Name: performing and processing fna of anterior fat pad for amyloid
Uploaded: 2010-10-30T17:00:00
Duration: 9 min 41 s
Description: Watch this Scientific Journal Video about Performing and Processing FNA of Anterior Fat Pad for Amyloid at JoVE.com

Agradecemos a Sra. Bonnie Phetteplace, RN para a assistência durante o vídeo da demonstração gráfica procedimento PAAF.

Introdução Amiloidose sistêmica é uma doença altamente variável que se refere à deposição extracelular de várias proteínas que são referidos coletivamente como amilóide. Deposição destas fibras de amilóide beta pregueada com configuração leva a várias apresentações clínicas dependendo dos órgãos envolvidos. As manifestações de maior relevância clínica de amiloidose sistêmica são observadas quando há envolvimento de órgãos importantes como coração, fígado e / ou renal. Historicamente, esses órgãos envolvidos seria biópsia para demonstrar a presença de amilóide. Estes procedimentos invasivos realizados moderadamente um risco significativo incluindo hemorragia. Aspiração almofada de gordura já foi mostrado para fornecer um método confiável e não-invasivos para a detecção de amilóide na amiloidose sistêmica 1. Este procedimento é essencialmente comparável à lipoaspiração da gordura subcutânea na parede abdominal anterior, sob anestesia local para recuperar o tecido fibro para avaliar depósitos de amilóide escassa nas paredes de pequenos vasos sanguíneos 2. O método mais utilizado para a identificação de amilóide no tecido continua a ser o padrão de birrefringência característica verde-maçã visto quando seções Congo manchada de vermelho são visualizadas em um microscópio de luz polarizada 3. Quando a aspiração almofada de gordura é realizada, manchas vermelho Congo pode ser feito em qualquer direta slides manchada ou preparação de um bloco de células do tecido adiposo aspirado. No entanto, pacientes em estágios iniciais de amiloidose têm depósitos de amilóide escassa, o que reduz a sensibilidade do vermelho Congo seções célula corada bloco 4,5. Avaliação ultra-estrutural dos aspirados almofada de gordura por microscopia eletrônica tem melhor reprodutibilidade e sensibilidade melhorada 4. Portanto, recomenda-se a submeter todas as aspira gordura abdominal para ambos preparação de um bloco de celas e para a realização de microscopia eletrônica de 2. Aspiração gordura abdominal é um custo relativamente baixo e método não invasivo para a obtenção de tecido para diagnosticar amiloidose sistêmica. Este artigo descreve procedimento de aspiração de gordura pad, juntamente com detalhes sobre o processamento da amostra para enviar amostra para ambos os coloração vermelho Congo e avaliação ultra-estrutural por microscopia eletrônica. Neste vídeo, demonstramos este procedimento reprodutível e simples para recuperar material de diagnóstico ideal. 1. Realizar a FNA de Pad Fat Anterior A. anestesiados da área local (Figura 1 e 2) <ol><li> Usando compressas de álcool ou o agente de limpeza de pele preferido por uma instituição particular, limpar a pele na área quadrante inferior do abdômen lateral à linha média e abaixo do umbigo (Figura 1). </li><li> Marca uma área em forma rombóide cerca de 2 x 2 polegadas, como mostrado na Figura 1 com uma caneta de marcação. </li><li> Aspirar cerca de 10 mL de lidocaína a 1% com agulha 18G. Anexar a 1 25G ½ polegadas de agulha para a seringa. Remover o ar tocando a seringa na posição vertical, enquanto pressiona o êmbolo até que o líquido é dispensado e não há bolhas de ar estão presentes. </li><li> Anestesiar ao longo das fronteiras da área rombóide já está marcado como mostrado na Figura 2. Comece por inserir a agulha 25G logo abaixo da pele no ponto A e empurre cuidadosamente a agulha por via subcutânea a ponto X. Puxe o êmbolo da seringa para garantir que você não está dentro de um vaso. Em seguida, empurre lentamente o êmbolo para se infiltrar até 2,5 mL de lidocaína (cerca de ¼ da lidocaína em seringa de 10 mL) ao retirar a agulha para o ponto A, sem deixar que a agulha sair da pele no ponto A (Figura 2A3). Com a agulha ainda sob a pele mudar a direção para o ponto Y (Figura 2A4) e empurre a agulha por via subcutânea a ponto Y (Figura 2b1). Como antes, confirmar que a agulha não está em um navio e dispensar cerca de 2,5 mL de lidocaína, enquanto lentamente retirar a agulha através do ponto A. (Figura 2b3 através 2B4) Repita os passos similares a partir do ponto B e lidocaína distribuição subcutânea a partir de pontos para B X e B para Y (Figura 2c1 através 2d4). Prevenir o sangramento do pica A e B pela aplicação firme da parte calibre estéril. </li><li> Você pode agora verificar que a área é anestesiada pelo tocando levemente a pele dentro do rombóide anestesiados por uma dica / canto de pedaço de gaze de algodão, comparando com a pele não anestesiados adjacentes. </li></ol> B. Realizar aspiração almofada de gordura (Figura 3) (Aplicação de anestesia local antes da realização de aspiração almofada de gordura pode ser ignorada, dependendo das preferências regionais e individuais. Corretamente realizada PAAF procedimento de aspiração de gordura pad anterior pode ser completado em uma picada. No entanto, dependendo do limiar de dor de cada paciente , manobras de agulha 18G e voltando para o tecido adiposo subcutâneo é relativamente distrescantar. O método descrito aqui anestesiados alcança o efeito em apenas duas picadas com agulha 25G e evita a dor com maior tolerância ao procedimento.) <ol><li> Montar 18G 1 ½ polegadas de agulha em uma seringa de 10 mL. Monte o conjunto da seringa de agulha nas garras seringa (&quot;grip PAAF / pistola&quot;) para a aplicação adequada e liberação de vácuo. </li><li> Insira a ponta da agulha para a gordura subcutânea dentro da área limpa e anestesiados rombóide (Figura 3a). </li><li> Retrair completamente o êmbolo da seringa com a agulha inserida no tecido subcutâneo para gerar vácuo (Figura 3b). </li><li> Manter o vácuo e manobrar a agulha e para trás em várias direções para a gordura subcutânea (Figura 3c através 3i). Cada curso deve ser tão longo quanto possível, com o comprimento da agulha selecionadas, sem deixar que a agulha sair da pele. Máxima de amostragem é conseguido através da mudança de direção com cada curso (Figura 3i). É importante manter a direção da agulha tangente ao serosa e paralelo à superfície da pele para evitar a perfuração da cavidade peritoneal. </li><li> O tecido fibro-adiposo se acumula na seringa. Fragmentos de tecido fibro uma vez o suficiente (até 1 mL de amostra de sangue rico fragmentos mistos) são recuperados, a liberação do vácuo completamente e retire a agulha (Figura 3k). </li><li> Que o paciente ou o assistente aplicar pressão firme sobre a área de processo com uma almofada de gaze para evitar o extravasamento de sangue. </li></ol> 2. Processamento de amostras <ol><li> Coloque pelo menos 5-6 fragmentos de tecido fibro-adiposo na solução de glutaraldeído para microscopia eletrônica (Figura 4B). </li><li> Dependendo do protocolo institucional, um esfregaço alguns dos fragmentos de tecido fibro-adiposo podem ser preparadas por espalhá-las entre dois slides (Figura 4A). </li><li> O restante do material é permitida a coagular na seringa (isso pode demorar 5-7 minutos, dependendo do tempo de coagulação) (Figura 4C). </li><li> Aspirar formalina a 10% dentro da seringa para que o material de tecido coagulado fibroadiposo é desalojado da parede da seringa e flutuantes livres (Figura 4C2). Retire o êmbolo da seringa (Figura 4C3) e transferir o tecido fibro-adiposo coagulado no recipiente formalina a 10% da extremidade aberta da seringa em frente à extremidade do bico (Figura 4C4). </li><li> Rotular os recipientes de forma adequada e submeter o glutaraldeído para microscopia eletrônica e do formol para a H &amp; E seção e coloração vermelho Congo para avaliação sob microscopia de polarização. Se esfregaços são preparados, eles podem ser processados ​​de acordo com o protocolo do laboratório individual. </li></ol><img src="/files/ftp_upload/1747/1747fig1.jpg" alt="Figura 1" /> Figura 1. Área a ser anestesiada para a PAAF de gordura abdominal anterior. <img src="/files/ftp_upload/1747/1747fig2.jpg" alt="Figura 2" /> Figura 2. Anestesiados da área local. <img src="/files/ftp_upload/1747/1747fig3.jpg" alt="Figura 3" /> Figura 3. Realizar PAAF de gordura abdominal anterior. <img src="/files/ftp_upload/1747/1747fig4.jpg" alt="Figura 4" /> Figura 4. Processamento de gordura abdominal anterior aspirar a ser submetida ao laboratório para detectar depósitos de amilóide. 3. Resultados representante <img src="/files/ftp_upload/1747/1747fig5.jpg" alt="Figura 5" /> Figura 5. Amilóide na parede dos pequenos vasos sanguíneos em fragmentos de tecido fibro-adiposo. Tecido fibro-adiposo foi formalina processados ​​parafina, incorporado, corados com vermelho Congo, e examinadas por microscopia de luz polarizante. Amilóide na parede dos pequenos vasos sanguíneos mostra birrefringência verde-maçã (seta branca). <img src="/files/ftp_upload/1747/1747fig6.jpg" alt="Figura 6" /> Figura 6. Tecido falta amiloidose. A maçã birrefringência verde é ausente em tecidos de um paciente diferente sem amiloidose. Birrefringência azul (seta branca) de fibras colágenas, geralmente estão presentes em quase todos os espécimes. <img src="/files/ftp_upload/1747/1747fig7.jpg" alt="Figura 7" /> Figura 7. Fibrilas amilóides consistente com na parede dos vasos sanguíneos. Micrografia eletrônica da reta, não-ramificadas, aleatoriamente espalhadas 80-10 nm de diâmetro fibrilas amilóides formado por na parede dos vasos sanguíneos.

<ol>
	<li>Westermark, P., Stenkvist, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+the+diagnosis+of+systemic+amyloidosis.">A new method for the diagnosis of systemic amyloidosis.</a> Arch Intern Med. 132, 522-523 (1973).</li><li>Devata, S., Hari, P., Markelova, N., Li, R., Shidham, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+AL+Amyloid+in+Abdominal+fat+pad+aspirates:+Is+Congo+red+sufficient?.">Detecting AL Amyloid in Abdominal fat pad aspirates: Is Congo red sufficient?.</a> Amyloid. , .</li><li>Merlini, G., Bellotti, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Mechanisms+of+Amyloidosis.">Molecular Mechanisms of Amyloidosis.</a> New Engl J Med. 349 (6), 583-596 (2003).</li><li>Shidham, V. B., Kumar, N., Cihlar, K., Varsegi, G., Markelova, N., Li, R., Hari, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fine+needle+aspiration+of+abdominal+fat+pad+for+diagnosis+of+early+amyloidosis:+How+can+the+clinical+role+of+the+test+be+improved?.">Fine needle aspiration of abdominal fat pad for diagnosis of early amyloidosis: How can the clinical role of the test be improved?.</a> Mod Pathol. 20, 1A-380A (2007).</li><li>Halloush, R., Lavrovskaya, E., Mody, D., Lager, D., Truong, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+and+typing+of+systemic+amyloidosis:+The+role+of+abdominal+fat+pad+fine+needle+aspiration.">Diagnosis and typing of systemic amyloidosis: The role of abdominal fat pad fine needle aspiration.</a> Cytojournal. 6, 24-24 (2010).</li><li>Hazenberg, I. I., Rijswijk, C. V. a. n., H, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+Accuracy+of+Subcutaneous+Abdominal+Fat+Tissue+Aspiration+for+Detecting+Systemic+Amyloidosis+and+Its+Utility+in+Clinical+Practice.">Diagnostic Accuracy of Subcutaneous Abdominal Fat Tissue Aspiration for Detecting Systemic Amyloidosis and Its Utility in Clinical Practice.</a> Arthritis Rheum. , 54-546 (2006).</li><li>Guy, C. D., Jones, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abdominal+fat+pad+aspiration+biopsy+for+tissue+confirmation+of+systemic+amyloidosis:+specificity,+positive+predictive+value,+and+diagnostic+pitfalls.">Abdominal fat pad aspiration biopsy for tissue confirmation of systemic amyloidosis: specificity, positive predictive value, and diagnostic pitfalls.</a> Diagn Cytopathol. 24, 181-185 (2001).</li><li>Westermark, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosing+Amyloidosis.">Diagnosing Amyloidosis.</a> Scand J Rheumatol Suppl. 24, 327-329 (1995).</li><li>Linke, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+Sensitive+Diagnosis+of+Amyloid+and+Various+Amyloid+Syndromes+Using+Congo+Red+Fluorescence.">Highly Sensitive Diagnosis of Amyloid and Various Amyloid Syndromes Using Congo Red Fluorescence.</a> Virchow Arch. 436, 439-448 (2000).</li><li>Giorgadze, T. A., Shiina, N., Baloch, Z. W., Tomaszewski, J. E., Gupta, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+detection+of+amyloid+in+fat+pad+aspiration:+an+evaluation+of+Congo+red+stain+by+fluorescent+microscopy.">Improved detection of amyloid in fat pad aspiration: an evaluation of Congo red stain by fluorescent microscopy.</a> Diagn Cytopathol. 31, 300-306 (2004).</li><li>DeMay, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fine+Needle+Aspiration+Biopsy.">Fine Needle Aspiration Biopsy.</a> The Art &#38; Science of Cytopathology. , 465-46 (1996).</li><li>Arbustini, E., Vrga, L., Concardi, M., Pallandini, G., Obici, L., Merlini, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+and+Immuno-electron+Microscopy+of+Abdominal+Fat+Identifies+and+Characterizes+Amyloid+Fibrils+in+Suspected+Cardiac+Amyloidosis.">Electron and Immuno-electron Microscopy of Abdominal Fat Identifies and Characterizes Amyloid Fibrils in Suspected Cardiac Amyloidosis.</a> Amyloid. 9, 108-114 (2002).</li></ol>

Diagnóstico de amiloidose é geralmente obtida com uma biópsia do tecido dos órgãos afetados, como o rim, fígado e / ou coração. Esta abordagem tem rendimento diagnóstico de alta, no entanto, é invasivo e pode estar associada a complicações, incluindo hemorragia 2. Retal, gengival e biópsias de medula óssea foram preferidas para o diagnóstico como abordagem relativamente menos invasivo em 1960 6,7,8. Pad aspiração de gordura abdominal foi relatada em 1973 como um cofre, procedimento minimamente invasivo, simples e menos dispendiosa para o diagnóstico do tecido de amiloidose sistêmica 1. No entanto, os detalhes do procedimento e abordagem ao processamento da amostra recuperada não é coerente relatados. Este vídeo eo artigo descrever o procedimento passo a passo. A abordagem para a detecção de amilóide na aspiração almofada de gordura, da mesma forma, também não é bem padronizado, com poucos estudos comparando e avaliando várias abordagens 1,5,6,7,8. Avaliação do anterior aspira gordura abdominal com coloração vermelho congo é menos sensível com reprodutibilidade menor interobservador especialmente nos casos iniciais de amiloidose com depósitos de amilóide escassos 5. Imuno-histoquímica realizada em formol fixa parafina bloco de celas seções e Congo fluorescência vermelha realizado em esfregaços citológicos têm sido relatadas para melhorar a sensibilidade diagnóstica 9,10. Microscopia eletrônica melhora a detecção e identificação de fibrilas amilóides escassa nas paredes de pequenos vasos sanguíneos no tecido fibro-adiposo em gordura pad aspirados 4, 5. Com base nessas informações, este artigo descreve o processamento da amostra para preparar blocos de células (para avaliação de vermelho Congo seções célula corada bloco sob microscópio de luz polarizada para o diagnóstico birrefringência verde maçã e também para imuno-histoquímica como indicado) e para microscopia eletrônica. No entanto, dependendo da abordagem escolhida para a avaliação da amostra para amilóide, que pode ser submetido a citologia preparação de esfregaço, a preparação de blocos de células, e processamento para microscopia eletrônica. Alguns laboratórios realizam os testes nos esfregaços preparados a partir dos fragmentos de tecido fibro aspirado e corados com vermelho Congo para estudar a 10 de fluorescência. Geralmente em uma amiloidose tarde, a microscopia óptica com vermelho Congo microscopia de polarização pode ser suficiente, no entanto, nos estágios iniciais da doença coloração vermelho Congo com microscopia de polarização em seções bloco de células é menos sensível e confiável para a gordura pad aspirados 4,5,11 . Outras colorações especiais incluindo Thioflavin T pode ser usado com limitações semelhantes. Outras abordagens para detectar amilóide incluir a avaliação do esfregaço de aspirado de amilóide com vermelho Congo microscopia de polarização e fluorescência 10. Em nossa experiência este método é menos reprodutíveis. Avaliação dos vasos sanguíneos na almofada de gordura para amilóide por microscopia eletrônica supera estas limitações 4,11. Caracterização mais de amilóide foi relatado pela imunomicroscopia 12, no entanto, estes métodos podem não ser amplamente disponível em um ambiente de cuidados não-superior. Agulhas usadas para a realização de procedimentos de aspiração podem ser classificados de acordo com seus tamanhos de bitola em multa (21-25G), intermediária (18-20G) e grandes (por exemplo-14G) 11. Anterior aspirações gordura abdominal foram relatados para ser executada usando calibres variáveis ​​que vão desde intermediária (18 a 20G) de multa (21 a 22G) 2,10. A maioria das lesões de massa são aspirados com agulhas mais finas calibre (como 25G) para obter boa esfregaços citológicos, juntamente com passagens adicionais com agulha maior calibre (como 18G) para recuperar amostra adicional para os blocos de células. Durante a aspiração almofada anterior de gordura, o tecido fibro coesa não aspirar bem com agulhas mais finas. Preparação celular bloco e microscopia eletrônica são importantes para a avaliação de paredes de pequenos vasos sanguíneos em fragmentos de tecido fibro no aspirado de gordura. Agulhas bitola mais larga (como 18G) são recomendados para produzir material adequado de diagnóstico 2,10. Como o procedimento é comparável com a PAAF convencional, a aspiração de gordura abdominal é referida como PAAF, embora agulhas bitola mais larga pode ser usado para executá-lo 5.

A correction was made to <a href="http://www.jove.com/index/details.stp?id=1747">Performing and Processing FNA of Anterior Fat Pad for Amyloid</a>. There was an error with an author's name. The author's last name had a typo and was corrected to:
Safwan S. Jaradeh
instead of:
Safwan S. Jardeh

Erratum: Performing and Processing FNA of Anterior Fat Pad for Amyloid

O algodão, as compressas de gaze, caneta de marcação, 10 mL seringas x2, lidocaína a 1% (anestésico local lidocaína-Se é usado sozinho, faz com que uma sensação de queimação inicial após a instilação. Isto pode ser evitado usando uma mistura 1:1 de 1% lidocaína e 1% de sódio bi-carbonato), calibre 18 (G) 1 ½ polegadas x2 agulhas (para a realização de PAAF), 25G 1 ½ polegadas de agulha (para injetar anestésico local), FNA titular seringa (&quot;arma&quot;), curativo, frasco de glutaraldeído (para microscopia eletrônica), e biópsia recipiente de formol a 10%.

performing and processing fna of anterior fat pad for amyloid

Historicamente, as biópsias coração, fígado e rim foram realizados para demonstrar os depósitos de amilóide na amiloidose. Como a apresentação clínica da doença é muito variável e não-específicos, os riscos associados dessas biópsias são grandes demais para o rendimento diagnóstico. Outros sites que têm um menor risco de biópsia, como a pele ou gengival, também são relativamente invasivo e caro. Além disso, estas biópsias não pode ter sempre os depósitos de amilóide suficiente para estabelecer um diagnóstico. Aspiração almofada de gordura tem demonstrado boa correlação clínica com baixo custo e mínima morbidade. No entanto, não existem protocolos padronizados para realizar este procedimento ou o processamento do espécime aspirado, o que leva a resultados variáveis ​​e nonreproducible. A modalidade mais utilizada para a detecção de amilóide no tecido é uma birrefringência verde-maçã em seções Congo manchada de vermelho usando um microscópio de luz polarizada. Esta técnica requer a preparação de células bloco de material aspirado. Infelizmente, pacientes em fase inicial de amiloidose têm quantidades mínimas de amilóide que reduz a sensibilidade do vermelho Congo seções célula corada bloco de aspirados gordura abdominal. Portanto, a avaliação ultra-estrutural dos aspirados almofada de gordura por microscopia eletrônica deve ser utilizado, dada a sua maior sensibilidade para a detecção de amilóide. Este artigo demonstra um procedimento simples e reprodutível para a realização de aspiração de gordura pad anterior para a detecção de amilóide utilizando tanto Congo coloração vermelha das seções bloco de células e microscopia eletrônica para identificação ultra-estrutural.

Watch this Scientific Journal Video about Performing and Processing FNA of Anterior Fat Pad for Amyloid at JoVE.com

Performing and Processing FNA of Anterior Fat Pad for Amyloid

Aspiração gordura abdominal é um preferido, abordagem de custo minimamente invasivo e de baixo, em comparação com outros métodos para detectar amilóide para o diagnóstico de amiloidose sistêmica. Este artigo vídeo demonstra um esquema processual para a realização de aspiração de gordura pad com o processamento adequado da amostra para o resultado ideal de diagnóstico.

Executar e Processamento de FNA Pad Fat Anterior para amilóide

Historically, heart, liver, and kidney biopsies were performed to demonstrate amyloid deposits in amyloidosis.  Since the clinical presentation of this ...

performing-and-processing-fna-of-anterior-fat-pad-for-amyloid

Research

JoVE Journal

Medicine

Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a large extracellular domain and a short cytosolic domain termed the APP intracellular domain (AICD). During maturation through the secretory pathway, APP can be cleaved by proteases termed &alpha;, &beta;, and &gamma;-secretases1. Sequential proteolytic cleavage of APP with &beta; and &gamma;-secretases leads to the production of a small proteolytic peptide, termed A&beta;, which is amyloidogenic and the core constituent of senile plaques. The AICD is also liberated from the membrane after secretase processing, and through interactions with Fe65 and Tip60, can translocate to the nucleus to participate in transcription regulation of multiple target genes2,3. Protein-protein interactions involving the AICD may affect trafficking, processing, and cellular functions of holo-APP and its C-terminal fragments. We have recently shown that AICD can aggregate in vitro, and this process is inhibited by the AD-implicated molecular chaperone ubiquilin-14. Consistent with these findings, the AICD has exposed hydrophobic domains and is intrinsically disordered in vitro5,6, however it obtains stable secondary structure when bound to Fe657. We have proposed that ubiquilin-1 prevents inappropriate inter- and intramolecular interactions of AICD, preventing aggregation in vitro and in intact cells4. While most studies focus on the role of APP in the pathogenesis of AD, the role of AICD in this process is not clear. Expression of AICD has been shown to induce apoptosis8, to modulate signaling pathways9, and to regulate calcium signaling10. Over-expression of AICD and Fe65 in a transgenic mouse model induces Alzheimer's like pathology11, and recently AICD has been detected in brain lysates by western blotting when using appropriate antigen retrieval techniques12. To facilitate structural, biochemical, and biophysical studies of the AICD, we have developed a procedure to produce recombinantly large amounts of highly pure AICD protein. We further describe a method for inducing the in vitro thermal aggregation of AICD and analysis by atomic force microscopy. The methods described are useful for biochemical, biophysical, and structural characterization of the AICD and the effects of molecular chaperones on AICD aggregation.

A method for large-scale purification of the APP intracellular domain (AICD) is described. We also describe methodology to induce in vitro AICD aggregation and visualization by atomic force microscopy. The methods described are useful for biochemical/structural characterization of the AICD and the effects of molecular chaperones on its aggregation.

Purificação e Agregação do Domínio proteína precursora de amilóide intracelular

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a ...

Medicina

Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture

Experimental examination of normal human mammary epithelial cell (HMEC) behavior, and how normal cells acquire abnormal properties, can be facilitated by in vitro culture systems that more accurately model in vivo biology. The use of human derived material for studying cellular differentiation, aging, senescence, and immortalization is particularly advantageous given the many significant molecular differences in these properties between human and commonly utilized rodent cells1-2. Mammary cells present a convenient model system because large quantities of normal and abnormal tissues are available due to the frequency of reduction mammoplasty and mastectomy surgeries.
The mammary gland consists of a complex admixture of many distinct cell types, e.g., epithelial, adipose, mesenchymal, endothelial. The epithelial cells are responsible for the differentiated mammary function of lactation, and are also the origin of the vast majority of human breast cancers. We have developed methods to process mammary gland surgical discard tissues into pure epithelial components as well as mesenchymal cells3. The processed material can be stored frozen indefinitely, or initiated into primary culture. Surgical discard material is transported to the laboratory and manually dissected to enrich for epithelial containing tissue. Subsequent digestion of the dissected tissue using collagenase and hyaluronidase strips stromal material from the epithelia at the basement membrane. The resulting small pieces of the epithelial tree (organoids) can be separated from the digested stroma by sequential filtration on membranes of fixed pore size. Depending upon pore size, fractions can be obtained consisting of larger ductal/alveolar pieces, smaller alveolar clusters, or stromal cells. We have observed superior growth when cultures are initiated as organoids rather than as dissociated single cells. Placement of organoids in culture using low-stress inducing media supports long-term growth of normal HMEC with markers of multiple lineage types (myoepithelial, luminal, progenitor)4-5. Sufficient numbers of cells can be obtained from one individual's tissue to allow extensive experimental examination using standardized cell batches, as well as interrogation using high throughput modalities.
Cultured HMEC have been employed in a wide variety of studies examining the normal processes governing growth, differentiation, aging, and senescence, and how these normal processes are altered during immortal and malignant transformation4-15,16. The effects of growth in the presence of extracellular matrix material, other cell types, and/or 3D culture can be compared with growth on plastic5,15. Cultured HMEC, starting with normal cells, provide an experimentally tractable system to examine factors that may propel or prevent human aging and carcinogenesis.

A method to process human mammary surgical discard material is described. Processed tissue, in the form of organoids, can be stored frozen indefinitely or placed in culture for long-term growth. This method enables experimental examination of normal human epithelial cell biology, and the effects of exogenous perturbations.

Processamento da mamoplastia redutora e tecidos humanos Mastectomia para cultura de células

Experimental  examination of normal human mammary epithelial cell (HMEC) behavior, and how  normal cells acquire abnormal properties, can be facilitated ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

Injeção Orthotopic de células cancerosas da mama na mamária Fat Pad de Ratos para o Estudo de crescimento tumoral.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft tissue filler as it is readily available, easily obtained, inexpensive, and inherently biocompatible.1 However, despite its burgeoning popularity, fat grafting is hampered by unpredictable results and variable graft survival, with published retention rates ranging anywhere from 10-80%. 1-3
To facilitate investigations on fat grafting, we have therefore developed an animal model that allows for real-time analysis of injected fat volume retention. Briefly, a small cut is made in the scalp of a CD-1 nude mouse and 200-400 &#181;l&#160;of processed lipoaspirate is placed over the skull. The scalp is chosen as the recipient site because of its absence of native subcutaneous fat, and because of the excellent background contrast provided by the calvarium, which aids in the analysis process. Micro-computed tomography (micro-CT) is used to scan the graft at baseline and every two weeks thereafter. The CT images are reconstructed, and an imaging software is used to quantify graft volumes.
Traditionally, techniques to assess fat graft volume have necessitated euthanizing the study animal to provide just a single assessment of graft weight and volume by physical measurement ex vivo. Biochemical and histological comparisons have likewise required the study animal to be euthanized. This described imaging technique offers the advantage of visualizing and objectively quantifying volume at multiple time points after initial grafting without having to sacrifice the study animal. The technique is limited by the size of the graft able to be injected as larger grafts risk skin and fat necrosis. This method has utility for all studies evaluating fat graft viability and volume retention. It is particularly well-suited to providing a visual representation of fat grafts and following changes in volume over time.

Fat grafting is an essential technique for reconstructing soft tissue deficits. However, it remains an unpredictable procedure characterized by variable graft survival. Our goal was to devise a mouse model that utilizes a novel imaging method to compare volume retention between differing techniques of fat graft preparation and delivery.

Avaliação da viabilidade de gordura humana injecção em ratinhos nu com Micro-Tomografia Computadorizada

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft ...

Transplantation Into the Mouse Ovarian Fat Pad

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic transplantation of ovarian surface and tubal epithelia include intraperitoneal and intrabursal administration of cells. The respective limitations of these methods include poorly defined location of injected cells and limited space volume. Furthermore, they are poorly suited for long-term structural preservation of transplanted organs. To address these challenges, we have developed an alternative approach, which is based on the introduction of cells and tissue fragments into the mouse fat pad. The mouse ovarian fat pad is located in the immediate vicinity of the ovary and uterine tube (aka oviduct, fallopian tube), and provides a familiar microenvironment for cells and tissues of these organs. In our approach fluorescence-labeled mouse and human cells, and fragments of the uterine tube are engrafted by using minimally traumatic dorsal incision surgery. Transplanted cells and their outgrowths are easily located in the ovarian fat pad for over 40 days. Long-term transplantation of the entire uterine tube allows correct preservation of all principle tissue components, and does not result in adverse side effects, such as fibrosis and inflammation. Our approach should be uniquely applicable for answering important biological questions such as differentiation, regenerative and neoplastic potential of specific cell populations. Furthermore, it should be suitable for studies of microenvironmental factors in normal development and cancer.

We describe an ovarian fad pad transplantation assay suitable for studies of normal and transformed epithelia of the female reproductive tract. The mouse fat pad allows transplantation of large tissue fragments, is easily accessible for surgery and imaging, and provides the most favorable native environment for tissues of M&#252;llerian origin.

Transplantação no Pad Mouse ovário Fat

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic ...

Confirmation of Myocardial Ischemia and Reperfusion Injury in Mice Using Surface Pad Electrocardiography

Many animal models have been established for the study of myocardial remodeling and heart failure due to its status as the number one cause of mortality worldwide. In humans, a pathologic occlusion forms in a coronary artery and reperfusion of that occluded artery is considered essential to maintain viability of the myocardium at risk. Although essential for myocardial recovery, reperfusion of the ischemic myocardium creates its own tissue injury. The physiologic response and healing of an ischemia/reperfusion injury is different from a chronic occlusion injury. Myocardial ischemia/reperfusion injury is gaining recognition as a clinically relevant model for myocardial infarction studies. For this reason, parallel animal models of ischemia/reperfusion are vital in advancing the knowledge base regarding myocardial injury. Typically, ischemia of the mouse heart after left anterior descending (LAD) coronary artery occlusion is confirmed by visible pallor of the myocardium below the occlusion (ligature). However, this offers only a subjective way of confirming correct or consistent ligature placement, as there are multiple major arteries that could cause pallor in different myocardial regions. A method of recording electrocardiographic changes to assess correct ligature placement and resultant ischemia as well as reperfusion, to supplement observed myocardial pallor, would help yield consistent infarct sizes in mouse models. In turn, this would help decrease the number of mice used. Additionally, electrocardiographic changes can continue to be recorded non-invasively in a time-dependent fashion after the surgery. This article will demonstrate a method of electrocardiographically confirming myocardial ischemia and reperfusion in real time.

During murine myocardial ischemia/reperfusion surgery, correct placement of the occluding ligature is typically confirmed by visible observation of myocardial pallor. Herein, a method of electrocardiographically confirming ischemia and reperfusion, to supplement observed myocardial pallor, is demonstrated in male C57Bl/6 mice.

A confirmação da Isquemia Miocárdica e reperfusão em ratos usando Pad Superfície Eletrocardiografia

Many animal models have been established for the study of myocardial remodeling and heart failure due to its status as the number one cause of mortality ...

Stromal Vascular Fraction-enriched Fat Grafting for the Treatment of Symptomatic End-neuromata

The purpose of this study was to methodically illustrate and highlight the crucial steps of stromal vascular fraction (SVF)-enriched fat grafting as a novel treatment of symptomatic end-neuromata of peripheral sensory nerves, and in this study, specifically of the superficial branch of the radial nerve (SBRN). Despite a multitude of existing treatments, persistent postoperative pain and common pain relapse are still very common, independent of the procedure assessed. The neuroma is microsurgically excised accordingly to standardized protocol. Instead of the relocation of the regenerating nerve stump in neighboring anatomical structures, such as muscle or bone, a fat graft is applied perifocally and acts as a mechanical barrier. In order to reduce the fat resorption rate and boost the regenerative potential of the graft, the highly concentrated SVF is integrated in the grafting. The SVF is isolated from subcutaneous fat by enzymatic and mechanic separation of the lipoaspirate by a specific commercial isolation system. The SVF-enriched fat graft provides both a mechanical barrier and various biological effects at the cellular level, including improving angiogenesis, inflammation, and fibrosis. Both mechanical and biologic effects help to reduce the disorganized axonal outgrowth of the nerve stump during nerve regeneration and hence prevent the recurrence of painful end-neuromata.

The purpose of the study is to illustrate the technical procedure of stromal vascular fraction (SVF)-enriched fat grafting as a novel treatment of symptomatic end-neuromata. This technique provides advantages of both the mechanical barrier and the biological action at the cellular level by the processed and concentrated SVF.

Do estroma Vascular fração enriquecida enxerto de gordura para o tratamento sintomático final-neuromata

The purpose of this study was to methodically illustrate and highlight the crucial steps of stromal vascular fraction (SVF)-enriched fat grafting as a ...

Methodology for Sputum Induction and Laboratory Processing

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which is interesting in various respiratory diseases like asthma, chronic obstructive pulmonary disease (COPD), chronic cough, or idiopathic pulmonary fibrosis. This technique is well tolerated, safe and non-invasive, but is currently limited to research services and specialized centers in clinical practice because it is technically demanding, time-consuming, and requires trained staff. The success rate of sputum induction and analysis is about 80%.
Here, we describe the induction and laboratory processing of sputum samples. Sputum is induced by inhalation of hypertonic or isotonic saline with salbutamol. For the processing, we use the whole sputum technique. Dithiothreitol (DTT) is used to allow mucolysis of sputum samples. The primary aim of sputum processing is to obtain a differential cell count to study the cell types present in the airway lumen. Additional analyses may also be performed on sputum supernatant and sputum cells, which may allow further investigation into inflammatory processes and immune mechanisms. Examples include studying mediators in sputum supernatant and performing a large spectrum of analysis on sputum cells such as flow cytometry, genomics, or proteomics.
Finally, representative results of sputum analysis in healthy controls, asthmatics, and COPD patients are presented.

Here we describe the technique of sputum induction. This protocol also explains the sputum processing to perform a differential cell count and to collect sputum supernatant and cells for further analysis.

Metodologia para a indução de escarro e laboratório de processamento

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which ...

Sampling Strategies and Processing of Biobank Tissue Samples from Porcine Biomedical Models

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of genetically modified pig models, and the often-limited number of available animals of these models, establishment of (biobank) collections of adequately processed tissue samples suited for a broad spectrum of subsequent analyses methods, including analyses not specified at the time point of sampling, represent meaningful approaches to take full advantage of the translational value of the model. With respect to the peculiarities of porcine anatomy, comprehensive guidelines have recently been established for standardized generation of representative, high-quality samples from different porcine organs and tissues. These guidelines are essential prerequisites for the reproducibility of results and their comparability between different studies and investigators. The recording of basic data, such as organ weights and volumes, the determination of the sampling locations and of the numbers of tissue samples to be generated, as well as their orientation, size, processing and trimming directions, are relevant factors determining the generalizability and usability of the specimen for molecular, qualitative, and quantitative morphological analyses. Here, an illustrative, practical, step-by-step demonstration of the most important techniques for generation of representative, multi-purpose biobank specimen from porcine tissues is presented. The methods described here include determination of organ/tissue volumes and densities, the application of a volume-weighted systematic random sampling procedure for parenchymal organs by point-counting, determination of the extent of tissue shrinkage related to histological embedding of samples, and generation of randomly oriented samples for quantitative stereological analyses, such as isotropic uniform random (IUR) sections generated by the &#34;Orientator&#34; and &#34;Isector&#34; methods, and vertical uniform random (VUR) sections.

The practical application and performance of methods for generation of representative tissue samples of porcine animal models for a broad spectrum of downstream analyses in biobank projects are demonstrated, including volumetry, systematic random sampling, and differential processing of tissue samples for qualitative and quantitative morphologic and molecular analyses types.

Estratégias de amostragem e processamento de amostras de tecido do Biobanco de suínos modelos biomédicos

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of ...

A Training and Testing System for Performing Vascular Reconstruction In Vitro

Manual vascular reconstruction training is essential for a beginner surgeon. However, an optimal training system for vascular reconstruction in vitro has yet to be developed. In this study, we introduce an in vitro training and testing system using a magnetic anchoring technique with which a trainee can practice manual vascular reconstruction individually. Additionally, this system can also be used to test the quality of the reconstruction. The described system includes a vascular reconstruction training machine, magnetic tractors, and a magnetic suture puller. In this manuscript, we detail an end-to-end vein anastomosis using porcine right and left iliac veins. To identify the potential damage caused by a magnetic suture puller on the suture, we created three groups with six segments of 4-0 polypropylene sutures each: a control group with no intervention on the polypropylene suture, a group in which the polypropylene suture is manually pulled with sterile gloves 20x, and a magnetic puller group in which the magnetic puller pulled the polypropylene suture 20x. These groups were tested by light microscopy and breaking strength tests, and the effect of reconstruction was assessed. In the light microscopy test, the control group was less likely to be damaged (p &#60; 0.05) and the number of damaged points of the manual group and magnetic puller group were similar (p &#62; 0.05). The results of the breaking strength test were compared across groups and no significant difference was observed (p &#62; 0.05). The end-to-end anastomosis of the porcine iliac veins was successfully performed using this training system, and the reconstructed veins could undergo 2.0 kPa perfusion pressure. Using this training and testing system the trainee can practice manual vascular reconstruction in vitro individually with the aid of magnetic tractors and a magnetic suture puller, and the quality of the reconstruction can be tested.

Here we present a training and testing system where a trainee can complete manual vascular reconstruction in vitro individually using a magnetic anchoring technique. The system can also be used to test the quality of reconstruction.

Um sistema de treinamento e teste para a realização de reconstrução vascular in vitro

Manual vascular reconstruction training is essential for a beginner surgeon. However, an optimal training system for vascular reconstruction in vitro has ...