Effects Of Hemodynamic Changes Induced By Proximal Stenosis On The Outcomes Of Abdominal Aortic Aneurysm In Rabbits

Background and ObjectiveAimed to establish a new type of abdominal aortic aneurysm?AAA?model capable of better mimicking human AAA disease by overcoming the phenomenon of self-healing in rabbit AAA model induced by elastase.Also aimed to observe the hemodynamic and pathological changes of AAA model before and after termination of the stenosis,and to compare the differences between these two AAA models.Materials and methods1.Rabbit AAA induced by absorbable suture ligation combined with Chinese elastase.Forty-two rabbits were randomly divided into two groups.Rabbits in experimental group were incubated with Chinese porcine pancreatic elastase solution,and absorbable suture was used for partial ligation of proximal abdominal aorta after incubation.Sham operation was performed with physiological saline incubation.The diameters were measured before induction and 1 week,3 weeks,7 weeks and 15weeks after operation,respectively.After 1 week,7 weeks and 15 weeks,every 8rabbits were sacrificed in each group and stained with hematoxylin-eosin,elastic van-Gieson?EVG?and immunohistochemical staining.2.Effects of hemodynamic changes induced by proximal stenosis on the outcomes of rabbit AAA.Seventy-two New Zealand white rabbits were randomly divided into 3 groups:aneurysm group,balloon dilation group and control group.Aorta segments in aneurysm group and balloon dilation group were incubated with 60?l of elastase?1unit/?l?for 10 min and partially ligated to establish rabbit AAA model.Absorbable suture was used in balloon dilation group,and balloon angioplasty was performed to terminate the stenosis 1 month after operation.The aneurysm group was partially ligated with unabsorbable suture.Computational fluid dynamics?CFD?analysis was performed after 3 and 15 weeks.Eight rabbits were killed in each group after 1 week,5 and 15 weeks for pathological analysis and quantitative study.3.A novel AAA modelA midline laparotomy was performed under sterile conditions after anesthesia by intravenous injection of 30 mg/kg sodium pentobarbital.A 1.5-cm segment of infrarenal abdominal aorta was carefully dissociated and the domestic pig pancreatic elastase solution with a concentration of 60?l?1 unit/?l?was soaked for 10 minutes,and the proximal abdominal aorta was ligated with absorbable suture.If there was no absorption after 2 months,balloon dilation was performed to relieve the stenosis.4.Intravenous digital subtraction angiographyRabbits underwent intravenous digital subtraction angiography?IVDSA?imaging after 1,3,7,and 15 weeks.Specifically,a 22-G angiocatheter was placed in the ear vein for 5s DSA scanning?Siemens Artis Zee,Germany?.Iodinated contrast material?10 ml?was injected into the ear vein to visualize the AAA.3D-DSA analysis and diameter measurement were performed with RadiAnti DICOM Viewer 3.4.2?Medixant Company,Poznan,Poland?.Successful AAA formation was defined as increase of at least 50 percent in the focal dilation ratio of the incubated aorta compared to its normal diameter.5.Computational fluid dynamics analysisComputational fluid dynamics analysis of the abdominal aorta,including streamline,velocity and pressure distribution,wall shear stress?WSS?,oscillatory shear index?OSI?,and relative residence time?RRT?,were performed at two time points during the progression of AAA.The relationship between the aforementioned hemodynamic parameters and the initiation and progression of AAA were investigated.Animal-specific computational fluid dynamics models were studied.6.Animal sacrifice and histopathologyAfter IVDSA examination,every 7 rabbits in each group were sacrificed for histopathology after 1,3,7,and 15 weeks,respectively.Animals were anesthetized again,and pressure perfusion was performed with 4%buffered paraformaldehyde to maintain the shape of AAA.Aortic tissues were embedded in paraffin and cut into5-?m sections.To study the overall morphology and distribution of elastic lamellae,hematoxylin-eosin?HE?and Elastin Van-Gieson?EVG?staining were performed.Paraffin sections were stained according to the manufacturer's instructions.7.Immunohistochemical analysis and immunofluorescenceTissue sections were deparaffinized in xylene and rehydrated through graded alcohol washes.Sections were incubated with 1%H₂O₂ in methanol and 10%goat serum for30 min to block endogenous peroxidase activity and non-specific binding,respectively.The primary antibodies to MMP2?1:200 dilution?,MMP9?1:300dilution?,and RAM11?1:100 dilution?were incubated overnight at 4?.According to the manufacture's protocol,sections were then incubated with biotinylated anti-mouse second antibody for 20 min followed by the SP staining method.Sections were visualized with diaminobenzidine tetrahydrochloride and counterstained with hematoxylin.For immunofluorescence staining,sections were incubated with mouse monoclonal anti-alpha-smooth muscle actin?1:150 diluted in PBS?at 4?overnight after blocking endogenous peroxidases.Aortic sections were subsequently incubated with fluorescein isothiocyanate-conjugated goat antimouse IgG?1:100 dilution?and to detect the change of smooth muscle cells?SMCs?.8.Real time PCR for MMP2,MMP9 and CD68The Applied Biosystems 7500?QuantStudio 6?was used for real time PCR according to the manufacturer's instructions.Reactions performed in a 10?l volume with 2?l cDNA and 1?l primers,as well as 5?l SYBR Green PCR Master mix?Applied Biosystems?.The typical protocol included a optimal AmpErase UNG enzyme activity step?2 min,50??and an activation AmpliTag Gold DNA polymerase step?10 min,95??,then 40 cycles with a denaturation for 30 s?95??and 1-min annealing,and extension at different annealing temperatures.The PCR products were normalized by?-actin expression to confirm amplification specificity.9.Western blot for MMP2,MMP9 and RAM11Aortic tissues were lysed with RIPA protein lysis buffer?Code P0013B,Beyotime Biotechnology,Shanghai,China?.Total protein was separated on 10%SDS-PAGE and then transferred to a polyvinylidene difluoride membrane of 0.45?m?Code IPVH00010,Millipore Corporation,Billerica,MA.,USA?.The membrane was blocked with 5%milk solution and then probed with primary antibodies?MMP2,MMP9 and RAM11?overnight at 48?.The membrane was incubated with an HRP-conjugated secondary antibody?dilution 1:50000?for 2 h at 37?after being washed 5±6 times.An enhanced chemiluminescence kit?Applygen Technologies Inc.,Beijing,China?was used to visualize the protein band.The?-actin was used as an internal control for all bots.10.Statistical analysisAll data were expressed as means±SD.One-way ANOVA and two-way ANOVA followed by Bonferroni post-hoc tests were used for statistical analysis?Prism 5.0,GraphPad Software,Inc.,SanDiego,CA.,USA?.Differences were considered statistically significant when p<0.05.Results1.All rabbits formed AAA in the experimental group,the AAA diameter increased gradually after 4 weeks after termination of the ligation.Histologically,the elastic fibers and SMCs in the experimental group were severely destroyed 1 week after operation.The expressions of MMP2 and MMP9 increased and inflammatory cell infiltration was found.The content of elastic fiber and SMCs increased thereafter.In the control group,no AAA formed and no obvious pathological changes were observed.2.Two rabbits died 8 weeks and 11 weeks after operation in the balloon dilation group,and the remained rabbits survived the operation.The higher velocity magnitude,intensified bulk flow and obvious vortex formation were found in the balloon dilation group 3 weeks after operation.The values of low WSS and high RRT increased significantly in aneurysm group at the 10th week,but the increase of high OSI value was not as high as that in the balloon dilation group.The aortic diameter in the balloon dilation group did not increase after 5 weeks,which was significantly lower than that in the aneurysm group?p<0.05?.The thickness of intima media in balloon dilation group was significantly increased at 15 weeks,which was higher than that in the aneurysm group.The destructions of elastic fiber and SMCs were obvious after 1 week in aneurysm groups.The expressions of MMP2,MMP9 and RAM11?macrophages?in the aneurysm group and balloon dilation group significantly increased after 15 weeks.Except for the stable expression of MMP2 in the experimental group,all others expressions showed a downward trend of decrease.Conclusions1.Absorbable suture ligation combined with incubation of 60?l Chinese elastase?1U/?1?can be capable to induce rabbit AAA formation.This AAA model enlarges progressively,with no self-healing phenomenon,and can mimicking human AAA disease comparatively more advantageous.2.The higher velocity magnitude,intensified bulk flow and obvious vortex formation induced by partial stenosis,as well as low wall shear stress and increased high relative residence time may related to enlargement of AAA.These factors inhibit the self-healing process in rabbit AAA model by controlling the regeneration process of elastin and SMCs.