Name: in vivo luminal measurement of distension-evoked urothelial atp release in rodents
Uploaded: 2022-09-07T13:20:00
Description: Watch this Scientific Journal Video about In Vivo Luminal Measurement of Distension-Evoked Urothelial ATP Release in Rodents at JoVE.com

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...

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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</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" /> 
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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. ...

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