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. 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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. 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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 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" /> 
<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

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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

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 ...

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 ...

Remote Limb Ischemic Preconditioning:  A Neuroprotective Technique in Rodents

Sublethal ischemia protects tissues against subsequent, more severe ischemia through the upregulation of endogenous mechanisms in the affected tissue. Sublethal ischemia has also been shown to upregulate protective mechanisms in remote tissues. A brief period of ischemia (5-10 min) in the hind limb of mammals induces self-protective responses in the brain, lung, heart and retina. The effect is known as remote ischemic preconditioning (RIP). It is a therapeutically promising way of protecting vital organs, and is already under clinical trials for heart and brain injuries. This publication demonstrates a controlled, minimally invasive method of making a limb &#8211; specifically the hind limb of a rat &#8211; ischemic. A blood pressure cuff developed for use in human neonates is connected to a manual sphygmomanometer and used to apply 160 mmHg pressure around the upper part of the hind limb. A probe designed to detect skin temperature is used to verify the ischemia, by recording the drop in skin temperature caused by pressure-induced occlusion of the leg arteries, and the rise in temperature which follows release of the cuff. This method of RIP affords protection to the rat retina against bright light-induced damage and degeneration.

Remote ischemic preconditioning (RIP) is a method of conditioning tissues against damaging stress. We have established a method of remote ischemia at the hind limb, by inflating a sphygmomanometer cuff for 5-10 min. The neuroprotective capabilities of RIP have been demonstrated in a model of retinal degeneration in rodents.

Sublethal ischemia protects tissues against subsequent, more severe ischemia through the upregulation of endogenous mechanisms in the affected tissue. ...

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 ...

On-Site Sampling and Extraction of Brain Tumors for Metabolomics and Lipidomics Analysis

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still in need. In recent years, mass spectrometry has become a method of choice for untargeted profiling of discriminatory compound as potential biomarkers of a disease. Biofluids are generally considered as preferable matrices given their accessibility and easier sample processing while direct tissue profiling provides more selective information about a given cancer. Preparation of tissues for the analysis via traditional methods is much more complex and time-consuming, and, therefore, not suitable for fast on-site analysis. The current work presents a protocol combining sample preparation and extraction of small molecules on-site, immediately after tumor resection. The sampling device, which is of the size of an acupuncture needle, can be inserted directly into the tissue and then transported to the nearby laboratory for instrumental analysis. The results of metabolomics and lipidomics analyses demonstrate the capability of the approach for the establishment of phenotypes of tumors related to the histological origin of the tumor, malignancy, and genetic mutations, as well as for the selection of discriminating compounds or potential biomarkers. The non-destructive nature of the technique permits subsequent performance of routinely used tests e.g., histological tests, on the same samples used for SPME analysis, thus enabling attainment of more comprehensive information to support personalized diagnostics.

The manuscript presents on-site sampling of human brain tumors with solid phase microextraction followed by their biochemical profiling towards the discovery of biomarkers.

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still ...