The overall goal of this procedure is to label bile acids with naturally-occurring, non-radioactive fluorine for measurement of their concentration in the gall bladder by MRI to access bile acid transport in live animals. This approach has a potential for identifying individuals with chronic diarrhea who should be treated for bile acid malabsorption. The main advantages of this approach are that it does not require exposure to ionizing radiation and that it has great translational potential for both clinical research and practice.
We first had the idea for this method when we realized that in the United States there are no practical clinical tests for detecting impaired intestinal bile acid transport. Begin by filling a one-milliliter syringe with the appropriate volume of fluorine-labeled bile acid stock solution and attached a 20-gauge, 1.5-inch, curved bulb-tipped gastric gavage needle. Then use the thumb and index finger to firmly grasp the mouse by the loose skin at the back of the neck and the remaining fingers to grasp the skin on the lower back and tail.
Holding the animal upright, pass the gavage needle along the side and roof of the mouth into the esophagus and down into the stomach. At the appropriate experimental endpoint, make a midline abdominal skin incision from the pubis to the xiphoid followed by a corresponding incision through the peritoneal lining to expose the abdominal organs without piercing the diaphragm. Next, use a five-inch clamp to lift the xiphoid process, exposing the upper abdominal cavity.
Using forceps and a blunt instrument, dissect and move the liver aside to reveal the gallbladder. Then place a four-inch clamp across the common bile duct and sever the ligament attaching the superior pole of the gallbladder to the diaphragm allowing the gallbladder to be moved to the right side of the abdomen. Take care and clamp the common bile duct as soon as possible after the laparotomy to avoid laceration of the liver or the inadvertent emptying of the gallbladder.
After exsanguination, transfer the harvested blood into a 1.5-milliliter heparinized tube for centrifugation. Precipitate the plasma with four parts of acetonitrile and centrifuge the blood sample again. Then analyze the supernatant by liquid chromatography and mass spectroscopy.
To free the gallbladder, blunt dissect the surrounding tissue followed by transection of the common bile duct below the clamp. Weigh the harvested gallbladder, then place the tissue in a 1.5-milliliter microcentrifuge tube and remove the liver. TO extract the tissues, add 75%acetonitrile and 25%water and homogenize approximately 100 milligrams of the liver and the entire gallbladder in individual-sized 21 glass tissue homogenizers on ice.
Transfer the samples to a 1.5-milliliter Eppendorf tube and centrifuge. Then dilute the extracts as appropriate and quantify the bile acid contents using liquid chromatography and mass spectroscopy. At the appropriate experimental endpoint, cleanse the inner left inner thigh of the anesthetized mouse and inset a 24-gauge by 0.75-inch needle catheter subcutaneously through the thigh into the abdominal cavity.
Remove the needle, leaving the intraperitoneal catheter in place. Transfer the mouse to the MRI scanner animal bed and attach a temperature-controlled thermal pad. Connect the catheter to a 72-inch piece of sterile tubing and connect the tubing to a sterile 1-milliliter syringe for maintenance of anesthesia throughout the procedure.
After confirming that no metals are near the scanner, use a fluorine proton dual-tuned linear volume MRI coil to transmit and receive the radio frequency signals. Then use a fast low angle shot sequence to obtain three-slice scalp images to calibrate the system and verify the position of the animal. To start the experiment, click the traffic light button to acquire multi-slice proton magnetic resonance images using the rapid acquisition with relaxation and enhancement sequence in the crossview of the sample.
Finally, click the GOP button in the spectrometer control tool window to acquire fluorine images using a second fast low angle shot sequence in the same region. Using this method, a highly-selective concentration of multi-fluorinated bile acid, or MFBA, can be measured in the gallbladder that is a thousandfold order of magnitude higher than the levels observed in the liver or the plasma, inconsistent with physiological levels of bile acids. As expected for the physiological connectics of bile acid enterohepatic circulation, peak gallbladder concentrations of MFBA are apparent four to seven hours after oral dosing.
Indeed, two and a half to four hours after MFBA oral gavage, fluorine signals emanating from what appears to be the common bile duct and a more robust signal emanating from the gallbladder are observed. By seven to eight and a half hours, the common bile duct signal is no longer detected and the gallbladder fluorine signal increases to the same intensity as the signal emanating from the adjacent MFBA phantom. In apical sodium-dependent bile acid transporter-deficient mice, which exhibit a reduced intestinal uptake of bile acids, in approximately 22-fold reduction in the concentration of MFBA occurs compared to controlled wild-type animals.
Indeed, whereas a robust fluorine signal is detected in the gallbladders of wild-type mice, there is no corresponding fluorine signal in apical sodium-dependent bile acid transporter-deficient animals, confirming both that MFBA are handled similarly to physiological bile acids and that MFBA MRI can be used to detect the impaired intestinal uptake of bile acids. Once mastered and performed properly, the laparotomy, blood draw and gallbladder and liver incision can be completed within 20 minutes. Signal acquisition during MRI will take one and half to two hours.
After it's development and commercialization, this technique has the potential for paving the way for the exploration of the role of intestinal bile acid transport in physiology and disease in both small animals and humans. After watching this video you should have a good understanding of how to administer foreign-labeled bile acids to small animals and to measure their uptake in the liver and gallbladder by MRI or organ incision.
