This protocol provides a guide for waterproofing the skin with cyanoacrylate to prevent urine absorption by fur and skin. It includes instructions for applying the glue to the skin, implanting a bladder catheter, and electrodes for cystometry and external urethral sphincter electromyography recordings in awake mice.
Accurate measurement of urinary parameters in awake mice is crucial for understanding lower urinary tract (LUT) dysfunction, particularly in conditions like neurogenic bladder post-traumatic spinal cord injury (SCI). However, conducting cystometry recordings in mice presents notable challenges. When mice are in a prone and restricted position during recording sessions, urine tends to be absorbed by the fur and skin, leading to an underestimation of voided volume (VV). The goal of this study was to enhance the accuracy of cystometry and external urethral sphincter electromyography (EUS-EMG) recordings in awake mice. We developed a unique method utilizing cyanoacrylate adhesive to create a waterproof skin barrier around the urethral meatus and abdomen, preventing urine absorption and ensuring precise measurements. Results show that after applying the cyanoacrylate, the sum of VV and RV remained consistent with the infused saline volume, and there were no wet areas observed post-experiment, indicating successful prevention of urine absorption. Additionally, the method simultaneously stabilized the electrodes connected with the external urethral sphincter (EUS), ensured stable electromyography (EMG) signals, and minimized artifacts caused by the movement of the awakened mouse and manipulation of the experimenter. Methodological details, results, and implications are discussed, highlighting the importance of improving urodynamic techniques in preclinical research.
The storage and release of urine are dependent on the coordinated activity of the urinary bladder and external urethral sphincter (EUS). In some pathologies such as neurogenic bladder, both the bladder detrusor muscles and the sphincter can become dysfunctional, leading to significant bladder problems, especially after traumatic spinal cord injury(SCI)1.
Small rodents are commonly used as an experimental model to study the preclinical function of the lower urinary tract (LUT)2. Filling Cystometry (FC) and EUS electromyography (EUS-EMG) recording techniques can provide precise objective information depending on the choice of methods, accurate measurement, and interpretation of results3. Urodynamic tests are commonly used to evaluate the voiding volume (VV), voiding efficiency (VE), and bladder capacity4. VE measures how effectively the bladder can empty itself. It is calculated by dividing the voided volume by the sum of voided and residual volumes (VV+RV). On the other hand, bladder capacity is calculated by adding the VV (the amount of urine expelled during urination) to the RV (the amount of urine left in the bladder after urination)5. Therefore, the measurement of VV and RV are the keys to deducing other parameters.
Precisely measuring VV in mice during urodynamic tests presents various challenges. The urine of rodents, when physically restrained in a prone position, tends to be drawn downwards through the ventral abdominal wall due to the influence of gravity6. This phenomenon can lead to the absorption of urine by the abdominal fur and skin, which, in turn, underestimates the volume of urine excreted. Considering the small amount of urine produced by mouse, the impact of this absorbance on the accuracy of results is even more pronounced7. Furthermore, in models of SCI, VV is often lower than in normal mice due to the impact of the detrusor sphincter dyssynergia (DSD), which increases the risk of leak point pressures and urine absorption by the fur8. These factors have a significant impact on the results. Therefore, accurate measurement of VV and RV during terminal urodynamic studies in mice is crucial9. Currently, there is a lack of details in the methodologies provided in published literature about how to measure urine volume accurately in mouse models.
Cyanoacrylate adhesive is a type of glue that is commonly used in surgical procedures in human and animal models due to its quick and effective bonding properties10,11,12. This adhesive is particularly useful for closing wounds and lacerations, as it forms a strong and flexible bond when applied to the skin13. Moreover, it can be a great barrier against urine and wetness that may come into contact with fur and wounds11.
In this article, we have developed a novel and cost-effective technique that utilizes cyanoacrylate adhesive to achieve precise results in cystometry and EUS-EMG recordings in awake mice. This method will be beneficial in understanding the underlying causes of bladder dysfunction and devising more effective treatments for LUT disorders.
The animal study protocol was approved by the Institutional Animal Care and Use Committee of Indiana University School of Medicine. Approval Code: 21098MD/R/MSS/HZ Approval Date: 29 September 2021.
1. Preparation of catheter
2. Preparation of electrodes
3. Preparation of animal
4. Anesthesia during surgery
5. Surgical preparation
6. Surgical procedure
7. Cystometry and EUS-EMG recording preparation
Cystometry and EUS-EMG activity tracings were used to analyze the data. The continuous cystometry method involves the infusion of saline into the bladder and simultaneously measuring the pressure and volume changes in the bladder. To measure VV, 0.4 mL of saline was infused at a speed of 0.01 mL/min, and urine was collected over 40 min in a cap. The post-void residual (PVR) can be obtained by aspirating the saline through the catheter. In normal mice without glue, the sum of VV and RV was often less than 0.4 mL. After the experiment, the fur in the abdomen and surrounding the meatus was wet due to the absorption of urine (Figure 3A). After applying a thin layer of glue to cover small furs, the sum of VV and RV was shown to be 0.4 mL, and there was no wet area (Figure 3B,C).
The resulting cystometry tracings provided a detailed analysis of various parameters, including maximum voiding bladder contraction pressure (27.2 cmH2O), contraction duration (16.26 s), and inter-contraction interval (4.48 min). At the same time, we had a good recording of intravesical pressure and EUS-EMG signals in mice, as shown in Figure 4.
Many mouse urodynamic measurements are performed under anesthesia14. Although this may seem like a convenient method to reduce the noise of electrical signals and loss of urine resulting from the animal's movement, it is essential to consider that the anesthetic drugs can affect the urinary flow, which may lead to inaccurate or unreliable results15. Therefore, urodynamic recording in awake animals is more popular to obtain results closer to the physiological condition. The urodynamic recording in awake animals usually begins after a 40-50 min period of recovery from isoflurane16. This process involves closely monitoring the mice to ensure that they are relaxed and comfortable without the need for anesthesia. It has been observed through several experiments that the movement of a conscious mouse can affect urodynamic signals5,14, leading to inaccurate measurements of specific parameters such as leak point pressure, VV, and VE17. As a result, we have implemented a method by partially restraining conscious mice to ensure more reliable urodynamic results. However, even with limited restraint, the conscious mice still struggle when they wake up immediately from the anesthesia, which can also cause detachment or unstable contact between the electrode hook and the EUS and create a significant noise in the EUS-EMG signals. As shown in Figure 3B, to minimize these artifacts, we have taken the approach of fixing the electrodes with glue at the exit point from the skin. This method has proven to be effective in minimizing the movement of electrodes and the subsequent artifacts that they can produce.
Figure 1: Displacement of the electromyography electrodes. Implantation of electrodes (yellow asterisk) bilaterally to the external urethral muscle (EUS; black arrows). Please click here to view a larger version of this figure.
Figure 2: Restraint of the awake mouse. After implantation of catheter and electrodes, the mouse was restrained on the plate for stability during urodynamic recording. Please click here to view a larger version of this figure.
Figure 3: Abdominal and meatus regions after urodynamic recording. (A) A large wet area (contoured by a red dash line) was seen in the abdomen and genital regions. (B) Dry, waterproof abdominal and genital areas were created with cyanoacrylate glue (contoured by a red dash line) after recording. (C) A urine drop (yellow arrow) formed at the meatus during urodynamic recording and stayed as a drop for a long time without being absorbed by the skin and fur. Please click here to view a larger version of this figure.
Figure 4: Representative traces of cystometry and external urethral sphincter electromyography (EUS-EMG) in an awake and restrained female mouse. (A) Trace A: Simultaneous recordings of continuous cystometrogram (CMG) and EUS-EMG (upper and lower traces, respectively). (B) Trace B is the expanded portion of trace A, indicated by a rectangular box with different time scales. During the voiding phase, intermittent voiding coincided with reductions in intravesical pressure in the CMG recording (top trace; arrows), which occurred during low tonic and reduction periods of EUS-EMG activity (bottom trace; arrows). Please click here to view a larger version of this figure.
This urodynamic technique describes an improved procedure for measuring urine volume and EUS- EMG signal in awake and restrained mice. The presence of fur around the urethral meatus and abdominal area can interfere with the accuracy of the VV measurement by absorbing urine. Although the fur surrounding the urethral meatus and abdomen had been carefully shaved before surgery, the remaining small furs within these areas and the skin still absorbed urine, usually leaving a wet area in the abdomen after recording. This issue is particularly noticeable in female rodents due to the extremely short distance between the urethral meatus and the surrounding skin18. In this technique, cyanoacrylate glue was applied on the abdominal and surrounding urethral skin to create a waterproof skin surface and provide a precise assessment of urinary volume during the urodynamic recording, allowing for a better understanding of bladder function. The glue was applied with precision, ensuring that it covered the skin surrounding the meatus and nearby. The purpose of applying the glue was to create a waterproof barrier that would prevent the furs from absorbing any urine. The glue was spread evenly, with care taken to avoid any clumping or blocking of the urethral meatus. The recorded results of the procedure confirmed that our objective had been completely achieved, as the sum of VV and RV remained constant at infusion volume, and no further wet areas were observed. To ensure the accuracy of measurements, we checked the bladder after the experiment, and it was found to be empty. This additional step of checking the bladder is crucial as it eliminates any possibility of urine retention, causing a discrepancy between the amount we withdrew through a syringe and the actual amount of RV.
This method has limitations: 1) it is unsuitable for longitudinal and multiple-time-point studies. 2) it cannot be applied to a freely moving mouse. 3) if the detachment of the electrodes from EUS occurs, it is difficult to open the abdomen and reinstall them. 4) While cyanoacrylate adhesives are a valuable tool in many surgical settings due to their ease of use and effectiveness, it is important to use them cautiously and follow proper safety protocols to minimize any potential risks. Cyanoacrylate is generally safe for the skin, but frequent contact with it should be avoided, and researchers should take appropriate personal protective measures. Cyanoacrylate adhesives can release toxic vapors if inhaled. To minimize the risk of inhaling these vapors, researchers should maintain higher levels of humidity and optimize room ventilation in the working environment19. Special air conditioning filters can also be used to further reduce the toxicity of the vapors.
Overall, this experiment provided important insights into the accuracy of measuring voided urine during the urodynamic recording and helped to identify potential sources of error that could have led to discrepancies in the total amount of VV and RV after infusion.
The authors have nothing to disclose.
This study was supported by NIH-NINDS (R21NS130241), IND DEPT HLTH (55051, 74247, 74244), and US ARMY (HT94252310700).
Accelerator | BOB SMITH INDUSTRIES | BSI-152 | |
Cyanoacrylate | TED PELLA, Inc | 14478 | |
Disposable base mold | TED PELLA, Inc | 27147-4 | |
Infusion pump | Harvard Apparatus PHD ULTRA | 70-3006 | |
Isoflurane | Henry Schein Inc | 1182097 | |
PIN | World Precision Instruments | 5482 | |
Polyethylene Tubing 30 | Braintree Scientific Inc | PE30 | |
Sterile Weighing Boat | HEATHROW SCIENTIFIC | 797CK2 | |
Windaq/Lite | DATAQ INSTRUMENTS | 249022 |
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