Gli autori ringraziano Brian Muntean, Shao Lo, Maki Takahashi e Blair Mell per la loro assistenza tecnica. Questo lavoro è stato sostenuto da premi dal NIH (DK080640) e The University of Translational Research Award stimolazione Toledo &amp; Promedica al dottor Surya Nauli.

1. Cella pretrattamento e la preparazione metafase <ol><li> Le cellule sono cresciute in modif Dulbeccòs di Medio di Eagle (DMEM) contenente 10-15% siero bovino fetale (FBS) e 1% penicillina / streptomicina a 37 ° C con 5% CO 2 incubatore, fino a raggiungere 70-80% confluenza. </li><li> Trattare le cellule con soluzione Colcemid a 0,05 ug / ml per 30-60 min. </li><li> Raccogliere il terreno contenente le cellule galleggianti da 50 ml sterili provette Falcon. Lavare le cellule sulla piastra con sterile1X-PBS. Dopo aver incubato con tripsina sterile per 1-2 min, raccolto e raccogliere le cellule rimanenti nel tubo stesso. </li><li> Centrifugare le provette a 1000 rpm per 5 min, aspirare il surnatante lasciando 0,5 ml e allentare pellet da sfogliando con un dito solo. </li><li> A seconda delle dimensioni pellet, aggiungere 5-10 ml di soluzione ipotonica dello 0,56% KCl in dH 2 O e incubare la sospensione a 37 ° C per 30-45 min. </li><li> Aggiungere una goccia di metanolo / acido acetico(3:1 vol / vol) per ml di sospensione cellulare ipotonico e invertire delicatamente il tubo per la miscelazione. </li><li> Centrifugare a 1200 rpm per 5 minuti e raccogliere il pellet come step 1.4, quindi aggiungere 5 ml di metanolo dolce / acido acetico (3:1 vol / vol) goccia a goccia la soluzione fissativa, mentre sfogliando il pellet continuamente. Questa procedura è critica così che i metafase non verrà intrappolato nella ciuffi delle cellule &#39;che comprometterebbero l&#39;esperimento. </li><li> Centrifugare nuovamente a 1200 rpm per 5 min e aggiungere 5-10 ml di ghiaccio-freddo metanolo / fissativo acido acetico lungo la parete del tubo. In questa fase, se necessario, le cellule possono essere conservate in soluzione fissativa in un tubo stretto e sigillato a -20 ° C per breve termine o -80 ° C per lungo termine (anni) per uso futuro. </li><li> Vetrini Pulizia in etanolo assoluto, quindi immergerlo in dH 2 O per approximately10X per formare una guaina di acqua sulla superficie del vetrino. Porre il vetrino su una piastra di vetro e calo del 15-20μl di sospensione cellulare (dal punto 1,8) Da 10 &quot;sopra il vetrino. Porre il vetrino in un bagnomaria regolato a 65-70 ° C per 1-2 minuti e lasciare asciugare. </li><li> Controllare il vetrino sotto un microscopio ottico con obiettivi a secco 10X e 40X assicurandosi che vi sia cromosomi in metafase e gli spread sono distribuiti uniformemente. Verifica della presenza citoplasma che circonda i cromosomi. Se citoplasma è presente procedere con pretrattamento diapositiva (digestione pepsina), se non citoplasma è presente e le cromosomi hanno buona morfologia, allora non c&#39;è bisogno di pretrattamento diapositiva. </li></ol> 2. Presentazione pretrattamento (pepsina trattamento) <ol><li> Applicare 120 ml di soluzione 1:200 RNasi (20 mg / ml) disciolto in 2X-SSC su un 24 mm coverglass microscopio x 60 mm e invertire la metafase faccia scivolare sul coverglass poi capovolgere delicatamente la faccia slitta metafase e incubare a 37 ° C per 45 min. </li><li> Rimuovere con cautela il vetrino coprioggetti senza graffiare il vetrino e lavare in SSC 2X-buffer in una vaschetta Coplin ogni 5 min per 15 mincon agitazione. </li><li> Aggiungere 5-15 pl di soluzione di archivio di pepsina (100 mg / ml in dH 2 O) in un bicchiere pulito e quindi aggiungere 100 ml di preriscaldato (37 ° C) 0.01M HCl. È importante che la pepsina viene aggiunto in un pulito becher prima e non direttamente nella soluzione di HCl, altrimenti non sarebbe sciogliere in soluzione. Incubare il vetrino in una vaschetta Coplin, contenente la HCl / pepsina soluzione a 37 ° C per 3-5 min. Questo passaggio è molto critica come digestione troppo causerà i cromosomi da overdigested e troppo digestione poco lascerà il citoplasma non digerito che potrebbe portare ad legame non specifico della sonda e interferire con il segnale di ibridazione. </li><li> Lavare il vetrino in una vaschetta Coplin contenente 100 ml 1X-PBS per 5 min due volte a temperatura ambiente. </li><li> Lavare il vetrino in una vaschetta Coplin contenente 100 ml 1X-PBS/MgCl 2 per 5 min a temperatura ambiente (50 ml di 1M MgCl 2 in 950 ml di 1X-PBS). </li><li> Mettere il vetrino in una vaschetta Coplin containing 100 ml di formaldeide 1% per 10 min a temperatura ambiente (1,7 ml del 37% formaldeide in 100 ml di 1X-PBS/MgCl 2). </li><li> Lavare il vetrino in una vaschetta Coplin contenente 100 ml 1X-PBS per 5 min. </li><li> Osservare il vetrino sotto un microscopio ottico 40X con lenti a secco per assicurare che i vetrini vengono digeriti correttamente e non citoplasma è presente e la morfologia dei cromosomi è conservato. Selezionare un&#39;area per l&#39;ibridazione con una penna di diamante. </li></ol> 3. Cromosoma e sonda denaturazione e l&#39;ibridazione <ol><li> Preparare la soluzione fresca denaturazione (70% formamide/2X SSC, pH 7.0) e preriscaldare a 70-80 ° C in una vaschetta Coplin posto in un luogo bagnomaria il vetrino nel vaso Coplin contenente la soluzione di denaturazione a bagnomaria a 70 ° C per i cromosomi del mouse e 80 ° C per i cromosomi umani per anni &#39;30-1.5 min. </li><li> Disporre il vetrino in ghiacciato etanolo al 70% per 3 min seguita da 80% e 100% di etanolo per 3 minuti ciascuna e lasciare asciugare all&#39;aria. Esaminare il vetrinoper morfologia cromosoma. Buona la morfologia dei cromosomi si indica con cromosomi scure e non &quot;phase-luce&quot; o halo cromosomi. </li><li> Riscaldare la sonda SkyPaint (vernice kit SKY; flaconcino # 1) a 37 ° C con agitazione per 20 min, vortex e centrifugare brevemente a 1000 rpm per pochi secondi. </li><li> Denaturare il sonda in un termociclatore programmato per un due-step ciclo a 85 ° C per 5 ciclo min seguita da 37 ° C per 60 min per consentire marcato-sonda di DNA per preannealing. </li><li> Applicare 10 pl della sonda denaturata sull&#39;area di ibridazione e coprire con un mm 22 x 40 millimetri coprioggetti facendo attenzione a non intrappolare bolle d&#39;aria. Sigillare i bordi del coverglass con cemento gomma e incubare in una camera umidificata a 37 ° C per 48-72 ore. </li></ol> 4. Sonda di rilevamento della fluorescenza <ol><li> Rimuovere il copri con cura e porre il vetrino in una vaschetta Coplin contenente preriscaldato (45 ° C) la soluzione di lavaggio I (preparata formamide 50% in 2X SSC). Lavare per 5 mintre volte a 45 ° C in un bagno d&#39;acqua agitando a 45 rpm </li><li> Lavare il vetrino nella soluzione di lavaggio II (1X SSC) a 45 ° C per 5 min due volte con agitazione. </li><li> Lavare il vetrino nella soluzione di lavaggio III (4X SSC/0.1% Tween 20) per 5 min a 45 ° C con agitazione. </li><li> Applicare 80 microlitri di reagente bloccante (paint kit SKY; flacone # 2), inserire un coprioggetto e incubare a 37 ° C per 30 min. </li><li> Rimuovere il vetrino e consentire al fluido di drenare. Applicare 80 microlitri di reagente di colorazione Cy5 (Concentrated Ricerca anticorpi CAD kit; flacone # 3), applicare il coprioggetti e incubare a 37 ° C per 40 min. </li><li> Lavare il vetrino con soluzione di lavaggio III a 45 ° C per 5 min tre volte con agitazione. </li><li> Applicare 80 microlitri di reagente Cy5.5 colorazione (Concentrated Ricerca anticorpi CAD kit; fiala # 4), posizionare il coprioggetti e incubare a 37 ° C per 40 min. </li><li> Lavare il vetrino con soluzione di lavaggio III a 45 ° C per 5 min tre volte con agitazione. </li><li> Inclinare la diapositiva e consentire il fluido<html> scolare. Applicare 20 l di anti-fade reattivo DAPI (paint kit SKY; flaconcino # 5) e mettere da 24 mm coverglass microscopio x 60 mm. Rimuovere con cura le bolle d&#39;aria che potrebbero essere formate. Le diapositive possono essere ripreso immediatamente oppure conservati a 4 ° C al buio per non più di 1 settimana. </li></ol> 5. Acquisizione di immagini e analisi <ol><li> Acquisizione Immagine è compiuta da visualizzando vetrini metafase usando un microscopio Olympus equipaggiata con un obiettivo 60X immersione in olio, un cubo Spectral (progettato su misura tripla banda filtro passa), un filtro DAPI e un modulo interferometro Sagnac con una telecamera CCD. </li><li> Spettrale cariotipi sono state effettuate utilizzando il software View SKY (Applied Version immagini spettrali 1,62) seguendo il manuale dell&#39;utente. </li><li> Dopo aver analizzato le immagini, i cromosomi possono essere visti come immagini a colori (con appositi colori fluorescenti), immagini a colori pseudo (con i colori per la classificazione) e invertiti immagini DAPI (specifico disegno a strisce). </li></ol>e_title &quot;&gt; 6. rappresentativi Risultati Una procedura di SKY completa dura solitamente circa una volta alla settimana (Figura 1). Ciò include l&#39;acquisizione delle immagini e l&#39;analisi a condizione che le cellule in metafase sono in quantità adeguata. Analisi Karyotyping rivela cariotipo normale topo (40, XY) di cellule provenienti da topi wild type (Figura 2a). In contrasto, cellule da PKD1 - / - topo (modello topo PKD) mostra un significativo aumento in numero dei cromosomi e anomalie strutturali, quali delezioni cromosomiche (cromosoma # 8) e le traslocazioni (cromosomi # 11 e 19) (Figura 2b). Abbiamo anche analizzato i tessuti vascolari dai pazienti ADPKD. Uno studio semplice contando i numeri cromosomiche indicato che non-ADPKD e alcuni campioni ADPKD vascolari avuto normali numeri cromosomiche di 23 coppie (Figura 2c). In generale, però, abbiamo osservato fallimento di segregazione cromosomica, con conseguente in 46 coppie di cromosomi in ACampioni DPKD invece di 23 coppie (figura 2d). <img alt="Figura 1" src="/files/ftp_upload/3887/3887fig1.jpg" /> Figura 1. Diagramma di flusso protocollo di SKY. Diagramma di flusso del protocollo di SKY illustra i passaggi per completare un esperimento a partire dal pre-trattamento delle cellule in metafase e preparati per l&#39;acquisizione delle immagini e analisi. Una approssimativa di una settimana timeline è presentato a sinistra con passo-passo le procedure per ogni giorno. <img alt="Figura 2" src="/files/ftp_upload/3887/3887fig2.jpg" /> Figura 2. Cariotipo spettrale del mouse su linee cellulari e immortalati appena isolato le cellule primarie umane. Colori e immagini capovolte DAPI di cromosomi singoli mostrato prima l&#39;ordinamento cromosomico. Dopo la cernita, i cromosomi sono presentati nella tabella &quot;classificazione&quot;. A) l&#39;immagine di SKY uno spread metafase dal topo wild-type contiene cariotipo normale (40, XY). B) </strong&gt; SKY immagine di uno spread metafase da PKD1 - / -. del mouse mostra il numero dei cromosomi anormali (68 invece di 40) e le anomalie cromosomiche come le delezioni cromosomiche (cromosoma # 8) e le traslocazioni cromosomiche (cromosomi # 11 e 19) c) SKY immagine di uno spread metafase dal tessuto vascolare di un non-ADPKD paziente ha cariotipo normale (46, XX). d) immagine SKY di uno spread metafase dal tessuto vascolare di un paziente ADPKD contiene cariotipo anormale (92, XXXX). Parti dei dati sono stati riportati in precedenza e riutilizzato con autorizzazione 14.

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Cariotipo spettrale (SKY) è una tecnica utilizzata nello studio di citogenetica composizioni genomiche e cromosomiche. Questa tecnica si avvale di sonde cromosoma pittura, e il rilevamento di queste sonde sono stati acquisiti tramite un interferometro di Sagnac. Il processo di SKY completo dura di solito circa una settimana, e coinvolge diversi passaggi chiave (Figura 1). La SKY utilizza un protocollo standard, che è stato descritto da Padilla-Nash et al 3. Il protocollo è stato p...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> DMEM </td><td> Cellgro Mediatech Inc. </td><td> 10-013-CV </td><td></td></tr><tr><td> Siero bovino fetale (FBS) </td><td> Hyclone </td><td> SH30088-03 </td><td></td></tr><tr><td> Penecillin / streptomicina </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 pg / 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 soluzione salina tamponata </td><td> Thermo Scientific </td><td> SH30256-01 </td><td></td></tr> &lt;tr&gt; <td> SKY paint kit sonda (Human) </td><td> Applicata Imaging Spectral </td><td> SKY000028 </td><td></td></tr><tr><td> SKY vernice sonda kit (mouse) </td><td> Applicata Imaging Spectral </td><td> SKY000030 </td><td></td></tr><tr><td> Concentrato kit per il rilevamento degli anticorpi </td><td> Applicata Imaging Spectral </td><td> SKY000033 </td><td></td></tr><tr><td> Tripsina </td><td> Thermo Scientific </td><td> SH30236.01 </td><td></td></tr><tr><td> Metanolo </td><td> Fisher Scientific </td><td> A433P-4 </td><td></td></tr><tr><td> Acido acetico </td><td> Fisher Scientific </td><td> A38-212 </td><td></td></tr><tr><td> RNasi 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> 37% Formaldeide </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> Formammide </td><td> Fluka </td><td> 47671 </td><td> Preparare al momento dell&#39;uso </td></tr><tr><td> Tween-20 </td><td> Fisher Scientific </td><td> BP337-500 </td><td></td></tr><tr><td> Microscopio diapositive di vetro </td><td> Fisher Scientific </td><td> 12-549 </td><td></td></tr><tr><td> Microscopio copertura in vetro 24x60mm </td><td> VWR Vista Vision </td><td> 16004-312 </td><td></td></tr><tr><td> Mastice </td><td> Elmer (locali negozio al dettaglio) </td><td></td><td></td></tr><tr><td> Ibridizzazione / humidifiedchamber / Tray </td><td> Simport </td><td> M920-2 </td><td> mettere gli asciugamani di carta bagnati in fondo </td></tr><tr><td> Thermocycler </td><td> Eppendorf </td><td> Epgradient S </td><td></td></tr><tr><td> Scuotendo la piattaforma / Agitatore orbitale </td><td> Bellco Biotecnologie </td><td></td><td></td></tr><tr><td> Scuotendo bagno / acqua </td><td> Rigore scientifico </td><td></td><td></td></tr><tr><td> DAPI filtro cubo </td><td> Tecnologia Chroma </td><td></td><td></td></tr><tr><td> SKY filtro cubo </td><td> Tecnologia Chroma </td><td></td><td></td></tr><tr><td> SpectraCube </td><td> Applicata Imaging Spectral </td><td></td><td></td></tr><tr><td> Inverted cella microscopio cultura </td><td> Nikon </td><td> Nikon Eclipse TS100 </td><td></td></tr><tr><td> Microscopio a fluorescenza </td><td> Olimpo </td><td> IX70 </td><td> Olio 60X </td></tr></tbody></table>

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

Metodo convenzionale per identificare e classificare i singoli cromosomi dipende dal modello unico bande di ciascun cromosoma in una determinata specie in fase di analisi 1, 2. Questa tecnica classica banding, tuttavia, non è affidabile nell&#39;identificare complesse aberrazioni cromosomiche come quelle associate con il cancro. Per superare i limiti della tecnica banding, cariotipo spettrale (SKY) viene introdotto per fornire molte informazioni affidabili sulle anomalie cromosomiche. SKY è una fluorescenza multicolor ibridazione in situ (FISH), tecnica per rilevare cromosomi in metafase, con microscopio spettrale 3, 4. SKY ha dimostrato di essere uno strumento prezioso per l&#39;analisi citogenetica di una vasta gamma di anomalie cromosomiche associate con un gran numero di malattie genetiche e tumori maligni 5, 6. SKY implica l&#39;uso di sonde di DNA multicolor marcato in fluorescenza preparati dal degenerato oligonucleprimers otide mediante PCR. Così, ogni cromosoma ha un colore univoco spettrale dopo in-situ ibridazione con sonde, che sono differenzialmente etichettati con una miscela di coloranti fluorescenti (Rodamina, Texas Red, Cy5, FITC e Cy5.5). Le sonde utilizzate per SKY contenere fino a 55 sonde cromosoma specifiche 7-10. La procedura per SKY prevede diversi passaggi (Figura 1). SKY richiede la disponibilità di cellule con elevato indice mitotico da tessuto normale o malato o di sangue. I cromosomi di una cellula singola da o una cella isolate di fresco primaria o una linea cellulare sono diffuse su un vetrino. Questo spread cromosoma è etichettato con una diversa combinazione di coloranti fluorescenti specifici per ciascun cromosoma. Per il rilevamento sonda e acquisizione delle immagini, il sistema di imaging spettrale consiste di interferometro Sagnac e una telecamera CCD. Questo permette misurazione dello spettro di luce visibile emessa dal campione e di acquisire una immagine spettrale frocromosomi m individuali. HiSKY, il software utilizzato per analizzare i risultati delle immagini catturate, fornisce una facile identificazione di anomalie cromosomiche. Il risultato finale è uno metafase e una immagine classificazione cariotipo, in cui ciascuna coppia di cromosomi ha un colore distinto (Figura 2). Questo permette una facile identificazione delle identità e le traslocazioni cromosomiche. Per ulteriori informazioni, visitare il sito Applied Spectral Imaging ( <a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a> ). SKY è stato recentemente utilizzato per l&#39;identificazione di difetti segregazione dei cromosomi e le anomalie cromosomiche negli esseri umani e nei topi con malattia autosomica dominante del rene policistico (ADPKD), una malattia genetica caratterizzata da una disfunzione in cilia primaria 11-13. Utilizzando questa tecnica, abbiamo dimostrato la presenza di segregazione dei cromosomi anormali e difetti cromosomici in pazienti ADPKD e modelli di topo 14. Ulteriori analisi basate SKY non solo ci ha permesso di identificare il numero cromosomico e identità, ma anche per rilevare con precisione le aberrazioni cromosomiche molto complessi come le delezioni e le traslocazioni cromosomiche (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

Cariotipo spettrale (SKY) è una tecnica avanzata citogenetica per individuare aberrazioni genomiche e cromosomiche. Questa tecnica si avvale di sonde cromosomiche pittura, che permettono la classificazione di tutti i cromosomi. SKY può anche identificare aberrazioni cromosomiche complesse e difetti segregazione nei topi e nell&#39;uomo con varie patologie, tra cui la malattia del rene policistico.

Cariotipo spettrale allo studio anomalie cromosomiche negli esseri umani e nei topi con malattia del rene policistico

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

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38.2K Views.  University of Toledo, College of Pharmacy and Pharmaceutical Sciences. Cariotipo spettrale (SKY) è una tecnica avanzata citogenetica per individuare aberrazioni genomiche e cromosomiche. Questa tecnica si avvale di sonde cromosomiche pittura, che permettono la classificazione di tutti i cromosomi. SKY può anche identificare aberrazioni cromosomiche complesse e difetti segregazione nei topi e nell'uomo con varie patologie, tra cui la malattia del rene policistico.

Video: 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.

Aortica trapianto ortotopico in Mouse per lo studio della malattia vascolare

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

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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 nefrectomia in combinazione con dieta di sale e di ossido nitrico sintasi Inibizione di indurre la malattia renale cronica nel ratto 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.

Valutazione della funzione vascolare nei pazienti con malattia renale cronica

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.

Un possibile modello Zebrafish della malattia del rene policistico: Knockdown di<em&gt; Wnt5a</em&gt; Provoca cisti in Zebrafish Reni

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.

L&#39;uso di ultra-alto campo MRI in piccolo roditore modelli di malattia del rene policistico per<em&gt; In Vivo</em&gt; Fenotipizzazione 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.

Simmetrica Bihemispheric Postmortem cervello taglio per studiare sano e patologico del cervello Condizioni di esseri umani

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).

Un modello per simulare ipossia clinicamente rilevanti negli esseri umani

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.

In Vivo Imaging dei topi Reporter Cx3cr1gfp/gfp con tomografia a coerenza ottica Spectral-domain e scansione 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.

Valutazione della funzione del rene in modelli murini di malattia glomerulare

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.

Rilascio di miRNA esogeno sintetizzato artificialmente al rene utilizzando nanoparticelle di polietilenimina in diversi modelli murini di malattie renali

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