Name: come to the light side: in vivo monitoring of pseudomonas aeruginosa biofilm infections in chronic wounds in a diabetic hairless murine model
Uploaded: 2017-10-10T13:00:00
Description: Watch this Scientific Journal Video about Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model at JoVE.com

Forfatterne vil gerne takke den amerikanske Diabetes Association til støtte for dette arbejde (Grant # #7-13-BS-180), Michigan State University forskning teknologi støtte facilitet til uddannelse og adgang til den i vivo imaging system og den Michigan State University undersøgende histopatologi Lab for forarbejdning mus biopsier for histopatologiske undersøgelse.

dyreforsøg blev godkendt af institutionelle Animal Care og brug Udvalget af Michigan State University.
 1. forberedelse af okklusiv dressinger og silikone afstandsstykker <ol><li>skære den gennemsigtige okklusiv dressing til at gøre firkanter ca 1 cm x 1 cm med saks.</li>
	<li>Skæres 10 mm cirkler på 0,5 mm tykt silikone ark ved hjælp af en 10 mm biopsi punch. Center en 5 mm biopsi punch i midten af 10 mm cirkel og tryk fast til at skabe et hul til at danne en &#34; doughnut &#34;-som...

<ol>
	<li>James, G. 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=Biofilms+in+chronic+wounds.">Biofilms in chronic wounds.</a> Wound Repair Regen. 16 (1), 37-44 (2008).</li><li>Gordois, A., Scuffham, P., Shearer, A., Oglesby, A., Tobian, J. 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+health+care+costs+of+diabetic+peripheral+neuropathy+in+the+US.">The health care costs of diabetic peripheral neuropathy in the US.</a> Diabetes Care. 26 (6), 1790-1795 (2003).</li><li>Reiber, G. E., McDonell, M. B., Schleyer, A. M., Fihn, S. D., Reda, D. 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H., 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=Photonic+detection+of+bacterial+pathogens+in+living+hosts.">Photonic detection of bacterial pathogens in living hosts.</a> Mol Microbiol. 18 (4), 593-603 (1995).</li><li>Rocchetta, H. L., 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=Validation+of+a+noninvasive,+real-time+imaging+technology+using+bioluminescent+Escherichia+coli+in+the+neutropenic+mouse+thigh+model+of+infection.">Validation of a noninvasive, real-time imaging technology using bioluminescent Escherichia coli in the neutropenic mouse thigh model of infection.</a> Antimicrob Agents Chemother. 45 (1), 129-137 (2001).</li><li>Kadurugamuwa, J. L., 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=Rapid+direct+method+for+monitoring+antibiotics+in+a+mouse+model+of+bacterial+biofilm+infection.">Rapid direct method for monitoring antibiotics in a mouse model of bacterial biofilm infection.</a> Antimicrob Agents Chemother. 47 (0066-4804), 3130-3137 (2003).</li><li>Anderl, J. N., Franklin, M. J., Stewart, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+antibiotic+penetration+limitation+in+Klebsiella+pneumoniae+biofilm+resistance+to+ampicillin+and+ciprofloxacin.">Role of antibiotic penetration limitation in Klebsiella pneumoniae biofilm resistance to ampicillin and ciprofloxacin.</a> Antimicrob Agents Chemother. 44 (7), 1818-1824 (2000).</li><li>Morton, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+approach+for+establishing+humane+endpoints.">A systematic approach for establishing humane endpoints.</a> ILAR J. 41 (2), 80-86 (2000).</li><li>Dunn, L., 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=Murine+model+of+wound+healing.">Murine model of wound healing.</a> J Vis Exp. (75), e50265 (2013).</li><li>Zhao, G., 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=Delayed+wound+healing+in+diabetic+(db/db)+mice+with+Pseudomonas+aeruginosa+biofilm+challenge+-+a+model+for+the+study+of+chronic+wounds.">Delayed wound healing in diabetic (db/db) mice with Pseudomonas aeruginosa biofilm challenge - a model for the study of chronic wounds.</a> Wound Repair Regen. 18 (5), 467-477 (2010).</li><li>Holley, A. K., Xu, Y., Noel, T., Bakthavatchalu, V., Batinic-Haberle, I., St. Clair, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manganese+superoxide+dismutase-mediated+inside-out+signaling+in+HaCaT+human+keratinocytes+and+SKH-1+mouse+skin.">Manganese superoxide dismutase-mediated inside-out signaling in HaCaT human keratinocytes and SKH-1 mouse skin.</a> Antioxid Redox Signal. 20 (15), 2347-2360 (2014).</li><li>Abbas, S., Alam, S., Pal, A., Kumar, M., Singh, D., Ansari, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UVB+exposure+enhanced+benzanthrone-induced+inflammatory+responses+in+SKH-1+mouse+skin+by+activating+the+expression+of+COX-2+and+iNOS+through+MAP+kinases/NF-&#312;B/AP-1+signalling+pathways.">UVB exposure enhanced benzanthrone-induced inflammatory responses in SKH-1 mouse skin by activating the expression of COX-2 and iNOS through MAP kinases/NF-&#312;B/AP-1 signalling pathways.</a> Food Chem Toxicol. 96, 183-190 (2016).</li><li>Watters, C., Everett, J. A., Haley, C., Clinton, A., Rumbaugh, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+treatment+modulates+the+host+immune+system+to+enhance+Pseudomonas+aeruginosa+wound+biofilms.">Insulin treatment modulates the host immune system to enhance Pseudomonas aeruginosa wound biofilms.</a> Infect Immun. 82 (1), 92-100 (2014).</li></ol>

Her beskriver vi en ny musemodel for studier af biofilm i diabetisk kroniske sår, der er mange fordele ved at oprette en reproducerbar, oversætbare og fleksibel model.
Den første nyhed er brugen af hairless mus. Andre musemodeller er blevet udviklet for at studere diabetisk kronisk sårheling10,11, men alle har påberåbt sig anvendelsen af hår mus der kræver fjernelse af pels af processer, der involverer enten voks eller hår kli...

Biofilm er komplekse samfund af mikroorganismer indkapslet i en matrix af polymere stoffer, der er blevet fremhævet som en medvirkende faktor for den dårlig opløsning af kroniske sår1. Studiet af disse meget organiseret, vedvarende mikrobielle populationer er særligt vigtigt for hvor dårligt blodomløb på lemmer og ændrede perifer sensorisk mekanismer føre til uopdaget læsioner2-diabetikere. I USA anslås det, at 15% af diabetespatienter vil udvikle mindst ét sår i løbet af deres liv. Dette svarer til en økonomisk udgift for omkring 28 milliarder dollars i behandling3,4, for ikke for at nævne de immensurable følelsesmæssige og sociale byrder. Forstå de faktorer, der giver mulighed for mikrobielle samfund at fremture i såret seng og disse biofilm indvirkning i de helbredende begivenheder er bydende nødvendigt at køre bedre pleje for ramte patienter og drive udviklingen af nye behandlingsmetoder. Etablering af reproducerbar og oversætbare i vivo modeller for at udforske bakteriel-værtssammenspil er derfor altafgørende.
Blevet udviklet murine modeller for at studere virkningerne af biofilm i kroniske sår. Disse modeller, men ofte udnytte hår arter og evaluere biofilm clearance af plade tæller for levedygtige bakterieceller skåret væv fra ofrede dyr, hvilket gør dem tidskrævende og dyrt.
Et biophotonic alternativ til slutpunktet prøveudtagning af dyr i evaluering infektion blev først foreslået af Contag mfl. (1995) 5 , der har udviklet en metode til at fange luminescence fra constitutively en bioluminescerende Salmonella typhimurium at måle antibiotisk behandling virkning. Andre undersøgelser, at drage fordel af bioluminescens energieffektiv bakterier fulgt. For eksempel Rochetta mfl. (2001) 6 valideret en infektion model for at studere Escherichia coli låret infektioner i mus ved at måle luminescence ved hjælp af en øget afgift - sammen enhed og senere, Kadurugamuwa mfl. (2003) 7 benyttede sig af den photon udsender egenskaber af en manipuleret stamme af Staphylococcus aureus at undersøge effekten af flere antibiotika i et kateter sår model i mus.
Metoden kendetegnet her præsenterer en enkel protokol for at fremkalde diabetes i hairless mus, producere og podes sår med præ-dannet en bioluminescerende biofilm af P. aeruginosa, og gennemføre biophotonic overvågning af infektionen ved hjælp af en i vivo imaging system. Det tilbyder en direkte, hurtig, i situ, non-invasiv og kvantitative proces at evaluere biofilm i kroniske sår og derudover giver mulighed for yderligere analyse, såsom mikroskopisk billeddannelse af healing sår, intermitterende blod samling for cytokin målinger, og terminal væv indsamling til histologi.

<table>
	<tbody>
		<tr>
			<td>Opsite</td>
			<td>Smith &#38; Nephew</td>
			<td>Model 66000041</td>
			<td>Smith &#38; Nephew Flexfix Opsite Transparent Adhesive Film Roll 4&#34; x 11yards</td>
		</tr>
		<tr>
			<td>SKH-1 mice Crl:SKH1-Hrhr</td>
			<td>Charles River Breeding Laboratories</td>
			<td>SKH1</td>
			<td>Hairless mice, 8 weeks old</td>
		</tr>
		<tr>
			<td>Streptozotocin (STZ)</td>
			<td>Sigma Aldrich</td>
			<td>S0130-1G</td>
			<td>Streptozocin powder, 1g</td>
		</tr>
		<tr>
			<td>AccuChek glucometer</td>
			<td>Accu-Chek Roche</td>
			<td>Art No. 05046025001</td>
			<td>ACCU-CHEK CompactPlus Diabetes Monitoring Care Kit</td>
		</tr>
		<tr>
			<td>Pseudomonas aeruginosa Xen 41</td>
			<td>Perkin Elmer</td>
			<td>119229</td>
			<td>Bioluminescent Pseudomonas aeruginosa</td>
		</tr>
		<tr>
			<td>Polycarbonate membrane filters</td>
			<td>Sigma Aldrich</td>
			<td>P9199</td>
			<td>Millipore polycarbonate membrane filters with 0.2 &#956;m pore size</td>
		</tr>
		<tr>
			<td>Dulbelcco phosphate buffer saline (DPBS)</td>
			<td>Sigma Aldrich</td>
			<td>D8537</td>
			<td>PBS</td>
		</tr>
		<tr>
			<td>Tryptic soy agar</td>
			<td>Sigma Aldrich</td>
			<td>22091</td>
			<td>Culture agar</td>
		</tr>
		<tr>
			<td>Meloxicam</td>
			<td>Henry Schein Animal Health</td>
			<td>49755</td>
			<td>Eloxiject (Meloxicam) 5mg/mL, solution for injection</td>
		</tr>
		<tr>
			<td>10% povidone-iodine (Betadine)</td>
			<td>Purdue Products LP</td>
			<td>301879-OA</td>
			<td>Swabstick, Betadine Solution. Antiseptic. Individ. Wrapped, 200/case</td>
		</tr>
		<tr>
			<td>4% paraformaldehyde</td>
			<td>Fisher Scientific</td>
			<td>AAJ61899AK</td>
			<td>Alfa Aesar Paraformaldehyde, 4% in PBS</td>
		</tr>
		<tr>
			<td>Capillary glass tube</td>
			<td>Fisher Scientific</td>
			<td>22-362-566</td>
			<td>Heparinized Micro-Hematocrit Capillary Tubes</td>
		</tr>
		<tr>
			<td>Silicone to make splints</td>
			<td>Invitrogen Life Technologies Corp</td>
			<td>P-18178</td>
			<td>Press-to-Seal Silicone Sheet, 13cm x 18cm, 0.5mm thick, set of 5 sheets</td>
		</tr>
		<tr>
			<td>Tryptic soy broth</td>
			<td>Sigma Aldrich</td>
			<td>22092</td>
			<td>Culture broth</td>
		</tr>
		<tr>
			<td>IVIS Spectrum</td>
			<td>Perkin Elmer</td>
			<td>124262</td>
			<td>In vivo imaging system</td>
		</tr>
		<tr>
			<td>IVIS Spectrum Isolation chamber</td>
			<td>Perkin Elmer</td>
			<td>123997</td>
			<td>XIC-3 animal isolation chamber</td>
		</tr>
		<tr>
			<td>HEPA filter</td>
			<td>Teleflex</td>
			<td>28022</td>
			<td>Gibeck ISO-Gard HEPA Light number 28022</td>
		</tr>
		<tr>
			<td>Biopsy punches</td>
			<td>VWR International Inc</td>
			<td>21909-142</td>
			<td>Disposable Biopsy Punch, 5mm, Sterile, pack of 50.</td>
		</tr>
		<tr>
			<td>Biopsy punches</td>
			<td>VWR International Inc</td>
			<td>21909-140</td>
			<td>Disposable Biopsy Punch, 4mm, Sterile, pack of 50.</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>J.T.Baker</td>
			<td>1916-01</td>
			<td>Dextrose, Anhydrous, Powder</td>
		</tr>
		<tr>
			<td>Citric acid</td>
			<td>Sigma Aldrich</td>
			<td>C2404-100G</td>
			<td>Citric Acid</td>
		</tr>
		<tr>
			<td>Mastisol</td>
			<td>Eloquest Healthcare</td>
			<td>HRI 0496-0523-48</td>
			<td>Mastisol Medical Liquid Adhesive 2/3 mL vial, box of 48</td>
		</tr>
		<tr>
			<td>Corning 96-well black plates</td>
			<td>Fisher Scientific</td>
			<td>07-200-567</td>
			<td>96-well clear bottom black polysterene microplates</td>
		</tr>
		<tr>
			<td>25 gauge 5/8 inch needle</td>
			<td>BD</td>
			<td>305122</td>
			<td>Regular bevel needle</td>
		</tr>
		<tr>
			<td>Bransonic M Ultrasonic Cleaning Bath</td>
			<td>Branson Ultrasonics</td>
			<td>N/A</td>
			<td>Ultrasonic Cleaner</td>
		</tr>
	</tbody>
</table>

come to the light side: in vivo monitoring of pseudomonas aeruginosa biofilm infections in chronic wounds in a diabetic hairless murine model

Tilstedeværelse af bakterier som struktureret biofilm i kroniske sår, især hos diabetikere, menes at hindre sårheling og opløsning. Kroniske sår musemodeller har været brugt til at forstå de underliggende samspil mellem mikroorganismer og værten. De modeller udviklet til dato er afhængige af brugen af hårede dyr og terminal samling af sår væv til bestemmelse af levedygtige bakterier. Mens betydelig indsigt er opnået med disse modeller, denne eksperimentelle procedure kræver et stort antal dyr og prøvetagning er tidskrævende. Vi har udviklet en roman murine model, der indeholder flere optimal nyskabelser for at evaluere biofilm progression i kroniske sår: en) det udnytter hairless mus, eliminerer behovet for hårfjerning; b) gælder præformerede biofilm på sår, gør det muligt for den umiddelbare vurdering af persistens og virkningen af disse Fællesskaber på vært; c) overvåger biofilm progression af kvantificere lys produktion af en genetisk manipuleret en bioluminescerende stamme af Pseudomonas aeruginosa, giver mulighed for tidstro overvågning af infektionen dermed reducere antallet af dyr kræves pr. undersøgelse. I denne model, er en enkelt fuld-dybde sår produceret på bagsiden af STZ-induceret diabetisk hairless mus og podes med biofilm af P. aeruginosa en bioluminescerende stamme Xen 41. Lyseffekten fra sårene registreres dagligt i en i vivo imaging system, giver mulighed for i vivo og i situ hurtige biofilm visualisering og lokalisering af biofilm bakterier inden for sår. Denne nye metode er fleksibel, da det kan bruges til at studere andre mikroorganismer, herunder genetisk modificerede arter og flere arter biofilm, og kan være af særlig værdi i anti-biofilm teststrategier herunder antimikrobiel okklusiv forbindinger.

I udviklingen af denne nye model, observeret vi mange fordele i at udnytte hairless SKH-1 over C57BL/6J mus, som vi har brugt i fortiden. Dyr underkastes STZ injektioner normalt opleve gradvis vægttab med udbruddet af diabetes; men i sår healing eksperimenter tidligere udført af vores laboratorier gengiver den model, præsenteret af Dunn mfl. (2012) 9 brug af C57BL/6J, drastiske vægttab blev observeret (figur 1). Derimod b...

Watch this Scientific Journal Video about Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model at JoVE.com

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

Kommer til lys Side: In Vivo overvågning af Pseudomonas aeruginosa Biofilm infektioner i kroniske sår i en diabetisk Hairless Murine Model

Her beskriver vi en roman diabetisk murine model udnytte hairless mus for real-time, ikke-invasiv overvågning af biofilm sårinfektioner af en bioluminescerende Pseudomonas aeruginosa. Denne metode kan tilpasses til at evaluere infektion af andre bakteriearter og genetisk modificerede mikroorganismer, herunder flere arter biofilm, og teste effekten af antibiofilm strategier.

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. ...

Research

JoVE Journal

Immunology and Infection

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

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Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological ...

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

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Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Brug af kunstig Sputum Medium til Test antibiotikum Effekt mod<em&gt; Pseudomonas aeruginosa</em&gt; I forhold mere relevante for cystisk fibrose Lung

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

Langtids kronisk<em&gt; Pseudomonas aeruginosa</em&gt; Luftvejsinfektion i mus

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

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Pseudomonas aeruginosa Induced Lung Injury Model

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. ...

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

At studere mikrobielle samfund<em&gt; In vivo</em&gt;: En model af Host-medieret samspil mellem<em&gt; Candida albicans</em&gt; Og<em&gt; Pseudomonas aeruginosa</em&gt; I luftvejene

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Visualisere virkningerne af sputum på Biofilm Development Ved hjælp af en Chambered dækglas Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

En forsinket vaccination Model af kronisk Pseudomonas aeruginosa Sårinfektion

Pseudomonas aeruginosa (P. aeruginosa) is a major nosocomial pathogen of increasing relevance to human health and disease, particularly in the setting of ...

Assessing Biofilm Dispersal in Murine Wounds

Vurdering biofilm spredning i Murine Sår

Biofilm-related infections are implicated in a wide array of chronic conditions such as non-healing diabetic foot ulcers, chronic sinusitis, reoccurring ...