Biology
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Cystometric and External Urethral Sphincter Measurements in Awake Rats with Implanted Catheter and Electrodes Allowing for Repeated Measurements
Chapters
Summary January 30th, 2018
This protocol first describes the surgical procedure of the permanent implantation of a urinary bladder catheter combined with external urethral sphincter electrodes, and second, the measurement of the function of the urinary bladder and external urethral sphincter in implanted awake animals.
Transcript
The overall goal of this surgical intervention is to observe the function of the urinary bladder in conscious rats during a cystometric measurement. This method can help answer key questions in the functional urology field such as changes in urinary bladder function due to a progressive disease or injury. The main advantage of this technique is that it allows for repetitive cystometric records in alert rats.
After anesthetizing the animal for surgery, shave its abdomen including the genital region and back region at the level of the scapulae. Then disinfect the abdominal and neck regions and the front and hind paws using 70%ethanol and povidone-iodine solution. Begin the surgery with a low midline laparotomy at the level of the third and fourth teat using a scalpel.
Make the incision 2 to 2.5 centimeters long, then use surgical scissors to cut the abdominal muscles. Next, expose the bladder by dissecting along the abdominal wall in the craniocaudal direction. Then, secure the bladder using the back of a forceps.
Now, place a purse-string suture around the bladder dome using a 6-0 non-absorbable monofilament suture with a taper tip needle. After placing the suture, make the opening for the catheter in the bladder dome within the purse-string. Use either the scalpel or an 18-gauge needle.
Next, insert the catheter pre-loaded with sterile physiological sodium chloride solution. Carefully retract the bladder catheter until its flared opening is just below the bladder dome. Now, secure the purse-string suture around the catheter and make a stop suture around the catheter body to further secure the catheter.
Before proceeding check for leaks around the bladder dome by slowly filling the bladder through the catheter with solution. To stop leaks, add another stop suture around the catheter. First, prepare the three electrodes for implantation.
Mark one electrode with permanent ink to identify it as the null electrode. Then, disinfect all three with 70%ethanol. To implant the electrodes, first use surgical scissors to extend the abdominal incision up to the pubic bone without cutting the public symphysis.
Next, identify the urethra and create blunt pockets on either side. Avoid causing trauma to the vessels or nerves while making the pockets. Then, identify the appropriate fat pouches to close the urethra.
Now, fix two electrodes bilaterally to the fat pouches using 6-0 non-absorbable monofilament sutures. Then suture the two electrodes to each other. The electrodes should be bilateral at the mid region of the urethra.
Next, suture the marked null electrode to the abdominal wall muscle of the urethra. Then, secure all three electrodes together with a single suture. To begin the tunneling procedure, make a small skin incision between the scapulae, then tunnel the device from the abdominal region to the neck.
Next, tunnel the electrode wires to the neck. Be certain to verify their position in relation to the electrodes. Next, tunnel the bladder catheter to the scapulae level.
While doing so be careful to hold the catheter just before entering the bladder dome to avoid twisting of the bladder. Next, close the abdominal muscles using continuous or interrupted stitches with 4-0 polyfilament absorbable suture. Then, close the skin incision using interrupted mattress sutures.
Now, stretch out the animal to its full length to find out how long the wires must be. Then, fix the catheter and electrode wires using a sunk-in suture to the shoulder muscle. Continue by using a single stitch of 4-0 suture to close the skin.
Next, pull a harness over the head of the rat and then fully pull its four limbs between the silicone strips. Now, tunnel the bladder catheter to the central hull of the harness. Then, pass the wires through the pre-drilled customized holes in the harness.
With the wires in place, adjust the harness by pulling the silicone strips. Tighten the harness as much as possible without impeded the rat's movements. Once adjusted, secure the strips using the cable strap.
Now, shorten the length of the bladder catheter to extend three centimeters above the harness. Then, connect the catheter to a 23-gauge stopper and fix it to the harness. Securely attaching the electrode wires to the harness is crucial.
First, cut three short lengths of small heat-shrink tubes, one with a unique color. Then, make two slightly larger lengths of heat-shrink tubes to hold all three wires. Now, adjust the length of the electrode wires, so they can be plugged in without being too loose or too short.
Then, strip two millimeters of insulation off each wire and twist the exposed threads. Next, feed the two larger heat-shrink tubes over all three wires and then feed one small heat-shrink tube over each wire using the unique color to identify the null electrode. Now, solder the electrodes to a male three-connection plug.
Place the null electrode in the middle and shrink the three small individual tubes above the soldering points. Collapse one of the larger tubes over the other heat-shrinks and collapse the other at the border with the male plug. Then, connect the male plug to the harness and secure the connection with water-repellent tape.
The success rate of the surgical procedure was around 80%Failed procedures were mainly due to detachment of the electrode wires from the plug. Seven days postoperative, the rats were usually ready for analysis. The recorded cystometric measurements usually included three consecutive voiding cycles per measurement, which took between 20 to 40 minutes depending on the anxiety and handling status of the rat.
Then, the main readout parameters include the baseline, threshold, average, and maximum detrusor pressure readings, the voided volume, flow and time, the compliance of the urinary bladder, and the external urethral sphincter EMG activity. After watching this video, you should have a good understanding of how to implant a permanent bladder catheter and external urethral sphincter electrodes for repetitive awake cystometric recordings. Once mastered, an implantation can be completed within 90 minutes if it is performed properly.
After its development, this technique paved the way for researchers in the field of urology to explore aspects of functional urology in alert rats.
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