Name: ultrasonography of the adult male urinary tract for urinary functional testing
Uploaded: 2019-08-14T13:30:00
Description: Watch this Scientific Journal Video about Ultrasonography of the Adult Male Urinary Tract for Urinary Functional Testing at JoVE.com

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. 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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. 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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>
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			<td height="21" style="height:21px;width:128px;">27 gauge needle</td>
			<td style="width:184px;">BD</td>
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			<td height="21" style="height:21px;width:128px;">DEFINITY</td>
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			<td height="63" style="height:63px;width:128px;">Graefe forceps (Serrated, Straight)</td>
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			<td style="width:119px;">11050-10</td>
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			<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>
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			<td style="width:184px;">Supera Anesthesia Innov</td>
			<td style="width:119px;">VAP3000</td>
			<td style="width:147px;"></td>
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			<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>
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			<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>
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		<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>
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		<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>
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			<td height="42" style="height:42px;width:128px;">Rebreathing Circuits</td>
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			<td style="width:119px;">CIR529</td>
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			<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>
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			<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>
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			<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>
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			<td style="width:119px;">309646</td>
			<td style="width:147px;">5mL</td>
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			<td height="21" style="height:21px;width:128px;">Vevo 770</td>
			<td style="width:184px;">Fujifilm VisualSonics Inc</td>
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			<td height="22" style="height:22px;width:128px;">VIALMIX</td>
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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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Research

JoVE Journal

Medicine

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 1. Because ageing is the most important risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson&#039;s Disease

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

Probe-based Confocal Laser Endomicroscopy of the Urinary Tract: The Technique

Probe-based confocal laser endomicroscopy (CLE) is an emerging optical imaging technology that enables real-time in vivo microscopy of mucosal surfaces during standard endoscopy. With applications currently in the respiratory1 and gastrointestinal tracts,2-6 CLE has also been explored in the urinary tract for bladder cancer diagnosis.7-10 Cellular morphology and tissue microarchitecture can be resolved with micron scale resolution in real time, in addition to dynamic imaging of the normal and pathological vasculature.7
The probe-based CLE system (Cellvizio, Mauna Kea Technologies, France) consists of a reusable fiberoptic imaging probe coupled to a 488 nm laser scanning unit. The imaging probe is inserted in the working channels of standard flexible and rigid endoscopes. An endoscope-based CLE system (Optiscan, Australia), in which the confocal endomicroscopy functionality is integrated onto the endoscope, is also used in the gastrointestinal tract. Given the larger scope diameter, however, application in the urinary tract is currently limited to ex vivo use.11 Confocal image acquisition is done through direct contact of the imaging probe with the target tissue and recorded as video sequences. As in the gastrointestinal tract, endomicroscopy of the urinary tract requires an exogenenous contrast agent&mdash;most commonly fluorescein, which can be administered intravenously or intravesically. Intravesical administration is a well-established method to introduce pharmacological agents locally with minimal systemic toxicity that is unique to the urinary tract. Fluorescein rapidly stains the extracellular matrix and has an established safety profile.12 Imaging probes of various diameters enable compatibility with different caliber endoscopes. To date, 1.4 and 2.6 mm probes have been evaluated with flexible and rigid cystoscopy.10 Recent availability of a &lt; 1 mm imaging probe13 opens up the possibility of CLE in the upper urinary tract during ureteroscopy. Fluorescence cystoscopy (i.e. photodynamic diagnosis) and narrow band imaging are additional endoscope-based optical imaging modalities14 that can be combined with CLE to achieve multimodal imaging of the urinary tract. In the future, CLE may be coupled with molecular contrast agents such as fluorescently labeled peptides15 and antibodies for endoscopic imaging of disease processes with molecular specificity.

Probe-based confocal laser endomicroscopy enables real-time microscopy of the human urinary tract during cystoscopy, providing dynamic, intravital imaging of pathological states such as bladder cancer with cellular resolution. Endomicroscopy may augment the diagnostic accuracy of standard white light endoscopy and provide intraoperative image guidance to improve surgical resection.

Probe-based  confocal laser endomicroscopy (CLE) is an emerging optical imaging technology  that enables real-time in vivo microscopy of mucosal surfaces ...

A Modified Precipitation Method to Isolate Urinary Exosomes

Identification of biomarkers that allow early detection of kidney diseases in urine and plasma has been an area of active interest for several years. Urinary exosome vesicles, 40-100 nm in size, are released into the urine under normal conditions by cells from all nephron segments and may contain protein, mRNA and microRNA representative of their cell type of origin. Under conditions of renal dysfunction or injury, exosomes may contain altered proportions of these components, which may serve as biomarkers for disease. There are currently several methods available for isolation of urinary exosomes, and we have previously conducted an experimental comparison of each of these approaches, including three based on ultracentrifugation, one using a nanomembrane ultrafiltration concentrator, one using a commercial precipitation reagent and one using a modification of the precipitation technique using ExoQuick reagent that we developed in our laboratory. We found the modified precipitation method produced the highest yield of exosome particles, miRNA, and mRNA, making this approach suitable for the isolation of exosomes for subsequent RNA profiling. We conclude that the modified exosome precipitation method offers a quick, scalable, and effective alternative for the isolation of exosomes from urine. In this report, we describe our modified precipitation technique using ExoQuick reagent for isolating exosomes from human urine.

This manuscript describes a protocol for isolating exosomes from human urine using a modified precipitation technique.

Identification of biomarkers that allow early detection of kidney diseases in urine and plasma has been an area of active interest for several years. ...

Urinary Bladder Distention Evoked Visceromotor Responses as a Model for Bladder Pain in Mice

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized by increased urgency and frequency of urination, as well as nocturia and general pelvic pain, especially upon bladder filling or voiding. Despite years of research, the cause of IC/BPS remains elusive and treatment strategies are unable to provide complete relief to patients. In order to study nervous system contributions to the condition, many animal models have been developed to mimic the pain and symptoms associated with IC/BPS. One such murine model is urinary bladder distension (UBD). In this model, compressed air of a specific pressure is delivered to the bladder of a lightly anesthetized animal over a set period of time. Throughout the procedure, wires in the superior oblique abdominal muscles record electrical activity from the muscle. This activity is known as the visceromotor response (VMR) and is a reliable and reproducible measure of nociception. Here, we describe the steps necessary to perform this technique in mice including surgical manipulations, physiological recording, and data analysis. With the use of this model, the coordination between primary sensory neurons, spinal cord secondary afferents, and higher central nervous system areas involved in bladder pain can be unraveled. This basic science knowledge can then be clinically translated to treat patients suffering from IC/BPS.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized in part by pelvic pain. In order to study nervous system contributions to the condition, a physiological model of urinary bladder pain is used in mice and rats.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition ...

Bladder Smooth Muscle Strip Contractility as a Method to Evaluate Lower Urinary Tract Pharmacology

We describe an in vitro method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties of the smooth muscle as well as changes induced by pathology. This method provides critical information for understanding bladder function while overcoming major methodological difficulties encountered in in vivo experiments, such as surgical and pharmacological manipulations that affect stability and survival of the preparations, the use of human tissue, and/or the use of expensive chemicals. It also provides a way to investigate the properties of each bladder component (i.e. smooth muscle, mucosa, nerves) in healthy and pathological conditions.
The urinary bladder is removed from an anesthetized animal, placed in Krebs solution and cut into strips. Strips are placed into a chamber filled with warm Krebs solution. One end is attached to an isometric tension transducer to measure contraction force, the other end is attached to a fixed rod. Tissue is stimulated by directly adding compounds to the bath or by electric field stimulation electrodes that activate nerves, similar to triggering bladder contractions in vivo. We demonstrate the use of this method to evaluate spontaneous smooth muscle contractility during development and after an experimental spinal cord injury, the nature of neurotransmission (transmitters and receptors involved), factors involved in modulation of smooth muscle activity, the role of individual bladder components, and species and organ differences in response to pharmacological agents. Additionally, it could be used for investigating intracellular pathways involved in contraction and/or relaxation of the smooth muscle, drug structure-activity relationships and evaluation of transmitter release.
The in vitro smooth muscle contractility method has been used extensively for over 50 years, and has provided data that significantly contributed to our understanding of bladder function as well as to pharmaceutical development of compounds currently used clinically for bladder management.

This manuscript presents a simple, yet powerful, in vitro method for evaluating smooth muscle contractility in response to pharmacological agents or nerve stimulation. Main applications are drug screening and understanding tissue physiology, pharmacology,&#160;and pathology.

We describe an in vitro method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties ...

Combined Intravital Microscopy and Contrast-enhanced Ultrasonography of the Mouse Hindlimb to Study Insulin-induced Vasodilation and Muscle Perfusion

It has been demonstrated that insulin's vascular actions contribute to regulation of insulin sensitivity. Insulin's effects on muscle perfusion regulate postprandial delivery of nutrients and hormones to insulin-sensitive tissues. We here describe a technique for combining intravital microscopy (IVM) and contrast-enhanced ultrasonography (CEUS) of the adductor compartment of the mouse hindlimb to simultaneously visualize muscle resistance arteries and perfusion of the microcirculation in vivo. Simultaneously assessing insulin's effect at multiple levels of the vascular tree is important to study relationships between insulin's multiple vasoactive effects and muscle perfusion. Experiments in this study were performed in mice. First, the tail vein cannula is inserted for the infusion of anesthesia, vasoactive compounds and ultrasound contrast agent (lipid-encapsulated microbubbles). Second, a small incision is made in the groin area to expose the arterial tree of the adductor muscle compartment. The ultrasound probe is then positioned at the contralateral upper hindlimb to view the muscles in cross-section. To assess baseline parameters, the arterial diameter is assessed and microbubbles are subsequently infused at a constant rate to estimate muscle blood flow and microvascular blood volume (MBV). When applied before and during a hyperinsulinemic-euglycemic clamp, combined IVM and CEUS allow assessment of insulin-induced changes of arterial diameter, microvascular muscle perfusion and whole-body insulin sensitivity. Moreover, the temporal relationship between responses of the microcirculation and the resistance arteries to insulin can be quantified. It is also possible to follow-up the mice longitudinally in time, making it a valuable tool to study changes in vascular and whole-body insulin sensitivity.

Insulin-induced vasodilation regulates muscle perfusion and increases the microvascular surface area (microvascular recruitment) available for solute exchange between blood and tissue interstitium. Combined intravital microscopy and contrast-enhanced ultrasonography is presented to simultaneously assess insulin&#39;s action at the larger vessels and the microcirculation in vivo.

It has been demonstrated that insulin's vascular actions contribute to regulation of insulin sensitivity. Insulin's effects on muscle perfusion regulate ...

Ultrasonography in Experimental Reproductive Investigations on Rats

With the development of assisted reproductive technology and the ethical limitations of research on humans, rat animal models have been widely used in reproductive medicine. In the past, the study of reproductive system development in rodents has been based on one-time histological examination of excised tissues. Recently, with the development of high-resolution transabdominal ultrasound, high-quality sonography can now be performed to evaluate the reproductive organs of rats, allowing a new method for studying the reproductive system. Images were obtained using a high-resolution ultrasonographic system. Gynecological ultrasonography was performed on 28 eight-week-old non-pregnant rats and 5 pregnant Sprague-Dawley rats. We describe how to recognize organs of the reproductive system and associated structures in typical views during different phases of the estrus cycle. Color flow Doppler was used to measure uterine artery blood flow and evaluate uterine blood flow pattern changes during different stages of pregnancy. We have demonstrated that ultrasound exploration is a useful method for evaluating changes in internal reproductive organs. Its use raises the possibility of conducting additional experiments, including medical or surgical procedures, and provides the ability to monitor sonographic changes to internal organs without sacrificing animals.

This manuscript describes the utility of ultrasound performed on female rats to design experimental models for reproductive and gynecological investigation. A step-by-step explanation of how to perform ultrasonographic evaluation is shown.

With the development of assisted reproductive technology and the ethical limitations of research on humans, rat animal models have been widely used in ...

Muscle Imbalances: Testing and Training Functional Eccentric Hamstring Strength in Athletic Populations

Many hamstring injuries that occur during physical activity occur while the muscles are lengthening, during eccentric hamstring muscle actions. Opposite of these eccentric hamstring actions are concentric quadriceps actions, where the larger and likely stronger quadriceps straighten the knee. Therefore, to stabilize the lower limbs during movement, the hamstrings must eccentrically combat against the strong knee-straightening torque of the quadriceps. As such, eccentric hamstring strength expressed relative to concentric quadricep strength is commonly referred to as the &#34;functional ratio&#34; as most movements in sports require simultaneous concentric knee extension and eccentric knee flexion. To increase the strength, resiliency, and functional performance of the hamstrings, it is necessary to test and train the hamstrings at different eccentric speeds. The main purpose of this work is to provide instructions for measuring and interpreting eccentric hamstring strength. Techniques for measuring the functional ratio using isokinetic dynamometry are provided and sample data will be compared. Additionally, we briefly describe how to address hamstring strength deficiencies or unilateral strength differences using exercises that specifically focus on increasing eccentric hamstring strength.

The hamstrings are a group of muscles that are sometimes problematic for athletes, resulting in soft tissue injury in the lower limbs. To prevent such injuries, functional training of the hamstrings requires intensive eccentric contractions. Additionally, hamstring function should be tested in relation to quadricep function at different contraction speeds.

Many hamstring injuries that occur during physical activity occur while the muscles are lengthening, during eccentric hamstring muscle actions. Opposite ...

Technical Modification of the Terminal Ureter During Total Transperitoneal Laparoscopic Nephroureterectomy for Upper Urinary Tract Urothelial Carcinoma

Upper tract urothelial carcinoma (UTUC) accounts for 5%&ndash;10% of all urothelial tumors. Radical nephroureterectomy is the standard treatment procedure. At present, different choices still exist for treating the ureteral end during laparoscopic ureteral bladder sleeve resection. Our center has adopted a new method for treating the ureteral end. This new method can increase the operating space and reduce the difficulty of the surgery compared with current methods.

Here, we present a protocol to increase the surgical field of view and reduce the difficulty of total transperitoneal laparoscopic nephroureterectomy surgery by precutting the umbilical ligament before treating the terminal ureter.

Upper tract urothelial carcinoma (UTUC) accounts for 5%&ndash;10% of all urothelial tumors. Radical nephroureterectomy is the standard treatment ...

Estimation of Urinary Nanocrystals in Humans using Calcium Fluorophore Labeling and Nanoparticle Tracking Analysis

Kidney stones are becoming more prevalent worldwide in adults and children. The most common type of kidney stone is comprised of calcium oxalate (CaOx) crystals. Crystalluria occurs when urine becomes supersaturated with minerals (e.g., calcium, oxalate, phosphate) and precedes kidney stone formation. Standard methods to assess crystalluria in stone formers include microscopy, filtration, and centrifugation. However, these methods primarily detect microcrystals and not nanocrystals. Nanocrystals have been suggested to be more harmful to kidney epithelial cells than microcrystals in vitro. Here, we describe the ability of Nanoparticle Tracking analysis (NTA) to detect human urinary nanocrystals. Healthy adults were fed a controlled oxalate diet prior to drinking an oxalate load to stimulate urinary nanocrystals. Urine was collected for 24 hours before and after the oxalate load. Samples were processed and washed with ethanol to purify samples. Urinary nanocrystals were stained with the calcium binding fluorophore, Fluo-4 AM. After staining, the size and count of nanocrystals were determined using NTA. The findings from this study show NTA can efficiently detect nanocrystalluria in healthy adults. These findings suggest NTA could be a valuable early detection method of nanocrystalluria in patients with kidney stone disease.

The objective of this study was to determine whether nanoparticle tracking analysis (NTA) could detect and quantify urinary calcium containing nanocrystals from healthy adults. The findings from the current study suggest NTA could be a potential tool to estimate urinary nanocrystals during kidney stone disease.

Kidney stones are becoming more prevalent worldwide in adults and children. The most common type of kidney stone is comprised of calcium oxalate (CaOx) ...