Our protocol provides an improved verification method for circulation chimerism and a method for successful parabiosis model setup for biomedical research. Compared with existing methods for the detection of blood chimerism in parabiosis that are often lethal or cumbersome, our protocol is safe and simple, allowing a quick and reliable verification. Although this method is specific to the detection of blood chimerism in parabiosis models, it can be applied to any biomedical studies involving parabiotic procedures.
The most challenging step is glucose injection. Therefore be sure to practice the tail vein injection prior to performing an experimental procedure, or use a venous visual mouse tail fixator. After confirming a lack of response to pedal reflex, place two 20 to 25-gram anesthetized C57 black 6 male mice in the supine position, and use an electric shaver to thoroughly shave the left side of one mouse and the right side of the second mouse from approximately one centimeter above the elbow to one centimeter below the knee.
Remove any remaining hair with depilatory cream before making longitudinal skin incisions in the cleared skin of each animal, starting from 0.5 centimeters above the elbow to 0.5 centimeters below the knee joint, and gently detach the skin from the subcutaneous fascia. Connect the olecranon and knee of the parabiont with 3-0 suture, and suture the shaved skin with a continuous 5-0 suture. Then subcutaneously inject 0.5 milliliters of 0.9%sodium chloride to prevent dehydration, and intramuscularly inject 10 milligrams of tramadol per 25 grams of mouse to relieve pain, into each mouse per day for three days.
To verify the successful construction of circulation between chimerism between parabionts by glucose fluctuation, on the 10th day after parabiosis surgery, secure the donor mice in a venous visual mouse tail fixator, and use a 70 to 75%ethanol-soaked cotton ball to rub the caudal veins in the flank of one parabiont tail. Insert a one-milliliter syringe needle into the caudal vein two to four centimeters from the tail tip and as parallel to the vein as possible, and inject one to two grams per kilogram of glucose in 100 microliters of saline solution into the donor on a period of 10 seconds. Next use scissors to remove the toes from the donor and recipient animals to allow the collection of a drop of blood at various time points of interest after the injection.
Then add a drop of each blood sample into the center of a glucometer test strip for glucose level detection. To verify the successful construction of circulation by Evans blue dye absorbance, inject 200 microliters of 0.5%Evans blue counterstain intraperitoneally into the donor mouse, and collect blood from both parabionts by cardiac puncture after two hours. Separate the serum by centrifugation, and transfer the serum into a conical tube for dilution at a one to 50 ratio in 0.9%sodium chloride.
Then measure the absorbance of the diluted serum samples at 620 nanometers on a spectrophotometer. In this representative analysis of the blood glucose levels in six donor mice, the blood glucose sharply increased to 26.5 micromolar per liter at an average of one minute after the injection of 100 microliters of glucose through the tail vein. Blood glucose levels then gradually decreased to 13.3 microliters per liter at 60 minutes.
In recipient mice, the blood glucose slowly increased after the injection, reaching the first peak level at 15 minutes. The concentration of Evans blue dye in the serum of parabionts also indicated a successful construction of circulation chimerism, and subcutaneous vascular junctions between the parabionts could be clearly observed. In parabionts that did not establish blood chimerism 15 days after parabiosis surgery, the recipient mice did not exhibit an increased blood glucose level within 60 minutes after glucose injection into the donor animal.
The blood concentration of Evans blue in the two recipients was also not elevated, demonstrating that the glucose fluctuation method is as sensitive as the Evans blue method. Moreover the blood insulin level was markedly decreased one hour after glucose injection because of the quickly increased glucose levels and recovered to normal levels by three hours, indicating that the effects of the injected glucose on insulin metabolism are restorable. Take care to make sure that the needle is inserted directly into the tail vein.
After confirming the blood chimerism of the parabionts by this method, physiopathological changes can be studied in live parabiotic animals.
