This study was funded by the Department of Surgery University of Vermont, Danish Council for Independent Research, and by the Odense University Hospital.

Animals were housed in the University of Vermont Animal Care Facility according to institutional guidelines. All animal experiments were carried out in accordance with the National Institutes of Health guide for the care and use of laboratory animals.
1. Intravesical Tube Implantation
<ol>
	<li>Preparation of tubing and instruments for the surgical procedure
	<ol>
		<li>Cut a 7-cm piece of PE10 tubing to make the catheter for implantation.</li>
		<li>Create a flare at one en...

<ol>
	<li>Perez, L. M., Webster, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+History+of+Urodynamics.">The History of Urodynamics.</a> Neurourol Urodyn. 11 (1), 1-21 (1992).</li><li>Fry, C. H., 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=Animal+models+and+their+use+in+understanding+lower+urinary+tract+dysfunction.">Animal models and their use in understanding lower urinary tract dysfunction.</a> Neurourol Urodyn. 29 (4), 603-608 (2010).</li><li>Maggi, C. A., Santicioli, P., Meli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nonstop+transvesical+cystometrogram+in+urethane-anesthetized+rats:+a+simple+procedure+for+quantitative+studies+on+the+various+phases+of+urinary+bladder+voiding+cycle.">The nonstop transvesical cystometrogram in urethane-anesthetized rats: a simple procedure for quantitative studies on the various phases of urinary bladder voiding cycle.</a> J Pharmacol Methods. 15 (2), 157-167 (1986).</li><li>Malmgren, A., 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=Cystometrical+evaluation+of+bladder+instability+in+rats+with+infravesical+outflow+obstruction.">Cystometrical evaluation of bladder instability in rats with infravesical outflow obstruction.</a> J Urol. 137 (6), 1291-1294 (1987).</li><li>Pandita, R. K., Fujiwara, M., Alm, P., Andersson, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cystometric+evaluation+of+bladder+function+in+non-anesthetized+mice+with+and+without+bladder+outlet+obstruction.">Cystometric evaluation of bladder function in non-anesthetized mice with and without bladder outlet obstruction.</a> J Urol. 164 (4), 1385-1389 (2000).</li><li>Chang, H. Y., Havton, 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=Differential+effects+of+urethane+and+isoflurane+on+external+urethral+sphincter+electromyography+and+cystometry+in+rats.">Differential effects of urethane and isoflurane on external urethral sphincter electromyography and cystometry in rats.</a> Am J Physiol Renal Physiol. 295 (4), F1248-F1253 (2008).</li><li>Matsuura, S., Downie, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+anesthetics+on+reflex+micturition+in+the+chronic+cannula-implanted+rat.">Effect of anesthetics on reflex micturition in the chronic cannula-implanted rat.</a> Neurourol Urodyn. 19 (1), 87-99 (2000).</li><li>DePaul, M. A., Lin, C. Y., Silver, J., Lee, Y. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+Nerve+Transplantation+Combined+with+Acidic+Fibroblast+Growth+Factor+and+Chondroitinase+Induces+Regeneration+and+Improves+Urinary+Function+in+Complete+Spinal+Cord+Transected+Adult+Mice.">Peripheral Nerve Transplantation Combined with Acidic Fibroblast Growth Factor and Chondroitinase Induces Regeneration and Improves Urinary Function in Complete Spinal Cord Transected Adult Mice.</a> PLoS One. 10 (10), e0139335 (2015).</li><li>Kadekawa, 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=Characterization+of+bladder+and+external+urethral+activity+in+mice+with+or+without+spinal+cord+injury--a+comparison+study+with+rats.">Characterization of bladder and external urethral activity in mice with or without spinal cord injury--a comparison study with rats.</a> Am J Physiol Regul Integr Comp Physiol. 310 (8), R752-R758 (2016).</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> J Vis Exp. (66), (2012).</li><li>Andersson, K. E., Soler, R., Fullhase, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+models+for+urodynamic+investigation.">Rodent models for urodynamic investigation.</a> Neurourol Urodyn. 30 (5), 636-646 (2011).</li><li>Smith, P. P., Kuchel, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+uroflow+cystometry+in+the+urethane-anesthetized+mouse.">Continuous uroflow cystometry in the urethane-anesthetized mouse.</a> Neurourol Urodyn. 29 (7), 1344-1349 (2010).</li><li>Aizawa, N., Homma, Y., Igawa, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+High+Fat+Diet+Feeding+for+20+Weeks+on+Lower+Urinary+Tract+Function+in+Mice.">Influence of High Fat Diet Feeding for 20 Weeks on Lower Urinary Tract Function in Mice.</a> Low Urin Tract Symptoms. 5 (2), 101-108 (2013).</li><li>Bjorling, D. E., 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=Evaluation+of+voiding+assays+in+mice:+impact+of+genetic+strains+and+sex.">Evaluation of voiding assays in mice: impact of genetic strains and sex.</a> Am J Physiol Renal Physiol. 308 (12), F1369-F1378 (2015).</li><li>Morikawa, 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=Effects+of+various+drugs+on+bladder+function+in+conscious+rats.">Effects of various drugs on bladder function in conscious rats.</a> Jpn J Pharmacol. 50 (4), 369-376 (1989).</li><li>Yaksh, T. L., Durant, P. A., Brent, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micturition+in+rats:+a+chronic+model+for+study+of+bladder+function+and+effect+of+anesthetics.">Micturition in rats: a chronic model for study of bladder function and effect of anesthetics.</a> Am J Physiol. 251 (6 Pt 2), R1177-R1185 (1986).</li><li>Cornelissen, L. L., Misajet, B., Brooks, D. P., Hicks, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+genetic+background+and+gender+on+bladder+function+in+the+mouse.">Influence of genetic background and gender on bladder function in the mouse.</a> Auton Neurosci. 140 (1-2), 53-58 (2008).</li><li>Lemack, G. E., Zimmern, P. E., Vazquez, D., Connell, J. D., Lin, V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+response+to+partial+bladder+outlet+obstruction+in+mice+lacking+inducible+nitric+oxide+synthase.">Altered response to partial bladder outlet obstruction in mice lacking inducible nitric oxide synthase.</a> J Urol. 163 (6), 1981-1987 (2000).</li></ol>

Optimal material and size of intravesical tubing
To determine the effect tubing diameter has on pressure recordings, we tested different microfluidic tubes; PE50 (0.58 mm ID), polyurethane PU027 (0.4 mm ID), PE25 (0.46 mm ID), and PE10 (0.28 mm ID). For each tube, pressure was recorded with the infusion pump running at 1 mL/h, while rapidly moving the tube vertically from 0 to 30 cm. Initial in vivo experiments attempted to use PE50 tubing, but were unsuccessful due to the size of tubing compared...

Filling cystometry (FC) is a diagnostic method that involves placing a catheter into the urinary bladder to record pressure during slow bladder filling. First introduced in 1927 as a clinical diagnostic method to evaluate lower urinary tract function, it has remained widely used.1 In research applications, FC can be used to test bladder function in healthy and diseased animal models and to study the effects of pharmacological agents. Rodent animal models are commonly used to investigate lower urinary tract function.2 In this group of mammals, FC was first developed for use in rats.3 Here, the methodology to implant a tube into the urinary bladder and perform FC has been well described and used by many researchers with an acceptable level of reproducibility.4 The availability of transgenic and knock out strains make mice a valuable species for numerous research areas, including the field of lower urinary tract dysfunction. The methodology used for performing mouse cystometry varies appreciably between laboratories, making it difficult to compare results.5
Compared to ex vivo models, FC preserves lower urinary tract anatomy, allowing the coordinated function between the bladder and its outlet during the storage and voiding phases of the micturition cycle to be assessed. Previous research shows that numerous, commonly used anesthetics suppress micturition contraction. Agents that preserve urinary bladder smooth muscle contraction (urethane, &#945;-chloralose, ketamine and xylazine), allowing the animal to micturate, still significantly reduce functional bladder capacity and suppress neurotransmission.6,7,8,9 Although technically more challenging, FC performed in awake ambulating animals preserves the functional integrity of the micturition reflex.
Lower urinary tract function is influenced by multiple factors, including post-operative bladder wall swelling, stress due to pain and discomfort, and environmental influences. Using a surgical technique that minimizes tissue damage during tube implantation and recording methods that reduce tube movement, while simultaneously allowing the animal to ambulate freely, are essential for obtaining accurate and reproducible recordings.
If performed adequately, in vivo FC in freely moving animals can provide data that reliably reflects physiological bladder function.10 FC in freely moving animals can provide data on the following parameters; Basal or baseline pressure: Minimum pressure between two micturitions. Intermicturition pressure: Mean pressure between two micturitions. Threshold pressure: Intravesical pressure immediately before micturition. Maximum pressure: Maximum bladder pressure during a micturition cycle. Spontaneous activity (or mean intermicturition oscillatory pressure): Intermicturition pressure minus basal pressure. Non-voiding contractions: Increase in intravesical pressure during the filling phase, not associated with the release of fluid. Bladder compliance: Bladder capacity divided by threshold pressure minus basal pressure. Micturition frequency: Number of micturitions per unit of time. Intermicturition interval: Period between two maximum voiding pressures. Bladder capacity: Infused volume divided by the number of micturitions. A detailed description of these parameters and standardized terminology has been previously published.11
FC can be performed using a continuous or single cycle intravesical infusion method. Continuous cystometry allows for recording of multiple micturition cycles and selecting representative data based on reproducibility. Its accuracy in measuring bladder capacity is limited due to the unknown residual volume. In addition, it is challenging to collect small voided volumes (which based on strain and sex vary between 30 and 184 &#181;L) in freely ambulating mice. Using this method to record voided volume is less accurate compared to an anesthetized preparation, but it is superior in that it avoids the suppressive effects of anesthetics on bladder function. Single cycle cystometry should be used to assess bladder capacity. In this method, the bladder is emptied by aspiration prior to infusion and capacity is calculated as a function of infusion rate multiplied by time to maximum pressure.
Although the technique of performing cystometry in small rodents has been published, it described the surgery performed in a rat and recommended that mouse cystometry should be performed under urethane anesthesia.10 The goal of this communication is to describe both the microsurgical techniques used to implant an intravesical tube into the dome of the urinary bladder and the experimental set-up used to record lower urinary tract function, in vivo, during continuous bladder filling and micturition in an awake freely moving mouse. In addition, experiments were performed to address how tubing length, diameter, and material, as well as the methodology for performing in vivo FC, affect the recording. This experimental protocol summarizes previously published techniques and proposes a number of modifications based on experimental results.

<table border="0" cellpadding="0" cellspacing="0" width="946">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Polyethylene (PE) 10 tubing</td>
			<td style="width:259px;">Instech</td>
			<td style="width:119px;">BTPE-10</td>
			<td style="width:191px;">Fits 30G connectors/plugs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Polyethylene (PE) 50 tubing</td>
			<td style="width:259px;">Instech</td>
			<td style="width:119px;">BTPE-50</td>
			<td>Fits 22G connectors/plugs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">22ga single channel stainless steel swivel</td>
			<td style="width:259px;">Instech</td>
			<td style="width:119px;">375/22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">High Carbon Steel Utility Extension Spring (9/64&#34; OD)</td>
			<td style="width:259px;">Grainger</td>
			<td style="width:119px;">1NAH1</td>
			<td>Protects PE50 tubing - Cut to length</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">22G connector</td>
			<td style="width:259px;">Instech</td>
			<td style="width:119px;">SP22/12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Yutaoz Professional Hot Melt Adhesive Glue Gun</td>
			<td style="width:259px;">Yutaoz</td>
			<td style="width:119px;"></td>
			<td>Use low temperature setting (100&#176;C) - Any hot melt glue gun with an adjustable temperature range will work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Surebonder DT-2010 all purpose glue stick</td>
			<td style="width:259px;">Surebonder</td>
			<td style="width:119px;"></td>
			<td>Any all purpose hot glue will work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Dumont #5 curved microforceps</td>
			<td style="width:259px;">World Precision Instruments</td>
			<td align="right">500232</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Dumont #7 curved microforceps</td>
			<td style="width:259px;">World Precision Instruments</td>
			<td align="right">14188</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mini dissecting scissors - straight</td>
			<td style="width:259px;">World Precision Instruments</td>
			<td align="right">503240</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro mosquito forceps (12.5cm)</td>
			<td style="width:259px;">World Precision Instruments</td>
			<td align="right">500451</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissecting scissors - straight</td>
			<td style="width:259px;">World Precision Instruments</td>
			<td align="right">14393</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Castroviejo Needle Holder</td>
			<td style="width:259px;">World Precision Instruments</td>
			<td align="right">503258</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Isoflurane, USP</td>
			<td style="width:259px;">Phoenix</td>
			<td style="width:119px;"></td>
			<td>2%, 1 L/min flow rate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Buprenorphine</td>
			<td style="width:259px;"></td>
			<td style="width:119px;"></td>
			<td>0.05mg/kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">0.9% Sodium Chloride Irrigation, USP</td>
			<td style="width:259px;">Baxter</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">6-0 Ethilon black monofilament, non-absorbable suture</td>
			<td style="width:259px;">Ethicon</td>
			<td style="width:119px;"></td>
			<td>Bladder tie</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">6-0 Vicryl violet braided, absorbable suture</td>
			<td style="width:259px;">Ethicon</td>
			<td style="width:119px;"></td>
			<td>Muscle suture, running</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">6-0 Prolene blue monofilament, non-absorbable suture</td>
			<td style="width:259px;">Ethicon</td>
			<td style="width:119px;"></td>
			<td>Skin suture, vertical mattress, buried interrupted</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">KD Legato 210 infuse/withdraw pump</td>
			<td style="width:259px;">KD Scientific</td>
			<td style="width:119px;"></td>
			<td>1.5ml/hr</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Disposable pressure transducer</td>
			<td style="width:259px;">Digitimer</td>
			<td style="width:119px;">NL108T2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Pressure Amplifier</td>
			<td style="width:259px;">Digitimer</td>
			<td style="width:119px;">NL108A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Power1401-3 data acquisition interface</td>
			<td style="width:259px;">Digitimer</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Spike2&#160;</td>
			<td style="width:259px;">Cambridge Electronic Design Limited</td>
			<td style="width:119px;"></td>
			<td>PC pressure recording software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Leica MZ6 surgical operating microscope (3.2-20X)</td>
			<td style="width:259px;">Leica Microsystems</td>
			<td style="width:119px;"></td>
			<td>Magnification</td>
		</tr>
	</tbody>
</table>

evaluating the procedure for performing awake cystometry in a mouse model

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

There was no significant difference between the tubing materials and diameters in the consistency of pressure rise and fall within the system during tube occlusion. Bladder wall swelling post intravesical tube implantation was significant for both polyethylene (PE) and polyurethane (PU) materials. On day 2, severe submucosal swelling developed. It occupied half the cross-section of the bladder, leading to obstruction of the lumen. On day 5, the edema resolved completely, leaving the submu...

Watch this Scientific Journal Video about Evaluating the Procedure for Performing Awake Cystometry in a Mouse Model at JoVE.com

Evaluating the Procedure for Performing Awake Cystometry in a Mouse Model

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

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

The overall goal of this microsurgical procedure is to implant an intravesical tube into the urinary bladder and to record the lower urinary tract function in an awake mouse. This method can help in the acquisition of reproducible experimental data from awake cystometry and the testing of bladder function in healthy and diseased mouse models. The main goal of this technique is to provide a standardized method based on experimental data for performing filling cystometry in a mouse model.To prepare the tube for implantation, first cut a seven centimeter piece of pe10 tubing, and slowly advance one end toward an open flame to create a flare, quickly withdrawing the tube as soon as the flare develops. Then use the low heat setting on a glue gun to apply three drops of all purpose hot glue at 4.5, 5, and 5.5 centimeters from the flared end of the tube. Use scissors to trim irregularities from the glue bubbles.Apply hot glue to one end of a 30 gauge needle, which will be used to plug the pe10 tubing after it is implanted. Next confirm the lack of response to toe pinch and apply ointment to the animal's eyes. After sterilizing the upper back and lower abdomen with alcohol and betadine, make a 1.5 centimeter incision between the scalpula, and place the animal in the supine position on top of a 37 degree celsius heating pad covered with sterile drapes under a dissecting microscope.Now make a 1.5 centimeter lower midline abdominal skin incision. Followed by a matching incision through the fascia along the linea alba and muscle to expose the dome and upper half of the urinary bladder. Keeping the abdominal viscera moist with warm physiological saline, rotate the animal onto it's side to access the incision on the nape of the neck.Push a narrow hemostat subcutaneously through the incision to start a subcutaneous channel on the back and continuing along the side of the animal. Once the tip of the instrument reaches the bottom of the ribcage, turn the tip towards the midline in the inside of the abdomen and continue advancing the hemostat until the tip is exposed at the abdominal incision under the muscular layer. Once the hemostat tip is inside the incision, pinch the skin above the shoulder and apply traction to help stabilize the animal until the instrument tip is seen at the abdominal incision.Now use the hemostat to grasp the non flared end of the tubing and slowly retract the hemostat, pulling the end of the tube out of the incision at the back of the neck and adjusting the flared end of the tubing so that it lies directly above the dome of the bladder. Stabilize the bladder with a small roll of lint free tissue. Place a loosely tied, not absorbable 6-O monofilament suture on top of the bladder dome.Using the non dominant hand and the Dumont number seven curved micro forceps, grasp the dome of the bladder and use a 21 gauge needle in the dominant hand to make a cystotomy in the dome apex. Gently probe the cystotomy with a closed pair of number five curved micro forceps to make sure the catheter can easily pass through the hole and place the flared end of the pe10 catheter into the bladder, pushing the flare down to the bladder neck so that it does not slip out while being secured. With the monofilament tie placed anterior to the tubing, tighten the 6-O suture around the dome of the bladder and tubing as high up on the bladder as possible to avoid artificially reducing the bladder capacity.Alternatively, place a loose purse-string suture on the dome of the bladder, make a cystotomy as just demonstrated, and insert the flared end of the catheter into the puncture. Then tie the suture around the dome and the tubing as demonstrated for the flat suture. In either case take care to secure the tubing without causing extensive injury to the bladder wall.If too much tissue is taken, the bladder capacity will be reduced. If too little is taken, the connection will leak. To test the seal and position of the tube in the bladder, attach a 0.5 milliliter insulin syringe equipped with a 30 gauge needle to the distal end of the tube and slowly fill the bladder with 0.1 to 0.2 milliliters of 0.9%sodium chloride until a drop appears at the urethral orifice.If the seal is incomplete, loop suture tails around the bladder and retie. Then empty the bladder by aspiration. If no leaks occur at the dome, brace the bladder with a pair of curved micro forceps and gently pull on the tubing until the flare is resting against the inside of the bladder dome.Retest the seal using the previously described method. Trim the tails of the suture, remove the tissue roll, and return the bladder to it's normal position. Then use a running 6-O suture to close the abdominal wall in two layers.And gently rotate the animal onto it's abdomen. Insert the subcutaneous portion of the metal anchor into the intrascopular incision, and use a 6-O monofilament to secure the tube and anchor with a vertical mattress suture. Confirm that a glue bubble remains above and below the skin to prevent the tube from pulling out and cut the tube approximately two centimeters above the skin.Then gently insert a 30 gauge plug into the end of the tube to prevent urine from leaking out. Before performing the reading, use pe50 tubing to connect the infusion pump, pressure transducer, and 22 gauge swivel. Then place the anesthetized experimental animal in the prone position and remove the 30 gauge plug.Connect the tether to the anchor, slide the bladder catheter into the end of the pe50, and use hot glue to form a water tight seal. Place the mouse onto the wire floor. And begin the recording.Then when the animal recovers from the anesthesia and the bladder pressure stabilizes, begin infusing 0.9%sodium chloride at 0.6 milliliters per hour rate. Typically, on day two after catheter insertion, severe submucosal swelling that occupies half of the cross section of the bladder develops leading to obstruction of the lumen. On day five the edema resolves completely leaving the submucosal areas infiltrated with inflammatory cells that partially invaded the muscularis.The greatest extent of the tissue swelling observed on days two and day three correlates with the corresponding behavioral voiding with the animals demonstrating a significantly impaired bladder function at these time points. As expected from the histologic data, the voiding frequency normalizes by post operative day five. Intravesical pressure in an awake freely moving animal with minimal movement artifacts is characterized by a baseline pressure of 10 to 15 centimeters of water which may remain unchanged or may gradually increase by no more than 10 centimeters of water during the filling cycle.Followed by a sudden pulse style pressure increase and drop during voiding. Once mastered, this technique can be completed in 30 minutes if it is performed properly. While attempting this procedure it is important to remember to minimize the damage to the bladder to preserve the physiology of the tissue.Following this procedure, other methods, like single cycle filling cystometry can be performed to answer additional questions about the functional bladder capacity. After watching this video, you should have a good understanding of how to microsurgically implant a tube into the urinary bladder and to use awake filling cystometey to evaluate lower urinary tract function in mice.

evaluating-procedure-for-performing-awake-cystometry-mouse

Research

JoVE Journal

Medicine

12.4K visualizações.  University of Vermont. The overall goal of this microsurgical procedure is to implant an intravesical tube into the urinary bladder and to record the lower urinary tract function in an awake mouse. This method can help in the acquisition of reproducible experimental data from awake cystometry and the testing of bladder function in healthy and diseased mouse models. The main goal of this technique is to provide a standardized method based on experimental data for performing filling cystometry in a mouse model.