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

Podsumowanie

The overall goal of this procedure is to successfully resect a portion of bone from the rib of a mouse. The procedure was developed as a model to study large-scale long bone repair.

Streszczenie

This protocol introduces researchers to a new model for large-scale bone repair utilizing the mouse rib. The procedure details the following: preparation of the animal for surgery, opening the thoracic body wall, exposing the desired rib from the surrounding intercostal muscles, excising the desired section of rib without inducing a pneumothorax, and closing the incisions. Compared to the bones of the appendicular skeleton, the ribs are highly accessible. In addition, no internal or external fixator is necessary since the adjacent ribs provide a natural fixation. The surgery uses commercially available supplies, is straightforward to learn, and well-tolerated by the animal. The procedure can be carried out with or without removing the surrounding periosteum, and therefore the contribution of the periosteum to repair can be assessed. Results indicate that if the periosteum is retained, robust repair occurs in 1 - 2 months. We expect that use of this protocol will stimulate research into rib repair and that the findings will facilitate the development of new ways to stimulate bone repair in other locations around the body.

Wprowadzenie

Debilitating skeletal injury, chronic osteoarthritis, and the severe problems associated with reconstructive surgery impact economic productivity, family well-being, and quality of life. While small breaks and lesions can heal fairly well, humans are not capable of repairing large defects and therefore must rely on reconstructive procedures to restore structure and function. Reconstruction may involve allogeneic or heterogeneic grafts, morcellized bone, implanted scaffolds, or distraction osteogenesis. Unfortunately, not only are there persistant morbidity factors associated with these treatments but the original strength of the repaired bone is rarely attained. Thus, new clinical approaches are needed.

One-way to develop innovative methods to treat segmental defects is to study situations in which large-scale repair occurs naturally. Amphibians famously can regenerate skeletal elements, while mammals are considered limited in this ability. However, since the early part of the 20^th century, a few reports of regeneration in the human rib have been published suggesting that humans may not be so limited^1-4 . Currently this phenomenon is best known by plastic surgeons who use rib material for jaw, face and ear reconstruction, but it is not more broadly appreciated⁵. In order to study this repair in more detail, we have developed a surgical model using the mouse. Using this protocol, researchers can identify the innate factors involved and use this information to facilitate skeletal healing in other locations.

There are many advantages to using the ribs as a model for studying skeletal repair. First, the surrounding ribs provide a natural fixator (as compared to resection of the femur^6,7). This decreases the morbidity risk of internal and external fixators and simplifies the surgical procedure. Secondly, the thin muscular layers of the chest wall provide for easy access and excellent visibility which make the assay comparable to the convenience of calvarial resections⁸. Thirdly, in contrast to the calvariae which form by intramembranous ossification, the ribs form by endochondral ossification and grow in length via extension at growth plates located at either end of a central diaphysis. Therefore, repair of the ribs may be more comparable to repair of the long bones of the appendicular skeleton. Furthermore, we have found that compared to the femur, the periosteum of the rib is thicker and can be more easily manipulated. Thus, investigators who wish to assay bone repair for the purpose of studying the periosteum or testing cell therapies, pharmacological agents, and/or tissue scaffolds may find this surgical model useful. In summary, this rib resection model provides a context within which to study natural large-scale bone repair in mammals as no such model in general use currently exists.

Protokół

NOTE: All procedures are in accordance with an animal protocol approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Southern California.

1. Preparation for Surgery

1. Lay out the necessary surgical tools and supplies (sterilized, items 1 - 18 in Materials list) and arrange a dissecting microscope and a fiber optics trans-illuminator in a hood or on a downdraft table.
2. Place a sexually mature mouse (20 - 30 g, 1 - 3 months old) in an induction chamber with 4% isoflurane to induce anesthesia.
3. Confirm anesthesia with a paw and/or tail pinch test.
4. Apply ointment to the mouse’s eyes to avoid eye dryness during the surgery.
5. Move the mouse to the microscope stage. Place the mouse into the nose cone of the maintenance hose and position laterally. Provide a warming pouch adjacent to the animal to help maintain body temperature.
   NOTE: Position of the mouse is dependent on personal preference and/or handedness of the surgeon., either left or right side is fine.
6. Adjust isoflurane to 2 - 3% for maintenance, and watch breathing rate.
7. Inject buprenorphine (0.05 mg/kg) subcutaneously (lateral flank of the leg) for post-surgery pain with a 25 G needle. The leg may twitch which is a normal response.

2. Opening Incision

1. Detect the desired rib by palpation, and clear the area of hair with a shaver.
   NOTE: Ribs 8-10 are recommended since ribs 1-7 are closer to the lungs and 11-13 are considerably shorter. Counting the ribs should begin with the most rostral rib as number 1.
2. Prepare the area with alternating povidone-iodine and isopropanol (70%) swabbing (3x is often recommended by animal use protocols).
3. Cut a 2 cm incision through the skin, directly above and parallel to the desired rib with medium sized microsurgery scissors. Incise through the underlying muscle and fat layers.
4. Place all 3 layers (skin, muscle, fat) into a retractor to expose the surgical area, while minimizing the size of the incision.

3. Excising the Rib

1. Cut through the intercostal muscles overlying the desired section of rib with a 5.0 mm scalpel. Position this incision around 5 mm proximal to the chondrocostal joint as this is where the rib is not as highly curved. Carefully separate the muscle from the bone with fine tip forceps.
2. To create a resection while retaining the periosteum in the animal, cut through the periosteum, along the length of rib with a 5.0 mm scalpel. Carefully separate the periosteum from the underlying bone laterally with fine tip forceps. Proceed with caution, as the periosteum is very delicate and has a gelatinous consistency.
3. Next make a cross-section through the bone at one end with fine micro-scissors. If needed, measure the resection with a reticle in the microscope or a ruler type gauge. Then carefully lift the bone out of the periosteum and cut the other end.
   NOTE: Proceed with caution. This is the most delicate step, as pulling up without excising the rib below will tear the pleural membrane and result in a pneumothorax. If the pleural membrane is torn this will be readily evident as the interior of the thoracic cavity will become visible.
4. If bleeding occurs as a result of cutting the bone, apply pressure at the cut end with a cotton-tipped swab for 4 - 5 sec to stop the bleeding.
   NOTE: On the rare occasion that bleeding continues, it may be necessary to stop and euthanize the animal as significant blood loss will compromise recovery (without fluid replacement, the maximum blood volume which can be safely be lost is 10% of the total blood volume or 7.7 - 8 µl/g. For a 25 g mouse, this is equivalent to around 180 - 200 µl⁹).
5. Immediately place the removed rib into 4% PFA for future analysis.
   NOTE: If the resection of both the bone and the periosteum is to be carried out, omit the periosteum incision and separation (step 3.2). Since the periosteum is very tightly attached to the pleural membrane proceed cautiously, teasing the rib away from the pleural membrane with forceps to avoid tearing.

4. Closing the Incision

1. Suture the intercostal muscles over the top of the remaining periosteal sleeve with 9-0 sutures (2 sutures are usually sufficient). Place the sutures directly above the cut ends of the rib, to act as indicators for surgery location.
2. Remove the retractor. Suture the overlying muscle and fat with 9-0 sutures (3 - 4 sutures are usually sufficient). In mice with particularly thick layers of muscle and/or fat, suture each layer separately (e.g., 1 layer of sutures for the muscular layer and 1 layer of sutures for the fatty layer).
3. Close the skin with 7-0 sutures (4 - 5 sutures are usually sufficient).
4. Secure the incision with suture glue, pinching the edges together with large forceps.
5. Slowly wean the mouse off isoflurane by first adjusting to 1% for few minutes and then turn off.
6. Place the mouse under a heat lamp and leave until consciousness is regained, 5 - 10 min. After consciousness is regained and throughout the healing period, the mouse should move and ambulate normally and show no signs of distress.

5. Recovery and Analysis

1. Provide post-operative pain management including the administration of buprenorphine in an oral gelatin form at 0.5 mg/kg every 12 hr for 48 hr. Oral administration does not require restraining the animal which might cause pain to the thoracic region.
2. Maintain the mouse in a cage with free access to food and water during the healing period. After the incision is dry, female animals can be co-housed while males must remain isolated to prevent fighting.
3. After the healing period, follow the approved procedure for euthanasia. Remove the rib cage for fixation and analysis.

Wyniki

Animals typically recover quickly from this procedure, the incision heals well, and the animals exhibit normal behavior. The procedure uses commercially available supplies and after practice, can be completed in under 1 hr. Before using this procedure to generate experimental data, it is important to analyze the resection at the day 0 time point to determine if any large bone fragments might remain within the resection zone. One way to assess proper procedure is to assess the animals by X-ray or microCT imaging postopera...

Dyskusje

When first learning this protocol, determining where to locate the initial incision can be challenging. However, practice on euthanized mice helps the surgeon learn where to place the initial incision and expose the desired rib to be resected. Working on cadavers also improves the fine-motor skills required to remove the rib portion with or without the periosteum. In addition, someone new to this procedure might find manipulating the fine tools and thin sutures to be difficult. While tying off, excess pulling on the sutu...

Materiały

Name	Company	Catalog Number	Comments
Medium sized micro-dissection scissors (Vannas-Tübingen Spring Scissors 5 mm)	Fine Science Tools	15003-08
Fine micro-dissection scissors (Vannas Spring Scissors - 2mm Cutting Edge)	Fine Science Tools	15000-04	curved tip is beneficial
Micro-scalpel 5.0 mm	Fine Science Tools	10315-12	other fine scalpels can be substituted
Dumont 55 forceps	Fine Science Tools	11295-51
Retractor	Fine Science Tools	17004-05	adjustability is convenient
Micro-needle holders	Fine Science Tools	12060-01
9.0 nylon sutures (Ethilon), taper point best	Ethicon	2819G or similar	taper point best but reverse cutting is also good
7.0 prolene sutures (Prolene)	Ethicon	8700H or similar	6-0 can be used too, needle point can vary
Large forceps (Adson Forceps)	Fine Science Tools	11006-12	other brands are fine
Lubricant Eye Ointment (Akwa Tears)	Akorn	17478-062-35
Suture glue (GLUture Topical Tissue Adhesive)	Abbot	32046-01	has excellent working time
Shaver	Wahl	9918-6171 or similar
Clamp lamp	Zoo Med	LF-5
Infrared Bulb, 75W	Zoo Med	RS-75
RC2 Rodent Anesthesia System	VetEquip	922100
IsoFlo (Isoflurane)	Abbot	05260-05
Buprenorphine (Buprenex)	Reckitt Benckiser	12496-0757-1
Betadine	Purdue Frederick	67618015017
Flavored Gelatin, raspberry	Jell-O	B000E1FYL0	made up firm, to the consistency of 'jigglers'