Name: quantifying single microvessel permeability in isolated blood-perfused rat lung preparation
Uploaded: 2014-06-30T14:00:00
Description: Watch this Scientific Journal Video about Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation at JoVE.com

Undersøgelserne blev støttet af NIH HL75503 til KP.

Alle eksperimenter udført på dyr var som godkendt af Institutional Animal Care og brug Udvalg University of Tennessee Health Science Center. 1.. Tubing for perfusing rottelunge Forberedelser <ol><li> Forbered et rørsystem med Tygon slanger (# 18) til blodperfusion som vist i figur 1.. Slut tryktransducere (P23XL) og placere slangen på mikroskopet scenen. </li><li> Indstil vandbadstemperatur på 37 ° C. </li><li> Forbind lunge ind-og udtag midlertidigt med Tygon slange...

<ol>
	<li>Ware, L. B., Matthay, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+acute+respiratory+distress+syndrome.">The acute respiratory distress syndrome.</a> N Engl J Med. 342, 1334-1349 (2000).</li><li>Matthay, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+acute+respiratory+distress+syndrome.">The acute respiratory distress syndrome.</a> J Clin Invest. 122, 2731-2740 (2012).</li><li>Bhattacharya, J., Matthay, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+and+repair+of+the+alveolar-capillary+barrier+in+acute+lung+injury.">Regulation and repair of the alveolar-capillary barrier in acute lung injury.</a> Annu Rev Physiol. 75, 593-615 (2013).</li><li>Parthasarathi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connexin+43+mediates+spread+of+Ca2+-dependent+proinflammatory+responses+in+lung+capillaries.">Connexin 43 mediates spread of Ca2+-dependent proinflammatory responses in lung capillaries.</a> J Clin Invest. 116, 2193-2200 (2006).</li><li>Parthasarathi, K., Bhattacharya, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localized+Acid+instillation+by+a+wedged-catheter+method+reveals+a+role+for+vascular+gap+junctions+in+spatial+expansion+of+Acid+injury.">Localized Acid instillation by a wedged-catheter method reveals a role for vascular gap junctions in spatial expansion of Acid injury.</a> Anat Rec (Hoboken). 294, 1585-1591 (2011).</li><li>Gorin, A. B., Stewart, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+permeability+of+endothelial+and+epithelial+barriers+to+albumin+flux.">Differential permeability of endothelial and epithelial barriers to albumin flux.</a> J Appl Physiol Respir Environ Exerc Physiol. 47, 1315-1324 (1979).</li><li>Boutoille, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FITC-albumin+as+a+marker+for+assessment+of+endothelial+permeability+in+mice:+comparison+with+125I-albumin.">FITC-albumin as a marker for assessment of endothelial permeability in mice: comparison with 125I-albumin.</a> Exp Lung Res. 35, 263-271 (2009).</li><li>Thorball, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FITC-dextran+tracers+in+microcirculatory+and+permeability+studies+using+combined+fluorescence+stereo+microscopy,+fluorescence+light+microscopy+and+electron+microscopy.">FITC-dextran tracers in microcirculatory and permeability studies using combined fluorescence stereo microscopy, fluorescence light microscopy and electron microscopy.</a> Histochemistry. 71, 209-233 (1981).</li><li>Stevens, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+molecular+heterogeneity+of+pulmonary+endothelial+cells.">Functional and molecular heterogeneity of pulmonary endothelial cells.</a> Proc Am Thorac Soc. 8, 453-457 (2011).</li><li>Ofori-Acquah, S. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+barrier+function+in+the+lung+reflects+diversity+in+endothelial+cell+junctions.">Heterogeneity of barrier function in the lung reflects diversity in endothelial cell junctions.</a> Microvasc Res. 75, 391-402 (2008).</li><li>Kandasamy, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+imaging+reveals+endothelium-mediated+leukocyte+retention+in+LPS-treated+lung+microvessels.">Real-time imaging reveals endothelium-mediated leukocyte retention in LPS-treated lung microvessels.</a> Microvasc Res. 83, 323-331 (2012).</li><li>Kandasamy, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipopolysaccharide+induces+endoplasmic+store+Ca2+-dependent+inflammatory+responses+in+lung+microvessels.">Lipopolysaccharide induces endoplasmic store Ca2+-dependent inflammatory responses in lung microvessels.</a> PloS One. 8, (2013).</li><li>Qiao, R. L., Bhattacharya, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segmental+barrier+properties+of+the+pulmonary+microvascular+bed.">Segmental barrier properties of the pulmonary microvascular bed.</a> J Appl Physiol. 71, 2152-2159 (1991).</li><li>Parthasarathi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+connexin43+mediates+acid-induced+increases+in+pulmonary+microvascular+permeability.">Endothelial connexin43 mediates acid-induced increases in pulmonary microvascular permeability.</a> Am J Physiol Lung Cell Mol Physiol. 303, (2012).</li><li>Wu, Y., Perlman, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+methods+for+assessing+alveolar+mechanics.">In situ methods for assessing alveolar mechanics.</a> J Appl Physiol 1985. 112, 519-526 (2012).</li><li>Kuebler, W. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+signaling+mechanism+between+gas+and+blood+compartments+of+the+lung.">A novel signaling mechanism between gas and blood compartments of the lung.</a> Journal Clin Invest. 105, 905-913 (2000).</li></ol>

Den isolerede lunge forberedelse blod-perfunderet kombineret med realtids billedbehandling giver et simpelt værktøj til bestemmelse af ændringer permeabilitet i enkelt lunge mikrokar. Vi anvendte denne fremgangsmåde til at definere ændringer permeabilitet som reaktion på infusion af LPS. Vores data tyder klart på, at LPS infusion forårsagede en stigning i den mikrovaskulære permeabilitet. Yderligere data viser også, at ændringer i permeabilitet induceret af LPS var ens i begge vener og kapillærer. Således e...

Øget mikrovaskulære permeabilitet i lungerne fører til udvikling af alveolær ødem og kompromitteret gasudveksling og er en stor karakteristisk for akut lungeskade (ALI) 1-3. Overslag over vaskulær permeabilitet er vigtige ved fastlæggelse af omfanget af lungeskader og effekt af de foreslåede terapeutiske indgreb. Gravimetrisk analyse såsom blod gratis lunge våd-til-tørre-forhold og mikrovaskulære filtrering koefficient er almindeligt anvendte metoder til at estimere permeabilitet 4,5. Andre metoder omfatter kvantificering af tilbageholdelse af radioaktivt eller fluorescerende prober i lungevæv 6-8. Men de ovennævnte metoder kræver postexperiment behandling af lunge vævsprøver mod belyse data permeabilitet. Eftersom et dyr kan kun anvendes til en enkelt behandling protokol, kan der være behov for en fuldstændig undersøgelse stort antal dyr. En fælles egenskab ved de ovennævnte fremgangsmåder er, at de bestemmer middelværdien vaskulær permeabilitet foralle blodkar i vævsprøven. Det er imidlertid velkendt, at pulmonale mikro-og makro-skibe er fænotypisk forskellige 9. Derfor kan svarene permeabilitet være heterogen blandt de forskellige skibstyper såvel 9,10. Således kvantificerer gennemsnitlige gennemtrængelighed af alle pulmonale fartøjer i en vævsprøve, kan ikke i tilstrækkelig grad afspejler denne heterogenitet. I den isolerede lunge forberedelse blod perfunderet kan blodkar på lungeoverfladen visualiseres ved en opretstående mikroskop 4,11,12. Dette gør det muligt kendetegner reaktioner i enkelte skibe, og dermed løse eventuelle heterogenitet i svarene 13. Desuden ved at udnytte fluorescensimagografi af mikrokar fluorescens assays kan inkorporeres. Endvidere kan en venstre atrial mikrokateter anvendes til at levere midler og fluorescens prober i blodkar 11,14. Mikrokateteret begrænser levering til en lille lunge regionen og således exkun at frembyde blodkarrene i regionen til de infused agenter og fluorophores. Dette tillader flere små områder inden for samme lunge skal bruges til separate eksperimenter, hvilket fører til en samlet reduktion i dyr, der er nødvendige for en undersøgelse. Real time scanning muliggør opsamling af dynamiske ændringer i vaskulær og ekstravaskulære fluorescens af enlige mikrokar af de isolerede forberedelse lunge. For hvert mikrokar i et billede felt, kan registreres ændringer i fluorescens under infusion af fluoroforer og afvaskningsstation og kvantificeret offline 14. Brug værdierne for det maksimalt og resterende vaskulær fluorescens, kan et indeks gennemtrængelighed for hver mikrokardannelse i Billedfeltet bestemmes. For at bestemme ændringer permeabilitet som reaktion på inflammation eller skadelige stoffer, kan den ønskede middel administreres først, og derefter indeksere permeabilitet bestemt. Desuden kan billedfeltet sættes overalt i lungen region infunderesmicrocatheter, således at en høj grad af fleksibilitet i valg af den ønskede vaskulære netværk. Således isolerede forberedelse lunge blod-perfunderet i tandem med realtid billedbehandling giver en attraktiv eksperimentel model til at kvantificere permeabilitet i enkelt lunge mikrokar.

<table border="0" cellpadding="0" cellspacing="0" width="740">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tygon Tubing</td>
			<td style="width:108px;">Fisher Scientific</td>
			<td style="width:119px;"></td>
			<td style="width:297px;">#18</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pressure Transducer</td>
			<td style="width:108px;">Data Sciences International</td>
			<td style="width:119px;">P23XL</td>
			<td>Need quantity 3</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Butterfly Needle</td>
			<td style="width:108px;">Greiner Bio-One</td>
			<td align="right" style="width:119px;">450081</td>
			<td>21G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Peristaltic pump</td>
			<td style="width:108px;">Cole Parmer</td>
			<td style="width:119px;">Masterflex L/S</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PE-90 tubing</td>
			<td style="width:108px;">Becton Dickinson</td>
			<td align="right" style="width:119px;">427421</td>
			<td>30 cm needed</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PE-10 tubing</td>
			<td style="width:108px;">Becton Dickinson</td>
			<td align="right" style="width:119px;">427401</td>
			<td>40 cm needed</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Syringe Pump</td>
			<td style="width:108px;">Braintree Scientific</td>
			<td style="width:119px;">BS8000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">O-ring</td>
			<td style="width:108px;"></td>
			<td style="width:119px;"></td>
			<td>Custom made with a 20 mm diamter hole&#160; and a handle&#160; to secure o-ring to holder</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Upright fluorescence microscope</td>
			<td style="width:108px;">Olympus America</td>
			<td style="width:119px;">BX61WI</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Image Acquisition Software</td>
			<td style="width:108px;">Molecular Devices</td>
			<td style="width:119px;">Metamorph</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FITC Dextran 20KD</td>
			<td style="width:108px;">Sigma Aldrich</td>
			<td style="width:119px;"></td>
			<td>0.5 mg/ml (A dextran of different molecular size can be selected, if trial experiments indicate its suitability based on the calculated permeability index values)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lipopolysaccharide</td>
			<td style="width:108px;">Sigma Aldrich</td>
			<td style="width:119px;"></td>
			<td>Serotype 0111:B4</td>
		</tr>
	</tbody>
</table>

quantifying single microvessel permeability in isolated blood-perfused rat lung preparation

Den isolerede lunge forberedelse blod-perfunderet er almindeligt anvendt til at visualisere og definere signalering i enkelt mikrokar. Ved at koble dette præparat med real time scanning, bliver det muligt at bestemme ændringer permeabilitet i de enkelte pulmonale mikrokar. Heri beskrives trin for at isolere rottelunger og perfundere dem med autologt blod. Så vi skitsere skridt til at indgyde fluorophores eller agenter via en microcatheter ind i en lille lunge region. Ved hjælp af disse procedurer, der er beskrevet, vi bestemt permeabiliteten vokser i rottelunge mikrokar reaktion på infusioner af bakteriel lipopolysaccharid. De data viste, at lipopolysaccharid øget lækage på tværs af både venular og kapillær mikrokardannelse segmenter. Således er denne metode gør det muligt at sammenligne svarene permeabilitet blandt vaskulære segmenter og dermed definere nogen heterogenitet i svaret. Mens almindeligt anvendte metoder til at definere lunge permeabilitet kræver efterbehandle af lungevæv prøver, denanvendelse af tidstro billeddannelse overflødiggør dette krav som det fremgår af den foreliggende fremgangsmåde. Således isolerede forberedelse lunge kombineret med real time scanning giver flere fordele i forhold til traditionelle metoder til at bestemme lunge mikrovaskulære permeabilitet, men alligevel er en enkel metode til at udvikle og implementere.

En isoleret lunge forberedelse blod perfunderet tilsluttet perfusionsslange og beslægtet udstyr er vist i fig. 2. Til demonstrationsformål, vi anvendte en Sprague Dawley rotte, selvom fremgangsmåderne beskrevet heri, kan anvendes med enhver rottearten. Infusioner gennem en venstre forkammer mikrokateter nå kun et lille område af lungen. Det infunderede region kan identificeres ved infusion-induceret misfarvning (figur 3). Fremstillingen lungen som positioneret for real-time imaging...

Watch this Scientific Journal Video about Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation at JoVE.com

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

Kvantificering Single mikrokardannelse Permeability i Isoleret Blood-perfunderet rottelunge Forberedelse

Den isolerede lunge forberedelse blod perfunderet gør det muligt at visualisere mikrokar net på lungeoverfladen. Her beskriver vi en metode til at kvantificere permeabilitet af enlige mikrokar i isolerede lunger ved hjælp af real tid fluorescens billeddannelse.

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

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