When a long bone, such as a femur or tibia is fractured, the injured muscle and soft tissue are exposed to damaged bone fragments and bone marrow. This is followed by a strong immune response to aid in tissue repair. A model for studying the effects of this exposure can be generated by harvesting both femurs and tibia.
From a donor mouse. The bones are crushed to create a bone solution, which is then injected into the injured thigh of a recipient Mouse. Immune responses are then monitored and remote organ injuries assessed to study the long-term effects of fracture induced trauma.
The main advantage of this technique over existing methods of peripheral tissue trauma, like the bilateral femur fracture model, is that this model, the pseudo fracture model, allows long-term survival without complications of prolonged anesthesia or intricate fracture fixation techniques, and hence permits the study of immune responses in the late term following trauma. Before beginning the procedure, prepare the following supplies an ice bucket, Betadine surgical instruments, GREs hemostats, a mortar and pestle forceps, 20 gauge needles, A one CC syringe, PBS anesthesia, and an isof fluorine nose cone. A single euthanized age and weight match.
Genic donor mouse should provide enough material for three recipients. Tape the donor to a plexiglass board, making sure to tape the lower extremities only at the tip of the foot. Carefully shave the lower extremities.
Then completely cover them in Betadine and alcohol for sterility to remove the long bones from the lower extremity, make a surgical incision into the skin in the inguinal region, and to continue to cut the skin along the length of the extremity down to the ankle, retract the skin and dissect it away from the subcutaneous facia and muscle, both on the medial and lateral aspects. Then insert one blade of the scissors under the muscles that lie anterior and lateral to the tibia. Slide the blade proximally and distally to lift the muscles cleanly away from the underlying bone.
Repeat this technique on the posterior and medial underside of the tibia to separate the muscles underneath the tibia. Minimal additional exertion should be used on the posterior side when sliding distally to snap the fibula away from the tibia at the proximal end of these leg muscles. Cut the tendons as close to their insertions as possible and retract the muscles distally while pulling on the muscle group together In a downward fashion, a small tug will release the distal attachments from the ankle joint using scissors cut through the ankle joint.
Do not detach the tibia from the femur at this point, this connection gives additional leverage that will aid in the dissection of the femur from the hip joint. Use a similar technique for release of the muscles from the femur. Perform this posteriorly and both medially and laterally if needed.
Cut the distal attachment of these muscle groups to release them from the knee joint. Extend the skin incision as needed to achieve adequate exposure and follow underneath the muscles. To dissect the end of the femur out of the hip joint, remove the femur and attach tibia from the donor mouse and place them on sterile gauze.
Separate the two bones at the knee joint by simply grasping each bone in a separate hand and gently twisting or rotating the two bones in opposite directions along their long AEs. To remove the remaining tendon attachments from the long bones, wrap a four by four piece of gauze around the length of the bone. Grasp tightly and scrape the full length of the bones several times to remove the remaining tissues from the surface.
Place the collected bones into a sterile 1.5 milliliter micro refused tube on ice. Transport the donor bones to a thoroughly disinfected biological safety cabinet. For preparation of the bone solution.
The hood should be prepared with a sterile field dressing, a sterile mortar and pestle, a sterile pair of forceps, a sterile eight milliliter tube sterile PBSA one milliliter pipet with respective pipette tips. A 100 microliter pipette man with respective tips, one milliliter syringes, and 20 gauge needles using the forceps, transfer the bones into the mortar and use the pestle to gently crush the bones. Pipette one milliliter of PBS into the mortar and continue to crush the remaining fragments using circular motions to ensure full resus suspension.
Then add another one milliliter of PBS and continue crushing to create the bone solution, which should have a pink hue. Slowly pour the solution out of the mortar into the eight milliliter tube while ensuring that any larger remnants remain behind. Transfer the bone solution to a one milliliter syringe.
With a 20 gauge needle, the bone solution should remain on ice. Pipette one microliter of the bone solution onto a Mac congar plate spreading evenly over the surface. Incubate the plate for 24 to 48 hours to ensure sterility.
Begin the surgical procedure for soft tissue injury and bone solution injection by assessing the depth of anesthesia of a penta barbital anesthetized mouse by toe.Pinch. Place the mouse in the supine position on a plexiglass board and immobilize the animal by loosely wrapping thin strips of tape sticky side out around the distal portion of each extremity using Oscar a five clippers with a size 40 blade. Shave the animal's abdominal, inguinal, and thigh regions.
Transfer the mouse to the sterile surgical field. Wipe the surgical area with Betadine. Then wipe with alcohol to sterilize it.
Place a nose cone on the mouse containing a piece of gauze with one milliliter of 99%isof fluorine. Once the animal's respirations begin to slow, remove the nose cone. If additional anesthesia is required throughout any part of the procedure, additional pentobarbital may be injected or isof fluorine administered.
Next fold, a sterile field dressing and drape it across the mouse to allow exposure of only the lower extremities and head. This soft tissue injury is a bilateral hind limb crush injury. Ensure that the mouse is positioned with the hind limbs slightly abducted and laterally rotated.
This helps in accessibility to the correct muscle. Group the knee flexors to perform the crush injury Clamp a large 18 centimeter hemostat around the posterior thigh musculature midpoint along the femur with its convex curve facing the femur. Lock the hemostat shut to the first click only for 30 seconds.
Insert the needle through the skin two to three millimeters as the tip of the bevel just touches against the femur. Inject 0.15 milliliter of the bone solution into the crushed thigh musculature on each side. Withdraw the needle and quickly place a sterile gloved finger over the injection site to stop any backflow of bone solution out of the wound.
Hold this finger there for several seconds. Place the mouse back into the cage and allow full freedom of movement directly as anesthesia subsides.Appropriate. Pain management must be administered as the mouse awakens.
This general schematic shows the biphasic immune response following trauma at the zero hour time point. Trauma is induced. This is followed by an early hyper inflammatory response that can be seen to peak at six hours and is measured through assessment of systemic inflammation and remote organ dysfunction.
The second component of the posttraumatic immune response is a delayed immunosuppression measured by immune cell dysfunction and is depicted as a trough around 48 hours. This is a representative diagram of the inflammatory cytokine response in plasma that occurs early after trauma and is an example of systemic inflammation. Remote organ dysfunction as measured through liver injury is found to be elevated within a similar timeframe.
This representative diagram is an example to highlight immune cell dysfunction late following trauma. While attempting this procedure, it is important to remember to ensure sterility and precise manipulation throughout the experiment. This is not a model of true fracture or of wound infection.
