Intestinal manipulation (Steps 1-12)
1. Induce and maintain anesthesia using isoflurane in oxygen.
2. Fix the mouse with its head away from the operator by taping its feet to the operating table.
3. Adjust direct halogen illumination to appropriate intensity for the operation.
4. Shave the abdomen using hair clippers and sterilize the skin with Kodan.
5. Make a 2 cm mid-line skin incision distally from the xiphisternum .
6. Enter the peritoneal cavity via an incision along the linea alba made using a straight forceps and a sterile small scissor.
7. Keep the abdomen open with 2 retractors and place sterile moist cotton pads around the incision.
8. Carefully eventrate the small intestine with two saline-moistened cotton buds onto the sterile cotton pads.
9. Carefully unfold the small bowel loops on the cotton pads and run it once with moderate compression with two moist cotton buds from the oral to aboral direction (Figure 1).
10. Carefully replace the intestine back into the abdomen with moist cotton buds.
11. Complete surgical closure of the peritoneal by two continuous sutures using the needle holder, straight forceps and suture material.
12. Terminate anesthesia and allow the animal to recover from the surgery under a heating lamp.
Complications are rare but might include torsion of the intestine, local intestinal hematoma and postoperative infection of the laparotomy wound. By strictly avoiding touching and compressing the mesentery, especially the blood vessels entering the bowel wall from the mesentery site, the risk of bleeding and severe complications can be minimized.
*Gastrointestinal (GIT) and colonic transit
GIT Part 1(Steps 13-15)*
13. After 22.5 hours inspect the animal and wound closure for signs of infection or other complications.
14. Replace the animals under anesthesia using isoflurane in oxygen and hyperextend its head.
15. Use a forceps to carefully pull out the tongue and feed forward an arterial catheter connected to a 1ml syringe into the mouth and push it forward into the stomach (Figure 2).
The initial process of marker application for GIT measurement is now finished. Further analysis is described in steps 19-33.
Colonic transit (Steps 16-18)
16. Carefully check patency of the colon by inserting a polished metal rod 3cm into the colon.
17. Pull out the rod and insert a 2mm glass ball transanally with a blunt surgical forceps and carefully feed it forward for 3cm into the colon with a polished metal rod.
18. Put the animal in a box allowing observation and measurement of the colonic transit time as the time from insertion until excretion of the glass ball.
GIT Part 2 (Step 19-33)
GIT will be analyzed by FITC-dextran distribution along the GI tract 90 minutes after oral administration. This analysis terminates the in vivo experimental part and additionally allows harvesting of any other organs of interest.
19. Replace the animals under deep anesthesia using isoflurane. Maintain anesthesia if any other analysis besides the GIT measurement requires that the animal stays alive (i.e. whole body perfusion). Otherwise kill the animal at this time point by cervical dislocation and terminate anesthesia.
20. Reopen the abdomen by using small scissors and straight forceps.
21. Remove the complete GI tract by subdiaphragmal transection of the esophagus and the colon at the most distal position accessible with a small scissor (sample any organ or tissue of interest for further studies).
22. Place the GI tract in chilled KHB and inspect it for integrity and the absence of hematomas.
23. Place the GI tract in a Sylgard dish with chilled KHB and transect attaching mesentery arcades using small scissors and straight forceps to completely unfold the organ.
24. Place the intestine onto a foil-covered polystyrene pad and avoid stretching of the intestine.
25. Measure full length of the small bowel and colon separately.Divide the length of the small bowel intestine into 10 equally-sized segments and mark the segments by pinning it onto the polystyrene pad with needles.
26. Proceed in the same manner with the colon but divide it into 3 segments.
27. Transect the intestine at the marked positions and flush the lumen of each segment twice with the same 1ml of KHB into 2ml centrifuge tubes. Flushed intestinal segments can be stored in cold KHB buffer for further analysis.
28. Transect the stomach and cecum longitudinally and put it completely in 2ml tube with 1ml KHB.
29. Vortex all tubes vigorously for at least 10 seconds.
30. Spin down at 1500xg for 5 min and transfer supernatants into new 2ml tubes.
31. Recentrifuge at 11.000xg for 5min and pipette 200µl duplicates of supernatants into a black 96-well plate.
32. Measure fluorescence with excitation/emission wavelength of 494/521nm and subtract blank values (KHB).
33. Calculate geometric center (GC) of FITC dextran distribution by the following formula:
GC = ∑ (% of total fluorescent signal per segment * segment number) / 100.