La fonte di finanziamento per la dimostrazione laboratorio era NIGMS concessione: GM077119. Ringraziamo il Dr. Joseph R. Haywood e il Dr. Peter Cobbett per il loro supporto Short Couse in Integrativa e organo Sistemi Farmacologia. Ringraziamo anche la signora Hannah Garver per il suo supporto tecnico della manifestazione laboratorio.

Prima di qualsiasi procedura animale, la cura degli animali e l&#39;uso comitato istituzionale (IACUC) deve approvare il protocollo. Questo protocollo è stato approvato dalla Michigan State University IACUC. 1. Preparazione Pre-lab di FITC-inulina Solution <ol><li> Warm 20 ml di soluzione salina a 70 ° C e agitare lentamente in 100 mg di FITC-inulina (5 mg / ml FITC-inulina) finché tutto inulina è disciolto. </li><li> Soluzione Raffreddare a RT e aggiungere 800 mg di albumina sierica bovina (40 mg / ml BSA, polvere liofilizzata, essenzialmente globulina, basso endotossina, ≥98% di purezza mediante elettroforesi su gel di agarosio). </li><li> Filtrare la soluzione di inulina-BSA con carta da filtro (grado 1). Mettere la soluzione filtrata in una siringa da 20 ml con un filtro a siringa punta (0,2 micron) e coprire con un foglio al riparo dalla luce. </li></ol> 2. Anestesia e Chirurgia <ol><li> Posizionare il ratto in una camera di induzione riempita con 5% isoflurano per indurre anestesia. Record bodpeso y (250-350 g) e posizionare il ratto su una piattaforma chirurgica riscaldato progettato per mantenere la temperatura corporea di 37 ° C tutto l&#39;esperimento. Garantire delicatamente il topo alla piattaforma con del nastro di laboratorio sulle zampe. Mantenere anestesia con 1-2% isoflurano con grado medico di 100% O 2 al flusso d&#39;aria di 0,8-1,0 L / min. </li><li> Inserire un catetere conica (OD punta intravascolare, 2.7F) nell&#39;arteria femorale per la pressione sanguigna e il monitoraggio della frequenza cardiaca, e il prelievo di sangue. </li><li> Inserire un catetere (PE-50) nella vena femorale per inulina infusione. Fissare il catetere al tessuto con 5-O seta intrecciata sutura chirurgica 6 circostante. </li><li> Fissare il catetere arterioso ad un trasduttore di pressione estensimetri. Registrare la pressione sanguigna e la frequenza cardiaca utilizzando software di acquisizione dei dati e la visualizzazione sullo schermo di un computer in tempo reale. Questa tecnica è illustrata in dettaglio in video 6. </li><li> Esporre la vescica attraverso un&#39;incisione sovrapubica. Tagliare un piccoloforo nella punta della vescica e inserire una cannula (PE-190) con un calore svasate punta all&#39;interno della vescica per la raccolta delle urine. Fissare la cannula alla vescica con una sutura a borsa di. </li></ol> 3. urine e il prelievo di sangue <ol><li> Posizionare la siringa di FITC-inulina in una pompa a siringa con portata regolata di 1 ml / h per 100 g di peso corporeo (3 ml / h per un topo peso 300 g). Attaccare la siringa al catetere vena femorale. Avviare l&#39;infusione di inulina e consentire un periodo di equilibrio 1-2 ore. Tenere la siringa ricoperto di un foglio al riparo dalla luce. </li><li> Determinare se il flusso urinario è stabile e adeguata per l&#39;analisi del campione (20 microlitri / min) per raccogliere un campione di urina in una fiala di raccolta pre-pesato per un periodo di 10 min. Determinazione del volume di urina metodo gravimetrico con una bilancia digitale. Un volume di urina adeguata per un periodo di raccolta 10 minuti è di 0,2 ml. Continuare a raccogliere campioni di urina fino a due collezioni consecutivi indicano un flusso di urina di 20 l / min o più. </li><li> Campioni Pre-droga <ol><li> Raccogliere un campione di urina nel corso di un periodo di 20 min. Raccogliere un campione di sangue (0,5 ml) dal catetere arterioso a metà del periodo di raccolta delle urine. Fare attenzione a cancellare completamente il catetere arterioso di soluzione fisiologica prima di raccogliere un campione di sangue in una fiala di raccolta contenente 1 U eparina. Utilizzare fiale raccolta con marcature volume per facilitare la raccolta di 0,5 ml di sangue arterioso. </li><li> Lavare il catetere arterioso con eparina-salina (20 U / ml) per cancellare il catetere di sangue (circa 0,1 ml.). La lunghezza del catetere arterioso deve essere il più breve possibile per limitare il volume di eparina-salina necessaria per irrigare. Nota: i campioni di sangue diluito producono calcoli imprecisi di GFR e l&#39;escrezione frazionale di Na e K. </li><li> Attendere 10 minuti, e ripetere la raccolta di una seconda urina pre-droga e campione di sangue. </li></ol></li><li> Dopo la raccolta di due campioni pre-droga, amministrare un dru diureticog, furosemide (10 mg / kg), tramite il catetere arterioso. Lavare il catetere arterioso con soluzione salina eparinizzata per cancellare il catetere di droga. Fare attenzione per evitare l&#39;immissione di aria attraverso il catetere arterioso. Registrare il tempo di iniezione furosemide. </li><li> Campioni post-farmaco: Ad ognuno dei 3 punti di tempo indicati, raccogliere un campione di urina durante un periodo di raccolta 10 min, e un campione di sangue (0,5 ml) a metà del periodo di raccolta delle urine. <ol><li> Per il campione post-Drug 1 - raccogliere cinque minuti dopo furosemide. </li><li> Per il campione post-Drug 2 - raccogliere dieci minuti dopo furosemide. </li><li> Per il campione post-Drug 3 - raccogliere quindici minuti dopo la furosemide. </li></ol></li><li> Dopo che tutti i campioni sono stati raccolti, eutanasia il ratto secondo procedure istituzionali da toracotomia e la rimozione del cuore. Rimuovere entrambi i reni. Decapsulate (rimuovere la membrana che circonda) e asciugare i reni per rimuovere il sangue in eccesso. Pesare i reni. </li></ol><ol><li> Misurare tutti i volumi di campione di urina con metodo gravimetrico con una bilancia digitale, e pesi record. </li><li> Campioni di centrifuga di sangue intero con una centrifuga da tavolo (1.800 xg) per separare il plasma. Trasferire campioni di plasma di piccole fiale etichettate. </li><li> Analizzare le concentrazioni di Na e K in campioni di urina e di plasma con un analizzatore di sodio / potassio. </li><li> Misura di FITC-inulina nel plasma e nelle urine <ol><li> Diluire urina pre-droga (da 1: 200 a 1: 400), e nelle urine post-droga (1,10) con tampone HEPES (500 mM, pH 7,4). </li><li> Aggiungere 40 ml di microlitri standard o campione e 60 di tampone HEPES in una piastra da 96 pozzetti (un campione per pozzetto) e lasciare mescolare per 10 minuti mentre coperto con un foglio di alluminio. </li><li> Generare una curva standard per FITC-inulina per le concentrazioni di 6,25, 12,5, 25, 50, 100, 200, 400 ug / ml (Figura 1). Determinare FITC-inulina fluorescenza in campioni e standard utilizzando un lettore di micropiastre con l&#39;excitazioni e di emissione lunghezze d&#39;onda di 485 e 538 nm, rispettivamente. </li><li> Montare i valori fluorescenti per gli standard di un 4 Parametro analisi di regressione funzione logistica. I parametri della funzione di regressione sono utilizzati per calcolare la concentrazione FITC-inulina in campioni di plasma ed urina (Tabella 1). </li></ol></li></ol> 5. Post-laboratorio Analisi dei risultati: Calcoli <ol><li> Calcola urina Portata (UV; ml / min): [volume di urina raccolto (ml)] ÷ [momento della raccolta (min)] </li><li> Calcola velocità di filtrazione glomerulare (GFR; ml / min): [concentrazione inulina Urina (mg / ml) x UV (ml / min)] ÷ [plasma inulina conc. (Mg / ml)] </li><li> Calcola filtrata carico di sodio (mmol / min): concentrazione di sodio al plasma (mmol / ml) x GFR (ml / min) </li><li> Calcola sodio escrezione Rate (U Na V; mmol / min): concentrazione di sodio Urina (mmol / ml) x UV (ml / min) </li><li> Calcola frazionale escrezione di sodio (Na FE;%): [U Na V (mmol / min)] ÷ [filtrato sodio Load (mmol / min)] x 100 </li><li> Calcola carico filtrato di potassio (mmol / min): la concentrazione di potassio del plasma (mmol / ml) x GFR (ml / min) </li><li> Calcola potassio escrezione Rate (U K V; mmol / min): concentrazione di potassio Urina (mmol / ml) x UV (ml / min) </li><li> Calcola frazionale escrezione di potassio (FE K;%): [U K V (mmol / min)] ÷ [filtrato carico di potassio (mmol / min)] x 100 </li></ol>

<ol>
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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assay+of+inulin+in+tissues+using+anthrone.">Assay of inulin in tissues using anthrone.</a> Clinical Biochemistry. 8 (1), 67-70 (1975).</li><li>Shalmi, M., Lunau, H. E., Petersen, J. S., Bak, M., Christensen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suitability+of+tritiated+inulin+for+determination+of+glomerular+filtration+rate.">Suitability of tritiated inulin for determination of glomerular filtration rate.</a> Americam Journal of Physiology. 260 (2 Pt 2), F283-F289 (1991).</li><li>Denton, K. M., Anderson, W. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glomerular+filtration+rate+determinations+in+conscious+type+II+diabetic+mice.">Glomerular filtration rate determinations in conscious type II diabetic mice.</a> Americam Journal of Physiology. 300 (3), F618-F625 (2011).</li><li>Rosen, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+anaesthesia+and+surgery+on+renal+hemodynamics.">Effects of anaesthesia and surgery on renal hemodynamics.</a> British Journal of Anesthesiology. 44, 252-258 (1972).</li><li>Cousins, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthesia+and+the+kidney.">Anesthesia and the kidney.</a> Anaesthesia and intensive care. 11 (4), 292-320 (1983).</li><li>Walter, S. J., Zewde, T., Shirley, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+anaesthesia+and+standard+clearance+procedures+on+renal+function+in+the+rat.">The effect of anaesthesia and standard clearance procedures on renal function in the rat.</a> Quarterly Journal of Experimental Physiology. 74, 805-812 (1989).</li><li>Rieg, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+method+for+measurement+of+glomerular+filtration+rate+in+conscious+mice.">A high-throughput method for measurement of glomerular filtration rate in conscious mice.</a> Journal of Visualized Experiments. (75), (2013).</li></ol>

Gli autori dichiarano di non avere interessi finanziari in competizione. I pareri o le affermazioni contenute nel presente documento sono le opinioni personali dell&#39;autore e non sono da intendersi come ufficiale o come riflesso le opinioni del Dipartimento dell&#39;Esercito o il Dipartimento della Difesa.

Un indicatore adeguato per la misura GFR devono soddisfare quattro criteri: essere liberamente filtrato a livello glomerulare, essere non legato alle proteine ​​plasmatiche, e né essere assorbiti né secreto nel nefrone. L&#39;inulina è un polimero di fruttosio che soddisfa questi criteri. Come risultato, la clearance renale dell&#39;inulina è considerato lo standard per misurare GFR 7. La tecnica dimostrata rappresenta l&#39;approccio tradizionale di determinare la clearance renale di inulina utilizzando raccolta delle urine temporizzate durante un&#39;infusione costante di inulina 8,9. Misurazioni inulina tradizionali sono state effettuate utilizzando il metodo antrone per produrre una determinazione colorimetrica quantitativa di inulina misurata allo spettrofotometro 10,11. Tuttavia, nel tentativo di facilitare la misurazione di inulina in piccoli volumi di urina e plasma, inulina è stato etichettato con radioattivi 12-14, e 15-17 etichette fluorescenti. La dimostrazione di laboratorio presentato in questo video used FITC etichettato inulina per la misurazione della funzionalità renale a causa della mancanza di rischio di esposizione a radiazioni umana e la facilità di misurare la fluorescenza FITC 15. Questa dimostrazione di laboratorio ha lo scopo di fornire una comprensione concettuale di come misurare la funzionalità renale a studenti con minime competenze di laboratorio. Pertanto, la preparazione pre-lab di soluzione FITC-inulina, e preparazione chirurgica degli animali sono effettuate da tecnici esperti prima dell&#39;inizio della manifestazione. Gli studenti arrivano per la manifestazione alla fine del periodo di inulina equilibrio 1-2 ore. A questo punto, gli studenti sono presentati con una panoramica Pre-laboratorio e informati delle procedure che sono stati condotti sugli animali. Due studenti sono assegnati a un esperimento animale, e istruiti su come raccogliere campioni di sangue e urine prima e dopo la somministrazione del farmaco diuretico. L&#39;analisi dei campioni di sangue e urine è condotta da experietecnici e risultati NCED vengono consegnati agli studenti per i calcoli di funzionalità renale. I risultati sono presentati nel corso di una discussione post-laboratorio che può essere programmata dopo la manifestazione. Ci sono diversi passaggi critici all&#39;interno del protocollo per assicurare risposte valide. In primo luogo, FITC inulina deve essere completamente sciolto e filtrato prima della somministrazione animale. Idealmente, FITC inulina deve essere dializzato in acqua per 48 ore a temperatura ambiente per eliminare residui di FITC non legato. In secondo luogo, i campioni di plasma devono essere privi di soluzione salina. Gli studenti sono incaricati di raccogliere un campione di sangue solo dopo che tutte le saline nel catetere arterioso sono stati espulsi e solo il sangue scorre fuori del catetere. I campioni di sangue che vengono diluiti con soluzione fisiologica fornirà valori accurati per inulina plasma, sodio e potassio. In terzo luogo, il flusso di urina deve essere costante e sufficiente per produrre abbastanza campione per l&#39;analisi. Una portata costante urine al basale è critica perché è un&#39;indicazioneuna preparazione sperimentale stabile. Se il flusso di urina è troppo bassa, la velocità di infusione di inulina può essere aumentata prima campionare collezioni. Tuttavia, l&#39;infusione di inulina deve essere costante durante il corso dell&#39;esperimento, cioè, inulina velocità di infusione non deve essere regolato durante l&#39;esperimento. Infine, la misura di inulina fluorescenza in campioni di plasma ed urina dal lettore di micropiastre è fondamentale per un esperimento riuscito. Poiché le specifiche del lettore di micropiastre determinerà se campioni richiedono diluizioni, si raccomanda un test del saggio inulina essere condotto prima della manifestazione laboratorio nel tentativo di ottimizzare le caratteristiche del lettore di micropiastre e garantiscono che i valori campione fluorescenza sono all&#39;interno del mid-range della curva standard. Mentre la valutazione della funzione renale in base alla clearance renale di inulina è considerato il gold standard, questa tecnica ha limiti, perché gli animali devono essere anestetizzati, estrumentato con vascolari e cateteri vescicali. Agenti Anesthethetic hanno dimostrato di influenzare l&#39;emodinamica renale e GFR 18,19; tuttavia isoflurano e inactin sono tipicamente utilizzati in esperimenti di funzionalità renale a causa dei loro effetti minimi sul rene 19,20. La tecnica di depurazione inulina richiede anche un&#39;infusione costante di campioni di inulina e di sangue e di plasma multiple che possono essere proibitivi in ​​animali più piccoli, come i topi. Modifiche di questa tecnica sono stati sviluppati per consentire la misurazione della clearance plasmatica da una singola iniezione di inulina in animali coscienti 21. Tali modifiche richiedono anche piccoli volumi di campioni di sangue per l&#39;analisi, e forniscono un metodo alternativo per valutare la funzione renale nei topi. La misura della funzione renale è applicabile a studi di fisiologia, patologia, tossicologia, farmacologia e la malattia gli stati. Gli studenti che partecipano al Function manifestazione renale impareranno tegli tecnica gold standard di clearance renale di inulina per valutare la funzionalità renale. Per padroneggiare questa tecnica, gli studenti comprendere i principi di funzionalità renale e consentire loro di applicare la tecnica per la propria attività di ricerca e di determinare se le modifiche alla tecnica sono adatti per i loro studi.

The most important function of the kidney is the homeostatic regulation of extracellular water and electrolyte content. The kidneys closely regulate extracellular water, sodium (Na) and potassium (K) to maintain normal physiological levels. Disturbances in renal function can result in serious metabolic disorders which can be fatal. The basic renal process occurs in the nephron and begins with the filtration of plasma at the glomerulus and ends with the excretion of urine. Other processes that determine the final concentration of water, Na and K in the urine are secretion and reabsorption within the nephron. Measurements of glomerular filtration rate (GFR) and the fractional excretion of Na and K are critical in assessing renal function in health and disease. The reader is referred to previously published review articles and textbooks for a more thorough discussion of kidney function1-4. 
GFR can be measured as the steady state renal clearance of inulin which is filtered at the glomerulus, but not secreted or reabsorbed along the nephron5. While this technique requires anesthesia, surgical preparation, and a terminal experiment, it is considered the gold standard of GFR measurement. Using inulin that is tagged with fluorescein-isothiocyanate (FITC), plasma and urine concentration of FITC-inulin can be easily measured in small volumes and used to calculate GFR during multiple time points of an experiment. The fractional excretion of Na and K can be determined from the concentration of Na and K in plasma and urine. 
The conceptual understanding of how to measure renal function can easily be demonstrated in a short lab designed to allow students to actively participate in some aspects of the experiment. This video depicts the pre-lab preparation, the renal function demonstration, and the post-lab evaluation of results. The surgical techniques necessary for making measurements of GFR are demonstrated in an anesthetized rat. In addition, example calculations for GFR, and the fractional excretion of Na and K are shown before and after administration of a diuretic drug.

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			<td>5-0 Braided Silk Surgical Suture</td>
			<td>Surgical Specialties Corp</td>
			<td>SP1033</td>
			<td></td>
		</tr>
		<tr>
			<td>Assay Plate, 96-Well</td>
			<td>Costar&#160;</td>
			<td>3922</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma Chemical Co</td>
			<td>A2934-25G</td>
			<td></td>
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		<tr>
			<td>Centrifuge</td>
			<td>Beckman Coulter</td>
			<td>MicroFuge 18, 357160</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical Sample Tubes</td>
			<td>Dot Scientific Inc.&#160;</td>
			<td>#711-FTG</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton Tipped Applicators</td>
			<td>Solon Manufacturing Co</td>
			<td>56200</td>
			<td></td>
		</tr>
		<tr>
			<td>Data Acquisition Software</td>
			<td>ADInstruments</td>
			<td>LabChart Pro 7.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital Scale&#160;</td>
			<td>Denver Instrument</td>
			<td>APX-4001</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC-Inulin</td>
			<td>Sigma Chemical Co</td>
			<td>F3272-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Gauze Sponges</td>
			<td>Covidien</td>
			<td>2146</td>
			<td></td>
		</tr>
		<tr>
			<td>Heated Surgical Bed</td>
			<td>EZ-Anesthesia</td>
			<td>EZ-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sagnet</td>
			<td>NDC 25021-402-10</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma Chemical Co</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Abbott Animal Health</td>
			<td>IsoFlo, 5260-04-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane Vaporizer</td>
			<td>EZ-Anesthesia</td>
			<td>EZ-190F</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Dissecting Forceps</td>
			<td>Biomedical Research Instruments Inc.</td>
			<td>70-1020</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate Reader - Fluoroskan</td>
			<td>ThermoScientific</td>
			<td>Ascent FL, 5210460</td>
			<td></td>
		</tr>
		<tr>
			<td>NOVA 5+ Sodium/Potassium Analyzer</td>
			<td>NOVA BioMedical</td>
			<td>14156</td>
			<td></td>
		</tr>
		<tr>
			<td>Olsen-Hegar Needle Holders with Scissors</td>
			<td>Fine Science Tools</td>
			<td>12002-12</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-190 (for bladder catheter)</td>
			<td>BD Medical</td>
			<td>427435</td>
			<td></td>
		</tr>
		<tr>
			<td>Pressure Transducer&#160;</td>
			<td>ADInstruments</td>
			<td>MLT1199</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyrex Culture Tubes</td>
			<td>Corning Inc.</td>
			<td>99445-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat Femoral Tapered Artery Catheter</td>
			<td>Strategic Applications Inc.</td>
			<td>RFA-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Salix Furosemide 5%</td>
			<td>Intervet</td>
			<td>#34-478</td>
			<td></td>
		</tr>
		<tr>
			<td>Strabismus Scissors</td>
			<td>Fine Science Tools</td>
			<td>14075-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Student Surgical Scissors</td>
			<td>Fine Science Tools</td>
			<td>91402-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Gloves</td>
			<td>Kimberly-Clark</td>
			<td>Sterling Nitrile Gloves</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Razel Scientific</td>
			<td>R99-E</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Forceps</td>
			<td>Fine Science Tools</td>
			<td>91121-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Scissors</td>
			<td>George Tiemann&#160; Co</td>
			<td>105-420</td>
			<td></td>
		</tr>
	</tbody>
</table>
5-0 Braided Silk Surgical Suture Surgical Specialties Corp SP1033 Assay Plate, 96-Well Costar&#160; 3922 Bovine Serum Albumin Sigma Chemical Co A2934-25G Centrifuge Beckman Coulter MicroFuge 18, 357160 Conical Sample Tubes Dot Scientific Inc.&#160; #711-FTG Cotton Tipped Applicators Solon Manufacturing Co 56200 Data Acquisition Software ADInstruments LabChart Pro 7.0 Digital Scale&#160; Denver Instrument APX-4001 FITC-Inulin Sigma Chemical Co F3272-1G Gauze Sponges Covidien 2146 Heated Surgical Bed EZ-Anesthesia EZ-212 Heparin Sagnet NDC 25021-402-10 HEPES Sigma Chemical Co H3375 Isoflurane Abbott Animal Health IsoFlo, 5260-04-05 Isoflurane Vaporizer EZ-Anesthesia EZ-190F Micro Dissecting Forceps Biomedical Research Instruments Inc. 70-1020 Microplate Reader - Fluoroskan ThermoScientific Ascent FL, 5210460 NOVA 5+ Sodium/Potassium Analyzer NOVA BioMedical 14156 Olsen-Hegar Needle Holders with Scissors Fine Science Tools 12002-12 PE-190 (for bladder catheter) BD Medical 427435 Pressure Transducer&#160; ADInstruments MLT1199 Pyrex Culture Tubes Corning Inc. 99445-12 Rat Femoral Tapered Artery Catheter Strategic Applications Inc. RFA-01 Salix Furosemide 5% Intervet #34-478 Strabismus Scissors Fine Science Tools 14075-11 Student Surgical Scissors Fine Science Tools 91402-12 Surgical Gloves Kimberly-Clark Sterling Nitrile Gloves Syringe pump Razel Scientific R99-E Tissue Forceps Fine Science Tools 91121-12 Tissue Scissors George Tiemann&#160; Co 105-420

physiology lab demonstration: glomerular filtration rate in a rat

Measurements of glomerular filtration rate (GFR), and the fractional excretion of sodium (Na) and potassium (K) are critical in assessing renal function in health and disease. GFR is measured as the steady state renal clearance of inulin which is filtered at the glomerulus, but not secreted or reabsorbed along the nephron. The fractional excretion of Na and K can be determined from the concentration of Na and K in plasma and urine. The renal clearance of inulin can be demonstrated in an anesthetized animal which has catheters in the femoral artery, femoral vein and bladder. The equipment and supplies used for this procedure are those commonly available in a research core facility, and thus makes this procedure a practical means for measuring renal function. The purpose of this video is to demonstrate the procedures required to perform a lab demonstration in which renal function is assessed before and after a diuretic drug. The presented technique can be utilized to assess renal function in rat models of renal disease.

Il diuretico usato nella dimostrazione laboratorio era furosemide che inibisce molto rapidamente il riassorbimento di Na e K filtrati dal rene con conseguente aumento Na, K, e l&#39;escrezione dell&#39;acqua entro minuti dalla somministrazione del farmaco. Con il suo meccanismo primario, furosemide dovrebbe avere effetti minimi sul GFR e il carico filtrato di Na e K, ma aumenterà il flusso di urina, e l&#39;escrezione frazionale di Na e K. I risultati rappresentativi in Tabella 3 mostrano che in un ratto anestetizzato, la media dei valori pre-farmacologici per GFR è stata di 3,2 ml / min, Na escrezione è 0,58 mmol / min (0,1% del carico filtrato), e K escrezione era 4.4 mmol / min (27% del carico filtrato). Cinque minuti dopo furosemide (post-droga 1), GFR e il carico filtrato di Na e K non sono stati influenzati. Tuttavia, l&#39;escrezione frazionata di Na aumentato al 11,5%, e l&#39;escrezione frazionata di K aumentato al 63% dei rispettivi carichi filtrati. Le misure di MAP e HR indicano che furosemide ha avuto effetti minimi sul MAP e HR (Tabella 2). Gli indici di funzionalità renale valutati nella dimostrazione laboratorio erano la GFR, definito come il tasso con cui il plasma viene filtrata dal rene; Na filtrato e K, definito come il tasso con cui Na e K sono filtrati dal rene; Na e K Escrezione Rate, definito come il tasso con cui Na e K sono escreti per via renale; e l&#39;escrezione frazionata di Na e K, definito come la percentuale di filtrato Na e K che è escreta dal rene <img alt="Figura 1" src="/files/ftp_upload/52425/52425fig1.jpg" /> Figura 1: valori. Inulina curva standard FITC fluorescenza sono mostrate per standard contenenti 6,25, 12,5, 25, 50, 100, 200 e 400 ug / ml di inulina. Un&#39;analisi funzione di regressione logistica a 4 Parametro genera la curva di best-fit. I parametri della funzione di regressione di questa curva sono stati usati per calcolare FIConcentrazione TC-inulina in campioni di plasma e nelle urine. <table border="0" cellpadding="0" cellspacing="0" height="515" width="592"><tbody><tr><td></td><td colspan="3"> FITC-inulina fluorescenza </td><td nowrap="nowrap"> Concentrazione </td><td></td><td> Risultato </td></tr><tr><td> Standard </td><td> replicare 1 </td><td> replicare 2 </td><td> Significare </td><td> ug / ml </td><td> Diluizione </td><td> ug / ml </td></tr><tr><td> Vuoto </td><td align="right"> 63,9 </td><td align="right"> 64.8 </td><td align="right"> 64,4 </td><td align="right"> 0.4 </td><td align="right"> 1 </td><td align="right"> 0.4 </td></tr><tr><td> 6.25 </td><td align="right"> 253,2 </td><td align="right"> 264,1 </td><td align="right"> 258,7 </td><td align="right"> 5.9 </td><td align="right"> 1 </td><td align="right"> 5.9 </td></tr><tr><td> 12.5 </td><td align="right"> 474.0 &lt;/ Td&gt; <td align="right"> 491,3 </td><td align="right"> 482,7 </td><td align="right"> 12.5 </td><td align="right"> 1 </td><td align="right"> 12.5 </td></tr><tr><td> 25 </td><td align="right"> 854,8 </td><td align="right"> 881,3 </td><td align="right"> 868,1 </td><td align="right"> 24.4 </td><td align="right"> 1 </td><td align="right"> 24.4 </td></tr><tr><td> 50 </td><td align="right"> 1.617,1 </td><td align="right"> 1.618,0 </td><td align="right"> 1.617,6 </td><td align="right"> 50.3 </td><td align="right"> 1 </td><td align="right"> 50.3 </td></tr><tr><td> 100 </td><td align="right"> 2.813,1 </td><td align="right"> 2.846,1 </td><td align="right"> 2.829,6 </td><td align="right"> 101.3 </td><td align="right"> 1 </td><td align="right"> 101.3 </td></tr><tr><td> 200 </td><td align="right"> 4.367,3 </td><td align="right"> 4.588,7 </td><td align="right"> 4.478,0 </td><td align="right"> 198,2 </td><td align="right"> 1<html></td><td align="right"> 198,2 </td></tr><tr><td> 400 </td><td align="right"> 6.258,0 </td><td align="right"> 6.650,0 </td><td align="right"> 6.454,0 </td><td align="right"> 401,6 </td><td align="right"> 1 </td><td align="right"> 401,6 </td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Urine Sample </td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Pre-droga 1 </td><td align="right"> 2.443,9 </td><td align="right"> 2.062,3 </td><td align="right"> 2.253,1 </td><td align="right"> 88.5 </td><td align="right"> 200 </td><td align="right"> 17700 </td></tr><tr><td> Pre-droga 2 </td><td align="right"> 2.266,5 </td><td align="right"> 1.707,0 </td><td align="right"> 1.986,8 </td><td align="right"> 76.3 </td><td align="right"> 200 </td><td align="right"> 15250 </td></tr><tr><td> Post-droga 1 </td>HT &quot;&gt; 1.208,9 </td><td align="right"> 1.391,2 </td><td align="right"> 1.300,1 </td><td align="right"> 44.7 </td><td align="right"> 10 </td><td align="right"> 447 </td></tr><tr><td> Post-droga 2 </td><td align="right"> 2.753,4 </td><td align="right"> 2.120,5 </td><td align="right"> 2.437,0 </td><td align="right"> 97.0 </td><td align="right"> 10 </td><td align="right"> 970 </td></tr><tr><td> Post-droga 3 </td><td align="right"> 2.888,3 </td><td align="right"> 3.178,0 </td><td align="right"> 3.033,2 </td><td align="right"> 124,4 </td><td align="right"> 10 </td><td align="right"> 1244 </td></tr></tbody></table> Tabella 1:. Risultati del campione di valori di fluorescenza inulina Assay FITC-inulina sono indicati per il bianco reagente, standard 7 e 5 campioni di urina. Norme e campioni sono stati analizzati in duplicato e diluiti, se necessario. La fluorescenza media per ciascun campione è stato utilizzato per calcolare la concentrazione di inulina.<html> Le concentrazioni di inulina in questi campioni di urina sono inclusi nella tabella di misurazioni (Tabella 2). <img alt="Tabella 2" src="/files/ftp_upload/52425/52425table2.jpg" /> Tabella 2: misurazioni registrate durante la funzione renale Lab Dimostrazione Le variabili registrate durante cinque periodi di tempo (due pre-droga e tre post-droga) della renale laboratorio funzione dimostrativa sono a destra ea sinistra il peso del rene, la pressione arteriosa media (MAP),. frequenza cardiaca (HR), tempo di campionamento, volume di urina, plasma ed urina di sodio (Na), potassio (K), e le concentrazioni di inulina. Le concentrazioni inulina urina sono stati determinati dal saggio inulina mostrato in Tabella 1. 25table3.jpg &quot;/&gt; Tabella 3:. Parametri di funzionalità renale in base a misurazioni registrate utilizzando le formule riportate nella Sezione protocollo 5, le variabili rilevate (tabella 2) sono usati per calcolare l&#39;urina portata, velocità di filtrazione glomerulare (GFR), peso GFR / g rene, tasso di escrezione , carico filtrato, e l&#39;escrezione frazionale di sodio (Na) e potassio (K) nei due pre-farmaco e tre periodi post-farmaco.

Watch this Scientific Journal Video about Physiology Lab Demonstration: Glomerular Filtration Rate in a Rat at JoVE.com

Dimostrazione in laboratorio di fisiologia: velocità di filtrazione glomerulare in un ratto | Biologia | Video

Physiology Lab Demonstration: Glomerular Filtration Rate in a Rat

The purpose of this protocol is to demonstrate the principles and techniques for measuring and calculating glomerular filtration rate, urine flow rate, and excretion of sodium and potassium in a rat. This demonstration can be used to provide students with an overall conceptual understanding of how to measure renal function.

Fisiologia Lab Dimostrazione: filtrazione glomerulare in un ratto

Measurements of glomerular filtration rate (GFR), and the fractional excretion of sodium (Na) and potassium (K) are critical in assessing renal function ...

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17.2K Views.  U.S. Army Institute of Surgical Research. The purpose of this protocol is to demonstrate the principles and techniques for measuring and calculating glomerular filtration rate, urine flow rate, and excretion of sodium and potassium in a rat. This demonstration can be used to provide students with an overall conceptual understanding of how to measure renal function. 

Video: Physiology Lab Demonstration: Glomerular Filtration Rate in a Rat

Large Volume (20L+) Filtration of Coastal Seawater Samples

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For this method, samples were collected from the deck of the HMS John Strickland operating in Saanich Inlet. This video documents large volume (&ge;20 L) filtration of microbial biomass, ranging between 0.22&mu;m and 2.7&mu;m in diameter, from the water column. Two 20L samples can be filtered simultaneously using a single pump unit equipped with four rotating heads. Filtration is done in the field on extended trips, or immediately upon return for day trips. It is important to record the amount of water passing through each sterivex filter unit. To prevent biofilm formation between sampling trips, all filtration equipment must be rinsed with dilute HCl and deionized water and autoclaved immediately after use. This procedure will take approximately 5 hours plus an additional hour for clean up.

Large Volume 20L+ Filtration of Coastal Seawater Samples

This video documents large volume (&ge;20 L) filtration of microbial biomass, ranging between 0.22&mu;m and 2.7&mu;m in diameter, from the water column.

Volume di grandi dimensioni (20L +) Filtrazione di campioni d&#39;acqua di mare costiero

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For this method, ...

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Small Volume (1-3L) Filtration of Coastal Seawater Samples

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For today s demonstration samples were collected from the deck of the HMS John Strickland operating in Saanich Inlet. This video documents small volume (~1 L) filtration of microbial biomass from the water column. The protocol is an extension of the large volume sampling protocol described earlier, with one major difference: here, there is no pre-filtration step, so all size classes of biomass are collected down to the 0.22 &mu;m filter cut-off. Samples collected this way are ideal for nucleic acid analysis. The set-up, filtration, and clean-up steps each take about 20-30 minutes. If using two peristaltic pumps simultaneously, up to 8 samples may be filtered at the same time. To prevent biofilm formation between sampling trips, all filtration equipment must be rinsed with dilute HCl and deionized water and autoclaved immediately after use.

Small Volume 1-3L Filtration of Coastal Seawater Samples

This video documents small volume (~1 L) filtration of microbial biomass from the water column.

Volume piccoli (1-3L) Filtrazione di campioni d&#39;acqua di mare costiero

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For today s ...

A Video Demonstration of Preserved Piloting by Scent Tracking but Impaired Dead Reckoning After Fimbria-Fornix Lesions in the Rat

Piloting and dead reckoning navigation strategies use very different cue constellations and computational processes (Darwin, 1873; Barlow, 1964; O&#x2019;Keefe and Nadel, 1978; Mittelstaedt and Mittelstaedt, 1980; Landeau et al., 1984; Etienne, 1987; Gallistel, 1990;
Maurer and S&#233;guinot, 1995). Piloting requires the use of the relationships between relatively stable external (visual, olfactory, auditory) cues, whereas dead reckoning requires the integration of cues generated by self-movement. Animals obtain self-movement information from vestibular receptors, and possibly muscle and joint receptors, and efference copy of commands that generate movement. An animal may also use the flows of visual, auditory, and olfactory stimuli caused by its movements. Using a piloting strategy an animal can use geometrical calculations to determine directions and distances to places in its environment, whereas using an dead reckoning strategy it can integrate cues generated by its previous movements to return to a just left location. Dead reckoning is colloquially called "sense of direction" and "sense of distance."

Although there is considerable evidence that the hippocampus is involved in piloting (O&#x2019;Keefe and Nadel, 1978; O&#x2019;Keefe and Speakman, 1987), there is also evidence from behavioral (Whishaw et al., 1997; Whishaw and Maaswinkel, 1998; Maaswinkel and Whishaw, 1999), modeling (Samsonovich and McNaughton, 1997), and electrophysiological (O&#x2019;Mare et al., 1994; Sharp et al., 1995; Taube and Burton, 1995; Blair and Sharp, 1996; McNaughton et al., 1996; Wiener, 1996; Golob and Taube, 1997) studies that the hippocampal formation is involved in dead reckoning. The relative contribution of the hippocampus to the two forms of navigation is still uncertain, however. Ordinarily, it is difficult to be certain that an animal is using a piloting versus a dead reckoning strategy because animals are very flexible in their use of strategies and cues (Etienne et al., 1996; Dudchenko et al., 1997; Martin et al., 1997; Maaswinkel and Whishaw, 1999). The objective of the present video demonstrations was to solve the problem of cue specification in order to examine the relative contribution of the hippocampus in the use of these strategies. The rats were trained in a new task in which they followed linear or polygon scented trails to obtain a large food pellet hidden on an open field. Because rats have a proclivity to carry the food back to the refuge, accuracy and the cues used to return to the home base were dependent variables (Whishaw and Tomie, 1997). To force an animal to use a a dead reckoning strategy to reach its refuge with the food, the rats were tested when blindfolded or under infrared light, a spectral wavelength in which they cannot see, and in some experiments the scent trail was additionally removed once an animal reached the food. To examine the relative contribution of the hippocampus, fimbria&#x2013;fornix (FF) lesions, which disrupt information flow in the hippocampal formation (Bland, 1986), impair memory (Gaffan and Gaffan, 1991), and produce spatial deficits (Whishaw and Jarrard, 1995), were used.

In a piloting scent tracking task, the ability of the rats to return to a refuge with food using visual an odor trail or using dead reckoning in infrared light, the integrated record of previous movements, demonstrates that the hippocampus is necessary for dead reckoning.

Una dimostrazione video di pilotaggio da parte di Conserve Monitoraggio Profumo ma deteriorate Dead Reckoning Dopo Fimbria-fornice lesioni nel ratto

Piloting and dead reckoning navigation strategies use very different cue constellations and computational processes (Darwin, 1873; Barlow, 1964; ...

Historical View and Physiology Demonstration at the NMJ of the Crayfish Opener Muscle

Here we present some of the key important discoveries made with the opener neuromuscular (NMJ) preparation of crustaceans and illustrate that there is still much to learn from this model preparation. In understanding the history one can appreciate why even today this NMJ still offers a rich playground to address questions regarding pre- and post-synaptic function and plasticity. The viability and ease of access to the terminal for intracellular as well as extracellular electrophysiology and imaging are significant advantages. The mechanisms behind the modulation of vesicular kinetics and fusion within the high- and low-output terminals are begging for investigation. The preparation also offers a testable model system for computational assessments and manipulations to examine key variables in theoretical models of synaptic function, for example calcium dynamics during short-term facilitation. The synaptic complexity of active zone and statistical nature of quantal release is also an open area for future investigation both experimentally and computationally.

The opener muscle of the crayfish leg is presented for its historical importance and experimental versatility in muscle phenotype, synaptic physiology and plasticity.

Vedi storici e dimostrazione di Fisiologia presso la NMJ del Muscolo Opener gamberi di fiume

Here we present some of the key important discoveries made with the opener neuromuscular (NMJ) preparation of crustaceans and illustrate that there is ...

Ovariectomy and 17&beta;-estradiol Replacement in Rats and Mice: A Visual Demonstration

Estrogens are a family of female sexual hormones with an exceptionally wide spectrum of effects. When rats and mice are used in estrogen research they are commonly ovariectomized in order to ablate the rapidly cycling hormone production, replacing the 17&beta;-estradiol exogenously. There is, however, lack of consensus regarding how the hormone should be administered to obtain physiological serum concentrations. This is crucial since the 17&beta;-estradiol level/administration method profoundly influences the experimental results1-3. We have in a series of studies characterized the different modes of 17&beta;-estradiol administration, finding that subcutaneous silastic capsules and per-oral nut-cream Nutella are superior to commercially available slow-release pellets (produced by the company Innovative Research of America) and daily injections in terms of producing physiological serum concentrations of 17&beta;-estradiol4-6. Amongst the advantages of the nut-cream method, that previously has been used for buprenorphine administration7, is that when used for estrogen administration it resembles peroral hormone replacement therapy and is non-invasive. The subcutaneous silastic capsules are convenient and produce the most stable serum concentrations. This video article contains step-by-step demonstrations of ovariectomy and 17&beta;-estradiol hormone replacement by silastic capsules and peroral Nutella in rats and mice, followed by a discussion of important aspects of the administration procedures.

Ovariectomy and 17&#946;-estradiol Replacement in Rats and Mice: A Visual Demonstration

Ovariectomy and subsequent replacement of 17&beta;-estradiol by means of silastic capsules and peroral Nutella are demonstrated in rats and mice.

Sostituzione ovariectomia e 17β-estradiolo in ratti e topi: A dimostrazione visiva

Estrogens are a family of female sexual hormones with an exceptionally wide spectrum of effects. When rats and mice are used in estrogen research they are ...

A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice

The measurement of glomerular filtration rate (GFR) is the gold standard in kidney function assessment. Currently, investigators determine GFR by measuring the level of the endogenous biomarker creatinine or exogenously applied radioactive labeled inulin (3H or 14C). Creatinine has the substantial drawback that proximal tubular secretion accounts for ~50% of total renal creatinine excretion and therefore creatinine is not a reliable GFR marker. Depending on the experiment performed, inulin clearance can be determined by an intravenous single bolus injection or continuous infusion (intravenous or osmotic minipump). Both approaches require the collection of plasma or plasma and urine, respectively. Other drawbacks of radioactive labeled inulin include usage of isotopes, time consuming surgical preparation of the animals, and the requirement of a terminal experiment. Here we describe a method which uses a single bolus injection of fluorescein isothiocyanate-(FITC) labeled inulin and the measurement of its fluorescence in 1-2 &mu;l of diluted plasma. By applying a two-compartment model, with 8 blood collections per mouse, it is possible to measure GFR in up to 24 mice per day using a special work-flow protocol. This method only requires brief isoflurane anesthesia with all the blood samples being collected in a non-restrained and awake mouse. Another advantage is that it is possible to follow mice over a period of several months and treatments (i.e. doing paired experiments with dietary changes or drug applications). We hope that this technique of measuring GFR is useful to other investigators studying mouse kidney function and will replace less accurate methods of estimating kidney function, such as plasma creatinine and blood urea nitrogen.

Measurement of glomerular filtration rate (GFR) is the gold standard for kidney function assessment. Here we describe a high-throughput method which allows the determination of GFR in conscious mice by using a single bolus injection, determination of fluorescein isothiocyanate (FITC)-inulin in plasma and calculation of GFR by a two-phase exponential decay model.

Un metodo ad alta velocità per la misura della velocità di filtrazione glomerulare in topi coscienti

The measurement of  glomerular filtration rate (GFR) is the gold standard in kidney function  assessment. Currently, investigators determine GFR by ...

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator&#8217;s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 1010 viable and respiration competent mitochondria from 0.18 &#177; 0.04 g (wet weight) tissue sample.

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is &#60;30 min compared to 60 - 100 min using alternative methods.

Isolamento rapida e purificazione di mitocondri per il trapianto da tessuto Dissociazione e Filtrazione Differenziale

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, most notably developmental arrest and reduced metabolism. Global gene expression profiling via RNA-Seq can provide important insights into the transcriptional mechanisms of photoperiodic diapause. The Asian tiger mosquito, Aedes albopictus, is an outstanding organism for studying the transcriptional bases of diapause due to its ease of rearing, easily induced diapause, and the genomic resources available. This manuscript presents a general experimental workflow for identifying diapause-induced transcriptional differences in A. albopictus. Rearing techniques, conditions necessary to induce diapause and non-diapause development, methods to estimate percent diapause in a population, and RNA extraction and integrity assessment for mosquitoes are documented. A workflow to process RNA-Seq data from Illumina sequencers culminates in a list of differentially expressed genes. The representative results demonstrate that this protocol can be used to effectively identify genes differentially regulated at the transcriptional level in A. albopictus due to photoperiodic differences. With modest adjustments, this workflow can be readily adapted to study the transcriptional bases of diapause or other important life history traits in other mosquitoes.

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

RNA-Seq analyses are becoming increasingly important for identifying the molecular underpinnings of adaptive traits in non-model organisms. Here, a protocol to identify differentially expressed genes between diapause and non-diapause Aedes albopictus mosquitoes is described, from mosquito rearing, to RNA sequencing and bioinformatics analyses of RNA-Seq data.

Un Sperimentale e Bioinformatica Protocollo per analisi di RNA-Seq di photoperiodic diapausa nella zanzara tigre,<em&gt; Aedes albopictus</em

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, ...

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular mechanisms regulating vascular permeability in cultured endothelial cell monolayers and the functional exchange properties of whole microvascular beds. A method to cannulate and perfuse venular microvessels of rat mesentery and measure the hydraulic conductivity of the microvessel wall is described. The main equipment needed includes an intravital microscope with a large modified stage that supports micromanipulators to position three different microtools: (1) a beveled glass micropipette to cannulate and perfuse the microvessel; (2) a glass micro-occluder to transiently block perfusion and enable measurement of transvascular water flow movement at a measured hydrostatic pressure, and (3) a blunt glass rod to stabilize the mesenteric tissue at the site of cannulation. The modified Landis micro-occlusion technique uses red cells suspended in the artificial perfusate as markers of transvascular fluid movement, and also enables repeated measurements of these flows as experimental conditions are changed and hydrostatic and colloid osmotic pressure difference across the microvessels are carefully controlled. Measurements of hydraulic conductivity first using a control perfusate, then after re-cannulation of the same microvessel with the test perfusates enable paired comparisons of the microvessel response under these well-controlled conditions. Attempts to extend the method to microvessels in the mesentery of mice with genetic modifications expected to modify vascular permeability were severely limited because of the absence of long straight and unbranched microvessels in the mouse mesentery, but the recent availability of the rats with similar genetic modifications using the CRISPR/Cas9 technology is expected to open new areas of investigation where the methods described herein can be applied.

The modified Landis technique enables paired measurement of the hydraulic conductivity of individual microvessels in the mesentery of normal and genetically modified rats under control and test conditions using microperfusion techniques. It provides a convenient method to evaluate mechanisms that regulate microvessel permeability and transvascular exchange under physiological conditions.

Microperfusione Tecnica per Indagare regolamento di microvasi permeabilità a Rat mesentere

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

L&#39;isolamento di filtrazione di acidi nucleici: DNA metodo di estrazione semplice e veloce

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Fisiologia Lab Dimostrazione: filtrazione glomerulare in un ratto

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