The overall goals for creating a comprehensive spatiotemporal map of ex vivo intestines is to study the response to luminal content by observing the detailed movement of the intestinal wall throughout an entire segment. The main advantage of this technique is that activity within different regions of the same tissue segment can be recorded and compared in space and time, allowing identification of complex motility patterns rather than simple contraction or relaxation. Though this method can provide insight into normal intestinal motility, it can also be applied to the ideedification of effects of various drugs and intraluminal agents, such as nutrients or motility patterns.
Generally, individuals new to this method will struggle because initially, spatiotemporal maps can be difficult to interpret. But with time, one can discern complex patterns at a glance. To begin, remove the colon or intestine from a euthanized guinea pig.
Pin it down in warm Krebs solution. In the dissection tray, remove all the intraluminal content by gently perfusing Krebs buffer through the lumen. Use a syringe with a blunt end catheter and avoid overly distending the colon segment during the perfusion.
Next, attached two, three millimeter long lengths of polyethylene tubing to two fire-polished glass tube catheters, which are three millimeter in diameter. In a heated dissection tray, insert one catheter into the oral end of the tissue preparation and tie a suture behind the small piece of tubing, surrounding the catheter using a surgeon's knot. Gently attempt to pull the catheter back out to ensure the knot is snug.
The small piece of polyethylene tubing prevents slippage of the suture. Repeat this process using the other catheter on the aboral end of the tissue. Then remove the mesentery from the tissue preparation.
With both catheters secured, transfer the preparation from the dissection tray into one of the GIMM organ baths and position the tissue with the oral end facing away from the user. Next, secure the glass tube catheters to the organ bath using modelers clay by attaching the end of the glass tube to the side of the bath or by using plastic clips to attach the catheters to the poles above the bath used to secure the cameras. Now attach a five centimeter piece of tubing to the open end of each catheter.
For the oral catheter, connect the short tubing piece to a longer piece of tubing with a luer lock attachment. Then connect the five milliliter syringe to the luer lock fitting. This will be used to inject buffer into the tissue preparation.
For the aboral catheter, use the tubing to direct the luminal outflow into a collection container. Now, using the attached syringe, slowly flush the tissue with warmed Krebs buffer to ensure there is good flow, as seen by liquid exiting the aboral catheter. Proceed by calibrating the video recording system while the tissue equilibrates for 30 minutes.
Calibrate the camera height placement, video settings for brightness and contrast and horizontal distance using the GIMM software. Click on the experiment tab for the camera to be calibrated. Then in the File menu, click Calibrate.
Once the video appears in the calibrate workstation window, set the camera at a height where the image includes the ends of the catheters inserted into the colonic lumen. Then calibrate the horizontal distance viewed in the image on screen using the translucent ruler sticker attached to each bath. In the same calibrate workstation window, set the red vertical guidelines so that they are 10 millimeters apart.
Then type this distance into the distance cursor's window and click the Cal button. Next, adjust the gain, brightness and shutter sliders within the calibrate workstation window to make the tissue segment appear as a dark silhouette on a light background. Then adjust the focus and aperture knobs on the camera itself to further improve the image.
To complete the image calibration, click Save followed by Yes in the pop-up screen. Finally, click Exit. After the half-hour equilibration period, begin the experiment.
Start with naming the experimental trial. Next, occlude the lumen of the tube protruding from the aboral catheter with the plastic clamp. Then inject approximately 7 milliliters of Krebs buffer into the proximal colonic lumen.
The goal is to provide enough distention to initiate propulsive contractions. Once the segment is distended by luminal fluid, start the video recording. In the software, double-click the named trial to open the experimental camera view and then click the toggle switch to the on position to view the camera field.
Collect a predetermined length of video data. At the end of this initial controlled distention trial, loosen or remove the clamp to allow the tissue segment to propel the distending liquid out of the lumen. Allowed the tissue to re-equilibrate for 10 to 20 minutes and then repeat the procedure using the treatment.
A single tissue preparation can go through as many as five, 10-minute treatments for the appropriate rest periods between tests. Be aware of mucosal sloughing, which signals degradation of the tissue. to analyze the recording, double-click the name of a specific trial to open the analysis window for construction of an ST map.
Within the video playback area, adjust the contrast and brightness sliders to make the image appropriate for analysis. Create a black tissue silhouette on a light background. Next, set the horizontal and vertical red guidelines within the video image window to isolate the tissue region for analysis and remove areas containing artifacts.
Then set the time for analysis. Click the green A button to select the start point, and click the yellow B button for the stop point when the time slider is in the correct positions. Next, select the Motor Analysis tab to open the ST map window, and then click the stopwatch button.
Begin ST map generation by clicking the crosshair cursor within the black silhouette of the tissue. The map generation may take a few minutes to complete. After the ST map is constructed, select the zoom feature to enlarge the image.
Then select the color option to see the image in color instead of grayscale. Next, select enable so the cursor can be used to select the pixel for analysis. For the tissue associated with the selected pixels, the change in luminal diameter overtime is analyzed and displayed as a grain diameter tracing to the right of the ST map.
After the analyses, select Exit. Now export the ST map for other purposes by selecting File and then Export Data, and then Metafile. Name the file and save it as a emf file by clicking Save.
The maps generated by this technique show changes in the luminal diameter of the tissue in both distance and in time. Horizontal distance is on the X axis, and time on the Y axis, with the starting time at the top and ending time at the bottom. In the upper right corner is a legend, which displays the minimum and maximum luminal diameters, as well as scaling for both the X and the Y axes.
Thus, different pixel shades within the grayscale image corresponds to different luminal diameters. Darker pixels correspond to wider diameters, and lighter pixels correspond to smaller diameters. Contractile waves of the circular muscle will appear as regions of lighter pixelation due to the reduction in luminal diameter.
In contrast, luminal distention due to circular muscle relaxation or a large bolus of fluid is shown with darker pixels. Besides waves of a propagating contraction, the mixing motility pattern known as segmentation can be visualized as well. Small stationary contractions occur in different areas at the same time.
Overtime, these small contractions move towards the aboral anal end. Mesentery remaining on the tissue can cause artifacts, which display as dark vertical lines. The horizontal movement of these artifactual vertical lines can be used to determine the timing of longitudinal muscle movement.
After watching this video, you should have a good understanding of how to prepare intestinal segments for video recording and how to generate the subsequent representation of intestinal motility as spatiotemporal maps. Following this procedure, different nutrients can be added to the luminal solution in order to determine how natural components induce specific motility patterns in order to facilitate digestion and absorption. The use of specific pharmacological agonists or antagonists of receptors for neural humoral agents can be used to determine the mechanisms by which motility is initiated and propagated.
This technique could help produce data useful for determining compounds useful for the treatment of motility disorders.
