Gadolinium decreases inflammation related to myocardial ischemia and reperfusion injury

Abstract

Background: The lanthanide cation, gadolinium (GdCl3) protects the myocardium against infarction following ischemia and reperfusion. Neutrophils and macrophages are the main leukocytes responsible for infarct expansion after reperfusion. GdCl3 interferes with macrophage and neutrophil function in the liver by decreasing macrophage secretion of inflammatory cytokines and neutrophil infiltration. We hypothesized that GdCl3 protects against ischemia and reperfusion injury by decreasing inflammation. We determined the impact of GdCl3 treatment for reperfusion injury on 1) circulating monoctye and neutrophil counts, 2) secretion of inflammatory cytokines, and 3) influx of monocytes and neutrophils into the myocardium.

Methods: Rats (n = 3-6/gp) were treated with saline or GdCl3 (20 mumol/kg) 15 min prior to a 30 min period of regional ischemia and 120 min reperfusion. Sham rats were not subject to ischemia. Blood was collected either after 30 min ischemia or 120 min reperfusion and hearts were harvested at 120 min reperfusion for tissue analysis. Blood was analyzed for leukocytes counts and cytokines. Tissue was analyzed for cytokines and markers of neutrophil and monocyte infiltration by measuring myeloperoxidase (MPO) and alpha-naphthyl acetate esterase (ANAE).

Results: GdCl3 did not affect the number of circulating neutrophils prior to ischemia. Two hours reperfusion resulted in a 2- and 3- fold increase in circulating monocytes and neutrophils, respectively. GdCl3 decreased the number of circulating monocytes and neutrophils during reperfusion to levels below those present prior to ischemia. Furthermore, after 120 min of reperfusion, GdCl3 decreased ANAE and MPO activity in the myocardium by 1.9-fold and 6.5-fold respectively. GdCl3 decreased MPO activity to levels below those measured in the Sham group. Serum levels of the major neutrophil chemoattractant cytokine, IL-8 were increased from pre-ischemic levels during ischemia and reperfusion in both control and GdCl3 treated rats. Likewise, IL-8 levels increased throughout the 3 hour time period in the Sham group. There was no difference in IL-8 detected in the myocardium after 120 min reperfusion between groups. In contrast, after 120 min reperfusion GdCl3 decreased the myocardial tissue levels of macrophage secreted cytokines, GM-CSF and IL-1.

Conclusion: GdCl3 treatment prior to ischemia and reperfusion injury decreased circulating monocytes and neutrophils, macrophage secreted cytokines, and leukocyte infiltration into injured myocardium. These results suggest GdCl3 decreased monoctye and neutrophil migration and activation and may be a novel treatment for inflammation during ischemia and reperfusion.

Figure 1

In vivo rat model of ischemia and reperfusion injury. A) Rats were treated with either saline or GdCl₃ (20 μmol/kg) 15 minutes prior to a 30 minute period of regional ischemia and 2 hours reperfusion.↑ Blood collection.↓ Harvest the free wall of the left ventricle. B) Measurement of infarct size as a percentage area at risk using this protocol.

Figure 2

Gadolinium chloride decreases circulating monocytes and neutrophils following ischemia and reperfusion. Rats were treated with either saline or GdCl₃ (20 μmol/kg) 15 minutes prior to a Sham procedure or a 30 minute period of regional ischemia and 2 hours reperfusion. A) Increase in monocytes. B) Increase in neutrophils. Data mean ± SD, n = 3-6/gp, + = p < 0.05, Sham control vs. Ischemia-Reperfusion (IR) control,* = p < 0.05, IR Control vs. IR GdCl₃, ± = p < 0.05, Sham Control vs. Sham GdCl₃, § = p < 0.05, IR GdCl₃l vs. Sham GdCl₃

Figure 3

Gadolinium chloride decreases Alpha naphthyl acetate esterase activity in the reperfused myocardium. Rats were either treated with vehicle or GdCl₃ either before 30 min ischemia and 120 min reperfusion or a Sham procedure. Alpha naphthyl acetate esterase activity was measured after 120 min reperfusion in IR Control and IR + GdCl₃ groups and at a comparable time point in Sham Control and Sham + GdCl₃ groups. Data is mean ± SD, n = 6/group, * = p < 0.05 vs. IR control.

Figure 4

Gadolinium chloride decreases myocardial tissue myeloperoxidase activity. Rats were either treated with vehicle or GdCl₃ either before 30 min ischemia and 120 min reperfusion or a Sham procedure. Myeloperoxidase activity was measured after 120 min reperfusion in IR Control and IR + GdCl₃ groups and at a comparable time point in Sham and Sham + GdCl₃ groups. Data is mean ± SD, n = 3/group, * = p < 0.05 vs. IR control; § = p < 0.05 vs. Sham control.

Figure 5

Gadolinium chloride does not decrease IL-8/CINC-1 production in the myocardium or serum during ischemia and reperfusion. Rats were either treated with vehicle or GdCl₃ either before 30 min ischemia and 120 min reperfusion or a Sham procedure. (Ischemia) and (Reperfusion) in the Sham groups is equal to the time period that correlates with Ischemia and Reperfusion in the Control and GdCl₃ groups. IL-8/CINC-1 was measured from the serum at 30 min ischemia and 120 min reperfusion and from the tissue at 120 min reperfusion. A) Quantitation of tissue IL-8/CINC-1 B) Quatitation of serum IL-8/CINC-1. Data mean ± SD, n = 3-4/gp, * = p < 0.05 vs. perfusion. No significant difference between IR vs. Sham groups or Control vs. GdCl₃ groups.

Figure 6

Gadolinium chloride decreases macrophage secreted cytokines after ischemia and reperfusion in rats. (A) Representative blots from cytokine arrays from control hearts vs. GdCl₃-treated hearts. The GM-CSF and IL-1 dots are indicated. Dots were quantitated and normalized to the positive control dots (lower left 4 dots) and results displayed above (B). Data mean ± SD, n = 3-4/gp, * = p < 0.05, GdCl₃ vs. control, += macrophage secreted cytokines, ‡ = cardiomyocyte secreted cytokines.