A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.     

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating important information on the pathogenesis of lesion formation and the action of novel therapies. Use of atherosclerosis-prone mice, surgical methods of lesion induction, and dietary modification has dramatically improved understanding of the mechanisms that contribute to disease development and the potential of new treatments.Classically, analysis of lesions is performed ex vivo using 2-dimensional histological techniques. This article describes application of optical projection tomography (OPT) to 3-dimensional quantitation of arterial lesions. As this technique is non-destructive, it can be used as an adjunct to standard histological and immunohistochemical analyses.Neointimal lesions were induced by wire-insertion or ligation of the mouse femoral artery whilst atherosclerotic lesions were generated by administration of an atherogenic diet to apoE-deficient mice.Lesions were examined using OPT imaging of autofluorescent emission followed by complementary histological and immunohistochemical analysis. OPT clearly distinguished lesions from the underlying vascular wall. Lesion size was calculated in 2-dimensional sections using planimetry, enabling calculation of lesion volume and maximal cross-sectional area. Data generated using OPT were consistent with measurements obtained using histology, confirming the accuracy of the technique and its potential as a complement (rather than alternative) to traditional methods of analysis.This work demonstrates the potential of OPT for imaging atherosclerotic and neointimal lesions. It provides a rapid, much needed ex vivo technique for the routine 3-dimensional quantification of vascular remodelling.     

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development^1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice^{1, 9, 10}. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse’s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.     

Impact of Body Mass Index on Changes in Common Carotid Artery Wall Thickness

Objective: To examine associations between changes in body mass index (BMI) and changes in carotid artery intima-media thickness (IMT) in a community-based sample. Research Methods and Procedures: Carotid artery IMT and BMI were assessed at baseline (between 1987 and 1990) and in three subsequent examinations at 3-year intervals in participants in the Atherosclerosis Risk in Communities cohort. The 9316 African-American and white men and women in the analysis were 45 to 64 years of age at baseline. Cross-sectional associations between BMI and IMT were assessed using general linear models. Longitudinal associations were examined using mixed models analysis. Results: Cross-sectional associations between BMI and IMT were confirmed. At baseline, a 1-kg/m² increase in BMI was associated with an increase in IMT that ranged from 2.5 to 7.5 μm among the ethnic-gender groups examined. Changes in BMI were not associated with changes in IMT in models that adjusted for aging and other covariates. Results were similar across ethnic-gender groups. Discussion: Among free-living, 45- to 64-year-old adults, changes in common carotid artery IMT associated with changes in BMI are either very small or absent.     

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound and aortic blood pressure is measured invasively with a solid-state pressure catheter. Blood pressure is raised then lowered incrementally by intravenous infusion of vasoactive drugs phenylephrine and sodium nitroprusside. Aortic diameter is measured for each pressure step to characterize the pressure-diameter relationship of the ascending aorta. Stiffness indices derived from the pressure-diameter relationship can be calculated from the data collected. Calculation of arterial compliance is described in this protocol.This technique can be used to investigate mechanisms underlying increased aortic stiffness associated with cardiovascular disease and aging. The technique produces a physiologically relevant measure of stiffness compared to ex vivo approaches because physiological influences on aortic stiffness are incorporated in the measurement. The primary limitation of this technique is the measurement error introduced from the movement of the aorta during the cardiac cycle. This motion can be compensated by adjusting the location of the probe with the aortic movement as well as making multiple measurements of the aortic pressure-diameter relationship and expanding the experimental group size.     

综述: 长链非编码RNA对动脉粥样硬化等疾病影响的研究新进展

动脉粥样硬化是一种以胆固醇等脂质代谢紊乱为主要特征的病理过程,严重影响人类健康．随着遗传学和生物信息学研究的发展,曾被认为无作用的非编码基因序列逐步受到研究者的关注．长链非编码RNA(lncRNA)通过表观遗传调控、转录调控和转录后调控等途径参与剂量补偿效应、基因组印记、细胞发育分化等重要生物学过程,从而影响人类的生长发育、代谢、衰老及疾病等进程．最新研究发现,lncRNA可参与血管内皮细胞的损伤与修复、血管平滑肌细胞的增殖与迁移、巨噬细胞胆固醇的流出与炎症反应、脂质的沉积与斑块的形成等过程,从而影响动脉粥样硬化及其他心血管疾病的发生与发展．     

Ultrasound-guided Transthoracic Intramyocardial Injection in Mice

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. Experiments involving myocardial injection are currently performed by direct surgical access through a thoracotomy. While convenient when performed at the time of another experimental manipulation such as coronary artery ligation, the need for an invasive procedure for intramyocardial delivery limits potential experimental designs. With ever improving ultrasound resolution and advanced noninvasive imaging modalities, it is now feasible to routinely perform ultrasound-guided, percutaneous intramyocardial injection. This modality efficiently and reliably delivers agents to a targeted region of myocardium. Advantages of this technique include the avoidance of surgical morbidity, the facility to target regions of myocardium selectively under ultrasound guidance, and the opportunity to deliver injectate to the myocardium at multiple, predetermined time intervals. With practiced technique, complications from intramyocardial injection are rare, and mice quickly return to normal activity on recovery from anesthetic. Following the steps outlined in this protocol, the operator with basic echocardiography experience can quickly become competent in this versatile, minimally invasive technique.     

瑞舒伐他汀治疗颈动脉粥样硬化斑块的临床研究

目的:研究瑞舒伐他汀对颈动脉粥样硬化斑块的治疗效果。方法:将在本院接受治疗的250例颈动脉粥样硬化斑块患者随机分成治疗组125例和对照组125例,治疗组服用瑞舒伐他汀10mg/晚,对照组行其他非瑞舒伐他汀药物治疗,进行为期6个月的观察对比。结果:治疗组治疗后总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白(LDL)水平显著下降,高密度脂蛋白(HDL)水平显著升高(P<0.05),颈动脉内膜-中层厚度(IMT)、斑块面积变小,与治疗前比较,差异有统计学意义(P<0.05);对照组治疗前后无显著性差异(P>0.05)。结论:瑞舒伐他汀对降低血脂、减缓不稳定型心绞痛早期动脉粥样硬化、稳定斑块和预防脑血管疾病起到非常重要的作用。     

冠心病患者颈动脉粥样硬化与血管内皮功能的临床研究价值

目的:探讨颈动脉粥样硬化与血管内皮功能与冠心病患者的相关性。方法:选取114例冠心病患者(54例单支病变和60例多支病变)为观察组和60例健康体检者为对照组,对两组患者动脉粥样硬化及血管内皮功能进行分析。结果:观察组患者TC、TG、HDL及血糖水平均高于对照组,观察组LDL水平显著低于对照组,两组比较差异有统计学意义(P<0.05);观察组患者颈动脉IMT、斑块积分及斑块数明显高于对照组(P<0.05),观察组FMD显著降低(P<0.05),多支病变组病变程度更严重(P<0.05)。结论:颈动脉粥样硬化与血管内皮功能可作为预测冠心病的重要指标,对预防和治疗冠心病具有重要意义。     

冠心病患者颈动脉粥样硬化超声相关参数与病变严重程度的关系

目的:研究冠心病患者颈动脉粥样硬化超声相关参数与病变严重程度的关系,为冠心病的诊断提供依据。方法:选取2015年1月至2016年6月我院收治的冠心病患者100例作为研究组,根据冠状动脉造影结果分为1支病变组(24例),2支病变组(44例)和3支病变组(32例)。根据Gensini评分分为轻度病变组(Gensini评分20分,n=26),中度病变组(20分≤Gensini评分40分,n=45)和重度病变组(Gensini评分≥40分,n=29)。另选取同期医院体检的健康体检者30例作为对照组。应用颈动脉超声多普勒检查各组颈动脉超声相关参数,采用Pearson相关性分析超声相关参数与病变严重程度的关系。结果:研究组颈动脉中膜厚度(IMT)、颈总动脉硬化度(β)、颈动脉顺应性(AC)、弹性系数(EP)、脉搏波传导速度(PEVβ)均显著高于对照组,差异有统计学意义(P0.05)。随着冠状动脉病变支数增加,受试者IMT、β、EP、AC、PEVβ升高,其中3支病变组2支病变组1支病变组对照组(P0.05)。随着冠状动脉病变程度增加,受试者IMT、β、EP、AC、PEVβ升高,其中重度病变组中度病变组轻度病变组对照组(P0.05)。经Pearson相关性分析显示:冠心病患者IMT、β、EP、AC、PEVβ与病变血管支数呈正相关(r=0.607,0.428,0.532,0.507,0.556,均P0.05),与Gensini评分呈正相关(r=0.624,0.432,0.517,0.521,0.543,均P0.05)。结论:冠心病患者颈动脉粥样硬化超声相关参数与患者病变程度相关,颈动脉超声检查可以作为冠心病的诊断手段。     

Implantation of Radiotelemetry Transmitters Yielding Data on ECG, Heart Rate, Core Body Temperature and Activity in Free-moving Laboratory Mice

The laboratory mouse is the animal species of choice for most biomedical research, in both the academic sphere and the pharmaceutical industry. Mice are a manageable size and relatively easy to house. These factors, together with the availability of a wealth of spontaneous and experimentally induced mutants, make laboratory mice ideally suited to a wide variety of research areas.In cardiovascular, pharmacological and toxicological research, accurate measurement of parameters relating to the circulatory system of laboratory animals is often required. Determination of heart rate, heart rate variability, and duration of PQ and QT intervals are based on electrocardiogram (ECG) recordings. However, obtaining reliable ECG curves as well as physiological data such as core body temperature in mice can be difficult using conventional measurement techniques, which require connecting sensors and lead wires to a restrained, tethered, or even anaesthetized animal. Data obtained in this fashion must be interpreted with caution, as it is well known that restraining and anesthesia can have a major artifactual influence on physiological parameters^{1, 2}.Radiotelemetry enables data to be collected from conscious and untethered animals. Measurements can be conducted even in freely moving animals, and without requiring the investigator to be in the proximity of the animal. Thus, known sources of artifacts are avoided, and accurate and reliable measurements are assured. This methodology also reduces interanimal variability, thus reducing the number of animals used, rendering this technology the most humane method of monitoring physiological parameters in laboratory animals^{3, 4}. Constant advancements in data acquisition technology and implant miniaturization mean that it is now possible to record physiological parameters and locomotor activity continuously and in realtime over longer periods such as hours, days or even weeks^{3, 5}.Here, we describe a surgical technique for implantation of a commercially available telemetry transmitter used for continuous measurements of core body temperature, locomotor activity and biopotential (i.e. onelead ECG), from which heart rate, heart rate variability, and PQ and QT intervals can be established in freeroaming, untethered mice. We also present pre-operative procedures and protocols for post-operative intensive care and pain treatment that improve recovery, well-being and survival rates in implanted mice^{5, 6}.     

Orthotopic Aortic Transplantation in Mice for the Study of Vascular Disease

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. This is largely true, but there are a number of important principles that can reduce the difficulty of these procedures and enhance reproducibility. Orthotopic aortic transplantation is an excellent procedure in which to learn these principles because it involves only two end-to-end anastomoses, but requires good suturing technique and handling of the vessels for consistent success. This procedure begins with the procurement of a length of abdominal aorta from a donor animal, followed by division of the native aorta in the recipient. The procured aorta is then placed between the divided ends of the recipient aorta and sutured into place using end-to-end anastomoses. To accomplish this objective successfully requires a high degree of concentration, good tools, a steady hand, and an appreciation of how easily the vasculature of a mouse can be damaged, resulting in thrombosis. Learning these important principles is what occupies most of the beginner''s time when learning microsurgery in small rodents. Throughout this protocol, we refer to these important points. This model can be used to study vascular disease in a variety of different experimental systems^1-8. In the context shown here, it is most often used for the study of post-transplant vascular disease, a common long-term complication of solid organ transplantation in which intimal hyperplasia occurs within the allograft. The primary advantage of the model is that it facilitates quantitative morphometric analyses and the transplanted vessel lies contiguous to the endogenous vessel, which can serve as an additional control⁹. The technique shown here is most often used for mice weighing 18-25 grams. We have accumulated most of our experience using the C57BL/6J, BALB/cJ, and C3H/HeJ strains.     

Heterotopic Mucosal Engrafting Procedure for Direct Drug Delivery to the Brain in Mice

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high molecular weight compounds from the bloodstream and into brain tissue. Some direct delivery success in humans has been achieved via implantation of transcranial catheters; however this method is highly invasive and associated with numerous complications. A less invasive alternative would be to dose the brain through a surgically implanted, semipermeable membrane such as the nasal mucosa that is used to repair skull base defects following endoscopic transnasal tumor removal surgery in humans. Drug transfer though this membrane would effectively bypass the BBB and diffuse directly into the brain and cerebrospinal fluid. Inspired by this approach, a surgical approach in mice was developed that uses a donor septal mucosal membrane engrafted over an extracranial surgical BBB defect. This model has been shown to effectively allow the passage of high molecular weight compounds into the brain. Since numerous drug candidates are incapable of crossing the BBB, this model is valuable for performing preclinical testing of novel therapies for neurological and psychiatric diseases.     

Murine Model of Femoral Artery Wire Injury with Implantation of a Perivascular Drug Delivery Patch

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. While these therapies lead to the rapid restoration of blood flow, these technologies remain limited by restenosis in the case of bare metal stents and angioplasty, or reduced healing and possibly enhanced risk of thrombosis in the case of drug eluting stents. A key pathophysiological mechanism in the formation of restenosis is intimal hyperplasia caused by the activation of vascular smooth muscle cells and inflammation due to arterial stretch and injury. Surgeries that induce arterial injury in genetically modified mice are useful for the mechanistic study of the vascular response to injury but are often technically challenging to perform in mouse models due to the their small size and lack of appropriate sized devices. We describe two approaches for a surgical technique that induces endothelial denudation and arterial stretch in the femoral artery of mice to produce robust neointimal hyperplasia. The first approach creates an arteriotomy in the muscular branch of the femoral artery to obtain vascular access. Following wire injury this arterial branch is ligated to close the arteriotomy. A second approach creates an arteriotomy in the main femoral artery that is later closed through localized cautery. This method allows for vascular access through a larger vessel and, consequently, provides a less technically demanding procedure that can be used in smaller mice. Following either method of arterial injury, a degradable drug delivery patch can be placed over or around the injured artery to deliver therapeutic agents.     

Mouse model of surgically-induced endometriosis by auto-transplantation of uterine tissue

Endometriosis is a chronic, painful disease whose etiology remains unknown. Furthermore, treatment of endometriosis can require laparoscopic removal of lesions, and/or chronic pharmaceutical management of pain and infertility symptoms. The cost associated with endometriosis has been estimated at 22 billion dollars per year in the United States. To further our understanding of mechanisms underlying this enigmatic disease, animal models have been employed. Primates spontaneously develop endometriosis and therefore primate models most closely resemble the disease in women. Rodent models, however, are more cost effective and readily available. The model that we describe here involves an autologous transfer of uterine tissue to the intestinal mesentery (Figure 1) and was first developed in the rat and later transferred to the mouse. The goal of the autologous rodent model of surgically-induced endometriosis is to mimic the disease in women. We and others have previously shown that the altered gene expression pattern observed in endometriotic lesions from mice or rats mirrors that observed in women with the disease. One advantage of performing the surgery in the mouse is that the abundance of transgenic mouse strains available can aid researchers in determining the role of specific components important in the establishment and growth of endometriosis. An alternative model in which excised human endometrial fragments are introduced to the peritoneum of immunocompromised mice is also widely used but is limited by the lack of a normal immune system which is thought to be important in endometriosis. Importantly, the mouse model of surgically induced endometriosis is a versatile model that has been used to study how the immune system, hormones and environmental factors affect endometriosis as well as the effects of endometriosis on fertility and pain.     

Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. The long-term clinical results showed excellent patency rates, however, with significant incidence of stenosis. To investigate the cellular and molecular mechanisms of vascular neotissue formation and prevent stenosis development in tissue engineered vascular grafts (TEVGs), we developed a mouse model of the graft with approximately 1 mm internal diameter. First, the TEVGs were assembled from biodegradable tubular scaffolds fabricated from a polyglycolic acid nonwoven felt mesh coated with ε-caprolactone and L-lactide copolymer. The scaffolds were then placed in a lyophilizer, vacuumed for 24 hr, and stored in a desiccator until cell seeding. Second, bone marrow was collected from donor mice and mononuclear cells were isolated by density gradient centrifugation. Third, approximately one million cells were seeded on a scaffold and incubated O/N. Finally, the seeded scaffolds were then implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. The implanted grafts demonstrated excellent patency (>90%) without evidence of thromboembolic complications or aneurysmal formation. This murine model will aid us in understanding and quantifying the cellular and molecular mechanisms of neotissue formation in the TEVG.     

ApoE基因缺失幼龄小鼠动脉粥样硬化相关基因的时序表达研究

利用RT-PCR技术检测apoE基因缺失(apoE-/-小鼠在1、2和3月龄3个年龄段主动脉以及14天、1、2和3月龄4个年龄段肝脏中动脉粥样硬化(atherosclerosis,AS)重要相关基因的时序表达特点,并通过血清牛化检测结合主动脉根部病理形态学特征的分析,探讨AS重要相关基因时序表达特点与AS早期病变的关系.在主动脉检测的9条AS相关基因中,apoE-/-与野生型(WT)小鼠相比,1、2和3月龄时IL-1β的mRNA表达水平均显著上调.1月龄时VCAM-1、IKB和TGF-β,2月龄时PDGF-α和CD36,3月龄时TNF-α和MM2的mRNA表达水平均显著上调,其他年龄段无显著变化.在肝脏检测的2条AS相关基因中,C反应蛋白(CRP)的mRNA表达水平在14天到2月龄时无显著变化,3月龄时显著上调.NF-kB在各年龄段apoE-/-小鼠主动脉和肝脏中的基因表达量与同龄WT小鼠相比均无显著差异.不同年龄段的apoE-/-小鼠血清TC、TG和LDL-C水平均明显高于同龄WT小鼠.2月龄apoE-/-小鼠主动脉内膜出现散在的脂质沉积,并随年龄增长逐渐加重.上述结果显示,时序表达上调的AS重要相关基因构成了以NF-kB为核心的复杂调控网络,它们之间相互作用,共同参与慢性炎症过程,在apoE-/-小鼠AS的早期发生发展中发挥重要作用.     

脑梗死患者颈动脉粥样硬化的相关因素分析

目的:探讨脑梗死患者颈动脉粥样硬化的危险因素。方法:选取148例脑梗死患者作为实验组和同期住院的80例非脑梗死疾病患者为对照组,检测并比较各组年龄、吸烟、血压、饮酒、糖尿病、血脂、同型半胱氨酸和磷脂酶A2等指标,然后进行卡方检验、相关性和Logistic回归分析。结果:实验组颈动脉粥样硬化检出率为82.43%,对照组为12.5%,其差异具有统计学意义(P0.05)。高龄、吸烟、高血压、高血脂、高同型半胱氨酸血症、高磷脂酶A2(LP-PLA2)血症是经动脉粥样硬化的危险因素,且血浆同型半胱氨酸水平、LP-PLA2水平、血压与颈动脉斑块稳定性有关(P0.05)。结论:颈动脉粥样硬化是脑梗死的病理基础,与高龄、吸烟、高血压、高血脂、高同型半胱氨酸血症、高磷脂酶A2(LP-PLA2)血症有关,对高同型半胱氨酸血症、LP-PLA2和高血压患者要关注其斑块的稳定性。     

Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The contractile characteristics can be evaluated independently of external influences such as vascular tonus or neurohumoral status. It depicts a scientific approach between single cell measurements with isolated cardiac myocytes and in vivo studies like echocardiography. Thus, papillary muscle preparations serve as an excellent model to study cardiac physiology/pathophysiology and can be used for investigations like the modulation by pharmacological agents or the exploration of transgenic animal models. Here, we describe a method of isolating the murine left anterior papillary muscle to investigate cardiac contractility in an organ bath setup. In contrast to a muscle strip preparation isolated from the ventricular wall, the papillary muscle can be prepared in toto without damaging the muscle tissue severely. The organ bath setup consists of several temperature-controlled, gassed and electrode-equipped organ bath chambers. The isolated papillary muscle is fixed in the organ bath chamber and electrically stimulated. The evoked twitch force is recorded using a pressure transducer and parameters such as twitch force amplitude and twitch kinetics are analyzed. Different experimental protocols can be performed to investigate the calcium- and frequency-dependent contractility as well as dose-response curves of contractile agents such as catecholamines or other pharmaceuticals. Additionally, pathologic conditions like acute ischemia can be simulated.