The gastrointestinal motility monitor provides a simple and reproducible method to monitor propulsive motility in isolated segments of Guinea pig distal colon. This procedure begins by removing the colon from the animal and bathing it in warmed oxygenated Krebs solution. Fecal pellets are then inserted in the oral end and propelled along the segment of colon video.
Recordings of the fecal pellet proceeding along the segment are captured, and the software tracks the progress of the fecal pellet. In addition to analysis of rates of propulsive motility, spatiotemporal maps can be constructed from captured video segments to assess spontaneous motor activity patterns. Ultimately, this system can be used to determine the effects of pharmacological compounds on propulsive motility patterns, as well as assessment of changes that result from pathophysiological conditions.
The main advantage of the gastrointestinal motility monitor over traditional methods of monitoring colonic motility is that this system allows for continuous quantitative evaluation of pate propulsion. In addition, digital videos of each trial in a given experiment are saved to the system and can be reanalyzed as Needed. This method can help answer key questions in the field of GI physiology, such as the role of various neurotransmitter systems on colonic motility through assessment of the effects of receptor agonists or antagonists on pellet propulsion, as well as study the effects of conditions such as stress or inflammation on propulsive motility patterns.
To prepare a segment of distal colon for the gastrointestinal motility monitor, place the isolated Guinea pig colon in ice. Cold crebs make a small incision in the oral end so it can be distinguished when the colon is later placed into the organ bath. The tissue may remain an iced crebs solution for up to two hours prior to experimentation.
Attempts to use this approach in rat and mouse intestines have been met with limited success, whereas consistent results can be obtained with the Guinea pig colon. When ready to start experimentation, transfer the colon to the organ bath. The next step is generally the biggest challenge to individuals.
New to this technique, the correct amount of tension must be applied while pinning the segment of colon in the organ bath. As the degree of tension on the preparation can affect motility through activation of stretch reflexes. For this reason, colonic segment should be pinned in the same manner by the same researcher for every experiment within a given set of experiments.
While keeping track of the oral versus anal ends pin a segment of at least five centimeters of distal colon on either end in the organ bath, allowing a small degree of slack so that the segment can move freely up to one centimeter in the middle. After pinning the colon clear away remaining masonary from the outer wall so that it does not interfere with the auto tracking software. The gastrointestinal motility monitor system consists of a perfusion chamber that is illuminated from beneath a digital video camera positioned above the chamber, a computer and peristaltic and heated water bath circulating pumps connect the inflow and outflow conduits to the organ bath, continuously perfused the organ bath with 37 degrees Celsius pre-war oxygenated kreb solution at a flow rate of 10 milliliters per minute.
The oral end of the colon, marked by the previously made incision should be positioned toward the researcher. For ease of placing the fecal pellet. Allow the preparation to equilibriate for at least 30 minutes before data acquisition.
Normally native pellets will be expelled during this time. To prepare the gastrointestinal motility monitor for data acquisition, first turn on the light illumination source, then turn on the custom software and set up a new experiment in the application. Start with a colonic segment from a healthy animal and without applying any drugs in order to obtain a control value for the rate of fecal pellet propulsion.
As the tension on the colon segment may change during warming of the tissue in the organ bath, reap in the segment so that the desired amount of slack is maintained. The coon should move freely up to one centimeter in the middle, trim excess tissue from the preparation as needed. Double click on a trial within a new experiment and turn the camera on by clicking the toggle switch.
Begin by inserting an epoxy coated fecal pellet into the oral end of the colonic segment. To initiate peristalsis, click on the record button on the computer to start recording. The movement of the pellet in the anal direction is recorded by the video camera, and the digital recordings are stored on the computer for later analysis.
When the pellet has reached the end of the colonic segment, click on the record switch to stop recording. Conduct three to five trials in a single preparation with a recovery period of five minutes between each run. To determine the effects of certain conditions or drugs on colonic motility, perform three to five trials in each preparation for each experimental condition.
In addition, perform each experiment on at least five different colons from at least five different animals. The rate of fecal pellet propulsion is calculated as the time it takes for appellate to traverse X centimeters of the colonic segment. Results are displayed in time distance graphs in which the Y axis represents the distance and millimeters traveled by a fecal pellet, and the x axis represents time in seconds as shown in this example from a control colon, the pellet progressed linearly at a rate of about two millimeters per second.
Resulting data can be exported as text files to excel for further analysis. In contrast to the control, the administration of certain drugs or inflammation in the colon can result in disrupted motility patterns such as halted motility and obstructed motility. The digital videos acquired from individual runs can be converted to spatiotemporal maps using the custom software to assess spontaneous motor activity patterns or to visually evaluate progress of a fecal pellet on the horizontal axis.
Changes in colonic diameter are plotted over time by converting the image of the colon in each video frame to a silhouette and calculating and converting the diameter along the entire length into gray scale. The end result is that the pellet in the areas of relaxation appear black, while areas of contraction appear white as illustrated by the spatiotemporal map of a control colon. The Y axis represents pellet position over time.
While the x axis represents the distance that the pellet progressed through the colon segment in contrast to the control, these spatiotemporal maps show halted and obstructed motility caused by the administration of certain drugs or inflammation in the colon. With this development, we can now assess motility along a segment of colon rather than simply determining the velocity over the entire segment, as well as assess spontaneous activity in the same preparation. Once trained a simple protocol, using this technique can be done in about two hours, and studies from more than one animal can be performed within a day following this procedure.
Other in vitro approaches such as electrophysiological recordings can be performed on the same tissue.
