Abstract
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Objectives: This study aimed to explore how colchicine administration after severe ischemia and reperfusion affects myocardial inflammatory response and macrophage infiltration using C-14-methionine autography and dual-immunofluorescence staining and subsequent ventricular dimensions and function.
Methods: The left coronary artery (LCA) was occluded for 30 min followed by reperfusion. In control rats, 3 (n=5) and 7 (n=7) days after reperfusion, C-14-methionine (0.74 MBq) was injected 20 min before sacrifice. 1 min before sacrifice the LCA was re-occluded and Tc-99m-MIBI was injected to verify the area at risk. In rats with colchicine, 0.4 mg/kg/day of colchicine was intraperitoneally administered every day from 1 day after reperfusion to the day before methionine injection (day 3 (n=6) and day 7 (n=6)). Dual-tracer autoradiography of the left ventricular short axis slices was performed. The 1st autoradiographic exposure on an imaging plate was performed for 15-20 min to visualize the area at risk expressed by Tc-99m-MIBI distribution. 3 days later the 2nd exposure was made for 1-2 weeks to visualize the methionine uptake. The methionine uptake ratio (MUR) in an ischemic area was calculated by dividing the count density in an ischemic area by that of a normally perfused area. For the analysis of ventricular function, gated Tc-99m-MIBI SPECT was performed at 2, 4, 8 weeks after reperfusion in both control (n=5) and colchicine treatment (n=5) rats (colchicine (0.4 mg/kg/day) was administered daily from day1 to 6 after reperfusion). A QGS software algorithm was used to assess the end diastolic volume (EDV), end systolic volume (ESV) and left ventricular ejection fraction (LVEF). For the pathological evaluation of macrophage infiltration into myocardium, dual-immunofluorescence staining (CD68, DAPI staining) was performed at 3 and 7 days after reperfusion in both control and colchicine treatment using the slices next to autoradiographies. The CD68 positive percentage in an ischemic area was calculated by dividing the CD68 positive cell counts by that of DAPI positive cell counts in the ischemic area.
Results: In control rats (n=12), MURs at day 3 and 7 were 1.87 ± 0.14 and 1.39 ± 0.12, respectively. With colchicine (n=12), MUR was reduced significantly at both day 3 (1.56 ± 0.26, p< 0.05) and at day 7 (1.23 ± 0.099, p< 0.05). At 2 weeks, EDV (μL), ESV (μL), LVEF (%) in control rats were 632 ± 135, 385 ± 130, 40.2 ± 8.3 and those in colchicine rats were 485 ± 50, 266 ± 54, 45.4 ± 6.8 (p = ns), respectively. At 4 weeks, those in control rats were 686 ± 132, 452 ± 107, 34.2 ± 6.3 and those in colchicine rats were 585 ± 86, 360 ± 85, 38.8 ± 7.9 (p = ns), respectively. At 8 weeks, those in control rats were 864 ± 115, 620 ± 100, 28.4 ± 2.5, and those in colchicine rats were 665 ± 75, 390 ± 97, 42.2 ± 8.5 (p = 0.022, 0.022, 0.012), respectively. In control rats, CD68 positive percentages at day 3 and 7 were 38.4 ± 1.9 % and 24.0 ± 2.4 %, respectively. With colchicine, CD68 positive percentages were reduced significantly at both day 3 (31.5 ± 2.0, p< 0.0001) and at day 7 (12.0 ± 1.6, p< 0.0001) than that of control.
Conclusions: Administration of colchicine after severe ischemia significantly reduced MUR and macrophage infiltration both at day 3 and day 7, while attenuated left ventricular dilatation and LVEF deterioration at only 8 weeks. The result suggested that early colchicine treatment after ischemia and reperfusion suppressed acute inflammatory response and attenuated subsequent excessive ventricular remodeling. We conclude that short-term colchicine treatment at acute phase after myocardial infarction is effective to prevent subsequent excessive ventricular remodeling and reserve left ventricular function. Methionine imaging and gated Tc-99m-MIBI would be feasible to monitor the effectiveness of the anti-inflammatory therapy and left ventricular function.