Horse serum was purchased from HyClone. Methylprednisolone was purchased from Pfizer Pharmaceuticals (Belgium). Penicillin injection was purchased from Harbin Pharmaceutical Group Holding Co. Sodium pentobarbital was purchased from Merk, Germany. The TUNEL kit was purchased from Roche, USA. Hematoxylin and eosin were purchased from Amresco, USA. DAPI were purchased from Themo, USA. Fluorescent sealants were purchased from Biyuntian Company, China.
Ultra-thin slicer: Leica EM UC6. Optical microscope (model 107JC): Shanghai Precision Instrument Factory. Fluorescence microscope: Nikon, Tokyo, Japan. Transmission electron microscope: FEI Tecnai Spirit. Magnetic resonance scanner: Signal 3.0T MRI produced by GE, USA. SPECT/CT scanner: Discovery NM / CT 670 SPECT/CT from United States GE Company.
The animals used in this experiment strictly complied with the “Guidelines for the Care and Use of Laboratory Animals” formulated by Inner Mongolia Autonomous Region, China, and approved by the Institutional Animal Care and Use Committee of Inner Mongolia Medical University(No.: 2013037).
New Zealand white rabbits (~5 months old, 2.65 ± 0.21 kg, male or female, n = 54) were purchased from Xi’an Dilepu Biological Resources Development Co., Ltd. (Qualification No.: SCXK (Shan) 2014–001). The experimental rabbits were given standard rabbit pellet feed and free drinking tap water, single cage feeding, full light and good ventilation.
The Establishment of Model Animals
Grouping: After one week of adaptive feeding, 24 of New Zealand white rabbits (n = 54) were assigned to the control group by random number table method, and the remaining 30 were assigned to the model group.
Model establishment: In the model group, model rabbits were established by using hormone combined with allogeneic serum. Briefly, a rabbit was injected with horse serum (10 ml/kg) via ear vein at the first time. After 2 weeks, the same rabbit was injected with horse serum (5ml/kg) every day for 2 days. After 2 weeks, methylprednisolone acetate (7.5 mg/kg) was injected at gluteal muscle, and injected once every 3 days for 6 weeks. In the control group, the same amount of physiological saline was intravenously injected through the ear vein of the rabbits twice a week for 6 weeks. During the modeling period, two groups of rabbits were intramuscularly injected with 800,000 units of penicillin, 2 times/week for 12 times.
Imaging Protocol for Experimental Rabbit
At 1 week before modeling and 2, 4, and 6 weeks after hormone injection, 10 rabbits from the model group and 8 rabbits from the control group were randomly selected to perform MRI imaging and 99mTc-Cys-Annexin V SPECT imaging. The rabbits were anesthetized with a 3% sodium pentobarbital solution (25–30 mg/Kg, Merk, Germany). The anesthetized rabbit was placed in the supine position on the orthopedic support frame to perform MRI imaging. After 2 days, the model rabbit was fixed at the same position to perform 99mTc-Cys-Annexin V SPECT imaging.
MRI (American GE 3.0 T MRI) was used to scan the bilateral femoral head of the rabbits. The scanning sequence was T1WI, T2WI and fat suppression imaging was added. The parameters for MRI imaging were: coronal T1WI scan parameters (TR/TE: 900/18.6 ms and matrix: 384 × 256), coronal T2WI scan parameters (TR/TE: 3400/85 ms, matrix: 320 × 256), fat suppression sequence (TR/ TE: 1456/70 ms), layer thickness: 3 mm, layer spacing: 3 mm, field of view: 18 cm, scan time 10–15 min.
The positive criteria for MRI diagnosis of femoral head necrosis: T1WI showed a point, thin line, flaky low signal, and (or) T2WI showed a point, thin line, flaky low signal or high signal. Joint effusion is considered an indirect sign.
According to the literature , 99mTc-Cys-Annexin V was prepared by direct reduction labeling method, and the radiochemical purity was over 95% by Radio-HPLC. 99mTc-Cys-Annexin V (18.5 MBq for each rabbit) was injected through rabbit ear vein. At 1 h after injection, the anesthetized rabbits were placed on a SPECT scanner for SPECT imaging. Image acquisition conditions: Planar static acquisition, acquisition time (6 min/each), magnification (1.0), energy peak (140 kV), window width (20), and matrix (256×256). Before SPECT imaging, the model rabbits were banned from water for 8 hours, and a catheter was used to urinate before imaging to avoid false positive results due to bladder filling.
Image Processing and Analysis: SPECT image processing was performed using a Xeleris post-processing system workstation. A combination of qualitative and quantitative methods was used as the diagnostic criteria. 1) Qualitative method: Abnormal radioactive concentration, sparseness and defects in the bilateral femoral head were abnormal. 2) Semi-quantitative method: Region of interest (ROI) of the femoral head and the ipsilateral femoral shaft (ie, target tissue and non-target tissue) were delineated. The semi-quantitative analysis was used to determine the radioactivity counts of target tissue and non-target tissue (T/NT), and the T/NT ratio was calculated.
Specimen Collection and Processing
At the end of the last time point, the model rabbits were sacrificed by injection of excess anesthetic (3% sodium pentobarbital), and the bilateral femoral heads were taken and cut along the coronal plane. Specimens for Hematoxylin-Eosin (HE) staining and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay were fixed in 10% formaldehyde solution and specimens for transmission electron microscopy(TEM) were fixed in 2.5% glutaraldehyde solution.
After 48 h, the femoral head was removed from the fixative and placed in 10% Ethylenediaminetetraacetic acid (EDTA) solution for decalcification for 2 months. After successful decalcification, the femoral head was rinsed in the water for 24 h, and then were dehydrated step by step, transparent with xylene, embedded in paraffin, and sliced (thickness 4 μm). HE staining was performed. Morphological changes of trabecular bone, bone cells, fat cells in bone marrow, hematopoietic cells, etc. were observed under light microscope.
Femoral head necrosis was determined according to the rate of empty bone lacuna: 10 fields were randomly selected, 50 bone lacuna were counted in each field, the number of empty bone lacuna was counted, and the rate of empty lacuna was calculated = (number of empty bone lacuna / 50)
The experimental procedure was carried out in accordance with the instructions. Paraffin sections were washed with xylene for 5 min × 2, washed with gradient alcohol (100, 95, 90, 80, 70%) for 3 min × 1, rinsed with PBS× 2, treated with Proteinase K working solution at room temperature for 15–30 min and rinsed with PBS × 3. After dried, the slides were added 50 μL of TUNEL reaction mixture (pre-configuration before use), incubated at 37 °C for 1 h in the dark, and then rinsed with PBS× 3. Finally, the slices were stained with DAPI solution, and examined under a fluorescent microscope. The cells, of which the nuclei showed green fluorescence under the microscopy, were TUNEL-positive apoptotic cells. Ten fields of view were randomly selected, 50 bone cells were counted in each field, the number of apoptotic cells was counted, and Apoptosis index (AI) (number of apoptotic cells/50) was calculated.
The decalcified specimen was cut into small bone pieces of about 1 mm3, rinsed with PBS for 15 min × 3, fixed at 1% citrate for 1 h, washed with PBS for 15 min × 3, dehydrated in gradient alcohol at room temperature for 30 min, soaked in epoxy resin embedding solution (1:1) for 2 h, polymerized in oven at 45 °C for 12 h and then in oven at 65 °C for another 48 h. Ultrathin sections (thickness 70 nm) were prepared, and the ultrastructure of bone tissue was observed by transmission electron microscopy using double staining of uranium acetate and lead citrate.
All experimental data were expressed as mean ± standard deviation (M ± SD) and statistical analysis was performed using SPSS 19.0 sotware (IBM, USA). The t-test of two independent samples was used for comparison between groups; the ANOVA test was performed within the group. If the variance was homogeneous, a post hoc test was performed; otherwise, the LSD-t test was used. P < 0.05 indicated that the difference was statistically significant.