1.1 Animal Model
Forty female Sprague dawley rats (12-week-old, specific-pathogen-free grade) were purchased from the weitonglihua Experimental Animal Technology Co., Ltd. (Beijing, China). Then they were assigned randomly to group for either bilaterally ovariectomy (n=20) and group which was only performed abdominal incision without ovariectomy (Sham group, n=20). The animals were maintained under standard conditions (relative humidity = 50–60%; ambient temperature = 20 ± 2 °C; 12h light/dark cycle) with free access to water and food, and allowed to acclimate for 1 week. At 0 (baseline), 4, 8 and 12 weeks after operation, 5 rats in each group were randomly selected for spectral CT Gemstone Spectral Imaging (GSI) and dual energy X-ray absorption (DXA) scanning under anesthesia (3ml of Urethane intraperitoneal injection, Shanghai Shanpu Chemical Co.,Ltd). Excessive anesthesia after examination, the fifth lumbar vertebra (L5) was separated and taken for pathological observation. All animal experiments were approved by the Committee of animal management and use institutions of Shandong First Medical University and carried out in accordance with the guidelines for the use of experimental animals of the National Institutes of Health. This study is reported in accordance with ARRIVE guidelines.
1.2 GSI Protocol and Analysis
At baseline, 4, 8 and 12 weeks after ovariectomy, all rats were taken from prone position for lumbar vertebra GSI scanning (GE Revolution CT, America) after anesthesia. Specific parameters: tube voltage 80-140KV, tube current 400mA, layer thickness 0.625mm, Stnd reconstruction mode and ASIR 30%. When scanning was finished, transfer the original data to AW4.6 workstation and select the Volume Viewer. Then measure the Hydroxyapatite (HAP)/fat value (mg/cm3) and 74KeV monoenergetic CT value (HU) of cancellous bone in the sagittal middle layer of L5 vertebral body in sagittal position (place the cursor on the central line of L5 vertebral body in coronal position as far as possible, meanwhile the sagittal position automatically corresponds to the middle plane). Due to the small vertebral body of rats, in order to avoid overlapping with bone cortex and ensure the accuracy of region of interest (ROI), as shown in Fig. 1, ROI is divided into two parts with a total size of about 5.0mm2. Two experienced radiologists measured it respectively for three times and took the average value to get the final results.
1.3 Measurement of BMD
The BMD (unit, g/cm2) of the L5 vertebral body was measured at baseline, 4, 8 and 12 weeks post-OVX surgery by DXA (Primus, osteosys Co.,Ltd.) under anesthesia. The rats were placed in the prone position on the DXA scanning table according to the instructions. As shown in Fig. 2, the ROI was analyzed with its own software.
1.4 Histological analysis
The L5 vertebral body was decalcified, dehydrated and embedded in paraffin prior to processing. Then the 5μm tissue sections were stained with hematoxylin and eosin (H&E) staining. As shown in Fig. 3, images of the L5 vertebral body sections were captured using the microscope (20X) equipped with a camera to observe the morphological structure of trabecular bone.
1.5 Statistical analysis
All results were presented as means ± standard deviation (SD) and N represents the number of animals in each group. Pearson correlation analysis was performed on HAP(fat) value, 74KeV monoenergetic CT value and BMD of L5 vertebral body in OVX group by the statistical software SPSS V22.0 (IBM, Armonk, NY, USA). The comparison between groups and intra groups were carried out by one-way ANOVA with subsequent LSD post-hoc test. P<0.05 were considered statistically signifificant.