WILLIAM KONDO
TRANSPLANTEDECÉLULAS-TRONCOMONONUCLEARESDAMEDULAÓSSEA NOTRATAMENTODAENDOMETRIOSEEXPERIMENTAL
CURITIBA 2011
WILLIAM KONDO
TRANSPLANTEDECÉLULAS-TRONCOMONONUCLEARESDAMEDULA ÓSSEANOTRATAMENTODAENDOMETRIOSEEXPERIMENTAL
Dissertação apresentada ao Programa de Pós-Graduação em Ciências da Saúde da Pontifícia Universidade Católica do Paraná, como requisito para obtenção do título de Mestre.
Orientadora: Profa. Dra. Vivian Ferreira do Amaral
Coordenador: Prof. Dr. Roberto Pecoits Filho
CURITIBA 2011
Dados da Catalogação na Publicação Pontifícia Universidade Católica do Paraná Sistema Integrado de Bibliotecas – SIBI/PUCPR
Biblioteca Central
Kondo, William
K82t Transplante de células-tronco mononucleares da medula óssea no
2011 tratamento da endometriose experimental / William Kondo ; orientadora, Vivian Ferreira do Amaral ; coordenador, Roberto Pecoits Filho. -- 2011.
68 f. ; il. ; 30 cm
Dissertação (mestrado) – Pontifícia Universidade Católica do Paraná, Curitiba, 2011
Inclui bibliografias
Texto em português e inglês
1. Transplante de células-tronco. 2. Endometriose. 3. Fator de necrose de tumor. 4. Medula óssea. I. Amaral, Vivian Ferreira do. II. Pecoits Filho, Roberto. III. Pontifícia Universidade Católica do Paraná. Programa de Pós- Graduação em Ciências da Saúde. IV. Título.
CDD 20. ed. – 610
AGRADECIMENTOS
À Profa. Dra. Vivian Ferreira do Amaral, orientadora e profunda conhecedora do assunto. Entusiasta e comprometida com a pesquisa em prol das mulheres portadoras de endometriose.
Ao Prof. Dr. Roberto Pecoits Filho, Coordenador do Programa de Pós-Graduação em Ciências da Saúde da PUC-PR, por seu comprometimento para com os discentes e com a Instituição.
Ao Prof. Dr. Luiz Cesar Guarita-Souza, um dos pioneiros no uso de células-tronco da medula óssea em modelos experimentais e clínicos.
Aos pesquisadores colaboradores e amigos Eduardo Andreazza dal Lago, Priscila Maestrelli e Carolina Chaves Ferreira, por seu incansável apoio durante toda a pesquisa.
Aos amigos, Julio Cesar Francisco, Rossana Baggio Simeoni, Lucia de Noronha, mestres e doutores da PUC-PR, por seu apoio e orientação durante o decorrer da pesquisa.
À Profa. Marcia Olandoski, por seu auxílio e sua paciência durante a análise estatística dos resultados da pesquisa.
Aos demais funcionários do Programa de Pós-Graduação em Ciências da Saúde, pelo empenho e dedicação aos alunos e à Instituição, possibilitando a realização das pesquisas e o crescimento dos alunos e da Universidade.
À minha família (Manabu Kondo, Satiko Utzumi Kondo e Ricardo Eiji Kondo), pelo amor incondicional e apoio constante, possibilitando minha formação e meu crescimento profissional, com uma base de apoio sólida.
À Monica Tessmann Zomer, minha companheira e mulher amada.
"Todo o futuro da nossa espécie, todo o governo das sociedades, toda a prosperidade moral e material das nações dependem da ciência, como a vida do homem depende do ar. Ora, a ciência é toda observação, toda exatidão, toda verificação experimental. Perceber os fenômenos, discernir as relações, comparar as analogias e as dessemelhanças, classificar as realidades, e induzir as leis, eis a ciência; eis, portanto, o alvo que a educação deve ter em mira. Espertar na inteligência nascente as faculdades cujo concurso se requer nesses processos de descobrir e assimilar a verdade, é o a que devem tender os programas e os métodos de ensino."
Rui Barbosa.
RESUMO
Introdução: A endometriose é uma doença ginecológica benigna que afeta cerca de 10% das mulheres em idade reprodutiva. A fisiopatologia da endometriose não é completamente compreendida, mas parece haver uma imunoreatividade aberrante induzida pela doença, resultando em um aumento dos níveis de citocinas, substâncias angiogênicas e fatores de crescimento. Recentemente, o transplante de células da medula óssea em modelos experimentais e em estudos clínicos demonstrou uma possível ação anti-inflamatória dessas células.
Objetivo: Avaliar o efeito do transplante de células-tronco mononucleares no tratamento da endometriose em um modelo experimental.
Métodos: Os implantes de endometriose foram induzidos cirurgicamente em 120 ratas fêmeas Wistar. Os animais com implante endometrial viável (maior do que 25 mm²) foram aleatoriamente divididos em três grupos para receber uma injeção de 0,2ml de solução salina (grupo C; n=30), uma injeção subcutânea de 1mg/kg de leuprolide (grupo L; n=34) ou uma injeção intraperitoneal de 5x106 células-tronco mononucleares derivadas da medula óssea (grupo CT; n=36). As ratas foram sacrificadas 21 dias após o tratamento para a avaliação do tamanho dos implantes e da expressão do receptor de fator de crescimento vascular endotelial (VEGF-R) e do fator de necrose tumoral alfa (TNF alfa).
Resultados: Houve redução da área de superfície do implante endometriótico no grupo tratado com leuprolide (grupo L) comparado com os grupos CT e C. A redução absoluta da área de superfície do implante foi de 16,5mm, 0mm e 0mm (p=0,007), respectivamente, e a redução percentual foi de 40,2%, 0% e 0% (p=0,001). A expressão do VEGF-R no implante endometriótico diminuiu após o tratamento nos grupos L e CT comparado com o grupo C (409,6µm2, 465µm2 e 920,9µm2, respectivamente; p=0,021). A expressão do TNF alfa também diminuiu nos grupos L e CT comparado com o grupo C (585,7µm2, 549,3µm2 e 2402,1µm2, respectivamente; p<0,001).
Conclusões: Houve menor expressão de VEGF-R e TNF alfa nos implantes de endometriose no grupo tratado com células-tronco mononucleares derivadas da medula óssea, mas não houve redução da área de superfície das lesões.
Palavras-chave: Endometriose; Células-tronco; Receptor do fator de crescimento vascular endotelial; Endometriose experimental; Fator de necrose tumoral alfa.
ABSTRACT
Introduction: Endometriosis is a common benign gynecologic disease that affects reproductive-aged women. The physiopathology of endometriosis is not completely understood, but there seems to be an aberrant immunoreactivity induced by the disease, resulting in an increase in the cytokines, angiogenic substances, and growth factors levels. Recently, transplantation of bone marrow cells in experimental models and in clinical studies showed a possible anti-inflammatory potential of these cells.
Objectives: To study the effect of mononuclear stem cells transplantation in the treatment of surgically-induced endometriosis in an experimental model.
Methods: Endometriotic implants were surgically induced in 120 female Wistar rats.
The animals with viable endometrial implant (larger than 25 mm2) were randomically divided into 3 groups to receive an intraperitoneal injection of 0.2 cc of saline solution (C group; n = 30), a subcutaneous injection of 1 mg/kg of leuprolide (L group; n = 34), or an intraperitoneal injection of 5x106 bone marrow derived-mononuclear stem cells (SC group; n = 36). They were sacrificed after 21 days to assess the implants’ size and the tissue expression of vascular endothelial growth factor receptor (VEGF-R) and tumor necrosis factor-alpha (TNF-alpha).
Results: Treatment with leuprolide decreased the surface area of the endometriotic implant compared to the SC group and the C group. The absolute reduction in the surface area of the implant was 16.5 mm, 0mm, and 0mm (p = 0.007), respectively, and the percent reduction was 40.2%, 0%, and 0% (p = 0.001). VEGF-R expression in the endometriotic implant decreased after treatment in the L and SC groups compared to the C group (409.6 µm2 vs. 465 µm2 vs. 920.9 µm2, respectively; p = 0.021). TNF-alpha expression also reduced in the L and SC groups compared to the C group (585.7 µm2 vs. 549.3 µm2 vs. 2402.1 µm2, respectively; p < 0.001).
Conclusions: There was less expression of VEGF-R and TNF-alpha in the endometriotic implant in the group treated with bone marrow derived-mononuclear stem cells but did there was no reduction in the surface area of the lesion.
Key-words: Endometriosis; Stem cells; Vascular endothelial growth factor receptor;
Experimental endometriosis; Tumor necrosis factor alpha.
LISTA DE ABREVIATURAS E SIGLAS
cc - Centímetro cúbico cm - Centímetro
cm2 - Centímetro quadrado
d - Densidade
g - Grama
g/m3 - Grama/metro cúbico
GnRH - Hormônio liberador de gonadotrofina
h - Hora
HE - Hematoxilina-eosina kg - Kilograma
m - Metro
mg - Miligrama
mg/mL - Miligrama/mililitro mL - Mililitro
mm - Milímetro
mm2 - Milímetro quadrado µm2 - Micrômetro quadrado
PUC-PR - Pontifícia Universidade Católica do Paraná TNF alfa - Fator de necrose tumoral alfa
UI - Unidade Internacional
VEGF - Fator de crescimento vascular endotelial
VEGF-R - Receptor de fator de crescimento vascular endotelial
NOTA EXPLICATIVA
Este trabalho será apresentado sob a forma de 2 artigos publicados, seguindo as normas editoriais dos respectivos periódicos.
1. O primeiro é um artigo original, publicado na Revista do Colégio Brasileiro de Cirurgiões (ISSN:0100-6991), intitulado “Development of an experimental model of endometriosis in rats” Rev. Col. Bras. Cir., v. 36, n. 3, p. 250-255, 2009.
2. O segundo é um artigo original, publicado na European Journal of Obstetrics &
Gynecology and Reproductive Biology (ISSN: 0301-2115), intitulado “Effect of the bone marrow derived-mononuclear stem cells transplantation growth, VEGF-R and TNF-alpha expression of endometrial implants in Wistar rats” Eur J Obstet Gynecol, 2011, in press.
A introdução é breve, pois está inserida no texto dos artigos originais. Ela resume os conceitos básicos da doença, sua fisiopatologia, suas manifestações clinicas, seu diagnóstico e seu tratamento. Revisa a importância dos modelos experimentais no estudo da endometriose, assim como embasa a pesquisa atual.
O desenvolvimento de um modelo experimental para o estudo da endometriose está descrito no primeiro artigo, que foi o projeto piloto da pesquisa atual e confirmou a possibilidade de se induzir cirurgicamente implantes endometrióticos em ratas, com uma alta taxa de sucesso.
O objetivo, o desenho metodológico, os resultados e a discussão estão inseridos no segundo artigo.
As conclusões e as considerações finais estão descritas após os artigos desta dissertação.
As referências bibliográficas utilizadas na introdução estão descritas após as considerações finais.
SUMÁRIO
1 INTRODUÇÃO ... 11
1.1 FISIOPATOLOGIA DA DOENÇA ... 11
1.2 FATORES DE RISCO ... 12
1.3 MANIFESTAÇÕES CLÍNICAS ... 12
1.4 DIAGNÓSTICO E TRATAMENTO ... 13
1.5 MODELOS EXPERIMENTAIS E ENDOMETRIOSE ... 14
1.6 EMBASAMENTO TEÓRICO PARA O ESTUDO ATUAL ... 15
1.6.1 Células-tronco ... 15
1.6.2 Propriedades das células-tronco mononucleares ... 16
1.6.3 Células-tronco, endométrio e endometriose ... 16
1.6.4 Marcadores inflamatórios e endometriose ... 17
2 OBJETIVO ………... 19
2.1 OBJETIVO GERAL ……… 19
2.1 OBJETIVOS ESPECÍFICOS ……… 19
3 DEVELOPMENT OF AN EXPERIMENTAL MODEL OF ENDOMETRIOSIS IN RATS ……….. 20
3.1 ABSTRACT ……….… 22
3.2 INTRODUCTION ……… 23
3.3 METHODS ……….. 24
3.3.1 Reoperation and histological analysis ………. 27
3.4 RESULTS ……….…….. 29
3.5 DISCUSSION ………...……. 30
3.6 REFERENCES ……….………. 32
4 EFFECT OF THE BONE MARROW DERIVED-MONONUCLEAR STEM CELLS TRANSPLANTATION GROWTH, VEGF-R AND TNF-ALPHA EXPRESSION OF ENDOMETRIAL IMPLANTS IN WISTAR RATS ……… 35
4.1 ABSTRACT ………. 37
4.2 INTRODUCTION…….……… 38
4.3 MATERIALS AND METHODS ………. 38
4.3.1 Surgical procedures ………... 39
4.3.1.1 Day 0 — first surgery ………. 39
4.3.1.2 Day 21 — collection of bone marrow derived-mononuclear stem cells (in the
morning) ………. 40
4.3.1.3 Day 21 — second surgery (in the afternoon) ………. 41
4.3.1.4. Day 42 — third surgery ………. 41
4.3.2 Histopathological examination ……….. 42
4.3.3 Statistical analysis ……….. 43
4.4 RESULTS ……… 44
4.5 COMMENT ………. 47
4.5.1 Stem cells ……… 48
4.5.2 Properties of mononuclear stem cells ……… 48
4.5.3 Stem cells, endometrium and endometriosis ……... 49
4.6 CURRENT STUDY ... 50
4.7 REFERENCES ... 54
5 CONCLUSÕES ... 57
6 CONSIDERAÇÕES FINAIS E PERSPECTIVAS FUTURAS ... 58
REFERÊNCIAS ... 59
ANEXOS ... 66
APROVAÇÃO PELO COMITÊ DE ÉTICA NO USO ANIMAL ... 67
1 INTRODUÇÃO
A endometriose é uma condição crônica caracterizada pela presença de tecido endometrial (glândulas e/ou estroma) fora da cavidade uterina (D’HOOGHE et al, 2004; KENNEDY et al, 2005; FARQUHAR, 2007). Estes implantes ectópicos de endométrio são mais encontrados nos ovários, no peritônio, nos ligamentos útero- sacros e no fundo-de-saco de Douglas, mas implantes extra-pélvicos também podem estar presentes (MOUNSEY et al, 2006).
A prevalência da doença nas mulheres em idade reprodutiva é estimada em 10% (ESKENAZI e WARNER, 1997). Em mulheres com dor pélvica crônica essa prevalência pode chegar a 82% (CARTER, 1994; LAUFER et al, 1997) e naquelas submetidas a investigação por infertilidade, a 20 a 50% (MAHMOOD e TEMPLETON, 1991; ESKENAZI e WARNER, 1997).
1.1 FISIOPATOLOGIA DA DOENÇA
Várias teorias têm sido sugeridas para explicar a patogênese desta doença (SELI et al, 2003). A teoria da menstruação retrógrada, também conhecida como teoria da implantação ou de Sampson propõe que tecido endometrial viável é refluído através das tubas durante a menstruação e se implanta na superfície peritoneal ou nos órgãos pélvicos (SAMPSON, 1927). A teoria da metaplasia celômica propõe que a endometriose se desenvolve da metaplasia de células que revestem o endométrio pélvico (METZGER e HANEY, 1989). MEYER (1919) sugeriu que estímulo infeccioso, hormonal ou outro estímulo indutor qualquer poderia induzir em metaplasia, o que poderia resultar em endometriose. A teoria da indução é uma extensão da teoria da metaplasia celômica e propõe que fatores bioquímicos e imunológicos endógenos podem induzir células indiferenciadas a se diferenciar em tecido endometrial (LEVANDER e NORMANN, 1955). Nos anos 1890, VON RECKLINGHAUSEN (1896) e RUSSELL (1899) introduziram a teoria dos restos embrionários que propõe que restos celulares de origem mülleriana poderiam ser ativados e se diferenciar em endométrio na presença de um estímulo específico. Nos
anos 1920, HALBAN (1924) e SAMPSON (1925) sugeriram que a endometriose também poderia ser resultado de disseminação linfática e hematogênica (teoria de metástases linfática e vascular). Embora a fisiopatologia da endometriose seja pouco conhecida, a habilidade dos implantes endometriais sobreviverem em localizações ectópicas pode estar relacionada à imunoreatividade aberrante das próprias lesões.
Evidências têm mostrado que os mediadores do sistema imune, tais como citocinas e quimiocinas, têm papel chave na patogênese da endometriose (LEBOVIC et al, 2002).
1.2 FATORES DE RISCO
As alterações menstruais como sangramento menstrual prolongado, hipermenorréia e menarca precoce são fatores de risco para o desenvolvimento da endometriose (CRAMER et al, 1986; DARROW et al, 1993; MOEN e MAGNUS, 1993; GRODSTEIN et al, 1994; PARAZZINI et al, 1994; ESKENAZI e WARNER, 1997). O sobrepeso e tabagismo estão associados a um menor risco de endometriose (CRAMER e MISSMER, 2002).
1.3 MANIFESTAÇÕES CLÍNICAS
A endometriose geralmente surge na idade reprodutiva, quando as lesões são estimuladas pelos hormônios ovarianos. Um número significativo de mulheres com endometriose é assintomática. Naquelas mulheres sintomáticas, as manifestações dolorosas tendem a ser mais intensas no período pré-menstrual e menstrual, melhorando após a parada da menstruação. A dor pélvica, na forma de dismenorréia, é o sintoma mais comum e não se correlaciona com o grau de endometriose visível (DEMCO, 1998). Outros sintomas que podem estar presentes são dor lombar, disquezia, dor durante a micção e dispareunia. A dispareunia profunda pode ser decorrente de fibrose dos ligamentos útero-sacros, nodularidade do septo reto-vaginal, obliteração do fundo de saco e/ou retroversão uterina. Tudo
isso pode levar a uma dor lombar baixa crônica. Estes sintomas são acentuados no período menstrual. Mulheres com infiltração profunda dos ligamentos útero-sacros têm maior comprometimento da função sexual (FERRERO et al, 2005). Além disso, a endometriose está associada à infertilidade por causa de aderências que distorcem a anatomia pélvica e causam dificuldade para a liberação e captura do óvulo. No entanto, a distorção tubária não é a única causa de infertilidade, porque pacientes com endometriose parecem ter pior reserva ovariana, com pior qualidade do oócito e do embrião (KONDO et al, 2009).
1.4 DIAGNÓSTICO E TRATAMENTO
O método padrão ouro para o diagnóstico da endometriose é a visão direta da lesão endometrial utilizando a cirurgia laparoscópica, seguida de confirmação histológica da presença de pelo menos dois dos seguintes achados: macrófagos contendo hemossiderina ou epitélio, glândulas ou estroma endometrial (MOUNSEY et al, 2006).
Os tratamentos clínicos padrões para pacientes com endometriose e sintomas dolorosos incluem analgésicos (anti-inflamatórios não esteróides ou acetaminofeno), anticoncepcionais orais, agentes androgênicos (danazol), progestágenos (acetato de medroxiprogesterona), análogos do GnRH (leuprolide, goserelina, triptorelina, nafarelina) e antiprogestágenos (gestrinona) (KENNEDY et al, 2005; MOUNSEY et al, 2006). Aparentemente, todos os tratamentos hormonais são igualmente efetivos em termos de alívio da dor, mas seus efeitos adversos e custos diferem significativamente (KENNEDY et al, 2005).
Embora os tratamentos medicamentosos tenham um papel substancial na melhora da dor nas pacientes com endometriose, o seu efeito é temporário (VERCELLINI et al, 2009). As mulheres muito sintomáticas normalmente necessitam de tratamento cirúrgico para completar o tratamento clínico (KONDO et al, 2011a;
KONDO et al, 2011c). As evidências sugerem que a excisão completa da endometriose oferece um bom alívio sintomático a longo-prazo, especialmente em mulheres com sintomas severos ou debilitantes. Para garantir a remoção completa da doença e para obter os melhores resultados em termos de qualidade de vida,
procedimentos complexos muitas vezes devem ser realizados, incluindo tratamento do ureter (passagem de cateter duplo J, ureterólise, ressecção ureteral segmentar ou reimplante ureteral), cirurgia retal (shaving retal, ressecção em disco ou ressecção segmentar e anastomose colo-retal), ressecção de fórnice vaginal posterior e ressecção de bexiga (KONDO et al, 2011a; KONDO et al, 2011c;
KONDO et al, 2011d). Esses procedimentos cirúrgicos estão associados a uma alta taxa de complicações intra e pós-operatórias (KONDO et al, 2011a), algumas das quais piora a qualidade de vida das pacientes (KONDO et al, 2011a; VALETON et al, 2010) e prejudica o futuro reprodutivo das mesmas (KONDO et al, 2011b).
Tendo em vista a complexidade e os riscos relacionados ao tratamento cirúrgico da endometriose, é de extrema importância a pesquisa de um tratamento clínico mais efetivo e durarouro do que os existentes atualmente.
1.5 MODELOS EXPERIMENTAIS E ENDOMETRIOSE
Um bom modelo experimental animal é necessário para a pesquisa, com o intuito de elucidação do mecanismo da doença e desenvolvimento de novas drogas terapêuticas. No entanto, a endometriose ocorre espontaneamente apenas em primatas, que são muito onerosos para uso experimental (HIRATA et al, 2005).
Os modelos experimentais de endometriose têm sido criados cirurgicamente em pequenos animais, tais como coelhos, ratos e camundongos (SCHENKEN e ASCH, 1980; ZAMAH et al, 1984; BERGQVIST et al, 1985; BRUNER et al, 1997;
VERNON e WILSON, 1985; SOMIGLIANA et al, 1999). Estes modelos são classificados em dois tipos: homólogos e heterólogos. Nos modelos homólogos, o endométrio é obtido do útero do animal e suturado ou disperso na cavidade peritoneal. No modelo heterólogo, fragmentos endometriais de humanos são injetados em ratas imunodeficientes por via intraperitoneal ou subcutânea. Em ambos os modelos, lesões endometriose-símiles, identificadas histologicamente, são formadas nos animais. O modelo murino proposto por JONES (1984) para a indução de endometriose é o mais amplamente utilizado em trabalhos experimentais, uma vez que a técnica cirúrgica é simples e a maioria dos implantes é viável; ou seja, a indução da endometriose é efetiva e reprodutível (SCHOR et al, 1999). Trata-se do
auto-transplante de um segmento uterino com a face endometrial voltada para a cavidade peritoneal. Recentemente, aplicamos esta técnica proposta por JONES (1984) em ratas e obtivemos implantes endometrióticos viáveis em 76,9% dos animais (AMARAL et al, 2009).
Embora o uso de não primatas seja uma vantagem com relação ao seu relativo baixo custo e sua habilidade de estabelecer lesões endometriose-símile, esses modelos têm desvantagens. As comparações são dificultadas pelo grande número de técnicas experimentais utilizadas nesses modelos e pelo grande intervalo filogenético entre os não primatas e os humanos. Mais importante, os não primatas não têm ciclos menstruais e não desenvolvem endometriose espontânea. A rata ovula espontaneamente, mas a fase lútea é mais curta que nos humanos (STORY e KENNEDY, 2004). Apesar dessas diferenças, os modelos experimentais são de extrema importância para o estudo da doença e a avaliação de novas modalidades terapêuticas.
1.6 EMBASAMENTO TEÓRICO PARA O ESTUDO ATUAL
1.6.1 Células-tronco
As células-tronco são células indiferenciadas que podem se replicar sem se diferenciar (habilidade de auto-renovação) e que, sob condições específicas, podem se diferenciar em vários tipos celulares especializados (WEISSMAN, 2000;
WAGERS e WEISSMAN, 2004; MENG et al, 2007). As células-tronco podem ser divididas em embrionárias e adultas. Aquelas isoladas da massa celular interna do embrião ou das células germinativas primordiais do feto são consideradas totipotentes, uma vez que têm a habilidade de colonizar um organismo por completo e dar origem a todos os tipos celulares. As células-tronco presentes nos organismos adultos e pós-natais são referidas como células-tronco adultas órgão ou tecido- específicas, e se encontram na maioria dos órgãos adultos. Elas são consideradas pluripotentes, uma vez que podem originar tipos celulares maduros e uma ou mais
linhagens celulares, mas não podem reconstituir um organismo como um todo (SHI et al, 2009).
As células-tronco pluripotentes humanas da medula óssea podem se diferenciar em células mononucleares (indiferenciadas) ou multinucleadas (FRIEDENSTEIN et al, 1968). As células-tronco mononucleares englobam as mesenquimais e as hematopoiéticas.
As células-tronco adultas têm sido isoladas da medula óssea, placenta, tecido adiposo, sangue do cordão umbilical, fluido amniótico e fígado, e parecem ser uma grande promessa como terapias biológicas para uma ampla gama de necessidades médicas ainda não descobertas (LEON et al, 2001).
1.6.2 Propriedades das células-tronco mononucleares
Vários estudos têm proposto que os benefícios funcionais observados após o transplante de células-tronco mononucleares derivadas da medula óssea em modelos animais de lesão cardíaca podem estar relacionados à secreção de fatores solúveis que, agindo de uma maneira autócrina nas células-tronco administradas ou de uma maneira parácrina nas células hospedeiras adjacentes, protegem o coração, atenuam o remodelamento ventricular patológico, induzem neovascularização e promovem reparo (KINNAIRD et al, 2004). Alguns autores ainda acreditam que as células-tronco mononucleares derivadas da medula óssea apresentam um efeito anti-inflamatório, identificado em modelos animais de doença de Chagas (SOARES et al, 2004). O racional para a administração da fração completa de células-tronco mononucleares derivadas da medula óssea é que qualquer efeito terapêutico depende do balanço entre os múltiplos tipos celulares, e que usando um grande painel de células precursoras, nenhum tipo celular com benefício em potencial seria omitido durante a terapia (LEBLOND et al, 2009).
1.6.3 Células-tronco, endométrio e endometriose
Especula-se que as células-tronco fetais possam persistir no útero adulto para substituir o epitélio e o estroma glandular que descama em cada ciclo menstrual (SASSON e TAYLOR, 2008). No entanto, estas células multipotentes no endométrio podem surgir de outra fonte e estudos recentes têm sugerido que a medula óssea pode ser a outra fonte de células-tronco endometriais. É possível que as células- tronco derivadas da medula óssea migrem para os tecidos danificados e que sejam responsáveis pela angiogênese e transdiferenciação em células do novo tecido (FIGUEIRA et al, 2011).
Em 2004, TAYLOR forneceu evidência de que existe regeneração endometrial após transplante de medula óssea. Células epiteliais e estromais endometriais derivadas dos doadores foram detectadas em amostras endometriais dos receptores, sugerindo que as células-tronco derivadas da medula óssea contribuíram para a repopulação do endométrio nos receptores.
Em um estudo experimental realizado por DU e TAYLOR (2007) utilizando camundongos fêmeas recebendo transplante de medula óssea de doadores machos, foi observado que células genotipicamente derivadas de machos se incorporaram ao endométrio. De forma semelhante, mulheres que receberam transplante de medula óssea incompatível mostraram fenótipo similar ao dos doadores (TAYLOR, 2004). Estes achados sugerem, portanto, que as células-tronco derivadas da medula óssea podem contribuir tanto para a homeostase do tecido normal e a patogênese da endometriose.
1.6.4 Marcadores inflamatórios e endometriose
O ambiente peritoneal de mulheres com endometriose é repleto de citocinas, substâncias angiogênicas e fatores de crescimento (HARADA et al, 2001) que são produzidos principalmente pelas células peritoneais imunes e pelas células de endometriose (KYAMA et al, 2003).
O fator de necrose tumoral alfa (TNF alfa) é uma citocina chave em uma variedade de processos inflamatórios e se acredita que tenha um papel na patogênese da endometriose (LEBOVIC et al, 2002). Vários investigadores têm demonstrado que as concentrações de TNF alfa estão elevadas no líquido peritoneal
de mulheres com endometriose (BULLIMORE, 2003; RICHTER et al, 2005;
GOGACZ et al, 2008) e que seu nível se correlaciona com o estádio da doença (EISERMANN et al, 1988). Além disso, vários artigos têm revisado o papel do fator de crescimento vascular endotelial (VEGF) na neovascularização de novos implantes de endometriose (TAYLOR et al, 1997; DONNEZ et al, 1998; LEBOVIC et al, 1999).
Esta correlação existente entre endometriose e aumento de citocinas abre a possibilidade para a investigação de novas abordagens terapêuticas da doença em modelos experimentais, particularmente com o uso de tratamentos anti-citocinas ou anti-inflamatórios.
Relatos preliminares usando células-tronco em camundongos chagásicos crônicos (SOARES et al, 2004) e em pacientes com doenças auto-imunes (EL- BADRI et al, 2000; MOORE et al, 2001; TRAYNOR et al, 2002; KIM et al, 2002) demonstraram um possível potencial anti-inflamatório dessas células. Talvez essas propriedades possam ser benéficas em mulheres com endometriose, o que desperta o interesse do estudo dessa linhagem celular em modelos experimentais de endometriose.
2 OBJETIVO
2.1 OBJETIVO GERAL
• Avaliação do efeito da terapia com células-tronco mononucleares derivadas da medula óssea em um modelo experimental de endometriose em ratas Wistar.
2.2 OBJETIVOS ESPECÍFICOS
• Avaliação semiquantitativa da persistência de células epiteliais nos implantes endometrióticos;
• Avaliação da alteração na área de superfície dos implantes endometrióticos após o tratamento;
• Avaliação da expressão do fator de necrose tumoral alfa (TNF alfa) nos implantes endometrióticos após o tratamento;
• Avaliação da expressão do receptor de fator de crescimento vascular endotelial (VEGF-R) nos implantes endometrióticos após o tratamento.
3 DEVELOPMENT OF AN EXPERIMENTAL MODEL OF ENDOMETRIOSIS IN RATS
Artigo original publicado na Revista do Colégio Brasileiro de Cirurgiões – Rev. Col.
Bras. Cir., v. 36, n. 3, p. 250-255, 2009.
ISSN:0100-6991
DEVELOPMENT OF AN EXPERIMENTAL MODEL OF ENDOMETRIOSIS IN RATS
Vivian Ferreira do AmaralI; Eduardo Andreazza Dal LagoII; William Kondo, ACBC- PRIII; Luiz César Guarita-SouzaIV; Júlio César FranciscoV
Study conducted for the Graduate Program in Health Sciences of the Pontifícia Universidade Católica do Paraná (PUCPR), Curitiba, PR, Brazil.
I Associate Professor of Gynecology and in the Graduate Program in Health Sciences, Pontifícia Universidade Católica do Paraná (PUCPR)
II Medical student, PUCPR
III Master's student in Health Sciences, PUC-PR – BR
IV Professor of Operative Technique and in the Graduate Program in Health Sciences, PUCPR-BR
V PhD student in Biotechnology, Universidade Federal do Paraná – BR
3.1 ABSTRACT
OBJCTIVE: To develop an experimental model of endometriosis in rats.
METHODS: Thirty adult female Wistar rats were used. The surgical technique consisted of median laparotomy with identification of the bicornuate uterus and resection of a 2-cm segment of the right uterine horn. A 0.25 cm2 flap was removed from that structure and sutured to the abdominal wall with the endometrial side facing the peritoneal cavity. The rats were randomly divided into two groups according to the reoperation date: group 1 (n=15) was reoperated in 30 days, and group 2 (n=15), in 60 days. On the occasion of the second laparotomy, the implants were evaluated macroscopically, resected and referred for microscopic analysis with hematoxylin- eosin and immunohistochemical staining (HEMA, AE1 and AE2).
RESULTS: The implants developed in 83.3% of group 1 and 71.4% of group 2. There was no statistically significant difference between the weights of the animals in the two groups. No statistically significant difference was found in the surface area of the induced lesions: in group 1, the mean was 0.37 cm2 and in group 2, 0.25 cm2. According to Keenan's semiquantitative histological classification (based on the preservation status of the epithelial layer of the endometrium), the mean for group 1 was 1.9 and for group 2, 2.4.
CONCLUSION: The technique used for inducing the development of endometriosis in rats was satisfactory.
Key words: Endometriosis. Rats. Models, animal. Endometrium.
3.2 INTRODUCTION
Endometriosis is a chronic condition characterized by the presence of endometrial tissue (glands and/or stroma) outside the uterine cavity1. These ectopic deposits of endometrium are more commonly found in the ovaries, peritoneum, uterosacral ligaments and rectovaginal pouch, but extrapelvic implants may also be present2.
The prevalence in women of reproductive age is estimated to be 10%3. In women presenting with chronic pelvic pain, that prevalence may reach up to 82%4,5, and in those undergoing investigation for infertility, between 20% and 50%3,6-8.
Several theories have been proposed to explain the pathogenesis of this disease9. Among them, the most relevant are the retrograde menstruation theory (implantation theory or Sampson's theory)10, the celomic metaplasia theory11-13 and the embryonic remnants theory14,15. However, none of them sufficed to elucidate the etiopathogeny of the disease. There is a current trend to associate those theories with each other and with aspects such as immunological, hormonal, genetic and environmental factors.
Endometriosis usually appears in the reproductive years, when the lesions are stimulated by ovarian hormones. A significant number of women remains asymptomatic. In those who are symptomatic, the events of pain tend to be more intense in the premenstrual period, improving after cessation of menstruation. Pelvic pain as dysmenorrhea is the most common symptom, and does not correlate with the visually assessed degree of endometriosis16 nor with the depth of tissue infiltration17,18. Other symptoms that may be present are lumbar pain, dyschezia, pain during micturition and dyspareunia. The latter, when deep, may be the result of fibrosis in the uterosacral ligaments, nodularity of the rectovaginal septum, obliteration of the pouch and/or uterine retroversion. In addition, endometriosis is associated with infertility as a result of adhesions that distort the pelvic anatomy and impair ovum release and capture. However, tubal distortion is not the only cause of infertility, since endometriosis patients seem to have worse ovarian reserve with lower oocyte and embryo quality2.
The gold standard for the diagnosis of endometriosis is direct visualization of the endometrial lesions via laparoscopy, accompanied by histological confirmation of
the presence of at least two of the following findings: macrophages containing hemosiderin or endometrial epithelium, glands, or stroma19.
The ideal treatment for this disease is yet to be discovered20. The available options are based on drugs, surgery and on the association of both. The therapeutic modality to be adopted is dependent on factors such as the stage of the disease, symptomatology, the patient's age and the desire for a future pregnancy.
The fact that endometriosis requires an invasive method for diagnosis hampers or even prevents the undertaking of controlled studies on the response of endometriosis implants to the various drugs21. Therefore, a good animal experimental model is required in order to elucidate the disease mechanism and test new therapeutic drugs. However, endometriosis occurs spontaneously only in primates, which are too costly for experimental use22. Consequently, experimental models have been surgically produced in small animals such as rabbits, rats and mice23-28. The murine model proposed by Jones29 in 1984 is the most widely employed, since the operative technique is simple and most implants are successful, that is, induction of endometriosis is effective and reproducible21.
The objective of this study is the development of an experimental model of endometriosis in rats according to the technique proposed by Jones29.
3.3 METHODS
The study was previously approved by Opinion 80/07 of the Animal Research Ethics Committee of the Pontifícia Universidade Católica do Paraná (PUC-PR) and followed the guidelines of the Colégio Brasileiro de Experimentação Animal (Brazilian College of Animal Experimentation, COBEA).
Female Wistar rats (Rattus norvegicus albinus), adult and virgin, were kept in appropriate cages with five animals each, under controlled temperature, humidity and environment luminosity. They received water and chow ad libitum. After a three-day acclimation period, the surgical procedure for induction of endometriosis was performed.
Over the period from December 2007 through January 2008, 30 female rats were operated on at the Operative Technique and Experimental Surgery Laboratory
of PUCPR. A preoperative fast was established for the 12 hours preceding the operation.
Prior to anesthesia, the animals were weighed for anesthetic dose calculation.
Anesthesia was achieved with an intraperitoneal injection of 0.2 mL/100 g body weight of the combination of 1 mL ketamine (50mg/mL) and 1 mL xylazine (20 mg/mL).
Following anesthesia, electric hair clippers were used to remove the hair from the abdominal wall of the animals. The rats were then tied to the surgery board in supine, with the limbs in abduction, and routine antisepsis was performed. The operation was initiated with a 3-cm median incision 2 cm above the pubis.
The procedure for inducing the development of an endometriotic lesion was carried out based on a published study29. The bicornuate uterus was identified and the blood vessels of the left uterine horn were ligated with 3-0 vicryl (Figures 1 and 2). A 2-cm segment of the middle-third of the left uterine horn was then resected.
This segment was immersed in saline 0.9% at 4º C for approximately two minutes, and then incised longitudinally, originating a flap, from which a 5 x 5 mm (0.25 cm2) section was removed. This section was sutured to the abdominal wall on the right flank of the rat using two simple 6-0 mononylon stitches, next to a blood vessel, and in such a way that the endometrial side was always facing the abdominal cavity (Figure 3). After ensuring hemostasis in the left uterine horn and the cleaning of the abdominal cavity, the abdominal wall was closed in layers. The musculoaponeurotic layer was sutured with continuous 3-0 vicryl suture and the skin with continuous 3-0 mononylon.
After the operation, the animals were taken to the PUC-PR breeding colony, where they stayed until reoperation date.
Figure 1 - Typical rat bicornuate uterus.
Figure 2 - Resection of a segment of the uterine horn.
Figure 3 - Endometrium implant on the abdominal wall.
3.3.1 Reoperation and histological analysis
Reoperation was performed one month (Group 1) and two months (Group 2) after the first operation in order to confirm the growth of the lesion macroscopically and to collect samples for histological analysis. After opening the abdominal wall, the lesions were identified and measurements were taken. Subsequently, those implants were resected (Figure 4), fixed in 10% formalin and processed for paraffin embedding and histological analysis at the Experimental Pathology Laboratory of PUC-PR.
Figure 4 - Endometrium implant in group 2.
The slides were stained with HE (hematoxylin-eosin) and analyzed under a light microscope (Olympus; Melville, NY) to confirm the presence of endometrial tissue. The manual tissue microarray technique was performed using the paraffin blocks. The prepared slides were analyzed by light microscopy to locate regions with the presence of endometrium, which were marked with a pen. Through the mirror system, the marked slide was used to pinpoint the region in the paraffin block, which was marked as well. A 2-mm skin biopsy punch was used to extract the cores from the paraffin block. In order to assist in locating the material, a map was made. After the removal of the sections, histological slides were prepared from all blocks using the markers HEMA, AE1 and AE2.
The persistence of epithelial cells in the implants was evaluated according to the classification proposed by Keenan30, as follows: well-preserved epithelial layer = score 3, moderately preserved epithelium with leukocyte infiltrate = score 2, poorly- preserved epithelium (occasional epithelial cells only) = score 1, no epithelium = score zero.
The results obtained in the study were expressed as means, medians, minimum and maximum values, and standard deviation, or as frequencies and percentages. Student's t test for independent samples or the non-parametric Mann- Whitney test were used, as appropriate, for the inter-group comparison regarding the quantitative variables. The groups were compared through Fisher's exact test for the dichotomous nominal variables. Values of p<0.05 indicated statistical significance.
3.4 RESULTS
Out of the 30 rats operated on, 4 died in the period between the first and the second operation: three in Group 1 and one in Group 2. Upon reoperation, the implantation sites of endometrial tissue were evaluated macroscopically. In four animals, it was not possible to identify that tissue (two animals in each group). In addition, an abscess was found at the implant site in two rats of Group 2. These two animals were counted as not having developed the lesion. The overall success rate was 76.9% (20/26) – 83.3% in group 1 (10/12) and 71.4% in Group 2 (10/14).
There was no statistically significant difference between the mean weights of the animals in groups 1 and 2 (271.6 g and 260 g, respectively; p = 0.147).
No statistically significant difference was found between the groups regarding the variables length, width and surface area of the surgically-induced endometriosis lesions (Table 1).
All implants were submitted to histological analysis; there was no difference between the two groups (Figure 5).
Figure 5 - Photomicrograph of a well-preserved epithelial cell (animal from Group 2).
3.5 DISCUSSION
The experimental models of endometriosis surgically produced in small animals are classified into two types: homologous and heterologous. In the homologous model, the endometrium is obtained from the animal's uterus and sutured or dispersed in the peritoneal cavity. In the heterologous model, human endometrial explants are injected intraperitoneally or subcutaneously into immunocompromised rats. In both cases, endometriosis-like lesions, histologically identified, are induced in the animals.
Although using models in non-primates is advantageous because they can develop endometriosis-like lesions and for the low cost, these models have various disadvantages. Comparisons are made difficult by the large number of experimental techniques used and by the wide phylogenetic gap between non-primates and humans. More importantly, non-primates have no menstrual cycles and do not develop spontaneous endometriosis. The rat ovulates spontaneously, but the luteal phase is shorter than in humans. The endometriotic lesions produced in rats are different from those in humans, as the former consist of cysts containing a clear serous fluid with no evidence of neoangiogenesis31. In spite of these differences, experimental models are very important for the study of the disease and the testing of new therapeutic modalities.
The choice for the experimental model with rats was based on the low cost, previous background from the literature and the resistance of these animals to infections. The technique described by Jones in 198429 in a murine model is the most widely used in the world literature, since the surgical procedure is simple and most implants develop successfully, i.e., endometriosis induction is effective and reproducible21,23. It is possible, therefore, to standardize implant size, which is indispensable to evaluate the efficacy of new therapeutic drugs.
In the present study, 4 out of the 30 rats submitted to the procedure were excluded, since they died between the first and the second operations, leaving 26 animals for the final analysis. The success rate in the macroscopic development of endometriosis lesions was 76.9% (20 out of 26). Sometimes the lesions are not clear enough to be distinguished from the adjacent normal tissue. This makes it difficult to determine the surface area and weight of the lesions, essential variables in the experiment32.
The main parameter analyzed in the present study was the surface area of the implants. Contrary to most studies, which show stability or growth of the lesions in the interval between the second and third operations22, regression of the implant area (0.37 cm2 to 0.25 cm2) was found, yet with no statistically significant difference.
According to the anatomopathological classification proposed by Keenan30, eight animals had a score of zero. Conceptually, the lesions in those animals could not be regarded as endometriosis, as the disease is characterized by the presence of glandular and/or stromal tissue outside the uterine cavity. However, if the animals with a score of zero were included, the mean of scores would be 1.3 and 1.4 in Groups 1 and 2, respectively, which is comparable to the mean of the control group scores in Keenan's study30.
Although the expression "experimental endometriosis" is used in the literature, strictly speaking, the present study involved fragments of normal endometrium on the abdominal wall of healthy rats. The actual correlation of these foci of endometrial tissue with human endometriosis is unknown33. Nevertheless, the widespread utilization of research protocols with experimental endometriosis in animal models is justified in that a thorough evaluation of the implants in humans is not feasible, since the diagnostic methods are invasive (videolaparoscopy or laparotomy).
The development of experimental endometriosis in rats according to Jones's model was satisfactory.
3.6 REFERENCES
1. Farquhar C. Endometriosis. BMJ. 2007; 334(7587):249-53.
2. Mounsey AL, Wilgus A, Slawson DC. Diagnosis and management of endometriosis. Am Fam Physician. 2006; 74(4):594-600.
3. Eskenazi B, Warner ML. Epidemiology of endometriosis. Obstet Gynecol Clin North Am. 1997; 24(2):235-58.
4. Laufer MR, Goitein L, Bush M, Cramer DW, Emans SJ. Prevalence of endometriosis in adolescent girls with chronic pelvic pain not responding to conventional therapy. J Pediatr Adolesc Gynecol. 1997; 10(4):199-202.
5. Carter JE. Combined hysteroscopic and laparoscopic findings in patients with chronic pelvic pain. J Am Assoc Gynecol Laparosc. 1994; 2(1):43-7.
6. Mahmood TA, Templeton A. Prevalence and genesis of endometriosis. Hum Reprod. 1991; 6(4):544-9.
7. Strathy JH, Molgaard CA, Coulam CB, Melton LJ 3rd. Endometriosis and infertility:
a laparoscopic study of endometriosis among fertile and infertile women. Fertil Steril.
1982; 38(6):667-72.
8. Verkauf BS. Incidence, symptoms, and signs of endometriosis in fertile and infertile women. J Fla Med Assoc. 1987; 74(9):671-5.
9. Seli E, Berkkanoglu M, Arici A. Pathogenesis of endometriosis. Obstet Gynecol Clin North Am. 2003; 30(1):41-61.
10. Sampson JA. Peritoneal endometriosis due to the menstrual dissemination of endometrial tissue into the peritoneal cavity. Am J Obstet Gynecol.1927; 14(4):422- 69.
11. Metzger DA, Haney AF. Etiology of endometriosis. Obstet Gynecol Clin North Am. 1989; 16(1):1-14.
12. Ridley JH. The histogenesis of endometriosis: a review of facts and fancies.
Obstet Gynecol Surv. 1968; 23(1):1-35.
13. Gardner G, Greene RR, Ranney B. The histogenesis of endometriosis; recent contributions. Obstet Gynecol. 1953; 1(6):615-37.
14. Von Recklinghausen F. Adenomyomas and cystadenomas of the wall of the uterus and tube their origin as remnants of the wolffian body. Wien Klin Wochenschr.
1896; 8:530.
15. Russell WW. Aberrant portions of the müllerian duct found in an ovary: ovarian cysts of müllerian origin. Bull John Hopkins Hospital. 1899; 10:8-10.
16. Demco L. Mapping the source and character of pain due to endometriosis by patient-assisted laparoscopy. J Am Assoc Gynecol Laparosc. 1998; 5(3):241-5.
17. Koninckx PR, Martin DC. Deep endometriosis: a consequence of infiltration or retraction or possibly adenomyosis externa? Fertil Steril. 1992; 58(5):924-8.
18. Koninckx PR, Oosterlynck D, D’Hooghe T, Meuleman C. Deeply infiltrating endometriosis is a disease whereas mild endometriosis could be considered a non- disease. Ann N Y Acad Sci. 1994; 734:333-41.
19. ACOG Committee on Practice Bulletins-Gynecology. ACOG practice bulletin.
Medical management of endometriosis. Number 11, December 1999 (replaces Technical Bulletin Number 184, September 1993). Clinical management guidelines for obstetrician-gynecologists. Int J Gynaecol Obstet. 2000; 71(2):183-
96.
20. Amaral VF, Sanson LT, Sepulcri RP. Novas perspectivas no tratamento da endometriose pélvica. Femina. 2006; 34(3):189-96.
21. Schor E, Freitas V, Soares Jr. JM, Simões MJ, Baracat ED. Endometriose:
modelo experimental em ratas. Rev Bras Ginecol Obstet. 1999; 21(5):281-4.
22. Hirata T, Osuga Y, Yoshino O, Hirota Y, Harada M, Takemura Y et al.
Development of an experimental model of endometriosis using mice that ubiquitously express green fluorescent protein. Hum Reprod. 2005; 20(8):2092-6.
23. Schenken RS, Asch RH. Surgical induction of endometriosis in the rabbit: effects on fertility and concentrations of peritoneal fluid prostaglandins. Fertil Steril. 1980;
34(6):581-7.
24. Zamah NM, Dodson MG, Stephens LC, Buttram VC Jr, Besch PK, Kaufman RH.
Transplantation of normal and ectopic human endometrial tissue into athymic nude mice. Am J Obstet Gynecol. 1984; 149(6):591-7.
25. Bergqvist A, Jeppsson S, Kullander S, Ljungberg O. Human endometrium transplanted into nude mice. Histologic effects of various steroid hormones. Am J Pathol. 1985; 119(2):336-44.
26. Vernon MW, Wilson EA. Studies on the surgical induction of endometriosis in the rat. Fertil Steril. 1985; 44(5):684-94.
27. Somigliana E, Viganò P, Rossi G, Carinelli S, Vignali M, Panina-Bordignon P.
Endometrial ability to implant in ectopic sites can be prevented by interleukin-12 in a murine model of endometriosis. Hum Reprod. 1999; 14(12):2944-50.
28. Bruner KL, Matrisian LM, Rodgers WH, Gorstein F, Osteen KG. Suppression of matrix metalloproteinases inhibits establishment of ectopic lesions by human endometrium in nude mice. J Clin Invest. 1997; 99(12):2851-7.
29. Jones RC. The effect of a luteinizing hormone releasing hormone (LRH) agonist (Wy-40,972), levonorgestrel, danazol and ovariectomy on experimental endometriosis in the rat. Acta Endocrinol. 1984; 106(2):282-8.
30. Keenan JA, Williams-Boyce PK, Massey PJ, Chen TT, Caudle MR, Bukovsky A.
Regression of endometrial explants in a rat model of endometriosis treated with the immune modulators loxoribine and levamisole. Fertil Steril. 1999; 72(1):135-41.
31. Story L, Kennedy S. Animal studies in endometriosis: a review. ILAR J. 2004;
45(2):132-8.
32. Nogueira Neto J, Torres OJM, Borges MOR, Coelho TM, Nascimento AGPAC, Nunes Jr JNN, Esteves GL. Modificações do volume e da histologia de focos de endometriose em ratas tratadas com sinvastatina. Rev Bras Ginecol Obstet. 2007;
29(8):396-402.
33. Rosa e Silva, JC. Desenvolvimento de um modelo experimental de endometriose em coelha [dissertação]. Ribeirão Preto (SP): Universidade de São Paulo; 2004.
Correspondence address:
William Kondo
E-mail: williamkondo@yahoo.com Received in 12/10/2008
Accepted for publication in 12/12/2008 Conflict of interest: None
Financial source: None
4 EFFECT OF THE BONE MARROW DERIVED-MONONUCLEAR STEM CELLS TRANSPLANTATION GROWTH, VEGF-R AND TNF-ALPHA EXPRESSION OF
ENDOMETRIAL IMPLANTS IN WISTAR RATS
Artigo original publicado na European Journal of Obstetrics & Gynecology and Reproductive Biology
ISSN: 0301-2115
EFFECT OF THE BONE MARROW DERIVED-MONONUCLEAR STEM CELLS TRANSPLANTATION GROWTH, VEGF-R AND TNF-ALPHA EXPRESSION OF
ENDOMETRIAL IMPLANTS IN WISTAR RATS
William Kondo*, Eduardo Andreazza dal Lago, Julio Cesar Francisco, Rossana Baggio Simeoni, Lucia de Noronha, Ana Paula Camargo Martins, Marina Luise Viola
de Azevedo, Carolina Chaves Ferreira, Priscila Maestrelli, Marcia Olandoski, Luiz Cesar Guarita-Souza, Vivian Ferreira do Amaral
Department Center for Health and Biological Sciences, Pontifical Catholic University of Parana (PUC-PR), Brazil
4.1 ABSTRACT
Objective: To study the effect of bone marrow derived-mononuclear stem cells transplantation in the growth, VEGF-R and TNF-alpha expression of surgically induced endometriosis in an experimental model.
Study design: This is an experimental study conducted in the Center for Health and Biological Sciences at the Pontifical Catholic University of Parana, Brazil.
Endometriotic implants were surgically induced in 120 female Wistar rats. The animals with viable endometrial implant (larger than 25 mm2) were randomically divided into 3 groups to receive an intraperitoneal injection of 0.2 cc of saline solution (C group; n = 30), a subcutaneous injection of 1 mg/kg of leuprolide (L group; n = 34), or an intraperitoneal injection of 5 x 106 bone marrow derived-mononuclear stem cells (SC group; n = 36). They were sacrificed after 21 days to assess the implants’
size and the tissue expression of vascular endothelial growth factor receptor (VEGF- R) and tumor necrosis factor-alpha (TNF-alpha).
Results: Treatment with leuprolide decreased the surface area of the endometriotic implant compared to the SC group and the C group. The absolute reduction in the surface area of the implant was 16.5 mm, 0mm, and 0mm (p = 0.007), respectively, and the percent reduction was 40.2%, 0%, and 0% (p = 0.001). VEGF-R expression in the endometriotic implant decreased after treatment in the L and SC groups compared to the C group (409.6 µm2 vs. 465 µm2 vs. 920.9 µm2, respectively; p = 0.021). TNF-alpha expression also reduced in the L and SC groups compared to the C group (585.7 µm2 vs. 549.3 µm2 vs. 2402.1 µm2, respectively; p < 0.001).
Conclusion: Bone marrow derived-mononuclear stem cells transplantation decreased the expression of VEGF-R and TNF-alpha in the endometriotic implant but did not reduce the surface area of the lesion.
Keywords: Endometriosis; Stem cells; Vascular endothelial growth factor receptor;
Experimental endometriosis; Tumor necrosis factor alpha
4.2 INTRODUCTION
Endometriosis is an important benign gynecologic disease that is pathologically defined as the presence of endometrial tissue, consisting of glandular epithelium and/or stroma, outside the uterine cavity. This condition is predominantly found in women of reproductive age and affects approximately 7–10% of all women, 71–87% of women with chronic pelvic pain and 38% of all infertile women [1].
Although the physiopathology of endometriosis is understood incompletely, the ability of endometrial implants to survive in ectopic locations may be related to aberrant immunoreactivity elicited by the lesions themselves [2]. The peritoneal environment of women with endometriosis contains a rich cocktail of cytokines, angiogenic substances, and growth factors [3] that are produced mainly by the peritoneal immune cells and endometriotic cells.
Tumor necrosis factor alpha (TNF-alpha) is a key cytokine in a variety of inflammatory processes, and it is likely to play a role in the pathogenesis of endometriosis [2]. Numerous investigators have shown that TNF-alpha concentrations are elevated in the peritoneal fluid of patients with endometriosis and that their levels correlate with the stage of the disease [4]. Also, several articles have reviewed the role of vascular endothelial growth factor (VEGF) in the neovascularization of nascent endometriotic implants [5].
This suggested link between endometriosis and increased cytokines opens the possibility of novel approaches to its medical treatment, particularly in the use of anti- cytokines or anti-inflammatory treatments.
Preliminary reports using stem cells in chronic chagasic mice [6] and in patients with autoimmune diseases [7,8] demonstrate a possible anti-inflammatory potential of these cells. Maybe these properties could be beneficial in women with endometriosis. The aim of this article is to study the effect of bone marrow derived- mononuclear stem cells transplantation in the treatment of surgically induced endometriosis in an experimental model.
4.3 MATERIALS AND METHODS
One hundred and twenty mature female Wistar-Albino rats (200–250 g) were used for the experiment. The animals were provided by the Animal Reproduction Center of the Pontifical Catholic University of Parana (PUC-PR), Brazil, and housed in the Animal Laboratory at the same University.
4.3.1 Surgical procedures
4.3.1.1 Day 0 — first surgery
The animals were anesthetized with an intraperitoneal administration of 50 mg/kg ketamine hydrochloric acid and 7 mg/kg xylazine hydrochloric acid.
Endometriosis was surgically induced using the method described by Jones [9] in 1984, and reproduced by us [10] (Fig. 1A–D). After surgery, all rats were observed for 21 days.
Fig. 1. (A) Using the aseptic technique, a ventral midline incision was made to expose the reproductive organs. (B) The left uterine horn was ligated at both the uterotubal junction and the cervical end using 3-0 vicryl and removed. A 10 mm segment of the excised horn was cut
and placed in sterile isotonic saline. (C) The endometrium was separated from the
myometrium and trimmed to 5 mm x 5 mm (25 mm2). (D) The trimmed section of the endometrium was then transplanted into the right side of the peritoneal cavity with the epithelial lining of the segment apposed to the ventrolateral body wall adjacent to a large
vessel using sterile 6-0 polypropylene. The midline abdominal incision was closed with 3-0 vicryl (muscle and aponeurosis) and 3-0 mononylon (skin) using continuous sutures.
(E and F) Measurement of the surface area of the endometriotic implant in the second and in the third surgeries.
4.3.1.2 Day 21 — collection of bone marrow derived-mononuclear stem cells (in the morning)
In the morning of the second surgery, the rats were randomly allocated into three groups:
• C group (control group);
• L group (leuprolide group): positive control group;
• SC group (bone marrow derived-mononuclear stem cells group).
Each rat was identified by a tattoo with pre-established numbers. The rats allocated into the SC group underwent an additional procedure to obtain these cells in the morning of the 21st day. Approximately 5 h before the second operation and with the rat under anesthesia following the same protocol described above, bone marrow was collected by the aspiration puncture technique. The procedure was performed in the Laboratory of Experimental Surgery of the PUC-PR. The animals were placed in lateral decubitus position with the superior leg flexed at the knee and the inferior leg extended. The bone marrow was collected by aspiration puncture at the posterior iliac crest using a 5 mL disposable syringe (BD-Plastipak®) containing 0.2 mL of heparin heparinized (5.000 UI/mL), with a 25 x 8 21 mm G1 (BD-Precision Glide®) needle. The volume of bone marrow obtained was around 1 mL. All syringes were identified according to the identification of each rat [11]. The Ficoll-Hypaque density gradient medium (d = 1.077 g/m3) (Ficoll-Hypaque, Sigma, St. Loius, MO) was used for the isolation of the bone marrow-derived mononuclear stem cells according to Böyum [12], in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco BRL) supplemented with culture medium containing 1% antibiotic (Penicillin-
Streptomicyn) and 20% fetal bovine serum (Gibco BRL, Grand Island, NY) [11,12].
The number of cells was counted using Neubauer chamber.
4.3.1.3 Day 21 — second surgery (in the afternoon)
Each rat was anesthetized and a midventral laparotomy was performed to determine the attachment and viability of endometrial implant. The surface area of the implant was measured (length x width) in millimeters and recorded (Area 2) (Fig.
1E and F). The endometrial implant was considered viable when it was larger than 25 mm2, meaning that it had grown macroscopically from the first surgery until the second procedure.
Rats from the C group received an intraperitoneal injection of 0.2 cc of saline solution (sterile 0.9% salt water) next to the implant under direct view, during the laparotomy. Rats from the L group were treated with GnRH agonist (GnRHa) with a single subcutaneous injection of leuprolide acetate depot formulation (1 mg/kg body weight; Lucrin; Abbott, Brazil), administered right after the closure of the laparotomy.
The leuprolide acetate dose was based on a previous study in which 1 mg/kg was found to be optimal for female rats [13]. Rats from the SC group received an intraperitoneal injection of a 0.2 cc solution containing 5 x 106 bone marrowderived- mononuclear stem cells next to the implant under direct view, during the laparotomy.
The number of cells was based on previous animal studies [14].
After treatment, the laparotomy was closed and all rats were observed for 21 days.
4.3.1.4. Day 42 — third surgery
Three weeks after the second surgery, the third laparotomy was performed.
The effect of the treatment was evaluated by measuring the surface area of the implant (Area 3) (Fig. 1E and F). The implants were then excised and fixed in 10%
formalin for histopathological examination in the Laboratory of Experimental
Pathology of the PUC-PR. After the procedure, the rats were euthanized by ketamine anaesthesia.
4.3.2 Histopathological examination
The pathologist assessing the samples was blinded to the treatment groups.
The formalin-fixed endometriotic foci were embedded in paraffin blocks, sectioned at 5 mm thickness (four sections per sample), stained with hematoxylin and eosin and examined under a light microscope. The persistence of epithelial cells in endometrial implants was evaluated semiquantitatively [15] (Fig. 2).
Fig. 2. Semiquantitative evaluation of the endometriotic implant (10x magnification): (A) Score 0 = no epithelium. (B) Score 1 = a poorly preserved epithelium (occasional epithelial cells only). (C) Score 2 =
a moderately preserved epithelium with leukocyte infiltrate. (D) Score 3 = a well-preserved epithelial layer.
The paraffin blocks were used to construct tissue microarrays [16]. The endometriotic lesion was identified under a light microscope and a 4-mm punch of the cystic wall was performed to remove tissue cores. These tissue cores were then inserted in a recipient paraffin block in a precisely spaced, array pattern. Sections from the tissue microarrays blocks were cut for immunohistochemical analysis (immunoperoxidase staining). The protocol for the immunoperoxidase assay was standardized with positive and negative controls. Each microscope slide contained 15 samples from different animals. The tissue expression of vascular endothelial growth factor receptor (VEGF-R) was assessed using a commercially available enzyme- linked immunosorbent assay kit (rabbit anti-human VEGF receptor 1, Sinapse Biotecnologia Ltda, São Paulo, SP, Brazil) as well as the tissue expression of tumor necrosis factor-alpha (TNF-alpha) (mouse anti-human TNF-alpha, Sinapse Biotecnologia Ltda, São Paulo, SP, Brazil).
The tissue expression of VEGF-R and TNF-alpha was counted using a digital morphometric evaluation. Images were digitalized using a color digital video camera (TV 0.45x Nikon lens, Tokyo, Japan) adapted to a microscope (Nikon Eclipse E600, Tokyo, Japan). The images were analyzed using the Image Pro Program (Media Cybernetics), so that the tissue expression of both cytokines was counted and integrated by area.
4.3.3 Statistical analysis
The statistical analysis was performed using STATISTICA version 8.0.
Statistical analysis was performed in order to compare the three groups using Kruskal–Wallis and chi-square tests, when necessary. Kruskal–Wallis nonparametric test was followed by post hoc analysis for multiple comparisons. If the overall chisquare test was significant, Fisher’s exact test was used for multiple comparisons.
p values of <0.05 were considered statistically significant. Variables were expressed as median (range).
The three groups were compared according to the following variables:
• Absolute reduction of the endometriotic implant, calculated by subtracting the surface area in the third surgery (post-treatment) from the surface area in the second surgery. A positive result indicated a reduction in the surface area and a negative result indicated an augmentation of the surface area.
• Percent reduction of the endometriotic implant, calculated by dividing the absolute reduction of the surface area by the surface area in the second surgery. The result was multiplied by 100. A positive result indicated a reduction in the surface area and a negative result indicated an augmentation of the surface area.
• Tissue expression of VEGF-R.
• Tissue expression of TNF-alpha.
Absolute and percent reductions of the surface area of the implant were considered for comparisons instead of the absolute area in the third surgery (Area 3) because the implants could have different growth patterns from the first surgery (5 mm x 5 mm) until the second procedure (Area 2). So, comparing only the absolute areas in the third surgery could lead us to an important bias.
4.4 RESULTS
In all rats the surface area of the implant was 25 mm2 (5 mm x 5 mm) in the first surgery. Surface areas of the implants were measured during the second (Area 2) and the third (Area 3) surgeries, and are described in Table 1. Four rats died in the period between the first and the second surgeries. Sixteen endometrial implants did not grow from the first until the second procedure and were excluded from the analysis. One hundred rats presented macroscopically viable implants in the second surgical procedure (C group = 30 rats, SC group = 36 rats, and L group = 34 rats).
They were found to be well vascularized and attached to the abdominal wall peritoneum at the second laparotomy. The median surface area of the implant in the second surgery was 51.5 mm2, 42 mm2 and 42 mm2 for the C group, SC group and L group, respectively (p = 0.035). Post hoc analysis for multiple comparisons
demonstrated that the surface area in the C group was statistically bigger than the SC group (p = 0.009).
In the third surgery (after treatment) (Table 1), the median surface area of the implant was 48 mm2, 35mm2 and 25 mm2 for the C group, SC group and L group, respectively (p < 0.001). The median surface area of the implant in the C group was larger than the SC group (48 mm2 vs. 35 mm2; p = 0.027) and the L group (48 mm2 vs. 25 mm2; p < 0.001). The median surface area of the implant in the SC group was bigger than the L group (35 mm2 vs. 25 mm2; p = 0.027).
Table 1. Surface area of the endometriotic implant in the second and third surgical procedures.
Area 2 (mm2) (range) Area 3 (mm2) (range) C group 51.5 (28-196) 48 (6-204)
L group 42 (28-120) 25 (2-100) SC group 42 (28-120) 35 (12-168)
p value 0.035 <0.001
Semiquantitative analysis of the persistence of epithelial cells in the endometrial implant is demonstrated in Table 2. Complete regression of epithelial cells (score 0) and poor epithelial preservation (score 1) were more common in the C and L group compared with the SC group (p = 0.017 and p = 0.032, respectively).
Table 2. Semiquantitative evaluation of the persistence of epithelial cells in endometrial implants according to Keenan et al [15].
Scores 0 and 1 Scores 2 and 3 Total C group 3 + 5 = 8 (26.7%) 7 + 15 = 22 (73.3%) 30 L group 4 + 4 = 8 (23.5%) 1 + 25 = 26 (76.5%) 34 SC group 2 + 0 = 2 (5.6%) 2 + 32 = 34 (94.4%) 36 p value = 0.049 (Chi-square test)
The implants of the stem cells-treated animals often retained normal epithelial morphology. There was no difference in the semiquantitative evaluation of the persistence of endometrial epithelial cells in the implants comparing leuprolide- treated rats with control rats (p = 0.77).
