Semiautomated Longitudinal Microcomputed Tomography-based Quantitative Structural Analysis of a Nude Rat Osteoporosis-related Vertebral Fracture Model

The overall goal of this surgical model and image analysis method is to observe the affect of your treatment on a preclinical osteoporosis-related vertebral compression fracture model. This method can help answer key questions in this glial regenerative medicine field such as what could be an effective treatment for osteoporosis-related vertebral compression fractures, and how would such a treatment aid in bone regeneration? The main advantage of this technique is that it involves nude rats so novel treatments can be tested in vivo without immune rejection concerns.

Furthermore the bone regeneration underlies this method presented here is semiautomated so it is more accurate and less user dependent than previously published. Demonstrating the procedure will be Doctor Wafa Tawackoli, a research scientist from our laboratory. First induce anesthesia in an osteoporotic rat using 5%isoflurane in 100%oxygen then transfer to a nose cone at two to 3%isoflurane for maintenance.

Shave the abdominal area using an electric shaver. Swab with iodine-based antiseptic in 0.5%chlorhexidine gluconate followed by 70%ethanol. Use vet ointment on the eyes to prevent dryness while under anesthesia.

Apply a toe pinch stimulus to ensure an adequate plane of anesthesia. If no response is noted, initiate the procedure. Place the anesthetized rat in dorsal recumbency on a heating pad set to 37 degrees Celsius and stretch the limbs using a magnetic fixator retraction system.

Inject the rat subcutaneously with carprofen before beginning the surgical procedure. After making a five to eight centimeter incision in the skin starting one centimeter below the xiphoid process and through the midline, use surgical scissors to make an incision of the aponeurosis through the linea alba to access the abdominal cavity. Expose the abdominal cavity using retractors.

Deflect the intestines to the right of the rat to expose the abdominal aorta and the left kidney. To avoid dehydration, use sterile soaked gauzes to wrap the internal organs. Use additional retractors as necessary to expose the aorta.

Palpate the lumbar spine then proceed to expose it. Use thermocautery to expose the anterior aspect of lumbar vertebral bodies L four to five and isolate them from the surrounding connective tissue and muscles. Use a cotton swab to remove blood and residual tissue from the L four vertebrae.

Next use a sterile, two centimeter diameter drill bit to drill a five millimeter deep bone defect in the center of the exposed anterior aspect of the vertebral body. Apply minimal pressure to drill through only the ventral cortex and underlying trabecular bone. Avoid drilling through the dorsal cortex.

Then drill the L five vertebrae in the same manner to create two defects per rat. When drilling is complete, return the intestines to the abdominal cavity and remove the retractors. Next use vicryl synthetic absorbable surgical suture in a continuous pattern to suture the aponeurosis.

Close the skin using a 4-0 monofilament nylon nonabsorbable suture in a simple interrupted pattern. Then apply 100 microliters of topical skin adhesive on top of and between the skin sutures to ensure the complete closure of the skin. After injecting the rat with 37 degree Celsius lactated ringer's solution to prevent hypothermia and dehydration and buprenorphine for post-operative pain relief, allow the animal to recover from anesthesia on the heating pad.

On the day following the surgical procedure induce anesthesia with isoflurane as before. Scan the rat using an in vivo micro-CT scanner. After scanning and recovery from anesthesia, return the rat to the home cage.

To separate the vertebrae data for analysis click on micro-CT evaluation program and select the sample from the menu. Then use the mouse to contour individual vertebrae on a slice. Use the Z bar to go to the next slice and contour the individual vertebrae in the same manner.

Save the contoured vertebra as a separate file by clicking on file, save GOBJ every couple of slices. Then after setting the reference vertebra, import it to a micro-CT environment. Apply the VOI defined for the reference vertebra to the registered target vertebra by clicking micro-CT evaluation program, file, load GOBJ and selecting the GOBJ previously created.

Finally send the VOI for evaluation using a micro-CT evaluation program. This frontal 3D image shows a representative vertebral defect image one day after defect generation. Bone formation in the void is indicated in red.

This is a sagittal 2D image of the same area, and finally an axial 2D image. As seen here bone formation occurs over time and is detected with longitudinal imaging. Bone volume density and apparent density were calculated and compared using a repeated measures two-way ANOVA with Bonferroni correction for multiple comparisons.

As seen here bone volume density and apparent density significantly increase from two weeks after the creation of the vertebral defect. Following this procedure other methods like histology and immunohistochemistry can be performed in order to answer additional questions like which cells are present and what proteins are expressed in the defect. After watching this video, you should have a good understanding of how to create multiple vertebral defects in nude rats and analyze bone regeneration over time in these defects.