Neuroinflammation and Neurodegeneration in Adult Rat Brain from Binge Ethanol Exposure: Abrogation by Docosahexaenoic Acid

Evidence that brain edema and aquaporin-4 (AQP4) water channels have roles in experimental binge ethanol-induced neurodegeneration has stimulated interest in swelling/edema-linked neuroinflammatory pathways leading to oxidative stress. We report here that neurotoxic binge ethanol exposure produces comparable significant effects in vivo and in vitro on adult rat brain levels of AQP4 as well as neuroinflammation-linked enzymes: key phospholipase A2 (PLA2) family members and poly (ADP-ribose) polymerase-1 (PARP-1). In adult male rats, repetitive ethanol intoxication (3 gavages/d for 4 d, ∼ 9 g/kg/d, achieving blood ethanol levels ∼ 375 mg/dl; Majchrowicz model) significantly increased AQP4, Ca+2-dependent PLA2 GIVA (cPLA2), phospho-cPLA2 GIVA (p-cPLA2), secretory PLA2 GIIA (sPLA2) and PARP-1 in regions incurring extensive neurodegeneration in this model--hippocampus, entorhinal cortex, and olfactory bulb--but not in two regions typically lacking neurodamage, frontal cortex and cerebellum. Also, ethanol reduced hippocampal Ca+2-independent PLA2 GVIA (iPLA2) levels and increased brain oxidative stress footprints (4-hydroxynonenal-adducted proteins). For in vitro studies, organotypic cultures of rat hippocampal-entorhinocortical slices of adult age (∼ 60 d) were ethanol-binged (100 mM or ∼ 450 mg/dl) for 4 d, which augments AQP4 and causes neurodegeneration (Collins et al. 2013). Reproducing the in vivo results, cPLA2, p-cPLA2, sPLA2 and PARP-1 were significantly elevated while iPLA2 was decreased. Furthermore, supplementation with docosahexaenoic acid (DHA; 22:6n-3), known to quell AQP4 and neurodegeneration in ethanol-treated slices, blocked PARP-1 and PLA2 changes while counteracting endogenous DHA reduction and increases in oxidative stress footprints (3-nitrotyrosinated proteins). Notably, the PARP-1 inhibitor PJ-34 suppressed binge ethanol-dependent neurodegeneration, indicating PARP upstream involvement. The results with corresponding models support involvement of AQP4- and PLA2-associated neuroinflammatory pro-oxidative pathways in the neurodamage, with potential regulation by PARP-1 as well. Furthermore, DHA emerges as an effective inhibitor of these binge ethanol-dependent neuroinflammatory pathways as well as associated neurodegeneration in adult-age brain

Effects of binge ethanol exposure on oxidative stress (4HNE-) protein footprint levels <i>in vivo</i>.

<p>Significant increases over respective controls (Cont) in levels of oxidative stress footprint, 4-hydroxynonenal (4HNE)-adducted proteins, in hippocampus (HC) and entorhinal cortex (EC) following neurotoxic binge ethanol treatment of adult male rats for 4 days (Majchrowicz model). Top: representative immunoblots of 4HNE-adducted proteins in HC and EC. Bottom: quantitation of immunoblots of 4HNE-adducted proteins in HC and EC. *p<0.01 vs. control (Cont); n = 4–7 rats per group.</p

Effect of PARP-1 inhibitor PJ-34 on ethanol-induced neurodegeneration in adult-age HEC slice cultures.

<p>Neurodegeneration (PI staining) due to 4 days of binge ethanol treatment (100 mM) of rat adult-age organotypic HEC slice cultures is suppressed by co-treatment with PARP-1 inhibitor, PJ-34 (10 µM). (Top) Images of representative PI-stained slices from C, PJ-34, E and E+PJ-34 slice cultures indicate increased neurodegeneration (E) that is reduced in E+PJ-34. (Bottom) Quantitation of PI staining demonstrates increased neurodegeneration due to binge ethanol (E) that is significantly suppressed by PJ-34 (E + PJ-34). **p<0.01 vs. C. ##p<0.01 vs. E.</p

Selective effects of binge ethanol exposure on brain AQP4 and PARP-1 levels <i>in vivo</i>.

<p>Significant elevations over respective controls (C) in levels of AQP4 and PARP-1 in those brain regions selectively incurring neurodamage due to binge ethanol (E) treatment in adult male rats for 4 days (Majchrowicz model). <u>Fig. 3A</u>: Significantly increased AQP4 levels in the HC, EC and OB, but not in CB and FC. <u>Fig. 3B</u>: Significantly increased PARP-1 levels in the HC, EC and OB, but not in CB and FC. *p<0.05 vs. C. n = 4–7 rats/group.</p

Selective effects of binge ethanol exposure on brain PLA2 levels <i>in vivo</i>.

<p>Significant alterations compared to respective controls (C) in protein expression (immunoblots) of PLA2 enzymes in brain regions selectively incurring neurodamage due to binge ethanol (E) treatment in adult male rats for 4 days (Majchrowicz model). <u>Fig. 2A</u>: Significantly increased levels of cPLA2 (top) and p-cPLA2 (bottom) in the hippocampus (HC), entorhinal cortex (EC) and olfactory bulb (OB), but not in cerebellum (CB) and frontal cortex (FC). <u>Fig. 2B</u>: Significantly increased sPLA2 levels in the HC, EC and OB, but not in CB and FC. <u>Fig. 2C</u>. Significantly decreased levels of iPLA2 in the HC. *p<0.05 vs. C. **p<0.01 vs. C. n = 4–7 rats/group.</p

Effects of binge ethanol treatment and DHA supplementation on PLA2 and PARP-1 levels in adult-age HEC slice cultures.

<p>Significant neuroinflammatory enzyme alterations compared to control (C) caused by 4 days of binge ethanol (E) treatment (100 mM) of adult-age rat HEC slice cultures, and prevention of the alterations by supplementation of cultures with DHA (25 µM). <u>Fig. 5A</u>: Increased levels of cPLA2 GIVA over control levels due to binge E and inhibition of the increase by DHA supplementation (E+ DHA). *p<0.05 vs. C; #p<0.05 vs. E. <u>Fig. 5B</u>: Increased levels of p-cPLA2 GIVA over control levels due to binge E and inhibition of the increase by DHA supplementation (E+DHA). *p<0.05 vs. C; #p<0.05 vs. E. <u>Fig. 5C</u>: Increased levels of sPLA2 GIIA over control levels due to binge E and inhibition of the increase by DHA supplementation (E+DHA). *p<0.05 vs. C; #p<0.05 vs. E. <u>Fig. 5D</u>: Reduced levels of iPLA2 GVIA with respect to control levels due to binge E and partial blockade of the reduction by DHA supplementation (E+DHA). *p<0.05 vs. C. <u>Fig. 5E</u>: Increased levels of PARP-1 over control levels due to binge E and inhibition of the increase by DHA supplementation (E+DHA). *p<0.05 vs. C; #p<0.05 vs. E.</p