A pesquisa em meu laboratório é apoiado pelo Departamento WKU de fundos de arranque Biologia, WKU Research Foundation RCAP-I conceder # 11-8032 e por uma concessão KBRIN-AREA financiado através de uma subvenção pai do Instituto Nacional de Ciências Médicas Gerais dos Institutos Nacionais da Saúde sob o número prêmio 5P20GM103436-13. Gostaria também de agradecer Dr. Tian Xu, em cujo laboratório fui apresentado a esta técnica e Dr. Raymond Pagliarini que primeiro estabeleceu esta técnica em laboratório Xu.

1. Indução de tumores benignos não-invasivos <ol><li> Use ações listadas na Tabela 2 para a indução de tumores benignos. </li><li> Prepare uma cultura de partida para o &quot;testador&quot; balanço da seguinte genótipo: y, w, ey-FLP1; Tub-Gal80, FRT40A; Act5C&gt; y +&gt; Gal4, UAS-GFP </li><li> Prepare uma cultura inicial para o estoque &quot;testado&quot; do seguinte genótipo: w; FRT40A, UAS-Ras V12 / cyo </li><li> Colete dez fêmeas virgens do estoque &quot;tester&quot; e dez homens do estoque &quot;testado&quot;. </li><li> Cruze os machos e fêmeas do &quot;testador&quot; e o estoque &quot;testado&quot;, colocando-os tanto em um frasco com alimentos mosca. </li><li> Coloque a cruz em uma incubadora de 25 ° C e permitir que os machos e as fêmeas para acasalar e pôr ovos por 24-48 horas. </li><li> Confira os frascos para a deposição de ovos e suficiente para a presença de larvas de primeiro instar certificando-se de que a cultura não está secando. <ol><li> Adicione algumas gotas de autoclavadoágua destilada à cultura para mantê-lo húmido (se a cultura está a secar para fora). </li><li> Continue a acompanhar a cultura e repita o passo 1.7.1, conforme necessário. </li></ol></li><li> Observe vagando larvas de terceiro estádio sob um microscópio estereoscópico fluorescência como descrito na secção 3. </li></ol> 2. Indução de tumores invasivos <ol><li> Use ações listadas na Tabela 2 para a indução de tumores invasivos. </li><li> Use a cultura de partida a partir do passo 1.2 acima da seguinte genótipo: y, w, ey-FLP1; Tub-Gal80, FRT40A; Act5C&gt; y +&gt; Gal4, UAS-GFP </li><li> Prepare uma cultura inicial para o estoque &quot;testado&quot; do seguinte genótipo: w; LGL 4, FRT40A, UAS-Ras V12 / cyo </li><li> Colete dez fêmeas virgens do estoque &quot;tester&quot; e dez homens do estoque &quot;testado&quot;. </li><li> Cruze os machos e fêmeas do &quot;testador&quot; e o estoque &quot;testado&quot;, colocando-os tanto em um frasco com a mosca food. </li><li> Coloque a cruz em uma incubadora de 25 ° C e permitir que os machos e as fêmeas para acasalar e pôr ovos por 24-48 horas. </li><li> Confira os frascos para a deposição de ovos e suficiente para a presença de larvas de primeiro instar certificando-se de que a cultura não está secando. <ol><li> Adicionar algumas gotas de água destilada esterilizada para a cultura para mantê-lo húmido (se a cultura está a secar para fora). </li><li> Continue a monitorar a cultura para a secura e repita o passo 2.7.1, conforme necessário. </li></ol></li><li> Observe larvas de terceiro estádio sob um microscópio estereoscópico fluorescência como descrito na secção 3. </li></ol> 3. Observação de tumores benignos e invasoras <ol><li> Use larvas de terceiro instar para isolamento tumor benigno e visualização. <ol><li> Molhar um pincel em água destilada, e utilizando esta pincel raspar algumas larvas de terceiro instar a partir da parede do frasco de cultura. </li><li> Coloque as larvas em um slide depressão com 1X PBS e em seguida, usando um pincel de pintura molhado sucataenviar um e fora de todo o material alimentar da epiderme larval. </li><li> Coloque as larvas em uma placa de Petri e deixou-se a -20 ° C congelador durante 30 minutos para imobilizar as larvas. <ol><li> Método alternativo para imobilizar larvas usando FLYNAP. </li><li> Coloque uma varinha FLYNAP mergulhado em FLYNAP para o frasco de 3.1.3.2 e depois ligar o let frasco frasco conectado com larvas representam de 30 a 40 min. </li></ol></li><li> Coloque a larva imobilizada em uma lâmina de vidro, adicione uma gota de óleo halocarbono luz, cubra com uma tampa de vidro e observar com um microscópio estereoscópico de fluorescência com a capacidade de visualizar Proteína Fluorescente Verde (GFP). </li><li> As larvas rolamento tumor benigno irá fluorescência verde na região anterior onde o complexo cefálica está presente. </li></ol></li><li> Selecione dia 10 larvas (após indução) a partir da cultura definido na etapa 2 acima para a observação de tumores invasivos. Dia 10 larvas são selecionados porque larvas rolamento tumor invasivo ter um l estendidaArval fase e geralmente não conseguem pupariate. </li><li> Siga os passos 3.1.1 a 3.1.4, exceto em vez de três dias uso larvas 10 larvas. <ol><li> As larvas dia 10 rolamento tumor invasivo fluorescência verde na região anterior onde o complexo cefálica está presente. </li></ol></li><li> Se o microscópio fluorescente é equipado com uma câmera digital, então tirar fotos das larvas rolamento tumor benigno e invasivo fluorescente. </li></ol> 4. Dissecção da Observação complexo e mais cefálica de tumores benignos e invasoras A epiderme translúcidas da larva torna difícil de observar o grau de invasão de tumores. Assim, a extensão da invasão é melhor visualizado por dissecar o complexo cefálica fora da larva. Os passos seguintes devem ser utilizados para dissecar o complexo cefálica. <ol><li> Usando um pincel molhado selecionar larvas de terceiro estádio (para tumores benignos) e dia 10 larvas (para tumores invasivos) from dos respectivos frascos de cultura. </li><li> Coloque as larvas em um poço de um prato dissecção contendo frio 1X PBS. Use pincel para raspar as partículas de alimentos da epiderme larval. <ol><li> Transferir larvas limpo para um novo bem do prato dissecção contendo frio 1X PBS. </li><li> Verifique se há presença de GFP fluorescência utilizando um microscópio estereoscópico capaz de detectar a fluorescência. Descarte larvas rolamento não-GFP. </li></ol></li><li> Adicionar 1,0 mL de PBS 1X frio para um novo bem no prato de dissecação e usando um par de fórceps transferir uma larva de rolamento, quer os tumores benignos ou invasivos (o protocolo de dissecção é o mesmo para estes dois tipos de tumores). </li><li> Segurar a larva para baixo usando um par de fórceps cerca 2/3RDS a partir da extremidade anterior. <ol><li> Use o outro par de fórceps e inteligentemente separar o 1/3rd posterior da larva e descartá-lo. </li><li> Deixe de lado o 2/3RDS anterior da larva. À medida que a pressão dentro da larva é libertado o conteúdo dolarva vai escorrer para fora da cavidade do corpo. </li><li> Utilizar um par de fórceps para remover o intestino, a gordura corporal e outros conteúdos interiores da larva que escorreu para fora da cavidade do corpo. </li><li> Use uma pinça para segurar o gancho boca da larva e empurrá-lo para dentro da epiderme larval e usar o outro par de fórceps inverter a larva completamente. </li><li> Use a pinça para suavemente e remova cuidadosamente o corpo de gordura, glândulas salivares, intestino, o complexo disco asa e quaisquer tubos traqueais. O complexo cefálica agora deve estar visível anexado ao gancho boca da larva invertido. Os hemisférios cerebrais e da medula nervosa ventral (VNC) seria ainda ser ligada à epiderme larvais através dos nervos que emanam do VNC. </li><li> Utilizar um par de pinças para quebrar suavemente as conexões entre os nervos cefálicos e epiderme larvares. </li><li> Como as ligações de nervos entre o VNC e epiderme larvas são quebrados e gordura corporal em excesso e outro tecido removido, o co cefálicamplex seria claramente visível e será anexado à epiderme larvas só nos ganchos na boca. </li><li> O complexo cefálica pode ser deixado ligado à epiderme larval se o complexo precisa ser corrigido para aplicações a jusante como a coloração de anticorpos ou para posterior visualização. </li><li> Se não há aplicações a jusante poderia ser realizada, em seguida, o complexo cefálica pode ser separado da epiderme larvais e colocados numa gota de glicerol a base meios de montagem para posterior observação e análise. Use Vectashield como a mídia de montagem. </li></ol></li></ol>

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Eu não tenho nada a divulgar.

O cancro é uma doença complexa, com uma muito melhor compreensão hoje do que no passado. No entanto, ainda há muito a ser aprendido e explicou, antes de ter um quadro completo dos mecanismos subjacentes. O protocolo simples aqui apresentada torna possível induzir geneticamente tumores benignos e invasivos num organismo inteiro e depois estudar a biologia associada com a progressão de tumores neste modelo. A maioria das técnicas existentes em Drosophila e outros organismos ou utilizar um sistema de cultur...

Progressão de tumores benignos de um para um estado metastático é um processo de passo sábio que é caracterizada por a evasão de mecanismos de protecção presentes no corpo 8. Por exemplo células tumorais no corpo deve ser capaz de evitar a apoptose e do sistema imunológico, avanço da matriz extracelular especializada (ECM) chamado Membrana Basal, e superar todos os controles sociais impostas pelas células vizinhas 8. É através de uma progressão sábio passo que as células cancerosas adquirem a capacidade de migrar e colonizar locais distantes em um processo chamado metástase. A nossa compreensão de como a célula tumoral supera as barreiras impostas pelo órgão ainda está em sua infância, no entanto, a imagem que emerge da pesquisa feita pontos, até agora, a um uso repetido de processos normais de desenvolvimento e vias de sinalização pelas células cancerosas 11-13. A mosca da fruta Drosophila melanogaster tem contribuído enormemente para a nossa understanding do desenvolvimento normal e doença através do uso de técnicas genéticas sofisticadas desenvolvidas ao longo das últimas décadas 14-17. Usando mutagênese e ferramentas superexpressão Chegamos a uma melhor compreensão de vários oncogenes e genes supressores de tumor 18-22. No entanto, a metástase tumoral é um resultado de uma cooperação entre várias lesões genéticas que tem sido estudado principalmente em modelos de cultura de células 23,24, bem como vários modelos de xenotransplante de 25-27. Estes modelos porém poderoso têm suas limitações, pois não reproduzem inteiramente as condições encontradas em um organismo vivo. Além disso, os modelos transgênicos disponíveis em ratos são pesados ​​e não contribuem para a análise genética de comportamento invasivo 28,29. Vários estudos têm tentado compreender a invasão das células tumorais em Drosophila 30,31. Estas técnicas utilizam principalmente transplante de tumores primários para anfitriões e, em seguida, contar com acompanhamento do tratumores nsplanted por invasão de tecidos vizinhos 32,33. Uma poderosa técnica chamada MARCM 10 foi adaptada por Pagliarini e Xu modelar invasão tumoral em Drosophila 9. Esta modelação genética elegante de invasão tumoral explorada a cooperação entre um oncogene activado e a perda da polaridade celular. O poder deste modelagem situa-se no facto de que os tumores invasivos são criados num organismo intacto contornando assim a necessidade de transplante de tecidos. Para provocar a cooperação oncogénica, um oncogene activado como Ras V12 é expressa em clones de células no disco olho-antenais de larvas. Como um resultado da técnica MARCM estes clones também foram marcados com a proteína verde fluorescente (GFP) para facilitar a visualização e são feitas homozigotos para mutantes de polaridade celular como larvas letal gigante, íntima, e discos grandes. O resultado é GFP marcado tumores invasivos no complexo cefálica. Neste relatório eudemonstrar como para induzir, e visualizar estes tumores invasivos, tanto no âmbito de um larvas intacta e em dissecados complexo cefálica. A indução de tumores aqui apresentada utiliza os reagentes no segundo cromossoma de Drosophila. Na Tabela 2, que fornecem um perfil de existências em X e 3 rd cromossomas que podem ser utilizados para o mesmo fim. Acredito que este protocolo simplificado vai fazer esta técnica de fácil acesso para pesquisadores interessados ​​em compreender a base molecular da progressão do tumor.

<table border="1">
	<tbody>
 <tr>
 <td>10X PBS (phosphate buffered saline) pH 7.2 stock solution</td>
 <td>Invitrogen, Sigma Aldrich</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Chilled 1X PBS pH7.2 working solution</td>
 <td>Invitrogen, Sigma Aldrich</td>
 <td></td>
 <td>Make fresh and refrigerate, can be used up to a week</td>
 </tr>
 <tr>
 <td>Flynap</td>
 <td>Carolina Biologicals</td>
 <td></td>
 <td>Fly anesthesia needed to anesthetize larvae</td>
 </tr>
 <tr>
 <td>Fixative</td>
 <td>0.1M PIPES, pH 7.2, 4% Paraformaldehyde</td>
 <td></td>
 <td>Needed to fix the dissected cephalic complex</td>
 </tr>
 <tr>
 <td>Ice Bucket</td>
 <td>Several</td>
 <td></td>
 <td>Maintain solutions on ice. Also, dissect cephalic complex in chilled 1X PBS and then place on ice in an Eppendorf tube</td>
 </tr>
 <tr>
 <td>1.7ml Eppendorf tube</td>
 <td>Various</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Glass slides, cover glass</td>
 <td>Fisher Scientific</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Vectashield Mounting Media or any other mounting media</td>
 <td>Vector Laboratories</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Halocarbon 200 or 700 Oil</td>
 <td>Polysciences Inc. or Halocarbon.com</td>
 <td></td>
 <td>Halocarbon 200 is used to mount the larvae for visualization on a fluorescence stereoscope</td>
 </tr>
 <tr>
 <td>Sally Hansen "Hard as Nails" nail polish</td>
 <td>Can be found at any general merchandise store</td>
 <td></td>
 <td>Needed to seal the edges of Coverslip</td>
 </tr>
 <tr>
 <td>A Leica MZ16.5 fluorescence stereomicroscope or any other fluorescence stereomicroscope</td>
 <td>Leica and others</td>
 <td></td>
 <td>Needed to observe the GFP fluorescence in larvae</td>
 </tr>
 <tr>
 <td>Dumont #5 forceps</td>
 <td>Fine Science Tools</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Pyrex 9 well spot plate or any other dissection dish</td>
 <td>Sigma Aldrich</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Paint Brush</td>
 <td>Can be found at any general merchandise store</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Table 1. Materials needed to perform the experimental protocol presented in this article.</td>
 </tr>
 </tbody>
 </table>

a protocol for genetic induction and visualization of benign and invasive tumors in cephalic complexes of drosophila melanogaster

Drosophila tem iluminado o nosso entendimento da base genética do desenvolvimento normal e da doença para as últimas décadas e hoje continua a contribuir imensamente para a nossa compreensão de doenças complexas 1-7. Progressão de tumores benignos de um para um estado metastático é um evento complexo 8 e foi modelado em Drosophila para nos ajudar a entender melhor a base genética da doença 9. Aqui eu apresento um protocolo simples para induzir geneticamente, observar e, em seguida, analisar a progressão de tumores em larvas de Drosophila. A técnica de indução de tumores baseia-se no sistema MARCM 10 e explora a cooperação entre um oncogene activado, Ras V12 e perda dos genes de polaridade celular (íntima, discos larvas grandes e letal gigante) para gerar tumores invasivos 9. I demonstram como esses tumores podem ser visualizados no larvas intacta eentão como estes podem ser dissecados para análise posterior. O protocolo simplificado aqui apresentados devem tornar possível que esta técnica para ser utilizado pelos investigadores interessados ​​na compreensão da função de um gene na invasão tumoral.

Como um resultado do protocolo apresentado aqui o utilizador irá ser capaz de induzir a superexpressão de tumores benignos por um oncogene activado no olho antenal disco imaginal larval. O utilizador também será capaz de induzir tumores invasivos no disco antenal olho por sobre-expressam um oncogene activado, em clones de células também para um mutante do gene da polaridade celular. Os tumores podem ser facilmente visualizadas com a ajuda de um estereomicroscópio fluorescente como o tecido &quot;fluorescente verd...

Watch this Scientific Journal Video about A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster at JoVE.com

A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster

Cooperação entre um oncogene ativado como RAS V12 e mutações em genes de polaridade celular como íntima, resultar em crescimento do tumor em Drosophila, onde as células tumorais também exibem comportamentos invasivos. Aqui, um protocolo simples para a indução e observação dos tumores benignos e invasoras é apresentado.

Um protocolo para indução genética e Visualização de tumores benignos e invasoras em Cephalic Complexos de<em&gt; Drosophila melanogaster</em

Drosophila has illuminated our  understanding of the genetic basis of normal development and disease for the  past several decades and today it continues ...

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Research

JoVE Journal

Medicine

A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster

9.4K visualizações.  Western Kentucky University.  Cooperação entre um oncogene ativado como RAS V12 e mutações em genes de polaridade celular como íntima, resultar em crescimento do tumor em Drosophila, onde as células tumorais também exibem comportamentos invasivos. Aqui, um protocolo simples para a indução e observação dos tumores benignos e invasoras é apresentado. 

Vídeo: A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

Estabelecimento e propagação de Tumores Humanos em Retinoblastoma Imune camundongos deficientes

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

Medicina

Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants

Perinatal brain injury remains a significant cause of infant mortality and morbidity, but there is not yet an effective bedside tool that can accurately screen for brain injury, monitor injury evolution, or assess response to therapy. The energy used by neurons is derived largely from tissue oxidative metabolism, and neural hyperactivity and cell death are reflected by corresponding changes in cerebral oxygen metabolism (CMRO2). Thus, measures of CMRO2 are reflective of neuronal viability and provide critical diagnostic information, making CMRO2 an ideal target for bedside measurement of brain health.
Brain-imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) yield measures of cerebral glucose and oxygen metabolism, but these techniques require the administration of radionucleotides, so they are used in only the most acute cases.
Continuous-wave near-infrared spectroscopy (CWNIRS) provides non-invasive and non-ionizing radiation measures of hemoglobin oxygen saturation (SO2) as a surrogate for cerebral oxygen consumption. However, SO2 is less than ideal as a surrogate for cerebral oxygen metabolism as it is influenced by both oxygen delivery and consumption. Furthermore, measurements of SO2 are not sensitive enough to detect brain injury hours after the insult 1,2, because oxygen consumption and delivery reach equilibrium after acute transients 3. We investigated the possibility of using more sophisticated NIRS optical methods to quantify cerebral oxygen metabolism at the bedside in healthy and brain-injured newborns. More specifically, we combined the frequency-domain NIRS (FDNIRS) measure of SO2 with the diffuse correlation spectroscopy (DCS) measure of blood flow index (CBFi) to yield an index of CMRO2 (CMRO2i) 4,5.
With the combined FDNIRS/DCS system we are able to quantify cerebral metabolism and hemodynamics. This represents an improvement over CWNIRS for detecting brain health, brain development, and response to therapy in neonates. Moreover, this method adheres to all neonatal intensive care unit (NICU) policies on infection control and institutional policies on laser safety. Future work will seek to integrate the two instruments to reduce acquisition time at the bedside and to implement real-time feedback on data quality to reduce the rate of data rejection.

We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain.

Não-invasivo de medição óptica do metabolismo cerebral e Hemodinâmica em Lactentes

Perinatal brain injury remains a significant cause of infant mortality  and morbidity, but there is not yet an effective bedside tool that can  accurately ...

Renal Capsule Xenografting and Subcutaneous Pellet Implantation for the Evaluation of Prostate Carcinogenesis and Benign Prostatic Hyperplasia

New therapies for two common prostate diseases, prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend critically on experiments evaluating their hormonal regulation. Sex steroid hormones (notably androgens and estrogens) are important in PrCa and BPH; we probe their respective roles in inducing prostate growth and carcinogenesis in mice with experiments using compressed hormone pellets. Hormone and/or drug pellets are easily manufactured with a pellet press, and surgically implanted into the subcutaneous tissue of the male mouse host. We also describe a protocol for the evaluation of hormonal carcinogenesis by combining subcutaneous hormone pellet implantation with xenografting of prostate cell recombinants under the renal capsule of immunocompromised mice. Moreover, subcutaneous hormone pellet implantation, in combination with renal capsule xenografting of BPH tissue, is useful to better understand hormonal regulation of benign prostate growth, and to test new therapies targeting sex steroid hormone pathways.

We describe the manufacture of compressed hormone pellets, as well as subcutaneous surgical implantation into mice. This strategy can be combined with the growth of cell and tissue xenografts under the renal capsule of athymic nude mice to evaluate hormonal carcinogenesis and regulation of benign prostate growth.

Renal Capsule xeno e subcutânea Pellet Implantação de Avaliação de próstata Carcinogênese e hiperplasia benigna da próstata

New therapies for two common prostate diseases,  prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend  critically on experiments ...

Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-&#947;)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.

An N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer model was developed in human mucin 1 (MUC1) transgenic mice for the purpose of testing MUC1-directed immunotherapy. After administering a MUC1-targeted peptide vaccine, a cytotoxic T lymphocyte response to MUC1 was confirmed by measuring serum cytokine levels and T-cell specific activity.

Indução de carcinoma da bexiga invasivo de células transicionais em imunológico intacto Humanos MUC1 Ratos transgênicos: um modelo de desenvolvimento Imunoterapia

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

Induction of Accelerated Atherosclerosis in Mice: The &quot;Wire-Injury&quot; Model

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Indução de Aterosclerose Acelerada em Camundongos: O Modelo de "Lesão por Fio"

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Intravital Microscopy and Thrombus Induction in the Earlobe of a Hairless Mouse

Thrombotic complications of vascular diseases are one leading cause of morbidity and mortality in industrial nations. Due to the complex interactions between cellular and non-cellular blood components during thrombus formation, reliable studies of the physiology and pathophysiology of thrombosis can only be performed in vivo. Therefore, this article presents an ear model in hairless mice and focuses on the in vivo analysis of microcirculation, thrombus formation, and thrombus evolution. By using intravital fluorescence microscopy and the intravenous (iv) application of the respective fluorescent dyes, a repetitive analysis of microcirculation in the auricle can easily be performed, without the need for surgical preparation. Furthermore, this model can be adapted for in vivo studies of different issues, including wound healing, reperfusion injury, or angiogenesis. In summary, the ear of hairless mice is an ideal model for the in vivo study of skin microcirculation in physiological or pathophysiological conditions and for the evaluation of its reaction to different systemic or topical treatments.

The ear model of the hairless SKH1-Hrhr mouse enables intravital fluorescence microscopy of microcirculation and phototoxic thrombus induction without prior surgical preparation in the examined microvascular bed. Therefore, the ear of the hairless mouse is an excellent in vivo model to study the complex interactions during microvascular thrombus formation, thrombus evolution, and thrombolysis.

Intravital Microscopia e trombo Indução no lóbulo da orelha de um rato calvo

Thrombotic complications of vascular diseases are one leading cause of morbidity and mortality in industrial nations. Due to the complex interactions ...

Technique of Minimally Invasive Transverse Aortic Constriction in Mice for Induction of Left Ventricular Hypertrophy

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure overload-induced left ventricular hypertrophy (LVH) and its progression to heart failure. In the majority of the reported investigations, this procedure is performed with intubation and ventilation of the animal which renders it demanding and time-consuming and adds to the surgical burden to the animal. The aim of this protocol is to describe a simplified technique of minimally invasive TAC without intubation and ventilation of mice. Critical steps of the technique are emphasized in order to achieve low mortality and high efficiency in inducing LVH.
Male C57BL/6 mice (10-week-old, 25-30 g, n=60) were anesthetized with a single intraperitoneal injection of a mixture of ketamine and xylazine. In a spontaneously breathing animal following a 3-4 mm upper partial sternotomy, a segment of 6/0 silk suture threaded through the eye of a ligation aid was passed under the aortic arch and tied over a blunted 27-gauge needle. Sham-operated animals underwent the same surgical preparation but without aortic constriction. The efficacy of the procedure in inducing LVH is attested by a significant increase in the heart/body weight ratio. This ratio is obtained at days 3, 7, 14 and 28 after surgery (n = 6 - 10 in each group and each time point). Using our technique, LVH is observed in TAC compared to sham animals from day 7 through day 28. Operative and late (over 28 days) mortalities are both very low at 1.7%.
In conclusion, our cost-effective technique of minimally invasive TAC in mice carries very low operative and post-operative mortalities and is highly efficient in inducing LVH. It simplifies the operative procedure and reduces the strain put on the animal. It can be easily performed by following the critical steps described in this protocol.

The aim of this protocol is to describe step-by-step the technique of minimally invasive transverse aortic constriction (TAC) in mice. By elimination of intubation and ventilation which are mandatory for the commonly used standard procedure, minimally invasive TAC simplifies the operative procedure and reduces the strain put on the animal.

Técnica de constrição da aorta minimamente invasiva transversal em camundongos para indução de Hipertrofia Ventricular Esquerda

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure ...

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively affect skeletal muscle. This can result in a loss of muscle mass (atrophy) and/or loss of muscle quality (reduced force per unit of muscle mass), both of which are prevalent in chronic disease, muscle-specific disease, immobilization, and aging (sarcopenia). Skeletal muscle function in animals can be evaluated by a range of different tests. All tests have limitations related to the physiological testing environment, and the selection of a specific test often depends on the nature of the experiments. Here, we describe an in vivo, non-invasive technique involving a helpful and easy assessment of force frequency-curve (FFC) in mice that can be performed on the same animal over time. This permits monitoring of disease progression and/or efficacy&#160;of a potential therapeutic treatment.

Measurement of rodent skeletal muscle contractile function is a useful tool that can be used to track disease progression as well as efficacy of therapeutic intervention. We describe here the non-invasive, in vivo assessment of the dorsiflexor muscles that can be repeated over time in the same mouse.

Avaliação não-invasiva da função de músculos levantadores em ratos

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively ...

Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated insulin release. Assessment of mitophagy is challenging and often requires genetic reporters or multiple complementary techniques not easily utilized in tissue samples, such as primary human pancreatic islets. Here we demonstrate a robust approach to visualize and quantify formation of key endogenous mitophagy complexes in primary human pancreatic islets. Utilizing the sensitive proximity ligation assay technique to detect interaction of the mitophagy regulators NRDP1 and USP8, we are able to specifically quantify formation of essential mitophagy complexes in situ. By coupling this approach to counterstaining for the transcription factor PDX1, we can quantify mitophagy complexes, and the factors that can impair mitophagy, specifically within beta cells. The methodology we describe overcomes the need for large quantities of cellular extracts required for other protein-protein interaction studies, such as immunoprecipitation (IP) or mass spectrometry, and is ideal for precious human islet&#160;samples generally not available in sufficient quantities for these approaches. Further, this methodology obviates the need for flow sorting techniques to purify beta cells from a heterogeneous islet population for downstream protein applications. Thus, we describe a valuable protocol for visualization of mitophagy highly compatible for use in heterogeneous and limited cell populations.

This protocol outlines a method for quantitative analysis of mitophagy protein complex formation specifically in beta cells from primary human islet samples. This technique thus allows analysis of mitophagy from limited biological material, which are crucial in precious human pancreatic beta cell samples.

Visualização de complexos de Mitophagy endógenos in situ em células beta pancreáticas humanas utilizando o ensaio de ligadura de proximidade

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

Um modelo pré-clínico do rato para o estudo da lesão da Isquemia-reperfusion na microcirurgia reconstrutiva

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...