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MINISTÉRIO DA EDUCAÇÃO
UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE
CENTRO DE CIÊNCIAS DA SAÚDE
PROGRAMA DE PÓS GRADUAÇÃO EM CIÊNCIAS DA SAÚDE
CONSEQUÊNCIAS DA COLECTOMIA ASSOCIADA À HEPATECTOMIA NO
METABOLISMO HEPÁTICO E NA FORMA E FUNÇÃO DE HEMÁCIAS EM RATOS
MARÍLIA DANIELA FERREIRA DE CARVALHO
NATAL/RN
2012
ii
MARÍLIA DANIELA FERREIRA DE CARVALHO
CONSEQUÊNCIAS DA COLECTOMIA ASSOCIADA À HEPATECTOMIA NO
METABOLISMO HEPÁTICO E NA FORMA E FUNÇÃO DE HEMÁCIAS EM RATOS
ORIENTADOR: Prof. Dr. ALDO DA CUNHA MEDEIROS
COORIENTADOR: Prof. Dr. IRAMI ARAÚJO FILHO
NATAL/RN
2012
Dissertação apresentada ao Programa de Pós-Graduação em Ciências da Saúde da Universidade Federal do Rio Grande do Norte como requisito para a obtenção do título de Mestre em Ciências da Saúde.
iii
MINISTÉRIO DA EDUCAÇÃO
UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE
CENTRO DE CIÊNCIAS DA SAÚDE
PROGRAMA DE PÓS GRADUAÇÃO EM CIÊNCIAS DA SAÚDE
Coordenadora do Programa de Pós-Graduação em Ciências da Saúde:
Prof.a Dra.: Ivonete Batista de Araújo
iv
MARÍLIA DANIELA FERREIRA DE CARVALHO MOREIRA
CONSEQUÊNCIAS DA COLECTOMIA ASSOCIADA À HEPATECTOMIA NO
METABOLISMO HEPÁTICO E NA FORMA E FUNÇÃO DE HEMÁCIAS EM RATOS
Aprovada em: ____/____/______
Banca Examinadora:
Presidente da Banca:
Prof. Dr. Aldo da Cunha Medeiros – UFRN
________________________________________
Membros da Banca:
Prof.ª Dra. Maria Goretti Freire de Carvalho – UnP
________________________________________
Prof. Dr. Geraldo Barroso Cavalcanti Júnior – UFRN
________________________________________
v
A Daniel, meu irmão, por me fazer
entender com a sua ausência o essencial
na vida, e por sempre ser uma referência
às minhas atividades intelectuais.
vi
AGRADECIMENTOS
A Deus, pela minha vida, minha saúde e minha família.
Ao meu orientador, Prof. Aldo da Cunha Medeiros, por ter despertado em mim
a vocação de ensinar e pesquisar, por ter me dado a oportunidade de estar ao seu
lado aprendendo com o seu exemplo de honestidade, determinação, ética, paciência
e cordialidade.
Ao Prof. Irami Araújo Filho, por ter me ensinado muito além da técnica
cirúrgica através de seu exemplo de simplicidade e dedicação aos nossos pacientes.
À Amália Cínthia Meneses do Rêgo, pelo exemplo de mulher, mãe,
professora e pesquisadora; pela orientação de toda parte laboratorial do nosso
estudo.
Ao estatístico Ítalo Medeiros Azevedo, pela orientação do nosso trabalho.
Aos meus pais, Antônio Martins e Fátima, pelo amor incondicional, principal
apoio na realização dos meus sonhos e por sempre estimularem minhas atividades
intelectuais.
Ao meu marido, Ricardo Moreira, por sua dedicação, compreensão, companheirismo
e incentivo constante à realização dos meus sonhos; por tornar minha vida completa
e feliz.
Às minhas irmãs, Diva e Michele, a quem tenho amor absoluto, por estarem
sempre ao meu lado me apoiando, dividindo comigo os momentos mais difíceis e
felizes.
Às minhas amigas, em especial à Mônica, a quem amo como uma irmã, que
sempre esteve próxima, mesmo agora distante, e sempre me motivou.
A minha sobrinha Júlia por preencher nossos corações com o seu sorriso.
vii
“Feliz aquele que transfere o que
sabe e aprende o que ensina.”
(Cora Coralina)
viii
RESUMO
Este trabalho trata de investigação sobre a influência da colectomia associada
à hepatectomia parcial, na biodistribuição do fitato-99mTcO4, na marcação e
morfologia de hemácias e parâmetros metabólicos da função hepática. Dezoito ratos
Wistar foram distribuídos em três grupos (seis animais cada), denominados:
colectomia, colectomia+hepatectomia e sham. No primeiro grupo os animais foram
submetidos a uma colectomia direita, no segundo foram submetidos ao mesmo
procedimento porém associou-se uma hepatectomia esquerda e no terceiro houve
apenas realização de uma laparotomia e leve manipulação de alças intestinais. No
trigésimo dia pós-operatório, foi feita injeção de 0,1 mililitro intravenoso de fitato-99mTcO4 (radioatividade 0,66 MBq) em todos os animais. Após quinze minutos, uma
amostra de fígado foi colhida e pesada. O percentual de radioatividade por grama de
tecido (%AIT/g) foi determinado no fígado e hemácias usando-se um contador gama
automático. Dosagem sérica de alanina aminotransferase (ALT), aspartato
aminotransferase (AST), fosfatase alcalina (FA), morfologia e marcação de
hemácias com pertecnetato foram determinadas. O %AIT/g no fígado e nas
hemácias foi menor nos animais dos grupos colectomia e colectomia+hepatectomia
do que no grupo sham (p<0,05; teste de Tukey). Nenhuma diferença foi detectada
comparando os grupos colectomia e colectomia + hepatectomia. A morfologia das
hemácias não diferiu entre os 3 grupos. Os níveis séricos de AST, ALT e FA foram
significativamente maiores no grupo colectomia+hepatectomia do que no grupo
colectomia (p<0,001). Em conclusão, a colectomia associada a hepatectomia
contribuiu para reduzir a captação de radiofármaco no fígado e hemácias de ratos,
coincidindo com alterações na atividade enzimática do fígado.
Descritores: Colectomia. Hepatectomia. Metabolismo. Biodisponibilidade.
Radiofármaco.
ix
ABSTRACT
This study investigated the influence of partial colectomy associated with
hepatectomy on the biodistribution of the 99mTc-phytate, on metabolic parameters, as
well as labeling and morphology of red blood cells. Wistar rats were distributed into
three groups (each with 6), nominated as colectomy, colectomy+hepatectomy and
sham. In the 30th postoperative day all rats were injected with 99mTc-phytate 0.1mL
i.v. (radioactivity 0.66 MBq). After 15 minutes, liver sample was harvested and
weighed. Percentage radioactivity per gram of tissue (%ATI/g) was determined using
an automatic gamma-counter. Serum AST, ALT, alkaline phosphatase and red blood
cells labeling were determined. The liver %ATI/g and red blood cells labeling were
lower in colectomy and colectomy+hepatectomy rats than in sham rats (p <0.05), and
no difference was detected comparing the colectomy and colectomy+hepatectomy
groups. Red blood cells morphology did not differ among groups. Serum levels of
AST, ALT and alkaline fosfatase were significantly higher in colectomy+hepatectomy
than in colectomy rats (p<0.001). Hepatectomy associated with colectomy lowered
the uptake of radiopharmaceutical in liver and in red blood cells in rats, coinciding
with changes in liver enzymatic activity.
Key words: Colectomy. Hepatectomy. Metabolism. Biological availability.
Radiopharmaceutical.
x
LISTA DE ABREVIATURAS E SIGLAS
UFRN- Universidade Federal do Rio Grande do Norte
PVPI- Polivinil Pirrolidona Iodo ou iodopovidona
CNEM- Comissão Nacional de Energia Nuclear
LIGA- Liga Norteriograndense Contra o Câncer
%AIT/g- Percentual de radioatividade por grama de tecido
ALT- Alanina aminotransferase
AST- Aspartato aminotransferase
FA- Fosfatase Alcalina
ANOVA- Análise de variância
Tb- Tempo de meia vida biológica
keV- Quiloelétron volt 99Mo/99mTc- sistema gerador de tecnécio a partir de molibdênio 99mTc- 99mTecnécio
Na99mTcO4- Pertecnetato de Sódio
SnCl2 - Cloreto Estanoso
CCR- Câncer colorretal
UTI- Unidade de Terapia Intensiva
CEP- Comitê de Ética em Pesquisa
MBq- Megabecquerel
HUOL- Hospital Universitário Onofre Lopes
xi
LISTA DE FIGURAS
FIGURA 1-Retirada das amostras hepáticas ............................................................14
FIGURA 2-Lavagem das amostras hepáticas ...........................................................14
FIGURA 3-Pesagem dos fragmentos hepáticos em balança de precisão.................15
FIGURA 4-Imagem do Contador Gama.....................................................................15
FIGURA 5-Fotomicrografia das hemácias nos três grupos estudados. (A) Grupo
colectomia, (B) Grupo colectomia + hepatectomia e (c) Grupo Sham. 1000x...........23
xii
LISTA DE TABELAS
Tabela 1- Distribuição do radioisótopo em atividade radiológica por grama de tecido
em cada grupo...........................................................................................................23
Tabela 2- Níveis séricos das enzimas hepáticas nos três grupos estudados............22
xiii
01
01
03
04
05
08
10
12
12
12
13
17
17
28
41
52
62
76
89
98
110
116
SUMÁRIO
1- INTRODUÇÃO…..……………………………………………………….…......................
1.1-Os Radiofármacos………….…………………………………………………………….
1.2-O Tecnécio………………………………………………………..……………………….
1.3-A Cintilografia………………..…………………………………………………………....
1.4-Colectomia e hepatectomia……………………………………..……………………….
1.5-Marcação e Morfologia das hemácias.....………..…………………………………….
2-JUSTIFICATIVA………..…………………………………………...…….….....................
3- OBJETIVO……………………………………………………………………....................
3.1-Objetivos gerais…………………………………………………………………………...
3.2-Objetivos específicos...............................................................................................
4- MÉTODO...................................................................................................................
5-ARTIGOS PRODUZIDOS...........................................................................................
5.1-Metabolic and hematologic consequences of colectomy associated to
hepatectomy in rats……………………………………………………………………………
5.2-Metabolism and gastric remnant changes after Roux-en-Y Gastric Bypass in
rats………………………………………………………………………………………….......
5.3-Diabetes and biodistribution of pertechnetate (Na99mTcO4) in rats………………..
5.4-Splenectomy changes the biodistribution of pertechnetate (99mTcO4-) in
rats.................................................................................................................................
5.5-Bacterial translocation in rats treated with simvastatin undergoing intestinal
ischemia and reperfusion……………………………………………………………………..
5.6-Ileal interposition for the treatment of diabetes in rats: repercussion on beta cells
mass…………………………………………………………………………………………….
5.7-Hepatectomy for benign and malign diseases in University Hospital.......................
5.8-Sildenafil as a vasodilatatory mediator in the treatment of abdominal
sepsis……………………………………………................................................................
6- COMENTÁRIOS, CRÍTICAS E SUGESTÕES...........................................................
7-REFERÊNCIAS...........................................................................................................
1
1. INTRODUÇÃO
Ao longo dos anos os avanços na área da saúde modificaram a forma como é
diagnosticada e tratada a grande maioria das doenças. Alguns métodos diagnósticos
possuem grande precisão porém, não são ao todo isentos de resultados
equivocados, principalmente quando se entende o paciente como um ser singular
exposto a situações e experiências distintas. Já é bem estabelecido na literatura que
algumas doenças, medicações, radioterapias e cirurgias podem falsear resultados
de exames cintilográficos1-10, visto que estas circunstâncias podem modificar a
capacidade de ligação entre os radiofármacos e as proteínas teciduais9.
Uma cirurgia de grande porte (um extenso procedimento cirúrgico
relativamente difícil, envolvendo órgãos vitais) pode resultar em importantes
alterações anatômicas e metabólicas e/ou complicações mutáveis e
imprevisíveis4,11,12. Existem poucos trabalhos na literatura que avaliam os efeitos
dessas intervenções cirúrgicas na biodistribuição de radiofármacos3.
1.1 Os radiofármacos
Radiofármacos são compostos químicos radioativos usados em procedimentos
diagnósticos e terapêuticos4,5,7,11,13-15. Na medicina nuclear quase 95% dos
radiofármacos são usados para fins de diagnóstico, enquanto os demais são
utilizados para tratamento, podendo este último causar danos aos tecidos. Quando
possuem função diagnóstica são utilizados para avaliar esvaziamento gástrico,
função hepática, função tireoidiana, doenças ósseas, metástases à distância,
sangramentos, entre outras aplicações3,4,7,11,16,17, além de distúrbios
hemodinâmicos18, avaliação da perfusão pulmonar19, função e anatomia renal20, e
estudos da perfusão miocárdica21. A distribuição de um radiofármaco num órgão é
proporcional ao seu grau de disfunção. Por exemplo, no fígado a biodistribuição
mantém correlação com o grau da gravidade da doença hepática, a severidade do
grau de fibrose na histologia e da disfunção hepática22. Ainda em relação ao fígado,
a cintilografia também é uma ferramenta no diagnóstico diferencial da esteato-
hepatite não alcoólica da simples esteatose. A captação hepática de 99mTc-fitato
fornece um índice prático da função hepática e da sua imagem, usando a
cintilografia como importante método diagnóstico das alterações da função e
2
morfologia23. Um radiofármaco tem dois componentes: um radionuclídeo e um
fármaco; a escolha de qual radiofármaco deverá ser usado é orientada pela sua
predileção por determinado órgão ou por determinado componente fisiológico que se
pretende ser avaliado14,24. É desejável que o radiofármaco se localize
preferencialmente no órgão objeto do estudo, uma vez que a atividade de áreas não
alvo podem obscurecer os detalhes estruturais da imagem do órgão alvo. As
radiações do radioisótopo devem ser facilmente detectadas através de
equipamentos nucleares, e a dose de radiação para o paciente deve ser mínima. Por
serem usados em humanos, essas substâncias devem ser estéreis e livres de
pirógenos, sendo submetidos a todas as medidas de controle de qualidade exigidas
para um medicamento convencional. A diferença entre um traçador radioativo e um
radiofármaco reside no fato de que o primeiro não pode ser administrado em
humanos, devido à falta de controles adequados como o de esterilidade e
apirogenicidade. Geralmente estas substâncias são administrados em pequena
quantidade24, em dose única e não devem produzir efeitos farmacológicos. Os
radiofármacos considerados ideais deverão apresentar rapidez de obtenção, fácil
disponibilidade e produção, baixo custo e meia-vida suficientemente curta para
diminuir a taxa de exposição do doente à radiação4,14 mas também longa o suficiente
para permitir finalizar o processamento da imagem. Um radiofármaco decai com
uma meia-vida definida que é caracterizada pelo radionuclídeo, denominada pela
abreviação Tp ou t1/2, independente das condições físico-químicas. Quando um
radiofármaco é administrado, o processo de biodistribuição se inicia e este consiste
de: absorção, distribuição, metabolismo e excreção; esta última etapa pode
acontecer através da eliminação fecal, urinária, transpiração ou outros mecanismos.
A biodistribuição de um radiofármacos baseia-se nos mesmos princípios
farmacocinéticos descritos para um agente terapêutico e pode ser alterada por
procedimentos cirúrgicos25. Há fatores que afetam a marcação de hemácias com 99mTc, entre eles: o tempo de estanhagem, níveis de hematócrito, método de injeção,
período de tempo que o radiofármaco é incubado com hemácias, volume de sangue
radiomarcado, a quantidade de íon estanoso usado, entre outros. Particularmente
com o advento do método in vivo de marcação das hemácias, problemas relativos à
interferência de vários medicamentos começaram a aparecer na literatura7-10 . Desse
modo, quando um paciente está usando um determinado medicamento, ou se foi
previamente submetido a um procedimento cirúrgico, a biodistribuição do
3
radiofármaco administrado por via intravenosa para a realização de cintilografia
poderia7-9,25,26 gerar a possibilidade de diagnósticos equivocados, levando à
repetição do exame com um aumento na dose de radiação4,5. Este fato não ocorre
somente no tecido traumatizado, mas também em outros órgãos não manipulados
pela intervenção cirúrgicas7. O decaimento biológico de um radiofármaco segue uma
lei exponencial semelhante ao do decaimento do radionuclídeo. Assim, cada
radiofármaco tem uma meia-vida biológica (Tb), sendo este o tempo necessário para
que a metade dele desapareça e, por conseguinte, está relacionada com uma
constante de decaimento, λb = 0.693/Tb. Portanto, em qualquer sistema biológico, a
perda de um radiofármaco é devida tanto à desintegração física do radionuclídeo
como pela eliminação biológica do radiofármaco. O decaimento pode ocorrer pela
emissão de partículas α e β, porém estas não devem ser usadas para fins
diagósticos, visto que causam danos aos tecidos, por este motivo prefere-se os raios
γ. Seja qual for o modo de decaimento, para estudos de diagnóstico do
radionuclídeo, este deve emitir radiação γ com uma energia entre 30 e 300 keV14.
Abaixo de 30 keV os raios γ são absorvidos pelo tecido e não são captados pelo
detector24.
1.2 O 99mTecnécio
O crescimento e a ampla aplicação da medicina nuclear diagnóstica tem sido
impulsionada pela fácil disponibilidade do tecnécio através do sistema gerador 99Mo/99mTc portátil e das propriedades quase ideais da radiação gama produzida por
este radionuclídeo que possibilitou seu uso clínico nos mais diversos tecidos26. O 99mtecnécio (99mTc) é um metal de transição de cor acinzentada pertencente ao
grupo VIIB da tabela periódica e tem o número atômico 43. Apresenta emissão de
raios gama numa energia de 140KeV11,27-29 com pequena emissão de radiação
corpuscular29, sendo obtido em gerador de molibidênio (99Mo/99mTc)14,15 que é o
gerador mais importante em medicina nuclear, o que levou à disponibilidade quase
ilimitada de 99mTc29. O tecnécio pode existir em oito estados de oxidação de -1 até
+7, que resultam da perda de um determinado número de elétrons. A estabilidade
destes estados de oxidação depende do tipo de ligantes e do ambiente químico,
sendo os estados +4 e +7 os mais estáveis, sob a forma de óxidos, sulfuretos,
halogenetos e pertecnetatos. Os estados de oxidação mais baixos, -1, 1+, +2, e +3,
4
são normalmente estabilizados por complexação com ligantes24. A forma química
utilizada para leitura em gama câmera é o pertecnetato de sódio (Na99mTcO4),
possui valência +7, e necessita de redução iônica para a marcação de substâncias,
células ou órgãos em estudo. Para tal processo, a substância mais utilizada é o
cloreto estanoso (SnCl2), sendo os componentes inicialmente dissolvidos em
solução, liofilizados e armazenados em atmosfera de azoto ou gás inerte (diminui a
oxidação do Sn2+)14.
O tecnécio é um dos radionuclídeos mais utilizados em medicina nuclear27,30,
corresponde a mais de 80% do total usado4,24,26 devido se ligar a uma grande
variedade de moléculas e células5,31. O Na99mTcO4 pode entrar ou sair do meio
intracelular livremente, num processo de difusão passiva32. Além disso, essa
substância preenche muitos dos critérios de um radionuclídeo ideal: possui radiação,
custo, impacto ambiental e energia de emissão muito baixos; é de fácil uso na
marcação de hemácias; tem facilidade de obtenção; possui 10% de conversão
interna e uma meia vida curta (6h), o que diminui a taxa de exposição dos doentes à
radiação2,11,15,24,26,33,34.
1.3 A cintilografia
As radiações gama (γ), são emissões de natureza eletromagnética, ou seja,
são fótons com massa desprezível e sem carga elétrica; tais características
conferem a esta radiação uma menor possibilidade de interação com a matéria
quando comparada à capacidade de interação das partículas alfa (α) e beta (β).
Assim, a radiação gama tem menor capacidade de ionização e alta energia, o que
permite que ela percorra longas trajetórias e atravesse corpos de maior espessura.
Esses fatores fizeram com que a medicina nuclear a elegesse como a radiação
usada nos exames de imagem, sendo o tecnécio o isótopo radioativo mais
frequentemente usado como seu emissor35.
Após a administração do radiofármaco, geralmente por via endovenosa,
podendo ser também via inalatória ou oral, os radioisótopos, ou os compostos aos
quais estão acoplados, têm um comportamento biológico que é idêntico ao de
similares não radioativos. Este comportamento biológico é determinado pelas
características físico-químicas do composto e também pelo estado funcional dos
diferentes tecidos ou células que podem estar envolvidos em sua manipulação5.
5
Essas radiações partem do interior do corpo e são captadas por equipamentos,
dotados de um cristal sensível, que capta cada radiação que chega produzindo uma
centelha ou cintilação para formar a imagem final, daí o nome cintilografia. O
aparelho que registra as radiações é chamado de gama câmara e seu componente
principal é a câmara de cintilação, onde se originam informações processadas por
um sistema complexo de computação que serão convertidas em imagem. Esta
câmara opera no mesmo princípio de um contador de radiação gama, mas é
consideravelmente mais complexa na sua construção e funcionamento35,36. A
biodistribuição pode ser medida por método invasivo, envolvendo o sacrifício de
animais e quantificação da radioatividade nos órgãos e tecidos de interesse, como
realizado no nosso estudo, ou por métodos não invasivos37. Esse exame pode ser
realizado em diversos órgãos como coração, pulmões, ossos, rins e outros. No caso
da cintilografia renal, o exame fornece imagens funcionais dos rins, ou seja, mostra
como as células estão desempenhando suas funções. Devido à permanência
prolongada do traçador no córtex renal, o exame fornece informações principalmente
relativas à concentração do mesmo no parênquima renal, morfologia e dimensões
dos rins38. Outros exemplos do uso da cintilografia com o tecnécio (99mTc-
pertecnetato de sódio) é o diagnostico da mucosa gástrica ectópica (pesquisa de
divertículo de Meckel) onde o pertecnetato é captado pela mucosa, além do uso na
pesquisa do sangramento intestinal39-41. A sensibilidade avançada dessa técnica
permite detectar alteração na função de diversos órgãos, muitas vezes superior à de
outros exames36.
1.4 Colectomia e hepatectomia O câncer colorretal corresponde à segunda causa de morte por câncer no
Estados Unidos e Reino Unido42-45 além de ser o terceiro mais prevalente no
mundo44 depois do câncer de pulmão e estômago46; aproximadamente 150.000
casos de câncer colorectal (CCR) ocorrem todos os anos nos Estados Unidos47 com
63.000 mortes anualmente45. No Brasil o cancer colorretal também é o terceiro mais
comum, no ano de 2012, esperam-se 14.180 casos novos de câncer do cólon e reto
em homens e 15.960 em mulheres48. Aproximadamente 40% dos pacientes com
CCR morrem devido à recorrência e às metástases. Durantes as duas últimas
6
décadas, testemunhamos melhorias substanciais na sobrevida de pacientes com
esta neoplasia que foi resultante de um diagnóstico mais precoce, maior eficiência
da quimioterapia e radioterapia, e os avanços nas técnicas cirúrgicas, melhorias na
área da anestesia e cuidados pós-operatórios, principalmente em UTIs, reduzindo a
morbi-mortalidade apesar da abordagem cirúrgica mais agressiva49. No entanto, a
melhor promessa de cura continua sendo a excisão cirúrgica adequada do
tumor43,44,50-53.
O fígado é o sítio mais frequente de metástases do CCR e estas podem ocorrer
em até 60% dos pacientes43,47,52-54 porque a drenagem venosa do cólon e reto
superior ocorre através da veia porta que drena diretamente para o fígado. Sabe-se
que 60-70% do câncer colorretal recorrente envolve o fígado e que este é o órgão
unicamente envolvido em 20-35% dos casos46-49. No momento do diagnóstico entre
15-25% dos pacientes têm metástases hepáticas clinicamente detectáveis41-44,52 e
em torno de 20%-45% dos pacientes vão desenvolver metástase hepática
metacrônica43,46,55, que são aquelas evidenciadas após 6 meses do diagnóstico.
Porém apenas 20% dos pacientes possuem doença ressecável44,46. As recidivas
após ressecção primária das lesões estão confinadas ao fígado, em
aproximadamente, 50% dos casos46.
As ressecções das metástases hepáticas caracterizavam um grande desafio
para os cirurgiões no passado, devido a sua alta taxa de mortalidade, mas
atualmente a taxa de mortalidade é de cerca de 1-5% em importantes centros
médicos46,56. Esses procedimentos se tornam cada vez mais seguros, podendo-se
ressecar até 80% do parênquima hepático57. Apesar desses avanços, a taxa de
ressecabilidade de metástases no momento do diagnóstico ainda é baixa,
representando a baixa proporção de pacientes que podem se beneficiar de uma
abordagem cirúrgica. Os principais obstáculos para a indicação da ressecção
hepatica são: a doença metastática bilobar, tumores volumosos e deixar parênquima
hepático residual funcional suficiente após a ressecção57.
As hepatectomias são procedimentos relacionados a grande morbi-mortalidade
devido a insuficiência hepática que guarda relação com o tamanho da ressecção e
se o paciente tem outras doenças hepáticas. Não há evidência de que o número de
lesões ou suas localizações no fígado comprometam a sobrevida desde que toda a
doença macroscópica seja ressecada. Tem sido argumentado que o fator limitante
para o número de lesões que podem ser ressecados é se é tecnicamente possível
7
remover todos os tumores43,49. Porém, os critérios de ressecção vêm sendo
contestados por diferentes grupos de pesquisadores ao longo da última década44,52;
além disso, o paradigma sobre o tratamento cirúrgico das metástases colorretais
sincrônicas também começou a mudar, com autores relatando bons resultados para
ressecção simultânea de cólon e metástases do fígado. Os defensores de uma
abordagem em dois tempos aprovam esta estratégia devido à preocupações com
aumento da morbidade e mortalidade associadas à ressecção simultânea do câncer
colorretal com metástases hepáticas58, porém tal fato culmina com uma abordagem
mais agressiva no tratamento de metástases hepáticas colorretais45. Embora o
câncer colorretal primário tenha um bom prognóstico quando ressecado com
margens livres e linfonodos negativos, o diagnóstico de metástases hepáticas é o
prenúncio de uma evolução pior. Até recentemente, os pacientes inicialmente
considerados como não-ressecáveis foram tratados através de quimioterapia
paliativa, com baixas chances de sobrevida em cinco anos51.
Estratégias que ampliaram as indicações de ressecção hepática incluindo a
quimioterapia locorregional44, embolização da veia porta, quimioterapia
neoadjuvante e os procedimentos ablativos; estes últimos apesar da alta taxa de
recorrência local podem ser considerados como opção terapêutica nos pacientes
sem indicação de quimioterapia loco-regional59. A recente adição de novos regimes de quimioterapia, tais como o irinotecano e
oxaliplatina46 e terapia com drogas específicas, tais como bevacizumab e cetuximab
demonstraram sobrevida mediana de até 20 meses para pacientes com câncer do
cólon, mas eles raramente fornecem uma cura a longo prazo. As taxas de resposta
obtidas com 5-fluorouracil e leucovorina foram significativamente aumentadas em
combinação com oxaliplatina, irinotecano. Isto permitiu o aumento da sobrevida do
paciente em um status paliativo, mas também ofereceu a possibilidade de cura de
pacientes previamente irressecáveis sendo estes considerados em reestadiamento
melhor que os anteriores. Por reconsiderar a irressecabilidade inicial de pacientes
que respondem fortemente à quimioterapia, a sobrevida poderia então ser alcançada
através da ressecção hepática em uma significativa proporção de pacientes51. Cerca
de 10-20% dos pacientes considerados irresecáveis passam a ser candidatos à
metastasectomia. A cirurgia oferece a melhor chance de cura em pacientes com
metástases hepáticas colorretais sendo as taxas de sobrevida após a ressecção, em
cinco anos, entre 25% e 58%46,49,52-56 com uma sobrevida mediana entre 28 e 40
8
meses em grandes séries. Resultados de dez anos de sobrevida foram
documentados em torno de 20% a 26%. Em contraste, a sobrevida mediana para
não-ressecados, com metástases hepáticas colorretais, é 5-10 meses54. Porém, nem
todos os pacientes possuem indicação cirúrgica e a decisão sobre ressecabilidade
depende das condições do paciente, extensão da doença e da função hepática.
Desse modo, apenas 10 a 20 % são candidatos à ressecção52,59. Apesar disto, as
opções para tratamento não-cirúrgico do câncer colorretal metastático é muito vasta.
Se a metástase hepática é diagnosticada juntamente ao tumor primário, e é
factível sua remoção, deve ser ressecada no mesmo ato operatório do tumor
primário, mas caso seja evidenciada posteriormente, ela é ressecada em outro
tempo cirúrgico. No presente trabalho optamos por submeter os animais a uma
colectomia direita e hepatectomia esquerda simulando que esses teriam um tumor
de cólon direito e metástases hepáticas apenas à esquerda (que caracterizaria uma
cirurgia de grande porte) para avaliar as possíveis alterações, em modelo animal, na
biodistribuição do radiofármaco e da morfologia das hemácias. Não foi objeto do
nosso estudo a carcinogênese , desse modo não foi induzido câncer nestes animais,
mas sim o impacto de uma cirurgia de grande porte nos parâmetros estudados. A
cirurgia em questão foi escolhida por ser um procedimento cirúrgico cada vez mais
indicado para o tratamento, com possibilidade curativa, de paciente com diagnóstico
de adenocarcinoma de cólon com metástase hepática, visto que sua incidência é
crescente ao longo das últimas décadas.
1.5 Marcação e Morfologia das hemácias
Desde o primeiro relato do uso do tecnécio como métodos de marcação de
hemácias, foram observadas melhorias que levaram a redução: do dano celular, da
eficiência de marcação e de rendimento, e do tempo gasto no procedimento60. Os
glóbulos vermelhos marcados com 99mTc são usados em muitos procedimentos de
medicina nuclear2,10,34,61, incluindo os estudos de localização do baço, do sistema
cardiovascular, da placenta7,24, além de localizar sítios de sangramento
gastrointestinal9,39. As hemácias marcadas com radiomarcadores podem ser usadas
in vivo, in vitro ou em uma combinação de ambos chamada in vivo/vitro7,34,62. Há
uma qualidade maior de imagem no método in vitro em comparação com as outras
técnicas31. Os passos sequenciais do processo de marcação intracelular das
9
hemácias incluem: (1) o transporte transmembranar de íons de cloreto estanoso7 e
pertecnetato no compartimento interno destas células, (2) redução de 99mTc
(99mTcO4-) pelo SnCl2 e (3) de ligação do 99mTc reduzido para hemoglobina2. A
banda-3 é o sistema de transporte do tecnécio para o interior das hemácias, e os
canais de cálcio são responsáveis pelo transporte do estanho33,34,63. A literatura
médica sobre o assunto em questão descreve o efeito de drogas artificiais e naturais
no processo de radiomarcação de células vermelhas do sangue e proteínas
plasmáticas com 99mTc. Qualquer droga pode modificar a biodisponibilidade de
radiofármacos, podendo alterar a marcação de constituintes sanguíneos atuando
como agente antioxidante, modificando a estrutura da membrana ou diminuindo a
eficiência do sistema de transporte transmembrana de íons estanoso e pertecnetato
nas células. Neste estudo avaliamos o impacto da cirurgia de grande porte na
morfologia e na marcação das hemácias61 com o tecnécio, a partir de um modelo
experimental, pois sabemos que ocorrem alterações nas mesmas em situações de
trauma, e quanto mais severo o trauma, mais intensas são as alterações64.
10
2. JUSTIFICATIVA
Já é bem estabelecido na área da medicina nuclear que determinadas
patologias, medicamentos, produtos naturais e determinados procedimentos
cirúrgicos podem alterar a biodistribuição do tecnécio e/ou fixação dos constituintes
sanguíneos7,8,9,26. Este fato não ocorre somente no tecido traumatizado, mas
também em outros órgãos não submetidos a intervenções cirúrgicas7. Este estudo
se propõe a avaliar se a biodistribuição de um determinado radiofármaco também
sofreria influência após a realização de uma cirurgia de grande porte, em modelo
murino, além de estimar se tais fatores exercem alguma alteração na função
hepática, bem como sobre a morfologia e marcação das hemácias.
O radiofármaco utilizado foi o tecnécio por ser facilmente disponibilizado e ter
propriedades quase ideais26. Devido ao CCR ser uma doença muito prevalente44, e
as metástases hepáticas associadas à este muito frequentes41-44; temos na cirurgia
o melhor tratamento para esta doença avançada43,44,51-53. Por isso, escolhemos o
procedimento cirúrgico colectomia direita com hepatectomia esquerda como
representante do grupo de cirurgia de grande porte pois, além de ser cada dia mais
realizado, por ser mais factível, este procedimento também revolucionou o
tratamento do câncer colorretal nas últimas décadas, elevando os pacientes
portadores do CCR com metástase hepática de uma situação de tratamento paliativo
a uma de potencial cura.
A cintilografia é um procedimento diagnóstico usado pela medicina nuclear
para avaliar diversas patologias como sangramentos digestivos, estudos de
perfusão, estudos funcionais renais. Esta pode ser utilizada em pacientes com
história prévia de câncer no cólon, com implantes metastáticos no fígado, que foram
submetidos à ressecção curativa através de colectomia associada à hepatectomia
para acompanhar um possível surgimento de metástase ou sangramentos em
diversos órgãos digestivos. Qualquer desvio na captação de radiofármacos por
órgãos vitais após procedimento cirúrgico pode acarretar interpretações indesejáveis
nos resultados de exames cintilográficos, conduzindo a diagnósticos equivocados de
doenças, com graves consequências ao acompanhamento dos pacientes. Além
disso, existindo a necessidade de repetição do exame teríamos, consequentemente,
um aumento na dose de radiação4,5 para o paciente e maior risco para os
profissionais envolvidos. Desse modo, o metabolismo hepático, a marcação de
11
hemácias e a biodistribuição do tecnécio podem estar alterados em órgãos
importantes, assim os exames cintilográficos poderão estar sujeitos a resultados
falso-positivos ou falso-negativos.
Pesquisa bibliográfica minuciosa em bases de dados eletrônicas revelou
ausência de estudos nessa linha de pesquisa: alteração na biodistribuição de
radiofármacos após colectomia com hepatectomia. Foram usados descritores como:
metabolismo, biodistribuição, tecnécio, isótopos radioativos versus colectomia
associada a hepatectomia. Desse modo, o estudo experimental teve a intenção de
dar contribuição importante para o conhecimento de aspectos ainda não
esclarecidos.
12
3. OBJETIVOS
3.1 Objetivo geral
Este trabalho objetiva avaliar se a realização de uma colectomia com hepatectomia
em ratos exerce influência na biodistribuição do radiofármaco 99mTc-fitato e na
função hepática.
3.2 Objetivos específicos
Avaliar se a biodistribuição do tecnécio, no fígado e hemácias de ratos, sofre
alteração após um colectomia direita com hepatectomia esquerda.
Avaliar se o procedimento cirúrgico descrito altera os valores das enzimas hepáticas:
TGO, TGP e FA.
Avaliar se tal intervenção cirúrgica altera a marcação e morfologia das hemácias.
13
4. MÉTODOS
O modelo animal escolhido para realização desta pesquisa experimental foi o
rato Wistar. Os 18 animais utilizados foram fornecidos pelo biotério do Centro de
Ciências da Saúde, com pesos no intervalo de 285±23 g. Foram alojados em gaiolas
individuais de polipropileno e mantidos sob condições controladas de temperatura e
ambiente com um ciclo claro-escuro de 12 horas. Foi permitido acesso ad libitum ao
alimento na forma de ração (Labina, Purina ®) e água.
Todos os procedimentos experimentais envolvendo os animais foram
realizados de acordo com o Código de Ética para a experimentação animal e as
diretrizes brasileiras para o uso científico de animais (Lei n º 11.794). O protocolo foi
aprovado pelo Comitê de Ética em Pesquisa (CEP) da Universidade Federal do Rio
Grande do Norte (UFRN).
No pré-operatório os animais permaneceram em jejum entre 12-18 horas. A
anestesia foi realizada com injeção intraperitoneal de 0,2 ml/100 g de uma solução
contendo 1 ml de cetamina (50 mg) e 1 ml de xilazina (20 mg) e então os animais
foram operados sob condições assépticas após tricotomia e antissepsia com álcool
70%. Além disso, foi mantida analgesia por via subcutânea imediatamente após a
cirurgia, e a cada 12 horas durante três dias com cloridrato de tramadol solução
contendo 1ml (100 mg) na dose de 20 mg/kg.
Os animais foram divididos, aleatoriamente, em três grupos com seis ratos
cada; o primeiro grupo chamado colectomia foi submetidos à laparotomia mediana e
realizou-se uma colectomia parcial direita. Foram ressecados o ceco, 10 cm de
cólon direito e 5 cm de íleo terminal. O trânsito intestinal foi reconstruído através de
anastomose término-terminal ileocólica com sutura em pontos separados usando fio
de polipropileno 6-0 (Ethicon®, Brasil), com auxílio um microscópio cirúrgico –
aumento 10x (DFVasconcelos, São Paulo, Brasil). A laparotomia foi fechada por
planos através de sutura contínua com fio mononylon 4-0 (Ethicon®, Brasil). No
segundo grupo, denominado colectomia+hepatectomia, os ratos foram submetidos à
colectomia parcial, com a mesma técnica descrita acima para o grupo colectomia,
associada à ressecção do lobo esquerdo do fígado (hepatectomia esquerda), sendo
a reconstrução do trânsito e o fechamento da cavidade realizado da mesma forma
citada para o grupo colectomia. No terceiro grupo, chamado sham, que caracteriza o
grupo controle, foi realizada laparotomia mediana e manipulação suave das alças do
14
intestino, sendo a parede abdominal fechada nas mesmas condições dos outros
grupos.
Os animais dos três grupos permaneceram em observação por 30 dias, em
gaiolas individuais nas condições ambientais previamente descritas, e nesse período
foram pesados semanalmente em balança digital (Filizola® São Paulo, Brasil) com
sensibilidade para variação a partir de 1 grama. Após esse periodo os animais foram
anestesiados e submetidos à administração de radiofármaco através da
cateterização da veia femoral numa alíquota de 0,1 ml de 99mTc-fitato,
correspondendo a uma atividade radioativa de 0,66 MBq. Decorridos quinze minutos
da administração do radiofármaco, foi colhida amostra de 5 ml de sangue por
punção cardíaca, os animais foram sacrificados com superdose de anestésico
(tiopental sódico 100mg/Kg intraperitoneal) e procedeu-se a retirada de amostra do
fígado para determinar a biodistribuição do fitato (Figura 1), a seguir estas foram
lavadas com solução salina 0,9% (Figura 2) e pesadas em balança de precisão
(Figura 3). Um contador gama automático Wizard 1470 (Perkin-Elmer®, Finlândia)
detectou a captação radioativa hepática e das hemácias, medidas em percentual de
radioatividade por grama de tecido (% ATI/g) (Figura 4).
Figura 1- Retirada das amostras hepáticas.
Figura 2- Lavagem das amostras.
15
Toda essa fase do experimento, que utiliza radiação, seguiu as normas de
radioproteção preconizados pela Comissão Nacional de Energia Nuclear (CNEM). O
radiofármaco utilizado foi fornecido pelo Serviço de Medicina Nuclear da Liga
Norteriograndense Contra o Câncer (LIGA) e os rejeitos radioativos foram
prontamente devolvidos para a LIGA em recipiente de chumbo, para tratamento
adequado.
Avaliação bioquímica As amostras de sangue, em volumes de 3 ml, foram inseridas em tubos de
ensaio sem anticoagulante, centrifugadas a 3000 rpm por 10 min e as alíquotas de
plasma estocadas a -40°C até análise. Os provas de função hepática como:
Aspartato aminotransferase (AST), alanina aminotransferase (ALT) e fosfatase
alcalina (FA) foram medidas usando um kit comercial e dosadas em auto-analisador
(Konelab, versão do software, 60i, Finlândia).
Morfologia das hemácias A partir de amostras de sangue total foram preparadas distensões sanguíneas
em lâminas, que foram coradas pelo método Leishman. As imagens das hemácias
foram capturadas por câmera digital (Samsung®) usando-se microscopia óptica
(Microscópio Olympus, modelo BX50, Japan, aumento 1000x,) para análise digital,
após a transferência da imagem do microscópio para a tela do computador. Para
Figura 3-Pesagem em balança de precisão.
Figura 4-O Contador Gama.
16
obtenção da forma das hemácias e das medidas morfométricas (relação
perímetro/área) foi utilizado programa específico (Image ProPlus Software, versão
6.0).
Estatística
Os dados obtidos foram expressos em média ± desvio padrão. A comparação
entre os grupos foi realizada pela análise de variância (ANOVA) e pelo teste de
comparações múltiplas de Tukey, usando-se significância de 0,05.
17
5. ARTIGOS PRODUZIDOS
5.1. O Artigo foi publicado no periódico Acta Cirúrgica Brasileira 2011;26(6):503-07.
ISSN 1678-2674. Que possui fator de impacto 0.2486, Qualis B1 da Capes para
Área Medicina III.
Metabolic and hematologic consequences of colectomy associated to hepatectomy in rats1
Consequências metabólicas e hematológicas da colectomia associada à
hepatectomia em ratos Marília Daniela Ferreira CarvalhoI, Irami Araújo-FilhoII, Ítalo Medeiros AzevedoIII, Amália Cínthia Meneses RêgoIV, Aldo Cunha MedeirosV
1 Research performed at Center of Experimental Surgery, Department of Surgery, UFRN, Natal-RN,
Brazil.
I Fellow Master Degree, Postgraduate Program of Health Sciences, UFRN, Natal, Brazil. Acquisition,
interpretation of data and collection of study informations. II PhD, Associate Professor, Department of Surgery, Federal University of Rio Grande do Norte
(UFRN), Natal, Brazil. Involved with technical procedures. III Fellow Master Degree, Department of Statistics, UFRN, Natal, Brazil.
Responsible for interpretation of data, statistical analysis. IV PhD, Associate Professor, Potiguar University, Natal, Brazil. Helped with technical procedures,
collection and processing of study informations V Chairman, Full Professor, Department of Surgery and Postgraduate Program of Health Sciences,
UFRN, Natal, Brazil. Responsible for conception, design, intellectual and scientific content of the
study.
ABSTRACT Purpose: This study investigated the influence of partial colectomy associated with
hepatectomy on the biodistribution of the 99mTc-phytate, on metabolic parameters, as
well as labeling and morphology of red blood cells. Methods: Wistar rats were
distributed into three groups (each with 6), nominated as colectomy,
18
colectomy+hepatectomy and sham. In the 30th postoperative day all rats were
injected with 99mTc-phytate 0.1mL i.v. (radioactivity 0.66 MBq). After 15 minutes, liver
sample was harvested and weighed. Percentage radioactivity per gram of tissue
(%ATI/g) was determined using an automatic gamma-counter. Serum AST, ALT,
alkaline phosphatase and red blood cells labeling were determined. Results: The
liver %ATI/g and red blood cells labeling were lower in colectomy and
colectomy+hepatectomy rats than in sham rats (p <0.05), and no difference was
detected comparing the colectomy and colectomy+hepatectomy groups. Red blood
cells morphology did not differ among groups. Serum levels of AST, ALT and alkaline
fosfatase were significantly higher in colectomy+hepatectomy than in colectomy rats
(p<0.001). Conclusion: Hepatectomy associated with colectomy lowered the uptake
of radiopharmaceutical in liver and in red blood cells in rats, coinciding with changes
in liver enzymatic activity.
Key words: Colectomy. Hepatectomy. Metabolism. Biological availability.
Radiopharmaceutical.
RESUMO Objetivo: Investigar a influência da colectomia associada à hepatectomia parcial, na
biodistribuição do fitato-99mTcO4, na marcação e morfologia de hemácias e em
parâmetros metabólicos. Métodos: Ratos Wistar foram distribuídos em três grupos
(6 animais cada), denominados: colectomia, colectomia+hepatectomia e sham. No
30º dia pós-operatório, em todos eles foi feita injeção de 0,1 mL i.v. de fitato-99mTcO4
(radioatividade 0,66 MBq). Após 15 minutos, uma amostra de fígado foi colhida e
pesada. O percentual de radioatividade por grama de tecido (%ATI/g) foi
determinado no fígado e hemácias usando-se um contador gama automático.
Dosagem sérica de AST, ALT, fosfatase alcalina, morfologia e marcação de
hemácias com pertecnetato foram determinadas. Resultados: O %ATI/g no fígado e
nas hemácias foi menor nos animais dos grupos colectomia e
colectomia+hepatectomia do que no grupo sham (p<0,05; teste de Tukey). Nenhuma
diferença foi detectada comparando os grupos colectomia e
colectomia+hepatectomia. A morfologia das hemácias não diferiu entre os 3 grupos.
Os níveis séricos de AST, ALT e fosfatase alcalina foram significativamente maiores
no grupo colectomia+hepatectomia do que no grupo colectomia (p<0,001).
Conclusão: A colectomia associada a hepatectomia contribuiu para reduzir a
19
captação de radiofármaco no fígado e hemácias de ratos, coincidindo com
alterações na atividade enzimática do fígado.
Descritores: Colectomia. Hepatectomia. Metabolismo. Biodisponibilidade.
Radiofármaco.
INTRODUCTION The liver is affected by metastases in 50% of patients with colorectal cancer and
metastatic disease remains the leading cause of cancer-related death. The
metastases may be single or multiple, affecting one or both hepatic lobes and are
synchronous with the primary tumor in 16 to 25% of cases, several months or years
after diagnosis1. The surgical resection of liver metastases is possible in 10 to 15%
of patients and it is considered the treatment of choice with potential for cure and
five-year survival in 22 to 65% of cases2.
One of the most widely used diagnostic tools in identifying diseases and
metabolic changes, uses radiopharmaceuticals. On nuclear medicine, pertechnetate
(99mTcO4-) is used in the diagnosis of gastric, kidney, liver, lung, intestinal, skeletal,
endocrine, heart, blood flow and postoperative disorders3. Used intravenously, this
radiopharmaceutical remains in the circulation long enough to analyse various
organic and functional parameters for the diagnosis of diseases. Gradually it comes
into equilibrium with the extracellular space, is taken up by tissues, and a small
percentage is eliminated by urinary excretion. The 99mTcO4- is used in approximately
85% of diagnosis procedures, has low cost and small environmental impact3. Some
recent studies have demonstrated significant changes in biodistribution of 99mTcO4-
on the postoperative of major surgery4-8.
In nuclear medicine, red blood cells can be labeled by 99mTcO4-, and used to
detect certain clinical disorders, digestive bleeding, blood flow, heart perfusion, etc9.
The labeling of blood constituents with 99mTcO4- has been used as a tool in
experimental studies in vitro and in vivo, in order to demonstrate the interaction
between drugs and radiopharmaceuticals18. The labeling of red blood cells involves
intracellular reaction between 99mTcO4- and hemoglobin. It is often used to evaluate
pulmonar perfusion10, digestive bleeding, liver tumors and hemodynamic disorders11.
It has been shown that changes on the morphology of red blood cells may arise in
the first hours after trauma, persists for several days and may interfere with the their
20
labeling. These changes are more severe in patients with postoperative septic
complications12.
Any deviation in the uptake of radiopharmaceuticals, on vital organs and red
blood cells after surgery, can result undesirable interpretations in the results of
scintigraphic examinations, leading to mistaken diagnoses, with serious
consequences to the patients. Repetition of the exams can determine an increase of
radiation dose for patients and risk for the involved professionals.
This work is part of a research field that has been studying the consequences
of major surgery performed in organs of the digestive system. We have
demonstrated relevant postoperative metabolic disorders after massive intestinal
resection4, total gastrectomy5,6, total colectomy7 and Roux-en-Y gastric bypass8, by
using this experimental model.
Based on the above concepts, this study aimed to examine in an animal model
if colectomy associated with hepatectomy modifies liver biodistribution of 99mTc-
phytate, metabolic parameters and labeling of red blood cells.
METHODS
Eighteen Wistar rats weighing 285±23g, were housed in polypropylene cages
and kept under controlled conditions of temperature in a clear-dark cycle of 12 hours
and allowed ad libitum access to food (Labina, Purina ®) and water. All experimental
procedures in animals were conducted according to the code of ethics for animal
experimentation of the Council for International Organization of Medical Sciences
and the Brazilian Law on the Scientific use of Animals (Law No. 11794). The protocol
was approved by the Institutional Research Ethics Committee. The anesthesia was
accomplished with intraperitonial injection of 0,2ml/100g of a solution containing 1ml
of ketamine (50mg) and 1 ml of xilazine (20 mg) and operated under aseptic
conditions. In addition, analgesia (tramadol 20 mg/kg body weight) was applied
subcutaneously immediately after surgery and every 12 hours for three days.
The animals from group colectomy underwent laparotomy and a subtotal
colectomy was performed, proceeding ileocolic anastomosis with 6-0 polypropilene
suture. Cecum, 5 cm of colon and 10 cm of ileum were resected. The laparotomy
was closed in layers with 4-0 mononylon. In the group colectomy+hepatectomy the
rats were submitted to partial colectomy associated to resection of the left lobe of the
21
liver. In the group sham, we performed medium laparotomy and soft manipulation of
the intestine.
The animals were weighed weekly and remained under observation. After 30
days they were anesthetized, the femoral vein was dissected and 0.1 mL of 99mTc-
phytate was injected i.v. (radioactivity 0.66 MBq). After 15 minutes, 5 mL of blood
were harvested by cardiac puncture and the rats were killed with an overdose of
anesthetic (sodium thiopental 100mg/Kg). A sample of liver was harvested to
examine the biodistribution of 99mTc-phytate.
After centrifugation of 2 mL of blood, red blood cells were separated for
examination of in vivo labeling with pertechnetate and morphology. The liver samples
were isolated, washed with 0.9% saline solution and weighed with a precision scale
(Bel-Mark 160-II-Italy). The detection of liver radioactive uptake was determined
using an automatic gamma counter, Wizard 1470 (PerkinElmer, Finland). The
percentage of radioactivity per gram of tissue (%ATI/g) was calculated dividing the
activity of the liver sample by the total activity administered to each animal. The
experiment with radiopharmaceutical was carried out in compliance with radiation
protection standards recommended by the Comissão Nacional de Energia Nuclear.
The radiopharmaceutical was provided by Department of Nuclear Medicine of Liga
Norte-Riograndense Contra o Câncer (LIGA) and the radioactive waste returned to
the LIGA for proper treatment.
Assay for biochemical levels Samples of blood (3 mL) were inserted into test tubes without anticoagulant,
centrifuged to 3000 rpm by 10 min and the serum was stocked to -40°C until
analysis. Serum levels of aspartate aminotransferase (AST), alanine
aminotransferase (ALT), and alkaline phosphatase were measured using a
commercial kit on the autoanalyzer (Konelab, Software Version, 60i, Finland).
Red blood cells morphology Blood strains were prepared on slides, them stained with Leishman. The
images of red blood cells were captured by digital camera using optical microscopy
(Olympus microscope, model BX50, Japan, 1000x,) for analysis. To take the shape
of red blood cells and the morphometric measurements we used the ImagePro Plus
Software, version 6.0.
22
Estatistics Data were expressed as mean±sd. The comparison between groups was
performed by analysis of variance (ANOVA) and by the multiple comparison test of
Tukey, using a 0.05 significance.
RESULTS The animals were randomly divided into three groups of six each. We
observed 8% weight loss in group colectomy+hepatectomy at the end of first week. A
progressive recovery of weight occured until day 30. In the other groups there was no
weight loss after surgery. At the end of the experiment there was no significant
difference in mean weights among groups.
Table 1 shows the results of the percentage of radioactivity per gram of tissue
(%ATI/g), found in red blood cells and liver, as well as the tests to investigate the
statistical differences among the groups colectomy, colectomy+hepatectomy and
sham.
Table 1 - 99mTc-phytate biodistribution studies for each group.
Groups Organ (%ATI/g)1
Red blood cells2 Liver2
Colectomy 0.21 ± 0.04 4.35 ±0.71b
Colectomy + hepatectomy 0.16 ± 0.02a 4.41 ±0.56a
Sham 0.22 ± 0.05a 5.70 ± 0.87ab
p-value 0.042 0.024
Mean±Standard deviation
1. Percent of radioactivity per gram of tissue
2. Dosages in each column, followed by the same letter differ significantly (Tukey test).
Rats from colectomy and colectomy+hepatectomy groups had significantly
lower 99mTc-phytate uptake in the liver than in the sham group rats (p=0.024). The 99mTc-phytate uptake in the liver of colectomy rats was higher than in the
colectomy+hepatectomy group rats, but the difference was not significant (p>0.05).
Concerning the erythrocytes labeling, we observed a significant difference in %ATI/g
23
when the colectomy+hepatectomy group rats were compared with sham rats
(p=0.042). These data are summarized in table 1.
Table 2–Serum levels of liver enzymes and alkaline phosphatase in colectomy,
colectomy+hepatectomy and sham rats.
Groups AST (u/L)1 ALT (u/L)1 Alkaline phosphatase (u/L)1
Colectomy 37,67 ± 1,37a 47,33 ± 2,50a 170,6± 22,91b
Colectomy +
hepatectomy 155,00 ± 13,67ab 76,33 ± 6,89a 232,3 ± 28,93ab
Sham 44,33 ± 4,72b 51,17 ± 3,19 180,7 ± 20,35a
p-value <0,001 <0,001 <0,001 Mean ± Standard deviation
1. Dosages in each column, followed by the same letter, differ significantly (Tukey test).
AST, aspartate animotransferase; ALT, alanine aminotraferase
Serum levels of AST, ALT and alkaline phosphatase were significantly higher
in colectomy+hepatectomy rats than in colectomy group rats (p<0.001), signaling that
hepactectomy contributed to these results. Comparing the serum levels of AST, ALT
and alkaline phosphatase from colectomy group with sham rats, the results were
similar (p>0.05) (Table 2).
The qualitative and quantitative evaluation of the shape of red blood cells
under optical microscopy was performed and alterations on the shape and
morphometric measures were not found, when compared the three groups (Figure
1).
Figure 1 – Photomicrography of red blood cells from (A) colectomy, (B) colectomy +hepatectomy and (C)
sham rats. 1000x.
A B C
24
DISCUSSION It is well established in the area of digestive surgery that surgical resection is
the most effective treatment for isolated liver metastasis in patients with colorectal
cancer13. Perioperative morbidity and mortality does not differ between simultaneous
resections and staged procedures for selected patients with liver metastases.
Meanwhile, patients undergoing simultaneous resections could expect a short
duration of surgery and postoperative hospitalization as well as less blood loss during
surgery13. Although the treatment for patients with synchronous colorectal cancer
liver metastases remains controversial, surgical resection of both the primary tumor
and liver metastases is the only option offering a potential cure13.
In the experimental model used in this work we did not include induction of
colon cancer, because our objective was to examine the repercussion of colectomy
associated partial hepatectomy without carcinogenesis. After the standardization of
the technique in the pilot study, the morbidity was low and there was not mortality
during the 30 days of observation of all rats. Despite the great surgical trauma, the
animals progressed well until the end of the experiments. Weight loss in the first
week was observed in the animals from colectomy+hepatectomy group, but they had
a satisfactory recovery in the three subsequent weeks. These findings corroborate
the statement that hepatectomy increased morbidity, but over time the animals had
satisfactory evolution.
However, at the end of the observation and collection of biological samples,
we detected significant changes in some parameters, when the groups were
compared. There was a significant reduction in 99mTc-phytate biodistribution in the
liver of colectomy+hepatectomy animals, compared with the sham group, but no
difference was observed comparing with colectomy group. This finding may be
interpreted based on the hypotesis that after 30 days liver regeneration was sufficient
to compensate any disfunction, normalizing the 99mTc-phytate uptake.
The distribution of radiocolloids in the liver has been shown to correlate well
with the severity of chronic liver diseases, the severity of histologic fibrosis,
prognosis, and hepatic function14. Thus, liver uptake of 99mTc-phytate colloid
provides a practical index of hepatic function and image, by using planar scintigraphy
techniques15. In a study from our laboratory it was demonstrated that in
splenectomized rats the biodistribution of 99mTc-phytate to the liver was higher than in
25
controls, suggesting that the operation favored the hepatic uptake of the
radiopharmaceutical. This result coincided with the improvement in liver function,
confirmed by the better alanine aminotransferase, aspartate aminotransferase and
lactic dehydrogenase activities in splenectomized rats, compared with controls16.
Otherwise, in the present study the lower liver 99mTc-phytate uptake coincided with a
lower alanine aminotransferase, aspartate aminotransferase and alkaline
phosphatase activities in colectomy+hepatectomy rats, than in sham rats.
Nuclear medicine procedures have proven increasingly effective imaging
modalities in the study of several disorders17. Besides the disease, these procedures
could be altered by medications, surgery and natural products that could change the
biodistribution of radiopharmaceuticals in a specific target or the fixation of 99mTc to
blood constituents16,18. In general, the labeling of blood constituents could decrease
by the action of drugs and trauma:(a) changing of the cellular membrane structure or
modifying the transport systems of pertechnetate ions into cells, (b) by direct
oxidation or generation of free radicals, (c) by direct inhibition (chelating action) of
pertechnetate ions, or (d) binding at the same sites on the blood constituents3. In this
study we observed that pertechnetate labeling of red blood cells of the
colectomy+hepatectomy rats was lower than in sham rats. This data is relevant,
because red blood cells labeling is an important method for diagnosis of digestive
and renal bleeding19. Interactions of some factors resulting from hepatic resection
may be involved with the ion transport systems of pertechnetate ions, decreasing the
labeling of red blood cells with pertechnetate. This could in part, explain the data
obtained.
CONCLUSION In conclusion, hepatectomy associated with colectomy lowered the liver and
erytrocytes uptake of pertechnetate in rats, coinciding with changes in hepatic
enzymatic activity.
26
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metastasis. Surg Oncol. 2007;16:33–45.
2. Lee WS, Yun SH, Chun HK, Lee WY, Kim SJ, Choi SH, Heo JS, Joh JW, Choi
D, Kim SH, Rhim H, Lim HK. Clinical outcomes of hepatic resection and
radiofrequency ablation in patients with solitary colorectal liver metastasis. J
Clin Gastroenterol. 2008;42:945–9.
3. Arano, Y. Recent advances in 99mTc radiopharmaceuticals. Ann Nucl Med
2002, 16:79-93.
4. Chacon DA, Araujo Filho I, Villarim Neto A, Medeiros AC. Biodistribuição do
radiofármaco pertecnetato de sódio (Na99mTcO4) em ratos submetidos a
ressecção extensa de intestino delgado. Acta Cir Bras. 2007;22: 430-5.
5. Medeiros AC, Filho IA, Medeiros VB, Pinheiro LA, Freire FH, Azevedo IM,
Brandão-Neto J. Comparing reconstruction with ileocecal graft to jejunal
interposition pouch after total gastrectomy in rats. J Invest Surg. 2007;20:41-8.
6. Rêgo AC, Villarim Neto A, Azevedo IM, Araújo-Filho I, Egito ES, Medeiros AC.
Biodistribution of Technetium-99m pertechnetate after total gastrectomy and
Roux-en-Y jejunal pouch. J Invest Surg. 2010;23:94-100.
7. Rêgo AC, Ramalho RA, Egito ES, Araújo-Filho I, Azevedo IM, Palestro CJ,
Medeiros AC. Biodistribution of technetium-99m pertechnetate after total
colectomy in rats. Appl Radiat Isot. 2010;68:2169-73.
8. Rêgo AC, Araújo-Filho I, Azevedo IM, Jácome DT, Ramalho R A, Medeiros
AC. Biodistribution of technetium-99m pertechnetate after Roux-en-Y gastric
bypass (Capella technique) in rats. Acta Cir Bras. 2010;25:9-12.
9. Zink SI, Ohki SK, Stein B, Zambuto DA, Rosenberg RJ, Choi JJ, Tubbs DS.
Noninvasive evaluation of active lower gastrointestinal bleeding: comparison
between contrast-enhanced MDCT and 99mTc-labeled RBC scintigraphy. Am
J Roentgenol. 2008;191:1107-14.
10. Hunt A, Frier M, Johnson R, Berezenko S, Perkins A C. Preparation of Tc-
99m-macroaggregated albumin from recombinant human albumin for lung
perfusion imaging. Euro J Pharm Biopharm. 2006; 62: 26-31.
11. Schneider A, Attaran M, Gratz K F, Bleck J S, Winkler M, Manns M P, Ott M.
Intraportal infusion of 99mtechnetium-macro-aggregrated albumin particles and
27
hepatocytes in rabbits: assessment of shunting and portal hemodyn changes.
Exp Transplant. 2003; 75: 296-302.
12. Berezina T L, Zaets S B, Machiedo G W. Alterations of red blood cell shape in
patients with severe trauma. J Trauma-Injury Infect Crit Care. 2004; 57: 82-7.
13. Jong M, van Vledder M, Ribero D, Hubert C, Gigot J, Choti MA, Schulick
RD, Capussotti L, Dejong CH, Pawlik TM. Therapeutic efficacy of combined
intraoperative ablation and resection for colorectal liver metastases: an
international, multi-institutional analysis. J Gastrointest Surg. 2011;15:336–44.
14. Hoefs JC, Wang F,Kanel G. Functional measurement of nonfibrotic hepatic
mass in cirrhotic patients. Am J Gastroenterol. 1997; 92:2054–8.
15. Kikuchi M, Tomita K, Nakahara T, Kitamura N, Teratani T, Irie R, Yokoyama
H, Suzuki T, Yokoyama T, Taguchi T, Tanaka S, Noguchi M, Ohkura T, Hibi T.
Utility of quantitative 99mTc-phytate scintigraphy to diagnose early-stage non-
alcoholic steatohepatitis. Scand J Gastroenterol. 2009;44:229-36.
16. Pereira KRSG, Açucena MKMT, Villarim-Neto A, Rêgo ACM, Bernardo-Filho
M, Azevedo IM, Medeiros AC. Biodistribution of the radiopharmaceutical
technetium-99m-sodium phytate in rats after splenectomy. Braz Arch Biol
Technol. 2008;51:203-7.
17. Lodge MA, Braess H, Mahmoud F, Suh J, Englar N, Geyser-Stoops S,
Jenkins J, Bacharach SL, Dilsizian V. Developments in nuclear cardiology:
transition from single photon emission computed tomography to positron
emission tomography-computed tomography. J Invasive Cardiol. 2005;17:491-
6.
18. Fonseca AS, Frydman JN, Rocha VC, Bernardo-Filho M. Acetylsalicylic acid
decreases the labeling of blood constituents with technetium-99M. Acta Biol
Hung. 2007; 2:187-98.
19. Howarth DM. The role of nuclear medicine in the detection of acute
gastrointestinal bleeding. Semin Nucl Med. 2006;36:133-46.
Correspondence: Aldo Cunha Medeiros ([email protected])
Av. Nilo Peçanha 620, Natal-RN, Brazil 59012-300 Conflict of interest: none.
Financial source: CNPQ
28
5.2. Artigo publicado no periódico Journal of Investigative Surgery. 2011;24:109-14.
(Qualis B1). ISSN 0894-1939
Metabolism and gastric remnant changes after Roux-en-Y Gastric Bypass in rats.
Aldo Cunha Medeiros, Amália Cínthia Meneses Rêgo, Ítalo Medeiros Azevedo, Marília D. Ferreira Carvalho, Vítor Brasil Medeiros, Irami Araújo-Filho. Department of Surgery, Postgraduate Program in Health Sciences, Federal University of Rio Grande
do Norte, Natal, Brazil.
ABSTRACT Background: The Roux-en-Y gastric bypass (RYGB) may affect gastric microbiology
as well as the histology of the excluded stomach. Therefore, this study aimed to
investigate these issues in rats. Methods: Twelve rats were randomly allocated to a
RYGB group (n=6) and nonoperated control group (n=6). After 30 postoperative
days, all rats were killed. Samples of the remnant stomach mucosa were harvested
for bacterial and fungal count such as colony-forming units/g and histology. Results:
The excluded stomach mucosa of RYGB rats showed higher bacterial and fungal
count, atrophy, intestinal metaplasia and neutrophilic polymorphonuclear
inflammation than in controls. Conclusions: This work showed that a model of murine
RYGB significantly modified the microbiota/histology of the excluded stomach.
Keywords: Bariatric surgery, Gastric microbiology, Histology, Roux-en-Y gastric
bypass, Rats
INTRODUCTION
Morbidity and mortality are strongly correlated to the level of obesity [1], and
the list of diseases with a causal factor for obesity continues to expand. The failure of
most current approaches to control morbid obesity has led to the development of
surgical techniques for the upper gastrointestinal tract designed to induce weight loss
29
(bariatric surgery). Patients have achieved effective weight loss after bariatric surgery
and most have had complete resolution or improvement of their diabetes,
hypertension, hyperlipidemia, and obstructive sleep apnea [2], in addition to reduced
long-term mortality [3]. The Roux-en-Y gastric bypass (RYGB) is the predominant
approach used in the United States [4] and Brazil [5]. After the adoption of RYGB,
occasional concern has arisen regarding bacterial overgrowth in the upper digestive
tract, especially in the excluded stomach. Gastric juice is an important barrier against
bacterial colonization and an essential component of the gut barrier, which protects
the organs from infection when vagal innervation is lost [6].
Although the procedure can be successful, it is associated with a number of
metabolic and structural complications. These complications are sometimes subtle
and difficult to diagnose early. Since these patients have limited physiologic reserve,
it is imperative that complications are identified early by image procedures and
appropriately managed [7]. One of the most widely used diagnostic methods in
identifying a great number of diseases and metabolic disorders is the use of
radiopharmaceuticals, radioactive compounds used in diagnostic procedures as
sources of radiation and tracers. Examination of gastric emptying, liver function,
thyroid function, bone disorders, etc, are often used in patients undergoing bariatric
surgery. Radiopharmaceutical biodistribution may provide important information
about their uptake to target organs and their function, but post-surgery data are
scarce [8]. It is important to investigate the biodistribution of technetium-99m
pertechnetate after RYGB, since it is highly secreted by gastric mucosa. After this
operation the stomach is mostly bypassed, the metabolism is affected, and changes
in biodistribution are expected. Drugs and surgery may interfere in the biological
behavior of radiopharmaceuticals used in scintigraphy and single photon emission computed tomography (SPECT). Technetium-99m pertechnetate is used in more
than 80% of scintigraphic examinations [9-11]. Thus, the biological effects of
technetium-99m pertechnetate and its uptake to various organs may be altered.
RYGB is a major surgery and may therefore result in important anatomical and
metabolic changes as well as unpredictable complications [7]. Accordingly,
scintigraphic studies may be needed in the postoperative period. Given that the
biodistribution of technetium-99m pertechnetate is expected to cause changes in
important organs and tissues due to bariatric surgery, scintigraphic examinations
30
may produce false-positive or false negative images, leading to repetition of nuclear
medicine procedures with unnecessary radiation exposure for patients.
The aim of this study was to analyze the effects of RYGB on gastric microbiology,
metabolism, histology and technetium-99m pertechnetate biodistribution in rats.
METHODS
The use of laboratory animals followed the Council for International Organization
of Medical Sciences Ethical Code for animal experimentation and the Brazilian
guidelines for the scientific use of animals (Law no. 11.794). The protocol was
approved by the Institutional Research Ethics Committee. Rats were observed in
individual polypropylene cages at room temperature of 24ºC, relative humidity of
45%, 12-hour light/dark cycles, with food and water ad libitum. For preoperative
procedures, rats were deprived of food for 16 to 18 hours and anesthetized with a
mixture of ketamine and xylazine (200 mg: 5 mg, 0. 8 ml/kg, intraperitoneally). For
postoperative pain control, 1.5 mg/kg of tenoxicam (Roche, Brazil) was administered
subcutaneously, once a day for 3 days.
Operative and laboratory procedures
Twelve Wistar rats (349.3 ±10.7 g) were equally divided into RYGB and control
groups. The rat abdomens were shaved and prepared with 70% alcohol. All the
surgical procedures were carried out by the same well-trained investigator,
experienced in animal surgery. Three previous series of experiments were conducted
in sequence to develop the RYGB model. A midline incision was made, exposing the
stomach and distal esophagus. The stomach was divided 2 cm below the esophagus
and both gastric ends were oversewn using a running 6-0 polypropylene suture. The
suture lines were then embrocated. The jejunum was divided 16 cm below the
ligament of Treitz, creating a 16 cm biliary-pancreatic limb. A 4 to 5 mm end-to-side
gastrojejunostomy was sewn using interrupted 6-0 polypropylene sutures on the
anterior surface of the gastric fundus. The stump of the proximal jejunum was closed
with a running suture. A 7 to 8 mm side-to-side jejunojejunostomy was sewn 10 cm
below the gastrojejunostomy. The procedure lasted approximately 30 minutes, after
31
which the abdomen was closed in layers, using a running 4-0 nylon suture. Rats
drank water and a 10% glucose plus 2% saline solution starting 24 hours after the
operation and for the first 3 days. This was followed by a solid diet (Labina-Purina®).
For the first three postoperative days, rats were hydrated with normal saline solution
(20ml) injected subcutaneously to prevent dehydration. A surgical microscope with
10x magnification (DF Vasconcelos, São Paulo, Brazil) was used for anastomosis.
The control group rats (n=6) were not operated.
The animals remained under observation for 30 days after surgery and under
anesthesia, injected with 0.1 ml of technetium-99m pertecnetate via the orbital
plexus, corresponding to radioactive activity of 0.66 MBq. Thirty minutes after
radiopharmaceutical administration, blood samples were obtained by cardiac
puncture to determine serum dosages. Furthermore, a midline laparotomy was
performed under sterile conditions. The defunctionalized stomach was opened and
intragastric material (including mucosa) was harvested for culture. Samples were
collected from the nonoperated stomach of control rats. Samples were then removed
from the liver, kidney, heart, lung, thyroid, stomach and femur. The tissue samples
were washed in 0.9% saline and weighed on a precision scale (Mark 160®, Bel
equipment, Italy). Radioactivity was determined in an automatic gamma counter
(Wizard 1470®, Perkin-Elmer, Finland). The results were shown in counts per minute
(CPM), corrected by disintegrations per minute (DPM). The efficiency of the gamma
counter was 86%, as specified by the manufacturer. The specific activity of each
sample was calculated by dividing absolute count in DPM by weight (DPM/g). The
percentage radioactivity of each sample (% ATI/g) was calculated by dividing its
specific activity (DPM/g) by the total radioactivity of each animal.
Laboratory tests
Blood samples were centrifuged immediately at 3000 rpm for 10 min. and serum
samples stored at -80°C until analysis. Serum albumin, calcium, alanine (ALT) and
aspartate aminotransferase (AST) levels were assayed using a Konelab 60i analyzer
(assay kit from Weiner, São Paulo, Brazil). The levels of free triiodothyronine (T3),
free thyroxin (T4) and parathyroid hormone (PTH) were determined by
radioimmunoassay using an [I125] and PTH assay kit (Diagnostic Products, Los
Angeles, USA). Sensitivity was determined to be 0.4 ng/dL.
32
Microbiological analysis
The excluded stomach was opened and antral specimens (5 mm punch-
biopsy) for bacterial quantification were obtained on day 30. In control rats, samples
were harvested from the antrum of the non operated stomach. They were then
individually weighed and homogenized in 1 ml of sterile 0.9% saline solution using a
sterile technique. Serial dilution was performed, and samples were plated onto
trypticase soy agar containing 5% sheep blood agar for colony counts of viable
aerobic bacteria. Aliquots were spread on blood/agar plates for Gram positives and
agar McConkey for Gram negative bacteria. Incubation proceeded at 37˚C for 48 h.
The number of colony-forming units (CFU)/g was counted in 100-µl aliquots of fluid.
Fungi were inoculated in acidified agar/dextrose/potato plates, and incubation
proceeded at 25˚C for 7 days. The number of colonies was computed by colony
counter, and all colony counts were expressed as the number of colony-forming units
per gram of tissue (CFU/g).
Statistical analysis
The data were expressed as mean ± standard deviation. Statistical analysis for
group comparison was performed using the Student t test, at a significance level of
0.05.
RESULTS
All animals survived the study. Table 1 summarizes the descriptive results of
percentage radioactivity (% ATI) in the organs of rats from groups RYGB and
control.
33
Table 1. Percentage radioactivity (% ATI/g) of each organ sample in animals
submitted to Roux-en-Y gastric bypass and controls.
Organs %ATI/g P
RYGB Control
Liver 0.32 ± 0.04 0.57 ± 0.06 0.023
Kidney 0.57 ± 0.21 0.43 ± 0.06 0.120
Heart 0.22 ± 0.05 0.26 ± 0.08 0.131
Lung 0.52 ± 0.17 0.39 ± 0.16 0.099
Tyroid 2.97 ± 0.98 3.42 ± 1.68 0.530
Stomach 0.53 ± 0.31 2.95 ± 1.64 0.005
Femur 0.13 ± 0.03 0.25 ± 0.07 0.026
The values appear as mean±standard deviation. RYGB, Roux-en-Y gastric bypass.
In the kidney and lung we observed an increase in radioactivity uptake in rats
submitted to RYGB, compared with controls, but the difference was not statistically
significant (p>0.05). However, in the RYGB rats a significant decrease in the
radioactivity uptake (%ATI/g) occurred in the stomach, liver and femur, compared
with controls (p<0.05). A comparison between radioactivity uptake in the heart and
thyroid of RYGB and control rats showed a tendency for a further decrease in %
ATI/g in RYGB animals, although it was not statistically significant (p>0.05).
Table 2 shows a significant reduction mean serum albumin and calcium levels in
animals undergoing RYGB surgery, compared with those of the control group (p
<0.05). Liver function tests (ALT and AST) revealed a significant difference between
the two groups. T3 and T4 levels were significantly lower in RYGB rats than in control
rats (p=0.002 and 0.016 respectively). However, PTH levels were significantly higher
in RYGB rats than in controls (p=0.009) (Table 2).
34
Table 2. Comparison of control and RYGB postoperative laboratory values.
Control RYGB p-valor
Albunin (g/dL) 3.2±0.7 2.3±0.2 0.048
Calcium (mg/dL) 8.2±1.4 5.3±1.1 0.031
ALT (U/L) 97.3±18.5 57.6±14.2 0.045
AST (U/L) 47.3±12.7 33.7±10.5 0.048
T3 (ng/dL) 28.7±4.10 16.3±3.06 0.002
T4(ng/dL) 31.4±2.01 20.1±2.13 0.016
PTH (pg/mL) 59.3±1.96 65.7±2.5 0.009 Results are shown in means ± SD. ALT, alanine aminotransferase; AST, aspartate
aminotransferase; T3, free triiodothyronine; T4, free thyroxin; PTH, parathyroid hormone; RYGB,
Roux-en-Y gastric bypass.
Quantification of microorganisms The homogenate fluid of the excluded stomach of RYGB rats showed a high
bacterial count on postoperative day 30, with 46033.33 ± 18369.29 CFU/g of tissue.
Control rats showed significantly lower counts (11100 ± 1678.09 CFU/g; p = 0.005),
compared to RYGB rats. On day 30 we detected 190.00 ± 19.75 CFU/g of fungi in
the homogenate fluid of the excluded stomach of RYGB rats. A lower fungi count of
38.23 ± 9.93 CFU/g was found in the control rats (p < 0.001). Microorganisms were
identified as Escherichia coli, Staphylococcus sp. and Klebsiela sp. Fungi were
predominantly Candida spp. These data are summarized in table 3.
Table 3. Comparison of control and RYGB postoperative gastric bacteria and fungal
counts.
Bacteria and fungi Group
p-valor(1)
RYGB Control
Bacteria (CFU/g) 46033.33 ±
18369.29 11100.00 ± 1678.09 0.005
Fungi (CFU/g) 190.00 ± 19.75 38.23 ± 9.93 <0.001
1 - P-valor, Student t test for independent data; Numbers indicate colony forming units per g (CFU/g) of
tissue.
35
Histology
We determined the effects of RYGB on the mucosa of the excluded stomach.
Histological examination revealed mucosal atrophy with reduced tissue and cell size.
Atrophy affected the glands, which were sparse and small (Figure 1). The difference
between the two groups was significant (p=0.01). Intestinal metaplasia occurred in
one RYGB rat. We found the simultaneous presence of intraepithelial mononuclear
cell infiltrate and neutrophilic polymorphonuclear inflammation in all the excluded
stomachs. Prominent numbers of eosinophils were observed in the antral mucosa of
two RYGB rats. Mean inflammatory cell density and mucosal thickness related to
these findings are summarized in table 4.
Table 4 – Histologic findings expressed as mean cell density and mucosal thickness.
Parameters Group
p-valor(1)
RYGB Control
Inflammation 20045± 97 4130 ±104 0.005
Mucosal length
(mµ) 95 ± 11 173 ± 14 0.01
1 - P-valor, Student t test for independent samples; Numbers indicate mean inflammatory cell density
and mucosal thickness (mµ).
DISCUSSION
Significant efforts have been made to obtain good results with surgical
techniques, and patients subjected to bariatric operations have achieved significant
36
weight loss [12]. Despite these promising results, bariatric surgery may lead to
anatomical and metabolic complications [13]. A literature survey shows that severe
disease can affect the bypassed stomach after RYGB. Following this surgery
conventional gastroscopy cannot assess the excluded stomach, which may result in
delayed diagnosis of gastric disease. Perforated peptic ulcer [14] and gastric cancer
have been reported [15].
The diagnosis of these disorders may require imaging examinations such as
radiography, endoscopy and scintigraphy. Technetium-99m is the most widely used
radionuclide in nuclear medicine and research studies. It has a short half-life (6h),
emits low radiation and requires small doses for diagnostic procedures [18]. Changes
in the biodistribution of technetium-99m pertecnetate in organs and tissues are well
identified in a number of clinical studies. It has been used in vivo and in vitro, in the
study of diseases, drugs and chemotherapy that interfere with its distribution [16,17].
However, few studies have focused on radiopharmaceutical biodistribution in the
postoperative period of major surgery. To understand and explore the relationship
between the RYGB and radiopharmaceutical biodistribution in organs and tissues,
we used a reproducible and well characterized animal model. Scintigraphic
examinations in the postoperative period are used to diagnose gastrointestinal tract
bleeding, gastroesophageal reflux and patency of anastomosis. Diagnosis of
postoperative changes in the kidneys, liver, lung, heart and other organs are
performed using scintigraphy and SPECT with radiotracers and technetium-99m
pertecnetato [18]. In this study, the RYGB did not affect the biodistribution of
technetium-99m pertecnetate in the heart, lungs, kidneys and thyroid.
The stomach is often examined in the postoperative period of bariatric surgery,
primarily for diagnosing the presence of fistulas, since escape from the anastomosis
may be an early or late complication of this operation. A case series of 63 patients
with leaks after RYGBP reports that most were not detected by CT imaging and that
most required surgery (63%), with morbidity of 53% and mortality of 10% [19]. In the
case of small fistulas, since the rate of escape of contrast medium is low, leaks may
be overlooked by radiography. It has been suggested that scintigraphy is highly
accurate in diagnosing gastroparesis and other postoperative complications of
postoperative bariatric surgery [20]. In this study the pertechnetate uptake was
significantly lower in the remnant functional stomach after RYGB. Therefore,
37
scintigraphic examination of this organ in the postoperative period of bariatric surgery
must be interpreted considering this finding.
Several different mechanisms may explain the changes in bone metabolism
observed after bariatric surgery. Poor mineral and vitamin absorption, including
calcium and vitamin D, has been documented [21]. In this study we detected low
levels of serum calcium in the RYGB rats. Consistent data have been reported
suggesting that secondary hyperparathyroidism occurs after gastric bypass. Elevated
and these levels persisted after 6 months of calcium and vitamin D supplementation
[22]. In our RYGB rats we demonstrated higher levels of PTH compared with
controls, and there is evidence that the increase in PTH may be progressive [23].
Higher turnover and reduced bone mineral density may occur as a physiological
adaptation to weight loss and changes in skeletal mechanics or as a result of
pathophysiological responses to surgery [24]. The metabolic changes in calcium and
PTH may, at least in part, explain the decreased biodistribution of technetium-99m
pertecnetate in the femur of animals submitted to RYGB compared to the control
group. In our study the uptake of technetium-99m pertecnetate by the liver of RYGB
rats was significantly lower than in controls, indicating that liver function may have
been affected, as suggested by our data. These results should be viewed with
caution. Indeed, some authors demonstrated significant improvement in
aminotransferase and gamma glutamyl transferase levels in the postoperative period
of patients submitted to RYGB [25], and others found transiently higher postoperative
aminotransferase values (five to eightfold) [26]. The rat model for RYBG, which
seems to be reasonably reliable, will enable continued research into metabolic
changes, aimed at validating postoperative metabolic findings.
Bacterial concentrations were consistently higher in the gastric biopsy
homogenate of the bypassed stomach than in the control organ. This finding is in
disagreement with the results described by Ishida et al. [27], who studied the
bacterial count of intragastric fluid endoscopically harvested in patients. However, our
data indicate that the assessment and quantification of bacteria in intragastric fluid
may reflect different results, when compared with the bacterial count of mucosal
biopsy homogenates. These data support the hypothesis that the use of intragastric
fluid for diagnosis of infection may be insufficient and if possible, should be replaced
by tissue biopsies. It has been demonstrated that Candida albicans is able to
colonize the murine alimentary tract, as well as adhere to and penetrate gastric
38
tissues [28]. In this study, we observed Candida spp colonization in the bypassed
stomach. This evoked a prominent, granulocyte-dominated, inflammatory response in
the gastric mucosal tissues. In the present study, antral mucosal atrophy affected
primarily the glands, which became sparse and small. In some specimens, mucosal
thinning was non-apparent, likely due to accompanying inflammation characterized
by the simultaneous presence of mononuclear and neutrophilic polymorphonuclear
infiltrate. The occurrence of chronic gastritis and intestinal metaplasia has been
confirmed in the excluded stomach [29,30], but the clinical significance of these
histological changes remains obscure. In conclusion, the current investigation demonstrated that a murine model of
RYGB modified the biodistribution pattern of technetium-99m pertechnetate, the
microbiota/histology of the excluded stomach and the metabolism.
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disease after eight years of Roux-en-Y gastric bypass. Obes Surg. 2008; 18:386–
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8. Chacon DA, Araújo-Filho I, Villarim-Neto A, et al. Biodistribution of the
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bowel resection in rats. Acta Cir Bras. 2007;22:430-435.
39
9. Moreno S R F, Carvalho J J, Nascimento A L, et al. Bioavailability of the sodium
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12. Hutter MM, Randall S, Khuri SF, et al. Laparoscopic versus open gastric bypass
for morbid obesity: a multicenter, prospective, risk-adjusted analysis from the
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13. Leea CW, Kellyb JJ, Wassef WY. Complications of bariatric surgery. Curr Opin
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14. Macgregor AM, Pickens NE, Thoburn EK. Perforated peptic ulcer following gastric
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15. Lord RV, Edwards PD, Coleman MJ. Gastric cancer in the bypassed segment
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17. Braga ACS, Oliveira MBN, Feliciano GD, et al. The Effect of Drugs on the
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20. Tarakji AM, Morales F, Rovito P. Hepatobiliary scintigraphy as a diagnostic
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21. Madan AK, Orth WS, Tichansky DS, Ternovits CA. Vitamin and trace mineral
levels after laparoscopic gastric bypass. Obes Surg. 2006;16:603-606.
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30. Safatle-Ribeiro AV, Kuga R, Iriya K, et al. What to Expect in the Excluded
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The authors declare that they have no conflict of interest.
41
5.3. O artigo foi publicado no periódico Journal of Surgical and Clinical Research
2010;1:13-21 (ISSN: 2179-7889)
Diabetes and biodistribution of pertechnetate (Na99mTcO4) in rats1
Diabetes e biodistribuição de pertecnetato de sódio (Na99mTcO4) em
ratos
Ítalo Medeiros Azevedo, Daniele Pimentel Fernandes, Ticiana Cabral da Costa, Irami Araújo-Filho, MD, PhD, Amália Cinthia Meneses Rêgo, PhD, Vítor Brasil Medeiros, Marília Daniela Ferreira Carvalho, Aldo Cunha Medeiros, MD, PhD. 1-Research performed at Nucleus for Experimental Surgery, Department of Surgery, Federal
University of Rio Grande do Norte (UFRN), Brazil. Financial support: CNPq, Brazil. Conflict of interest:
None.
Correspondence address: Ítalo Medeiros Azevedo, Department of Surgery, Federal University of Rio
Grande do Norte, at Ave Nilo Peçanha 620, Natal, RN, Brazil, Email: [email protected]
Submitted: 25 November 2010. Accepted, after review: 27 December 2010.
ABSTRACT
Purpose: This study aimed to clarify if diabetes induced in rats changes the
biodistribution of the radiopharmaceutical sodium pertechnetate (Na99mTcO4) and,
consequently, the accuracy of the scintigraphic exams. Methods: We used 14 male
Wistar rats, randomly allocated in 2 group: the diabetic group (D) rats (n=7) were
submitted to the induction of diabetes with streptozotocin, and 7 non diabetic rats
were used as controls (C). After 7 days, in the 14 animals it was administered 0,1 mL
of Na99mTcO4 (0.66 MBq) through orbital plexus and, after 30 minutes, the
radiopharmaceutical sodium pertechnetate was evaluated, being compared the
uptske of this in the several studied organs of diabetic animals and controls. Results: The biodistribution of Na99mTcO4 was significantly higher in the liver and smaller in
the bladder, thyroid and stomach of diabetic mice when compared to the control
group rats. Conclusion: Diabetes induced in rats alters the biodistribution of
Na99mTcO4, and this finding could have clinical implications on scintigraphic exams.
42
Key words: Bioavailability. Tc 99m Pertechnetate. Diabetes. Streptozotocin. Rats.
RESUMO Objetivo: Contribuir para esclarecer se o diabetes induzido em ratos altera a
biodistribuição do radiofármaco pertecnetato de sódio (Na99mTcO4). Métodos: Estudo do tipo experimental com utilização de 14 ratos machos Wistar, divididos
aleatoriamente em 2 grupos, um grupo submetido à indução do diabetes com
estreptozotocina, e o outro somente observado. Após 7 dias, nos 14 animais foi
administrado 0,1 mL de Na99mTcO4 (0.66 MBq) via plexo venoso orbital e, após 30
minutos, foi avaliada a biodistribuição do radiofármaco, comparando-se a captação
deste nos diversos órgãos estudados de animais diabéticos e controles.
Resultados: A biodistribuição do Na99mTcO4 foi significativamente maior no fígado e
menor na bexiga, tireóide e estômago de ratos diabéticos quando comparados aos
ratos do grupo controle. Conclusão: O diabetes induzido em ratos altera a
biodistribuição do radiofármaco Na99mTcO4, podendo esses achados ter implicações
clínicas na interpretação de exames cintilográficos.
Descritores: Biodisponibilidade. Pertecnetato. Diabetes. Estreptozotocina. Ratos.
INTRODUCTION
The radionuclides are employed in many fields of knowledge. In health sciences
they contribute to the improvement of diagnosis and treatment, enabling advances in
clinical and experimental research. Most diagnostic tests like scintigraphy, reveals
the uptake of radioisotopes in several organs and tissues1-4. The most used
radioisotopes are gamma radiation emitting, as a source of radiation or tracers1.
Since the 60s, the 99mTechnetium (99mTc) is used in the biomedical area because
it is easily obtained from molybdenum/technetium (99Mo/99mTc) generators, it has
short half-life (6h), low emission energy, it is easy to label red blood cells, cellular
structures or molecular, it has low cost and low environmental impact5-8. These
features make 99mTc the most widely used radioisotope, administered in the form of
sodium pertechnetate (Na99mTcO4) or attached to other molecules. Their
biodistribution is evaluated by scintigraphy or other systems to detect radioativity6.
43
The scintigraphic image reflects morphology and organ function. Thus, different
radioactive compounds can be used to study the physiology of organs and tissues.
Therefore, a diseased tissue may contain higher or lower uptake of
radioactivity, depending on its disfunction8. Scintigraphic examinations are used in
the diagnosis of tumors, metastasis, gastric emptying, esophageal motility, thyroid
nodules, bleeding, and in the monitoring of systemic diseases and their
consequences, among them, diabetes9-15.
The metabolic imbalance associated with diabetes causes secondary disorders
in multiple organ systems, which can cause alterations in biodistribution of
radioisotopes. Changes in scintigraphic examination results can generate false
positive images, repetition of exams and increased patient exposure to radiation.
Some studies advocate the use of streptozotocin to induce experimental diabetes,
because it is a cytotoxic chemical agent, specific for pancreatic beta cells, causing
severe primary insulin insuficiency and high glucose levels, followed by the
establishment of diabetes in the subsequent 24 hours. This model, although with a
high mortality rate is relatively easy, destroying the endocrine cells with preservation
of exocrine function of pancreas16-18. Considering such principles, the objective of
this study was to evaluate the biodistribution of sodium pertechnetate (Na99mTcO4)
in different organs of rats with diabetes.
METHODS We used 14 three months old Wistar rats, weighing 265g ± 31g, provided by the
vivarium of the Health Sciences Center, Federal University of Rio Grande do Norte,
Brazil. All animals were weighed, placed in individual cages with water and food
(Purina ® Labina) ad libitum and acclimated in the laboratory for 7 days. They were
kept under controlled temperature (21 degrees), humidity (60-70%), lighting (12/12 h)
light / dark cycle and handled in accordance with the Ethical Code for Animal
Experimentation (Council for International Organization of Medical Sciences) and the
rules of the Brazilian College of Animal Experimentation. They were distributed
randomly into two groups: diabetes (D, n = 7) and control group (C, n = 7). The
animals in group D were subjected to induction of diabetes by injecting streptozotocin
at the dose 80mg/kg, intraperitoneally (IP). Seven days after the injection of
streptozotocin, glucose was measured by collecting blood from the dorsal vein of the
44
tail, using the equipment Accu-Chek Advantage, Roche Diagnostics, Mannheim,
Germany (2003). We considered diabetic the animals with fasting glucose above 200
mg/dL. The C animals were not subjected to any procedure, only observed.
On day 7, all animals were anesthetized with thiopental (20mg/kg-IP) and Ketamine
(20mg/Kg-IM), administered 0, l ml Na99mTcO4 intravenously into the orbital plexus,
and radioactivity dose was 0.68 MBq. After 30 minutes, the animals were killed with a
lethal dose of anesthetic thiopental (100mg/kg), intracardiac, and samples were
harvested from pancreas, brain, thyroid, lung, heart, stomach, liver, kidney, bladder
and right femur. The samples were washed in 0.9% NaCl, weighed on a digital
precision balance (Bel-Mark 160-II-Italy) and taken for detection of radioactivity
through the 1470 Auto Gamma counter, WizardTM-Perkin-Elmer (Finland) with
automatic correction of decay. The percentage of radioactivity per gram (% ATI/g) of
each organ was calculated by dividing the activity per gram of tissue by the total
radioactivity administered to each animal. The experiment was completely
randomized with statistical analysis by Student t test. The level of significance for the
test was 5% (95% CI), ie, p<0.05 was considered statistically significant.
RESULTS Table 1 shows the results of %ATI/g in group D and C. In group D rats we
observed a significant increase of the biodistribution of Na99mTcO4 in the liver and
reduction in the uptake of thyroid, bladder and stomach when compared with group C
rats. (p <0.05). The other organs did not show difference in %ATI/g, comparing with
controls.
45
DISCUSSION Research shows the interference of anesthetic drugs, chemotherapy and
herbals on the biodistribution of sodium pertechnetate (Na99mTcO4). Recently,
studies from our laboratory demonstrated changes in the uptake of
radiopharmaceuticals in the postoperative of major surgeries involving experimental
models of short bowel syndrome and bariatric surgery19-23.
Diabetes mellitus and associated metabolic disturbances interfere with
homeostasis, which may have caused changes in the biodistribution of pertechnetate
on this study. We detected an increase in radioisotope uptake in the liver and a
reduction in the stomach, bladder and thyroid gland of diabetic rats compared to
controls. Bertin et al24 argued that the control of gastric emptying involves
neurological, hormonal and metabolic factors and that the role of diabetes on gastric
motility is little known. However El-Shaldy et al25 attributed the delay in gastric
emptying to the inhibition of the hormone motilin by hiperglicemia. Theoretically, it
was expected to obtain a higher uptake of gastric pertechnetate in rats of the
experimental group, given the prior knowledge of several other trials on diabetic
gastroparesis associated with autonomic neuropathy. However, we observed the
opposite. This may affect the results of scintigraphy for the study of gastric emptying
in diabetic patients commonly performed during the search of dispeptic-
symptoms14,26-31.
Diabetes and biodistribution of pertechnetate (Na99mTcO4) in rats Azevedo, IM, et al
J Surg Cl Res Vol. 1 (1) 2010:13-21 15
samples were harvested from pancreas, brain, thyroid, lung, heart, stomach, liver, kidney, bladder and right femur. The samples were washed in 0.9% NaCl, weighed on a digital precision balance (Bel-Mark 160-II-Italy ) and taken for detection of radioactivity through the 1470 Auto Gamma counter, WizardTM-Perkin-Elmer ( Finland) with automatic correction of decay. The percentage of radioactivity per gram (% ATI/g) of each organ was calculated by dividing the activity per gram of tissue by the total radioactivity administered to each animal. The experiment was completely randomized with statistical analysis by Student t test. The level of significance for the test was 5% (95% CI), ie, p<0.05 was considered statistically significant. Results
Table 1 shows the results of %ATI/g in group D and C. In group D rats we
observed a significant increase of the biodistribution of Na99mTcO4 in the liver and reduction in the uptake of thyroid, bladder and stomach when compared with group C rats. (p <0.05). The other organs did not show difference in %ATI/g, comparing with controls. Table 1. Biodistribution of Na99mTcO4 in organs and respective groups
Organs % ATI/g
p-valor1
Diabetes Control Brain 0.02 ± 0.008 0.01 ± 0.003 0.093109 Thyroid 0.46 ± 0.154 3.89 ± 1.447 0.00075*
Lung 0.41 ± 0.157 0.39 ± 0.141 0.803036 Heart 0.23 ± 0.067 0.29 ± 0.061 0.223256 Stomach 0.43 ± 0.373 3.33 ± 1.593 0.004118*
Liver 2.97 ± 1.745 0.34 ± 0.050 0.009782*
Kidney 0.27 ± 0.161 0.42 ± 0.074 0.106062 Bladder 0.19 ± 0.055 0.34 ± 0.094 0.015865*
Femur 0.15 ± 0.058 0.14 ± 0.032 0.709645 Pancreas 0.15 ± 0.047 0.13 ± 0.046 0.518932 Mean±SD. *Significant diference p<0.05. 1- p-valor of Student t test.
46
Another autonomic dysfunction in diabetes is the neurogenic bladder. Many
researchers adopt as diagnostic of this pathology an increased tracer retention in the
bladder during the performance of radionuclide cystography. The bladder dysfunction
would result from neurological, muscular and urothelial injuries, uriginated on
hiperglicemia32,33.
We suppose that the low bladder uptake of sodium pertechnetate observed in
this study was due to a marked polyuria in the experimental group rats, which may
have contributed to greater intravesical radioactive energy dissipation. In relation to
thyroid disorders associated with diabetes, it is a phenomenon widely reported in the
current literature. It is estimated that diabetes is associated with hypothyroidism,
Hashimoto's thyroiditis and nontoxic goiter in about 20-56% of cases involving mainly
patients with type II diabetes34-36. It is therefore of great importance for thyroid
evaluations. In this sense, thyroid scintigraphy is used in complementing clinical data
and ultrasound. The use 99mTc displays advantages over iodine 131, because it
labels the gland, allows its morpho-functional evaluation, with less patient exposure
to radiation without interfering with the glandular function36.
There was a lower thyroid uptake of sodium pertechnetate (Na99mTcO4) in our
diabetic animals due to a probable subclinical hypothyroidism, as evidenced by
Akbar et al, studying thyroid dysfunction in patients with type II diabetes37. Passos et
al38 stated that a reduction in the synthesis of thyroglobulin precursor results in a
lower uptake of sodium pertechnetate by the gland, since there is not enough protein
to be marked. Thus, we alert to possible changes in thyroid scintigraphy in diabetic
patients.
A final analysis concerns the greater hepatic uptake of pertechnetate in the rats
of the experimental group. Clinical trials point to the diabetes in the pathogenesis of
liver diseases, with emphasis on non-alcoholic steatohepatitis, cirrhosis and
hepatocellular carcinoma, including transplanted patient39-45. The liver participates
directly in the glucose metabolism, raising or lowering the glucose uptake as vary
their organic levels. It is postulated that a normalization of blood glucose, would
result in regression of these pathological processes in inicial stage46. In this study, a
likely steatohepatitis could have caused an increased uptake of sodium
pertechnetate in the liver of diabetic rats, because once present in the circulation, the
radiopharmaceuticals label not only erythrocytes but also leukocytes, showing a
higher bioavailability in inflammed sites47,48. This phenomenon could alter the results
47
of liver scintigraphy when searching for primary tumors, liver cirrhosis and functional
assessments in detecting metastases.
CONCLUSION Diabetes altered the biodistribution of sodium pertechnetate (Na99mTcO4) in
the liver, stomach, thyroid and urinary bladder of rats, emphasizing the need for a
careful analysis of the results, if scintigraphic examinations are to be performed in
patients with this disease.
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21. Oliveira JF, Avila AS, Braga AC, de Oliveira MB, Boasquevisque EM, Jales RL et
al. Effect of extract of medicinal plants on the labeling of blood elements with
Technetium-99m and on the morphology of red blood cells: I--a study with Paullinia
cupana. Fitoterapia. 2002;73:305-12.
22. Chacon DA, Araújo-Filho I, Villarim-Neto A, Rêgo AC, Azevedo IM, Bernardo-
Filho M et al. Biodistribution of the radiophamarceutical sodium pertechnetate
(Na99mTcO4) after massive small bowel resection in rats. Acta Cir Bras.
2007;22:430-5.
23. Araújo-Filho I, Rêgo ACM, Brandão-Neto J, Villarim-Neto A, Egito EST, Azevedo
IM et al . Biodistribution of the radiopharmaceutical sodium pertechnetate after
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26. Malmud LS, Fisher RS, Knight LC, Rock E. Scintigraphic evaluation of gastric
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31. Yoshimura N, Chancellor MB, Andersson KE, Christ GJ. Recent advances in
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52
5.4. O Artigo foi publicado no periódico Journal of Surgical and Clinical Research.
2010:1(1):46-53. (ISSN 2179-7889)
Splenectomy changes the biodistribution of pertechnetate (99mTcO4
-) in rats
A esplenectomia altera a biodistribuição do pertecnetato (Tc99mo4-) em
ratos Ítalo Medeiros Azevedo, Ana Paula Fernandes Neves, Danilo Veras Lobo de Paiva, Jurema Samara Fonsêca Véras, Larissa de Castro Tomasi, Amália Cínthia Meneses Rêgo, PhD, Marília Daniela Ferreira Carvalho, Vítor Brasil Medeiros, Irami Araújo- Filho, MD, PhD, Aldo Cunha Medeiros, MD, PhD. From the Department of Surgery and Postgraduate Program in Health Sciences, Federal University of
Rio Grande do Norte, Brazil Financial support: CNPq, Brazil. Conflict of interest: None.
Correspondence address: Ítalo Medeiros Azevedo, Department of Surgery, Federal University of Rio
Grande do Norte, at Ave Nilo Peçanha 620, Natal, RN, Brazil. Email: [email protected]
Submitted: 06 December 2010. Accepted, after review: 27 December 2010.
ABSTRACT Purpose: To assess if splenectomy alters the biodistribution of sodium pertechnetate
in organs and tissues of rats. Methods: Twelve Wistar rats were randomly allocated
into two groups, A (splenectomized) and B (control), anesthetized with ketamine (50
mg / kg, IM, and sodium thiopental (20 mg / kg-IP) and operated under aseptic
conditions. Group A (n = 6) rats underwent laparotomy for splenectomy and group B
rats (n = 6) were only anesthetized. Both remained under postoperative observation
and after 10 days they were injected with 0.1 mL of sodium pertechnetate (0.66MBq)
via orbital plexus. After 30 min, the rats were killed by an overdose of anesthetic and
samples of stomach, liver, heart, lung, thyroid, bladder, kidney, brain and femur were
harvested. Detection of radioactivity was determined by an automated gamma
counter, Wizard Gamma Counter Perkin-Elmer. Data were expressed as
mean±standard deviation and Student t test was used for independent samples,
considering p<0.05 as significant. Results: There was a lower uptake of
pertechnetate in group A than group B in kidney, heart, lung, bladder and femur
53
(p<0.05), when compared with controls. The liver of the splenectomized animals
showed radioactive uptake significantly higher than in controls. Conclusion:
According to the experimental model, we conclude that totalsplenectomy in rats
resulted in alteration in the biodistribution of pertechnetate in vital organs.
Key words: Bioavailability. Tc 99m Pertechnetate. Splenectomy. Rats.
RESUMO Objetivo: Avaliar se a esplenectomia altera a biodistribuição do radiofármaco
pertecnetato de sódio nos órgãos e tecidos de ratos. Métodos: Doze ratos Wistar
foram distribuídos aleatoriamente em 2 grupos, A (esplenectomizados) e B
(controle), anestesiados com ketamina (50 mg/Kg-IM, e tiopental sódico (20 mg/Kg-
IP), operados sob condições assépticas. Os animais do grupo A (n=6) foram
submetidos a laparotomia mediana e esplenectomia total e grupo B (n=6) apenas
foram anestesiados. Ambos permaneceram sob observação pós- operatória e após
10 dias foram submetidos à administração de 0,1 ml de pertecnetato de sódio
(0,66MBq) via plexo orbital. Após 30 min, foram mortos com superdose de
anestésico e submetidos à retirada de segmentos do estômago, fígado, coração,
pulmão, tireóide, bexiga, rim, fêmur e cérebro. A detecção da radioatividade foi
determinada através de contador gama automático, Wizard Gama Counter Perkin-
Elmer®. Dados foram expressos em média±desvio padrão e o teste t de Student para amostras independentes foi usado, considerando as diferenças significantes
com p<0,05. Resultados: Observou-se menor captação do pertecnetato nos
animais do grupo A em relação ao grupo B, no rim, coração, pulmão, bexiga e fêmur
com p<0,05. O fígado dos animais esplenectomizados apresentou captação
radioativa significativamente maior do que nos controles. Conclusão: De acordo
com o modelo experimental utilizado, pode-se concluir que a esplenectomia total em
ratos resultou em alteração na biodistribuição do pertecnetato de sódio em órgãos
vitais.
Descritores: Biodisponibilidade. Pertecnetato. Esplenectomia. Ratos.
INTRODUCTION
54
The use of radionuclides has contributed to important advances in health
sciences. Diagnostic evaluations are possible by using radiopharmaceuticals labeled
with 99m technetium (99mTc)1-6. The widespread use of 99mTc is due to a number
of chemical, physical, economic and ambiental characteristics7. 99mTc is an artificial
radionuclide originating from the disintegration of 99molibdenium, an isotope from the
nuclear fission of uranium. Its main features are: half-life of six hours, emission of
gamma (g) rays and radiation energy of 140 Kev. By its high availability, easy for
connecting to the red blood cells, biological species, cellular structures, molecular,
low cost and negligible environmental impact, it has become the most used
radionuclide in nuclear medicine under sodium pertechnetate (Na99mTc04)8. It is
known that some drugs can interfere with the biodistribution of radiofarmaceuticals9-
12. However, little is known about the impact of organ resection on the biodistribution
of Na99mTc04 and publications are unavailable in the literature regarding the effects
of splenectomy on biodistribution13.
If splenectomy interferes with the biodistribution of radiopharmaceuticals,
scintigraphic exams may result in images of dubious accuracy, culminating in
repeated examinations and unnecessary exposure of patients to radiation. In recent
decades, traditional indications for splenectomy have been discarded due to high
incidence of deaths due to postoperative sepsis14. The emergence of alternative
management replaced splenectomy. However, serious cases such as pancytopenia,
hematological diseases with high risk of systemic or intracranial bleeding,
hypersplenism, gastric carcinoma, portal hypertension and trauma are still indications
for esplenectomy14. The aim of this study was to evaluate if the excision of the spleen
alters the biodistribution of Na99mTc04 in organs and tissues of operated rats.
METHODS In this experimental study we used 12 Wistar rats weighing 295±23g. The rats
were from Vivarium of Center of Health Sciences, Federal University of Rio Grande
do Norte, Brazil. They were randomly allocated into 2 groups: A (Esplenectomized)
and B (Control), of 6 rats each. The investigational protocol was approved by the
Institutional Animal Care Committee, and the research was performed in accordance
with the guidelines of the Brazilian College of Animal Experimentation.
55
The rats were observed in individual cages with food and water ad libitum; They
were anesthetized with ketamine (50 mg/Kg) i.m. and sodium thiopental (20 mg/Kg)
i.p, operated under aseptic conditions. Splenectomy group animals underwent
midline laparotomy and subsequent splenectomy. The laparotomy was closed in
layers with mononylon 4-0 suture. Hydration was done with normal saline (10
mL/100g weight) injected subcutaneously in the rats for the first 2 postoperative
days. Postoperative pain was treated with tenoxicam (Roche Pharm., Brazil); 0.5
mg/kg was injected i.m. once a day for 3 days. In the control group the rats were only
anesthetized. Both groups remained under observation for 10 postoperative days,
during which the rats were weighed dayly. The animals were again anesthetized with
the above-mentioned anesthetic association and injected with 0.1 ml of Na99mTc04
via the orbital plexus, producing a radioactivity of 0.66 MBq. After 30 minutes, the
rats were killed by an overdose of anesthetic (thiopental 100mg/Kg) and samples of
stomach, liver, heart, lung, thyroid, bladder, right kidney, right femur and brain were
harvested. The samples were washed with 0.9% saline solution, and weighed on a
precision scale. The detection of radioactivity in each sample was determined using
an automatic gamma counter, Wizard Gamma Counter® Perkin-Elmer, Finland. The
percentage of radioactivity of each organ (%ATI/g) was calculated by dividing the
radioactivity of each organ by the total activity administered to each animal. Data
were expressed as mean±SD. The comparison between groups was performed by
Student t test for independent samples, using a 0.05 significance.
RESULTS
After analysis of the percentages of radioactive uptake (%ATI/g) from the
organs samples, comparing the control and splenectomized groups, it was observed
that there was lower uptake of pertechnetate in group A rats than group B in the
kidney, heart, lung, bladder and femur. These differences were statistically significant
(p<0.05). The liver of the splenectomized animals showed significantly higher
radioactive uptake than in controls (p=0.0239). The other organs showed no
significant change in the uptake of compared splenectomy and control groups (Table
1).
56
Table 1 - Values of % radioactivity per gram of tissue (%ATI/g) from rats organs.
Mean±SD
* p≤0.05, comparing splenectomy group and control.
1. P-valor after analysis by t test for independent samples.
DISCUSSION 99mTechnetium (99mTc) is of great diagnostic utility. The labeling of tissues is
based on the reduction capacity of stannous chloride (SnCl2), which acts on the 99mTc in the form of Na99mTc04. Several factors influence the biodistribution of
different radiopharmaceuticals, among them are surgical procedures13,32-34. Changing
in biodistribution can lead to repeat examinations for patients undergoing surgery,
resulting in unnecessary irradiation. The human spleen is located in the left
hypochondrium and has the dimensions 12 cm long by 8 cm wide and 3 cm thick. His
weight without blood varies from 75 to 90g; in vivo, the spleen weight varies between
150 and 250g15. Despite its size, it is irrigated with 350 liters of blood per day, at a
speed of 200 ml/min, accounting for 40% of portal vein flow16. Through its great
amount of macrophages, spleen has one quarter of the lymphoid tissue of the body,
and is able to remove foreign particles from the blood and abnormal cells17. This
function is very important because these macrophages can phagocyte even without
the presence of opsonins. This peculiarity gives great power to the spleen as a
defense organ in cases of acute infection. However, this paper only begins to
develop over the years, since in childhood the spleen is an organ histologically and
57
physiologically imature18.
Splenectomy is indicated in several situations, among them, traumatic causes14.
It is also held in non-traumatic cases, such as the surgical treatment of gastric
carcinoma, portal hypertension, idiopathic thrombocytopenic purpura and splenic
cysts19-21. As there are no published reports of research involving the study of the
biodistribution of Na99mTc04 after splenectomy, this study has sought to fill this gap
and contribute to clarify this issue. The distribution, fixation and disposal of
radiopharmaceuticals in the organs and tissues depend on the flow circulation,
metabolism and binding to tissue and blood elements22. However, it is known that red
blood cells are remodeled in the spleen, passing from spherical to biconcave discs; it
clears substances deposited on their surface, such as proteins, Howell- Jolly bodies
(nuclear remnants), Heinz bodies (denatured hemoglobin) and Pappenheimer
corpuscles (siderotic granules).
The spleen is also active on the destruction of aging red blood cell, poorly
formed or coated with antibodies14. Therefore, the spleen is of fundamental
importance in the formation of functional red blood cells and destruction of aged red
blood cells. Splenectomy can interfere with labeling with radiopharmaceuticals,
thereby altering the biodistribution of Na99mTc04. Some studies have related
splenectomy as a source of repercussions on the monocytic fagocitosis23 lipidic
profile24 limph nodes25, showing that the asplenic state carries systemic
consequences. In our study there was a change in the Na99mTc04 biodistribution in
various organs. There was lower uptake in kidney, heart, lung, bladder, femur and
brain of splenectomized animals than in controls. This finding confirms the
hypothesis that removal of the spleen significantly alters the biodistribution of
Na99mTc04 in rats, which may be related to the effects of asplenia in the formation of
red blood cells or other systems. It has been reported that spleen interferes with
hepatic function26. In fact, in our study certainly the most relevant finding was the
observation that the radioactive uptake in the liver was found to be higher than in
controls, indicating that the absence of the spleen increased the uptake of Na99mTc04
in liver. If this is true, we can infer that there are clinical implications after
splenectomy, i.e., the liver can undergo changes in their physiology, affecting the
uptake of radiopharmaceuticals for diagnostic or therapeutic use.
Splenectomy might somewhat promote hepatic regeneration26,27, prevent liver
fibrosis to a certain degree28, reduce serum bilirubin concentration and improve liver
58
function29,30. In a clinical study, serum total bilirubin concentration was promptly
decreased to normal range or pre-operative level on the 7th day in splenectomy
group, though it was raised for a transient time. Serum total bilirubin concentration
increased on 10 to 14 days in non-splenetomy group. The total bilirubin concentration
was significantly lower in the former than in the latter on the 7th day after operation,
obviously reflecting the effect of splenectomy on reducing the burden of hepatocyte
bilirubin31.
CONCLUSION According to the experimental model used in this study, we can conclude that
total splenectomy in rats resulted in alteration in the biodistribution of Na99mTc04 in
vital organs.
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62
5.5. O Artigo foi publicado no periódico Journal of Surgical and Clinical Research.
2010;1:54-65. (ISSN 2179-7889).
Bacterial translocation in rats treated with simvastatin undergoing intestinal ischemia and reperfusion.
Translocação bacteriana em Ratos tratados com sinvastatina
submetidos a isquemia intestinal e reperfusão.
Ítalo Medeiros Azevedo, Irami Araújo Filho, MD, PhD, Amália Cinthia Meneses Rêgo, PhD, Vítor Brasil Medeiros, Marília Daniela Ferreira Carvalho, MD, Eryvaldo Sócrates Tabosa Egito, PhD, Aldo Cunha Medeiros, MD, PhD. Research performed at Nucleus for Experimental Surgery and Postgraduate Program in Health
Sciences, Federal University of Rio Grande do Norte (UFRN), Brazil. Financial support: CNPq, Brazil.
Conflict of interest: None.
Correspondence address: Ítalo Medeiros Azevedo, Department of Surgery, Federal University of Rio
Grande do Norte, at Ave. Nilo Peçanha 620, Natal, RN, Brazil, Email: [email protected]
Submitted: 10 December 2010. Accepted, after review: 27 December 2010.
ABSTRACT Purpose: To evaluate the anti-inflammatory effect of simvastatin in an experimental
model of intestinal ischemia/reperfusion, as well as in the prevention of bacterial
translocation. Methods: We used Wistar rats, randomly allocated in 5 groups: C
(n=10) controls; S (N=10) Shan-operated; I/R (n=10) intestinal ischemia and
reperfusion; S+Sim (n=7) sham treated with simvastatin and I/R+Sim (n=7)
ischemia/reperfusion treated with simvastatin. In the group S, a laparotomy and
manipulation of intestinal loops were performed. In the groups I/R and I/R+Sim, the
superior mesenteric artery was occluded with a vascular microclamp, the laparotomy
was closed and reopened 60 minutes after for pull back the clamp. The reperfusion
was confirmed by the return of the pulsation of the mesenteric arcade. The animals
were sacrificed after 120 minutes of reperfusion. Simvastatin microemulsion
(10mg/kg) was administered (gavage) 18 hs and 2 hours before the surgical
procedure. Blood was collected by cardiac puncture for measurement of TNF-α, IL-
63
1β, IL-6 and IL-10. One gram of spleen, liver and mesenteric lymph nodes was
harvested for culture in selective means for Gram (-) and Gram (+) bacteria. A
sample of terminal ileum of each animal was harvested, fixed in formalin 10% and
included in paraffin. Slices were stained with hematoxilin-eosin for morphometric
measurement. The damages of the intestinal samples were examined in a blind way
by an experienced pathologist, in agreement with microscopic criteria for levels of
aggressions based previously on a grade system. ANOVA and the post-hoc Tukey
test were used, considering p<0,05 as significant. Results: We observed bacterial
translocation to mesenteric lymph nodes, spleen, liver and blood in all animals
submitted to I/R, being smaller in the group I/R treated with simvastatin than in
controls. In the I/R group rats the values of pro-inflammatory cytokines were
significantly higher, when compared to the I/R+Sim group rats. The I/R+Sim group
rats showed higher levels of IL-10, when compared with the other groups (p<0.05).
The ileal segments presented macroscopic dilation and intramural hemorrhage. The
microscopy revealed intense mucosa lesion in the group I/R compared to the other
groups. The histopathologic findings of the I/R+Sim group rats were similar to that
found in the groups C and S. Conclusion: The simvastatin contributed to reduce the
bacterial translocation, the values of pro-inflammatory cytokine and to increase the
levels of anti-inflammatory cytokine, preserving the integrity of the intestinal
epithelium in an experimental model of ischemia/reperfusion.
Key words: Simvastatin. Ischemia/Reperfusion. Bacterial Translocation. Sepsis. Rat.
RESUMO Objetivo: Avaliar o efeito anti-inflamatório da sinvastatina em modelo experimental
de isquemia/reperfusão intestinal, assim como na prevenção da translocação
bacteriana. Métodos: Ratos Wistar foram distribuídos aleatoriamente em 5 grupos:
C (n=10) controle; S (n=10) simulação (sham); I/R (n=10) isquemia intestinal e
reperfusão; S+Sin (n=7) sham tratado com sinvastatina e I/R+Sin (n=7)
isquemia/reperfusão tratado com sinvastatina. No grupo S, procedeu-se laparotomia
e manipulação atraumática de alças intestinais. Nos grupos I/R e I/R+Sin, a artéria
mesentérica superior foi ocluída com microclamp vascular por 60 minutos. A
laparotomia foi fechada e reaberta após 60 minutos para retirada do clamp e a
reperfusão foi confirmada pelo retorno da pulsação da arcada mesentérica. Os
64
animais foram sacrificados após 120 minutos de reperfusão. Microemulsão de
sinvastatina (10mg/kg) foi administrada por gavagem 18 horas e 2 horas antes do
procedimento cirúrgico. Sangue total foi coletado por punção cardíaca, para
dosagem de TNFa, IL-1b, IL-6 e IL-10. Um grama de baço, fígado e linfonodos
mesentéricos foram removidos para cultura bacteriana em meios seletivos para
Gram (-) e Gram (+). Segmento de íleo terminal de cada animal foi fixado em
formalina a 10% e embebido em parafina. Secções coradas com hematoxilina-
eosina para usadas para medidas morfométricas. Os danos intestinais foram
avaliados de acordo com critérios microscópicos para níveis de lesões baseados em
um sistema de graduação previamente descrito. Foram utilizados os testes ANOVA
e Tukey, considerando p<0,05 estatisticamente significante. Resultados: Observou-
se translocação bacteriana para linfonodos mesentéricos, baço, fígado e sangue em
todos os animais submetidos a I/R, sendo menor no grupo I/R+ Sin, tratado com
sinvastatina. No grupo I/R, os valores de citocinas pró-inflamatórias foram
significativamente maiores, quando comparados ao grupo I/R+Sin. Os animais do
grupo I/R+Sin apresentaram os maiores níveis de IL-10, em relação aos demais
grupos (p<0,05). Os segmentos de íleo apresentaram dilatação e hemorragia
intramural macroscópicas. A microscopia revelou lesão intensa de mucosa nos ratos
do grupo I/R comparado aos demais grupos. Os achados histopatológicos do grupo
I/R+Sin foram semelhantes aos encontrados nos grupos C e S. Conclusão: A
sinvastatina contribuiu para a redução da translocação bacteriana, dos valores de
citocinas pró-inflamatórias e elevou os níveis de citocina anti-inflamatória,
preservando a integridade do epitélio intestinal em modelo experimental de isquemia
e reperfusão.
Descritores: Sinvastatina; Isquemia/Reperfusão; Translocação Bacteriana; Sepse;
Rato.
INTRODUCTION Intestinal ischemia-reperfusion (I-R) injury is a severe condition resulting from
acute mesenteric ischemia, small bowel transplantation, abdominal aortic aneurysm,
hemorrhage, trauma, septic shock, or severe burns1,2. Various chemical and cellular
mediators have been implicated in the pathogenesis of intestinal
65
ischemia/reperfusion, such as reactive oxygen, cytokines, endotoxins, and
neutrophils. Following adhesive interactions among neutrophils and endothelial cells,
neutrophil accumulation in the intestinal mucosa contributes to intestinal
ischemia/reperfusion injury via production of reactive oxygen metabolites and
proteases3. Leukocyte accumulation is a complex phenomenon that also involves
endothelium-based adhesion molecules as well as leukocyte chemotaxis factors
such as interleukin-8 (IL-8). Intercellular adhesion molecules are normally expressed
at a low basal level, but their expression can be enhanced by several inflammatory
cytokines such as IL-1β and tumor necrosis factor-α (TNF-α). A variety of cytokines,
including TNF-α, interferon-γ and IL-1β, are released from post-ischemic tissues4.
On 1976, in independent publications, Endo et al. e Brown et al, described the
first statin able to inhibit the HMG-CoA redutase. These drugs attracted the attention
of researchers because of their capacity to reduce serum cholesterol. Then, many
other statins were isolated5-7. Increasing number of evidence suggests that 3-
hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, or statins,
exert pleiotropic effects which are independent from their cholesterol-lowering
action8. One of these effects appears to be protection against ischemia/reperfusion
injury. Several studies dealing with ischemia/reperfusion have shown that statins
significantly reduce infarct size not only in heart but also in brain9,10. Statins have
been shown to elevate the expression of endothelial nitric oxide synthase (eNOS);
hence enhancing the basal and stimulated production of NO and improving
endothelium dependent vasorelaxation besides promoting antiinflammatory
processes11. They are involved in diverse cellular functions, including actin
cytoskeleton organization, cell adhesion and motility, gene expression, and
inflammation. Thus, inhibition of Rho kinases may contribute to some of the
cholesterol-independent beneficial effects of statin therapy. It has been postulated
that their antiinflammatory effects may be associated with modulation of both
adhesion molecule and cytokine production12,13.
Maintenance of bacteria and their products in the intestinal lumen are made by
mucin and epithelial cells, essential for survival. The enterocytes in constant division,
form an impermeable barrier to the intestinal contents. Because they are
metabolically active, they are susceptible to hypoxia, reducing the absorptive function
with loss of basement membrane integrity, leading to the bacterial translocation 14,15.
Berg and Garlington (1979) described the translocation as the passage of viable
66
bacteria through the intestinal mucosa to mesenteric lymph nodes, tissues and
órgãos16. It is postulated that ischemia and reperfusion promote translocation,
bacteremia and toxemia, initiating an inflammatory response and activating
inflammatory mediators, including cytokines17. In sepsis, the mortality ratio is directly
proportional to the intensity of the inflammatory response18-20. Based on these
principles, the objective of this study was to evaluate the anti- inflammatory effect of
simvastatin in an experimental ischemia/reperfusion model, as well as bacterial
translocation.
METHODS Animals Fifty male Wistar rats weighing 265±32g (from Nucleus of Experimental
Surgery, Federal University of Rio Grande do Norte-UFRN, Brazil) were used. Rats
were housed in polypropilene cages and maintained under controlled temperature
conditions on a 12h light-dark cycle and allowed ad libitum access to commercially
available rat chow and water. The experimental protocol was approved by the
Research Ethics Committee of the Federal University of Rio Grande do Norte, Brazil,
and adhered to the Guide for the Care and Use of Laboratory Animals, US National
Research Council, 1996.
Experimental design The rats were randomly assigned to 5 groups (n=7 in each group) as described
below. The control group (C) received only chow and water; Group sham (S) served
as a normal control, and a laparotomy was performed and the rats received only
chow and water. The ischemia/reperfusion rats (I/R) were submitted to mesenteric
ischemia/reperfusion; the sham/simvastatin (S+Sim) and the Ischemia/reperfusion
(I/R+Sim) group rats were treated with simvastatin. Treatment with simvastatin or
normal saline (0.9%) was done according to the protocol. Simvastatin rats received
20 mg/Kg of simvastatin microemulsion via gavage for 5 days before surgery.
Animals were fasted 12 hr before the experiment and anesthetized with an
intramuscular injection of 50mg of ketamine/Kg of body weight and thiopental
20mg/Kg IP. In groups I/R and I/R+Sim, under sterile conditions, a laparotomy was
performed and the superior mesenteric artery (SMA) was occluded with a
67
microvascular clamp for 60 minutes. In order to block any collateral blood supply, the
right colic and proximal jejunal arteries were also clamped. The laparotomy incision
was then closed, to be opened later for removal of the clamps after 60 minutes of
ischemia. Reperfusion was confirmed by the return of the mesenteric arcade
pulsation. The incision was closed again and the animals were killed by anesthetic
overdose (thiopental 100mg/Kg) after 120 minutes of reperfusion. The sham-
operated rats received the same surgical procedure as the other groups without
being subjected to the ischemia- reperfusion protocol.
Measurement of bacterial translocation At the end of the procedures (time = 180 minutes), a midline laparotomy was
performed under aseptic conditions and biopsies were aseptically obtained for
bacterial colony counts. One gram of MLN complex, liver and lung was removed for
culture. Tissues were homogenized and aseptically solubilized after addition of 0.5
mL of 0.9% saline. Aliquots of 0.2mL were processed and cultured on selective
MacConkey's agar and blood agar for detection of gram- negative and gram-positive
bacteria, respectively. The agar plates were incubated at 37 oC and examined for
growth after 24 and 48 hours. Any growth in the plates of bacteria of the same
biotype as cultured was considered positive and expressed as colony-forming units
per gram of tissue (CFU/g).
Histological study Ileum specimens were fixed in 10% buffered formalin and embedded in paraffin.
Sections cut at a thickness of 4mm were stained with hematoxylin and eosin for
morphometric measurements using an image analyzer (Image-Pro Plus, Media
Cyber®). The damage of the intestinal specimens was assessed in a blinded manner
by an experienced pathologist according to microscopic criteria for degree of damage
based on a grading system previously described [17]: normal mucosa, 0;
subepithelial space at the viluus tip, 1; more extended subepithelial space, 2;
epithelial lifting along villus, 3; denuded villi, 4; loss of villus tissue, 5; crypt layer
infarction, 6; transmucosal infarction, 7; transmural infarction, 8.
Cytokine assays Portal blood samples were collected and used for measurement of tumor
68
necrosis factor-alfa (TNFa), interleukin-1b (IL-1b), interleukin-6 (IL-6) and interleukin-
10 (IL-10), determined using enzyme-linked immunoassay kits (all from PeproTech,
Rocky Hill, NJ, USA), according to the manufacturer’s recommended protocols. The
fluorescence was measured by a Bio-Tec Instruments EL 808 ultra microplate
reader, using KC4-V3.0 analysis software. Sensitivity of detection was 30 pg/ml for
cytokines.
Statistics Data analysis was performed using the BioEstat 2.0 program. Differences
between the microbiological samples as measured by positive cultures were
evaluated by a test for differences between proportions. The results were tabulated
and compared by ANOVA using post hoc analysis with Tukey test. P<0.05 was
considered significant.
RESULTS We observed bacterial translocation to mesenteric lymph nodes, spleen, liver
and blood in all animals subjected to I / R. However, in I/R group rats treated with
simvastatin, translocation to these organs was significantly lower than in I/R
untreated (Table 1). In the control group, there was no bacterial translocation as well
as in the sham group rats, except in the mesenteric lymph nodes. In group S+Sin,
paradoxically, there was bacteremia at the expense of Gram-positive
(Staphylococcus aureus).
Table 1- Bacterial Translocation in groups treated and not treated with simvastatin
(colony-forming units per gram of tissue - CFU/g).
*p < 0.01 compared with groups C, S, IR/Sim.
Cytokines were not detected in group C rats. In group S+Sin, we observed
69
lower levels of proinflammatory cytokines (TNF-a, IL-1 b, IL-6) and a higher level of
antiinflammatory-cytokine (IL-10), when compared with group S (Table 2). In I/R
group rats, the levels of pro-inflammatory cytokines were significantly higher when
compared to I/R+Sim. This group had the highest values of IL-10 when compared
with all other groups (p<0.05).
Table 2- Serum levels of cytokines in groups with and without simvastatin treatment.
*p<0.01 compared with I/R, S, S+Sim
Macroscopically, the ileum segments studied showed dilation and intramural
hemorrhage, with higher intensity in I/R group rats, compared with the other groups.
The microscopic findings revealed marked mucosal injury after ischemia and
reperfusion injury; we observed most intense injury in I/R group rats compared with
the other groups. The lesions most often found were: thinning of the mucosa,
transmural infarction, infiltration of leukocytes in the lamina propria and mucosa.
(Figures 1,2,3). Histopathological findings of I/R+Sim were comparable to those
found in groups C and S.
70
DISCUSSION The HMG-CoA reductase inhibitors, including simvastatin, are used in the
treatment of hypercholesterolemia, because of their effects on cholesterol
biosynthesis, mevalonate pathway. Some studies have reported that statins preserve
endothelial function in the absence of hypercholesterolemia, raising eNOS22,23. The
activity of eNOS (nitric oxide synthase) inhibits leukocyte- endothelial interactions in
microcirculation24, reducing injury during ischemia and reperfusion of myocardium25
indeed "stroke protection"26. Nitric oxide suppresses the expression of several
endothelial adhesion molecules, including P-selectin, VCAM-1 and ICAM-12728.
In consequence, the present study used an experimental model of ischemia and
reperfusion, to verify the effect of simvastatin on intestinal injury and bacterial
translocation. Some authors consider the bacterial factor, crucial in the pathogenesis
of sepsis and multiple organ failure. In surgery and intensive care, intestinal
obstruction and intestinal ischemia are the most associated pathological conditions29-
31. This study showed that pretreatment with simvastatin attenuated the translocation
71
of bacteria to the liver, spleen and lymph nodes of rats. We used a simvastatin dose
of 10 mg/kg via gavage 18 hours and 2 hours before ischemia and reperfusion,
based on findings of Pruefer et al, who concluded that these are the periods with the
best results of the antiinfective effects of simvastatin32. Naito et al found that
pretreatment with rosuvastatin inhibited bacterial translocation, reducing the levels of
cytokines in maintaining the integrity of epithelial mucosa33. A similar phenomenon
occurred in the trial of Ozacmak et al. They demonstrated that atorvastatin not only
preserved the ileal mucosa but also the muscle contractility after ischemia and
reperfusion34.
OuvirLer In the sham group without simvastatin treatment it was observed
bacterial translocation only in mesenteric lymph nodes, but in Group S + Sin, some
animals showed Staphylococcus aureus in the blood. As we observed growing
exclusively of Gram positive bacteria, we interpreted it as a probable contamination
in the isolate. The I/R+Sim group rats showed levels of bacterial translocation
significantly lower in lymph nodes and liver, compared with group I/R. In the study of
Pirat et al, simvastatin reduced the neutrophil infiltration and severity of injury in lung
tissue in similar experimental model35. Other research found that treatment with
simvastatin reversed inflammatory alterations in mice subjected to sepsis by cecal
ligation and puncture, attributing these effects to the reduction of monocyte adhesion
to endotelium36,37.
It is known that the balance between anti-and pro-inflammatory cytokines is
important in the development of bacterial translocation and septic shock. Activated
leukocytes produce TNF-a and IL-6, which activate additional neutrophils to
propagate inflammation32. In our study the levels of proinflammatory cytokines (TNF-
a, IL-1 b, IL-6) were significantly lower in I/R+Sin rats than in other groups; however,
the levels of the anti-inflammatory IL-10 were higher in I/R+Sim rats than in other
groups. It is attributed to IL-10 the inhibition of tissue factors released by activated
monocytes via endotoxins, and reduction of expression of TNF-a by monocytes38,39.
Ando et al argued that cerivastatin was able to reduce serum levels of TNF-a, IL-1 b
in rats with sepsis induced by endotoxin, improving survival41. Finally, Waehre et al
researching the effect of statins in patients with coronary artery disease,
demonstrated significantly reduced serum levels of IL-1 in treated patients compared
to controls42. In this regard, we observed that simvastatin exerted marked anti-
inflammatory effects, a phenomenon found in other studies38,40.
72
CONCLUSION The use of simvastatin in rats subjected to intestinal ischemia and reperfusion
reduced bacterial translocation and the levels of pro-inflammatory cytokines and
increased levels of anti-inflammatory cytokine, influencing the integrity of the
intestinal epithelium.
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5.6. O Artigo foi publicado no periódico Journal of Surgical and Clinical Research.
2011:2:03-14. (ISSN: 2179-7889).
Ileal interposition for the treatment of diabetes in rats: repercussion on beta cells mass
Tratamento do diabetes pela interposição ileal em ratos: Repercussão
na massa de células beta Layra Ribeiro de Sousa Leão, Irami Araújo Filho, MD, PhD, Amália Cínthia Meneses Rêgo, PhD, Ítalo Medeiros Azevedo, Marília Daniela Ferreira Carvalho, MD, Aldo Cunha Medeiros, MD, PhD. Research performed at University Hospital Onofre Lopes, Department of Surgery, Federal University of Rio Grande do Norte (UFRN), Brazil. Financial support: CNPq, Brazil. Conflict of interest: None. Correspondence address: Aldo Cunha Medeiros, Department of Surgery, Federal University of Rio Grande do Norte, at Ave. Nilo Peçanha 620, Natal, RN, Brazil, Email: [email protected] Submitted: 08 July 2011. Accepted, after review: 11 August 2011.
ABSTRACT Purpose: Research with the aim of studying the effect of ileum interposition in
hyperglycemia and pancreatic beta cell mass in nonobese diabetic rats. Methods: We used 18 Wistar rats randomly divided into 3 groups of 6 each. A non-diabetic
control group, a diabetic control group, and diabetic with ileal interposition group.
Induction of diabetes was underwent with streptozotocin. The beta cell mass was
quantified by an indirect method by dividing the serum C-peptide (ng/mL) for blood
glucose (mg / dL). We used variance (ANOVA) and Tukey's test for analysis of
specific differences, considering the significance p <0.05. Results: The blood
glucose of diabetic animals with ileal interposition was significantly lower (94.5 ±
5.6mg/dL) than in diabetic control rats (245 ± 4.8 mg / dL) (p <0.05). The C-peptide
levels showed significantly higher in diabetes interposition group (0.58 ± 0.06 ng /
mL) than in control diabetes group (0.42 ± 0.03 ng /mL), suggesting a greater
response to pancreatic stimulation in that group (p <0.05). The ratio of C-peptide and
glucose levels showed a higher beta cells mass in diabetic interposition animals
(0.61) than in control diabetes (0.004), and the difference was significant (p<0.05).
Conclusion: The results showed that the ileal interposition contributed to the
reduction of blood glucose and to increase the mass of functioning beta cells in
diabetic rats.
77
Key words: Diabetes. Ileum interposition. Streptozotocin. Beta cells. Rats
RESUMO Objetivo: Pesquisa com o objetivo de estudar a ação da interposição do íleo
terminal na hiperglicemia e na massa de células beta pancreáticas em ratos
diabéticos não obesos. Métodos: Foram utilizados de 18 ratos Wistar, divididos
aleatoriamente em 3 grupos de 6 cada. Um grupo controle não diabético, um grupo
(controle diabético) submetido à indução do diabetes com estreptozotocina, e outro
grupo diabetes submetido à interposição ileal. A massa de células beta foi
quantificada por método indireto através da divisão dos níveis séricos de peptídeo-C
(ng/mL) pela glicemia (mg/dL). Usados os testes de variância (ANOVA) e Tukey,
para análise de diferenças específicas, considerando-se significância de p<0.05.
Resultados: A glicemia dos animais diabéticos com interposição ileal foi
significativamente reduzida (94,5±5.6mg/dL), quando comparada com o grupo
controle diabético (245±4,8mg/dL) (p<0,05). O peptídeo-C revelou níveis
significativamente maiores nos animais do grupo diabetes interposição
(0,58±0,06ng/mL) do que no grupo controle diabetes (0,42±0,03ng/mL), sugerindo
maior resposta à estimulação pancreática neste grupo (p<0,05). A razão entre
peptídeo-C e glicemia revelou diferença significante (p<0,05), observando-se maior
massa e função de células beta nos animais diabetes interposição (0,61) do que nos
controle diabetes (0,004). Conclusão: Os resultados obtidos permitem concluir que
a interposição ileal contribuiu para a redução da glicemia e para o aumento da
massa de células beta funcionantes em ratos diabéticos.
Descritores: Diabetes. Interposição. Íleo. Estreptozotocina. Células beta. Ratos.
INTRODUCTION Diabetes mellitus (DM) has great importance in Brazil, because it affects 7.6%
of individuals between 30 and 69 years1,2, with increasing prevalence particularly in
urbanized population. The increasing prevalence is a worldwide phenomenon in the
United States and a study of 18,825 adults aged >20 years showed a prevalence of
5.1% of diagnosed DM, and 2.7% of non diagnosed DM3. The DM causes
cardiovascular, neurological, renal and eye complications and is among the 10
78
leading causes of morbidity and mortality in Brazil4, resulting in high costs to the
health system5. The correct treatment decreases morbidity and mortality, improves
quality of life, and reduce costs6,7.
Type 2 diabetes is the most common form of diabetes in humans and is a
combination of genetic and acquired factors that impair the function of pancreatic
beta cells and tissue sensitivity to insuline8. There is evidence that beta-cell
dysfunction is crucial for the development and progression of this form of diabetes9,10.
Studies in patients and in isolated pancreatic islets have demonstrated quantitative
and qualitative defects in insulin secretion, stimulated by glucose. Thus, there has
been growing interest in the possibility of preservation of beta cells to prevent
diabetes, or to prevent the progressive deterioration of glycemic control observed
after diagnosis of the disease over the years10.
Assuming that the beta-cell dysfunction is the key to the development and
progression of type 2 diabetes, there is evidence that the beginning and triggering of
disease can be slowed and the blood glucose control can be affected by certain
therapeutic measures. Ideally, these beneficial effects are associated, at least in part,
to the maintenance of beta cell function. For this to occur, strategies must be
developed through drugs, cells and islets transplantation, or other appropriate
measures. In addition, efforts should be undertaken to better understand what
changes in the beta cells are present11.
Obesity and diabetes mellitus (DM) have reached epidemic proportions, and the
scientific world has studied new methods of treating these metabolic disorders12.
Remission of diabetes has been observed after surgical procedures that affect the
entero-insular axis, as is the case of bariatric surgery13,14, with improvement in
glucose homeostasis independent of weight loss15,16. Bariatric surgery offers the
control of diabetes and co-morbidities, leading to a change in lifestyle, having a role
of surgery metabólica17. With the increasing number of bariatric surgeries, technical
modifications have been explored to minimize the morbidity and associated
malabsorption. However, the improvement of diabetes after bariatric surgery may be
related to other factors that do not occur in non obese diabetic individuals. This
applies to the large reduction in calorie intake and very fast reduction of fat tissue
mass. The possibility of surgical intervention interfere with the beta cells mass has
been studied18.
The ileal interposition involves alteration of the intestinal tract through the
79
transposition of a segment of distal ileum for the zone of proximal jejunum. This
surgery, together with the sleeve gastrectomy, has resulted in post-operative glucose
control. As it does not involve bypass of small bowel and nutrients, changing in food
absorption is small18,19. The hypothesis inherent in the model, initially proposed by
Mason, is that the early stimulation of the ileum is an important component in the
mediation and release of hormones that can improve type 2 diabetes19. Some studies
have demonstrated improved glucose homeostasis in diabetic patients and in
postoperative of animal models of ileal interposition18,20.
Possible factors involved include the increase of hormones produced by the
ileum, such as glucagon-like peptide-1 (GLP-1), which is significantly elevated after
ileal interposition20. Since GLP-1 and its receptor antagonists promote the
proliferation of pancreatic beta cells and reduce apoptosis, is likely that ileal
interposition results in proliferation of beta cells mass21,22,23.
Treatment with streptozotocin results in the destruction of beta cells and causes
severe hyperglycemia in rats24. In the present study we used a streptozotocin dose
enough to partial destruction of beta cells25. Assuming that the ileal interposition
results in increased secretion of GLP-1, the protocol for this project worked with the
hypothesis that the interposition will extend the beta cell mass and glucose tolerance
in rats. The incretins, including GLP-1, are hormones secreted in the digestive tract
and make the production of insulin more efficient20. The gastrointestinal bypass
prevents and cures diabetes type II, stimulating the release of GLP-1 from the
terminal ileum when glucose and fat come to this intestinal region25.
There is no consensus about which test is best to monitor beta cell mass and
function in patients with diabetes and after islet transplantation26. Many tests give
variable results, are time consuming and difficult to perform. The low variability, high
reproducibility and close relationship between the serum C-peptide with insulin
secreted into the portal system, makes it an important tool to monitor beta cell
function27. The measurement of C-peptide has been routinely performed before and
after transplantation of pancreatic islets to document the survival of transplanted
islets28. Plasma levels of C-peptide values depend on glucose, so that they may
indicate good allograft function if the glucose is normal, but may be too low if glucose
is high. Calculations involving C-peptide values in relation to blood glucose in type 2
diabetes, as well as in transplants of the pancreas and islets have been made with
measurements in urine and serum29-31. In this study we used the ratio between C-
80
peptide and glucose (CP/G), which corrects the calculations according to their
glycemic control32.
Based on the above concepts and the fact that this is an important issue, and
that this is an issue poorly understood, in this study we induced diabetes in rodents
and examened the action of the ileum transposition in the mass of pancreatic beta
cells and treatment of hyperglycemia in non-obese rats.
OBJETIVE To examine the effect of ileal interposition in the evolution of glycemia in
diabetic rats. Through an indirect method, to quantify the beta cells mass before and
after ileal interposition.
METHODS Animals and diet Non-diabetic Wistar rats 3 months old, were housed in individual polypropylene
cages and maintained with free access to water and food (Labina - Purina ®). The
protocol followed all the precepts of the Brazilian Law No. 11.794/08, which
addresses the use of animals in research.
Experimental protocol After acclimatization in the laboratory for 7 days, the animals were weighed. An
initial dosage of glicemia was performed and the animals were randomly distributed
according to the protocol in three groups: non-diabetic control group (n = 6), diabetic
control (n = 6); diabetic interposition (n = 6).
Diabetes induction The groups of diabetic rats were treated with a single dose of streptozotocin
(STZ) (Sigma-Aldrich ®) (35 mg / kg dissolved in citrate buffer pH 4.5) to induce
hyperglycemia. With this dose, not all beta cells were destroyed and theoretically this
model did not create insulin-dependent diabetes. 48 hours after the use of STZ,
blood glucose was measured by manual Glucometer (Accu-chek, Roche ®,
Germany).
81
Surgery After defined hyperglycemia between 200 and 300 mg/dL, the rats fasted for 12
hours and they were anesthetized with thiopental (20 mg/kg) and ketamine (30
mg/kg) IM. Using aseptic technique, 3 cm midline laparotomy was performed in all of
them, the cecum was exposed and the terminal was ileum identified. In the diabetes
interposition group, the ileum was cut to 2 cm and 12 cm from the ileocecal valve,
and an ileal loop 10 cm in length was prepared, keeping the blood supply intact. Ileo-
ileal anastomosis was performed to reconstruct intestinal transit. The jejunum was
transected 10 cm from the Treitz angle, the previously isolated ileal loop was
interposed between the two ends of the jejunum by an isoperistaltic anastomosis,
using 6-0 Prolene ® (Ethicon), with the aid of surgical microscope 10x (DFV, São
Paulo, Brazil). Rats body weight was determined and recorded once a week after
surgery and all animals were observed for 30 days.
Quantification of indirect functional beta cells mass Completed 30 days of evolution, glucose concentrations were determined by
the Accu-Chek (Roche, Germany). Measurement of C-peptide was performed by
radioimunoassay. The ration between C-peptide and glucose levels was performed
using the following formula:
C-peptide/glucose ratio= C-Peptide (ng/ml) x 100
Glucose (mg/ml)
All calculations were performed using measurements of C-peptide and serum
glucose obtained from the same blood sample.
Morbidity and Mortality All rats were examined daily from the clinical point of view during the
experiment. Diarrhea, pain signals, water and food intake and weight control were
measured.
Statistical Analysis The data were submitted to analysis of variance (ANOVA). The Tukey test was
82
applied to analyze specific differences. Data were expressed as mean±standard
deviation, considering the significant differences at p<0.05.
RESULTS There was a significant difference in body weight among the 3 groups. All 18
rats survived for 30 days. Group control diabetes animals had a mean weight loss of
85±15.1 g and the diabetes interposition group had a mean weight loss of 73±13.4 g
over the 30 days (p<0.05). Non diabetes control group gained a mean of 65±6.2 g.
Table 1 shows the results of serum glucose and C-peptide, as well as tests to
investigate the statistically significant differences between diabetic and diabetic
control ileal transposition, as well as between the control non-diabetic and diabetic
control rats.
Table 1 – Effect of ileal interposition in serum biochemical parameters in non-diabetic
control rats, diabetic control and diabetic rats undergoing ileal interposition.
Data expressed as mean±standard deviation * Values significantly different compared with the
diabetic control group * P <0.05, ** Values with significant difference compared with control non-
diabetic and diabetic control group ** P <0.05.
The analysis of Table 1 to means that the blood glucose, in diabetic animals which
ileal interposition was performed, was significantly reduced, when compared with
non-operated diabetic animals (p<0.05). The same profile was observed in relation to
determination. Significantly higher levels of C-peptide were observed in the diabetic
interposition group than in diabetic control rats, suggesting increased pancreatic
stimulation response (p<0.05).
Evaluation of beta cells mass Table 2 – Effect of ileal interposition on functional beta cell mass, calculated by the
C- peptide/glucose ratio in non diabetic control, diabetic control and diabetic ileal
83
interposition rats.
*P<0.05. Values significantly different comparing with diabetic control group.
The C-peptide and glucose ratio was higher in diabetic ileal interposition group
(0.61), when compared with diabetic control rats (0.004), and the difference was
significant (p<0.05). Thus, we can infer that the ileal interposition contributed to
increase the beta cell mass (Table 2).
DISCUSSION The bariatric and metabolic surgery emerged to control morbid obesity and to
treat comorbidities such as type 2 diabetes mellitus. It is known that these surgical
techniques imply on restriction of food intake, malabsorption and increased release
of intestinal glucagon20. On early times the improvement of metabolic alterations after
bariatric surgery was exclusively attributed to weight loss. However, studies have
shown that most obese and diabetic patients undergoing surgical treatment had an
improvement in type 2 diabetes long before a significant weight loss33,34. The origin of
this observation is related to increased release of incretins by intestine L cells,
especially GLP-1. L cells are found mainly in the ileum and proximal colon and they
are part of the enteroendocrine system, which is involved in the mechanism of
hunger/satiety control, gastrointestinal motility, peripheral insulin sensitivity, glucose
and lipid metabolism35. Anatomical abnormalities resulting from the ileal interposition
stimulate the release of some hormones, mainly GLP-1, because of the early
perfusion of nutrients in the distal ileum36.
Thus, the ileal interposition promotes the release of incretins, without causing
disabsorptive syndrome. The present study found significant improvement in blood
glucose in the diabetic group after ileal interposition, compared with diabetic control
group. This fact corroborates the findings of Culnan et al37, in which the early
stimulation of the ileum after ileal interposition improved glucose tolerance, insulin
sensitivity and muscle glucose uptake in Zucker obese rats, without changing the
absorption of nutrients. Similarly, clinical study interposing a segment of 50 cm ileum
84
distal to the Treitz angle, showed the same endocrine stimulation, with reduction in
serum glucose38.
Strader et al39 demonstrated weight reduction associated with increased levels
of GLP-1 in rats. This effect is possibly related to the anorectic effect of this hormone
and peptide YY, an incretin hormone also produced by L cells.
Regarding the measurement of indirect effects of incretins on the mass of
functioning beta cells of animals, dosage of C-peptide in the systemic circulation has
been used. C-peptide is produced by beta cells and secreted into the bloodstream.
For along time, the C-peptide was considered important in the biosynthesis of insulin,
but it has minimal biological activity40. However, it remains an excellent parameter to
evaluate the function of pancreatic beta cells. It is equimolecular with insulin
secretion, has longer half life than insulin and a negligible hepatic clearance. Many
researchers prefer C-peptide concentrations instead of insulin, to detect changes in
insulin secretion by beta cells27,32,40.
The ileal interposition used in our study to treat diabetes in the animal model
used in this study revealed a positive effect on the measurement of C-peptide. The
animals of the diabetes interposition group showed levels of this peptide significantly
higher than the diabetic control rats. Likewise, the analysis of the C-peptide/glicemia
ratio showed a significant difference between groups ileal interposition diabetes and
diabetes control. This finding validates the finding that ileal interposition results in
increased beta cell mass in rats21-23.
The results of this study, which is part of a research line, corroborated with the
literature, demonstrate the great importance of the issue, in view of the effective
potential of this surgical technique in treating diabetes and preventing its
complications. Other parameters of immunogenetics, cell proliferation and hormone
examination are part of further research to validate the preliminary results of this
work.
CONCLUSION The results showed that the ileal interposition was beneficial for the evolution of
blood glucose and contributed to increased beta cell mass in diabetic rats.
85
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89
5.7. O Artigo foi publicado no periódico Journal of Surgical and Clinical Research.
2011;2:42-50. (ISSN: 2179-7889).
Hepatectomy for benign and malign diseases in University Hospital
Hepatectomia para doenças benignas e malignas em Hospital
Universitário
Marilia Daniela Ferreira Carvalho, MD; Irami Araújo-Filho, MD, PhD; Amália Cínthia Meneses Rêgo, PhD; Ítalo Medeiros Azevedo, Aldo Cunha Medeiros, MD, PhD.
Research performed at University Hospital Onofre Lopes, Department of Surgery, Federal University
of Rio Grande do Norte (UFRN), Brazil. Financial support: None. Conflict of interest: None.
Correspondence address: Marília Daniela Ferreira Carvalho, Department of Surgery, Federal
University of Rio Grande do Norte, at Ave. Nilo Peçanha 620, Natal, RN, Brazil, Email:
[email protected]. Submitted: 01 December 2010.Accepted, after review: 27 July 2011.
ABSTRACT The hepatectomy is a surgical modality devoted to the treatment of benign and
malignant conditions of the liver. This retrospective study aims to analyze the main
clinical and technical aspects concerning to 33 Resections performed from July 2006
to July 2010, at the Hospital Universitário Onofre Lopes. Showed agreement
between the sexes, most patients in the interior of our state, major comorbid
hypertension and complication fistula and mortality of 12%, surgery was performed
over the right trissegmentectomy, 21% of pacientes had gallbladder cancer and 12%
metastatic colon cancer. The age ranged 25-84th, 51st average. 45% had no
comorbidities, 30% were hypertensive. The operative time ranged from 110-660min,
mean 314.6 min, length of stay 02-69 days, average 16d. Hepatic surgery has
become more feasible in recent years because of the best preoperative evaluation
which allowed the control of postoperative complications.
Keywords: hepatectomy, liver surgery, liver resections.
90
INTRODUCTION
Liver resections characterized a major challenge for the surgeon in the past due
to its high mortality, but currently the mortality rate is around 5% in important medical
centers1. Today it is a surgical procedure for the treatment of benign hemangiom,
hepatocellular adenoma and malignant diseases as the primary hepatocellular
carcinoma and hepatoblastoma in childhood, in addition to metastasis of colorectal
carcinomas and rare tumors gastrinomas and carcinoids2. Hepatocellular carcinoma
(HCC) is the sixth most common cancer in the world and the third most common
cause of cancer-related death3. Patients at the early stage of disease are those who
present with an asymptomatic single HCC with the nodule < 5 cm in diameter or ≤ 3
in number. Patients exceeding these limits, but free of cancer-related symptoms and
vascular invasion or extrahepatic spread, are considered at the intermediate stage.
The patients with the cancer-related symptoms and vascular invasion or extrahepatic
spread are deemed at the advanced stage. HCC is frequently diagnosed at the late
stage and has a high mortality rate4. This increase of cases occurs because of the
spread of hepatitis viruses B and C5,6.
The liver is a frequent site of metastasis of solid tumors. The treatment of liver
metastases of colorectal tumors is already well established, but colorectal tumors for
therapy is not well defined7. Currently 15 to 25% of patients diagnosed with colorectal
tumors have liver metastasis and 25 to 50% of them will develop metastases within
three years. Surgical resection of colorectal metastases is the only curative option
and this procedure can guarantee the survival of 25 to 50% in 5 years8. Surgical
resection is a potentially curative therapy for HCC. However, only 10%-30% of
patients with HCC are eligible for curative hepatectomy9. Radical resection is still the
first choice for treatment of HCC10, even at the intermediate or advanced stage11. If
radical resection is impractical, palliative resection combined with comprehensive
therapy can significantly prolong patients’ survival time12. Despite medical advances
with new modalities of treatment for liver, resection is still the best curative
therapy8,13.
The aim of this work was to analyze the epidemiological data and surgical
treatment performed on all the patients submitted to hepatectomy in a University
Hospital during a five years interval.
91
METHODS
Based on the information stored in the program in 2000 MV ®, developed by VM
software and consulting, we selected the surgeries registered as hepatectomies
performed between July 2006 and July 2010, at the Onofre Lopes University
Hospital, and found 41 procedures, but when performed after reading the charts, only
33 were included. These eight charts were disregarded as they were non-surgical
treatments consistent with the proposed surgical or medical records with incomplete
information. The data of interest for the work were organized in tables, and these
were worked in the Microsoft Office Excel ® 2003 for the construction of statistical
data and graphs.
RESULTS
Demographic characteristics of 33 patients are presented in Figure 1. Female
patients predominated (n=17; 51%), and the median age was 51 years (range, 25–
84). 64% of patients were from provincial small towns and 36% from the capital
(Figure 2).
Figure 1-Distribution by gender.
92
Figure 2 - Distribution by city origin.
With regard to co-morbidities, 45% of patients had any associated diseases,
30% were hypertensive, 12% were smokers, 9% alcoholics,12% had a history of
diabetes mellitus treatment before surgical resection, and 6% had chronic
pancreatitis.The percentage of patients with chronic renal failure, cardiac arrhythmia
and Crohn's disease was 3% each (Figure 3).
Figure 3 - Distribution of comorbidities.
With regard to diseases that originated the hepatectomy, gallbladder cancer
occurred in 21% of cases, hepatic metastasis of colon cancer in 12% and GIST in
93
3% of cases. Klatskin tumor, adjacent tumor invasion, metastasis from
neuroendocrine tumors, tumors of undetermined origin, benign cysts and
hemangiomas contributed with 9%; hepatocellular carcinoma occurred in 6% of
patients and iatrogenic lesions of the bile duct in 3% of cases (Figure 4).
Figure 4 - Distribution of diseases that originated resections
The operative time ranged from 110 to 660 minutes (mean of 314.6
minutes).The length of hospital stay was from 2 to 69 days (average 16 days). The
stay in the intensive care unit ranged from 0 to 13 days, with an average of 3 days.
60.6% of patients were transfused during surgery or during hospitalization, and 6%
had autotransfusion during surgery; 33.3% were not transfused. Only 9% of patients
received i.v. human albumin.
Anatomical resection predominated (n=25; 75.7%). Seven of them (21.2%)
underwent major resection exceeding hemihepatectomy, and 4 underwent extended
bisegmentectomy. Eight patients (24.3%) underwent non-anatomical (atypical)
resections. These data are summarized in Table 1.
94
Table 1-Types of resections.
The most common complication was fistula (18%), followed by abscess (15%).
Nine percent of patients developed sepsis, acute renal failure occurred in 6% and
intra- abdominal hematoma in 6%. Other complications such as stroke, bilioma,
empyema, pleural fistula, liver failure, coagulopathy, atrial fibrillation, liver failure and
pneumonia totalized 3% each (Figure 5). Four patients (12%) died during
hospitalization.
Figure 5 - Postoperative complications.
95
DISCUSSION
Liver resection is a complex surgery to be performed by experienced surgeons
in specialized centers. For many years it was associated with increasing morbidity
and mortality. After the study of functional anatomy and physiology of liver
regeneration, performed by Couinaud, major advances in this area led to the current
period that witnessed satisfactory results14. As surgical techniques improved over the
last recent decades, after a good understanding of modern liver anatomy15, hepatic
resection is now considered a safe procedure and the gold standard of curative
treatment. Nevertheless, surgical resection is often contraindicated due to
deterioration of hepatic function and excessive tumor burden, both of which may
result from delayed diagnosis. Fortunately, early diagnosis of HCC has recently
increased because of regular examination of high-risk patients and advancement of
radiological diagnostic techniques. One of the most relevant factor of successfull
hepatectomy is the advancement of pre-operative radiological workup for the
diagnosis and study of injuries, often in early stage, including the intraoperative
ultrasonography. This modernization of radiology is reflected not only in diagnosis but
also in therapy1. Despite the decrease in surgery-related mortality and the increase in
possible surgical approaches, long-term outcome of surgical resection remains
unsatisfactory in many cases due to early recurrence16.
Analyzing our data with regard to gender, there was a similarity between them.
We found that most of our patients came from provincial small towns (interior) of Rio
Grande do Norte State, Brazil. This is understandable, since HUOL is a reference
public regional hospital. The age had a very wide range (25-84 years). One of the
deaths occurred in a young patient (25 years) with colon carcinoma and extensive
liver metastasis. In our series there was still a death in a patient with an initial
indeterminate diagnosis. The definitive diagnosis revealed benign disease. The
mortality in our series was 12% and is consistent with the literature of important
centers, around 3-15%2. Most patients who died had malignant disease, jaundice
and malnutrition. It was noted high morbidity and mortality, length of intensive care
unit and hospital stay in patients undergoing liver resection due to cancer of the bile
ducts.
Some patients stayed for a long time in intensive care unit because of the need
for ventilatory assistance and sepsis. Co-morbidities were conveniently evaluated
96
and treated to minimize postoperative complications. The main comorbidity found
was hypertension, which is consistent with the literature, followed by smoking and
alcoholism2. The hepatectomy was more frequently performed in cases of gallbladder
cancer. This certainly is due to the high prevalence of biliary tract diseases operated
in our hospital, not infrequently faced with a pathological diagnosis of malignancy.
The metastasis of colon cancer was the cause of our second indication of
hepatectomy. Only three patients were operated with benign diseases. Abscess and
biliary fistula were the main postoperative complications.
Based on these informations we emphasize the real need to encourage studies like
this, revealing the particular aspects of our service, for the knowledge of our reality.
These findings may guide our future steps in surgical liver diseases. In conclusion,
these data showed that morbidity and mortality posthepatectomy was compatible
with literature and that radiological and functional assessment permitted hepatectomy
including anatomic resection and optimal pathologic margins. Measures to minimize
postoperative morbidity were implemented.
REFERENCES 1. Marrero JA, Welling T. Modern diagnosis and management of hepatocellular
carcinoma. Clin Liver Dis. 2009;13:233-47.
2. Araújo GF, Costa OM, Santos MF et al. Hepatectomias: Análise crítica
retrospectiva de 21 casos. Rev Bras Cir. 2002;29:161-5.
3. Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J
Clin. 2005; 55: 74-108.
4. Rampone B, Schiavone B, Martino A, Viviano C, Confuorto G. Current
management strategy of hepatocellular carcinoma. World J Gastroenterol 2009; 15:
3210-6.
5. Zhou Y, Sui C, Li B et al. Repeat hepatectomy for recurrent hepatocellular
carcinoma: a local experience and systematic review. World J Surg Oncol. 2010,8:
55.
6. WaechterFL, SampaioJA, PintoRDetal. Utilização das hepatectomias centrais nas
cirurgias hepatobiliares. Rev Col Bras Cir. 2000;27:413-21.]
7. Choi EA, Abdalla EK. Patient selections and outcomes of hepatectomy for non
colorectal non-neuroendocrine liver metastases. Surg Oncol Clin N Am.
97
2007:16;557-7.
8. Lupinacci R, Penna C, Nordlinger B. Hepatectomy for respectable colorectal
cancer metastases-Indicators of prognosis, definition of respectability, thecniques
and outcomes. Surg Oncol Clin N Am. 2007:16;493-06.
9. Yau T, Chan P, Epstein R, Poon RT. Evolution of systemic therapy of advanced
hepatocellular carcinoma. World J Gastroenterol. 2008; 14: 6437-41.
10. Liu JH, Chen PW, Asch SM, Busuttil RW, Ko CY. Surgery for hepatocellular
carcinoma: does it improve survival? Ann Surg Oncol. 2004; 11: 298-303.
11. Wakabayashi H, Ushiyama T, Ishimura K, Izuishi K, Karasawa Y, Masaki T,
Watanabe S, Kuriyama S, Maeta H. Significance of reduction surgery in
multidisciplinary treatment of advanced hepatocellular carcinoma with multiple
intrahepatic lesions. J Surg Oncol. 2003; 82: 98-103.
12. Lin DX, Zhang QY, Li X, Ye QW, Lin F, Li LL. An aggressive approach leads to
improved survival in hepatocellular carcinoma patients with portal vein tumor
thrombus. J Cancer Res Clin Oncol.2011; 137: 139-49.
13. Jaeck D, Oussoultzoglou E, Rosso E. Hepatectomy for colorectal metastases in
presence of extrahepatic disease. Surg Oncol Clin N Am. 2007;16:507-23.
14. Chen J, Huang K, Wu J, Zhu H, Shi Y, Wang Y, Zhao G. Survival after anatomic
resection versus non anatomic resection for hepatocellular carcinoma: a meta-
analysis. Dig Dis Sci. 2011;56:1626-33.
15. Trivino T, Abib SC. Anatomia cirúrgica do fígado. Acta Cir Bras. 2003;18:407-14.
16. Agrawal S, Belghiti J. Oncologic resection for malignant tumors of the liver. Ann
Surg. 2011;253:656-65.
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5.8. O Artigo foi publicado no periódico Journal of Surgical and Clinical Research.
2011;2:78-86. ISSN: 2179-7889.
Sildenafil as a vasodilatatory mediator in the treatment of abdominal sepsis
Sildenafil como um mediador vasodilatador no tratamento de sepse
abdominal
Marília Daniela Ferreira Carvalho, Ingrid Tinôco Silvestre, Amanda Jayne Guedes Risuenho, Amália Cínthia Meneses Rêgo, Irami Araújo-Filho, Ítalo Medeiros Azevedo, Aldo Cunha Medeiros
Research performed at Department of Surgery, Federal University of Rio Grande do Norte (UFRN),
Brazil. Financial support: CNPq, Brazil. Conflict of interest: None. Correspondence address: Marília
Daniela Ferreira Carvalho, Department of Surgery, Federal University of Rio Grande do Norte, at Ave.
Nilo Peçanha 620, Natal, RN, Brazil, Email: [email protected]
Submitted: 08 October 2011. Accepted, after review: 11 December 2011.
ABSTRACT Objective: To analyse the effects of previous treatment with sildenafil in rats with
abdominal sepsis induced by cecal ligation and puncture (CLP). Methods: Wistar
rats were randomly allocated in 3 groups of 6 each. Sham group (SG): rats were
subjected to laparotomy and no induction of sepsis; CLP/sil: rats subjected to cecal
ligation and puncture, treated with sildenafil 1mg/Kg via gavage, 60 min before
sepsis induction; CLP group: rats with sepsis, no sildenafil. After anesthesia under
aseptic technique, we underwent a laparotomy and CLP. Postoperative pain was
controlled with tenoxicam 3mg/Kg, i.m. The rats were observed for 24 hs, and
examined on the late stage of polymicrobial abdominal sepsis. Body weight,
leukogram, C-reactive protein, TNF-α, IL-1β, IL-6, IL-10. Statistical analysis was
done by ANOVA and Tukey test, with significance p<0.05. Results: Group CLP rats
had their weight reduced in 12% when compared with sham rats (p<0.05). However,
comparing the sham and CLP/sil rats the diference on weight loss was not
99
significant. Leukocytes and neutrophils counts were significantly elevated in group
CLP rats compared with sham (p<0.05). In CLP/sil group rats a decreased total
leukocyte and neutrophil counts were detected, compared with the CLP group
(p<0.05). Reduced levels of C-reactive protein in the LPC/sil group were observed,
compared with CLP rats (p<0.05). Serum levels of TNF-α, IL-1β and IL-6 were lower
in the CLP/sil rats, compared with untreated CLP animals (p<0.05). Increased IL-10
in the CLP/sil rats, compared with CLP rats, demonstrated a protective effect on the
vasodilatory sepsis. Conclusion: The data revealed that the pretreatment of
abdominal sepsis with the vasodilator sildenafil favorably influenced the evolution of
inflammation and immune response in rats.
Keywords: Abdominal Sepsis. Treatment. Sildenafil. Vasodilation. Cytokines. Rats.
RESUMO Objetivo: Analisar os efeitos do tratamento prévio com sildenafil em ratos com
sepse abdominal induzida por ligadura e punção do ceco. Métodos: Ratos Wistar
foram distribuídos aleatoriamente em 3 grupos de 6 animais cada. Grupo Sham
(GS): Animais submetidos a laparotomia sem indução da sepse; grupo LPC/sil:
Ratos submetidos à ligadura e punção do ceco (LPC) tratados com sildenafil 1mg/kg
via oral por gavagem, 60 minutos antes da indução da sepse; grupo LPC: indução
de sepse, sem uso do sildenafil. Após anestesia e com técnica asséptica, foi
realizada laparotomia mediana e LPC. Dor pós-operatória controlada tenoxicam i.m.
3 mg/Kg. Os animais foram observados até completar 24 horas, estágio tardio e
hipodinâmico da sepse abdominal polimicrobiana. Foram analisados: Peso dos
animais, leucograma, dosagem sérica de proteína C reativa, TNF-α, IL-1β, IL-6 e IL-
10. Análise estatística pelo ANOVA e teste de Tukey, significância p<0,05.
Resultados: Redução de 12% do peso dos animais do grupo LPC quando
comparado com grupo sham (p<0,05). Comparação entre o grupo sham e LPC/sil
não revelou diferença significante. A quantificação de leucócitos totais e neutrófilos
revelou aumento significante nos animais do grupo LPC quando comparado com o
grupo sham (p<0,05). No grupo LPC/sil houve redução de leucócitos totais e
neutrófilos, comparando-se com o grupo LPC (p<0,05). Houve redução dos níveis de
proteína C reativa no grupo LPC/sil, em comparação com o grupo LPC (p<0,05).
Redução significante da expressão de TNF-α, IL-1β e IL-6 no grupo LPC/sil em
100
comparação com os animais não tratados (p<0,05). O aumento de IL-10 no grupo
LPC/sil, comparado com o grupo LPC sugeriu efeito protetor do vasodilatador sobre
o quadro séptico. Conclusão: Os dados permitem afirmar que o pré-tratamento da
sepse abdominal com o vasodilatador sildenafil influenciou a evolução favorável da
inflamação e da resposta imune em ratos.
Descritores: Sepse abdominal. Tratamento. Sildenafil. Vasodilatação. Citocinas.
Ratos.
INTRODUCTION Sepsis is a disease whose pathophysiology is linked to an imbalance between
anti-inflammatory and pro-inflammatory substances that will mediate a response to
damage to organs caused by infection. The infection process may be initiated by
gram-negative, gram-positive and anaerobic bacteria, fungi, protozoa and/or virus,
the latter being in smaller proportions. Gram-negative bacteria are the main cause of
sepsis1. In spite of increasing advance on the treatment of sepsis, this is still a major
cause of death in intensive care units, has a high cost, as diagnosed in the last two
decades, especially in polytraumatized patients in use of catheters, mechanical
ventilation, steroid-dependent patients and those with pneumonia2.
By using video-microscopy in vivo, investigators have demonstrated in rats
models that sepsis is characterized by decreased rate of flow in the microcirculation,
increased heterogeneity of flow, increased vascular stasis and decreased capillary
perfusion3.
The ideal therapy for improving the microcirculation must modulate endothelial
function prematurely and cause vasodilation of low flow units. Treatment with
vasodilators (prostacyclin) and a mixture of inotropes/vasodilators (dobutamine) have
been used for this purpose and have demonstrated an increase in systemic oxygen
consumption, suggesting improvement in microcirculation4-6.
Recently there has been considerable interest in investigating the role of sildenafil in
protecting ischemic and reperfusion injury in animals7. This is a new class of
vasoactive drugs that have been developed for the treatment of erectile dysfunction.
Its mechanism of action involves active inhibition of type 5 phosphodiesterase
enzyme that increases cGMP, nitric oxide (NO) and improves endothelial
101
dysfunction8.
Based on these aspects of interaction between microcirculation and its effects
on sepsis and in the vasoactive effects of sildenafil, the present experimental model
aims to analyze the effects of previous use of sildenafil in abdominal sepsis induced
by cecum ligation and puncture in rats.
METHODS Rats sample We used 18 Wistar rats weighing 280±18g, from the Center for Health
Sciences, Federal University of Rio Grande do Norte, Brazil. The experimental
protocol and the care of the animals were in accordance with the Animal Welfare Act
and Brazilian Law 11,794/2009. This project was approved by the Institutional Animal
Care and Use Committee of UFRN, Brazil. The animals were kept in an adaptive
period of seven days and throughout the experiment they were observed at the
Nucleus for Experimental Surgery in individual cages with water and standard food
for rodents (Labina-Purina ®) ad libitum, subjected to light-dark cycle of 12 hours with
humidity and noise control.
Experimental design The animals were randomly distributed into 3 groups of 6 animals each, with the
following characteristics: Sham group (SG) - Sham-operated rats (control) underwent
laparotomy and the cecum was neither ligated nor punctured. Group CPL /sil – The
rats underwent cecal ligation and puncture (CLP) and were treated with sildenafil.
CPL Group - Induction of sepsis, without sildenafil treatment.
Sildenafil treatment In groups SG and CLP/sil the rats were treated with sildenafil (Pfizer, Brazil)
suspension 1mg/kg by gavage, 60 minutes before sepsis induction.
Operative procedures Rats were fasted overnight (16 h) before the induction of sepsis but allowed
water adlibitum. They were anesthetized with ketamine 50mg/kg body weight and
xilazine 20 mg/kg (intramuscular), their abdomens were shaved, and a 4-cm ventral
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midline incision was made. The cecum was exposed and isolated by ligation with a 3-
0 cotton ligature just distally to the ileocecal valve to avoid intestinal obstruction. The
cecum was punctured twice at opposite ends with an 18-gauge needle and
confirmation of the punctures was established by forcing a small amount of the cecal
contents out of the cecum. The ligated and punctured cecum was then returned into
the abdominal cavity. The abdominal incision was then closed in two layers using a
4-0 nylon suture and the animals received 3 mL per 100 g body wt of normal saline
solution subcutaneously (i.e., fluid resuscitation).
Postoperative examinations The animals were observed for 24 hours, considering that 24 hours after CPL
represents the late stage of hypodinamic polymicrobial abdominal sepsis1. The
following parameters were analyzed: 1) Weighing of the animals at the beginning and
end of the experiment on a digital scale. 2) Collection of blood through cardiac
puncture at the end of the observation period for leukocytes and neutrophils count in
automatic cell counter (AbbottCell-Dyn 3500R-CD 3500 5L, USA). Blood serum was
separated by centrifugation at 2000rpm and stored at -40°C for subsequent dosage.
C-reactive protein was analyzed by autoanalyzer (BT Plus WeinerLab 3000). TNF-α,
IL-1β, IL-6 and IL-10 were assayed by ELISA (enzyme-linked-immunosorbent-
assay), an analytical methods based on antigen-antibody interaction used to
determine specific amounts of protein in tissue samples and body fluids. The
reagents were from PeproTech, NJ, USA. Observation of animal behavior (presence
or absence of hair bristling, lethargy and dark circles around the eyes), morbidity,
mortality and weight control were done.
Data were statistically analyzed by ANOVA and the Tukey test, considering
differences significant when p <0.05.
RESULTS There was a 12% weight loss on the CLP animals compared with sham group
(p<0.05). When the comparison was made between the sham group and the CLP/sil
group, no significant difference was observed. All animals in LPC group showed
lethargy, hair bristling and dark circles around the eyes after 24 hours. These findings
were not observed in animals from other groups.
103
The leukocytes and neutrophils counts showed significant increase in CLP
group animals when compared to the sham group (p<0.05). Treatment with sildenafil
(CLP sil group) reduced the levels of leukocytes and neutrophils, compared with the
CLP group (p<0.05). We observed a decrease in the levels of C-reactive protein in
the LPC/sil group rats, indicating a lower inflammatory response in this group
compared with CLP group (p <0.05), whose numerical data are shown in Table 1.
Table 1 - Results of leukocytes, neutrophils counts and measurement of C-reactive
protein.
*p<0.05 vs. grupos CLP, CLP/Sil; ** p<0.05 vs. grupos Sham, CLP /sil.
Inflammatory cytokines The serum levels of proinflammatory cytokines TNF-α, IL-1β and IL-6 showed a
significant reduction (p<0.05) in the group previously treated with sildenafil (CLP/sil)
compared with untreated animals. The serum level of the anti-inflammatory cytokine
L-10 was significantly higher in CLP rats than in CLP/sil animals, meaning a
protective effect on the vasodilator sepsis. Numerical data are summarized in Table
2.
Table 2 – Values of serum cytokines on each group rats.
*p<0.05 vs. CLP, CLP/Sil groups; ** p<0.05 vs. Sham, CLP groups.
DISCUSSION Sepsis has a high mortality rate, with estimated values between 20 and 50%
104
and several studies have been developed in the search for more effective therapies
to reverse sepsis2,9-11. In the present study, we verified the effectiveness of sildenafil
as a protector against the effects of sepsis caused by CLP. The pathophysiology of
this disease is related to an inflammatory response to infection. There is an
exacerbation in the release of inflammatory modulators and excessive activation of
inflammatory cells, meaning that the patients own defense can’t control the disease.
This may evolve into a severe sepsis until the failure of multiple organs, leading to
death1.
The presence of gram-negative bacteria in the peritoneal cavity triggers the
release of endotoxins and exotoxins that stimulate the expression of primary
modulators through activation of macrophages and monocytes. TNF-α and IL-1β are
some of these modulators, particularly TNF-α has been shown to be the most
involved in the development of septic shock. In the current study the pretreatment
with the vasodilator sildenafil interfered on the level of these pro-inflammatory
cytokines by reducing them in significant amounts in comparison with the untreated
group. This reduction was important to demonstrate that pretreatment with sildenafil
may act in the attenuation of the inflammatory response during sepsis. Cadirci et al
demonstrated that sildenafil attenuated exacerbated release of the pro-inflammatory
cytokine TNF-α in a study in which sildenafil was also used in rats subjected to
CLP12. IL-6 is another cytokine that appears to be the an efficient promoter on
hepatic production of acute phase proteins as the C-reactive protein, a very sensitive
marker of systemic inflammation. Damas et al13 demonstrated that IL-6 acts as a
second messenger released by macrophages, endothelial cells, fibroblasts and other
cells in response to sepsis. As seen in the quantitative evaluation of the present
study, pretreatment with sildenafil resulted in a significant reduction in IL-6 and C-
reactive protein levels, suggesting that these two mediators are correlated during
development of the sepis.
The exaggerated inflammatory response that occurs in sepsis is
counterbalanced by the early and sustained expression of potent anti-inflammatory
cytokines such as IL-10. In sepsis, IL-10 has been identified as a modulator of the
production of frequently lethal proinflammatory cytokines. Neutralization of IL-10
results in increased expression of proinflammatory cytokines and death, while the
administration of its recombinant form provides therapeutic protection14. Thus, the
increase in IL-10 in rats of CLP/sil group was certainly an important moderating and
105
neutralizing factor on the intense inflammatory process induced by sepsis.
Happening pro-inflammatory stimuli, such as IL-1, IL-6 and TNF-α, or oxidative
stress15, endothelial activation occurs, leading to a procoagulant environment, pro-
adhesive cell surfaces, dysregulation of vasomotor tone, and compromised barrier
function. This inflammatory environment is further propagated by the release of
additional cytokines directly from endothelial cells, which leads to local microvascular
damage, disrupted tight junctions, edema, and tissue hypoxia1,15.
In the presence of infectious focus, endotoxins and cytokines will activate
cellular and humoral immune response. The first line of defense against infections
after natural barriers are the monocyte-macrophages. Furthermore, the presence of
TNF-α, IL-1β and IL-6 also activates other cells or blood components such as the
polymorphonuclear cells1. In the present study we detected an increased migration
of leukocytes to circulation and to the infectious focus in the CLP rats, showing that
there was an intense attraction to the infected site by chemotactic factors. The use of
sildenafil, however, by reducing the intensity of the infectious process, required a
smaller amount of immune cells during sepsis. This was validated by the reduction of
total leukocytes and neutrophils in the rats of CLP/sil group compared to the CLP
group.
Kukreja et al demonstrated that sildenafil induces cardioprotective effect against
ischemia and reperfusion in dogs, rabbits, rats and mice. The effect was attributed to
the pharmacological preconditioning mechanism16. Lledo-Garcia et al demonstrated
a beneficial effect of sildenafil after renal ischemia. The aim of their experimental
study was to determine the hemodynamic, biochemical, and histological effects of
sildenafil as a preconditioning vasodilator before a period of warm ischemia17. A
similar effect was demonstrated in our laboratory7.
Tissue hypoperfusion can be present even in the normal blood pressure and
adequate cardiac output, a state sometimes referred to as cryptic shock. This
hypoperfusion may be related to preferential maldistribution of blood flow at the
regional or microvascular level18,19. Derangements of small vessel perfusion are
largely a function of intrinsic events in the microcirculation. The causes of
microcirculatory flow alterations in sepsis are multifactorial and include endothelial
cell dysfunction, increased leukocyte and platelet adhesion, fibrin deposition,
erythrocyte stiffness, altered local perfusion pressures due to regional redistribution
of blood flow, and functional shunting20,21. Although research on septic shock is
106
classically focused on macrocirculatory hemodynamics that reflect the distribution of
blood flow globally throughout the body, a functioning microcirculation is another
critical component of the cardiovascular system that is necessary for effective oxygen
delivery to tissues.
In the present study we used the pretreatment with sildenafil, a potent
vasodilator, in rats with abdominal sepsis, with significant changing in leukocytes,
cytokines and reactive C protein. Regardless of the cause, it seems that an early and
aggressive hemodynamic intervention can impart the best opportunity to limit the
damage caused by tissue hypoperfusion, including attenuating the inflammatory
response and endothelial injury22.
An ideal therapy to improve microcirculation would modulate endothelial
function and vasodilate low-flow units. Pharmacotherapies such as vasodilators
(prostacyclin) and mixed inotropes/vasodilators (dobutamine) have been used to this
end and have demonstrated an increase in systemic oxygen consumption suggesting
microcirculatory recruitment23. De Backer et al demonstrated increases in capillary
perfusion independent of systemic hemodynamic effects with dobutamine and
recombinant human activated protein C (rhAPC). These effects are likely to be due to
the vasodilatory and rheologic action of dobutamine and perhaps to modulation of the
leukocyte-endothelial cell interaction by rhAPC6,24.
Seen through the lens of the microcirculation, sepsis-induced increases in nitric
oxide (NO) may actually be an adaptive response that is an attempt to restore blood
flow at the level of capillaries. Contrary to previous lines of investigation, exogenous
NO may be viewed as an attractive therapeutic agent in sepsis if it is able to augment
microcirculatory flow. NO fulfills the requirement of being a potent vasodilator and
modulator of leukocyte-endothelial reactions, as seems to be the sildenafil used in
the present experimental model25.
Administration of exogenous vasodilator sildenafil, prior to the induction of
sepsis, demonstrated important protection against inflammation. One obvious
concern about the use of exogenous sildenafil in sepsis is the potential for
exacerbation of arterial hypotension, which could attenuate its positive effect on the
microcirculation. Studies about this are lacking. Therefore, at present, whether or not
exogenous sildenafil administration to patients with sepsis is efficacious and safe is
unknown and the subject of ongoing clinical investigation. We are sure that, as
promising as targeting the microcirculation in sepsis may appear, the microcirculation
107
is only one of many pathophysiologic factors that contribute to the overall picture of
circulatory failure and, ultimately, cellular dysfunction.
In conclusion, the data of our study support the statement that pretreatment of
abdominal sepsis with the vasodilator sildenafil influenced the favorable evolution of
inflammation and immune response in rats.
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3. Fries M, Weil MH, Sun S, et al. Increases in tissue Pco2 during circulatory shock
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6. De Backer D, Creteur J, Dubois MJ, et al. The effects of dobutamine on
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systemic effects. Crit Care Med. 2006;34:403–8.
7. Medeiros PJ, Villarim Neto A, Lima FP, Azevedo IM, Leão LR, Medeiros AC. Effect
of sildenafil in renal ischemia/reperfusion injury in rats. Acta Cir Bras. 2010;25:490-5.
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9. Lin SM, Huang CD, Lin HC, et al. A modified goal-directed protocol improves
clinical outcomes in intensive care unit patients with septic shock: a randomized
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10. Jones AE, Brown MD, Trzeciak S, et al. The effect of a quantitative resuscitation
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11. Otero RM, Nguyen HB, Huang DT, et al. Early goal-directed therapy in severe
sepsis and septic shock revisited: concepts, controversies, and contemporary
findings. Chest. 2006;130:1579–95.
12. Cadirci E, Halici Z , Odabasoglu F , Albayrak A , Karakus E , Unal D , Atalay F,
Ferah I andUnal B. Sildenafil treatment attenuates lung and kidney injury due to
overproduction of oxidant activity in a rat model of sepsis: a biochemical and
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6. COMENTÁRIOS, CRÍTICAS E SUGESTÕES
O câncer colorretal é o terceiro tipo de neoplasia maligna mais frequente no
mundo44,46,65-69 e as metástases hepáticas estão presentes em 15% a 25% dos
pacientes no momento do diagnóstico44,46,65,70-73. Cerca de 50% dos pacientes
desenvolverão metástases hepáticas na evolução da doença53,67-69,73-77 estas são
responsáveis por, no mínimo, dois terços das mortes68,72,74,78. O tratamento cirúrgico
das metástases hepáticas em pacientes com câncer colorretal já é bem estabelecido
na literatura como o que proporciona a maior chance potencial de cura dos
pacientes, apesar de todas as outras modalidades terapêuticas44,51,68,72,77-83 como
embolização vascular, radioterapia, ablação por radiofrequência, quimioterapia81,84.
Porém, apenas 10-25% dos pacientes com metástases hepáticas receberão
indicação de cirurgia para ressecção44,46,70,72,76,78-80,83. Esta consiste na capacidade
de remover todas as metástases hepáticas com margens livres e de preservar um
fígado remanescente futuro de no mínimo 20-30% do volume total em pacientes com
fígado saudável67. Na ausência de doença extra-hepática irressecável, este
remanescente deve ainda proporcionar fluxo arterial e portal adequados, bem como
drenagem biliar e retorno venoso78.
Não há diferença importante na morbi-mortalidade perioperatória entre as
ressecções simultâneas e em dois tempos, porém há o benefício de uma duração da
cirurgia menor e de internação pós-operatória mais curta no grupo operado
simultaneamente, bem como menor perda de sangue durante a cirurgia44. Quando
um paciente é submetido apenas a cirurgia, a sobrevivência após esta é estimada
em várias séries entre 20-58% em 5 anos e 20% aos 10 anos44,49,54,66-70,73,74,76,78,81-
83,86,87. A recorrência tem o fígado como o local mais comumente acometido49,79,88.
O objetivo desde trabalho foi identificar se há alteração na biodistribuição do
radiofármaco fitato-99mtecnécio, usado para realização de cintilografias, bem como
avaliar a marcação e morfometria das hemácias, de roedores submetidos a um
procedimento cirúrgico de grande porte. Elegemos como representante deste grupo
a colectomia associada à hepatectomia pela importância do tratamento e por se
tratar de um procedimento que vem crescendo em indicações nos pacientes que
possuem CCR com metástase hepática, pelos motivos explicados acima. Este
procedimento revolucionou o tratamento do câncer colorretal nas últimas décadas,
como descrito previamente, devolvendo em alguns casos a chance de cura aos
111
pacientes com doença avançada. Não foi objetivo deste estudo a carcinogênese do
câncer colorretal, logo esta não foi induzida nos animais. Apesar do grande trauma
cirúrgico a que estes animais do grupo colectomia+hepatectomia foram submetidos,
eles evoluíram bem até o trigéssimo dia do experimento. Na primeira semana este
grupo apresentou uma perda de peso importante quando comparado aos outros dois
grupos, porém tal perda foi recuperada satisfatoriamente nas três semanas
subsequentes. Estes achados colaboram com o fato de que a hepatectomia
aumenta a morbidade neste grupo e a regeneração hepática deve compensar as
perdas.
Ao final deste experimento, quando as amostras obtidas foram analisadas,
observou-se que existiam diferenças importantes em alguns parâmetros quando os
três grupos foram comparados, como na captação do fitato-pertecnetato pelo fígado
do grupo colectomia+hepatectomia que foi significativamente menor que a do grupo
sham, porém quando o grupo colectomia+hepatectomia foi comparado ao grupo
colectomia não foram observadas divergências. Tal fato pode ser explicado pela
hipótese que ao final de 30 dias ocorreu regeneração hepática suficiente para
corrigir qualquer disfunção, normalizando a captação do radiofármaco. A literatura
refere que a regeneração hepática no rato ocorre muito rapidamente, completando-
se em aproximadamente uma semana89,90.
A biodistribuição do radiofármaco pelo fígado guarda relação com a intensidade
da doença que o acomete, com a fibrose, o prognóstico e a função hepática22,90-93.
Desse modo, a captação do fitato-pertecnetato pelo fígado é um índice indireto e
prático da função hepática23,94-96.
Os nossos estudos em laboratório, que seguem esta linha de pesquisa,
conseguiram demonstrar que quando a cirurgia realizada é a esplenectomia13 a
distribuição do radiofármaco no fígado é maior que nos grupos controles, sugerindo
que o procedimento cirúrgico favorece a captação. Este aumento da captação deve
resultar da melhora da função hepática, que no estudo foi confirmada pelos
melhores níveis de enzimas hepáticas nos ratos esplenectomizados do que nos
controles. No presente estudo, a menor absorção de fitato-pertecnetato pelo fígado
coincidiu com aumento das enzimas hepáticas (ALT,AST,FA) em ratos colectomia +
hepatectomia parcial quando comparados aos ratos sham. Várias patologias são
estudadas através de procedimentos de medicina nuclear, que cada vez mais se
tornam mais eficazes21 e menos invasivos23,97,98.
112
Além de doenças, outras condições também podem alterar a biodistribuição do
pertecnetato ou fixação dos constituintes sanguíneos como: cirurgias, medicações,
produtos naturais1-5,11. A alteração da marcação de constituintes sanguíneos no
geral é resultado da ação de medicamentos e trauma (cirurgias) devido à: (1)
mudança da estrutura da membrana celular ou modificação nos sistemas de
transporte de íons de pertecnetato em células, (2) oxidação direta ou geração de
radicais livres, (3) inibição direta (ação quelante) dos íons pertecnetato, ou (4)
ligação nos mesmos locais nos elementos do sangue27. O estudo em questão
observou que a marcação das hemácias com pertecnetato no grupo
colectomia+hepatectomia foi menor do que em ratos sham. Tal fato é importante
devido à marcação de hemácias ser um procedimento utilizado no diagnóstico de
sangramentos digestivos e renais39,41. Os dados obtidos nesta pesquisa podem ser
explicados, em parte, através de interações de alguns fatores que resultam da
ressecção hepática com os sistemas de transporte de íons pertecnetato, o que
culmina com a diminuição da marcação das hemácias pelo pertecnetato.
Ao ingressar no Programa de Pós-graduação em Ciências da Saúde da UFRN
evidenciei que as diversas disciplinas em carácter multiprofissional contribuíram para
diversificar meus conhecimento antes só restritos à área da medicina, pois neste tive
contato com alunos e professores de diversas áreas da saúde. Através das
disciplinas obrigatórias e complementares, aprendi sobre como tornar a escrita da
minha dissertação mais simples, o passo-a-passo da metodologia minimizando os
riscos de erros, sobre como pesquisar artigos e avaliar a sua relevância, e pude ter
também contato com conceitos avançados em estatística. Ao longo dessa jornada
aperfeiçoei a técnica da elaboração de artigos através dos meus artigos publicados,
participei de eventos científicos que enriqueceram meus conhecimentos e fizeram
com que eu pudesse ter contato com outros pesquisadores. Cada uma dessas
etapas foi importante no meu crescimento na pesquisa e ampliou minha visão crítica
na prática da minha área de atuação.
O conhecimento adquirido neste Mestrado contribuiu para o desenvolvimento de
modelos experimentais em outras pesquisas, bem como avançar nesse mesmo
modelo, para a verificação de outros resultados. Uma constante no delineamento e
execução deste projeto foi a preocupação exaustiva de evitar riscos, buscando
seguir rigorosamente as condições metodológicas, para permitir a reprodutibilidade
integral de nossos estudos pela comunidade científica objetivando estimular a
113
obtenção de outros resultados que tragam maiores contribuições ao meio
acadêmico. Para isso procuramos descrever a metodologia de forma simples e
clara, para que todos os pesquisadores possam reproduzir e desenvolver novos
modelos experimentais que contribuam com a consolidação de novos
conhecimentos nesta área.
Durante o desenvolvimento dessa pesquisa pude compreender a importância
da pesquisa básica, no sentido de criar subsídios para que a prática clínica na
Medicina Nuclear evolua, contribuindo para uma melhor compreensão dos efeitos
das intervenções cirúrgicas na funções dos órgãos operados e de outros órgãos à
distância que não sofreram intervenções. Tal entendimento sobre esse tema em
particular minimizaria erros e interpretações equivocadas de exames que podem
prejudicar o diagnóstico e prognóstico de um paciente. Dessa forma estaríamos
auxiliando os profissionais da área de Medicina Nuclear com novos conhecimentos,
até agora não amplamente elucidados no que diz respeito aos efeitos da colectomia
e hepatectomia.
A inspiração para realizar este estudo surgiu em primeiro lugar, do contato
com meu orientador, cuja linha de pesquisa especulava a influência de cirurgias e
outros fatores como medicamentos, plantas medicinais, entre outros nos exames de
medicina nuclear. O mesmo já tinha estudado as consequências da colectomia total
nesta área da radiologia. A pergunta a ser respondida dizia respeito aos efeitos
dessa intervenção cirúrgica associada à hepatectomia parcial, aumentando o porte
cirúrgico, na biodistribuição do fitato-99mTcO4, na marcação e morfologia de
hemácias e em parâmetros metabólicos. Entretanto repercussões da colectomia
associada à hepatectomia têm sido pouco estudadas, como foi constatado em prévio
levantamento bibliográfico amplo realizado no início do projeto. Outro aspecto que
motivou a realização deste trabalho foi a possibilidade de utilizar os recursos da
Medicina Nuclear, em estudos dessa natureza. Houve grande facilidade de
manipulação e emprego do radiofármaco, bem como compreensão da sua utilidade
para elucidar as alterações na captação da atividade radioativa pelo órgão alvo da
pesquisa.
Após concluído este trabalho, é continuo o desejo de prosseguir com os
estudos nesta área de atuação, desenvolvendo um doutorado nesta mesma base de
pesquisa, estudando algo que não foi contemplado neste estudo como os efeitos da
colectomia na regeneração hepática, parâmetro de suma importância na cirurgia do
114
fígado. Para prosseguir nesta carreira tenho o total empenho e suporte do meu
orientador que é chefe do Núcleo de Cirurgia Experimental, e que já evidenciou ter
disponibilidade de continuar minha orientação nesta trajetória. Durante todo tempo
em que estive envolvida com essa dissertação trabalhei também,
concomitantemente, como professora substituta da Disciplina de Técnica Operatória
que faz parte da grade do Curso de Medicina, da qual meu orientador é Professor
Titular com dedicação exclusiva. Além disso pude participar de eventos científicos
onde meus artigos escritos ao longo deste Mestrado puderam ser apresentados à
comunidade científica. Outro aspecto importante foi poder participar como
orientadora de Trabalhos de Conclusão de Curso dos alunos da graduação de
Medicina.
De grande relevância para confecção deste estudo foi o suporte dado pelo
Setor de Medicina Nuclear da Liga Norteriograndense Contra o Câncer fornecendo
os radiofármacos, e pelo serviço de patologia do Hospital Universitário Onofre Lopes
(HUOL)-Universidade Federal do Rio Grande do Norte (UFRN). Além destes,
destaca-se a estrutura do Núcleo de Cirurgia Experimental da UFRN, o laboratório
onde toda esta pesquisa foi idealizada e executada, e dos funcionários que
trabalham neste núcleo de pesquisa. Estes apoios foram fundamentais para a
realização dessa dissertação. Sem a participação efetiva de todas essas instituições
e seus respectivos serviços e funcionários, não teria sido possível concluir esta
dissertação. Os diferentes serviços que contribuíram para realização desta
dissertação comprovam o seu carácter multidisciplinar.
O rato Wistar foi escolhido como modelo experimental para o estudo, por ser
de fácil aquisição, manutenção e manipulação no laboratório. Além disso, trata-se de
um modelo animal previamente testado em um número considerável de estudos na
área de cirurgia experimental, que suporta muito bem atos anestésicos e
procedimentos operatórios como o realizado neste estudo e guarda boa correlação
com os objetivos do estudo, realizado de forma segura e satisfatória.
Os resultados obtidos neste estudo sugerem que a colectomia associada à
hepatectomia alterou a captação do fitato-pertecnetato no fígado e hemácias de
ratos, e a atividade enzimática hepática. O estudo mostrou-se relevante, pois além
de contribuir sobremaneira para minha formação, ainda elucidou dados até então
pouco explorados na literatura
115
Através deste estudo acreditamos haver contribuído para que futuros
trabalhos nesta área disponham de parâmetros sobre os efeitos da colectomia e
hepatectomia sobre a biodistribuição de radiofármacos. Deseja-se também que as
informações levantadas por esta pesquisa sirvam de ponto de partida para novos
estudos sobre a avaliação das consequências da colectomia associada à
hepatectomia, com potencial para implicações clínicas para pacientes que venham a
ser submetidos a esta intervenção cirúrgica e venham a realizar exames de
medicina nuclear.
Finalmente, esta pesquisa experimental me proporcionou um grande
enriquecimento intelectual, me introduziu nesta fascinante área da investigação
científica e abriu horizontes para a aplicabilidade clínica de conhecimentos de
radiofarmácia adquiridos em laboratório.
116
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