A cynomolgus monkey model of carotid atherosclerosis induced by puncturing and scratching of the carotid artery combined with a high-fat diet.

Cardio-cerebrovascular disease is one of the three major causes of mortality in humans and constitutes a major socioeconomic burden. Carotid atherosclerosis (CAS) is a very common lesion of the arterial walls, which leads to narrowing of the arteries, in some cases occluding them entirely, increasing the risk of cardiovascular events. The aim of the present study was to evaluate a cynomolgus monkey model of carotid atherosclerosis (CAS) induced by puncturing and scratching combined with a high-fat diet. A total of 12 cynomolgus monkeys were randomly divided into four groups: A, puncturing and scratching carotid artery intimas + high-fat diet (n=3); B, puncturing and scratching carotid artery intimas + regular diet (n=3); C, high-fat diet only (n=3); and D, regular diet only (n=3). Blood was harvested at weeks 4, 6 and 8 and plasma lipid levels were assessed. At week 8, monkeys were sacrificed and carotid arteries were harvested for hematoxylin and eosin (H&E) staining to observe pathological changes. The results revealed that a high-fat diet led to increased plasma lipid levels and accelerated plaque formation. Carotid color Doppler ultrasonography was performed and, along with H&E staining, revealed plaque formation in group A. In summary, the results of the present study suggest that a cynomolgus monkey model of CAS model may be successfully constructed by puncturing and scratching of the carotid artery intimas in combination with a high-fat diet.

Introduction

Cardio-cerebrovascular disease is a major cause of mortality in humans and constitutes a major socioeconomic burden. According to epidemiological surveys, the mean incidence of acute brain cardiovascular stroke (BCS) events is 116/100,000, with a mortality rate of 81/100,000 (1). Ischemic cerebrovascular diseases account for >70% of all cerebrovascular diseases and the incidence increases annually (2). In 2005, 5.7 million patients succumbed to BCS, 87% of whom resided in low-income countries (3). The World Health Organization (WHO) predicts that the number of cardiovascular-associated mortalities will reach 20.5 million annually by 2020 (4).

Carotid atherosclerosis (CAS) is a common lesion of the arterial walls, which leads to artery narrowing and occlusion and is the basis for the development of cardiovascular events (5). CAS mainly comprises the thickening, hardening and restructuring of the arterial walls (6–9). CAS is predominantly observed in great or middle arterial walls and typically presents with a build up of lipid, cells, cellular matrix and calcium salt deposition on the arterial intima, followed by intimal thickening, structural failure, arterial wall deformation and narrowing of the arterial lumen (10,11). A number of mechanisms of CASE have been suggested, including the thrombosis theory, lipid infiltration theory, monoclonal theory, damage reaction hypothesis, oxidative stress hypothesis, stem cell formation hypothesis, immune dysfunction hypothesis, homocysteine and arginine hypothesis and the inflammatory reaction theory (12–14). The damage reaction hypothesis (15–18) suggests that the inflammatory response following vascular endothelial cell damage is an important factor promoting the pathogenesis of atherosclerosis as it is associated with a series of vascular intimal changes, including endothelial cell damage, increased endothelial cell permeability and adhesion, increased blood coagulation and cytokine release and increased growth factors, ultimately resulting in CAS development. However, researchers supporting the lipid infiltration theory (6,19–21) reported that the occurrence and progression of CAS are associated with abnormal lipid metabolism, mainly the accumulation of cholesterol, low-density lipoprotein cholesterol (LDL-C) in particular, in endothelial cells.

In summary, vascular intimal damage combined with elevated plasma lipid levels eventually lead to the formation of CAS, followed by vascular stenosis and occlusion. At present, the lack of an optimal and effective CAS model limits our ability to study its pathogenesis and potential treatments; research is therefore required to develop an efficient CAS model that is easy to construct, exhibits the typical pathological changes observed in CAS and is suitable for intervention treatment.

A number of CAS models have been developed, including those utilizing high-fat diets (20,21), mixed diets (22–24), endothelial damage (16–18), inflammation and the immune response (25–27), the hemodynamic method (28–30) and the genetic engineering method (31–33); however, each method comes with its own pros and cons. The high-fat diet method is simple, cost-effective and may induce atherosclerosis in the coronary arteries and brain, but the modeling time is long (3–5 months) and the development of CAS is not guaranteed. It is difficult to form CAS plaques similar to those observed in humans and secondary infections could develop, affecting the stability of the experiment (34–37). This may be associated with the luminal structures of local arteries, hemodynamic characteristics and endothelial cell function. In addition, the balloon injury method combined with the use of a high-fat diets, although it may shorten CAS modeling time, is controversial (38–40), due to the complex surgery, plaque instability and risk of thrombosis; in addition, the balloon size was poorly controlled, which may cause arterial rupture and increase the risk of model failure. With regards to the genetic engineering method, it was demonstrated (41) that, although rabbits with natural defects or genetic modifications by artificial cultivation could develop CAS, constructing a CAS model was high-cost, time-consuming and difficult to apply. The choice of experimental animals is one of the key considerations in CAS modeling. Rats, rabbits and pigs are currently the most commonly used experimental animals for the study of CAS (42–44); however, due to their physiological and structural differences with humans, optimal efficacy often cannot be achieved. Cynomolgus monkeys are primates that are physiologically and structurally similar to humans; furthermore, they are easily fed, sensitive to a high-fat diet and readily develop CAS (45). Therefore, cynomolgus monkeys were selected as the experimental animals in the present study.

Based on the aforementioned theories and objectives, puncturing and scratching of the carotid artery intima combined with a high-fat diet were used in the present study to develop a CAS model in cynomolgus monkeys. This method shortens the time required for modeling and improves the model quality; furthermore, this model may accurately imitate the plaque formation process that occurs in humans under hyperlipidemic conditions.

Materials and methods

Ethical statement

All procedures were approved by the Ethical Inspection Committee of Animal Experiments of Yunnan Yinmore Biological Technology Co., Ltd. (no. YBT1602; Xishuangbanna, Yunnan, China).

Animals

A total of 12 male cynomolgus monkeys (age, 4.0–5.0 years; weight, 6.0–7.0 kg) were purpose-bred and purchased from Yunnan Yinmore Biological Technology Co., Ltd., which is an Association for Assessment and Accreditation of Laboratory Animal Care International (Jinghong City, China) accredited animal research facility.

Monkeys were housed at the Laboratory Animal Breeding Center of Yunnan Yinmore Biological Technology Co., Ltd. in stable cages. For sleeping, feeding and rest, cages measured 1.5×2×1.5 m. The housing conditions were as follows: 12 h light/dark cycle at 22–24°C with a relative humidity of 45–65%. Water was available ad libitum via water bottles. Monkeys were moved out of cages to an activity room measuring 4×12.5×8 m for 6–8 h per day.

Monkeys were randomly divided into four groups (n=3): A, puncturing and scratching of carotid artery intimas + high-fat diet (500 g/day); B, puncturing and scratching of carotid artery intimas + with a regular diet (500 g/day); C, high-fat diet (500 g/day); and D, blank control with a regular diet (500 g/day) (n=3). Prior to the experiment, monkeys were adaptively fed for 1 month; individuals in groups A and C were fed with a regular diet (Yunnan yinmore Biotechnology Co., Ltd.), containing 100% ordinary particles feed), monkeys in groups B and D were fed with a high-fat diet (Yunnan yinmore Biotechnology Co., Ltd.), containing 2% cholesterol, 10% lard and 88% ordinary particles feed).

Animal modeling

In groups A and B, the CAS model was constructed in the left carotid artery by puncturing and scratching the carotid artery intima. Monkeys were denied access to food and water prior to surgery and were anesthetized using 5 mg/kg Zoletil 50 (Virbac, Carros, France). Monkeys were fixed in a dorsal position, the neck was shaved and the skin was disinfected. A 4-cm longitudinal incision was made at 1 cm laterally from the laryngeal prominence, followed by stepwise separation of the skin and subcutaneous tissues. In the space between the sternocleidomastoid muscle and the laryngeal prominence, the common carotid artery was isolated and fixed with a surgical suture. A 5-ml syringe needle (30 mm long, 0.6 mm diameter) was used to pierce the artery and repeatedly scratch the arterial walls, taking caution not to perforate the artery (Fig. 1). The needle was retracted and gauze was applied to the puncture point, following which the wound was washed and sutured.

Figure 1.

The cynomolgus monkey model of carotid atherosclerosis was constructed by puncturing the carotid artery and scratching the intima. The white arrow indicates the point at which the carotid artery was pierced with a needle.

All surgeries were completed by the same group of surgeons. On postoperative day 3, levofloxacin hydrochloride and sodium chloride injection (Jiangsu hausen Pharmaceutical Group Co., Ltd., Jiangsu, China; 8 mg/kg twice daily intravenously) were administered to prevent infection and the wound was closely observed; tramadol hydrochloride (Shijiazhuang Pharmaceutical Group Co., Ltd, Hebei, China; 2 mg/kg, once daily intramuscularly) was administered for pain relief.

Following surgery, monkeys in group A were fed a high-fat diet (containing 2% cholesterol, 10% lard and 88% regular granulated feed), whereas the monkeys in group B were fed a regular diet for 8 weeks. During this period, the wound condition, eating and swallowing were observed. Furthermore, blood was harvested and plasma lipid levels were measured at weeks 4, 6 and 8 postoperatively. Color Doppler ultrasonography was performed at week 8 monkeys were euthanized. The carotid arteries were harvested to perform hematoxylin and eosin (H&E) staining and evaluate the pathological changes.

Examination of plasma lipid levels

Plasma lipid levels were measured in all groups prior to surgery and at weeks 4, 6 and 8 postoperatively. A total of 2–3 ml fasting venous blood was collected, the serum was separated by centrifugation (radius, 18 cm) at a speed of 1,814.4 × g for 5 min at room temperature and stored at −80°C. Plasma lipid levels were measured using an automatic biochemical analyzer (Selectra-E; ELITechGroup, Paris, France). The measured indicators included triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C).

Carotid color Doppler ultrasonography examination

At week 8, all monkeys were anesthetized with 5 mg/kg Zoletil and placed in a supine position with their arms at the sides and the neck was fully exposed. CAS was assessed using a color Doppler ultrasonic diagnostic instrument (Philips Medical Systems B.V., Eindhoven, The Netherlands). The scan evaluated the characteristics of the CAS plaques (low-, mixed- and high-echo plaques) and the rate of vascular stenosis (cross-sectional area of the plaque/cross-sectional area of the whole vessel ×100) (46).

According to the standard of the Radiological Society of North America in 2003 (47), carotid stenosis can be classified as mild (rate of vascular stenosis: <50%), moderate (rate: 50–69%), severe (rate: 70–99%) or total occlusion (rate: 100%).

Histology

Following carotid color Doppler ultrasonography examination all monkeys were sacrificed. Part of the carotid artery was harvested, rinsed with isotonic saline, fixed at room temperature for 24 h in 4% paraformaldehyde and dehydrated in a graded series of alcohol. Tissues were embedded in paraffin and 5 µm sections were prepared using a rotary microtome (Leica Microsystems GmbH, Wetzlar, Germany). H&E staining was performed on tissue sections at room temperature for 3 min. The typical atherosclerotic histological changes were examined using an optical microscope (magnification, ×8).

Statistical analysis

All data are presented as the mean ± standard error. In order to determine whether the data were normally distributed, the Kolmogorov-Smirnov and Shapiro-Wilk tests were conducted. A repeated measures analysis of variance was performed with Fisher's post hoc test. Statistical analysis was using SPSS version 20.0 (IBM Corp., Armonk, NY, USA) and P<0.05 was considered to indicate a statistically significant difference.

Results

Changes in plasma lipid levels

Prior to intervention, all plasma lipid levels were within the normal range (Normal ranges: TC, 5.23–5.69 M/l; TG, 0.56–1.7 M/l; HDL-C, >1 mmol/l; LDL-C, <3.12 mmol/l.) and no statistically significant differences were observed between the groups (Fig. 2). However, plasma TG was significantly increased at weeks 6 and 8 compared with week 4 in groups A and C (P<0.05; Fig. 3A). In groups A and C, TC and HDL-C levels were significantly increased at weeks 6 and 8 compared with week 4 (P<0.05; Fig. 3B and C). However, no significant differences in TC and HDL-C were observed between weeks 6 and 8 in groups A and C. No significant differences in TG, TC or HDL-C were observed between any time points in groups B and D (Fig. 3A-C). TG, TC and HDL-C levels in groups A and C were significantly increased compared with group D at all time points (P<0.05; Fig. 3A-C). However, no significant difference in TG, TC and HDL-C were observed between groups A and C or between groups B and D at any time point (Fig. 3A-C). No significant differences in LDL-C levels were observed between groups or within groups at different time points (Fig. 3D).

Figure 2.

Plasma lipid levels in each group prior to intervention. No statistically significant differences were observed. TG, triglycerides; TC, total cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol.

Figure 3.

Plasma (A) TG, (B) TC, (C) HDL-C and (D) LDL-C levels in each group at different time points. #P<0.05 vs. 6 weeks in the same group; $P<0.05 vs. 8 weeks in the same group; %P<0.05 vs. group B at the same time; &P<0.05 vs. group C at the same time; *P<0.05 vs. group D at the same time. TG, triglycerides; TC, total cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol.

Carotid color Doppler ultrasonography

In group A, color Doppler ultrasound revealed mild stenosis (rate: 5–7%) and high-echo plaques, which were irregular with a rough surface, with discontinuous linear high-echo plaques (fibrous cap) on the arterial walls (Fig. 4). No plaque formation was observed in groups B, C or D.

Figure 4.

Color Doppler ultrasound of the carotid artery. Images of (A) an artery transect and (B) a longitudinal section were obtained. White arrows indicate plaque formation.

Histological observations

In groups A and B, the intima was obviously injured and thickened, with sloughing off of the endothelial cells (Fig. 5A-D). However, in group A, the plaques on the vessel wall were covered with fibrous tissue, with obvious hyperplasia of the lipid foam cells underneath the intima (Fig. 5A and B). In groups C and D, the arterial wall was complete and the intima was composed of a monolayer of endothelial cells adherent to the elastic plate (Fig. 5E-H). The endothelial cell layer maintained its integrity and the diameter of the lumen was uniform.

Figure 5.

Hematoxylin and eosin staining revealed the morphological characteristics of carotid atherosclerosis. (A) Transect and (B) longitudinal section of the carotid artery in group A. (C) Transect and (D) longitudinal section of the carotid artery in group B. (E) Transect and (F) longitudinal section of the carotid artery in group C. (G) Transect and (H) longitudinal section of the carotid artery in group D. Black arrows indicate scratch injuries on the carotid intima. White arrows indicate the location of atherosclerotic plaques. In groups C and D, the arterial wall was complete, the endothelial cell layer maintained its integrity and the diameter of the lumen was uniform.

Discussion

There are certain associations between CAS plaque formation, brain cardiovascular stroke and coronary heart disease. The basic pathological changes observed in CAS include intimal deposition of lipids, intimal focal fibrosis and plaque formation, which result in hardening of the vessel wall, luminal stenosis and ischemic changes in the corresponding organs (6,10,48,49). The risk factors of CAS mainly include hyperlipidemia, hypertension, high blood glucose (diabetes), smoking and age (50). CAS may be caused by a variety of factors, including thrombosis, lipid infiltration, injury response, oxidative stress, the stem cell formation theory, immune dysfunction, the homocysteine and arginine hypothesis and the inflammatory reaction theory (12–14,51). However, due to limitations associated with medical technology, social and environmental complexity, researchers have yet to fully elucidate the pathogenesis of CAS (52,53). Therefore, a highly efficient optimal modeling method of CAS is crucial for studying the pathogenesis and treatment of cardio-cerebrovascular disease. The model must be easy to construct, must mimic the formation of CAS plaques in humans under hyperlipidemic conditions and must be suitable for interventional treatment.

In the present study, on the basis of carotid artery intimal injury, changes in plasma lipids were associated with CAS. Lipid TG levels in groups A and C increased over time, indicating that TG may be used as a specific indicator of plaque formation and the development of CAS. TC and HDL-C increased at week 4 and remained stable at weeks 6 and 8, which suggests that TC and HDL-C increases occur during the early stages of CAS development. However, LDL-C levels were not associated with CAS formation in the present study. TG, TC and HDL-C levels were greater in groups A and C compared with group D; furthermore, no significant changes in plasma lipid levels were observed in groups B and D over time. These results suggest that a high-fat diet may promote increases in plasma lipid levels and accelerate plaque formation.

In the present study, the carotid artery intima was injured by puncturing and scratching with a needle, which results in platelets and leukocytes adhering to the intima and intimal hyperplasia (54). When intimal injury was coupled with a high-fat diet, CAS plaque formation was observed; however, puncturing and scratching the intima alone, or a high-fat diet alone, did not result in the formation of CAS plaques. In addition, the pathological changes observed during the development of CAS were similar to those reported in humans, including early degeneration of endothelial cells, deposition of foam cells, endothelial proliferation, gradual thickening of the endothelium, thinning of the membrane, lipid deposition, fibrous plaques and the formation of atheromatous plaques (55,56). As such, the cynomolgus monkey model of CAS induced by puncturing and scratching combined with a high-fat diet was deemed to be an optimal, highly efficient, simple and feasible modeling method.

Several details are crucial for the success of the model: i) The modeling method must include intimal injury and a high-fat diet to accelerate the formation of CAS; ii) scratching of the artery wall must be performed with caution to avoid perforating the artery; iii) differences in animal weight should be kept to a minimum; iv) deep anesthesia should be avoided and local anesthesia used instead where possible; v) separation of the skin and subcutaneous connective tissues must be performed with caution to avoid damaging vessels and peripheral nerves, while fully exposing the carotid artery; vi) needle retraction must be followed by gauze oppression hemostasis to prevent arterial hemorrhage; and vii) following surgery, the condition of the wound, swallowing and eating must be monitored.

The modeling method used in the present study has several advantages. Firstly, the procedure is simple and has a low animal mortality rate. Secondly, the duration required for CAS formation is short and the model is easily constructed, as well as stable and reliable. Thirdly, it is possible to assess whether CAS formation is associated with changes in plasma lipid levels. Using color Doppler ultrasound to visualize CAS allows for accurate assessment of stenosis and plaque. Finally, the pathological changes associated with CAS are similar in monkeys and humans; as such, this model may be suitable for studying the pathogenesis of CAS and potential drug interventions. Together, these advantages make this model suitable for studying the prevention and control of CAS and may be more widely applied as a novel modeling method.

However, the present study has some limitations. Firstly, the sample size was relatively small. Due to the cost of obtaining cynomolgus monkeys and funding limitations, only 12 experimental animals were used as research subjects in the present study. However, the sample size was sufficient for basic requirements. Secondly, only H&E staining was performed for histopathology and the histological and Doppler analyses were not repeated. Meanwhile, high magnification images were not captured. Thirdly, arteries should have been perfusion fixed in order to better assess lumen area changes. Finally, due to the scratch-method used there was some variation in injury and the subsequent degree of plaque formation between individuals. To address the abovementioned limitations, the authors aim to gradually improve the scientific nature of this model for future studies.