动物实验,动脉粥样硬化 z-bo 2020-09-04 453

　　Background: Atherosclerosis and its complications are the main cause of death in most countries. Although many systemic factors, including abnormal glucose metabolism, hyperlipidemia, hypertension, and smoking, have been identified as risk factors, turbulence is more likely to play a pivotal role in the occurrence and development of atherosclerosis. The random priority mode is characterized by low shear stress and oscillating wall shear stress (SS) of the branch or curved artery wall. More and more evidences show that SS is the chief culprit in vascular injury. . With the application of intravascular ultrasound (IVUS) and computational fluid dynamics, researchers have demonstrated that SS can be used to predict intimal formation caused by in-stent restenosis and evaluate the progress of vascular reconstruction after balloon angioplasty. Studies have shown that high SS is related to in-stent restenosis and revascularization of target lesions, even in successful balloon angioplasty. However, the relationship between neointimal hyperplasia and neointimal hyperplasia after balloon dilation needs to be further confirmed. Vascular endothelial cells (EC) are an important part of the blood vessel wall lining and are directly exposed to turbulent blood flow. At the same time, endothelial cells may be affected by various chemical and mechanical stimuli, and ultimately regulate homeostasis. EC dysfunction is an important pathological factor in vascular diseases, including atherosclerosis and thrombosis. In recent years, a variety of in vitro models have been established to regulate the shear flow of endothelial cells in order to simulate the characteristics of the endothelial cell flow environment in vivo. These models help researchers to explore its response to SS, including its effects on EC morphology, cytoskeleton organization, proliferation, migration, permeability and connexin, EC signaling and gene expression. A number of studies have explored the effect of SS on vascular structure. However, the role of SS in vascular remodeling and neointima formation has not been fully elucidated, especially the disturbed flow in the body. An appropriate model should be established to explore this issue. Therefore, we used a cylindrical cannula to accept the abdominal aorta coarctation of the rabbit. It simulates the hemodynamic characteristics of atherosclerosis-induced stenosis, and explores the effect of SS on vascular remodeling after balloon injury. We hypothesize that this constrictive vascular model, similar to atherosclerotic plaque, will cause varying degrees of intimal hyperplasia upstream and downstream of the constricted blood vessel, regardless of whether it is accompanied by balloon injury.

　　Method: Animals: 40 New Zealand white rabbits, weighing 2.2–2.6 kg, male, randomly divided into 5 groups (n = 8 each group), raised in a single cage, ad libitum in eating and drinking, and adapted for at least 1 week before surgery. The rabbit was anesthetized with 3% sodium pentobarbital (1 ml/kg) and then the right femoral artery was intubated with four introducer sheaths and all catheters were then introduced into the abdominal aorta through the sheath. . Heparin sodium (200 IU/kg) is injected intraarterially. , Median sagittal incision is about 1 cm in the middle of the renal artery and common iliac artery, waiting for the next grouping decision. The abdominal aorta of each rabbit was taken for baseline angiography and IVUS examination. All operations are in a sterile state.

　　Surgical operation: Group 1, only injured the aortic balloon, which is just upstream of the separated artery, and sutured without ligation as a control. . In group 2, not only the balloon injured the aorta, but the artery was also upstream of the separated artery. The isolated artery (approximately 2 mm in diameter and 5 mm in length) was partially contracted with a cylindrical cannula and ligated with surgical sutures. Group 3: Only the aortic balloon was injured below the separated artery, and surgical suture without ligation was used as a control. Group 4: Injured aortic balloon, located downstream of the separated artery, partially isolated the narrowed artery with a cylindrical cannula, and ligated with surgical sutures. Group 5: Only the cylindrical cannula connected with surgical sutures partially separated the narrowed artery. At the level of balloon dilation injury, the dilation rate of the aorta is between 1.2 and 1.3. According to the basic IVUS diameter data, the digital subtraction angiography (DSA) and IVUS images are recorded twice at 10 intervals at the same place. After the operation, the incision was sutured aseptically, and antibiotics were injected intravenously to prevent infection. The animals were allowed to resume their normal diet. Four weeks later, the rabbits were examined with DSA and IVUS. Overdose pentobarbital sodium was euthanized, the aorta was dissected from the renal artery to the iliac bifurcation, and the aorta was perfused and fixed with 10% neutral formaldehyde overnight. The fixed aorta was embedded in paraffin blocks for morphometric and immunohistochemical studies.

　　Morphology: The cross-section is obtained from two different parts, namely the upstream and downstream parts of the systolic artery and the false artery. The specimen was cut into a continuous 5 μm section for general histological staining and hematoxylin-eosin (HE). The Image-Pro Plus 6 image analysis system was used to determine the lumen area, the area of the inner elastic layer and the area of the neointima. Perform statistical analysis on the average area of the corresponding segment (N = 3).

　　Immunohistochemistry: the sections were deparaffinized and hydrated. The sections were treated with PBS 3% hydrogen peroxide for 10 min to inhibit the activity of peroxidase. Citrate buffer (pH 6) is used for antigen retrieval. Incubate overnight with CD31 monoclonal antibody at 4°. After washing with PBS, the slices were incubated with goat anti-mouse IgG at room temperature for 20 min, and then incubated with the SABC complex and washed with PBS again, stained with DAB, counterstained with hematoxylin, dehydrated, and fixed. The control group performed the same procedure, which was mainly replaced by non-immune serum antibodies.

　　Results: Establishment of rabbit model: Four rabbits (two in group 2 and one in group 3 and group 4) died during the experiment due to excessive anesthesia, thrombosis, hemorrhagic shock, and balloon injury. The rest The rabbits all completed the research. The average baseline weight was 2.36±0.25 kg, and the weight after four weeks was 3.14±0.16 kg. There was no significant difference in body weight among the five groups, indicating that the model was successfully established. Evaluation of hemodynamics confirmed the change of vascular SS after partial abdominal aorta narrowing. These routine measurements are evaluated to determine the variable SS model.

　　The stenotic upstream SS reduces the loss of lumen after balloon injury: Four weeks later, intravascular ultrasound is used to measure the diameter of the upper and downstream vessels of the separated artery to evaluate balloon injury and/or stenosis of the vascular remodeling. In the upstream of the isolated artery, the balloon injury significantly reduced the lumen diameter of the balloon injury group and the balloon injury group compared with the other three groups without balloon injury and/or stenosis. In addition, arterial contraction did not lead to a further decrease in the inner diameter secondary to balloon injury, but weakened the loss of the upstream vessel lumen. At the same time, compared with other groups, single artery stenosis did not change significantly compared with normal blood vessel diameter. In the downstream of the separated artery, single balloon injury can significantly reduce the lumen diameter of group 3, suggesting that the normal vascular lumen of group 2 and group 5 is dilated due to contraction. In addition, balloon injury makes the expansion trend more and more obvious, and group 4 causes There is excessive dilation of the vessel lumen.

　　The upstream SS of

　　stenosis induces normal vascular intimal hyperplasia and reduces the hyperplasia after balloon injury: the degree of intimal hyperplasia was evaluated by morphology and quantitative four weeks after operation. Ascending in the separated artery, similar to IVUS, the area of neointima was significantly increased after balloon injury in groups 1 and 2 compared with other groups without IU balloon injury. Compared with group 1, the hemodynamic changes caused by arterial contraction did not aggravate the intimal hyperplasia secondary to balloon injury but inhibited the intimal hyperplasia group. In the downstream of the separated artery, a simple contraction will cause a significant neointimal hyperplasia group in the downstream artery, but the lumen diameter increases with the increase of intravascular ultrasound. However, in group 4, contraction did not aggravate the intimal hyperplasia and almost completely disappeared. Compared with the other groups, the inner diameter of the lumen was excessively expanded.

　　The stenotic upstream SS accelerates the re-endothelialization after balloon injury, but the effect of the downstream SS is opposite: to explore the effect of low ESS on the re-endothelialization of the injured blood vessel, and detect the vascular endothelial cell marker (CD31) after CD31 antibody immunohistochemical staining. The number of CD31-positive cells covered on the luminal surface was calculated and then endothelialized, and CD31-positive staining was observed along the luminal surface of the vessel wall. The upstream SS accelerates the re-endothelialization of the balloon-injured artery, and the downstream SS inhibits the repair of damaged endothelial cells.

　　Conclusion: Appropriate low unidirectional SS is conducive to the inhibition of intimal hyperplasia after angioplasty and is a potential therapeutic target for stent stenosis.