Establishing an animal model to investigate depression with coronary heart disease

To the Editor: Coronary heart disease (CHD) and depression are common in patients worldwide and have a high comorbidity rate. Researches on depression have shown that it has been linked with long-term risk for CHD.^[1] The incidence and mortality of depression with CHD were higher, and the prognosis was worse.^[2] The association between CHD and depression is well established and is suggested to be bidirectional.^[3] Therefore, to provide possible conditions for the study of depression with CHD in the future, establishing a reliable animal model of depression with CHD is necessary. Chronic unpredictable mild stress (CUMS) is the most widely used to establish an animal model of depression for the past few years. Feeding a high-fat diet (HFD) to rats is a widely used method for establishing a model for atherosclerosis and CHD. Research reported that female cynomolgus monkeys that were housed in single cages and consumed an atherogenic diet could develop depression combined with CHD model.^[4] Another paper reported that CUMS combined with HFD in rats was similar to the clinical manifestation of vascular depression in humans.^[5] The former demonstrated that housing in single cages and feeding an atherogenic diet in monkeys could establish a depression combined with CHD model, the latter demonstrated that CUMS combined with HFD in rats was similar to vascular depression. It remains unclear whether CUMS combined with HFD in rats could establish a complex rat model of depression with CHD. Consequently, the aim of this study was to explore whether CUMS combined with HFD in rats could establish a representative, reliable rat model of depression with CHD and to provide possible conditions for an in-depth study of depression with CHD.

After a week of adaptation, Wistar rats were randomly assigned to three groups: the CUMS with normal feed group (CUMS, n = 8), the CUMS combined with HFD group (CUMS + HFD, n = 8), and the normal control group (NC, n = 8). The rats in the CUMS group were caged alone and subjected to different types of stressors: cage tilting for 24 h, wet bedding for 24 h, swimming in 5°C cold water for 3 min, swimming in 45°C hot water for 5 min, fasting for 24 h, water deprivation for 24 h, level shaking for 10 min, tail nip for 1 min, and inversion of the light/dark cycle for 24 h. These nine stressors were applied for 21 days, during which each stressor was applied 2 to 3 times. Rats received one of these stressors per day, and the same stressor was not applied consecutively over 2 days so that the animals could not predict the occurrence of the stimulation. All rats were maintained on the schedule for 8 weeks. The rats in the CUMS + HFD were caged alone and subjected to different types of stressors. In addition to CUMS, rats were administered a HFD, which was composed of 3% cholesterol, 0.5% sodium cholate, 0.2% propylthiouracil, 5% sugar, 10% lard, and 81.3% normal feed. Before the feed was given, a vitamin D3 injection was also administered into the rat's abdominal cavity at 600,000 U/kg only once. All rats were maintained on the schedule for 8 weeks. After 8 weeks of modeling, rats were subjected to an open field test and a forced swimming test; pathological sectioning of the heart, cardiac and cerebral vessels was performed; and serum lipid levels and brain tissue expression of brain-derived neurotrophic factor (BDNF) were assessed. All tests were performed using the SPSS Graduate Pack 17.0 statistical software package (SPSS, Chicago, IL, USA). Data were expressed as the mean ± standard deviation. In the animal study, one-way analysis of variance was used to analyze differences in the parameters. A P < 0.05 was considered to be statistically significant.

Compared with the NC group, the CUMS (1603.2 ± 361.0 vs. 821.3 ± 303.2 cm, P < 0.01) and CUMS + HFD (1603.2 ± 361.0 vs. 806.6 ± 347.0 cm, P < 0.01) groups had a shorter total distance in the open field test, and increased immobility times (9.7 ± 7.9 vs. 23.9 ± 10.47 s, 9.7 ± 7.9 vs. 24.4 ± 11.1 s, respectively, P < 0.01) in the forced swimming test [Figure 1A]. The CUMS (0.9 ± 0.1 vs. 0.8 ± 0.1, P < 0.01) and CUMS + HFD (0.9 ± 0.1 vs. 0.7 ± 0.1, P < 0.01) groups displayed a marked decrease in BDNF expression by Western blot, compared with the NC group. The results indicated a successful CUMS model [Figure 1B]. We observed pathological sections of cardiac and cerebral vessels by oil red O-staining to investigate whether CUMS + HFD induced pathological changes associated with CHD. Photographs of cardiac and cerebral vessels in the NC, CUMS, and CUMS + HFD groups are shown in Figure 1C. We observed that the NC and CUMS groups had no obvious abnormalities in cardiac and cerebral vessel structure. In the CUMS + HFD group, distributions of diffuse intimal thickening and focal fat deposition in cardiac and cerebral vessels were observed. To investigate whether CUMS + HFD induced pathological changes associated with CHD, we observed pathological sections of the heart by hematoxylin-eosin staining. Photographs of myocytes in the NC, CUMS, and CUMS + HFD groups are shown in Figure 1C. In the CUMS + HFD group, evidence for myocyte welling, rupture, degeneration, necrosis, and inflammatory cell infiltration was observed. The NC and CUMS groups showed no obvious changes under the microscope. To investigate whether CUMS + HFD induced pathological changes associated with CHD, we measured serum lipid levels. Compared with the NC and CUMS groups, the CUMS + HFD group showed increased serum levels of triglycerides (TC) (1.1 ± 0.2 vs. 1.6 ± 0.3 mmol/L; 1.1 ± 0.3 vs. 1.6 ± 0.3 mmol/L, respectively, P < 0.01), total cholesterol (TG) (0.4 ± 0.1 vs. 1.1 ± 0.2 mmol/L; 0.5 ± 0.2 vs. 1.1 ± 0.2 mmol/L, respectively, P < 0.01), as shown in Figure 1D. The results revealed that the CUMS + HFD group-exhibited hyperlipidaemia.

Figure 1.

CUMS + HFD have the symptoms of depression, atherosclerosis of cardiac and cerebral vessels, myocardial damage, and hyperlipidemia in rats. (A) CUMS + HFD induced depression-like behavior. CUMS and CUMS + HFD groups shortened total distance (cm) in open field test. CUMS and CUMS + HFD groups increased immobility time (s) in forced swimming test (^†P < 0.01 CUMS + HFD vs. NC group; ^∗P < 0.01 CUMS vs. NC group, n = 8). (B) CUMS and CUMS + HFD decreased BDNF expression in the hippocampus. (^†P < 0.01 CUMS + HFD group vs. NC group; ^∗P < 0.01 CUMS group vs. NC group, n = 8). (C) CUMS + HFD induced atherosclerosis of cardiac and cerebral vessels (Oil red O-staining, original magnification ×400). The arrow in cardiac and cerebral vessels of CUMS + HFD group points to focal fat deposition. Atherosclerosis was observed in cardiac and cerebral vessels. The arrow in myocytes points to myocyte swelling, rupture, degeneration (HE, original magnification ×400). Myocardial damage was observed in CUMS + HFD group. (D) CUMS + HFD induced hypercholesterolemia. CUMS + HFD group increased serum levels of TC, TG (^†P < 0.01 CUMS + HFD vs. NC group, ^∗P < 0.01 CUMS + HFD vs. CUMS group, n = 8). CUMS: Chronic unpredictable mild stress; HE: Hematoxylin-eosin; HFD: High-fat diet; NC: Normal control; TC: Triglycerides; TG: Total cholesterol.

Different from previous simple CUMS or HFD modeling, we used CUMS combined with HFD to establish a composite model of CHD with depression in rats. In the present study, we demonstrated that CUMS combined with HFD could establish a model of depression with CHD in rats. Compared with the NC group, the CUMS and CUMS + HFD groups had a shorter total distance in the open field test, an increased immobility time in the forced swimming test, and decreased BDNF expression. Compared with the NC and CUMS groups, the CUMS + HFD group had not only atherosclerosis of cardiac and cerebral vessels and myocardial damage but also increased serum levels of TC and TG. These findings provide evidence that CUMS + HFD induced symptoms of depression, atherosclerosis of cardiac and cerebral vessels, myocardial damage, and hyperlipidaemia. The establishment and characteristics of the rat model induced by CUMS + HFD provided a better experimental tool for basic research and drug development of CHD with depression.

In conclusion, the experimental results showed that CUMS combined with HFD in rats provided good similarity to the clinical manifestations of depression with CHD. The findings of this study may provide an ideal animal model for future studies of CHD with depression. However, the animal model needs further verification, and the relationship between CHD and depression needs to be studied in greater depth.

Acknowledgement

The authors thank Prof. Xi Huang for his support in preparing the work.

Funding

The study was approved by the grants from the National Natural Science Foundation of China (No. 81560730), and the Training Project for Young and Middle-aged Chinese Medicine Leaders in Yunnan Province.

Conflicts of interest

None.