The overall goal of these surgical methods is to observe the effects of trauma on multi-organ dysfunction, specifically parabiosis will be used to anastomosis of a young and old mouse together resulting in a shared blood supply. One of the mice within this model will then be fractured to observe the regenerative effects and the effect on cognitive function in relation to the shared system of the young and old mouse. These methods can help answer key questions related to the field of immunology, aging, tissue regeneration, and cognitive function.
The main advantage of the orthopedic procedure is the clinical applicability of these findings to common complications following surgery, for example delirium and post-operative cognitive dysfunction. Prior to the experiment, keep mice in an air conditioned environment with 12-hour light and dark cycles and proper access to standard food and water. House no more than five litter mates per cage and avoid conditions that could encourage fighting.
Use one female mouse at three months of age and one old mouse at 17 months of age. For parabiosis, acclimate two mice together for at least two weeks before surgery. Check the body condition and overall appearance of mice daily.
To prepare for surgery, weigh the two mice. After administering general anesthesia, place the mice on a heated pad in the supine position. Finally, maintain correct level of anesthesia for each mouse.
Next, shave each of the two mice along a continuous line from the elbow, the flank, and the knee on the side to be joined. Disinfect with iodine plus 70%alcohol skin scrub over three alternating cycles in preparation for incision. Begin by administering analgesia after induction and before surgical manipulation.
Inject 0.25%bupivacaine at the flanks just before opening. On each mouse, use a scalpel to make a skin incision along the flank ranging proximal to the knee to just proximal to the elbow and without disturbing the muscles underneath the skin. Join the triceps of the animals with two interrupted sutures.
Then join the body walls along the flanks with a running continuous suture of seven to nine passes. Next, join the quadriceps of the animal with two interrupted sutures and close the skin of the two parabionts with interrupted sutures. Complete interrupted sutures with mouse in prone position.
Allow the mice to awaken in ambient air. House each pair individually and be sure to monitor the pairs daily by performing body condition scoring twice weekly and weighing the pairs twice weekly. Lastly, allow four weeks of recovery time to ensure shared circulation between parabionts if performing tibial fracture surgery.
Next, shave the medial aspect of the right hind limb of the mouse to expose the surgical area and disinfect with iodine plus 70%alcohol skin scrub over three alternating cycles. Begin by administering analgesia after induction and before surgical manipulation. Inject 0.25%bupivacaine at the surgical site proximal to the knee just before the opening.
Maintain a sterile surgical field throughout the procedure by using autoclaved instruments. Make an incision using scissors proximal to the knee along the medial aspect of the right hind limb down to the midshaft of the tibia on the left mouse. Expose the midshaft of the tibia and visually locate the diaphysis.
Flex the knee and visualize the tibial plateau using the patellofemoral ligament as a landmark. Visualize the patellar tendon. Then manually drill a hole in the intramedullary canal using a 25 gauge needle using a rolling motion of the finger and thumb.
Next, insert a 0.38 millimeter stainless steel pin through the hole about 15 millimeters into the medullary cavity until resistance is felt and cut flesh with the tibial plateau using a wire cutter. Then use straight bond scissors to fracture the tibia midshaft. Visually observe the fracture site and adjacent tissues to inspect for stabilization of the fracture site.
Then close the skin using dermal staples. Finally, inject one milliliter pre-warmed normal saline at 37 degrees Celsius subcutaneously in each mouse for fluid replacement. Place the mice on heated pads to recover before returning them to a clean home cage.
Check mice daily for signs of lameness, infection, or bleeding. Radiographs of midshaft fractured tibia indicated more tissue deposition in the fracture calluses of young mice than in the fracture calluses of aged mice. Further, fracture calluses of young mice contained more bone and less fibrotic tissue than fracture calluses from aged mice.
Tibial fracture surgery induced greater hippocampal neuroinflammation in 20-month-old aged mice compared to four-month-old mice. Additionally, mice after surgery also display impairments in hippocampal-dependent memory function, for example using fear conditioning behavioral assessment. Exposure to a youthful circulation enhanced bone repair with earlier union, increased bone deposition, and decreased fibrosis.
This rejuvenation of bone regeneration occurred independent of the endogenous osteocalcin positive osteoblasts, but rather relied on CD45 positive cells that migrated from the young parabiont. Once mastered, the parabiosis technique can performed within 1-1/2 hours and the tibial fracture model can be performed within 15 minutes. If needed, the two procedures can also be performed independent of one another.
While attempting the tibial fracture procedure, it's important to maintain a sterilized surgical field and ensure proper placement of the pin and proper osteotomy. Finally, remember to carefully monitor mice throughout the life of the study. After watching this video, you should have a good understanding of how to perform the parabiosis model and the tibial fracture surgery.
