Ethanol preconditioning protects against ischemia/reperfusion-induced brain damage: role of NADPH oxidase-derived ROS

Abstract

Ethanol preconditioning (EtOH-PC) refers to a phenomenon in which tissues are protected from the deleterious effects of ischemia/reperfusion (I/R) by prior ingestion of ethanol at low to moderate levels. In this study, we tested whether prior (24 h) administration of ethanol as a single bolus that produced a peak plasma concentration of 42-46 mg/dl in gerbils would offer protective effects against neuronal damage due to cerebral I/R. In addition, we also tested whether reactive oxygen species (ROS) derived from NADPH oxidase played a role as initiators of these putative protective effects. Groups of gerbils were administered either ethanol or the same volume of water by gavage 24 h before transient global cerebral ischemia induced by occlusion of both common carotid arteries for 5 min. In some experiments, apocynin, a specific inhibitor of NADPH oxidase, was administered (5 mg/kg body wt, i.p.) 10 min before ethanol administration. EtOH-PC ameliorated behavioral deficit induced by cerebral I/R and protected the brain against I/R-induced delayed neuronal death, neuronal and dendritic degeneration, oxidative DNA damage, and glial cell activation. These beneficial effects were attenuated by apocynin treatment coincident with ethanol administration. Ethanol ingestion was associated with translocation of the NADPH oxidase subunit p67(phox) from hippocampal cytosol fraction to membrane, increased NADPH oxidase activity in hippocampus within the first hour after gavage, and increased lipid peroxidation (4-hydroxy-2-nonenal) in plasma and hippocampus within the first 2 h after gavage. These effects were also inhibited by concomitant apocynin treatment. Our data are consistent with the hypothesis that antecedent ethanol ingestion at socially relevant levels induces neuroprotective effects in I/R by a mechanism that is triggered by ROS produced through NADPH oxidase. Our results further suggest the possibility that preconditioning with other pharmacological agents that induce a mild oxidative stress may have similar therapeutic value for suppressing stroke-mediated damage in brain.

Figure 1

Changes of plasma ethanol concentrations in gerbils after ethanol administration by gavage (n=6 for each time point). The volume (in µl) of 95% ethanol instilled in each animal was calculated as follows: [body weight (in g) × 0.6] + 0.3), and was mixed in 0.3 ml sterile distilled water just before administration as a single bolus by gavage. *Values that are statistically different from corresponding values obtained during the control period (before ethanol gavage) and 2 hrs after ethanol ingestion at p < 0.05.

Figure 2

Immunoreactivities (IR) of p47^phox and p67^phox in normal gerbil brain. In cerebral cortex, IR of p47^phox and p67^phox were observed in all layers, but were especially prominent in layer V (Panel A and E). In the hippocampus, p47^phox and p67^phox IR were prominently observed in the pyramidal cell layer of the CA1 (Panel B and F) and CA3 (Panel C and G) regions and in both the molecular and polymorphic layers of dentate gyrus (DG) (Panel D and F). Arrows highlight individual cells that are positive for p47^phox and p67^phox IR. (Magnification, 200x).

Figure 3

Moderate ethanol administration by gavage increased lipid peroxidation in plasma (Panel A) and hippocampus (Panel B), as assessed by changes in 4-hydroxy-2-nonenal (HNE) levels (n=6 for each time point and each sample type), and effects of apocynin on plasma (Panel C) and hippocampal (Panel D) lipid peroxidation after ethanol administration by gavage. Data depicted in Panels C and D illustrate the HNE levels in plasma and hippocampus in water control, ethanol ingestion alone (EtOH-PC), and i.p. injection of apocynin 10 min prior to ethanol gavage (Apo+EtOH-PC). * and # indicate values that were statistically different from corresponding values in control and EtOH-PC groups at p < 0.05, respectively.

Figure 4

Effects of apocynin on translocation of p67^phox (Panel A, B and C) and on NADPH oxidase activity in hippocampus (Panel D) after ethanol administration by gavage. Data in Panels A to C illustrate the effects of control, ethanol ingestion alone (EtOH-PC), and i.p. injection of apocynin 10 min prior to ethanol gavage (Apo+EtOH-PC) on p67^phox and gp91^phox expression in membrane and cytosolic fractions. Data in Panel D show NADPH oxidase activity in hippocampus. See details in Method for the assay protocols. Samples were collected 1h after administration of ethanol or distilled water vehicle. Western blotting results are expressed as fold of control for three independent experiments (Panel B and C). * and # indicate values that are statistically different from corresponding values in control and EtOH alone groups at p < 0.05, respectively.

Figure 5

Effects of ethanol preconditioning and apocynin pretreatment on the spontaneous locomotor activity after cerebral I/R. Data represent mean (± S.E.M.) distance traveled (in cm) (n = 7 – 9 gerbils/group). Gerbils were placed in an automated locomotor activity monitor 24 hrs after cerebral I/R and locomotor activity was measured for 30 min. Statistical analysis revealed a significant main effect of session time (p <0.001) and treatment group (p < 0.001), but the session time by treatment group interaction was not significant (p = 0.65).

Figure 6

Representative micrographs depicting I/R-induced delayed neuronal death (DND) (cresyl violet) and neuronal (Fluoro-Jade B) and dendritic degeneration (MAP-2) in the hippocampal CA1 subfield of gerbils subjected to sham operation (Panels A, E, and I, respectively), 5 min occlusion of the common carotid arteries (ischemia) followed by 4 days reperfusion (I/R) (Panels B, F, and J, respectively), EtOH-PC 24 hrs prior to I/R (EtOH-PC+I/R) (Panels C, G, and K, respectively), and apocynin treatment coincident with ethanol administration 24 hrs prior to I/R (Apo+EtOH-PC+I/R) (Panels D, H, and L, respectively). (Magnification, 200x).

Figure 7

Quantification of viable neurons (cresyl violet, Panel A) and neuronal (Fluoro-Jade B, Panel B) and dendritic (MAP-2, Panel 3) degeneration in the hippocampal CA1 region of gerbils subjected to sham, I/R alone, ethanol preconditioning 24 hrs prior to I/R (EtOH-PC+I/R), and apocynin treatment coincident with ethanol administration 24 hrs prior to I/R (Apo+EtOH-PC+I/R). *, ^#, and Δ = values statistically different from corresponding values in sham, I/R, and EtOH-PC+I/R groups, respectively, at p < 0.05.

Figure 8

Representative micrographs depicting I/R-induced activation of astrocytes (GFAP, Panel A), microglial cells (isolectin-B4, Panel B) and DNA oxidation (8-OHdG, Panel C) in the hippocampal CA1 subfield of gerbils subjected to sham (Panels A, E, and I, respectively), I/R (Panels B, F, and J, respectively), ethanol preconditioning 24 hrs prior to I/R (EtOH-PC+I/R) (Panels C, G, and K, respectively), and apocynin treatment coincident with ethanol administration prior to I/R (Apo+EtOH-PC+I/R) (Panels D, H, and L, respectively). (Magnification, 200x).

Figure 9

Quantification of astrocytic (GFAP, Panel A) and microglial activation (Isolectin-B4, Panel B) and DNA oxidation (8-OhdG, Panel C) in gerbils subjected to sham, 5 min occlusion of the common carotid arteries followed by 4 days of reperfusion (I/R), ethanol preconditioning 24 hrs prior to I/R (EtOH-PC+I/R), and apocynin treatment coincident with ETOH-PC 24 hrs prior to I/R (Apo+EtOH-PC+I/R). *, ^#, and Δ = values statistically different from corresponding values in sham, I/R, and EtOH-PC + I/R groups, respectively, at p < 0.05.