Name: urinary tract infection in a small animal model: transurethral catheterization of male and female mice
Uploaded: 2017-12-01T13:00:00
Description: Watch this Scientific Journal Video about Urinary Tract Infection in a Small Animal Model: Transurethral Catheterization of Male and Female Mice at JoVE.com

Ringraziamo il Dr Matthieu Rousseau per lettura critica del manoscritto e il laboratorio di immunologia-dendritiche per loro spunti utili durante lo sviluppo di questo progetto e protocollo. Questo lavoro è stato in parte sostenuto da finanziamenti dall'Unione europea settimo quadro programma Marie Curie azione (PCIG11-GA-2012-3221170 e la Immuno-oncologia LabEx (MAI).

Mouse esperimenti sono stati condotti in conformità con l'approvazione di numero di protocollo 2012-0024 dalla Comité d' ' éthique en expérimentation animale Paris Centre et Sud (comitato etico per la sperimentazione animale), in applicazione del europeo direttiva 2010/63 DELL'UNIONE EUROPEA.
1. preparazione dei cateteri
<ol><li>Preparare una cannula di accesso endovenoso pediatrica per ogni gruppo di topi infetti. Utilizzando il meccanismo a molla integrato, cedere ogni cannula del suo ...

<ol>
	<li>Hooton, T. M., Stamm, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+and+treatment+of+uncomplicated+urinary+tract+infection.">Diagnosis and treatment of uncomplicated urinary tract infection.</a> Infect Dis Clin North Am. 11 (3), 551-581 (1997).</li><li>Harper, M., Fowlis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Management+of+urinary+tract+infections+in+men.">Management of urinary tract infections in men.</a> Trends in Urology, Gynaecology &amp; Sexual Health. 12 (1), 30-35 (2007).</li><li>Foxman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epidemiology+of+urinary+tract+infection.">The epidemiology of urinary tract infection.</a> Nat Rev Urol. 7 (12), 653-660 (2010).</li><li>Lipsky, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urinary+tract+infections+in+men.+Epidemiology,+pathophysiology,+diagnosis,+and+treatment.">Urinary tract infections in men. Epidemiology, pathophysiology, diagnosis, and treatment.</a> Ann Intern Med. 110 (2), 138-150 (1989).</li><li>Conway, L. J., Carter, E. J., Larson, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+Factors+for+Nosocomial+Bacteremia+Secondary+to+Urinary+Catheter-Associated+Bacteriuria:+A+Systematic+Review.">Risk Factors for Nosocomial Bacteremia Secondary to Urinary Catheter-Associated Bacteriuria: A Systematic Review.</a> Urol Nurs. 35 (4), 191-203 (2015).</li><li>Markle, J. G., Fish, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SeXX+matters+in+immunity.">SeXX matters in immunity.</a> Trends Immunol. 35 (3), 97-104 (2014).</li><li>Yu, C. Y., Whitacre, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex,+MHC+and+complement+C4+in+autoimmune+diseases.">Sex, MHC and complement C4 in autoimmune diseases.</a> Trends Immunol. 25 (12), 694-699 (2004).</li><li>Castelao, J. E., 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=Gender-+and+smoking-related+bladder+cancer+risk.">Gender- and smoking-related bladder cancer risk.</a> J Natl Cancer Inst. 93 (7), 538-545 (2001).</li><li>Donsky, H., Coyle, S., Scosyrev, E., Messing, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+differences+in+incidence+and+mortality+of+bladder+and+kidney+cancers:+national+estimates+from+49+countries.">Sex differences in incidence and mortality of bladder and kidney cancers: national estimates from 49 countries.</a> Urol Oncol. 32 (1), 40-e31 (2014).</li><li>Garg, T., 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=Gender+Disparities+in+Hematuria+Evaluation+and+Bladder+Cancer+Diagnosis:+A+Population-Based+Analysis.">Gender Disparities in Hematuria Evaluation and Bladder Cancer Diagnosis: A Population-Based Analysis.</a> J Urol. , (2014).</li><li>Dobruch, J., 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=Gender+and+Bladder+Cancer:+A+Collaborative+Review+of+Etiology,+Biology,+and+Outcomes.">Gender and Bladder Cancer: A Collaborative Review of Etiology, Biology, and Outcomes.</a> Eur Urol. , (2015).</li><li>Hsu, J. W., 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=Decreased+tumorigenesis+and+mortality+from+bladder+cancer+in+mice+lacking+urothelial+androgen+receptor.">Decreased tumorigenesis and mortality from bladder cancer in mice lacking urothelial androgen receptor.</a> Am J Pathol. 182 (5), 1811-1820 (2013).</li><li>El Behi, 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=An+essential+role+for+decorin+in+bladder+cancer+invasiveness.">An essential role for decorin in bladder cancer invasiveness.</a> EMBO Mol Med. 5 (12), 1835-1851 (2013).</li><li>Foxman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+of+urinary+tract+infections:+incidence,+morbidity,+and+economic+costs.">Epidemiology of urinary tract infections: incidence, morbidity, and economic costs.</a> Am J Med. 113, 05S-13S (2002).</li><li>Ingersoll, M. A., Kline, K. A., Nielsen, H. V., Hultgren, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G-CSF+induction+early+in+uropathogenic+Escherichia+coli+infection+of+the+urinary+tract+modulates+host+immunity.">G-CSF induction early in uropathogenic Escherichia coli infection of the urinary tract modulates host immunity.</a> Cell Microbiol. 10 (12), 2568-2578 (2008).</li><li>Mora-Bau, 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=Macrophages+Subvert+Adaptive+Immunity+to+Urinary+Tract+Infection.">Macrophages Subvert Adaptive Immunity to Urinary Tract Infection.</a> PLoS Pathog. 11 (7), e1005044 (2015).</li><li>Oliveira, P. 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=Technical+Report:+Technique+of+Bladder+Catheterization+in+Female+Mice+and+Rats+for+Intravesical+Instillation+in+Models+of+Bladder+Cancer.">Technical Report: Technique of Bladder Catheterization in Female Mice and Rats for Intravesical Instillation in Models of Bladder Cancer.</a> (Conference Title)39th Scand-LAS and ICLAS Joint Meeting. 36, 5-9 (2009).</li><li>Seager, C. 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=Intravesical+delivery+of+rapamycin+suppresses+tumorigenesis+in+a+mouse+model+of+progressive+bladder+cancer.">Intravesical delivery of rapamycin suppresses tumorigenesis in a mouse model of progressive bladder cancer.</a> Cancer Prev Res (Phila). 2 (12), 1008-1014 (2009).</li><li>Hagberg, 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=Ascending,+unobstructed+urinary+tract+infection+in+mice+caused+by+pyelonephritogenic+Escherichia+coli+of+human+origin).">Ascending, unobstructed urinary tract infection in mice caused by pyelonephritogenic Escherichia coli of human origin).</a> Infect Immun. 40 (1), 273-283 (1983).</li><li>Olson, P. D., Hruska, K. A., Hunstad, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Androgens+Enhance+Male+Urinary+Tract+Infection+Severity+in+a+New+Model.">Androgens Enhance Male Urinary Tract Infection Severity in a New Model.</a> J Am Soc Nephrol. , (2015).</li><li>Knipper, J. 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=Interleukin-4+Receptor+alpha+Signaling+in+Myeloid+Cells+Controls+Collagen+Fibril+Assembly+in+Skin+Repair.">Interleukin-4 Receptor alpha Signaling in Myeloid Cells Controls Collagen Fibril Assembly in Skin Repair.</a> Immunity. 43 (4), 803-816 (2015).</li><li>Hung, C. S., Dodson, K. W., Hultgren, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+murine+model+of+urinary+tract+infection.">A murine model of urinary tract infection.</a> Nat Protoc. 4 (8), 1230-1243 (2009).</li><li>Wright, K. J., Seed, P. C., Hultgren, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+intracellular+bacterial+communities+of+uropathogenic+Escherichia+coli+depends+on+type+1+pili.">Development of intracellular bacterial communities of uropathogenic Escherichia coli depends on type 1 pili.</a> Cell Microbiol. 9 (9), 2230-2241 (2007).</li></ol>

Transuretrale instillazione di topo maschio offre molte nuove opportunità di ricerca sull'influenza del sesso sui malattia mucosa della vescica, ma anche presenta varie sfide. Innanzitutto, una limitazione è che l'instillazione inizialmente può risultare per essere tecnicamente difficile, con conseguente eccessiva infiammazione durante l'inserimento del catetere. Miglioramento nella tecnica può essere raggiunto tramite l'instillazione di topi morti con una soluzione colorata, come colorante blu di Evans. Per conferma...

Infezioni del tratto urinario (UTI) sono una delle infezioni più comuni nei paesi sviluppati1. Tassi di infezione sono simili tra femmine e maschi tra i neonati e gli anziani2. Donne di età premenopausa, tuttavia, hanno un'incidenza notevolmente aumentata di comunità-acquistata UTI rispetto a uomini2,3. Dato che questa malattia influisce principalmente donne, ricerca fondamentale e clinica è prevalentemente concentrata sulla UTI nelle femmine. Tuttavia, UTI negli uomini è una sfida significativa e understudied assistenza sanitaria4. Infatti, poiché UTI negli uomini sono associati con la morbosità più alto, queste infezioni sono ordinariamente definite come complicato4,5.
Come si evolve la nostra comprensione del ruolo centrale delle polarizzazioni di sesso in fisiologia e patologia, nuovi metodi sono necessari per esplorare questo aspetto precedentemente trascurato della malattia. Le differenze del sesso gioca un ruolo essenziale nell'immunità e infezione; le femmine hanno la più alta incidenza della malattia autoimmune, mentre i maschi sono più suscettibili alle infezioni, come la tubercolosi, la malaria e HIV6,7. Cancro alla vescica, un'altra patologia urologica, è sensibilmente più prevalente negli uomini che nelle donne, e diversi studi hanno dimostrato un ruolo per gli androgeni nello sviluppo di malignità8,9,10,11 ,12. In particolare, tuttavia, indagine intravesical terapie per cancro alla vescica è realizzata esclusivamente in animali di sesso femminile, a causa dell'incapacità di cateterismo ripetutamente topo maschio13.
Lo studio della patogenesi UTI si basa pesantemente su modelli del roditore di infezione (per esempio, topi e ratti). Modelli murini di UTI possono impiegare uropathogens originariamente isolato da infezioni umane, come ad esempio uropathogenic Escherichia coli (UPEC), Klebsiella, enterococco, stafilococcoo Proteus14. In genere, i batteri vengono introdotti nella cateterizzazione della vescica tramite l'uretra. Dopo l'infezione, vesciche e reni può essere rimosso per valutare i parametri specifici di infezione, come colonizzazione batterica, danni ai tessuti o host risposta immunitaria15,16. Universalmente, tuttavia, solo gli animali femminili sono utilizzati per la ricerca UTI. Infatti, molti studi hanno notato che, per ragioni anatomiche, la cateterizzazione di topo maschio non è possibile13,17,18,19. Più recentemente, è stato descritto un approccio chirurgico per instillazione maschile, in cui l'addome è aperto, la vescica è spostata esternamente applicando leggera pressione all'apertura nell'addome e batteri vengono iniettati nella vescica20. Questo approccio consente a infezione maschio, a costo di intervento chirurgico. Così, un avvertimento importante di questo approccio include l'influenza della infiammazione, sul risultato di infezione, come il potenziale di indurre una risposta antinfiammatoria di guarigione all' incisione21. Come i nostri interessi vi sono la comprensione bias di sesso in risposta alla malattia, abbiamo sviluppato un metodo di instillazione intravescicale batterica in di topo maschio che rispecchia maggiormente l'approccio transuretrale non chirurgica consolidata utilizzato in femminile roditori22 .
Il nostro modello si basa su una metodologia consolidata e offre la possibilità di confrontare direttamente la risposta immunitaria a UTI in animali maschili e femminili. Questo metodo verrà permesso di dissezione delle differenze basate sul sesso nell'infezione e potenzialmente offrire indizi molecolari e cellulari delle pronunciate differenze nella suscettibilità e risposta all'infezione tra i sessi. Inoltre, questo modello ha valore di là di studi UTI, permettendo la creazione di modelli per studiare altre malattie di vescica-collegate, ad esempio cancro alla vescica, prostatite, sindrome della vescica a sotto - o sovra - attivo e cistite interstiziale.

<table border="0" cellpadding="0" cellspacing="0" width="808">
	<tbody>
		<tr height="45">
			<td height="45" style="height:45px;width:283px;">BD Insyte Autoguard Shielded IV Catheters 24G, 0.7 mm external diameter, 14 mM long</td>
			<td style="width:131px;">BD Medical</td>
			<td style="width:135px;">381811 or 381411*</td>
			<td style="width:260px;">* catalog number is country-dependent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">inoculating loop</td>
			<td style="width:131px;">Greiner Bio-One</td>
			<td style="width:135px;">731171</td>
			<td style="width:260px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">LB Miller broth</td>
			<td style="width:131px;">Difco</td>
			<td style="width:135px;">244620</td>
			<td style="width:260px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">LB Miller agar</td>
			<td style="width:131px;">Difco</td>
			<td style="width:135px;">244520</td>
			<td style="width:260px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Cuvettes</td>
			<td style="width:131px;">Bio-rad</td>
			<td style="width:135px;">223-9955</td>
			<td style="width:260px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">syringes</td>
			<td style="width:131px;">B Braun</td>
			<td style="width:135px;">9166017V</td>
			<td style="width:260px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Thumb (Adson) forceps</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:135px;">11006-12</td>
			<td style="width:260px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">5 mL tubes, polypropylene</td>
			<td style="width:131px;">Falcon</td>
			<td style="width:135px;">352063</td>
			<td style="width:260px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Tissue Ruptor disposable probes</td>
			<td style="width:131px;">Qiagen</td>
			<td style="width:135px;">990890</td>
			<td style="width:260px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">15 mL flip cap tubes</td>
			<td style="width:131px;">Thermo Scientific</td>
			<td style="width:135px;">362694</td>
			<td style="width:260px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">DNAse</td>
			<td style="width:131px;">Invitrogen</td>
			<td style="width:135px;">18047019</td>
			<td style="width:260px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Liberase TM</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:135px;">5401119001</td>
			<td style="width:260px;">should be aliquotted and stored at -20 &#176;C to avoid repeated freeze-thaw</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">100 &#181;M MACS SmartStrainers&#160;</td>
			<td style="width:131px;">Miltenyi</td>
			<td style="width:135px;">130-098-463</td>
			<td style="width:260px;">compatible with 15 mL tubes, preferred</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">100 &#181;m cell strainers&#160;</td>
			<td style="width:131px;">Falcon</td>
			<td style="width:135px;">352360</td>
			<td style="width:260px;">compatible with 50 mL tubes, if MACS Smart Strainers are not available</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">96 well plate</td>
			<td style="width:131px;">Falcon</td>
			<td style="width:135px;">353077</td>
			<td style="width:260px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Fc block</td>
			<td style="width:131px;">BD Pharmingen</td>
			<td style="width:135px;">553142</td>
			<td style="width:260px;">anti-CD16/CD32</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">anti-mouse CD45 antibody</td>
			<td style="width:131px;">BD Biosciences</td>
			<td style="width:135px;">561487</td>
			<td style="width:260px;">many different fluorescent conjugation options are available</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">5 mL tubes polystyrene with filter cap</td>
			<td style="width:131px;">Falcon</td>
			<td style="width:135px;">352235</td>
			<td style="width:260px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">insulin syringe</td>
			<td style="width:131px;">Terumo</td>
			<td style="width:135px;">BS05M2913</td>
			<td style="width:260px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">hand held homogenizer</td>
			<td style="width:131px;">Qiagen</td>
			<td style="width:135px;">9001272</td>
			<td style="width:260px;">TissueRuptor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">flow cytometer</td>
			<td style="width:131px;">BD Biosciences</td>
			<td style="width:135px;">N/A</td>
			<td style="width:260px;">Fortessa SORP&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Flow cytometry software</td>
			<td style="width:131px;">BD Biosciences</td>
			<td style="width:135px;">N/A</td>
			<td style="width:260px;">Diva</td>
		</tr>
	</tbody>
</table>

urinary tract infection in a small animal model: transurethral catheterization of male and female mice

Infezioni del tratto urinario (UTI) sono estremamente comuni in tutto il mondo, incorrere in spese associate all'assistenza sanitaria e morbosità significativa. Piccoli modelli animali, che riflettano la progressione e l'istituzione di malattia, consentono la dissezione delle interazioni ospite-patogeno e generazione di immunità all'infezione. Nei topi, instillazione intravescicale di uropathogenic Escherichia coli, l'agente causativo in più dell'85% della Comunità acquisito UTI, ricapitola molte delle fasi di infezione osservati in esseri umani. Fino a poco tempo, tuttavia, UTI potrebbe solo essere modellati in animali di sesso femminile. Questa limitazione ha ostacolato lo studio delle differenze sesso-correlate in UTI, così come altre patologie della vescica, come il cancro. Qui, descriviamo un metodo per infondere di topo maschio che permette il confronto diretto tra gli animali femminili e maschili e fornire un protocollo dettagliato per valutare il tessuto della vescica mediante citometria a flusso come un mezzo per comprendere meglio la risposta dell'ospite all'infezione. Insieme, questi approcci saranno di aiuto nell'identificazione di fattori dell'ospite che contribuiscono a pregiudizi di sesso osservati in UTI e altre malattie della vescica-collegate.

Lo sviluppo del presente protocollo, coorti dei topi C57Bl/6 maschili e femminili all'età di 6-8 settimane sono stati infusi e batterici onere valutato in vesciche a intervalli di tempo che vanno da 1 ora a 30 giorni. I risultati di questi studi sono dettagliati altrove (Zychlinsky Scharff et al., in sospeso). Qui, presentiamo i dati rappresentativi da infezioni di 24 ore. In particolare, la carica batterica era equivalente tra topi maschi e femmine alle 24 ore post-infezione (<...

Watch this Scientific Journal Video about Urinary Tract Infection in a Small Animal Model: Transurethral Catheterization of Male and Female Mice at JoVE.com

Urinary Tract Infection in a Small Animal Model: Transurethral Catheterization of Male and Female Mice

Il modello stabilito di cateterizzazione transuretrale dei topi permette lo studio delle patologie della vescica, compreso l'infezione delle vie urinarie, ma può essere eseguito solo nelle femmine. Un nuovo modello di maschio instillazione transuretrale, qui presentato, consentirà la ricerca in un'area segnata da forti differenze cliniche ed epidemiologiche tra i sessi.

Infezione di apparato urinario in un modello animale piccolo: cateterizzazione transuretrale di topi maschi e femmine

Urinary tract infections (UTI) are extremely common worldwide, incurring significant morbidity and healthcare-associated expenses. Small animal models, ...

The overall goal of this experimental approach is to investigate the immune response to urinary tract infection, and to prevent direct comparison between male and female animals. This method can help answer key questions of mucosal immunology related to how males and females respond to urinary tract infection. The main advantage is that infection is established via the natural root.We first had the idea for this method after noting how often it is stated, that the installation of the bladder of the male mice is impossible. Though this method will provide insight into urinary tract infection, it can also be applied to other disease studies, such as bladder cancer. To begin, prepare one pediatric intravenous access canula for each group of mice to be infected.Using the inbuilt spring mechanism, divest each canula of its needle as instructed by the manufacturer. Discard the needles, preserving only the plastic intravenous canula. Sterilize the catheters in a laminar flow hood for one UV cycle of approximately 25 to 30 minutes.After going in overnight culture of uropathogenic e coli, or UPEC, according to the text protocol, spin the bacterial suspension in a table-top microcentrifuge at 17, 000 times g for one minute, and resuspend the resulting bacterial pellet at two times 10 to the eighth CFU per milliliter in PBS. Serially dilute an aliquot of suspension, and plate on LB agar with antibiotics if appropriate, to determine the exact inoculum for each infection. Draw the bacterial inoculum into a one milliliter syringe, and attach the catheter to the end of the syringe.Tap the syringe to remove any air, and depress the plunger to fill the dead air space in the catheter before beginning the instillation. After anesthetizing a female mouse according to an approved protocol, place the animal supine, and apply medium pressure to the lower abdomen to empty the bladder of urine. Full bladders feel like a pea under the skin, between the iliac crests.When the bladder is empty, using the non-dominant hand, place the thumb on the tail and a finger of the same hand on the abdomen of the mouse, and apply gentle pressure in opposing directions to hold the mouse firmly in place. Next, place the tip of the catheter perpendicular to the mouse at the urethral orifice, then, with gentle pressure, slide the catheter into the urethra until the hub meets the urethral orifice, while simultaneously lowering the syringe so that it is parallel to the working surface. Once the catheter is in place, use a finger from the non-dominant hand that's resting on the mouse abdomen to very gently pull the abdominal skin towards the head of the mouse.Note that the urethral orifice will not move. However, if the catheters is in the vagina, the tissue will move up and away from the catheter. With the hub of the catheter against the urethral orifice, slowly dispense 15 microliters of the bacterial inoculum.A slow installation rate minimizes vesicoureteral reflux into the kidney. Slowly remove the catheter to prevent leakage. Then place the animal in its cage in a supine position.To inoculate male mice, place two thumb forceps cranially and caudally to the mouses external genitalia. Retract the prepuce to fully expose the gland's penis. Then, once the penis is externally positioned, release the thumb forceps.Reposition the forceps to stabilize the protruding penis perpendicular to the animal, holding the organ gently but tautly. Then through the small opening in the tip of the urethral meatus, carefully introduce the catheter and gently guide it into the penis using forceps to gently maintain tension. Once the hub of the catheters meets the tip of the penis, very slowly dispense 50 microliters of the inoculum, while maintaining the position of the penis.Note the quality of the instillation according to a predetermined instillation quality score, such as the one shown here. Retract the catheter slowly over five seconds to prevent leakage of the inoculum. Place the mouse in its cage in a supine position.The animal should begin to recover 30 to 45 minutes following administration of the anesthetic. After sacrificing mice according to an approved protocol, use 70%Epinal to moisten the abdomen thoroughly to minimize contamination by the fur. Using scissors, make a two centimeter incision across the lower third of the mouses abdomen, and aseptically remove the bladder and any other desired organs.Transfer the organs into five milliliter polypropylene snap-cap tubes, containing one milliliter of sterile PBS and keep all samples on ice. With a hand-held homogenizer, homogenize the organs until almost no solid tissue remains. Discard any shiny, beige-white fat tissue that does not homogenize well.Then, use Epinal, followed by PBS to wash the homogenizer between each experimental or organ group. After preparing serial dilutions of homogenized organ suspension, plate dilutions on LB agar plates, with antibiotics when appropriate, and incubate at 37 degrees Celsius overnight. To carry out flow cytometry on bladder tissue, after isolating the bladder as demonstrated earlier in this video, use scissors to mince the tissue well.Add one minced bladder per tube containing digestion buffer. Then shake the tube to wash the minced tissue in the digestion buffer, and keep it on ice until all the bladders are dissected and minced. Incubate the minced bladders at 37 degrees Celsius, for 60 to 75 minutes with vigorous shaking by hand for five seconds, every 15 minutes.When the tissue has a glassy transparent appearance resembling wet tissue paper, digestion is complete. Inactivate the digestion enzymes by adding two to three milliliters of ice-cold flow cytometry buffer, and gently mix. Then place the tubes on ice.To ensure a single-cell suspension, and to remove any connective tissue, pass the contents of the tube through a 100 micrometer filter placed in a 15 milliliter conical tube. Then, with the end of a syringe plunger, gently press any remaining tissue through the filter. Wash the filter with an additional two milliliters of flow cytometry buffer, and keep the samples on ice.The wash the samples by centrifugation at 200 times g and four degrees Celsius, for seven minutes. Resuspend the pellets in 100 microliters of flow cytometry buffer containing Fc block, diluted to five micrograms per milliliter, and transfer to a 96 Well round bottom plate. After 10 to 15 minutes add 100 microliters of the desired antibody cocktail to each sample.Then incubate the samples on ice protected from light for 30 to 45 minutes. Wash the samples by centrifugation at 200 times g and four degrees Celsius for seven minutes. Finally, resuspend the cell pellets in 200 microliters of flow cytometry buffer, and pass the samples through a 40 micrometers cell strainer on a five milliliter polystyrene tube, just prior to acquisition on a flow cytometer.This figure shows representative data from mice infected for 24 hours. Bacterial burden was equivalent between male and female mice at 24 hours post infection. To determine whether loss of inoculum at the time of infection impacted the establishment of infection each instillation was scored on a scale of one to five, with one being the most optimal.Bacterial colonization was determined 24 hours post infection. No statistically significant differences were found among the CFU's obtained from animals with different instillation scores as assessed by a nonparametric Kruskal-Wallis test, comparing the mean rank of each column with the mean rank of every other column, and correcting for multiple comparisons using a Dunn's test. It can be concluded that suboptimal instillations, resulting in substantial leakage of bacterial inoculum during infection, do not significantly impact bacterial colonization at 24 hours.Importantly, the frequency of suboptimal instillation will decrease with practice. Finally, while the number of CD45 positive immune cells, present in naive animals was not different between female and male mice, there was a statistically-significant increase in infiltration into the bladders of infected female mice. While attempting catheterization, it is important to use slow, gentle movements.With its development, this technique has helped us to explore sex-based differences in immunity in the bladder of male and female animals.

Research

JoVE Journal

Immunology and Infection

Transurethral Induction of Mouse Urinary Tract Infection

Induzione transuretrale di infezioni del tratto urinario mouse

Uropathogenic bacterial strains of interest are grown on agar. Generally, uropathogenic E. coli (UPEC) and other strains can be grown overnight on ...

Ricerca

Immunologia e infezioni

Diagnosis of Ecto- and Endoparasites in Laboratory Rats and Mice

La diagnosi di Ecto-e endoparassiti in ratti e topi di laboratorio

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized ...

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

4D imaging multimodale di<em&gt; Citrobacter rodentium</em&gt; Infezioni in topi

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

Transurethral Instillation Procedure in Adult Male Mouse

Procedura transuretrale instillazione in di topo maschio adulto

Transurethral instillation can be used to deliver different solutions with active ingredients (e.g., drugs, chemicals, bacteria, and viruses) locally into ...

Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

Isolamento di cellule dendritiche dal tratto riproduttivo femminile umano per studi fenotipici e funzionali

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low ...

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Intracranica Subarachnoidal itinerario dell'infezione per indagare ruoli di biofilm di Streptococcus suis nella meningite in un modello di infezione del topo

Streptococcus suis is not only a major bacterial pathogen of pigs worldwide but also an emerging zoonotic agent. In humans and pigs, meningitis is a major ...

Isolation of Single Intracellular Bacterial Communities Generated from a Murine Model of Urinary Tract Infection for Downstream Single-cell Analysis

Isolamento di singole comunità batteriche intracellulari generati da un modello murino di infezione delle vie urinarie per analisi unicellulare a valle

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected ...

A Non-Coding Small RNA MicC Contributes to Virulence in Outer Membrane Proteins in Salmonella Enteritidis

A Non-Coding Small RNA MicC Contributes to Virulence in Outer Membrane Proteins in Salmonella Enteritidis

Un piccolo RNA non codificante MicC contribuisce alla virulenza nelle proteine della membrana esterna in Salmonella enteritidis

A non-coding small RNA (sRNA) is a new factor to regulate gene expression at the post-transcriptional level. A kind of sRNA MicC, known in Escherichia ...

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Infezione ricorrente del tratto urinario da Escherichia coli innescata dall'esposizione alla vescica di Gardnerella vaginalis nei topi

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of ...

Visualizing Lung Cellular Adaptations during Combined Ozone and LPS Induced Murine Acute Lung Injury

Visualizzazione degli adattamenti cellulari polmonari durante l'ozono combinato e la lesione polmonare acuta murina indotta da LPS

Lungs are continually faced with direct and indirect insults in the form of sterile (particles or reactive toxins) and infectious (bacterial, viral or ...