Name: a method for generating pulmonary neutrophilia using aerosolized lipopolysaccharide
Uploaded: 2014-12-15T13:00:00
Description: Watch this Scientific Journal Video about A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide at JoVE.com

Vi vil gerne takke Kerstin Thim (AstraZeneca, Lund, Sverige), Benita Dahlberg og Dr. Anders Eklund (Karolinska Instituttet, Stockholm, Sverige) samt Dr. Martin Stampfli (McMaster University, Hamilton, ON, Canada) for dygtige assistance og ekspertrådgivning.

Undersøgelserne dyr blev godkendt af Nordlige Stockholm dyrevelfærd etiske udvalg. De eksperimentelle procedurer blev udført i overensstemmelse med den svenske lovgivning. 1. Generering en LPS Aerosol <ol><li> 0,5 g oprenset P. Opløs aeruginosa LPS i 50 ml sterilt saltvand under forsigtig omrøring og kontrollere opløsning. Fortyndes 1 ml opløst LPS i 9 ml sterilt saltvand til en slutkoncentration på 1 mg / ml. Beskyt mod lys med alufolie og opbevares ved -20 ° C....

<ol>
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Aerosoliseret LPS genererer et inflammatorisk respons i luftvejene, kendetegnet ved, neutrofiler i epitel submucosa, rum omkring de ledende luftveje, samt de alveolære rum. Dette er, sammen med den øgede totale proteinindhold i BALF, indikerer plasmalækage, repræsentant for patologien af ​​akut lungeskade. Som LPS inducerer en steril inflammation, reaktionen er uafhængig af det adaptive immunrespons, og der er begrænsninger for relevans for bakterielle infektioner. Teknikken kan dog anvendes til at dissekere i...

Lipopolysaccharid (LPS) er en cellevægsbestanddel af gramnegative bakterier 1. Challenge LPS er en veldokumenteret model for akut lungeskade, et syndrom karakteriseret ved akut neutrofil inflammation og ødem 2. Desuden pulmonal neutrofili er også et kendetegn for kronisk obstruktiv lungesygdom (COPD) 3, og LPS-provokationen i mennesker er blevet anvendt til at modellere KOL forværringer 4. Således eksperimentelle modeller af LPS eksponering er klinisk relevante og værdifulde værktøjer til at forstå den menneskelige patologi. Formålet med den pulmonale levering af aerosoliserede LPS beskrevet her er at generere en neutrofil inflammatorisk respons i den ledende og luftvejene, uden systemisk involvering. Adskillige teknikker til LPS-provokationen er blevet beskrevet tidligere. Intravenøs injektion af LPS er den mest almindeligt anvendte indgivelsesvej. Selv om denne teknik er let tilgængelig, than primære skade på endotelet, med sekundær ødelæggelse af lungeepitelet efter neutrofil migration til lungen. Intravenøs administration inducerer også systemisk inflammation 2, som kan komplicere det kliniske billede i dyremodeller. Systemisk inflammation er i modsætning ikke observeret med intratracheal administration. Denne teknik er imidlertid arbejdskrævende og kræver anæstesi samt en betydelig træning 5, 6. Desuden pulmonal deposition af denne administrationsvej afhænger af vejrtrækning 7. Således lungeaflejring påvirket af dybden af ​​bedøvelse er nødvendige for kan observeres intra administration luftrør og variabel aflejring i luftvejene. I modsætning hertil pulmonal levering med aerosoliserede LPS kræver minimal træning, og kan let opnås på et stort antal dyr med lille eller ingen variation mellem individer 5, 8.En nylig undersøgelse viser, at aerosol levering er overlegen i forhold til intratracheal rute med hensyn til aflejring, og at mere relevante doser af LPS inducerer neutrofil inflammation med denne model 8. Tidligere undersøgelser har vist, at udfordring for aerosoliseret Pseudomonas aeruginosa LPS genererer en markant inflammatorisk reaktion i luftvejslumen og lungeparenkym, herunder alveolære rum 9, 10. Inflammationen er kendetegnet ved en overvægt af neutrofiler og tilstedeværelsen af ​​lungeødem, og kan således anvendes til at behandle patogenesen af ​​akut lungeskade og få yderligere kendskab til de mekanismer, der bidrager til sygdommen patologi.

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	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:307px;">Name of the material/equipment&#160;</td>
			<td style="width:244px;">Company</td>
			<td style="width:145px;">Catalog number</td>
			<td style="width:235px;">Comments/Description</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:307px;">Purified Pseudomonas aeruginosa LPS&#160;</td>
			<td style="width:244px;">Sigma-Aldrich</td>
			<td style="width:145px;"></td>
			<td style="width:235px;">Harmful. Recomended purification. LPS purified from other bactria may be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Pari LC sprint star nebulizer</td>
			<td style="width:244px;">PARI Respiratory Equipment Inc.&#160;</td>
			<td style="width:145px;">023G1250</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">TSI mass flowmeter 4040</td>
			<td style="width:244px;">TSI</td>
			<td style="width:145px;">4040</td>
			<td style="width:235px;">Alternative product from supplier may be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Saint-Gobain 15.9 mm Tygon tube</td>
			<td style="width:244px;">Sigma-Aldrich</td>
			<td style="width:145px;">Z685704</td>
			<td style="width:235px;">Recomended brand.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">Plexiglas boxes with removable lids</td>
			<td style="width:244px;">Custom built</td>
			<td style="width:145px;">N/A</td>
			<td style="width:235px;">150 x 163 x 205 mm (a 2 mm hole on the side).&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">3M Half Facepiece Reusable Respirator</td>
			<td style="width:244px;">3M</td>
			<td style="width:145px;">7503</td>
			<td style="width:235px;">Recomended brand.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">3M Advanced Particulate Filters (P100)&#160;</td>
			<td style="width:244px;">3M</td>
			<td style="width:145px;">2291</td>
			<td style="width:235px;">Recomended brand.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Sissors</td>
			<td style="width:244px;">VWR</td>
			<td style="width:145px;">233-1104</td>
			<td style="width:235px;">Preferred scissors may be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Forceps&#160;</td>
			<td style="width:244px;">VWR</td>
			<td style="width:145px;">232-1313</td>
			<td style="width:235px;">Preferred forceps may be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Intramedic PE50 polyethylene tube</td>
			<td style="width:244px;">BD</td>
			<td style="width:145px;">427411</td>
			<td style="width:235px;">Recomended brand.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Ethicon 2-0 Perma-hand silk tread&#160;</td>
			<td style="width:244px;">VWR</td>
			<td style="width:145px;">95056-992</td>
			<td style="width:235px;">Recomended brand.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">26 &#189;&#160; gage needle&#160;</td>
			<td style="width:244px;"></td>
			<td style="width:145px;"></td>
			<td style="width:235px;">Alternative suppliers exist.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">1 mL BD slip-tip syringe, non-sterile</td>
			<td style="width:244px;">BD</td>
			<td style="width:145px;"><a href="http://catalog.bd.com/bdCat/viewProduct.doCustomer?productNumber=301025">301025</a></td>
			<td style="width:235px;">Alternative suppliers exist.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">60 mL BD Luer-Lok syringe, non-sterile, polypropolene&#160;</td>
			<td style="width:244px;">BD</td>
			<td style="width:145px;">301035</td>
			<td style="width:235px;">Alternative suppliers exist.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Fluka Hematoxylin-Eosin</td>
			<td style="width:244px;">Sigma-Aldrich</td>
			<td style="width:145px;">3972</td>
			<td style="width:235px;">Alternative suppliers exist.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">T&#252;rk&#39;s solution</td>
			<td style="width:244px;">Merck Millipore</td>
			<td style="width:145px;">109277</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Table top centrifuge</td>
			<td style="width:244px;"></td>
			<td style="width:145px;"></td>
			<td style="width:235px;">Alternative manufacturers exist.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Cytospin 4 cytocentrifuge</td>
			<td style="width:244px;">Thermo Scientific</td>
			<td style="width:145px;">A78300003</td>
			<td style="width:235px;">Alternative centrifuge can be used.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">HEMA-3 stat pack</td>
			<td style="width:244px;">Fisher Scientific</td>
			<td style="width:145px;">23-123-869</td>
			<td style="width:235px;">Alternative staining kits exists.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:307px;">Formalin solution, neutral buffered, 10%</td>
			<td style="width:244px;">Sigma-Aldrich</td>
			<td style="width:145px;">HT501128&#160;</td>
			<td style="width:235px;">Alternative suppliers exist.</td>
		</tr>
	</tbody>
</table>

a method for generating pulmonary neutrophilia using aerosolized lipopolysaccharide

Akut lungeskade (ALI) er en alvorlig sygdom karakteriseret ved alveolær neutrofili, med begrænsede behandlingsmuligheder og høj dødelighed. Eksperimentelle modeller af ALI er nøglen til at øge vores forståelse af sygdommen patogenese. Lipopolysaccharid (LPS) fra gram-positive bakterier inducerer neutrofil inflammation i luftvejene og lungeparenkym af mus. Effektiv pulmonal levering af forbindelser, såsom LPS er imidlertid vanskeligt at opnå. I den fremgangsmåde der er beskrevet her, er pulmonal tilførsel i mus opnås ved udfordring for aerosoliseret Pseudomonas aeruginosa LPS. Opløst LPS blev aerosoliseret af en forstøver forbundet til trykluft. Mus blev udsat for en kontinuerlig strøm af LPS aerosol i en plexiglas kasse i 10 minutter, efterfulgt af 2 minutters konditionering efter aerosolen blev afbrudt. Trakealintubation og efterfølgende bronchoalveolær lavage, efterfulgt af formalin perfusion blev dernæst udført, hvilket tillader karakterisering af den sterile pulmonary inflammation. Aerosoliseret LPS genererer en pulmonal inflammation karakteriseret ved alveolær neutrofili, detekteret i bronchoalveolær udskylning og histologisk vurdering. Denne teknik kan sættes op på en lille pris med få apparater, og kræver minimal træning og ekspertise. Eksponeringen Systemet kan således rutinemæssigt på ethvert laboratorium, med potentiale til at øge vores forståelse af lunge patologi.

Udfordring til aerosoliseret P. aeruginosa LPS giver normalt en markant inflammatorisk reaktion i luftvejslumen og alveolære rum, kendetegnet ved en overvægt af neutrofiler på både tidlige og sene tidspunkter. Aerosoliseret LPS inducerer pulmonal neutrofili C57BL / 6by og BALB / c-mus blev udsat for aerosoliseret P. aeruginosa LPS eller alene køretøjet og neutrofiler blev optalt i BALF. Det samlede celleantal i BALF af C57BL / 6by mus udsat f...

Watch this Scientific Journal Video about A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide at JoVE.com

A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide

En fremgangsmåde til frembringelse Pulmonal Neutrofili Brug Aerosolized Lipopolysaccharid

Vi beskriver en fremgangsmåde til at inducere neutrofil pulmonal inflammation ved udfordring aerosoliseret lipopolysaccharid ved forstøvning, at modellere akut lungeskade. Desuden er grundlæggende kirurgiske teknikker til lunge isolation, tracheal intubation og bronchoalveolær lavage også beskrevet.

Acute lung injury (ALI) is a severe disease characterized by alveolar neutrophilia, with limited treatment options and high mortality. Experimental models ...

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