Sixty-four healthy male F344 rats were purchased from Silaike Experimental Animal Co., Ltd. (Shanghai, China), with gestational age of 6–8 months and body weight of 250±15 g. The rats were kept in cages and given unrestricted access to food and water, with 12-h light/dark cycle, a temperature of 20±3°C, and humidity of 53±5%.

According to the Olivette method, the rats were fixed in supine position after being anesthetized with ether and anesthesia was maintained by intraperitoneal injection of ketamine (100 mg/kg). ALC-V8 ventilator (Alcott Biotechnology Co., Ltd., Shanghai, China) was used to support respiration and an ECG instrument was used for ECG monitoring. The rats were operated with chest median longitudinal incision, and tissues were separated by layer separation. Thoracotomy was conducted along the fourth intercostal space, and the pericardium was separated to expose the heart. A needle was inserted in the junction below the edge of the atrioventricular, left atrial appendage and conus and 6-0. Prolene was used for ligature of the left anterior descending branch. The left ventricular anterior wall lost its original luster, and had a pale appearance, with a weak pulse and the ECG showed ST segment and Q wave changes, indicating that AMI models were successfully manufactured. The chest was observed continually for 3–5 min until a stable cycle was obtained. The rats in the sham operation group had only thread in the corresponding position without ligation.

The rats were randomly divided into the sham operation, model, SDF-1 intervention and SDF-1 antibody groups, with 16 rats in each group. On day 1 after the model was successfully established, the rats in the SDF-1 intervention group were injected with 10 µl recombinant SDF-1 (400 ng/ml; Peprotech, Inc., Rocky Hill, NJ, USA) in five regions including the myocardial infarction area and the four surrounding areas. The rats in the model group were injected with 10 µl normal saline including the myocardial infarction area and the four surrounding areas, and those in the SDF-1 antibody group were injected with 1 ml SDF-1 antibody (2 µg/ml; United States Biological, Swampscott, MA, USA). Four rats were sacrificed (5% isoflurane followed by cervical dislocation) 1, 3, 7 and 14 days after the intervention, and the analysis was carried out.

TUNEL method was used to count in situ labeling apoptotic cells. According to the instructions of the kit procedures, the heart was fixed with 4% polyformaldehyde phosphate solution, dehydrated and paraffin-embedded, and sectioned (4 µm). The interval to the long axis was 100 µm perpendicular. After the conventional xylene dewaxing and gradient ethanol hydration, the terminal deoxynucleotidyl transfer enzyme-mediated method was used. TUNEL in situ was used to mark myocardial cell apoptosis. Normal myocardial nuclei were blue, while apoptotic-positive myocardial nuclei were brown. The proportion of the number of apoptotic-positive cardiac muscle cells to the total number of nuclei in 5 random high fields of view (×40) were counted under an optical microscope (Olympus, Tokyo, Japan), and the mean value was obtained.

CD34 immunohistochemical staining measurement for microvessel density (MVD) was counted using the Image-Pro Plus 5.0 system (Media Cybernetics, Inc., Rockville, MD, USA) with reference to the method used by Weidner (8). First, optical microscope at a magnification of ×20 scanned the entire section, and five regions of highest vascular density as ‘hot spots’ around the infarction area were selected. The number of vessels was counted with dyed brown inside the hot spots under a magnification of ×200 using the optical microscope. The endothelial cells, the cluster of endothelial cells, and cabled endothelial cells were counted as one blood vessel without taking the lumen into account or if there were any red blood cells. The vessel with a luminal diameter of >20 µm or thick muscle layer was not included. Each sample was selected for five views (×200) counting the number of microvessels.

HP SONOS 5500 animal ultrasonic echocardiography (Hewlett-Packard, Palo Alto, CA, USA) was used to measure the left ventricular end-diastolic diameter (LVEDd), left ventricular end-systolic diameter (LVESd), left ventricular fractional shortening (FS) and ejection fraction (EF) value. The probe was 11.5 MHz and placed in the left parasternal intercostal 4 for 5 of the rats. According to the American Heart Association ultrasound recommended guidelines (9), the measurement was completed within at least three consecutive cardiac cycles. The maximum and minimum of ventricular volume for the diastolic and systolic ends were chosen, and the capacity measurements were performed using the modified Simpson's method. Ketamine (50 mg/kg) was used for sedation prior to the examination.

The expression level of Toll-like receptor (TLR)-4 and nuclear factor-κB (NF-κB) were selected. WIP tissue cell lysis was used for cell protein extraction (Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China). After measurement of protein concentration using the BCA protein determination kit (Beyotime Institute of Biotechnology, Shanghai, China), 30 µg protein sample was added with the sample buffer and boiled for denaturation at 95°C for 5 min. Ten percent of SDS-PAGE gel was used for electrophoresis, and the protein was transferred to a PVDF membrane (both from Solarbio, Beijing, China). Then, 5% milk was used to block in a shaker at 25°C for 2 h. Rabbit anti-mouse TLR-4 polyclonal antibody (1:1,000; Affinity Biosciences, Inc., Cincinnati, OH, USA; catalog no. AF7017), rabbit anti-mouse NF-kBp65 polyclonal antibody (1:200; Wuhan Boster Biological Engineering Co., Ltd., Wuhan, China; catalog no. PB0321) and negative control rabbit anti-mouse GAPDH IgG antibody (1:1,000; ZSGB-BIO Technology Co., Ltd., Beijing, China; catalog no. TA309157) were used for incubation at 4°C overnight. TBST buffer was used for washing, followed by incubation of the membrane with horseradish peroxidase-labeled goat anti-rabbit IgG (1:5,000; ZSGB-BIO Technology Co., Ltd.; catalog no. ZDR-5306) at 25°C in the shaker for 2 h. After washing the membrane with TBST buffer, Super ECL Plus sensitive luminous liquid was added and placed in the dark (Applygen Technologies, Inc., Beijing, China). After exposure, the image was recorded using the SensiAnsys gel imaging system. With the gray value, the corresponding gray level ratio of target protein bands with reference protein bands was used to calculate each protein relative expression level.

SPSS software (Chicago, IL, USA) was used for data analysis and processing. Mean ± standard deviation was used for quantitative data. Single-factor ANOVA analysis was used for group comparison, and the number of cases or percentage were determined for the qualitative data, and χ² test was used for comparison between the groups. P<0.05 was considered statistically different.

There was no obvious apoptosis in the sham operation group. The number of apoptotic cells increased with the extension of time in the model group. The number of apoptotic cells of the SDF-1 treatment group was significantly lower at each time-point than the other groups, and the number of apoptotic cells reached their peak on the 3rd day. The number of apoptotic cells were reduced further after 7 and 14 days. The number of apoptotic cells in the SDF-1 antibody group was significantly higher (p<0.05) (Table I and Fig. 1).

The blood vessel density in the SDF-1 intervention group was significantly higher than that in the remaining groups. The vessel density of the SDF-1 antibody group was significantly smaller at each time-point (p<0.05), (Table II).

LVEDd and LVESd values of each time-point in the model group were significantly higher than those in the sham operation group, and significantly increased with time. The values of each time-point in the SDF-1 intervention group were smaller compared with those of the model group, but larger than those in the sham operation group and decreased with time. The values in the SDF-1 antibody group were significantly largest in each time-point and increased with time (p<0.05). The FS and EF values in model group were significantly smaller than those in the sham-operated group and decreased over time. Those in the SDF-1 treatment group at each time-point were higher than those of the model group, but smaller than those of the sham operation group and increased over time. The FS and EF values in the anti-SDF-1 antibody group at each time-point were significantly minimum and decreased with time extended (p<0.05) (Fig. 2).

The expression levels of TLR-4 and NF-κB in the SDF-1 intervention group were significantly higher than those in the other groups at each time-point and increased with time (p<0.05). In other groups, the differences were not statistically significant (p>0.05), as shown in Table III and Fig. 3.