Abstract
Objective
To explore the relationship between abdominal aortic aneurysm development and inflammation in the rabbit through the establishment of a rabbit infrarenal abdominal aortic aneurysm model and the use of 18F-FDG PET/CT imaging.
Methods
Twenty male New Zealand rabbits were administered an elastase intracavity perfusion to induce an infrarenal abdominal aortic aneurysm model. Prior to surgery, the rabbits underwent abdominal aorta ultrasonic testing and blood collection from the ear veins. Of the original 20 rabbits, 10 rabbits were euthanized two weeks after the operation following ultrasonic testing, PET/CT scanning and blood collection, and their arterial tissue samples were prepared for pathological and immunohistochemical staining. The remaining 10 rabbits were euthanized four weeks after the operation following ultrasonic testing, PET/CT scanning and blood collection, and the arterial tissue samples were prepared for pathological and immunohistochemical staining.
Results
Compared with the preoperative measurement, the maximum growth rate of the aneurysm diameter is 89.21 ± 0.02% (the absolute increase in diameter is 2.040 ± 0.376 mm) two weeks after the operation. Compared with the two-week postoperative value, the maximum growth rate of the aneurysm diameter is 15.8 ± 0.01% (the absolute increase in diameter is 0.684 ± 0.115 mm) four weeks after the operation. Compared with the preoperative values, the blood MMP-2 and MMP-9 levels significantly increase two weeks after surgery, P < 0.05. Compared with the two-week postoperative values, the blood MMP-2 and MMP-9 levels significantly decrease after four weeks post-surgery, P < 0.05. At two weeks after the operation, the SUVmax and the TBR of the 18F-FDG PET/CT of the AAA wall are 0.90 ± 0.03 and 1.19 ± 0.09, respectively. At four weeks after the operation, the SUVmax and the TBR of the 18F-FDG PET/CT of the AAA wall are 0.35 ± 0.05 and 1.15 ± 0.12, respectively. Compared with two weeks after the operation, the SUVmax significantly decreases at four weeks after the operation, P < 0.05. Compared with two weeks after the operation, there is no significant difference in the TBR at four weeks after the operation, P > 0.05. Immunohistochemical staining shows that the CD68-positive cell rate at four weeks after the operation significantly decreases (P < 0.05) compared with the CD68-positive cell rate at two weeks after the operation.
Conclusion
In the early stages of abdominal aortic aneurysm development, the inflammatory response of the arterial wall is significant, the local metabolic activity is strengthened, the SUVmax value of 18F-FDG is high, and the abdominal aortic aneurysm diameter experiences rapid growth. In the later stages of abdominal aortic aneurysm development, the diameter continues to increase; however, there are decreases in the wall inflammatory response, the local metabolic activity, and the SUVmax value of 18F-FDG. Thus, inflammation plays an important role in the early development of abdominal aortic aneurysm.
Abdominal aortic aneurysm (AAA) refers to the local sustained expansion of the original normal abdominal aortic artery diameter by at least 50%. 1 A ruptured AAA is often fatal. 2 Clinical data indicate that there is no inherent connection between the AAA diameter and AAA rupture. Many AAAs with smaller diameters can rupture, indicating that the size of the AAA cannot be used to predict aneurysm rupture. Therefore, treatment decisions based solely on the size of the AAA are not appropriate.2,3 The use of 18F-FDG PET/CT imaging, which was developed in recent years, shows that the uptake rate of 18F-FDG by the AAA wall clearly increases, suggesting local inflammatory activity. 4 The current study is a clinical cross-sectional study; however, there is limited basic research on the relationship between inflammation and progression of AAAs. In this study, we established a rabbit AAA model and performed a systematic PET/CT in vivo imaging study to investigate the relationship between inflammation and progression of AAAs in rabbits.
Materials and methods
Materials
Twenty healthy male New Zealand white rabbits (Beijing Haidian Xingwan Animal Breeding Farm, License No. SCXK (Beijing) 2006–0006) that weighed approximately 2.50 ± 0.20 kg were used in this study. The rabbits were allowed free access to water and food and were reared in separate cages with an ambient temperature of 23–25°C. The housing environment was dry with good ventilation, adequate lighting, and easy drainage. The cage was 60 cm in width and 50 cm in height, and one animal was fed per cage. The feed formulation included alfalfa powder, wheat bran, flour, corn, soybean meal, fish meal, bone meal, yeast powder, salt, and fish liver oil. Clean tap water was provided as drinking water. The following materials were used: a 100 u/ml pig pancreas elastic protease solution (elastase at pH 8.8, 37.0°C) (45124, Sigma-Aldrich, St. Louis, Missouri, USA); 0.9% sodium chloride (products from Shandong Hualu Pharmaceutical Co., Ltd); 4F Forgaty catheter with double-cavity saccule (Beijing Life Oasis Science and Technology Co., Ltd); EVG staining, Mason staining, and CD68 staining kits (Wuhan Guge Biotech Company); pentobarbital sodium (Bioszune Company, USA); heparin sodium injection (Beijing Double-Crane Pharmaceutical Co., Ltd); GE Discovery ST16 type PET/CT system (American GE Company); color ultrasonic equipment (Netherlands Philips Company); upright optical microscope (Nikon in Japan); and MMP-2 and MMP-9 ELISA kits (Shanghai Lanpai Biotech Co., Ltd, China).
Methods
This study was performed in strict accordance with the recommendations described in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of Anzhen Hospital.
Modeling
Twenty New Zealand white rabbits underwent elastase intracavity perfusion to induce an infrarenal AAA model according to the following steps: ① All operations were aseptically performed on experimental animals. In order to prevent thrombosis, we injected heparin through the ear vein before operation. ② Approximately 0.5–1 cm of the common femoral artery was dissected. ③ The abdominal cavity was opened and approximately 1.5–2.0 cm of the infrarenal abdominal aorta was dissected. ④ The catheter was placed into the dissected aortic segment through the femoral artery, the proximal abdominal aorta was blocked with an artery clamp and rubber filter lines, the distal part was blocked with rubber filter lines, and a closed lumen was formed with the catheter. ⑤ Then, 100 mmHg of pressure was provided to inject 0.1 ml of 100 IU/ml pig pancreatic elastase protease solution into the arterial lumen through a double-cavity catheter. After 7 min, the solution was withdrawn and heparin and saline were injected through a vice catheter to wash the artery lumen three times. ⑥ The aortic blood flow was restored and the abdomen was closed. ⑦ Penicillin (800,000 U) was injected intraperitoneally into each rabbit for three consecutive days following surgery.
Doppler ultrasound examination
Doppler ultrasound examination was performed as follows: the sagittal and horizontal positions were examined, the maximum diameter of the lumen expansion was measured, the average values of the dimeters of the experimental animal tumors were obtained, and the abdominal aorta expansion rate was calculated. The rate of expansion at two weeks = (the diameter of the AAA at two weeks – the diameter of the normal abdominal aorta)/the diameter of the normal abdominal aorta. The rate of expansion at four weeks = (the diameter of the AAA at four weeks – the diameter of the AAA at two weeks)/the diameter of the AAA at two weeks.
Serum MMP-2 and MMP-9 detection
For this procedure, blood was collected prior to the operation, two weeks after the operation and four weeks after the operation, the serum was separated, and serum MMP-2 and MMP-9 were detected with ELISA kits.
18F-FDG PET/CT scanning and post-processing
For imaging, food and water were withheld from the animals 4 h prior to scanning and inject 18F-FDG（1 mCi/kg) via an ear vein. Intraperitoneal anesthesia was administered 120 min after the injection of 18F-FDG. CT plain scans were completed to locate the aortic lumen and PET image collection was subsequently initiated. Then, TBR＝AAA SUVmean/thoracic aorta SUVmean was calculated, based on the body weight of the rabbit, the 18F-FDG injection quantity, the mean standardized uptake value (SUVmean), and the maximal standardized uptake value (SUVmax) of the drug metabolism time for the selected region.
Tissue pathological staining
After paraffin embedding and tissue biopsies, CD68 macrophage staining, EVG staining and Masson staining were applied. NIS-Elements AR Analysis was used to calculate the number of macrophages.
Statistical analysis
SPSS 17.0 was used for the statistical analyses. The means ± standard deviation is used to show the measurement data. The groups were compared, and if the variances were equal, independent samples t-tests were used; if the variances were not equal, correction t-tests of independent samples of two groups were used. Pearson’s correlation was used to analyze the data. Inspection was set to a two-sided test, with P < 0.05 for statistical significance.
Results
Ultrasound results
Compared with the pre-surgical measurement, the arterial aneurysm lumen diameter growth is 89.21 ± 0.02% (the absolute increase in diameter is 2.040 ± 0.376 mm) two weeks after the operation, P<0.05, and the difference is statistically significant. Compared with the measurement from two weeks after the operation, the aneurysm lumen maximum diameter growth rate significantly decreases at four weeks after the operation; its growth rate is 15.8 ± 0.01% (the absolute increase in diameter is 0.684 ± 0.115 mm), P<0.05, and the difference is statistically significant (Figure 1).
Ultrasound results. (a) The transverse and longitudinal sections of the abdominal aorta vascular ultrasound at different periods are shown, and the arrows in the figure indicate the abdominal vascular lumen. The preoperative blood vessel lumen diameter is smaller; however, it significantly increases two weeks and four weeks after the operation. (b)The comparison of the AAA lumen diameter results at different periods is shown. Compared with the preoperative measurement, the AAA lumen diameter growth is 89.21 ± 0.02% two weeks after the operation, and the difference is statistically significant, P<0.05. Compared with the measurement from two weeks after the operation, the aneurysm tumor maximum diameter growth is 15.8 ± 0.01% four weeks after the operation, and the difference is statistically significant, P<0.05.
Serum MMP-2 and MMP-9 levels
Compared with the preoperative measurements, the serum MMP-2 and MMP-9 levels are significantly elevated two weeks after the operation, 50.91 ± 2.00 and 95.54 ± 3.21, respectively, P<0.05, and the difference is statistically significant. Compared with the measurements from two weeks after the operation, MMP-2 and MMP-9 are both decreased four weeks after the operation, 35.27 ± 1.12 and 65.84 ± 2.25, respectively, P<0.05, and the difference is statistically significant (Figure 2).
Comparison of serum MMP-2 and MMP-9 levels at different periods. Compared with the preoperative measurements, the MMP-2 and MMP-9 levels significantly increase two weeks after the operation, P<0.05, and the difference is statistically significant. Compared with two weeks after the operation, the MMP-2 and MMP-9 levels are decreased four weeks after the operation, P<0.05, and the difference is statistically significant.
Histopathological results
At two weeks after the operation, Masson staining shows increases in the collagen fiber content in the tunica media and adventitia areas of the aneurysm wall tissue; however, the tunica media and adventitia structures in the wall are clear. EVG staining shows that the elastic fiber content is damaged, and the hierarchical structure is not sufficiently clear; however, the continuity of part of the elastic fiber content is complete and maintains its curvature. At four weeks after the operation, Masson staining shows increases in the collagen fiber content in the tunica media and adventitia areas of the aneurysm wall tissue, with substantial blurring. EVG staining shows that the hierarchical structure of the elastic fiber content is not clear, and it has no curvature, indicating the occurrence of breaking, thinning, and fragmentation. CD68 immunohistochemistry staining shows that the CD68-positive cell staining percentage is 4.21 ± 0.53% two weeks after the operation, whereas the percentage is 0.87 ± 0.11% four weeks after the operation. The difference between the two groups is statistically significant (P<0.05) (Figure 3).
At two weeks, Masson staining shows increases in the collagen fiber content in the tunica media and adventitia areas. EVG staining indicates that the elastic fiber content is damaged, and the hierarchical structure is not clear. At four weeks, Masson staining shows increases in the collagen fiber content in the tunica media and adventitia area. EVG staining indicates that the hierarchical structure of the elastic fiber content is not clear. CD68 staining shows that CD68-positive cells are widely distributed with large numbers at two weeks, whereas the CD68 cells are distributed with smaller numbers at four weeks.
PET/CT imaging results
The PET/CT imaging results are shown in Figure 4. The PET/CT quantitative and statistical analyses show that two weeks after the operation, the SUVmax is 0.90 ± 0.03, and the TBR is 1.19 ± 0.09. At four weeks after the operation, the SUVmax is 0.35 ± 0.05, and the TBR is 1.15 ± 0.12. Compared with two weeks after the operation, the SUVmax and TBR at four weeks after the operation significantly decreases, P<0.05, and the difference is statistically significant (Figure 5).
PET/CT imaging results: A presents PET/CT images (sagittal) two weeks after the operation. A represents the CT image; b represents the PET image; c represents the PET/CT fusion image. B shows the PET/CT images (sagittal) four weeks after the operation. A represents the CT image; b represents the PET image; c represents the PET/CT fusion image. The arrows indicate the AAA. As indicated in Figure A(a) and Figure B(a), the diameter of the AAA increases, and the expansion is clear at two and four weeks. Figure A(b) and Figure B(b) are the 18F-FDG absorption images of the AAA. Compared with Figure B(b), in Figure A(b), the 18FFDG maximum absorption (SUVmax) was significantly increased, and the AAA site absorption had color deepening.
Comparison of SUVmax and TBR values at different periods after the operation. Compared with two weeks after the operation, the SUVmax value decreases four weeks after the operation, and the difference is statistically significant (P<0.05); however, the TBR value does not change between two weeks after the operation and four weeks after the operation (P > 0.05).
Correlation analysis. (a) Two weeks after the operation, the 18F-FDG PET SUVmax value of the AAA wall is positively correlated with the growth rate of the AAA, r = 0.459; P = 0.042. (b) Four weeks after the operation, the 18F-FDG SUVmax value of the AAA wall is negatively correlated with the aneurysm growth, r = −0.531, P= 0.016. (c) Two weeks after the operation, the 18F-FDG PET SUVmax value of the AAA wall is positively correlated with the area percentage of CD68-positive cells, r = 0.468; P = 0.037. (d) Four weeks after the operation, the 18F-FDG SUVmax value of the AAA wall is negatively correlated with the area percentage of stained macrophages, r=−0.459, P = 0.042.
Correlation analysis
Two weeks after the operation, the 18F-FDG PET SUVmax value of the AAA wall is positively correlated with the growth rate of the AAA, r = 0.459; P=0.042 (Figure 6(a)). Two weeks after the operation, the 18F-FDG PET SUVmax value of the AAA wall is positively correlated with the area percentage of CD68-positive cells, r=0.468; P=0.037 (Figure 6(c)). Four weeks after the operation, the 18F-FDG SUVmax value of the tumor wall is negatively correlated with the aneurysm growth rate, r = −0.531, P = 0.016 (Figure 6(b)). Four weeks after the operation, the 18F-FDG SUVmax value of the tumor wall is negatively correlated with the area percentage of the stained macrophages, r=−0.459, P=0.042 (Figure 6(d)).
Discussion
Through the establishment of the rabbit AAA model and the application of PET/CT imaging and ultrasound as follow-up methods, we show the AAA development, local inflammatory reaction and histological changes at different periods and analyze the relationship between the AAA 18F-FDG PET/CT uptake values and aneurysm diameter growth rates, as well as the inflammatory response at each period. This study indicates that in the early stages of AAA development, the 18F-FDG SUVmax value is positively correlated with the expansion rate of the AAA and CD68-positive cell percentage. It shows that at the early stages of AAA development, the inflammatory reaction is evident, the partial metabolic activity of the wall is enhanced, the 18F-FDG PET/CT SUVmax value is high, and the AAA growth rate is rapid. At the later stages of AAA development, the 18F-FDG SUVmax value is negatively correlated with the expansion rate of the AAA and the CD68-positive cell percentage. The results indicate that the AAA diameter increases; however, the partial metabolic activity, inflammatory reaction, and 18F-FDG SUVmax uptake decrease. Our results indicate that inflammation plays an important role in AAA progression during the early stages.
The occurrence and development of AAAs are associated with various factors. In recent years, the study of the pathogenesis of AAAs has focused on MMP activation, inflammatory cell infiltration, SMC apoptosis, and other processes, of which the inflammatory reaction is thought to be an important component. Research indicates that various pathogenic factors participate in the occurrence and development of AAAs through activated macrophages.5–8 The main mechanism of inflammatory cells is the release of matrix metalloproteinases (MMPs), and MMP-2 and MMP-9 are the most important MMPs.9,10 These MMPs may cause the degradation of the elastic fiber content of the arterial wall, decrease the SMC content, and result in a relative increase in collagen fiber content, which ultimately thins the AAA wall thin.11,12 Our experimental results show that the serum MMP-2 and MMP-9 levels at two weeks after the operation are higher than the preoperative levels, which indicates that MMP-2 and MMP-9 are involved in AAA progression. Over the same period, immunohistochemical detection shows that the CD68-positive cell percentage results are consistent with these results; thus, the CD68-positive cell percentage increases two weeks after the operation. When the numbers of inflammatory cells increase, the release of MMP-2 and MMP-9 increases, which is consistent with the study by Matthew Longo et al., 13 These authors report that the MMP-2 and MMP-9 levels in the rabbit AAA model increase, and they suggest that both MMPs play important roles in AAA development.
Histologically, elastin degradation is essential for aneurysm formation. 14 EVG staining displays damage to elastic fibers in the abdominal aortic wall. Mason staining displays changes in the collagen fiber content in the AAA wall. In our study, we show that two weeks after the operation, Masson staining indicates an increase in the collagen fiber content in the tunica media and adventitia areas of the aneurysm wall tissue; however, the tunica media and adventitia structures in the wall are clear. EVG staining shows that the elastic fiber content is damaged, and the hierarchical structure is not sufficiently clear; however, the continuity of part of the elastic fiber content is complete and maintains its curvature. The immunohistochemical results indicate that the CD68-positive cell percentage is higher two weeks after the operation. At this stage, the inflammatory reaction is evident. The elastic fibers in the AAA wall are destroyed, and the collagen fibers form by the action of matrix MMPs secreted by inflammatory cells. At four weeks after the operation, Masson staining shows an increase in the collagen fiber content in the tunica media and adventitia areas of the aneurysm wall tissue, with substantial blurring. EVG staining shows that the hierarchical structure of the elastic fibers is not clear, and it has no curvature, which indicates the occurrence of breaking, thinning, and fragmentation. The immunohistochemical results show that the CD68-positive cell percentage is substantially lower than that at two weeks after the operation. At this stage, the inflammatory reaction weakens, which may be related to the large destruction of vascular wall elastic fibers and smooth muscle by matrix MMPs together with the widespread formation of collagen fibers.
Inflammation and tunica media cell death are important biological activities involved in aneurysm growth.15–17 PET imaging can help locate and measure the metabolic activity of cells. PET/CT belongs to the composite imaging technologies that combine function and form, demonstrate the state of metabolic activity and provide the anatomical structure. In recent years, it has been used in the clinical detection of inflammatory states of diseases, such as atherosclerosis and AAA.18–21 Zhao et al. 22 established the rabbit model for atherosclerosis plaque; using PET/CT imaging to investigate the relationship between 18F-FDG uptake and the macrophage content in the atherosclerosis plaque, they identified a positive correlation. Nahrendorf et al. 21 established an AAA model in mice and used PET/CT imaging to observe 18F-FDG uptake in the AAA, as well as to judge the inflammatory reaction. The results show that the inflammatory reaction in part of the rat AAA wall is significantly enhanced compared with the non-AAA part of the wall. Reeps et al. 23 conducted a clinical study involving AAA patients. All hospitalized patients were scanned with 18F-FDG PET/CT prior to surgery. The scan results were used in a correlation analysis with AAA specimens obtained during the operation. The results show that the 18-FDG SUV uptake value is positively correlated with the degree of infiltration by inflammatory cells. Nchimi 24 obtained similar results in clinical studies involving AAA patients. However, several studies indicate the opposite conclusions. Tegler et al. 25 performed resections of AAA tissue in patients and observed the tissues for pathological findings. They report that the infiltration of inflammatory cells in the wall of patients with larger AAAs is not obvious, and the 18F-FDG SUV uptake value is not increased. Therefore, they believe that the 18F-FDG PET/CT SUV uptake value does not reflect the inflammatory state and AAA progression. There is no correlation between the occurrence and development of AAA and inflammation.
The different conclusions obtained by previous studies suggest that the AAA course may be more complex. The different stages of the AAA course must be discussed in greater detail. We established the rabbit AAA model using elastase intracavity perfusion and observed the development of the AAA, the local inflammatory reaction and the histological changes at different periods through the application of PET/CT imaging and ultrasound as follow-up methods; we subsequently analyzed the relationship between the AAA 18F-FDG PET/CT uptake value and the growth rate of the aneurysm diameter, as well as the inflammatory response at each period. According to the results, at the early postoperative stage, namely, at two weeks after the operation, the AAA diameter is substantially larger than the diameter before the operation with a growth rate close to 90%. The 18F-FDG SUVmax value of the AAA wall is higher, and the correlation analysis shows that these two values are positively related. The immunohistochemistry of this stage indicates that the percentage area of macrophages determined via CD68-positive cell staining and the SUVmax value are also positively correlated. We suggest the inflammation of the AAA wall at this stage is evident, leading to the increasing release of MMPs, such as MMP-2 and MMP-9, and damage to the elastic fibers and smooth muscle structure in the AAA wall, as well as wall expansion. Our results are consistent with the results of Xu et al. 26 who showed that the progression and rupture of the abdominal aorta were closely related to the maximum uptake of the 18F-FDG SUVmax, the inflammation of the vascular wall and MMP expression. Thus, they conclude that 18F-FDG PET/CT imaging may be used as a novel technology for AAA diagnosis and may help predict the risk of AAA rupture.
At four weeks after the operation, the AAA diameter continues to increase with a growth rate close to 16% compared with the AAA diameter at two weeks after the operation. The 18F-FDG SUVmax value of the AAA wall is lower, and the correlation analysis indicates that the two variables are negatively related. The immunohistochemistry of this stage shows that the percentage area of macrophages via CD68-positive cell staining decreases, and it is negatively correlated with the SUVmax. We suggest that at the later stage of aneurysm development, heavy damage to the vascular wall structure, a decrease in local inflammation and weakening of the local metabolic response of the wall affect the SUVmax uptake. These findings are consistent with the results of a clinical study by Marini et al. 27 They suggest that for patients with advanced AAAs, the heavy damage to the arterial wall structure affects the 18F-FDG uptake. When the arterial wall structure is severely damaged, the FDG uptake may not correspond to the inflammation. Our results also indicate that regardless of the postoperative period of two weeks or four weeks, the TBR value has no correlation with the AAA diameter growth rate or the percentage area of macrophages as shown by CD68 staining.
Conclusion
Our results confirm that in the early stages of AAA development, the 18F-FDG SUVmax value is positively correlated with the expansion rate of the AAA and the CD68-positive cell percentage. In the later stages of AAA development, the 18F-FDG SUVmax value is negatively correlated with the expansion rate of the AAA and the CD68-positive cell percentage. The results indicate that inflammation plays an important role in AAA progression in the early stages of development; however, the role of inflammation in AAA progression at the later stage is not clear. One limitation of our research is that this paper only discusses inflammation and does not include other factors that may affect AAT development, such as blood pressure, or mechanical factors that may influence the progress and rupture of AAAs.
Footnotes
Acknowledgments
Thanks to all the teachers who gave me support in the course of experiment and thesis writing!
Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by National Natural Science Foundation of China (81370437).