This work was supported by a grant from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) to JMB (DK117884).

All procedures carried out in rodents must adhere to the applicable guidelines and be approved by the local institutional ethics review committee. The experiments performed for this manuscript were carried out in accordance with the National Research Council&#39;s Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Pittsburgh School of Medicine. See Figure 1 for a modified version of the standard rodent cystometry setup used in this protocol.
1. Laboratory animals
<ol>
	<li>Maintain the rats in social housing (multiple rodents in one cage) with a 12 h light/dark cycle and ad libitum access to water and food pellets.</li>
</ol>
2. Anesthesia and ganglionic block
<ol>
	<li>Induce initial anesthesia by placing the animal in a closed box gassed with 4%-5% isoflurane in O2 (1 L/min).</li>
	<li>Anesthetize the animal using urethane.
	<ol>
		<li>Inject urethane subcutaneously bilaterally (1/2 dose on each side of the animal) at a dose of 1.2 g/kg. Place the animal in a cage to allow the urethane to take effect, which generally takes 2 h.</li>
		<li>Alternatively, administer urethane intraperitoneally (i.p.) by injecting the full dose in two separate doses ~10 min apart.</li>
	</ol>
	</li>
	<li>After waiting for the appropriate time for the urethane to take effect (s.c.: 2 h, i.p.: 30 min), test for a proper plane of anesthesia by pinching the foot of the animal using forceps. If a reflex is observed, administer an additional dose of urethane (0.05-0.1 mL i.p.), wait for 15 min, and test again.&#160;Continue to monitor the animal for the proper plane of anesthesia throughout the procedure.</li>
	<li>To prevent a contraction of the bladder during distension, inject the animal with a ganglionic blocking agent, such as hexamethonium (20 mg/kg, i.p.).</li>
	<li>Apply ophthalmic ointment to the animal&#39;s eyes to prevent drying during the experiment.</li>
</ol>
3. Surgical procedure-suprapubic bladder catheterization
<ol>
	<li>Shave the abdomen of the animal and perform a midline laparotomy to expose the urinary bladder.</li>
	<li>Prepare a catheter by flaring one end of a short (~10-15 cm) length of PE50 intramedic tubing using a flame. Place a 22 G needle into the other end of the tubing and fill with Krebs solution (see Table of Materials for the composition).</li>
	<li>Place a small loop of 3-0 silk suture over the dome of the bladder and perform a small cystostomy (using fine scissors or an 18 G needle) large enough to insert the flared end of the catheter made above. With one hand holding the catheter in place, use the other hand to tighten the loop of the suture to secure the catheter in place. Finish securing the catheter by tying two knots in the suture and pulling the catheter back until the flared head is in contact with the bladder wall.
	<ol>
		<li>Alternatively, secure the catheter using a classic purse-string suture technique, as previously described for cystometry12. 
		NOTE: It is imperative not to introduce air bubbles into the bladder during this procedure.</li>
	</ol>
	</li>
	<li>Test the setup for leaks by infusing a small amount of Krebs solution through the catheter. If the fluid leaks out of the cystostomy, resecure the catheter with additional suture around the cystostomy.</li>
</ol>
4. Transurethral catheterization
<ol>
	<li>Dip the end of a 20 G x 1&#34; I.V. catheter (with the needle removed) in surgical lubricant.</li>
	<li>Hold the external urethral meatus gently with a pair of forceps and insert the tip of the catheter into the urethral orifice in the direction of the tail until the tip causes the wall of the adjacent vaginal opening to deform. Rotate the catheter 90&#176; (bringing the Luer-Lock end of the catheter toward the tail) and gently advance. Fully insert the catheter until the Luer-Lock hub is approximately 5 mm distal from the external urethral opening. 
	NOTE: Do not insert the catheter too far, which may cause the tip to poke the inside wall of the bladder. If resistance is felt while advancing the catheter, stop and begin again or risk puncturing the urethra. See the discussion section on tips to increase successful catheterization.</li>
	<li>Secure the catheter and prevent leakage around the catheter by looping a short length of 3-0 silk suture around the external urethral meatus and tie it off tightly. Secure the catheter to the tail with tape to prevent it from accidentally being pulled out.</li>
	<li>Once catheterized, gently infuse Krebs solution into the bladder through the suprapubic bladder catheter and confirm that the fluid flows out of the urethral catheter and not around it. If necessary, retie the suture around the external urethral orifice.</li>
	<li>Close the abdominal incision over the bladder using a 3-0 silk suture.</li>
</ol>
5. Experimental setup
<ol>
	<li>Secure the animal on a board capable of being inclined to aid in the draining of intravesical fluid through the urethral catheter. Place a heating pad and absorbent underpad between the animal and the board to maintain body heat and absorb fluid draining from the urethral catheter.</li>
	<li>Connect the suprapubic catheter to a three-way stopcock, which connects the catheter to a syringe pump and a pressure transducer. Connect the pressure transducer to a computer by way of an amplifier and a data acquisition system. 
	NOTE: Care should be taken to prevent air bubbles from forming in the tubing connecting the syringe pump, transducer, and bladder catheter.</li>
	<li>Calibrate the bladder pressure recording using the procedure suggested by the manufacturer of the pressure transducer and/or data acquisition software.</li>
	<li>Infuse Krebs solution through the suprapubic catheter at a rate of 0.1 mL/min and allow the fluid to drain from the urethral catheter for 1 h to wash out any residual ATP released during the catheter implantations.</li>
	<li>After this washout period, cap the urethral catheter using a Luer-Lock plug and measure the pressure in the bladder. Look for a slow rise in the intravesical pressure to a pressure of 30 cm H2O without a sharp increase in pressure, which would indicate a bladder contraction (see Figure 2). Remove the plug from the urethral catheter when the pressure reaches 30 cm H2O to prevent damage to the bladder. 
	&#8203;NOTE: If bladder contractions continue to occur after 1 h, give an additional dose of hexamethonium (5 mg/kg dose i.p.).</li>
</ol>
6. Collection of samples
<ol>
	<li>Infuse the bladder at 0.1 mL/min and collect the eluate from the urethral catheter. Test 100 &#181;L aliquots of the eluate immediately for ATP (see below) or freeze for later batch quantification.</li>
	<li>To test the effect of bladder distension on luminal ATP concentrations, cap the urethral catheter with the plug and monitor bladder pressure until it reaches the desired level. Then, uncap the urethral catheter and collect the eluate for ATP measurement or freezing, as described above.</li>
	<li>After each distension, allow the bladder to rest and wash out for 10-15 min before taking additional samples. Take a total of 3-5 predistension samples and 3-5 samples at each desired distention pressure to demonstrate repeatability.</li>
	<li>To test the effect of drugs on the release of ATP, switch the Krebs solution infusing the bladder to Krebs containing the drug of choice. Perfuse at 0.1 mL/min for 10-15 min for the drug to have an effect, and then collect the samples from non-distended and distended bladders as described in steps 6.1 and 6.2.</li>
</ol>
7. Quantifying ATP from collected samples
<ol>
	<li>Quantify ATP in the collected 100 &#181;L samples using a commercially available luciferin/luciferase assay kit following the manufacturer&#39;s instructions and a luminometer.
	<ol>
		<li>To quantify ATP, combine 100 &#181;L samples of perfusate with 50 &#181;L of the assay mix and place them in the luminometer for reading. To convert the Relative Light Units (RLUs) reported by the luminometer to a concentration of ATP, make serial dilutions of ATP in Krebs solution ranging from 1 &#181;M to 10 pM in 10-fold dilutions to create a standard curve and read them in the luminometer. Plot the resulting readings on a graph and perform a non-linear (quadratic) regression to extrapolate concentrations from the samples taken from the animal. 
		&#8203;NOTE: It is important to make ATP standards for any drug solution tested in the experiment, as many drugs interfere with the luciferin/luciferase reaction, which must be corrected for.</li>
	</ol>
	</li>
</ol>
8. Euthanasia of animals
<ol>
	<li>When the experiment is completed and all the samples are collected, humanely euthanize the animal according to USDA guidelines and the National Research Council&#39;s Guide for the Care and Use of Laboratory Animals.
	<ol>
		<li>Remove the anesthetized animal from the experimental setup, place it in a closed box, and gas it with 100% CO2. Ensure that the fill rate is equal to 30%-70% of the chamber volume per minute (e.g., 3-7 L/min for a box with a 10 L volume). Continue the CO2 flow for at least 1 min after respiration ceases.</li>
	</ol>
	</li>
	<li>Use a secondary form of euthanasia to ensure death.
	<ol>
		<li>Perform a thoracotomy as a secondary form of euthanasia by grasping the small flap of skin at the caudal end of the sternum and cutting a small hole in the skin and musculature at the diaphragm with a sharp pair of scissors. Complete the thoracotomy by inserting the scissors into the opening and cutting rostrally through the rib cage and exposing the thoracic cavity. 
		NOTE: The euthanized animal should be disposed of&#160;according to institutional guidelines.</li>
	</ol>
	</li>
</ol>

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	<li>Burnstock, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purinergic+signalling+in+the+urinary+tract+in+health+and+disease.">Purinergic signalling in the urinary tract in health and disease.</a> Purinergic Signalling. 10 (1), 103-155 (2014).</li><li>Birder, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urothelial+signaling.">Urothelial signaling.</a> Autonomic Neuroscience: Basic &amp; Clinical. 153 (1-2), 33-40 (2010).</li><li>Silva-Ramos, 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=Urinary+ATP+may+be+a+dynamic+biomarker+of+detrusor+overactivity+in+women+with+overactive+bladder+syndrome.">Urinary ATP may be a dynamic biomarker of detrusor overactivity in women with overactive bladder syndrome.</a> PLoS One. 8 (5), 64696 (2013).</li><li>Sun, Y., Chai, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Augmented+extracellular+ATP+signaling+in+bladder+urothelial+cells+from+patients+with+interstitial+cystitis.">Augmented extracellular ATP signaling in bladder urothelial cells from patients with interstitial cystitis.</a> American Journal of Physiology - Cell Physiology. 290 (1), 27-34 (2006).</li><li>Gill, K., 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=Urinary+ATP+as+an+indicator+of+infection+and+inflammation+of+the+urinary+tract+in+patients+with+lower+urinary+tract+symptoms.">Urinary ATP as an indicator of infection and inflammation of the urinary tract in patients with lower urinary tract symptoms.</a> BMC Urology. 15 (1), 7 (2015).</li><li>S&#228;ve, S., Persson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+ATP+and+P2Y+receptor+activation+induce+a+proinflammatory+host+response+in+the+human+urinary+tract.">Extracellular ATP and P2Y receptor activation induce a proinflammatory host response in the human urinary tract.</a> Infection and Immunity. 78 (8), 3609-3615 (2010).</li><li>Munoz, A., Smith, C. P., Boone, T. B., Somogyi, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overactive+and+underactive+bladder+dysfunction+is+reflected+by+alterations+in+urothelial+ATP+and+NO+release.">Overactive and underactive bladder dysfunction is reflected by alterations in urothelial ATP and NO release.</a> Neurochemistry International. 58 (3), 295-300 (2011).</li><li>Beckel, J. 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=Pannexin+1+channels+mediate+the+release+of+ATP+into+the+lumen+of+the+rat+urinary+bladder.">Pannexin 1 channels mediate the release of ATP into the lumen of the rat urinary bladder.</a> The Journal of Physiology. 593 (8), 1857-1871 (2015).</li><li>Zhang, Y. 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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+infection+in+a+small+animal+model:+Transurethral+catheterization+of+male+and+female+mice.">Urinary tract infection in a small animal model: Transurethral catheterization of male and female mice.</a> Journal of Visualized Experiments. (130), e54432 (2017).</li><li>Uvin, P., 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=The+use+of+cystometry+in+small+rodents:+a+study+of+bladder+chemosensation.">The use of cystometry in small rodents: a study of bladder chemosensation.</a> Journal of Visualized Experiments. (66), e3869 (2012).</li><li>Leitao, J. M., Esteves da Silva, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Firefly+luciferase+inhibition.">Firefly luciferase inhibition.</a> Journal of Photochemistry and Photobiology B. 101 (1), 1-8 (2010).</li><li>Auld, D. S., Thorne, N., Nguyen, D. T., Inglese, 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+specific+mechanism+for+nonspecific+activation+in+reporter-gene+assays.">A specific mechanism for nonspecific activation in reporter-gene assays.</a> ACS Chemical Biology. 3 (8), 463-470 (2008).</li><li>Harvey, 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=Studies+on+the+oxidation+of+luciferin+without+luciferase+and+the+mechanism+of+bioluminescence.">Studies on the oxidation of luciferin without luciferase and the mechanism of bioluminescence.</a> Journal of Biological Chemistry. 78 (2), 369-375 (1928).</li><li>Wang, J., Jackson, D. G., Dahl, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+food+dye+FD&amp;C+Blue+No.+1+is+a+selective+inhibitor+of+the+ATP+release+channel+Panx1.">The food dye FD&amp;C Blue No. 1 is a selective inhibitor of the ATP release channel Panx1.</a> The Journal of General Physiology. 141 (5), 649-656 (2013).</li><li>Avazpour, M., Shiri, S., Delpisheh, A., Abbasi, A. 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The authors have no conflicts of interest to disclose.

The majority of research into urothelial ATP release is conducted in cultured cells, using either immortalized cell lines or primary cultures of rodent urothelial cells. While these models have the benefit of being relatively high throughput (i.e., one culture/passage can make many plates/dishes of cells), their physiological relevance is diminished due to: 1) the inability of urothelial cells to grow polarized unless they are grown on special supports and 2) the difficulty in exposing cultured cells to physiological lev...

Urinary ATP is thought to play a significant sensory role in the control of micturition1. For example, it is thought that ATP is released from the urothelium in response to distension where it can act on receptors on bladder afferent nerves to increase their excitability, leading to sensations of fullness2. Thus, it is also thought that urinary ATP could be an important player in the development of bladder pathologies. In support of this hypothesis, urinary ATP concentrations are significantly increased in patients suffering from overactive bladder (OAB)3, bladder pain syndrome/interstitial cystitis (BPS/IC)4, or a urinary tract infection (UTI)5,6, all conditions characterized by increased urgency, frequency and, sometimes, pain. Conversely, patients suffering from underactive bladder (UAB), which is characterized by an inability to empty one's bladder and can sometimes include a decreased ability to sense bladder fullness, have been shown to have decreased urinary ATP concentrations7. Experimentally, manipulation of urinary ATP concentrations can alter bladder reflexes in the rat; increasing ATP concentrations by blocking endogenous ATPases in the bladder lumen can increase voiding frequency, while decreasing ATP concentrations by instilling exogenous ATPases into the bladder reduces voiding frequency8. Thus, the importance of urinary ATP to bladder function is clear.
Given the apparent importance of urinary ATP to bladder pathology, accurate measurement of urothelial ATP release is an important step in understanding the mechanisms that control release. Many studies have been completed using different experimental models to measure urothelial ATP release. Foremost among these are cell cultures, either primary cultures or cell lines. However, the use of cultured urothelial cells is complicated by the fact that urothelial cells do not take on their physiological polarized morphology unless they are grown on special permeable membranes (such as Transwell technology [well inserts])9. Thus, it is difficult to relate any ATP release measured to physiology. Urothelial cells grown on well inserts can polarize and form a barrier akin to what is seen in vivo; however, the growth of a fully differentiated urothelium can take days or weeks. Additionally, while it is possible to mount well inserts into an Ussing chamber and apply pressure to the apical side to cause stretch, it is difficult to apply enough pressure to mimic conditions inside the bladder during pathology (i.e., pressures of 30 cm H2O or above). Whole bladder tissue can also be mounted in an Ussing chamber for stretch experiments, but this removes the bladder from the organism along with the trophic factors maintaining urothelial cell health and, hence, urothelial barrier function. Therefore, the most physiologically relevant way to study the release of ATP from the urothelium in response to stretch or pressure is in vivo. The surgical techniques needed to set up the experiment are identical to those commonly used in animal cystometry and, therefore, should be easily performed by anyone familiar with that technique.
In this protocol, we will describe the technique used to examine luminal ATP in female Sprague Dawley rats weighing approximately 200-250 g, as the transurethral catheterization described below is much easier in females; however, transurethral catheterization can also be performed in male rodents10. As transurethral catheterization has now been performed in mice of both sexes as well11, these experiments can easily be adapted for mice or rats of either sex or of varying sizes, depending on the needs of the research team.

<table><tbody><tr><td>amplifier</td><td>World Precision Instruments (WPI)</td><td>SYS-TBM4M</td><td></td></tr><tr><td>ATP assay kit</td><td>Sigma-Aldrich, Inc.</td><td>FLAA-1KT</td><td></td></tr><tr><td>data acquisition system/ software</td><td>DataQ Instruments</td><td>DI-1100</td><td>Software included, requires Windows-based computer</td></tr><tr><td>Hexamethonium bromide</td><td>Sigma-Aldrich, Inc.</td><td>H0879</td><td>20 mg/kg dose</td></tr><tr><td>Isoflurane</td><td>Covetrus North America</td><td>29404</td><td></td></tr><tr><td>lidocaine</td><td>Covetrus North America</td><td>2468</td><td></td></tr><tr><td>Luer Lock plugs</td><td>Fisher Scientific</td><td>NC0455253</td><td></td></tr><tr><td>luminometer (GloMax 20/20)</td><td>Promega</td><td>E5311</td><td></td></tr><tr><td>Polyethylene (PE50) tubing</td><td>Fisher Scientific</td><td>14-170-12B</td><td></td></tr><tr><td>Pump 33 DDS syringe pump</td><td>Harvard Apparatus</td><td>703333</td><td></td></tr><tr><td>pressure transducers</td><td>World Precision Instruments (WPI)</td><td>BLPR2</td><td></td></tr><tr><td>surgical instruments (scissors, hemostats, forceps, etc.)</td><td>Fine Science Tools</td><td>multiple numbers</td><td></td></tr><tr><td>surgical lubricant</td><td>Fisher Scientific</td><td>10-000-694</td><td></td></tr><tr><td>Sur-Vet I.V. catheter&amp;nbsp;</td><td>Covetrus North America</td><td>50603</td><td>20 G x 1 inch</td></tr><tr><td>tiltable surgical table (Plas Labs)</td><td>Fisher Scientific</td><td>01-288-30A</td><td></td></tr><tr><td>Tubing connectors</td><td>Fisher Scientific</td><td>14-826-19E</td><td>allows Luer-Lock connectors to attach to tubing</td></tr><tr><td>Urethane</td><td>Sigma-Aldrich, Inc.</td><td>U2500</td><td>0.5 g/mL conc., 1.2 g/kg dose</td></tr></tbody></table>

in vivo luminal measurement of distension-evoked urothelial atp release in rodents

ATP, released from the urothelium in response to bladder distension, is thought to play a significant sensory role in the control of micturition. Therefore, accurate measurement of urothelial ATP release in a physiological setting is an important first step in studying the mechanisms that control purinergic signaling in the urinary bladder. Existing techniques to study mechanically evoked urothelial ATP release utilize cultured cells plated on flexible supports or bladder tissue pinned into Ussing chambers; however, each of these techniques does not fully emulate conditions in the intact bladder. Therefore, an experimental setup was developed to directly measure ATP concentrations in the lumen of the rodent urinary bladder.
In this setup, the bladders of anesthetized rodents are perfused through catheters in both the dome of the bladder and via the external urethral orifice. Pressure in the bladder is increased by capping the urethral catheter while perfusing sterile fluid into the bladder through the dome. Measurement of intravesical pressure is achieved using a pressure transducer attached to the bladder dome catheter, akin to the setup used for cystometry. Once the desired pressure is reached, the urethral catheter's cap is removed, and fluid collected for ATP quantification by luciferin-luciferase assay. Through this experimental setup, the mechanisms controlling both mechanical and chemical stimulation of urothelial ATP release can be interrogated by including various agonists or antagonists into the perfusate or by comparing results between wildtype and genetically modified animals.

The described protocol allows for the accurate measurement of urothelial ATP release in vivo from the lumen of the bladder, using a modified version of the standard rodent cystometry setup (see Figure 1). This allows the researcher to examine the effects of drugs on stretch-mediated ATP release in a physiological setting.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64227/64227fig01.jpg" /> 
<s...

Watch this Scientific Journal Video about In Vivo Luminal Measurement of Distension-Evoked Urothelial ATP Release in Rodents at JoVE.com

In Vivo Luminal Measurement of Distension-Evoked Urothelial ATP Release in Rodents

This protocol describes the procedure for measuring ATP concentrations in the lumen of the bladder in an anesthetized rodent.

In Vivo Luminal Measurement of Distension-Evoked Urothelial ATP Release in Rodents

ATP, released from the urothelium in response to bladder distension, is thought to play a significant sensory role in the control of micturition. ...

in-vivo-luminal-measurement-distension-evoked-urothelial-atp-release

Research

JoVE Journal

Medicine

1.5K Views.  University of Pittsburgh School of Medicine. This protocol describes the procedure for measuring ATP concentrations in the lumen of the bladder in an anesthetized rodent.

Video: In Vivo Luminal Measurement of Distension-Evoked Urothelial ATP Release in Rodents

Evaluation of Biomaterials for Bladder Augmentation using Cystometric Analyses in Various Rodent Models

Renal function and continence of urine are critically dependent on the proper function of the urinary bladder, which stores urine at low pressure and expels it with a precisely orchestrated contraction. A number of congenital and acquired urological anomalies including posterior urethral valves, benign prostatic hyperplasia, and neurogenic bladder secondary to spina bifida/spinal cord injury can result in pathologic tissue remodeling leading to impaired compliance and reduced capacity1. Functional or anatomical obstruction of the urinary tract is frequently associated with these conditions, and can lead to urinary incontinence and kidney damage from increased storage and voiding pressures2. Surgical implantation of gastrointestinal segments to expand organ capacity and reduce intravesical pressures represents the primary surgical treatment option for these disorders when medical management fails3. However, this approach is hampered by the limitation of available donor tissue, and is associated with significant complications including chronic urinary tract infection, metabolic perturbation, urinary stone formation, and secondary malignancy4,5.
Current research in bladder tissue engineering is heavily focused on identifying biomaterial configurations which can support regeneration of tissues at defect sites. Conventional 3-D scaffolds derived from natural and synthetic polymers such as small intestinal submucosa and poly-glycolic acid have shown some short-term success in supporting urothelial and smooth muscle regeneration as well as facilitating increased organ storage capacity in both animal models and in the clinic6,7. However, deficiencies in scaffold mechanical integrity and biocompatibility often result in deleterious fibrosis8, graft contracture9, and calcification10, thus increasing the risk of implant failure and need for secondary surgical procedures. In addition, restoration of normal voiding characteristics utilizing standard biomaterial constructs for augmentation cystoplasty has yet to be achieved, and therefore research and development of novel matrices which can fulfill this role is needed.
In order to successfully develop and evaluate optimal biomaterials for clinical bladder augmentation, efficacy research must first be performed in standardized animal models using detailed surgical methods and functional outcome assessments. We have previously reported the use of a bladder augmentation model in mice to determine the potential of silk fibroin-based scaffolds to mediate tissue regeneration and functional voiding characteristics.11,12 Cystometric analyses of this model have shown that variations in structural and mechanical implant properties can influence the resulting urodynamic features of the tissue engineered bladders11,12. Positive correlations between the degree of matrix-mediated tissue regeneration determined histologically and functional compliance and capacity evaluated by cystometry were demonstrated in this model11,12. These results therefore suggest that functional evaluations of biomaterial configurations in rodent bladder augmentation systems may be a useful format for assessing scaffold properties and establishing in vivo feasibility prior to large animal studies and clinical deployment. In the current study, we will present various surgical stages of bladder augmentation in both mice and rats using silk scaffolds and demonstrate techniques for awake and anesthetized cystometry.

Surgical stages of bladder augmentation are described using 3-D scaffolds in murine and rat models. To test the efficacy of biomaterial configurations for use in bladder augmentation, techniques for both awake and anesthetized cystometry are presented.

Renal function and continence of urine are critically dependent on the proper function of the urinary bladder, which stores urine at low pressure and ...

Bioengineering

The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation

The lower urinary tract (LUT) functions as a dynamic reservoir that is able to store urine and to efficiently expel it at a convenient time. While storing urine, however, the bladder is exposed for prolonged periods to waste products. By acting as a tight barrier, the epithelial lining of the LUT, the urothelium, avoids re-absorption of harmful substances. Moreover, noxious chemicals stimulate the bladder's nociceptive innervation and initiate voiding contractions that expel the bladder's contents. Interestingly, the bladder's sensitivity to noxious chemicals has been used successfully in clinical practice, by intravesically infusing the TRPV1 agonist capsaicin to treat neurogenic bladder overactivity1. This underscores the advantage of viewing the bladder as a chemosensory organ and prompts for further clinical research. However, ethical issues severely limit the possibilities to perform, in human subjects, the invasive measurements that are necessary to unravel the molecular bases of LUT clinical pharmacology. A way to overcome this limitation is the use of several animal models2. Here we describe the implementation of cystometry in mice and rats, a technique that allows measuring the intravesical pressure in conditions of controlled bladder perfusion.
	After laparotomy, a catheter is implanted in the bladder dome and tunneled subcutaneously to the interscapular region. Then the bladder can be filled at a controlled rate, while the urethra is left free for micturition. During the repetitive cycles of filling and voiding, intravesical pressure can be measured via the implanted catheter. As such, the pressure changes can be quantified and analyzed. Moreover, simultaneous measurement of the voided volume allows distinguishing voiding contractions from non-voiding contractions3.
	Importantly, due to the differences in micturition control between rodents and humans, cystometric measurements in these animals have only limited translational value4. Nevertheless, they are quite instrumental in the study of bladder pathophysiology and pharmacology in experimental pre-clinical settings. Recent research using this technique has revealed the key role of novel molecular players in the mechano- and chemo-sensory properties of the bladder.

Cystometry is an efficient technique to measure bladder function of small animals in vivo. The bladder is continuously infused at rates controlled through an intravesical catheter, whereas the urethra is left free for micturition. This allows for repetitive filling and emptying of the bladder, while intravesical pressure and voided volume are recorded.

	The lower urinary tract (LUT) functions as a  dynamic reservoir that is able to store urine and to efficiently expel it at a  convenient time. While ...

Urinary Bladder Distention Evoked Visceromotor Responses as a Model for Bladder Pain in Mice

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized by increased urgency and frequency of urination, as well as nocturia and general pelvic pain, especially upon bladder filling or voiding. Despite years of research, the cause of IC/BPS remains elusive and treatment strategies are unable to provide complete relief to patients. In order to study nervous system contributions to the condition, many animal models have been developed to mimic the pain and symptoms associated with IC/BPS. One such murine model is urinary bladder distension (UBD). In this model, compressed air of a specific pressure is delivered to the bladder of a lightly anesthetized animal over a set period of time. Throughout the procedure, wires in the superior oblique abdominal muscles record electrical activity from the muscle. This activity is known as the visceromotor response (VMR) and is a reliable and reproducible measure of nociception. Here, we describe the steps necessary to perform this technique in mice including surgical manipulations, physiological recording, and data analysis. With the use of this model, the coordination between primary sensory neurons, spinal cord secondary afferents, and higher central nervous system areas involved in bladder pain can be unraveled. This basic science knowledge can then be clinically translated to treat patients suffering from IC/BPS.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized in part by pelvic pain. In order to study nervous system contributions to the condition, a physiological model of urinary bladder pain is used in mice and rats.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition ...

Real-Time Void Spot Assay

Normal voiding behavior is the result of the coordinated function of the bladder, the urethra, and the urethral sphincters under the proper control of the nervous system. To study voluntary voiding behavior in mouse models, researchers have developed the void spot assay (VSA), a method that measures the number and area of urine spots deposited on a filter paper lining the floor of an animal&#8217;s cage. Although technically simple and inexpensive, this assay has limitations when used as an end-point assay, including a lack of temporal resolution of voiding events and difficulties quantifying overlapping urine spots. To overcome these limitations, we developed a video-monitored VSA, which we call real-time VSA (RT-VSA), and which allows us to determine voiding frequency, assess voided volume and voiding patterns, and make measurements over 6 h time windows during both the dark and light phases of the day. The method described in this report can be applied to a wide variety of mouse-based studies that explore the physiological and neurobehavioral aspects of voluntary micturition in health and disease states.

This article describes a new method to study mouse voiding behavior by incorporating video monitoring in the conventional void spot assay. This approach provides temporal, spatial, and volumetric information on the voiding events and details of mouse behavior during the light and dark phases of the day.

Normal voiding behavior is the result of the coordinated function of the bladder, the urethra, and the urethral sphincters under the proper control of the ...

Behavior

Evaluating the Procedure for Performing Awake Cystometry in a Mouse Model

Awake filling cystometry has been used for a long time to evaluate bladder function in freely moving mice, however, the specific methods used, vary among laboratories. The goal of this study was to describe the microsurgical procedure used to implant an intravesical tube and the experimental technique for recording urinary bladder pressure in an awake, freely moving mouse. In addition, experimental data is presented to show how surgery, as well as tubing type and size, affect lower urinary tract function and recording sensitivity. The effect of tube diameter on pressure recording was assessed in both polyethylene and polyurethane tubing with different internal diameters. Subsequently, the best performing tube from both materials was surgically implanted into the dome of the urinary bladder of male C57BL/6 mice. Twelve-hour, overnight micturition frequency was recorded in healthy, intact animals and animals 2, 3, 5, and 7 days post-surgery. At harvest, bladders were assessed for signs of swelling using gross observation and were subsequently processed for pathological analysis. The greatest extent of bladder swelling was observed on day 2 and 3, which correlated with behavioral voiding data showing significantly impaired bladder function. By day 5, bladder histology and voiding frequency had normalized. Based on the literature and evidence provided by our studies, we propose the following steps for in vivo recording of intravesical pressure and voided volume in an awake mouse: 1) Perform the surgery using an operating microscope and microsurgical tools, 2) Use polyethylene-10 tubing to minimize movement artifacts, and 3) Perform cystometry on post-operative day 5, when bladder swelling resolves.

This study describes the surgical procedures and experimental techniques for performing awake cystometry in a freely moving mouse. In addition, it provides experimental evidence to support its optimization and standardization.

Awake filling cystometry has been used for a long time to evaluate bladder function in freely moving mice, however, the specific methods used, vary among ...

Longitudinal Follow-Up of Urinary Tract Infections and Their Treatment in Mice using Bioluminescence Imaging

Urinary tract infections (UTI) rank among the most common bacterial infections in humans and are routinely treated with empirical antibiotics. However, due to increasing microbial resistance, the efficacy of the most used antibiotics has declined. To find alternative treatment options, there is a great need for a better understanding of the UTI pathogenesis and the mechanisms that determine UTI susceptibility. In order to investigate this in an animal model, a reproducible, non-invasive assay to study the course of UTI is indispensable.
For years, the gold standard for the enumeration of bacterial load has been the determination of Colony Forming Units (CFU) for a particular sample volume. This technique requires post-mortem organ homogenates and serial dilutions, limiting data output and reproducibility. As an alternative, bioluminescence imaging (BLI) is gaining popularity to determine the bacterial load. Labeling pathogens with a lux operon allow for the sensitive detection and quantification in a non-invasive manner, thereby enabling longitudinal follow-up. So far, the adoption of BLI in UTI research remains limited.
This manuscript describes the practical implementation of BLI in a mouse urinary tract infection model. Here, a step-by-step guide for culturing bacteria, intravesical instillation and imaging is provided. The in vivo correlation with CFU is examined and a proof-of-concept is provided by comparing the bacterial load of untreated infected animals with antibiotic-treated animals. Furthermore, the advantages, limitations, and considerations specific to the implementation of BLI in an in vivo UTI model are discussed. The implementation of BLI in the UTI research field will greatly facilitate research on the pathogenesis of UTI and the discovery of new ways to prevent and treat UTI.

This manuscript describes the intravesical administration of uropathogenic bacteria with a lux operon to induce a urinary tract infection in mice and subsequent longitudinal in vivo analysis of the bacterial load using bioluminescence imaging.

Urinary tract infections (UTI) rank among the most common bacterial infections in humans and are routinely treated with empirical antibiotics. However, ...

Immunology and Infection

Ultrasonography of the Adult Male Urinary Tract for Urinary Functional Testing

The incidence of clinical benign prostatic hyperplasia (BPH) and lower urinary tract symptoms (LUTS) is increasing due to the aging population, resulting in a significant economic and quality of life burden. Transgenic and other mouse models have been developed to recreate various aspects of this multifactorial disease; however, methods to accurately quantitate urinary dysfunction and the effectiveness of new therapeutic options are lacking. Here, we describe a method that can be used to measure bladder volume and detrusor wall thickness, urinary velocity, void volume and void duration, and urethral diameter. This would allow for the evaluation of disease progression and treatment efficacy over time. Mice were anesthetized with isoflurane, and the bladder was visualized by ultrasound. For non-contrast imaging, a 3D image was taken of the bladder to calculate volume and evaluate shape; the bladder wall thickness was measured from this image. For contrast-enhanced imaging, a catheter was placed through the dome of the bladder using a 27-gauge needle connected to a syringe by PE50 tubing. A bolus of 0.5 mL of contrast was infused into the bladder until a urination event occurred. Urethral diameter was determined at the point of the Doppler velocity sample window during the first voiding event. Velocity was measured for each subsequent event yielding a flow rate. In conclusion, high frequency ultrasound proved to be an effective method for assessing bladder and urethral measurements during urinary function in mice. This technique may be useful in the assessment of novel therapies for BPH/LUTS in an experimental setting.

We describe the use of high frequency ultrasound with contrast imaging as a method to measure bladder volume, bladder wall thickness, urine velocity, void volume, void duration, and urethral diameter. This strategy can be used to assess voiding dysfunction and treatment efficacy in various mouse models of lower urinary tract dysfunction (LUTD).

The incidence of clinical benign prostatic hyperplasia (BPH) and lower urinary tract symptoms (LUTS) is increasing due to the aging population, resulting ...

A Decentralized (Ex Vivo) Murine Bladder Model with the Detrusor Muscle Removed for Direct Access to the Suburothelium during Bladder Filling

Previous studies have established the release of chemical substances from flat bladder mucosa sheets affixed in Ussing chambers and exposed to changes in hydrostatic pressure or mechanical stretch and from cultured urothelial cells upon hydrostatic pressure changes, stretch, cell swelling, or drag forces, and in bladder lumen at end of filling. Such findings led to the assumption that these mediators are also released in suburothelium (SubU)/lamina propria (LP) during bladder filling, where they affect cells deep in the bladder wall to ultimately regulate bladder excitability. There are at least two obvious limitations in such studies: 1) none of these approaches provide direct information about the presence of mediators in SubU/LP, and 2) the stimuli used are not physiological and do not recapitulate authentic filling of the bladder. Here, we discuss a procedure that enables direct access to the suburothelial surface of the bladder mucosa in the course of bladder filling. The murine detrusor-free preparation we created closely resembles filling of the intact bladder and allows pressure-volume studies to be performed on the bladder in the absence of confounding signaling from spinal reflexes and detrusor smooth muscle. Using the novel detrusor-free bladder model, we recently demonstrated that intravesical measurements of mediators cannot be used as a proxy to what has been released or present in the SubU/LP during bladder filling. The model enables examination of urothelium-derived signaling molecules that are released, generated by metabolism and/or transported into the SubU/LP during the course of bladder filling to transmit information to neurons and smooth muscle of the bladder and regulate its excitability during continence and micturition.

A Decentralized Ex Vivo Murine Bladder Model with the Detrusor Muscle Removed for Direct Access to the Suburothelium during Bladder Filling

The detrusor-free bladder model enables direct access to the suburothelium to study local mechanisms for regulation of biologically active mediator availability in suburothelium/lamina propria during storage and voiding of urine. The preparation closely resembles filling of an intact bladder and allows pressure-volume studies to be performed without systemic influences.

Previous studies have established the release of chemical substances from flat bladder mucosa sheets affixed in Ussing chambers and exposed to changes in ...

Ex Vivo Analysis of Mechanically Activated Ca2+ Transients in Urothelial Cells

Mechanically activated ion channels are biological transducers that convert mechanical stimuli such as stretch or shear forces into electrical and biochemical signals. In mammals, mechanically activated channels are essential for the detection of external and internal stimuli in processes as diverse as touch sensation, hearing, red blood cell volume regulation, basal blood pressure regulation, and the sensation of urinary bladder fullness. While the function of mechanically activated ion channels has been extensively studied in the in vitro setting using the patch-clamp technique, assessing their function in their native environment remains a difficult task, often because of limited access to the sites of expression of these channels (e.g., afferent terminals, Merkel cells, baroreceptors, and kidney tubules) or difficulties applying the patch-clamp technique (e.g., the apical surfaces of urothelial umbrella cells). This protocol describes a procedure to assess mechanically evoked Ca2+ transients using the fluorescent sensor GCaMP5G in an ex vivo urothelial preparation, a technique that could be readily adapted for the study of mechanically evoked Ca2+ events in other native tissue preparations.

Ex Vivo Analysis of Mechanically Activated Ca2+ Transients in Urothelial Cells

This protocol describes a methodology to assess the function of mechanically activated ion channels in native urothelial cells using the fluorescent Ca2+ sensor GCaMP5G.

Mechanically activated ion channels are biological transducers that convert mechanical stimuli such as stretch or shear forces into electrical and ...

Cystometric and External Urethral Sphincter Measurements in Awake Rats with Implanted Catheter and Electrodes Allowing for Repeated Measurements

Lower urinary tract function is mainly assessed by means of cystometric bladder function analysis in rodents. Conventional cystometries are usually performed as terminal analysis under urethane anesthesia. It is well known that anesthetic drugs can influence bladder function. Hence, the aim of this technique is to perform cystometric measurements of the urinary bladder and external urethral sphincter in lightly restrained awake rats. For this purpose, a bladder catheter is implanted into the bladder dome. Subsequently, two electrodes are implanted bilateral to the external urethral sphincter and a ground electrode is sutured to a non-responsive skeletal muscle. The bladder catheter and the three electrodes are finally tunneled subcutaneously to the neck region and affixed to a harness. With this technique, the lower urinary tract can be measured at multiple time points in the same animal to assess lower urinary tract function. The main application of this technique is the follow-up of simultaneous urinary bladder and external urethral sphincter function in awake healthy rats and after induction of a disease or injury. Moreover, subsequent lower urinary tract monitoring can be performed during evaluation of the disease/injury and to monitor treatment efficacy.

This protocol first describes the surgical procedure of the permanent implantation of a urinary bladder catheter combined with external urethral sphincter electrodes, and second, the measurement of the function of the urinary bladder and external urethral sphincter in implanted awake animals.

Lower urinary tract function is mainly assessed by means of cystometric bladder function analysis in rodents. Conventional cystometries are usually ...

In Vivo Luminal Measurement of Distension-Evoked Urothelial ATP Release in Rodents

Summary

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Chapters in this video

Evaluation of Biomaterials for Bladder Augmentation using Cystometric Analyses in Various Rodent Models

The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation

Urinary Bladder Distention Evoked Visceromotor Responses as a Model for Bladder Pain in Mice

Real-Time Void Spot Assay

Evaluating the Procedure for Performing Awake Cystometry in a Mouse Model

Longitudinal Follow-Up of Urinary Tract Infections and Their Treatment in Mice using Bioluminescence Imaging

Ultrasonography of the Adult Male Urinary Tract for Urinary Functional Testing

A Decentralized (Ex Vivo) Murine Bladder Model with the Detrusor Muscle Removed for Direct Access to the Suburothelium during Bladder Filling

Ex Vivo Analysis of Mechanically Activated Ca²⁺ Transients in Urothelial Cells

Cystometric and External Urethral Sphincter Measurements in Awake Rats with Implanted Catheter and Electrodes Allowing for Repeated Measurements