Animal procedures

The protocol was approved by the Animal Care and Use Committee at Shanghai Jiaotong University and conformed to the Chinese Medical Association guidelines for the use of animals in research.

On day 1, minipigs (23–30 kg) were sedated with ketamine (15 mg/kg intramuscularly) and anaesthetized with an intravenous infusion of sodium pentobarbital (10 mg/kg initially, then 1 mg/kg as needed), then intubated and mechanically ventilated. Through a right femoral arterial sheath, a 6-F guiding catheter (Amplatz Left 1) was placed into the ostium of the left main coronary artery. The LAD was occluded for 2 h using an intracoronary balloon catheter (15 × 2.5 mm balloon) at the site distal to the second diagonal branch. After reperfusion for 2 h, intravenous anaesthesia was stopped. Sedation with midazolam (1 mg/kg intramuscularly, as needed) was continued for 24 h, during which the right femoral artery and vein remained cannulated for MCE and microsphere injection at 24 h of reperfusion. Blood pressure and heart rate were continuously monitored. During coronary intervention, pigs were heparinized (5000 U bolus followed by 100 U/kg/hour) and then oral aspirin (300 mg) was given daily until euthanization.

On day 8 and day 15 (7 and 14 days after reperfusion, respectively), under sedation with midazolam, the carotid artery was cannulated for microsphere injection, while the carotid vein was used for MCE contrast injection.

After 14 days of reperfusion, the LAD was reoccluded and 2% Evans Blue dye was injected into the LAD via a carotid venous sheath, to identify the risk area. The animals were then euthanized and the heart was rapidly removed and cut into five short-axis slices, perpendicular to the ventricular septum from the base to the apex of the heart (6–8 mm thick; labelled as S1 to S5, respectively). All five slices were weighed and recorded.

Study groups

After LAD occlusion, pigs were randomized into the following groups: Group I (saline control), normal saline without salvianolate; Group II, 5 mg/kg salvianolate diluted with 100 mL saline and given intravenously at a rate of 100 mL/hour, starting 30 min before reperfusion; the same dose was repeated once a day for 7 days; Group III, as for Group II but using 10 mg/kg salvianolate; Group IV, in addition to the same intravenous dose as Group III, 20 mg salvianolate in 2 mL saline given through an over-the-wire balloon catheter just after reperfusion.

Drug under investigation

Salvianolate is derived from the dried root of cultivated Radix salviae miltiorrhizae and manufactured by Greenvalley Pharmacia (Shanghai, China), using the technique described previously . The manufacture and clinical application of salvianolate was granted by the State Food and Drug Administration of China in 2005. The major active component, magnesium lithospermate B (content > 85%), is detected for quality control during production. The other components of salvianolate consist of different phenol salts, including magnesium lithospermate, dipotassium lithospermate, sodium rosmarinate, potassium Danshensu, dipotassium isolithospermate B and magnesium salvianolate G.

Salvianolate is recommended for use at a dose of 200 mg/day (3.3 mg/kg/day for an adult weighing 60 kg) in the treatment of chronic stable ischaemic heart disease . The equivalent dose for a minipig is approximately 5 mg/kg according to the exchange coefficient determined by surface area. In the present study, we selected an equivalent standard dosage (5 mg/kg/day) and a higher dosage (10 mg/kg/day) for investigation.

Myocardial contrast echocardiography examination

In order to detect MBF, real-time MCE was performed with a Sequoia 512 machine (Acuson, Mountain View, CA, USA) at different time points, including baseline, 2 h after LAD occlusion, and 2 h, 24 h, 7 days and 14 days after reperfusion, using the methodology described previously . Briefly, after gain settings were optimized, real-time perfusion images were acquired from the short-axis papillary muscle view (mechanical index of 0.17), while SonoVue (Bracco Diagnostics, Inc., Geneva, Switzerland) was infused at a rate of 1 mL/minute via a femoral or carotid venous sheath. After high-energy flash frames (mechanical index of 1.9) lasting for 10 cardiac cycles were manually triggered to destroy the microbubbles within the myocardium, continuous refilling sequences of 15 cardiac cycles were obtained and recorded on magnetic optical disks.

The risk area was identified as the region of opacification defect during LAD occlusion. The perfusion defect area was manually traced for the final three end-diastolic images of the 15-cycle refilling sequence. The risk area was expressed as percentage of left ventricle and the reperfusion defect area was expressed as percentage of risk area.

For quantitative analysis, the sample volume was placed in the risk area (region of interest) avoiding both the epicardium and the endocardium. Using Cardio U/S Quantification software (Version 1.4; Siemens Healthcare, Erlangen, Germany), quantitative perfusion data were obtained by fitting intensity data to an exponential function: y = A (1−e ^−βt ) + C, where y is the signal intensity at any given time; A is the plateau signal intensity that reflects the microvascular cross-sectional area; β is the rate of signal intensity rise that reflects myocardial microbubble velocity; t is the time after the high-energy flash frames; and C is the intercept at the origin reflecting the background intensity level. The product (A × β) correlates with MBF. Because the value of A can be affected by various factors, including gain and attenuation, it was normalized to the A value derived from the adjacent LV cavity .

Measurement of myocardial blood flow

MBF was also quantified using 15 μm coloured microspheres (Dye-Trak, Triton Technology, Inc., South Easton, MA, USA) as described previously . Via the femoral or carotid artery sheath, a pigtail catheter was introduced into LV cavity. After being thoroughly vibrated for optimal mixing, an adequate amount of microspheres was injected into the LV cavity for 10 s. A 10 mL bolus of warm saline was immediately injected to wash out the catheter. A reference arterial blood sample was continuously aspirated from 10 s before microsphere injection until 60 s after the injection, at a rate of 5.8 mL/minute. The microsphere injection was repeated at different time points with different colours. After the animal was euthanized after 14 days reperfusion, the anterior wall on the S3 heart tissue slice was used for quantification analysis. Microspheres were recovered by a sedimentation process. The microsphere dye was then extracted and MBF (mL/minute/g) was calculated.

Evaluation of the effects of myocardial contrast echocardiography and myocardial blood flow examination on haemodynamics

To ensure that MCE and microsphere examination had no side effects on microcirculation, a pilot study was done on four normal minipigs without any intervention. MCE and microsphere injection was done at baseline, and 2 and 4 h after anaesthesia on days 1, 2, 8 and 15; time course changes in microcirculation and cardiac function were quantified.

Myocardial function measurement by transthoracic echocardiography

While the animal was sedated for MCE examination at each time point, as described above, transthoracic echocardiography was performed, using a Sequoia 512 machine with a 1.75–3.5 MHz transducer. LV dimensions and wall thickness were measured from the parasternal short-axis view at papillary muscle level. Wall thickening and fractional area change were calculated and expressed as percentages . LV volume was measured from the apical four-chamber view and LV ejection fraction was determined by Simpson’s method.

Histological assessment

For infarct size quantification, only three slices (S1, S3 and S5) were incubated in 1% triphenyltetrazolium chloride at 37 °C for 15 minutes. The infarct size (stained in the pale area) and risk area (delineated with Evans Blue dye) were measured by planimetry (ImageJ 1.36, National Institutes of Health, Bethesda, MD, USA) at both the apical and basal sides of the three slices. The infarct areas or risk areas for slices S1, S3 and S5 were the average of the apical and basal values of each slice; the values for slice S2 were the average of the basal side of S3 and the apical side of S1; and the values for slice S4 were the average of the basal side of S5 and the apical side of S3. Summation of the infarct weights of all slices was obtained and the infarct size was expressed as a percentage of the total weight of the left ventricle and as the total weight of the risk area.

The heart tissue slices S2 and S4 were used for immunohistochemistry, protein expression and oxidative stress measurement. For the histochemistry examination, the tissue was fixed with 4% paraformaldehyde, paraffin embedded and then sliced at a thickness of 5 μm. Endothelial cells were stained using rabbit antihuman vWF antibody (Dako Denmark A/S, Glostrup, Denmark). Capillary density was calculated in 20 random high-power fields (400 ×) and expressed as the number per mm ² .

Terminal deoxynucleotide transferase-mediated dUTP nick end labelling and endothelial cell staining

TUNEL staining was performed using a commercially available kit (Nanjing KeyGen Biotech. Co. Ltd., Nanjing, China). The cell nuclei were labelled with 4’6-diamidino-2-phenylindole (DAPI), while endothelial cells were labelled with antibody against vWF. For each animal, at least 10 randomly selected high-magnification fields (400 ×) from five different sections were analysed. The apoptotic index was calculated as the ratio of TUNEL-positive cells to total cells, while the endothelial cell apoptotic index was calculated as the ratio of TUNEL-positive endothelial cells to total endothelial cells.

Assessment of tissue concentrations of superoxide dismutase, glutathione, malondialdehyde and nitric oxide

The tissue levels of superoxide dismutase enzyme activity and tissue concentrations of glutathione, malondialdehyde and nitric oxide were all determined spectrophotometrically using appropriate kits purchased from the Jiancheng Bioengineering Institute (Nanjing, China), according to the manufacturer’s instructions. The protein concentration of heart tissue homogenates was determined with the bicinchoninic acid (BCA) assay (BioTime Inc., Haimen, China).

Thioredoxin activity assessment

Using the same method as described previously , the activity of thioredoxin was determined for heart tissue from the infarct area using an insulin reduction assay, where insulin is reduced by thioredoxin with NADPH in the presence of excess thioredoxin reductase. Briefly, after tissue homogenization, 40 μg protein in a volume of 68 μL plus 2 μL DTT activation buffer (composed of 50 mM HEPES [pH 7.6], 1 mM EDTA, 1 mg/mL BSA and 2 mM DTT) were incubated at 37 °C for 15 min to reduce thioredoxin, then 40 μL of the reaction mixture (containing 250 mM HEPES [pH 7.6], 10 mM EDTA, 2.4 mM NADPH and 6.4 mg/mL insulin) were added. The reaction began with the addition of 10 μL of rat thioredoxin reductase (90.899 A412 U/mL; Sigma-Aldrich Inc., St. Louis, MO, USA) followed by incubation for 20 min at 37 °C. The reaction was stopped by the addition of 0.5 mL of 6 M guanidine-HCl and 1 mM DTNB (3-carboxy-4-nitrophenyl disulfide). For each sample, the absorbance at 412 nm was adjusted by subtracting the absorbance reading without thioredoxin reductase. One unit of activity was calculated using the following formula: A ₄₁₂ × 0.62/(13.6 × 2).

Western blotting

Tissue homogenates was separated by 12% SDS-PAGE and then transferred to a polyvinylidene-difluoride membrane (Millipore, Billerica, MA, USA). After blocking with 5% skimmed milk, membranes were incubated with a first antibody targeting Bcl-2 and Bax (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), followed by the corresponding HRP-conjugated second antibody. An enhanced chemiluminescence reagent western blotting detection kit (Pierce Biotechnology, Inc., Rockford, IL, USA) was finally used to measure immunoreaction with a light-sensitive film (Kodak, Rochester, NY, USA). The band for each protein was then quantified by densitometry with ImageJ software.

Statistical analysis

All data are presented as means ± standard deviations. Intergroup differences were determined by one-way analysis of variance and when statistical significance was found in a group, pairwise comparisons were made using the Student-Newman-Keuls post-hoc test. A value of P < 0.05 was considered statistically significant.

Time course changes in haemodynamics: pilot study

In the pilot study, during repeated MCE examination and microsphere injection, no significant changes were detected in heart rate, blood pressure and LV systolic function, or in A, β, (A × β) and MBF ( Table 1 ).

Table 1

Haemodynamics, microvascular blood flow and left ventricular systolic function in the pilot study ( n = 4).

	Baseline	2 h*	4 h*	24 h*	7 days †	14 days †
Heart rate (beats/minute)	116 ± 6	117 ± 9	115 ± 9	114 ± 4	112 ± 7	118 ± 4
Systolic blood pressure (mmHg)	151 ± 2	151 ± 5	151 ± 3	152 ± 2	153 ± 5	149 ± 3
Diastolic blood pressure (mmHg)	113 ± 3	113 ± 1	112 ± 2	112 ± 2	111 ± 3	111 ± 4
MCE in anterior wall
A	0.57 ± 0.05	0.58 ± 0.04	0.60 ± 0.06	0.58 ± 0.02	0.58 ± 0.02	0.58 ± 0.04
β (sec −1 )	0.47 ± 0.05	0.44 ± 0.12	0.43 ± 0.04	0.40 ± 0.05	0.49 ± 0.06	0.45 ± 0.04
A × β (sec −1 )	0.268 ± 0.016	0.260 ± 0.082	0.259 ± 0.029	0.237 ± 0.035	0.288 ± 0.044	0.263 ± 0.029
MBF by microspheres (mL/g/minute)	1.74 ± 0.23	1.94 ± 0.42	1.86 ± 0.63	1.90 ± 0.57	1.53 ± 0.43	1.75 ± 0.70
LV systolic function
Anterior wall thickening (%)	53.36 ± 6.27	56.22 ± 7.25	54.01 ± 5.98	55.36 ± 5.04	55.54 ± 5.98	53.56 ± 7.27
FAC (%)	60.95 ± 1.98	60.06 ± 2.63	60.30 ± 2.08	61.20 ± 1.18	60.90 ± 1.62	59.86 ± 2.05
LV ejection fraction (%)	64.37 ± 6.86	64.18 ± 2.45	64.03 ± 1.16	65.21 ± 4.43	64.41 ± 1.88	63.67 ± 1.38

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