The overall goal of this procedure is to perform para biotic joining of two mice to study a shared circulatory system. This is accomplished by first performing an incision to the adjacent sides of the animals. After the fascia are separated, the two mice are firmly connected at the elbow and knee joints.
The final step is to connect the skin of the two animals by a continuous suture. Ultimately, flow cytometric analysis can show blood chimerism within 10 to 14 days after the procedure. The main advantage of this technique over existing method, like bone marrow transplantation or cell injection, is that it does not require immunosuppression and also allows the maintenance of CX circulation for a long period of time.
Demonstrating the procedure will be punctu jaw. The animal surgeon from my laboratory At least two weeks in advance, set up several cages of mouse pairs of the same sex, preferably female, same genetics, and similar size and weight. Once a harmonious pair has been selected, perform the parabiosis in a clean animal surgery room.
Be sure all contact surfaces tools and gloves are sterile. Anesthetize the mice in a PAI seal induction chamber connected to an iso fluorine vaporizer. Once anesthesia is induced, maintain it throughout the procedure.
Using a nose cone, delivering a lower concentration of isof fluorine, now shave their abdominal fur on opposite sides and apply ophthalmic ointment. Then aseptically. Prepare the shaved areas by thoroughly wiping them with betadine soaked wipes followed by alcohol wipes.
Next, transfer the mice to a heated pad covered by a sterile pad. For analgesia, use carprofen and buprenorphine. Position the mice on their sides back to back with the shaved areas upwards.
Then cover the mice with a sterile drape, exposing only the operation area. Using a sharp scissor, perform longitudinal skin incisions to the sides of each animal, starting half a centimeter below the knee joint, and finishing half of a centimeter above the elbow. Following the incision, gently detached the skin from the subcutaneous fascia by holding the skin up with a pair of curved forceps and separating the fascia with a second pair.
To create half a centimeter of free skin, the LECs and knee joints are clearly distinguishable. Following the skin incision, begin joining the left and right LECs. First bend the elbow of the first mouse and pass the needle of the 3.0 suture under Theon.
Second, bend the elbow of the second mouse and pass the same suture under it. Attach the joints tightly by a double surgical knot. Next, connect the knee joints following the same procedure.
Following the attachment of the joints. Connect the skin of the two animals with a continuous absorbable five oh Vicryl suture. Starting ventrally from the elbow to prevent skin rupture and separation.
Perform tight suture closures of the skin in the area around the elbows and knees. Connecting the skin mice is a critical step of this procedure. It is important to ensure that the mice are tightly joined to avoid rupture and separation after the surgery.
Therefore, every four to five months, we confirm that the skin is tightly joined. Once the ventral skin attachment has been completed, place the mice in the prone position and continue the suture dorsally ending with a double surgical knot. Verify the continuity of the suture and confirm that it lacks openings.
Finish the surgery by administering half a milliliter of 0.9%Sodium chloride subcutaneously to each animal to prevent dehydration. Keep the animals on the heated pad until they recover. House each para biotic pair in a clean cage with moistened food pellets to minimize the strain of reaching for food while adjusting to para biotic existence.
Place the food pellets on the cage floor. Also, be sure to provide nesting material over the first 48 postoperative hours. Administer carprofen every 24 hours and administer buprenorphine every 12 hours daily for two weeks.
Monitor the animals for signs of pain and distress, such as shaking lethargy, tail chewing, arched back, lack of grooming, and so forth. To prevent bacterial infections over the first 10 days post-op, treat the mice with an oral suspension of sulfamethoxazole and trimethoprim in their water bottle. Blood cism will occur 10 to 14 days following the surgery.
So after two weeks, draw blood from their tail veins to confirm that this has occurred. Use flow cytometry to analyze the blood. Dilute two to three drops in 500 microliters of 10 millimolar EDTA.
Then thoroughly mix the dilution with 500 microliters of 2%Dextrin in PBS. Incubate the blood mixture at 37 degrees Celsius for 30 minutes, and then spin it down to collect the supernatant. For in practice.
Probiotic pairs can be kept together for nine months and separated without any complications. Perform the reverse procedure on a sterile surgical surface. Use the same anesthesia protocol as previously described, and remove the hair from the area surrounding the initial suture.
After aseptically, preparing the shaved skin, administer the analgesics and cover the surgical area with a drape using sharp scissors. Separate the mice through a longitudinal incision along the lateral suture to separate the joints, cut the knots of the suture connecting them on each mouse. Trim off the skin along the incision to smoothen the edges.
Then reattach the skin with an absorbable continuous five oh coated Vicryl suture to prevent dehydration, administer half a milliliter of 0.9%sodium chlorides subcutaneously, and then keep the animals on a heated pet until they recover from the anesthesia. Use the same analgesic and antibacterial postoperative treatments as previously described, the anticipated outcome of a parabiosis of two organisms is the equal contribution of each animal circulatory system to a common blood circulation. Equilibration of the blood from Parabios Wild type and GFP positive mice was investigated by flow cytometric analysis consistent with previous studies.
Blood from the wild type mouse revealed presence of chimerism as indicated by approximately half GFP positive and half wild type blood cells Once mastered. This technique can be done in 45 to 60 minutes if it performed properly.
