Name: intracavernosal pressure recording to evaluate erectile function in rodents
Uploaded: 2018-06-06T12:30:00
Description: Watch this Scientific Journal Video about Intracavernosal Pressure Recording to Evaluate Erectile Function in Rodents at JoVE.com

This work was supported by the Fundamental Research Funds for the Central Universities (020814380018, 020814380077), The China Scholarship Council (CSC, No. 201606195024), Natural Science Foundation of Jiangsu Province (BK20160138), and Key Project supported by Science and Technology Development Foundation, Nanjing Medical University (2014NJMUZD053).

Three-month-old and 18-month-old Sprague-Dawley rats were used in the present study. All animals were handled in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals. Procedures involving animal subjects were approved by the local Institutional Animal Care and Ethics Committee, with an effort to minimize animal suffering. The protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at Nanjing Tech University (Nanjing, China).
The rats were div...

<ol>
	<li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Consensus+Conference.+Impotence.+NIH+Consensus+Development+Panel+on+Impotence.">NIH Consensus Conference. Impotence. NIH Consensus Development Panel on Impotence.</a> JAMA. 270 (1), 83-90 (1993).</li><li>Chung, E., De Young, L., Brock, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigative+models+in+erectile+dysfunction:+a+state-of-the-art+review+of+current+animal+models.">Investigative models in erectile dysfunction: a state-of-the-art review of current animal models.</a> J Sex Med. 8 (12), 3291-3305 (2011).</li><li>Lue, T. F., Takamura, T., Schmidt, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamics+of+erection+in+the+monkey.">Hemodynamics of erection in the monkey.</a> J Urol. 130 (6), 1237-1241 (1983).</li><li>Carati, C. J., Creed, K. E., Keogh, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autonomic+control+of+penile+erection+in+the+dog.">Autonomic control of penile erection in the dog.</a> J Physiol. 384, 525-538 (1987).</li><li>Steers, W. D., Mallory, B., de Groat, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+study+of+neural+activity+in+penile+nerve+of+the+rat.">Electrophysiological study of neural activity in penile nerve of the rat.</a> Am J Physiol. 254 (6 Pt 2), R989-R1000 (1988).</li><li>Kapoor, M. S., Khan, S. A., Gupta, S. K. <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+of+erectile+dysfunction.">Animal models of erectile dysfunction.</a> J Pharmacol Toxicol Methods. 76, 43-54 (2015).</li><li>Burnett, A. L., Lowenstein, C. J., Bredt, 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=Nitric+oxide:+A+physiologic+mediator+of+penile+erection.">Nitric oxide: A physiologic mediator of penile erection.</a> Science. 257 (5068), 401-403 (1992).</li><li>Oh, T. Y., Kang, K. K., Ahn, B. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erectogenic+effect+of+the+selective+phosphodiesterase+type+5+inhibitor,+DA-8159.">Erectogenic effect of the selective phosphodiesterase type 5 inhibitor, DA-8159.</a> Arch Pharm Res. 23 (5), 471-476 (2000).</li><li>Ouyang, B., Sun, X., Han, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+urine-derived+stem+cells+alone+or+genetically-modified+with+FGF2+Improve+type+2+diabetic+erectile+dysfunction+in+a+rat+model.">Human urine-derived stem cells alone or genetically-modified with FGF2 Improve type 2 diabetic erectile dysfunction in a rat model.</a> PLoS One. 9 (3), e92825 (2014).</li><li>Pan, F., Xu, J., Zhang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+the+MicroRNA+profile+in+aging+rats+with+erectile+dysfunction.">Identification and characterization of the MicroRNA profile in aging rats with erectile dysfunction.</a> J Sex Med. 11 (7), 1646-1656 (2014).</li><li>Cho, M. C., Park, K., Kim, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+of+erectile+function+by+suppression+of+corporal+apoptosis,+fibrosis+and+corporal+veno-occlusive+dysfunction+with+rho-kinase+inhibitors+in+a+rat+model+of+cavernous+nerve+injury.">Restoration of erectile function by suppression of corporal apoptosis, fibrosis and corporal veno-occlusive dysfunction with rho-kinase inhibitors in a rat model of cavernous nerve injury.</a> J Urol. 193 (5), 1716-1723 (2015).</li><li>Hannan, J. L., Matsui, H., Sopko, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspase-3+dependent+nitrergic+neuronal+apoptosis+following+cavernous+nerve+injury+is+mediated+via+RhoA+and+ROCK+activation+in+major+pelvic+ganglion.">Caspase-3 dependent nitrergic neuronal apoptosis following cavernous nerve injury is mediated via RhoA and ROCK activation in major pelvic ganglion.</a> Sci Rep. 6, 29416 (2016).</li><li>Heaton, J. P., Varrin, S. J., Morales, 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+characterization+of+a+bio-assay+of+erectile+function+in+a+rat+model.">The characterization of a bio-assay of erectile function in a rat model.</a> J Urol. 145 (5), 1099-1102 (1991).</li><li>Mehta, N., Sikka, S., Rajasekaran, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+as+an+animal+model+for+male+erectile+function+evaluation+in+sexual+medicine+research.">Rat as an animal model for male erectile function evaluation in sexual medicine research.</a> J Sex Med. 5 (6), 1278-1283 (2008).</li><li>Quinlan, D. M., Nelson, R. J., Partin, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rat+as+a+model+for+the+study+of+penile+erection.">The rat as a model for the study of penile erection.</a> J Urol. 141 (3), 656-661 (1989).</li><li>Albersen, M., Orabi, H., Lue, T. F. <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+erectile+dysfunction+in+the+aging+male:+A+mini-review.">Evaluation and treatment of erectile dysfunction in the aging male: A mini-review.</a> Gerontology. 58 (1), 3-14 (2012).</li><li>Hedlund, P., Matsumoto, K., 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=Animal+Models+of+Erectile+Dysfunction.">Animal Models of Erectile Dysfunction.</a> Curr Prot Pharmacol. 29, 5.41.1-5.41.22 (2005).</li><li>Giuliano, F., Bernabe, J., Rampin, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telemetric+monitoring+of+intracavernous+pressure+in+freely+moving+rats+during+copulation.">Telemetric monitoring of intracavernous pressure in freely moving rats during copulation.</a> J Urol. 152 (4), 1271-1274 (1994).</li><li>Bernabe, J., Rampin, O., Giuliano, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracavernous+pressure+changes+during+reflexive+penile+erections+in+the+rat.">Intracavernous pressure changes during reflexive penile erections in the rat.</a> Physiol Behav. 57 (5), 837-841 (1995).</li><li>Bernabe, J., Rampin, O., Sachs, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracavernous+pressure+during+erection+in+rats:+an+integrative+approach+based+on+telemetric+recording.">Intracavernous pressure during erection in rats: an integrative approach based on telemetric recording.</a> Am J Physiol. 276 (2 Pt 2), R441-R449 (1999).</li><li>Adachi, H., Kodama, K., Ishihara, H. <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+erectile+response+by+continuous+measurement+of+penile+diameter+in+rats.">Evaluation of erectile response by continuous measurement of penile diameter in rats.</a> J Pharmacol Toxicol Methods. 41 (4), 147-152 (1999).</li></ol>

As a direct measure of erectile function, ICP is a reliable method14. It allows for the acquisition of data on basal ICP, peak ICP, plateau ICP, time to erection and detumescence time, duration of response, etc. Besides these direct measured parameters, there are some other index parameters: (1) &#34;T80&#34;, the time to reach 80% of peak ICP; (2) &#34;D20&#34;, the time to decrease to 20% of peak ICP; (3) &#34;&#916;T80&#34;, the rate of increase in pressure (per second) at T80; and (4)...

ED is defined as the inability to attain or keep a penile erection, and has become a common male sexual disorder1. Experimental animals are used and provide reproducible models to investigate erectile function2. For a long time, several larger animal models have been employed for investigating erectile function3,4,5. Although rodents are relatively small compared to other animals, they are also used for the study of male erectile dysfunction due to exhibiting several advantages6. First, the morphological and functional sexual characteristics of humans are recapitulated in rodents. Second, compared to larger animals used in ED studies, rodents are more economical to purchase, house, and maintain. Third, genetically modified rodent models provide advantages in reproducible and subsequent behavioral as well as neurophysiological studies. Therefore, rodents have quickly become the primary animals used in the study of male erectile dysfunction.
Benefiting from a pure genetic background and consistent culture conditions, rodent models have provided consistently reproducible data5,6,7,8. Among the numerous available studies related to many aspects of erectile functions, the apomorphine (APO)-induced erectile response test and the electric-stimulation-induced ICP response test are the most widely used methods that reliably reflect erectile function9,10,11,12. The APO-induced erectile function test, developed by Heaton et al.13, is a bio-assay that utilizes the phenomenon that administration of apomorphine to rats elicits erections and yawns. As an easy, noninvasive, and stable bio-assay to evaluate erectile function, the APO-induced erectile function test is widely used in many studies. However, this assay does not adequately reflect the quality of erections or the dynamic changes in blood flow associated with an erectile response14. ICP measurements were initially developed by Quinlan et al.15. In this method, a catheter is placed into the carotid artery to measure systemic blood pressure, and another catheter is inserted into the crus corpus cavernosum to record the ICP. Before or during the ICP recording, a vasoactive agent and/or electrical field stimulation of the major pelvic ganglion (MPG) or CN were often given to the rats14. This assay has been a reliable tool for evaluating the therapies and medicines for ED, and will likely be used as a vital evaluation method in the future6.
Compared to the easily-performed APO-induced erectile function test, successful ICP recordings require attention to detail, practice, and adherence to the operation method. Therefore, here, we provide a detailed description of how to perform ICP recording.

<table border="0" cellpadding="0" cellspacing="0" style="width:1099px;" width="1098">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:128px;">Animal</td>
			<td style="width:289px;"></td>
			<td style="width:336px;"></td>
			<td style="width:345px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rats</td>
			<td></td>
			<td></td>
			<td>Strain: Sprague-Dawley 
			Age: 2-3 month</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rats</td>
			<td></td>
			<td></td>
			<td>Strain: Sprague-Dawley 
			Age: 15-18 month</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Reagents</td>
			<td></td>
			<td></td>
			<td>formula</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:128px;">Saline</td>
			<td style="width:289px;">Sigma-Aldrich, S7653</td>
			<td style="width:336px;"></td>
			<td style="width:345px;">dissolve 8.5 gram sodium chloride in distilled water</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:128px;">Pentobarbital sodium solution</td>
			<td style="width:289px;">Sigma-Aldrich, P3761</td>
			<td style="width:336px;"></td>
			<td style="width:345px;">dissolve 1 gram in 100 ml saline</td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:128px;">Povidone-iodine</td>
			<td>BTP Pharmaceutical Co. Limited</td>
			<td></td>
			<td style="width:345px;">10% (V/V)</td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:128px;">Ethanol</td>
			<td style="width:289px;">China National Pharmaceutical Group Corporation (SINOPHARM)</td>
			<td style="width:336px;"></td>
			<td style="width:345px;">70% (V/V)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:128px;">Heparin</td>
			<td style="width:289px;">Sigma-Aldrich, H3149</td>
			<td style="width:336px;"></td>
			<td style="width:345px;">dissolve 20000U heparin in 100 ml saline</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Materials</td>
			<td></td>
			<td colspan="2"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:128px;">Hypodermic needle</td>
			<td style="width:289px;">Shandong Weigao Group Medical Polymer Co., Ltd.&#160;</td>
			<td>23G</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:128px;">Syringe</td>
			<td style="width:289px;">Shandong Weigao Group Medical Polymer Co., Ltd</td>
			<td>10 ml</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:128px;">Three-way stopcock</td>
			<td style="width:289px;">Chengdu Instrument factory</td>
			<td>TSK 01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:128px;">Electrode</td>
			<td style="width:289px;">Chengdu Instrument factory</td>
			<td>JST-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:128px;">Catheter tube</td>
			<td style="width:289px;">Chengdu Instrument factory</td>
			<td>PE-10, PE-50</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:128px;">Operating scissors</td>
			<td>Shanghai operation equipment factory</td>
			<td>J22010, J22020</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:128px;">Ophthalmic operating scissors</td>
			<td>Shanghai operation equipment factory</td>
			<td>Y00010, Y00020</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:128px;">Ophthalmic forceps</td>
			<td>Shanghai operation equipment factory</td>
			<td>JD1010, JD1020</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:128px;">MicroScissors</td>
			<td>World Precision Instruments</td>
			<td>WAA260</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:128px;">silk suture</td>
			<td style="width:289px;">Shandong Weigao Group Medical Polymer Co., Ltd.&#160;</td>
			<td>5-0</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Equipment</td>
			<td></td>
			<td colspan="2"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:128px;">Stimulator</td>
			<td style="width:289px;">Nanjing medease science and technology co. ltd (model 4C501H)</td>
			<td style="width:336px;"></td>
			<td style="width:345px;">15 Hz, 5 ms pulse, 5 V, 60 s duration and 5 minutes interval</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:128px;">Multichannel signal collection processing system</td>
			<td style="width:289px;">Nanjing medease science and technology co. ltd (model 4C501H)</td>
			<td style="width:336px;"></td>
			<td>Blood pressure model</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:128px;">Pressure transducer</td>
			<td style="width:289px;">Beijing Xin Hang Xing Ye Technology Trading Company Limited (model YP100)</td>
			<td style="width:336px;"></td>
			<td>40KPa</td>
		</tr>
	</tbody>
</table>

intracavernosal pressure recording to evaluate erectile function in rodents

Erectile dysfunction (ED) is defined as the inability to attain or keep an erection of the penis, and this has become a prevalent male sexual disorder. Rodents are employed by many studies to research the physiology/pathology of erectile function. Erectile function in rodents can be evaluated by measuring the intracavernosal pressure (ICP). In practice, ICP can be monitored following electrical stimulation of the cavernous nerves (CNs). The arterial pressure of the carotid artery (the mean arterial pressure) is used as the reference for ICP. Using ICP recording protocols, many key parameters of erectile function can be measured from the ICP response curve. The ICP measurement provides more information than the apomorphine-induced penile erection test, and is cheaper than telemetric monitoring of the corpus spongiosum penis, making this method the most popular one to evaluate erectile function. However, compared to the easily-performed APO-induced erectile function test, successful ICP recordings require attention to detail, practice, and adherence to the operation method. In this work, an introduction to ICP recording in rats is provided to complement the procedure efficiently.

Numerous studies have shown that erectile dysfunction in aged males is becoming a common problem. However, medical treatment is limited in the management of aging-related ED16. In rodent models of aging-related ED, many therapies are tested on the erectile function of aged rats. As we have introduced above, the ICP recording test could be used to distinguish the ED animals from the total population of experimental animals, which is also valuable to quantify the eff...

Watch this Scientific Journal Video about Intracavernosal Pressure Recording to Evaluate Erectile Function in Rodents at JoVE.com

Intracavernosal Pressure Recording to Evaluate Erectile Function in Rodents

Intracavernosal pressure recording (ICP) is an important method to evaluate the erectile function of experimental animals. Here, a detailed protocol is demonstrated for the recording procedure of ICP by catheterizing the crura penis and then electrically stimulating the cavernous nerves in rats.

Erectile dysfunction (ED) is defined as the inability to attain or keep an erection of the penis, and this has become a prevalent male sexual disorder. ...

The overall goal of this procedure is to test erectile function of rats by catheterizing the crus corpus cavernosum and recording intracavernous pressure after stimulation of the cavernous nerves. This method can help answer key questions in the male erectile dysfunction field, such as, for evaluating the therapies and medicines for erectile dysfunction. So one advantage of this technique is that it provides a more accurate evaluation of erection function and reflexes, the dynamic change in blood flow associated with erectile response.Visual demonstration of this method is critical as catheterization is difficult to learn because the small area of the white tunica albuginea of crus corpus cavernosum makes it difficult to insert the needle into the penis crura. To prepare for the surgery, make a pair of bipolar electrodes for intracavernosal pressure recordings. Slightly bend the ends of the electrodes and adjust the distance between them to one to two millimeters.Then connect the electrodes to the stimulator using two crocodile clamps. Next, assemble the catheter system. First, use tubing to connect a hypodermic 23-gauge needle to a three-way stopcock.Then connect the stopcock to a pressure transducer. Next attach a 10 milliliter syringe loaded with heparin saline to the third position of the stopcock. Then fill the whole system with heparin saline and check for leaks.Next lift the needle 20 centimeters and calibrate the recording system to 20 centimeters of water. After the calibration, adjust the height of the needle and check the accuracy of the recording system. As needed, repeat the calibration until the accuracy is confirmed.Next, transfer the rats from the animal facility to the surgery room and allow them to acclimate to the room for at least 30 minutes. Meanwhile, sterilize the surgical tools with 70%ethanol and prepare the necessary surgical materials. After 30 minutes prepare the first rat for surgery.Anesthetize it with sodium pentobarbital and wait for five to 10 minutes. Then pinch the rats toes and check for an absence of reflex to confirm a proper anesthetization. Next, shave the fur from the abdomen and neck and place the rat supine on a surgical board.Now wipe the exposed skin with three alternating scrubs with 10%povidone iodine followed by 70%ethanol. Also, apply ophthalmic ointment to prevent the eyes from drying out. Then proceed with the surgery.Before isolating the cavernous nerve, catheterize the left carotid artery according to the text protocol. Start the nerve isolation by lifting the skin and muscle of the abdomen with a pair of forceps. Then, with scissors, make a midline incision from the lower abdomen to the penis.Next, gently move aside the intestines into the upper part of the abdominal cavity. Then grasp the bladder with a pair of forceps and pull it out of the abdominal cavity. Now find the ventral lobes of the prostate which are located on the ventral portion of the urethra.Next, expose the dorsal lobe of the prostate by pulling out the ventral lobes, seminal vesicle, and vas deferens. Now find the point of adhesion of the vas deferens and prostate. Then separate the space between the prostate and vas deferens.Proceed by carefully exposing the fibrous capsule located posterior to the junction point of the prostate and vas deferens. Then find the major pelvic ganglion and cavernous nerves located on the surface of the prostate. Now carefully dry the cavernous nerves area with a swab or tissue paper, and then carefully isolate and hook the right cavernous nerve with the bipolar electrodes.To continue the surgery, cut a small incision in the skin of the penis with dissecting scissors and then carefully denude the skin of the penis shaft. Then expose enough tissue to dissect the striated penile musculature. Now find the upper branch of pubis bone.Then expose the bulbospongiosus muscle which covers the spongios bulb. Next, using curved forceps, divide the bulbospongiosus muscle from the ischiocavernosus muscle. Proceed by carefully isolating the ischiocavernosus muscle.Then cut the ischiocavernosus muscle to expose the white tunica albuginea of the crus corpus cavernosum. Now perform the catheterization. Carefully follow the anatomical direction of the crus corpus cavernosum and gently insert the needle through the white tunica albuginea into the cavernosum.The location of the needle is crucial to the operation's success, so as a test, inject a small amount of heparinized saline. This should result in a slight penile tumescence if the needle has been inserted correctly. Next, without repositioning the needle, or disrupting the connecting tube, carefully release the needle.Then inspect the area for any leakage. If none is found, proceed with stimulating the nerve and analyzing the data. The described ICP recording test was used to distinguished aged rats with erectile disfunction to help quantify the effect of potential treatment or drugs on erectile function.A typical ICP response curve of the erectile disfunction group was much lower than the control group's curve. Usually the highest ICP was chosen for statistical analysis. After calculating the ratio of ICP to mean arterial pressure, the pooled data from five rats showed that the ratio in the aged group decreased significantly compared to that of the control groups.Besides these two key parameters, the peak ICP, the plateau ICP, the detumescence time, the duration of response, and the area under the curve, all significantly decreased in aged rats, thus, data is full of quantitative measurements that reflect erectile function. After watching this video, you should have a good understanding of how to efficiently perform the ICP recording on animals. Don't forget that while attempting this procedure, it's important to remember to use heparin in order to ensure the canula tube remains unobstructed.Once mastered, this technique can be done in about two hours if it is performed properly.

intracavernosal-pressure-recording-to-evaluate-erectile-function

Research

JoVE Journal

Biology

Survivable Stereotaxic Surgery in Rodents 

The ability to measure extracellular basal levels of neurotransmitters in the brain of awake animals allows for the determination of effects of different systemic challenges (pharmacological or physiological) to the CNS. For example, one can directly measure how the animal's midbrain dopamine projections respond to dopamine-releasing drugs like d-amphetamine or natural stimuli like food. In this video, we show you how to implant guide cannulas targeting specific sites in the rat brain, how to insert and implant a microdialysis probe and how to use high performance liquid chromatography coupled with electrochemical detection (HPLC-EC) to measure extracellular levels of oxidizable neurotransmitters and metabolites. Local precise introduction of drugs through the microdialysis probe allows for refined work on site specificity in a compound s mechanism of action. This technique has excellent anatomical and chemical resolution but only modest time resolution as microdialysis samples are usually processed every 20-30 minutes to ensure detectable neurotransmitter levels. Complementary ex vivo tools (i.e., slice and cell culture electrophysiology) can assist with monitoring real-time neurotransmission.

Survivable Stereotaxic Surgery in Rodents

The monitoring of extracellular neurotransmitter levels in distinct brain regions of freely moving animals offers insights on the link between neurotransmitter release and behavior. In vivo microdialysis coupled with electrochemical detection provides excellent anatomical and chemical resolution; and information on how basal neurotransmission is altered by pharmacological or physiological manipulations.

The ability to measure extracellular basal levels of neurotransmitters in the brain of awake animals allows for the determination of effects of different ...

Pressure-polishing Pipettes for Improved Patch-clamp Recording

Pressure-polishing is a method for shaping glass pipettes for patch-clamp recording. We first developed this method for fabricating pipettes suitable for recording from small (&lt;3 m) neuronal cell bodies. The basic principal is similar to glass-blowing and combines air pressure and heat to modify the shape of patch pipettes prepared by a conventional micropipette puller. It can be applied to so-called soft (soda lime) and hard (borosilicate) glasses. Generally speaking, pressure polishing can reduce pipette resistance by 25% without decreasing the diameter of the tip opening (Goodman and Lockery, 2000). It can be applied to virtually any type of glass and requires only the addition of a high-pressure valve and fitting to a microforge. This technique is essential for recording from ultrasmall cells (&lt;5 m) and can also improve single-channel recording by minimizing pipette resistance. The blunt shape is also useful for perforated-patch clamp recording since this tip shape results in a larger membrane bleb available for perforation.

This is a guide to modifying the shape of glass micropipettes. Specifically, by using heat and air pressure the taper is widened without increasing the tip opening, leading to lower pipette resistance. This is critical to obtain low noise recordings of small cells but is useful in many applications.

Pressure-polishing is a method for shaping glass pipettes for patch-clamp recording.  We first developed this method for fabricating pipettes suitable for ...

Measuring Blood Pressure in Mice using Volume Pressure Recording, a Tail-cuff Method

The CODA 8-Channel High Throughput Non-Invasive Blood Pressure system measures the blood pressure in up to 8 mice or rats simultaneously. The CODA tail-cuff system uses Volume Pressure Recording (VPR) to measure the blood pressure by determining the tail blood volume. A specially designed differential pressure transducer and an occlusion tail-cuff measure the total blood volume in the tail without the need to obtain the individual pulse signal. Special attention is afforded to the length of the occlusion cuff in order to derive the most accurate blood pressure readings. VPR can easily obtain readings on dark-skinned rodents, such as C57BL6 mice and is MRI compatible. The CODA system provides you with measurements of six (6) different blood pressure parameters; systolic and diastolic blood pressure, heart rate, mean blood pressure, tail blood flow, and tail blood volume. Measurements can be made on either awake or anesthetized mice or rats. The CODA system includes a controller, laptop computer, software, cuffs, animal holders, infrared warming pads, and an infrared thermometer. There are seven different holder sizes for mice as small as 8 grams to rats as large as 900 grams.

The CODA 8-Channel High Throughput Non-Invasive Blood Pressure system measures the blood pressure in up to 8 mice or rats simultaneously. This tail-cuff system uses Volume Pressure Recording (VPR) to measure the blood pressure by determining the tail blood volume.

The CODA 8-Channel High Throughput Non-Invasive Blood Pressure system measures the blood pressure in up to 8 mice or rats simultaneously. The CODA ...

Exploring Arterial Smooth Muscle Kv7 Potassium Channel Function using Patch Clamp Electrophysiology and Pressure Myography

Contraction or relaxation of smooth muscle cells within the walls of resistance arteries determines the artery diameter and thereby controls flow of blood through the vessel and contributes to systemic blood pressure. The contraction process is regulated primarily by cytosolic calcium concentration ([Ca2+]cyt), which is in turn controlled by a variety of ion transporters and channels. Ion channels are common intermediates in signal transduction pathways activated by vasoactive hormones to effect vasoconstriction or vasodilation. And ion channels are often targeted by therapeutic agents either intentionally (e.g. calcium channel blockers used to induce vasodilation and lower blood pressure) or unintentionally (e.g. to induce unwanted cardiovascular side effects).
Kv7 (KCNQ) voltage-activated potassium channels have recently been implicated as important physiological and therapeutic targets for regulation of smooth muscle contraction. To elucidate the specific roles of Kv7 channels in both physiological signal transduction and in the actions of therapeutic agents, we need to study how their activity is modulated at the cellular level as well as evaluate their contribution in the context of the intact artery.
The rat mesenteric arteries provide a useful model system. The arteries can be easily dissected, cleaned of connective tissue, and used to prepare isolated arterial myocytes for patch clamp electrophysiology, or cannulated and pressurized for measurements of vasoconstrictor/vasodilator responses under relatively physiological conditions. Here we describe the methods used for both types of measurements and provide some examples of how the experimental design can be integrated to provide a clearer understanding of the roles of these ion channels in the regulation of vascular tone.

Measurements of Kv7 (KCNQ) potassium channel activity in isolated arterial myocytes (using patch clamp electrophysiological techniques) in parallel with measurements of constrictor/dilator responses (using pressure myography) can reveal important information about the roles of Kv7 channels in vascular smooth muscle physiology and pharmacology.

Contraction  or relaxation of smooth muscle cells within the walls of resistance arteries  determines the artery diameter and thereby controls flow of ...

Assessing Murine Resistance Artery Function Using Pressure Myography

Pressure myograph systems are exquisitely useful in the functional assessment of small arteries, pressurized to a suitable transmural pressure. The near physiological condition achieved in pressure myography permits in-depth characterization of intrinsic responses to pharmacological and physiological stimuli, which can be extrapolated to the in vivo behavior of the vascular bed. Pressure myograph has several advantages over conventional wire myographs. For example, smaller resistance vessels can be studied at tightly controlled and physiologically relevant intraluminal pressures. Here, we study the ability of 3rd order mesenteric arteries (3-4 mm long), preconstricted with phenylephrine, to vaso-relax in response to acetylcholine. Mesenteric arteries are mounted on two cannulas connected to a pressurized and sealed system that is maintained at constant pressure of 60 mmHg. The lumen and outer diameter of the vessel are continuously recorded using a video camera, allowing real time quantification of the vasoconstriction and vasorelaxation in response to phenylephrine and acetylcholine, respectively.
	To demonstrate the applicability of pressure myography to study the etiology of cardiovascular disease, we assessed endothelium-dependent vascular function in a murine model of systemic hypertension. Mice deficient in the &alpha;1 subunit of soluble guanylate cyclase (sGC&alpha;1-/-) are hypertensive when on a 129S6 (S6) background (sGC&alpha;1-/-S6) but not when on a C57BL/6 (B6) background (sGC&alpha;1-/-B6). Using pressure myography, we demonstrate that sGC&alpha;1-deficiency results in impaired endothelium-dependent vasorelaxation. The vascular dysfunction is more pronounced in sGC&alpha;1-/-S6 than in sGC&alpha;1-/-B6 mice, likely contributing to the higher blood pressure in sGC&alpha;1-/-S6 than in sGC&alpha;1-/-B6 mice. 
	Pressure myography is a relatively simple, but sensitive and mechanistically useful technique that can be used to assess the effect of various stimuli on vascular contraction and relaxation, thereby augmenting our insight into the mechanisms underlying cardiovascular disease.

In pressure myography, an intact small segment of a vessel is mounted onto two small cannulas and pressurized to a suitable luminal pressure. Here, we describe the method to measure vasorelaxation response of the mouse 3rd order mesenteric arteries in c57 and sGC&alpha;1-/- mice using pressure myography.

Pressure myograph systems are  exquisitely useful in the functional assessment of small arteries, pressurized  to a suitable transmural pressure. The  ...

Force Measurement During Contraction to Assess Muscle Function in Zebrafish Larvae

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional measures of muscle health. In this video article, we demonstrate a method to measure force generation during contraction of zebrafish larval trunk muscle. Force measurements are accomplished by placing an anesthetized larva into a chamber filled with a salt solution. The anterior end of the larva is tied to a force transducer and the posterior end of the larva is tied to a length controller. An isometric twitch contraction is elicited by electric field stimulation and the force response is recorded for analysis. Force generation during contraction provides a measure of overall muscle health and specifically provides a measure of muscle function. Although we describe this technique for use with wild-type larvae, this method can be used with genetically modified larvae or with larvae treated with drugs or toxicants, to characterize muscle disease models and evaluate treatments, or to study muscle development, injury, or chemical toxicity.

Force measurements can be used to demonstrate changes in muscle function due to development, injury, disease, treatment or chemical toxicity. In this video, we demonstrate a method to measure force during a maximal contraction of zebrafish larval trunk muscle.

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Photodiode-Based Optical Imaging for Recording Network Dynamics with Single-Neuron Resolution in Non-Transgenic Invertebrates

The development of transgenic invertebrate preparations in which the activity of specifiable sets of neurons can be recorded and manipulated with light represents a revolutionary advance for studies of the neural basis of behavior. However, a downside of this development is its tendency to focus investigators on a very small number of &#8220;designer&#8221; organisms (e.g., C. elegans and Drosophila), potentially negatively impacting the pursuit of comparative studies across many species, which is needed for identifying general principles of network function. The present article illustrates how optical recording with voltage-sensitive dyes in the brains of non-transgenic gastropod species can be used to rapidly (i.e., within the time course of single experiments) reveal features of the functional organization of their neural networks with single-cell resolution. We outline in detail the dissection, staining, and recording methods used by our laboratory to obtain action potential traces from dozens to ~150 neurons during behaviorally relevant motor programs in the CNS of multiple gastropod species, including one new to neuroscience &#8211; the nudibranch Berghia stephanieae. Imaging is performed with absorbance voltage-sensitive dyes and a 464-element photodiode array that samples at 1,600 frames/second, fast enough to capture all action potentials generated by the recorded neurons. Multiple several-minute recordings can be obtained per preparation with little to no signal bleaching or phototoxicity. The raw optical data collected through the methods described can subsequently be analyzed through a variety of illustrated methods. Our optical recording approach can be readily used to probe network activity in a variety of non-transgenic species, making it well suited for comparative studies of how brains generate behavior.

This protocol presents a method for imaging neuronal population activity with single-cell resolution in non-transgenic invertebrate species using absorbance voltage-sensitive dyes and a photodiode array. This approach enables a rapid workflow, wherein imaging and analysis can be pursued over the course of a single day.

The development of transgenic invertebrate preparations in which the activity of specifiable sets of neurons can be recorded and manipulated with light ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...