Protective Effect of Low-Dose Alcohol Consumption against Post-Ischemic Neuronal Apoptosis: Role of L-PGDS

Abstract

Ischemic stroke is one of the leading causes of permanent disability and death in adults worldwide. Apoptosis is a major element contributing to post-ischemic neuronal death. We previously found that low-dose alcohol consumption (LAC) protects against neuronal apoptosis in the peri-infarct cortex following transient focal cerebral ischemia. Lipocalin-type prostaglandin D2 synthase (L-PGDS), which is mainly localized in the central nervous system (CNS), was previously shown to inhibit neuronal apoptosis. Therefore, we determined whether L-PGDS is involved in the protective effect of LAC against post-ischemic neuronal apoptosis. Wild-type (WT), CaMKIIα^CreERT2/+/L-PGDS^+/+, and CaMKIIα^CreERT2/+/L-PGDS^flox/flox mice on a C57BL/6J background were gavage fed with ethanol or volume-matched water once a day for 8 weeks. Tamoxifen (2 mg/day) was given intraperitoneally to CaMKIIα^CreERT2/+/L-PGDS^+/+ and CaMKIIα^CreERT2/+/L-PGDS^flox/flox mice for 5 days during the fourth week. AT-56 (30 mg/kg/day), a selective inhibitor of L-PGDS, was given orally to AT-56-treated WT mice from the fifth week for four weeks. Cerebral ischemia/reperfusion (I/R) injury, TUNEL-positive neurons, and cleaved caspase-3-positive neurons were measured at 24 h of reperfusion after a 90 min unilateral middle cerebral artery occlusion (MCAO). We found that 0.7 g/kg/day but not 2.8 g/kg/day ethanol significantly upregulated L-PGDS in the cerebral cortex. In addition, 0.7 g/kg/day ethanol diminished cerebral ischemia/reperfusion (I/R) injury and TUNEL-positive and cleaved caspase-3-positive neurons in the peri-infarct cortex in WT and CaMKIIα^CreERT2/+/L-PGDS^+/+ mice. Furthermore, the neuroprotective effect of 0.7 g/kg/day ethanol was alleviated in AT-56-treated WT and CaMKIIα^CreERT2/+/L-PGDS^flox/flox mice. Our findings suggest that LAC may protect against cerebral I/R injury by suppressing post-ischemic neuronal apoptosis via an upregulated L-PGDS.

Keywords: L-PGDS; apoptosis; brain; ethanol; ischemic stroke.

Figure 1

Effect of chronic ethanol consumption on protein expression of L-PGDS in the cerebral cortex. (A) Representative Western blots. (B) Values are means ± SEM for 4–5 mice in each group. * p < 0.05. Analyzed using one-way ANOVA with Dunnett’s post hoc test.

Figure 2

Effect of AT-56 on cerebral I/R injury. (A) Representative brain sections stained with cresyl violet. (B) Total infarct volume. (C) Neurological deficit score. Values are means ± SEM for 4 mice in each group. * p < 0.05. ** p < 0.005. Analyzed using two-way ANOVA followed by Tukey’s test.

Figure 2

Effect of AT-56 on cerebral I/R injury. (A) Representative brain sections stained with cresyl violet. (B) Total infarct volume. (C) Neurological deficit score. Values are means ± SEM for 4 mice in each group. * p < 0.05. ** p < 0.005. Analyzed using two-way ANOVA followed by Tukey’s test.

Figure 3

Effect of forebrain neuron-specific L-PGDS knockdown on cerebral I/R injury. (A) PCR-based genotyping. The homozygous (fl/fl) mice with Cre positive were considered to be forebrain neuron-specific L-PGDS knockdown mice following tamoxifen treatment. (B) Western blots of L-PGDS in the cerebral cortex of tamoxifen-treated CaMKIIα^CreERT2/+/L-PGDS^+/+ and CaMKIIα^CreERT2/+/L-PGDS^flox/flox mice. (C) Representative brain sections stained with cresyl violet. (D) Total infarct volume. (E) Neurological deficit score. Values are means ± SEM for 5–6 mice in each group. * p < 0.05. ** p < 0.005. Analyzed using two-way ANOVA followed by Tukey’s test.

Figure 4

Effect of AT-56 on TUNEL-positive neurons in the cerebral cortex following transient focal cerebral ischemia. (A) Representative double staining of NeuN and TUNEL in the peri-infarct cortex of the temporal lobe at the section 0.23 mm caudal to bregma. Scale bar = 20 μm. (B) Values of TUNEL-positive neurons are means ± SEM for 4 mice in each group. * p < 0.05. Analyzed using two-way ANOVA followed by Tukey’s test.

Figure 5

Effect of forebrain neuron-specific L-PGDS knockdown on TUNEL-positive neurons in the cerebral cortex following transient focal cerebral ischemia. (A) Representative double staining of NeuN and TUNEL in the peri-infarct cortex of the temporal lobe at the section 0.23 mm caudal to bregma. Scale bar = 20 μm. (B) Values of TUNEL-positive neurons are means ± SEM for 5–6 mice in each group. * p < 0.05. Analyzed using two-way ANOVA followed by Tukey’s test.

Figure 6

Effect of AT-56 on cleaved caspase-3-positive neurons in the cerebral cortex following transient focal cerebral ischemia. (A) Representative double staining of NeuN and cleaved caspase-3 in the peri-infarct cortex of the parietal lobe at the section 0.23 mm caudal to bregma. Scale bar = 100 μm. (B) Values of cleaved caspase-3-positive neurons are means ± SEM for 4 mice in each group. * p < 0.05. Analyzed using two-way ANOVA followed by Tukey’s test.

Figure 7

Effect of forebrain neuron-specific L-PGDS knockdown on the cleaved caspase-3-positive neuron in the cerebral cortex following transient focal cerebral ischemia. (A) Representative double staining of NeuN and TUNEL in the peri-infarct cortex of the parietal lobe at the section 0.23 mm caudal to bregma. Scale bar = 100 μm. (B) Values of cleaved caspase-3-positive neurons are means ± SEM for 5–6 mice in each group. * p < 0.05. Analyzed using two-way ANOVA followed by Tukey’s test.