Selective blockade of PGE2 EP1 receptor protects brain against experimental ischemia and excitotoxicity, and hippocampal slice cultures against oxygen-glucose deprivation

Abstract

Cyclooxygenase-2 (COX-2) enzyme increases abnormally during excitotoxicity and cerebral ischemia and promotes neurotoxicity. Although COX-2 inhibitors could be beneficial, they have significant side effects. We and others have shown that the EP1 receptor is important in mediating PGE2 toxicity. Here, we tested the hypothesis that pretreatment with a highly selectiveEP1 receptor antagonist, ONO-8713, would improve stroke outcome and that post-treatment would attenuate NMDA-induced acute excitotoxicity and protect organotypic brain slices from oxygen-glucose deprivation (OGD)-induced toxicity. Male C57BL/6 mice were injected intracerebroventricularly with ONO-8713 before being subjected to 90-min middle cerebral artery occlusion (MCAO) and 96-h reperfusion.Significant reduction in infarct size was observed in groups given 0.1 (25.9 +/- 4.7%) and 1.0 nmol(27.7 +/- 2.8%) ONO-8713 as compared with the vehicle-treated control group. To determine the effects of ONO-8713 post-treatment on NMDA induced excitotoxicity, mice were given a unilateral intrastriatal NMDA injection followed by one intraperitoneal injection of 10 microg/kg ONO-8713, 1 and 6 h later. Significant attenuation of brain damage (26.6 +/-4.9%) was observed at 48 hin the ONO-8713-treated group. Finally, brain slice cultures were protected (25.5 +/- 2.9%) by the addition of ONO-8713 to the medium after OGD.These findings support the notion that the EP1receptor propagates neurotoxicity and that selective blockade could be considered as a potential preventive and/or therapeutic tool against ischemic/hypoxic neurological conditions.

FIGURE 1

Effect of ONO-8713 pretreatment on MCAO-induced brain damage. Mice were treated with 0.1, 1.0, or 10 nmol of the EP1 antagonist ONO-8713 and subjected to 90-min MCAO. At 96 h, the mice were sacrificed and brain sections were stained with TTC to analyze the brain infarction. (A) Representative photographs of coronal sections of brains from vehicle-treated (left) and ONO-8713 (1.0 nmol)-treated (right) mice showing infarction due to MCAO. (B) Analysis of the TTC-stained brain sections revealed that 0.1 and 1.0 nmol ONO-8713 rescued the brain from ischemia, whereas 10 nmol ONO-8713 had no protective effect. *P <0.01, when compared with vehicle-treated mice.

FIGURE 2

Effect of ONO-8713 post-treatment on NMDA-induced brain lesion. Anesthetized mice were given a single intrastriatal injection of 15 nmol NMDA (in 0.3 μl) to induce acute excitotoxicity. After 1 h and 6 h, mice were given a 10 μg/kg injection of ONO-8713 i.p. Mice were allowed to survive for 48 h, and brain sections were stained with cresyl violet to analyze the brain lesion. (A) Representative photographs of coronal sections of the brains from vehicle-treated (left) and ONO-8713-treated (right) mice; areas of brain injury are encircled by dashed lines. (B) Analysis of the brain sections revealed a significant neuroprotective effect from ONO-8713 post-treatment. *P <0.05, when compared with the vehicle-treated group.

FIGURE 3

Effect of ONO-8713 on OGD-induced cell death in the CA1 region of cultured hippocampal slices. Organotypic mouse hippocampal slice cultures were placed in an air-tight chamber that was flushed with anoxic gas (5% CO₂, 95% N₂) for 1 h at 37°C. Then the cultures were transferred to normal culture medium with or without 1 μM ONO-8713 and incubated for 24 h. The PI fluorescence was obtained at 24 h after OGD, and the maximal PI fluorescence was obtained by stimulating the cultures for 24 h with a lethal amount of NMDA (100 μM). (A) Fluorescence images (4X magnification) showing PI staining in hippocampal slices subjected to OGD for 1 h. Images were taken before OGD (Base), 24 h after OGD (OGD), and 24 h after NMDA incubation (MAX). (B) Histogram representing CA1 neuronal damage measured by PI fluorescence intensity. The neuronal damage was significantly lower in ONO-8713-treated slices (n=27) than in vehicle-treated slices (n=24) after OGD-induced injury. ***P <0.001 as compared with the vehicle-treated group.