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

Agradecemos o Dr Matthieu Rousseau pela leitura crítica do manuscrito e o Laboratory of Immunobiology dendríticas para suas introspecções úteis durante o desenvolvimento do presente protocolo e projeto. Este trabalho foi financiado em parte por fundos da União Europeia sétimo programa Marie Curie acção-quadro (PCIG11-GA-2012-3221170 e o LabEx imuno-Oncologia (AMI).

Mouse experimentos foram conduzidos em conformidade com a aprovação do protocolo número 2012-0024 pelo Comitê d'éthique en expérimentation animale Paris Centre et Sud (Comitê de ética para experimentação animal), em aplicação da Directiva 2010/63 Europeu UE.
1. preparação de cateteres
<ol><li>Prepare uma cânula de acesso intravenosa pediátrica para cada grupo de ratos para ser infectado. Usando o mecanismo de mola embutido, alienar cada cânula de sua agulha, conforme as inst...

<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>

Instilação transuretral de ratos masculinos oferece muitas novas oportunidades de pesquisa para a influência do sexo sobre a doença da mucosa da bexiga, mas também apresenta vários desafios. Acima de tudo, uma limitação é que a instilação inicialmente pode provar para ser tecnicamente difícil, resultando em inflamação excessiva durante a inserção do cateter. Melhoria na técnica pode ser conseguida a instilação de ratos mortos com uma solução colorida, como corante de azul de Evans. Para confirmar que...

Infecções do trato urinário (ITU) são uma das infecções mais comuns em países desenvolvidos1. Taxas de infecção são semelhantes entre as fêmeas e os machos entre os recém-nascidos e os idosos2. Mulheres adultas na pré-menopausa, no entanto, têm uma incidência muito maior de ITU adquirida na Comunidade em comparação com os homens,2,3. Dado que esta doença afeta principalmente as mulheres, de investigação fundamental e clínica centrou-se esmagadoramente na UTI nas fêmeas. No entanto, a ITU em homens é um desafio de saúde significativos e pouco estudado4. Com efeito, porque a ITU em homens estão associadas a maior morbidade, estas infecções rotineiramente são definidas como complicado4,5.
Como nosso entendimento do papel central de enviesamentos de sexo na fisiologia e patologia evolui, novos métodos são necessários para explorar este aspecto anteriormente negligenciado da doença. Diferenças de sexo desempenham um papel essencial na imunidade e infecção; as fêmeas têm maior incidência de doença auto-imune, enquanto os machos são mais suscetíveis a determinadas infecções, como tuberculose, malária e HIV6,7. Câncer de bexiga, outra patologia urológica, é significativamente mais prevalente em homens do que nas mulheres, e vários estudos têm demonstrado um papel de andrógenos no desenvolvimento de malignidade8,9,10,11 ,12. Nomeadamente, no entanto, investigação de terapias intravesical para câncer de bexiga é realizada exclusivamente em fêmeas, devido à incapacidade de cateterismo repetidamente camundongos machos13.
O estudo da patogênese da ITU confia pesadamente em modelos de roedores de infecção (por exemplo, ratos e ratazanas). Murino modelos de UTI podem empregar uropathogens originalmente isolados de infecções humanas, tais como uropathogenic e. coli (UPEC), Klebsiella, Enterococcus, Staphylococcusou Proteus14. Normalmente, as bactérias são introduzidas o bexiga através de cateterismo da uretra. Após a infecção, bexiga e rins podem ser removido para avaliar parâmetros específicos de infecção, tais como a colonização bacteriana, dano tecidual ou anfitrião resposta imune15,16. Universalmente, no entanto, apenas fêmeas são utilizadas para pesquisa de ITU. De fato, muitos estudos observaram que, devido a razões anatômicas, cateterismo de ratos masculinos não é possível13,17,18,19. Mais recentemente, tem sido descrita uma abordagem cirúrgica para instilação masculina, no qual o abdome é aberto, a bexiga é deslocada externamente aplicando uma pressão suave para a abertura no abdômen e bactérias são injetadas a bexiga20. Essa abordagem permite que a infecção masculina, ao custo de intervenção cirúrgica. Assim, uma ressalva importante desta abordagem inclui a influência da inflamação, sobre o resultado da infecção, como o potencial para induzir uma resposta anti-inflamatórios-cicatrização da incisão21. Como nossos interesses incluem compreender o viés sexual em resposta à doença, desenvolvemos um método de instilação de intravesical bacteriana em ratos masculinos que mais se aproxima da abordagem de transuretral não-cirúrgico bem estabelecida usada em roedores femininos22 .
Nosso modelo baseia-se uma metodologia estabelecida e fornece a capacidade de comparar diretamente a resposta imune do hospedeiro à ITU em animais do sexo femininos e masculinos. Este método irá permitir a dissecação das diferenças baseadas no sexo, na infecção e potencialmente oferecer pistas moleculares e celulares para as pronuncia-se diferenças na susceptibilidade e resposta à infecção entre os sexos. Além disso, este modelo tem valor para além de estudos UTI, permitindo a criação de modelos para investigar outras doenças associadas a bexiga, como o câncer de bexiga, próstata, sob ou sobre active síndrome da bexiga e cistite intersticial.

<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

Infecções do trato urinário (ITU) são extremamente comuns em todo o mundo, incorrer em despesas associadas a cuidados de saúde e Morbidade significativa. Modelos animais pequenos, que refletem com precisão a progressão e o estabelecimento da doença, permitam a dissecação das interações patógeno-hospedeiro e geração de imunidade à infecção. Em ratos, instilação intravesical de uropathogenic e. coli, o agente causador em mais de 85% da Comunidade adquiriu UTI, recapitula a muitos dos estágios de infecção observada em humanos. Até recentemente, no entanto, UTI pode somente ser modelado em fêmeas. Essa limitação tem dificultado o estudo das diferenças relacionadas ao sexo na UTI, bem como outras patologias de bexiga, como o câncer. Aqui, descrevemos um método para incutir ratos masculinos que permite a comparação direta entre animais masculinos e femininos e fornecer um protocolo detalhado para avaliar o tecido da bexiga por citometria de fluxo como meio para compreender melhor as respostas do hospedeiro à infecção. Juntas, estas abordagens ajudarão na identificação de fatores do hospedeiro que contribuem para sexo enviesamentos observados em UTI e outras doenças associadas a bexiga.

No desenvolvimento do presente protocolo, coortes de camundongos C57Bl/6 de feminino e masculinos com idade entre 6 a 8 semanas eram encargos inculcado e bacterianos avaliados em bexigas em pontos de tempo variando de 1 hora a 30 dias. Os resultados destes estudos são detalhados em outro lugar (Zychlinsky Scharff et al., pendente). Aqui, apresentamos dados representativos de infecções 24 hr. Notavelmente, a carga bacteriana foi equivalente entre ratos masculinos e femininos em...

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

O modelo estabelecido de transuretral cateterismo de ratos permite o estudo das patologias de bexiga, incluindo a infecção do trato urinário, mas só pode ser realizado nas fêmeas. Um novo modelo de instilação transuretral masculino, apresentado aqui, permitirá a pesquisa em uma área marcada por fortes diferenças clínicas e epidemiológicas entre os sexos.

Infecção do trato urinário em um modelo Animal de pequeno porte: transuretral cateterismo de ratos machos e fêmeas

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

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