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

Summary

The established model of transurethral catheterization of mice allows the study of bladder pathologies, including urinary tract infection, but can only be performed in females. A new model of male transurethral instillation, presented here, will enable research in an area marked by strong clinical and epidemiological differences between the sexes.

Abstract

Urinary tract infections (UTI) are extremely common worldwide, incurring significant morbidity and healthcare-associated expenses. Small animal models, which accurately reflect disease establishment and progression, permit dissection of host-pathogen interactions and generation of immunity to infection. In mice, intravesical instillation of uropathogenic E. coli, the causative agent in more than 85% of community acquired UTI, recapitulates many of the stages of infection observed in humans. Until recently, however, UTI could only be modeled in female animals. This limitation has hindered the study of sex-related differences in UTI, as well as other bladder pathologies, such as cancer. Here, we describe a method to instill male mice that allows direct comparison between female and male animals and provide a detailed protocol to assess bladder tissue by flow cytometry as a means to better understand host responses to infection. Together, these approaches will aid in the identification of host factors that contribute to sex biases observed in UTI and other bladder-associated diseases.

Introduction

Urinary tract infections (UTI) are one of the most common infections in developed countries¹. Infection rates are similar between females and males among neonates and the elderly². Premenopausal adult women, however, have a greatly increased incidence of community-acquired UTI compared to men^2,3. Given that this disease primarily impacts women, fundamental and clinical research has overwhelmingly focused on UTI in females. However, UTI in men is a significant and understudied health care challenge⁴. Indeed, because UTI in men are associate....

Protocol

Mouse experiments were conducted in accordance with approval of protocol number 2012-0024 by the Comité d'éthique en expérimentation animale Paris Centre et Sud (the ethics committee for animal experimentation), in application of the European Directive 2010/63 EU.

1. Preparation of Catheters

1. Prepare one pediatric intravenous-access cannula for each group of mice to be infected. Using the inbuilt spring mechanism, divest each cannula of its needle, as instructed by the manufacturer. Discard the needles, preserving only the plastic intravenous cannula.
2. Sterilize catheters in a laminar flow hood for one....

Results

In the development of this protocol, cohorts of female and male C57Bl/6 mice aged 6 to 8 weeks were instilled, and bacterial burden evaluated in bladders at time points ranging from 1 hr to 30 days. The results from these studies are detailed elsewhere (Zychlinsky Scharff et al., pending). Here, we present representative data from 24 hr infections. Notably, bacterial burden was equivalent between male and female mice at 24 hr post-infection (Figure 1). Little var.......

Discussion

Transurethral instillation of male mice offers many new research opportunities into the influence of sex on bladder mucosal disease, but also presents several challenges. Foremost, one limitation is that the instillation may initially prove to be technically difficult, resulting in excessive inflammation during catheter insertion. Improvement in technique can be achieved by the instillation of dead mice with a colored solution, such as Evans blue dye. To confirm that the instillation is successful, bladders should be vis.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BD Insyte Autoguard Shielded IV Catheters 24 G, 0.7 mm external diameter, 14 mM long	BD Medical	381811 or 381411*	* catalog number is country-dependent
inoculating loop	Greiner Bio-One	731171
LB Miller broth	Difco	244620
LB Miller agar	Difco	244520
Cuvettes	Bio-rad	223-9955
syringes	B Braun	9166017V
Thumb (Adson) forceps	Fine Science Tools	11006-12
5 ml tubes, polypropylene	Falcon	352063
Tissue Ruptor disposable probes	Qiagen	990890
15 ml flip cap tubes	Thermo Scientific	362694
DNAse	Invitrogen	18047019
Liberase TM	Sigma-Aldrich	5401119001	should be aliquotted and stored at -20 °C to avoid repeated freeze-thaw
100 µM MACS SmartStrainers	Miltenyi	130-098-463	compatible with 15 ml tubes, preferred
100 µm cell strainers	Falcon	352360	compatible with 50 ml tubes, if MACS Smart Strainers are not available
96 well plate	Falcon	353077
Fc block	BD Pharmingen	553142	anti-CD16/CD32
anti-mouse CD45 antibody	BD Biosciences	561487	many different fluorescent conjugation options are available
5 ml tubes polystyrene with filter cap	Falcon	352235
insulin syringe	Terumo	BS05M2913
hand held homogenizer	Qiagen	9001272	TissueRuptor
flow cytometer	BD Biosciences	N/A	Fortessa SORP
Flow cytometry software	BD Biosciences	N/A	Diva