Os autores gostariam de agradecer Hong Jiang, Ph.D. e Deepa Parathasarthy, MPH, BDS para a assistência técnica.

1. Coleta de Sangue Total <ol><li> Coletar sangue venoso de humanos ou de animais experimentais em ELm / tubos contendo EDTA. </li><li> Imediatamente girar sangue em uma centrífuga de bancada a 14.300 rcf (força centrífuga relativa) por 7 minutos para o preparo do plasma e dos glóbulos vermelhos pelota. </li><li> Preparar amostras de plasma para cromatografia líquida de alta eficiência (HPLC) e detecção por quimioluminescência (CLD) análise. HPLC: Adicionar 01:01 volume de metanol frio para o plasma, vortex e centrifugar a 13.200 rpm durante 10 minutos para precipitar as proteínas plasmáticas. Recolher o sobrenadante para análise por HPLC. Cale: alíquota de uma amostra e pré-incubar com sulfanilamida e cloreto mercúrico a nitrosotióis especificamente ensaio. </li><li> Prepare células vermelhas do pellet para HPLC e análise CLD. HPLC: 01:04 Adicionar vermelho pellet celular a uma solução de lise hipotónico contendo 10 mM de NEM, 2,5 mM de EDTA e 10 mM de ferricianeto. Vortex completamente e em seguida, adicione 1:1 metanol, vórtice e centrifEGD em 13.200 rpm durante 10 minutos para a proteína precipitado. Recolher o sobrenadante para análise por HPLC. CLD. Adicionar 01:04 vermelho pellet celular a uma solução de lise hipotónico contendo 10 mM de EDTA, 2,5 mM de EDTA e 10 mM de ferricianeto. Partes alíquotas para tubos contendo cloreto de sulfanilamida e mercúrico para a detecção específica de nitrosotióis. </li></ol> 2. Extração de Tecidos e Preparação <ol><li> Para determinar os níveis de tecido de metabólitos, é primeiro necessário para colher tecido sanguíneo livre para a preparação da amostra, como descrito acima. Uma troca de sangue completo terá lugar através da infusão de tampão fisiológico através do vértice do ventrículo esquerdo. Uma vez que todo o sangue é removido, os tecidos de interesse pode então ser colhido. </li><li> Homogeneizar amostras de tecido e preparar amostras como descrito acima para HPLC e análise CLD. </li></ol> 3. Isolamento anéis de aorta para a função endotelial Banho de órgãos de tecido <ol><li> Ratos será Anesthetized com éter dietílico até já não respondem ao toe pitada, e passam por deslocamento cervical antes da cirurgia. Um toracotomia é realizada para expor aorta torácica e abdominal. Uma seringa de calibre 25 é inserido no ápice do ventrículo esquerdo e perfundidos livre de sangue oxigenado com tampão de Krebs Henseleit. </li><li> O átrio direito é cortado para proporcionar uma saída para o sangue. Aorta abdominal será removido e limpo de adventícia. </li><li> Anéis irá ser cortado em segmentos longos 2 mm e montado sobre um banho de órgãos de quatro canais de tecido (DMT 720MO, instrumentos ad) banhadas em solução de tampão fisiológico. </li><li> Anéis de vasos são mantidos em banhos de órgãos de 10 ml com tampão de Krebs oxigenada (95% de O2 e 5% de CO 2), a 37 ° C. Um grama pretensão é colocado em cada anel aórtico (tensão de partida apropriado para a função vasomotora óptima, tal como determinado em experiências anteriores). Um oito canais octal ponte (Powerlab) e de aquisição de dados de software (Gráfico versão 5.2.2) umaestá acostumado a gravar todas as medidas de força. </li><li> Os anéis são deixadas equilibrar durante 80 minutos com o tampão em cada banho de órgãos mudado cada 20 min. Após o equilíbrio por 80 min, 1 mM de fenilefrina é adicionado a cada anel para a contração submáxima. </li><li> Após estabilização, agonista endotelial como a acetilcolina será adicionado para determinar a produção de NO e grau de relaxamento do vaso. Após a resposta à dose para a acetilcolina, banhos serão lavadas e recontracted e depois tratada com uma fonte exógena de óxido nítrico, nitroprussiato de sódio para determinar a resposta do músculo liso e para obter um valor para o relaxamento de 100%. </li><li> NO scavengers pode ser adicionado ao banho para ilustrar claramente libertação de NO e de inibição de relaxamento do vaso. </li></ol> 4. Os resultados representativos A utilização do ENO-20 dedicado HPLC fornece um método mais fácil de usar com rendimento elevado para a detecção específica e sensível de nitrito e nitrato namatrizes biológicas. O princípio de detecção e um cromatograma original é mostrada na Figura 1. Este método pode ser usado para qualquer amostra biológica para a determinação de nitrito e nitrato. Detecção baseada em CLD de metabólitos do requer um passo de derivatização química para determinar a fonte molecular do NO. A configuração experimental para desnitrosação oxidativo simultânea e desnitrosação redutiva para o detector de quimioluminescência é mostrado na Figura 2. O vaso de reacção do lado direito é preenchido com ferricianeto de 800mm em PBS pH 7,4 e do vaso de reacção do lado esquerdo é preenchido com a mistura de iodeto / iodeto de potássio em ácido acético para desnitrosação redução (Figura 2A). Toda a configuração é mostrada na Figura 2B. A Figura 3 ilustra ensaios de grupos específicos desnitrosação que podem detectar e quantificar nitrito, nitrosotióis, nitrosaminas, bem como produtos de heme nitrosilo. Este método tem sido previamente descricama e validado 4,5. Estas análises bioquímicas importantes pode facilmente ser correlacionada com os estudos funcionais em anéis de aorta isolados para determinar a produção de NO endotelial em animais experimentais. Este experimento clássico farmacológico pode facilmente e com precisão avaliar a função endotelial de NO e de produção. Medindo reactividade vaso aos agonistas endoteliais, tais como a acetilcolina pode determinar directamente a produção de NO endotelial que pode então ser correlacionada com os biomarcadores bioquímicos detectados no sangue e nos tecidos dos animais experimentais. Uma representação típica de uma resposta à dose para a acetilcolina é mostrado na Figura 4. Camundongos de controle saudáveis ​​com função endotelial normal responder a acetilcolina, relaxando. Os ratos com disfunção endotelial (ratos hipercolesterolêmicos) mostram relaxamento reduzida devido à redução da produção ao mesmo estímulo. <img alt="A Figura 1" src="/files/ftp_upload/3722/3722fig1.jpg" /> FigoA Figura 1. Princípio da detecção de nitrito e nitrato por ENO-20 e cromatograma da amostra. (Top) Esquema de ENO-20 método de detecção de nitrito e nitrato. (Inferior) chromotogram padrão de 10 pmol de nitrito e nitrato injectado ENO-20 (100 uL de solução 100 nM de nitrito e nitrato). Sensibilidade de 1 nM para cada anião com 100 ul volume de injecção. Não há interferência com a proteína ou espécies coloridas. <img alt="A Figura 2" src="/files/ftp_upload/3722/3722fig2.jpg" /> Figura 2. Configuração CLD experimental usando tanto desnitrosação oxidativo por desnitrosação ferricianeto e redutora usando iodeto / iodo ensaio com detecção de fase gasosa de gás de óxido nítrico purgado. <img alt="A Figura 3" src="/files/ftp_upload/3722/3722fig3.jpg" /> Figura 3. (Painel A) de detecção de quimioluminescência de nitrito, RSNO, RNNO no ensaio desnitrosação redutora por pré-incubação da amostra com o grupo de chemic específicoai reagentes. Subtração das áreas dos picos permitir a detecção de nitrito e RSNOs. (Painel B) de detecção quimioluminescente de espécies heme nitrosilo usando solução de ferricianeto de desnitrosação oxidativo. Este método é específico para o NO heme-produtos sem reactividade cruzada com RSNOs (GSNO ou SNO-albumina), ou RNNO (NO-pirrolidina e N-nitroso-albumina). <img alt="A Figura 4" src="/files/ftp_upload/3722/3722fig4.jpg" /> Figura 4. Ex banho de órgãos in vivo em anéis de aorta isolados fornece uma medida directa da produção de NO endotelial que pode então ser correlacionada com os biomarcadores bioquímicos detectados por HPLC e CLD. Esta figura ilustra o relaxamento reduzido nos ratinhos com disfunção endotelial devido à diminuição da produção de NO.

<ol>
	<li>Kelm, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide+metabolism+and+breakdown.">Nitric oxide metabolism and breakdown.</a> Biochimica et Biophysica Acta. 1411, 273-289 (1999).</li><li>Furchgott, R. F., Zawadzki, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+obligatory+role+of+endothelial+cells+in+the+relaxation+of+arterial+smooth+muscle+by+acetycholine.">The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetycholine.</a> Nature. 288, 373-376 (1980).</li><li>Ignarro, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelium-derived+relaxing+factor+produced+and+released+from+artery+and+vein+is+nitric+oxide.">Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide.</a> Proc. Natl. Acad Sci. U.S.A. 84, 9265-9269 (1987).</li><li>Feelisch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concomitant+S-,+N-,+and+heme-nitrosylation+in+biological+tissues+and+fluids:+implications+for+the+fate+of+NO+in+vivo.">Concomitant S-, N-, and heme-nitrosylation in biological tissues and fluids: implications for the fate of NO in vivo.</a> FASEB J. 16, 1775-1785 (2002).</li><li>Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+nitric+oxide+levels+in+the+red+cell:+validation+of+tri-iodide-based+chemiluminescence+with+acid-sulfanilamide+pretreatment.">Measurement of nitric oxide levels in the red cell: validation of tri-iodide-based chemiluminescence with acid-sulfanilamide pretreatment.</a> J. Biol. Chem. 281, 26994-27002 (2006).</li><li>Angelo, M., Singel, D. J., Stamler, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+S-nitrosothiol+(SNO)+synthase+function+of+hemoglobin+that+utilizes+nitrite+as+a+substrate.">An S-nitrosothiol (SNO) synthase function of hemoglobin that utilizes nitrite as a substrate.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 8366-8371 (2006).</li><li>Bryan, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bound+NO+in+human+red+blood+cells:+fact+or+artifact.">Bound NO in human red blood cells: fact or artifact.</a> Nitric Oxide. 10, 221-228 (2004).</li><li>Feelisch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+Processing+of+Nitrite+in+Hypoxia:+An+Intricate+Interplay+of+Nitric+Oxide-Generating+and+-Scavenging+Systems.">Tissue Processing of Nitrite in Hypoxia: An Intricate Interplay of Nitric Oxide-Generating and -Scavenging Systems.</a> J. Biol. Chem. 283, 33927-33934 (2008).</li><li>Bryan, N. S., Grisham, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+detect+nitric+oxide+and+its+metabolites+in+biological+samples.">Methods to detect nitric oxide and its metabolites in biological samples.</a> Free Radic. Biol. Med. 43, 645-657 (2007).</li><li>Lundberg, J. O., Weitzberg, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NO+generation+from+inorganic+nitrate+and+nitrite:+Role+in+physiology,+nutrition+and+therapeutics.">NO generation from inorganic nitrate and nitrite: Role in physiology, nutrition and therapeutics.</a> Arch. Pharm. Res. 32, 1119-1126 (2009).</li><li>Lundberg, J. O., Weitzberg, E., Gladwin, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nitrate-nitrite-nitric+oxide+pathway+in+physiology+and+therapeutics.">The nitrate-nitrite-nitric oxide pathway in physiology and therapeutics.</a> Nat. Rev. Drug. Discov. 7, 156-157 (2008).</li></ol>

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Os métodos descritos aqui para a quantificação de metabólitos do NO relevantes em vários compartimentos biológicos permitirá para o fingerprinting de NO biologia na saúde e na doença que podem ser correlacionados com as medições funcionais de NO pelo endotélio. Estes métodos requerem preparação de amostras simples, com potencial de adaptação para a produção elevada. A quantidade relativa destas moléculas pode ajudar a compreender a produção de NO e sua transformação metabólica num certo número ...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> N-etilmaleimida </td><td> Thermo Scientific </td><td> 23030 </td></tr><tr><td> EDTA </td><td> Sigma-Aldrich </td><td> E7889 </td></tr><tr><td> Ferricianeto de potássio </td><td> Fluka </td><td> 60299 </td></tr><tr><td> HPLC </td><td> Eicom Corp </td><td> ENO-20 </td></tr><tr><td> Autosampler </td><td> Alcott </td><td></td></tr><tr><td> DMT Myograph </td><td> Instrumentos AD </td><td></td></tr><tr><td> PowerLab </td><td> Instrumentos AD </td><td></td></tr><tr><td> Quimioluminescente </td><td> EcoPhysics </td><td> CLD 88Y </td></tr><tr><td> Centrifugar </td><td> Eppendorf </td><td> 5415D </td></tr><tr><td> A acetilcolina </td><td> Sigma-Aldrich</td><td> A6625 </td></tr><tr><td> R-(-) Fenilefrina </td><td> Sigma-Aldrich </td><td> P6126 </td></tr></tbody></table>

analytical techniques for assaying nitric oxide bioactivity

O óxido nítrico (NO) é um radical livre diatômico que é extremamente curta duração em sistemas biológicos (menos de 1 segundo no sangue circulante) 1. NO pode ser considerada uma das moléculas de sinalização mais importantes produzidos no nosso corpo, regulando as funções essenciais, incluindo mas não limitado a regulação da pressão arterial, a resposta imune e comunicação neural. Portanto, sua detecção precisa e quantificação, em matrizes biológicas é crucial para entender o papel do NO na saúde e na doença. Com tal meia vida um curto fisiológica de NO, estratégias alternativas para a detecção dos produtos de reacção de NO bioquímica têm sido desenvolvidos. A quantificação dos metabólitos do NO relevantes em vários compartimentos biológicos fornece informação valiosa no que diz respeito ao método in vivo a produção de NO a biodisponibilidade, e metabolismo. Basta provar um único compartimento, tais como sangue ou plasma nem sempre pode fornecer uma avaliação precisa do bo todody NO estado, particularmente em tecidos. A possibilidade de comparar o sangue com tecidos selecionados em animais de experimentação ajudará a ponte entre a ciência básica ea medicina clínica, na medida do utilitário de diagnóstico e prognóstico de NO biomarcadores na saúde e na doença. Portanto, a extrapolação do plasma ou do sangue para os tecidos estado NO específicos de interesse não é mais uma abordagem válida. Como resultado, os métodos de continuar a ser desenvolvido e validado que permitem a detecção e quantificação de NO e NO-produtos afins / metabolitos em múltiplos compartimentos de animais experimentais in vivo. O paradigma estabelecido de NO bioquímica da produção de NO sintases a ativação de guanilato ciclase solúvel (GCs) à oxidação eventual a nitrito (NO 2 -) e nitrato (NO 3 -) pode representar apenas parte de NO de efeitos in vivo. A interacção de metabolitos de NO e NO-derivada com tióis proteicos, aminas secundárias, e metais para formar S-nitrosothióis (RSNOs), N-nitrosaminas (RNNOs), e nitrosilo-heme representam respectivamente cGMP-independentes efeitos de NO e são susceptíveis tão importante como fisiologicamente activação de sGC pelo NO. Uma compreensão verdadeira de NO na fisiologia é derivado a partir de experiências in vivo de amostragem compartimentos múltiplos simultaneamente. O óxido nítrico metodologia (NO) é uma ciência complexa e muitas vezes confuso e o foco de muitos debates e discussão sobre NO bioquímica. A elucidação de novos mecanismos e vias de sinalização envolvendo NÃO depende de nossa capacidade de, especificamente, de forma seletiva e sensível detectar e quantificar todas as informações relevantes e NÃO NO produtos e metabólitos em matrizes biológicas complexas. Aqui, nós apresentamos um método para a análise rápida e sensível de nitrito e nitrato por HPLC, bem como a detecção de NO livre em amostras biológicas utilizando in vitro de quimioluminescência ozono com base com derivitazation química para determinar a fonte molecular de NO, bem como ex vivo comMiografia banho de órgãos.

Watch this Scientific Journal Video about Analytical Techniques for Assaying Nitric Oxide Bioactivity at JoVE.com

Analytical Techniques for Assaying Nitric Oxide Bioactivity

A produção endógena de óxido nítrico (NO) regula uma grande variedade de funções biológicas. Está se tornando cada vez mais claro que a ruptura ou desregulagem de sinalização NO base está envolvida em muitas doenças humanas. Os métodos para quantificar relevante metabólitos podem fornecer novos biomarcadores de diagnóstico ou de prognóstico para a doença humana.

Técnicas Analíticas para ensaiar Bioatividade Óxido Nítrico

Nitric oxide (NO) is a diatomic free radical that is extremely short lived in biological systems (less than 1 second in circulating blood)1. NO may be ...

analytical-techniques-for-assaying-nitric-oxide-bioactivity

Research

JoVE Journal

Medicine

17.9K visualizações.  University of Texas Health Science Center at Houston. A produção endógena de óxido nítrico (NO) regula uma grande variedade de funções biológicas. Está se tornando cada vez mais claro que a ruptura ou desregulagem de sinalização NO base está envolvida em muitas doenças humanas. Os métodos para quantificar relevante metabólitos podem fornecer novos biomarcadores de diagnóstico ou de prognóstico para a doença humana.

Vídeo: Analytical Techniques for Assaying Nitric Oxide Bioactivity

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 ...

Medicina

Techniques for Processing Eyes Implanted With a Retinal Prosthesis for Localized Histopathological Analysis

With the recent development of retinal prostheses, it is important to develop reliable techniques for assessing the safety of these devices in preclinical studies. However, the standard fixation, preparation, and automated histology procedures are not ideal. Here we describe new procedures for evaluating the health of the retina directly adjacent to an implant. Retinal prostheses feature electrode arrays in contact with eye tissue. Previous methods have not been able to spatially localize the ocular tissue adjacent to individual electrodes within the array. In addition, standard histological processing often results in gross artifactual detachment of the retinal layers when assessing implanted eyes. Consequently, it has been difficult to assess localized damage, if present, caused by implantation and stimulation of an implanted electrode array. Therefore, we developed a method for identifying and localizing the ocular tissue adjacent to implanted electrodes using a (color-coded) dye marking scheme, and we modified an eye fixation technique to minimize artifactual retinal detachment. This method also rendered the sclera translucent, enabling localization of individual electrodes and specific parts of an implant. Finally, we used a matched control to increase the power of the histopathological assessments. In summary, this method enables reliable and efficient discrimination and assessment of the retinal cytoarchitecture in an implanted eye.

Techniques for visualizing retinal cytoarchitecture directly adjacent to individual electrodes within a retinal stimulator.

Técnicas de Processamento de Olhos implantados com uma prótese de retina para análise histopatológica localizada

With  the recent development of retinal prostheses, it is important to develop  reliable techniques for assessing the safety of these devices in ...

Experimental and Imaging Techniques for Examining Fibrin Clot Structures in Normal and Diseased States

Fibrin is an extracellular matrix protein that is responsible for maintaining the structural integrity of blood clots. Much research has been done on fibrin in the past years to include the investigation of synthesis, structure-function, and lysis of clots. However, there is still much unknown about the morphological and structural features of clots that ensue from patients with disease. In this research study, experimental techniques are presented that allow for the examination of morphological differences of abnormal clot structures due to diseased states such as diabetes and sickle cell anemia. Our study focuses on the preparation and evaluation of fibrin clots in order to assess morphological differences using various experimental assays and confocal microscopy. In addition, a method is also described that allows for continuous, real-time calculation of lysis rates in fibrin clots. The techniques described herein are important for researchers and clinicians seeking to elucidate comorbid thrombotic pathologies such as myocardial infarctions, ischemic heart disease, and strokes in patients with diabetes or sickle cell disease.

In this manuscript, experimental techniques, including blood preparation, confocal microscopy, and lysis rate analysis, to examine the morphological differences between normal and abnormal clot structures due to diseased states are presented.

Técnicas Experimentais e de imagem para o exame de Estruturas do coágulo de fibrina em normais e doentes Unidos

Fibrin is an extracellular matrix protein that is responsible for maintaining the structural integrity of blood clots. Much research has been done on ...

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models. 
Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&amp;E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

Análise otimizada de<em&gt; In Vivo</em&gt; e<em&gt; In Vitro</em&gt; A esteatose hepática

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

Synthesis, Characterization, and Application of Superparamagnetic Iron Oxide Nanoprobes for Extrapulmonary Tuberculosis Detection

A molecular imaging probe comprising superparamagnetic iron oxide (SPIO) nanoparticles and Mycobacterium tuberculosis surface antibody (MtbsAb) was synthesized to enhance imaging sensitivity for extrapulmonary tuberculosis (ETB). An SPIO nanoprobe was synthesized and conjugated with MtbsAb. The purified SPIO-MtbsAb nanoprobe was characterized using TEM and NMR. To determine the targeting ability of the probe, SPIO-MtbsAb nanoprobes were incubated with Mtb for in vitro imaging assays and injected into Mtb-inoculated mice for in vivo investigation with magnetic resonance (MR). The contrast enhancement reduction on magnetic resonance imaging (MRI) of Mtb and THP1 cells showed proportional to the SPIO-MtbsAb nanoprobe concentration. After 30 min of intravenous SPIO-MtbsAb nanoprobe injection into Mtb-infected mice, the signal intensity of the granulomatous site was enhanced by 14-fold in the T2-weighted MR images compared with that in mice receiving PBS injection. The MtbsAb nanoprobes can be used as a novel modality for ETB detection.

To improve serological diagnostic tests for Mycobacterium tuberculosis antigens, we developed superparamagnetic iron oxide nanoprobes to detect extrapulmonary tuberculosis.

Síntese, Caracterização e Aplicação de Nanoprobes superparamagnéticos de óxido de ferro para detecção de tuberculose extrapulmonar

A molecular imaging probe comprising superparamagnetic iron oxide (SPIO) nanoparticles and Mycobacterium tuberculosis surface antibody (MtbsAb) was ...

Platelet-based Detection of Nitric Oxide in Blood by Measuring VASP Phosphorylation

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent vasoactive agents, nitric oxide (NO), can also act as a powerful inhibitor of platelet aggregation. Direct NO detection in blood is very challenging due to its high reactivity with cell-free hemoglobin that limits NO half-life to the millisecond range. Currently, NO changes after interventions are only estimated based on measured changes of nitrite and nitrate (members of the nitrate-nitrite-NO metabolic pathway). However precise, these measurements are rather difficult to interpret vis a vis actual NO changes, due to the naturally high baseline nitrite and nitrate levels that are several orders of magnitude higher than expected changes of NO itself. Therefore, the development of direct and simple methods that would allow one to detect NO directly is long overdue. This protocol addresses a potential use of platelets as a highly sensitive NO sensor in blood. It describes initial platelet rich plasma (PRP) and washed platelet preparations and the use of nitrite and deoxygenated red blood cells as NO generators. Phosphorylation of VASP at serine 239 (P-VASPSer239) is used to detect the presence of NO. The fact that VASP protein is highly expressed in platelets and that it is rapidly phosphorylated when NO is present leads to a unique opportunity to use this pathway to directly detect NO presence in blood.

Here, we present a protocol to address the potential use of platelets as a highly sensitive nitric oxide sensor in blood. It describes initial platelet preparation and the use of nitrite and deoxygenated red blood cells as nitric oxide generators.

Detecção baseada em plaquetas de óxido nítrico no sangue, medindo-se a fosforilação VASP

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent ...

Cardiac Magnetic Resonance for the Evaluation of Suspected Cardiac Thrombus: Conventional and Emerging Techniques

We present the conventional cardiac magnetic resonance (CMR) protocol for evaluating a suspected thrombus and highlight emerging techniques.&#160;The appearance of a mass on certain magnetic resonance (MR) sequences can help differentiate a thrombus from competing diagnoses such as a tumor. T1 and T2 signal characteristics of a thrombus are related to the evolution of hemoglobin properties. A thrombus typically does not enhance following contrast administration, which also helps differentiation from a tumor. We also highlight the emerging role of T1 mapping in the evaluation of a thrombus, which can add another level of support in diagnosis. Prior to any CMR exam, patient screening and interviews are critical to ensure safety and to optimize patient comfort. Effective communication during the exam between the technologist and the patient promotes proper breath holding technique and higher quality images. Volumetric post processing and structured reporting are helpful to ensure that the radiologist answers the ordering services&#39; question and communicates these results effectively. Optimal pre-MR safety evaluation, CMR exam execution, and post exam processing and reporting allow for delivery of high quality radiological service in the evaluation of a suspected cardiac thrombus.

The goal of this article is to describe how cardiac magnetic resonance can be used for the evaluation and diagnosis of a suspected cardiac thrombus. The method presented will describe data acquisition as well as the pre-procedure and post-procedure protocol.

Ressonância magnética cardíaca para avaliação de suspeita de trombo cardíaco: técnicas convencionais e emergentes

We present the conventional cardiac magnetic resonance (CMR) protocol for evaluating a suspected thrombus and highlight emerging techniques.&#160;The ...

Tracking Superparamagnetic Iron Oxide-labeled Mesenchymal Stem Cells using MRI after Intranasal Delivery in a Traumatic Brain Injury Murine Model

Stem cell-based therapies for brain injuries, such as traumatic brain injury (TBI), are a promising approach for clinical trials. However, technical hurdles such as invasive cell delivery and tracking with low transplantation efficiency remain challenges in translational stem-based therapy. This article describes an emerging technique for stem cell labeling and tracking based on the labeling of the mesenchymal stem cells (MSCs) with superparamagnetic iron oxide (SPIO)&#160;nanoparticles, as well as intranasal delivery of the labeled MSCs. These nanoparticles are fluorescein isothiocyanate (FITC)-embedded and safe to label the MSCs, which are subsequently delivered to the brains of TBI-induced mice by the intranasal route. They are then tracked non-invasively in vivo by real-time magnetic resonance imaging (MRI). Important advantages of this technique that combines SPIO for cell labeling and intranasal delivery include (1) non-invasive, in vivo MSC tracking after delivery for long tracking periods, (2) the possibility of multiple dosing regimens due to the non-invasive route of MSC delivery, and (3) possible applications to humans, owing to the safety of SPIO, non-invasive nature of the cell-tracking method by MRI, and route of administration.

Presented here is a protocol for non-invasive mesenchymal stem cell (MSC) delivery and tracking in a mouse model of traumatic brain injury. Superparamagnetic iron oxide&#160;nanoparticles are employed as a magnetic resonance imaging (MRI) probe for MSC labeling and non-invasive in vivo tracking following intranasal delivery using real-time MRI.

Rastreamento de células-tronco mesenchymal rotuladas por óxido de ferro superparamagnético usando ressonância magnética após parto intranasal em um modelo de urina de urina de lesão cerebral traumática

Stem cell-based therapies for brain injuries, such as traumatic brain injury (TBI), are a promising approach for clinical trials. However, technical ...

Near Infrared Photoimmunotherapy for Mouse Models of Pleural Dissemination

The efficacy of photoimmunotherapy can be evaluated more accurately with an orthotopic mouse model than with a subcutaneous one. A pleural dissemination model can be used for the evaluation of treatment methods for intrathoracic diseases such as lung cancer or malignant pleural mesothelioma.
Near-infrared photoimmunotherapy (NIR-PIT) is a recently developed cancer treatment strategy that combines the specificity of tumor-targeting antibodies with toxicity caused by a photoabsorber (IR700Dye) after exposure to NIR light. The efficacy of NIR-PIT has been reported using various antibodies; however, only a few reports have shown the therapeutic effect of this strategy in an orthotopic model. In the present study, we demonstrate an example of efficacy evaluation of the pleural disseminated lung cancer model, which was treated using NIR-PIT.

Near-infrared photoimmunotherapy (NIR-PIT) is an emerging cancer therapeutic strategy that utilizes an antibody-photoabsorber (IR700Dye) conjugate and NIR light to destroy cancer cells. Here, we present a method to evaluate the antitumor effect of NIR-PIT in a mouse model of pleural disseminated lung cancer and malignant pleural mesothelioma using bioluminescence imaging.

Fotoimunoterapia infravermelha próxima para modelos de mouse de disseminação pleural

The efficacy of photoimmunotherapy can be evaluated more accurately with an orthotopic mouse model than with a subcutaneous one. A pleural dissemination ...

A Novel Inhalation Mask System to Deliver High Concentrations of Nitric Oxide Gas in Spontaneously Breathing Subjects

Nitric Oxide (NO) is administered as gas for inhalation to induce selective pulmonary vasodilation. It is a safe therapy, with few potential risks even if administered at high concentration. Inhaled NO gas is routinely used to increase systemic oxygenation in different disease conditions. The administration of high concentrations of NO also exerts a virucidal effect in vitro. Owing to its favorable pharmacodynamic and safety profiles, the familiarity in its use by critical care providers, and the potential for a direct virucidal effect, NO is clinically used in patients with coronavirus disease-2019 (COVID-19). Nevertheless, no device is currently available to easily administer inhaled NO at concentrations higher than 80 parts per million (ppm) at various inspired oxygen fractions, without the need for dedicated, heavy, and costly equipment. The development of a reliable, safe, inexpensive, lightweight, and ventilator-free solution is crucial, particularly for the early treatment of non-intubated patients outside of the intensive care unit (ICU) and in a limited-resource scenario. To overcome such a barrier, a simple system for the non-invasive NO gas administration up to 250 ppm was developed using standard consumables and a scavenging chamber. The method has been proven safe and reliable in delivering a specified NO concentration while limiting nitrogen dioxide levels. This paper aims to provide clinicians and researchers with the necessary information on how to assemble or adapt such a system for research purposes or clinical use in COVID-19 or other diseases in which NO administration might be beneficial.

This simple and highly adaptable system device for the inhalation of high-concentration nitric oxide (NO) gas does not require mechanical ventilators, positive pressure, or high gas flows. Standard medical consumables and a snug-fitting mask are used to safely deliver NO gas to spontaneously breathing subjects.

Um novo sistema de máscara de inalação para fornecer altas concentrações de gás óxido nítrico em sujeitos de respiração espontânea

Nitric Oxide (NO) is administered as gas for inhalation to induce selective pulmonary vasodilation. It is a safe therapy, with few potential risks even if ...