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  <front>
    <journal-meta>
      <journal-id journal-id-type="publisher-id">abc</journal-id>
      <journal-title-group>
        <journal-title>Arquivos Brasileiros de Cardiologia</journal-title>
        <abbrev-journal-title abbrev-type="publisher">Arq. Bras. Cardiol.</abbrev-journal-title>
      </journal-title-group>
      <issn pub-type="ppub">0066-782X</issn>
      <issn pub-type="epub">1678-4170</issn>
      <publisher>
        <publisher-name>Sociedade Brasileira de Cardiologia - SBC</publisher-name>
        <publisher-loc>S&#227;o Paulo, SP, Brazil</publisher-loc>
      </publisher>
    </journal-meta>
    <article-meta>
      <article-id pub-id-type="publisher-id" specific-use="scielo-v3">FsQDwL7kPkRPdqPcXqsfffj</article-id>
      <article-id pub-id-type="other">00002</article-id>
      <article-id pub-id-type="publisher-id" specific-use="scielo-v2">S0066-782X2008000600002</article-id>
      <article-id pub-id-type="publisher-id" specific-use="scielo-v1">S0066-782X(08)09000602</article-id>
      <article-id pub-id-type="doi">10.1590/S0066-782X2008000600002</article-id>
      <article-categories>
        <subj-group subj-group-type="heading">
          <subject>Artigos Originais</subject>
        </subj-group>
      </article-categories>
      <title-group>
        <article-title>Nova t&#233;cnica cir&#250;rgica de preparo da veia safena para revasculariza&#231;&#227;o do mioc&#225;rdio sem manipula&#231;&#227;o direta - no-touch</article-title>
      </title-group>
      <contrib-group>
        <contrib contrib-type="author">
          <name>
            <surname>Rueda</surname>
            <given-names>Fabio de</given-names>
          </name>
          <xref ref-type="aff" rid="aff01"/>
        </contrib>
        <contrib contrib-type="author">
          <name>
            <surname>Souza</surname>
            <given-names>Domingos</given-names>
          </name>
          <xref ref-type="aff" rid="aff02"/>
        </contrib>
        <contrib contrib-type="author">
          <name>
            <surname>Lima</surname>
            <given-names>Ricardo de Carvalho</given-names>
          </name>
          <xref ref-type="aff" rid="aff01"/>
        </contrib>
        <contrib contrib-type="author">
          <name>
            <surname>Menezes</surname>
            <given-names>Alexandre</given-names>
          </name>
          <xref ref-type="aff" rid="aff01"/>
        </contrib>
        <contrib contrib-type="author">
          <name>
            <surname>Johansson</surname>
            <given-names>Benny</given-names>
          </name>
          <xref ref-type="aff" rid="aff02"/>
        </contrib>
        <contrib contrib-type="author">
          <name>
            <surname>Dashwood</surname>
            <given-names>Michael</given-names>
          </name>
          <xref ref-type="aff" rid="aff03"/>
        </contrib>
        <contrib contrib-type="author">
          <name>
            <surname>Th&#233;</surname>
            <given-names>Emmanuel</given-names>
          </name>
          <xref ref-type="aff" rid="aff01"/>
        </contrib>
        <contrib contrib-type="author">
          <name>
            <surname>Gesteira</surname>
            <given-names>M&#225;rio</given-names>
          </name>
          <xref ref-type="aff" rid="aff01"/>
        </contrib>
        <contrib contrib-type="author">
          <name>
            <surname>Escobar</surname>
            <given-names>Mozart</given-names>
          </name>
          <xref ref-type="aff" rid="aff01"/>
        </contrib>
        <contrib contrib-type="author">
          <name>
            <surname>Vasconcelos</surname>
            <given-names>Frederico</given-names>
          </name>
          <xref ref-type="aff" rid="aff01"/>
        </contrib>
      </contrib-group>
      <aff id="aff03">
        <addr-line>
          <named-content content-type="city">Londres</named-content>
          <named-content content-type="state">Reino Unido</named-content>
        </addr-line>
        <institution content-type="orgname">University College Medical School</institution>
        <institution content-type="orgdiv1">Royal Free Hospital </institution>
        <institution content-type="orgdiv2">Divis&#227;o de Patologia Molecular e Bioqu&#237;mica</institution>
      </aff>
      <aff id="aff01">
        <addr-line>
          <named-content content-type="city">Recife</named-content>
          <named-content content-type="state"/>
        </addr-line>
        <institution content-type="orgname">Universidade de Pernambuco</institution>
        <institution content-type="orgdiv1">Hospital Oswaldo Cruz </institution>
        <institution content-type="orgdiv2">Divis&#227;o de Cirurgia Cardiotor&#225;cica</institution>
        <country country="BR">Brazil</country>
      </aff>
      <aff id="aff02">
        <addr-line>
          <named-content content-type="state"/>
        </addr-line>
        <institution content-type="orgname">&#214;rebro University Hospital</institution>
        <institution content-type="orgdiv1">Divis&#227;o de Cirurgia Cardiotor&#225;cica </institution>
        <country country="SE">Sweden</country>
      </aff>
      <pub-date date-type="collection" publication-format="electronic">
        <month>06</month>
        <year>2008</year>
      </pub-date>
      <volume>90</volume>
      <issue>6</issue>
      <fpage>388</fpage>
      <lpage>395</lpage>
      <history>
        <date date-type="rev-recd">
          <day>05</day>
          <month>09</month>
          <year>2007</year>
        </date>
        <date date-type="received">
          <day>14</day>
          <month>02</month>
          <year>2007</year>
        </date>
        <date date-type="accepted">
          <day>16</day>
          <month>10</month>
          <year>2007</year>
        </date>
      </history>
      <permissions>
        <license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by-nc/4.0/" xml:lang="en">
          <license-p>This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.</license-p>
        </license>
      </permissions>
      <abstract>
        <p>FUNDAMENTO: Otimiza&#231;&#227;o da veia safena na revasculariza&#231;&#227;o mioc&#225;rdica. OBJETIVO: Apresentar a t&#233;cnica no-touch de preparo da veia safena. Essa t&#233;cnica consiste na retirada da veia safena do seu leito, com um ped&#237;culo de tecido adiposo, protegendo-a contra espasmos, sendo desnecess&#225;rio distend&#234;-la. M&#201;TODOS: Estudo prospectivo e randomizado, incluindo 156 pacientes submetidos a cirurgia de revasculariza&#231;&#227;o mioc&#225;rdica. Compara&#231;&#227;o da t&#233;cnica no-touch com duas outras t&#233;cnicas: convencional e intermedi&#225;ria. Procedeu-se &#224; avalia&#231;&#227;o da morfologia endotelial, utilizando a microscopia. A perviabilidade das pontes foi determinada com exame angiogr&#225;fico num per&#237;odo m&#233;dio de 18 meses ap&#243;s a opera&#231;&#227;o. A enzima &#243;xido n&#237;trico sintetase endotelial (eNOS) foi identificada por meio do estudo imunohistoqu&#237;mico. RESULTADOS: A avalia&#231;&#227;o morfol&#243;gica mostrou integridade endotelial de 97% nas veias do grupo no-touch; enquanto quase metade da superf&#237;cie endotelial das veias tratadas pelas outras t&#233;cnicas exibiu aus&#234;ncia de c&#233;lulas endoteliais. A angiografia revelou perviabilidade de 95,4% para as pontes do grupo no-touch, 88,9 e 86,2% para as pontes do grupo convencional e intermedi&#225;ria, respectivamente. O estudo imunohistoqu&#237;mico revelou a presen&#231;a da eNOS nas tr&#234;s camadas que comp&#245;em a parede da veia no grupo no-touch e redu&#231;&#227;o dessa enzima no grupo convencional. CONCLUS&#195;O: A integridade endotelial e a atividade da eNOS foram melhor preservadas com o uso da t&#233;cnica no-touch. A prote&#231;&#227;o mec&#226;nica fornecida pelo tecido gorduroso circundante &#224; veia e a atividade vasodilatadora e bloqueadora da agrega&#231;&#227;o plaquet&#225;ria causada pelo &#243;xido n&#237;trico podem ser respons&#225;veis pela prote&#231;&#227;o da veia contra o espasmo, como tamb&#233;m por sua alta perviabilidade imediata.</p>
      </abstract>
      <kwd-group xml:lang="pt" kwd-group-type="author-generated">
        <kwd>Veia safena</kwd>
        <kwd>endot&#233;lio vascular</kwd>
        <kwd>revasculariza&#231;&#227;o mioc&#225;rdica</kwd>
      </kwd-group>
      <pub-date publication-format="electronic" date-type="pub">
        <day>26</day>
        <month>06</month>
        <year>2008</year>
      </pub-date>
    </article-meta>
  </front>
  <body>
    <p>ARTIGO ORIGINAL </p>
    <p> CIRURGIA CARD&#205;ACA - ADULTOS</p>
    <p>Nova t&#233;cnica cir&#250;rgica de preparo da veia safena para revasculariza&#231;&#227;o do mioc&#225;rdio sem manipula&#231;&#227;o direta &#150; <italic><bold>no-touch</bold></italic></p>
    <p>
      <bold>Novel <italic>no-touch</italic> technique of harvesting the saphenous vein for coronary artery bypass grafting</bold>
    </p>
    <p>
      <bold>Fabio de Rueda<sup>I</sup>; Domingos Souza<sup>II</sup>; Ricardo de Carvalho Lima<sup>I</sup>; Alexandre Menezes<sup>I</sup>; Benny Johansson<sup>II</sup>; Michael Dashwood<sup>III</sup>; Emmanuel Th&#233;<sup>I</sup>; M&#225;rio Gesteira<sup>I</sup>; Mozart Escobar<sup>I</sup>; Frederico Vasconcelos<sup>I</sup></bold>
    </p>
    <p><sup>I</sup>Divis&#227;o de Cirurgia Cardiotor&#225;cica, Hospital Oswaldo Cruz, Universidade de Pernambuco, Recife </p>
    <p><sup>II</sup>Brasil; Divis&#227;o de Cirurgia Cardiotor&#225;cica, University Hospital of &#214;rebro, Su&#233;cia </p>
    <p><sup>III</sup>Divis&#227;o de Patologia Molecular e Bioqu&#237;mica, Royal Free Hospital &amp; University College Medical School, Londres, Reino Unido</p>
    <p>
      <xref ref-type="corresp" rid="end">Correspond&#234;ncia</xref>
    </p>
    <p>
      <bold>RESUMO</bold>
    </p>
    <p><bold>FUNDAMENTO:</bold> Otimiza&#231;&#227;o da veia safena na revasculariza&#231;&#227;o mioc&#225;rdica. </p>
    <p><bold>OBJETIVO:</bold> Apresentar a t&#233;cnica <italic>no-touch</italic> de preparo da veia safena. Essa t&#233;cnica consiste na retirada da veia safena do seu leito, com um ped&#237;culo de tecido adiposo, protegendo-a contra espasmos, sendo desnecess&#225;rio distend&#234;-la. </p>
    <p><bold>M&#201;TODOS:</bold> Estudo prospectivo e randomizado, incluindo 156 pacientes submetidos a cirurgia de revasculariza&#231;&#227;o mioc&#225;rdica. Compara&#231;&#227;o da t&#233;cnica <italic>no-touch</italic> com duas outras t&#233;cnicas: convencional e intermedi&#225;ria. Procedeu-se &#224; avalia&#231;&#227;o da morfologia endotelial, utilizando a microscopia. A perviabilidade das pontes foi determinada com exame angiogr&#225;fico num per&#237;odo m&#233;dio de 18 meses ap&#243;s a opera&#231;&#227;o. A enzima &#243;xido n&#237;trico sintetase endotelial (eNOS) foi identificada por meio do estudo imunohistoqu&#237;mico. </p>
    <p><bold>RESULTADOS:</bold> A avalia&#231;&#227;o morfol&#243;gica mostrou integridade endotelial de 97% nas veias do grupo <italic>no-touch</italic>; enquanto quase metade da superf&#237;cie endotelial das veias tratadas pelas outras t&#233;cnicas exibiu aus&#234;ncia de c&#233;lulas endoteliais. A angiografia revelou perviabilidade de 95,4% para as pontes do grupo <italic>no-touch</italic>, 88,9 e 86,2% para as pontes do grupo convencional e intermedi&#225;ria, respectivamente. O estudo imunohistoqu&#237;mico revelou a presen&#231;a da eNOS nas tr&#234;s camadas que comp&#245;em a parede da veia no grupo<italic> no-touch</italic> e redu&#231;&#227;o dessa enzima no grupo convencional. </p>
    <p><bold>CONCLUS&#195;O:</bold> A integridade endotelial e a atividade da eNOS foram melhor preservadas com o uso da t&#233;cnica <italic>no-touch</italic>. A prote&#231;&#227;o mec&#226;nica fornecida pelo tecido gorduroso circundante &#224; veia e a atividade vasodilatadora e bloqueadora da agrega&#231;&#227;o plaquet&#225;ria causada pelo &#243;xido n&#237;trico podem ser respons&#225;veis pela prote&#231;&#227;o da veia contra o espasmo, como tamb&#233;m por sua alta perviabilidade imediata. </p>
    <p><bold>Palavras-chave:</bold> Veia safena; endot&#233;lio vascular; revasculariza&#231;&#227;o mioc&#225;rdica.</p>
    <p>
      <bold>SUMMARY</bold>
    </p>
    <p><bold>BACKGROUND: </bold>Optimization of the saphenous vein for myocardial revascularization. </p>
    <p><bold>OBJECTIVE: </bold>To present the no-touch technique of the saphenous vein preparation. This technique consists of harvesting the vein with a pedicle of surrounding tissue, which protects the vein from spasms, obviating the need for distension. </p>
    <p><bold>METHODS: </bold>A prospective, randomized study with 156 patients who underwent artery bypass grafting was performed comparing three saphenous vein harvesting techniques: conventional, intermediate, and no-touch. A morphological study of the endothelium was carried out using scanning microscopy. An angiographic assessment of the vein graft patency was performed at a mean follow-up time of 18 months. Also, an immunohistochemical assessment was carried out to identify the endothelial enzyme nitric oxide synthase (eNOS) in the vein wall </p>
    <p><bold>RESULTS: </bold>The preservation of the endothelial cell integrity was greater in the no-touch technique than in the other procedures. At angiographic follow-up, the patency for the no-touch group was 95.4%, 88.9% for the grafts of the conventional technique group, and 86.2% for the grafts performed in the intermediate technique group. The immunohistochemical assessment revealed eNOS in all three layers of the vein wall in the no-touch group and reduction of this enzyme in the conventional group. </p>
    <p><bold>CONCLUSION: </bold>The endothelial integrity and eNOS activity were better preserved when using the no-touch technique for vein graft harvesting. The mechanical protection provided by the cushion of surrounding tissue in the no-touch group, the vasorelaxation and thromboresistant activities of nitric oxide may be responsible for the reduction of vasospasms and improved patency rate. </p>
    <p><bold>Key words: </bold>Saphenous vein; endothelium, vascular; myocardial revascularization.</p>
    <p>
      <bold>Introdu&#231;&#227;o</bold>
    </p>
    <p>A doen&#231;a coronariana ateroscler&#243;tica &#233; a principal causa de morbi-mortalidade em pa&#237;ses industrializados. Anualmente, mais de um milh&#227;o de pessoas morrem por doen&#231;a coronariana ateroscler&#243;tica no mundo<sup>1</sup>. A revasculariza&#231;&#227;o mioc&#225;rdica por meio de enxertos de veia safena (VS), iniciada na d&#233;cada de 60, alterou profundamente o perfil da doen&#231;a coronariana isqu&#234;mica, modificando a hist&#243;ria natural da mesma, com melhora na qualidade de vida. Por&#233;m, a falha do enxerto proporciona angina recorrente<sup>2</sup>, sendo uma das muitas raz&#245;es de reopera&#231;&#245;es. Tem sido demonstrado que 30% dos enxertos de safena ocluem no primeiro ano p&#243;s-revasculariza&#231;&#227;o<sup>3</sup> e, em 10 anos, essas cifras ultrapassam 50%.</p>
    <p>Recentemente, o emprego de enxertos arteriais tem dado melhores resultados em longo prazo, apesar disso, o enxerto de safena continua sendo amplamente utilizado na cirurgia de revasculariza&#231;&#227;o do mioc&#225;rdio (CRM)<sup>4,5</sup>. Conseq&#252;entemente, algumas medidas v&#234;m sendo tomadas para aumentar a pat&#234;ncia em longo prazo de enxertos de VS. A prepara&#231;&#227;o da VS desempenha um papel muito importante, uma vez que a manipula&#231;&#227;o inadequada da VS causa danos morfol&#243;gicos e funcionais ao enxerto<sup>4</sup>.</p>
    <p>O objetivo deste trabalho &#233; avaliar a t&#233;cnica no-touch (dissec&#231;&#227;o da VS com m&#237;nima manipula&#231;&#227;o, mantendo tecido adiposo circundante e sem distens&#227;o do enxerto)<sup>6</sup>, incluindo a import&#226;ncia cl&#237;nica na prepara&#231;&#227;o da VS.</p>
    <p>
      <bold>M&#233;todos</bold>
    </p>
    <p>
      <bold>Sele&#231;&#227;o de pacientes</bold>
    </p>
    <p>Seguindo a aprova&#231;&#227;o do comit&#234; de &#233;tica e com autoriza&#231;&#227;o por escrito, 156 pacientes foram submetidos a CRM na Divis&#227;o de Cirurgia Cardiotor&#225;cica do Hospital Universit&#225;rio de &#214;rebro, Su&#233;cia. Os pacientes foram alocados aleatoriamente em tr&#234;s grupos em fun&#231;&#227;o das diferentes t&#233;cnicas de prepara&#231;&#227;o da VS. O crit&#233;rio de inclus&#227;o dos pacientes selecionados objetivou a possibilidade de controle em longo prazo. As opera&#231;&#245;es foram realizadas no per&#237;odo de junho de 1993 a abril de 1997.</p>
    <p>
      <bold>T&#233;cnicas de prepara&#231;&#227;o da veia safena</bold>
    </p>
    <p><italic>Grupo C (t&#233;cnica convencional)</italic> - A VS foi obtida por meio de incis&#227;o longitudinal na perna. A camada advent&#237;cia foi retirada, e os ramos foram ligados com fio de algod&#227;o 3-0. A veia foi removida do leito imediatamente ap&#243;s a dissec&#231;&#227;o e distendida com solu&#231;&#227;o salina injetada com aux&#237;lio de uma seringa, mantendo-se uma press&#227;o de 300 mmHg durante 1 minuto. Ap&#243;s o procedimento, a VS foi armazenada em solu&#231;&#227;o salina na temperatura ambiente da sala.</p>
    <p><italic>Grupo I (t&#233;cnica intermedi&#225;ria)</italic> - A VS foi dissecada como no grupo C. Inicialmente, n&#227;o foi distendida, sendo submetida aos seguintes procedimentos: deixada <italic>in situ</italic> e coberta com uma compressa embebida de solu&#231;&#227;o salina contendo papaverina (1 mg/ml), mantendo-se a perfus&#227;o da VS. Ap&#243;s a remo&#231;&#227;o, a VS foi armazenada em sangue obtido da c&#226;nula introduzida na aorta. Mesmo com o uso da papaverina, todas as veias precisaram ser distendidas devido a espasmos.</p>
    <p><italic>Grupo NT (t&#233;cnica no-touch)</italic> - A VS foi exposta por incis&#227;o cont&#237;nua na perna. Utilizo-se fio de algod&#227;o 3-0 para ligar todas as colaterais vis&#237;veis aproximadamente a 0,5 cm da parede da safena. Subseq&#252;entemente, a VS foi isolada do leito e deixada <italic>in situ</italic> juntamente com o ped&#237;culo de gordura, mantendo-se a sua perfus&#227;o nativa e cobrindo-a com uma compressa embebida em solu&#231;&#227;o salina pura. Ap&#243;s ser retirada do leito da perna, a VS foi armazenada em sangue obtido da c&#226;nula arterial (<xref ref-type="fig" rid="fig01">Figura 1</xref>).</p>
    <fig id="fig01">
      <graphic xlink:href="1678-4170-abc-90-06-0388-00002-gf01.jpg"/></fig>
    <p>Em rela&#231;&#227;o &#224; forma de preparo, na t&#233;cnica convencional, a VS foi desnudada e distendida; na t&#233;cnica intermedi&#225;ria, a VS foi desnudada, mas n&#227;o foi distendida; e, na t&#233;cnica <italic>no-touch</italic>, a veia n&#227;o foi desnudada, nem distendida.</p>
    <p>
      <bold>Aspectos cir&#250;rgicos</bold>
    </p>
    <p>A utiliza&#231;&#227;o de circula&#231;&#227;o extracorp&#243;rea (CEC) e de cardioplegia a 4&#186;C foi rotineira. O di&#226;metro das art&#233;rias coron&#225;rias foi mensurado com aux&#237;lio de sonda espec&#237;fica. Em todos os grupos, a anastomose distal foi a primeira a ser confeccionada.</p>
    <p>Para verificar poss&#237;veis sangramentos da anastomose e de ramos colaterais nos grupos NT e I, a por&#231;&#227;o proximal da VS foi conectada &#224; c&#226;nula arterial da CEC rapidamente. No grupo C, a VS foi testada manualmente, usando uma seringa com solu&#231;&#227;o salina acoplada a um transdutor de press&#227;o. Todas as art&#233;rias tor&#225;cicas internas (ATI) foram anastomosadas &#224; art&#233;ria descendente anterior (DA). A anastomose proximal foi feita ap&#243;s desclampeamento da art&#233;ria aorta. As caracter&#237;sticas da VS, sua qualidade e origem (proximal, mediana e distal) foram consideradas e registradas. O fluxo sang&#252;&#237;neo dentro dos enxertos foi mensurado por estudo ultra-sonogr&#225;fico (pesquisador de medidas de fluxo, Transonic System Inc., EUA).</p>
    <p>
      <bold>Avalia&#231;&#227;o morfol&#243;gica</bold>
    </p>
    <p>A avalia&#231;&#227;o morfol&#243;gica do endot&#233;lio foi executada com aux&#237;lio de microscopia eletr&#244;nica (ME) e microscopia &#243;ptica (MO) de segmentos de VS obtidos dos pacientes de cada grupo. O controle foi obtido da por&#231;&#227;o distal da veia imediatamente ap&#243;s a retirada. A amostra controle, tamb&#233;m denominada de amostra prim&#225;ria, n&#227;o foi dilatada nem estocada, por&#233;m, foi fixada imediatamente. Ap&#243;s terminar a anastomose proximal, outra amostra, denominada secund&#225;ria, foi obtida da veia e imediatamente fixada. A fixa&#231;&#227;o foi feita por imers&#227;o da amostra em paraformalde&#237;do a 2% e glutaralde&#237;do a 2,5% na solu&#231;&#227;o tamponada Millonig.</p>
    <p>
      <bold>Avalia&#231;&#227;o imunohistoqu&#237;mica</bold>
    </p>
    <p>Em dez outros pacientes, a por&#231;&#227;o proximal da VS foi preparada com a t&#233;cnica NT, j&#225; a parte distal foi tratada com a t&#233;cnica convencional. Ambos os segmentos forma estocados em sangue com heparina at&#233; o momento da anastomose distal. Uma amostra de excesso de VS foi obtida ap&#243;s realiza&#231;&#227;o da anastomose proximal, e uma amostra controle foi obtida de veia desnudada que n&#227;o sofreu dilata&#231;&#227;o.</p>
    <p>Os m&#233;todos imunohistoqu&#237;micos cl&#225;ssicos foram utilizados para identificar o endot&#233;lio e o col&#225;geno. As amostras de VS foram fixadas em acetona, e a inativa&#231;&#227;o s&#233;rica de enzimas ativas endoteliais foi realizada em ordem para bloquear poss&#237;veis altera&#231;&#245;es na colora&#231;&#227;o. A seguir, o tecido foi incubado, juntamente com anticorpos CD31, para identificar as c&#233;lulas endoteliais e, com anticorpos monoclonais de ratos, para identificar o col&#225;geno. Uma solu&#231;&#227;o de 3,3'-tetrahidrocloreto de diaminobenzidina foi empregada como substrato. A sec&#231;&#227;o foi mantida com hematoxilina Mayer e preparada para exame microsc&#243;pico. Uma an&#225;lise com NADPH-diaforase (Sigma- Aldrich, Poole, Dorset, Reino Unido) foi realizada para localizar a enzima &#243;xido n&#237;trico sintetase endotelial (eNOS), que &#233; precursora do &#243;xido n&#237;trico.</p>
    <p>Uma auto-radiografia <italic>in vitro</italic> com nitroarginina (NOARG), marcada com um is&#243;topo radioativo, foi utilizada para localizar os s&#237;tios da eNOS no endot&#233;lio. As l&#226;minas foram pr&#233;-incubadas em solu&#231;&#227;o tamponada de HCl e, posteriormente, incubadas em uma solu&#231;&#227;o contendo 10 nM de NOARG radioativo. Ap&#243;s incuba&#231;&#227;o, foram enxugadas e secas ao ar. A seguir, as l&#226;minas foram p&#243;s-fixadas em paraformalde&#237;do, mergulhadas em uma emuls&#227;o nuclear e mantidas dessa forma durante toda a noite. Finalmente, as l&#226;minas foram acondicionadas em caixas &#224; prova de luz e armazenadas a 4&#186;C por 12 semanas. Ap&#243;s esse procedimento, os tecidos foram preparados com hematoxilina-eosina Mayer e examinados ao microsc&#243;pio sob uma ilumina&#231;&#227;o negra com campo de vis&#227;o brilhante.</p>
    <p>Este estudo foi realizado dessa forma para evitar poss&#237;veis suspeitas de que tiv&#233;ssemos utilizado as melhores veias para a t&#233;cnica NT. Nossa inten&#231;&#227;o foi verificar os efeitos imunohistoqu&#237;micos em dois segmentos da mesma VS, tratados com duas t&#233;cnicas diferentes.</p>
    <p>
      <bold>Avalia&#231;&#227;o angiogr&#225;fica</bold>
    </p>
    <p>Aproximadamente 18 meses ap&#243;s a cirurgia, foi realizado um controle angiogr&#225;fico de acordo com a t&#233;cnica de Judkins<sup>7</sup>. Vispaque, uma subst&#226;ncia de baixa osmolaridade, foi utilizada como contraste (Nycomed Amersham AB, Estocolmo, Su&#233;cia). A avalia&#231;&#227;o angiogr&#225;fica foi realizada por dois radiologistas aleatoriamente. O enxerto foi visibilizado em duas proje&#231;&#245;es. A raz&#227;o de pat&#234;ncia e o grau de les&#245;es localizadas e difusas foram determinados. A oclus&#227;o foi identificada por localiza&#231;&#227;o do pesco&#231;o remanescente do enxerto ap&#243;s inje&#231;&#227;o seletiva de contraste dentro da anastomose proximal e pelo tipo de opacifica&#231;&#227;o dos enxertos ap&#243;s a inje&#231;&#227;o de 50 ml de contraste na aorta ascendente.</p>
    <p>
      <bold>M&#233;todo estat&#237;stico</bold>
    </p>
    <p>O teste n&#227;o-pareado Kruskal-Wallis foi utilizado em an&#225;lise retrospectiva para otimizar uma compara&#231;&#227;o histol&#243;gica do endot&#233;lio entre os tr&#234;s grupos, como descrito por Siegel e Castellan<sup>8</sup>.</p>
    <p>A pat&#234;ncia dos enxertos foi avaliada com o m&#233;todo de regress&#227;o l&#243;gica. O fator explanat&#243;rio m&#233;dio foi os tr&#234;s diferentes m&#233;todos de prepara&#231;&#227;o das veias: convencional, intermedi&#225;rio e <italic>no-touch</italic>. Fatores adicionais foram avaliados individualmente. Os fatores que apresentaram uma associa&#231;&#227;o significativa nos resultados e que tiveram distribui&#231;&#227;o irregular entre as tr&#234;s diferentes t&#233;cnicas foram selecionados e utilizados no modelo final.</p>
    <p>
      <bold>Resultados</bold>
    </p>
    <p>
      <bold>Caracter&#237;sticas dos pacientes</bold>
    </p>
    <p>N&#227;o houve diferen&#231;a significativa com rela&#231;&#227;o &#224;s caracter&#237;sticas dos pacientes entre os grupos (<xref ref-type="table" rid="tab01">Tabela 1</xref>). Aqueles que estavam sendo tratados devido a altas taxas de colesterol ou que tiveram valores de colesterol acima do normal (2,6-5,0 &#181;mol/l) foram considerados como portadores de dislipidemia. Em rela&#231;&#227;o &#224; press&#227;o arterial, a hipertens&#227;o foi definida como press&#227;o diast&#243;lica acima de 90 mmHg e/ou press&#227;o sist&#243;lica acima de 140 mmHg. A fun&#231;&#227;o ventricular foi determinada pela fra&#231;&#227;o de eje&#231;&#227;o. </p>
    <table-wrap id="tab01">
      <graphic xlink:href="1678-4170-abc-90-06-0388-00002-gt01.jpg"/></table-wrap>
    <p>O uso de aspirina foi iniciado no primeiro dia p&#243;s-operat&#243;rio com uma dose de 160 mg/dia. Durante o per&#237;odo de exames angiogr&#225;ficos, 42 pacientes do grupo C receberam uma dose de 160 mg/dia, tr&#234;s receberam 75 mg/dia e um paciente n&#227;o recebeu nenhum agente antiagregante plaquet&#225;rio. No grupo I, 35 pacientes receberam 160 mg/dia de aspirina, quatro receberam 75 mg/dia e dois n&#227;o fizeram uso de nenhuma droga antiplaquet&#225;ria. No grupo NT, 35 pacientes receberam 160 mg/dia, sete pacientes receberam 75 mg/dia e tr&#234;s n&#227;o receberam nenhum agente antiplaquet&#225;rio.</p>
    <p>
      <bold>Achados morfol&#243;gicos</bold>
    </p>
    <p>As amostras secund&#225;rias do grupo NT apresentavam integridade celular semelhante ao grupo controle. Como observado, quase a metade da superf&#237;cie endotelial do grupo C n&#227;o apresentava c&#233;lulas endoteliais. As amostras do grupo I apresentavam integridade endotelial que variava entre a superf&#237;cie endotelial do grupo C e do grupo NT.</p>
    <p>Comparando as amostras dos tr&#234;s grupos, observamos uma diferen&#231;a significativa na integridade endotelial, com resultados apresentando os seguintes valores de p: controle versus amostra convencional = p &lt; 0,01; controle versus amostra intermedi&#225;ria = p &lt; 0,05; e controle versus amostra NT = p &lt; 0,05. As <xref ref-type="fig" rid="fig02">Figuras 2</xref> e <xref ref-type="fig" rid="fig03">3</xref> demonstram o grau de dano endotelial em uma amostra venosa NT e uma amostra venosa do grupo convencional.</p>
    <fig id="fig02">
      <graphic xlink:href="1678-4170-abc-90-06-0388-00002-gf02.jpg"/></fig>
    <fig id="fig03">
      <graphic xlink:href="1678-4170-abc-90-06-0388-00002-gf03.jpg"/></fig>
    <p>
      <bold>Achados imunohistoqu&#237;micos</bold>
    </p>
    <p>O estudo imunohistoqu&#237;mico para a identifica&#231;&#227;o de fibras de col&#225;geno demonstrou uma camada adventicial intacta contendo microvasos no grupo de veias NT. Esses achados n&#227;o foram encontrados nas VS tratadas com a t&#233;cnica convencional (<xref ref-type="fig" rid="fig04">Figura 4</xref>).</p>
    <fig id="fig04">
      <graphic xlink:href="1678-4170-abc-90-06-0388-00002-gf04.jpg"/></fig>
    <p>A eNOS foi identificada por estudo auto-radiogr&#225;fico, atrav&#233;s de incuba&#231;&#227;o dos tecidos em uma solu&#231;&#227;o contendo o marcador NOARG. Notamos a presen&#231;a de uma depend&#234;ncia direta entre a liga&#231;&#227;o do NOARG no l&#250;men do vaso e a propor&#231;&#227;o de &#225;rea intacta do endot&#233;lio, o que revelou que as veias do grupo NT mantiveram a estrutura morfol&#243;gica preservada. A liga&#231;&#227;o mostrou-se reduzida nos enxertos do grupo C (<xref ref-type="fig" rid="fig05">Figura 5</xref>).</p>
    <fig id="fig05">
      <graphic xlink:href="1678-4170-abc-90-06-0388-00002-gf05.jpg"/></fig>
    <p>A an&#225;lise feita com NADPH-diaforase revelou uma redu&#231;&#227;o de 19,5% na concentra&#231;&#227;o da eNOS nas VS do grupo I (controle) quando comparadas com as VS do grupo NT, ou seja, as veias foram desnudadas, mas n&#227;o dilatadas. A redu&#231;&#227;o foi muito maior para as VS do grupo C, com uma raz&#227;o maior que 35%.</p>
    <p>
      <bold>Achados cir&#250;rgicos</bold>
    </p>
    <p>A dura&#231;&#227;o m&#233;dia do clampeamento da art&#233;ria aorta e da CEC foi de 121 (59 a 187) e 64 (34 a 95) minutos, respectivamente, para o grupo C; 126 (90 a 180) e 69 (41 a 117) minutos, respectivamente, para o grupo I; e 139 (88 a 195) e 72 (41 a 113) minutos, respectivamente, para o grupo NT. Os pacientes dos tr&#234;s grupos receberam enxertos de VS e ATI.</p>
    <p>Um total de 127 enxertos de VS e 41 de ATI foram implantados no grupo C. No grupo I, 116 enxertos de VS e 35 de ATI foram utilizados; e, no grupo NT, foram utilizados 124 enxertos de VS e 42 de ATI. No grupo C, foram utilizados 107 enxertos livres, 16 duplos seq&#252;enciais e quatro triplos seq&#252;enciais. No grupo I, foram utilizados 100 enxertos simples, 14 duplos seq&#252;enciais e dois triplos seq&#252;enciais; e, no grupo NT, foram utilizados 109 simples e 15 duplos seq&#252;enciais. A ATI n&#227;o foi considerada como bom enxerto em cinco pacientes do grupo C, em cinco pacientes do grupo I e em tr&#234;s pacientes do grupo NT. Realizou-se tromboendarterectomia (TEA) em duas descendentes posteriores (DP) no grupo C e em uma no grupo I, n&#227;o sendo necess&#225;ria no grupo NT.</p>
    <p>A taxa de mortalidade foi de 0% nos tr&#234;s grupos. O infarto agudo do mioc&#225;rdio (IAM) perioperat&#243;rio ocorreu em dois pacientes do grupo C. Um paciente do grupo NT foi reoperado por sangramento do leito da ATI. Complica&#231;&#245;es menores foram associadas com infec&#231;&#227;o do leito da VS na perna, tais como celulite e infec&#231;&#227;o superficial, as quais representaram 10% dos pacientes de cada grupo.</p>
    <p>
      <bold>Pat&#234;ncia dos enxertos</bold>
    </p>
    <p>Cento e trinta e dois pacientes foram submetidos a controle angiogr&#225;fico: 46 do grupo C, 41 do grupo I e 45 do grupo NT. Seis pacientes do grupo C, 11 do grupo I e sete do grupo NT recusaram-se a se submeter ao exame. A m&#233;dia de tempo de seguimento angiogr&#225;fico foi de 17,2 &#177; 4,4 meses para o grupo C, 16 &#177; 2,6 meses para o grupo I e 16,3 &#177; 2,7 para o grupo NT.</p>
    <p>A pat&#234;ncia dos enxertos de VS nos grupos NT, C e I foram de 118/124 (95,4%), 113/127 (88,9%) e 100/116 (86,2%), respectivamente. A pat&#234;ncia da ATI foi de 108/118 (91,5%).</p>
    <p>Os enxertos com baixo fluxo e que foram anastomosados a veias de pequeno calibre simultaneamente tiveram uma alta incid&#234;ncia de oclus&#227;o nos tr&#234;s grupos (<xref ref-type="table" rid="tab02">Tabela 2</xref>). A qualidade das art&#233;rias coron&#225;rias n&#227;o influenciou na pat&#234;ncia dos enxertos em nenhum dos grupos. No grupo C, ambos os enxertos que foram anastomosados em DP ap&#243;s TEA funcionaram bem. A taxa de oclus&#227;o de veias de baixa qualidade foi 4/10 (44,4%) no grupo C, 6/19 (31,6%) no grupo I e 3/29 (10,3%) no grupo NT.</p>
    <table-wrap id="tab02">
      <graphic xlink:href="1678-4170-abc-90-06-0388-00002-gt02.jpg"/></table-wrap>
    <p>Todos os enxertos seq&#252;enciais dos grupos NT e I funcionaram adequadamente. Por&#233;m, no grupo C, dois enxertos seq&#252;enciais estavam totalmente oclu&#237;dos, e outros dois estavam parcialmente oclu&#237;dos. Portanto, 47/51 (92,2%) de todos os enxertos estavam completamente abertos.</p>
    <p>A an&#225;lise estat&#237;stica por regress&#227;o log&#237;stica de fatores muito importantes que afetaram os resultados da taxa de pat&#234;ncia dos enxertos apresentou uma diferen&#231;a estat&#237;stica melhor para o grupo NT comparado ao grupo C (p = 0,025). Os piores resultados foram encontrados no grupo I (<xref ref-type="table" rid="tab02">Tabela 2</xref>).</p>
    <p>Entre a oclus&#227;o dos enxertos e os fatores cl&#237;nicos cl&#225;ssicos que poderiam alterar os resultados, tais como idade, IAM pr&#233;-operat&#243;rio, n&#237;veis de colesterol, fumo e hipertens&#227;o arterial sist&#234;mica (HAS), nenhuma correla&#231;&#227;o foi encontrada.</p>
    <p>A <xref ref-type="fig" rid="fig06">Figura 6</xref> demonstra um enxerto NT que foi anastomosado a uma coron&#225;ria de 1 mm. Quando a veia &#233; protegida pela camada de gordura, o <italic>kinking</italic> n&#227;o ocorre (<xref ref-type="fig" rid="fig07">Figura 7</xref>).</p>
    <fig id="fig06">
      <graphic xlink:href="1678-4170-abc-90-06-0388-00002-gf06.jpg"/></fig>
    <fig id="fig07">
      <graphic xlink:href="1678-4170-abc-90-06-0388-00002-gf07.jpg"/></fig>
    <p>
      <bold>Discuss&#227;o</bold>
    </p>
    <p>Por muitas raz&#245;es, a VS e a ATI continuam sendo amplamente utilizadas na CRM. A obten&#231;&#227;o de melhores resultados de pat&#234;ncia dos enxertos de VS &#233; o grande avan&#231;o da cirurgia cardiovascular. O uso indiscriminado de enxertos arteriais, utilizando a ATI com outros enxertos, pode n&#227;o ser totalmente justificado em provas cient&#237;ficas como as que se apresentam em estudos controle atualmente<sup>5</sup>.</p>
    <p>&#201; poss&#237;vel que as diferen&#231;as estruturais entre art&#233;rias e veias expliquem os melhores resultados dos enxertos arteriais<sup>9</sup>. Contudo, o dano &#224; parede da VS, que ocorre durante a prepara&#231;&#227;o do enxerto, pode contribuir para os piores resultados dos enxertos venosos. O endot&#233;lio da ATI &#233; protegido durante toda a opera&#231;&#227;o. O endot&#233;lio da VS, por outro lado, sofre grandes les&#245;es durante o procedimento, tornando-se trombog&#234;nico<sup>10</sup>. A prolifera&#231;&#227;o do m&#250;sculo liso da camada m&#233;dia &#233; similar na veia n&#227;o lesada e na ATI<sup>11</sup>. Geralmente, a prolifera&#231;&#227;o torna-se muito acentuada nas veias que sofreram les&#227;o endotelial durante a opera&#231;&#227;o. Isso demonstra que a grande causa de hiperplasia da camada &#237;ntima, detectada na VS, pode n&#227;o ser resultante de diferen&#231;as intr&#237;nsecas da musculatura lisa entre art&#233;rias e veias, mas da grande sensibilidade da veia ao trauma.</p>
    <p>Muitas estrat&#233;gias t&#234;m sido utilizadas para impedir a oclus&#227;o da veia. Boa parte dessas estrat&#233;gias vem sendo aprimorada constantemente e origina-se de terapia m&#233;dica, agentes farmacol&#243;gicos<sup>12</sup> e terapia gen&#233;tica<sup>13</sup>. N&#227;o obstante, um importante aspecto &#233; a melhoria de t&#233;cnicas cir&#250;rgicas para obten&#231;&#227;o de VS sem les&#245;es importantes na parede venosa durante a opera&#231;&#227;o.</p>
    <p>A remo&#231;&#227;o da VS juntamente com o ped&#237;culo de tecido circundante protege a veia de espasmos e, conseq&#252;entemente, evita a necessidade de dilata&#231;&#227;o. Al&#233;m disso, o ped&#237;culo possibilita uma meticulosa manipula&#231;&#227;o da VS quando sua remo&#231;&#227;o ocorrer no momento da implanta&#231;&#227;o.</p>
    <p>A t&#233;cnica <italic>no-touch</italic> demonstra uma excelente manuten&#231;&#227;o da integridade endotelial, que &#233; confirmada por ME<sup>14,15</sup>. O ped&#237;culo de gordura cont&#233;m grande amontoado de fibras col&#225;genas que podem oferecer uma prote&#231;&#227;o externa &#224; parede da veia contra os efeitos delet&#233;rios da press&#227;o a&#243;rtica. A altera&#231;&#227;o na distribui&#231;&#227;o da eNOS na VS preparada pela t&#233;cnica NT foi comparada com veias tratadas pela t&#233;cnica convencional<sup>16</sup>. Observamos que ocorreu a preserva&#231;&#227;o dessa enzima no endot&#233;lio, na camada m&#233;dia e na advent&#237;cia das veias do grupo NT. Em ambos os casos, a quantidade da eNOS foi reduzida quando a desnuda&#231;&#227;o e a distens&#227;o da veia foram efetuadas, sugerindo que essas medidas contribuem para a redu&#231;&#227;o da eNOS nas veias preparadas pela t&#233;cnica convencional. A distens&#227;o tem tido um grande papel na redu&#231;&#227;o da concentra&#231;&#227;o da eNOS no endot&#233;lio e na camada m&#233;dia, pois quando se desnuda a veia, remove-se grande quantidade da eNOS da advent&#237;cia. A preserva&#231;&#227;o da eNOS indica que o &#243;xido n&#237;trico dispon&#237;vel, usualmente reduzido na VS tratada pela t&#233;cnica convencional<sup>17</sup>, pode estar em concentra&#231;&#245;es elevadas nas veias tratadas pela t&#233;cnica NT. Essa pode, portanto, ser uma das muitas raz&#245;es que levam aos melhores resultados com a t&#233;cnica NT.</p>
    <p>A pat&#234;ncia inicial da VS tratada pela t&#233;cnica NT foi muito alta (95,4%) mesmo nos enxertos com baixo fluxo<sup>18</sup>. Para investigar se o ped&#237;culo de gordura teve import&#226;ncia na melhora da pat&#234;ncia dos enxertos venosos, comparamos os resultados angiogr&#225;ficos dos enxertos tratados pela t&#233;cnica NT com aqueles tratados pelas t&#233;cnicas I e C. Os resultados demonstraram que a pat&#234;ncia foi melhor quando a t&#233;cnica NT foi utilizada (p = 0,02).</p>
    <p>Quando a veia &#233; desnudada do tecido circundante, o ajuste do tamanho do enxerto tem que ser cuidadoso, para n&#227;o correr o risco de <italic>kinking</italic> e posterior comprometimento funcional. Muitas t&#233;cnicas t&#234;m sido recomendadas para impedir esse tipo de complica&#231;&#227;o<sup>19</sup>, e isso pode simplesmente ser evitado se a VS &#233; separada do seu leito com o ped&#237;culo de tecido adiposo.</p>
    <p>
      <bold>Conclus&#245;es</bold>
    </p>
    <p>A integridade endotelial apresentou um melhor resultado com o uso da t&#233;cnica <italic>no-touch</italic>. A camada advent&#237;cia e outras estruturas contidas no tecido gorduroso possuem propriedades mec&#226;nicas e funcionais que protegem a VS de espasmo e isquemia. A atividade da enzima precursora do &#243;xido n&#237;trico, eNOS, no endot&#233;lio e nos microvasos da camada advent&#237;cia sugere que a disponibilidade do &#243;xido n&#237;trico &#233; mantida nos enxertos tratados pela t&#233;cnica NT. Conseq&#252;entemente, o &#243;xido n&#237;trico pode ser um fator respons&#225;vel pelo aumento da pat&#234;ncia inicial dos enxertos de VS no grupo NT. Adicionalmente, o tecido circundante protege os enxertos excessivamente longos contra a forma&#231;&#227;o de tor&#231;&#245;es.</p>
    <p>
      <bold>Potencial Conflito de Interesses</bold>
    </p>
    <p>Declaro n&#227;o haver conflito de interesses pertinentes.</p>
    <p>
      <bold>Fontes de Financiamento</bold>
    </p>
    <p>O presente estudo n&#227;o teve fontes de financiamento externas.</p>
    <p>
      <bold>Vincula&#231;&#227;o Acad&#234;mica</bold>
    </p>
    <p>Este artigo &#233; parte de tese de Doutorado de Domingos S&#225;vio Ramos de Souza pela Acta Universitatis Upsaliensis &#150; UPPSALA &#150; Faculty of Medicine.</p>
    <p>
      <fn id="end" fn-type="corresp">
        <label>
          <bold>Correspond&#234;ncia:</bold>
        </label>
        <p> Fabio Gon&#231;alves de Rueda </p>
        <p> Rua Doutor Jos&#233; Fulco, 81 - Tamarineira </p>
        <p> 52111-010, Recife, PE - Brasil </p>
        <p> E-mail: </p>
        <p>
          <email xlink:href="fgrueda@yahoo.com.br">fgrueda@yahoo.com.br</email>
        </p>
      </fn>
    </p>
    <p>Artigo recebido em 14/02/07; revisado recebido em 05/09/07; aceito em 16/10/07.</p>
  </body>
  <back>
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  <sub-article article-type="translation" id="TRen" xml:lang="en">
    <front-stub>
      <article-categories>
        <subj-group subj-group-type="heading">
          <subject>Original Articles</subject>
        </subj-group>
      </article-categories>
      <title-group>
        <article-title xml:lang="en">Novel no-touch technique of harvesting the saphenous vein for coronary artery bypass grafting</article-title>
      </title-group>
      <abstract xml:lang="en">
        <p>BACKGROUND: Optimization of the saphenous vein for myocardial revascularization. OBJECTIVE: To present the no-touch technique of the saphenous vein preparation. This technique consists of harvesting the vein with a pedicle of surrounding tissue, which protects the vein from spasms, obviating the need for distension. METHODS: A prospective, randomized study with 156 patients who underwent artery bypass grafting was performed comparing three saphenous vein harvesting techniques: conventional, intermediate, and no-touch. A morphological study of the endothelium was carried out using scanning microscopy. An angiographic assessment of the vein graft patency was performed at a mean follow-up time of 18 months. Also, an immunohistochemical assessment was carried out to identify the endothelial enzyme nitric oxide synthase (eNOS) in the vein wall RESULTS: The preservation of the endothelial cell integrity was greater in the no-touch technique than in the other procedures. At angiographic follow-up, the patency for the no-touch group was 95.4%, 88.9% for the grafts of the conventional technique group, and 86.2% for the grafts performed in the intermediate technique group. The immunohistochemical assessment revealed eNOS in all three layers of the vein wall in the no-touch group and reduction of this enzyme in the conventional group. CONCLUSION: The endothelial integrity and eNOS activity were better preserved when using the no-touch technique for vein graft harvesting. The mechanical protection provided by the cushion of surrounding tissue in the no-touch group, the vasorelaxation and thromboresistant activities of nitric oxide may be responsible for the reduction of vasospasms and improved patency rate.</p>
      </abstract>
      <kwd-group xml:lang="en">
        <kwd>Saphenous vein</kwd>
        <kwd>endothelium, vascular</kwd>
        <kwd>myocardial revascularization</kwd>
      </kwd-group>
    </front-stub>
    <body>
      <p>
        <bold>ORIGINAL ARTICLE</bold>
      </p>
      <p><undefined id="tx-body2"/>Novel no-touch technique of harvesting the saphenous vein for coronary artery bypass grafting</p>
      <p>
        <bold>Fabio de Rueda<sup>I</sup>; Domingos Souza<sup>II</sup>; Ricardo de Carvalho Lima<sup>I</sup>; Alexandre Menezes<sup>I</sup>; Benny Johansson<sup>II</sup>; Michael Dashwood<sup>III</sup>; Emmanuel Th&#233;<sup>I</sup>; M&#225;rio Gesteira<sup>I</sup>; Mozart Escobar<sup>I</sup>; Frederico Vasconcelos<sup>I</sup></bold>
      </p>
      <p><sup>I</sup>Divis&#227;o de Cirurgia Cardiotor&#225;cica, Hospital Oswaldo Cruz, Universidade de Pernambuco, Recife, Brasil</p>
      <p><sup>II</sup>Divis&#227;o de Cirurgia Cardiotor&#225;cica, University Hospital of &#214;rebro, Su&#233;cia</p>
      <p><sup>III</sup>Divis&#227;o de Patologia Molecular e Bioqu&#237;mica, Royal Free Hospital &amp; University College Medical School, Londres, Reino Unido</p>
      <p>Mailing address</p>
      <p>
        <bold>SUMMARY</bold>
      </p>
      <p><bold>BACKGROUND:</bold> Optimization of the saphenous vein for myocardial revascularization.</p>
      <p><bold>OBJECTIVE:</bold> To present the no-touch technique of the saphenous vein preparation. This technique consists of harvesting the vein with a pedicle of surrounding tissue, which protects the vein from spasms, obviating the need for distension.</p>
      <p><bold>METHODS:</bold> A prospective, randomized study with 156 patients who underwent artery bypass grafting was performed comparing three saphenous vein harvesting techniques: conventional, intermediate, and no-touch. A morphological study of the endothelium was carried out using scanning microscopy. An angiographic assessment of the vein graft patency was performed at a mean follow-up time of 18 months. Also, an immunohistochemical assessment was carried out to identify the endothelial enzyme nitric oxide synthase (eNOS) in the vein wall.</p>
      <p><bold>RESULTS:</bold> The preservation of the endothelial cell integrity was greater in the no-touch technique than in the other procedures. At angiographic follow-up, the patency for the no-touch group was 95.4%, 88.9% for the grafts of the conventional technique group, and 86.2% for the grafts performed in the intermediate technique group. The immunohistochemical assessment revealed eNOS in all three layers of the vein wall in the no-touch group and reduction of this enzyme in the conventional group.</p>
      <p><bold>CONCLUSION:</bold> The endothelial integrity and eNOS activity were better preserved when using the no-touch technique for vein graft harvesting. The mechanical protection provided by the cushion of surrounding tissue in the no-touch group, the vasorelaxation and thromboresistant activities of nitric oxide may be responsible for the reduction of vasospasms and improved patency rate.</p>
      <p><bold>Key words:</bold> Saphenous vein; endothelium, vascular; myocardial revascularization.</p>
      <p>
        <bold>Introduction</bold>
      </p>
      <p>Atherosclerotic coronary disease is the major cause of morbidity and mortality in industrialized countries. Over a million people die every year in the world due to atherosclerotic coronary disease.<sup>1</sup> Myocardial revascularization using saphenous vein grafts, developed in the 1960s, deeply affected the outcomes and natural history of ischemic coronary disease and improved patient quality of life. However, graft failure may lead to recurrent angina<sup>2</sup>, one of the several causes of redo surgeries. About 30% of saphenous vein grafts occlude in the first year after revascularization<sup>3</sup>, and this rate reaches over 50% at 10 years.</p>
      <p>The recent use of arterial grafts has yielded better long-term results, but saphenous vein grafts are still widely used in coronary artery bypass grafting (CABG)<sup>4,5</sup>. Several procedures have been adopted to increase the long-term patency of saphenous vein (SV) grafts. The preparation of the SV plays a very important role in outcomes because inadequate manipulation of the SV may cause morphological and functional damage to the graft<sup>4</sup>.</p>
      <p>This study evaluated a no-touch technique to dissect the SV with surrounding tissue, which ensures very little manipulation and avoids graft distention<sup>6</sup>, and analyzed the clinical importance of this technique in the preparation of the saphenous vein.</p>
      <p>
        <bold>Methods</bold>
      </p>
      <p><italic>Patient selection </italic><bold>- </bold>After approval by the Ethics Committee and written informed consent from participants, 156 patients underwent CABG in the Cardiothoracic Surgery Division of the University Hospital of &#214;rebro, Sweden. Patients were randomly assigned to three groups according to the techniques used for saphenous vein preparation. Inclusion criteria ensured that patients would be available for long-term follow-up. Surgeries were performed from June 1993 to April 1997.</p>
      <p>
        <bold>Saphenous vein preparation techniques.</bold>
      </p>
      <p><italic>Conventional technique - C group</italic><bold> - </bold>The saphenous vein was harvested through a longitudinal incision in the leg. The adventitial layer was removed, and tributaries were ligated with 3-0 cotton thread suture. The vein was removed immediately after dissection, distended with saline solution injected with a syringe at a constant pressure of 300 mmHg for one minute, and then stored in saline solution at room temperature.</p>
      <p><italic>Intermediate technique - I group</italic><bold> -</bold>The saphenous vein was dissected as in the C group, but was not immediately distended. It was left in situ and covered with a compress soaked in saline solution containing papaverine (1 mg/ml), and perfusion was maintained. After removal, the SV was stored in blood obtained from the cannula introduced in the aorta. Although papaverine was used, all veins had to be distended because of spasm.</p>
      <p><italic>No-touch technique - NT group</italic><bold> - </bold>A continuous incision in the leg exposed the saphenous vein. All visible tributaries were ligated at about 0.5 cm from the SV wall with 3-0 cotton thread suture. The SV and a surrounding tissue pedicle were isolated and left in situ; perfusion was maintained, and the vein was covered with a compress soaked in pure saline solution. After removal from the leg, the SV was stored in blood obtained from the arterial cannula. (<xref ref-type="fig" rid="fig01-body2">Fig. 1</xref>)</p>
      <fig id="fig01-body2">
        <graphic xlink:href="1678-4170-abc-90-06-0388-00002-gf01-en.jpg"/></fig>
      <p>In summary, the SV was stripped and distended in the conventional technique; in the intermediate technique, it was stripped but not distended; and in the no-touch technique, it was neither stripped nor distended.</p>
      <p><italic>Surgical aspects</italic> - Cardiopulmonary bypass (CPB) and cardioplegia at 4&#186; C were routinely used, and the diameter of coronary arteries was measured with a probe. </p>
      <p>In all groups, distal anastomosis was the first to be made. The proximal portion of the SV was rapidly connected to the arterial cannula of the CPB unit to check possible bleeding of anastomosis or tributaries in the NT and I groups. In the C group, SV was manually tested with a syringe containing saline solution connected to a pressure transducer. All internal thoracic arteries (ITA) were anastomosed to the anterior descending artery (ADA). The proximal anastomosis was made after aortic declamping. The SV characteristics, as well as its quality and origin (proximal, medial or distal) were recorded. Blood flow through the grafts was measured using ultrasound (Flow Measure Probe, Transonic System Inc., US).</p>
      <p><italic>Morphological evaluation</italic> - Endothelial morphology was evaluated using scanning electron microscopy (SEM) and light microscopy (LM) of SV segments obtained from ten consecutive patients in each group. Control segments were obtained from the distal portion of the vein immediately after removal. The control sample, also called the primary sample, was immediately fixed, and was not distended or stored. After proximal anastomosis was completed, another vein sample, named secondary, was obtained and immediately fixed.</p>
      <p>Samples were fixed by immersion in 2% paraformaldehyde and 2.5% glutaraldehyde in Millonig phosphate buffer.</p>
      <p><italic>Immunohistochemical evaluation</italic><bold> - </bold>In ten other patients, the proximal portion of the SV was prepared according to the NT technique, whereas the distal portion was treated using the conventional technique. Both segments were stored in heparinized blood up to the moment of distal anastomosis. Samples of SV excess were obtained after proximal anastomosis and the control sample was obtained from the stripped vein that was not distended.</p>
      <p>Conventional immunohistochemical methods were used to detect endothelium and collagen. The SV samples were fixed in acetone, and serum activity of endothelial enzymes was inactivated to block possible changes in color. Samples were incubated with anti-CD31 antibodies to detect endothelial cells, and with rat monoclonal antibodies to detect collagen. A 3.3&#146;-diaminovenxidine tetrahydrochloride solution was used as substrate; the sections were stored in Mayer hematoxylin and prepared for microscopy. Analysis with NADPH-diaphoresis (Sigma- Aldrich, Poole, Dorset, UK) was used to detect endothelial nitric oxide synthetase (eNOS), which is a precursor of nitric oxide.</p>
      <p>In-vitro autoradiography and nitroarginine (NOARG) marked with a radioactive isotope were used to detect eNOS in endothelium. Slides were pre-incubated in buffered HCl solution and later incubated in a solution of 10 nM radioactive NOARG. After incubation, they were wiped and let to dry in open air. The slides were post-fixed in paraformaldehyde and kept in nuclear emulsion overnight. They were then stored in light-tight slide boxes and stored at 4&#186;C for 12 weeks. After that, tissues were stained with Mayer hematoxylin-eosin and examined using bright-field microscopy under black light.</p>
      <p>This procedure was conducted as described above to rule out suspicions that the better veins were used for the NT technique. Our purpose was to investigate immunohistochemical effects on two segments of the same SV treated according to two different techniques.</p>
      <p><italic>Angiographic evaluation</italic> - Angiographic control using the Judkins technique was performed about 18 months after surgery<sup>7</sup>. Visipaque, a low osmolarity medium, was used as contrast (Nycomed Amersham AB, Stockholm). Angiographic evaluations were randomly performed by two radiologists. The grafts were visualized in two planes, and patency ratios, as well as the degree of localized and diffuse lesions, were determined. Occlusion was identified by locating the remaining graft neck after selective contrast injection in the proximal anastomosis and by the type of graft opacification after injection of 50 ml of contrast in the ascending aorta.</p>
      <p><italic>Statistical analysis </italic>- The unpaired Kruskal-Wallis test was used for the retrospective comparison of endothelial histological findings between the three groups, as described by Siegel &amp; Castellan<sup>8</sup>.</p>
      <p>Logistic regression was used to evaluate graft patency. The main explanatory factor was the three different methods of vein preparation: conventional, intermediate and no touch. Additional factors were evaluated individually. Factors that were statistically significant and irregularly distributed between the three different techniques were selected and used in the final model.</p>
      <p>
        <bold>Results</bold>
      </p>
      <p><italic>Patient characteristics</italic><bold> - </bold>No significant patient differences were found between groups (<xref ref-type="table" rid="tab01-body2">Table 1</xref>). Patients treated because of high cholesterol levels or whose cholesterol values were above normal (2.6 &#150; 5.0 &#181;mol/l) were classified as patients with dyslipidemia. Hypertension was defined when diastolic pressure was greater than 90 mmHg and/or systolic pressure was greater than 140 mmHG. Ventricular function was determined according to ejection fraction. </p>
      <table-wrap id="tab01-body2">
        <graphic xlink:href="1678-4170-abc-90-06-0388-00002-gt01-en.jpg"/></table-wrap>
      <p>The use of aspirin was initiated on the first postoperative day at a dose of 160 mg per day. At the time of angiographic examinations, 42 patients in the C group, 35 in the I group, and 35 in the NT group were receiving 160 mg/day; 3 patients in the C group, 4 in the I group, and 7 in the NT group, were receiving 75 mg/day; and one patient in the C group, 2 in the I group, and 3 in the NT group were not taking any antiplatelet drugs.</p>
      <p><italic>Morphological findings</italic> - Secondary samples from the NT group showed that cell integrity was similar to that found in the control group. Almost half of the endothelial surface of samples in the C group had no endothelial cells, and the endothelial integrity of samples in the I group varied between that of the C and of the NT group. </p>
      <p>The comparison of samples of the three groups showed a significant difference in endothelial integrity, and p values for the comparisons were: control <italic>versus </italic>conventional samples - p&lt;0.01; control <italic>versus</italic> intermediate samples - p&lt;0.05; and control <italic>versus</italic> NT samples - p&lt;0.05. <xref ref-type="fig" rid="fig02-body2">Figures 2</xref> and <xref ref-type="fig" rid="fig03-body2">3</xref> show the degree of endothelial damage in samples of the NT and C groups.</p>
      <fig id="fig02-body2">
        <graphic xlink:href="1678-4170-abc-90-06-0388-00002-gf02-en.jpg"/></fig>
      <fig id="fig03-body2">
        <graphic xlink:href="1678-4170-abc-90-06-0388-00002-gf03-en.jpg"/></fig>
      <p><italic>Immunohistochemical findings - </italic>Immunohistochemistry to identify collagen fibers revealed an intact adventitial layer with microvessels in the NT group. No similar findings were seen in SV treated with the conventional technique (<xref ref-type="fig" rid="fig04-body2">Fig. 4</xref>).</p>
      <fig id="fig04-body2">
        <graphic xlink:href="1678-4170-abc-90-06-0388-00002-gf04-en.jpg"/></fig>
      <p>Endothelial nitric oxide synthase (eNOS) was identified by autoradiography of tissues incubated in a solution containing the NOARG marker. There was a direct association between NOARG binding to the lumen of vessels and area of intact endothelium, and the veins in the NT group had their morphological structure preserved. Such binding was reduced in the grafts of the C group (<xref ref-type="fig" rid="fig05-body2">Fig. 5</xref>).</p>
      <fig id="fig05-body2">
        <graphic xlink:href="1678-4170-abc-90-06-0388-00002-gf05-en.jpg"/></fig>
      <p>The analysis with NADPH-diaphoresis revealed a reduction of 19.5% in eNOS concentration in the samples of the I group (control), that is, the veins were stripped, but not distended, when compared with the sample of the NT group. The reduction was over 35% greater in the veins in the C group.</p>
      <p><italic>Surgical findings</italic><bold> - </bold>Mean duration of aortic clamping and CPB was 121 (59 to 187) minutes and 64 (34 to 95) minutes for the C group; 126 (90 to 180) and 69 (41 to 117) for the I group, and 139 (88 to 195) and 72 (41 to 113) for the NT group. Patients in the three groups received saphenous vein and left internal thoracic artery (ITA) grafts.</p>
      <p>A total of 127 SV grafts and 41 ITA grafts were implanted in the C group, 116 SV and 35 ITA grafts in the I group, and 124 SV and 42 ITA grafts in the NT group. In the C group, 107 free, 16 double sequential and 4 triple sequential grafts were used; In the I group, 100 simple, 14 double sequential and 2 triple sequential grafts were used; and in the NT groups, 109 simple and 15 double sequential grafts were implanted. The ITA was not considered a good graft in 5 patients of the C group, 5 of the I group, and in 3 of the NT group. Thromboendarterectomy (TEA) was performed in two posterior descending (PD) coronary arteries in the C group, and in one in the I group, but was not necessary in the NT group.</p>
      <p>Mortality rate was 0% in the 3 groups. Perioperative acute myocardial infarction (AMI) occurred in two patients in the C group. One patient in the NT group was re-operated due to bleeding in the vascular bed of ITA. Minor complications were associated with leg wound, such as cellulites and superficial infection, and affected about 10% of the patients in each group.</p>
      <p><italic>Graft patency</italic><bold> - </bold>One hundred thirty-two patients underwent angiographic control, 46 from the C group, 41 from the I group, and 45 from the NT group. Six patients in the C group, 11 in the I group, and 7 in the NT group refused the examination. Mean time of angiographic follow-up was 17.2 &#177; 4.4 months for the C group, 16 &#177; 2.6 months for the I group, and 16.3 &#177; 2.7 for the NT group.</p>
      <p>SV graft patency in the NT, C and I groups was 118/124 (95.4%), 113/127 (88.9%) and 100/116 (86.2%). ITA patency was 108/118 (91.5%).</p>
      <p>Grafts with low blood flow and anastomosed to small caliber veins had a high incidence of occlusion in the three groups (<xref ref-type="table" rid="tab02-body2">Table 2</xref>). The quality of coronary arteries did not affect graft patency in any of the groups. The two grafts in the C group that were anastomosed to PD after TEA worked well. Occlusion rates of low-quality veins were 4/10 (44.4%) in the C group, 6/19 (31.6%) in the I group, and 3/29 (10.3%) in the NT group.</p>
      <table-wrap id="tab02-body2">
        <graphic xlink:href="1678-4170-abc-90-06-0388-00002-gt02-en.jpg"/></table-wrap>
      <p>All sequential grafts in the NT and I groups worked adequately. In the C group, however, two sequential grafts were totally occluded, and two were partially occluded. Therefore, 47/51 (92.2%) of all grafts were fully patent.</p>
      <p>Logistic regression analysis of factors that substantially affected results of graft patency rates showed a better statistical difference in the NT group than in the C group (p=0.025). The poorest results were found in the I group (<xref ref-type="table" rid="tab02-body2">Table 2</xref>).</p>
      <p>No correlation was found between graft occlusion and classical clinical factors that may affect results, such as age, preoperative AMI, cholesterol levels, smoking and hypertension.</p>
      <p><xref ref-type="fig" rid="fig06-body2">Figure 6</xref> shows a NT graft that was anastomosed to a 1 mm coronary artery. When the vein is protected by a fat pedicle, kinking does not occur (<xref ref-type="fig" rid="fig07-body2">Fig. 7</xref>).</p>
      <fig id="fig06-body2">
        <graphic xlink:href="1678-4170-abc-90-06-0388-00002-gf06-en.jpg"/></fig>
      <fig id="fig07-body2">
        <graphic xlink:href="1678-4170-abc-90-06-0388-00002-gf07-en.jpg"/></fig>
      <p>
        <bold>Discussion</bold>
      </p>
      <p>For several reasons, SV and ITA are still widely used in CABG. Better SV graft patency is the greatest advance in cardiovascular surgery. The indiscriminate use of ITA grafts together with other grafts does not seem to be fully justified by scientific findings of controlled studies<sup>5</sup>.</p>
      <p>Structural differences between arteries and veins may explain the better results of arterial grafts<sup>9</sup>. At the same time, the damage to the SV wall, which occurs during graft preparation, may contribute to the poor results obtained with venous grafts. The ITA endothelium is protected during all the operation. The SV endothelium, however, suffers severe lesions during the procedure, and becomes thrombogenic<sup>10 </sup>The proliferation of smooth muscle of the medial layer is similar in the uninjured vein and in the ITA<sup>11</sup>. Proliferation is greater in the veins that suffer endothelial lesion during operation. Therefore, the major cause of intimal layer hyperplasia, detected in the SV, may be a result not of intrinsic differences in the smooth muscles between arteries and veins, but of the greater sensitivity of veins to trauma.</p>
      <p>Several strategies have been used to avoid vein occlusion. Many advances have come from medical therapy, pharmacological agents<sup>12</sup>, and genetic therapy<sup>13</sup>, and have undergone constant improvement. Surgical techniques to obtain the SV without important damage to the venous wall during the operation may represent an important improvement.</p>
      <p>Removal of the SV with a pedicle of surrounding tissue protects the vein from spasms and, consequently, avoids the need for distension. Moreover, the pedicle can be useful for the careful manipulation of the vein from its removal to the time of implantation.</p>
      <p>The no-touch technique results in excellent preservation of endothelial integrity, which is confirmed by electron microscopy<sup>14,15</sup>. The fat pedicle contains numerous collagen fibers that may provide external protection to the vein wall against the deleterious effects of aortic pressure. Changes in the distribution of eNOS in the SV prepared using the NT technique were compared with those of veins treated with the conventional technique<sup>16</sup>. We found that eNOS was preserved in the endothelium, both in the medial and adventitial layers, in the veins of the NT group. The amount of eNOS was reduced when stripping and distention were used, which suggests that these procedures contribute to a reduction of eNOS in the veins prepared with the conventional technique. Distention plays an important role in the reduction of eNOS concentration in the endothelium and in the medial layer; and a large amount of eNOS is removed from the adventitia when the vein is stripped. The presence of eNOS indicates that available nitric oxide, usually reduced in the SV treated with the conventional technique<sup>17</sup>, may be found in high concentrations in the veins treated with the NT technique. This may be one of the many explanations for the better results of the NT technique.</p>
      <p>Initial patency of SV treated with the NT technique was very high (95.4%) even in grafts with low blood flow<sup>18</sup>. To investigate whether the fat pedicle had a role in the improved patency of venous grafts, the angiographic results were compared between grafts treated with the NT technique and those treated with the I and C techniques. Results showed that patency was better when the NT technique was used (p=0.02).</p>
      <p>When the vein is stripped of surrounding tissue, graft size has to be carefully adjusted to avoid the risk of kinking and later functional impairment. Several techniques have been recommended to avoid kinking<sup>19</sup>, but our study shows that it may be simply avoided if the SV is removed with a fat tissue pedicle.</p>
      <p>
        <bold>Conclusions</bold>
      </p>
      <p>Endothelial integrity was better when the no-touch technique was used. The adventitial layer and other structures in the fat tissue have mechanical and functional properties that protect the SV from spasms and ischemia. The activity of a nitric oxide precursor (eNOS) in the endothelium and also in the microvessels of the adventitial layer, suggests that the availability of nitric oxide is preserved in the grafts treated with the NT technique. Consequently, nitric oxide may be one of the factors responsible for the increase in initial patency of the SV grafts in the NT group. In addition, surrounding tissue protects excessively long grafts against kinking.</p>
      <p>
        <bold>Potential Conflict of Interest</bold>
      </p>
      <p>No potential conflict of interest relevant to this article was reported.</p>
      <p>
        <bold>Sources of Funding</bold>
      </p>
      <p>There were no external funding sources for this study.</p>
      <p>
        <bold>Study Association</bold>
      </p>
      <p>This article is part of the thesis of Doctoral submitted by Domingos S&#225;vio Ramos de Souza, from Acta Universitatis Upsaliensis &#150; UPPSALA &#150; Faculty of Medicine.</p>
      <p>
        <bold>References</bold>
      </p>
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  </sub-article>
</article>
