A Surgical Procedure for Resecting the Mouse Rib: A Model for Large-Scale Long Bone Repair

The goal of this procedure is to create a large scale resection in the bony portion of a rib as a model for skeletal repair. This is accomplished by first creating and opening an incision through the skin on the lateral side of the animal. The second step is to expose the rib bone that is to be removed.

Next, a portion of the rib bone is removed either along with the surrounding connective tissue sheath or the periosteum, or leaving the periosteum behind. The final step is to close the incisions and allow the animal to recover. Ultimately, this model for bone repair can be used to show the cellular and molecular events that occur during large scale skeletal repair.

In a mammal Visual demonstration of this method is critical as manipulating the surgical tools requires precision and successful surgery involves knowing where the different tissues are located. Demonstrating the procedure will be Marissa Roar and Nikita Tripper and any both graduate students in my laboratory. After preparing the surgical area and the necessary sterile tools and supplies, induce anesthesia by placing a ture mouse into an induction chamber with 4%ice of fluorine.

Once the mouse is anesthetized, apply ointment to the eyes of the mouse and confirm the depth of anesthesia. By performing a toe pinch, there should be no reaction to the toe pinch. Place the mouse on or adjacent to a heat pad on the stage of a dissecting microscope with the nose cone of the anesthesia maintenance hose placed over the nose of the mouse.

Ensure that the mouse is in a lateral position. Adjust the isof fluorine to two to 3%for maintenance and watch the breathing rate. Then inject buprenorphine subcutaneously via a 25 gauge needle.

For preemptive post-surgery pain, the leg may twitch, which is a normal response. First, select the desired rib by palpating from the most rostral rib, which is counted as number one. In this video, a portion of rib number 10 will be removed.

Shave the area over the rib and then clean with povidone iodine, followed by isopropanol three times After creating a two centimeter incision through the skin, muscle and fat layers above the rib to be excised, place all three layers into a retractor to expose the surgical area while minimizing the size of the incision. Then use a five millimeter scalpel to cut through the intercostal muscles above the rib at a location, five millimeter proximal to the chocos joint where the rib is not as highly curved. Then carefully separate the muscle from the bone with fine tip forceps.

Next to create a resection while retaining the periosteum in the animal, cut through the periosteum along the length of the rib. Then use fine tip forceps to very carefully separate the delicate periosteum laterally from the underlying bone. If needed, measure the resection with a ridicule in the microscope or a ruler type gauge.

Then use fine micro scissors to make a cross-section through the bone. At one end, carefully lift the bone out of the periosteum and cut the other end Immediately. Place the removed rib into 4%paraldehyde for future analysis.

If bleeding occurs as a result of cutting the bone, apply pressure at the cut end with a cotton tipped swab for four to five seconds to stop the bleeding. To close the incision, suture the intercostal muscles over the top of the remaining periosteal sleeve with three or 4 9 0 discontinuous sutures. Place the sutures directly above the cut ends of the rib to act as indicators for surgery location.

Remove the retractor and then suture the overlying muscle and fat with nine zero discontinuous sutures. Suturing each layer separately in mice with particularly thick layers of muscle or fat. Close the skin with four to 5 7 0 sutures, and then secure the incision with tissue adhesive.

Pinching the edges together with large forceps. Slowly wean the mouse off isof fluorine by adjusting the concentration to 1%for a few minutes, and then turn off the isof fluorine. Place the mouse under a heat lamp and leave until consciousness is regained.

After consciousness is regained and throughout the healing period, the mouse should move and ambulate normally and show no signs of distress. Once the incision is dry, house the mouse in a clean cage with free access to food and water. Females can be co housed, but males must be housed alone to prevent fighting, provide postoperative pain management, including the administration of buprenorphine in an oral genin form at 0.5 milligrams per kilogram every 12 hours for 48 hours.

Oral administration does not require restraining the animal, which might cause pain to the thoracic region. This image shows a 4.5 millimeter rib resection collected immediately after surgery. No bone fragments are left behind.

The sample was stained with a zarin red and cleared in 80%glycerol. The arrowhead indicate the location of the cut ends. This is the removed rib portion from the rib cage in the previous image without the periosteum attached.

The histological section stained with hematin and eoin shown here illustrates that there is minimal periosteum attached to the bone as indicated by the yellow arrowhead on the left and no periosteum to the right. The yellow asterisk indicates the bone marrow cavity. This image shows a removed rib portion with the periosteum still attached, and this histological section shows the intact periosteum overlying the bone as indicated by yellow bracket.

The yellow asterisk again indicates the bone marrow cavity. This image shows a skeletal preparation after two months of healing following resection with the intact periosteum inside the mouse, complete repair of the region can be observed. This histological section of the skeletal preparation just shown demonstrates a restoration of the lamella bone walls and the bone marrow cavity.

This image shows a skeletal preparation following three months of healing. After removing both a rib portion and the surrounding periosteum, there was only minimal repair at the cut end shown at the left of the panel. The yellow arrowhead points to the chocos joint.

This histological section of the previously shown skeletal preparation shows granulation, tissue adipose and muscle cells filling in the site of resection. Once mastered, this technique can be performed in under one hour if done properly following this procedure. Other methods like creating a resection in the cartilage portion of the rib can be performed in order to answer additional questions like, how does large scale repair of cartilage occur?

After watching this video, you should have a good understanding of how to create a resection in the bone portion of the rib in order to study skeletal repair.I.