The effects of apoptosis vulnerability markers on the myocardium in depression after myocardial infarction

Male Sprague-Dawley rats (N = 36, weighing 250 g ± 20 g) were used for the experimental procedures. Rats were allowed to adapt to the surroundings for one week prior to the commencement of the experiment. Each rat was housed singly at room temperature (22 to 25°C), humidity (40 to 50%), the light period was 12 h from 8 a.m., and food and water were freely available. Rats were divided into four groups: (i) sham group (N = 8), (ii) depression group (N = 8), (iii) MI group (N = 13), (iv) post-MI depression group (N = 7). The rats in all four groups performed the same open field and sucrose preference behavioral tests. In the depression and post-MI depression rats, there were significantly decreased movements and reduced total sucrose consumption, modeling behavioral deficits and a depressive-like anhedonic state [25]. The model of post-MI depression, in which decreased movement and an anhedonic- state occurred, was observed in 7 out of 20 myocardial infarction rats in our experiment. Animals were managed in accordance with the American Psychological Association (APA) ethical standards in the treatment of rats and National Institute of Health and Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23) revised in 1996, and the study was approved by the ethics Committee on the Guidelines for Animal Experiment at Guiyang Medical University. Efforts were made to minimize the number of animals used and their suffering.

In this study, we used the myocardial infarction model of ischemia described by Wu et al., which is a validated animal model of myocardial infarction [26]. Surgeries were performed in the morning. Twenty- two rats were anesthetized by 10% chloral hydrate (0.3 ml/100 g), intubated, and maintained on a ventilator (R: 60 times/minute). A thoracic incision of about 2.0 cm was made, and the base of the heart was exposed by retractors in the central region of the rib cage. The myocardial infarction model was induced by ligating the left anterior descending coronary artery 2 mm from the tip of the left auricle by polypropylene suture for 40 minutes. Rats were monitored by ECG (electrocardiogram) before and after coronary artery ligation. Ischemia was confirmed by ST-T segment elevation in ECG recordings performed on the rats (Figure 1) [27], and after the myocardial surface became blanched, the animal was returned to its home cage after the ligation was removed and the thorax sutured for 14 days. In the sham group, eight rats were operated on using the same protocol, except the coronary artery was not ligated. After the surgery, all rats were administered an analgesic (2 mg/kg butorphanol tartrate, s.c. every 8 h during the 24 h after surgery) and an antibiotic (10,000 IU penicillin G, i.m.). The rats did not show any signs of cardiac failure nor arrhythmias during the myocardial infarction procedure. The percentage of rats surviving the MI induction procedure was 90%.

In this study, the chronic mild stress model was used as a validated animal model of depression [28]. Chronic mild unpredictable stress and separation were used in the depression group for 21 days. Eight rats were given the following stressors [25, 29], consisting each week of repeated periods of confinement to a small (38 × 20 ×16 cm) cage, restraint (1 h), water deprivation (24 h), food deprivation (24 h), isolation (24 h), flashing light (3 h), forced cold water swimming (10 minutes) and were group-housed in a soiled cage overnight. Individual stressors and length of time used each day are listed in Table 1. Stressors were used daily in a random and unpredictable order for 21 days.

The behavior and anhedonic-like state of all rats were detected by the open field test [30] and sucrose consumption test [31]. The behavioral response to a new environment and activating behavior of rats was detected by the open field test [32], which has been used as an indicator of emotional state. This includes assessment of horizontal movements (the total number of crossing squares) and vertical movements (grooming and rearing) during a five-minute period. The former can be used as a proxy of emotional activity; the latter is regarded as a measure of exploratory activity to novel environments [33]. A special white square (80 × 80 × 40 cm), which has 25 sectors with black stripes on the ground, was used. Animals were separately put in the same central sector, and their activity was recorded during a five-minute period by an installed video camera. Observers analyzed the results of videotapes. Rats in both the depression and post-MI depression groups showed significantly lower scores of both horizontal and vertical movement compared with the sham and MI groups (Figure 2A), suggesting similarly decreased behavioral indices in the post-MI depression and depression groups.

Sucrose consumption is a rat model of an anhedonia-like state [34]. Every cage was offered a bottle of water and a bottle of 1% sucrose water. Total sucrose consumption was measured after 60 minutes. The test was begun 23 h after water and food deprivation, and the experiment was begun, and on day 15 after surgery [23] and the day after the end of the chronic mild unpredictable stress procedure [28]. Before the experiment, there were similar levels of sucrose water consumption between the four groups; however, after the experiment, the total consumptions of sucrose was significantly lower in the depression and post-MI depression groups compared to the sham and MI groups (Figure 2B), suggesting the post-MI depression group was similar to the depression group in terms of developing an anhedonic-like state.

The rats were sacrificed by decapitation 24 h after completion of the behavioral tests, prior to which horizontal movements, vertical movements and sucrose consumption tests were recorded according to the experimental protocol. The heart was removed, and the aorta was cannulated and washed with saline, the left anterior descending coronary artery was ligated again at the same site, and the aorta was infused with 2 ml of 0.5% Evan's blue to determine the extent of the non-colored ischemic risk area, then the myocardium was bisected into two parts from the apex to the base along the left anterior descending coronary artery, which was frozen at -80°C for five minutes, and then sliced into 2 mm transverse sections and stained using 2,3,5-triphenyl tetrazolium chloride (1.5%TTC) and myocardial infarction size (×2) was thus confirmed [21]).

The rest of the myocardium was washed using DEPC (diethylpyrocarbonate) H₂O. The anterior myocardial regional tissue sample (50 mg), which was non-colored tissue using Evan's blue was separated from the edge of the myocardial infarct risk area of the left ventricle. Tissue was frozen using liquid nitrogen and stored at -80°C for future real time RT-PCR use; the remainder was put into 4% paraformaldehyde for 12 h, paraffin embedded, and sectioned (thickness of 4 μm) for immunohistochemical staining.

Total RNA was extracted from the samples by the TRIZOL reagent (Shanghai ShengGong Biological Engineering Services Co., LTD, Shanghai, China). Primers were synthesized (Shanghai ShengGong Biological Engineering Services Co., LTD, Shanghai, China). cDNA was synthesized (Invitrogen Corp., San Diego, California USA) by combining the components (5× VILO™ Reaction Mix 4 μl, 10× Superscript Enzyme Mix 2 μl, RNA (up to 2.5 μg) Xμl and DEPC-treated water to 20 μl). Tube contents were gently mixed and incubated at 25°C for 10 minutes. Tubes were incubated at 42°C for 60 minutes. The reaction was terminated at 85°C at 5 minutes. cDNA was amplified with Power SYBR Green PCR Master Mix (Roche Molecular Biochemicals, Basel, Switzerland) for each gene, by the Applied Biosystems StepOne™ and Stepone Plus™ real-time PCR System (Foster City, California, USA): pre-denaturation 95°C for 10 minutes, denaturation 95°C for 15 s, annealing 60°C for 1 minute, extending 72°C for 1 minute, denaturation and annealing for a total of 40 cycles, extending again 72°C for 10 minutes. PCR products were analyzed by quantitative high-resolution DNA melting analysis. β-actin was used as an internal standard. Table 2 shows Primer series of Bax, Bcl-2 and β-actin, caspase-3 gene.

For Bax and Bcl-2 protein determination [35], immunohistochemical staining kits for Bax, Bcl-2 were provided by Wuhan Boster Bioengineering Co., Ltd. (Wuhan, China). The sections (thickness of 4 μm) were de-waxed and rehydrated with freshly distilled water. At room temperature, samples underwent inactivation of endogenous peroxidase for 10 minutes and were washed with distilled water for 2 minutes (three times), then immersed in 0.1 mol/L citrate buffer solution (pH = 6.0) and heated in a microwave oven until boiling (twice, with a 5-minute interval). After refrigerated flushing three times with phosphate buffered saline (PBS) for 2 minutes, antigen-retrieval buffers were added for 10 minutes and tissues were then flushed three times with PBS for 2 minutes. At room temperature, samples were non-specifically blocked with normal goat serum for 20 minutes. After removal of the goat serum, rabbit anti-Bax, Bcl-2 antibody was added (incubated for 60 minutes at 37°C). Biotinylated goat anti-rabbit antibody immunoglobulin G was applied for 20 minutes and flushed with PBS for 2 minutes (three times), then the tissues were placed in a streptavidin-biotin-enzyme complex reagent, incubated for 20 minutes at 37°C, after flushing with PBS for 5 minutes (four times), samples were stained with diaminobenzidine for 25 minutes and washed for 3 minutes. Then samples were stained with hematoxylin, and were dehydrated and mounted for microscopic examination. Images of the sections were obtained (400 X) using the Image-Pro Plus 4.5 software (Media Cybernetics, Silver. Spring, USA) with brown cytoplasmic staining under light microscopy indicating a positive reaction of Bax, Bcl-2. The myocardial infarction border zone was chosen using a 10 high power fields (400×), in which the mean number of bax and bcl-2 positive cells were obtained. The Bax:Bcl-2 ratio was also confirmed.

The analyses were conducted using IBM SPSS 19.0 analysis software (SPSS Inc, Chicago, Illinois, USA). One-way ANOVA test was performed to examine the data among groups (mean ± SEM) with the Bonferroni post-hoc tests for multiple comparisons. There was a significant difference (P < 0.05) and all the statistical tests were two-tailed.

The myocardial infarct risk area of the left ventricular area was 65 ± 2% (mean ± SEM) indicated by Evan Blue staining. The myocardial infarct size in the myocardial infarct risk area was similar in both groups: (MI group 43.2 ± 1.9%; post-MI depression group 45.6 ± 2.6%). No difference was seen between the MI and post-MI depression groups.

The ratio of Bax:Bcl-2 (mean ± SEM) in the depression, MI and post-MI depression groups was statistically significantly larger than in the sham group (P < 0.05). The ratio of Bax-Bcl-2 was significantly larger in MI group than the depression group (P < 0.05), and this ratio was significantly greater in the post-MI depression group compared with the MI group (P < 0.05) (Table 3).

There was a significantly higher ratio of Bax:Bcl-2 (mean ± SEM) in the myocardium in the depression, MI and post-MI depression groups than in the sham group (P < 0.01). Furthermore, in the post-MI depression group there was a significantly higher ratio of Bax:Bcl-2 than in the depression and MI groups (P < 0.01), and in the MI group there was a significantly higher ratio of Bax:Bcl-2 than in the depression group (P < 0.01) (Table 4).

No difference was seen in caspase-3 levels among the sham, depression, MI and post-MI depression groups (Table 3).