2.1 Research objects
Thirty male wistar rats were obtained from Experimental Animal breeding Co., Ltd.(China, Jinan). The rats were 7 weeks old and weighed 170-200g. All rats were acclimatized in a Specific Pathogen Free(SPF) environment for one week before the experiment. At 8, 13, 18, 23, 28 and 33 weeks, 5 rats were randomly selected for MRI examination under anaesthesia (3ml of Urethane intraperitoneal injection), then euthanized and sent for histology acquisition.Urethane was purchased from Shanghai Shanpu Chemical Co., Ltd. A 0.2g/mL solution of urethane was prepared using saline (total of 10ml/kg) with intraperitoneal injection.The study was approved by the Institutional Animal Care and Use Committee and was performed in accordance with the National Institutes of Health guidelines for the use of laboratory animals.
2.2 MR Imaging Techniques
All MR imaging was performed using a 3.0-T MR (GE discovery MR750) and utilizing a matched eight-channel animal coil (Wankang Medical Technology Co., Ltd, China). Four standard MR sequences were performed.(A)Axial T2 fat saturated(FS) images[echo time(TE)/repetition time (TR),96.1ms/3000ms; echo train length,16];(B)Coronal T1 FS fast spin echo (FSE)(TE/TR, 13.5ms/500ms; echo train length, 3); (C)IVIM-DWI: repetition time/echo time (TR/TE) 4000/66.8 ms, slice thickness 3.0mm, matrix 64×64,FOV 140mm×112mm, spatial resolution 3.8mm2. a total of 12 b-values were used: 10, 20, 30, 50, 80, 100, 200, 300, 600, 800, 1000, 1500s/mm2. (D)DCE-MRI: fat saturated contrast enhanced T1 images with liver acquisition with volume acceleration(LAVA) sequence, repetition time/echo time (TR/TE) 5.6/1.9 ms, slice thickness 2.0 mm, matrix 128×128,FOV 200mm×160mm. A total of 80 phases were acquired, with a spatial resolution of 2.0mm2. 1ml of gadopentetate dimeglumine contrast agent(BeiLu Pharmaceutical Co., Ltd, Beijing, China) was administered intravenously(tail vein) at a rate of 0.1ml/s, followed by a 2ml saline(0.9%, Shandong Qidu Pharmaceutical Co., Ltd) flush by hand. The concentration of gadopentetate dimeglumine is 0.5g/ml.After the acquisition of seven baseline dynamic scans, 960 images were collected totally with 80 phases for approximately 5 minutes of scanning; Function tool software of GE MR Advantage Workstation 4.6 was used to perform the measurements of DCE-MRI and IVIM-DWI.
2.3 Image Analysis
2.3.1 Analysis of IVIM Parameters
The DWI signal follows the biexponential model to calculate the signal attenuation IVIM, as:
Where Sb is the signal intensity in the pixel with diffusion gradient b, S0 is the signal intensity without diffusion gradient, D (×10-4 mm2/s) is the water diffusion coefficient in the tissue, f is the perfusion fraction related to microcirculation(flowing blood fraction) and D* (×10-4 mm2/s) is the pseudo-diffusion coefficient which represents perfusion-related diffusion.
2.3.2 Analysis of DCE-MRI Parameters
For DCE-MRI analysis, all data were quantitatively analyzed using image processing software. By manually drawing different regions of interest (ROIs), we obtained time-intensity curves (TICs).
Enhancement factor Fenh (%) = ( SImax –SI0) ×100% /SI0;
Enhancement slope Senh (%/s) = ( SImax-SI0) 100 / ( SI0×Tmax);
where SI0 (baseline Signal Intensity before contrast injection) approaches the SImax (maximum Signal Intensity) exponentially in time. SImax is determined as the maximum SI during the DCE-MRI examination and Tmax is the time point at SImax .
2.3.3 Data analysis
The original images were processed using the Advantage Workstation(ADW 4.6 version, GE, US) and post-processed by Functool workstation. Two observers with 15 years and 20 years of experience in MRI were blinded to the information and individually measured the resulting parameter maps. Disagreement of both radiologists was resolved in consensus. All data were measured for 3 times and the average of 3 times is taken to reduce the bias caused by measurement error. On the IVIM-DWI and DCE-MRI series,an ovoid region of interest (ROI)was placed within the bilateral sacral or iliac bone marrow.The ROI of the joint space was placed at the lower third of the cartilaginous portion of the SIJ.All ROIs were 2-4mm2. Then the bilateral average values were taken.Care was taken to avoid cortex, venous plexus,ligaments, or any imaging-related artifacts(Figure 1, 2, 3).
2.4 Histological assessment
After MR examnation, all rats were weighed and each rat was euthanized with
1% pentobarbital(Sigma company) 100mg/kg intraperitoneal injection.When heartbeats had not been detected for five minutes, samples of the SIJ were cut across the midline and removed, fixed in 10% formalin(200ml per sample) for one-two day, acid-decalcified with 10% methanoic acid for one week, embedded in paraffin, cut after dehydration in graded ethanol(75% ethanol 15s, 85% ethanol 10s, 95% ethanol 10s, absolute ethanol 1min, absolute ethanol 1min,) and stained with hematoxylin(8min) and eosin(2min).The histological changes of the SIJ were observed under microscope (MODEL BX53F,OLYMPUS, Tokyo,Japan)finally.
2.5 Statistical Analysis
SPSS Statistics version 19.0 was used for statistical analysis. The homogeneity of variances was tested using the Levene’s tests. All the parameter values were compared by one-way ANOVA. The measured parameters were expressed as means ± standard deviation (SD). P values of less than 0.05 were considered as statistically significant.