A3 adenosine receptor agonist reduces brain ischemic injury and inhibits inflammatory cell migration in rats

Abstract

A3 adenosine receptor (A3AR) is recognized as a novel therapeutic target for ischemic injury; however, the mechanism underlying anti-ischemic protection by the A3AR agonist remains unclear. Here, we report that 2-chloro-N(6)-(3-iodobenzyl)-5'-N-methylcarbamoyl-4'-thioadenosine (LJ529), a selective A3AR agonist, reduces inflammatory responses that may contribute to ischemic cerebral injury. Postischemic treatment with LJ529 markedly reduced cerebral ischemic injury caused by 1.5-hour middle cerebral artery occlusion, followed by 24-hour reperfusion in rats. This effect was abolished by the simultaneous administration of the A3AR antagonist MRS1523, but not the A2AAR antagonist SCH58261. LJ529 prevented the infiltration/migration of microglia and monocytes occurring after middle cerebral artery occlusion and reperfusion, and also after injection of lipopolysaccharides into the corpus callosum. The reduced migration of microglia by LJ529 could be related with direct inhibition of chemotaxis and down-regulation of spatiotemporal expression of Rho GTPases (including Rac, Cdc42, and Rho), rather than by biologically relevant inhibition of inflammatory cytokine/chemokine release (eg, IL-1β, TNF-α, and MCP-1) or by direct inhibition of excitotoxicity/oxidative stress (not affected by LJ529). The present findings indicate that postischemic activation of A3AR and the resultant reduction of inflammatory response should provide a promising therapeutic strategy for the treatment of ischemic stroke.

Figure 1

LJ529 reduces cerebral infarct volume and ED-1 immunoreactivity. A: Cerebral infarct and edema volume. Rats were exposed to MCAO for 1.5 hours and subsequent reperfusion (R) for 24 hours. LJ529 [1 or 2 mg/kg, respectively marked as LJ(1) or LJ(2)] was administered intraperitoneally twice, at 2 and 7 hours after onset of ischemia, in the absence or presence of either MRS1523 (1 mg/kg) or SCH58261 (70 μg/kg). Representative tetrazolium chloride staining images (left) of brain sections (for LJ529, 2 mg/kg, twice). Quantitative data (right) are reported as means ± SEM. n = 4 to 14 per group. **P < 0.01 and ***P < 0.001 versus MCAO/R group; ^†††P < 0.001 versus LJ529 (2 mg/kg, twice) group [LJ (2)]. B and C: ED-1 immunoreactivity. Brain tissues were stained with anti-ED-1 antibody at 24 hours after MCAO/R (B) or LPS injection into corpus callosum (C). ED-1-postive cells per 0.1 mm² were counted in the cortex or striatum of ipsilateral regions. n = 8 per group. **P < 0.01 versus MCAO/R or LPS-treated group.

Figure 2

LJ529 decreases release of inflammatory cytokines/chemokines. Microglial cells were stimulated with LPS in the absence or presence of LJ529 or Cl-IB-MECA. A–C, left: Six hours later, the amounts of IL-1β (A), TNF-α (B), or MCP-1 (C) released into bathing medium were measured using enzyme-linked immunosorbent assay kits. n = 4 per group. *P < 0.05, **P < 0.01, and ***P < 0.001, untreated versus drug-treated groups. A–C, right: To measure effects of A3A or A2AAR antagonists, microglial cells were pretreated with either MRS1523 (1 μmol/L) or SCH58261 (100 nmol/L) for 30 minutes and then stimulated with LPS in the absence or presence of 10 μmol/L LJ529. n = 3 per group. ^†P < 0.05 and ^††P < 0.01 versus corresponding LPS/LJ529-treated groups.

Figure 3

Inhibition of MCP-1-induced microglial chemotaxis by LJ529. A: Time-lapse microscopy (see Supplemental Videos S1 and S2 at http://ajp.amjpathol.org). Microglia were pretreated with 100 nmol/L LJ529 or vehicle for 15 minutes and then stimulated with 100 ng/mL MCP-1 for 20 minutes. B: MCP-1-evoked migration of microglia was quantified using a chemotaxis chamber. n = 6. ***P < 0.001 versus MCP-1-treated microglia in the absence of LJ529. C: Microglia were pretreated with MRS1523 (1 μmol/L) or SCH58261 (100 nmol/L) for 20 minutes and the effect of either LJ529 (10 nmol/L) or Cl-IB-MECA (10 nmol/L) on chemotactic movement was determined in the absence or presence of MRS1523 or SCH58261. n = 4. ***P < 0.001 versus MCP-1-treated group. ^†P < 0.05 and ^†††P < 0.001 versus MCP-1/LJ529-treated group. D: Time-lapse microscopy (see Supplemental Videos S3 and S4 at http://ajp.amjpathol.org). Arrows indicate protruding edges toward tip, and arrowheads indicate retracting edges. 100 nmol/L LJ529 inhibited chemotactic sensing and movement to the point of MCP-1 source. n = 3.

Figure 4

Inhibition of activity and expression of Rho GTPases. A–C: Microglia were pretreated with 100 nmol/L LJ529 for 15 minutes and then stimulated with 100 ng/mL MCP-1 for the indicated times. Active forms of each Rho GTPase were pulled down from microglial lysates by using a pull-down kit and then subjected to Western blotting analysis. n = 4 per group. **P < 0.01 and ***P < 0.001 versus untreated group. D: Double staining of GTPases (Red) and filamentous actin (Green) in migrating cells. Microglia were stimulated with gradient MCP-1 for 5 minutes in the presence or absence of 100 nmol/L LJ529 and then fixed for immunocytochemistry. Asterisks indicate the tip point of the micropipette containing 100 ng/mL MCP-1. Data are representative of four separate experiments.

Figure 5

No blockade by LJ529 of neuronal injury evoked by OGD/R or NMDA. A and B: Although neuronal cell death induced by OGD/reoxygenation or NMDA was significantly inhibited by MK-801, an NMDA receptor blocker, it was not inhibited by LJ529. Cortical neurons were exposed to OGD for 1 hour (A) or 100 μmol/L NMDA for 10 minutes (B) in the absence or presence of LJ529. Another 6 hours later, lactate dehydrogenase released into bathing medium was determined. C: Depolarization of plasma membrane potential. NMDA-induced plasma membrane potential depolarization was not inhibited by LJ529. Cells were loaded with DiBAC₄(3) and then LJ529 was added. Ten minutes later, cells were exposed to 100 μmol/L NMDA and the fluorescence intensity was measured. Fluorescence intensity values were corrected by subtracting autofluorescence [ie, fluorescence in cells not loaded with DiBAC₄(3)]. n = 4 at each time point.

Figure 6

ROS scavenging effect or inhibitory effect of ROS release by LJ529. A: Dihydrorhodamine 123 fluorescence. LJ529 did not directly scavenge hydrogen peroxide or peroxynitrite. The fluorescence intensity of the control cells was assigned to 1. n = 3 per group. B: DCF fluorescence. LJ529 did not reduce the release of ROS in activated microglial cells. DCF fluorescence was measured after LPS treatment for 18 hours in the presence or absence of LJ529. Catalase or superoxide dismutase (SOD) was applied for 30 minutes before fluorescence measurement. n = 4 per group. C: Nitrite assay. LJ529 did not reduce the release of NO in LPS-treated microglial cells. The amount of nitrite was obtained from the supernatant of microglia treated with LPS for 18 hours in the presence or absence of LJ529. n = 4 per group.