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

Summary

The goal of this protocol is to generate a nude rat osteoporosis-related vertebral compression fracture model that can be longitudinally evaluated in vivo using a semiautomated microcomputed tomography-based quantitative structural analysis.

Abstract

Osteoporosis-related vertebral compression fractures (OVCFs) are a common and clinically unmet need with increasing prevalence as the world population ages. Animal OVCF models are essential to the preclinical development of translational tissue engineering strategies. While a number of models currently exist, this protocol describes an optimized method for inducing multiple highly reproducible vertebral defects in a single nude rat. A novel longitudinal semiautomated microcomputed tomography (µCT)-based quantitative structural analysis of the vertebral defects is also detailed. Briefly, rats were imaged at multiple time points post-op. The day 1 scan was reoriented to a standard position, and a standard volume of interest was defined. Subsequent µCT scans of each rat were automatically registered to the day 1 scan so the same volume of interest was then analyzed to assess for new bone formation. This versatile approach can be adapted to a variety of other models where longitudinal imaging-based analysis could benefit from precise 3D semiautomated alignment. Taken together, this protocol describes a readily quantifiable and easily reproducible system for osteoporosis and bone research. The suggested protocol takes 4 months to induce osteoporosis in nude ovariectomized rats and between 2.7 and 4 h to generate, image, and analyze two vertebral defects, depending on tissue size and equipment.

Introduction

More than 200 million people worldwide suffer from osteoporosis¹. The underlying pathological decrease in bone mineral density (BMD) and altered bone microarchitecture increase bone fragility and, consequently, the relative risk of fracture². Osteoporosis is so prevalent and detrimental to health that the WHO has defined it a major public health concern. Furthermore, as the world's population is expected to age, osteoporosis is expected to become even more common.

Osteoporotic vertebral compression fractures are the most common fragility fractures, estimated at more than 750,000 a year in the US. They are associated with significant morbidity and as much as a nine-times higher mortality rate³. In clinical trials, currently available surgical interventions, such as vertebroplasty and kyphoplasty, were found to be no more effective than a sham treatment^4,5, leaving only pain management available to these patients. Since current OVCF treatments are limited, it is imperative to develop an animal model that can replicate the disorder^6,7,8. Such animal models could facilitate both the investigation of current treatment methods and the development of novel therapies that will translate into clinical practice. Osteoporosis has been induced and sustained in model animals through the administration of a low-calcium diet (LCD) in conjunction with ovariectomy^{1,9,10,11,12,13,14,15}. To further model the bone loss associated with OVCFs, vertebral bone defects were established in osteoporotic immunocompetent rats ^{16,17,18,19,20,21,22,23,24}. In this work, a vertebral defect model of immunocompromised rats with modeled osteoporosis is presented. This novel model can be used to assess cell-based therapies involving stem cells derived from various sources and species for the repair of challenging fractures, such as OVCFs.

Bone imaging is a crucial part of the evaluation of fractures and bone diseases. Advanced imaging methods were developed for the accurate assessment of structural bone changes and regeneration strategies²⁵. Among them, µCT imaging has emerged as a non-invasive, easy-to-use, and inexpensive method that provides high-resolution 3D images. µCT imaging has several advantages over other modalities in evaluating osteoporosis patients, as it offers high-resolution 3D bone microarchitecture²⁶ that can then be quantitatively analyzed. The latter can then be used to compare the therapeutic effects of proposed treatments. Indeed, in vivo µCT imaging is a gold standard for vertebral defect regeneration monitoring^1,16,27. However, few publications^28,29,30,31 have employed automated registration tools to minimize the user-dependency, interpolation bias, and precision error of µCT imaging-based analysis. Recently, we were the first to use a registration procedure to improve the analysis of bone regeneration in a standardized bone void, as explained in this protocol³² .

The method described here can be used to study the effect of novel cell therapies for OVCFs, unhindered by host T-cell responses that might reject xenogeneic or allogeneic cells. Osteoporosis is induced in young rats through ovariectomy (OVX) and 4 months of an LCD. The young age of the OVX rats, combined with the LCD allowed, us to reach a low peak bone mass, mimicking postmenopausal osteoporosis by leading to irreversible bone loss. This can be explained partly by the fact that, during the LCD and at around 3 months of age, the rats transition from the bone modeling to remodeling phase at the lumbar vertebrae³³, thereby increasing the likelihood of maintaining the osteoporosis over time. Using young animals makes this model more cost effective, as they cost less. Nonetheless, it is limited by inherently not accounting for the biological changes in the aging animal.

Protocol

All animal experiments were performed under a protocol approved by the Institutional Animal Care and Use Committee (IACUC) of Cedars-Sinai Medical Center (Protocol # 3609). Anesthesia was administered for all imaging and surgical procedures. All animals were housed in accordance with approved IACUC protocols.

NOTE: The experimental design of this protocol is shown in Figure 1. Purchase six-week-old rats with their ovaries surgically removed and feed them an LCD consisting of 0.01% calcium and 0.77% phosphate. After a period of 4 months of an LCD, drill a critical-size vertebral defect in the fourth and fifth lumbar vertebral bodies (L4-L5). Following surgery, image the rats on day 1 and weeks 2, 4, 8, and 12 after defect establishment. Locate defect margins on the day 1 scan, reorient to a standard position, and define a cylindrical volume of interest (VOI). Automatically register the subsequent µCT scans (i.e., for weeks 2, 4, 8, and 12) of each rat to the standard position defined for the corresponding day 1 scan. Apply the day 1 predefined VOI to the registered scans. Assess the bone volume density and apparent density of the VOIs.

1. Induction of Osteoporosis

1. Put six-week-old athymic ovariectomized rats on 4 months of an LCD consisting of 0.01% calcium and 0.77% phosphate.
2. Switch back to a normal diet.
   NOTE: These rats will be referred to as "osteoporotic rats" hereafter.

2. Vertebral Defect Model

NOTE: The timing is 40 - 50 min per animal.

1. Autoclave all surgical tools prior to surgery.
2. In the case of multiple surgeries, sterilize all surgical tools.
   1. Wash the tools and place them in a sonicator bath for 5 min. Place them in a hot bead sterilizer set to 250 °C for 20 s. Allow the tools to cool down for 5 min.
3. Induce anesthesia.
   1. Place the osteoporotic rat in the induction chamber attached to 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. Use vet ointment on the eyes to prevent dryness while under anesthesia.
   2. Apply a toe-pinch stimulus to ensure adequate plane of anesthesia. If no response is noted, initiate the procedure.
4. Place the anesthetized rat in dorsal recumbency on a heating pad (37 °C) and stretch the limbs using a magnetic fixator retraction system (Figure 2A).
   NOTE: The temperature of the heating pad is important for the prevention of hypothermia, since an anesthetized rat is unable to regulate its body temperature.
5. Shave the abdominal area using an electric shaver. Swab it with iodine-based antiseptic and chlorhexidine gluconate 0.5% followed by 70% ethanol.
6. Inject the rat with carprofen (5 mg/kg bodyweight (BW), subcutaneous (SQ)) before beginning the surgical procedure.
7. Use a sterile scalpel to cut the skin. Begin the incision 1 cm below the xiphoid process and cut through the midline (~5 - 8 cm) (Figure 2B).
8. Use surgical scissors to make an incision of the aponeurosis through the linea alba to access the abdominal cavity (Figure 2C).
9. Expose the abdominal cavity using retractors (Figure 2D).
10. Deflect the intestines to the right of the rat to expose the abdominal aorta and the left kidney (Figure 2E). Palpate the lumbar spine before proceeding to expose it. To avoid dehydration, use sterile soaked gauzes with sterile saline solution to wrap the internal organs.
11. Use thermocautery to expose in layers the anterior aspect of lumbar vertebral bodies L4-5 and isolate them from the surrounding connective tissue and muscles (Figure 2F-G).
    NOTE: Thermocautery should be used to control bleeding during the dissection.
12. Use a sterile cotton swab saturated with sterile saline to remove blood and residual tissue from the L4 vertebrae. Use a sterile Trephine drill bur (~2 mm in diameter) to drill a 5 mm-deep bone defect in the center of the exposed anterior aspect of the vertebral body (Figure 2H-I).
    NOTE: Apply minimal pressure to drill through only the ventral cortex and underlying trabecular bone; avoid drilling through the dorsal cortex. Note that the vertebrae of osteoporotic rats are very fragile. Use a cotton swab to clean the defect and apply pressure to stop bleeding, if present.
13. Repeat step 2.11 on the L5 vertebra to create a total of 2 defects per rat (Figure 2J).
14. Return the intestines to the abdominal cavity.
15. Use a vicryl synthetic absorbable surgical suture (3-0 vicryl undyed 27" SH taper) in a continuous pattern to suture the aponeurosis (Figure 2K).
16. Close the skin using a 4-0 monofilament nylon non-absorbable suture in a simple interrupted pattern (Figure 2L).
17. Apply 100 µL of topical skin adhesive on top of the skin sutures and between them to ensure the complete closure of the skin.
18. Inject the rat with warm (37 °C) lactated ringer's solution (1CC/100 g BW, SQ) to prevent hypothermia and dehydration.
19. Inject the rat with buprenorphine (0.5 mg/kg BW, SQ) prior to the surgery and every 8 - 12 h for post-op pain relief as needed.
20. Do not leave the animal unattended until it has regained sufficient consciousness to maintain sternal recumbency. Also, do not return an animal that has undergone surgery to the company of other animals until it has fully recovered.
21. After the animal has recovered on the heating pad, return it to its cage.
    NOTE: House the rats individually (i.e., 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 h post-surgery for pain relief every 24 h as needed.
24. Remove the skin sutures while the animal is under 2% isoflurane anesthesia 10 - 14 days post-operation.

3. MicroCT Scanning

NOTE: The timing is 30 - 40 min per animal.

1. On the day following the surgical procedure, place the osteoporotic rat in the induction chamber attached to 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.
2. Scan the rat using an in vivo µCT scanner. Repeat scanning for the longitudinal analysis of bone regeneration.
   NOTE: Make sure that all animals are scanned using the same settings (i.e., X-ray energy, scanning medium, intensity, voxel size, and image resolution) and in a similar orientation. For example: X-ray energy, 55 kVP; current, 145 µA; voxel size, 35 µm; increments, 115 µm; and integration time, 200 ms; with the samples in PBS. Refer to Bouxtein et al.³⁴ for further explanations and considerations involved in rodent µCT scanning for an assessment of bone microstructure. Ideally, the highest scan resolution available would be used for all scans; however, higher-resolution scans require longer acquisition times, generate large data sets, and expose the animals to more ionizing radiation. The latter may introduce unwanted effects, including decreased fracture healing.Therefore, the tradeoff between additional data and scan time should be carefully considered.

4. Vertebral Separation

NOTE: The timing is 20 - 30 min per sample.

1. Contour the vertebra of interest, as demonstrated in Figure 3A-I. Make sure to include all parts of the vertebra while excluding parts that belong to adjacent vertebrae.
   1. Click on "µCT evaluation program" and select the sample from the menu.
   2. Contour each slice using the mouse.
   3. Use the "Z" bar to go to the next slice.
2. Save the contoured vertebra as a separate file (Figure 3J-K) by clicking on "File" → "Save GOBJ" every couple of slices.

5. Definition of the VOI for Longitudinal Quantitative Evaluation

NOTE: The following steps depend on whether the scan is from day 1 after surgery (reference vertebra) or from the subsequent time points (target vertebrae).

1. Reference vertebra.
   NOTE: The timing is 20 - 30 min per sample.
   1. For Z-rotation, measure the angle of the margins using an XY-slice from the center of the defect (Figure 4A-B).
      1. On the Z-plane, go to the area of the vertebra where the defect is most clear and screen capture the vertebra.
      2. In a presentation software, prepare a rectangle-shaped object that will fit into the defect.
      3. Rotate the image of the vertebra such that the defect faces upwards and the defect margins are parallel to the sides of the rectangle.
      4. Measure the angle of rotation (right-click on the image → "Format Picture" → "Size").
      5. Use the measured angle to rotate the vertebra (Figure 4C).
         1. Open a new DECterm window ("Session manager" → "Applications" → "DECterm").
         2. Run "ipl":
         3. Ipl> turn3d
         4. -input [in]>
         5. -output [out]>
         6. -turnaxis_angles [0.000 90.000 90.000]> 90 90 0
         7. -turnangle [0.000]> measured angle
         8. -img_interpol_option [1]>
   2. For X-rotation, measure the angle of the margins using a YZ-slice from the center of the defect (Figure 4D-E). Use the measured angle to rotate the vertebra (Figure 4F).
      1. Click on "YZ" in "uCT evaluation program" and repeat steps 5.1.1.1-5.1.1.5.2.
      2. Ipl> isq
      3. -aim_name [in]>
      4. -isq_filename [default_file_name]> Insert the ISQ file directory (e.g., "DK0:[MICROCT.DATA.GAZIT.MAXIM.80.DAY1]Z2102970.ISQ")
      5. -pos [0 0 0]>
      6. -dim [-1 -1 -1]>
   3. Flip the rotated vertebra by changing the XY-plane to the ZX-plane.
      1. Open a new DECterm window ("Session manager" → "Applications" → "DECterm").
      2. Run "ipl":
      3. Ipl> flip
      4. -input [in] > out
      5. -input [out] > out2
      6. -new_xydir [yz] > zx
   4. Define the VOI.
      1. Draw a circular contour of the defect using a slice from the center of the defect by selecting the circular contour icon in "uCT evaluation program" (Figure 6A). Copy that contour and paste it on all slices in the defect (Figure 6B).
         NOTE: Since all defects were created using the same procedure, analyze the same number of slices and, subsequently, the total volume (TV) for all samples.
2. Target vertebra.
   NOTE: The timing is 10 - 20 min per sample.
   1. Load the DICOM files of both the target and the reference vertebrae to the main window of the image analysis software.
      NOTE: To avoid grayscale value changes, define the same output data type as the original DICOM files in the load menu.
   2. Register to the reference vertebra.
      1. 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 (Figure 5).
   3. Save the registered file using the same data type and import it to a µCT environment.
   4. Apply the VOI.
      1. Apply the VOI defined for the reference vertebra to the registered target vertebra by clicking "uCT evaluation program" → "File" → "Load GOBJ" and selecting the GOBJ previously created. Check that the VOI and defect are concentric.

6. MicroCT Analysis

NOTE: The timing is 10 - 20 min per sample.

1. Send the VOI for evaluation using a µCT evaluation program (Figure 6).
   NOTE: 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. If a radiopaque biomaterial is used, a number of strategies could be used to analyze bone formation. If there is a difference in density between the biomaterial and bone tissue, the biomaterial could be segmented out^35,36. Otherwise, the investigators could qualitatively evaluate the differences in bone formation between experimental groups.

7. Euthanasia

1. PPlace the osteoporotic rat in the induction chamber attached to an anesthesia machine. Induce anesthesia using 5% isoflurane in 100% oxygen.
2. Maintain anesthesia via nose cone and perform euthanasia by incising the chest cavity to produce a bilateral pneumothorax³⁷.

Results

Using this protocol, one can image and quantify the regeneration of n = 8 modeled osteoporotic vertebral defects across different time points. The anatomic match obtained by the registration procedure allows for the analysis of the same VOI at all time points. This results in a highly accurate longitudinal 3D histomorphometric analysis, even when the margins of the original defect are no longer recognizable. We used five time points (day 1, week 2, week 4, week 8, and week 12) as an examp...

Discussion

Osteoporosis is the most prevalent cause of vertebral compression fractures caused by an increased load on the spine and that result in the collapse of the vertebral body. However, it is practically impossible to generate an injury in a rodent that authentically replicates a similar vertebral collapse. Instead, researchers create a cylindrical void in the center of the vertebral body to mimic OVCFs^16,17,18,

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Isoflurane	MWI Animal Health, Pasadena, CA	501017
BetadineSolution	MWI Animal Health, Pasadena, CA	4677
Chlorhexidine Gluconate 2% scrub	MWI Animal Health, Pasadena, CA	510083
Isopropyl Alcohol 70%-quart	MWI Animal Health, Pasadena, CA	501044
Carprofen	MWI Animal Health, Pasadena, CA	26357
Buprenorphine 0.3 mg/mL	MWI Animal Health, Pasadena, CA	56163
Ovariectomized Athymic nude rats	Harlan Laboratories, Indianapolis, IN	Hsd:RH-Foxn1 rnu
Low calcium food	Newco Distributors, Inc., CA	1814948 (5AV8 AIN-93M w/low calcium)
Phosphate Buffered Saline	Life Technologies Corporation	14190250
Dermabond	J AND J ETHICON	DHVM12
Anesthesia machine	Patterson Scientific	TEC 3EX
Slide Top Induction Chambers	Patterson Scientific	78917833
ProStation Heated Workstation	Patterson Scientific	78914731
Surgical drape	HALYARD HEALTH INC	89101
Magnetic fixator retraction system	Fine Science Tools, Inc., CA	18200-50
Dissecting Scissors, 10 cm, Curved, SS	World Precision Instruments, FL	14394
Iris Scissors, 11.5 cm, 45 °Angle, Serrated, Sharp/Sharp	World Precision Instruments, FL	503225
Forceps, no. 5	World Precision Instruments, FL	555048FT
Micro Mosquito Hemostatic Forceps	World Precision Instruments, FL	503360
Sterile cotton gauze	Medtronic, MINNEAPOLIS, MN	9024
Absorption Spears - Mounted/Sterile	Fine Science Tools, CA	18105-01
Syringe, 1 mL	TERUMO TERUMO MED	SS-01T
Needle, 25 gauge	BD MED SYS INJECTION SYS	305127
Laminar flow hood	Baker	SterilGARD e3-Class II Type A2 Biosafety Cabinet
Thermal Cautery Unit	World Precision Instruments, FL	501292
Micro-Drill OmniDrill115/230V	World Precision Instruments, FL	503598
Trephines for Micro Drill, 2 mm diameter	Fine Science Tools, CA	18004-20
3-0 Vicryl undyed 27” SH taper	J AND J ETHICON	1663G
4-0 Ethilon black 18” PC3 conventional cutting	J AND J ETHICON	1954G
Conebeam in vivo microCT (vivaCT 40)	Scanco Medical	vivaCT 40
SCANCO Medical microCT systems software suite	Scanco Medical	vivaCT 40
Analyze software	Biomedical Imaging, Mayo Clinic, Rochester, MN	Analyze 12	Image analysis software
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