We would like to thank Emily Ricke, Kristen Uchtmann, and the Ricke lab for their assistance with animal husbandry and feedback on this manuscript. We would like to thank the NIDDK and NIEHS for their financial support for these studies: U54 DK104310 (WAR, JAM, PCM, CMV, DEB), R01 ES001332 (WAR, CMV), K12 DK100022 (TTL, AR-A, DH). The content is the sole responsibility of the authors and does not represent the official views of the NIH.

Procedures involving animal subjects have been approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Wisconsin &#8211; Madison.
1. Animal preparation
<ol>
	<li>Place a 24-month-old, C57Bl6/J male mouse in a pre-charged chamber with 3-5% isoflurane until the righting reflex is lost and the breathing rate slows.</li>
	<li>If necessary, use clippers to shave the abdominal hair from the animal for surgery and/or imaging. Remove all remaining hair using a depila...

<ol>
	<li>Kirby, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+natural+history+of+benign+prostatic+hyperplasia:+what+have+we+learned+in+the+last+decade.">The natural history of benign prostatic hyperplasia: what have we learned in the last decade.</a> Urology. 5, 3-6 (2000).</li><li>Berry, S. J., Coffey, D. S., Walsh, P. C., Ewing, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+human+benign+prostatic+hyperplasia+with+age.">The development of human benign prostatic hyperplasia with age.</a> Journal of Urology. 132 (3), 474-479 (1984).</li><li>Lotti, F., 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=Elevated+body+mass+index+correlates+with+higher+seminal+plasma+interleukin+8+levels+and+ultrasonographic+abnormalities+of+the+prostate+in+men+attending+an+andrology+clinic+for+infertility.">Elevated body mass index correlates with higher seminal plasma interleukin 8 levels and ultrasonographic abnormalities of the prostate in men attending an andrology clinic for infertility.</a> Journal of Endocrinological Investigation. 34 (10), 336-342 (2011).</li><li>Lotti, F., 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=Metabolic+syndrome+and+prostate+abnormalities+in+male+subjects+of+infertile+couples.">Metabolic syndrome and prostate abnormalities in male subjects of infertile couples.</a> Asian Journal of Andrology. 16 (2), 295-304 (2014).</li><li>Chute, C. G., 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+prevalence+of+prostatism:+a+population-based+survey+of+urinary+symptoms.">The prevalence of prostatism: a population-based survey of urinary symptoms.</a> Journal of Urology. 150 (1), 85-89 (1993).</li><li>Isaacs, J. T., Coffey, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Etiology+and+disease+process+of+benign+prostatic+hyperplasia.">Etiology and disease process of benign prostatic hyperplasia.</a> Prostate Supplemental. 2, 33-50 (1989).</li><li>Kortt, M. A., Bootman, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+economics+of+benign+prostatic+hyperplasia+treatment:+a+literature+review.">The economics of benign prostatic hyperplasia treatment: a literature review.</a> Clinical Therapeutics. 18 (6), 1227-1241 (1996).</li><li>Abrams, 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=Evaluation+and+treatment+of+lower+urinary+tract+symptoms+in+older+men.">Evaluation and treatment of lower urinary tract symptoms in older men.</a> Journal of Urology. 181 (4), 1779-1787 (2009).</li><li>Roehrborn, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Benign+prostatic+hyperplasia:+an+overview.">Benign prostatic hyperplasia: an overview.</a> Reviews Urology. 7, 3-14 (2005).</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> Neurourology and Urodynamics. 30 (5), 636-646 (2011).</li><li>Nicholson, T. 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=Estrogen+receptor-alpha+is+a+key+mediator+and+therapeutic+target+for+bladder+complications+of+benign+prostatic+hyperplasia.">Estrogen receptor-alpha is a key mediator and therapeutic target for bladder complications of benign prostatic hyperplasia.</a> Journal of Urology. 193 (2), 722-729 (2015).</li><li>Nicholson, T. 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=Testosterone+and+17beta-estradiol+induce+glandular+prostatic+growth,+bladder+outlet+obstruction,+and+voiding+dysfunction+in+male+mice.">Testosterone and 17beta-estradiol induce glandular prostatic growth, bladder outlet obstruction, and voiding dysfunction in male mice.</a> Endocrinology. 153 (11), 5556-5565 (2012).</li><li>Ricke, W. 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=In+Utero+and+Lactational+TCDD+Exposure+Increases+Susceptibility+to+Lower+Urinary+Tract+Dysfunction+in+Adulthood.">In Utero and Lactational TCDD Exposure Increases Susceptibility to Lower Urinary Tract Dysfunction in Adulthood.</a> Toxicological Sciences. 150 (2), 429-440 (2016).</li><li>Bell-Cohn, A., Mazur, D. J., Hall, C. C., Schaeffer, A. J., Thumbikat, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uropathogenic+Escherichia+coli-Induced+Fibrosis,+leading+to+Lower+Urinary+Tract+Symptoms,+is+associated+with+Type-2+cytokine+signaling.">Uropathogenic Escherichia coli-Induced Fibrosis, leading to Lower Urinary Tract Symptoms, is associated with Type-2 cytokine signaling.</a> American Journal of Physiology Renal Physiology. , (2019).</li><li>Wegner, K. 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=Void+spot+assay+procedural+optimization+and+software+for+rapid+and+objective+quantification+of+rodent+voiding+function,+including+overlapping+urine+spots.">Void spot assay procedural optimization and software for rapid and objective quantification of rodent voiding function, including overlapping urine spots.</a> American Journal of Physiology Renal Physiology. , (2018).</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> American Journal of Physiology Renal Physiology. 308 (12), 1369-1378 (2015).</li><li>Leung, Y. Y., Schwarz, E. M., Silvers, C. R., Messing, E. M., Wood, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uroflow+in+murine+urethritis.">Uroflow in murine urethritis.</a> Urology. 64 (2), 378-382 (2004).</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> Neurourology and Urodynamics. 29 (4), 603-608 (2010).</li><li>Khoo, S. W., Han, D. C. <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+ultrasound+in+vascular+procedures.">The use of ultrasound in vascular procedures.</a> Surgical Clinics of North America. 91 (1), 173-184 (2011).</li><li>Hunter, L. E., Simpson, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prenatal+screening+for+structural+congenital+heart+disease.">Prenatal screening for structural congenital heart disease.</a> Nature Reviews Cardiology. 11 (6), 323-334 (2014).</li><li>Hammoud, G. M., Ibdah, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+of+endoscopic+ultrasound+in+patients+with+portal+hypertension.">Utility of endoscopic ultrasound in patients with portal hypertension.</a> World Journal of Gastroenterology. 20 (39), 14230-14236 (2014).</li><li>Sikes, R. A., Thomsen, S., Petrow, V., Neubauer, B. L., Chung, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+experimentally+induced+mouse+prostatic+hyperplasia+by+castration+or+steroid+antagonist+administration.">Inhibition of experimentally induced mouse prostatic hyperplasia by castration or steroid antagonist administration.</a> Biology of Reproduction. 43 (2), 353-362 (1990).</li><li>Mizoguchi, S., 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+Estrogen+Receptor+beta+Stimulation+in+a+Rat+Model+of+Non-Bacterial+Prostatic+Inflammation.">Effects of Estrogen Receptor beta Stimulation in a Rat Model of Non-Bacterial Prostatic Inflammation.</a> Prostate. 77 (7), 803-811 (2017).</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> Journal of Urology. 164 (4), 1385-1389 (2000).</li></ol>

The authors have nothing to disclose

Current techniques for evaluating the lower urinary tract of rodents are limited by their ability to directly correlate changes in voiding physiology with changes in prostatic histology consequent to disease progression. Void spot assays and uroflowmetry can be used to assess spontaneous urination events in rodents, and these techniques can be used to evaluate changes over a period of time15,16,17. However, for both techniques, ...

Benign prostatic hyperplasia (BPH) is a disease that develops in men as they age and affects nearly 90% of men over 80 years of age1,2. Although the development of BPH is generally associated with aging, other factors including obesity and metabolic syndrome can lead to BPH in relatively younger men3,4. Many men with BPH develop lower urinary tract symptoms (LUTS) that significantly decrease their quality of life, and some experience complications that may include bleeding, infection, bladder outlet obstruction (BOO), bladder stones, and renal failure. The cost of treatment for BPH exceeds $4 billion annually5,6,7. Diagnosis of LUTS caused by BPH generally relies on the use of the AUA symptom index (AUASI) score, uroflowmetry, and assessment of prostate size8. The etiology of BPH/LUTS is complex and multifactorial, and disease development and progression has been associated with prostatic hyperplasia (prostate proliferation), smooth muscle contractility, and fibrosis. Current treatments include the use of &#945;-adrenergic blockers to regulate smooth muscle tone within the bladder and prostate to alleviate LUTS and/or 5&#945;-reductase inhibitors to decrease androgen metabolism and decrease prostate size. Better disease models, murine and other, to allow the accurate study of the effects of varied causative and therapeutic factors in this disease process over time is highly desirable9.
Rodent models have been extensively used to study urodynamics; however, most studies are focused on female micturition and disease10. In order to fully examine all aspects of male LUTS, rodent models have been developed and used to study different aspects of BPH including changes in cellular proliferation, smooth muscle function, collagen deposition, and inflammation11,12,13,14. However, rodent and human prostate anatomy differ. While the human prostate is compact and encased by a condensed fibromuscular layer, the rodent prostate is lobular; and these differences complicate direct comparisons of disease progression and treatment efficacy. Additionally, LUTS are difficult to assess in mice, since it is not possible to directly measure bother. Instead, current methods for studying disease correlate histological features with physiological features (i.e., bladder volume and wall thickness with uroflowmetry, void spot assays, and cystometry endpoint data) that compare the level of urinary dysfunction between BPH model and control animals12,15,16,17,18. Physiological features are frequently evaluated as post-mortem necropsy endpoints, and there is an inability within the same animal to observe BOO across time. Recently, we have identified a subdivision of the pelvic urethra (the prostatic urethra) where exogenous hormone implants cause a narrowing based on post-mortem necropsy assessments12. Current methods do not allow for the direct, in vivo assessment of urethral narrowing during voiding.
Ultrasound is a non-invasive diagnostic and evaluation technique that has successfully been used in other disease models. It is used to quantify organ volume and assess vascular flow19,20,21. Ultrasound is also used to visualize and guide microinjections, allowing for targeted injections of stem cells or other drugs, and to evaluate systolic and diastolic cardiac function.
This protocol describes the use of high frequency ultrasound to evaluate lower urinary tract anatomy and assess urinary physiology in anesthetized mice. We describe the use of ultrasound for measuring bladder volume and wall thickness. We also describe the use of contrast-enhanced ultrasound to measure urine velocity, urine volume, void duration, and urethra diameter. The use of ultrasound provides a more comprehensive understanding of the lower urinary tract in vivo, determines how disease alters normal voiding function, and gives us the tools to better evaluate the effectiveness of new therapeutic options. Currently, the non-contrast imaging protocol is non-terminal, while the current contrast-enhanced imaging protocol is a terminal procedure.

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			<td height="42" style="height:42px;width:128px;">21mm Clear Tubing</td>
			<td style="width:184px;">Supera Anesthesia Innov</td>
			<td style="width:119px;">301-150</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">27 gauge needle</td>
			<td style="width:184px;">BD</td>
			<td style="width:119px;">Z192376</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">4 port Manifold</td>
			<td style="width:184px;">Supera Anesthesia Innov</td>
			<td style="width:119px;">RES536</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">DEFINITY</td>
			<td style="width:184px;">Lantheus Medical Imaging</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">F/AIR Canister</td>
			<td style="width:184px;">Supera Anesthesia Innov</td>
			<td style="width:119px;">80120</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:128px;">Graefe forceps (Serrated, Straight)</td>
			<td style="width:184px;">F.S.T.</td>
			<td style="width:119px;">11050-10</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:128px;">Inlet/Outlet Fittings</td>
			<td style="width:184px;">Supera Anesthesia Innov</td>
			<td style="width:119px;">VAP203/4</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Isoflurane</td>
			<td style="width:184px;">Midwest Vet Supply</td>
			<td style="width:119px;">193.33161.3</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:128px;">Isoflurane Vaporizer</td>
			<td style="width:184px;">Supera Anesthesia Innov</td>
			<td style="width:119px;">VAP3000</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">MV707 probe</td>
			<td style="width:184px;">Fujifilm VisualSonics Inc</td>
			<td style="width:119px;"></td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Oxygen Flowmeter</td>
			<td style="width:184px;">Supera Anesthesia Innov</td>
			<td style="width:119px;">OXY660</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:128px;">Polyethylene 50 tubing</td>
			<td style="width:184px;">BD</td>
			<td style="width:119px;">427516</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:128px;">Pressure Reg/Gauge</td>
			<td style="width:184px;">Supera Anesthesia Innov</td>
			<td style="width:119px;">OXY508</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:128px;">Rebreathing Circuits</td>
			<td style="width:184px;">Supera Anesthesia Innov</td>
			<td style="width:119px;">CIR529</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:128px;">Small Mice Nose Cone</td>
			<td style="width:184px;">Supera Anesthesia Inov</td>
			<td style="width:119px;">ACC526</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Sterile saline</td>
			<td style="width:184px;">Midwest Vet Supply</td>
			<td style="width:119px;">193.74504.3</td>
			<td style="width:147px;">NaCl 0.9%, Injectable</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:128px;">Straight Sharp/Blunt Scissors</td>
			<td style="width:184px;">Fine Scientific Tools (F.S.T)</td>
			<td style="width:119px;">14054-13</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Syringe</td>
			<td style="width:184px;">BD</td>
			<td style="width:119px;">309646</td>
			<td style="width:147px;">5mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Vevo 770</td>
			<td style="width:184px;">Fujifilm VisualSonics Inc</td>
			<td style="width:119px;"></td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:128px;">VIALMIX</td>
			<td style="width:184px;">Lantheus Medical Imaging</td>
			<td style="width:119px;">VMIX</td>
			<td style="width:147px;"></td>
		</tr>
	</tbody>
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ultrasonography of the adult male urinary tract for urinary functional testing

The incidence of clinical benign prostatic hyperplasia (BPH) and lower urinary tract symptoms (LUTS) is increasing due to the aging population, resulting in a significant economic and quality of life burden. Transgenic and other mouse models have been developed to recreate various aspects of this multifactorial disease; however, methods to accurately quantitate urinary dysfunction and the effectiveness of new therapeutic options are lacking. Here, we describe a method that can be used to measure bladder volume and detrusor wall thickness, urinary velocity, void volume and void duration, and urethral diameter. This would allow for the evaluation of disease progression and treatment efficacy over time. Mice were anesthetized with isoflurane, and the bladder was visualized by ultrasound. For non-contrast imaging, a 3D image was taken of the bladder to calculate volume and evaluate shape; the bladder wall thickness was measured from this image. For contrast-enhanced imaging, a catheter was placed through the dome of the bladder using a 27-gauge needle connected to a syringe by PE50 tubing. A bolus of 0.5 mL of contrast was infused into the bladder until a urination event occurred. Urethral diameter was determined at the point of the Doppler velocity sample window during the first voiding event. Velocity was measured for each subsequent event yielding a flow rate. In conclusion, high frequency ultrasound proved to be an effective method for assessing bladder and urethral measurements during urinary function in mice. This technique may be useful in the assessment of novel therapies for BPH/LUTS in an experimental setting.

Ultrasound can be used with or without contrast enhancement depending on the experimental design and endpoint measurement. Mice are anesthetized with isoflurane and shaved and all traces of hair removed with a depilatory cream. Anesthetized animals are placed on a heated platform with the ultrasound probe positioned along the long axis of the bladder (Figure 1).
Figure 2 shows representative ultrasound images of a mouse bladder acquir...

Watch this Scientific Journal Video about Ultrasonography of the Adult Male Urinary Tract for Urinary Functional Testing at JoVE.com

Ultrasonography of the Adult Male Urinary Tract for Urinary Functional Testing

We describe the use of high frequency ultrasound with contrast imaging as a method to measure bladder volume, bladder wall thickness, urine velocity, void volume, void duration, and urethral diameter. This strategy can be used to assess voiding dysfunction and treatment efficacy in various mouse models of lower urinary tract dysfunction (LUTD).

The incidence of clinical benign prostatic hyperplasia (BPH) and lower urinary tract symptoms (LUTS) is increasing due to the aging population, resulting ...

This protocol uses high-frequency ultrasonography to evaluate the changes in the bladder and lower urinary tract in mice. This technique allows a longitudinal examination of the urinary tract in vivo for anatomical and physiological measurements and is complementary to other techniques for measuring urinary flow. The ability to assess in vivo voiding changes in rodent models that recapitulate aspects of human urological diseases can provide insight into disease progression and treatment efficacy.Begin by connecting a probe with a center frequency of 30 megahertz to the active port of the ultrasound system and presetting the application to abdominal imaging. For optimum contrast enhancement, shake an appropriate contrast agent in a vortex mixer for 45 seconds to encapsulate the microbubbles in solution. After confirming a lack of response to toe pinch, place an anesthetized 24-week-old C57 black 6 male mouse in the supine position on a heated platform and shave the hair from the abdomen.Remove any remaining hair with depilatory cream and position the ultrasound probe parallel to the long axis of the bladder. Then using the XY manipulator to move the mouse as necessary, obtain long and short axis images of the bladder. Before imaging, make a midline incision through the skin and abdominal wall to expose the bladder and pre-fill a piece of flexible polyethylene tubing with saline.Then connect a syringe equipped with a 27 gauge needle to the piece of tubing and insert the needle into the bladder. To confirm correct placement of the needle, instill 10 microliters of saline into the bladder. Replace the saline syringe with a syringe containing the vortexed contrast agent and collect another B-mode image of the catheterized bladder.To facilitate measurement of the urethra diameter, instill a 0.5 milliliter bolus of microbubbles over a three-second period into the bladder until a urination event occurs. To facilitate measurement of the velocity, correct the Doppler sample window angle until it is parallel to the urine flow and instill a second bolus of microbubbles into the bladder. Then collect a 3D image of the full bladder.When all of the images have been obtained, trace the outside to inside edges of the bladder wall and use the linear distance measurement tool to measure the bladder. Use the volumetric tool to trace the inside of the bladder walls to create a contour. Multiple contours can then be generated through the thickness of the bladder to calculate the volume of the bladder in the 3D acquisition mode.For the first voiding event, use the linear distance tool to measure the urethral diameter edge to edge at the point of the Doppler velocity sample window. For the first voiding event, use the velocity time integral tool to measure the urethral velocity. Here, representative ultrasound images of a mouse bladder acquired without contrast agent are shown.The bladder wall is echogenic and the bladder wall thickness can be measured using a software measurement package. A bladder size and shape can be rendered in 3D to determine bladder volume. Here, a representative image of a microbubble filled echogenic bladder is shown.After low frequency ultrasonic burst, the bubbles are destroyed and the bladder becomes transiently echogenic before the bubbles reform confirming the structure as the bladder. During a urination event, the urethral lumen diameter can be acquired during urination along with the flow velocity of the urine passing through that region of the urethra. Using contrast imaging, diameter measurements can be made throughout the entire visible length of the urethra.From these measurements, further calculations can be made to assess the urinary flow and bladder compliance. Activating the contrast agent correctly is essential to the success of this protocol. We can reconstruct rodent urinary tracts with MRI and microcomputed tomography to confirm the ultrasound flowmetry as well as introduce new complex models of urologic flow dynamics and 3D reconstruction.We have begun using this technique to quantitatively examine models of lower urinary tract disease, the effects of aging, and the efficacy of therapeutic treatments on urinary function.

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JoVE Journal

Medicine

18.8K 조회수.  University of Wisconsin-Madison. 이 프로토콜은 고주파 초음파 를 사용하여 방광의 변화를 평가하고 쥐의 요로를 낮춥니다. 이 기술은 해부학적 및 생리적 측정을 위해 생체 내의 요로의 세로 검사를 허용하고 요로 흐름을 측정하기위한 다른 기술에 보완적입니다. 인간 비뇨기과 질환의 측면을 재구성 하는 설치류 모델의 생체 무효 변화를 평가 하는 능력은 질병 진행 및 치료 효능에 대 한 통찰력을 제공할 ...