Moderate blood alcohol and brain neurovulnerability: Selective depletion of calcium-independent phospholipase A2, omega-3 docosahexaenoic acid, and its synaptamide derivative as a potential harbinger of deficits in anti-inflammatory reserve.

BACKGROUND: Repetitive, highly elevated blood alcohol (ethanol) concentrations (BACs) of 350 to 450 mg/dl over several days cause brain neurodegeneration and coincident neuroinflammation in adult rats localized in the hippocampus (HC), temporal cortex (especially the entorhinal cortex; ECX), and olfactory bulb (OB). The profuse neuroinflammation involves microgliosis, increased proinflammatory cytokines, and elevations of Ca+2 -dependent phospholipase A2 (cPLA2) and secretory PLA2 (sPLA2), which both mobilize proinflammatory ω-6 arachidonic acid (ARA). In contrast, Ca+2 -independent PLA2 (iPLA2) and anti-inflammatory ω-3 docosahexaenoic acid (DHA), a polyunsaturated fatty acid regulated primarily by iPLA2, are diminished. Furthermore, supplemented DHA exerts neuroprotection. Given uncertainties about the possible effects of lower circulating BACs that are common occurring during short- term binges, we examined how moderate BACs affected the above inflammatory events, and the impact of supplemented DHA. METHODS AND RESULTS: Young adult male rats sustaining upper-moderate BACs (~150 mg/dl) from once-daily alcohol intubations were sacrificed with appropriate controls after 1 week. The HC, ECX and OB were quantitatively examined using immunoblotting, neurodegeneration staining, and lipidomics assays. Whereas neurodegeneration, increases in cPLA2 IVA, sPLA2 IIA, and ARA, and microglial activation were not detected, the HC and ECX regions demonstrated significantly reduced iPLA2 levels. Levels of DHA and synaptamide, its anti-inflammatory N-docosahexaenoylethanolamide derivative, also were lower in HC, and DHA supplementation prevented the iPLA2 decrements in HC. Additionally, adult mice maintaining upper-moderate BACs from limited alcohol binges had reduced midbrain iPLA2 levels. CONCLUSIONS: The apparently selective depletion by moderate BACs of the metabolically linked anti-inflammatory triad of hippocampal iPLA2, DHA, and synaptamide, and of iPLA2 in the ECX, potentially indicates an unappreciated deficit in brain anti-inflammatory reserve that may be a harbinger of regional neurovulnerability.

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.

Alcohol, phospholipase A2-associated neuroinflammation, and ω3 docosahexaenoic acid protection.

Chronic alcohol (ethanol) abuse causes neuroinflammation and brain damage that can give rise to alcoholic dementia. Insightfully, Dr. Albert Sun was an early proponent of oxidative stress as a key factor in alcoholism-related brain deterioration. In fact, oxidative stress has proven to be critical to the hippocampal and temporal cortical neurodamage resulting from repetitive "binge" alcohol exposure in adult rat models. Although the underlying mechanisms are uncertain, our immunoelectrophoretic and related assays in binge alcohol experiments in vivo (adult male rats) and in vitro (rat organotypic hippocampal-entorhinal cortical slice cultures) have implicated phospholipase A(2) (PLA(2))-activated neuroinflammatory pathways, release of pro-oxidative arachidonic acid (20:4 ω6), and elevated oxidative stress adducts (i.e., 4-hydroxynonenal-protein adducts). Also, significantly increased by the binge alcohol treatments was aquaporin-4 (AQP4), a water channel enriched in astrocytes that, when augmented, may trigger brain (esp. cellular) edema and neuroinflammation; of relevance, glial swelling is known to provoke increased PLA(2) activities or levels. Concomitant with PLA(2) activation, the results have further implicated binge alcohol-elevated poly (ADP-ribose) polymerase-1 (PARP-1), an oxidative stress-responsive DNA repair enzyme linked to parthanatos, a necrotic-like neuronal death process. Importantly, supplementation of the brain slice cultures with docosahexaenoic acid (22:6 ω3) exerted potent suppression of the induced changes in PLA(2) isoforms, AQP4, PARP-1 and oxidative stress footprints, and prevention of the binge alcohol neurotoxicity, by as yet unknown mechanisms. These neuroinflammatory findings from our binge alcohol studies and supportive rat binge studies in the literature are reviewed.

Docosahexaenoic acid (DHA) prevents binge ethanol-dependent aquaporin-4 elevations while inhibiting neurodegeneration: experiments in rat adult-age entorhino-hippocampal slice cultures.

Repetitive binge intoxication with ethanol (alcohol) in adult rats, mimicking chronic ethanol abuse in alcoholics, causes trauma-like brain edema and relatively selective neurodegeneration of hippocampal dentate granule cells and pyramidal neurons in the temporal cortex (especially entorhinal cortex). We have now modeled the aspects of this type of acquired brain damage in vitro with rat entorhino-hippocampal slice cultures of adult brain age (62 ± 3 days). When sequentially treated (four 16-h overnight exposures) with 100 mM ethanol, the slices display elevated levels of aquaporin-4 (AQP4) water channels accompanied by significant neurodegeneration. Increased AQP4 has been associated with neuroinflammatory responses including edema, pro-inflammatory cytokine elevations, arachidonic acid release, and oxidative stress. Co-treatment of ethanol-binged slice cultures with docosahexaenoic acid (DHA), an omega-3 fatty acid known to suppress brain damage from other insults, prevents both the AQP4 elevations and the neurodamage. Surmising that AQP4 augmentation is a causative neuroinflammatory component in this model, we are investigating several possibilities to explain the protective actions of the omega-3 fatty acid. Since the worldwide incidence of cognitive dysfunction and dementia from ethanol abuse and alcoholism is not inconsequential, DHA supplementation with chronic alcoholics could emerge to be a rational approach to potentially lessening brain disabilities.

Binge ethanol-induced neurodegeneration in rat organotypic brain slice cultures: effects of PLA2 inhibitor mepacrine and docosahexaenoic acid (DHA).

Using rat organotypic hippocampal-entorhinal cortical (HEC) slice cultures, we examined whether phospholipase A2 (PLA2) activity is involved in binge alcohol (ethanol)-induced neurodegeneration, and whether docosahexaenoic acid (DHA; 22:6n-3), a fish oil-enriched fatty acid that is anti-inflammatory in brain damage models, is neuroprotective. Assessed with propidium iodide and lactate dehydrogenase (LDH) leakage, neurodamage from ethanol (6 days 100 mM ethanol with four withdrawal periods) was prevented by the PLA2 pan-inhibitor, mepacrine. Also, ethanol-dependent neurodegeneration-particularly in the entorhinal region-was significantly ameliorated by DHA supplementation (25 microM); however, adrenic acid, a 22:4n-6 analog, was ineffective. Consistent with PLA2 activation, [(3)H] liberation was approximately fivefold greater in [(3)H]arachidonic acid-preloaded HEC slice cultures during ethanol withdrawal compared to controls, and DHA supplementation suppressed [(3)H] release to control levels. DHA might antagonize PLA2 activity directly or suppress upstream activators (e.g., oxidative stress); however, other DHA mechanisms could be important in subdueing ethanol-induced PLA2-dependent and independent neuroinflammatory processes.