Induction of osteoporosis
1) Put six week-old athymic ovariectomized rats on a 4 months of low calcium diet (LCD) consisting of 0.01% calcium and 0.77% phosphate. Then, switch back to normal diet. These rats will be referred to as “osteoporotic rats” hereafter.
CAUTION: Research protocols involving the use of animals should be reviewed by the investigator’s institutional ethical review board to avoid any unnecessary discomfort or pain to the animals and to determine whether alternatives exist to animal research. All animal experiments should be performed in accordance with relevant guidelines and regulations of protocols approved by the investigator’s institutional animal research review committee. Personnel should be trained in animal handling.
Vertebral defect model
2) Anesthesia. Place the osteoporotic rat in an induction chamber of an anesthesia machine with a central scavenging system. Induce anesthesia using 5% isoflurane in 100% oxygen and maintain via nose cone at 2-3% isoflurane.
3) Apply toe pinch stimulus to ensure adequate plane of anesthesia. If no response is noted, the procedure can be initiated.
4) Preoperative care. Place the rat in dorsal recumbency on a heating pad (37 °C).
5) CRITICAL STEP: The temperature of the heating pad is important to prevent hypothermia since an anesthetized rat is unable to regulate its body temperature. Stretch the rat limbs using a magnetic fixator retraction system (Fig. 2A)
6) Shave the abdominal area and swab it with Betadine and Chlorhexidine Gluconate 0.5% followed by 70% ethanol.
7) Inject the rat with Carprofen (5 mg/kg BW, SQ) before beginning the surgical procedure.
8) Surgical procedure. Use sterile surgical scissors to cut the skin. Begin the incision 1cm below the xiphoid process and cut through the midline (~5-8 cm) (Fig. 2B).
CAUTION: Avoid using a scalpel for abdominal incisions. Using surgical scissors, as opposed to a scalpel, reduces bleeding and damage to underlying tissues.
CAUTION: All surgical tools must be autoclaved a day before surgery. In case of multiple surgeries, appropriate sterilization must be applied to all surgical tools. Between surgeries, wash the tools and place them in a sonicator bath for 5 minutes. Then, spray the surgical tools with Isopropyl Alcohol 70% and dry them. Next, place them for 20 seconds in a hot bead sterilizer set to 250°C. Allow the tools to cool down for 5 minutes. Now, the surgical tools can be used for the next surgery.
9) Use surgical scissors to make an incision of the aponeurosis through the linea alba to access the abdominal cavity. (Fig. 2C).
10) Use retractors to expose the abdominal cavity (Fig. 2D).
11) Deflect the intestines to the rat’s right to expose the abdominal aorta and the left kidney (Fig. 2E). Palpate the lumbar spine before proceeding to expose it.
CAUTION: To avoid dehydration, use sterile saline soaked gauzes to wrap the internal organs.
12) Use thermocautery to expose the anterior aspect of lumbar vertebral bodies and isolate them from surrounding connective tissue and muscles (Fig. 2F-G).
CAUTION: Use a cotton swab saturated with 3% hydrogen peroxide to remove blood and residual tissue from the vertebrae.
13) Use a sterile drill bit (~2mm in diameter) to drill a 5mm deep bone defect in the center of the vertebral body (Fig. 2H-I).
CRITICAL STEP: Apply minimal pressure to drill through only the ventral cortex and underlying trabecular bone and avoid drilling through the dorsal cortex. Note that the vertebrae of osteoporotic rats are very fragile.
CAUTION: Use a cotton swab to clean the defect and apply pressure to stop bleeding if present.
14) Repeat step 13 on an adjacent vertebra, to create a total of 2 defects per rat (Fig. 2J).
15) Return the intestines to the abdominal cavity.
16) Use a vicryl synthetic absorbable surgical suture (3-0 Vicryl undyed 27" SH taper) in a continuous pattern to suture the aponeurosis (Fig. 2K).
17) Close the skin using a monofilament nylon non-absorbable suture (4-0 Ethilon black 18" PC-3 conventional cutting) in a simple interrupted pattern (Fig. 2L).
18) Apply 100ul of Dermabond on top of the skin sutures and between them to ensure complete closure of the skin.
19) Postoperative care. Inject the rat with warm (37°C) lactated ringer’s solution (1CC/100g BW, SQ) to prevent hypothermia and dehydration.
20) Inject the rat with Buprenorphine (0.5 mg/kg, SQ) for post-op pain relief.
21) After the animal is recovered on the heating pad, return it to its cage.
CAUTION: House the rats individually, in separate cages, to prevent rat-to-rat mutilation of the sutures and wound.
22) Place chow soaked in water in a petri dish on the cage floor for a few days post-op to help the rats reach the food.
23) Administer Carprofen (5 mg/kg BW, SQ) 24 hours post-surgery for pain relief.
24) Remove skin sutures while animal is under 2% isoflurane anesthesia 10-14 days post-op.
25) One day after the surgical procedure, place the osteoporotic rat in an induction chamber of an anesthesia machine with central scavenging system. Induce anesthesia using 5% isoflurane in 100% oxygen and maintain via nose cone at 2-3% isoflurane.
26) Scan the rat using an in-vivo microCT scanner. Repeat scanning for longitudinal analysis of bone regeneration.
CRITICAL STEP: Make sure that all animals are scanned using the same settings (X-ray energy, scanning medium, intensity, voxel size and image resolution) and in a similar orientation. Refer to Bouxtein et al for further explanations and considerations involved in rodent microCT scanning for assessment of bone microstructure.
27) Contour the vertebra of interest, as demonstrated in Fig. 3A-I. Make sure to include all parts of the vertebra while excluding parts that belong to adjacent vertebrae.
28) Save the contoured vertebra as a separate file (Fig. 3J-K).
Definition of the VOI for longitudinal quantitative evaluation
29) The following steps depend on whether the scan is from day 1 after surgery (reference vertebra) or from the subsequent time points (target vertebrae).
(A) Reference vertebra
\(i) Z rotation. Measure the angle of the margins using a xy slice from the center of the defect \(Fig. 4A-B). Use the measured angle to rotate the vertebra \(Fig. 4C).
\(ii) X rotation. Measure the angle of the margins using a yz slice from the center of the defect \(Fig. 4D-E). Use the measured angle to rotate the vertebra \(Fig. 4F).
\(iii) Flip. Flip the rotated vertebra by changing the xy plane to the zx plane.
\(iv) VOI definition. Draw a circular contour of the defect using a slice from the center of the defect \(Fig. 6A). Then, copy that contour and paste it on all the slices in the defect \(Fig. 6B).
CRITICAL STEP: Since all defects were created using the same procedure, the same number of slices and subsequently total volume (TV) should be analyzed for all samples.
(B) Target vertebra
\(i) Load the DICOM files of both the target and the reference vertebrae to the main window of the “Analyze” software.
CRITICAL STEP: To avoid gray scale value changes, define the same output data type as the original DICOM files in the load menu.
\(ii) Registration to reference vertebra. Launch the “3-D Voxel Registration” module, and input the reference vertebra as the “Base Volume” and the target vertebra as the “Match Volume”. Click “Register” to register the vertebrae \(Fig. 6).
\(iii) Save the registered file using the same data type and import it to your microCT environment.
\(iv) Application of VOI. Apply the VOI defined for the reference vertebra to the registered target vertebra. Check that the VOI and defect are concentric.
30) Send the VOI for evaluation using your microCT evaluation program.
CRITICAL STEP: Make sure to use the same parameters when analyzing all VOIs. Make sure the threshold is set high enough to omit background noise with minimal loss of bone.