Clinical And Experimental Study On Coronary Microvascular Dysfunction

The coronary slow flow phenomenon (CSFP) was defined as abnormally slow antegrade progression of contrast during coronary arteriography in the normal or near-normal coronary arteries. Although the phenomenon is not rarely found in coronary angiography, CSFP patients have poor life qualities with recurrent chest pain and adverse cardiac events, and CSFP may be one of the diseases of coronary microvascular dysfunction (CMD). The CSFP was ignored usually by cardiologists, and until now, we know little about CSFP. Therefore, it needs further study.The aim of this study was to assess the benefit of intracoronary administration of nitroglycerin and verapamil in the coronary flow in patients with CSFP according to the TIMI frame count (TFC).Sixty-four patients (mean age 57.43±10.87, male 75%) with coronary slow flow and no stenotic lesions during diagnostic coronary angiography because of chest pain were enrolled and divided into the nitroglycerin group (n=35) and verapamil group (n=29), and 29 patients with normal coronary flow were selected as control. CSFP was defined as four or more beats for the contrast media to opacify the distal vasculature. Intracoronary injection of 100-400μg nitroglycerin or verapamil through the diagnostic catheter was not given in patients with CSFP until the coronary flow improved. The coronary blood flow was evaluated by Thrombolysis In Myocardial Infarction (TIMI) frame count (TFC) method in this study.Positive treadmill test was observed in some of patients in nitroglycerin group and verapamil group with CSFP (P>0.05) as compared with none of the 29 patients in the control group. There was no difference regarding the other clinical characteristics among the three groups. Each patient had 2.14±0.79 slow flow coronary arteries with (262.06±88.29)μg nitroglycerin in nitroglycerin group and 1.97±0.89 slow flow coronary arteries with (248.57±110.80)μg verapamil in verapamil group, respectively, and there was no difference in patients with CSFP. The basic TFCs of left anterior descending artery (LAD), left circumflex artery (LCX) and right coronary artery (RCA) were 78.28±19.40,57.24±14.58,56.87±12.47 in the verapamil group, and were 70.84±21.66,55.33±12.52,51.05±15.35 in the nitroglycerin group, respectively, which were significantly higher than those in the normal controls (LAD 29.15±4.42, LCX 23.14±3.48 and RCA 19.72±1.75, respectively). There was no difference in the basic TFCs in the CSFP patients (P＞0.05). After the administration of drugs, the TFCs of LAD, LCX and RCA were 42.32±8.88,36.65±6.78,30.32±5.94 respectively (vs. base, all P＜0.05) in the nitroglycerin group and 37.68±9.31,31.50±11.30,24.58±4.40 respectively (vs. base, all P<0.05) in the verapamil group. The TFCs in both of groups after administration of drugs were still higher than those in normal controls (all P<0.05). The TFCs in the verapamil group had larger decrease than those in the nitroglycerin group (P<0.05).CSFP may be the result of CMD, which may refer to cardiologists complaint of chest pain and other symptoms of coronary artery diseases once and again and most of them was ignored. TFC is the useful method to study CSFP. Intracoronary administration of verapamil could obtain more improvement of the coronary flow in patients with CSFP than nitroglycerin, although the coronary flow were still slower than normal. The rupture debris of atherosclerotic plaque and thrombus resulted in coronary microembolization (CME) with the dysfunction of coronary microvessels, which was the manifestation of CMD. Until now, there is no gold standard on the detection of CME. Cardiac magnetic resonance imaging (MRI) has unique advantages in the diagnosis of cardiovascular diseases and became to be applied in clinical settings and models of CME. The contrast enhanced first-pass perfusion imaging of cardiac MRI could reveal the hypoenhanced zone which is the sign of myocardial hypoperfusion caused by coronary obstruction and edema, and the delayed contrast enhancement of cardiac MRI could reveal the abnormal hyperenhanced zone which is the sign of local myocardial necrosis and edema.There was little simple CME in the clinic that were usually accompanied by coronary artery diseases, and therefore, the CME models were built to study by injection microspheres into coronary arteries. It's hard to evaluate the conclusions of all the studies on this problem because of different CME animals with different methods.In this study, we built the swine CME models. Cardiac MRI, including cine MRI, first-pass perfusion imaging and delayed contrast enhancement was used to observe the microinfarct area and left ventricular wall motions and evaluate the left ventricular end-systolic volume (LVESV), left ventricular end-diastolic volume (LVEDV) and left ventricular ejection fraction (LVEF) to assess the value of cardiac MRI in detection of CME.CME models were made by injection of inertia plastic microspheres (diameter 42μm) into left anterior descending coronary in eighteen swines. According to the doses of microspheres, the swines were divided to three groups, three for group A with 50000 microspheres, eight for group B with 120000 microspheres and seven for group C with 150000 microspheres. Cardiac MRI was performed at base, six hours and one week after CME using 1.5T system (Magnetom Avanto, Siemens AG, Erlangen, Germany). Cine MRI was acquired to observe the motion of ventricular wall. After the cine MRI images were obtained, the swines received intravenous bolus of 0.05 mmol/kg Gd-DTPA (Magnevist) at a rate of 4mL/s for the first-pass perfusion images. Then, a second bolus of 0.15mmol/kg Gd-DTPA was given, and after ten minutes, delayed contrast images were acquired. Results were analyzed with Argus software to observe the abnormal signal regions, the left ventricular wall motions and calculate the left ventricular volume and ejection fraction. After experiment finished one week later, swines were sacrificed and the hearts were taken out for NBT staining to observe the infarction.Two swines died before the end of study in group C. No abnormality was found at base for all the sixteen swines. Hypoenhanced region on first-pass perfusion imaging was only observed in group C at papillary level six hours after CME and it became less obvious one week later. Delayed enhanced areas were found on short-axis sections in all the swines six hours after CME, and the areas in group A were less than the other two groups which existed only in anterior wall at apical level in group A and in anterior and anterior septal wall from papillary to apical levels in group B and group C. However, the delayed enhanced areas disappeared one week later in group A and group B on repeated MR imaging. And in group C the hyperenhanced regions diminished and only existed at papillary level one week after CME, which were consistent with NBT pathologic findings. Systolic function of anterior and anterior septal wall were impaired in all the swines and improved slightly one week later.The left ventricular volume of all the swines was enlarged gradually, but the LVEF decreased six hours after CME and recovered one week later. In group A, LVEDV was (32.53±1.13) ml, (33.24±2.11) ml and (38.95±2.67) ml (one week vs. at base and six hours,P<0.05, respectively) at base, six hours and one week later, respectively. LVESV was (16.24±1.14) ml, (19.83±1.58) ml and (20.29±1.52) ml (one week vs. at base, P＜0.05) at base, six hours and one week later, respectively. LVEF was 0.50±0.02,0.40±0.02 and 0.48±0.01 (six hours vs. at base and one week, P<0.05, respectively) at base, six hours and one week later, respectively. In group B, LVEDV was (39.82±4.65) ml, (40.40±4.79) ml and (49.03±3.95) ml for the three different time (one week vs. at base and six hours, P＜0.05, respectively), and LVESV was (20.61±2.42) ml, (24.22±2.69) ml and (26.29±3.10) ml (at base vs. six hours and one week, P<0.05, respectively), and LVEF was 0.48±0.06,0.40±0.07,0.46±0.06 (six hours vs. base, P＜0.05; six hours vs. one week, P＜0.05), respectively. In group C, LVEDV was (39.03±4.89) ml, (40.71±4.22) ml and (47.48±5.53) ml for the three time (one week vs. base, P<0.05; one week vs. six hours, P＜0.05), and LVESV was (18.63±1.93) ml, (23.62±2.41) ml and (24.73±2.07) ml (base vs. six hours, P<0.05; base vs. one week, P＜0.05), and LVEF was 0.52±0.05,0.42±0.05 and 0.48±0.05 (all P<0.05 for each two time).At base, LVESV of group A was less than group B (P＜0.05) and there was no difference for other parameters for different time. Six hours after CME, there was no difference for other parameters among the three groups. One week later, LVEDV and LVESV of group A was less than that of group B (P<0.05), and there was no difference for other parameters among the three groups.Myocardial microinfarction were observed only at papillary muscle level in the swines of group C at NBT staining which was consistent with the MRI findings.First-pass perfusion imaging and delayed contrast enhancement cardiac MRI could detect myocardial microinfarction, which were related to the amount of microspheres and the areas of myocardial infarction, and the larger the necrosis, the more easily it was found. Systolic function of myocardial wall was impaired after CME, and it decreased six hours later and recovered one week later. The change of LVEF was accompanied by the dilation of ventricular cavity, which indicated the procedure of ventricular remodeling was not consistent with the change of systolic function. The change of function of left ventricle was not associated with the dose of microspheres or the infarct sizes. Cardiac MRI is useful for the detection of CME. The coronary microembolization (CME) could result in myocardial ischemia and necrosis with cardiac remodeling and dysfunction. And the morbidity of adverse coronary events increases in CME patients with worse prognosis. The mechanism of cardiac remodeling and dysfunction remains unknown. Previous studies demonstrated that the inflammation with over-expression of tumor necrosis factorα(TNF-α) played an important role in the change of structure and function of heart with CME. Toll-like receptors (TLRs) family and nuclear factor kappa B (NF-κB) are the upstream signal of TNF-α, which participated in many inflammatory diseases. But it's unclear whether the TLR4/NF-κB signal transduction system is involved in the cardiac remodeling and dysfunction after CME.In this study, we detected TLR4 and NF-κB expression in the CME model to preliminarily study the role of TLR4/NF-κB system in the progress remodeling and inflammation after CME.The piglet CME model was built according to Part Two. Six swines were enrolled in group C with 150000 microspheres, and each swine in group A with 50000 microspheres and group B with 120000 microspheres. One swine was selected as control with sham operation (the sham operation group was built by intracoronary injection of same doses of normal saline). One week later, the hearts were removed. TLR4 were detected in myocardial tissue of anterior wall and posterior wall using Western-Blot and Real Time PCR. Western-Blot was used to detect NF-κB expression and electrophoretic mobility shift assay (EMSA) was used to detect NF-κB DNA-binding activity of myocardial tissue of anterior wall and posterior wall. One swine died when operation for group C. Compared to control group, the expression of TLR4 and NF-κB and activity of NF-κB of posterior wall were unchanged, which were enhanced in the anterior wall in group C. And in group C of CME, the expression of TLR4 and NF-κB and activity of NF-κB of anterior wall were elevated than those of posterior wall P＜0.05), but it seemed that there were no difference of TLR4 and NF-κB in anterior wall among all the CME groups.One week after CME, the expressions of TLR4 and NF-κB in the affected myocardial tissue were elevated. The expression of TLR4 in the abnormal and normal myocardial tissue before and after CME were consistent with NF-κB, which suggested that TLR4/NF-κB signal transduction system may participate in the inflammation and cardiac remodeling after CME.