Perfusion imaging with 320-slice spiral computed tomography and color-coded digital subtraction angiography for assessing acute skeletal muscle ischemia-reperfusion injury in a rabbit model

Fifty New Zealand white rabbits (male, 2.0–2.5 kg) were obtained from the Huadong Xinhua Laboratory Animal Center (Guangzhou, China), and randomly assigned to the IR (n = 40) or control group (n = 10). All rabbits were raised at the Animal Laboratory Center of Jinan University, and housed with five mice per cage. After 3 h of right hindlimb ischemia, the IR group were assessed at 0, 6, 12, or 24 h (n = 10, for each time point) after reperfusion. A sham operation was performed to rabbits in the control group. For all of the IR groups, CTPI (CTPI subgroup, n = 5) and color-coded DSA (DSA subgroup, n = 5) were used to image the bilateral limbs of each animal, respectively.

The rabbits were anesthetized with an intraperitoneal injection of 1% pentobarbital sodium (40 mg/kg of body weight). Using 1% pentobarbital sodium (30 mg/kg of body weight), subsequent anesthesia was maintained every 3 h through the ear-rim auricular vein injection. In the IR groups, an anterior abdominal wall incision was made after sterilization. The right iliolumbar artery, right internal iliac artery and right common iliac artery were exposed and ligated with a 3–0 silk suture (Jinhuan, Shanghai, China). The blood supply of the tail was blocked using a tight elastic band to prevent the collaterals to the hindlimb during the ischemia. After confirming successful ischemia induction, the abdomen was closed. At 3 h post-surgery, the abdomen was reopened and the ligatures were removed. The red color of vessels with a visible pulsation indicated that the reperfusion was successful. In the control group, the abdomen of rabbits was only opened and closed without ligation of the arteries. A flowchart of this study is shown as Fig. 1.

In the CTPI subgroup of IR, the Aquilion One 320 volumetric CT scanner (Toshiba, Japan) was used to scan the bilateral hindlimbs of the experimental rabbits at 0, 6, 12, or 24 h post-reperfusion depending on the group (CT-0, CT-6, CT-12, or CT-24, respectively). After sufficient anesthesia, the rabbits were fixed at the center of the CT scanner in a supine position. The scan range included the full-length of the hindlimbs. Before scanning, 3 mL of iodixanol (32 mg/mL) was injected with a high-pressure syringe at the rate of 1 mL/s. Following the iodixanol, 8 mL of physiological saline was injected at the same rate through the ear vein. Then 3 s later, the delayed x-ray scanning was performed. The data collection time was 60.5 s which included 3 parts. In the initial 22.5 s, data was collected every 1.5 s, and the exposure time was 0.5 s. The collection was performed 12 times during this procedure. In the middle 18 s, data was collected every 2.5 s, and the exposure time was 0.5 s. The collection was performed six times during this procedure. In the last 20 s, data was collected every 4.5 s, and the exposure time was 0.5 s. The collection was performed four times during this procedure. The scanning parameters consisted of the detector arranged by 320 × 0.5 mm with a thickness of 0.5 mm, 0.5 s/ring, 80 kV, and 40 mA.

Similar to the CTPI subgroup, rabbits in the DSA subgroup were fixed onto the operation table in supine position. After supplemental anesthesia and skin preparation, the right main carotid artery was exposed and punctured. Depending on the group (IR-0, IR-6, IR-12, or IR-24), angiography of the lower abdominal aorta was performed to assess changes in the blood flow changes of the right hindlimb using a 2.7 F microcatheter (Terumo, Japan) through the carotid artery at 0, 6, 12, or 24 h. The DSA equipment used in this study was Artis Zeego (Siemens, Erlangen, Germany). Iodixanol (320 mg/mL) was injected with a velocity of 3 mL/s through the microcatheter using a power injector. The injection pressure was 200 psi and the total dose of the contrast agent was 9 mL. The DSA acquisition frame rate was six frames per sec and each acquisition process lasted until the inferior vena cava was visualized.

The imaging data collected from CT and DSA were sent to their respective post-processing workstations, respectively (CT: Vitrea, Toshiba, Japan; DSA: Leonardo, Siemens, Germany). The CTPI and color-coded DSA images were automatically generated by the software. Based on the images, the 50-mm region of interest (ROI) was selected in the bilateral vastus lateralis muscle from all the rabbits by two practicing interventional radiologists independently. The ROI was chosen in the soft tissue, yet away from the skeleton and vessels in both the CTPI and DSA groups.

Using the post-processing workstation, perfusion parameters, including blood flow (AF), blood volume (BV), and contrast agent clearance (C) values of the right and left limb ROI (AF-R, AF-L; BV-R, BV-L; C-R, C-L, respectively) in the CTPI subgroup and the maximum contrast enhancement (peak) of the right and left limb ROI (peak-R, peak-L, respectively) in the DSA subgroup were obtained. To eliminate individual differences among the rabbits in this study, the ratios of AF, BV, C, and the peak were calculated during the post-processing (AF-R/L, BV-R/L, C-R/L and peak-R/L).

Prior to the CT and DSA scanning in each rabbit, 3 mL of blood was taken from the external jugular vein. After centrifugation of the blood samples, the separated serum was stored at − 20 °C. The samples were used to measure SOD, CK, LDH and MDA. CK and LDH using an automatic biochemical analyzer. In addition, MDA was measured using the thialbarbital sodium method, while SOD was measured via the xanthine oxidase method.

After the experiment, all rabbits were sacrificed by injecting 10 mL of air through the ear-rim auricular vein.

SPSS 16.0 (IBM, Chicago, IL, USA) was used for the statistical analysis. A single factor variance analysis was used for comparison between groups. The independent-samples t-test was used for comparison between data obtained from the control group and IR group. The correlation of AF-R/L, BV-R/L, C-R/L and peak-R/L between various biochemical indicators, including SOD, CK, LDH and MDA, were observed by the Pearson’s correlation analysis. The statistical data are expressed as standard error of the mean and p-values < 0.05 were considered significant.

The surgical procedure was successful in all 45 rabbits (Fig. 2). CTPI and color-coded DSA were successfully achieved in each group (Figs. 3 and 4). There were no deaths during this study.

The detailed CTPI and color-coded DSA parameters are shown in Tables 1 and 2. For CTPI, a significant difference existed between each experimental group and the control group in AF-R/L (p < 0.001). When compared with the control group, significant differences only existed in the 12 h and 24 h groups in BV-R/L and C-R/L (p < 0.001). Using a single factor variance analysis, significant differences in AF-R/L were detected between each group when compared with the control group (F = 6.206, p = 0.002). The AF-R/L value gradually decreased over time, yet this decrease was not significant between 0 h and 24 h. The BV-R/L and C-R/L from each group showed no significant differences between each other. For the color-coded DSA, there was a significant difference in Peak-R/L between the control group and all IR groups (p < 0.001). Extension of IRI time resulted in a gradual decrease of the Peak-R/L.

As shown in Table 3, SOD in the IR group was lower than in the control group. However, CK, LDH and MDA in the IR groups were higher than those of the sham group (p < 0.001). The variance analysis showed that the differences in CK and LDH among all groups were statistically significant (p < 0.001). CK and LDH increased gradually with the IR time. However, there were no significant differences in MDA or SOD among the groups (p > 0.05).

In the present study, AF-R/L was positively correlated with SOD (r = 0.57, p < 0.001) and negatively correlated with CK, LDH, and MDA (r = − 0.60, p < 0.001; r = − 0.44, p < 0.001; r = − 0.62, P < 0.001; respectively). LDH was negatively correlated with BV-R/L (r = − 0.45, p < 0.001), and no correlation existed between any of the biochemical biomarkers and C-R/L.

The Peak-R/L was positively correlated with SOD (r = 0.59, p < 0.001) but negatively correlated with CK, LDH and MDA (r = − 0.68, p < 0.001; r = − 0.71, p < 0.001; r = − 0.66, p < 0.001; respectively). The Peak-R/L was positively correlated with AF-R/L in the color-coded DSA and CTPI (r = 0.70, p < 0.001).