This protocol investigates the biaxial mechanical properties of the reproductive organs elucidating the contribution from the smooth muscle cells and the passive matrix. This technique maintains organ geometry and native smooth muscle cell to matrix interactions permitting subjection of the organ to a physiologic range of pressures and axial extensions. Investigating the organ biaxial function under physiologically relevant loads may aid in a better understanding of the etiology of reproductive pathologies.
Biaxially testing the cervix and vagina provides insight into the fields of women's health and biomechanics. Similar methods are utilized to study blood vessels, the esophagus and large intestines. When working with a new organ, it may take time to finalize the technical details such as how to best suture the tissue or how to determine the unloaded length.
To collect the reproductive system from the experimental animal, place the mouse on an absorbent pad under a dissecting microscope and use angled tweezers and scissors to lift the skin around the abdomen. Make an initial incision at the base of the abdomen above the pubic bone shallow enough to not puncture the abdominal muscle wall and continue the incision superiorly toward the rib cage and deep through the abdominal muscles. After removing the superficial fat and carefully cutting the pubic symphysis, use straight tweezers to grasp the bladder to create tension and use blunt dissection to separate the surrounding tissue from the vagina.
Then cut the base of the reproductive system to remove the tissues from the body cavity. After determining the appropriate size cannula for the harvested system, mount the cervical side on the force transducer portion of the cannulation device and mount the opposite end of the organ on the micrometer portion of the device. Then tighten both ends with sutures.
To find the unloaded length of the mounted tissue, first stretch the organ so that the wall is not in tension. For the vagina, observe the grooves on the vaginal wall. For the cervix, cut immediately below the ink dots located above and below the central cervix mark.
Use calipers to measure the length of the system from suture to suture and increase the pressure of the myograph system from zero to 10 millimeters of mercury in one millimeter of mercury increments. The pressure in which the organ is no longer collapsed can be determined as the largest jump in the outer diameter at a given pressure. After recording the pressure and the outer diameter, note these data as the first point wherein the organ is not collapsed and zero the force.
To find the experimental in vivo stretch, adjust the organ to the estimated in vivo length while at the unloaded pressure and click start to assess the pressure versus force values for the pressure values ranging from the unloaded pressure to the maximum pressure. Then click stop and save the file. For pressure diameter pre-conditioning, set the pressure to zero millimeters of mercury, the length to the experimental in vivo length, and the gradient to 1.5 millimeters of mercury per second.
Run a sequence that takes the pressure from zero millimeters of mercury to the maximum pressure plus the unloaded pressure, hold for 30 seconds and returns to zero millimeters of mercury with an additional 30-second hold period. After repeating this sequence for a total of five cycles, click stop and save the file. For force length pre-conditioning, enter 1/3 of the maximum pressure plus the unloaded pressure for both the inlet and outlet pressures and adjust the organ to minus 2%of the in vivo length.
Click start and adjust the length to plus 2%in vivo length and back down to minus 2%at a 10 micrometer per second rate. Repeat the axial extension for a total of five cycles before clicking stop and saving the file. For pressure diameter testing of the in vivo length, click start and adjust the organ to the in vivo length, set the pressure to zero millimeters of mercury, and set the gradient to 1.5 millimeters of mercury per second.
Increase the pressure from zero millimeters of mercury to the maximum pressure before bringing the pressure back down to zero millimeters of mercury with a 20-second hold period. Then repeat the test for five cycles before stopping the system and saving the data. For force length testing of 1/3 of the maximum pressure plus the unloaded pressure, set the pressure to 1/3 of the maximum pressure plus the unloaded pressure and adjust the organ to minus 2%of the in vivo length.
After clicking start, stretch the organ to plus 2%of the in vivo length and back to minus 2%of the in vivo length at a rate of 10 micrometers per second. Then repeat the test for a total of three cycles before stopping the system and saving the data. After the last test, remove the KRB testing medium and wash the organ with calcium-free KRB medium.
After the wash, incubate the organ with fresh calcium-free KRB solution supplemented with two millimolar EGTA for 30 minutes before replacing the treatment solution with fresh calcium-free KRB. At the completion of the basal tone testing, for pressure diameter pre-conditioning, click start and set the pressure to zero millimeters of mercury, the length as the estimated in vivo length, and the gradient to 1.5 millimeters of mercury per second. Begin running a sequence that takes the pressure from zero millimeters of mercury to the maximum pressure and back to zero millimeters of mercury.
Repeat this process through five cycles with a 30-second hold time. For force length pre-conditioning, adjust the organ to the in vivo length and manually enter the unloaded pressure for both pressures. Click start and set the gradient to 1.5 millimeters of mercury and the pressure to 1/3 of the maximum.
Stretch the organ up to plus 2%and back down to minus 2%stretch at 10 micrometers per second and repeat the cycle for a total of five times. For pressure diameter testing, with the organ at minus 2%of the experimentally determined in vivo length and the pressure at zero millimeters of mercury, click start and increase the pressure from zero millimeters of mercury to the maximum pressure and back to zero millimeters of mercury. Hold the zero millimeters of mercury step for 20 seconds and repeat the cycle five times.
For force length testing, set the pressure to a nominal pressure and adjust the organ to minus 2%of the in vivo length. Stretch the organ up to plus 2%of the in vivo length and back to minus 2%of the in vivo length at a rate of 10 micrometers per second. After a total of three cycles, save the data and repeat the force length testing for 1/3 of the maximum pressure, 2/3 of the maximum pressure, and at the maximum pressure.
For a successful analysis of the mechanical properties of the female reproductive organs, it is imperative to explant the uterine horns to the vagina without any defects. Depending on the organ type, the cannula size will vary. The cannulation must be performed so that the organ cannot move during the experiment but without damaging the wall of the organ during the procedure.
The pressure myograph system can be used to monitor various aspects of the organ as it undergoes mechanical testing and the ultrasound system can be used to measure the thickness of the organs in the unloaded state with and without basal tone. After mechanical testing, the tangent moduli may be calculated for the circumferential and axial directions. Both basal tone testing and passive testing yield key mechanical properties of the reproductive tract with and without the contractile contribution of smooth muscle cells.
Scaling between the organs requires a few adjustments to the protocols as the cervix and vagina experience different loads in vivo. It is key to perform the dissection quickly to maintain the viability of smooth muscle cells but carefully to not puncture the desired organ. Opening angle experiments which measure the residual strain of the organ may be performed after mechanical testing procedures as well as various histological and biochemical assays.
After its development and investigation of the smooth muscle cell basal contribution, this technique also permits evaluating the reproductive system's smooth muscle maximum contractile response under physiologically relevant loads.
