Establishment of new transurethral catheterization methods for male mice

Abstract

Transurethral catheterization in mice is multifaceted, serving essential functions such as perfusion and drug delivery, and is critical in the development of various urological animal disease models. The complex anatomy of the male mouse urethra presents significant challenges in transurethral catheterization, leading to a predominance of research focused on female specimens. This bias limits the utilization of male mice in lower urinary tract disease studies. Our research aims to develop new reliable methods for transurethral catheterization in adult male mice, thereby expanding their use in relevant disease research. Experiments were conducted on adult male C57BL/6J mice. Utilizing a PE10 catheter measuring 4.5–5 cm in length, the catheter was inserted into the bladder via the mouse’s urethra under anesthesia. The intubation technique entailed regulating the insertion force, ensuring the catheter's lubrication, using a trocar catheter, modifying the catheter’s trajectory, and accommodating the curvature of the bladder neck. Post-catheter insertion, ultrasound imaging was employed to confirm the catheter's accurate positioning within the bladder. Subsequent to catheterization, the bladder was perfused using trypan blue. This method was further validated through its successful application in establishing an acute urinary retention (AUR) model, where the mouse bladder was infused with saline to a pressure of 50 or 80 cm H₂O, maintained steadily for 30 min. A thorough morphological assessment of the mouse bladder was conducted after the infusion. Our study successfully pioneered methods for transurethral catheterization in male mice. This technique not only facilitates precise transurethral catheterization but also proves applicable to male mouse models for lower urinary tract diseases, such as AUR.

Introduction

Breakthroughs in biomedical research frequently arise from investigations using animal models, particularly mice, which are favored due to their diminutive size, cost-effective breeding, and evolutionary parallels with humans, especially in terms of tissue, organ structure, and cellular function [1]. The completion of the mouse genome sequencing project further underscores their significance [2], paving the way for their widespread use in various disease model studies. Among the myriad health issues globally, urinary tract disorders, such as lower urinary tract symptoms [3], are notably prevalent. While primates share even more physiological and anatomical resemblances with humans, research into experimental urinary system diseases in primates or humans remains constrained due to ethical considerations and associated costs. Consequently, mice have ascended as the primary organism for urinary system disease research [4–6].

Certain urinary system diseases manifest differently based on gender. Take urinary tract infections (UTIs) for instance. Epidemiological data reveal that adult women experience UTIs far more frequently than men [7, 8]. Consequently, most foundational and clinical research revolves around female UTIs. Yet, men and women exhibit notable disparities in UTI characteristics and treatment. Specifically, UTIs in men are often classified as complex UTIs due to their intricate urinary tract anatomy, leading to an increased chance of complications and a higher risk of treatment failure [9–11]. Moreover, the lifetime prevalence of UTIs in men stands at 13.7% [12]. This presents a pressing query: How can we more effectively emulate UTIs in males to delve deeper into its underlying mechanisms and therapeutic strategies? The anatomy of male mice, particularly their urethra—with its innate convoluted structure and strictures—poses considerable challenges for transurethral intubation, catheterization, and perfusion. This technical obstacle impedes comprehensive UTIs research in male mice and stymies the creation of other male urinary system disease models, such as bladder cancer and prostatitis. Notably, the male incidence rate of bladder cancer is quadruple that of females [13–15]. However, in establishing mouse models of bladder cancer, many researchers typically resort to female mice, inserting a catheter through the urethra to introduce cancer cells [16, 17]. Additionally, prostatitis predominantly affects middle-aged and elderly men. In fact, nearly 80% of prostate tissue biopsies in men over 60 reveal inflammation [18, 19]. Given that most acute and chronic prostatitis models stem from transurethral catheterization [20, 21], it accentuates the need to resolve the intubation challenge in male mice.

In prior studies, Olson et al. developed a minimally invasive surgical bladder inoculation technique, involving an abdominal incision and direct bladder puncture using a syringe for bacterial injection [22]. However, this method led to direct bladder trauma, potentially causing inflammation and fibrosis, which could significantly impact the bladder's physiological and pathological functions, thereby influencing experimental outcomes. In contrast, Lee et al. faced challenges with catheters of varying sizes for urethral intubation in mice [23]. The soft PE10 catheter presented difficulties in directional control post-pubic bone passage into the bladder. Conversely, the PE50 catheter, having an outer diameter of 0.96 mm, encountered resistance when inserted through the urethra into the bladder, given that the minimum urethral stricture in adult male mice is <0.96 mm. While female mice are often favored for urinary system disease models due to the relative ease of transurethral Intubation, it is imperative to recognize the significance of male mouse models in urinary system research.

Although male mice pose challenges for transurethral intubation, these obstacles are not insurmountable. In response, we undertook an investigation of the male mouse urinary tract, culminating in the development of specialized catheterization methods and techniques. These approaches facilitate experiments like male mouse transurethral intubation and instillation, paving the way for more accurate mouse models of male-centric urinary system diseases.

Materials and methods

Animals

All male mice used in this study were specific pathogen-free in the C57BL/6J background and were 3–4 months old of age and purchased from Gempharmatech (Chengdu, China). The mice were housed in standard polycarbonate cages and five mice per cage. The mice house environment was maintained on a 12:12-h light–dark cycle at 25°C with humidity between 40% and 70%. The mice in cages had free access to standard laboratory food and water. All animal care and experimental procedures were performed in adherence to the National Institutes of Health Guidelines for the Care and Use Committee of Southwest Medical University (swmu20220301-008).

Catheter design and preparation

Based on the anatomical characteristics of adult male mice, specifically the length of their urethra and the diameter of the stricture, we designed a catheter using 4.5–5 cm segments of PE10 tubing (Fig. 2A), ensuring two blunt ends. While PE50 tubing is commonly used in transurethral catheterization, the PE10 variant was chosen here because of its reduced outer diameter and softer. This design ensures its unobstructed passage through multiple strictures in male mice, significantly lowering the risk of urethral injury. However, one inherent limitation of the PE10 is its softness, which can make it challenging to control the insertion direction, especially at the distal end near the bladder neck. To counteract this issue and provide additional rigidity, a trocar is incorporated into the catheter. This trocar’s diameter is intentionally smaller than the inner diameter of the PE10, facilitating a smooth insertion. However, to mitigate the risk of bladder damage, the trocar is kept about 1–2 mm shorter than the catheter. Before application, both the catheter and trocar are thoroughly disinfected with alcohol and subsequently rinsed with saline.

Transurethral catheterization

Male mice were subjected to inhalation anesthesia using 2% isoflurane, which was delivered through a vaporizer and nose cone. Once foot reflexes were no longer observable, the mice were positioned supine with their lower abdomens elevated by 1 cm. Depilatory cream was applied to remove fur from the lower abdomen, followed by disinfection using 75% ethanol and iodophor. The mouse's limbs were secured with tape before commencing with transurethral intubation. After lubricating the catheter, insertion was initiated at the urethral opening. Upon navigating past the pubic bone from the urethral opening, the trocar catheter technique was utilized to guide the catheter's distal insertion direction. Concurrently, gentle finger pressure was applied on the mouse's abdomen to modulate the curvature of the bladder neck, facilitating the catheter's entrance into the bladder. For enhanced visualization and control during this procedure, a 0.5 cm midline incision at the bladder neck was considered. After blunt dissection of the surrounding tissues, the bladder and bladder neck were exposed. Then, using forceps, the curvature of the bladder neck was adjusted to direct the catheter's insertion pathway, ensuring the catheter's gentle progression into the bladder. A detailed protocol including a step-by-step surgical procedure and intubation protocol can be found in the Supplementary data.

Male mouse bladder perfusion

Following the successful noninvasive insertion of the catheter into the bladder, blunt forceps or cotton swabs were used to gently depress the mouse's abdomen, facilitating the evacuation of urine from the bladder. Subsequently, an insulin syringe was employed to infuse the bladder with trypan blue. Post-infusion, for those not intubated using minimally invasive techniques, an incision in the abdomen was made to visually assess trypan blue distribution. In mice intubated through minimally invasive techniques, the perfusion of trypan blue was directly visualized without the need for an additional incision.

Bladder ultrasonography

Transabdominal bladder ultrasonography was conducted on mice using the Vevo 3100 Imaging System (Visual Sonics, Toronto, Canada). Each mouse was positioned supine on the ultrasound operation panel, with its limbs secured using tape. A medical ultrasound coupling agent was applied to the mouse's lower abdomen, ensuring the elimination of air between the ultrasound probe and the skin, and preventing the formation of bubbles. The ultrasound probe was then placed perpendicular to the mouse. By scanning the sagittal plane of the mouse's lower abdomen, adjustments to the probe's direction and angle allowed for observation of the catheter's placement.

Male mouse acute urinary retention model and morphological analysis

Acute urinary retention (AUR) was conducted as previously described [24]. Briefly, following the completion of transurethral catheterization, to simulate the excessive bladder wall pressure characteristic of AUR, we applied pressures of 50 and 80 cm H₂O to the bladder lumen. A water column of the designated height (50 or 80 cm) was then maintained to keep the pressure constant for 30 min, ensuring the bladder was subjected to the intended stress during this period. Bladder pressure was continuously monitored using a pressure transducer (and a syringe pump via a side arm) coupled with data-acquisition devices (Trans bridge, AD Instruments, CO, USA) and a computerized recording system (Lab Chart software, AD Instruments). Post-AUR treatment, bladders from the mouse model were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned transversely into 5 μm slices. For histological examination, Hematoxylin and Eosin staining was performed. The slides were examined under a KFBIO KF-PRO-002 brightfield microscope, and images were captured using K-Viewer software on a computer.

Statistical analyses

Statistical analysis was conducted using GraphPad Prism 8.0 software. Quantitative data were represented as box plots, where the center line indicates the median, and the boxes encompass the interquartile range, representing 75% of the data. The whiskers of the box plot extend to the maximum and minimum values of the dataset. For parametric data, one-way ANOVA was utilized to test for statistical significance. A P < .05 was considered to indicate statistical significance.

Results

Anatomical challenges in male mouse lower urinary tract catheterization

The intricacies of the male mouse's lower urinary tract anatomy present challenges for catheterization, with two primary regions posing difficulty. Acquiring this anatomy, along with proficient operational skills and tool selection, is paramount in ensuring successful intubation. The male mouse's lower urinary tract predominantly comprises the bladder and urethra. As shown in Fig. 1A, the male mouse's urethra traverses beneath the pubic bone encountering the bulbourethral gland in its path (Fig. 1B and C). Here, the urethra undergoes a bend, accompanied by a narrowing, marking the first intubation obstacle. The pubic bone and the bulbourethral gland can exert pressure on the catheter during intubation, heightening the risk of urethral injury and subsequent catheterization failure. To circumvent this, we advocate employing a moderately rigid catheter and ensuring lubrication during insertion. Implementing a gentle rotational movement for the catheter can further enhance intubation success rates. The mouse bladder, characterized by its ovoid structure (Fig. 1A), comprises distinct sections: the bladder apex, base, body, and neck. The bladder neck, the juncture at which the bladder connects with the urethra, is ensconced by the prostate (Fig. 1B and C), resulting in a constricted region (Fig. 1D). A fulfilled bladder manifests a pronounced bend at the bladder neck (Fig. 1A and C), constituting the second anatomical challenge for intubation. To overcome this, we suggest applying gentle abdominal pressure to optimize bladder positioning, aiming to alleviate the pronounced curvature of the bladder neck. Concurrently, using a stiffer catheter can ameliorate the intubation challenges at the bladder neck.

Noninvasive transurethral catheterization and perfusion in male mice

Once the mouse has been anesthetized, elevate its abdomen by approximately 1∼2 cm to arch its body (Fig. 2B). Next, expose the mouse's penile body (Fig. 2C), and insert the lubrication catheter through the urethral opening below the baculum (Fig. 2D). Upon inserting the catheter to a depth of around 1 cm, it encounters resistance upon reaching the pubic bone. To address this, realign the catheter to achieve an insertion angle nearly parallel to the mouse's body (Fig. 2E). Should there be an obstruction, it is advisable to re-lubricate the catheter and gently rotate it to facilitate continued insertion. At an approximate depth of 2.5 cm, the catheter approaches the bladder neck. Here, manipulate the bladder's position by delicately pressing on the mouse's abdomen, which helps reduce the curvature at the bladder neck, easing intubation (Fig. 2F). For smoother navigation through the bladder neck, we introduce the trocar catheter technique (Fig. 2G). After the catheter successfully navigates past the pubic bone and bulbourethral gland (Fig. 2H), insert a needle into the catheter (Fig. 2I and J). Following this, apply gentle abdominal pressure to further advance the trocar catheter into the bladder. Once the trocar catheter has been fully introduced into the bladder, retract the trocar (Fig. 2K). Subsequent ultrasound assessments of the mouse's bladder demonstrated a clear visual of the intravesical catheter (Fig. 2L). After ensuring the catheter's successful placement in the bladder, exert a mild pressure on the abdomen to achieve complete urinary evacuation (Fig. 3A and B). Thereafter, infuse trypan blue into the bladder via the catheter, resulting in a perceptible abdominal distension in the mouse (Fig. 3C). To authenticate the perfusion outcome, we dissected the mouse's abdomen, revealing a distended, blue-tinted bladder (Fig. 3D).

Minimally invasive transurethral catheterization and perfusion in male mice

To improve the success rate of intubation, and avoid frequent abdominal finger compression and its associated bladder position adjustment during noninvasive intubation, we further designed a minimally invasive surgical catheterization method. After the mice were anesthetized, a small ∼5 mm incision was made in the abdomen of the mice to expose the bladder and its neck (Fig. 4A and B). Next, proceeding with the prior noninvasive intubation method, the catheter was inserted into the urethra (Fig. 4C). When the catheter reaches the bladder neck, its position becomes visually discernible through the incision (Fig. 4D and E). At this time, use medical forceps to accurately adjust the curvature angle of the bladder neck so that the catheter can be inserted into the bladder easily and smoothly. Considering the softness of the catheter used, directional control as it passes through the bladder neck is a challenge. To solve this problem, we combined the trocar catheter technique: after the catheter has successfully passed through the pubic bone and bulbourethral gland, a needle is inserted into the catheter (Fig. 4D). In this way, while the forceps are used to adjust the curvature of the bladder neck, the trocar provides additional support and orientation for the catheter, ensuring smooth entry into the bladder (Fig. 4E). After the catheter is completely inserted into the bladder, the skin is sutured, and a subsequent ultrasound examination of the mouse bladder reveals that the catheter within the bladder is clearly visible (Fig. 4F). Subsequently, gently press the bladder to allow complete drainage of urine (Fig. 5A and B). After ensuring complete emptying of urine, slowly inject trypan blue into the bladder through the catheter so that the bladder gradually fills and appears bright blue (Fig. 5C and D).

Pressurized bladder causes significant injury and edema in bladder tissue

We successfully established an AUR model in male mice using the developed transurethral intubation method. As depicted in Fig. 6, pressurization significantly damaged the bladder tissue. There was an increase in bladder wall thickness following saline infusion (Fig. 6A, D, E, and I). The urothelium was notably stretched and thinned, with observable damage to the umbrella cells (Fig. 6B, F, H, and J). Additionally, there was severe congestion and edema in the lamina propria (Fig. 6E, F, L, I, and J). The smooth muscle layer also exhibited stretching and thinning, with muscle bundles appearing damaged and torn, leading to a substantial increase in the gaps (Fig. 6C, G, K, and M).

Discussion

The selection of the appropriate catheter is paramount in the bladder of male mice catheterization experiments. Given that the urethral length in adult male C57BL/6J mice typically ranges between 3 and 4 cm, the catheter should exceed this length to ensure its full insertion. As such, a recommended catheter length lies between 4.5 and 5 cm. Mclean's study utilized nuclear magnetic resonance to gage the dimensions of the lower urinary tract in adult male mice. They determined that the diameter of the urethra's constricted segment fluctuates between 0.85 and 1.48 mm [25]. While this does not denote the narrowest portion of the urethra, it offers a useful reference. In response, we opted for the PE10 conduit—the slimmest in the PE series—with an outer diameter of 0.61 mm and an inner diameter of 0.28 mm.

Two primary anatomical challenges arise during lower urinary tract catheterization in male mice. To enhance catheterization success rates, a comprehensive understanding of these challenges and the strategies to address them is essential. First, the urethra's positioning beneath the pubic bone presents the initial challenge. This section of the urethra exhibits a distinct curvature and constricted form. To prevent potential perforation from excessive force during intubation, we suggest elevating the mouse's lower abdomen by 1–2 cm, forming an arch in the body, and maintaining the catheter parallel to the mouse's body. Adjust the catheter direction progressively until minimal resistance is perceived. For deep insertion into the urethra, opting for a supple catheter and ensuring its consistent lubrication is pivotal. Second, the bladder neck, encased by the prostate, is the subsequent hurdle due to its innate narrowness. The bladder neck may exhibit enhanced curvature when the bladder is full. For improved bladder visualization and positioning, we recommend shaving the mouse's abdominal fur. This allows for rudimentary bladder visualization and permits minor finger adjustments to align the bladder neck and urethra. If resistance emerges during intubation, subtle rotation or angle modification of the catheter proves beneficial. To ensure directional precision during intubation, enhancing the catheter's rigidity can be beneficial. A trocar catheter can amplify its stiffness. However, it is vital to ensure that the trocar's length does not surpass that of the catheter, preventing unintentional urethral punctures. Furthermore, due to the challenges and extensive practice required for noninvasive intubation technology, an alternative approach was adopted to improve intubation success rates and eliminate the need for recurrent abdominal pressing for bladder positioning. This method involves making an incision to expose the bladder and its neck, thereby facilitating direct observation and timely adjustment of the catheter's direction. This ensures more reliable and successful catheter entry into the bladder.

The noninvasive catheter method, while demonstrating a lower success rate compared to the minimally invasive approach, offers the benefit of not causing damage to mice. This technique is versatile and applicable to both acute and chronic animal models, including acute UTI, acute urinary tract obstruction models, and chronic urinary retention (CUR) models. In contrast, the minimally invasive method, with its higher success rate, does induce trauma to the mouse. This makes it particularly suitable for chronic disease models such as CUR models, chronic infectious models, and chronic prostatitis models. Our advancements in these two catheterization technologies have effectively overcome the challenges traditionally faced in intubating male mice. By selecting the most appropriate intubation method tailored to the specific research model, the success rate and stability of the model can be significantly improved, thus ensuring more reliable results for further research.

Lower urinary tract diseases often manifest differently across genders. Despite this, the predominant focus of current research centers on female mice, leaving male mice relatively underexplored. This research trajectory is largely driven by anatomical considerations. Female mice possess a more simplified structure of the lower urinary tract, with their urethra measuring only about 1 cm in length. Consequently, intubation procedures in female mice are comparatively straightforward, facilitating the establishment of diverse disease models. In contrast, male mice present a more intricate lower urinary tract anatomy, characterized by numerous bends and strictures in the urethra [26]. These complexities escalate intubation challenges and hinder the construction of specific disease models. For instance, when modeling in-situ bladder cancer, researchers typically instill cancer cells into the female mice's bladder via urethral intubation. Yet, epidemiological evidence suggests that men have a higher bladder cancer incidence than women [13–15]. Similarly, in UTI studies, researchers predominantly utilize female mice for instilling urinary pathogenic bacteria into the bladder through urethral intubation [6, 7, 10]. Paradoxically, men experience a heightened vulnerability to complicated UTIs [9, 11]. In the study on bladder infection in male mice, Olson et al. resorted to direct surgical bladder exposure, instilling Escherichia coli via catheterization for infection [22]. However, such an approach might amplify the bladder's infection and inflammatory response due to the surgical incision, potentially skewing the experimental outcomes [27]. Addressing the intubation challenges associated with male mice can pave the way for more representative and meaningful studies using male specimens.

While our introduced method does not guarantee a 100% success rate, adhering to the procedures and techniques delineated in this study can notably elevate the intubation success rate for male mice. Emphasizing gentleness throughout the procedure is paramount. If resistance is encountered, operators must exercise caution, refraining from excessive force to prevent urethral injury or inadvertent punctures. In such instances, it is prudent to delicately retract the catheter, consider re-lubrication, or subtly adjust the insertion angle and direction to pinpoint the optimal route into the bladder. Successful catheter insertion then paves the way for subsequent procedures like catheterization and infusion. Leveraging this method, disease models tailored for male mice can be established, enabling the exploration and application of relevant therapeutic strategies.

Acknowledgements

The authors acknowledge funding received from the National Natural Science Foundation of China (82270813); Sichuan Science and Technology Program (2022YFS0617, and 2022YFS0632); Southwest Medical School (2021ZKZD006, and 2021ZKZD004); Neijiang First People's Hospital (2021NJXNYD05).

Author contributions

Xi Duan (Data curation [equal], Methodology [equal], Software [equal], Writing—original draft [equal]), Zhibin Chen (Methodology [equal]), Zhean Zhan (Software [equal]), Langhui Li (Data curation [equal], Methodology [equal]), Xianying Lei (Methodology [equal]), Yang Long (Methodology [equal]), Xie Xiang (Writing—original draft [equal], Writing—review & editing [equal]), and Huan Chen (Conceptualization [equal], Methodology [equal], Writing—original draft [equal], Writing—review & editing [equal]).

Supplementary data

Supplementary data is available at Biology Methods and Protocols online.

Conflict of interest statement. None declared

Data availability

The data underlying this article will be shared on reasonable request to the corresponding author.

Institutional review board statement

All procedures were approved by the SWMU Animal Care and Use Committee (swmu20220301-008).

References

Author notes