage)
doses of 50 mg/kg.
Fluvoxamine (Abott®, New Jersey, USA), in a 50 mg/kg dose were dissolved in distilled
water and given by oral gavage.
Experimental procedure
The study design was based on a depression mouse model involving exogenous
corticosterone administration (ZHAO et al., 2008), where repeated corticosterone injections
increase depression-like behavior in mice after up to 1 week.
The animals were divided in four experimental groups (n = 12 animals/group, on
average): (1) Control; (2) corticosterone (Cort); (3) corticosterone + riparin IV (Cort + Rip IV)
and (4) corticosterone + fluvoxamine (Cort + Flu). Groups (1), (2) and (3) were administrated
subcutaneously (SC) with 20mg/kg corticosterone in a saline vehicle for twenty-one days, while
the control group (1) was administrated only with saline vehicle.
In the last seven days of treatment, each group was administrated with tested drugs
riparin IV (50mg/kg) (group 3), fluvoxamine (50mg/kg) (group 4) or distilled water vehicle
(distilled water emulsified with 2% Tween® 80) (groups 1 and 2), per gavage, with a 1-h interval
between corticosterone treatment injections (Figure 2).
45
Figure 2. Schematic overview of the experimental design. SC: subcutaneous; PO: per oral gavage; FST: Forced Swimming Test; TST: Tail Suspension Test; OFT: Open Field Test; EPM: Elevated Plus Maze Test; SPT: Sucrose Preference Test; BDNF: Brain-derived Neurotrophic Factor.
Behavioral determinations were registered sixty minutes after the last drug
administration and the hippocampus was removed for BDNF levels evaluation. One group of
40 animals, divided in 4 groups was tested in OFT, FST and SPT behavioral tests. Other group
of 40 animals, divided as explained above, was submitted to other behavioral tests and their
hippocampus used to BDNF analysis.
Behavioral tests
Forced swimming test
The procedure used was based on that described by Porsolt, Bertin and Jalfre (1977)
with a minimum modification. Mice were placed individually in the cylinders tank filled with
water (25 ± 1 °C) to a depth of 25 cm, dimensions the mice will not be able to touch the bottom
of the tank, either with their feet or their tails, during the swimming test. Animal behavior was
analyzed by an independent researcher who did not know the experimental groups. The
immobility time during a five minute period was recorded. Immobility was defined as the
46
animal floating in the water with the absence of any movement except for those necessary for
keeping the nose above water. An increase in the duration of immobility is indicative of
depressed-like behavior (YANKELEVITCH-YAHAV et al., 2015).
Tail suspension test
The procedure followed in this study was previously described by Steru et al. (1985).
Mice were suspended 50cm above the floor by adhesive scotch tape placed around 1 cm from
the tip of the tail. Immobility time was measured using a chronometer by an observer during a
six minute period.
Open Field Test
The test was performed in a soundproof in an air-conditioned chamber under dim light.
The apparatus used was a cube of transparent acrylic with a black floor (30 × 30 × 15 cm) and
divided into nine equal square grids clearly drawn on the surface. After 60 minutes of last
treatment, the animals were placed on the central quadrant to begin the test. The outcome
measured during the 5 minute test was: number of line crosses contacted with all four legs
(spontaneous movement), number of grooming (licking the paws, washing movements over the
head, fur licking and/or tail/genitals cleaning) and rearing, which is the number of times an
animal stood erect on its hind legs with forelegs in the air against the wall (ARCHER, 1973).
Elevated plus maze
The elevated plus maze is a plus-shaped apparatus with four arms at right angles to each
other as described by Handley and Mithani (1984). Sixty minutes after oral treatment, the
animal was placed at the center of the plus maze facing one of the enclosed arms, and observed
for 5 minutes, according to the following parameters: number of entries into the open and closed
arms and time of permanence in each of them. The criterion for arm visit was considered only
when the animal decisively moved all its four limbs into an arm. The percentage of time spent
in the arms and the number of entries into the arms was calculated using the following formula:
Timespentinopenarms(%) =timespentinopenarms
timespentinopenarm + closedarms x100
47
Entriesinopenarms(%) =numberofentriesintoopenarms
totalnumberofentries x100
Sucrose preference test
Sucrose preference is considered to be an index of anhedonia (WANG et al., 2014). The
test was performed as described previously by Strekalova et al. (2004), with minor
modifications. In this model, a mouse is given free choice between two solutions to drink: water
or a sucrose solution. Usually, mice show a clear preference for the sweetened water, while
depressed animals demonstrate less interest. Before the test, the mice were trained to adapt to
1% (w/v) sucrose solution by placing two bottles of 1% sucrose solution in each cage for
eighteen hours. After adaptation, mice were housed in individual cages for eighteen hours and
exposed to two identical bottles, one filled with 1% sucrose solution and the other filled with
water. The beginning of the test started with the onset of the dark (active) phase of the animals’
cycle. No previous food or water deprivation was applied before the test. After eighteen hours,
sucrose and water consumption were recorded and the sucrose preference was calculated by the
following formula:
Sucrosepreference(%) =Sucroseconsumption
Waterconsumption + Sucroseconsumption x100
Neurochemical tests
BDNF
After twenty-one days of treatment, the animals were euthanized by decapitation to
remove the skulls. The hippocampi were dissected and stored in a freezer at -80 °C for posterior
biochemical analysis. The content of BDNF protein was measured using a commercially
available enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems Inc, Minneapolis,
Minnesota) according to the manufacturer’s instructions. The amount of BDNF was determined
by absorbance in 450 nm and expressed as pg per g of wet tissue. The standard curve
demonstrates a direct relationship between optical density and BDNF concentration.
STATISTICAL ANALYSIS
48
The data were analyzed with GraphPad Prism 7.0a (San Diego, CA, USA). Statistical
analysis of the data was performed by one-way ANOVA, followed by Student-Newman-Keuls
post hoc test. Data are expressed as mean ± SEM and differences were considered significant
when p ≤ 0.05.
RESULTS AND DISCUSSION
Mice were treated with subcutaneous corticosterone injections at a dose of 20 mg/kg,
daily, and behavioral tests were assessed on the twenty-first day by using the forced swimming
test (FST), tail suspension test (TST), sucrose preference test (SPT), elevated plus maze (EPM)
and open field test (OFT).
Stress induced by repeated corticosterone administration was chosen to induce chronic
depression because it can control over increases in circulating glucocorticoids, different than
other stress models such as chronic mild stress exposure and repeated restraint stress (GREGUS
et al., 2005; HERRERA-PÉREZ et al., 2016). Animals can differ in stress responses (quality
and quantity) and HPA axis stimulation and this may result in differentiating corticosterone
levels between different animals exposed to the same stressor, which in turn could lead to
increased experimental variability (MARKS; FOURNIER; KALYNCHUK, 2009; ZHAO et
al., 2008).
Results have showed that exogenous corticosterone administration for twenty-one days
can produce depression-like behavior in the forced swimming test [F (3, 54) = 36.74] and tail
suspension test [F (3, 58) = 39.08] (Figure 3). Administration of riparin IV and fluvoxamine for
7 days has decreased immobility time compared to corticosterone administration only
(p<0.0001).
49
Figure 3. Immobility time analysis in forced swimming test after 21 days of treatment with corticosterone
and tested drugs Rip IV (50mg/kg) and Fluvoxamine (50mg/kg). Data are expressed as mean ± SEM of
immobility time during 5 minutes’ test. Statistical analysis was performed by one-way ANOVA, followed
by Student-Newman-Keuls’ post hoc test. aaaap<0.0001 vs control; bbbbp<0.0001 vs stressed group.
Animals were submitted to tail suspension test and showed a significant difference
between groups [F (3, 58) = 39.08]. Results (Figure 4) showed that corticosterone treated mice
spent a significantly greater percentage of time immobile during the TST than did the control
group (p<0.0001). Riparin IV and fluvoxamine were able to decrease immobility time caused
by stress conditions (p<0.0001).
50
Figure 4. Effect of Rip IV (50 mg/kg) and Flu (50mg/kg) on immobility time of mice in tail suspension test.
Data are expressed as mean ± SEM of immobility time during 6 minutes’ test. Statistical analysis was
performed by one-way ANOVA, followed by Student-Newman-Keuls’ post hoc test. Significant values:
aaaap<0.0001 vs control; bbbbp<0.0001 vs Cort group.
Animal models of depression are typically based on exposure of animals under a
stressful condition (a real or a potentially dangerous situation) and use a specific test for
measuring behavioral and physiological reactions. The forced swimming and tail suspension
tests are two of the most widely used models for assessing antidepressant-like activity in mice
(BERGNER et al., 2016; CAN et al., 2012; CRYAN et al., 2005; CRYAN; MARKOU; LUCKI,
2002; KRISHNAN; NESTLER, 2011; PALANZA, 2001) and they are highly sensitive to all
major classes of antidepressant drugs and treatments including MAO inhibitors, tricyclics,
serotonin-specific reuptake inhibitors, atypical antidepressants, and electroconvulsive shock
(CASTAGNÉ et al., 2011). The increased immobility behavior in the FST and TST is
considered to be an indicator of despair and is widely used to investigate the acute and chronic
effects of antidepressant drugs (BERGNER et al., 2016).
High cortisol levels could be associated to psychotic depression, where people with
severe depression may also develop psychotic symptoms (hallucinations and/or delusions),
most commonly thematically consistent with the negative, self-blaming cognitions and low
mood. This subtype of depression proves difficult to treat and the pharmacotherapy includes
the combination of antidepressants and antipsychotics associated with many adverse effects
(IIJIMA et al., 2010).
Clinical studies proved that monotherapy using the fluvoxamine, a selective serotonin
reuptake inhibitor (SSRI), was effective against both the psychotic and depressive symptoms
of this disorder (FURUSE; HASHIMOTO, 2009). Zanardi and collaborators (2000) conducted
a double-blind study comparing fluvoxamine and venlafaxine monotherapy for six weeks. In
twenty-eight hospitalized patients diagnosed with major depression and severe psychotic
features, fluvoxamine showed efficacy as the treatment of psychotic depression. These results
motivated the choice of fluvoxamine as the reference drug for this work.
51
The stress response is meant to maintain the stability or homeostasis of the organism.
Long-term activation of the stress system can cause pathological states, or exacerbate pre-
existing or latent morbid states such as obesity and cardiovascular diseases (PEREIRA-
FIGUEIREDO et al., 2017; ROHLEDER; WOLF; WOLF, 2010). Stressful events also underlie
to various pathophysiological processes associated with mood disorders, such as unipolar or
bipolar depression, as well as posttraumatic stress disorder (PTSD) (MORRIS; COMPAS;
GARBER, 2012) or anxiety (KIYOHARA; YOSHIMASU, 2009).
Nearly one-half of those diagnosed with depression are also diagnosed with an anxiety
disorder. Therefore, it was decided to study the effect of riparin IV in the open field and elevated
plus maze, two sensitive tests to evaluate anxious-like behavior (KORTE; DE BOER, 2003).
In the OFT, groups treated orally with riparin IV and fluvoxamine decreased the number
of rearing [F (3, 31) = 5.97] and grooming [F (3, 31) = 6.481] as compared to Cort group. No
alteration was observed in the number of crossing [F (3, 35) = 2.003] (Figure 5).
52
Figure 5. Number of squares crossed (crossings) (a), groomings (b) and rearings (c) by mice treated with
vehicle (control), Cort (20mg/kg), Rip IV (50 mg/kg) and Flu (50mg/kg) in an open field test. Results were
expressed as mean ± SEM (n=8 per group). Statistical analysis was performed by one-way ANOVA,
followed by Student-Newman-Keuls’ post hoc test. Significant values: aap<0.01 vs control; bp<0.05 and bbp<0.01 vs stressed group.
The open field test is used to measure not only anxiety-like behaviors but also activity
or even sedation (PRUT; BELZUNG, 2003). Our findings show that the corticosterone, riparin
IV and fluvoxamine treatment didn’t change the locomotor activity in animals, but chronic
corticosterone administration increased grooming and rearing, and the treatment with riparin
IV and fluvoxamine significantly decrease these parameters.
53
According to van Erp et al. (1994) and Kalueff and Tuohimaa (2004), stress can induce
grooming in rodents and this innate behavior it might be related to endocrine hypothalamus-
pituitary adrenal (HPA) axis. Riparin IV was able to decrease grooming suggesting that
treatment may alter cortisol homeostasis. Treatment with riparin IV was able to decrease mice
immobility time caused by chronic stress and also preserved locomotor activity in OFT,
suggesting that its antidepressant effect in this predictive model is specific and not related to an
increase in motor activity of the animals.
In the elevated plus maze (EPM), the number of entries into and the time spent in the
open arms were taken as indices of anxiety. Treatment significantly increased in number and
time spent in the open arm compared to Cort group. Comparing percentage time spent in the
open arm to the closed arm, the animals treated with riparin IV (p<0.0001) and fluvoxamine
(p<0.01) spent significantly more time in the open arm than compared to Cort and control
groups. These parameters were expressed as a percentage of the total entries into and the total
time spent in any arm during the 5 minute test session (Table 1).
Groups Number of entries % of entries in open
arms
% time spent in
open arms
Control 8.500 ± 0.8303 36.81 ± 2.963 43.20 ± 2.386
Cort 3.545 ± 0.4545 aaa 24.03 ± 2.152aa 26.15 ± 1.757 aaaa
Cort + Rip IV 11.60 ± 1.122 bbbb 45.83 ± 1.661bbb 53.34±2.651 bbbb/aa
Cort + Flu 6.357 ± 0,7603 bb 38,78 ± 2.182bb 34.46 ± 1.656 bb/aa
Table 1. Each value represents the mean ± S.E.M. Significant values: aap<0.01, aaap<0.001 and aaaap<0.0001
as compared to control, bbp<0.01, bbbp<0.001 and bbbbp<0.0001 as compared to stressed animals. Statistical
analysis was performed by one-way ANOVA followed by Newman–Keuls as the post hoc test.
In the animal’s models, the results found by Iijima et al. (2010) showed a different
conclusion. Rats received corticosterone injections (20 mg/kg, subcutaneously), once a day for
21 consecutive days prior to the forced swimming test. A day prior to the behavior test, animals
orally received fluvoxamine (3mg/kg), imipramine (10.0 mg/kg) and a combination of
risperidone (0.1 mg/kg) and fluvoxamine (3.0 mg/kg). Acute treatment with fluvoxamine and
imipramine monotherapy failed to decrease the immobility time but a combination of
54
antidepressant and antipsychotic drugs could decrease immobility time in the forced swimming
test when administered once. Our behavioral findings show that fluvoxamine also reversed
stress symptoms associated with depression and anxiety in a higher dosage (50mg/kg) and after
several days of administration.
Results from several previous studies have indicated that repeated corticosterone
treatments can influence rodent behavior and induce depressive symptoms (IIJIMA et al., 2010;
LUSSIER et al., 2013; MARKS; FOURNIER; KALYNCHUK, 2009; MURRAY; SMITH;
HUTSON, 2008; SILVA et al., 2016; SKÓRZEWSKA et al., 2014; ZHAO et al., 2008, 2009),
including anhedonia (ABELAIRA; RÉUS; QUEVEDO, 2013; GUPTA; RADHAKRISHNAN;
KURHE, 2015; LI et al., 2015; VASCONCELOS et al., 2015). In the evaluation of the sucrose
preference parameter (Figure 6), the Cort-treated group had a lower sucrose consumption when
compared to other groups, while riparin IV (p<0.05) and fluvoxamine (p<0.0001) treatment
was able to recover the sucrose preference after the corticosterone exogenous administration [F
(3, 27) = 9.704].
Figure 6. Effect of Rip IV (50 mg/kg) and Flu (50mg/kg) on the percentage of sucrose consumption of mice
induced to chronic corticosterone stress model. Data are expressed as mean ± SEM and statistical analysis
was performed by one-way ANOVA, followed by Student-Newman-Keuls’ post hoc test. Significant values:
aap<0.01 vs control; b p<0.05, bbbp<0.001 and bbbp<0.001 vs stressed group.
55
Anhedonia is a key symptom of all forms of depression and it can influence many of its
symptoms (BOGDAN; PIZZAGALLI, 2006). The reduction of the ability to experience
pleasure caused by stressful events tends to be long-lasting and its operationally defined by a
decreased preference for sweetened solutions (STREKALOVA et al., 2004). Corticosterone
administration led to a reduction of sucrose consumption and a seven-day treatment with Rip
IV normalize sucrose intake similar to fluvoxamine. This finding is very relevant because some
types of antidepressants and anxiolytics drugs are ineffective in reversing chronic stress-
induced anhedonia (PATEL, 2016; TREADWAY; ZALD, 2011; WILLNER; MUSCAT;
PAPP, 1992).
The neurochemical analysis, Figure 7 shows the significant effect of riparin IV and
fluvoxamine treatment on BDNF protein levels in the hippocampus [F (3, 28) = 18.52].
Corticosterone administration significantly decreased BDNF protein levels in the hippocampus
of mice, as compared to control. Riparin IV and fluvoxamine treatment significantly increased
the BDNF protein levels when compared to stressed animals.
Figure 7. Effect of Riparin IV (50mg/kg) and Fluvoxamine (50mg/kg) ztreatment on BDNF protein levels in
the hippocampus of chronic corticosterone stress model exposed mice. Data are expressed as mean ± SEM
and statistical analysis was performed by one-way ANOVA, followed by Student-Newman-Keuls’ post hoc
test. Significant values: aaaap<0.0001 vs control; bbbbp<0.0001 vs Cort group.
56
Brain Derived Neurotrophic Factor (BDNF) is a member of the nerve growth factor
family. BDNF, signaling in the mesolimbic pathway, play a role in survival mechanism in the
central nervous system, such as neurogenesis, neuronal growth, cellular differentiation and
survival of neurons. This type of neurotrophin also influences dendritic connectivity and
neuroplasticity (BANERJEE et al., 2014; BRAMHAM; MESSAOUDI, 2005; CASTRÉN;
RANTAMÄKI, 2010b).
However, under stress, the gene for BDNF is repressed, leading to atrophy and possible
apoptosis of neurons in the hippocampus. These events, in turn, lead to depression and
susceptibility to social anhedonia followed by social stress (HUANG; LIN, 2015; KIYOHARA;
YOSHIMASU, 2009; KUPFERBERG; BICKS; HASLER, 2016). Furthermore, the
hippocampus is particularly susceptible to the damaging effects of prolonged stress, (IHARA
et al., 2016; NOVKOVIC; MITTMANN; MANAHAN-VAUGHAN, 2015) evidenced by
decreased hippocampal neurogenesis and hippocampal glucocorticoid receptor (GR) mRNA
expression (STERNER; KALYNCHUK, 2010). This GR decrease could lead to a stimulation
of the HPA axis and increase glucocorticoid serum levels and creating even more hippocampal
damage.
Many antidepressant drugs acutely increase monoamine levels, but in order to achieve
success in therapy it is necessary to lead long-term adaptation such as regulation of
neurotrophins, as BDNF (KOZISEK; MIDDLEMAS; BYLUND, 2008). Studies have shown,
in depressed patients and animal models of stress, that the efficacy of antidepressants in causing
alterations behavioral symptoms of depression depends on their ability to increase BDNF levels
(BANERJEE et al., 2014; CASTRÉN; RANTAMÄKI, 2010a; DELTHEIL et al., 2008;
SOUSA et al., 2015).
Data suggests that chronic corticosterone administration reduces BDNF levels in the
hippocampus (GREGUS et al., 2005; JACOBSEN; MØRK, 2006; SOUSA et al., 2015;
VASCONCELOS et al., 2015; WARNER-SCHMIDT; DUMAN, 2006) and riparin IV shows
a significant effect in BDNF levels which is not a regular finding in all antidepressant treatment.
Jacobsen and Mørk (2004) found different changes in antidepressant treatment in BDNF
protein level, where escitalopram (a selective serotonin reuptake inhibitor [SSRI]) decreased
BDNF protein in the hippocampus and desipramine (a tricyclic antidepressant that inhibit the
reuptake of noradrenaline) did not affect the BDNF protein level.
57
As previously mentioned, chronic stress induced by corticosterone is a model of
depression involving psychotic symptoms and turn into a persistent kind of depression
(LORENZETTI et al., 2009). Psychotic depression is difficult to treat and normally involves
the administration of the combination of an antidepressant and an antipsychotic, which
increases the risk of adverse effects (HAMODA; OSSER, 2008). The evidence that riparin IV
could reverse the psychotic depression induced in animals as a monotherapy incites its
importance in depression management.
CONCLUSION
Depression is a common mental disorder associated with debilitating symptoms; it can
also co-exist with another mental disorders such as anxiety and psychosis and affect populations
around the globe. There is a lack of effective pharmacological treatments and 10-30% of
patients did not respond to regular antidepressant treatments (AL-HARBI, 2012). Treating a
resistant depression causes socioeconomic impact and the development of new strategies are
extremely necessary.
Animal models are utilized to provide knowledge of the neurobiological basis of several
disorders, which could ultimately produce improved treatment options for the patient with
depression. We have reviewed the findings of preclinical research demonstrating that riparin
IV prevents the effects of corticosterone induced stress on behavior. The ability to increase
BDNF levels is also a critical finding and could lead to riparin IV as a potential treatment
strategy in the future.
Funding
This study was financied in part by Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior – Brasil (CAPES) – Finance Code: 001; by Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq-Brazil; Process numbers: nº 12/2017, nº 306746/2013-1, nº
446120/2014-6 and nº 407567/2013-5), and Fundação Cearense de Apoio à Pesquisa
(FUNCAP-Ceará-Brazil).
58
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3.2 Capítulo II
THE NEUROPROTECTIVE EFFECT OF RIPARIN IV ON OXIDATIVE STRESS
AND NEUROINFLAMMATION RELATED TO COGNITIVE IMPAIRMENT
CHRONIC STRESS
Raquell de Castro Chavesa; Auriana Serra Vasconcelos Mallmann1; Natália Ferreira Oliveiraa;
Victor Celso Cavalcanti Capibaribea; Daniel Moreira Alves da Silvaa; Iardja Stéfane Lopesa;
José Tiago Valentima; Giovanna Riello Barbosab; Alyne Mara Rodrigues de Carvalhoa; Marta
Maria de França Fontelesc; Stanley Juan Chavez Gutierrezd; José Maria Barbosa Filhoe;
Francisca Cléa Florenço de Sousaa
a Neuropharmacology Laboratory, Drug Research and Development Center, Department of
Physiology and Pharmacology, School of Medicine, Federal University of Ceará, Fortaleza,
Ceará, Brazil. b Multi-User Facility, Drug Research and Development Center, Federal University of Ceará c Department of Pharmacy, Federal University of Ceará, Fortaleza, Brazil.
d Laboratory Chemistry of Bioactive Natural and Synthetic Products, Federal University of
Piauí, Teresina, Piauí, Brazil. e Laboratory of Pharmaceutics Technology, Federal University of Paraiba, João Pessoa-Paraiba,
Brazil.
ABSTRACT
Stress is one of the precipitating factors in the development of depression disorders and may be
related to hypothalamic–pituitary–adrenal (HPA) axis disruption. Cognitive impairment is
identified as one of the diagnostic criterion for major depressive disorder (MDD) and can
extensively affect the quality of life of those patients. Based on these findings, this study aimed
to investigate the possible effects of Riparin IV on cognition impairment induced by chronic
administration of corticosterone in mice. Female Swiss mice were divided into four groups:
control (Control), corticosterone (Cort), Riparin IV (Cort + Rip IV), and Fluvoxamine (Cort +
Flu). Three groups were administrated subcutaneously (SC) with corticosterone (20 mg/kg)
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during the 22-day study, while the control group received only vehicle. After the 14th day, the
groups were administrated tested drugs: Riparin IV (Rip IV), fluvoxamine (Flu), or distilled
water, by gavage, one hour after the subcutaneous injections. After the final treatment, animals
were exposed to behavioral models such as the Y-maze test (YMT), the step-down inhibitory
avoidance test (SDIT), the social interaction test (SIT), and the prepulse inhibition test (PPI).
The animals were euthanized, and the prefrontal cortex, the hippocampus, and the striatum were
removed for evaluation of oxidative stress and cytokine content. The results revealed the
development of cognitive impairment in mice treated with Cort, an increase in brain
malondialdehyde (MDA) and nitrite/nitrate levels, and a decrease in reduced glutathione (GSH)
content, catalase (CAT), and superoxide dismutase (SOD) activities as compared to the control.
Cort-treated mice also exhibited a an neuroinflammatory profile with increased pro-
inflammatory cytokines TNF-a, IFN-g and IL-2 and decreased anti-inflammatory IL-4, but no
significant alteration in IL-6 and IL-10. Rip IV treatment, like that of the antidepressant Flu,
significantly ameliorated cognitive deficit behavior induced by Cort. The antidepressant-like
ability of Rip IV treatment against the chronic Cort-induced stress may be due to its potential
to mitigate inflammatory damage and oxidative stress. This antioxidant and anti-inflammatory
effect observed indicates Rip IV as a possible drug in the antidepressant treatment of non-
responsive patients related to severe and cognitive symptoms.
Keywords: riparin; depression; corticosterone; oxidative stress; Th1-Th2 Balance.
INTRODUCTION
Stress is characterized by physiological changes that occur in response to novel or
threatening stimuli. The hypothalamic–pituitary–adrenal (HPA) axis is the final common
pathway in the mediation of the stress response, which subsequently causes the cortisol release
(BAO; MEYNEN; SWAAB, 2008). Glucocorticoids are released in response to physical,
emotional and/or metabolic stressors, but intense stimulation of the HPA axis may lead to
maladaptive responses (SKÓRZEWSKA et al., 2014; TAFET; BERNARDINI, 2003). Some
studies claim that chronic glucocorticoid administration decreases during neurogenesis, which
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may be responsible for memory deficits and mood disturbance (SKÓRZEWSKA et al., 2006;
ZHAO et al., 2008).
Chronic stress or even isolated traumatic experiences may alter cognitive functions,
such as learning and memory (QUERVAIN et al., 2009; SCHILLING et al., 2013;
WALESIUK; TROFIMIUK; BRASZKO, 2006). The literature suggests that exposure to stress
may be a precipitating factor in the development of depression. In fact, some studies show that
up to 85% of patients experience significant stressful events before the onset of depressive
symptoms (SAVEANU; NEMEROFF, 2012).
The Diagnostic and Statistical Manual of Mental Disorders (DSM-V) identifies
cognitive impairment (poor concentration, memory deficit, indecision) as a diagnostic criterion
for major depressive disorder (MDD). Cognitive complaints during the symptomatic and
remission phases of MDD are common when compared to other serious mental disorders such
as schizophrenia and bipolar disorder, all of which extensively affect the quality of life for those
patients (DARCET et al., 2016; MCINTYRE et al., 2013).
Riparin IV was first synthetized in 1990 by Barbosa-Filho, da Silva and Bhattacharyya
(Figure 1). Our previous studies (DIAS, 2012) and others (NASCIMENTO et al., 2016) showed
that it has anti-inflammatory and antinociceptive activities on mice. Considering its structure
has a molecule of tyramine, with recognized central effect, and a derivative of benzoic acid, the
trimethyl ether of gallic acid , the aim of this study is to investigate the possible effects of
Riparin IV on cognition impairment induced by chronic administration of corticosterone in
mice.
Figure 1. Chemical structure of Riparin IV.
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MATERIAL AND METHODS
Animals
Female Swiss mice (22–25 g; age: 8–10 weeks) were used in this study and were
randomly distributed into specified experimental groups. Their environments were maintained
on a 12/12 h light/dark cycle, with lights off at 18:00h. They were also maintained at a constant
temperature of 25±1 °C and the animals were given free access to food and tap water.
Experiments were performed in accordance with the current laws and the National Institute of
Health Guide for the Care and Use of Laboratory Animals and under the consent and
surveillance of the Ethics Committee from the Department of Physiology and Pharmacology of
the Federal University of Ceará (Protocol number 112/2014).
Drugs
Corticosterone (Sigma®, St Louis, MO, USA), administered in a dose of 20 mg/kg,
subcutaneously (SC), was dissolved in a 0.9% saline solution containing 0.1% polysorbate
(Tween®) 80 (VETEC™, USA) and 0.1% dimethyl sulfoxide (DMSO) (VETEC™, USA), at a
constant volume of 0.1 ml/10 g of body weight.
The Riparin IV, a total of three batches provided by Laboratory Chemistry of Bioactive
Natural and Synthetic Products (Federal University of Piaui, Teresina, PI, Brazil), was
emulsified with 2% Tween® 80 and administered in intragastric (oral gavage – p.o) doses of 50
mg/kg. Fluvoxamine (Abott®, New Jersey, USA), 50 mg/kg, and ketamine 20 mg/kg (Konig,
Brazil) diluted in distilled water, were administered by oral gavage.
Experimental procedure
The study aimed to evaluate the effects of Riparin IV treatment against chronic
corticosterone induced depressive-like behavior in mice cognitive function. The experimental
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design and chose of reference drug were previously described (LOPES et al., 2018;
VASCONCELOS et al., 2015).
The animals (5-10 per group) were divided in four experimental groups: (1) vehicle or
control group (Control), (2) corticosterone group (Cort), (3) corticosterone + Riparin IV (Cort
+ Rip IV) and corticosterone + Fluvoxamine (Cort + Flu). Mice in groups (2), (3) and (4) were
administrated subcutaneously (s.c.) with corticosterone (20mg/kg) in a saline vehicle (0.9%
NaCl + 0.1% DMSO + 0.1% Tween-80) during 22 days, while the control group (1) were
administrated only with saline vehicle. After the 14th day, groups received Cort for 22 days
and within them treatment groups received either Riparin IV (50mg/kg) (3), fluvoxamine
(50mg/kg) (4) and distilled water vehicle (distilled water emulsified with 2% Tween-80)(1)(2),
per oral, with a 1-h interval between corticosterone treatment injections (Figure 2). Sixty
minutes after the last drug administration, behavioral tests were performed to verify cognitive
function, such as Y-maze test (YMT), step-down inhibitory avoidance test (SDIT), social
interaction test (SIT) and prepulse inhibition test (PPI). Animals were euthanized and prefrontal
cortex, hippocampus and striatum were removed for oxidative stress evaluation and cytokine
content. This timeline was taken twice so the animals were divided between different behavioral
tests and neurochemical tests.
Figure 2. Experimental design of the study. Each group received daily subcutaneous injections of
corticosterone (Cort), 20 mg/kg, or saline vehicle, for 22 days. From the 14th day of treatment onward, in
addition to the stressor hormone, animals also received a daily, per gavage, administration of distilled water
vehicle, Riparin IV (Rip IV), or fluvoxamine (Flu), both 50 mg/kg, for 8 consecutive days. At the end of
schedule, behavioral tests were conducted. Animals was them sacrificed and brain areas removed for
70
neurochemical assays. YMT = Y-maze test; SDIT = step-down inhibitory avoidance test; SIT = social
interaction test; PPI = prepulse inhibition test.
Behavioral tests
Y-maze test
The working memory was analyzed on Y-maze, which allows to evaluate spatial
working memory performance (YAMADA et al., 1996). The maze consists of three identical
arms 40cm long, 25cm high and 3cm wide, each converging at an equal angle. Each mouse was
placed at end of one of the three arm of the apparatus and allowed to explore the freely for 8
minutes. The total number of arm entries was recorded visually and an alternation was
considered correct if the animal visited a new arm and did not return to the previously visited
arm. Thus, the percentage of alternations was calculated using following formula:
%𝑎𝑙𝑡𝑒𝑟𝑛𝑎𝑡𝑖𝑜𝑛𝑠 = 𝑠𝑢𝑐𝑒𝑠𝑠𝑖𝑣𝑒𝑎𝑙𝑡𝑒𝑟𝑛𝑎𝑡𝑖𝑜𝑛𝑠
(𝑡𝑜𝑡𝑎𝑙𝑛𝑢𝑚𝑏𝑒𝑟𝑜𝑓𝑒𝑛𝑡𝑟𝑖𝑒𝑠 − 2)𝑥100
Step-down inhibitory avoidance test
The step-down inhibitory avoidance is based on negative reinforcement and is used to
evaluate short-term memory (STM) and long-term memory (LTM). The apparatus consisted of
a box (31 cm long, 27 cm high, and 24 cm wide) with a frontal glass wall. The floor consisted
of a series of parallel stainless-steel bars spaced 0.8 cm apart and connected to a generator. The
left extremity of the grid was covered by a high formic non-conductive platform (safety
platform).
Training was performed in two similar sessions. First, each mouse was placed on the
safety platform. The amount of time for the animal stepdown with its four paws on the grid was
recorded (step-down latency time). Then, a shock (0.5 mA: 1s) was applied and the animal was
removed from the apparatus. The second session was carried out 90 min after the first test.
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The retention test was carried out in the similar manner, except in the retention test
animals were not submitted to footshock, and the step-down latency time (SDL) was recorded
(JOSHI; PARLE, 2006). For the evaluation of the STM and LTM, the animals were submitted
to the test session at 90 min (second session) and at 24 h (retention) after the training session,
respectively, with an upper cutoff time of 300 s.
Social Interaction
The testing apparatus consisted of a 60 x 40 x 22 cm Plexiglas box divided into three
chambers with rectangular openings allowing access into each chamber. Two iron cages were
placed in each side of the chambers where an unfamiliar, same-sex mouse from the same
experimental group, was placed in one of two restraining cages (MOY et al., 2004). Test mice
were placed in the center chamber and were allowed 5 min of exploration time in the box. The
time spent in each of the three chambers was manually recorded, and social preference was
defined as follows:
(%𝑡𝑖𝑚𝑒𝑠𝑝𝑒𝑛𝑡𝑖𝑛𝑡ℎ𝑒𝑠𝑜𝑐𝑖𝑎𝑙𝑐ℎ𝑎𝑚𝑏𝑒𝑟) −(%𝑡𝑖𝑚𝑒𝑠𝑝𝑒𝑛𝑡𝑖𝑛𝑡ℎ𝑒𝑜𝑝𝑝𝑜𝑠𝑖𝑡𝑒𝑐ℎ𝑎𝑚𝑏𝑒𝑟)
Prepulse inhibition (PPI)
Prepulse inhibition of the startle test is based on the fact that under specific conditions,
a weak stimulus, or "prepulse" stimulus, may inhibit the effects of a subsequent severe and
intense (pulse) stimulus. This test is widely used in translational models to understand the
biology of the brain based on inhibitory mechanisms and its deficiency in neuropsychiatric
disorders (SWERDLOW et al., 2009).
The test was performed as previously described by Levin et al. (2011) where individual
mice were placed in small metal cages equipped with a movable platform floor attached to a
sensor that recorded vertical movements of the floor (Insight, São Paulo, Brazil). Startle reflexes
were evoked by acoustic stimuli delivered from a loudspeaker that was suspended above the
cage and connected to an acoustic generator. For prepulse inhibition tests, 74
pseudorandomized protocol assays were divided into eight categories, where the 120 dB / 50
72
ms startle pulse was applied alone or preceded by a prepulse stimulus of 70, 75 or 80 dB of
intensity (300Hz frequency /20 milliseconds duration). An interval of 100 milliseconds with
background noise was employed between each pre-pulse and pulse stimulus. The amplitude of
the startle response was defined as the difference between the maximum force detected during
a recording window and the force measured immediately before the start of the stimulus.
Amplitudes were ascertained for each individual animal, separately for both types of assays
[i.e., isolated pulse (P) or stimulus preceded by a prepulse (PP + P)]. Prepulse inhibition was
calculated as the percentage of the startle amplitude reduction using the following formula:
%𝑃𝑃𝐼 = 100 − [𝑃𝑃 + 𝑃𝑃 𝑥100]
Determination of oxidative stress parameters
Following behavioral tests, the animals were euthanized and their prefrontal cortex
(PFC), hippocampus (HC), and striatum (ST) were quickly dissected, frozen and stored at −70
°C. The samples were subjected to biochemical estimation such as oxidative stress, as described
below.
Determination of reduced glutathione (GSH) levels
Reduced glutathione levels in cerebral areas were evaluated to estimate endogenous
defenses against oxidative stress and were measured based on non-protein thiol content. The
method was based on Ellman’s reagent (DTNB) reaction with free thiol groups. Homogenates
10% (w/v) in EDTA 0.02M were added to a 50% trichloroacetic acid solution. After
centrifugation (3000 rpm for 15 min), samples were mixed with a 0.4 M Tris-HCl buffer that
had a pH of 8.9 and 0.01M DTNB. The color reaction was measured at 412 and expressed as
μg of GSH/g of wet tissue (SEDLAK; LINDSAY, 1968).
Measurements of lipid peroxidation
73
Malondialdehyde (MDA) content, a measure of lipid peroxidation, was assayed in the
form of Thiobarbituric Acid Reacting Substances (TBARS) according to Draper and Hadley
(1990). The samples were briefly mixed with 50 mM potassium phosphate monobasic buffer
of pH 7.4. Meanwhile, 63 μL of the homogenate was mixed with 100 μL of 35% perchloric
acid. Then these were centrifuged (5000 rpm during 10 min) and 150 μL of the supernatant was
retrieved and mixed with 50μL of 1.2% thiobarbituric acid and heated in a boiling water bath
for 30 min. After cooling, the lipid peroxidation was determined by the amount of absorbance
at 535 nm. The calibration curve was developed and TBARS levels were calculated and
expressed as μg of MDA/g of wet tissue.
Nitrite and nitrate determination
In order to assess the effects of treatments with respective drugs on nitric oxide (NO)
production, nitrite/nitrate levels were determined in the mouse brain homogenates immediately
after decapitation in all groups. After centrifugation (10000 rpm for 10 min), the homogenate
supernatant was collected and the production of NO was determined based on the Griess
reaction (GREEN et al., 1982). Briefly, 100 μL of supernatant was incubated with the same
volume of Griess reagent at room temperature for 10 min. The absorbance was measured at 546
nm via a microplate reader. The amount of nitrite was calculated from a NaNO2 standard curve
and it was expressed as nmol/g of wet tissue.
Determination of Catalase (CAT) activity
The CAT activity was based on the ability of catalase to induce disappearance of
hydrogen peroxide (H2O2) (CHANCE; MAEHLY, 1955). The experimental procedure used
980 μL of the Reaction Medium (H2O2, 1 M Tris-HCl buffer, 5 mM EDTA, pH 8.0 in Milli-Q
water) with 20 μL of homogenate. The enzymatic action is measured by following the decrease
in absorbance per minute for 6 minutes and the results were expressed in U/mg protein at 230
nm. The protein concentration was determined by the method of Bradford (1976). One enzyme
unit (U) is defined as the amount of enzyme decomposing 1 mM H2O2 per minute at 25°C.
Determination of Superoxide dismutase (SOD) activity
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The SOD amount was assessed by measuring its ability to inhibit the photochemical
reduction of Nitro Blue Tetrazolium (NBT), previously described by Beauchamp and Fridovich
(1971). Briefly, 1 μM riboflavin and 750 μM NBT were added to the homogenate in a reaction
medium (50 mM Phosphate Buffer, 9.5 mM methionine, 0.1 mM EDTA). The reading was
taken after exposure of the material to fluorescent light for 15 min in a spectrophotometer at
560 nm. The enzymatic activity was expressed in U/g of wet tissue. One unit of SOD activity
induced approximately 50% inhibition of the auto-oxidation of NBT.
Determination of cytokine levels
The dissected brain areas (PFC, HC and ST) were homogenized with Assay Diluent,
and then centrifuged (10000 rpm, 5 min, 4 °C). The concentrations of the cytokines tumor
necrosis factor-α (TNF-α), interferon -γ (IFN-γ), interleukin-2 (IL-2), interleukin-6 (IL-6),
interleukin-4 (IL-4), and interleukin-10 (IL-10) were determined by flow cytometry with the
Th1 / Th2 / Th17 CBA cytokine kit (BD Cytometric Bead Array, San Jose, CA, USA) according
to the manufacturer and expressed in pg/ml. The results were extracted using the FCAP Array
software.
STATISTICAL ANALYSIS
The data were analyzed with GraphPad Prism 7.0a (San Diego, CA, USA). The
Shapiro–Wilk test was used to confirm normality. Statistical analysis of the data was performed
by one-way ANOVA, followed by Tukey as a post hoc test (parametric) or Kruskal-Wallis
followed by Dunn’s test (non-parametric). Data are expressed as mean ± standard error of mean
(SEM) and differences were considered significant when p ≤ 0.05.
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RESULTS Mice were treated with corticosterone injections over 22 days and a behavioral
neurochemical test was performed to evaluate memory impairment.
Effect of Rip IV on induced cognitive deficit behaviors in mice
The working memory was evaluated in Y-maze by the number of correct alternations
between the arms of the apparatus. As can be observed in Figure 3, the parameter was decreased
by the chronic administration of Cort (p =0.0013), when compared to the control, and this
decrease was reversed by the treatments with Rip IV and Flu (p = 0.0138) [F (3, 36) = 6.053].
Figure 3. Effect of the administration of Rip IV (50 mg/kg, p.o.) and Flu (50 mg/kg, p.o.) against the Cort-induced chronic stress, on the percentage of correct alternations in the Y-maze Test. Results were expressed as mean ± SEM (n=10 per group). Statistical analysis was performed by one-way ANOVA, followed by Tukey post hoc test. Significant values: aap<0.01 vs control (vehicle); bp<0.05 vs Cort group.
In the step-down avoidance test, Cort treatment profoundly decreased SDL on the
second training session (p = 0.0003) and retention test (p=0.0052), indicating impairment of
memory. Rip IV treatment increased SDL and after 24 hours (p=0.0038), similar to Flu, after
22 days of corticosterone administration (Figure 4).
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Figure 4. Effect of the administration of Rip IV (50 mg/kg, p.o.) and Flu (50 mg/kg, p.o.) against the Cort-induced chronic stress, on the step-down latency during a training session (A) and retention session (B). Data are expressed as mean ± SEM (n=10 per group) during 5 minutes’ test. Statistical analysis was performed by Kruskal-Wallis, followed by Dunn’s post hoc test. aap<0.01; aaaap<0.0001 vs control (vehicle); bp<0.05; bbp<0.01 bbbp<0.001 vs Cort group.
In social interaction test, corticosterone administration decreases the duration of social
interaction between two mice (p = 0.0030), and Riparin IV (p = 0.0354) and fluvoxamine (p =
0.0002) treated animals spent more time in active social interaction (Figure 5) [F (3, 36) =
7.851].
Figure 5. Effect of the administration of Rip IV (50 mg/kg, p.o.) and Flu (50 mg/kg, p.o.) against the Cort-induced chronic stress in social interaction test. Data are expressed as mean ± SEM (n=10 per group) of interaction time between animals during 5 minutes’ test. Statistical analysis was performed by one-way ANOVA, followed by Tukey post hoc test. aap<0.01 vs control (vehicle); bp<0.05; bbbp<0.001 vs Cort group.
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In the prepulse inhibition test (PPI), Cort mice demonstrated a significant decrease in
PPI for the 70 dB prepulse sound level followed by 120 dB startle stimulus compared to vehicle
mice (p = 0.0058; Figure 6) while mice with Riparin IV and fluvoxamine treatment exhibited
normal startle response, indicating the ability of the treatment to restore normal levels of PPI in
mice.
Figure 6. Effect of the administration of Rip IV (50 mg/kg, p.o.) and Flu (50 mg/kg, p.o.) against the Cort-induced chronic stress in prepulse inhibition test. Data are expressed as mean ± SEM (n=8 per group) of percent prepulse inhibition of the startle response following presentation of prepulse of 70, 75 and 80 dB. Statistical analysis was performed by two-way ANOVA, followed by Tukey post hoc test. ap<0.05; aap<0.01 vs control (vehicle); bp<0.05; bbp<0.01; bbbp<0.001 vs Cort group; **p<0.01; ***p<0.001 vs ketamine group.
Effect of Riparin IV on oxidative stress parameters
The oxidative stress evaluation was performed in the prefrontal cortex (PFC), the
hippocampus (HC), and the striatum (ST) that were isolated from mice exposed to Riparin IV
(50 mg/kg) and fluvoxamine (50 mg/kg), and were evaluated regarding lipid peroxidation
levels, nitrite/nitrate content, reduced glutathione (GSH) levels, catalase activity (CAT) and
superoxide dismutase (SOD) activity.
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The effect of Cort, Rip IV, and Flu on pro-oxidant and antioxidant markers is illustrated
in Table 1. Corticosterone administration induced oxidative load in the prefrontal cortex, the
hippocampus, and the striatum with a significant increase in pro-oxidant markers (lipid
peroxidation and nitrite/nitrate) and a decrease of antioxidant marker such as GSH, SOD and
CAT. Riparin IV treatment was able to ameliorate those parameters in the brain areas similar
to fluvoxamine.
Control Cort Cort + RipIV Cort + Flu
PFC
TBARS
(µg/g tissue)
17.97 ±
4.135
88.72
±11.49aaaa
43.61 ±
5.729bbb
11.39 ±
1.731bbbb
Nitrite/Nitrate (nM/g tissue)
2.293 ± 0.221
6.565 ± 0.977aaa
1.578 ± 0.263bbbb 3.969 ± 0.801b
GSH (µg/g tissue)
653.8 ± 40.66 520.5 ± 14.26 737.7 ±
73.48bb 527.1 ± 24.43
CAT (µM/min/mg
protein)
24.34 ± 1.447 14.09 ± 0.91a 31.62 ±
3.38bbbb 32.42 ±
1.661bbbb
SOD (U/g tissue)
2.007 ± 0.005
1.924 ± 0.024aa 1.981 ± 0.003 2.002 ±
0.003bb
HC
TBARS
(µg/g tissue)
16.18 ±
0.6865
27.77 ±
1.873aa 17.78 ± 3.536 17.32 ± 1.471
Nitrite/Nitrate (nM/g tissue) 2.14 ± 0.254 4.211 ±
0.605aa 2.396 ± 0.226b
2.226 ± 0.422bb
GSH (µg/g tissue)
974.7 ± 84.97 539 ± 23.24a 1312 ±
137.6bbb 1012 ± 133.7b
CAT (µM/min/mg
protein)
23.07 ± 2.206 23.08 ± 1.242 25.03 ± 1.642 30.57 ± 2.533
SOD (U/g tissue)
2.001 ±0.002
1.86 ± 0.043aaa
1.981 ± 0.004bbb 2 ±0.003bbb
ST
TBARS
(µg/g tissue)
18.43 ±
2.564
33.12 ±
4.913a 23.99 ± 4.616
10.73 ±
1.172bbb
Nitrite/Nitrate (nM/g tissue)
3.478 ± 0.569 6.561 ± 1.21 2.696 ± 0.403 1.367 ±
0.128bbbb
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GSH (µg/g tissue) 1179 ± 108 651.1 ±
22.34aaa 1014 ± 87.6b 968.1 ± 82.04b
CAT (µM/min/mg
protein)
22.17 ± 1.812 15.59 ± 2.085 29.51 ±
1.657bb 28.14 ± 1.66b
SOD (U/g tissue)
1.991 ±0.009
1.872 ± 0.055a
1.989 ± 0.005b 1.994 ± 0.007b
Table 1. Effects of corticosterone (20mg/kg), Riparin IV (50 mg/kg) and fluvoxamine (50 mg/kg) in lipid peroxidation levels (TBARS), nitrite/nitrate content, reduced glutathione levels (GSH), catalase (CAT) and superoxide dismutase (SOD) activities. Results were expressed as mean ± SEM (n= 6-8 per group) (ANOVA and Tukey as post hoc test or Kruskal-Wallis and Dunn’s as post hoc test). ap<0.05; aap<0.01; aaap<0.001; aaaap<0.0001 when compared with control (vehicle); bp<0.05; bbp<0.01; bbbp<0.001; bbbbp<0.0001 when compared with Cort group; PFC= prefrontal cortex; HC = hippocampus; ST = striatum;
Effect of Rip IV on cytokine levels in brain areas.
The results of the cytokine assays in mice exposed to corticosterone treatment are shown
accordingly in the brain areas: the prefrontal cortex (Figure 7), the hippocampus (Figure 8) and
the striatum (Figure 9).
Results shows the ability of corticosterone administration in alter cytokine levels in
brain tissue. Th1-related cytokines (TNF-a, IFN-g and IL-2) showed the same increasing trend
under corticosterone exposure in PFC (Figure 7A-C) (TNF-a: p = 0.03; F (3, 16) = 10.83; IFN-
g: p = 0.008; F (3, 16) = 5.658; IL-2: p = 0.0001; F (3, 16) = 16.2) whereas no alteration occur
on the Th2-related cytokines (IL-4, IL-6 and IL-10) in the same area ( Figure 7D-F) (IL-4: F
(3, 16) = 4.391; IL-6: F (3, 16) = 1.178; IL-10: F (3, 16) = 15.86). Riparin IV treatment was
able to decrease Th1-related cytokines (TNF-a: p = 0.0002; IFN-g: p = 0.0389; IL-2: p <
0.0001) and increase IL-4 and IL-10 (IL-4: p = 0.0185; IL-10: p < 0.0001).
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Figure 7. Effect of the administration of Rip IV (50 mg/kg, p.o.) and Flu (50 mg/kg, p.o.) against the Cort-induced chronic stress on cytokine levels in mice prefrontal cortex. Data are expressed as mean ± SEM (n=5 per group) (one-way ANOVA, followed by Tukey post hoc test. ap<0.05; aap<0.01; aaap<0.001 vs control (vehicle); bp<0.05; bbp<0.01; bbbp<0.001; bbbbp<0.0001 vs Cort group. Tumor Necrosis Factor-α (TNF-α), Interferon -γ (IFN-γ), Interleukin-2 (IL-2), Interleukin-6 (IL-6), Interleukin-4 (IL-4), Interleukin-10 (IL-10).
In the hippocampus, Cort exposure increased only IL-2 levels (p = 0.0397) (TNF-a: F
(3, 16) = 4.48; IFN-g: F (3, 16) = 1.216; IL-2: F (3, 16) = 12.46; IL-6: F (3, 16) = 4.594; IL-4:
F (3, 16) = 8.665; IL-10: F (3, 16) = 5.262) and riparin IV decreased IL-2 (p = 0.0002), TNF-a
(p = 0.0139) and IL-6 (p = 0.0289) and was able to significantly increase IL-4 (p = 0.0023) and
IL-10 ( p = 0.0069) (Figure 8).
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83
Figure 8. Effect of the administration of Rip IV (50 mg/kg, p.o.) and Flu (50 mg/kg, p.o.) against the Cort-induced chronic stress in cytokine levels in mice hippocampus. Data are expressed as mean ± SEM (n=5 per group) (one-way ANOVA, followed by Tukey post hoc test. ap<0.05 vs control (vehicle); bp<0.05; bbp<0.01; bbbp<0.001 vs Cort group. Tumor Necrosis Factor-α (TNF-α), Interferon -γ (IFN-γ), Interleukin-2 (IL-2), Interleukin-6 (IL-6), Interleukin-4 (IL-4), Interleukin-10 (IL-10).
Figure 9 shows the effect of stress in the striatum, where Cort exposure altered IL-2 (p
= 0.0079 ) and IL-4 (p = 0.0119) levels (TNF-a: F (3, 16) = 2.843; IFN-g: F (3, 16) = 3.12; IL-
2: F (3, 16) = 8.861; IL-6: F (3, 16) = 1.772; IL-4: F (3, 16) = 5.382; IL-10: F (3, 16) = 5.5) and
riparin IV treatment was able to normalize those cytokine levels (p = 0.0020 and p = 0.0204,
respectively).
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Figure 9. Effect of the administration of Rip IV (50 mg/kg, p.o.) and Flu (50 mg/kg, p.o.) against the Cort-induced chronic stress in cytokine levels in mice striatum. Data are expressed as mean ± SEM (n=5 per group) (one-way ANOVA, followed by Tukey post hoc test. ap<0.05; aap<0.01 vs control (vehicle); bp<0.05;
85
bbp<0.01 vs Cort group. Tumor Necrosis Factor-α (TNF-α), Interferon -γ (IFN-γ), Interleukin-2 (IL-2), Interleukin-6 (IL-6), Interleukin-4 (IL-4), Interleukin-10 (IL-10).
DISCUSSION
The presented data demonstrate that 22 days of corticosterone administration
induced marked amnesic effects in mice, evidenced as deficiencies in both short- and long-term
memory. This treatment induced a decrease in spontaneous alternation behavior in the Y-maze,
an index of spatial working memory, and alternated behavior was significantly ameliorated by
the administration of Riparin IV and fluvoxamine. The step-down type passive avoidance task
was also used to examine short- and long-term memory. Similar results, as for alternated
behavior, were obtained indicating that the Cort affected both memory storages, whereas
Riparin IV and fluvoxamine treatment for 7 days reestablished this impairment.
Several studies indicate that animals undergoing chronic stress frequently exhibit
diminished or suppressed HPA responses after reexposure to the same stressor (BAI et al., 2018;
ZENI; CAMARGO; DALMAGRO, 2017; ZHAO et al., 2008). It is well known that stress and
stress hormones have and large impact on memory encoding, consolidation, and retrieval
(SCHWABE; WOLF; OITZL, 2010).
In situations of emotional arousal, glucocorticoids play a crucial role in enabling the
significance of an experience to regulate the strength of memory, in enhancing memory
consolidation (SCHWABE; WOLF; OITZL, 2010), and is strongly related to glucocorticoid
interaction with basolateral amygdala modulation (BENDER et al., 2018; SMEETS et al.,
2008). However, these stress effects may be related to the intensity of the stressor and are time-
dependent. Additionally, they have the opposite effects on different memory processes. While
they can enhance consolidation, but they also impair the retrieval of dependent-hippocampal
memory (BARIK et al., 2013; QUERVAIN; SCHWABE; ROOZENDAAL, 2017).
Symptoms related with high levels of chronic glucocorticoids are usually associated
with decreased cognitive abilities. These disadvantages are thought to result from a cumulative
and long-lasting burden on the function and morphology of the hippocampus (DARCET et al.,
2014; OLESCOWICZ et al., 2018; QUERVAIN et al., 2009). Recently, however, it became
clear that memory deficits observe evidence that stress may also alter non-hippocampal, and in
86
particular, striatal memory processes such as in navigation memory (GUENZEL; WOLF;
SCHWABE, 2013; QI et al., 2018).
Learning and memory are behavioral elements related to plasticity of the central nervous
system (CNS). Evidence indicates that widespread cognitive deficits such as impairments in
executive functioning, attention, and memory, are associated with depressive patients
(BAUNE; RENGER, 2014; TERFEHR et al., 2011; WINGENFELD; WOLF, 2015), and may
be remitted in different types of antidepressant treatment for major depressive disorder (MDD)
(BAUNE; LI; BEBLO, 2013; SOLÉ et al., 2015).
In this way, the memory deficit observed in Cort-treated mice in behavior tests
corroborates with different studies that demonstrate that repeated corticosterone administration
can induce memory impairment in inhibitory avoidance (OLIVEIRA, 2017; RASHIDY-POUR
et al., 2004), novel object recognition (DARCET et al., 2014), the Y-maze (GRECH et al.,
2018; LOPES et al., 2018), the Morris water maze (DARCET et al., 2014; WORKMAN;
CHAN; GALEA, 2015) and in fear conditioning tests (DARCET et al., 2014; MARKS et al.,
2015).
Previously we demonstrated that Riparin IV presented antidepressant effects and
increased BDNF levels in the mouse hippocampus (CHAVES et al., 2019). In this way, its
beneficial effects on memory, demonstrated in the present work by normalization of memory
deficit induced by Cort, may be attributed to this ability of Riparin IV to increase hippocampal
neurogenesis. Interestingly, in another study conducted by Martel, Jaffard, and Guillou (2010)
observed that contextual retrieval deficit is supported by the hippocampus but also reveals that
others neural systems such as the amygdala and/or the striatum additionally mediate stimulus–
response learning.
In addition to this, individual social status stands out as an important impairment to the
quality of life of patients with depression. Based on this fact, the social interaction test was
conducted, which evaluates the tendency of an individual to spend time with a new animal.
Barik and col. (2013) showed that rodents exhibit social aversion triggered by chronic social
stress related to hyperactivation of the HPA axis and high corticosterone levels. This could be
a possible mechanism implicated in their resilience to stress and their modulation of emotional
and social behavior.
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Riparin IV was able to reverse the social isolation induced by corticosterone in a manner
like fluvoxamine. This finding is important because, despite the fact that these aspects of these
cognitive impairment can be resolved after successful treatment in some patients, it is often
persists beyond illness remission (AL-HARBI, 2012; BORA et al., 2013; DARCET et al., 2016;
GROVES; DOUGLAS; PORTER, 2018; MCINTYRE et al., 2013; SOLÉ et al., 2015).
Herrera-Guzmán and coworkers (2009) evaluated the cognitive symptoms of patients
with MDD who received duloxetine and escitalopram, a serotonin norepinephrine inhibitor
(SNRI) and a selective serotonin reuptake inhibitor (SSRI) respectively. Patients presented
improvement in attention, in executive function, and in mental processing after 24 weeks of
treatment with the drugs. However, duloxetine showed a superior response regarding the
improvement of episodic and working memory as compared to the response to escitalopram.
Several brain areas are activated by social cognition tasks in rodent and humans, such
as social recognition (the fusiform area, superior temporal gyrus, and accessory olfactory bulb),
social motivation (the ventral tegmental area, ventral pallidum, and nucleus accumbens),
context evaluation (the amygdala, and the temporal and prefrontal cortices) and the execution
of social behaviors execution (the hypothalamus, and the brainstem motor and autonomic
pathways) (KUPFERBERG; BICKS; HASLER, 2016; SANDI; HALLER, 2015). Studies on
rodents rely on the concept that the ‘social brain’, an interconnected network which interacts to
produce social and emotional behaviors, is governed by homologous brain networks in the
human social brain (FILE; SETH, 2003; KAS et al., 2014). Any functional improvement in
those connections correlates with the improvement of social and family relationships, an
increase in levels of pleasure with daily activities, and an increase in work capacity of depressed
patients.
Stress is a strong modulator of central nervous system structure and function, and most
of the brain areas that are particularly vulnerable to stress and high glucocorticoid levels (such
as the prefrontal cortex, the amygdala, the hippocampus and the mesolimbic system) exhibit
functional and/or structural alterations in patients with depression and/or abnormal social
behaviors (CHATTARJI et al., 2015; LIU et al., 2017).
Mechanisms within the CNS normally serve to inhibit responses to sequential or
repetitive stimuli. Prepulse inhibition (PPI) is unlearned attenuation in startle reflex magnitude
that occurs when a startling stimulus (a pulse) is closely preceded by a week sensory event (a
prepulse or prestimulus) (SWERDLOW, 2009). This inhibition reflects the basic inhibitory
88
process which regulates the input of sensory stimuli to the brain (such as visual and tactile
stimuli), thereby preventing sensory overload and cognitive fragmentation. This process is
interpreted as an operational measure of sensorimotor gating that involves cortico-striato-
pallido-pontine circuitry (DULEY et al., 2007; ROHLEDER et al., 2016; STURM, 2014).
At a clinical level, a number of neuropsychiatric disorders are characterized by the
failure of inhibition such as in the case of schizophrenia. In people at a high clinical risk for
psychosis and in some anxiety disorders, dysfunction of this gating processes has been linked
conceptually to the inability to suppress or gate intrusive or irrelevant sensory, motor, or
cognitive information (DULEY et al., 2007; MENA et al., 2016; SÁNCHEZ-MORLA et al.,
2016; SWERDLOW et al., 2009).
Studies show evidence of a relationship between cognitive processing and the PPI, in
which a disruption to the PPI may lead to a prognosis involving complex disturbances to
cognitive functioning, specifically in attentional and executive function (MICOULAUD-
FRANCHI et al., 2016; SCHOLES; MARTIN-IVERSON, 2009).
Although our findings do not show a significant reduction in startle reflex in all pulses
in corticosterone treated animals compared to controls, it is important to notice the trend. Dirks
et al. (2002) related the alterations in the HPA axis due to corticotropin-releasing factor (CRF)
hyperactivity to robust impairments of prepulse inhibition in transgenic mice, corroborating our
data that chronic stress is related to several brain alterations in function, behavior, and
neurogenesis (CHAVES et al., 2019; LOPES et al., 2018; SOUSA et al., 2015;
VASCONCELOS et al., 2015).
De La Casa, Mena and Ruiz-Salas (2016) conducted a clinical trial to analyze the effect
of induced stress and attentional load on PPI in volunteers. Their results indicate that stress can
reduce PPI, and that startle reflex intensity is reduced when attention is directed away from the
auditory stimulus that induces the reflex.
In this way, the ability of Riparin IV and fluvoxamine to reverse the reduction in
prepulse inhibition by Cort-treatment is very relevant since, several studies showed that a
variety of antidepressant drugs, including tricyclics, serotonin-selective reuptake inhibitors, and
norepinephrine-selective uptake inhibitors, appear to have minimal or no effect on PPI in either
animals or humans (BRAFF; GEYER; SWERDLOW, 2001; PEREIRA-FIGUEIREDO et al.,
89
2015, 2017; PHILLIPS et al., 2000). This data can contribute to improving our understanding
of the pathophysiology of the neurocognitive deficit in some neuropsychiatric disorder related
to stress.
Stress has been implicated in some volumetric changes in the brains of depressed
patients. It has been reported that the HPA axis dysregulation and subsequent changes in
glucocorticoid secretion may result in neuronal cell death in limbic and frontal regions, which
may result in volumetric changes and subsequent pathological functioning seen in patients with
depression (CHATTARJI et al., 2015). Fundamentally, the hippocampus, the prefrontal cortex
and the amygdala express mineralocorticoid and glucocorticoid receptors, which become
targets of cortisol action and are therefore particularly susceptible to stress-induced neuronal
atrophy or hypertrophy (LIU et al., 2017).
It has been observed that chronic exposure to stress can increase the generation of free
radicals, alter neurotransmission and neuronal function, and is one of the key pathogenic
mechanisms of depression (BAI et al., 2018; BOUAYED; RAMMAL; SOULIMANI, 2009;
FILHO et al., 2015; ZENI; CAMARGO; DALMAGRO, 2017). Studies have demonstrated that
various classes of antidepressants can reduce levels of oxidative stress in animals (GUPTA;
RADHAKRISHNAN; KURHE, 2015; KV et al., 2018; LOPES et al., 2018; SOUSA et al.,
2015; VASCONCELOS et al., 2015) and humans (MOYLAN et al., 2014; WIGNER et al.,
2018).
Based on that, we have examined whether the treatment with Cort, Rip IV and Flu can
alter the lipid peroxidation level, nitrite/nitrate and reduced glutathione content, superoxide
dismutase, and catalase activities in PFC, HC, and ST during corticosterone administration in
mice.
The brain is more vulnerable to oxidative damage. The simultaneous presence of high
levels of poly-unsaturated fatty acids and iron makes it susceptible to peroxidation, which
results in a decrease in membrane fluidity and damage (KIM et al., 2016; MOYLAN et al.,
2014). We have recorded the rise in lipid peroxidation levels in the mouse brain, which is
reflected by a rise in TBARS and oxide nitric metabolite levels. This may also be related to the
high production of reactive oxygen and nitrogen species that results in membrane damage. In
addition, the administration of Cort altered enzymatic and non-enzymatic antioxidant defense
decreases GSH concentration and therefore, activities of SOD and CAT.
90
Besides the CNS, a response to stress is also manifested in the peripheral organ systems.
Stanić et al. (2016) observed an expressive imbalance in the production and efficiency of
antioxidant defenses in rats that were chronically treated with corticosterone and expressed a
suppression of DNA repair of peripheral blood cells. Zafir and Banu (2009a, 2009b) also found
that oxidative stress markers significantly increased in the brain, the liver, and the heart.
Alterations induced by chronic stress were also observed in different animals (BIRNIE-
GAUVIN et al., 2017; LOPES et al., 2018; STIER et al., 2009; VASCONCELOS et al., 2015)
and humans (FLANAGAN et al., 2018; MYINT et al., 2017; PRASAD et al., 2016;
VERÍSSIMO; BAST; WESELER, 2018). The oxidative damage caused by exposure to Cort
may lead to neuronal loss, suppressing neurogenesis and tissue atrophy. It may also be
positively correlated with the depressive and cognitive behavior observed in the mice through
behavioral tests.
Interestingly, Rip IV, like Flu ameliorated Cort-induced oxidative loading in the brain.
This is in line with the earlier reports that several antidepressants reverse oxidative damage in
the brain. This is one of the important pathogenic causes of major depression, which indicates
the possible mechanism of antidepressant-like effect of Riparin IV. Thus, the antioxidant effect
observed may be related to the presence of unstable hydrogen in the Riparin IV molecular
structure, like the gallic acid structure.
Gallic acid is an polyphenolic compound (3,4,5-trihydroxybenzoic acid) that exhibits
high antioxidant activity (YILMAZ; TOLEDO, 2004). It has been recognized to have diverse
therapeutically beneficial characteristics, such as antimicrobial activity, immunomodulatory
activities, and even anticancer qualities (BADHANI; SHARMA; KAKKAR, 2015). Although
it has potential, Pereira and collaborators (2018) evaluated the effect of gallic acid treatment on
diabetic rats. These treatments presented anxiolytic-, but not antidepressant-like effects after
long-term administration. In this way, we may plausibly suggest that the mode of action for the
Riparin IV antidepressant effect may not only be related to its similarities in structure to gallic
acid.
Neuroinflammation is a known factor in the pathogenesis of neurodegenerative diseases
and psychiatric illnesses, such as depression. It has also been implicated in causing diminished
cognition and memory (SINGHAL et al., 2014). The hyperactivation of the HPA axis, under
prolonged stress conditions, stimulates the immunological cells relevant to the systemic
response to stress (BORTOLATO et al., 2015; GOLD, 2015), as well as promotes the release
91
of pro-inflammatory cytokines IL-1b, IL-6 and TNF-a, by macrophages and microglial cells,
which contributes to the direct stimulation of the HPA axis and glucocorticoid resistance
(KOMORI, 2017; MALEK et al., 2015; NIKKHESLAT; PARIANTE; ZUNSZAIN, 2018;
YOUNG; BRUNO; POMARA, 2014; ZUNSZAIN et al., 2011).
Based on the neuroinflammatory hypothesis of depression, this study evaluated the
effect of Riparin IV on cytokine content in various brain areas. Cort-treated mice exhibited a
neuroinflammatory profile with increased pro-inflammatory cytokines TNF-a and IL-2, and
decreased anti-inflammatory IL-4, but had no significant effect on IL-6 or IL-10.
In situations of chronic activation of the HPA axis and failed glucocorticoid
compensatory mechanisms (DUMBELL; MATVEEVA; OSTER, 2016), a neuroinflammation
process was observed. The imbalance between pro-inflammatory and anti-inflammatory
activities in the brain determines the consequent detrimental results, in which pro-inflammatory
cytokines, such as IL-1β, IL-6, and TNF-α, have a crucial role in the pathophysiology of MDD
(BORTOLATO et al., 2015; DOWLATI et al., 2010; KIM et al., 2016; MAES, 2011;
MANIKOWSKA et al., 2014; SINGHAL et al., 2014).
TNF-α, in addition to the IL-1 cytokine family, has been primarily implicated in
neuroinflammation. It has been well established that both TNF-α and IL-1β stimulate each
other’s secretion and exhibit overlapping and synergistic effects, such as promoting apoptosis,
and stimulating inflammasome activation, and stimulating others cytokine’s release (MAES,
2011; SINGHAL et al., 2014). Elevated levels of TNF-α in particular have been shown to cause
a reduction in hippocampal volumes through the neurodegenerative tumor necrosis factor
receptor (TNFR)-1 pathway. This results in severe neuroinflammation, neurodegeneration, and
in consequence, functional and cognitive sequelae (BAUNE; LI; BEBLO, 2013; SINGHAL et
al., 2014; WIGNER et al., 2018). IL-2, IL-4 and TNF-α inhibit GR function through different
pathways (KIM et al., 2016). In this way, the ability of Riparin IV to attenuate
neuroinflammation, in some situation better then fluvoxamine, might be another significant
aspect of antidepressant drug action.
In addition, cytokines have pleiotropic effects on the central nervous system. Not only
do they influence inflammation centrally, but they also play key roles in neurotransmitter
function, neuroendocrine regulation, neuroplasticity, and neurotrophic support (BORTOLATO
et al., 2015; GOLD, 2015; LOTRICH; ALBUSAYSI; FERRELL, 2013; NIKKHESLAT;
92
PARIANTE; ZUNSZAIN, 2018). Another mechanism relating proinflammatory cytokines to
mood is their capacity to stimulate indoleamine 2,3- dioxygenase (IDO) in glial cells to deviate
tryptophan from serotonin synthesis to the kynurenine pathway, which is then transformed into
neurotoxic quinolinic acid inside the brain (DEAN; KESHAVAN, 2017; MAES,
2011). Quinolinic acid binds to N-methyl-D-aspartate (NMDA) receptors, perturb
neurotransmission along glutamatergic pathways, and may lead to hippocampal neuron damage
and apoptosis (DOWLATI et al., 2010; YOUNG; BRUNO; POMARA, 2014). This excitotoxic
mechanism may also contribute to the symptoms of major depression and to hippocampal
volume loss (DOWLATI et al., 2010). Cytokines can also alter other neurotransmitters, such as
serotonin, dopamine, and noradrenaline, relating to the largest role in MDD (KUBERA et al.,
2005; LOTRICH, 2015; SCHWARCZ et al., 2012). In summary, complex neural mechanisms
are thought to be involved in depressive disorders.
Moreover, studies have also shown elevated levels of proinflammatory cytokines in the
plasma of patients with MDD who had attempted suicide and in patients with suicidal ideation.
This suggests that the inflammatory mechanisms may be positively associated with different
subsets of depressive disorders (NIKKHESLAT; PARIANTE; ZUNSZAIN, 2018; WIGNER
et al., 2018; YOUNG; BRUNO; POMARA, 2014) and with the development of comorbid
systemic illnesses (BANUELOS; LU, 2016; JÖRGENS; AROLT, 2018; MARTINO et al.,
2012; NIKKHESLAT et al., 2015).
Several studies found that antidepressants can decrease the pro-inflammatory/anti-
inflammatory cytokine ratio and the Th1-Th2 imbalance cytokine may impair the modulation
of cellular responses in the brain during psychological stress and depression, specially
treatment-resistant patients (MYINT et al., 2005; O’BRIEN et al., 2007; SUTCIGIL et al.,
2007; THOMAS; KHANAM; VOHORA, 2016). Nevertheless, actual findings are inconsistent
with the theory that treatment of depressive disorders with antidepressants can suppress
immune (KOMORI, 2017; MAES, 2011; MARTINO et al., 2012). (THOMAS; KHANAM;
VOHORA, 2016)(THOMAS; KHANAM; VOHORA, 2016)(THOMAS; KHANAM;
VOHORA, 2016)(THOMAS; KHANAM; VOHORA, 2016)(Thomas et al., 2016)Thomas,
Khanam and Vohora (2016) noticed that venlafaxine (SNRI) was able to reverse the elevated
serum levels of IL-1β and IL-6 caused by chronic stress in mice while agomelatine, atypical
antidepressant, failed to show such a reversal. A clinical study conducted by Sutcigil and co-
workers (2007) investigated the effects of sertraline (SSRI) therapy in unipolar depressive
93
patients and its administration might have exertd immunomodulatory effects through a decrease
in the proinflammatory cytokine IL-12, an increase in the anti-inflammatory IL-4 and TGF-β1
but no alterations in IL-2 and TNF-α. Otherwise, a robust study evaluated the variations in
circulating cytokine levels during 52 week course of different SSRI drugs and concluded that
was an increase in Th1 cytokines, such as IL-1β, IL-2 and IFN-γ, and decrease Th2 cytokines,
such as IL-4, IL-10 and IL-13.
In conclusion, divergent effects of antidepressant compounds in cytokine content
suggests a possible explanation for the differences in efficacy of treating different depressive
subtype and resistance. The capability of Riparin IV in alter the Th1/Th2 balance towards a Th2
shift, combined to its antioxidant effect, shows an anti-inflammatory profile. This result is very
important as a choice of specific antidepressant in specific clusters of symptoms will lead to a
successful and efficient treatment.
Thus, depression and cognitive impairment correlate positively with levels of circulating
cytokines, a finding that confirms once again the involvement of cytokines in the mediation of
emotional and cognitive responses to chronic stress conditions. Immune challenges are capable
of provoking oxidative stress (MOYLAN et al., 2014; WIGNER et al., 2018), increasing the
levels of other inflammatory cytokines (ROOMRUANGWONG et al., 2017), and decreasing
neurotrophic factor in the animal hippocampus (BORTOLATO et al., 2015) and in humans
(LOTRICH; ALBUSAYSI; FERRELL, 2013; WIGNER et al., 2018). Additionally, immune
challenges can stimulate the HHA axis (KIM et al., 2016; KOMORI, 2017), can alter
neurotransmitter levels (LOTRICH, 2015), and can inhibit long-term potentiation (ZUNSZAIN
et al., 2011), while impairing learning and memory, which are factors frequently affected in
depressive disorders.
CONCLUSION
Taken together, the effect of Riparin IV in ameliorate corticosterone cognitive and
memory impairment can be attributed, at least in part, to the decrease in oxidative stress and
neuroinflammation-induced neuronal damage, demonstrated as decreases in pro-oxidant
markers, increases in antioxidant defense systems, and the modulation cytokines levels in the
brain. This antioxidant and anti-inflammatory effects put Riparin IV on an interesting level as
94
a possible drug in the antidepressant treatment of non-responsive patients related to severe and
cognitive symptoms.
In conclusion, the results obtained regarding the neuroprotective role of Riparin IV in
cognition and memory are interesting. Therefore further studies are still necessary to explain
this mechanism.
ACKNOWLEDGMENT
This research was funded by Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior – Brasil (CAPES) (Finance Code: 001), Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq - Brazil; Process numbers: 12/2017, 306746/2013-1,
446120/2014-6 and 407567/2013-5) and Fundação Cearense de Apoio à Pesquisa (FUNCAP –
Ceará - Brazil). The authors would like to thank the Multi-User Facility of Drug Research and
Development Center of Federal University of Ceará for technical support.
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4 CONSIDERAÇÕES FINAIS
A depressão é uma séria condição psiquiátrica crônica e incapacitante que afeta cerca
de 322 milhões de pessoas no mundo e que representa um problema de saúde pública. Esta
doença é um fenômeno complexo e de etiologia multifatorial que é alvo de diversos estudos,
essenciais para o desenvolvimento de fármacos com ação mais rápida e eficaz.
O modelo de estresse induzido pela administração de corticosterona permitiu avaliar o
efeito da riparina IV em inúmeras alterações comportamentais e neuroquímicas induzidas pelo
estresse crônico, incluindo o comprometimento cognitivo. Esta disfunção interfere na
capacidade produtiva e impacto psicossocial, prejudicando relações familiares e sociais,
podendo não ser revertida com alguns tipos de tratamentos antidepressivos.
Dessa forma, a riparina IV mostrou-se capaz de reverter o comprometimento cognitivo
e de memória em animais submetidos a estresse crônico, reduzindo o estresse oxidativo e
padrão inflamatório, além de restaurar vias neurotróficas mediadas por BDNF em regiões
cortico-límbicas.
Estes resultados colocam a riparina IV como um potencial alternativa terapêutico no
tratamento de distúrbios depressivos, entretanto, estudos pré-clínicos e clínicos utilizando a
riparina IV para o tratamento da depressão maior e outros distúrbios psiquiátricos precisam ser
encorajados.
109
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ANEXO A – SUBMISSÃO DE ARTIGO CIENTÍFICO A REVISTA
Pharmacology, Biochemistry and Behavior
