( PDF ) Estudo das alterações comportamentais e neuroquímicas induzidas pela sepse em modelo animal: possível papel terapêutico de antioxidantes

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Universidade Federal do Rio Grande do Sul

Instituto de Ciência Básicas da Saúde

Departamento de Bioquímica

Curso de Pós-Graduação em Ciências Biológicas: Bioquímica

Estudo das alterações comportamentais e neuroquímicas

induzidas pela sepse em modelo animal: possível papel

terapêutico de antioxidantes

TATIANA BARICHELLO

Orientador:

Prof. Dr. Felipe Dal-Pizzol

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Universidade Federal do Rio Grande do Sul

Instituto de Ciência Básicas da Saúde

Departamento de Bioquímica

Curso de Pós-Graduação em Ciências Biológicas: Bioquímica

Estudo das alterações comportamentais e neuroquímicas

induzidas pela sepse em modelo animal: possível papel

terapêutico de antioxidantes

TATIANA BARICHELLO

Orientador:

Prof. Dr. Felipe Dal-Pizzol

Tese apresentada ao Programa de Pós-Graduação em Ciências Biológicas –

Bioquímica como requisito parcial para a obtenção do título de Doutor em

Ciências Biológicas: Bioquímica

PORTO ALEGRE 2007

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III

“O sistema límbico é a zona limite onde a psiquiatria encontra a neurologia”

“The limbic system is the border zone where psychiatry meets neurology”

Michael S. Mega et al. (1997)

Agradecimentos

Ao Prof. Dr. Felipe Dal-Pizzol pelo modelo de pesquisador, cuja busca incansável da

verdade científica é a força propulsora que motiva um grande número de

pesquisadores nos estudos da sepse em modelos animais e em estudos clínicos.

Além disso, pela confiança em mim depositada quando aceitou a minha orientação

e, mais recentemente, quando me convidou para integrar sua equipe de trabalho na

Universidade.

Ao Prof. João Quevedo pela colaboração no desenvolvimento dos desenhos

experimentais, na discussão dos resultados e no auxílio na preparação dos

manuscritos.

Aos colaboradores Márcio Rodrigo Martins Adalisa Reinke, Gustavo Feier, Cristiane

Ritter, Samira Valvassori, Gislaine Ziili Réus, Larissa Constantino, Roberta Machado,

Ângeles Vitali, José Cláudio Moreira e Fabrícia Petronilho pelo companherismo e

dedicação na realização dos diversos experimentos.

Aos orgãos de fomento (CNPq, FAPESC, UNESC e Instituto Cérebro e Mente) pelo

fundamental auxílio financeiro necessário a execução dos experimentos que

compõem essa Tese.

SUMÁRIO

Parte I.........................................................................................................................01

Resumo......................................................................................................................02

Abstract......................................................................................................................03

Lista de Abreviaturas..................................................................................................04

Introdução……………………….................……………...............................................05

Sepse…....…………………………………………………………………………………..05

Sepse e Sistema Nervoso Central.............................................................................07

Sepse e Cognição......................................................................................................09

Sepse e Estresse Oxidativo.......................................................................................12

Antioxidantes e Tratamento da Sepse.......................................................................15

Modelo Animal de Sepse............................................................................................18

Objetivos...............................……………………………………………………………...19

Parte II……………………………………………………………………………………….21

Capítulo 1. Cognitive impairment in sepsis survivors from cecal ligation and

perforation…………………………………………………………………………………...22

Capítulo 2. Behavioral deficits in sepsis-surviving rats induced by cecal ligation and

perforation …………………………………………………………………………….…….26

Capítulo 3. Long-Term Cognitive Impairment in Sepsis Survivors Behavioral deficits

in sepsis-surviving rats induced by cecal ligation and

perforation…………………………………………………………………………………...34

Capítulo 4. Oxidative variables in the rat brain after sepsis induced by cecal ligation

and perforation……………………………………………………………………………...36

Capítulo 5. Antioxidant treatment reverses late cognitive impairment in an animal

model of sepsis……………………………………………………………………………..41

Parte III………………………………………………………………………………………63

Discussão...................................................................................................................64

Dano cognitivo em ratos sobreviventes a sepse........................................................64

Medidas de estresse oxidativo no cérebro de ratos sobreviventes a sepse..............69

Tratamento com antioxidantes e dano cognitivo........................................................73

Limitações do estudo e perspectivas futuras.............................................................77

Referências Bibliográficas..........................................................................................79

Parte I

RESUMO

(Tatiana Barichello - Estudo das alterações comportamentais e neuroquímicas

induzidas pela sepse em modelo animal: possível papel terapêutico de

antioxidantes) – Este trabalho apresenta a compilação de 5 experimentos

carreados ao longo de 2004-2007. Na parte 1 apresenta sucintamente o marco

teórico dos quatro trabalhos. Inicialmente são discutidos aspectos gerais da sepse.

Após, são descritos alguns mecanismos conhecidos do envolvimento do sistema

nervoso central com a sepse, como a permeabilidade diminuída da barreira

sanguínea cerebral, alteração de alguns neurotransmissores, apoptose neuronal,

ativação de algumas cascatas imunológicas, enfim, o processo inflamatório no

sistema nervoso central. Os danos cognitivos ocasionados pela encefalopatia

séptica também foram relatados, e por fim o estresse oxidativo e as defesas

antioxidantes como um mecanismo que possa estar mediando os danos cognitivos

observados em pacientes sobreviventes as sepse. Os experimentos (Parte II)

comportamentais realizados 10 dias após a indução da sepse em ratos ocupam os

Capítulos 1 e 2. No capítulo 1 é apresentado um corpo de resultados que permite

observar os déficits cognitivos nos testes de Habituação ao Campo Aberto, Esquiva

Inibitória e Esquiva Inibitória de Múltiplos Treinos em ratos sobreviventes a sepse

após 10 dias da indução pelo modelo animal de CLP. Estes dados demonstram

uma incapacidade cognitiva no aprendizado e na memória dos animais sépticos. O

Capítulo 2 apresenta os resultados dos testes comportamentais de Reconhecimento

de Objeto, Labirinto de Cruz Elevada e Teste de Natação Forçada, onde foi possível

demonstrar que os ratos sépticos apresentaram uma incapacidade na memória e

comportamento compatível com “depressão”, mas não com ansiedade. O Capítulo 3

estende os achados comportamentais observados no Capítulo 1, realizando os

testes comportamentais de Habituação ao Campo Aberto, Esquiva Inibitória e

Esquiva Inibitória de Múltiplos Treinos após 30 dias da indução de sepse em ratos

pelo procedimento CLP. Os dados obtidos confirmam que a incapacidade de

aprendizado e memória demonstrados no Capítulo 1 persiste após 30 dias da

cirurgia de CLP. A mensuração do estresse oxidativo no hipocampo, cerebelo,

estriado e córtex de ratos submetidos à sepse foram descrito no Capítulo 4. Neste

experimento foi observado que nas horas iniciais após a indução de sepse em ratos,

houve danos oxidativos, avaliados pelo TBARS e carbonil em diversas regiões do

cérebro. Entretanto, a exceção do estriado, foi demonstrado um aumento na

atividade de SOD sem um proporcional aumento na atividade de CAT, com aumento

conseqüente na relação de SOD e CAT, também nos períodos iniciais após a

indução de sepse nos ratos (6 horas). O Capítulo 6 aborda os resultados do

experimento realizado com administração de antioxidantes NAC e DFX antes do

Teste de Esquiva Inibitória, Esquiva Inibitória de Múltiplos Treinos após 10 e 30 dias

da indução da sepse e Habituação ao Campo Aberto após 10 dias da cirurgia de

CLP, onde foi demonstrando que, DFX e NAC associado, porém não isoladamente,

revertem os danos cognitivos observados em ratos submetidos aos testes acima. A

discussão geral (parte III) busca integrar esses achados descritos nos capítulos

anteriores dentro de uma nova perspectiva para esclarecer os mecanismos

neurobiológicos do dano cognitivo em sobreviventes a sepse. Além disso, abre

discussões acerca de possíveis novas possibilidades de pesquisas.

ABSTRACT

(Tatiana Barichello – Investigation of behavioural and neurochemical sequelae in rats

submitted to sepsis: possible therapeutic role for antioxidant agents) - This work

presents the compilation of 5 experiments enters throughout 2004-2007. The part I

presents lightly the theoretical landmark of the four works. Initially general aspects of

sepsis are argued. After, some known mechanisms of the involvement of the central

nervous system with sepsis are described, as the diminished permeability of the brain

blood barrier, alteration of some neurotransmitters, neuronal apoptosis, activation of

some inflammatory cascades, at last, the inflammatory process in the central nervous

system. The cognitive damage caused by the septic encephalopathy had been also

discussed, and finally it stress oxidative and the antioxidants defenses as a

mechanism that can be causing the cognitive deficit in sepsis survivors patients. The

main experiments (part II) carried through 10 days after the induction of sepsis in rats

occupy chapters 1 and 2. In chapter 1 a body of results is presented that allows

observing the cognitive incapacities to the tests of Habituation to the Open Field,

Inhibitory Avoidance and Continuous Multiple Trials Step-down Inhibitory Avoidance

in rats surviving sepsis after 10 days of the induction for the animal model of CLP.

These data demonstrate a cognitive incapacity in the memory and the learning of the

septic animals. chapter 2 presents the results of the complementary behavioural

tests: Object Recognition, Elevated Plus-maze and Forced Swimming Test, where it

was possible to demonstrate that the septic rats had presented incapacity in the

memory and symptoms of depression, but not of anxiety. chapter 3 extends the

observed findings in chapter 1, carrying through the tests of Habituation to the Open

Field, Continuous Multiple Trials Step-down Inhibitory Avoidance after 30 days of the

induction of sepsis in rats for procedure CLP. The gotten data confirm that the

incapacity of demonstrated learning and memory in chapter 1 after persists 30 days

of the surgery of CLP. The measure of it stress oxidative in hippocampus,

cerebellum, striatum and cortex of rats submitted to sepsis was described in chapter

4. In this experiment it was observed that in the initial hours after the induction of

sepsis in rats, had oxidative damages, evaluated for the TBARS and carbonyl in

diverse regions of the brain. However, the exception of the striatum, was

demonstrated an increase in the activity of SOD without a proportional increase in the

activity of CAT, with consequent increase in the relation of SOD and CAT, also in the

initials periods after induction of sepsis in the rats (6 hours). chapter 5 approaches

the results of the experiment carried through with antioxidants substance

administration NAC and DFX before the Test of Inhibitory Avoidance and Continuous

Multiple Trials Step-down Inhibitory Avoidance after 10 and 30 days of the induction

of sepsis and Habituation to the Open Field after 10 days of the surgery of CLP,

where it was demonstrating that, DFX and NAC associated, but not separately,

above revert the observed cognitive damages in rats submitted to the tests. The

general quarrel (part III) searches to integrate these described findings in the

previous chapters inside of a new perspective to clarify the neurobiological

mechanisms underlying brain damage in sepsis survivors. Moreover, it opens

possible quarrels concerning new possibilities of research.

LISTA DE ABREVIATURAS

CAT - Catalase

CLP – Cecal Ligation and Perforation (Ligação e Perfuração Cecal)

CREB – (cAMP response element-binding)

DFX – Deferoxamina

DNA – Ácido Desoxirribonucléico

EAO – Espécies Reativas de Oxigênio

FAD - Flavina adenina dinucleotídeo

GABA – Ácido gama amino Butino

GPx – Glutationa Peroxidase

GSH – Glutationa

•

••

•

- Hidroperoxila

IL-1 – Interleucina 1

IL-6 – Interleucina 6

−

−−

−

•

••

•

– Superóxido

OH - Hidroxila

NAC – N-acetilcisteína

NO – Óxido Nítrico

SOD – Peróxido Dismutase

TBARS – Ácido Tiobarbitúrico

TNF – Fator de Necrose Tumoral

TNFα

αα

α – Fator α de necrose tumoral

VAChT – Vesículas Transportadoras de Acetilcolina

INTRODUÇÃO

Sepse

A Sepse é caracterizada por uma resposta inflamatória decorrente da

reação do sistema imunológico à infecção, sendo assim uma doença com

implicações clínicas relevantes (Vandijck et al, 2006). A incidência da sepse está

aumentando continuamente e esta tendência epidemiológica reflete o

envelhecimento da população e o aumentando do número de pacientes

cronicamente doentes. Uma resistência crescente dos agentes infecciosos que

causam a sepse também possui um papel importante no processo. O progresso no

diagnóstico e no tratamento da sepse foi crescente nas últimas décadas, porém

influenciando o prognóstico da doença somente ligeiramente. Um papel importante

na patogenia da sepse é realizado pela resposta inflamatória, que pode causar

danos aos tecidos que conduzem à falência orgânica. Esta reação é controlada pela

resposta antiinflamatória, que pode ser conduzida exageradamente e assim,

aumentando às infecções secundárias e levando a síndrome da respostas

antiinflamatória compensatória (Holub, 2007).

A síndrome da resposta inflamatória sistêmica é um estado fisiopatológico

multifatorial agressivo com uma taxa elevada de mortalidade de até 51% e a 10ª

causa principal de morte nas unidades de terapia intensiva (Martin et al, 2005). O

diagnóstico inclui uma exigência para dois ou mais dos seguintes sintomas: febre ou

hipotermia, taquicardia, e leucocitose ou leucopenia (Bone,1992). A diferença

principal entre a síndrome da resposta inflamatória sistêmica e a sepse é que a

sepse é produzida por microorganismos infecciosos, visto que a síndrome da

resposta inflamatória sistêmica pode ocorrer na ausência de uma fonte documentada

de infecção (Takala et al, 2002). Embora a causa seja difícil de verificar em doenças

médicas complexas, há ampla evidência clínica e experimental para suportar o

conceito da severidade da resposta inflamatória clínica e o resultado na

sobrevivência (Sasse et al, 1995). No fato, a resposta inflamatória produzida durante

a sepse pode estar dentro do espectro da resposta inflamatória sistêmica e envolve

a amplificação rápida dos sinais e respostas além do tecido invadido (Takala et al,

2002).

A Sepse e suas complicações conduzem à mortalidade em cerca de 10-

50% das ocorridas em unidades de terapia intensiva. Entretanto, os pacientes que

sobrevivem a sepse podem ter a função comprometida de alguns órgãos, que

podem resultar em sintomas tais como dispnéia, fadiga, depressão e alterações

funcionais (Brun-Buisson, 2000; Friedman et al, 1998; Hotchkiss e Karl, 2003; Tran

et al, 1990).

Um dos componentes principais que envolvem a fisiopatologia da sepse é

a exacerbada ativação da resposta imune inata. O papel central do sistema imune

inato durante a síndrome da resposta inflamatória sistêmica e a sepse é

documentado pelo aumento dos fatores pró-inflamatórios após a infecção (Williams

et al, 2000; Mastronardi et al, 2005; Mastronardi et al, 2001; Mastronardi et al, 2000;

Wong et al, 1997; Wong et al, 1996; Mastronardi et al, 2001; v et al, 2003), com

conseqüente aumento de citocinas pró-inflamatórias tais como o IL-1 e o TNF- para

o sistema nervoso central (Takala et al, 2002). Como a maioria dos estudos

envolvendo sepse focalizou órgãos periféricos como citados previamente, a

participação do cérebro durante este processo ainda não está muito clara (Young et

al, 1990).

Sepse e Sistema Nervoso Central

Os efeitos biológicos causados pela liberação de citocinas refletem no

sistema nervoso central como febre (Dinarello, 2004; Maier et al, 1998), anorexia

(Maier et al, 1998), e ativação do eixo hipotâlamo-pituitária-adrenal, tendo por

resultado o aumento da produção dos corticóides adrenais (Licinio e Wong, 1997).

As interações recíprocas entre o sistema nervoso central e sistema imunológico são

considerados como os componentes principais da resposta inflamatória à sepse, o

que acaba causando alterações nos sistemas neuroendócrino, autonômico

(Chrousos, 1995), comportamental (Gordon et al, 2004) e distúrbios em quaisquer

funções adaptáveis como as respostas imuno-inflamatórias e hemodinâmica

(Sharshar et al, 2003; Sharshar et al, 2004; Spyer, 1989; Saper e Breder, 1994).

No caso da sepse e da síndrome da resposta inflamatória sistêmica há

alteração na permeabilidade da barreira cerebral sanguínea e o processo

inflamatório acaba afetando os sistemas de controle do sistema nervoso central,

causando alteração as funções fisiológicas cruciais à homeostase levando a

encefalopatia (Chrousos e Gold,1992). A encefalopatia séptica pode ocorrer em 8-

70% dos pacientes sépticos, dependendo dos critérios de inclusão empregados

(Sprung et al, 1990; Young et al, 1990), sendo a encefalopatia mais comum nas

unidades de terapia intensiva (Bleck et al, 1993). O conceito de encefalopatia séptica

como uma entidade que não possa ser explicada pela disfunção, hipotensão, ou

pela hipóxia hepática ou renal, é relativamente novo, porém já está claro que a

sepse e suas reações podem ser associadas a um largo espectro de danos e

disfunções cerebrais (Papadopoulos et al, 2000). A disfunção da barreira

hematoencefálica parece ter efeito central na fisiopatologia da encefalopatia séptica,

uma vez que permite a passagem para o SNC de endotoxinas que parecem

influenciar diversos aspectos do metabolismo cerebral (Orlikowsk et al, 2003), além

de alterar a função de células endoteliais, astrócitos e neurônios (Papadopoulos et

al, 2000).

Além disto, o dano na estrutura da barreira hematoencefálica provoca

uma alteração severa dos fluxos diferenciais de numerosas substâncias entre o

plasma e o liquido cefalorraquidiano. Este fenômeno contribui para aumentar a

relação entre os aminoácidos aromáticos e os aminoácidos ramificados diminuindo

as concentrações intracerebrais de noradrenalina, dopamina e serotonina, enquanto

que as concentrações de GABA são inalteradas (Descamps et al, 2003; Freund et al,

1985). Nos doentes sépticos, acredita-se que a alteração do estado mental seja por

um excesso de ácidos aminos aromáticos, procedentes da parte proteolítica

muscular e pela diminuição dos ácidos aminos ramificados (Basler et al, 2002;

Komatsubara et al, 1995). A isto se associa a um aumento dos receptores

benzodiazepínicos em ratos sépticos (Soejima et al, 1990).

Além das alterações de permeabilidade da barreira hemato-encefálica as

modificações do débito sanguíneo cerebral e a sua auto-regulação (Bowie et al,

2003) foram implicadas na patogênese e a hipotensão foi associada

significativamente ao desenvolvimento de dano isquêmico cerebral (Bowton et al,

1989, Booke et al, 2003).

A análise histológica demonstra, em casos fatais, várias alterações como

proliferação de astrócitos e microglia no córtex, infartos cerebrais, púrpura cerebral,

múltiplas hemorragias pequenas em substância branca e disseminação de

microabsessos (Jackson et al, 1985). Nos sobreviventes, ao contrário, observam-se

alterações reversíveis tais como a redução do fluxo sanguíneo cerebral, constrição

capilar, e disfunção da barreira hematoencefálica como já mencionado anteriormente

(Bowton et al, 1989; Voigt et al, 2002).

Marcadores de danos cerebrais durante a sepse poderiam ter um valor

considerável, ambos para esclarecer os mecanismos envolvidos e quantificar o grau

de dado do cérebro. No estudo por Larsson e colaboradores (2005) a concentração

plasmática da proteína S-100β, como possível marcador de danos gliais, encontra-

se aumentada.

De qualquer maneira, a fisiopatologia da encefalopatia séptica está longe

ser conhecida perfeitamente e vários aspectos merecem ser esclarecidos, em

especial o início do desenvolvimento da encefalopatia. Tardiamente, as disfunções

ou as insuficiências dos órgãos contribuem para o seu agravamento. Além disso,

numerosos fatores de origem iatrogênicos também podem agravar o quadro.

Neste contexto, além dos estudos que envolvem humanos, os modelos de

ligação e perfuração cecal (CLP) são clinicamente relevantes, pois produzem uma

inflamação generalizada similar àquela observada durante a síndrome da resposta

inflamatória sistêmica e a sepse (Ritter et al, 2004).

Sepse e Cognição

Recentemente, diversos estudos foram realizados mostrando que os

sobreviventes de unidades de terapia Intensiva apresentam incapacidade cognitiva

em longo prazo, incluindo alterações na memória, atenção, na concentração e ou na

perda global da função cognitiva (Gordon et al, 2004; Granja et al, 2004; Hopkins et

al, 2005; Hough e Curtis, 2005; Jackson et al, 2004).

O termo incapacidade cognitiva resulta de anormalidades clínicas

significativas em uma ou mais funções do cérebro incluindo memória, atenção,

função executiva, anormalidades espaciais e visuais, e a função intelectual. A

incapacidade cognitiva pode ser suave, moderada, ou severa e pode limitar a

habilidade de um indivíduo de pensar, raciocinar, e executar tarefas diárias. O termo

declínio cognitivo relaciona-se a deterioração das habilidades cognitivas e não é

necessariamente sinônimo de incapacidade cognitiva, pois não implica em um nível

absoluto do funcionamento. Entretanto, este tipo de declínio pode causar

incapacidades significativas na vida diária de uma pessoa que deseja executar

níveis elevados da cognição em áreas ocupacionais e vocacionais. Alternativamente,

o rápido declínio cognitivo em uma pessoa com coeficiente de inteligência abaixo da

média poderia resultar no diagnóstico de incapacidade cognitiva, porém com impacto

menor na função diária (Gordon et al, 2004).

Estudos precedentes que envolveram pacientes sobreviventes das

unidades de terapia intensiva demonstraram algum grau de incapacidade cognitiva

(Gordon et al, 2004; Granja et al, 2004; Hopkins et al, 2005; Hough e Curtis, 2005;

Jackson et al, 2004). Em estudos em longo prazo a maioria dos pacientes mostrou

uma melhora na função cognitiva total, porém algumas habilidades cognitivas, tais

como a memória, não melhoraram completamente (Angus et al, 2001; Granja et al,

2005; Hopkins et al, 1994). É bem caracterizada a participação de vias inflamatórias

apoptóticas e danos neuronais secundários a encefalopatia séptica (Messaris et al.,

2004).

Muitos pacientes criticamente doentes possuem incapacidades

neurocognitivas crônicas significativas em 2 meses (Jones et al, 2006), 6 meses

(Weinert et al, 1997; Jackson et al, 2003b), 9 meses (Hopkins et al, 2006), 1 ano

(Hopkins et al, 1999), 2 anos (Hopkins et al, 2005), e em até 6 anos (Suchyta et al,

2004) após a alta hospitalar. As incapacidades neurocognitivas melhoram durante os

primeiros 6 a 12 meses da saída dos pacientes do ambiente hospitalar podendo ser

permanente ou não, e associados com as incapacidades na função diária, na baixa

qualidade de vida, e em uma inabilidade de retornar ao trabalho (Sukantarat et al,

2005; Jackson et al, 2004).

Estudos concluíram que sobreviventes da sepse que tiveram

incapacidades neurocognitivas na alta hospitalar, somente 45% mantiveram as

incapacidades neurocognitivas após 1 ano (Hopkins et al, 1999). Não havia

nenhuma melhoria adicional nas seqüelas neurocognitivas após 2 anos da alta

hospitalar (Hopkins et al, 2003).

Outros significativos sintomas que sobreviventes de unidades de terapia

intensiva apresentam são a depressão e a ansiedade (Scragg et al, 2001). A

prevalência e a severidade dos transtornos afetivos incluindo sintomas de depressão

e ansiedade variam de 10% a 58% (Jackson et al, 2003; Hopkins et al, 1999; Skozol

e Vender, 2001, Al-Saidi et al, 2003; Schelling et al, 1998). A depressão foi relatada

em até 30% dos sobreviventes (Jackson et al, 2003), e estima-se que 47% têm

ansiedade clinicamente significativa (Scragg et al, 2001). Certamente, taxas

elevadas de depressão entre sobreviventes de unidades de terapia intensiva sejam

relacionadas à incapacidade cognitiva. Em alguns casos a depressão severa pode

imitar sintomas de incapacidade cognitiva, embora as diferenças existam entre estas

circunstâncias. No geral, os indivíduos com depressão retêm a habilidade de

aprender, não se esquecem rapidamente e não indicam decréscimos significativos

na linguagem (Hart et al, 1997; McGlynn e Schacter, 1989; Jones et al, 1992).

A neurobiologia da depressão pode ser explicada na maior parte na

desregulação do eixo hipotalâmico-pituitário-adrenal e da sua ação no hipocampo

que implicam no fator liberador de corticotrofinas, glucocorticoides, fator neurotrofico

derivado do cérebro, e o CREB. Outras áreas do cérebro além do hipocampo

provavelmente também estão envolvidas. Por exemplo, os núcleos acumbens, a

amígdala, e o núcleo hipotalâmico são críticos na regulação da motivação como

comer, dormir, nível de energia, ritmo circadiano, e em respostas aos estímulos de

recompensa e aversivos, que estão anormais em pacientes deprimidos (Nestler et al,

2002)

Os sintomas de dano cognitivo como a memória, a depressão e a

ansiedade devem ser melhor entendidos para posteriormente justificar os

mecanismos que envolvem o dano cognitivo em sobreviventes de sepse. Neste

contexto, devido a grande incidência, associado à gravidade da sepse seria por si só

o suficiente para justificar o aprofundamento no conhecimento de sua fisiopatologia

e a tentativa de novas possibilidades terapêuticas, pois como exposto nos

parágrafos acima, a maioria dos sobreviventes de unidades de terapia intensiva

apresentam algum dano cognitivo em longo prazo, portanto faz-se necessário a

realização de estudos que possam investigar as possíveis incapacidades cognitivas

apresentadas em curto e médio prazo em um modelo animal de ligação e perfuração

cecal (CLP).

Sepse e Estresse Oxidativo

Diversos mecanismos de inflamação e dano celular são implicados na

fisiopatologia da sepse, choque séptico e disfunção orgânica relacionada à sepse,

entre eles a geração de espécies ativas do oxigênio (EAO). O elemento oxigênio

existe na atmosfera na forma diatômica O

, com exceção de certos microorganismos

unicelulares anaeróbios e aeróbios tolerantes, todos os animais, plantas e bactérias

necessitam de O

para eficiente produção de energia através do uso do oxigênio

dependente da cadeia transportadora de elétrons nas mitocôndrias de eucariontes

(Halliwell & Gutteridge,1999).

A necessidade do O

obscurece o fato de que é um gás tóxico e

mutagênico oferecendo sérios riscos, as espécies aeróbicas sobrevivem devido às

defesas antioxidantes que as protegem. Os efeitos dos danos em organismos

aeróbicos variam consideravelmente com o tipo de organismo, a idade, estado

fisiológico e a dieta. A toxicidade do oxigênio é influenciada pela presença na dieta

de variadas quantidades de vitaminas A, E, e C, metais tais como Zinco, Cobre e

Ferro, antioxidantes sintéticos e ácidos graxos poliinsaturados (Halliwell &

Gutteridge, 1999).

Para formar o oxigênio molecular O

, os dois elétrons do subnível p de um

elemento oxigênio fazem intercâmbio com os dois elétrons de outro elemento

oxigênio, formando um composto estável com 12 elétrons na última camada (L).

Quando no metabolismo normal ocorrer uma redução do oxigênio molecular O

, este

ganhará um elétron, formando o radical superóxido (O

−

•

) (Mello et al.,1983). Em

condições fisiológicas do metabolismo celular aeróbico, o O

sofre redução

tetravalente, com aceitação de quatro elétrons, resultando na formação de água,

durante este processo são formados intermediários reativos como os radicais

superóxido (O

−

•

), hidroperoxila (HO

•

••

•

), hidroxila (OH), e o peróxido de hidrogênio

) (Ferreira et al., 1997, Cuzzocrea et al,2001). Normalmente a redução

completa do O

ocorre na mitocôndria, e a reatividade das EAO é neutralizada pela

entrada de quatro elétrons (Cohen, 1989). Entre as espécies reativas de oxigênio

formadas o radical superóxido ocorre em quase todas as células aeróbicas e é

produzido durante a ativação máxima de neutrófilos, macrófagos, monócitos e

eosinófilos (Halliwell & Gutteridge, 1990). O radical superóxido reage com alvos

biológicos, sendo que o efeitos nos tecidos é resultado da formação secundaria de

novos radicais livres em adição a reação do superóxido com lipídios, catecolaminas

(Macarthur et al.,2000), e Dna (Dix et al.,1996).

Entre as EAO o oxigênio singlet que é a forma excitada do oxigênio

molecular e não possui elétrons desemparelhados na ultima camada (Halliwell &

Gutteridge, 1990) é reconhecido como um possível contribuinte para o estresse

oxidativo nos sistemas vivos, altamente energético e mutagênico, sendo capaz de

oxidar moléculas biológicas (Cuzzocrea et al.,2001; Ravanat et al.,1992).

O H

é capaz de atravessar camadas lipídicas, podendo reagir com a

membrana eritrocitária e com proteínas ligadas ao ferro, sendo assim é altamente

tóxico para as células, podendo ser aumentada esta toxicidade em presença de ferro

(Eaton,1991).

O radical mais reativo proposto muitos anos atrás é o HO

•

••

•-

, produzido

pela interação do O

−

•

e H

pela reação química conhecida como Haber-Weiss,

traços do metal ferro na forma de íons, reage com H

produzindo o radical

hidroxil, o íon ferro não esta presente em vivo, mas os íons são produzidos pela

ação do superóxido sobre os íons ferro armazenados em proteínas (Cuzzocrea et

al.,2001). A liberação do ferro intracelular, a baixa capacidade liquórica de ligação

ferro-proteína e a deficiência de enzimas antioxidantes no sistema nervoso central

ampliam o risco de lesão induzida pelo trauma com a liberação de ferro (Ferreira et

al.,1997). Estas espécies ativas de oxigênio são capazes de reagir

indiscriminadamente com qualquer tipo de molécula orgânica, extraindo elétrons e

gerando novos radicais livres em reação em cadeias altamente citotóxicas (Ames et

al.,1993).

Quando existe um aumento na produção ou diminuição das defesas

antioxidantes existe uma condição chamada de estresse oxidativo, em que os

radicais livres em excesso começam a produzir danos a lipídios, proteínas, DNA,

carboidratos (Halliwell & Gutteridge, 1999), inibição das enzimas da cadeia

respiratória mitocondrial, inativação do gliceraldeido-3-fosfato dehidrogenase,

inibição da atividade da ATPase na membrana sódio/potássio, inativação da

membrana no canal de sódio (Cuzzocrea et al., 2001).

A capacidade das células em diminuir os efeitos do estresse oxidativo é

determinada pelo balanço entre as quantidades de espécies oxidantes geradas, e, a

capacidade dos processos metabólicos de produzir antioxidantes (Beutler et al.,

1989).

Antioxidantes e tratamento de sepse

Intervenções que reduzem a produção das EAO exercem efeitos

benéficos em diversos modelos de endotoxemia e choque séptico. Estas

intervenções incluem a N-acetilcisteina (NAC) (Atis et al 2006, Victor et al 2003,

Ozdulger et al 2003), α-tocoferol (Durant et al 2004), alopurinol (Xiang et al 2003),

deferoxamina (DFX) (Messaris et al 2004b), catalase (Supinski et al 1993),

superoxide dismutase (Supinski e Callahan 2006), miméticos de superoxide

dismutase (Salvemini e Cuzzocrea 2003), magnolol (Kong et al 2000) e tempol

(Matejovic et al 2005). Geralmente estas intervenções são administradas antes ou

imediatamente após a indução da sepse o que pode limitar sua relevância clínica.

Entre os mais estudados antioxidantes no tratamento da sepse

encontra-se a NAC é bem conhecida como precursora artificial de glutationa e

utilizada clinicamente como droga mucolítica e no tratamento da intoxicação por

paracetamol, com raros efeitos adversos. O NAC é um scavenger de peróxido de

hidrogênio, ácido hipoclórico e radical hidroxil e por estas ações inibe a liberação de

TNFα, a ativação de citocinas pró-inflamatórias e apoptose celular.

As evidências sugerem que a expressão do gene TNF é controlada

pela transcrição do NF-kB, cuja a atividade pode ser induzida pelo peróxido de

hidrogênio. NAC mostrou inibir a atividade do NF-kB em várias linhagens celulares,

inclusive em macrófagos peritoneais de ratos (Pahan et al 1998). O peróxido de

hidrogênio diretamente ou indiretamente através de sua redução a radical hidroxil via

reação de Fenton, age como um mensageiro na síntese e ativação de mediadores

inflamatórios. O NAC como scavenger destes radicais mostrou inibir a liberação

destes mediadores.

Por estas razões é reconhecido o papel antioxidante da NAC na sepse,

mas quando utilizada antes da indução da sepse e não depois. Em contraste alguns

estudos demonstram um aumento no estresse oxidativo e mortalidade por sepse

após uso de altas doses da NAC, possivelmente relacionado à sua capacidade para

reduzir o ferro para sua forma catalicamente ativa (Sprong et al 1998), favorecendo a

reação de Fenton.

O DFX é um quelante de ferro empregado com segurança no tratamento

de várias doenças hematológicas. Experimentalmente, já foi citada em alguns

estudos, como uma droga que diminuiu a injúria oxidativa, quando usada antes e

não depois da indução da sepse, melhorando mortalidade em um modelo animal de

sepse abdominal (Messaris et al 2004).

As propriedades pró-inflamatórias dos EAO incluem dano às células

endoteliais, formação de fatores quimiotáticos, recrutamento de neutrófilos, oxidação

e peroxidação de lipídeos, dano ao DNA, liberação de TNF-α e IL-1β e formação de

peroxinitrito (Azevedo et al 2006, Protti e Singer 2006). A hiperprodução de EAO e a

falha nos mecanismos de “scavengers” naturais são implicados no dano endotelial,

alterações miocárdicas e falência orgânica múltipla.

Os monócitos e polimorfonucleares sofrem alterações, descritas como

ativação de leucócitos, em resposta a estimulação por TNF e interleucinas (IL), com

um conseqüente aumento na produção de superóxido (O

) por estas células.

Primariamente o superóxido (O

) tem um efeito pró-inflamatório, que é perpetuado

pela formação de peroxinitrito (reação de superóxido com óxido nítrico). O

peroxinitrito possui vários efeitos citotóxicos e pró-inflamatórios independentes que

levam ao dano celular irreversível, como evidenciado no choque séptico (Alvarez e

Evelson 2007).

O choque séptico é caracterizado por severa hipotensão e diminuição da

perfusão tecidual em decorrência da hiporreatividade vascular a catecolaminas

endógenas e exógenas, que pelo menos em parte é explicado pelo grande aumento

na produção de óxido nítrico que ocorre na sepse (Fernandes et al 2006, Cuzzocrea

et al , 2006).

O peróxido de hidrogênio (H

), apesar de ser considerado um oxidante

estável, conta com um papel importante na fisiopatologia da sepse. O H

pode ser

metabolizado por duas enzimas antioxidantes, a glutationa peroxidase e a catalase,

mas em presença de metais de transição, ele é decomposto em radical hidroxil via

reação de Fenton, um radical altamente tóxico e reativo. Os danos às células

musculares e acidose aumentam a quantidade de ferro liberado da mioglobina e

hemoglobina, facilitando esta reação. Recentemente foi demonstrado que alterações

do metabolismo de ferro podem estar relacionadas com mortalidade em modelos

animais de sepse (Wizorek et al 2003).

Associada a grande mortalidade envolvida, os gastos direto e indireto com

a sepse atinge altas cifras em nosso país. A possibilidade de diminuir a mortalidade

por sepse e reduzir os gastos com internação em Unidades de Terapia Intensiva

(alto custo com antibióticos de largo espectro, recursos técnicos, humanos e

tecnológicos associados com o manejo do paciente) justificam a necessidade de

maior investimento no estudo desta patologia. Outrossim, são pouco conhecidos os

efeitos da sepse no Sistema Nervoso Central e, através da determinação do

Estresse Oxidativo, poderemos identificar e melhor compreender estes danos.

Modelo animal de sepse

Estudos de sepse em humanos são difíceis devido a severidade da

doença, a necessidade de intervenções terapêuticas imediatas, a heterogeneidade

dos pacientes. Assim, modelos animais têm sido usados extensivamente para

explorar a patogênese e gerarem dados pré-clínicos de intervenções terapêuticas.

Para estas propostas, deve-se utilizar um modelo animal que reproduza a

vasodilatação, hipotensão, aumento do débito cardíaco, resposta ao tratamento e

mortalidade vistos em pacientes sépticos. Tem-se utilizado para isto modelo de

sepse abdominal, sepse cutânea, sepse induzida pela administração de

lipopolissacarídeo (LPS) ou fator de necrose tumoral. Porém os modelos que

induzem peritonite são mais amplamente usados. A peritonite pode ser induzida por

inoculação direta de bactérias ou de conteúdo fecal na cavidade peritoneal.

Entretanto o modelo mais aceito na literatura, e que parece simular mais

adequadamente o quadro clínico de sepse, é o chamado CLP. A CLP se baseia na

ligação do ceco logo abaixo da válvula ileo-cecal (mantendo desta maneira o trânsito

intestinal), perfuração do ceco com tamanho padronizado e liberação de conteúdo

fecal para a cavidade peritoneal, conforme classicamente descrito por Wichterman e

cols (1980). Desta maneira além da peritonite se induz isquemia mesentérica

simulando as grandes síndromes clínicas de sepse abdominal (p.ex. apendicite,

isquemia mesentérica). Recentemente este modelo foi modificado para melhor

simular as características clínicas dos pacientes com sepse abdominal, introduzindo

desta maneira a ressucitação volêmica e emprego de antibióticos de amplo espectro

(Hollenberg et al 2001).

2. OBJETIVOS

2.1 Objetivo Geral

Avaliar as alterações comportamentais e neuroquímicas induzidas pela sepse em

modelo animal com um possível papel terapêutico de antioxidantes.

2.2 Objetivos Específicos:

• Avaliar os efeitos da sepse após 10 e 30 dias sobre o aprendizado e

memória;

• Avaliar os efeitos da sepse leve e grave após 10 dias sobre a ansiedade;

• Avaliar os efeitos da sepse leve e grave após 10 dias sobre sintomas

depressivos em ratos de diferentes idades;

•

Determinar uma relação temporal entre a indução da sepse e parâmetros

de estresse oxidativo nas estruturas cerebrais dissecadas: hipocampo,

estriado, córtex e cerebelo em um modelo animal de sepse.

• Avaliar os efeitos do tratamento com antioxidantes na incapacidade

cognitiva precoce e tardia e no estresse oxidativo em hipocampo.

Parte II

Capítulo 1.

Cognitive impairment in sepsis survivors from cecal ligation

and perforation

Tatiana Barichello, Márcio R. Martins, Adalisa Reinke, Gustavo Feier,

Cristiane Ritter, João Quevedo, Felipe Dal-Pizzol

Critical Care Medicine (2005) 33:221-223

Brief Reports

Cognitive impairment in sepsis survivors from cecal ligation and

perforation*

Tatiana Barichello, PharmD, MSc; Márcio R. Martins, BSc; Adalisa Reinke, BSc; Gustavo Feier, Bsc;

Cristiane Ritter, MD, MSc; João Quevedo, MD, PhD; Felipe Dal-Pizzol, MD, PhD

espite major improvements

in intensive care and antibi-

otic therapy, the mortality

and morbidity rates due to

severe sepsis and septic shock remain

high. Critical illness survivors present

long-term cognitive impairment, includ-

ing alterations in memory, attention,

concentration, and/or global loss of cog-

nitive function (1– 4). The development

of an animal model that mimics the cog-

nitive alterations observed in patients is

of great value. In this context, murine

models of cecal ligation and perforation

(CLP) are clinically relevant since they

induce a polymicrobial sepsis that mim-

ics human sepsis (5). CLP models have

contributed to elucidate the pathogenesis

and to determine new therapies in sepsis

(5, 6). In this article, we evaluated learn-

ing and memory performance in rats af-

ter sepsis induced by CLP compared with

sham-operated rats.

MATERIALS AND METHODS

Under anesthesia (ketamine, 80 mg/kg,

and xylazine, 10 mg/kg), 105 male Wistar rats

(300 –350 g) underwent CLP (sepsis group)

and 40 rats underwent sham operation (con-

trol group) as previously described (5). After

surgery, the sepsis group received “basic sup-

port” (saline at 50 mL/kg immediately and 12

hrs after CLP plus ceftriaxone at 30 mg/kg and

clindamycin at 25 mg/kg every 6 hrs over a

total of 3 days). The sham-operated group re-

ceived only saline, 50 mL/kg, immediately and

12 hrs after surgery, and the volume of saline

corresponded to antibiotic administration.

Survival in the sham group was 100%, and in

the sepsis group, it was 40% (40 rats). The

number of survivals is in accordance with our

previous reports (5, 6). Ten days after surgery,

the animals separately underwent three be-

havioral tasks: a) the step-down inhibitory

avoidance task (single-training); b) continu-

ous multiple-trials step-down inhibitory

avoidance task; and c) the open-ﬁeld task. The

behavioral tests were performed by the same

person, who was blinded as to group (sham or

CLP). All experimental procedures involving

animals were performed in accordance with

the National Institute of Health Guide for the

Care and Use of Laboratory Animals and with

the approval of the local ethics committee.

The step-down inhibitory avoidance appa-

ratus and procedures have been described in

previous reports (7). Brieﬂy, the training ap-

paratus was a 50 ϫ 25 ϫ 25-cm acrylic box

(Albarsch, Porto Alegre, Brazil) whose ﬂoor

consisted of parallel caliber stainless steel bars

(1-mm diameter) spaced 1 cm apart. A 7-cm-

wide, 2.5-cm-high platform was placed on the

ﬂoor of the box against the left wall. In the

training trial, animals were placed on the plat-

form and their latency to step down on the

grid with all four paws was measured with an

automatic device. Immediately after stepping

down on the grid, the animals received a 0.4-

mA, 2.0-sed foot shock and returned to their

home cage. A retention test trial was per-

formed 24 hrs after training. The retention

test trial was procedurally identical to train-

ing, except that no foot shock was presented.

The retention test step-down latency (maxi-

*See also p. 262.

From the Laboratório de Fisiopatologia Experimen-

tal (TB, AR, CR, FD-P), the Laboratório de Neurotoxi-

cologia (MRM, GF, JQ), Universidade do Extremo Sul

Catarinense, Criciúma, Brazil.

Supported, in part, by UNESC (Brazil), CNPq (Bra-

zil), FUNCITEC (Brazil). None of the authors have any

ﬁnancial interests to disclose.

Address requests for reprints to: Felipe Dal-Pizzol,

MD, PhD, Laboratório de Fisiopatologia Experimental,

Universidade do Extremo Sul Catarinense, Criciúma,

SC, Brazil, Avenida Universitária, 1105, 88006–000.

E-mail: [email protected]

Medicine and Lippincott Williams & Wilkins

DOI: 10.1097/01.CCM.0000150741.12906.BD

Objective: Critical illness survivors present long-term cognitive

impairment, including problems with memory and learning. We

evaluated cognitive performance in rats that survived from sepsis

induced by cecal ligation and puncture (CLP).

Design: Prospective, controlled experiment.

Setting: Animal basic science laboratory.

Subjects: Male Wistar rats, weighing 300 –350 g.

Interventions: The rats underwent CLP (sepsis group) with

“basic support” (saline at 50 mL/kg immediately and 12 hrs after

CLP plus ceftriaxone at 30 mg/kg and clindamycin at 25 mg/kg 6,

12, and 18 hrs after CLP) or sham-operated (control group).

Measurements and Main Results: Ten days after surgery, the

animals underwent three behavioral tasks: a) inhibitory avoidance

task; b) habituation to an open ﬁeld; and c) continuous multiple-

trials step-down inhibitory avoidance task (CMSIA). In the habit-

uation to an open-ﬁeld task, there were no differences in the

number of crossings and rearings. The sepsis group showed

signiﬁcantly decreased performance in latency retention com-

pared with the sham group in inhibitory avoidance. Furthermore,

when tested by the habituation to an open-ﬁeld task, the sepsis

group did not show any difference between training and test,

indicating memory impairment. In the CMSIA, the sepsis group

showed a signiﬁcant increase in the number of training trials

required to reach the acquisition criterion.

Conclusion: Our data provide the ﬁrst experimental demon-

stration that survivors from CLP show learning and memory

impairment after complete physical recovery from sepsis. (Crit

Care Med 2005; 33:221–223)

EY WORDS: sepsis; survivors; cecal ligation and puncture;

learning; memory; rat

221Crit Care Med 2005 Vol. 33, No. 1

mum, 180 secs) was used as a measure of

inhibitory avoidance retention.

Habituation to an open ﬁeld was carried

out in a 40 ϫ 60-cm open ﬁeld surrounded by

50-cm high walls made of brown plywood with

a frontal glass wall. The ﬂoor of the open ﬁeld

was divided into 12 equal rectangles by black

lines. Animals were gently placed on the left

rear quadrant, and left to explore the arena for

5 mins (training session). Immediately follow-

ing this, the animals were taken back to their

home cage, and 24 hrs later submitted again

to a similar open-ﬁeld session (test session).

Crossing of the black lines and rearing per-

formed in both sessions were counted. The

decrease in the number of crossings and rear-

ings between the two sessions was taken as a

measure of the retention of habituation (8).

Continuous multiple-trials step-down in-

hibitory avoidance task was performed in the

same step-down inhibitory avoidance appara-

tus described above. However, in the training

session, the animal was placed on the platform

and immediately after stepping down on the

grid, received a 0.3-mA, 2.0-sec foot shock.

This procedure continued until the rat re-

mained on the platform for 50 secs. The ani-

mal was then returned to the home cage. The

number of training trials required to reach the

acquisition criterion of 50 secs on the plat-

form was recorded. The retention test was

performed 24 hrs later.

Data from the inhibitory avoidance task

and retention test latencies from continuous

multiple-trials step-down inhibitory avoidance

were expressed as median and interquartile

ranges. Statistical signiﬁcances were deter-

mined by the Mann-Whitney U test, p Ͻ .05.

Data from the open-ﬁeld task and the number

of training trials from continuous multiple-

trials step-down inhibitory avoidance were ex-

pressed as mean Ϯ

SEM. Statistical signiﬁ-

cances were determined by paired samples

Student’s t-test, p Ͻ .05.

RESULTS AND DISCUSSION

After 10 days, the animals seemed to

be free from infection. We performed

blood cultures that were all negative in

this period. The animals recovered their

weight and grooming habits, blood

counts returned to control levels, and

polymerase chain reaction values were

negative. In addition, we had previously

demonstrated that 5 days after CLP, or-

ganic oxidative stress, mitochondrial dys-

function, and neutrophil inﬁltration re-

turn to control levels in the present CLP

model (6).

No differences between groups were

demonstrated in the inhibitory avoidance

training session. In the test session, the

step-down latency was signiﬁcantly de-

creased in the sepsis group compared

with the sham group (p Ͻ .01) (Fig. 1A).

In the habituation to the open-ﬁeld train-

ing session, no difference in motor and

exploratory activity was demonstrated be-

tween groups, e.g., in the number of

crossings and rearings. This ﬁnding sup-

ports our hypothesis that no active infec-

tion remained in the CLP group.

In the open-ﬁeld task, there were no

differences in the number of crossings

and rearings between groups in the ha-

bituation to the open-ﬁeld training ses-

sion, demonstrating no difference in mo-

tor and exploratory activity between

groups, which reinforces the idea of no

active infection in the CLP group (Fig.

1B). In the test session, there was a sig-

niﬁcant reduction in both crossings and

rearings of the sham group compared

with the sepsis group (p Ͻ .001) (Fig.

1B). In addition, the sepsis group did not

show a difference in crossings and rear-

ings between training test sessions, dem-

onstrating memory impairment in this

group (Fig. 1B). On the other hand, the

sham group showed a decreased number

of crossings (p Ͻ .005) and rearings (p Ͻ

.005), indicating habituation to the task

environment (Fig. 1B).

The continuous multiple-trials step-

down inhibitory avoidance showed a signif-

icant increase in the number of training

trials required to reach the acquisition cri-

terion (50 secs on the platform) in the

sepsis group compared with the sham

group (p Ͻ .05) (Fig. 2A). In the reten-

tion test, there was no difference between

groups (Fig. 2B). The results of this task

showed that the sepsis group required

approximately two times more stimulus

to reach the acquisition criterion com-

pared with the sham group, indicating a

learning impairment (acquisition of new

knowledge) in the sepsis group.

These results do not appear to be sec-

ondary to antibiotic administration since

we performed the open-ﬁeld and inhibi-

tory avoidance in a sham-operated group

with antibiotic administration and did

Figure 1. A, inhibitory avoidance task. Data are

expressed as median (interquartile ranges) train-

ing and test session latencies, in seconds. *Sig-

niﬁcant difference compared with Sham group

(Mann-Whitney U test, p Ͻ .01). Both groups (n

ϭ 14 in the sham group and n ϭ 13 in the sepsis

group) showed signiﬁcant training test differ-

ences (Wilcoxon’s test, p Ͻ .01). B, Open-ﬁeld

task. Data are expressed as mean Ϯ SEM of cross-

ings and rearings of training (white columns)

and test (gray columns) session (n ϭ 14 in the

sham group and n ϭ 17 in the sepsis group).

*Signiﬁcant difference between training test

(paired samples Student’s t-test, p Ͻ .005). #Sig-

niﬁcant difference between groups in training

and test sessions (Student’s t-test, p Ͻ .0001).

Figure 2. A, continuous multiple-trials step-down

inhibitory avoidance. Data are expressed as mean

Ϯ SEM of the number of training trials required to

reach acquisition criterion (50 secs on the plat-

form). *Signiﬁcant difference between groups

(Student’s t-test, p Ͻ .0001). B, retention test

latencies, expressed as median (interquartile

ranges). No difference was observed between

groups (n ϭ 12 per group).

222 Crit Care Med 2005 Vol. 33, No. 1

not detect signiﬁcant differences in com-

parison with the sham-operated group

(data not shown).

Previous reports describing human in-

tensive care unit sepsis survivors demon-

strated that the vast majority of such

patients experienced cognitive impair-

ment at hospital discharge (1, 4). At 1-yr

follow-up, most patients showed im-

provement in overall cognitive function;

however, some cognitive skills, such as

memory, did not completely improve (1,

2, 4). Our data provide the ﬁrst experi-

mental demonstration in a clinically rel-

evant model of animal sepsis. The rats

showed impairment in learning and

memory. In this context, Shimizu et al.

demonstrated that 24 hrs after CLP, ani-

mals showed learning impairment in pas-

sive avoidance retention (9). Our results

are of greater signiﬁcance since 10 days

after CLP, but not 24 hrs, the animals are

fully recovered, with no signs of infection

or motor alterations (5, 6). In addition,

our survival study design using ﬂuids and

antibiotics replicates more closely the

supportive therapy performed in the clin-

ical setting, when compared with the

study of Shimizu et al. (9). The deﬁcits

demonstrated here mimics, at least in

part, the cognitive alterations observed in

patients surviving sepsis, particularly

memory impairment. In this way, we be-

lieve that the CLP model of sepsis will

help us to investigate the biological

mechanisms involved in the cognitive

deﬁcits associated with sepsis and to de-

termine therapeutic approaches to this

problem.

REFERENCES

1. Hopkins RO, Weaver LK, Pope D, et al: Neu-

ropsychological sequelae and impaired health

status in survivors of severe acute respiratory

distress syndrome. Am J Respir Crit Care Med

1999; 161:50–56

2. Angus DC, Musthafa AA, Clermont G, et al:

Quality-adjusted survival in the ﬁrst year after

the acute respiratory distress syndrome. Am J

Respir Crit Care Med 2001; 163:1389–1394

3. Granja C, Dias C, Costa-Pereira A, et al: Qual-

ity of life of survivors from severe sepsis and

septic shock may be similar to that of others

who survive critical illness. Crit Care 2004;

8:R91–R98

4. Heyland DK, Hopman W, Coo H, et al: Long-

term health-related quality of life in survivors

of sepsis. Short Form 36: a valid and reliable

measure of health-related quality of life. Crit

Care Med 2000; 28:3599–3605

5. Ritter C, Andrades ME, Reinke A, et al: Treat-

ment with N-acetylcysteine plus deferoxamine

protects rats against oxidative stress and im-

proves survival in sepsis. Crit Care Med 2004;

32:342–349

6. Ritter C, Andrades M, Frota Junior ML, et al:

Oxidative parameters and mortality in sepsis

induced by cecal ligation and perforation. In-

tensive Care Med 2003; 29:1782–1789

7. Quevedo J, Vianna MR, Roesler R, et al: Two

time windows of anisomycin-induced amnesia

for inhibitory avoidance training in rats: Pro-

tection from amnesia by pretraining but not

pre-exposure to the task apparatus. Learn

Mem 1999; 6:600 – 607

8. Vianna MR, Alonso M, Viola H, et al: Role of

hippocampal signaling pathways in long-term

memory formation of a nonassociative learn-

ing task in the rat. Learn Mem 2000;

7:333–340

9. Shimizu I, Adachi N, Liu K, et al: Sepsis facil-

itates brain serotonin activity and impairs

learning ability in rats. Brain Res 1999; 830:

94 –100

ur data provide

the ﬁrst experi-

mental demon-

stration that survivors from

cecal ligation and puncture

show learning and memory

impairment after complete

physical recovery from sepsis.

223Crit Care Med 2005 Vol. 33, No. 1

Capítulo 2.

Behavioral deficits in sepsis-surviving rats induced by

cecal ligation and perforation

Tatiana Barichello, Márcio Rodrigo Martins, Adalisa Reinke, Larissa S.

Constantino, Roberta Albino Machado, Samira S. Valvassori, José

Cláudio F. Moreira, João Quevedo, Felipe Dal-Pizzol

Brazilian Journal of Medical and Biological Research (2007) 40: 831-

831

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Sepsis and cognition deficits

www.bjournal.com.br

Brazilian Journal of Medical and Biological Research (2007) 40: 831-837

ISSN 0100-879X

Behavioral deficits in sepsis-surviving

rats induced by cecal ligation and

perforation

Laboratório de Fisiopatologia Experimental,

Laboratório de Neurociências, Universidade do Extremo Sul Catarinense,

Criciúma, SC, Brasil

Centro de Estresse Oxidativo, Departamento de Bioquímica,

Instituto de Ciências Básicas da Saúde,

Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brasil

T. Barichello

M.R. Martins

A. Reinke

L.S. Constantino

R.A. Machado

S.S. Valvassori

J.C.F. Moreira

J. Quevedo

and

F. Dal-Pizzol

Abstract

Sepsis and its complications are the leading causes of mortality in

intensive care units, accounting for 10-50% of deaths. Intensive care

unit survivors present long-term cognitive impairment, including

alterations in memory, attention, concentration, and/or global loss of

cognitive function. In the present study, we investigated behavioral

alterations in sepsis-surviving rats. One hundred and ten male Wistar

rats (3-4 months, 250-300 g) were submitted to cecal ligation and

puncture (CLP), and 44 were submitted to sham operation. Forty-four

rats (40%) survived after CLP, and all sham-operated animals sur-

vived and were used as control. Twenty animals of each group were

used in the object recognition task (10 in short-term memory and 10 in

long-term memory), 12 in the plus-maze test and 12 in the forced

swimming test. Ten days after surgery, the animals were submitted

individually to an object recognition task, plus-maze and forced

swimming tests. A significant impairment of short- and long-term

recognition memory was observed in the sepsis group (recognition

index 0.75 vs 0.55 and 0.74 vs 0.51 for short- and long-term memory,

respectively (P < 0.05). In the elevated plus-maze test no difference

was observed between groups in any of the parameters assessed. In

addition, sepsis survivors presented an increase in immobility time in

the forced swimming test (180 vs 233 s, P < 0.05), suggesting the

presence of depressive-like symptoms in these animals after recovery

from sepsis. The present results demonstrated that rats surviving

exposure to CLP, a classical sepsis model, presented recognition

memory impairment and depressive-like symptoms but not anxiety-

like behavior.

Correspondence

F. Dal-Pizzol

Laboratório de Fisiopatologia

Experimental

Universidade do Extremo Sul

Catarinense

88006-000 Criciúma, SC

Brasil

E-mail: [email protected]

Research supported by FAPESC,

CNPq and UNESC.

Received October 4, 2006

Accepted April 18, 2007

Key words

• Sepsis survivors

• Recognition memory

• Plus-maze test

• Forced swimming test

• Cecal ligation and puncture

• Cognitive impairment

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Introduction

Sepsis and its complications are a lead-

ing cause of mortality, accounting for 10-

50% of deaths on intensive care units (1-4).

Several studies have been performed to in-

vestigate the role of peripheral organs such

as lungs, liver, gut, and kidneys in sepsis

development (5), but the participation of the

central nervous system during sepsis has

been studied less. Septic encephalopathy rep-

resents brain dysfunction due to sepsis or the

systemic inflammatory response syndrome,

and has been reported to occur in 8-70% of

septic patients depending on the inclusion

criteria employed (6-8).

Survivors of critical care, including sep-

tic patients, may have persistenty compro-

mised organ function, which may result in

symptoms such as dyspnea, fatigue, depres-

sion, and impaired functional status. Re-

cently, several studies have demonstrated

that critical care survivors present long-term

cognitive impairment, including alterations

in memory, attention, concentration, and/or

global loss of cognitive function (9-17).

However, the neurobiological mechanisms

involved in this cognitive impairment re-

main unclear.

A recent study evaluating apoptosis and

vulnerability of different brain regions in-

duced by systemic inflammation concluded

that the hippocampus is the most vulnerable

region during experimental sepsis (18). In

this context, murine models of cecal ligation

and perforation (CLP) are clinically relevant

since they induce a polymicrobial sepsis that

mimics human sepsis (19-22). The CLP

model has contributed to the elucidation of

the pathogenesis and to the determination of

new therapies in sepsis (19,20). Shimizu et

al. (23) demonstrated that 24 h after CLP

animals presented learning impairment in

passive avoidance retention. Moreover, we

recently reported that CLP-induced sepsis

survivors presented learning, aversive and

spatial memory impairment when submitted

to behavioral tasks 10 days after CLP (21)

and learning and aversive memory impair-

ment lasting up to 30 days after CPL (22).

Thus, CLP seems to be a good model for the

study of cognitive and emotional alterations

as late manifestation of sepsis.

Therefore, the objective of the present

study was to determine changes in recogni-

tion memory and the presence of anxiety- or

depressive-like symptoms in severe sepsis-

surviving rats.

Material and Methods

Subjects

Male Wistar rats (3-4 months, 220-310

g) were obtained from our breeding colony

(UNESC). The animals were housed 5 to a

cage with food and water available ad libi-

tum and were maintained on a 12-h light/

dark cycle (lights on at 7:00 am). All experi-

mental procedures involving animals were

performed in accordance with the NIH Guide

for the Care and Use of Laboratory Animals

and the Brazilian Society for Neuroscience

and Behavior (SBNeC) recommendations

for animal care.

Cecal ligation and perforation surgery

The animals were subjected to CLP as

previously described (19-22). Briefly, rats

were anesthetized with a mixture of keta-

mine (80 mg/kg) and xylazine (10 mg/kg),

given intraperitoneally. Under aseptic con-

ditions, a 3-cm midline laparotomy was per-

formed to allow exposure of the cecum with

the adjoining intestine. The cecum was tightly

ligated with a 3.0-silk suture at its base,

below the ileocecal valve, and was perfo-

rated once with a 14-gauge needle. The cecum

was then gently squeezed to extrude a small

amount of feces from the perforation site

returned to the peritoneal cavity, and the

laparotomy was closed with 4.0-silk sutures.

Animals were resuscitated with normal sa-

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line (50 mL/kg subcutaneous) immediately

and 12 h after CLP. All animals were re-

turned to their cages with free access to food

and water. In this model septic rats become

bacteremic with Gram-negative enteric or-

ganisms. In the sham-operated group the

rats were submitted to all surgical proce-

dures but the cecum was neither ligated nor

perforated.

Treatment protocols

After surgery, the sepsis group received

“basic support” (50 mL/kg saline immedi-

ately and 12 h after CLP plus 30 mg/kg

ceftriaxone and 25 mg/kg clindamycin every

6 h for a total of 3 days). The sham-operated

group received only 50 mL/kg saline imme-

diately and 12 h after surgery and the vol-

ume of saline corresponding to antibiotic

administration. To perform behavior experi-

ments 44 animals were sham-operated and

the survival in this group was 100%; 10

animals were used as control to the short-

term memory in the object recognition task,

10 animals were used as control to the long-

term memory in the object recognition task,

12 animals were used as control to the plus-

maze test, and 12 animals were used as

control to the forced swimming test. One

hundred and ten animals were submitted to

CLP and 40% of these animals survived to

perform behavioral tests (N = 44). These

animals were divided as follows: 10 animals

were used in the short-term memory object

recognition task, 10 animals were used in the

long-term memory object recognition task,

12 animals were used in the plus-maze test,

and 12 animals were used in the forced

swimming test. The number of survivors

agreed with previous reports from our group

(19-20).

Behavioral tests

Ten days after surgery the animals were

submitted individually to the object recogni-

tion task, elevated plus-maze or forced swim-

ming tests. All behavioral procedures were

conducted between 13:00 and 16:00 h in a

sound-isolated room. All behavioral tests

were recorded by an observer who was blind

to the animal group.

Object recognition

The apparatus and procedures for the

object recognition task have been described

elsewhere (24,25). Briefly, the task took place

in a 40 x 50-cm open field surrounded by 50-

cm high walls made of plywood with a fron-

tal glass wall. The floor of the open field was

divided into 12 equal rectangles by black

lines. All animals were submitted to a ha-

bituation session where they were allowed

to freely explore the open field for 5 min. No

objects were placed in the box during the

habituation trial. Crossings of the black lines

and rearings performed in this session were

evaluated as locomotor and exploratory ac-

tivity, respectively.

Twenty-four hours after habituation,

training was conducted by placing individual

rats for 5 min in the field, in which two

identical objects (objects A1 and A2; both

being cubes) were positioned in two adja-

cent corners, 10 cm from the walls. In a

short-term recognition memory test given

1.5 h after training, the rats explored the

open field for 5 min in the presence of one

familiar (A) and one novel (B, a pyramid

with a square-shaped base) object. All ob-

jects had similar textures (smooth), colors

(blue), and sizes (weight 150-200 g), but

distinctive shapes. A recognition index cal-

culated for each animal is reported as the

ratio TB/(TA + TB) (TA = time spent explor-

ing the familiar object A; TB = time spent

exploring the novel object B). Between trials

the objects were washed with 10% ethanol

solution. In a long-term recognition memory

test given 24 h after training, the same rats

were allowed to explore the field for 5 min in

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the presence of the familiar object A and a

novel object C (a sphere with a square-

shaped base). Recognition memory was

evaluated as done for the short-term memo-

ry test. Exploration was defined as sniffing

(exploring the object 3-5 cm away from it) or

touching the object with the nose and/or

forepaws.

Elevated plus-maze

The elevated plus-maze task used in ani-

mal models of anxiety has been described in

detail elsewhere (26,27). Briefly, the appa-

ratus consisted of two open arms (50 x 10

cm) and two enclosed arms (50 x 10 x 40 cm)

arranged in such a way that the two arms of

each type were opposite to each other, and a

central platform (5 x 5 cm). The maze’s

height was 50 cm and the tests were con-

ducted under dim red light. Animals were

exposed for 5 min to the red light in their

own home cages before the testing proce-

dure. Next, they were placed individually on

the central platform of the plus-maze facing

an open arm. During a 5-min test period the

following measurements were recorded by

two observers: the number of entries, the

time spent in the open and closed arms, and

the total number of arm entries.

Forced swimming test

The forced swimming test was conducted

according to previous reports (28-30).

Briefly, the test involves two exposures to a

cylindrical water tank in which rats cannot

touch the bottom or from which they cannot

escape. The tank is made of transparent

Plexiglas and is 80 cm tall, 30 cm in diam-

eter, and filled with water (22-23ºC) to a

depth of 40 cm. Water in the tank was

changed after each rat. For the first expo-

sure, rats were placed in the water for 15 min

(pre-test session). Twenty-four hours later

the rats were placed in the water again for a

5-min session (test session). Behavior was

videotaped for later analysis, and the periods

of immobility, swimming, and struggling

time were recorded. The rats were judged to

be immobile whenever they stopped swim-

ming and remained floating in the water,

with their head just above water level.

Statistical analysis

Data for recognition indexes are reported

as median and interquartile ranges. Com-

parisons among groups were performed us-

ing Mann-Whitney U-tests. Recognition in-

dexes within individual groups were ana-

lyzed by Wilcoxon tests. The data for the

elevated plus-maze and forced swimming

tests are reported as means ± SEM and were

analyzed by the Student t-test. In all com-

parisons, P < 0.05 indicated statistical sig-

nificance.

Results

Object recognition

In the habituation session, no significant

differences were observed in the number of

crossings or rearings (P = 0.56; Figure 1A).

Figure 1. Object recognition

task. No significant difference

was observed in the numbers of

crossings and rearings in the

habituation session (Student t-

test). Data are reported as

means ± SEM (A). The sepsis

group presented a significant

impairment of novel object rec-

ognition memory compared to

the sham group (B). Results are

reported as median (interquar-

tile ranges) recognition indexes

in training, short-term (STM) and

long-term memory (LTM) reten-

tion test trials. N = 10 animals

per group. *P < 0.05 compared

to the sham group (Mann-Whit-

ney U-test).

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In Figure 1B, Wilcoxon tests showed

that the sham group, but not the sepsis group,

spent a significantly higher percentage of

time exploring the novel object during either

short- or long-term retention test sessions in

comparison with the training trial. In addi-

tion, the sepsis group presented a significant

reduction in the recognition index in short-

and long-term recognition retention tests

compared to the sham group (Mann-Whit-

ney U-test, P < 0.05). The results indicate

that sepsis survivors presented an impair-

ment of novel object recognition memory.

Elevated plus-maze

No statistically significant difference was

observed in the number of entries (P = 0.65)

or in the time spent in the arms (P = 0.51)

between groups.

Forced swimming test

In the test session (5 min), 24 h after the

pretest session (15 min), we observed a sig-

nificant increase in the immobility time in

the sepsis group compared to the sham group

(P < 0.05), as shown in Figure 2.

Discussion

Previous reports involving intensive care

unit survivors demonstrated cognitive im-

pairment at discharge from the hospital (9-

17). In long-term follow-up studies most

patients showed improvement in overall cog-

nitive function; however, some cognitive

skills, such as memory, were not completely

recovered (9,13,14). The mechanisms in-

volved in these cognitive impairments re-

main unclear. In recent reports we demon-

strated that CLP, a clinically relevant model

of sepsis in rats, presented impairment in

learning and memory. Our results were clini-

cally relevant since 10 and 30 days after

CLP the animals had fully recovered with no

signs of infection or motor alterations (21,22).

We have recently reported aversive and

spatial memory impairments in severe sep-

sis-surviving rats (21,22). The present study

investigated this issue in rats trained in an

object recognition task. This task, originally

developed by Ennaceur and Delacour (31),

is based on the tendency of rodents to ex-

plore a novel object more than a familiar

one. Because no rewarding or aversive stim-

ulation is used during training, the learning

occurs under conditions of relatively low

stress or arousal (31). Here we found that

sepsis-surviving rats presented significant

impairment of novel object recognition

memory. These findings are relevant since the

novel object recognition task in rodents is a

nonspatial, nonaversive memory test, in con-

trast to our previous reports (21,22). In addi-

tion, the object recognition task has also been

increasingly used as a powerful experimental

tool to assess drug effects on memory and to

investigate the neural mechanisms underlying

learning and memory (24,25,32-35).

In the elevated plus-maze, a validated

test to evaluate anxiety-like behavior (26),

no differences were demonstrated between

groups, indicating that sepsis survivors did

not presented anxiety-like symptoms after

recovery from disease.

Sepsis survivors presented depressive-

like symptoms assessed in the forced swim-

ming test. The time of immobility was sig-

nificantly longer in the sepsis group. The

original view of the forced swimming test

offered by Porsolt (28) was that of a model

of depression with features similar to those

Figure 2. Forced swimming test.

The sepsis group showed a sig-

nificant increase in the time of

immobility compared to the

sham group. Data are reported

as means ± SEM for N = 12 ani-

mals per group. *P < 0.05 com-

pared to the sham group (Stu-

dent t-test).

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of the learned helplessness model but tech-

nically easier to produce. The internal affec-

tive state of rodents after exposure to the

initial swim in the forced swimming test was

labeled as ‘behavioral despair’. The pretest

swim induction procedure has been proposed

to be similar to the initial session that in-

duces learned helplessness by exposing rats

to inescapable stress. Induction of learned

helplessness produces broad-ranging behav-

ioral deficits in affect, cognition, sleep, and

motor performance that closely resemble

many of the symptoms of depression (36).

Additionally, as described above, the sepsis-

surviving group did not present locomotor

activity impairment, supporting the idea that

the longer immobility time in the sepsis

group was related to depressive-like symp-

toms. This finding agrees with clinical stud-

ies that show depressive symptoms in survi-

vors of severe diseases such as sepsis and

septic shock (9,11-13,15).

In summary, our results demonstrated

that survivors of CLP, a classical sepsis

model, presented recognition memory im-

pairment and depressive-like symptoms but

not anxiety-like behavior. These findings,

together with our previous reports (21,22),

indicate that the CLP model could be a good

research tool for the study of the biological

mechanisms involved in the behavioral al-

terations secondary to sepsis.

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In: Nemeroff C, Schatzberg AS (Editors), Textbook of psychophar-

macology. New York: American Psychiatric Association Press; 1998.

p 88-123.

Capítulo 3.

Long-Term Cognitive Impairment in Sepsis

Survivors

Tatiana Barichello, Márcio R. Martins, Adalisa Reinke, Gustavo Feier,

Cristiane Ritter, João Quevedo, Felipe Dal-Pizzol

Critical Care Medicine (2005) 33:1671

Letters to the Editor

Would Patients with More Subtle

Signs of Coagulopathy Have Beneﬁted

from Treatment with Activated

Protein C?

To the Editor:

We read with great interest the article

by Dhainaut and colleagues (1) published

in the February issue of Critical Care Med-

icine. In this study including 840 patients

from the placebo arm of the PROWESS

trial (2), the authors nicely showed that

abnormalities in coagulation parameters

occurring early in the course of sepsis are

associated with an increased risk of organ

dysfunction in the following days and with

higher 28-day mortality, regardless of the

severity of illness as assessed by the Acute

Physiology and Chronic Health Evaluation

(APACHE) II score. Moreover, the authors

found a linear correlation between some

coagulation parameters, such as prothrom-

bin time and D-dimer levels, and 28-day

mortality, suggesting that subtle change in

these parameters, even within the normal

ranges, already may identify patients who

are at risk of developing subsequent organ

dysfunction. Dhainaut et al. subsequently

classiﬁed their patient population into

three groups according to increasing sever-

ity of septic coagulopathy by using a Com-

posite Coagulopathy Score based upon fac-

tors identiﬁed by logistic regression, with

notable outcome differences between the

groups. Since the authors wanted to assess

the natural evolution over time between

coagulation parameters within the 24 hrs

of sepsis and the subsequent development

of organ dysfunction in the following days,

unfortunately only the placebo arm of the

PROWESS trial was included in this study.

It would have been interesting to assess the

efﬁcacy of activated protein C treatment

across the different groups with increasing

coagulopathy score. In particular, it would

have been relevant to know whether pa-

tients with the more subtle signs of coagu-

lopathy have beneﬁted from this treatment.

Recombinant activated protein C has

been approved for the treatment of sepsis in

patients with two or more organ dysfunc-

tions in Europe and an APACHE II score of

more than 25 in the United States. Consid-

ering that activated protein C exerts its

effect through an anticoagulopathic rather

than antiinﬂammatory effect, septic pa-

tients with one organ failure and coagu-

lopathy may be more likely to beneﬁt from

such a treatment than, for example, pneu-

monia patients with progressive renal dys-

function and hypotension (e.g., because of

dehydration and/or a low cardiac output)

without measurable coagulopathy. This is

also supported by data coming from a sub-

group analysis of the PROWESS trial (3),

which revealed that activated protein C was

only efﬁcacious in the subset of patients

with overt diffuse intravascular coagulopa-

thy. Mortality in the treatment vs. placebo

arm was 32% vs. 46.2% (p Ͻ .05) among

patients with overt diffuse intravascular co-

agulopathy (n ϭ 378) and 22.6% versus

26.5% (p Ͼ .05) in patients without it (n ϭ

1,312). However, the deﬁnition of diffuse

intravascular coagulopathy used in this

study is less sensitive (4) than the Compos-

ite Coagulopathy Score identiﬁed by Dhain-

aut et al. (1). Should septic patients with

minor or intermediately severe coagulopa-

thy beneﬁt from activated protein C, or

should its use be restricted to patients with

major coagulopathy or all patients with more

than two organ failures, regardless of the se-

verity of coagulopathy? By including the

treatment arm of the PROWESS trial in their

analysis, Dhainaut et al. might have been able

to address this intriguing question.

Dominique D. Benoit, Pieter O. Depuydt,

Jan J. De Waele, Eric A. Hoste, Kirsten E.

Colpaert, Vanessa D. Van Hende, Johan M.

Decruyenaere, Department of Intensive

Care Medicine, Ghent University Hospital,

Belgium

REFERENCES

1. Dhainaut JF, Shorr AF, Macias WL, et al: Dy-

namic evolution of coagulopathy in the ﬁrst day

of severe sepsis: Relationship with mortality and

organ failure. Crit Care Med 2005; 33:341–347

2. Bernard GR, Vincent JL, Laterre PF, et al:

Efﬁcacy and safety of recombinant human ac-

tivated protein C for severe sepsis. N Engl

J Med 2001; 344:699–709

3. Ely EW, Laterre PF, Angus DC, et al: Drotreco-

gin alfa (activated) administration across clin-

ically important subgroups of patients with

severe sepsis. Crit Care Med 2003; 31:12–19

4. Taylor FB Jr, Toh CH, Hoots WK, et al: Scientiﬁc

and standardization committee communica-

tions: Towards a deﬁnition, clinical and labora-

tory criteria, and a scoring system for dissemi-

nated intravascular coagulation. 2001. Available

online at: http://www.med.unc.edu/isth/dicdef.

htm. Accessed January 15, 2002

DOI: 10.1097/01.CCM.0000166875.23234.95

The authors reply:

We appreciate the comments of Dr.

Benoit and colleagues regarding our

study. Our goal in studying the placebo

population of PROWESS was to examine

the natural history of severe sepsis with-

out the inﬂuence of rhAPC. Speciﬁcally,

we wanted to explore the potential con-

tributions of sepsis-induced coagulopathy

on organ dysfunction and mortality.

Dr. Benoit and associates request infor-

mation regarding the PROWESS groups

treated with recombinant human activated

protein C (rhAPC) and their mortality in

relation to increasing coagulopathy score.

First of all, we need to point out that per-

forming such an analysis is not as straight-

forward as evaluating subgroups based

solely on baseline characteristics. This dif-

ﬁculty arises because the Composite Co-

agulopathy Score utilizes three biomarkers

(antithrombin, D-dimer, prothrombin

time) measured at baseline and on the ﬁrst

day post-baseline. Therefore, a comparison

of the rhAPC- versus placebo-treated pa-

tients is confounded by the use of post-

baseline measures. Nonetheless, there is a

trend toward lower Composite Coagulopa-

thy Scores (Mantel-Haenszel chi-square

test, p ϭ .10) in rhAPC patients, indicating

a trend for improving coagulopathy. This

improvement was observed even though

rhAPC patients tend to have an increase in

prothrombin time (1), a pharmacodynamic

effect of the drug. There was no signiﬁcant

evidence of a differential survival beneﬁt

attributable to rhAPC among the three co-

agulopathy classes (Breslow-Day test, p ϭ

.70).

Dr. Benoit and colleagues suggest that

rhAPC was efﬁcacious in PROWESS only

among the patients with DIC. However, we

are underpowered to make conclusions

about the interaction of Xigris and baseline

DIC on the basis of PROWESS. The infor-

mation that they reference (2) indicates

that the treatment effects of both DIC and

non-DIC patients were generally consistent

with the overall treatment effect, in that

both of their 95% conﬁdence intervals for

the treatment effect encompass the overall

PROWESS treatment effect. In addition, a

recent article summarizes the ﬁndings with

Medicine and Lippincott Williams & Wilkins

1670 Crit Care Med 2005 Vol. 33, No. 7

an improved deﬁnition of DIC for the

PROWESS study (3). This publication indi-

cates a 19% relative risk reduction with

rhAPC for non-DIC patients and a 29%

relative risk reduction for DIC patients. The

Breslow-Day test indicates no statistical ev-

idence that the odds ratios differ (p ϭ .26).

However, as with patients having higher

Composite Coagulopathy Scores, the abso-

lute mortality reduction is higher among

DIC patients, because the placebo mortality

rate is higher among them, whereas the

relative risk reductions are similar between

patients with and without DIC.

Jean-Francois Dhainaut, MD, PhD,

Department of Intensive Care, Cochin

Hospital, AP-HP, Cochin Institute,

Cochin Port-Royal Medical School,

Paris V University, Paris, France;

William L. Macias, MD, Lilly Research

Laboratories, Eli Lilly and Company,

Indianapolis, IN; David R. Nelson, MS,

Lilly Research Laboratories, Eli Lilly

and Company, Indianapolis, IN

REFERENCES

1. Dhainaut JF, Yan SB, Margolis BD, et al: Dro-

trecogin alfa (activated) (recombinant human

activated protein C) reduces host coagulopa-

thy response in patients with severe sepsis.

Thromb Haemost 2003; 90:642–653

2. Ely EW, Laterre PF, Angus DC, et al: Drotreco-

gin alfa (activated) administration across clini-

cally important subgroups of patients with se-

vere sepsis. Crit Care Med 2003; 31:12–19

3. Dhainaut J-F, Yan SB, Joyce DE, et al: Treat-

ment effects of drotrecogin alfa (activated) in

patients with severe sepsis with or without

overt disseminated intravascular coagulation.

J Thromb Haemost 2004; 2:1924 –1933

DOI: 10.1097/01.CCM.0000166889.11063.07

Long-Term Cognitive Impairment in

Sepsis Survivors

To the Editor:

In the January issue of Critical Care

Medicine, Dr. Soriano wrote an editorial (1)

regarding our published article (2) on cog-

nitive deﬁcits of survivors from cecal liga-

tion and perforation (CLP). Dr. Soriano (1)

stresses the major bias associated with our

results: the time after sepsis chosen to per-

form the cognitive parameters. As Dr. So-

riano (1) stated, “The 10 days after CLP

reported in the study may not be a sufﬁ-

ciently long enough period for the animals

to be free from the systemic septic alter-

ations.” After 10 days, the animals seemed

to be free from infection. We performed

blood cultures that were all negative in this

period. The animals recovered their weight

and grooming habits, blood counts re-

turned to control levels, and reactive pro-

tein C values were negative. Because we

believe that the described model will be an

important tool in the study of these cogni-

tive deﬁcits, we extended the results to a

later time point after CLP. Using the same

model described in the article on which Dr.

Soriano commented (2), we demonstrate in

Figure 1A that rats 30 days after the CLP

presented a signiﬁcantly decreased step-

down latency in the inhibitory avoidance

task in the test session. No differences be-

tween groups were demonstrated in the

inhibitory avoidance training session (data

not shown). In addition, 30 days after the

CLP, the continuous multiple-trials step-

down inhibitory avoidance showed a signif-

icant increase in the number of training

trials required to reach the acquisition cri-

terion (50 secs on the platform) in the sep-

sis group compared with the sham group

(Fig. 1B). In the retention test, as demon-

strated previously (2), there was no differ-

ence between groups (data not shown).

These results indicate that the memory and

learning impairment demonstrated 10 days

after CLP (2) persist 30 days after CLP. The

persistency of the alterations 30 days after

CLP reinforces that this model could be an

important tool in the study of the late se-

quela of sepsis survivors. We hope that with

these extended results our work can be a

reference to the development of several

studies designed to understand the factors

and possible treatment options to the long-

term sequela of sepsis survivors.

Tatiana Barichello, PharmD, MSc,

Márcio R. Martins, BSc, Adalisa Reinke,

BSc, Gustavo Feier, Bsc, Cristiane

Ritter, MD, MSc, João Quevedo, MD,

PhD, Felipe Dal-Pizzol, MD, PhD,

Laboratório de Fisiopatologia Experi-

mental (TB, AR, CR, FD-P) and the

Laboratório de Neurociência (MRM,

GF, JQ), Universidade do Extremo Sul

Catarinense, Criciúma, Brazil

REFERENCES

1. Soriano FG: Quality of life: Late sequela in

sepsis. Crit Care Med 2005; 33:262–263

2. Barichello T, Martins MR, Reinke A, et al:

Cognitive impairment in sepsis survivors from

cecal ligation and perforation. Crit Care Med

2005; 33:221–223

DOI: 10.1097/01.CCM.0000170192.54682.C1

Table 1. Data on PROWESS placebo versus rhAPC-treated groups in relation to mortality and

increasing coagulopathy score

CCS

PLC

n (% of PLC)

rhAPC

n (% of rhAPC)

28-Day Mortality

Relative Risk

Reduction,

%PLC, % rhAPC, %

0–1 168 (24.9) 215 (29.9) 14.3 12.6 12

2 327 (48.4) 323 (44.9) 26.9 22.6 16

3–4 180 (26.7) 181 (25.2) 45.6 35.4 22

Mantel-Haenszel p ϭ .10 Breslow-Day p ϭ .70

CCS, composite coagulopathy score; PLC, placebo; rhAPC, recombinant human activated protein C.

Figure 1. A, inhibitory avoidance task. Data are

expressed as median (interquartile ranges) train-

ing and test session latencies, in seconds. *Sig-

niﬁcant difference compared with sham group

(Mann-Whitney U test, p Ͻ .01). Both groups (n

ϭ 14 in the sham group and n ϭ 13 in the sepsis

group) showed signiﬁcant training test differ-

ences (Wilcoxon’s test, p Ͻ .01). B, continuous

multiple-trials step-down inhibitory avoidance.

Data are expressed as mean Ϯ SEM of the number

of training trials required to reach acquisition

criterion (50 secs on the platform). *Signiﬁcant

difference between groups (Student’s t-test, p Ͻ

.001).

1671Crit Care Med 2005 Vol. 33, No. 7

Elevated Body Temperature in the

Neuroscience Intensive Care Unit

To the Editor:

Elevated body temperature is a com-

monly encountered phenomenon in the

neuroscience intensive care unit and is

associated with worsened outcome. The

treatment of these temperature eleva-

tions is frequently suboptimal; thus, I

read with great interest the recent article

by Mayer and colleagues (1) regarding the

use of the Arctic Sun water-circulating

system for treatment of fever in these

patients. The authors are to be com-

mended for recognizing and attempting

to address this clinically signiﬁcant and

often time-consuming problem. The au-

thors concluded that that the Arctic Sun

system was found to be “superior to con-

ventional water-circulating cooling blan-

ket methodology for reducing fever bur-

den in febrile neurocritical care patients.”

However, these results need to be in-

terpreted with caution. As pointed out in

the accompanying commentary (2), the

“conventional methodology” used as the

standard for comparison was not evi-

dence-based practice. As water-circulat-

ing cooling blankets rely on conduction

for heat exchange, the increased surface

area provided by two blankets provides

increased contact (3) and is used in most

institutions as the standard of care. In

addition, the authors report that they set

the SubZero water blanket temperature

at the lowest possible temperature of 4°C.

This is of signiﬁcant concern as Caruso et

al. (3) compared four different water

blanket temperatures from 7 to 24°C in

reducing fever and promoting patient

comfort. They reported that the warmer

water blanket temperatures were as effec-

tive in reducing temperature as colder

temperatures and were better tolerated

by the patients. Unfortunately, the use of

lower water blanket temperatures to

manage fever is a frequent misconception

among clinicians and in patient care pro-

tocols. Due to the increased temperature

gradient at lower blanket temperatures,

patients are more likely to shiver (4),

leading to ineffective therapy. Therefore,

I must wonder if the lack of shivering

reported (and also the lack of effect) of

the standard therapy group could be re-

lated to use of the single overlay blanket.

The authors are to be praised for their

systematic shivering protocol as this aer-

obic activity can have a high metabolic

toll on the neurologically vulnerable pa-

tient. However, their protocol could have

gone a step further as there has long been

evidence supporting the use of protective

wraps on the hands and feet for preven-

tion of shivering (5), not just an interven-

tion to manage it when using conductive

cooling. In addition, although the use of

meperidine for control of shivering/rigors

is well established (5), its use in this pa-

tient population is of particular concern

due to the need for accurate serial neu-

rologic examinations and the increasing

numbers of older adults presenting with

neurologic insults. Lastly, the authors

point to lack of set-point elevation due to

neurogenic hyperthermia as a potential

cause of the lack of shivering seen in

some patients. However, the pioneering

work of Ackerman and Rudy (6) previ-

ously found experimental neurogenic hy-

perthermia to be a highly regulated tem-

perature response, which included

thermogenesis via shivering; thus, active

cooling in these patients should result in

shivering.

The authors can conclude that the

Arctic Sun demonstrated efﬁcacy in re-

ducing fever burden in the neurologically

critically ill patient; however, they did not

demonstrate its superiority to conven-

tional therapy as the comparison protocol

was ﬂawed.

Hilaire J. Thompson, PhD, RN, Uni-

versity of Washington, Seattle, WA

REFERENCES

1. Mayer SA, Kowalski RG, Presciutti M, et al:

Clinical trial of a novel surface cooling system

for fever control in neurocritical care patients.

Crit Care Med 2004; 32:2508 –2515

2. Polderman KH: Keeping a cool head: How to

induce and maintain hypothermia. Crit Care

Med 2004; 32:2558 –2560

3. Caruso CC, Hadley BJ, Shukla R, et al: Cooling

effects and comfort of four cooling blanket

temperatures in humans with fever. Nurs Res

1992; 41:68–72

4. Sund-Levander M, Wahren LK: Assessment

and prevention of shivering in patients with

severe cerebral injury: A pilot study. J Clin

Nurs 2000; 9:55– 61

5. Holtzclaw B: The febrile response in critical

care: State of the science. Heart Lung 1992;

21:482–501

6. Ackerman D, Rudy TA: Thermoregulatory

characteristics of neurogenic hyperthermia in

the rat. J Physiol 1980; 307:59–70

DOI: 10.1097/01.CCM.0000170193.61598.89

Chlorhexidine Body Washing Plus

Topical Polymyxin/tobramycin

To the Editor:

We read with interest the article

published in Critical Care Medicine by

Camus and colleagues (1) reporting the

beneﬁt of a decontamination regimen

associating nasal mupirocin with chlo-

rhexidine body washing plus topical

polymyxin/tobramycin on acquired in-

fections. However, the potential risk of

both immediate and delayed hypersen-

sitivity reactions due to chlorhexidine

should be kept in mind.

The occurrence of life-threatening

immediate hypersensitivity reactions

has been described with chlorhexidine

after topical application, intraurethral

use, exposure to a disinfectant spray

containing chlorhexidine, and after in-

sertion of central catheters impreg-

nated with chlorhexidine. In the UK

during 1965–1996, the Committee on

Safety of Medicines received 182 re-

ported reactions to products containing

chlorhexidine, most of these being cu-

taneous or mucosal eruptions (2). In

Japan, between 1967 and 1989, 15 re-

ported cases of anaphylactic shock were

related to chlorhexidine, of which 13

cases followed application of the drug

to mucous membranes. In 1984, the

Japanese Ministry of Welfare recom-

mended a prohibition on the use of

chlorhexidine on mucous membranes.

Taken together, more than 60 pub-

lished case reports have conﬁrmed the

diagnosis of anaphylaxis due to chlo-

rhexidine (3). In these reported cases,

diagnosis of anaphylaxis was supported

by the onset delay of the reaction (gen-

erally within a few minutes after the

exposure), by the clinical symptoms ob-

served (which were mainly severe, with

hypotension, cardiovascular collapse,

or circulatory arrest sometimes associ-

ated to a bronchospasm), and by the

positivity of the skin tests with chlo-

rhexidine. The severity of the reported

or published cases due to the topical or

intra-urethral use of chlorhexidine or

with chlorhexidine-impregnated cathe-

ters prompted the United States Food

and Drug Administration to issue in

1998 an alert to the medical commu-

nity about the potential for serious hy-

persensitivity reactions to chlorhexi-

dine. This alert stated that because of

reports of anaphylaxis (including one

potentially associated death), chlo-

1672 Crit Care Med 2005 Vol. 33, No. 7

rhexidine-impregnated medical devices

had been banned in Japan (4).

Furthermore, chlorhexidine is also

known to elicit contact dermatitis, occur-

ring predominantly after prolonged or re-

peated application (5). A few cases have

been published, and diagnosis in these

cases was supported by the positivity of the

skin tests with chlorhexidine.

Therefore, the prescription of chlo-

rhexidine should include consideration of

the potential threat of an anaphylactic

reaction and potential event of delayed

hypersensitivity reaction.

Pascale Dewachter, MD, PhD, Claudie

Mouton-Faivre, MD, Paul Michel

Mertes, MD, PhD, CHU Hôpital Cen-

tral, Nancy, France

REFERENCES

1. Camus C, Bellissant E, Sebille V, et al: Preven-

tion of acquired infections in intubated pa-

tients with the combination of two decontam-

ination regimens. Crit Care Med 2005; 33:

307–314

2. Chisholm DG, Calder I, Peterson D, et al:

Intranasal chlorhexidine resulting in anaphy-

lactic circulatory arrest. BMJ 1997; 315:785

3. Dewachter P, Mouton-Faivre C, Mertes PM:

Preventing complications of central venous

catheterization. N Engl J Med 2003; 348:

2684 –2686

4. FDA Public Health Notice: Potential Hyper-

sensitivity Reactions to Chlorhexidine-Im-

pregnated Medical Devices. March 11, 1998.

US Food and Drug Administration, Center for

Devices and Radiological Health, Department

of Health and Human Services. Available at:

http://www.fda.gov/cdrh/chlorhex.html. Ac-

cessed May 16, 2005

5. Ebo DG, Stevens WJ, Bridts CH, et al: Contact

allergic dermatitis and life-threatening ana-

phylaxis to chlorhexidine. J Allergy Clin Im-

munol 1998; 101:128 –129

DOI: 10.1097/01.CCM.0000170194.67542.1C

The authors reply:

Dr. Dewachter and colleagues empha-

size the potential risk of delayed and im-

mediate hypersensitivity reactions due to

chlorhexidine, especially anaphylaxis.

No case of anaphylaxis was reported

during the study (1). The toilet of pa-

tients was made by aid-nurses and con-

sisted of normally washing, then carefully

rinsing with water and wiping, the un-

covered skin and genitalia. Most reported

cases of chlorhexidine-associated anaphy-

laxis occurred after usual disinfection of

wounds, burns, mucous membranes, or

normal skin before surgery, which does

not include rinsing. The risk of this very

rare but potentially life-threatening com-

plication must be weighed against the

much greater risk of severe acquired in-

fections, which are also life-threatening.

Clearly, the overall beneﬁt achieved by

the combination of polymyxin/tobramy-

cin and mupirocin/chlorhexidine favors

the use of this procedure.

Contact sensitivity to chlorhexidine is

less exceptional and is usually a mild ad-

verse event. Irritant contact dermatitis is

a common adverse reaction to chlorhexi-

dine in medical staff. A sensitization rate

of about 2% has been reported in other

studies (2). This rate is consistent with

the 1.9% rate of allergy reported in the

259 patients who were washed with chlo-

rhexidine (Hibiscrub) in our study but

was not different from the 1.2% rate of

allergy in the 256 patients who were

washed with the conventional liquid soap

without chlorhexidine (p ϭ .72). Body

washing with the study medication was

discontinued in all these cases. Chlo-

rhexidine should not be used for body

washing in patients with a history of sen-

sitization to chlorhexidine. We agree with

Dr. Dewachter and colleagues that all cli-

nicians should keep in mind the possibil-

ity of delayed or immediate hypersensi-

tivity reactions after skin application of

chlorhexidine and remove the drug in

any case of allergy with no other obvious

cause.

Christophe Camus, MD, Rémi Thomas,

MD, Service de Maladies Infectieuses

et Réanimation Médicale, Hôpital Pon-

tchaillou, Rennes, France

REFERENCES

1. Camus C, Bellissant E, Sébille V, et al: Preven-

tion of acquired infections in intubated pa-

tients with the combination of two decontam-

ination regimens. Crit Care Med 2005; 33:

307–314

2. Krautheim AB, Jerman TH, Bircher AJ: Chlo-

rhexidine anaphylaxis: Case report and review

of the literature. Contact Dermatitis 2004;

50:113–116

DOI: 10.1097/01.CCM.0000170184.33302.AC

Simpson’s Paradox

To the Editor:

The data reported by Topeli and col-

leagues (1) in the February 2005 issue of

Critical Care Medicine provides a classic

example of the statistical phenomenon

known as Simpson’s paradox. In their

study, the institution of a closed intensive

care unit system, run by an intensivist,

decreased hospital mortality for intu-

bated patients and likewise decreased

hospital mortality for nonintubated pa-

tients. When these samples are com-

bined, however, the remarkable result is

that the outcome is reversed, with an

apparent increase in mortality with the

closed intensive care unit system (Table

1, adapted from original article). The ex-

planation of this apparent paradox lies in

examination of the unequal sample sizes

within the subgroups.

In the open period, aggregate mortality

is weighted toward the large sample of pa-

tients not receiving mechanical ventilation,

a group with lower expected mortality to

begin with. In contrast, during the late

closed period, aggregate mortality is

weighted toward the large sample of pa-

tients treated with mechanical ventilation,

a group with higher expected mortality.

These unequal sample sizes result in aggre-

gate data essentially comparing mortality

between the mechanically ventilated pa-

tients and the nonmechanically ventilated

patients. Thus, although each subgroup ex-

periences improved mortality, data aggre-

gation obscures this trend and, in fact,

completely reverses it, with a highly signif-

icant p Ͻ .001 comparing the open period

with the late closed period.

Table 1. Combined results showing an apparent increase in mortality with the closed intensive care

unit system

Open Period

Early Closed

Period

Late Closed

Period Conclusion

Hospital mortality if mechanically

ventilated (%)

19/21 (90.5) 38/67 (56.7) 79/134 (59.0) Closed better

Hospital mortality if not

mechanically ventilated (%)

32/179 (17.9) 7/82 (8.5) 10/76 (13.2) Closed better

Combined total (%) 51/200 (25.5) 45/149 (30.2) 89/210 (42.4) Closed worse

Summary table adapted from Topeli et al (1).

1673Crit Care Med 2005 Vol. 33, No. 7

This statistical phenomenon, al-

though recognized for many years, was

brought to widespread attention by E. H.

Simpson in 1951 (2) and is thus popularly

referred to as Simpson’s paradox. It oc-

curs in the setting of unequal group sizes

and an unaccounted (or lurking) variable

in the aggregate data—in this case, intu-

bation status. Once the authors identiﬁed

this important variable and analyzed

their data by subgroups, they arrived at

the correct conclusion—implementation

of an intensivist-run, closed intensive

care unit system improved patient sur-

vival in this study—and thus avoided fall-

ing victim to Simpson’s paradox.

Ken Kunisaki, MD, University of Min-

nesota, Minneapolis, MN

REFERENCES

1. Topeli A, Laghi F, Tobin MJ: Effect of closed

unit policy and appointing an intensivist in a

developing country. Crit Care Med 2005; 33:

299 –306

2. Simpson EH: The interpretation of interaction

in contingency tables. J R Stat Soc B 1951;

13:238 –241

DOI: 10.1097/01.CCM.0000170197.42215.F6

The authors reply:

We thank Dr. Kunisaki for taking the

time to respond to our article (1). He has

taken into consideration an important,

yet often overlooked, methodologic issue

and has comprehensively discussed its

potential effects on study results.

In our study, the association between

the intensive care unit system (open or

closed) and intensive care unit mortality

was found to be different among sub-

groups when we stratiﬁed for use of me-

chanical ventilation. Unequal proportions

for use of mechanical ventilation in

closed and open systems led to different

mortality rates in the overall populations.

We agree that data aggregation obscures

the “real” association between intensive

care unit system and mortality. It even

reverses it, and it leads to a paradox when

ventilator use is overlooked.

The mixing of the effect of an extrane-

ous variable with the effects of exposure

(or disease) of interest is termed con-

founding (2). Confounding is not an all-

or-none property of an extraneous vari-

able, and it may occur to varying degrees

in different studies. Sometimes, it is pos-

sible that the confounding variable

changes the direction of an association.

This situation is termed Simpson’s para-

dox (3). As expressed by Rothman (4),

Simpson’s paradox is not really a paradox

but the logical consequence of failing to

recognize the presence of confounding

variables. A confounding variable, if ex-

pected before a study, can be considered in

designing a study and controlled for by

either a) restricting the sample to limited

levels of the potential confounder or b) by

matching the confounder variable (as in a

matched case-control study). In a situation

in which the confounder is detected in

analysis, as was the case in our study, con-

founding can be controlled for by a) strat-

iﬁcation (Table 3 of our original study (1):

the association between the system and

mortality was studied for different sub-

groups of mechanical ventilation) or b)

multivariate analysis techniques (Table 2 of

our original study (1): mechanical ventila-

tion was included in the system, and con-

trolled for as a potential confounder), in

order to obtain valid conclusions (4, 5).

As pointed out by Dr. Kunisaki, use of

stratiﬁed analysis in evaluating the effect

of the intensive care unit system on mor-

tality prevented our study from falling

victim to Simpson’s paradox.

Arzu Topeli, Medical Intensive Care

Unit, Hacettepe University Faculty of

Medicine, Ankara, Turkey; Franco

Laghi, Division of Pulmonary and

Critical Care Medicine, Edward Hines

Jr. Veterans Administration Hospital,

and Loyola University of Chicago

Stritch School of Medicine, Hines, IL;

Banu Cakir, MD, PhD, Department of

Public Health, Hacettepe University

Faculty of Medicine, Ankara, Turkey;

Martin J. Tobin, Division of Pulmo-

nary and Critical Care Medicine,

Edward Hines Jr. Veterans Adminis-

tration Hospital, and Loyola University

of Chicago Stritch School of Medicine,

Hines, IL

REFERENCES

1. Topeli A, Laghi F, Tobin MJ: Effect of closed

unit policy and appointing an intensivist in a

developing country. Crit Care Med 2005; 33:

299 –306

2. Greenberg RS, Daniels SR, Flanders WD, et al:

Medical Epidemiology. Third Edition. Stam-

ford, CT, Lange Medical Books, McGraw-Hill,

2001, pp 149 –150

3. Last J (Ed): A Dictionary of Epidemiology.

Fourth Edition. New York, Oxford University

Press, 2001, p 168

4. Rothman KJ: A pictorial presentation of con-

founding in epidemiologic studies. J Chronic

Dis 1975; 28:101–108

5. Rothman KJ, Greenland S: Modern Epidemi-

ology. Second Edition. Philadelphia, Lippin-

cott-Raven Publishers, 1998, pp 253–255

DOI: 10.1097/01.CCM.0000170185.05101.BE

Critical Illness Polyneuropathy

To the Editor:

We read with interest the article by

Garnacho-Montero and colleagues (1)

that added signiﬁcantly to the under-

standing of critical illness polyneurop-

athy (CIP). This study found that patients

with CIP (diagnosed by neurophysiologic

studies) spent more time on the ventila-

tor in the liberation phase—the time af-

ter recovery of the acute insult leading to

respiratory failure. Because management

of mechanical ventilation is a complex

process, we believe that several questions

remain to properly interpret the conclu-

sions of the study. It is unclear what

practices were in place in the primary

ICU to evaluate patients for ventilator lib-

eration. Although the general criteria

provided for being eligible for ventilator

liberation are consistent with consensus

guidelines (2), it is unclear how uni-

formly these were applied in practice. For

example, those with CIP reached candi-

dacy for “weaning” 5 days later than those

without CIP. This might be due to differ-

ences in the severity of illness between

the groups, a delay in recovery due to

CIP, or differences in management strat-

egies. It is unclear if the clinicians mak-

ing ventilator management decisions re-

mained blinded to the results of the

neurodiagnostic testing. Being alert to

the presence of CIP could alter the ag-

gressiveness of ventilator management

and discontinuation by practitioners be-

cause of a perceived risk of failed ventila-

tor liberation. It might also prompt ear-

lier or more frequent consideration of

tracheostomy. We also wonder if ICU re-

admission rates were different between

patients with CIP and those without.

More than half of hospital deaths oc-

curred outside of the ICU and were more

common in the CIP patients. If ICU read-

mission rates were similar between the

groups, then the increased mortality of

CIP patients in the post-ICU period might

reﬂect a bias toward less aggressive care

(e.g., less likely to readmit to the ICU) in

these patients.

In addition to questions about pro-

cesses of care, we wonder about the

relative utility of routine neurophysio-

logic studies compared with a neuro-

1674 Crit Care Med 2005 Vol. 33, No. 7

muscular physical examination. Detec-

tion of subclinical neurologic dys-

function may explain why Garnacho-

Montero and colleagues (1) reported a

prevalence of CIP (53%) twice that of

ICU-acquired weakness (25%) observed

by De Jonghe et al. (3) when using

bedside neuromuscular exam as the

clinical discriminator. Some studies

suggest that ICU-acquired weakness is

often a combined pathology involving

both muscle and nerve (4). If a thor-

ough physical examination provides

data as useful as the more complex testing

employed by Garnacho-Montero and col-

leagues (1), it would allow clinicians to be

attentive to ICU-acquired weakness without

requiring routine neurophysiologic testing

in all patients with sepsis.

In conclusion, we feel this study raises

important questions about the natural

history of ICU-acquired weakness that de-

serve further clariﬁcation. Advancing our

understanding of the long-term conse-

quences of sepsis is critical for us to prop-

erly plan for patients’ return to their

home and workplaces.

Naeem A. Ali, MD, James O’Brien, MD,

Stephen Hoffmann, MD, The Ohio

State University Medical Center, Co-

lumbus, OH

REFERENCES

1. Garnacho-Montero J, Amaya-Villar R, Garcia-

Garmendia JL, et al: Effect of critical illness

polyneuropathy on the withdrawal from me-

chanical ventilation and the length of stay in

septic patients. Crit Care Med 2005; 33:349 –354

2. MacIntyre NR, Cook DJ, Ely EW Jr, et al:

Evidence-based guidelines for weaning and

discontinuing ventilatory support: a collective

task force facilitated by the American College

of Chest Physicians; the American Association

for Respiratory Care; and the American Col-

lege of Critical Care Medicine. Chest 2001;

120(6 Suppl):375S–395S

3. De Jonghe B, Sharshar T, Lefaucheur JP, et al:

Paresis acquired in the intensive care unit: A

prospective multicenter study. Groupe de Re-

ﬂexion et d’Etude des Neuromyopathies en

Reanimation. JAMA 2002; 288:2859–2867

4. Bednarik J, Lukas Z, Vondracek P: Critical

illness polyneuromyopathy: The electrophysi-

ological components of a complex entity. In-

tensive Care Med 2003; 29:1505–1514

DOI: 10.1097/01.CCM.0000170196.25242.E6

The authors reply:

We appreciate the interesting com-

ments expressed by Dr. Ali and colleagues

regarding our recent contribution to

Critical Care Medicine. The main conclu-

sion of our study is that critical illness

polyneuropathy (CIP) signiﬁcantly

lengthens the duration of mechanical

ventilation and is an independent risk

factor for weaning failure in a cohort of

critically ill septic patients (1). This ﬁnd-

ing is in agreement with another recent

publication that reported similar results

in a heterogeneous cohort of mechani-

cally ventilated patients (2).

Apart from the presence of CIP, pro-

longed mechanical ventilation might be

due to other factors, such as the severity

of illness, the presence of co-morbidities,

or the total doses of sedatives adminis-

tered to the patients. These variables

were analyzed in our study, and we found

no differences between patients with CIP

and without CIP or between patients with

and without weaning failure according to

our reported deﬁnition. It is worth noting

that these variables were not recorded in

clinical trials comparing different wean-

ing modes, adding an extraordinary value

to our results. Obviously, prolonged me-

chanical ventilation can be explained by

the fact that the physicians in charge of

the patients were not blinded to the re-

sults of the neurophysiologic evaluation

(the investigator who performed all these

evaluations was unaware of the patient’s

medical condition). Nevertheless, daily,

the attending physicians assessed patient

readiness for liberation from mechanical

ventilation following an updated protocol

as it is done in clinical trials evaluating

different weaning approaches (3, 4).

It is true that more than half of the

deaths occurred after discharge from the

intensive care unit. This may be ex-

plained by the fact that these patients are

usually elderly patients with severe weak-

ness, which makes them extremely vul-

nerable in the post–intensive care unit

period (5). Interestingly, there is a lack of

information available in the medical lit-

erature about the evolution of patients

with CIP after being discharged from the

intensive care unit.

Finally, a very high rate of CIP has

been reported in adults with sepsis and

multiple organ dysfunction syndrome.

Witt et al. (6) carried out the ﬁrst pro-

spective study in a cohort of 43 patients

with sepsis and multiple organ dysfunc-

tion syndrome, and 70% of these patients

were diagnosed with CIP. Subsequent

prospective studies have reported a wide

prevalence (0–85%), depending on the

group of critically ill patients evaluated,

the timing of the electrophysiologic in-

vestigation, and the deﬁnitions used for

identifying neuropathy (7). Very recently,

clinically relevant paresis was found in

60% of the patients in the recovery of an

episode of acute respiratory distress syn-

drome. The neurophysiologic evaluation

was consistent with CIP in all except two

of these patients (8).

To summarize, there is compelling ev-

idence that CIP can inﬂuence the man-

agement and course of critically ill pa-

tients. In fact, mechanical ventilation is

prolonged by the development of this

neurologic complication. Because of the

harmful consequences that this may cause,

further studies are warranted to assess var-

ious interventions (different weaning strat-

egies, early tracheostomy, early use of non-

invasive after extubation) that could help to

improve patient outcome.

Jose Garnacho-Montero, MD, PhD,

Rosario Amaya-Villar, MD, Carlos

Ortiz-Leyba, MD, PhD, Intensive Care

Unit, Hospital Universitario Virgen del

Rocío, Sevilla, Spain

REFERENCES

1. Garnacho-Montero J, Amaya-Villar R, García-

Garmendía JL, et al: Effect of critical illness

polyneuropathy on the withdrawal from me-

chanical ventilation and the length of stay in

septic patients. Crit Care Med 2005; 33:349 –354

2. De Jonghr B, Bastuji-Garin S, Sharshar T, et

al: Does ICU-acquired paresis lengthen wean-

ing from mechanical ventilation. Intensive

Care Med 2004; 30:1117–1121

3. Esteban A, Frutos F, Tobin MJ, et al: A com-

parison of four methods of weaning patients

from mechanical ventilation. N Engl J Med

1995; 332:345–350

4. Esteban A, Alia I, Tobin MJ, et al: Effect of

spontaneous breathing trial duration on out-

come of attempts to discontinue mechanical

ventilation. Am J Respir Crit Care Med 1999;

159:512–518

5. Garnacho-Montero J, Madrazo-Osuna J,

García Garmendia JL, et al: Critical illness

polyneuropathy: Risk factors and clinical con-

sequences. A cohort study in septic patients.

Intensive Care Med 2001; 27:1288 –1296

6. Witt NJ, Zochodne DW, Bolton CF, et al: Pe-

ripheral nerve function in sepsis and multiple

organ failure. Chest 1991; 99:176 –184

7. Garnacho-Montero J, Madrazo-Osuna J,

García Garmendia JL, et al: Neuromuscular

disorders of the critically ill patient. Clin Pulm

Med 2001; 8:354 –359

8. Bercker S, Weber-Carstens S, Deja M, et al:

Critical illness polyneuropathy and myopathy

in patients with acute respiratory syndrome.

Crit Care Med 2005; 33:711–715

DOI: 10.1097/01.CCM.0000170407.44717.89

1675Crit Care Med 2005 Vol. 33, No. 7

Mixing Up Old Data

To the Editor:

In their secondary analysis of an inter-

national study of mechanical ventilation

practices, Ferguson and colleagues (1)

demonstrated considerable interpatient

variability in tidal volumes. Indeed, mean

tidal volume was 8.8 mL/kg measured

body weight with an

SD of 2.2 mL/kg

measured body weight. Several com-

ments can and must be made regarding

the usefulness and adequacy of this new

analysis of old data.

First, it is questionable whether all

practitioners were aware of the role of

tidal volume settings in ventilator-

induced lung injury at the moment of

collecting data. Indeed, almost all studies

on lower tidal volumes have been pub-

lished at or directly after that time (2–5).

It would be far more interesting to see

how tidal volumes are set nowadays, sev-

eral years after the landmark study by the

Acute Respiratory Distress Syndrome

Network (6). Several studies (7–9) show

that tidal volumes are presently still too

large.

Second, concerns arise on the absence

of data on height of the studied subjects,

making it only possible to express tidal

volumes in milliliters per kilogram of ac-

tual body weight. Although actual body

weight exceeded predicted body weight by

approximately 20% in one study (6), it is

not correct to simply state that “mean

tidal volume of 8.8 mL/kg actual body

weight might be equivalent to 10 –11

mL/kg predicted body weight” in the

present analysis because weight was not

normally distributed among the several

groups. Indeed, signiﬁcantly higher

weight values were registered in patients

mechanically ventilated with lower tidal

volumes, whereas weight values were sig-

niﬁcantly lower in those mechanically

ventilated with higher tidal volumes. Did

actual body weight exceed predicted body

weight evenly in all groups?

This also raises a third concern. Were

the heavier patients just taller, or did

they have a higher body mass index? High

body mass index is associated with mor-

tality (10). Interestingly, from the present

analysis, one might also conclude that

patients with a higher risk of death (those

that were heavier) were mechanically

ventilated with a more lung-protective

strategy than patients with a lower risk of

death.

Thus, although the investigators were

able to examine patterns of tidal volume

use, it is hard to draw ﬁrm conclusions

because important information (height)

is missing.

Marcus J. Schultz, MD, PhD, Esther K.

Wolthuis, MD, MSc, Rogier M.

Determann, MD, MSc, Department of

Intensive Care Medicine, Academic

Medical Center, Amsterdam, The

Netherlands

REFERENCES

1. Ferguson ND, Frutos-Vivar F, Esteban A, et

al: Airway pressures, tidal volumes, and mor-

tality in patients with acute respiratory dis-

tress syndrome. Crit Care Med 2005; 33:

21–30

2. Brochard L, Roudot-Thoraval F, Roupie E, et

al: Tidal volume reduction for prevention of

ventilator-induced lung injury in acute respi-

ratory distress syndrome: The Multicenter

Trial Group on Tidal Volume reduction in

ARDS. Am J Respir Crit Care Med 1998;

158:1831–1838

3. Stewart TE, Meade MO, Cook DJ, et al: Eval-

uation of a ventilation strategy to prevent

barotrauma in patients at high risk for acute

respiratory distress syndrome: Pressure- and

Volume-Limited Ventilation Strategy Group.

N Engl J Med 1998; 338:355–361

4. Amato MB, Barbas CS, Medeiros DM, et al:

Effect of a protective-ventilation strategy on

mortality in the acute respiratory distress

syndrome. N Engl J Med 1998; 338:347–354

5. Brower RG, Shanholtz CB, Fessler HE, et al:

Prospective, randomized, controlled clinical

trial comparing traditional versus reduced

tidal volume ventilation in acute respiratory

distress syndrome patients. Crit Care Med

1999; 27:1492–1498

6. Ventilation with lower tidal volumes as com-

pared with traditional tidal volumes for acute

lung injury and the acute respiratory distress

syndrome: The Acute Respiratory Distress

Syndrome Network. N Engl J Med 2000; 342:

1301–1308

7. Weinert CR, Gross CR, Marinelli WA: Impact

of randomized trial results on acute lung

injury ventilator therapy in teaching hospi-

tals. Am J Respir Crit Care Med 2003; 167:

1304 –1309

8. Young MP, Manning HL, Wilson DL, et al:

Ventilation of patients with acute lung injury

and acute respiratory distress syndrome: Has

new evidence changed clinical practice? Crit

Care Med 2004; 32:1260–1265

9. Wolthuis EK, Korevaar JC, Spronk P, et al:

Feedback and education improve physician

compliance in use of lung-protective me-

chanical ventilation. Intensive Care Med

2005; 31:540–546

10. Goulenok C, Monchi M, Chiche JD, et al: In-

ﬂuence of overweight on ICU mortality: A Pro-

spective study. Chest 2004; 125:1441–1445

DOI: 10.1097/01.CCM.0000170198.14613.F4

The authors reply:

We thank Dr. Schultz and colleagues

for their interest in our article that ex-

amined ventilation practices and out-

come associations in patients with acute

respiratory distress syndrome (1). They

point out that most of the clinical studies

of lung-protective ventilatory strategies

were published at or after the time the

data for this report were collected. We

agree wholeheartedly that determining

whether there has been a temporal

change in tidal volume prescriptions dur-

ing the intervening 7-yr period is an in-

teresting and important future research

question. Our data from the current pub-

lication may serve as a reference point

against which to judge this potential

change.

We also agree that it is difﬁcult to

draw ﬁrm conclusions about the effect of

different tidal volumes without the ability

to standardize these for height. This is

why we instead focused on the observed

variability in tidal volume. We would

point out that when we stated that 8.8

mL/kg actual body weight might be

equivalent to 10 –11 mL/kg predicted

body weight, we qualiﬁed that this ex-

trapolation should be done cautiously. In

addition, we were referring to the global

mean tidal volume for all patients, in

which the distribution was approximately

normal (Fig. 1 in the original article), not

to individual tidal volume groups as Dr.

Schultz and colleagues imply.

Finally, in the absence of height data,

any statements regarding the inﬂuence of

body mass index are purely speculative.

Now that height-based predicted body

weights have become widely used (which

was not the case in 1998), this will be an

important variable to consider in future

studies.

Again, we thank Dr. Schultz and col-

leagues for the opportunity to clarify the

issues above and to reassert the conclu-

sions we drew from this work: 1) tidal

volumes varied considerably during the

study period, 2) late-onset acute respira-

tory distress syndrome and low levels of

positive end-expiratory pressure were in-

dependently associated with increased

mortality, and 3) no evidence of increased

mortality was observed at lower inspira-

tory pressures.

Niall D. Ferguson, MD, FRCPC, MSc,

Thomas E. Stewart, MD, FRCPC, In-

terdepartmental Division of Critical

Care Medicine, University Health Net-

1676 Crit Care Med 2005 Vol. 33, No. 7

work and Mount Sinai Hospital, Uni-

versity of Toronto, Toronto, Ontario,

Canada; Fernando Frutos Vivar, MD,

Andrés Esteban, MD, PhD, Unidad de

Cuidados Intensivos, Hospital Univer-

sitario de Getafe, Madrid, Spain

REFERENCES

1. Ferguson ND, Frutos Vivar F, Esteban A, et al:

Airway pressures, tidal volumes, and mortality

in patients with the acute respiratory distress

syndrome. Crit Care Med 2005; 33:21–30

DOI: 10.1097/01.CCM.0000170945.82570.FF

Clinical Relevance of the LRINEC

(Laboratory Risk Indicator for

Necrotizing Fasciitis) Score for

Assessment of Early Necrotizing

Fasciitis

To the Editor:

Early diagnosis of necrotizing fasciitis

with subsequent operative debridement

has been shown in many studies to im-

prove survival (1, 2). However, delayed

diagnosis is frequently seen because early

in the evolution of this disease, it is often

clinically indistinguishable from other

more benign soft-tissue infections such

as cellulitis. We developed the Laboratory

Risk Indicator for Necrotizing Fasciitis

(LRINEC) score (3) with the hope that

routine laboratory parameters for the as-

sessment of severe soft-tissue infection

may yield diagnostic clues of the presence

of necrotizing soft-tissue infection, even

early in the evolution of the disease when

clinical ﬁndings were nondiagnostic.

The LRINEC score was developed ret-

rospectively by comparing the laboratory

parameters (complete blood count, elec-

trolytes, erythrocyte sedimentation rate,

and C-reactive protein) of patients with

proven necrotizing fasciitis vs. patients

with severe soft-tissue infections. On val-

idation with our own data, at a cutoff of a

LRINEC score of Ն6, the score has a

positive predictive value of 92.0% and

negative predictive value of 96.0% (3).

One weakness of this study we acknowl-

edge is its retrospective nature that pre-

disposes the study to selection bias (4).

Prospective validation of the score is

needed before routine application can be

recommended.

Wang and Hung (5) recently per-

formed a prospective study using tissue

saturation monitoring for diagnosing ne-

crotizing fasciitis. A total of 234 consec-

utive patients who fulﬁlled the United

States Centers for Disease Control and

Prevention criteria of soft-tissue infec-

tions were enrolled into the study. Of

these 234 patients, 19 were later con-

ﬁrmed to have necrotizing fasciitis and

215 patients had cellulitis. Routine pa-

rameters for evaluation of soft-tissue in-

fection were taken at admission. In this

study, the majority of cases were early

necrotizing fasciitis. Early diagnosis was

possible because of a rigorous protocol

that combined tissue oxygen saturation

monitoring, computed tomographic

scanning, and tissue biopsy. As a second-

ary analysis, Dr. Wang (6) used the LRI-

NEC score in his study subjects and

found a positive predictive value of 40%

and a negative predictive value of 95%.

The performance of the LRINEC score

in this prospective model shows a high

speciﬁcity but a low sensitivity. The im-

plications are two-fold. First, the majority

Dr. Wang’s patients had early necrotizing

fasciitis. A negative predictive value of

95% (a low false-negative rate) shows

that the LRINEC score will not miss these

cases (early necrotizing fasciitis). Second,

from a clinical standpoint, speciﬁcity is

more important than sensitivity. To this

end, the LRINEC score seems to fulﬁl the

purpose for which it was originally de-

vised. When the score is Ͻ6, necrotizing

fasciitis is quite unlikely. From Dr.

Wang’s data of a positive predictive value

of 40%, a high false-positive rate is ex-

pected for early necrotizing fasciitis. For

patients classiﬁed as high risk based on

the LRINEC score, urgent further evalu-

ation is needed. The use of additional

tools, such as computed tomography,

magnetic resonance imaging, tissue bi-

opsy, or tissue oxygen monitoring can

exclude or conﬁrm necrotizing fasciitis.

The LRINEC score therefore functions as

tool to limit and more importantly target

the use of these expensive (but more sen-

sitive) modalities to such high-risk pa-

tients. We hope that early diagnosis and

reduction in mortality is possible with

such an approach.

Chin-Ho Wong, MD, MRCS(Ed), De-

partment of Plastic, Reconstructive

and Aesthetic Surgery, Singapore

General Hospital, Singapore; Lay-Wai

Khin,MD, MSC, Clinical Trials and Ep-

idemiology Research Unit, National

Medical Research Council (NMRC),

Ministry of Health, Singapore

REFERENCES

1. Wong CH, Chang HC, Pasupathy S, et al: Ne-

crotizing fasciitis: Clinical presentation, mi-

crobiology and determinants of mortality.

J Bone Joint Surg (Am) 2003; 85-A:1454 –1460

2. McHenry CR, Piotrowski JJ, Petrinic D, et al:

Determinants of mortality in necrotizing soft

tissue infections. Ann Surg 1995; 221:

558 –563

3. Wong CH, Khin LW, Heng KS, et al: The

LRINEC (Laboratory Risk Indicator for Necro-

tizing Fasciitis) score: A tool for distinguish-

ing soft tissue infection from other soft tissue

infections. Crit Care Med 2004; 32:1535–1541

4. Barie PS: The laboratory risk indicator for

necrotizing fasciitis (LRINEC) score: Useful

tool or paralysis by analysis? Crit Care Med

2004; 32:1618–1619

5. Wang TL, Hung CR: Role of tissue oxygen

saturation monitoring in diagnosing necrotiz-

ing fasciitis of the lower limbs. Ann Emerg

Med 2004; 44:222–228

6. Wang TL: Tissue oxygen saturation monitor-

ing in the diagnosing necrotizing fasciitis: A

valuable tool but only for a select few. Reply.

Ann Emerg Med 2005; 45:462– 463

DOI: 10.1097/01.CCM.0000170199.43624.B8

1677Crit Care Med 2005 Vol. 33, No. 7

Capítulo 4.

Oxidative variables in the rat brain after sepsis induced

by cecal ligation and perforation

Tatiana Barichello, Jucélia J. Fortunato, Ângeles M. Vitali, Gustavo

Feier, Adalisa Reinke, José Cláudio F. Moreira, João Quevedo, Felipe

Dal-Pizzol

Critical Care Med (2006) 34:886-889

Brief Report

Oxidative variables in the rat brain after sepsis induced by cecal

ligation and perforation

Tatiana Barichello, PharmD, MSc; Jucélia J. Fortunato, MSc;

Angeles M. Vitali, BSc; Gustavo Feier, BSc;

Adalisa Reinke, BSc; José Cláudio F. Moreira, PhD; João Quevedo, MD, PhD; Felipe Dal-Pizzol, MD, PhD

epsis and septic shock have be-

come some of the most fre-

quent causes of morbidity and

mortality in intensive care

units. Neurologic abnormalities during

sepsis development include agitation,

confusion, disorientation, lethargy, and

coma, which are early characteristic ﬁnd-

ings in these patients (1). The concept of

septic encephalopathy as an entity that

cannot be explained by hepatic or renal

dysfunction, hypotension, or hypoxia is

relatively new and has been reported to

occur in a range of 8 –70% of septic pa-

tients, usually associated with a poor out-

come (1). In addition, sepsis survivors

present long-term cognitive deﬁcits that

could be secondary to central nervous

system alterations during sepsis develop-

ment (2). The underlying mechanisms of

the changes in mental status, septic en-

cephalopathy, and long-term cognitive

symptoms in sepsis survivors have only

been deﬁned in part (1).

The systemic inﬂammation resulting

from infection or other causes appears to

be the cause of septic encephalopathy (1).

Inﬂammatory mediators released by leu-

kocytes (such as tumor necrosis factor

and reactive oxygen species) in sepsis

have profound effects on endothelial

cells, astrocytes, and neurons; damage to

these cells results in impaired central

nervous system function (1).

Studies of sepsis in humans are difﬁ-

cult because the seriousness of the dis-

ease mandates immediate intervention

and because the heterogeneity of pa-

tients’ presentations imposes substantial

limitations on clinical trials. Thus, ani-

mal models have been used extensively to

explore the pathogenesis of sepsis and to

generate preclinical data for therapeutic

interventions. In this way we recently

demonstrated that oxidative stress might

have a major role in the development of

sepsis and severe hepatic failure (3, 4). In

addition, we demonstrated that oxidative

damage in the rat brain might be relevant

to the outcome after hepatic failure in an

animal model (4). To the best of our

knowledge there is no report that de-

scribes oxidative variables in different

brain regions after sepsis induction.

Thus, the aim of this study was to

investigate the temporal variation of dif-

ferent oxidative variables in several brain

structures after sepsis induction in rats.

MATERIALS AND METHODS

In vivo studies were performed in accor-

dance with National Institutes of Health

guidelines and approved by the Ethics Com-

mittee of Universidade do Extremo Sul Catari-

nense.

Cecal Ligation Puncture (CLP) Model.

Male Wistar rats 2–3 months old, weighing

250 –350 g, were subjected to CLP (14-gauge

From Laboratório de Neurociências (TB, JJF, AMV,

GF, JQ) and Laboratório de Fisiopatologia Experimental

(AR, JCFM, FD-P), Universidade do Extremo Sul Ca-

tarinense, Criciúma, SC, Brazil; and Centro de Estudos

em Estresse Oxidativo (JCFM), Universidade Federal do

Rio Grande do Sul, Porto Alegre, RS, Brazil.

Supported, in part, by grants from UNESC, CNPq,

and FUNCITEC.

Address requests for reprints to: Felipe Dal Pizzol,

MD, PhD, Laboratório de Fisiopatologia Experimental,

Universidade do Extremo Sul Catarinense, Criciúma,

SC, Brazil, Avenida Universitária, 1105, 88006-000.

E-mail: [email protected]

Medicine and Lippincott Williams & Wilkins

DOI: 10.1097/01.CCM.0000201880.50116.12

Objective: The underlying mechanisms of the changes in men-

tal status, septic encephalopathy, and long-term cognitive symp-

toms in sepsis survivors have only been deﬁned in part. The

present study was undertaken to assess different variables of

oxidative stress in several brain structures after cecal ligation and

perforation in the rat.

Design: Prospective animal study.

Setting: Animal basic science laboratory.

Subjects: Male Wistar rats, weighing 250 –350 g.

Interventions: Rats were subjected to cecal ligation and per-

foration (sepsis group) with saline resuscitation (at 50 mL/kg

immediately and 12 hrs after cecal ligation and perforation) or

sham operation (control group).

Measurements and Main Results: Oxidative damage, assessed

by the thiobarbituric acid reactive species and the protein car-

bonyl assays, occurred early (after 6 hrs) in the course of sepsis

development in the hippocampus, cerebellum, and cortex. At

longer times after sepsis induction (12–96 hrs), there was no

evidence of oxidative damage in all analyzed structures. Except

for the striatum, earlier in sepsis development (6 hrs) we dem-

onstrated an increase in superoxide dismutase activity without a

proportional increase in catalase activity with a consequent in-

crease in the relation of superoxide dismutase/catalase. The

balance between these enzymes was restored in the studied

structures 12–96 hrs after sepsis induction.

Conclusions: The short-term oxidative damage demonstrated

here could participate in the development of central nervous

system symptoms during sepsis development, or even septic

encephalopathy. The alterations in the superoxide dismutase/

catalase relation were temporally related to the occurrence or not

of oxidative damage in the central nervous system. (Crit Care Med

2006; 34:886–889)

EY WORDS: septic shock; oxidative stress; free radicals; rat

brain; septic encephalopathy

886 Crit Care Med 2006 Vol. 34, No. 3

Figure 1. Oxidative variables in the rat brain during sepsis development. Rats were submitted to cecal ligation and perforation (CLP) (14-gauge puncture)

or sham operation. Animals were killed, and brain structures (hippocampus, striatum, cortex, and cerebellum) were isolated 0 (immediately after), 6, 12,

24, 48, and 96 hrs after CLP to determine (A) thiobarbituric acid reactive species (TBARS), (B) protein carbonyls, (C) superoxide dismutase (SOD) activity,

and (D) catalase (CAT) activity as described in “Material and Methods.” Values are expressed as mean Ϯ SD (n ϭ 5 for each group). Oxidative variables were

not statistically different between the sham and 0-hr group (data not shown). Oxidative variables were not statistically different between sham groups when

comparing all time point analyzed; thus we presented in the graphs only one sham group. *Different from sham operated (p Ͻ .05).

887Crit Care Med 2006 Vol. 34, No. 3

needle) as previously described (3). A sham

operation (laparotomy and cecal exposure

without any more manipulation) was per-

formed as control. The sham and CLP groups

were allocated randomly during the proce-

dure. To avoid hypotension, animals were re-

suscitated immediately after CLP with normal

saline (50 mL/kg of body weight subcutane-

ously, bolus injection every 12 hrs in the ﬁrst

day) (3). Several times (0, 6, 12, 24, 48, and 96

hrs) after CLP ﬁve rats were killed by decapi-

tation, and brain structures (cerebellum, hip-

pocampus, striatum, and cortex) were imme-

diately isolated and stored at Ϫ80°C for

posterior analyses. Sham animals (n ϭ 5) were

killed at the same times after operation. Four-

teen animals died during the 96 hrs and were

excluded from the oxidative variable analyses.

All animals presented signs of encephalopathy

at 6 hrs after sepsis (lethargy, mild ataxia, lack

of spontaneous movement, loss of righting

reﬂex). Animals gradually returned to their

normal awake status 24 –36 hrs after CLP, but

some remained lethargic up to 96 hrs.

Measurement of Thiobarbituric Acid Reac-

tive Species (TBARS). As an index of lipid

peroxidation, we used the formation of TBARS

during an acid-heating reaction as previously

described (3). Brieﬂy, the samples were mixed

with 1 mL of trichloroacetic acid 10% and 1

mL of thiobarbituric acid 0.67% and then

heated in a boiling water bath for 15 mins.

TBARS were determined by the absorbance at

535 nm.

Measurement of Protein Carbonyls. The

oxidative damage to proteins was assessed by

the determination of carbonyl groups based

on the reaction with dinitrophenylhidrazine

as previously described (4). Brieﬂy, proteins

were precipitated by the addition of 20%

trichloroacetic acid and redissolved in dini-

trophenylhidrazine, and the absorbance was

read at 370 nm.

Measurement of Catalase (CAT) and Su-

peroxide Dismutase (SOD) Activity. To deter-

mine CAT activity, samples were sonicated in

50 mM phosphate buffer, and the resulting

suspension was centrifuged at 3000 ϫ g for 10

mins. The supernatant was used for enzyme

assay. CAT activity was measured by the rate of

decrease in hydrogen peroxide absorbance at

240 nm (3). SOD activity was assayed by mea-

suring the inhibition of adrenaline auto-

oxidation, as previously described (3).

Protein Measurements. All the results were

normalized by protein concentration mea-

sured by the Lowry assay.

Reagents. Thiobarbituric acid, catalase, su-

peroxide dismutase, dinitrophenylhidrazine,

adrenaline, hydrogen peroxide, luminal, and

succinate were purchased from Sigma Chem-

ical (St. Louis, MO). We purchased 2,2'-azobis

(2-28 methylpropionamidine) dihydrochloride

from Aldrich Chemical (Milwaukee, WI).

Statistical Analysis. All data are presented

as mean Ϯ

SD. Data were analyzed by two-way

analysis of variance, and multiple comparisons

were performed by Newman-Keuls’ test. To

determine whether data were normally dis-

tributed, we performed Bartlett’s test for ho-

mogeneity of variance. We considered p Ͻ .05

to be signiﬁcant.

RESULTS

We demonstrate in Figure 1, A and B,

that oxidative damage, assessed by

TBARS and the protein carbonyl assays,

occurred early in the course of sepsis

development in several brain regions.

The TBARS levels were elevated in the

hippocampus, the cortex, and the cere-

bellum 6 hrs after sepsis induction (p Ͻ

.05; Fig. 1A). In contrast, carbonyl levels

were increased only in the hippocampus

6 hrs after sepsis induction (p Ͻ .05; Fig.

1B). These suggest that oxidative damage

was more consistent in lipids compared

with proteins. In longer times after sepsis

induction (12–96 hrs), there was no evi-

dence of oxidative damage in all analyzed

structures. In contrast, both TBARS and

protein carbonyls levels tended to be

lower from 12 to 96 hrs after sepsis in-

duction.

To determine enzymatic antioxidant

status during sepsis development in the

rat brain, we determined the activity of

the main central nervous system antiox-

idant enzymes (CAT and SOD). We had

Table 1. Superoxide dismutase/catalase relation in the rat brain during sepsis development

Cerebellum Hippocampus Striatum Cortex

Sham 7.50 ϩ 1.2 12.39 ϩ 2.3 62.94 ϩ 5.4 31.62 ϩ 3.8

6 hrs 147.63 ϩ 17

133.18 ϩ 23

24.28 ϩ 4.7 172.42 ϩ 28

12 hrs 14.88 ϩ 1.8 13.60 ϩ 2.3 83.71 ϩ 6.9 56.87 ϩ 9.8

24 hrs 23.09 ϩ 3.9 31.82 ϩ 3.8 16.94 ϩ 1.3

30.65 ϩ 4.5

48 hrs 6.97 ϩ 2.1 1.57 ϩ 0.12

69.75 ϩ 6.3 44.47 ϩ 5.7

96 hrs 21.19 ϩ 6.7 26.47 ϩ 4.5 28.22 ϩ 4.8 13.99 ϩ 4.6

Different from sham (p Ͻ .05).

Figure 1. Continued.

888 Crit Care Med 2006 Vol. 34, No. 3

previously described that an imbalance

between SOD and CAT activities in heart,

kidney, lung, and diaphragm is, in part,

responsible for the oxidative damage and

outcome in the CLP model (3). Except for

the striatum, earlier in sepsis develop-

ment (6 hrs) we demonstrated an in-

crease in SOD activity without a propor-

tional increase in CAT activity (p Ͻ .05;

Fig. 1C and 1D) with consequent increase

in the relation of SOD and CAT (Table 1).

Thus, an imbalance in SOD and CAT ac-

tivity occurred at the same time and in

the same structures in which we demon-

strated oxidative damage. This early oxi-

dative damage and imbalance in antioxi-

dant enzymes seemed not to be secondary

to renal or hepatic failure since in this

early time we had no alterations in plas-

matic markers of renal and hepatic func-

tion (data not shown). In addition, it

seemed not to be secondary to hypoten-

sion since the study design with ﬂuid

resuscitation was not associated with hy-

potension at 6 hrs after sepsis induction.

DISCUSSION

We demonstrate for the ﬁrst time that,

differently from other organs involved in

septic response, central nervous system

oxidative stress is restricted to earlier

times after sepsis induction. These re-

sults are intriguing since oxidative dam-

age occurs in lung, liver, heart, and kid-

ney for longer times after sepsis

induction (3). Abd El-Gawad and Khalifa

(5) demonstrated that as early as 2 hrs

after lipopolysaccharide administration

there was an increase in free radicals gen-

eration in the rat brain, but these authors

did not analyze oxidative variables at

longer times after lipopolysaccharide ad-

ministration. In contrast, it was demon-

strated that oxidative stress occurred un-

til 48 hrs after sepsis induction the rat

brain (6, 7). However, in these reports

ﬂuid resuscitation was administered only

once, and this could induce late hypoten-

sion (6, 7). Supporting our results, Mes-

saris et al. (8) recently demonstrated mi-

tochondrial-mediated apoptosis in the rat

brain early (6 –12 hrs), but not later, in

sepsis development. These mitochondrial

alterations could be one of the mecha-

nisms associated with brain oxidative

stress. In addition, the impairment of as-

trocytic clearance of dehydroascorbic

acid from the extracellular ﬂuid and con-

sequent decrease of intracellular ascor-

bate concentration could be associated

with central nervous system oxidative

stress (9). This idea is reinforced by the

decrease in cerebrospinal ﬂuid ascorbate

levels in patients with septic encephalop-

athy (10). Furthermore, sepsis induces

inducible nitric oxide synthase and inhib-

its glutamate uptake by astrocytes

through mechanisms that can be modu-

lated by intracellular ascorbate (9). The

protection of the central nervous system

from oxidative damage seems to be an

adaptation to minimize dysfunction and

could be related to central nervous sys-

tem antioxidant defenses adaptation,

diminution of central nervous system

metabolism, or several other unstudied

factors.

We believe that, as we demonstrated

for other organs (3), an imbalance be-

tween SOD and CAT could be responsible,

in part, for the occurrence of oxidative

damage in the rat brain. The balance be-

tween these enzymes was restored in the

studied structures 12–96 hrs after sepsis

induction. In these times we demon-

strated that both SOD and CAT activity

did not change or increase proportionally

or that the increase in CAT activity was

higher than SOD activity increase (Fig.

1C and 1D). In these later times we could

not demonstrate an increase in the rela-

tion of SOD and CAT (Table 1) and oxi-

dative damage in all analyzed structures.

Thus, the alterations in the SOD/CAT re-

lation were temporally related to the oc-

currence or not of oxidative damage in

the central nervous system. To the best of

our knowledge there are no reports in the

literature that describe SOD and CAT

modulation in the rat brain after sepsis

induction.

We cannot ascertain that the short-

term oxidative damage demonstrated

here can lead to central nervous system

symptoms during sepsis development, or

even septic encephalopathy. Further

studies must determine the relation be-

tween the demonstrated short-term oxi-

dative damage and the development of

septic encephalopathy or long-term cog-

nitive impairments in sepsis survivors.

REFERENCES

1. Papadopoulos MC, Davies DC, Moss RF, et al:

Pathophysiology of septic encephalopathy: A

review. Crit Care Med 2000; 28:3019–3024

2. Barichello T, Martins MR, Reinke A, et al:

Cognitive impairment in sepsis survivors

from cecal ligation and perforation. Crit Care

Med 2005; 33:221–223

3. Ritter C, Andrades ME, Reinke A, et al: Treat-

ment with N-acetylcysteine plus deferox-

amine protects rats against oxidative stress

and improves survival in sepsis. Crit Care

Med 2004; 32:342–349

4. Ritter C, Reinke A, Andrades M, et al: Pro-

tective effect of N-acetylcysteine and deferox-

amine on carbon tetrachloride-induced acute

hepatic failure in rats. Crit Care Med 2004;

32:2079 –2083

5. Abd El-Gawad HM, Khalifa AE: Quercetin,

coenzyme Q10, and L-canavanine as protec-

tive agents against lipid peroxidation and ni-

tric oxide generation in endotoxin-induced

shock in rat brain. Pharmacol Res 2001; 43:

257–263

6. Sener G, Toklu H, Kapucu C, et al: Melatonin

protects against oxidative organ injury in a

rat model of sepsis. Surg Today 2005; 35:

52–59

7. Czapski GA, Cakala M, Kopczuk D, et al:

Inhibition of nitric oxide synthase prevents

energy failure and oxidative damage evoked

in the brain by lipopolysaccharide. Pol

J Pharmacol 2004; 56:643–646

8. Messaris E, Memos N, Chatzigianni E, et al:

Time-dependent mitochondrial-mediated

programmed neuronal cell death prolongs

survival in sepsis. Crit Care Med 2004; 32:

1764 –1770

9. Korcok J, Wu F, Tyml K, et al: Sepsis inhibits

reduction of dehydroascorbic acid and accu-

mulation of ascorbate in astroglial cultures:

Intracellular ascorbate depletion increases

nitric oxide synthase induction and gluta-

mate uptake inhibition. J Neurochem 2002;

81:185–193

10. Voigt K, Kontush A, Stuerenburg HJ, et al:

Decreased plasma and cerebrospinal ﬂuid

ascorbate levels in patients with septic en-

cephalopathy. Free Radic Res 2002; 36:

735–739

he short-term oxi-

dative damage

demonstrated here

could participate in the de-

velopment of central nervous

system symptoms during

sepsis development, or even

septic encephalopathy.

889Crit Care Med 2006 Vol. 34, No. 3

Capítulo 5.

Antioxidant treatment reverses late cognitive impairment

in an animal model of sepsis

Tatiana Barichello, Roberta A. Machado, Larissa Constantino, Samira S.

Valvassori, Gislaine Z. Reus, Fabricia Petronilho, Cristiane Ritter, João

Quevedo, Felipe Dal-Pizzol

Critical Care Med (2007) no prelo

For Peer Review

Antioxidant treatment reverses late cognitive impairment in an animal

model of sepsis

Journal: Critical Care Medicine

Manuscript ID: CCM-00016-2007

Manuscript Type: Original Articles - Laboratory Investigations

Date Submitted by the

Author:

04-Jan-2007

Complete List of Authors: Barichello, Tatiana; UNESC

Machado, Roberta; UNESC

Constantino, Larissa; UNESC

Valvassori, Samira; UNESC

Reus, Gislaine; UNESC

Petronilho, Fabricia; UNESC

Ritter, Cristiane; UNESC

Quevedo, Joao; UNESC

Dal-Pizzol, Felipe; Universidad do Extremo Sul Catarinese,

Department of Medicine

Key Words: septic shock, oxidative stress, memory, central nervous system

Critical Care Medicine

For Peer Review

Antioxidant treatment reverses late cognitive

impairment in an animal model of sepsis

Tatiana Barichello

,PharmD, MSc, Roberta Albino Machado

,BSc,

Larissa Constantino

,BSc, Samira S. Valvassori

,BSc, Gislaine Z. Réus

, BSc,

Fabricia Petronilho

,MSc, Cristiane Ritter

MD, MSc,

João Quevedo

, MD, PhD Felipe Dal-Pizzol

, MD, PhD

Laboratório de Fisiopatologia Experimental, Universidade do Extremo Sul

Catarinense, Criciúma, SC, Brazil.

Laboratório de Neurociências, Universidade do Extremo Sul Catarinense, Criciúma,

SC, Brazil.

Address for reprints: Felipe Dal-Pizzol. Laboratório de Fisiopatologia Experimental,

Universidade do Extremo Sul Catarinense, Criciúma, SC, Brazil. Avenida Universitária,

1105, 88806-000. phone: 55 48 4312641, fax: 55 48 431 2750, e-mail:piz@unesc.net

This work was supported by grants from UNESC and CNPq.

Key Words: septic shock, oxidative stress, memory, central nervous system

Page 1 of 20 Critical Care Medicine

For Peer Review

Abstract

Objective: Assess the effect of antioxidant treatment on late cognitive impairment and

early hippocampus oxidative stress after cecal ligation and perforation (CLP) in the rat.

Design: Prospective, controlled experiment.

Setting: Animal basic science laboratory.

Subjects: Male Wistar rats, weighing 300–350 g.

Interventions: Rats subjected to CLP were treated with either N-acetylcysteine (NAC)

plus deferoxamine (DFX) or vehicle with or without “basic support” (saline at 50 mL/kg

immediately and 12 hrs after CLP plus ceftriaxone at 30 mg/kg and clindamycin 25

mg/kg every 6 hrs).

Measurements and Main Results: Ten and thirty days after surgery, the animals

underwent three behavioral tasks: a) inhibitory avoidance task; b) habituation to an open

field; and c) continuous multiple trials step-down inhibitory avoidance task. The sepsis

group showed significantly decreased performance in latency retention compared with

the sham group in inhibitory avoidance. In the open-field task the sepsis group

presented memory impairment after sepsis. In the continuous multiple trials step-down

inhibitory avoidance task, the sepsis group showed a significant increase in the number

of training trials required to reach the acquisition criterion. All these cognitive

impairments were reversed by NAC plus DFX treatment, but not its isolate use. In

addition, the combined use of antioxidants attenuated oxidative damage in hippocampus

6 hours after sepsis induction.

Conclusions: antioxidant treatment could significantly attenuate late cognitive deficits in

sepsis survivors from cecal ligation and perforation. This was associated with

attenuation on hippocampus oxidative damage in early periods of sepsis development.

Page 2 of 20Critical Care Medicine

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Introduction

Advances in critical care medicine have led to improved survival rates among

those patients admitted to the ICU. Critical illness often results in multiple system organ

dysfunctions, including neurological dysfunction, and is associated with poor

neurological outcomes (1). Investigations of the effects of critical illness on neurological

dysfunction have been relatively neglected compared to the effects on other organ

systems, mainly neurocognitive outcomes in survivors of critical illness (2-6).

The mechanisms associated to neuropsychological and intellectual impairments

secondary to direct brain injury is better understood when compared to the

neurocognitive impairment secondary to the general critical illness. It is well

characterized the participation of inflammatory and apoptotic pathways in neuronal

damage secondary to brain injury (7-9). But, to the best of our knowledge there is no

published study describing the mechanisms associated to the long-term cognitive deficit

in acute inflammatory diseases.

We had previously described oxidative damage in different brain regions in two

different models of acute inflammatory disease (10,11), and it is well known that

oxidative stress could participate in the development of several central nervous system

diseases (12). Thus we here determine the role of hippocampal oxidative stress in the

development of long-term cognitive impairment in sepsis survivors, using an animal

model recently described by our group (13,14).

Methods

In vivo studies were performed in accordance with National Institutes of Health

guidelines and approved by the Ethics Committee of Universidade do Extremo Sul

Catarinense.

Page 3 of 20 Critical Care Medicine

For Peer Review

Animal model of long-term congnitive impairment: this model was described in

detail elsewhere (13,14). Briefly, after sepsis induction using the cecal ligation and

perforation procedure, the sepsis group received “basic support” (saline at 50 mL/kg

immediately and 12 hrs after CLP plus ceftriaxone at 30 mg/kg and clindamycin at 25

mg/kg every 6 hrs over a total of 3 days). The sham-operated group received only

saline, 50 mL/kg, immediately and 12 hrs after surgery, and the volume of saline

corresponded to antibiotic administration. Survival in the sham group was 100%, and in

the sepsis group was 40%. The number of survivals is in accordance with our previous

reports (13,14). Ten or 30 days after surgery, different cohorts of animals each time and

each task underwent three behavioral tasks: a) the step-down inhibitory avoidance task

(single-training); b) continuous multiple-trials step-down inhibitory avoidance task; and c)

the open-field task. The behavioral tests were performed by the same person who was

blinded to the experimental group (sham or CLP). All experimental procedures involving

animals were performed in accordance with the National Institute of Health Guide for the

Care and Use of Laboratory Animals and with the approval of the local ethics committee.

The step-down inhibitory avoidance apparatus and procedures have been

described in previous reports (15). Briefly, the training apparatus was a 50x25x25-cm

acrylic box (Albarsch, Porto Alegre, Brazil) whose floor consisted of parallel caliber

stainless steel bars (1-mm diameter) spaced 1 cm apart. A 7-cmwide, 2.5-cm-high

platform was placed on the floor of the box against the left wall. In the training trial,

animals were placed on the platform and their latency to step down on the grid with all

four paws was measured with an automatic device. Immediately after stepping down on

the grid, the animals received a 0.4-mA, 2.0-sed foot shock and returned to their home

cage. A retention test trial was performed 24 hrs after training. The retention test trial

was procedurally identical to training, except that no foot shock was presented. The

retention test step-down latency (maximum, 180 seconds) was used as a measure of

Page 4 of 20Critical Care Medicine

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inhibitory avoidance retention. Habituation to an open field was carried out in a 40x60-

cm open field surrounded by 50-cm high walls made of brown plywood with a frontal

glass wall. The floor of the open field was divided into 12 equal rectangles by black lines.

Animals were gently placed on the left rear quadrant, and left to explore the arena for 5

min (training session). Immediately following this, the animals were taken back to their

home cage, and 24 hrs later submitted again to a similar open-field session (test

session). Crossing of the black lines and rearing performed in both sessions were

counted. The decrease in the number of crossings and rearings between the two

sessions was taken as a measure of the retention of habituation (16). Continuous

multiple-trials step-down inhibitory avoidance task was performed in the same step-down

inhibitory avoidance apparatus described above. However, in the training session, the

animal was placed on the platform and immediately after stepping down on the grid,

received a 0.3-mA, 2.0-sec foot shock. This procedure continued until the rat remained

on the platform for 50 seconds. The animal was then returned to the home cage. The

number of training trials required to reach the acquisition criterion of 50 seconds on the

platform was recorded. The retention test was performed 24 hrs later.

Antioxidant effects on the development of long-term cognitive impairment in

sepsis survivors: in the above described model 10 days after sepsis animals presented

deficits in the three cognitive skills (step-down inhibitory avoidance, open field and

continuous multiple-trials step-down inhibitory avoidance) (13), and 30 days in two of

three cognitive skills (step-down inhibitory avoidance and continuous multiple-trials step-

down inhibitory avoidance) (14). In addition we had previously determined that early

after sepsis development oxidative damage occurs, mainly in the hippocampus (11).

Thus, to assess the participation of oxidative damage in the observed cognitive deficits,

we determine the effects of early antioxidant administration on long-term cognitive tasks

and on early markers of oxidative damage in the hippocampus. Using an antioxidant

Page 5 of 20 Critical Care Medicine

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treatment protocol described in detail by our group elsewhere (17) we determined the

effects of the administration of N-acetylcysteine (NAC, administered 20 mg/kg, 3 hrs, 6

hrs, 12 hrs, 18 hrs, and 24 hrs after CLP, subcutaneously), deferoxamine (DFX, 20

mg/kg, 3 hrs and 24 hrs after CLP, subcutaneously) or both in the above described

parameters. To asses the effects of antioxidant administration on long-term cognitive

deficits in sepsis survivors we divided animals in six different groups: 1) sham-operated;

2) CLP - received antibiotics and fluid resuscitation as described above; 3) CLP plus

NAC - as the CLP group with the administration of NAC as described above; 4) CLP plus

DFX - as the CLP group with the administration of DFX as described above; 5) CLP plus

NAC and DFX - as the CLP group with the administration of both NAC and DFX as

described above; 6) NAC plus DFX - septic animals that did not receive antibiotics, but

only NAC and DFX as described above. To assess the participation of early

hippocampal oxidative damage on the long-term cognitive deficits we used, in a

separate cohort of animals, these six groups. Six hours after sepsis induction animals

were sacrificed by decapitation, hippocampus was removed to the determination of

thiobarbituric acid reactive substances (18) and protein carbonyls (19) as described in

detail elsewhere (11).

Data from the inhibitory avoidance task and retention test latencies from

continuous multiple-trials step-down inhibitory avoidance were expressed as median and

interquartile ranges. Statistical significances were determined by the Mann-Whitney U

test, p <.05. Data from the open-field task and the number of training trials from

continuous multiple trials step-down inhibitory avoidance were expressed as mean +

S.D.. Statistical significances were determined by paired samples Student’s t-test, p <

.05. Data from oxidative damage parameters are presented as means +

S.D. Statistical

significances were determined by 2-way ANOVA and multiple comparisons were

performed by a Newman-Keuls test, p < .05.

Page 6 of 20Critical Care Medicine

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Results

Effects of early antioxidant administration on the inhibitory avoidance test: we

had previously described that in the CLP model sepsis survivors presented alterations in

the inhibitory avoidance test 10 and 30 days after sepsis induction. No differences

between groups were demonstrated in the inhibitory avoidance training session. In the

test session, the step-down latency was significantly decreased in the sepsis group

compared to the sham group, and this alteration was not reversed with the isolate use of

NAC or DFX 10 (Figure 1A) or 30 (Figure 1B) days after sepsis induction. In contrast,

both 10 and 30 days after sepsis the combined use of NAC and DFX, with or without

antibiotics reversed the observed alteration (Figures 1A and 1B).

Effects of early antioxidant administration on the continuous multiple-trials step-

down inhibitory avoidance test: As demonstrated previously, sepsis survivors presented

an increase in the number of training trials required to reach the acquisition criterion (50

secs on the plataform) in the continuous multiple-trials step-down inhibitory avoidance

test compared to the sham group 10 (Figure 2A) and 30 (Figure 2B) days after sepsis.

As demonstrated previously, in the retention test, there was no difference between

groups (data not shown). Nor NAC or DFX could reverse the observed deficit in both

times tested (Figures 2A and 2B), but as demonstrated to the inhibitory avoidance the

combined use of NAC and DFX reversed the observed alteration (Figures 2A and 2B).

Effects of early antioxidant administration on the open-field task: In the open-field

task, there were no differences in the number of crossings and rearings between groups

in the habituation to the open-field training session, demonstrating no difference in motor

and exploratory activity between groups (Figure 3A and 3B), as we previously

demonstrated (13). As we previously demonstrated, the sepsis group did not show a

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difference in crossings and rearings between training test sessions, demonstrating

memory impairment in this group (Figure 3A and 3B). The use of NAC plus DFX, but not

its isolated use, reversed the memory impairment in the open-field task 10 days after

sepsis induction. We did not investigate the effects of antioxidant administration 30 days

after sepsis in the open-field task since we demonstrated previously that at this period

sepsis survivors did not differ significantly from sham animals in this task (14).

Effects of antioxidant administration on hippocampus oxidative damage

parameters after sepsis: We demonstrated previously the occurrence of oxidative

damage 6 hours after sepsis induction in this model of rodent sepsis (11). Since

hippocampus is a major SNC structure responsible to memory formation we determined

if the reversal of late cognitive deficits in sepsis survivors was associated to the

attenuation of early oxidative damage. We demonstrated in figure 4A and 4B that NAC

plus DFX, but no other treatment reversed oxidative damage assessed by two different

oxidative damage parameters, and this correlates to an improve in cognitive

performance in sepsis survivors.

Discussion

We demonstrated for the first time that antioxidant treatment could significantly

attenuate late cognitive deficits in sepsis survivors from cecal ligation and perforation.

This was associated with attenuation on hippocampus oxidative damage in early periods

of sepsis development.

The evidence from 10 ongoing cohorts suggests that 25 to 78% of ICU survivors

experience cognitive impairments (for an excellent review see 20). In these patients

generally memory is the most frequently observed deficit, followed by executive function

and attention deficit (20). The mechanisms underlying these alterations are still obscure.

There is probably not a single cause of these impairments, but rather a number of

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several factors that interact dynamically with premorbid variables and result in adverse

outcomes (20). Several methodological issues limit the clear distinction between these

factors in the clinical setting. Thus, using a clinically relevant animal model of sepsis we

demonstrated that early hippocampus oxidative damage seems to be important in the

development of late cognitive deficits in sepsis survivors. We supposed that this early

alteration initiate a complex cascade of events that leads ultimately to SNC dysfunction

and late cognitive deficits observed in CLP survivors.

Some few studies were published in the intent to determine the molecular

mechanisms associated with late cognitive deficits in the context of critical care

medicine. Using an animal model of meningitis based on S. pneumoniae Irazuzta et al.

(21) reported that the dexamethasone treatment of rats with bacterial meningitis leads to

decreased activation of caspase 3 but not of caspase 1 and these was associated with a

preservation of neurobehavioral performance, but we need to be very cautious when

evaluating learning and memory performance in subjects treated with dexamethasone

(22). In addition, the administration of hydrocortisone during septic shock in a dosage

similar to the endogenous production rate was associated with a lower incidence of the

so-called posttraumatic stress disorder in long-term survivors (23). Irazuzta group

demonstrated that although hypothermia can reduce the inflammatory response and

biomarkers of brain injury it fails to improve neurobehavioral performance in an animal

model of bacterial meningitis (24). It is well known that neuronal and cerebral endothelial

cells undergone apoptosis via activation of toll-like receptor 2 (25,26), but its relation to

acute and chronic neurologic deficits is unknown. In the same way, LPS could trigger

CNS inflammation and neuronal death during chronic neurodegeneration (27), but the

relevance of this to chronic neurological deficits was never studied.

Since oxidative stress is associated to the development of neurodegenerative

disease (12) and is important to the development of multiple organ dysfunction

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syndrome during sepsis (28) is reasonable suppose that it could contribute to long-term

cognitive deficits in sepsis survivors. Oxidative stress was demonstrated previously in

different animal models of critical care disease. We had demonstrated the occurrence of

oxidative damage in an animal model of sepsis (11) and acute hepatic failure (10). Sener

et al described oxidative damage and reduce antioxidant content in a murine model of

sepsis and determined an antioxidant effect to melatonin in this setting (29). None of

these previous studies correlate brain oxidative stress and long-term cognitive deficits in

survivors from critical care diseases.

The evaluation of molecular mechanisms and signaling cascades which underlie

the events that ultimately lead to brain damage during sepsis will improve our

pathophysiological knowledge of this disease. More importantly, it will also promote the

development of new and more efficient therapeutic strategies, especially to prevent

neurological sequelae. In this context our results suggested, for the first time, a role to

early SNC oxidative damage in the development of long-term cognitive deficits. In

addition we demonstrated a new role to antioxidant treatment in an animal model of

sepsis.

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Figure Legends

Figure 1 - Inhibitory avoidance task. Rats were sham-operated or submitted to cecal

ligation and puncture (CLP). CLP animals were assigned to receive basic support (Sup),

Sup plus N-acetylcysteine (NAC), Sup plus deferoxamine (DFX), Sup plus NAC and

DFX or only NAC and DFX as described in Materials and Methods. Survivors from

sepsis were submitted to the inhibitory avoidance task A. 10 days or B. 30 days after

sepsis. Data are expressed as median (interquartile ranges) training and test session

latencies, in seconds. n = 15 each group. *Significant difference compared with Sham

group (Mann-Whitney U test, p < .01). Sup = basic support, NAC = N-acetylcysteine,

DFX = Deferoxamine

Figure 2 - Continuous multiple-trials step-down inhibitory avoidance. Rats were

sham-operated or submitted to cecal ligation and puncture (CLP). CLP animals were

assigned to receive basic support (Sup), Sup plus N-acetylcysteine (NAC), Sup plus

deferoxamine (DFX), Sup plus NAC and DFX or only NAC and DFX as described in

Materials and Methods. Survivors from sepsis were submitted to the continuous multiple-

trials step-down inhibitory avoidance task A. 10 days or B. 30 days after sepsis. Data

are expressed as mean +

SD of the number of training trials required to reach

acquisition criterion (50 secs on the platform). n = 15 each group *Significant difference

compared with Sham groups (Student’s t-test, p< .01). Sup = basic support, NAC = N-

acetylcysteine, DFX = Deferoxamine

Figure 3 - Open-field task. Rats were sham-operated or submitted to cecal ligation and

puncture (CLP). CLP animals were assigned to receive basic support (Sup), Sup plus N-

acetylcysteine (NAC), Sup plus deferoxamine (DFX), Sup plus NAC and DFX or only

NAC and DFX as described in Materials and Methods. Survivors from sepsis were

submitted to the open-field task 10 days after sepsis. Data are expressed as mean +

of A. crossings and B. rearings of training (white columns) and test (gray columns)

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session. n = 15 each group. *Significant difference between training test (paired samples

Student’s t-test, p < .01). # Significant difference between groups in training and test

sessions (Student’s t-test, p < .01). *Significant difference compared with Sham group

Sup = basic support, NAC = N-acetylcysteine, DFX = Deferoxamine

Figure 4 - Early oxidative damage in the hippocampus from septic rats. CLP

animals were assigned to receive basic support (Sup), Sup plus N-acetylcysteine (NAC),

Sup plus deferoxamine (DFX), Sup plus NAC and DFX or only NAC and DFX as

described in Materials and Methods. Six hours after sepsis induction animals were

sacrificed and hippocampus isolated to the determination of A. thiobarbituric acid

reactive substances and B. protein carbonyls. Data are expressed as mean +

SD n = 5

each group *Significant difference compared with Sham groups (ANOVA followed by

Newman-Keuls test, p< .01). Sup = basic support, NAC = N-acetylcysteine, DFX =

Deferoxamine

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Figure 1A

Figure 1B

120

150

180

Sham Sup Sup + Nac Sup + DFX Sup + Nac +

DFX

NAC + DFX

Seconds

Training

Test

Sham Sup Sup + Nac Sup + DFX Sup + Nac +

DFX

NAC + DFX

Seconds

Training

Test

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Figure 2A

Figure 2B

Trials

Sham Sup Sup + Nac Sup + DFX Sup + Nac +

DFX

Nac + DFX

Number of trials

Trials

Sham Sup Sup+NAC Sup+DFX Sup + NAC +

DFX

NAC + DFX

Number of trials

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Figure 3A

Figure 3B

Sham Sup Sup+NAC Sup+DFX Sup+NAC+DFX NAC+DFX

Number of crossings

Training

Test

Sham Sup Sup+NAC Sup+DFX Sup+NAC+DFX NAC+DFX

Number of rearings

Training

Test

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Figure 4A

Sham CLP + Sup CLP + Sup +

NAC

CLP + Sup +

DFX

CLP + Sup +

NAC + DFX

MDA equivalents (nm/mg protein)

Figure 4B

Sham CLP + Sup CLP + Sup +

NAC

CLP + Sup +

DFX

CLP + Sup +

NAC + DFX

Protein carbonyls (nm/mg protein)

Page 20 of 20Critical Care Medicine

DISCUSSÃO

Dano cognitivo em ratos sobreviventes a sepse

Os resultados dos experimentos do Capítulo 1 e 2 demonstraram que

ratos sobreviventes a sepse após 10 dias da indução nos testes de habituação ao

campo aberto, esquiva inibitória, esquiva inibitória de múltiplos treinos,

reconhecimento de objeto, labirinto de cruz elevada e natação forçada apresentaram

incapacidade na aprendizagem, na memória, e sintomas de depressão.

No Teste de Habituação ao Campo Aberto, os ratos submetidos à sepse

não apresentaram alteração na atividade locomotora e exploratória quando

comparados ao grupo sham, porém demonstraram incapacidade na habituação ao

local, sugerindo uma incapacidade na memória e aprendizagem.

No teste de esquiva inibitória o tempo de latência foi significativamente

menor no grupo sepse, demonstrando que estes possuem uma incapacidade na

memória aversiva. O teste de esquiva inibitória envolve vários estímulos, incluindo

percepção espacial e visual, sensibilidade à dor, acompanhados de um componente

emocional amplamente modulado por hormônios relacionados ao estresse (GoId,

1986). Os ratos submetidos á sepse que foram submetidos ao teste de esquiva

inibitória de múltiplos treinos mostraram um significativo aumento no número de

treinamentos requeridos para alcançar o critério de aquisição (50 segundos na

plataforma) e no teste de retenção não houve diferença entre os grupos. Os

resultados desta tarefa mostraram que o grupo sepse requereu aproximadamente

duas vezes mais estímulos para alcançar o critério da aquisição quando comparado

com o grupo sham, indicando uma incapacidade de aprendizagem (aquisição de

novo conhecimento).

A tarefa de reconhecimento de objetos, desenvolvida originalmente por

Ennaceur e por Delacour (Ennaceur e Delacour, 1988), é baseada na tendência dos

roedores explorarem o objeto novo. Nenhuma estimulação, recompensa ou

aversividade são utilizadas durante o treinamento, a aprendizagem ocorre sob

circunstâncias de estresse ou de medo relativamente baixo (Ennaceur e Delacour

1988). Os dados encontrados mostraram que os ratos sobreviventes a sepse

apresentaram incapacidade significativa da memória de reconhecimento do objeto

familiar. Estes achados são relevantes desde que à tarefa de reconhecimento do

objeto familiar nos roedores seja um teste não espacial e não aversivo para

memória. Além disso, a tarefa do reconhecimento do objeto está sendo usada

também cada vez mais como uma ferramenta experimental poderosa para avaliar os

efeitos de droga na memória e investigar a aprendizagem dos mecanismos neurais e

memórias subjacentes (Rosa et al, 2003; Schröder et al, 2003; Baker e Kim, 2002;

Okuda et al, 2004; Rampon et al, 2000).

No teste do labirinto de cruz elevada, um teste validado para avaliar

comportamento ansioso (Pellow et al, 1985), nenhuma diferença foi demonstrada

entre os grupos, indicando que os sobreviventes de sepse não apresentam sintomas

de ansiedade após a recuperação da doença.

Os sobreviventes de sepse apresentaram sintomas ligados à depressão

avaliados no teste forçado da natação. O tempo de imobilidade foi significativamente

mais longa no grupo dos sobreviventes de sepse. A origem do teste de natação

forçado relatada por Porsolt (1979) era aquela de um modelo da depressão com as

características similares àquelas do modelo instruído do comportamento

desamparado, mas tecnicamente mais fácil de reproduzir. O estado afetivo interno

dos roedores após a exposição ao nado inicial no teste de natação forçada é

caracterizado como comportamento de desespero. O teste preliminar ao

procedimento da indução da natação foi proposto para ser similar à sessão inicial

que induz o comportamento desesperado instruído para expor os ratos ao estresse

de não poder escapar daquela situação. A Indução do comportamento de desespero

produz um largo aspecto comportamental afetando a cognição, o sono e o

desempenho motor que se assemelham muitos dos sintomas de depressão (Weiss e

Kilts, 1998). Adicionalmente, como descrito acima, o grupo de sobreviventes de

sepse não apresentou incapacidade motora, suportando a idéia que o longo tempo

de imobilidade no grupo séptico foi relacionado aos sintomas ligados a depressão.

Estes resultados estão de acordo com estudos clínicos sobre sintomas depressivos

nos quais sobreviventes de doenças severas tais como o sepse e choque séptico

apresentam (Angus et al, 2001; Granja et al, 2004; Granja et al, 2005; Heyland et al,

2000 ; Hopkins et al, 2005).

Quando realizado os testes de esquiva inibitória, esquiva inibitória de

múltiplos treinos e habituação ao campo aberto após 30 dias, ratos sobreviventes a

sepse ainda demonstraram incapacidade de aprendizagem e memória, estes

resultados são apresentados no Capítulo 3

Estes resultados indicam que a incapacidade de memória e aprendizagem

demonstrada 10 dias após a indução da sepse, persistem após 30 dias do CLP. A

persistência das alterações 30 dias após CLP reforça a idéia que este modelo

poderia ser uma ferramenta importante no estudo das seqüelas cognitivas em

sobreviventes de sepse.

Os dados deste estudo fornecem a primeira demonstração experimental

em um modelo animal de sepse clinicamente relevante para demonstrar a

incapacidade na aprendizagem e memória. Neste contexto, Shimizu e colaboradores

(1999) demonstraram que 24 horas após CLP, os animais mostraram incapacidade

de aprendizagem na esquiva inibitória. Os resultados deste estudo podem ter um

significado maior, pois foram realizados os experimentos 10 dias e 30 dias após

CLP, e não 24 horas. Os animais após 10 dias da cirurgia estão recuperados

inteiramente, sem nenhum sinal de infecção ou alterações motoras (Ritter et al,

2004; Ritter et al, 2003). Além disso, este estudo utilizou reposição hídrica e

antibióticos para replicar o mais próximo a terapia de suporte executada no ajuste

clínico, quando comparado com o estudo de Shimizu e colaboradores (1999).

Em um modelo animal de sepse induzido por LPS, foi encontrado

mecanismos conhecidos de apoptoses no hipocampo após 24 horas após a indução,

sendo assim, o mais severamente afetado após a indução do sepsis (Semmler et al.,

2005).

Semmler e colaboradores (2007) mostraram que ratos submetidos à

sepse pelo modelo de LPS (injeções de polissacarídeos) apresentaram após 3

meses, alterações comportamentais, dano neuronal em sub-regiões hipocampais e

redução da inervação colinérgica em áreas corticais.

As incapacidades cognitivas parecem estar relacionadas diretamente aos

processos dependentes do hipocampo, danificando a memória (Squire e Zola-

Morgan, 1991; Jarrard, 1995). Similarmente, como indicou Semmler e colaboradores

(2007) em seu estudo, caracterizado por uma redução significativa na densidade dos

neurônios CA1/CA2 do hipocampo. Adicionalmente foi encontrado uma densidade

reduzida de células neuronais no córtex pré-frontal, onde as lesões podem conduzir

aos distúrbios de orientação e aos problemas amenésicos em tarefas complexas

(Heidbreder e Groenewegen, 2003; Mogensen et al., 2005). Além disso, foi

demonstrado uma densidade diminuída de VAChT após a indução da sepse

experimental, indicando uma inervação colinérgica diminuída nas áreas citadas

acima no estudo de Semmler e colaboradores (2007) , provavelmente relacionado

também as incapacidades cognitivas observadas, como mudanças na densidade

parietal de VAChT com incapacidades na memória foram encontrados também 1

ano após a indução experimental de trauma cerebral (Dixon et al., 1999).

Entretanto, ainda citando o estudo de Semmler e colaboradores (2007) a

VAChT é expressada excepcionalmente nos neurônios colinérgicos e concentrado

em seus terminais axônicos (Gilmor et al., 1996; Arvidsson et al., 1997), a redução

da imunoreatividade de VAChT dentro o córtex parietal junto com uma densidade

inalterada dos neurônios do córtex parietal indica reduzida inervação colinérfica

nessa área. Além disso, a maioria da inervação colinérgica hipocampal e cortical são

originadas fora do hipocampo e do córtex (Mesulam et al., 1983). Isto sugere que a

diminuição da imunoreatividade de VAChT reflete principalmente a retirada da fibra

colinérgica dos neurônios, como os neurônios colinérgicos representam 5- 10% dos

neurônios corticais e hipocampais (Gage et al., 1983; Eckenstein et al., 1988). Os

neurônios gabaérgicos e glutamatérgicos, a somatostatina e o neuropeptideo Y são

mais representativos nestas áreas.

As incapacidades observadas neste estudo, pelo menos parte delas,

como as alterações cognitivas são observadas em sobreviventes de sepse,

particularmente a incapacidade na memória. Nesta maneira, o modelo de CLP para

indução da sepse pode ajudar a investigar os mecanismos biológicos envolvidos na

incapacidade cognitiva associada à sepse e para determinar ações terapêuticas

para este problema. Estudos precedentes que descreveram os sintomas de

pacientes sobreviventes de sepse na unidade de terapia intensiva demonstram que

a maioria destes pacientes apresentou alguma incapacidade cognitiva na alta

hospitalar (Hopkins et al, 1999; Angus et al, 2001; Granja et al, 2004; Heyland et al,

2000). Após 1 ano, a maioria dos pacientes mostrou uma melhoria na função

cognitiva total; entretanto, algumas habilidades cognitivas, como a memória, não

melhorou completamente (Hopkins et al, 1999; Angus et al, 2001; Heyland et al,

2000). Entretanto os mecanismos envolvidos na incapacidade cognitiva ainda

permanecem obscuros.

Medidas de estresse oxidativo no cérebro de ratos sobreviventes a sepse

No Capítulo 4 foi realizada a mensuração do estresse oxidativo em ratos

submetidos à sepse para tentar esclarecer os mecanismos neurobiológicos

envolvidos na incapacidade cognitiva.

Foi demonstrado que, os danos oxidativos, avaliados pelo TBARS e

carbonil, e ocorrem precocemente no desenvolvimento do curso da sepse em

diversas regiões do cérebro. Os níveis de TBARS foram elevados no hipocampo,

córtex, e cerebelo 6 horas após a indução da sepse. Em contraste, níveis de carbonil

foram elevados somente no hipocampo 6 horas após a indução da sepse. Estes

sugerem danos oxidativos mais consistentes nos lipídios em comparação com

proteínas.

Para determinar a atividade enzimática antioxidante durante o

desenvolvimento da sepse no cérebro dos ratos, foi determinada a atividade

antioxidante enzimática do sistema nervoso central (CAT e SOD). À exceção do

estriado, foi demonstrado um aumento na atividade de SOD sem um proporcional

aumento na atividade de CAT, com aumento conseqüente na relação de SOD e

CAT. Assim, um desequilíbrio na atividade de SOD e CAT ocorrido ao mesmo tempo

e dentro das mesmas estruturas em que foi demonstrado o dano oxidativo. Foi

descrito previamente por Ritter e colaboradores (2004) que um desequilíbrio entre as

atividades de SOD e CAT no coração, fígado, pulmão, e diafragma são, em parte,

responsável pelos danos oxidativos em um modelo de. Os danos oxidativos e

desequilíbrio nas enzimas antioxidantes no início do curso da sepse pareceram não

ser secundários à falha renal ou hepática, pois no início não foi relatada nenhuma

alteração em marcadores plasmáticos da função renal e hepática. Além disso, o

dano não parece ser secundário a hipotensão, pois foi utilizado líquido de

ressuscitação não foi associado com a hipotensão em 6 horas após a indução de

sepse.

Foi demonstramos pela primeira vez que, diferentemente de outros

órgãos envolvidos na resposta séptica, no sistema nervoso central o estresse

oxidativo é restringido a períodos mais iniciais após a indução da sepse. Estes

resultados são intrigantes, pois os danos oxidativos no pulmão, no fígado, no

coração, e no rim ocorrem em períodos mais distantes após a indução da sepse

(Ritter et al, 2004). Abd El-Gawad e Khalifa (2001) demonstraram que 2 horas após

a administração de lipopolisacarideos havia um aumento na geração de radicais

livres no cérebro de ratos, porém estes autores não analisaram variáveis oxidativas

em períodos mais distantes após a administração de lipopolisacarideos. Em

contraste, demonstrou-se que o estresse oxidativo ocorreu até 48 horas após a

indução da sepse no cérebro dos ratos (Sener et al, 2005; Czapski et al, 2004).

Entretanto, nestes estudos o fluído para ressuscitação foi administrado somente uma

vez, e isto poderia induzir posteriormente a uma hipotensão (Sener et al, 2005;

Czapski et al, 2004). Suportando os dados apresentados neste estudo, Messaris e e

colaboradores (2004) demonstraram recentemente a apoptose mediada pela

mitocôndria no cérebro de ratos em períodos iniciais após a indução da sepse (6-12

horas), porém não em períodos mais tardis. Estas alterações mitocondriais poderiam

ser um dos mecanismos associados ao estresse oxidativo no cérebro.

Estudos do grupo do Dr Boveris demonstraram que a produção de NO

durante a sepse leva a inativação transitória da atividade da cadeia de transporte de

elétrons, provavelmente complexo II e IV seriam os alvos principais. Esta inativação

levaria a produção de superóxido mitocondrial e, conseqüentemente, de peroxinitrito

que inativaria irreversivelmente a cadeia de transporte de elétrons levando efeito em

cascata (Boczkowski et al 1999).

Outra possibilidade é a de que os diferentes mediadores da resposta

inflamatória da sepse possam levar à disfunção mitocondrial e esta a liberação de

espécies reativas de oxigênio. Entre estes, o TNF-α tem capacidade de levar as

alterações celulares, inclusive morte, mediadas por vias de sinalização intracelulares

dependente de ativação do seu receptor. A ativação de receptores de TNF ativa

esfingomielinases, levando a um aumento intracelular de ceramida e fosfocolina. A

ceramida pode levar a formação do poro de permeabilidade transitória mitocondrial

(MPT), com conseqüente liberação de citocromo C (Siskind et al 2002), ativação de

caspase-3 (Von Haefen et al 2002) e apoptose. Em concentrações de milimolar

ceramida inibe a fosforilação oxidativa e leva a produção de EAO (Garcia-Ruiz et al

1997).

Além disso, a incapacidade da limpeza dos astrócitos de ácido

desidroascórbico do líquido extracelular e da diminuição conseqüente do ascorbato

intracelular, a concentração poderia ser associada com o estresse oxidativo do

sistema nervoso central (Korcok et al, 2002). Esta idéia é reforçada pela diminuição

do ascorbato nos níveis do fluído cerebrospinal nos pacientes com encefalopatia

séptica (Voigt et al, 2002). Entretanto, a sepse induz a óxido nítrico sintase e inibe a

recaptação de glutamato pelos astrocitos através dos mecanismos que podem ser

modulados pelo ascorbato intracelular (Korcok et al, 2002).

A proteção do sistema nervoso central aos danos oxidativos parece ser a

adaptação para minimizar a disfunção e também poderia estar relacionado a

adaptação das defesas antioxidantes do sistema nervoso central, diminuição do

metabolismo do sistema nervoso central, ou outros diversos fatores ainda não

esclarecidos. Os dados deste estudo demonstram que, como em outros órgãos

(Ritter et al, 2004), um desequilíbrio no meio SOD e CAT poderia ser responsáveis,

em parte, pela ocorrência de danos oxidativos no cérebro dos ratos. Em

contrapartida estas enzimas normalizaram-se nas estruturas estudadas 12-96 horas

após a indução da sepse. Neste período (12-96 horas após a indução da sepse) foi

demonstrado que a atividade da SOD e da CAT não mudou nem aumentou

proporcionalmente ou que, o aumento na atividade da CAT foi mais elevada do que

o aumento da atividade da SOD. Assim, as alterações na relação de SOD/CAT

foram relacionadas temporariamente à ocorrência ou não de danos oxidativos dentro

do sistema nervoso central. Em relação ao exposto acima, não há nenhum estudo

que descreve a modulação da SOD e da CAT no cérebro de ratos após a indução da

sepse. Porém, não foi possível verificar em curto prazo os danos oxidativos

demonstrados neste estudo se podem conduzir ao sistema nervoso central sintomas

durante o desenvolvimento da sepse, ou mesmo a encefalopatia séptica.

Tratamento com antioxidantes e dano cognitivo

Baseado nos resultados apresentados no Capítulo 1, 2 e 4, foi realizada

a avaliação dos efeitos do tratamento com antioxidantes NAC e DFX na reversão da

incapacidade cognitiva demonstradas nos testes de habituação ao campo aberto,

esquiva inibitória, esquiva inibitória de múltiplos treinos e no estresse oxidativo em

hipocampo após a indução de sepse em ratos sobreviventes para tentar esclarecer

os possíveis mecanismos neurobiológicos que levam ao dano cognitivo.

Nos testes comportamentais citados acima, após 10 e 30 dias da indução

de sepse, DFX e NAC quando administrados concomitantemente reverteram à

incapacidade cognitiva apresentada nos Capítulo 1 e 3.

Foi demonstrada previamente no Capítulo 4, a ocorrência de dano

oxidativo 6 horas após a indução da sepse no hipocampo de cérebros de ratos

sépticos. O hipocampo é a estrutura principal do SNC responsável pela formação da

memória, devido a isso, a reversão dos danos cognitivos tardios em sobreviventes

de sepse poderia estar associada à atenuação dos danos oxidativos precoces. Foi

demonstrado que NAC e DFX administrados concomitantemente, mas não com o

seu uso isolado, reverteram os danos oxidativos avaliados por dois parâmetros

oxidativos diferentes de danos. Foi demonstrado pela primeira vez que o tratamento

com antioxidantes pode significativamente reverter danos cognitivos tardios em

sobreviventes de sepse em um modelo animal de CLP. Isto foi associado com a

atenuação nos danos oxidativos do hipocampo em períodos iniciais do

desenvolvimento da sepse.

A grande maioria dos estudos em animais (Andrades et al 2005, Jao et al

2005, Koksal et al 2004) e em humanos (Goode et al 1995, Cighetti et al 2005,

Mishra et al 2005, Winterbourn et al 2000) demonstram uma relação entre estresse

oxidativo e disfunção de múltiplos órgãos durante a sepse, mas não em SNC. Neste

sentido, diversos estudos utilizam antioxidantes para o tratamento de sepse em

animais (Victor et al 2003, Matejovic et al 2005, Supinski et al 2006, Carlson et al

2006) e humanos (Spapen et al 2005, Emet et al 2004, Hein et al 2004, Heller et al

2001, Rank et al 2000, Ortolani et al 2000, Spapen et al 1998, Angstwurm et al

2007). Tanto a NAC quanto a DFX já foram testados isoladamente para o tratamento

de sepse em animais. A NAC é efetiva em diferentes modelos de sepse, incluindo

endotoxemia (Hsu et al 2006) e CLP (Ozdulger et al 2003). Em modelo de CLP,

Messaris e colaboradores (2004) demonstraram que o uso profilático de DFX reduz

apoptose e mortalidade.

As evidências de 10 coortes sugerem que 25 a 78% dos sobreviventes de

centros de terapia intensiva têm experiências de danos cognitivos (Hopkins e

Jackson, 2006). Nestes pacientes geralmente a memória é o dano mais

freqüentemente observado, seguido pela função executiva e o dano na atenção

(Hopkins e Jackson, 2006).

Os mecanismos subjacentes a estas alterações ainda são obscuros. Há

provavelmente várias causa para estas incapacidades, mas sim diversos de fatores

que interagem dinamicamente com variáveis e resultados de pré morbidagem

(Hopkins e Jackson, 2006). Assim, usando um modelo animal clinicamente relevante

de sepse foi possível demonstrar que os danos oxidativos do hipocampo em

períodos inicias parecem ser importantes no desenvolvimento das incapacidades

cognitivas tardias em sobreviventes de sepse.

Este estudo supõe que haja uma alteração em cascatas de complexos

eventos que conduzam finalmente a disfunção do SNC e danos cognitivos tardios

observados em ratos sobreviventes de CLP. Alguns poucos estudos foram

publicados na intenção de determinar os mecanismos moleculares associados os

danos cognitivos tardios no contexto da medicina do cuidado crítico. Usando um

modelo animal de meningite baseado em S. pneumoniae Irazuzta e colaboradores

(2005) relataram que o tratamento com dexametasona em ratos com meningite

bacteriana conduz a menor ativação da caspase 3 mas não da caspase 1 e estes

foram associados com a preservação do desempenho neurocomportamental, porém

neste estudo necessita-se ser muito cauteloso quando o desempenho da avaliação

da aprendizagem e da memória tratados com dexametasona (Martins et al, 2005).

Porém, a administração de hidrocortisona durante o choque séptico em uma

dosagem similar à taxa de produção endogenosa foi associada com uma menor

incidência de alterações pós-traumáticas de estresse em sobreviventes em longo

prazo (Schelling et al, 2001).

O Grupo de Irazuzta tem demonstrado isto, embora a hipotermia possa

estar reduzindo a resposta inflamatoria e biomarcadores de ferimento do cérebro

não melhoram o desempenho neurocomportamental em um animal modelo de

meningite bacteriana (Irazuzta et al, 2002).

Desde que o estresse oxidativo é associado ao desenvolvimento de

doenças neurodegenerativas (Halliwell, 2006) sendo importante para o

desenvolvimento da síndrome da disfunção dos múltiplos órgãos durante a sepse

(Salvemini e Cuzzocrea, 2002) e suponha-se que poderia contribuir a longo prazo

para os danos cognitivos em sobreviventes de sepse. O estresse Oxidativo foi

demonstrado previamente dentro de modelos animais diferentes de doença crítica.

Sener e colaboradores (2005) relataram que os danos oxidativos descritos reduzem

o índice antioxidante em um modelo animal de sepse e determinando um efeito

antioxidante no ajuste. Nenhum estudo precedente correlacionam o estresse

oxidativo do cérebro e danos cognitivos a longo prazo de sobreviventes de doenças

críticas. A avaliação dos mecanismos e das cascatas moleculares sinalizadoras dos

quais induzem eventos que conduzem finalmente aos danos no cérebro durante a

sepse aperfeiçoaram os conhecimentos sobre a fisiopatologia da sepse e, mais

importante, promoverá também o desenvolvimento de estratégias terapêuticas mais

eficientes, para impedir as seqüelas neurológicas.

Os antioxidantes utilizados podem interferir com NO e seus metabólitos ou

com espécies reativas de oxigênio que devem ter ligação com a disfunção

mitocondrial da sepse. NAC pode atuar como scavenger direto de NO e peroxinitrito

(Halliwell e Gutteridge 2007), além de o uso de NAC e DFX, como descrito

anteriormente, pode interferir na geração de EAO e com isto diminuir o estresse

oxidativo e preservar a função mitocondrial.

O uso de antioxidantes pode apresentar uma dupla face na sepse. Por um

lado pode reduzir a resposta inflamatória e minimizar o dano oxidativo, mas a

resposta inflamatória e necessária para a erradicação do patógeno, reparo dos

tecidos envolvidos e ativação do sistema imune adquirido. Neste sentido cada vez

mais se caracteriza e determina a importância da resposta antiinflamatória na sepse

(Hotchkiss RS et al 2003). Por isso o uso de antioxidantes não pode ser visto como

isento de risco. Reduzir a resposta inflamatória através de estratégias

farmacológicas específicas já se mostrou ineficaz em estudos clínicos (Eichacker et

al 2002). Portanto o tempo e a dose da administração de antioxidantes devem ser

fundamentais na eficácia desta terapia, para que exista redução do dano oxidativo e

mitocondrial e atenuação da resposta inflamatória, sem prejuízo da erradicação dos

patógenos.

Neste contexto os resultados deste estudo sugerem uma incapacidade

cognitiva em curto e longo prazo incluindo alterações do aprendizado e memória,

além de sintomas ligados a depressão e, pela primeira vez, um papel dos danos

oxidativos em períodos iniciais no SNC no desenvolvimento do dano cognitivo em

curto e longo prazo. A associação de NAC e DFX pode ter um papel terapêutico

para o dano oxidativo em hipocampo. Entretanto, é difícil com nossos dados

determinar o exato mecanismo que leva ao estresse oxidativo e ao tratamento

adequado com antioxidantes para reverter e prevenir este processo.

Limitações do estudo e perspectivas futuras

Os modelos de doenças inflamatórias empregados apresentam algumas

limitações. O modelo de sepse, CLP, certamente é o mais empregado na literatura

por mimetizar sepse abdominal de humanos, ainda mais quando se utiliza reposição

volêmica e antibióticos, além de administrar as drogas de estudo após o

desenvolvimento de sepse como em nosso estudo. Entretanto, diferente da prática

clínica, em nosso modelo não removemos o foco infeccioso cirurgicamente por

dificuldades na realização de tal procedimento nestes animais. Existe modelo

recentemente desenvolvido, peritonite fecal por cateterização do cólon ascendente,

que facilita a remoção cirúrgica do foco infeccioso (Lustig et al 2007). Entretanto,

este modelo ainda não é bem estabelecido e o perfil de citocinas parece ser

diferente do CLP (Maier et al 2004), sendo uma das possibilidades de expansão de

nossos resultados no futuro.

A compreensão, mesmo que ainda não definitiva, dos fenômenos

neurobiológicos envolvidos no dano cognitivo em sobreviventes a sepse, evoluiu

sobremaneira nesta última década. Os conceitos, contudo, não foram alterados;

ocorreu, de fato, um aprofundamento do entendimento de seu substrato biológico.

Os resultados compilados nesta tese compreendem uma pequena fração desse

corpo de novos conhecimentos sobre os mecanismos que levam a incapacidade

cognitiva. O novo desafio é compreender os mecanismos que levam ao dano

cognitivo em sobreviventes a sepse e o surgimento de novas terapias que possam

impedir este dano a curto e longo-prazo.

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