Name: evaluating the procedure for performing awake cystometry in a mouse model
Uploaded: 2017-05-20T13:00:00
Description: Watch this Scientific Journal Video about Evaluating the Procedure for Performing Awake Cystometry in a Mouse Model at JoVE.com

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

Intra-Operative Behavioral Tasks in Awake Humans Undergoing Deep Brain Stimulation Surgery

Deep brain stimulation (DBS) is a surgical procedure that directs chronic, high frequency electrical stimulation to specific targets in the brain through implanted electrodes. Deep brain stimulation was first implemented as a therapeutic modality by Benabid et al. in the late 1980s, when he used this technique to stimulate the ventral intermediate nucleus of the thalamus for the treatment of tremor 1. Currently, the procedure is used to treat patients who fail to respond adequately to medical management for diseases such as Parkinson's, dystonia, and essential tremor. The efficacy of this procedure for the treatment of Parkinson's disease has been demonstrated in well-powered, randomized controlled trials 2. Presently, the U.S. Food and Drug Administration has approved DBS as a treatment for patients with medically refractory essential tremor, Parkinson's disease, and dystonia. Additionally, DBS is currently being evaluated for the treatment of other psychiatric and neurological disorders, such as obsessive compulsive disorder, major depressive disorder, and epilepsy. 
DBS has not only been shown to help people by improving their quality of life, it also provides researchers with the unique opportunity to study and understand the human brain. Microelectrode recordings are routinely performed during DBS surgery in order to enhance the precision of anatomical targeting. Firing patterns of individual neurons can therefore be recorded while the subject performs a behavioral task. Early studies using these data focused on descriptive aspects, including firing and burst rates, and frequency modulation 3. More recent studies have focused on cognitive aspects of behavior in relation to neuronal activity 4,5. This article will provide a description of the intra-operative methods used to perform behavioral tasks and record neuronal data with awake patients during DBS cases. Our exposition of the process of acquiring electrophysiological data will illuminate the current scope and limitations of intra-operative human experiments.

Deep brain stimulation surgery offers a unique opportunity to examine information encoding in the awake human brain. This article will describe intra-operative methods used to perform cognitive and behavioral tasks while simultaneously acquiring physiological data such as EMG, single-unit neuronal activity and/or local field potentials.

Deep brain stimulation (DBS) is a surgical procedure that directs chronic, high frequency electrical stimulation to specific targets in the brain through ...

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 ...

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis (ALS)

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 years for the sporadic disease and around 50 years for the familial disease. Due to its progressive course, 50% of the patients die within 30 months of symptom onset. In order to evaluate novel treatment options for this disease, genetic mouse models of ALS have been generated based on human familial mutations in the SOD gene, such as the SOD1 (G93A) mutation. Most important aspects that have to be evaluated in the model are overall survival, clinical course and motor function. Here, we demonstrate the clinical evaluation, show the conduction of two behavioural motor tests and provide quantitative scoring systems for all parameters. Because an in depth analysis of the ALS mouse model usually requires an immunohistochemical examination of the spinal cord, we demonstrate its preparation in detail applying the dorsal laminectomy method. Exemplary histological findings are demonstrated. The comprehensive application of the depicted examination methods in studies on the mouse model of ALS will enable the researcher to reliably test future therapeutic options which can provide a basis for later human clinical trials.

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis ALS

A mouse model for amyotrophic lateral sclerosis (ALS) is examined clinically and behaviorally. As a prerequisite for an accompanying immunohistological analysis the preparation of the spinal cord is depicted in detail.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 ...

Intranasal Administration of CNS Therapeutics to Awake Mice

Intranasal administration is a method of delivering therapeutic agents to the central nervous system (CNS). It is non-invasive and allows large molecules that do not cross the blood-brain barrier access to the CNS. Drugs are directly targeted to the CNS with intranasal delivery, reducing systemic exposure and thus unwanted systemic side effects1. Delivery from the nose to the CNS occurs within minutes along both the olfactory and trigeminal neural pathways via an extracellular route and does not require drug to bind to any receptor or axonal transport2. Intranasal delivery is a widely publicized method and is currently being used in human clinical trials3.
Intranasal delivery of drugs in animal models allows for initial evaluation of pharmacokinetic distribution and efficacy. With mice, it is possible to administer drugs to awake (non-anesthetized) animals on a regular basis using a specialized intranasal grip. Awake delivery is beneficial because it allows for long-term chronic dosing without anesthesia, it takes less time than with anesthesia, and can be learned and done by many people so that teams of technicians can dose large numbers of mice in short periods. Efficacy of therapeutics administered intranasally in this way to mice has been demonstrated in a number of studies including insulin in diabetic mouse models 4-6 and deferoxamine in Alzheimer's mouse models. 7,8
The intranasal grip for mice can be learned, but is not easy and requires practice, skill, and a precise grip to effectively deliver drug to the brain and avoid drainage to the lung and stomach. Mice are restrained by hand using a modified scruff in the non-dominant hand with the neck held parallel to the floor, while drug is delivered with a pipettor using the dominant hand. It usually takes 3-4 weeks of acclimating to handling before mice can be held with this grip without a stress response. We have prepared this JoVE video to make this intranasal delivery technique more accessible.

A method to intranasally administer drugs to awake mice for the purpose of targeting the brain is described. This method allows for repeat dosing over long periods using intranasal administration of drug without anesthesia, and nose-to-brain delivery with minimal systemic exposure.

Intranasal administration is a method of delivering  therapeutic agents to the central nervous system (CNS). It is  non-invasive and allows large ...

Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment epithelium (RPE) cells. RPE provide critical support for photoreceptors and death or dysfunction of RPE cells is characteristic of age-related macular degeneration (AMD), the leading cause of permanent vision loss in people age 55 and older. While no cure for AMD has been identified, implantation of healthy RPE in diseased eyes may prove to be an effective treatment, and large numbers of RPE cells can be readily generated from pluripotent stem cells. Several interesting questions regarding the safety and efficacy of RPE cell delivery can still be examined in animal models, and well-accepted protocols used to inject RPE have been developed. The technique described here has been used by multiple groups in various studies and involves first creating a hole in the eye with a sharp needle. Then a syringe with a blunt needle loaded with cells is inserted through the hole and passed through the vitreous until it gently touches the RPE. Using this injection method, which is relatively simple and requires minimal equipment, we achieve consistent and efficient integration of stem cell-derived RPE cells in between the host RPE that prevents significant amount of photoreceptor degeneration in animal models. While not part of the actual protocol, we also describe how to determine the extent of the trauma induced by the injection, and how to verify that the cells were injected into the subretinal space using in vivo imaging modalities. Finally, the use of this protocol is not limited to RPE cells; it may be used to inject any compound or cell into the subretinal space.

Here we present a community accepted protocol in multimedia format for subretinally injecting a bolus of RPE cells in rats and mice. This approach can be used for determining rescue potentials, safety profiles, and survival capacities of grafted RPE cells upon implantation in animal models of retinal degeneration.

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment ...

A Surgical Procedure for Resecting the Mouse Rib: A Model for Large-Scale Long Bone Repair

This protocol introduces researchers to a new model for large-scale bone repair utilizing the mouse rib. The procedure details the following: preparation of the animal for surgery, opening the thoracic body wall, exposing the desired rib from the surrounding intercostal muscles, excising the desired section of rib without inducing a pneumothorax, and closing the incisions. Compared to the bones of the appendicular skeleton, the ribs are highly accessible. In addition, no internal or external fixator is necessary since the adjacent ribs provide a natural fixation. The surgery uses commercially available supplies, is straightforward to learn, and well-tolerated by the animal. The procedure can be carried out with or without removing the surrounding periosteum, and therefore the contribution of the periosteum to repair can be assessed. Results indicate that if the periosteum is retained, robust repair occurs in 1 - 2 months. We expect that use of this protocol will stimulate research into rib repair and that the findings will facilitate the development of new ways to stimulate bone repair in other locations around the body.

The overall goal of this procedure is to successfully resect a portion of bone from the rib of a mouse. The procedure was developed as a model to study large-scale long bone repair.

This protocol introduces researchers to a new model for large-scale bone repair utilizing the mouse rib. The procedure details the following: preparation ...

A Quick Phenotypic Neurological Scoring System for Evaluating Disease Progression in the SOD1-G93A Mouse Model of ALS

The SOD1-G93A transgenic mouse is the most widely used animal model of amyotrophic lateral sclerosis (ALS). At ALS TDI we developed a phenotypic screening protocol, demonstrated in video herein, which reliably assesses the neuromuscular function of SOD1-G93A mice in a quick manner. This protocol encompasses a simple neurological scoring system (NeuroScore) designed to assess hindlimb function. NeuroScore is focused on hindlimb function because hindlimb deficits are the earliest reported neurological sign of disease in SOD1-G93A mice. The protocol developed by ALS TDI provides an unbiased assessment of onset of paresis (slight or partial paralysis), progression and severity of paralysis and it is sensitive enough to identify drug-induced changes in disease progression. In this report, the combination of a detailed manuscript with video minimizes scoring ambiguities and inter-experimenter variability thus allowing for the protocol to be adopted by other laboratories and enabling comparisons between studies taking place at different settings. We believe that this video protocol can serve as an excellent training tool for present and future ALS researchers.

This video protocol describes a sensitive, reliable, and quick method for evaluating the neuromuscular deficits in a transgenic mouse model of amyotrophic lateral sclerosis.

The SOD1-G93A transgenic mouse is the most widely used animal model of amyotrophic lateral sclerosis (ALS). At ALS TDI we developed a phenotypic screening ...

A Novel Murine Model of Arteriovenous Fistula Failure: The Surgical Procedure in Detail

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the exact pathophysiology remains unknown. In order to unravel the pathophysiology a murine model of AVF-failure was developed in which the configuration of the anastomosis resembles the preferred situation in the clinical setting. A model was described in which an AVF is created by connecting the venous end of the branch of the external jugular vein to the side of the common carotid artery using interrupted sutures. At a histological level, we observed progressive stenotic intimal lesions in the venous outflow tract that is also seen in failed human AVFs. Although this procedure can be technically challenging due to the small dimensions of the animal, we were able to achieve a surgical success rate of 97% after sufficient training. The key advantage of a murine model is the availability of transgenic animals. In view of the different proposed mechanisms that are responsible for AVF failure, disabling genes that might play a role in vascular remodeling can help us to unravel the complex pathophysiology of AVF failure.

Here we present a murine model of arteriovenous fistula (AVF) failure in which a clinically relevant anastomotic configuration is incorporated. This model can be used to study the pathophysiology and to test possible therapeutic interventions.

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the ...

The Monoiodoacetate Model of Osteoarthritis Pain in the Mouse

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The current treatments for OA pain such as NSAIDS or opiates are neither sufficiently effective nor devoid of detrimental side effects. Animal models of OA are being developed to improve our understanding of OA-related pain mechanisms and define novel pharmacological targets for therapy. Currently available models of OA in rodents include surgical and chemical interventions into one knee joint. The monoiodoacetate (MIA) model has become a standard for modelling joint disruption in OA in both rats and mice. The model, which is easier to perform in the rat, involves injection of MIA into a knee joint that induces rapid pain-like responses in the ipsilateral limb, the level of which can be controlled by injection of different doses. Intra-articular injection of MIA disrupts chondrocyte glycolysis by inhibiting glyceraldehyde-3-phosphatase dehydrogenase and results in chondrocyte death, neovascularization, subchondral bone necrosis and collapse, as well as inflammation. The morphological changes of the articular cartilage and bone disruption are reflective of some aspects of patient pathology. Along with joint damage, MIA injection induces referred mechanical sensitivity in the ipsilateral hind paw and weight bearing deficits that are measurable and quantifiable. These behavioral changes resemble some of the symptoms reported by the patient population, thereby validating the MIA injection in the knee as a useful and relevant pre-clinical model of OA pain.
The aim of this article is to describe the methodology of intra-articular injections of MIA and the behavioral recordings of the associated development of hypersensitivity with a mind to highlight the necessary steps to give consistent and reliable recordings.

Osteoarthritis (OA), or degenerative joint disease, is a debilitating condition associated with pain that remains only partially controlled by available analgesics. Animal models are being developed to improve our understanding of OA-related pain mechanisms. Here we describe the methodology for the monoiodoacetate model of OA pain in the mouse.

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The ...

Topical Airway Anesthesia for Awake-endoscopic Intubation Using the Spray-as-you-go Technique with High Oxygen Flow

A patient&#39;s willingness to cooperate is an absolute precondition for successful awake intubation of the trachea. Whilst drug-sedation of patients can jeopardize their spontaneous breathing, topical anesthesia of the airway is a popular technique. The spray-as-you-go technique represents one of the simplest opportunities to anesthetize the airway mucosa. The application of local anesthetic through the working channel of the flexible endoscope is a widespread practice for anesthetists as well as pulmonologists. There is neither need for additional devices nor special training as a pre-requisite to perform this technique. However, a known clinical problem is the coughing and gagging reflex that may occur when the liquid anesthetic strikes the airway mucosa and other sensitive structures like the vocal cords. This can be avoided by the use of oxygen applied through the working channel with the aim of fogging the local anesthetic into finer particles. Furthermore, the oxygen flow provides a higher oxygen supply and contributes to a better view, dispersing mucus secretions and blood away from the lens. Using an atomizer with a high oxygen flow of 10 L/min we maximized these benefits, caused less coughing and had more satisfied and therefore cooperative patients. Possible, but very rare complications of using oxygen flow including gastric insufflation, organ rupture or barotrauma did not arise. We attribute the complication-free use of high oxygen flow to the design of the set, which permits flow and pressure release.

The topical anesthetic lidocaine was atomized using a high oxygen flow through the working channel of a flexible intubating endoscope to achieve topical airway anesthesia for awake endotracheal intubation. We prefer this modified spray-as-you-go technique for endoscopic intubation to classical bolus application because of higher patient satisfaction and better compliance.

A patient&#39;s willingness to cooperate is an absolute precondition for successful awake intubation of the trachea. Whilst drug-sedation of patients can ...