The overall goal of this procedure is to determine the effects of various agonists, antagonists, or novel agents on ex vivo human myometrial contractions to investigate myometrial physiology and to validate pharmacological data from novel research probes or drug leads. This method can help answer key questions in the field of myometrial physiology and pathophysiology including preterm birth and dysfunctional labor. The main advantage of this technique is that once mastered, it's a relatively simple technique that can provide useful information about the functional responses of novel compounds in vivo.
Before beginning the procedure position one peristaltic feeder tube per tissue bath around the rollers of the peristaltic pump head. Secure the tubes with the retaining stops by tightening the compression cams and locking the keys around the tubes. Place the free ends of the peristaltic feeder tubes into a one liter container of physiological saline or PSS and start the pump to allow the PSS to continually perfuse into the tissue baths.
Place the pregnant human myometrium biopsy sample into a clear silastic based dissection dish filled with PSS and place the dish under the microscope. Carefully orientate the biopsy so that the serosa and decidua edges can be visualized. Secure the tissue to the dish space with dissection pins.
Using the 10X objective, identify the regions of myometrium free of scar tissue, serosa, decidua, and adherent fetal membranes. Then use large scissors to blunt dissect the sample until the two adjacent tissue layers are separated revealing two planes of muscle. Secure each corner of the tissue pieces with additional pins and inspect the sheets of muscle for fibers that run in parallel.
Grasping the tissue along the initial cut edge, use small dissection scissors to harvest strips of tissue along the longitudinal axis and aligned with the direction of the muscle fibers until the appropriate number of strips have been acquired. Pin each strip at both ends to straighten and secure the specimens to the dish, taking care not to overstretch the tissues. Then attach aluminum tissue clips to each strip end so that the tissues between the clips are approximately five millimeters long and carefully cut away any excess tissue.
To mount the tissues, transfer the strips to the experimental tissue baths and attach one end of each strip by the clip to the force transducer and the other end to a fixed hook within the tissue chamber. Ensure that the strips are at the base of each hook in the bath and open the recording software. Right click on the y-axis.
Select set scale and enter 10 for the maximum scale value and zero for the minimum value and select okay. Repeat for each channel of recording. Click record to begin live recording and turn the micromanipulators attached to each transducer to manually stretch each tissue following the upward tissue movement from baseline until the tension reaches 0.2 grams.
Reaffirm that the strips are at the base of each hook. Then allow the tissues to equilibrate for about two hours until spontaneous contractions arise. To test the effects of specific compounds of interest on muscle strip contractibility, place the feeder tubing for the first tissue bath into a glass laboratory bottle containing the lowest concentration of the experimental reagent diluted in PSS and record the time of application.
It's important to accurately note the time of application of each experimental reagent both in the recording software itself during the experiment and in a laboratory notebook to help with later data analysis. Repeat the process for each increasing concentration by sequentially placing the feeder tubing into a glass laboratory bottle containing the next concentration in the series until all of the concentrations have been applied. After the last concentration has been tested, return the feeder tubes to a bottle of PSS to wash out the system and click stop to end the live recording.
Save the data to the appropriate folder and export a version as a dot mat file. Then import the dot mat file into the appropriate analysis software and plot the data as an x-y coordinate graph. After a period of stable activity under oxytocin in this representative experiment, the oxytocin receptor antagonist atosiban was applied in increasing logarithmic concentrations and the amplitude and area under the curve for each concentration applied were calculated, revealing the expected indirect correlation between the concentration of this known antagonist and the contractibility of the muscle.
Exposure of the human myometrial strips to a novel compound under the same experimental conditions resulted in no overall change in muscle contractibility. Pre-equilibration in arginine vasopressin, however, demonstrated a concentration-dependent decrease in human myometrial contractions with each increase in concentration of the novel compound, similar to the response observed to increasing concentrations of the known antagonist, SR 49059, suggesting a selectivity of the novel compound for the vasopressin receptor over the oxytocin receptor. Once mastered and performed properly, this technique can be completed and the data can be generated in six to eight hours.
While attempting this procedure, it's important to remember to keep the tissue samples hydrated and to maintain the temperature of the tissue baths between 36 and 37 degrees Celsius. Following this procedure other methods like Western blotting, immunohistochemistry, or quantitative polymerase chain reaction can be performed to answer additional questions about changes in protein or gene expression within the tissue strips. After watching this video, you should have a good understanding of how to appropriately dissect and use strips of human myometrium to determine the functional and biological responses of a given compound on myometrial contraction.
Don't forget that working with human tissues can be hazardous. Standard precautions such as using personal protective equipment should always be taken while performing this procedure. Additionally, the appropriate ethical and institutional review board approvals must be sought before starting.
