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>

<ol>
	<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. Y., Ludwikowski, B., Hurst, R., Frey, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+and+long-term+culture+of+differentiated+normal+rat+urothelial+cells+in+vitro.">Expansion and long-term culture of differentiated normal rat urothelial cells in vitro.</a> In Vitro Cellular &amp; Developmental Biology. Animal. 37 (7), 419-429 (2001).</li><li>Lee, S., Carrasco, A., Meacham, R. B., Malykhina, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transurethral+instillation+procedure+in+adult+male+mouse.">Transurethral instillation procedure in adult male mouse.</a> Journal of Visualized Experiments. (129), e56663 (2017).</li><li>Zychlinsky Scharff, A., Albert, M. L., Ingersoll, M. 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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+determination+of+brilliant+blue+FCF+and+carmoisine+in+food+samples+by+aqueous+two-phase+system+and+spectrophometric+detection.">Simultaneous determination of brilliant blue FCF and carmoisine in food samples by aqueous two-phase system and spectrophometric detection.</a> Journal of Basic Research in Medical Sciences. 1 (1), 56-65 (2014).</li></ol>

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 levels of stretch/pressure. One way to deal with these limitations is by removing the bladder from a rodent, cutting it open, and placing it in an Ussing chamber, inserting the bladder tissue between two half chambers filled with physiological solution. This allows the researcher to subject the urothelium, in its native polarized state, to distension by adding volume to the urothelial side of the chamber, causing stretch of the bladder tissue in the chamber. However, it is difficult to relate this stretch to the distension seen by the urothelium in vivo. Moreover, distension-evoked ATP in the Ussing chamber is released into the large volume of physiological fluid contained in the chamber, diluting the concentration of ATP several-fold. Using this procedure, bladder pressure is measured directly, so that distension can be kept in a range relevant for physiology or pathophysiology. Moreover, since we are measuring ATP concentration directly from the luminal fluid and not from an arbitrary and excessive amount of fluid used to maintain tissue in vitro, the concentration measured does not have to be corrected. Thus, the procedure described in this paper allows the researcher to examine ATP release in the bladder lumen under conditions that much more closely resemble physiological or pathophysiological conditions of the bladder.
The technique described is very similar to standard cystometry techniques used to study the effects of drugs on reflex bladder activity and should be easily carried out by anyone who routinely carries out those experiments. For those researchers new to bladder catheterization, however, there are a few caveats to be aware of. First, transurethral catheterization can be difficult for the inexperienced due to the curvature of the rodent urethra. This can also be made more difficult by the tightness of the external urethral sphincter, even under urethane anesthesia. It is imperative that the researcher does not try to force the catheter into the urethra, as it will most likely cause increased inflammation and swelling, which will in turn make the urethra even harder to successfully catheterize. Additionally, it is possible to poke the catheter through the wall of the urethra, ruining the entire experiment.
We have developed some tips and tricks for increasing the rate of success. For example, it is sometimes necessary to give the animal another short dose of inhaled isoflurane (0.5%-1.0%) to relax the urethral sphincter. This should be performed sparingly and with care, as the combination of urethane and isoflurane can result in respiratory depression. Another trick is to dip the tip of the catheter into a 2% lidocaine solution before inserting it into the external urethral orifice; this will often cause the sphincter to relax after a few minutes. Emptying the bladder by gently pressing on the abdomen of the animal can also help relax the urethral sphincter. Finally, it may be necessary to use a smaller gauge catheter; we sometimes use a 24 G catheter, especially with smaller animals. Keep in mind, however, that it may be necessary to reduce the infusion rate into the bladder when using a smaller gauge transurethral catheter, to match the slower drainage from the urethral catheter. Failure to do so would cause bladder pressure to rise, despite the urethral catheter not being capped.
It should be noted that urethane is used as an anesthetic in this protocol. Urethane is commonly used in laboratories studying the lower urinary tract, since it spares the micturition reflex whereas other anesthetics do not. Because of this, distension of the bladder above a pressure of ~7-10 cm H2O under urethane anesthesia will trigger a micturition reflex. Since we are interested in ATP release in response to passive distension, this protocol calls for blocking bladder contractions using the ganglionic blocker hexamethonium. This allows the measurement of ATP release in response to the higher intravesical pressures (i.e., 30 cm H2O) commonly seen in bladder pathology involving outlet obstruction. Therefore, the use of a urethane/hexamethonium allows for the measurement of distension-evoked luminal ATP release for long periods of time (the duration of action of both drugs is measured in hours). However, some labs may prefer to use an anesthetic that does not spare the micturition reflex, such as ketamine, and eliminate the need for a ganglionic blocker. This will also work well for this procedure, although it will require more frequent (or continuous) administration due to the shorter duration of action of the anesthetic.
One noteworthy difference between this technique to measure ATP and the common cystometry setup urology researchers may be familiar with is the use of buffered Krebs solution as a perfusate instead of the standard isotonic saline solution. ATP is a rather labile molecule and is stabilized somewhat in a buffered solution. Additionally, the composition of Krebs solution is much closer to that of urine than saline, which further adds clinical relevance to these experiments. This is an important consideration, as the control of urothelial ATP release has been shown to be modulated by calcium permeable channels, and the lack of calcium in normal saline may negatively influence the ATP release. The instability of ATP in solution is also why we suggest that samples be immediately quantified using the luciferin-luciferase reaction. However, if the researcher is unable to read the samples immediately, the samples should be frozen as quickly as possible to limit the degradation of ATP.
One limitation of this technique is that it only measures luminal ATP release and cannot measure ATP release from the serosal side of the urothelium. It is believed that ATP is released from the serosal side to directly influence afferent excitability; however, direct measurement of this release is difficult. In this case, using cells grown on permeable membrane supports such as Transwell plates or surgically removing the urothelium for Ussing chamber experiments is the preferred technique. However, given the apparent importance of luminal ATP in bladder pathology, this experimental model is a useful tool to elucidate the cellular mechanisms controlling urothelial ATP release during pathology.

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" /> 
Figure 1: Experimental setup. (A) Image showing the experimental setup, with the various equipment labeled. The area of the animal outlined in a red rectangle is shown in B. (B) Photograph depicting the abdomen of the rat following surgery. Note the suprapubic catheter inserted and tied into the bladder dome, as well as the urethral catheter inserted through the external orifice. The urethral catheter is plugged in the photo to show that bladder pressure is increased during perfusion through the bladder catheter. During the experiment, the abdominal opening would be sutured shut, but was left open here to allow visualization of the urinary bladder. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64227/64227fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Of primary importance for the successful measurement of stretch-mediated ATP release in the rodent bladder is ensuring that the micturition reflex is blocked, allowing the bladder to be distended beyond the threshold pressure normally sufficient to activate the micturition reflex. As shown in Figure 2A, when the urethral catheter is plugged, infusion of the solution into the bladder causes intravesical pressure to rise sharply as the bladder contracts. As we are interested in the effects of passive stretch, not contraction, on urothelial ATP release, the animal is treated with a ganglionic blocker, hexamethonium, to prevent the micturition reflex. As evidenced in Figure 2B, when hexamethonium is administered, instillation of the solution into the bladder with the urethral catheter blocked results in a much more gradual rise in intravesical pressure, allowing pressures to rise as high as 30 cm H2O without contraction. This allows for measurement of luminal ATP release at pressures high enough to be relevant to pathology, such as those observed in an underactive bladder, partial bladder outlet obstruction, or during detrusor-sphincter dyssynergia after spinal cord injury. While it may require a supplemental dose of hexamethonium to completely block the micturition reflex, care should be taken to not administer enough to result in an overdose. This could lead to significantly decreased cardiac output and compromise the quality of the experiment. It should be noted that the same inhibitory effect on the bladder reflex can be obtained by either cutting the pelvic nerves bilaterally or severing the spinal cord at level L4 or above. This, of course, increases the difficulty of the experimental setup and the time required to complete the experiment, significantly.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64227/64227fig02.jpg" /> 
Figure 2: Pressure recordings from rats. Before (A) and after (B) ganglionic block.&#160;Arrows indicate when the urethral catheter was plugged. Notice that the pressure in the top trace rapidly increases when pressure reaches the threshold to induce micturition, while the pressure in the bottom trace gradually rises. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64227/64227fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Another very important consideration for these experiments is to ensure proper conversion of the luminometer readings (normally expressed in RLUs) to a concentration of ATP using standard curves. The luciferin/luciferase reaction is very susceptible to interfering agents13. This interference can come from a direct interaction with luciferin/luciferase. For example, a number of compounds can act as competitive or non-competitive inhibitors of luciferase; one study suggested that ~3% of compounds in the Molecular Libraries Small Molecule Repository could inhibit firefly luciferase at concentrations below 11 &#181;M14. Moreover, luciferin is susceptible to oxidizing agents, reducing the amount of substrate available for the luminescence-producing reaction15. It is also possible to interfere with the luciferin/luciferase reaction by altering the availability of the reaction&#39;s co-factor, magnesium. For example, one common technique for mimicking mechanical stretch in in vitro ATP experiments is to induce cell swelling by reducing the osmolarity of the perfusing solution. However, many researchers accomplish this reduction by diluting the solution with water, reducing the concentration of magnesium available to act as a co-factor in the luciferin/luciferase reaction. Additionally, some drugs are sold as magnesium salts, which can greatly increase the amount of magnesium in the solution, also leading to measurable differences in luminescence readings. Finally, it is possible for a drug solution to interfere with the ability to accurately measure the light produced by the luciferin/luciferase reaction. Brilliant Blue FCF (BB-FCF, also known as erioglaucine or FD&#38;C Blue dye #1) is a specific inhibitor of pannexin-mediated ATP release16. At effective doses, Brilliant Blue FCF solution has a distinct blue hue, which absorbs light at a peak of around 600 nm17. This is within the range of wavelengths of light that is emitted by firefly luciferase; therefore, light emission in experiments with BB-FCF is greatly reduced. Thus, it is imperative that standard curves using known quantities of ATP dissolved in solutions containing each experimental drug are used to quantify the ATP release during the experiment. As shown in Figure 3, BB-FCF (100 &#181;M) significantly decreases the slope of the standard curve, which is sufficient to significantly alter the calculated values for the concentration of ATP in the sample. As shown at the bottom of Figure 3, using the Krebs standard to calculate the concentration of ATP in a sample containing BB-FCF can result in an underestimation of the ATP concentration by ~50%. In some instances, using the wrong standard curve could obscure a drug-mediated change in ATP release, or make it erroneously appear as if a change has occurred. In the experiment depicted in Figure 3, for example, using the Krebs standard for all samples would have made it appear that Brilliant Blue FCF reduced ATP release by ~67% (16.6 to 5.5 nM) when, in actuality, it had only decreased ~42% (16.6 to 9.6 nM).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64227/64227fig03.jpg" /> 
Figure 3: Effects of experimental drugs on ATP standard curve. Note that the pannexin channel antagonist, Brilliant Blue, significantly alters the RLUs measured for any given concentration of ATP, which would result in an underestimation of the ATP measured, if not corrected for. Abbreviation: RLUs = Relative Light Units; BB-FCF = Brilliant Blue FCF. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64227/64227fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
One other consideration to keep in mind is that ATP is commonly released from tissues as a result of damage, and that these surgical procedures and urethral catheter insertion will result in abnormally high ATP readings if taken too soon after setup. This procedure describes a 1 h washout period after the experiment is set up, which should not be skipped. We have found that after 1 h, ATP concentrations measured in samples taken when the bladder is not distended are relatively low (~10-30 nM) while samples taken before the washout period can read an order of magnitude higher. Short, 5 min washouts should also be performed between distensions as ATP levels can remain elevated for a short period. A good rule of thumb would be to measure ATP in multiple samples at the beginning of the experiment and after each distension and only proceeding when ATP readings level out. In Figure 4, we show a representation of a typical experiment with measurements taken at various time points. Note the high readings taken pre-washout (samples #1 and #5) versus those taken post washout (samples #2 and #6).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64227/64227fig04.jpg" /> 
Figure 4: Representation of a typical experiment. A stylized representation of a typical pressure recording. Each number represents a timepoint when a sample was taken and measured for ATP: 1) immediately after setup is completed, before the washout period, 2) after a 1 h washout period, 3) immediately preceding a distension, 4) during distension, 5) immediately following a distension, and 6) after a short washout period following the distension. The inset table shows representative RLU values and ATP concentrations for each sample. Abbreviation: RLUs = Relative Light Units. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64227/64227fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure 5 shows representative graphs of experimental results examining the mechanism of ATP release from the urothelium. As shown in Figure 5A, increasing pressure causes a significant increase in the concentration of ATP in the lumen of the bladder, with pressures of 30 cm of water increasing concentrations almost 2.5x of non-distended controls. Intravesical perfusion of 2 U/mL of apyrase, an enzyme that catalyzes the hydrolysis of ATP, significantly decreases the measured concentration of ATP in the bladder lumen. Conversely, intravesical perfusion of ARL67156, an inhibitor of NTPDases present on the urothelium, significantly increases luminal ATP concentrations. Figure 5B depicts the experiments into the mechanism of stretch-induced ATP release from the urothelium. Perfusion with the pannexin channel antagonists Brilliant Blue FCF (BB-FCF, 100 &#181;M) or carbenoxolone (CBX, 100 &#181;M) significantly reduces the release of ATP into the bladder lumen at all pressures. Luminal ATP concentrations are not diminished when the connexin blocker 18&#945;-glycyrrhetinic acid is perfused, indicating that distension-evoked ATP release is mediated by pannexin channels and not connexin channels.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64227/64227fig05.jpg" /> 
Figure 5: Representative measurements of distension-evoked ATP release in the lumen of the urinary bladder. (A)&#160;Distention of the bladder increases luminal ATP concentrations, which can be diminished in the presence of the NTPDase apyrase (2 U/mL) or increased when endogenous NTPDasesare inhibited with ARL67156 (10 &#181;M). (B) Distension-evoked ATP release is blocked by the pannexin channel antagonists Brilliant Blue FCF (BB-FCF, 100 &#181;M) and carbenoxolone (CBX, 100 &#181;M) but not the connexin channel antagonist 18&#945;-glycyrrhetinic acid (18&#945;-GA, 50 &#181;M). Data are presented as the mean with standard error bars. ** p &#60; 0.05 between the two bars indicated by the line, ## p &#60; 0.05 as compared to Krebs controls at the same distension. This figure is reprinted with permission from Beckel et al.8. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64227/64227fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Nanyang Technological University

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

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 ...

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly hydrated, forming a substantial endothelial surface layer (ESL) that contributes to the maintenance of endothelial function. As the endothelial glycocalyx is often aberrant in vitro and is lost during standard tissue fixation techniques, study of the ESL requires use of intravital microscopy. To best approximate the complex physiology of the alveolar microvasculature, pulmonary intravital imaging is ideally performed on a freely-moving lung. These preparations, however, typically suffer from extensive motion artifact. We demonstrate how closed-chest intravital microscopy of a freely-moving mouse lung can be used to measure glycocalyx integrity via ESL exclusion of fluorescently-labeled high molecular weight dextrans from the endothelial surface. This non-recovery surgical technique, which requires simultaneous brightfield and fluorescent imaging of the mouse lung, allows for longitudinal observation of the subpleural microvasculature without evidence of inducing confounding lung injury.

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx/endothelial surface layer is ideally studied using intravital microscopy. Intravital microscopy is technically challenging in a moving organ such as the lung. We demonstrate how simultaneous brightfield and fluorescent microscopy may be used to estimate endothelial surface layer thickness in a freely-moving in vivo mouse lung.

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly ...

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these diseases. Chronic retinal diseases such as retinal oedema or neovascularization usually require multiple intraocular injections to effectively treat the condition. However, the risks associated with these injections increase with repeated delivery of the drug. Therefore, alternative delivery methods need to be established in order to minimize the risks of reinjection. Several other investigations have developed methods to deliver drugs over extended time, through materials capable of releasing chemicals slowly into the eye. In this investigation, we outline the use of Elvax 40W, a copolymer resin, to act as a vehicle for drug delivery to the adult rat retina. The resin is made and loaded with the drug. The drug-resin complex is then implanted into the vitreous cavity, where it will slowly release the drug over time. This method was tested using 2-amino-4-phosphonobutyrate (APB), a glutamate analogue that blocks the light response of the retina. It was demonstrated that the APB was slowly released from the resin, and was able to block the retinal response by 7 days after implantation. This indicates that slow-release drug delivery using this copolymer resin is effective for treating the retina, and could be used therapeutically with further testing.

This paper details how Elvax 40W can be used as a slow-release method for drug delivery to the adult rat retina. The protocol for preparing, loading, and delivering the drug-resin complex to the eye is described.

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

Transcutaneous Assessment of Renal Function in Conscious Rodents

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and time-consuming. In addition, serial blood or urine samples are required, with the associated stress for the experimental animals. A recent technique significantly reduces the investment in time and resources, minimizing the invasiveness and the animal stress, but being equally valid as the traditional approaches. The method measures transcutaneously renal function. Using an optical device and the exogenous renal marker fluorescein isothiocyanate (FITC)-sinistrin, this technique is capable of measuring the elimination kinetics of the marker through the skin. With neither blood nor urine samples nor the associated laboratory assays needed, the results of the transcutaneous measurement are almost instantaneously available. The method has been already validated in different species and successfully applied in several models of renal pathology. Moreover, due to its minimally invasive characteristics, it is suitable for sequential measurements within the same animal. Here is provided a detailed protocol to carry out the transcutaneous assessment of renal function in rodents.

Determination of glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional procedures to measure this parameter are cumbersome and require a large investment of time. Here we describe a faster and minimally invasive method to determinate GFR transcutaneously.

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular diseases, such as diabetic retinopathy, hypertensive retinopathy, and possibly glaucoma. Therefore, retinal microvascular preparations are pivotal tools for physiological and pharmacological studies to delineate the underlying pathophysiological mechanisms and to design therapies for the diseases. Despite the wide use of mouse models in ophthalmic research, studies on retinal vascular reactivity are scarce in this species. A major reason for this discrepancy is the challenging isolation procedures owing to the small size of these retinal blood vessels, which is ~ &#8804; 30 &#181;m in luminal diameter. To circumvent the problem of direct isolation of these retinal microvessels for functional studies, we established an isolation and preparation technique that enables ex vivo studies of mouse retinal vasoactivity under near-physiological conditions. Although the present experimental preparations will specifically refer to the mouse retinal arterioles, this methodology can readily be employed to microvessels from rats.

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Many vision-threatening ocular diseases are associated with dysfunctional retinal microvessels. Therefore, the measurement of retinal arteriole responses is important to investigate the underlying pathophysiological mechanisms. This article describes a detailed protocol for mouse retinal arteriole isolation and preparation to assess the effects of vasoactive substances on vascular diameter.

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular ...

Using a Chemical Biopsy for Graft Quality Assessment

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated with an increased survival rate and greater quality of patient&#8217;s life when compared to conventional dialysis. Regrettably, transplantology suffers from a lack of reliable methods for organ quality assessment. Standard diagnostic techniques are limited to macroscopic appearance inspection or invasive tissue biopsy, which do not provide comprehensive information about the graft. The proposed protocol aims to introduce solid phase microextraction (SPME) as an ideal analytical method for comprehensive metabolomics and lipidomic analysis of all low molecular compounds present in kidneys allocated for transplantation. The small size of the SPME probe enables performance of a chemical biopsy, which enables extraction of metabolites directly from the organ without any tissue collection. The minimum invasiveness of the method permits execution of multiple analyses over time: directly after organ harvesting, during its preservation, and immediately after revascularization at the recipient&#8217;s body. It is hypothesized that the combination of this novel sampling method with a high-resolution mass spectrometer will allow for discrimination of a set of characteristic compounds that could serve as biological markers of graft quality and indicators of possible development of organ dysfunction.

The protocol presents utilization of the chemical biopsy approach followed by comprehensive metabolomic and lipidomic analysis for quality assessment of kidney grafts allocated for transplantation.

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated ...

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

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Chapters in this video

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

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

In vivo ¹⁹F MRI for Cell Tracking

Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

Transcutaneous Assessment of Renal Function in Conscious Rodents

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Using a Chemical Biopsy for Graft Quality Assessment