Os autores gostariam de agradecer Brian Muntean, Shao Lo, Maki Takahashi e Blair Mell para a sua assistência técnica. Este trabalho foi financiado por concessões do NIH (DK080640) e da Universidade de Toledo &amp; ProMedica Prêmio Estímulo à Pesquisa Translacional Dr. Surya Nauli.

1. Pré-tratamento de célula e preparação metáfase <ol><li> As células são cultivadas em alteraç de Dulbecco de Medium de Eagle (DMEM) contendo 10-15% de soro fetal bovino (FBS) e 1% de penicilina / estreptomicina a 37 ° C, com 5% de CO 2 incubadora, até que eles chegar a 70-80% de confluência. </li><li> Trate as células com solução de Colcemid em 0,05 ug / ml para 30-60 min. </li><li> Recolher o meio contendo as células de flutuação em tubos estéreis de 50 ml de centrífuga de falcão. Lavar as células sobre a placa com sterile1X-PBS. Depois de incubar com tripsina estéril para 1-2 min, colheita e coletar as células restantes para dentro do tubo mesmo. </li><li> Spin os tubos a 1000 rpm durante 5 min, aspirar o sobrenadante deixando 0,5 ml e afrouxe pellet por flicking com o dedo somente. </li><li> Dependendo do tamanho do pellet, adicione 5-10 ml de solução hipotônica de KCl 0,56% em dH 2 O e incubar a suspensão a 37 ° C por 30-45 min. </li><li> Adicionar uma gota de metanol / ácido acético(3:1 vol / vol) por ml de suspensão de células hipotônica e inverta suavemente o tubo para a mistura. </li><li> Centrifugar a 1200 rpm durante 5 min e coletar pellet como passo 1,4, em seguida, adicionar 5 ml de metanol fresco / ácido acético (3:1 vol / vol) gota a gota solução fixadora enquanto flicking o pellet continously. Este procedimento é crítico de modo a que os spreads metafásicas não vai ser preso em clumps as células &#39;que comprometeriam o experimento. </li><li> Centrifugar novamente a 1200 rpm durante 5 min e adicione 5-10 ml de gelo-frio de metanol / fixador de ácido acético ao longo da parede do tubo. Na presente fase, se necessário, as células podem ser armazenadas em solução fixadora em um tubo de apertados e selado à temperatura de -20 ° C para o termo curto ou -80 ° C durante a longo prazo (anos) para uso futuro. </li><li> Slides limpas em etanol absoluto, em seguida, mergulhar o slide no dH 2 O para approximately10X, a fim de formar uma bainha de água sobre a superfície do slide. Coloque o slide sobre uma placa de vidro e gota 15-20 uL de suspensão de células (a partir do passo 1,8) A partir de 10 &quot;acima o slide. Coloque o slide em um banho de água fixado em 65-70 ° C durante 1-2 min e permitir que para secar. </li><li> Verifique se o slide sob um microscópio de luz, utilizando 10X e 40X objetivos secos certificando-se há cromossomos metafásicos e os spreads são uniformemente espaçados. Verifique a presença citoplasma ao redor dos cromossomos. Se citoplasma é presente proceder com pré-tratamento de slide (digestão pepsina), se nenhum citoplasma está presente e os cromossomas tem boa morfologia, em seguida, não há nenhuma necessidade para pré-tratamento slide. </li></ol> 2. Pré-tratamento de slides (pepsina tratamento) <ol><li> Aplicar 120 uL de 1:200 solução de RNase (20 mg / ml) dissolvido em 2X-SSC sobre um milímetro 24 lamínula microscópio x 60 mm e inverta o rosto de slide metáfase para baixo sobre o lamínula, em seguida, inverter cuidadosamente o rosto de slide metáfase-se e incubar a 37 ° C durante 45 min. </li><li> Retire cuidadosamente a lamínula sem arranhar a lâmina e lavar com 2X SSC-tampão em uma jarra de Coplin cada 5 min para 15 mincom agitação. </li><li> Adicionar 5-15 uL de pepsina solução estoque (100 mg / ml em dH 2 O), para um copo limpo e em seguida, adicione 100 ml de pré-aquecido (37 ° C) 0,01 M de HCl. É importante que a pepsina é adicionado em um limpo proveta de primeira e não diretamente para dentro da solução HCl, caso contrário ele não iria se dissolver em solução. Incubar o slide em uma jarra de Coplin contendo a solução de HCl / pepsina nas concentrações de 37 ° C durante 3-5 min. Este passo é muito crítico como a digestão demasia fará com que os cromossomos a ser overdigested e demasiado pouca digestão vai deixar o citoplasma não digerido que poderá conduzir a ligação não-específica da sonda e interferir com o sinal de hibridação. </li><li> Lave o slide em uma jarra de Coplin contendo 100 ml 1X-PBS durante 5 min duas vezes à temperatura de quarto. </li><li> Lave o slide em uma jarra de Coplin contendo 100 ml 1X-PBS/MgCl 2 para 5 min à temperatura ambiente (50 ml de MgCl 1M n º 2 em 950 ml de 1X-PBS). </li><li> Coloque o slide em uma jarra de Coplin containing 100 formaldeído ml 1% para 10 min à temperatura ambiente (1,7 ml de formaldeído a 37% em 100 ml de 1X-PBS/MgCl 2). </li><li> Lave o slide em uma jarra de Coplin contendo 100 ml 1X-PBS durante 5 min. </li><li> Observar o slide ao abrigo de um microscópio de luz usando lente seca de 40X para assegurar de que os slides são digeridos corretamente e nenhuma citoplasma está presente e a morfologia cromossomo é preservada. Selecione uma área para a hibridização usando uma caneta de diamante. </li></ol> 3. Cromossoma sonda e desnaturação e hibridização <ol><li> Prepare a solução de desnaturação fresco (70% formamide/2X SSC, pH 7,0) e Pré-aquecer a 70-80 ° C em uma jarra de Coplin colocado em um Lugar banho de água o slide no jarra de Coplin contendo a solução de desnaturação em um banho de água a 70 ° C para os cromossomos do rato e em 80 ° C para os cromossomos humanos para a 30s-1.5 min. </li><li> Imediatamente coloque o slide em etanol de gelo-frio 70% para 3 min seguido por 80% e etanol a 100% por 3 min cada um e de ar seco. Examine o slidede morfologia cromossômica. Morfologia cromossômica Bom é denotada por cromossomos escuros e não &quot;fase de luz&quot; ou halo cromossomos. </li><li> Aqueça a sonda de SkyPaint (kit de pintura SKY; frasco para injectáveis ​​# 1) a 37 ° C com agitação durante 20 min vórtice, e centrifugar brevemente a 1000 rpm durante alguns segundos. </li><li> Desnaturar a sonda em um termociclador programado para um ciclo de dois-passo de cada 85 ° C durante 5 ciclo de min seguido por 37 ° C por 60 min para permitir que sonda marcada com-de DNA para preannealing. </li><li> Aplicar 10 ul da sonda desnaturada para a área de hibridação e tampa com uma 22 mm x 40 mm lamela tomando cuidado para não prender as bolhas de ar. Selar as bordas da lamínula com cimento de borracha e incubar em uma câmara de umidificado a 37 ° C durante 48-72 hrs. </li></ol> 4. Detecção de sonda fluorescente <ol><li> Remova a lamínula cuidadosamente e coloque o slide em uma jarra de Coplin contendo pré-aquecido (45 ° C) de lavar roupa solução que eu (preparado de fresco formamida a 50% em 2X SSC). Lave por 5 mintrês vezes a 45 ° C em um banho de água agitação a 45 rpm </li><li> Lave o slide em uma solução de lavagem II (1X SSC) a 45 ° C por 5 min duas vezes com a tremer. </li><li> Lave o slide em uma solução de lavagem III (% SSC/0.1 4X de Tween 20) durante 5 min a 45 ° C com agitação. </li><li> Aplicar 80 uL de reagente de bloqueio (kit de pintura SKY; frasco para injectáveis ​​# 2), coloque uma lamela e incubar a 37 ° C durante 30 min. </li><li> Remova o slide e permita que o fluido para drenar. Aplicar 80 uL de Cy5 reagente de coloração (Concentrada anticorpo de detecção CAD kit; frasco para injectáveis ​​# 3), aplique uma lamínula e incubar a 37 ° C durante 40 min. </li><li> Lave o slide com uma solução de lavagem III a 45 ° C por 5 min três vezes com a tremer. </li><li> Aplicar 80 uL de Cy5.5 reagente de coloração (Concentrada anticorpo de detecção CAD kit; frasco para injectáveis ​​# 4), coloque uma lamínula e incubar a 37 ° C durante 40 min. </li><li> Lave o slide com uma solução de lavagem III a 45 ° C por 5 min três vezes com a tremer. </li><li> Incline o slide e permitir que o fluido a<html> escorrer. Aplicar 20 ul do reagente DAPI anti-fade (kit de pintura SKY; frasco # 5) e colocar uma lamela de microscópio 24 mm x 60 mm. Retire cuidadosamente as bolhas de ar que são formadas. Slides pode ser imaged imediatamente ou armazenado a 4 ° C no escuro por nenhuma mais tempo do que 1 semana. </li></ol> 5. Aquisição e análise de imagem <ol><li> De aquisição de imagem é realizado por que visualizaram o slides metáfase usando um microscópio Olympus equipado com uma lente de 60X óleo de imersão, um cubo Espectral (personalizado projetado triplo band-pass filtro), um filtro de DAPI e um módulo de interferômetro de Sagnac com uma câmera CCD. </li><li> Spectral-cariótipos foram realizadas usando o software SKY Ver (Versão imagem Aplicada espectral 1,62) Seguindo o manual do usuário. </li><li> Depois de analisar as imagens, os cromossomos podem ser visualizadas como imagens de cores específicas (com cores fluorescentes), imagens a cores (com pseudo cores para a classificação) e imagens invertidas DAPI (padrão de bandas específico). </li></ol>e_title &quot;&gt; 6. resultados representativos Um procedimento de SKY completa geralmente leva cerca de uma tempo de semanas (Figura 1). Isso inclui aquisição de imagem e de análise, desde que as células em metafase, estão em oferta adequada. Análise de cariotipagem revela cariótipo mouse normal (40, XY) de células de camundongos do tipo selvagem (Figura 2a). Em contraste, as células a partir de Pkd1 - / - do mouse (PKD modelo do rato) mostra um aumento significativo no número de cromossomos e anormalidades estruturais, tais como as deleções cromossômicas (cromossomo # 8) e translocações (cromossomos # 11 e 19) (Figura 2b). Analisamos também os tecidos vasculares dos pacientes DPRAD. Um estudo simples por meio da contagem os números de cromossômicas indicaram que não-ADPKD e algumas amostras DPRAD vasculares teve normais números de cromossômicas de 23 pares (Figura 2c). Em geral, porém, observou-se falha de segregação cromossômica, resultando em 46 pares de cromossomos em UmAmostras de DPKD em vez de 23 pares (Figura 2d). <img alt="Figura 1" src="/files/ftp_upload/3887/3887fig1.jpg" /> Figura 1. SKY fluxograma protocolo. Fluxograma do protocolo SKY ilustra passos para completar uma experiência a partir de pré-tratamento das células e preparações metáfase para aquisição e análise de imagem. Um cronograma de uma semana aproximada é apresentado à esquerda com o passo-a-passo os procedimentos para cada dia. <img alt="Figura 2" src="/files/ftp_upload/3887/3887fig2.jpg" /> Figura 2. Cariotipagem espectral em linhas mouse imortalizados celulares e recém-isoladas células humanas primárias. Cores e imagens invertidas DAPI de cromossomos individuais são mostrados antes a classificação cromossômica. Após a triagem, os cromossomos são apresentados na &quot;classificação&quot; da tabela. Uma imagem SKY) de um spread metáfase de rato do tipo selvagem contém cariótipo normal (40, XY). B) Pkd1 - / -. mouse mostra o número de cromossomos anormais (68 em vez de 40) e alterações cromossômicas, como deleção do cromossomo (cromossomo # 8) e translocações cromossômicas (cromossomos 11 e # 19) c) SKY imagem de um spread metáfase do tecido vascular de um paciente não tem ADPKD cariótipo normal (46, XX). d) SKY imagem de um spread metáfase do tecido vascular de um paciente ADPKD contém cariótipo anormal (92, XXXX). Parte dos dados foram previamente relatados e reutilizado com permissão 14.

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Cariotipagem espectral (SKY) é uma técnica utilizada em citogenética estudar composições genômicas e cromossômicas. Essa técnica leva vantagem das sondas de pintura de cromossomos, ea detecção destas sondas são adquiridas através de um interferômetro de Sagnac. O processo de SKY completa geralmente leva cerca de uma semana, e ele, envolve várias etapas-chave (Figura 1). A SKY usa um protocolo padrão, que foi primeiramente descrita por Padilla-Nash et al 3. O protocolo ...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de Catálogo </td><td> Comentários </td></tr><tr><td> DMEM </td><td> Cellgro Mediatech Inc. </td><td> 10-013-CV </td><td></td></tr><tr><td> Fetal de soro de bovino (FBS) </td><td> Hyclone </td><td> SH30088-03 </td><td></td></tr><tr><td> Penecillin Estreptomicina / </td><td> Thermo Scientific </td><td> SV30010 </td><td></td></tr><tr><td> Colcemid </td><td> Roche Diagnostics </td><td> 10 295 892 001 </td><td> 10 ug / ml </td></tr><tr><td> HCl </td><td> Fisher Scientific </td><td> A144-500 </td><td></td></tr><tr><td> KCl </td><td> Fisher Scientific </td><td> S77375-1 </td><td></td></tr><tr><td> Fosfato de solução salina tamponada com </td><td> Thermo Scientific </td><td> SH30256-01 </td><td></td></tr> &lt;tr&gt; <td> SKY pintura sonda kit (Humana) </td><td> Aplicado imagem espectral </td><td> SKY000028 </td><td></td></tr><tr><td> SKY pintura sonda kit (Mouse) </td><td> Aplicado imagem espectral </td><td> SKY000030 </td><td></td></tr><tr><td> Kit de detecção de Concentrada anticorpo </td><td> Aplicado imagem espectral </td><td> SKY000033 </td><td></td></tr><tr><td> Tripsina </td><td> Thermo Scientific </td><td> SH30236.01 </td><td></td></tr><tr><td> Metanol </td><td> Fisher Scientific </td><td> A433P-4 </td><td></td></tr><tr><td> O ácido acético </td><td> Fisher Scientific </td><td> A38-212 </td><td></td></tr><tr><td> RNase A </td><td> Roche Diagnostics </td><td> 10 109 169 001 </td><td></td></tr><tr><td> Pepsina </td><td> Sigma-Aldrich </td><td> P6887-5G </td><td></td></tr><tr><td> MgCl 2 </td><td> Fluka </td><td> 63069-500ML </td><td></td></tr><tr><td> Formaldeído 37% </td><td> Mallinckrodt Baker </td><td> 2106-02 </td><td></td></tr><tr><td> 20X SSC </td><td> Promega </td><td> V4261 </td><td></td></tr><tr><td> Formamida </td><td> Fluka </td><td> 47671 </td><td> preparar pouco antes de usar </td></tr><tr><td> Tween-20 </td><td> Fisher Scientific </td><td> BP337-500 </td><td></td></tr><tr><td> Vidro lâminas de microscópio </td><td> Fisher Scientific </td><td> 12-549 </td><td></td></tr><tr><td> Microscópio tampa de vidro 24x60mm </td><td> VWR Vista Visão </td><td> 16004-312 </td><td></td></tr><tr><td> Borracha cimento </td><td> Elmer (loja de varejo local) </td><td></td><td></td></tr><tr><td> Hibridação humidifiedchamber / / Bandeja </td><td> Simport </td><td> M920-2 </td><td> colocar toalhas de papel molhadas no fundo </td></tr><tr><td> Thermociclista </td><td> Eppendorf </td><td> Epgradient S </td><td></td></tr><tr><td> Plataforma de agitação / agitador orbital </td><td> Bélico Biotecnologia </td><td></td><td></td></tr><tr><td> Agitando banheira / água </td><td> Precisão científica </td><td></td><td></td></tr><tr><td> DAPI cubo filtro </td><td> Tecnologia Chroma </td><td></td><td></td></tr><tr><td> SKY cubo filtro </td><td> Tecnologia Chroma </td><td></td><td></td></tr><tr><td> SpectraCube </td><td> Aplicado imagem espectral </td><td></td><td></td></tr><tr><td> Microscópio invertido cultura de células </td><td> Nikon </td><td> Nikon Eclipse TS100 </td><td></td></tr><tr><td> Microscópio de fluorescência </td><td> Olimpo </td><td> IX70 </td><td> Óleo de 60X </td></tr></tbody></table>

spectral karyotyping to study chromosome abnormalities in humans and mice with polycystic kidney disease

Método convencional para identificar e classificar cromossomos individuais depende do padrão único de bandagem de cada cromossomo em uma espécie específicos que estão sendo analisados ​​1, 2. Esta técnica clássica de bandagem, no entanto, não é confiável na identificação de complexos aberrações cromossômicas, tais como aqueles associados com o câncer. Para superar as limitações da técnica de anilhamento, cariotipagem espectral (SKY) é introduzido para fornecer informações confiáveis ​​tanto em anormalidades cromossômicas. SKY é uma fluorescência multicolor hibridização in-situ (FISH) técnica para detectar cromossomos em metafase, com microscópio espectral 3, 4. SKY tem sido comprovada para ser uma ferramenta valiosa para a análise citogenética de uma ampla gama de anormalidades cromossômicas associados com um grande número de doenças genéticas e doenças malignas 5, 6. SKY envolve o uso de sondas de DNA multicolor fluorescentemente-com o Rótulo preparados a partir de o oligonucle degenerarprimers otide por PCR. Assim, cada cromossoma tem uma cor exclusiva espectral depois de hibridização in-situ com sondas, que são diferencialmente rotulados com uma mistura de corantes fluorescentes (Rodamina, Texas Vermelho, Cy5, FITC e Cy5.5). As sondas utilizadas para SKY consistir de até 55 sondas cromossômicas específicas 7-10. O procedimento para a SKY envolve várias etapas (Figura 1). SKY exige a disponibilidade de células com alto índice mitótico a partir de tecido normal ou doente ou sangue. Os cromossomos de uma única célula a partir de qualquer célula de um recém-isolado primária ou uma linha de células estão espalhados em um slide de vidro. Esse spread cromossomo é rotulado com uma combinação diferente de corantes fluorescentes específicos para cada cromossomo. Para a detecção da sonda e de aquisição de imagem, o sistema de imagem espectral consiste de interferômetro de Sagnac e uma câmera CCD. Este permite a medição do espectro de luz visível emitida a partir da amostra e para adquirir uma imagem espectral frocromossomos m individuais. HiSKY, o software usado para analisar os resultados das imagens capturadas, proporciona uma fácil identificação de anomalias cromossômicas. O resultado final é uma metáfase e uma imagem de classificação cariótipo, no qual cada par de cromossomos tem uma cor distinta (Figura 2). Isso permite a fácil identificação de identidades cromossômicas e translocações. Para mais detalhes, visite o site da Imagem Aplicada (Spectral <a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a> ). SKY foi recentemente usada para a identificação de defeitos cromossômicos de segregação e anormalidades cromossômicas em humanos e camundongos com doença renal policística autossômica dominante (ADPKD), uma doença genética caracterizada por uma disfunção em cílios 11-13. Usando esta técnica, foi demonstrada a presença de segregação cromossômica anormal e defeitos cromossômicos em pacientes DPRAD e modelos de mouse 14. Outras análises usando SKY não só nos permitiu identificar o número cromossômico e identidade, mas também para detectar com precisão muito complexos aberrações cromossômicas, como translocações e deleções cromossômicas (Figura 2).

Watch this Scientific Journal Video about Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease at JoVE.com

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Cariotipagem espectral (SKY) é uma técnica avançada citogenética para identificar as aberrações cromossômicas e cromossômicas. Essa técnica leva vantagem das sondas cromossômicas de pintura, que permitem a classificação de todos os cromossomos. SKY também pode identificar aberrações cromossômicas e defeitos complexos de segregação em camundongos e humanos, com várias doenças, incluindo doença renal policística.

Cariotipagem espectral para o Estudo de Anomalias cromossômicas em humanos e camundongos com doença renal policística

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being ...

spectral-karyotyping-to-study-chromosome-abnormalities-humans-mice

Research

JoVE Journal

Medicine

38.2K visualizações.  University of Toledo, College of Pharmacy and Pharmaceutical Sciences. Cariotipagem espectral (SKY) é uma técnica avançada citogenética para identificar as aberrações cromossômicas e cromossômicas. Essa técnica leva vantagem das sondas cromossômicas de pintura, que permitem a classificação de todos os cromossomos. SKY também pode identificar aberrações cromossômicas e defeitos complexos de segregação em camundongos e humanos, com várias doenças, incluindo doença renal policística.

Vídeo: Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Orthotopic Aortic Transplantation in Mice for the Study of Vascular Disease

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. This is largely true, but there are a number of important principles that can reduce the difficulty of these procedures and enhance reproducibility. Orthotopic aortic transplantation is an excellent procedure in which to learn these principles because it involves only two end-to-end anastomoses, but requires good suturing technique and handling of the vessels for consistent success. This procedure begins with the procurement of a length of abdominal aorta from a donor animal, followed by division of the native aorta in the recipient. The procured aorta is then placed between the divided ends of the recipient aorta and sutured into place using end-to-end anastomoses. To accomplish this objective successfully requires a high degree of concentration, good tools, a steady hand, and an appreciation of how easily the vasculature of a mouse can be damaged, resulting in thrombosis. Learning these important principles is what occupies most of the beginner's time when learning microsurgery in small rodents. Throughout this protocol, we refer to these important points. This model can be used to study vascular disease in a variety of different experimental systems1-8. In the context shown here, it is most often used for the study of post-transplant vascular disease, a common long-term complication of solid organ transplantation in which intimal hyperplasia occurs within the allograft. The primary advantage of the model is that it facilitates quantitative morphometric analyses and the transplanted vessel lies contiguous to the endogenous vessel, which can serve as an additional control9. The technique shown here is most often used for mice weighing 18-25 grams. We have accumulated most of our experience using the C57BL/6J, BALB/cJ, and C3H/HeJ strains.

We describe a technique in which a section of the abdominal aorta from a mouse is transplanted orthotopically to just below the renal arteries in an allogeneic or syngeneic recipient. This technique can be useful in studies in which transplantation of large arteries of uniform size is deemed advantageous.

Transplante ortotópico de aorta em camundongos para o Estudo da Doença Vascular

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. ...

Medicina

5/6th Nephrectomy in Combination with High Salt Diet and Nitric Oxide Synthase Inhibition to Induce Chronic Kidney Disease in the Lewis Rat

Chronic kidney disease (CKD) is a global problem. Slowing CKD progression is a major health priority. Since CKD is characterized by complex derangements of homeostasis, integrative animal models are necessary to study development and progression of CKD. To study development of CKD and novel therapeutic interventions in CKD, we use the 5/6th nephrectomy ablation model, a well known experimental model of progressive renal disease, resembling several aspects of human CKD. The gross reduction in renal mass causes progressive glomerular and tubulo-interstitial injury, loss of remnant nephrons and development of systemic and glomerular hypertension. It is also associated with progressive intrarenal capillary loss, inflammation and glomerulosclerosis. Risk factors for CKD invariably impact on endothelial function. To mimic this, we combine removal of 5/6th of renal mass with nitric oxide (NO) depletion and a high salt diet. After arrival and acclimatization, animals receive a NO synthase inhibitor (NG-nitro-L-Arginine) (L-NNA) supplemented to drinking water (20 mg/L) for a period of 4 weeks, followed by right sided uninephrectomy. One week later, a subtotal nephrectomy (SNX) is performed on the left side. After SNX, animals are allowed to recover for two days followed by LNNA in drinking water (20 mg/L) for a further period of 4 weeks. A high salt diet (6%), supplemented in ground chow (see time line Figure 1), is continued throughout the experiment. Progression of renal failure is followed over time by measuring plasma urea, systolic blood pressure and proteinuria. By six weeks after SNX, renal failure has developed. Renal function is measured using 'gold standard' inulin and para-amino hippuric acid (PAH) clearance technology. This model of CKD is characterized by a reduction in glomerular filtration rate (GFR) and effective renal plasma flow (ERPF), hypertension (systolic blood pressure&gt;150 mmHg), proteinuria (&gt; 50 mg/24 hr) and mild uremia (&gt;10 mM). Histological features include tubulo-interstitial damage reflected by inflammation, tubular atrophy and fibrosis and focal glomerulosclerosis leading to massive reduction of healthy glomeruli within the remnant population (&lt;10%). Follow-up until 12 weeks after SNX shows further progression of CKD.

A two-stage method to establish chronic kidney disease (CKD) in the Lewis rat by surgically removing 5/6th of renal mass is described. Combination of the surgical procedure, NOS-inhibition and a high-salt diet leads to a model resembling human CKD, allowing study of causal mechanisms and development of novel therapeutic interventions.

5/6th Nephrectomy em combinação com uma dieta alta de sal e Óxido Nítrico Sintase inibição para induzir o Doença Renal Crônica no Rato Lewis

Chronic kidney disease (CKD) is a global problem. Slowing CKD progression is a major health priority. Since CKD is characterized by complex derangements ...

Assessment of Vascular Function in Patients With Chronic Kidney Disease

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this is only partially explained by traditional CVD risk factors. Vascular dysfunction is an important non-traditional risk factor, characterized by vascular endothelial dysfunction (most commonly assessed as impaired endothelium-dependent dilation [EDD]) and stiffening of the large elastic arteries. While various techniques exist to assess EDD and large elastic artery stiffness, the most commonly used are brachial artery flow-mediated dilation (FMDBA) and aortic pulse-wave velocity (aPWV), respectively. Both of these noninvasive measures of vascular dysfunction are independent predictors of future cardiovascular events in patients with and without kidney disease. Patients with CKD demonstrate both impaired FMDBA, and increased aPWV. While the exact mechanisms by which vascular dysfunction develops in CKD are incompletely understood, increased oxidative stress and a subsequent reduction in nitric oxide (NO) bioavailability are important contributors. Cellular changes in oxidative stress can be assessed by collecting vascular endothelial cells from the antecubital vein and measuring protein expression of markers of oxidative stress using immunofluorescence. We provide here a discussion of these methods to measure FMDBA, aPWV, and vascular endothelial cell protein expression.

The degree of vascular dysfunction and contributing physiological mechanisms can be assessed in patients with chronic kidney disease by measuring brachial artery flow-mediated dilation, aortic pulse-wave velocity, and vascular endothelial cell protein expression.

Avaliação da função vascular em pacientes com doença renal crônica

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this ...

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The molecular mechanisms leading to cyst formation in PKD remain somewhat unclear, but many genes are thought to be involved. Wnt5a is a non-canonical glycoprotein that regulates a wide range of developmental processes. Wnt5a works through the planar cell polarity (PCP) pathway that regulates oriented cell division during renal tubular cell elongation. Defects of the PCP pathway have been found to cause kidney cyst formation. Our paper describes a method for developing a zebrafish cystic kidney disease model by knockdown of the wnt5a gene with wnt5a antisense morpholino (MO) oligonucleotides. Tg(wt1b:GFP) transgenic zebrafish were used to visualize kidney structure and kidney cysts following wnt5a knockdown. Two distinct antisense MOs (AUG - and splice-site) were used and both resulted in curly tail down phenotype and cyst formation after wnt5a knockdown. Injection of mouse Wnt5a mRNA, resistant to the MOs due to a difference in primary base pair structure, rescued the abnormal phenotype, demonstrating that the phenotype was not due to &#8220;off-target&#8221; effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a function in the kidney.

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

We describe a method of generating a possible zebrafish model of polycystic kidney disease. We used Tg(wt1b:GFP) fish to visualize kidney structure. Knockdown of wnt5a was by morpholino injection. Pronephric cyst formation after wnt5a knockdown was observed in this GFP transgenic zebrafish.

Um Modelo Possível Zebrafish da doença renal policística: Knockdown de<em&gt; Wnt5a</em&gt; Faz com cistos no peixe-zebra Rins

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The ...

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular mechanisms responsible for the disease, and are very convenient for rapid drug screening and testing of promising therapies. A limiting factor in these studies is often the lack of efficient non-invasive methods for sequentially analyzing the anatomical and functional changes in the kidney. Magnetic resonance imaging (MRI) is the current gold standard imaging technique to follow autosomal dominant polycystic kidney disease (ADPKD) patients, providing excellent soft tissue contrast and anatomic detail and allowing Total Kidney Volume (TKV) measurements.A major advantage of MRI in rodent models of PKD is the possibility for in vivo imaging allowing for longitudinal studies that use the same animal and therefore reducing the total number of animals required. In this manuscript, we will focus on using Ultra-high field (UHF) MRI to non-invasively acquire in vivo images of rodent models for PKD. The main goal of this work is to introduce the use of MRI as a tool for in vivo phenotypical characterization and drug monitoring in rodent models for PKD.

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

The use of ultra-high field MRI as a non-invasive way to obtain phenotypic information of rodent models for polycystic kidney disease and to monitor interventions is described. Compared with the traditional histological approach, MRI images can be acquired in vivo, allowing for longitudinal follow-up.

Uso de ultra-alta MRI campo em pequeno roedor modelos de doença renal policística para<em&gt; In Vivo</em&gt; Fenotipagem and Drug Monitoring

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular ...

Symmetric Bihemispheric Postmortem Brain Cutting to Study Healthy and Pathological Brain Conditions in Humans

Neuropathologists, at times, feel intimidated by the amount of knowledge needed to generate definitive diagnoses for complex neuropsychiatric phenomena described in those patients for whom a brain autopsy has been requested. Although the advancements of biomedical sciences and neuroimaging have revolutionized the neuropsychiatric field, they have also generated the misleading idea that brain autopsies have only a confirmatory value. This false idea created a drastic reduction of autopsy rates and, consequently, a reduced possibility to perform more detailed and extensive neuropathological investigations, which are necessary to comprehend numerous normal and pathological aspects yet unknown of the human brain. The traditional inferential method of correlation between observed neuropsychiatric phenomena and corresponding localization/characterization of their possible neurohistological correlates continues to have an undeniable value. In the context of neuropsychiatric diseases, the traditional clinicopathological method is still the best possible methodology (and often the only available) to link unique neuropsychiatric features to their corresponding neuropathological substrates, since it relies specifically upon the direct physical assessment of brain tissues. The assessment of postmortem brains is based on brain cutting procedures that vary across different neuropathology centers. Brain cuttings are performed in a relatively extensive and systematic way based on the various clinical and academic contingencies present in each institution. A more anatomically inclusive and symmetric bi-hemispheric brain cutting methodology should at least be used for research purposes in human neuropathology to coherently investigate, in depth, normal and pathological conditions with the peculiarities of the human brain (i.e., hemispheric specialization and lateralization for specific functions). Such a method would provide a more comprehensive collection of neuropathologically well-characterized brains available for current and future biotechnological and neuroimaging techniques. We describe a symmetric bi-hemispheric brain cutting procedure for the investigation of hemispheric differences in human brain pathologies and for use with current as well as future biomolecular/neuroimaging techniques.

Organized brain cutting procedures are necessary to correlate specific neuropsychiatric phenomena with definitive neuropathologic diagnoses. Brain cuttings are performed differently based on various clinico-academic contingencies. This protocol describes a symmetric bihemispheric brain cutting procedure to investigate hemispheric differences in human brain pathologies and to maximize current and future biomolecular/neuroimaging techniques.

Symmetric Bihemispheric Postmortem Cérebro de corte para o Estudo de Saúde e Condições Patológicas cerebral em humanos

Neuropathologists, at times, feel intimidated by the amount of knowledge needed to generate definitive diagnoses for complex neuropsychiatric phenomena ...

A Model to Simulate Clinically Relevant Hypoxia in Humans

In case of apnea, arterial partial pressure of oxygen (pO2) decreases, while partial pressure of carbon dioxide (pCO2) increases. To avoid damage to hypoxia sensitive organs such as the brain, compensatory circulatory mechanisms help to maintain an adequate oxygen supply. This is mainly achieved by increased cerebral blood flow. Intermittent hypoxia is a commonly seen phenomenon in patients with obstructive sleep apnea. Acute airway obstruction can also result in hypoxia and hypercapnia. Until now, no adequate model has been established to simulate these dynamics in humans. Previous investigations focusing on human hypoxia used inhaled hypoxic gas mixtures. However, the resulting hypoxia was combined with hyperventilation and is therefore more representative of high altitude environments than of apnea. Furthermore, the transferability of previously performed animal experiments to humans is limited and the pathophysiological background of apnea induced physiological changes is poorly understood. In this study, healthy human apneic divers were utilized to mimic clinically relevant hypoxia and hypercapnia during apnea. Additionally, pulse-oximetry and Near Infrared Spectroscopy (NIRS) were used to evaluate changes in cerebral and peripheral oxygen saturation before, during, and after apnea.

Hypoxia simulation in humans has usually been performed by inhaling hypoxic gas mixtures. For this study, apneic divers were used to simulate dynamic hypoxia in humans. Additionally, physiological changes in desaturation and re-saturation kinetics were evaluated with non-invasive tools such as Near-Infrared-Spectroscopy (NIRS) and peripheral oxygenation saturation (SpO2).

Um modelo para simular hipóxia clinicamente relevantes em humanos

In case of apnea, arterial partial pressure of oxygen (pO2) decreases, while partial pressure of carbon dioxide (pCO2) increases. To avoid damage to ...

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the present protocol, mice expressing green fluorescent protein (gfp) under the promoter of Cx3cr1 (BALB/c-Cx3cr1gfp/gfp) were used to image microglia cells in vivo in the retina. Microglia are resident macrophages of the retina and have been implicated in several retinal diseases1,2,3,4,5,6. This protocol provides a detailed approach for generation of retinal B-scans, with SD-OCT, and imaging of microglia cell distribution in Cx3cr1gfp/gfp mice with SLO in vivo, using an ophthalmic imaging platform system. The protocol can be used in several reporter mouse lines. However, there are some limitations to the protocol presented here. First, both SLO and SD-OCT, when used in the high-resolution mode, collect data with high axial resolution but the lateral resolution is lower (3.5 &#181;m and 6 &#181;m, respectively). Moreover, the focus and saturation level in SLO is highly dependent on parameter selection and correct alignment of the eye. Additionally, using devices designed for human patients in mice is challenging due to the higher total optical power of the mouse eye compared to the human eye; this can lead to lateral magnification inaccuracies7, which are also dependent on the magnification by the mouse lens among others. However, despite that the axial scan position is dependent upon lateral magnification, the axial SD-OCT measurements are accurate8.

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

This protocol describes how high-resolution imaging techniques such as spectral domain optical coherence tomography and scanning laser ophthalmoscopy can be utilized in small rodents, using an ophthalmic imaging platform system, to obtain information on retinal thickness and microglial cell distribution, respectively.

Na Vivo Imagem de Cx3cr1gfp/gfp repórter ratos com a tomografia de coerência óptica espectral-domínio e digitalização Laser Oftalmoscopia

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

Avaliação da função renal em modelos do rato da doença Glomerular

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

Delivery of Exogenous Artificially Synthesized miRNA Mimic to the Kidney Using Polyethylenimine Nanoparticles in Several Kidney Disease Mouse Models

microRNAs&#160;(miRNAs), small noncoding RNAs (21-25 bases) that are not translated into proteins, inhibit lots of target messenger RNAs (mRNAs) by destabilizing and inhibiting their translation in various kidney diseases. Therefore, alternation of miRNA expression by exogenous artificially synthesized miRNA mimics is a potentially useful treatment option for inhibiting the development of many kidney diseases. However, because serum RNAase immediately degrades systematically administered exogenous miRNA mimics in vivo, delivery of miRNA to the kidney remains a challenge. Therefore, vectors that can protect exogenous miRNA mimics from degradation by RNAase and significantly deliver them to the kidney are necessary. Many studies have used viral vectors to deliver exogenous miRNA mimics or inhibitors to the kidney. However, viral vectors may cause an interferon response and/or genetic instability. Therefore, the development of viral vectors is also a hurdle for the clinical use of exogenous miRNA mimics or inhibitors. To overcome these concerns regarding viral vectors, we developed a nonviral vector method to deliver miRNA mimics to the kidney using tail vein injection of polyethylenimine nanoparticles (PEI-NPs), which led to significant overexpression of target miRNAs in several mouse models of kidney disease.

Here, we deliver exogenous artificially synthesized miRNA mimics to the kidney via tail vein injection of a nonviral vector and polyethylenimine nanoparticles in several kidney disease mouse models. This led to significant overexpression of target miRNA in the kidney, resulting in inhibited progression of kidney disease in several mouse models.

Entrega de miRNA exógeno sintetizado artificialmente ao rim usando nanopartículas de polietilenimina em vários modelos de doença renal em camundongos

microRNAs&#160;(miRNAs), small noncoding RNAs (21-25 bases) that are not translated into proteins, inhibit lots of target messenger RNAs (mRNAs) by ...