Establishment Of Carotid Artery Bifurcation Aneurysm Model In The Canine And Its Morphological, Hemodynamic,Histophathological Study

Part1 Establishment of carotid bifurcation aneurysm modelObjective: To explore the feasibility of establishing the canine carotid artery bifurcation aneurysm model, and further clarify the occurrence and growth mechanism of aneurysm.Methods:New branch points in the common carotid artery were created in 18 dogs, which wererandomly divided into elastase-treated bifurcation groups（EBG, n = 9）, the control bifurcation group（CBG, n = 9）,with 6 female and 3 male dogs in each group. Elastase（3.0U/_L） was applied for 15 minutes to the apex of the reconstructedarterial bifurcation(approximately 4 * 4 mm²) in 9 animals inthe EBG by dripping it from a curved blunt syringe needle into atrapezoidal plastic tube, to prevent its flowing onto other portionsof the arteries; the same volume of saline was applied in 9 animalsas controls by using the method identical to that in the elastasemodel in the CBG. Before elastase or saline was applied, as muchof the tunica adventitia at the apex as possible was removed tofacilitate the penetration.. Before elastase or saline was applied, as muchof the tunica adventitia at the apex as possible was removed tofacilitate the penetration.Angiographic and hemodynamic analysis was performed immediately, 12 and 24 weeks post-surgery; histological response was evaluated at 12 and 24 weeks. Meanwhile, 3 dogs with both straight CCAs surgically exposed received elastase insult as an elastasetreatedstraight group（ESG）. The peak velocity of blood flowin the feeding artery at the systolic phase was measured beforeand after reconstruction surgery by using Doppler ultrasound.Results:The arterial bifurcation model was successfully reconstructed inall animals. Ultrasonography or angiography revealed no stenosisat the anastomoses or arterial occlusion postsurgery or duringfollow-up in any animal. Angiography indicated that the vessellumen at the arterial bifurcation apex herniated to form an aneurysmin 5/9 animals, with 4 aneurysms in female dogs（66.7%） and1 aneurysm in a male dog（33.3%; P > 0.05） from the elastasetreatedgroup, and 2D-DSA and 3D-DSA revealed that bubbleswere located at the apices, with a tent shape（mean sac diameter,3.2 ± 0.4 mm; mean neck diameter, 6.7± 2.3 mm）.However,the aneurysms were still at their unruptured stage until 24weeks’ follow-up, with only slight saccular enlargement, comparedwith 12 weeks in the EBG（3.5± 0.3 mm versus 3.0± 0.2mm; P=0.076）. Hematoxylin-eosin and Masson trichrome staining revealedthinning of media at the bifurcation, a reduced numbers of SMCs,and loss of fibronectin in the elastase-treated group. only 3 animals from the CBG displayed intima layerdisruption with mild internal elastic lamina discontinuity at thearterial bifurcation apices.Conclusion:We used the canine model which was more resemble to human blood vessels structureto simulate vascular rebuilding in human body. weinduced arterial wall degenerationvia internal elastic lamina and media elastic fiberinsult by using elastase to induce muscular layer degenerationafter blood flow action. aneurysms were successfully induced in the EBG and were confirmedby both angiography and histopathology.. This modelcould be helpful to clarify the pathophysiological mechanisms of the occurrence and development of the aneurysm, and we preliminarily found a relatively important linkthatplaying a pivotal role in the occurrence and development of the aneurysm. This canine model has the potential for the identificationand study of the genes, proteins, or conduction channels responsiblefor aneurysm generation, development, and rupture.Part 2 Histopathology of a Carotid Arterial Bifurcation in anAneurysm ModelPurpose:On the basis of the establishment of the an carotid arterial bifurcation in a canine model, we research whether arterial wall degeneration, inflammation and cellular immune response lead to the generation and growth of aneurysms in a canine model.Methods:Arterial bifurcations with a new branch point were surgically created in the CCA of 18 dogs, and then were divided into an elastase-treated bifurcation group（EBG, n=9） and a control bifurcation group（CBG, n=9）.Meanwhile, three dogs were compared who received an elastase insult to both straight CCAs（elastase-treated straight group, ESG=3）.Reconstructed arterial bifurcations were harvested 12 weeks（n=6） and 24 weeks（n=3） in the EBG and CBG, while straight vessel samples（n=6） were harvested at 24 weeks after surgery.CCA samples from the surgical models were stained with HE, Masson’s trichrome and elastic fiber stains（Verhoeff Van-Gieson and Orcein methods）.Serial longitudinal sections of the reconstructed arterial bifurcations were taken to assess tissue alternations in relationship to the consistent hemodynamic action.CCA samples were also immunostained using mouse anti–proliferating cell nuclear antigen（PCNA） antibody（1:100; Neo Markers, Thermo Fisher Scientific Inc. Fremont, CA, USA）, mouse monoclonal smooth muscle α-actin antibody（1:50; Santa Cruz Biotechnology Inc., CA, USA）, mouse monoclonal CD45 antibody（Leukocyte Common Antigen; clone RP2/18; 1:50; Invitrogen Inc., Carlsbad, CA, USA）, Mouse monoclonal anti-macrophage antibody（clone MAC387; 1:50; Abcam Inc., Cambridge, MA, USA）, mouse monoclonal MMP-2 antibody（clone CA-4001; 1:30; Invitrogen Inc.） and mouse monoclonal MMP-9 antibody（clone EP127; 1:50; Invitrogen Inc.） using the Envision immunohistochemical technique. Double immunofluorescent staining was performed using anti-CD45 for pan-leukocytes and anti-MAC387 for macrophages to assess whether macrophages were included in leukocytes. The SMC proliferation rate（percentage of PCNA-positive SMCs in the media）, and elastic layer and α-actin – positive SMC layer thickness were measured. The inflammatory cell infiltration index was defined as the percentage of CD45-positive cells in the media of the arterial bifurcation apices. The MMP-2 and MMP-9 expression indexes were defined as the percentage MMP-2/9-positive area in the media of the arterial bifurcation apices.Statistical analysis of the histopathological images was performed with Image-Pro Plus Version 6.0 software（Media Cybernetics, Inc., Bethesda, MD, USA） in at least 20 randomly selected high-power（×400） tubulointerstitial fields from each section. Every pathological index in each section was scored independently in a blinded manner by three pathologists.Graph Pad Prism 5.0 software（San Diego, CA, USA） was used for statistical analysis. Data were expressed as the mean ± standard deviation for continuous variables, and as counts or proportions（%） for categorical variables. Fisher’s exact test was used to compare categorical data. Grouped t-tests were used to compare the PCNA proliferation index, elastic layer and media α-actin-positive layer thickness, inflammatory cell infiltration index, and MMP-2 and MMP-9 indexes.All tests were two tailed and statistical significance was defined as P < 0.05.Results:In the five arterial bifurcation samples with aneurysms on angiography, biopsy confirmed the entire vessel wall was thinned with a semitransparent appearance and visible blood flow underneath, which enabled herniation and aneurysm formation at the arterial bifurcation apices.HE and Masson’s trichrome staining revealed thinning of media at the bifurcation, reduced numbers of SMCs and loss of fibronectin in the elastase-treated group. Elastic fiber staining revealed IEL discontinuity and elastic fiber disruption/insult in all elastase-treated animals in the ESG and EBG; only three animals from the CBG displayed intima layer disruption with mild IEL discontinuity at the arterial bifurcation apices. Elastase-treatment reduced the media elastic layer thickness in the EBG（21.2 ± 15.3 μm） and ESG（71.28 ± 19.47 μm） compared to that in the CBG（119.4 ± 29.9 μm, P < 0.001）.Immunohistochemical staining revealed increased numbers of PCNA-positive SMCs in the media of elastase-treated arterial bifurcation apices in the EBG（42.0 ± 15.7% vs. 7.3 ± 3.8% in ESG and 8.0 ± 6.3% in the CBG; P<0.001）.The number of α-SMA-positive SMCs also decreased and the SMC layer was thinner at elastase-treated arterial bifurcation apices in the EBG（31.3 ± 16.7 μm vs. 136.5 ± 25.5 μm in the ESG and 133.9 ± 26.1 μm in the CBG, P < 0.001）. The inflammatory cell infiltration index in the media of the aneurysm wall was 38.4 ± 10.6% in the EBG（vs. 5.1 ± 2.1% in the ESG and 2.9 ± 2.4% in the CBG; P < 0.001）. Macrophage staining showed macrophage infiltration into the aneurysmal wall with a distribution similar to that of leukocytes. Furthermore, double staining with anti-CD45 and anti-MAC387 revealed that some of leukocytes in media of aneurysm wall in this model were macrophages. The MMP-2 and MMP-9 expression indexes in the media of the aneurysm wall were 21.0 ± 8.7%（vs. 1.2 ± 1.4% in the ESG and 0.8 ± 1.2% in the CBG; P < 0.001） and 13.6 ± 5.6%（vs. 0.9 ± 0.8% in the ESG and 0.4 ± 0.6% in the CBG; P < 0.001）, respectively.Conclusion: During 24-weeks’ follow-up, aneurysms were successfully inducedin 5/9 elastase-treated animals in the EBG and were confirmedby both angiography and histopathology. Histologic analysis revealed internal elastic lamina discontinuity, elastic fiber disruption, a thinner muscular layer, reducedsmooth-muscle cell proliferation, increased inflammatory cell（macrophage） infiltration, and expression of matrix metalloproteinase-2 andmatrix metalloproteinase-9 in the media of the elastase-treated bifurcation group compared with that in either the control bifurcationgroup or the elastase-treated straight group（P<0.001）. Aneurysmgeneration or enlargement is a type of arterial wallremodeling, which may potentially involve increased inflammatorycell infiltration, increased MMP activity, extracellularmatrix protein synthesis, and SMC apoptosis, to counteract themechanical forces induced by altered blood flow. Inflammatorycell infiltration was detected in the media of the aneurysmwalls by pan-leukocyte staining by using an anti-CD45 antibody, consistent with observations in animal andhuman intracranial aneurysms. MMP-2 and MMP-9 were alsoexpressed in the walls of the aneurysms, both of which possess gelatinase and collagenase activity and can degenerate the importantextracellular matrix components in the walls of aneurysms. Taken together,these results indicate that MMP may be released from the infiltratingmacrophages and may play an important role in theprogression of aneurysms.According to the experimental data collected herein, we speculate that the aneurysmal occurrence and development are correlated with vascular structural degeneration, and the immune responses play a pivotal role in the aneurysmal formation and growth.Part 3 Morphology and Hemodynamics at the Apex of a CarotidArterial Bifurcation in a Canine ModelPurpose:On the basis of the establishment of the an carotid arterial bifurcation in a canine model,we investigate whether specific hemodynamic insult in combination with morphological changeleads to the generation and growth of aneurysms in a canine model.Methods:Arterial bifurcations with a new branch point were surgically created in the CCA of 18 dogs, and then were divided into an elastase-treated bifurcation group（EBG, n=9） and a control bifurcation group（CBG, n=9）.Meanwhile, three dogs were compared who received an elastase insult to both straight CCAs（elastase-treated straight group, ESG=3）. Angiographic and hemodynamic analysis was performed immediately, 12 and 24 weeks post-surgery. If an aneurysm-like bubble was observed, its neck and maximum body width was measured using the best 3D-DSA projection, and parent artery（PA） diameter and the angulation between the PAs were determined.Using in vivo 3D-DSA rotational angiography（Syngo AXIOM-Artis, Siemens, Germany）, the 3D lumen of the bifurcation was reconstructed, and together with the measured flow rates in the vessel branches, served as the boundary conditions for computational fluid dynamics simulations（Mimics 10.0; Materialise, Leuven, Belgium）.WSS（horizontal pressure）, velocity field, streamline, surface relative pressure field（vertical pressure） and total pressure field were analysed. WSS spatial gradient（WSSG） wascalculated along the flow streamline. The WSS and WSSG of bifurcationsof EBG are received quantitative analysis.Results: The arterial bifurcation model was successfully reconstructed in all animals. Angiography revealed nascent aneurysms（mean 3.2 ± 0.4 mm） at the arterial bifurcation apices in 5/9 models of the EBG（vs. 0 in the CBG and ESG）, without any observed aneurysm rupture. However, the aneurysms were still at their unruptured stagy until 24 weeks follow-up, with only slightly sac enlargement with a comparison to at 12 weeks in the EBG（3.5 ± 0.3 mm vs. 3.0 ± 0.2 mm; P=0.076）.The mean PA diameter and angulation were 4.3 ± 0.4 mm and 112.9 ± 36.1° in the CBG, and were 4.3 ± 0.4 mm and 120.3 ± 44.2° in the EBG, respectively. However, the average PA angulation of the 5/9 animals with aneurysms in the EBG was 146.8 ± 40.84°, with a comparison to that were 87.25 ± 18.87° in models with no aneurysm occurrence（P=0.032）.Hemodynamic parameters include WSS, blood velocity, streamline field, surface relative pressure field and total pressure field. Flow rates in the feeding artery of the de novo bifurcation increased from 85.3 ± 7.5 cm/s to 128.8 ± 13.1 cm/s（approximately 51 %） due to ligation of the contralateral CCA. CFD analysis immediately after surgery revealed the feeding artery wall experienced increased WSS in the CBG and EBG, whereas the bifurcation periapical regions were exposed to a more complex hemodynamic environment. The apex from the arterial bifurcation apex to the bilateral vessel wall experienced extremely low WSS and flow velocity, which increased to a maximum and then decreased further downstream to a similar level as the feeding artery. This acceleration and deceleration created a WSSG positive region（before maximum WSS） and negative region（after maximum WSS）. The apex also experienced the highest relative pressure and total pressure, which decreased to normal values towards both branch arms.Hemodynamic parameters were similar immediately after surgery and 24 weeks after surgery in the ESG, CBG and the 4/9 animals in the EBG without morphological changes. In the 5/9 elastase-treated animals with aneurysms in the EBG, WSS and flow velocity beside the apex decreased significantly during the 24 weeks after surgery, in a similar manner to the distal outflow vessel, due to arterial remodelling（aneurysm sac formation）. In these animals, the relative pressure and total pressure at the apex also decreased during follow-up. In the 5/9 elastase treatedanimals with aneurysms in the EBG, quantitative analysis of the WSS and WSSG values, including at the apex and equidistant points（1, 2, and 3 mm） on one of the affected sides, indicated an obvious decrease from immediately after surgery to 24 weeks after surgery（P≤01）, in a manner similar to that in the distal outflow vessel, due to arterial remodeling leading to the formation of an aneurysm sac. In these animals, the flow velocity, relative pressure, and total pressure at the apex also decreased during follow-up. Further quantitative analysis of the WSS and WSSG values immediately after surgery in the EBG also indicated a higher WSS（often 2mmbesides the apex） and WSSG（often 0–2 mm besides the apex） insult to the arterial wall in models with an aneurysm than in those without one. The arterial walls subjected to high WSS and WSSG values were confirmed to show remodeling by both angiographic and histologic observations. Considering that models that developed an aneurysm often had larger bifurcation angles, the arterial wall adjacent to the apex would be subjected to higher WSS and WSSG values.Conclusions:In this study, despite all models havingsimulated elastic fiber insults（arterial wall deterioration）, nascentaneurysms were generated in only 5 of 9 models. We hypothesizethat the bifurcation angle is an important factor because T-shapedbifurcations（146.8±40.84°） were more likely to induce thegeneration of aneurysms than Y-shaped bifurcations（87.25±18.87°）. Small bifurcation angles are associated with high bloodflow velocities and dispersed vortex intensities, while increasedbifurcation angles result in more concentrated vortex intensitiesnext to the apex and turbulent/disturbed laminar flow, whichgenerates various mechanical stimuli, such as WSS and WSSG.The quantitative analysis of the WSS and WSSG valuesat the bifurcation sites in our study proved that the arterialwall next to the apex was subjected to higher WSS and WSSGvalues before the generation of aneurysms in models with largebifurcation angles. Elevated WSS or WSSG levels disrupt the normalfunction of endothelial cells, stimulate gene transcription,and activate ion channels and the consequent reorganization ofthe cellular cytoskeleton.