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Comparative Study
. 2004 Jan 7;24(1):257-68.
doi: 10.1523/JNEUROSCI.4485-03.2004.

Neuroprotective function of the PGE2 EP2 receptor in cerebral ischemia

Louise McCullough  1 , Liejun Wu, Norman Haughey, Xibin Liang, Tracey Hand, Qian Wang, Richard M Breyer, Katrin Andreasson
Affiliations
Comparative Study

Neuroprotective function of the PGE2 EP2 receptor in cerebral ischemia

Louise McCullough et al. J Neurosci. .

Abstract

The cyclooxygenases COX-1 and COX-2 catalyze the first committed step of prostaglandin synthesis from arachidonic acid. Previous studies in rodent stroke models have shown that the inducible COX-2 isoform promotes neuronal injury, and the administration of COX-2 inhibitors reduces infarct volume. We investigated the function of PGE2, a principal prostaglandin product of COX-2 enzymatic activity, in neuronal survival in cerebral ischemia. PGE2 exerts its downstream effects by signaling through a class of four distinct G-protein-coupled EP receptors (for E-prostanoid: EP1, EP2, EP3, and EP4) that have divergent effects on cAMP and phosphoinositol turnover and different anatomical distributions in brain. The EP2 receptor subtype is abundantly expressed in cerebral cortex, striatum, and hippocampus, and is positively coupled to cAMP production. In vitro studies of dispersed neurons and organotypic hippocampal cultures demonstrated that activation of the EP2 receptor was neuroprotective in paradigms of NMDA toxicity and oxygen glucose deprivation. Pharmacologic blockade of EP2 signaling by inhibition of protein kinase A activation reversed this protective effect, suggesting that EP2-mediated neuroprotection is dependent on cAMP signaling. In the middle cerebral artery occlusion-reperfusion model of transient forebrain ischemia, genetic deletion of the EP2 receptor significantly increased cerebral infarction in cerebral cortex and subcortical structures. These studies indicate that activation of the PGE2 EP2 receptor can protect against excitotoxic and anoxic injury in a cAMP-dependent manner. Taken together, these data suggest a novel mechanism of neuroprotection mediated by a dominant PGE2 receptor subtype in brain that may provide a target for therapeutic intervention.

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Figures

Figure 4.
Figure 4.
Neuroprotective effect of EP2 receptor activation. A, RT-PCR of the EP2 receptor in hippocampal neurons at 14 d in vitro. Left lane (-) indicates the negative control in which no reverse transcriptase was added, and the right lane (+ RT) demonstrates EP2 receptor band (arrow). B, A 200× fluorescent microscopy confirms expression of EP2 receptor in hippocampal neurons at DIV 14. Double immunofluorescent staining with anti-EP2 and anti-NeuN antibodies confirms the neuronal expression of the EP2 receptor in these cultures. Control cultures (no primary antibody) did not show staining (data not shown). C, EP2 receptor activation by addition of the selective EP2 agonist butaprost (10 nm) protects hippocampal neurons from glutamate toxicity. Glutamate (50 μm) ± butaprost or vehicle was added overnight, and nuclei were stained with Hoechst and counted. The percentage of apoptotic nuclei was quantified as a percentage of the total number of nuclei counted (#p < 0.001; *p < 0.001; representative experiment of three experiments, n = 4-6 wells per condition). D, In organotypic hippocampal cultures, EP2 receptor activation protects CA1 neurons treated with NMDA (10 μm for 1 hr). Percentage of maximal PI fluorescence was calculated (see Materials and Methods) in slices treated with NMDA ± butaprost (0 nm to 1 μm); mean ± SE; **p < 0.01; *p < 0.05. Unlike the PGE2 experiments (Fig. 2), addition of butaprost and activation of the EP2 receptor result in neuroprotection in both dispersed and organotypic cultures. E, EP2 receptor activation also protects CA1 neurons in hippocampal slices subjected to OGD for 1 hr. Slices were subjected to 1 hr of OGD and then incubated with vehicle or butaprost (1 nm to 1 μm) for 24 hr. Butaprost at concentrations >10 nm significantly protected neurons, as determined by percentage of maximal PI fluorescence, *p < 0.05; **p < 0.01. F, EP2 receptor activation by butaprost significantly increases cytosolic levels of cAMP in 5-10 min; n = 3; mean ± SD; **p < 0.01. DIV 14 hippocampal neurons were stimulated with 10 nm butaprost, and cells were collected at 5 min intervals. G, The protective effects of EP2 receptor activation by butaprost are abolished with treatment with the PKA inhibitor H89 (1 μm) in hippocampal neurons treated with glutamate (50 μm for 18 hr). #p < 0.001 control versus glutamate; **p < 0.001 glutamate versus glutamate plus butaprost; p < 0.001 glutamate-butaprost versus glutamate-butaprost-H89. H, Administration of a second PKA inhibitor, KT 5720, also reverses EP2 neuroprotection in hippocampal neurons treated with glutamate (50 μm for 18 hr); **p < 0.01 control versus glutamate; #p < 0.02 glutamate versus glutamate-butaprost; *p < 0.05 comparing glutamate-butaprost to glutamate-butaprost-KT 5720 (0.6 and 1.2 μm).
Figure 1.
Figure 1.
COX-2 inhibition protects cultured neurons and hippocampal slices from glutamate-mediated toxicity and OGD. A, PGE2 levels in control (c), glutamate (gl; 100 μm), and glutamate + the COX-2 inhibitor NS 398 (gl/ns; NS 398 10 μm)-treated hippocampal cultures 24 hr after stimulation (c vs gl: *p < 0.001; gl/ns vs gl: #p < 0.001). B, Cell death was assayed by LDH release in hippocampal and cortical cultures 24 hr after glutamate stimulation (100 μm) ± NS 398 (10 and 50 μm; *p < 0.01). C, LDH assay demonstrates neuroprotection mediated by a second COX-2 inhibitor SC 58236 (0.01-10 μm) in hippocampal neurons stimulated with glutamate (50 μm; *p < 0.01). After subtraction of basal LDH release, cell death was calculated as the percentage of the LDH value from cultures treated with glutamate alone. D, The COX-2 inhibitor SC58236 protects neurons in hippocampal slices. Representative panels of sequential fluorescent images from control slice (a-c, NMDA + vehicle), and slice treated with 10 nm SC58236 (d-f, NMDA + SC 58236). Basal fluorescence images (a, d) do not show spontaneous neuronal degeneration in CA1 (outlined in white); 24 after 1 hr treatment of 20 μm NMDA, there is a significant increase in neuronal death in the control (b) slice, but an attenuated amount in slice cotreated with SC58236 (e). Maximal fluorescence images are obtained after complete killing of neurons with a further 24 hr treatment with NMDA (c, f). E, Neuroprotection in the CA1 region of organotypic hippocampal slices at 1 nm SC 58236, with increasing protection with higher doses, up to 1 μm. Slices were challenged at 14 DIV with NMDA 20 μm for 1 hr (n = 10-15 slices per condition). ANOVA followed by Newman-Keuls post hoc test shows differences between control and all NMDA groups (*p < 0.001), differences between treatment groups and NMDA group (#p < 0.05; ## p < 0.005). F, COX-2 inhibition protects against 1 hr OGD in hippocampal slices. Neuronal death (PI fluorescence) was quantified 24 hr after OGD. Slices were administered 2 mm 2-deoxy-d-glucose (DG) +/- 100 nm SC58236 (difference between OGD +/- inhibitor, *p < 0.01; difference between control and OGD, #p < 0.01).
Figure 2.
Figure 2.
PGE2 is neuroprotective in cultured hippocampal neurons but not in organotypic hippocampal slices. A, PGE2 administration at submicromolar concentrations promotes protection in hippocampal cultures stimulated with glutamate (100 μm) for 24 hr (n = 4 wells per condition; *p < 0.02; **p < 0.01; representative of 3 experiments). B, Increasing concentrations of PGE2 had no effect on neuronal viability when assayed in organotypic hippocampal slices treated with 1 hr NMDA (10 μm), suggesting a negative effect of PGE2 at the level of astrocytes in neutralizing the protective effects of PGE2 on pure hippocampal cultures. Neuronal death in CA1 is measured by percentage of maximal PI fluorescence (n = 10-15 slices per condition; n = 6 experiments). Concentrations of PGE2 were used at submicromolar concentrations to avoid the cross-activation of other PG receptors at higher concentrations (Breyer et al., 2001).
Figure 3.
Figure 3.
Immunocytochemical distribution of the EP2 receptor. A, Macroscopic distribution of EP2 receptor immunoreactivity at 100× magnification in a sagittal section showing hippocampus and overlying cerebral cortex immunostained with anti-EP2 antibody (brown) and counterstained with cresyl violet (stains nuclei blue). Note parenchymal distribution of EP2 receptors, with sparing of white matter tracts (VI and V: layers VI and V of cerebral cortex). B, A 400× magnification of the CA3 region of hippocampus shows abundant perinuclear (thin arrow) and dendritic (large arrow) staining in CA3 pyramidal neurons, and diffuse astrocytic staining. C, A 400× magnification of layer V pyramidal neurons in cerebral frontal cortex, demonstrating staining of neuronal perikarya and apical dendrites (thick arrow). D, A 400× magnification of EP2 staining in striatal neurons shows abundant staining in perinuclear distribution (thin arrows).
Figure 5.
Figure 5.
Deletion of the EP2 receptor results in marked increase in infarct volume. A, Macroscopic analysis of cerebral arterial vasculature does not show differences between genotypes. There are no differences in the circle of Willis and major cerebral arteries. B, Cortical perfusion during ischemia as estimated by laser Doppler flowmetry (%LDF) shows that the percentage of reduction in perfusion during ischemia and recovery of blood flow during reperfusion do not differ between genotypes. LDF measured over ischemic cortex indicates a rapid decrease in flow to 11.2% of baseline for wild-type and 11.4% for -/- mice after insertion of the filament. Flow is stable during ischemia and recovers to baseline after the filaments are withdrawn. C, The volume of infarcted brain tissue after 90 min of ischemia and 22.5 hr of reperfusion was measured and expressed as the percentage of corrected infarct in WT+/+ and EP2-/- male mice. Deletion of the EP2 receptor results in significantly larger stroke volumes in cerebral cortex, caudate-putamen (CP), and hemisphere (hemi). There is a 0.88-, 0.51-, and 0.91-fold increase in stroke volume in EP2-/- mice over wild-type for cerebral cortex, caudate-putamen, and hemisphere, respectively (*p < 0.001, 0.01, and 0.05, respectively). D, Percentage of corrected cortical infarct volume was measured over four coronal levels in wild-type and EP2-/- mice at 22.5 hr after MCAO and shows a significant increase in percentage of corrected cortical volume (p < 0.0005, 0.005, and 0.005 for slice number 2, 3, and 4, respectively). E, Representative TTC-stained coronal sections at the level of the striatum shows large infarct in EP2-/- mouse. F, End-ischemic CBF in WT (+/+) and EP2-/- (-/-) male mice. Mice were subjected to 90 min of MCAO, and CBF was measured at end-ischemia with [14C] iodoantipyrine autoradiography. rCBF measurements within the infarcted and noninfarcted hemispheres (infarcted hemi and noninfarcted hemi) are regionally quantified in cerebral cortex and striatum (FCx and PCx: frontal and parietal cortex, respectively; LSt and MSt: lateral and medial striatum, respectively) in WT and EP2-/- mice. No significant differences were seen in the level of rCBF reduction between genotypes (n = 4 WT; 5 EP2-/-) in the ischemic infarcted hemisphere, indicating an equivalence of vascular tone during MCAO. In the noninfarcted hemisphere, there is an increase in CBF in the EP2-/- as compared with WT; *p < 0.01. G, Brain volumes at incremental levels of absolute regional cerebral blood flow in EP2-/- versus wild-type male mice. Mice were subjected to 90 min of MCAO, and CBF was measured at end-ischemia with [14C] iodoantipyrine autoradiography. There were no differences between groups at any flow increment, suggesting that the loss of the EP2 receptor does not alter the distribution of tissue volume with low to near-zero perfusion during middle cerebral artery occlusion. H, Colorized autoradiograms from a pair of representative WT and EP2-/- brains are shown with sections taken at -2, -1, 0, +1, and +2 mm from the bregma showing changes in CBF at 90 min of MCAO.
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