The overall goal of this technique is to surgically create a partial bladder outlet obstruction in small female rodents that spares bladder outlet innervation with a low-animal morbidity, virtually no mortality, and little variability in the degree of obstruction. This method can help answer key questions about obstruction induced bladder remodeling, such as, what are the gene expression patterns and epigenetic changes associated with outlet obstruction? The main advantages of this technique are that it is reliable, and that it avoids the need for dissection around the bladder neck, thus sparing bladder outlet innervation.
The implication of this technique extend toward the therapy of bladder obstruction because the phase after the relief and recovery phase can be assessed in a reproducible manner. Bladder obstruction recovery is unpredictable in humans. Using this technique, the obstruction can be removed, allowing assessment of the cell and tissue responses associated with recovery from obstruction.
We elaborated this method when we noted that traditional, more proximal obstruction, was associated with significant undesirable bladder dysfunction and dysmorphism, after sham dissections with no actual surgical obstruction. Begin by placing 200 gram female rat onto a heating pad, and securing the limbs and tail with tape. Then disinfection and place sterile towels without covering the lower abdomen.
Then use a 20 gauge angiocatheter to catheterize the bladder, and secure the catheter with tape. Next, under a dissecting microscope, use a scalpel to make an eight millimeter longitudinal skin incision from the palpable pubic synthesis towards the urethral meatus. Lift off the skin on either side of the incision to facilitate its later closure, and use fine scissors to dissect bluntly the longitudinal spreading to identify the stunted urethra.
Place fine self-retaining skin hooks to enhance the exposure of the urethra, and use analogous blunt dissection to lift the ventrally running neurovascular bundle. Using curved scissors and gentle longitudinal spreading, develop the plane between the urethra and the vagina, dorsal to the urethra. Now bluntly pass a 4-0 silk suture behind the urethra, and prepare the double throw of surgeon's knot.
Then remove the angiocatheter, and place a 0.9 millimeter metal rod parallel to the urethra. Tighten the knot around the urethra and metal rod, so that the rod can still slide out easily, and secure the knot with three more gentle throws. After the last throw, remove the rod, and cut the suture ends to about three to four millimeters long, using a varied single stitch of absorbable braided 4-0 suture, to approximate the paraurethral glands.
Then close the skin with a varied horizontal mattress stitch, using the same absorbable suture, and inject the appropriate analgesia. For nerve-sparing mid-urethral obstruction, or NeMO in mice, place an 18 gram female C-57 black-6 mouse onto a heating pad in a supine position, and secure the limbs with tape. Then disinfect and place sterile towels without covering the lower abdomen.
Next, under a dissecting microscope, catheterize the bladder with a 24 gauge angiocatheter, and secure the catheter with tape. Under a dissecting microscope, use a scalpel to make a six millimeter longitudinal skin incision, from the palpable pubic synthesis towards the urethral meatus. Then dissect bluntly with fine scissors to identify the stunted urethra.
Using gentle longitudinal spreading, dorsal to the urethra, develop the plane between the urethra and vagina. Then bluntly pass a 5-0 non-absorbable braided suture behind the urethra, and prepare the double throw of surgeon's knot. Now connect a one milliliter syringe to the angiocatheter, and inject 0.1 milliliters of normal saline into the bladder.
When all of the saline has been delivered, use the knot to gently anchor a 26 gauge cannula, parallel to the urethra, while pulling back the angiocath. Secure the knot with three more gentle throws, followed by removal of the cannula, and apply gentle pressure to the bladder to test for the appearance of urine at the meatus. Cut the suture ends to about three millimeters.
Then use a 5-0 braided absorbable suture to close the skin with a varied horizontal mattress stitch, and inject the appropriate analgesia before placing the mouse into a recovery cage. The average relative bladder to body mass ratio in NeMO sham operated rats, is 0.33%two weeks after the procedure, while for the obstructed rats, it is 0.6%an 85%increase in the relative bladder mass in these animals. NeMO sham operated rats exhibit virtually no residual urine that can be aspirated by bladder puncture at the time of organ harvest.
Obstructed animals, by contract, demonstrate a significantly greater mean volume of residual urine, of 0.42 milliliters. Overall, mouse mortality in sham and NeMO operated animals, increases as the size of the place-holding cannula decreases. Further, the use of a 25 gauge cannula does not effectively increase the bladder to body mass ratio in mice, while the use of a 26 gauge cannula results in an over two-fold increase in the relative bladder mass.
When a 27 gauge placeholder is applied, an over 60%increase in the relative bladder mass is observed two weeks after the procedure. In addition, partial bladder outlet obstruction with the 25 or 26 gauge cannula along the urethra, does not impact the residual urine volume over the two week experimental period. Although animals with a 27 gauge placeholder demonstrate a three-fold mean residual urine volume increase, compared to their respective sham-operated peers.
After some practice, both the NeMO and NeMO sham procedures can be completed in 10 to 15 minutes. When performing NeMO in mice, it is important to remember to rule out the possibility of a complete obstruction, by gently expressing some urine before closing the animal. Following this procedure, other methods, like the inclusion of proximal sham animals, can be performed to answer additional questions about the relative contribution or association of local neurogenic outlet stimuli to result in bladder dysfunction, dysmorphism, and gene dysregulation.
This technique is expected to pave the way for researchers in the field of obstructive bladder myopathy to explore how anatomic and neurogenic bladder outlet disruptions contribute to the generation of and recoverability from outflow obstruction.
