Establishment and evaluation of a mouse model of affective disorder combined with atherosclerosis

Shuang Geng, Feng-Rong Wang, Shuai Wang, Fan-Yu He, Yu-Hang Guan

1. Liaoning University of traditional Chinese medicine, Shenyang 110847, China

2. Affiliated Hospital of Liaoning University of traditional Chinese medicine, Shenyang 110032, China

Keywords:Model Affective disorder Atherosclerosis Chronic unpredictable mild stress Blood lipid level HPA axis

ABSTRACT Objective: To establish a mouse model of affective disorder complicated with atherosclerosis(AS) by high fat feeding and chronic mild unpredictable stimulation (CUMS), and to provide an animal model for the later study of the prevention and treatment of affective disorder complicated with atherosclerosis by traditional Chinese medicine. Methods: 10 C57BL /6J mice were used as blank group, and 20 ApoE-/- mice were randomly divided into AS group and AS+CUMS group. After one week of adaptive feeding, except for the blank group, the other two groups were fed with high fat diet. Meanwhile, the AS+CUMS group was given chronic unpredictable mild stress. The model was evaluated after 16 weeks of modeling.During the experiment, the body weight, food intake, excitability, hair color and other general morphology of mice in each group were observed and recorded. Behavioral indexes(Sucrose preference tests and Open Field test) were detected in each group.The levels of 5-hydroxytryptamine (5-HT) and hypothalamic-pituitary-adrenal (HPA) axis hormones,including adrenocorticotropin (ACTH) and corticosterone (CORT), were detected by ELISA.Serum lipid levels, including total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C), were detected by ELISA. HE staining was used to observe the pathological condition of aorta. Results:Compared with blank group, the excitability and food intake of AS+CUMS group were significantly decreased. There were no significant differences in sugar water consumption and activity capacity of mice in AS group, while sugar water consumption and activity capacity of mice in AS+CUMS group were significantly decreased (P < 0.01). The serum 5-HT levels in AS group and AS+CUMS group were decreased, and the levels of ACTH and CORT in AS+CUMS group were significantly decreased (P < 0.01), and the levels of ACTH and CORT in AS+CUMS group were significantly increased (P < 0.01). The serum TC, TG and LDL-C levels of mice in AS group and AS+CUMS group were significantly increased (P < 0.01),while the HDL-C level of mice in both groups was significantly decreased (P < 0.01). HE staining results showed that the area and degree of plaques in the active vascular lumen of AS group and AS+CUMS group were larger and heavier. Conclusion: High fat feeding combined with CUMS was successful in establishing a mouse model of emotional disorder combined with atherosclerosis.

1. Introduction

Atherosclerosis is a chronic pathological state involving multiple processes such as vascular endothelial dysfunction, inflammatory cell infiltration, abnormal vascular smooth muscle proliferation and migration, lipid peroxidation deposition, and vascular matrix changes [1]. The pathological basis of cerebrovascular diseases,atherosclerotic cardiovascular disease (ASCVD) has become one of the main causes of death in my country [2]. With the gradual intensification of contemporary social environmental factors and social pressures, depression has become a common mental disorder that endangers the physical and mental health of the global population, and the incidence is gradually increasing [3], which is characterized by continuous depression, lack of pleasure, fatigue,If you like to stay still, do not move, etc., and have high morbidity,disability, high cost, and even risk of suicide [1]. Now more and more epidemiological studies and clinical observations have found that mental and psychological disorders such as depression and anxiety increase the risk of coronary heart disease in cardiovascular patients, and studies have shown that mental and psychological disorders are independent risks of cardiovascular disease. Among them, depression, anxiety, and cardiovascular disease are particularly closely related [4-6]. Patients with cardiovascular disease will also be accompanied by mental and psychological disorders, and epidemiological studies have confirmed that atherosclerosis and depression are common The disease rate is very high and each is a risk factor. This is the "double heart medicine" proposed by Professor Hu Dayi [7]. Modern medicine for the treatment of mental and psychological disorders with atherosclerosis is mostly the combined application of antidepressants and cardiovascular-related drugs. Studies have found that the combined application of the two types of drugs has certain risks and adverse reactions, especially in elderly patients. The safety and risk degree should be analyzed in detail before [8].However, TCM believes that atherosclerosis belongs to the category of "phlegm and blood stasis", and depression and anxiety belong to the category of emotional disorder, which belongs to the categories of "Depression syndrome" and "hysteria",etc.Clinical treatment of double heart disease starts from the overall concept, through the combination of disease and syndrome, and treatment based on syndrome differentiation, the clinical efficacy is quite good, and basically no adverse reactions [9, 10], but the specific molecular mechanism is not clear. In order to further study the molecular mechanism of Chinese medicine, it is inseparable from the establishment of an effective animal model. This experiment aims to establish a combination of emotional disorders The atherosclerosis mouse model provides an ideal and reliable animal model for the later study of the molecular mechanism of TCM prevention and treatment of emotional disorders and atherosclerosis.

2. Materials and methods

2.1 Experimental animals

10 SPF C57BL/6J mice and 20 APolipoprotein E Knockout(apoE-/-) mice, aged 4 weeks and weighing 16 ～ 20g, were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., license number: SCXK (Beijing) 2016-0006, purchased and raised in SPF animal Laboratory of Liaoning University of Traditional Chinese Medicine. This experiment has been approved by animal Experiment Ethics Committee of Liaoning University of Traditional Chinese Medicine (License number:21000042020014).

2.2 Reagents

Total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C),adrenal corticotropic hormone (ACTH), corticosterone (CORT)and 5-hydroxytryptamine (5-HT) ELISA kits were purchased from Shanghai Enzyme-linked Biotechnology Co., LTD. HE stain was purchased from Solebo (G1120).

2.3 Main Instruments

High-speed refrigerated centrifuge (Thermo Fisher Technology Co., Ltd., model Thermo ST16R), multifunctional enzyme label analyzer (Thermo Fisher Technology Co., Ltd., model Multiskan FC), automatic tissue dehydrator (Leica, Germany, model atherosclerosisP6025), paraffin embedding machine (Leica,Germany, model EG1150H+C), paraffin microtome (Leica,Germany, model RM2235), optical microscope (Olympus, Japan,model BX41)

2.4 Grouping and modeling method

Ten C57 mice were used as the blank group, and 20 ApoE-/-mice were randomly divided into AS group and AS+CUMS group.The animals in the 3 groups were all adapted to room temperature(22±2℃; humidity: 55±5%) Sexual feeding for one week, starting from the second week, except for the blank group to be fed with ordinary basic feed, the other two groups were fed with high-fat feed(basic feed plus 2% cholesterol, 10% lard, 0.5% sodium cholate).And the AS+CUMS group was given CUMS at the same time until the end of the experiment for a total of 16 weeks [11]. During the experiment, observe and record the weight status, food intake,excitement, hair color and other general morphologies of each group of mice every week.

The chronic unpredictable mild stimulation program of the AS+CUMS group was slightly modified according to the literature[12-14] (the specific changes are as follows). The mice were stimulated by the stressor weekly according to the random principle(can be combined), and the other two groups of mice They were housed in a separate animal room. Except for no stimulation, other experimental conditions were the same as those of the AS+CUMS group. The specific stimulus items scheduled every week are:

Day and night reversed 2 times/week (lighting at night for 12h);squirrel cage tilted 3 times/week (tilt angle of 45°, lasting 7h); fear stimulation 2 times/week (changed to rat scented squirrel cage,continued 1h); Orphan rearing combined with restricted activities 2 times/week (put each mouse into a transparent plastic milk tea cup for 2h); empty bottle stimulation 1 time/week (last 12h); humid environment 1 time/week (in Spray 100ml of water with sawdust bedding in the squirrel cage for 24h); water-free once/week (for 12h)

2.5 Index detection

2.5.1 Sucrose preference tests (SPT)Before the start of CUMS stimulation, all animals were trained to adapt to sugar water (double bottle training). The time was about 4 days. Two bottles of 1% sugar water were placed in the squirrel cage for 24 hours, and one bottle of sugar water was replaced with purified water for 24 hours (sugar water). On the left side of the squirrel cage, purified water on the right), and then fasting for 24 hours, the single mouse single cage is tested for sugar water preference between 8:30-9:30, and a bottle of 150ml is placed on each squirrel cage. The sugar water (left) and a bottle of 150ml purified water (right), change the order of the water bottles at half an hour intervals (to prevent the mice from producing position memory drinking water), allow the mice to drink freely for 1 hour, and then measure the amount of sugar water consumed by the mice , The amount of purified water, the total amount of liquid, calculate the preference rate of sugar water (sugar preference rate = sugar water consumption / total liquid consumption × 100%). After 16 weeks of model building, the same method was used to calculate the sugar water preference rate of each group of mice [15].

2.5.2 Open Field Test(OFT)

Prepare a 50cm×50cm×50cm black self-made open box, and divide the bottom surface into 25 equilateral squares. Choose to carry out this experiment in a quiet room between 9:30 and 12:30 in the morning. Place the mouse in the center of the open box and record its motion score (horizontal score + vertical score) within 3 minutes,that is, the number of grids ( The square in which all four claws enter can be counted as a horizontal score) and the number of times the hind limbs are upright (the front paw is empty, washes the face,or clings to the wall of the box, is a vertical score). After each test,clean the bottom surface thoroughly with alcohol to eliminate the deviation that may be caused by the odor left by the previous mice.

2.5.3 Detection of body weight, excitement and hair color of mice in each group

Body weight was measured and recorded at 0, 2, 8, 12 and 16 weeks, and food intake, excitability and hair color were observed.

2.5.4 Histopathological observation of aorta in each group

Hematoxylin-eosin (HE) staining, take out the aorta fixed in 4%paraformaldehyde, embed and slice after fully automatic gradient dehydration, the thickness of the slice is 5μm, perform dewaxing staining according to the instructions, after mounting Observe under an optical microscope.

2.5.5 Detection of 5-HT, ACTH, CORT content in serum of each group of mice

After 16 weeks of modeling, blood was collected by removing the eyeballs after completing the sugar-water preference experiment and the open field experiment. The blood was collected at room temperature for 30 minutes, and then centrifuged at 3 000 r·min-1 for 30 minutes. The serum was separated by ELISA. Detect the content of ACTH, CORT, 5-HT in the serum, and the specific operation steps are carried out in accordance with the kit instructions.

2.5.6 Detection of blood lipid levels of mice in each groupDetect the content of TC, TG, LDL-C, HDL-C in serum with the same method as above.

2.6 Statistical analysis

SPSS 22.0 statistical software was used for analysis. All experimental values are described by±s. One-way analysis of variance is used for data comparison between multiple groups, and LSD test is used for pairwise data comparison between groups. If P<0.05, it is considered The difference was statistically significant.

3. Results

3.1 Behavioral results of mice in each group

3.1.1 Sucrose preference testsThe sugar water preference of mice in the AS+CUMS group was(35.32±1.37) %, significantly lower than that in the blank group(79.90±1.03) % (P<0.01), and the difference was statistically significant.The partial sucrose preference of mice in AS group was(80.21±1.73)%, which was not significantly different from that in blank group, as shown in Figure 1.

3.1.2 Open Field Test

The exercise score of the AS+CUMS group was 50.9±14.7,significantly lower than that of the blank group 125.5±17.3 (P<0.01),and the difference was statistically significant. The exercise score of the AS group was 116.3±17.9, showing no significant difference compared with the blank group, as shown in Figure 2.

Figure 1 Sucrose preference of mice in each group

Figure 2 Open Field Test score of mice in each group

Figure 3 Changes in body weight of mice in each group

3.2 Comparison of weight, excitement, and hair color of mice in each group

The body weight of mice in AS group at 16 weeks was 34.61±0.45,significantly higher than 29.50±0.63 in blank group (P<0.01), and 28.96±0.94 in AS+CUMS group was significantly lower than that in AS group (P<0.01). The excitability of mice in blank group and AS group was normal, while the activity of mice in AS+CUMS group was significantly decreased, showing drowsiness, quietness, less food intake and water intake, and poor hair color.

Table1 Comparison of body weight of mice in each group(n=10,g,x±s)

3.3 Results of 5-HT, ACTH and CORT levels in serum of mice in each group

The 5-HT content in the serum of mice in the AS group was 183.55±5.08, which was significantly lower than that in the blank group, 191.98±9.31 (P<0.05), and the 5-HT content in the serum of mice in the AS+CUMS group was 153.15±7.31, which was significantly lower than that in the blank group. Group (P<0.01);serum ACTH and CORT levels in the AS group were 52.81±2.13,157.90±2.50, respectively, which were significantly higher than the blank group 49.25±1.81, 153.38±4.15 (P<0.05), the AS+CUMS group was small The contents of ACTH and CORT in rat serum were 65.06±4.22, 176.38±4.33, which were significantly higher than those in the blank group (P<0.01), and the differences were statistically significant, as shown in Figure 4.

Figure 4 The content of 5-HT, ACTH and CORT in each group of mice

3.4 Results of blood lipid levels of mice in each group

The serum TG, TC, and LDL-C levels of mice in the AS group were 2.06±0.32, 6.70±0.57, 7.11±0.58, respectively. The serum TG, TC, and LDL-C levels of the mice in the AS+CUMS group were 2.43±0.33, 7.34±, respectively. 0.59, 7.47±0.50, which were significantly higher than the blank group 1.25±0.13, 4.56±0.47,5.01±0.51 (P<0.01); the serum HDL-C levels of mice in the AS group and AS+CUMS group were 1.11±0.07, 1.06, respectively±0.06, which were significantly higher than 1.43±0.03 in the blank group (P<0.01), and the differences were statistically significant, as shown in Figure 5.

Figure 5 The contents of TG, TC, LDL-C, HDL-C in each group of mice

3.5 HE staining of the aorta of each group of mice

The HE stained pathological sections of mouse arteries showed that the intima of the aorta in the blank group was relatively smooth and complete, without inflammatory cell aggregation, protrusion or plaque formation; compared with the blank group, the aortic lumen of the AS group and AS+CUMS group changed Narrow, intimal damage has obvious proliferative protrusions, contains foam cells,plaques are formed with a large area and a heavier degree, as shown in Figure 6.

Figure 6 Aortic histopathology of mice in each group (HE staining, ×100)

4. Discussion

There are a variety of animal depression models, including behavioral despair (BD), learned helplessness (LH), CUMS, orphan,olfactory bulb resection (OBX), and novel environmental induction Type, social frustration stress type, glucocorticoid type, electrical induction type, drug induction type, transgenic type, menopausal depression type, etc. [16-18]. Through comparative analysis, this experiment adopts the method of chronic, unpredictable and gentle stimulation, and reforms it on the basis of traditional stimulation methods. The mechanism of accelerating its development is closer to [19], and at the same time avoiding the use of stimuli that cause physiological trauma to the body, such as electric shock, tailclamping, hanging, swimming in strong cold water, scorching,etc., and the methods used in this experiment and The modeling tools are simple and easy to operate. This model is highly effective,sustainable, and reproducible. While simulating the symptoms of mental and psychological disorders, it also simulates its important characteristics, namely, anhedonia, depression, calmness, etc., and is tested by anhedonia (Decrease in sugar water consumption and sugar water preference) as the key to whether the model is successfully constructed [20], the reason why ApoE-/- mice is selected is because they can not only spontaneously develop into atherosclerosis, but are currently studied in atherosclerosis. The most commonly used animal model [21, 22], and is very sensitive to chronic stressor stimulation,especially the stress hormone level disorder, cardiovascular response and atherosclerosis pathological changes in the stress process are similar to humans, and are currently the main domestic and foreign One of the animal models to study chronic psychological stress and atherosclerosis [23]. In today's society, the pressure of the social environment is getting heavier and people's living and eating habits are changing. This experiment uses the combination of CUMS and high-fat feeding to simulate the current living conditions of contemporary people.

As for the pathogenesis of depression, one of the most studied hypotheses is HPA axis hypothesis and 5-HT hypothesis [24, 25].HPA axis hypothesis holds that the HPA axis function is abnormally hyperactive under the stimulation of chronic stressors, resulting in the disorder of hormone level. When the function of HPA axis is abnormally hyperactive, adrenotropin releasing hormone (CRH)will be released in the small cell area of the paravicular nucleus of hypothalamus (PVN), which will stimulate the production and secretion of ACTH in the anterior pituitary gland, and induce the adrenal gland to synthesize and secrete a large amount of CORT(cortisol in humans), and the long-term high hormone level will eventually lead to depression [26]. The 5-HT hypothesis believes that the disorder of 5-HT, known as the "happy neurotransmitter", will affect the functional areas of the brain that regulate emotions, and the continuous low level of 5-HT will cause depression and other negative emotions, thus leading to depression [25]. Studies have found that hPA-axis related hormone levels are up-regulated and 5-HT content is decreased in depressed animal models [26, 27], which also confirmed the above hypothesis. Therefore, when the body is depressed, it can be manifested as hPA-axis related hormone levels and 5-HT levels.

In this experiment, compared with the blank group, the blood lipid levels of mice in the AS+CUMS group were significantly changed(increased TG, TC, LDL-C and down-regulated HDL-C), and aortic pathology showed narrowing of lumen, intima damage accompanied by obvious bulging and plaque formation, indicating that atherosclerosis had formed. Behavioral changes included decreased sugar water preference and open field exercise score, as well as the disorder of hormone levels in vivo, which was reflected in the imbalance of HPA axis (high expression of serum ACTH and CORT)and the down-regulation of 5-HT expression. These results indicated that the model of emotion disorder combined with atherosclerosis was successfully established. This experiment has also set up a single pathogenic factors AS group, is to want to compared with the AS + CUMS group, verify the psychological stress will further promote the development of the atherosclerosis, and in addition to atherosclerosis in the AS mice, the hormone levels also change, at the same time the claim is verified both each other risk factors, its specific mechanism needs further research.

There are still some shortcomings in this experiment, for example,the sample size is not large enough, so the sample size will be appropriately increased in the next study. And although the experiment process is relatively complicated, for a long time, but can be a very good simulation of contemporary life present situation,and effectively simulate the characteristics of the double heart, can be considered as an ideal animal model for emotional disorders combined with atherosclerosis, this lays the foundation for the next study of the molecular mechanism of Chinese medicine in reventing and treating emotional disorders combined with atherosclerotic diseases.