Questo lavoro è stato sostenuto dal National Institutes of Health (CA142664).

1. Rilevamento di ROS mediante carbossi-H 2 DCFDA Carbossi-H 2 DCFDA non è fluorescente ma in presenza di ROS, quando questo reattivo viene ossidato, diventa verde fluorescente. <ol><li> Immediatamente prima dell&#39;uso, preparare una soluzione fresca stock di carbossi-H 2 DCFDA in sterili dimetilsolfossido (DMSO) o 100% di etanolo. Evitare di più disgelo / cicli di congelamento del colorante. </li><li> Lavare le cellule con soluzione salina tamponata HEPES (HBSS) o tampone fosfato salino (PBS) per rimuovere le tracce del mezzo originale. </li><li> Caricare le cellule con il colorante (e la tintura di controllo, sezione nota cf). In questo test abbiamo utilizzato Jurkat, una linea di cellule di leucemia umana. Usa carbossi-H 2 DCFDA ad una concentrazione finale di 1μM nel terreno di coltura regolari con ridotta siero (2%). </li><li> Incubare le culture per 30 minuti al buio, in un incubatore tradizionale (37 ° C, 5% CO2). Eliminare tutte le soluzioni coloranti utilizzati. </li><li> Rimuovere carbossi-H 2 DCFDA contenente medie e lavare due volte con HBSS o PBS. Da questo passo in avanti, proteggere le cellule dalla luce. </li><li> Aggiungere mezzo fresco contenente il farmaco di scelta per regione carbossi-H 2 DCFDA-caricato cellule e incubare come desiderato. Per questo esempio usiamo H 2 O 2 (0,03%) per 1 ora. </li><li> Valutare ROS da subito analizzando le cellule mediante citometria di flusso utilizzando il canale FL1 (fluorescenza verde), o da lettore di piastre fluorescenza, o al microscopio a fluorescenza. La fluorescenza può essere rilevato usando lunghezze d&#39;onda di eccitazione e di emissione che sono appropriate per fluorescenza verde. </li></ol> Nota: I controlli devono comprendere carbossi-H 2 cellule non trattate DCFDA-caricati e senza macchia cellule non trattate. Carbossi-H 2 DCFDA è noto per rilevare perossidi, ma potrebbe anche essere ossidato da altri ROS. Questo reagente può anche essere modificata con altri mezzi, colorante controllo insensibile ossidazione come 5 - (e-6)-carbossi-2 &#39;, 7&#39;-dichlordiacetato ofluorescein (carbossi-DCFDA) dovrebbero pertanto essere inclusi nel test. 2. Misurazione di ossido nitrico (NO) Avrete bisogno Sulfanilamide e N-1-napthylethylenediamine dicloridrato (NED), soluzioni e standard di nitrito. Questo test è chiamato il saggio di Griess. NED soluzione: creare una soluzione di 0,1% di N-1-napthylethylenediamine dicloridrato diluito in soluzione sterile water.Sulfanilamide: Fai una soluzione all&#39;1% di sulfanilamide diluito in acido fosforico 5%. Nitriti standard: diluire lo stock 0.1M standard di nitrito di sodio per 100μM nel medio sterile, fare una diluizione seriale del medium stesso. Condizioni di stoccaggio: Conservare i prodotti chimici come indicato dal costruttore a temperatura ambiente. Una volta ricostituito, NED e soluzioni Sulfanilamide vengono memorizzati subito dopo l&#39;uso a 4 ° C, al buio, e per un massimo di 3 mesi. <ol><li> Celle di coltura in una piastra da 96 pozzetti,uso triplica per ogni condizione, e includono i controlli proprio in base alle proprie sperimentazioni. </li><li> Curare le cellule per indurre la produzione di NO. Nel nostro esperimento usiamo lipopolisaccaride (LPS) (100 ng / ml) e IL-4 ricombinante per curare le nostre cellule. In questo protocollo, abbiamo utilizzato RAW 264.7, una linea di topi cellule macrofagi (Mouse leucemiche monociti linea cellulare di macrofagi). </li><li> Il giorno del test, portare entrambi i reagenti a temperatura ambiente. </li><li> Gira il tuo piatto, raccogliere sovranatante cellulare e trasferire 50μl a un nuovo 96 pozzetti. Preparare la limitazione pozzi diluizione della soluzione madre a fare una curva standard. </li><li> Aggiungere 50μl di soluzione Sulfanilamide per ogni campione e di controllo dei pozzi e mescolare bene. </li><li> Incubare a temperatura ambiente per 10 minuti al buio. </li><li> Aggiungere 50μl di N-1-napthylethylenediamine soluzione dicloridrato per ogni campione e di controllo dei pozzi e mescolare bene. </li><li> Incubare a temperatura ambiente per 10 minuti al buio. </li><li> Misura Absorbance immediatamente utilizzando un lettore di piastre con filtro di lunghezze d&#39;onda tra 520 nm e 550 nm. </li></ol> Se si utilizzano diversi piatto / piatto dimensioni, utilizzare 1/1/1 volume per ogni soluzione e surnatante campione. Un colore viola apparirà nel pozzetti positivi. I risultati ottenuti con lo standard vi aiuterà a verificare la stabilità delle soluzioni. 3. Rilevamento di ROS azione: le proteine ​​ossidate Un altro metodo per identificare la produzione di ROS è guardare i risultati finali rilevando l&#39;ossidazione delle proteine. Infatti, ROS modificare glutatione, un antiossidante che si esprime nella maggior parte delle cellule e svolge un ruolo protettivo contro i ROS. A seguito di ossidazione da ROS, la modifica del glutatione ridotto (GSH) risultati nella sulfidrilici (tioli), gruppo di suoi cisteina essere collegata a un glutatione un secondo da un ponte disolfuro. Questo porta alla formazione di una proteina dimeri (GSSG proteine ​​ossidate). GSH può essere ripristinata tramite la modifica del GSSG dal reduttasi enzima glutatione. L&#39;aumento del GSSG / GSH rapporto riflette lo stress ossidativo. Il saggio che segue si basa sul rilevamento e la quantificazione di queste proteine ​​ossidate. Questo metodo non è selettivo per specifiche ROS, ma rileva piuttosto gli effetti di NO, H 2 O 2, O 2 - e di altri ROS. Qui si misura la quantità totale di ossidato (GSSG) e ridotto (GSH) glutatione utilizzando segnali bioluminescenti. <ol><li> Semi di cellule in una piastra da 96 pozzetti (bianco / trasparente, fondo piatto) come al solito. Per il nostro esperimento abbiamo usato 1x10 4 cellule per pozzetto di aderenti Raw 264,7 e A549 cellule e le cellule Jurkat sospensione. A seconda delle dimensioni delle cellule questi numeri potrebbero essere regolati. Si consiglia di cellule aderenti piatto il giorno prima del test per permettere loro di allegare alla piastra. Abbastanza pozzi dovrebbero essere pronti per la rilevazione delle proteine ​​ossidate e ridotte così come per &quot;solo medium&quot; ecellule non trattate come controlli. Usa triplica per tutte le condizioni. </li><li> Trattare le cellule con il farmaco per il tempo necessario. Qui abbiamo trattato le cellule Jurkat e A549 cellule per 1 ora con H 2 O 2 (a 5 mm e 2,5 mm rispettivamente). 264,7 prime cellule sono state trattate con 200ng/ml di LPS per 16 h. Durante questo periodo di incubazione, portare tutti i reagenti a temperatura ambiente (RT). Rendere tutti i reagenti non più di 30 minuti prima di eseguire il test. La tabella seguente mostra i volumi di reagenti per pozzetto. Quando possibile, è importante rimuovere i supporti contenenti il ​​riducente prima di procedere con il test. </li></ol><table border="1"><tbody><tr><td rowspan="2"></td><td colspan="2"> Cellule aderenti </td><td colspan="2"> Sospensione cellule </td></tr><tr><td> Totale Glu Lysis </td><td> Ossidato Glu Lysis </td><td> Totale Glu Lysis </td><td> Ossidato Glu Lysis </sTrong&gt; </td></tr><tr><td> NEM, 25mM </td><td> nessuno </td><td> 0.5μl </td><td> nessuno </td><td> 0.5μl </td></tr><tr><td> Luciferina-NT </td><td> 1 ml </td><td> 1 ml </td><td> 1 ml </td><td> 1 ml </td></tr><tr><td> Lysis Buffer passiva, 5X </td><td> 10μl </td><td> 10μl </td><td> 10μl </td><td> 10μl </td></tr><tr><td> Acqua distillata </td><td> 39.0μl </td><td> 38.5μl </td><td> 14μl </td><td> 13.5μl </td></tr><tr><td> Volume finale per pozzo </td><td> 50μl </td><td> 50μl </td><td> 25μl </td><td> 25μl </td></tr></tbody></table><ol start="3"><li> Rimuovere media da pozzi con cellule aderenti. Non rimuovere medie pozzetti con cellule in sospensione. </li><li> Aggiungi ridotto reagente di lisi o glutatione ossidato reagente di lisi glutatione nei pozzetti corrispondenti e agitare per 5 minuti a temperatura ambiente. </li><li> Aggiungere il reagente generazione Luciferin e incubate per 30 minuti a temperatura ambiente. </li><li> Aggiungere il reagente di rilevamento Luciferin e incubare per 15 minuti a temperatura ambiente. </li><li> Leggi il segnale bioluminescente in tempo di integrazione di 0,25-1 secondo per bene con un luminometro lettore di piastre. </li></ol><table border="1"><tbody><tr><td></td><td> Cellule aderenti </td><td> Sospensione cellule </td></tr><tr><td> Numero di cellule per pozzetto </td><td> 1x10 4 </td><td> 1x10 4 </td></tr><tr><td> Sospensione cellulare per ben + di droga </td><td> 100 l, da rimuovere prima di 3,4 </td><td> 25μl, non deve essere rimosso </td></tr><tr><td> Il glutatione ridotto Lysis Reagent </td><td> 50 microlitri </td><td> 25μl </td></tr><tr><td> Reagente di lisi glutatione ossidato </td><td> 50 microlitri </td><td> 25μl </td></tr><tr><td> Luciferina Generazione reagente </td><td> 50 microlitri </td><td> 50 microlitri </td></tr><tr><td> Luciferina di rilevamento dei reagenti </td><td> 100 ul </td><td> 100 ul </td></tr></tbody></table> 4. Rappresentante dei risultati: <img alt="Figura 1" src="/files/ftp_upload/3357/3357fig1.jpg" /> Figura 1 Rilevazione dei ROS con carbossi-H2DCFDA colorante. Cellule Jurkat (linea di cellule umane di leucemia) trattati con H 2 O 2 sono stati confrontati con le cellule non trattate. ROS induce la modifica di carbossi-H 2 DCFDA che diventa fluorescente verde come rilevato mediante citometria di flusso, il picco fluorescente in H 2 O 2 turni rispetto cellule trattate ai picchi nei controlli (H 2 O 2 cellule trattate colorati con coloranti ossidazione insensibile e non trattate con cellule colorate con carbossi-H 2 DCFDA). Risultati confermano la presenza di ROS nelle cellule trattate. <img alt="Figura 2" src="/files/ftp_upload/3357/3357fig2.jpg" /> Figura 2 Rilevamento of NO utilizzando reagenti di Griess. RAW 264,7 cellule (mouse macrofagi) sono stati trattati con LPS e IL-4. Un aumento significativo della produzione di NO è stata rilevata in cellule trattate rispetto al controllo cellule non trattate. <table border="1"><tbody><tr><td> Linee cellulari </td><td> Non trattati </td><td> Trattati </td></tr><tr><td> Raw 264,7 </td><td> 13,0 </td><td> 8,3 </td></tr><tr><td> A549 </td><td> 21,6 </td><td> 10,5 </td></tr><tr><td> Jurkat </td><td> 5,2 </td><td> 2,8 </td></tr></tbody></table> Tabella 1 Rilevazione dei ROS mediata ossidazione delle proteine. RAW 264,7 cellule sono state trattate con LPS, Jurkat e A549 (cancro ai polmoni umani), le cellule sono state trattate con H 2 O 2. I risultati sono espressi come rapporto ridotto (GSH) / ossidato (GSSG) glutatione. Rapporti più bassi di glutatione (GSH) / (GSSG) sono stati rilevati nelle cellule trattate rispetto alcontrollo cellule non trattate, rivelando che le proteine ​​erano più ossidato in cellule trattate.

<ol>
	<li>Guzik, T. J., Korbut, R., Adamek-Guzik, T. <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+and+superoxide+in+inflammation+and+immune+regulation.">Nitric oxide and superoxide in inflammation and immune regulation.</a> J Physiol Pharmacol. 54, 469-487 (2003).</li><li>Perl, A., Gergely, P., Banki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dysfunction+in+T+cells+of+patients+with+systemic+lupus+erythematosus.">Mitochondrial dysfunction in T cells of patients with systemic lupus erythematosus.</a> Int Rev Immunol. 23, 293-313 (2004).</li><li>Valko, M., Leibfritz, D., Moncol, J., Cronin, M. T., Mazur, M., Telser, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+radicals+and+antioxidants+in+normal+physiological+functions+and+human+disease.">Free radicals and antioxidants in normal physiological functions and human disease.</a> Int J Biochem Cell Biol. 39, 44-84 (2007).</li><li>Droge, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+radicals+in+the+physiological+control+of+cell+function.">Free radicals in the physiological control of cell function.</a> Physiol Rev. 82, 47-95 (2002).</li><li>Nakamura, K., Yube, K., Miyatake, A., Cambier, J. C., Hirashima, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+CD4+D3-D4+membrane+proximal+extracellular+domain+for+the+inhibitory+effect+of+oxidative+stress+on+activation-induced+CD4+down-regulation+and+its+possible+role+for+T+cell+activation.">Involvement of CD4 D3-D4 membrane proximal extracellular domain for the inhibitory effect of oxidative stress on activation-induced CD4 down-regulation and its possible role for T cell activation.</a> Mol Immunol. 39, 909-921 (2003).</li><li>Los, M., Droge, W., Stricker, K., Baeuerle, P. A., Schulze-Osthoff, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+peroxide+as+a+potent+activator+of+T+lymphocyte+functions.">Hydrogen peroxide as a potent activator of T lymphocyte functions.</a> Eur J Immunol. 25, 159-165 (1995).</li><li>Deem, T. L., Cook-Mills, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+cell+adhesion+molecule+1+(VCAM-1)+activation+of+endothelial+cell+matrix+metalloproteinases:+role+of+reactive+oxygen+species.">Vascular cell adhesion molecule 1 (VCAM-1) activation of endothelial cell matrix metalloproteinases: role of reactive oxygen species.</a> Blood. 104, 2385-2393 (2004).</li><li>Pacher, P., Beckman, J. S., Liaudet, L. <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+and+peroxynitrite+in+health+and+disease.">Nitric oxide and peroxynitrite in health and disease.</a> Physiol Rev. 87, 315-424 (2007).</li><li>Griendling, K. K., Sorescu, D., Lassegue, B., Ushio-Fukai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+protein+kinase+activity+and+gene+expression+by+reactive+oxygen+species+and+their+role+in+vascular+physiology+and+pathophysiology.">Modulation of protein kinase activity and gene expression by reactive oxygen species and their role in vascular physiology and pathophysiology.</a> Arterioscler Thromb Vasc Biol. 20, 2175-2183 (2000).</li><li>Loh, K., Deng, H., Fukushima, A., Cai, X., Boivin, B., Galic, S., Bruce, C., Shields, B. J., Skiba, B., Ooms, L. M., Stepto, N., Wu, B., Mitchell, C. A., Tonks, N. K., Watt, M. J., Febbraio, M. A., Crack, P. J., Andrikopoulos, S., Tiganis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species+enhance+insulin+sensitivity.">Reactive oxygen species enhance insulin sensitivity.</a> Cell Metab. 10, 260-272 (2009).</li><li>Goldstein, B. J., Mahadev, K., Wu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redox+paradox:+insulin+action+is+facilitated+by+insulin-stimulated+reactive+oxygen+species+with+multiple+potential+signaling+targets.">Redox paradox: insulin action is facilitated by insulin-stimulated reactive oxygen species with multiple potential signaling targets.</a> Diabetes. 54, 311-321 (2005).</li></ol>

Abbiamo ricevuto l&#39;appoggio di Promega per questa pubblicazione.

Diverse situazioni patologiche come le malattie infiammatorie, tumori, l&#39;ischemia / riperfusione, ed anche trattamenti come la radioterapia o la chemioterapia (cisplatino cioè) inducono sovrapproduzione ROS. Così, il rilevamento e la misurazione dei livelli di ROS è importante in molte di base, gli studi pre-clinici e clinici. Tuttavia, ROS hanno tempi di dimezzamento molto breve e potrebbe essere complicata da rilevare. Qui, proponiamo semplici test che vengono utilizzati di routine e ampiamente accettato per la...

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti (opzionale) </th></tr><tr><td> 5 - (e-6)-carbossi-2 &#39;, 7&#39;-diclorofluoresceina diacetato (carbossi-DCFDA) </td><td> Molecular Probes </td><td> C369 </td><td> controllo </td></tr><tr><td> carbossi-H 2 DCFDA </td><td> Molecular Probes </td><td> C400 </td><td></td></tr><tr><td> Sulfanilamide </td><td> Sigma </td><td> S9251-100G </td><td></td></tr><tr><td> N-1-napthylethylenediamine dicloridrato </td><td> Sigma </td><td> N9125-10G </td><td></td></tr><tr><td> Nitriti di serie </td><td> Sigma </td><td> 237213-100G </td><td></td></tr><tr><td> GSH / GSSG-Glo Assay </td><td> Promega </td><td> V6612 </td><td> Per quantificare ossidato, ridotto o ossidato / glutatione ridotto </td></tr></tbody>
</table>

production and detection of reactive oxygen species (ros) in cancers

Specie reattive dell&#39;ossigeno comprendono un certo numero di molecole che danno al DNA e RNA e ossidare le proteine ​​e lipidi (perossidazione lipidica). Queste molecole contengono reattive di ossigeno e comprendono H 2 O 2 (perossido di idrogeno), NO (ossido nitrico), O 2 - (anione ossido), il perossinitrito (ONOO -), acido hydrochlorous (HOCl), e radicale ossidrile (OH -) . Specie ossidativo sono prodotte non solo in situazioni patologiche (cancro, riperfusione ischemica /, patologie neurologiche e cardiovascolari, malattie infettive, malattie infiammatorie 1, malattie autoimmuni 2, ecc ...) ma anche in situazioni fisiologiche (non patologico), come il metabolismo cellulare 3 , 4. Infatti, ROS svolgono un ruolo importante in molti pathway di segnale cellulare (proliferazione, l&#39;attivazione delle cellule 5, 6, 7 migrazione ecc.). ROS può essere dannoso (è poi indicato come&quot;Stress ossidativo e nitrosativo&quot;) se prodotti in grandi quantità nel compartimento intracellulare e delle cellule generalmente rispondono a ROS da upregulating antiossidanti come la superossido dismutasi (SOD) e catalasi (CAT), glutatione perossidasi (GPx) e glutatione (GSH) che protegge li convertendo pericolosi radicali liberi in molecole innocue (cioè acqua). Vitamine C ed E sono anche stati descritti come spazzini ROS (antiossidanti). I radicali liberi sono utili in quantità bassa 3. Macrofagi e neutrofili-mediata risposte immunitarie coinvolgere la produzione e il rilascio di NO, che inibisce i virus, agenti patogeni e la proliferazione del tumore 8. NO reagisce anche con altri ROS e, quindi, ha anche un ruolo come disintossicante (ROS scavenger). Infine NO agisce sui vasi di regolare il flusso di sangue che è importante per l&#39;adattamento del muscolo per l&#39;esercizio fisico prolungato 9, 10. Diverse pubblicazioni hanno anche dimostrato che i ROS sono coinvolti in sens insulinaitivity 11, 12. Numerosi metodi per valutare la produzione di ROS sono disponibili. In questo articolo proponiamo alcuni test semplice, veloce e conveniente, questi test sono state convalidate da molte pubblicazioni e sono abitualmente utilizzati per rilevare ROS o dei suoi effetti in cellule di mammifero. Mentre alcuni di questi saggi di rilevare molteplici ROS, altri rilevano un solo ROS.

Watch this Scientific Journal Video about Production and Detection of Reactive Oxygen Species ROS in Cancers at JoVE.com

Production and Detection of Reactive Oxygen Species ROS in Cancers

Qui vi proponiamo semplici metodi per testare e valutare la presenza di specie reattive dell&#39;ossigeno nelle cellule.

Produzione e di rilevamento di specie reattive dell&#39;ossigeno (ROS) in Cancro

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

production-and-detection-of-reactive-oxygen-species-ros-in-cancers

Research

JoVE Journal

Medicine

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

69.8K Views.  Baylor College of Medicine. Qui vi proponiamo semplici metodi per testare e valutare la presenza di specie reattive dell'ossigeno nelle cellule.

Video: Production and Detection of Reactive Oxygen Species ROS in Cancers

Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of the hepatic blood supply is poorly oxygenated portal vein blood derived from the gastrointestinal tract and spleen. Oxygen is delivered to hepatocytes by blood flowing from a terminal branch of the portal vein to a central venule via sinusoids, and this makes an oxygen gradient in hepatic lobules. The oxygen gradient is an important physical parameter that involves the expression of enzymes upstream and downstream in hepatic microcirculation, but the lack of techniques for measuring oxygen consumption in the hepatic microcirculation has delayed the elucidation of mechanisms relating to oxygen metabolism in liver. We therefore used FITC-labeled erythrocytes to visualize the hepatic microcirculation and used laser-assisted phosphorimetry to measure the partial pressure of oxygen in the microvessels there. Noncontact and continuous optical measurement can quantify blood flow velocities, vessel diameters, and oxygen gradients related to oxygen consumption in the liver. In an acute hepatitis model we made by administering acetaminophen to mice we observed increased oxygen pressure in both portal and central venules but a decreased oxygen gradient in the sinusoids, indicating that hepatocyte necrosis in the pericentral zone could shift the oxygen pressure up and affect enzyme expression in the periportal zone. In conclusion, our optical methods for measuring hepatic hemodynamics and oxygen consumption can reveal mechanisms related to hepatic disease.

An optical system was developed to visualize hepatic microcirculation with FITC-labeled erythrocytes and to measure the partial pressure of oxygen in the microvessels with laser-assisted phosphorimetry. This method can be used to investigate physiological and pathological mechanisms by analyzing microvascular structure, diameter, blood flow velocity, and oxygen tension.

Visualizzazione e analisi del flusso ematico e consumo di ossigeno nella microcircolazione epatica: Applicazione di un modello di epatite acuta

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of ...

Ricerca

Medicina

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

In molecular imaging, positron emission tomography (PET) and optical imaging (OI) are two of the most important and thus most widely used modalities1-3. PET is characterized by its excellent sensitivity and quantification ability while OI is notable for non-radiation, relative low cost, short scanning time, high throughput, and wide availability to basic researchers. However, both modalities have their shortcomings as well. PET suffers from poor spatial resolution and high cost, while OI is mostly limited to preclinical applications because of its limited tissue penetration along with prominent scattering optical signals through the thickness of living tissues.
 Recently a bridge between PET and OI has emerged with the discovery of Cerenkov Luminescence Imaging (CLI)4-6. CLI is a new imaging modality that harnesses Cerenkov Radiation (CR) to image radionuclides with OI instruments. Russian Nobel laureate Alekseyevich Cerenkov and his colleagues originally discovered CR in 1934. It is a form of electromagnetic radiation emitted when a charged particle travels at a superluminal speed in a dielectric medium7,8. The charged particle, whether positron or electron, perturbs the electromagnetic field of the medium by displacing the electrons in its atoms. After passing of the disruption photons are emitted as the displaced electrons return to the ground state. For instance, one 18F decay was estimated to produce an average of 3 photons in water5. 
 Since its emergence, CLI has been investigated for its use in a variety of preclinical applications including in vivo tumor imaging, reporter gene imaging, radiotracer development, multimodality imaging, among others4,5,9,10,11. The most important reason why CLI has enjoyed much success so far is that this new technology takes advantage of the low cost and wide availability of OI to image radionuclides, which used to be imaged only by more expensive and less available nuclear imaging modalities such as PET.
 Here, we present the method of using CLI to monitor cancer drug therapy. Our group has recently investigated this new application and validated its feasibility by a proof-of-concept study12. We demonstrated that CLI and PET exhibited excellent correlations across different tumor xenografts and imaging probes. This is consistent with the overarching principle of CR that CLI essentially visualizes the same radionuclides as PET. We selected Bevacizumab (Avastin; Genentech/Roche) as our therapeutic agent because it is a well-known angiogenesis inhibitor13,14. Maturation of this technology in the near future can be envisioned to have a significant impact on preclinical drug development, screening, as well as therapy monitoring of patients receiving treatments.

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.

Luminescenza Imaging Cerenkov (CLI) per il monitoraggio Cancer Therapy

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

Isolating and Using Sections of Bovine Mesenteric Artery and Vein as a Bioassay to Test for Vasoactivity in the Small Intestine

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that system. Changes in blood flow to the small intestine can result in effects on the absorptive functions of the organ. Particular interest in toxins liberated from feedstuffs through fermentative and digestive processes has developed in ruminants as an area where productive efficiencies could be improved. The video associated with this article describes an in vitro bioassay developed to screen compounds for vasoactivity in isolated cross-sections of bovine mesenteric artery and vein using a multimyograph. Once the blood vessels are mounted and equilibrated in the myograph, the bioassay itself can be used: as a screening tool to evaluate the contractile response or vasoactivity of compounds of interest; determine the presence of receptor types by pharmacologically targeting receptors with specific agonists; determine the role of a receptor with the presence of one or more antagonists; or determine potential interactions of compounds of interest with antagonists. Through all of this, data are collected real-time, tissue collected from a single animal can be exposed to a large number of different experimental treatments (an in vitro advantage), and represents vasculature on either side of the capillary bed to provide an accurate picture of what could be happening in the afferent and efferent blood supply supporting the small intestine.

The small intestine is frequently exposed to toxins that can influence blood flow and negatively impact nutrient absorption. Using a multimyograph and mesenteric artery and vein isolates, compounds or toxins of interest can be screened for vasoactivity.

Isolare e utilizzo Sezioni di mesenterica bovina arteria e vena come un saggio biologico per verificare Vasoactivity nel piccolo intestino

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that ...

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitro prion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host&#8217;s species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host&#8217;s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

Valutare l&#39;encefalopatia spongiforme trasmissibile Specie Barriere con un<em&gt; In Vitro</em&gt; Prion Protein Assay Conversion

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they ...

Detection of Disease-associated &#945;-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated &#945;-synuclein

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated &#945;-synuclein (&#945;SD) in experimental models of synucleopathies. A transgenic mouse line (M83) over-expressing the human A53T &#945;S and spontaneously developing a dramatic clinical phenotype between eight and 22 months of age, characterized by symptoms including weight loss, prostration, and severe motor impairment, was used in this study. For molecular analyses of &#945;SD (disease-associated &#945;S) in these mice, an ELISA was designed to specifically quantify &#945;SD in sick mice. Analysis of the central nervous system in this mouse model showed the presence of &#945;SD mainly in the caudal brain regions and the spinal cord. There were no differences in &#945;SD distribution between different experimental conditions leading to clinical disease, i.e., in uninoculated and normally aging transgenic mice and in mice inoculated with brain extracts from sick mice. The specific detection of &#945;SD immunoreactivity using an antibody against Ser129 phosphorylated &#945;S by ELISA essentially correlated with that obtained by Western blot and immunohistochemistry. Unexpectedly, similar results were observed with several other antibodies against the C-terminal part of &#945;S. The propagation of &#945;SD, suggesting the involvement of a &#8220;prion-like&#8221; mechanism, can thus be easily monitored and quantified in this mouse model using an ELISA approach.

Detection of Disease-associated &amp;#945;-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated &amp;#945;-synuclein

An ELISA offering a novel quantitative approach is described. It specifically detects disease-associated &#945;-synuclein (&#945;SD) in a transgenic mouse model (M83) of synucleinopathy using several antibodies against either the Ser129 phosphorylated &#945;S form or the C-terminal part of the protein.

Il rilevamento della malattia associata α-sinucleina da avanzato ELISA nel cervello di topi transgenici A53T umano Mutato α-sinucleina

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated &#945;-synuclein (&#945;SD) ...

Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

Following injury to the CNS, astrocytes undergo a broad range of biochemical, morphological, and molecular changes collectively referred to as reactive astrogliosis. Reactive astrocytes exert both inflammatory and protective effects that inhibit and promote, respectively, neural repair. The mechanisms underlying the diverse functional properties of reactive astrogliosis are not well understood. Achieving a greater understanding of these mechanisms is critical to developing therapeutic strategies to treat the injured CNS. Here we demonstrate a method to trigger reactive astrogliosis in the adult mouse forebrain using a forebrain stab lesion. This lesion model is simple, reliable, and requires only a stereotaxic device and a scalpel blade to produce the injury. The use of stab lesions as an injury model in the forebrain is well established and amenable to studies addressing a broad range of neuropathological outcomes, such as neuronal degeneration, neuroinflammation, and disruptions in the blood brain barrier (BBB). Thus, the forebrain stab injury model serves as a powerful tool that can be applied for a broad range of studies on the CNS response to trauma.

Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

This article describes a detailed protocol to produce a forebrain stab injury in adult mice. The stab injury induces severe reactive gliosis and glial scar formation which can be subsequently examined by standard immunohistochemistry methods.

Attivazione reattiva Gliosi<em&gt; In Vivo</em&gt; Da un Forebrain Stab Injury

Following injury to the CNS, astrocytes undergo a broad range of biochemical, morphological, and molecular changes collectively referred to as reactive ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

In Vitro e In Vivo rilevamento di Mitophagy in cellule umane, C. Eleganse topi

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

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.

Rilevazione basata su piastrine di ossido nitrico nel sangue misurando VASP fosforilazione

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

Quantitative Mapping of Specific Ventilation in the Human Lung using Proton Magnetic Resonance Imaging and Oxygen as a Contrast Agent

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of the fresh gas entering a lung region divided by the region&#8217;s end-expiratory volume &#8213; in the human lung, using only inhaled oxygen as a contrast agent. Regional quantification of specific ventilation has the potential to help identify areas of pathologic lung function. Oxygen in solution in tissue shortens the tissue&#8217;s longitudinal relaxation time (T1), and thus a change in tissue oxygenation can be detected as a change in T1-weighted signal with an inversion recovery acquired image. Following an abrupt change between two concentrations of inspired oxygen, the rate at which lung tissue within a voxel equilibrates to a new steady-state reflects the rate at which resident gas is being replaced by inhaled gas. This rate is determined by specific ventilation. To elicit this sudden change in oxygenation, subjects alternately breathe 20-breath blocks of air (21% oxygen) and 100% oxygen while in the MRI scanner. A stepwise change in inspired oxygen fraction is achieved through use of a custom three-dimensional (3D)-printed flow bypass system with a manual switch during a short end-expiratory breath hold. To detect the corresponding change in T1, a global inversion pulse followed by a single shot fast spin echo sequence was used to acquire two-dimensional T1-weighted images in a 1.5&#160;T MRI scanner, using an eight-element torso coil. Both single slice and multi-slice imaging are possible, with slightly different imaging parameters. Quantification of specific ventilation is achieved by correlating the time-course of signal intensity for each lung voxel with a library of simulated responses to the air/oxygen stimulus. SVI estimations of specific ventilation heterogeneity have been validated against multiple breath washout and proved to accurately determine the heterogeneity of the specific ventilation distribution.

Specific ventilation imaging is a functional magnetic resonance imaging technique that allows for quantification of regional specific ventilation in the human lung, using inhaled oxygen as a contrast agent. Here, we present a protocol to collect and analyze specific ventilation imaging data.

Mappatura quantitativa della ventilazione specifica nel polmone umano utilizzando l'imaging a risonanza magnetica protonica e l'ossigeno come agente di contrasto

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of ...

Characterization of Sickling During Controlled Automated Deoxygenation with Oxygen Gradient Ektacytometry

In sickle cell disease (SCD), a single point mutation in the gene coding for beta-globin causes the production of abnormal hemoglobin S (HbS). When deoxygenated, HbS can polymerize, forming rigid rods of hemoglobin, resulting in the sickling of red blood cells (RBCs). These sickled RBCs have significantly reduced deformability, causing vaso-occlusion, which leads to numerous SCD-related clinical complications, including pain, stroke, and organ damage. RBC deformability is also reduced by RBC dehydration, resulting in dense red blood cells that are more likely to sickle. To date, there is not a single widely available, rapid, and reproducible laboratory assay capable of predicting the disease severity or directly monitoring the treatment effects for novel, non-fetal hemoglobin inducing therapies. In this study, we describe a protocol to measure RBC deformability as a function of pO2 that allows for the quantitation of sickling behavior in SCD patients. Oxygen gradient ektacytometry measures RBC deformability, expressed as the elongation index (EI), as a function of pO2. RBCs are exposed to a fixed shear stress of 30 Pa during one round of deoxygenation and reoxygenation. Six readout parameters are produced. Of these, the point of sickling (PoS), defined as the pO2 at which maximum EI (EImax) shows a 5% decrease, and minimum EI during deoxygenation (EImin) are the most informative, reflecting an individual patient&#8217;s pO2 at which sickling starts and the minimal deformability of a patient&#8217;s red blood cells, respectively. PoS is associated with an individual patient&#8217;s hemoglobin affinity for oxygen, whereas EImin shows a strong correlation with fetal hemoglobin levels. We conclude that oxygen gradient ektacytometry is a promising technique to monitor the treatment of patients with SCD, as a biomarker for anti-sickling agents in clinical and preclinical trials, and an important tool to study sickling behavior of RBCs from individuals with SCD and sickle cell traits.

Here, we present oxygen gradient ektacytometry, a rapid and reproducible method to measure red blood cell deformability in samples from patients with sickle cell disease under controlled deoxygenation and reoxygenation. This technique provides a way to study red blood cell sickling and to monitor sickle cell disease treatment efficacy.

Caratterizzazione della falciatura durante la deossigenazione automatizzata controllata con Ektacytometria gradiente di ossigeno

In sickle cell disease (SCD), a single point mutation in the gene coding for beta-globin causes the production of abnormal hemoglobin S (HbS). When ...