Linking binge alcohol-induced neurodamage to brain edema and potential aquaporin-4 upregulation: evidence in rat organotypic brain slice cultures and in vivo.
ABSTRACT Brain edema and derived oxidative stress potentially are critical events in the hippocampal-entorhinal cortical (HEC) neurodegeneration caused by binge alcohol (ethanol) intoxication and withdrawal in adult rats. Edema's role is based on findings that furosemide diuretic antagonizes binge alcohol-dependent brain overhydration and neurodamage in vivo and in rat organotypic HEC slice cultures. However, evidence that furosemide has significant antioxidant potential and knowledge that alcohol can cause oxidative stress through non-edemic pathways has placed edema's role in question. We therefore studied three other diuretics and a related non-diuretic that, according to our oxygen radical antioxidant capacity (ORAC) assays or the literature, possess minimal antioxidant potential. Acetazolamide (ATZ), a carbonic anhydrase inhibitor/diuretic with negligible ORAC effectiveness and, interestingly, an aquaporin-4 (AQP4) water channel inhibitor, prevented alcohol-dependent tissue edema and neurodegeneration in HEC slice cultures. Likewise, in binge alcohol-intoxicated rats, ATZ suppressed brain edema while inhibiting neurodegeneration. Torasemide, a loop diuretic lacking furosemide's ORAC capability, also prevented alcohol-induced neurodamage in HEC slice cultures. However, bumetanide (BUM), a diuretic blocker of Na(+)-K(+)-2Cl(-) channels, and L-644, 711, a nondiuretic anion channel inhibitor--both lacking antioxidant capabilities as well as reportedly ineffective against alcohol-dependent brain damage in vivo--reduced neither alcohol-induced neurotoxicity nor (with BUM) edema in HEC slices. Because an AQP4 blocker (ATZ) was neuroprotective, AQP4 expression in the HEC slices was examined and found to be elevated by binge alcohol. The results further indicate that binge ethanol-induced brain edema/swelling, potentially associated with AQP4 upregulation, may be important in consequent neurodegeneration that could derive from neuroinflammatory processes, for example, membrane arachidonic acid mobilization and associated oxidative stress.
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ABSTRACT: 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.PLoS ONE 07/2014; 9(7):e101223. · 3.53 Impact Factor
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ABSTRACT: 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 A2 (PLA2)-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 PLA2 activities or levels. Concomitant with PLA2 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 PLA2 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.Molecular Neurobiology 04/2014; · 5.29 Impact Factor
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ABSTRACT: Alcohol abuse can result in significant alterations to the structure of the brain and ultimately to behavioral dysfunctions. Epidemiological studies have shown that alcoholism is closely associated with impaired memory and judgment. However, the degree of deficit (brain injury) depends on factors such as the age of onset, duration of heavy drinking, continuous versus periodic (binge) drinking and the typical amount consumed per session. In recent years, neuroinflammation has been proposed as one of the alcoholism-induced neuropathological mechanisms, since increased levels of microglial markers are observed in the brains of both post-mortem human alcoholics and various alcohol-treated animals, from new-born or adolescent rodents to adult rodents. Many studies have investigated how microglia modulate alcohol-induced behavioral changes such as cognitive deficits, abnormal locomotor activity, motor impairment and mood disturbance. Importantly, we try to characterize and compare the distinct features in different ethanol (EtOH)-induced neurodegenerative disease (NDD) models. Moreover, mounting evidence indicates that in response to certain environmental toxins, microglia can become over-activated under oxidative stress, releasing pro-inflammatory mediators that cause central nervous system (CNS) disease. The molecular mechanisms involve free radical formation and the release of pro-inflammatory cytokines that are detrimental to neighboring neurons and interfere with the molecules regulating cell-cell interactions. The identification and understanding of the cellular and molecular mechanisms of microglial activation are described, as well as multiple downstream targets, in different alcohol-treated animal models. This review might contribute to the development of treatments and/or therapeutic agents that can reduce or eliminate the deleterious effects of alcohol-induced NDD.Pharmacology [?] Therapeutics 07/2014; · 7.75 Impact Factor
Linking Binge Alcohol-Induced Neurodamage to Brain Edema
and Potential Aquaporin-4 Upregulation: Evidence
in Rat Organotypic Brain Slice Cultures and In Vivo
Kumar Sripathirathan,1James Brown III,2Edward J. Neafsey,2and Michael A. Collins2
Brain edema and derived oxidative stress potentially are critical events in the hippocampal-entorhinal cortical
(HEC) neurodegeneration caused by binge alcohol (ethanol) intoxication and withdrawal in adult rats. Edema’s
role is based on findings that furosemide diuretic antagonizes binge alcohol–dependent brain overhydration and
neurodamage in vivo and in rat organotypic HEC slice cultures. However, evidence that furosemide has signif-
icant antioxidant potential and knowledge that alcohol can cause oxidative stress through non-edemic pathways
has placed edema’s role in question. We therefore studied three other diuretics and a related non-diuretic that,
according to our oxygen radical antioxidant capacity (ORAC) assays or the literature, possess minimal antioxi-
dant potential. Acetazolamide (ATZ), a carbonic anhydrase inhibitor=diuretic with negligible ORAC effectiveness
and, interestingly, an aquaporin-4 (AQP4) water channel inhibitor, prevented alcohol-dependent tissue edema
and neurodegeneration in HEC slice cultures. Likewise, in binge alcohol–intoxicated rats, ATZ suppressed brain
edema while inhibiting neurodegeneration. Torasemide, a loop diuretic lacking furosemide’s ORAC capability,
also prevented alcohol-induced neurodamage in HEC slice cultures. However, bumetanide (BUM), a diuretic
blocker of Na+-K+-2Cl?channels, and L-644, 711, a nondiuretic anion channel inhibitor—both lacking antioxidant
capabilities as well as reportedly ineffective against alcohol-dependent brain damage in vivo—reduced neither
alcohol-induced neurotoxicity nor (with BUM) edema in HEC slices. Because an AQP4 blocker (ATZ) was
neuroprotective, AQP4 expression in the HEC slices was examined and found to be elevated by binge alcohol. The
results further indicate that binge ethanol-induced brain edema=swelling, potentially associated with AQP4
upregulation, may be important in consequent neurodegeneration that could derive from neuroinflammatory
processes, for example, membrane arachidonic acid mobilization and associated oxidative stress.
Key words: acetazolamide; brain damage; diuretic; ethanol; furosemide; neurotoxicity
alcohol’s neurocellular and neurovascular effects; in some
cases, deficiencies of thiamine and other micronutrients may
with animal models, the specific mechanisms underlying
unclear. One reason may be that, depending on the exposure
model used and perhaps age and even gender, one mecha-
nism might predominate over others and hence could involve
different brain regions, cells, and signal transduction path-
hronic alcohol (ethanol) abuse causes brain neuro-
degeneration, which is believed to result primarily from
ways. Two general in vivo paradigms have been used in adult
rodents to simulate human alcoholic central nervous system
(CNS) damage: (a) continuous alcohol intake, usually in liq-
uid diets but sometimes in water, for periods of months or
more to produce a low-to-moderate blood alcohol concen-
tration (BAC) (Walker et al., 1980; Riikonen et al., 1999), and
(b) binge alcohol treatment, typically via gastric intubation
over a subchronic period (4–10 days, but sometimes longer), a
trauma-like paradigm that generates episodically high BACs
et al., 2000). In addition, a variety of different in vitro (culture)
models have been used to study neurodegeneration due to
either sustained chronic or binge alcohol exposure.
1CoMentis Inc., Oklahoma City, Oklahoma.
2Department of Cell Biology, Neurobiology & Anatomy, Loyola University Stritch School of Medicine, Maywood, Illinois.
JOURNAL OF NEUROTRAUMA 26:261–273 (February 2009)
ª Mary Ann Liebert, Inc.
Perhaps the most widely disseminated hypothetical mech-
based on synaptic excitatory glutamate receptors and ele-
vated intraneuronal Ca2þ(Lovinger, 1993; Tsai and Coyle,
1998). However, although chronic alcohol exposure has been
shown to increase expression of brain ionotropic glutamate
receptors and Ca2þchannels (Hoffman, 2003), and pharma-
cological results in developing brain cultures indicate a role
for the NMDA receptor (NMDAR) in alcohol withdrawal-
dependent neurotoxicity (Prendergast et al., 2004), pharma-
alcohol-induced neurodegeneration in vivo have failed. Ex-
periments with binge-intoxicated adult rats using NMDAR
and Ca2þchannel antagonists have not supported a gluta-
matergic receptor-mediated mechanism (Zou et al., 1996;
Collins et al., 1998; Corso et al., 1998), and the lack of effect of
NMDAR inhibitors was recently confirmed by others (Hame-
link et al., 2005). Spurred by indications that alcohol can in-
duce cellular edema in astroglial and hypothalamic cultures
(Sato et al., 1991; Snyder, 1996; Aschner et al., 2001), we con-
sidered the possible role of brain (particularly astroglial)
edema in the binge alcohol models. Indeed, brain edema is
implicated clinically in the neurodamaging sequelae of trau-
ma, status epilepticus, stroke, and hepatic failure (Lassmann
et al., 1984; Unterberg et al., 2004; Heo et al., 2005; Albrecht
and Norenberg, 2006) and has been postulated to be impor-
tant in alcohol abuse (Lambie, 1985). We found that brains of
adult rats binge-intoxicated daily with alcohol for *1 week
furosemide blocked the edema while significantly reducing
entorhinal cortical and hippocampal dentate neurodegen-
eration. Furosemide also suppressed binge alcohol–induced
cytotoxicity in organotypic hippocampal-entorhinal cortical
(HEC) slice cultures (Collins et al., 1998).
Based on these results, we proposed that brain edema has a
causative role in the brain neurotoxicity engendered by re-
petitive (binge) intoxication combined with withdrawal, via
its promotion of neuroinflammatory-related oxidative stress,
possibly accompanied in vivo by pressure necrosis. However,
brain edema’s essentiality as precursor for oxidative stress
from binge alcohol exposure has been questioned (Hamelink
et al., 2005), since—as antioxidants can neuroprotect in binge
alcohol–intoxicated rats—furosemide was determined to be a
potent antioxidant. To further study edema’s role in alcohol-
dependent brain damage, we first compared the antioxidant
capabilities of furosemide and selected other diuretics to the
vitamin E analog, Trolox, using the well-known oxygen rad-
ical absorbance capacity (ORAC) assay. We then examined
the extent of neurodegeneration and brain tissue edema in
binge alcohol–treated rat HEC slice cultures using those
diuretics and a related compound that have negligible anti-
oxidant capabilities. The compounds studied were acetazol-
amide (ATZ), a carbonic anhydrase inhibitor; torasemide, a
pyridine-sulfonylurea loop diuretic resembling furosemide
but with potassium-sparing ability; bumetanide (BUM), an-
other loop-type diuretic; and L-644, 711, a nondiuretic anion
channel inhibitor. Neurodegeneration in HEC slices was de-
termined with propidium iodide (PI) staining and=or lactate
dehydrogenase (LDH) release. Also, since ATZ potently in-
hibits aquaporin-4 (AQP4) (Huber et al., 2007), the effect of
binge alcohol on expression of this principal brain water
channel in the HEC slices was examined. ATZ actions were
further tested in binge alcohol–intoxicated adult rats; similar
to our published experiments with furosemide, edema was
determined in fresh brain portions and entorhinal cortical=
dentate gyrus neurodegeneration was assessed in fixed, cu-
pric silver–stained brain sections (Collins et al., 1996; Corso
et al., 1998). Overall, the findings were consistent with the
development of binge alcohol–induced neurodegeneration
being linked to brain edema, possibly involving AQP4 upre-
gulation. As such, the neurodamage could ultimately result
from oxidative stress arising in part from pro-inflammatory,
edema-dependent processes such as phospholipase 2 (PLA2)
activation and arachidonic acid (AA) mobilization (Crews
et al., 2004; Brown et al., 2008).
Furosemide, b-phycoerythrin (ß-PE), and dimethyl sulfox-
ide (DMSO) were obtained from Sigma Chemical (St. Louis,
MO). Torasemide was obtained from the Loyola University
Hospital Pharmacy, 2,20-Azobis (2-amidinopropane) dihy-
drochloride (AAPH) was purchased from Polyscience Co.
(Warrington, PA), and Trolox (6-hydroxy-2,5,7,8-tetrameth-
ylchroman-2-carboxylic acid) was obtained from Aldrich
Chemical Co. (Milwaukee, WI). Modified Eagles’ medium
(MEM) media, Hanks’ buffer, and horse serum were obtained
from Gibco (Gaithersburg, MD). Tissue culture inserts and
plasticware were from Fisher Scientific (Pittsburgh, PA).
Oxygen radical antioxidant capacity assay
Antioxidant activities of diuretics were determined with
the oxygen radical antioxidant capacity (ORAC) assay (Cao
et al., 1993), using ß-PE as indicator protein, AAPH as a per-
oxyl radical generator, and Trolox, a water-soluble vitamin E
analog, as a standard. The reaction mixture, prepared fresh in
triply-distilled deionized water, contained 1.67?10?8M ß-PE
and 3?10?3M AAPH in 7.5?10?2M phosphate buffer, pH
7.0, in a final volume of 2ml. Into each sample tube, 20ml of
blank (1% dimethylsulfoxide [DMSO] or water) or the di-
uretics (1, 2, and 4mM) were added in 1% DMSO. After ad-
well, the loss of fluorescence was measured every 5min until
zerofluorescence occurred, usingaPerkin-Elmer fluorescence
spectrophotometer at 565-nm emission and 540-nm excita-
tion. Trolox was assayed during each run, and the ORAC
value (U=ml) of the sample was calculated using the Trolox
standard curve. One ORAC unit has been assigned the net
protection area (S) provided by 1mM Trolox final concentra-
tion. The ORAC value was calculated as (Ssample-Blank)=
(STrolox- SBlank), where S is area under the quenching curve.
cortical slice cultures
Adult rats and rat pups were acquired and cared for in
accordance withthe guidelines publishedin the NIH Guidefor
the Care and Use of Laboratory Animals (National Institutes of
Health publication no. 85-23) and the principles presented in
the "Guidelines for the Use of Animals in Neuroscience Re-
search" by the Society for Neuroscience. All protocols have
been approved by Loyola University Institutional Animal
Care and Use Committee. Sprague-Dawley rat pups (7 days
262SRIPATHIRATHAN ET AL.
old; maternal source, Zivic-Miller, Portersville, PA)wereused
to prepare organotypic HEC slice cultures (Stoppini et al.,
1991; Collins et al., 1998). Pups were cold-anesthetized and
then were decapitated. The HEC was removed and trans-
verse slices (350m) were placed on Millipore 0.4-m Millicel tis-
sue culture inserts in six-well Falcon plates with covers; they
were culturedinanatmosphere of95%O2=5%CO2at378C on
MEM media containing 25% horse serum, 25% Hanks buffer,
20mM HEPES, and 6.5mg=ml glucose. Each treatment group
contained six wells containing three to four slices per well.
The media waschanged every 3 days. Slices wereperiodically
examined, and those appearing unhealthy under the phase
contrast microscope (darkened appearance) were discarded.
After 14–16 days of culture and maturation, plates with
HEC slices were treated with alcohol, and=or vehicle (0.1–1%
DMSO)?diuretic at the indicated concentrations over a pe-
riod of 6 successive days, depending on the experiment. They
were kept in the incubator in separate closed Tupperware
brand containers; alcohol culture containers also had a small
open dish of 1.8% v=v alcohol=water to maintain alcohol
levels in culture wells, and controls had a similar dish with
water only. One of two alcohol treatment modifications was
used: slices were exposed daily to alcohol in culture media
(initial concentration, 100mM, but 150mM for torasemide
and L-644, 711 experiments) for 15h (incubation), and then all
transferred to alcohol-free media for 9h (withdrawal), re-
sulting in six withdrawal episodes; alternatively, slices were
given one extended 3-dayalcohol orcontrol media incubation
period, followed by the first 9-h withdrawal, and then three
15-h incubation days with three more withdrawals, for a total
of four withdrawal episodes. Media alcohol concentrations,
which dropped moderately (15–20%, unpublished data)
during incubation periods, were re-established by addition of
alcohol at each incubation period. In either modification,
complete media changes were done after 3 days. When used,
ATZ, torasemide, BUM, or L-644, 711, dissolved in di-
methylsulfoxide and diluted appropriately into media, were
present in both incubation and withdrawal mediathroughout
the 6 days; control slices received changes with media only,
the last three withdrawal periods were analyzed for LDH
with a Sigma diagnostic kit. When required, tissue protein
was determined in centrifuged extracts of slice homogenates
with a bicinchoninic acid (BCA)–based method (Pierce Bio-
technology, Rockford, IL).
Detection of neurodegeneration in HEC slices
with propidium iodide
Degenerating neurons were detected in HEC slices with PI
(5mg=ml media), added during the final hour of the last alco-
hol withdrawal period. PI uptake in each slice was assessed
by capturing 8–12-sec exposures with a DS-5M Nikon color
an Epi-fluorescence attachment. Using Image J version 1.36
(NIH, Bethesda, MD), the entire slice was outlined, and the
percentage of the entire slice area that fluoresced red was
recorded and used for overall statistical analysis.
Western blot analysis of aquaporin 4
Following 4 days of binge alcohol treatment, HEC slices
by pipette action in radioimmunoprecipation assay buffer
(50mM Tris-HCl, 150mM NaCl, 1% NP-40, 0.5% sodium
deoxycholate, 2mM NaF, 2mM EDTA, 0.1% SDS, and pro-
tease inhibitor cocktail), centrifuged, and protein content de-
termined by the BCA method. Samples of equal protein
content (10–15mg) were resolved by 10% SDS-PAGE, trans-
ferred to Millipore Immobilon-P membranes, and blotted
with AQP4 primary antibody (Genetex, San Antonio, TX) at a
dilution of1:1000.Peroxidase-conjugated secondary antibody
was used at a dilution of 1:3000. Bound antibodies were vi-
sualized using an ECL detection kit (Pierce Biotechnology)
and exposed to Kodak X-Omat film (Kodak, Rochester, NY).
Binge alcohol intoxication experiments
Adult male Sprague-Dawley rats, 280–320g, obtained
from Zivic-Miller Laboratories (Portersville, PA), were accli-
matized in the animal facilities. Water and lab chow were
provided ad libitum, and the animals were maintained in a
temperature-controlled (258C), light-controlled (12:12h), and
humidity-controlled (50–55%) room. Rats were divided into
four experimental groups (8–10rats=group): vehicle (50:50
water=Vanilla Ensure, 355cal=8 fl oz; Ross Laboratories), al-
cohol, ATZ alone (10mg=kg=day in 1% DMSO=H2O), and
alcoholþATZ. They were intubated intragastrically with a
single morning dose of 25–35% alcohol (w=v) in vehicle, or
depending on intoxication state and BAC, in a procedure
extensively detailed previously (Collins et al., 1998). Im-
portantly, the 8-day dose average, *5g=kg, did not differ
significantly between the alcohol-treated groups. The daily
intraperitoneal dose of ATZ was based on our above-cited
studies conducted with furosemide and on ATZ’s estimated
potency and plasma half-life.
Blood alcohol concentration assays
Tailblood samples were taken daily with 50-mlheparinized
micro-hematocrit capillary tubes 2h after alcohol adminis-
tration, placed in 2ml of 3.5% HClO4, and centrifuged for
5min at 48C. An 80-ml aliquot of the supernatant was mixed
with 2ml of 0.5M Tris buffer (pH 8.8) containing 5% NAD
and 0.005% alcohol dehydrogenase (Boerhinger Mannheim,
Indianapolis, IN). After incubation for 15min at 378C, the
absorbance for NADH was read at 340nm, and BAC assays
were calculated from a standard curve.
Brain water determination
Brain and brain slice edema measurements applied a basic
wet weight=dry weight procedure (Olson et al., 1990). With in
vivo studies, at 9a.m. the morning following 8 days of alcohol
or vehicle intubation with and without ATZ co-treatment,
rats were lightly anesthetized with sodium pentobarbital
(60mg=kg i.p.) and decapitated. The brain cortices were
rapidly removed, and wet weights were obtained using pre-
weighed aluminum foil. The brain tissues in foil were heated
at 1028C for 48h, and the dry weights were obtained. The
percent of brain slice water was calculated according to 100?
(wet weight minus dry weight)=(wet weight). For HEC slices,
fresh slices were carefully severed from insert membranes,
pooled on pre-weighed foil, and weighed before and after
heating as above to obtain wet weight=dry weight ratios.
ALCOHOL NEURODAMAGE AND BRAIN EDEMA263
Detection and quantitation of in vivo neurodegeneration
(De Olmos cupric silver staining)
At 24h following the last alcohol=vehicle dose, rats were
anesthetized with sodium pentobarbital (80mg=kg ip) and
perfused intracardially with 4% paraformaldehyde in 0.4M
phosphate (pH 7.4) buffer. Brains were removed and kept 2
days in a solution of buffered paraformaldehyde containing
30% sucrose (w=v). Frozen brain sections (40 m) cut in the
horizontal plane were taken at every 0.5-mm level, stained
using the cupric silver technique for degenerating (argyr-
ophilic) neurons (De Olmos et al., 1981), and mounted on
glass slides. The mean number was obtained from counting
and averaging the number of argyrophilic neurons in all
brain sections=sides=regions, as described elsewhere (Collins
et al., 1996).
Data were expressed as mean?SEM, and the test for sig-
of variance (ANOVA). Subsequent pair-wise comparisons
were performed using Tukey LSD and Bonferroni post-hoc
tests. Experiments were usually replicated at least three times.
Mean BACs were analyzed with post-hoc Student t-test. The
mean number of argyrophilic neurons counted in brain sec-
tions (n¼8–9 at each successive level chosen) from rats trea-
ted with or without alcohol and=or diuretic was compared by
the non-parametric Wilcoxon Rank Sum test, because the
distributions were not normal.
The antioxidant capabilities of three diuretics—ATZ, tor-
asemide, and furosemide—were compared with the ORAC
assay to Trolox, a vitamin E analog and well-established an-
the assay). Figure 1A compares the effects of Trolox and in-
creasing concentrations of furosemide over time on the rela-
tive fluorescence of b-PE indicator protein; the two other
diuretics were similarly compared. Figure 1B summarizes
ORAC values versus concentrations for the three diuretics. It
confirms furosemide’s potency and demonstrates similar
dose-dependent increases for furosemide and Trolox with
respect to antioxidant capabilities. However, ATZ and tor-
asemide exhibited initially low ORAC values relative to
Trolox that changed only minimally with increasing concen-
trations. The lack of increase with concentration indicates that
ATZ and torasemide possess no discernible antioxidant ac-
tivities. Although not examined here, BUM and L-644, 711
have been judged to have negligible ‘‘biological’’ antioxidant
potencies using other assays (Hamelink et al., 2005).
With regard to binge treatment of HEC slices with alcohol
(100mM), Figure 2A shows that such treatment causes sta-
tistically significant brain tissue edema, consistent with find-
ings in intact binge alcohol–intoxicated rats (Collins et al.,
1998). Slice edema was apparent at 4 days, prior to evident
neurotoxicity. Furthermore, whereas daily treatment with
ATZ, a diuretic with minimal antioxidant capability, did not
significantly change control slice water content, it blocked
binge alcohol–dependent brain tissue edema. At the 4-day
duration of alcohol treatment and withdrawals, expression
levels of AQP4 water channel in the HEC slices were exam-
ined by Western blot analysis. Quantitation of AQP4 blots
(representative images in Fig. 2B) revealed that binge alcohol
treatment demonstrably increased AQP4 about 2.5-fold over
its expression levels in control slices.
Neuronal death in HEC slices that were binge-exposed to
alcohol and the effect of ATZco-treatment were assessed with
the vital dye, PI. As shown in Figure 3A, representative con-
trol andATZ-treated slicesshowedlittle PIlabeling,butbinge
alcohol–treated slices showed relatively intense PI fluores-
cence due to neurodegeneration that was prominent in the
entorhinal cortex. It was also clearly evident in the hippo-
campal dentate gyrus, but less so in CA1 and CA3 regions.
ATZ co-treatment largely prevented the binge alcohol–
induced PI labeling in the dentate and greatly suppressed
labeling in the entorhinal cortical and CA regions. Figure 3B
shows quantitation of total slice PI labeling in control and
Relative fluorescence of Beta-PE
FUR 2 µM
FUR 4 µM
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75
ORAC value (U/ml)
says. (A) Comparison of the effects of Trolox and increasing
concentrations of furosemide on the relative fluorescence
of b-phycoerythrin (ß-PE). (B) Relative ORAC values dem-
onstrating dose-related increases for the established anti-
oxidant, Trolox, and furosemide, while acetazolamide and
torasemide showed minimal basal activities with no signifi-
cant dose-dependent increases. ORAC values are means?
standard error of the mean (SEM); F (3, 8)¼5.20. Standard
errors for acetazolamide and torasemide are too small to be
seen in the figure.
Oxygen radical antioxidant capacity (ORAC) as-
264SRIPATHIRATHAN ET AL.
binge-exposed HEC slices?ATZ co-treatment. Neuronal PI
fluorescence in slices treated with ATZ alone did not differ
significantly from that in control slices. The density of PI la-
beling in slices episodically exposed to 100mM alcohol for 6
days was significantly greater than in control slices. ATZ co-
treatment during binge alcohol exposure completely abro-
gated the increased PI density of degenerating neurons. Also
consistent with this PI data, Figure 3C shows that 6 days of
binge alcohol treatment of HEC slice cultures increased LDH
leakage (180% of control), which is a measure of neurotoxic
damage in organotypic slices (Bruce et al., 1996). ATZ, hav-
ing no effect on LDH release=leakage itself, significantly
hippocampal-entorhinal cortical (HEC) slices induced by
repetitive binge alcohol exposure (100mM) and withdrawal
for 4 days, and prevention of the increased edema by co-
treatment with acetazolamide (ATZ, 1.25mM). Vehicle (0.1%
dimethyl sulfoxide [DMSO] final concentration) had no effect
on basal tissue water content. N¼10–15 slices=sample. F (3,
54)¼4.92, p¼0.004; *p<0.01 compared to control slices. (B)
Representative Western blot analysis of aquaporin-4 (AQP4)
in protein extracts of control (left lane) and binge-exposed
(right lane) HEC slices. Bottom: Induction of AQP4 expres-
sion levels in HEC slices by binge alcohol (100mM) for 4
days. N¼3=group; t¼?2.3; *p<0.05 compared to control.
(A) Increase in water content in rat organotypic
tion in hippocampal-entorhinal cortical (HEC) slice cultures
binge-exposed to alcohol (100mM, 6 days). (A) Repres-
entative photomicrographs of propidium iodide (PI) labeling
in HEC slices: Cont (0.1% dimethyl sulfoxide [DMSO]
vehicle-treated) slices with normal background fluorescence
and minimal cellular labeling; ATZ treatment (1.25mM) only,
with no degenerating neurons above control; binge alcohol
(Alc)–treated slices showing bright cellular fluorescence due
to PI uptake by degenerating neurons in the hippocampal
areas and entorhinal cortex; AlcþATZ co-treatment dem-
onstrating evident suppression of PI-labeled neurons com-
pared to alcohol. (B) Quantitation of PI labeling density in
HEC slices from cultures represented in photomicrographs
of A. N¼6–12 wells=group. F (3, 40)¼20.01; *p<0.01 com-
pared to control. (C) Increase in withdrawal media lactate
dehydrogenase (LDH) activity in HEC slice cultures binge-
exposed to alcohol as in A, and its prevention by co-
treatment with acetazolamide (ATZ, 1.25mM). Vehicle (0.1%
DMSO) had no effect on LDH activity. N¼6–9 wells=group.
F (3, 16)¼14.46, p¼0.0008; *p<0.05 compared to control.
Effect of acetazolamide (ATZ) on neurodegenera-
ALCOHOL NEURODAMAGE AND BRAIN EDEMA265
prevented the elevation in media LDH when co-administered
throughout the binge alcohol treatment.
Figure 4 shows the results of two further compounds, tor-
asemide and L-644, 711, which were screened for neuropro-
tection using LDH leakage from binge alcohol–treated HEC
slice cultures. Torasemide, the second diuretic in Figure 1B
with negligible ORAC antioxidant potential, had no effect on
basal LDH release; however, torasemide co-treatment com-
pletely suppressed binge alcohol–induced LDH leakage (Fig.
4A), indicating effective neuroprotection. On the other hand,
affect neurotoxicity in binge-intoxicated rats (Hamelink et al.,
2005), also had no effect on LDH release in alcohol-exposed
HEC slice cultures, confirming that it lacks neuroprotective
effects in these binge alcohol models. Neither compound was
examined further with PI labeling.
In Figure 5A, representative PI uptake results in HEC slices
during binge alcohol treatment are shown with BUM, a loop
diuretic that, unlike furosemide, was neither an effective
‘‘biological’’ antioxidant nor a neuroprotectant in binge-
intoxicated rats (Hamelink et al., 2005). In our HEC slice ex-
periments, BUM appeared to cause a modest degree of neu-
rotoxicity in the slices at 10mM, and higher concentrations
unpublished data). BUM at 10mM also failed to noticeably
reduce binge alcohol–induced PI labeling in any region of the
slice. In Figure 5B, quantitation of the PI fluorescence in re-
gions of binge-exposed HEC slices co-treated with 10mM
BUM confirms the interpretation of representative PI images
in Figure 5A, since there was a tendency for 10mM BUM alone
to potentiate PI staining, and a 10-fold increase in PI fluores-
cence over control due to binge alcohol that was not reduced
or changed by 10mM BUM co-treatment. We speculated that
the inability of BUM to reduce neurodegeneration in this
model might indicate failure to counter alcohol-induced brain
slice edema. In support of this speculation, Figure 5C un-
mistakably shows that binge alcohol–dependent edema in
HEC slices was not significantly suppressed by co-treatment
ATZ was examined in vivo in the once-daily binge intoxi-
cation and neurodegeneration protocol with adult male rats.
et al., 1998), neither the daily mean 2-h BAC values from a
single alcohol intubation for 8 days nor the averages of these
means differed significantly between the alcohol?ATZ
groups. In Figure 6B, the daily binge alcohol treatment pro-
voked brain edema, significantly increasing brain water con-
tent above control rat brain values. Daily ATZ co-treatment in
control rats did not significantly affect brain water content;
however, as evident in Figure 6B, ATZ completely inhibited
the alcohol-induced brain edema.
With regard to in vivo neurodegeneration, De Olmos cupric
silver stainingoffixedbrain sections fromeither the controlor
ATZ-treated rats revealed little or no argyrophilia, in agree-
ment with our published studies (Collins et al., 1998), and are
therefore omitted from Figure 6B. Cupric silver–stained
sections from binge-exposed rats (not shown) revealed ar-
gyrophilic degenerating neurons in the entorhinal cortex,
particularly layer III, and to a lesser extent in the dentate
gyrus, consistent with our results and those of others using
either bingeintoxication procedure(Collinsetal.,1998;Crews
et al., 2000; Hamelink et al., 2005). Scattered argyrophilic
pyramidal neurons were also apparent in other regions of the
temporal cortex of alcohol-intoxicated rats, as expected from
previous studies in our laboratory and in others (Collins
et al., 1996). Quantification of mean argyrophilic cell counts in
the entorhinal cortex (EC) and dentate gyrus (DG) of binge
alcohol–intoxicated rats (Fig. 6C) showed that binge alcohol–
induced neurodegeneration in the EC and DG was signifi-
cantly inhibited (>85%) by ATZ co-treatment. Thus ATZ, a
diuretic lacking in antioxidant capability, significantly pre-
vented brain edema concomitant with neurodegeneration
in both binge alcohol–treated HEC slices as well as in binge
We have proposed that alcohol-induced degeneration of
entorhinal cortical pyramidal neurons and dentate granule
cells in adult rats that occurs due to repetitive bouts of ex-
posure and withdrawal is linked to a significant extent to
nonsynaptic cellular (especially glial) swelling phenomena.
Control TorasemideAlcohol Torasemide
LDH/mg protein + sem
LDH/mg protein + sem
drogenase (LDH) release induced by binge alcohol treatment
of hippocampal-entorhinal cortical (HEC) slice cultures. (A)
Prevention by co-treatment with torasemide (1mM) of in-
creases in LDH in withdrawal media during binge alcohol
treatment (150mM, 6 days) and withdrawal in HEC slice
cultures. N¼6–9 wells=group. F (3, 25)¼39.58; *p<0.05
compared to control. (B) Failure of co-treatment with L-
644, 711 (1mM) to prevent increases in LDH in withdrawal
media due to binge alcohol treatment (150mM, 6 days) and
withdrawal in HEC slice cultures. N¼6–9 wells=group. F (3,
24)¼9.83; *p<0.05 compared to control.
Effects of torasemide or L-644, 711 on lactate dehy-
266SRIPATHIRATHAN ET AL.
This proposal was based on the suppression of both brain
edema and neurodegeneration in vivo and in vitro was
achieved with the diuretic furosemide, a potent Kþ-Cl?co-
transport inhibitor (Collins et al., 1998; Corso et al., 1998); the
present study is concerned mainly with ATZ and, to a lesser
extent, torasemide and BUM. Among the key results herein
neurotoxic binge alcohol exposure in vitro, and that ATZ, a
diuretic lacking antioxidant potency—unlike furosemide—
and also a potent inhibitor of AQP4 activity, suppresses binge
alcohol–dependent brain edema and neurodegeneration in
vitro and in vivo.
To verify furosemide’s antioxidant potential and to deter-
mine whether or not ATZ and torasemide possessed similar
capabilities, we utilized the ORAC assay, a widely accepted
standard tool for antioxidant activity measurements in the
pharmaceutical and food industries (Huang et al., 2002). Like
all antioxidant estimations, the ORAC assay has its short-
comings, but its use of peroxyl (or hydroxyl) radicals as pro-
oxidants makes it different from assays that involve oxidants
that are not necessarily pro-oxidants (Prior et al., 2005).
Nevertheless, as emphasized elsewhere (Hamelink et al.,
2005), for a variety of reasons a single in vitro parameter of
antioxidant activity does not necessarily predict biological
activity in vivo.
For in vitro assessments of binge alcohol neurotoxicity, the
organotypic HEC slice culture tends to replicate the regional
brain degeneration pattern observed in vivo, with some dif-
NMDAR involvement. We suspect that these differences are
related to the adolescent age of the HEC slices in culture (*4
weeks of age overall) relative to adult brain, but this requires
further study. Nevertheless, in retaining much of the neuron-
glia relationships and neuronal connectivity of intact matur-
ing brain, brain slice cultures have distinct advantages for
neurotoxicity experiments over dispersed (usually fetal)
hippocampal or cortical cultures (Diekmann et al., 1994;
Holopainen, 2005). Alcohol-induced neurodegeneration in
brain slice cultures has generally required subchronic expo-
sure to concentrations approaching *100mM, combined
with withdrawals (Collins et al., 1998; Prendergast et al.,
2004). However, such concentrations are not uncommon in
binging chronic alcoholics (Lindblad and Olsson, 1976; Urso
et al., 1981; Minion et al., 1989). Determination of neurode-
generation in the slice cultures used PI, a vital stain that labels
dying neurons (Vornov et al., 1991), and LDH release, a
general measure of neurotoxicity in brain slice cultures that
correlates well with PIlabeling (Bruce et al.,1996; Noraberg et
al., 1999). With regard to media conditions in our brain slice
experiments, we acknowledge they are likely to be hyper-
osmolar to varying degrees during the alcohol exposure
(consistent with the plasma of alcoholics during intoxication
(Snyder et al., 1992; Purssell et al., 2001)), but iso-osmolar
during withdrawal periods.
iodide (PI) labeling of degenerating neurons and tissue
edema in hippocampal-entorhinal cortical (HEC) slice cul-
tures binge-exposed to alcohol (100mM). (A) Representative
photomicrographs of propidium iodide (PI) labeling in HEC
slices: Cont (vehicle-treated) slices with normal background
fluorescence and no cellular labeling; BUM treatment only,
with indications of a detectable modest increase in degen-
erating neurons above control; slices treated with alcohol
(Alc) for 6 days showing increased cellular fluorescence due
to PI uptake by degenerating neurons in the hippocampus
and entorhinal cortex; slices treated with AlcþBUM for 6
days showing no apparent suppression of PI-labeled neurons
compared to alcohol alone. (B) Quantitation of PI labeling
densities in A, showing failure of BUM co-treatment to re-
duce neurodegeneration in HEC slice cultures due to binge
alcohol exposure (100mM, 6 days) and withdrawals. N¼
6–12slices=group. F (3, 48)¼50.21; *p<0.01 compared to
control slices. (C) Increase in water content induced in or-
ganotypic HEC slices by repetitive binge alcohol exposure
and withdrawal for 4 days, as in Figure 2, and the lack of
significant suppression by co-treatment with BUM. Vehicle
(0.1% DMSO final concentration) had no effect on brain water
content. N¼10–15 slices=sample. F (3, 60)¼9.82; *p<0.01
compared to control slices.
Effect of bumetanide (BUM, 10mM) on propidium
ALCOHOL NEURODAMAGE AND BRAIN EDEMA267
Turning to the in vivo methodology in this report, our
earlier binge alcohol–induced brain neurodegeneration stud-
ies (Corso et al., 1990; Collins et al., 1996) utilized the original
approach to induce alcohol withdrawal seizures (Majchro-
wicz, 1975) that was first noted to promote degeneration of
pyramidal neurons in limbic (especially entorhinal) cortical
regions and granule cells of the dentate gyrus (Switzer et al.,
1982). This entailed alcohol intubations three to four times
daily (9–12g=kg=day) for a 4-day duration to generate epi-
sodically high average BAC values (360–450mg=dl), but with
a mortality rate sometimes approaching 40%. We modified
the model to a less severe treatment (Collins et al., 1998) of a
single daily alcohol intubation (*5g=kg) for 7–10 days,
yielding average 2-h BAC values of *250mg=dl—still con-
sidered clinically severe intoxication (Lowenstein et al.,
1990)—and lower (*20%) mortality rates; this modification
was used here with ATZ. The fact that in this study the daily
and aggregate 2-h BAC values did not differ between alcohol-
and alcoholþATZ-treated rats (Fig. 6A) obviates the possi-
bility that lower brain alcohol concentrations might explain
the diuretic’s anti-edemic and neuroprotective actions.
The less severe single daily subchronic binge generates
regional distributions of degenerating (argyrophilic) neurons
that are indistinguishable from—but less intense than—those
in the original Majchrowicz (1975) procedure. No significant
neuroprotection is achieved by NMDAR inhibition in either
intoxication protocol. The degree of induced brain edema
is similar in both binge models; in that regard, the brain
water increase in Figure 6B, although a seemingly low per-
centage (*0.6%), represents nearly 2.5% brain swelling
(Elliott and Jasper, 1949). In brief, there is no indication that
the original Majchrowicz intoxication procedure and its
once-daily modification differ in the cellular mechanisms re-
sponsible for alcohol-induced brain edema and neurodegen-
Concomitant with inhibiting neurodamage, furosemide
suppressed the brain edema in adult rats due to repetitive
once-daily intoxication=withdrawal—an effect consistent
with the diuretic’s blockade of Ca2þ-independent astroglial
swelling in epileptogenic hippocampal slices (Hochman et al.,
1995). Lack of neuroprotection in the binge intoxication
models by MK-801, 6,7-dinitroquinoxaline-2,3-dione (non-
NMDAR glutamate [ionotropic] receptor antagonist), nimo-
dipine, ornitric oxidesynthaseinhibitors providesnosupport
for a central role for glutamate receptor-dependent ex-
citotoxicity, extracellular Ca2þuptake, or nitric oxide gener-
ation (Zou et al., 1996; Collins et al., 1998; Corso et al., 1998).
Also, the facts that binge alcohol–induced neurodegeneration
in rats was not reduced by the noncompetitive NMDAR an-
tagonist, memantine (Hamelink et al., 2005), nor was it ac-
companied by increased brain NMDAR as ascertained with
[3H]MK-801 binding (Rudolph et al., 1997), further argue
against a prominent excitotoxic mechanism. It is still possible,
as suggested by studies of ionotropic glutamate receptors in
alcoholics (Preuss et al., 2006) and reviewed by others (Tsai
and Coyle, 1998), that excessive glutamatergic transmission
could be involved in alcohol withdrawal seizures and dis-
turbed autonomic activation. However, in binge alcohol–
intoxicated adult rats, the density of neurodegeneration
reaches a maximum considerably earlier than the time of
greatest seizure activity (Majchrowicz, 1975) and does not
increase throughout a 36-h withdrawal period (Collins et al.,
Day of treatment
BAC mg/dl + sem
(247.2 + 17.2)
(253.6 + 12.8)
Control ATZAlcohol ATZ+Alcohol
Percent Whole Brain Water+ sem
Entorhinal CortexDentate Gyrus
Cell Counts + sem
kg=days i.p.) on 2-h BAC, brain edema, and regional neuro-
degeneration in adult rats binge-intoxicated once daily with
alcohol (3.5–6.5g=kg=days) for 8 successive days. (A) No
significant differences in the daily 2-h BAC means (?SEM)
between alcohol, and alcoholþATZ rats. Parentheses contain
means?SEM averaged from the 8 days, F (7, 8)¼4.53. (B)
Suppression of brain edema induced by once-daily binge al-
cohol intoxication for 8 days by ATZ co-treatment. N¼6–8
rats=group. F (3, 25)¼23.88; *p<0.05 compared to control.
(C) Significant reduction by ATZ co-treatment of argyrophilia
(mean degenerating neuronal cell counts) in the entorhi-
nal cortex and dentate gyrus of adult rats binge alcohol–
intoxicated once daily for 8 days. N¼6 rats=group. *p<0.05
compared to alcohol by individual nonparametric Wilcoxon
Rank Sum tests (for entorhinal cortex, W¼32, *p¼0.015; for
dentate gyrus, W¼30.5, *p¼0.027).
Effect of acetazolamide (ATZ) co-treatment (10mg=
268SRIPATHIRATHAN ET AL.
1996)—suggesting that seizure propensity and neurodamage
are not directly related.
Table 1 summarizes the effects of the three diuretics on
findings with their antioxidant potentials. Our HEC slice
culture results as well as the reported in vivo studies with
BUM and L-644, 711 are included. It also includes reported
results with several anti-oxidants already alluded to, which
will be discussed below. The ORAC assays verified that fu-
rosemide is an effective antioxidant, being at least equipotent
with the vitamin E–related Trolox. Based on furosemide’s
activity, as well as positive results with several established
antioxidants and negative results with L-644, 711 and BUM,
Hamelink et al. (2005) suggested that furosemide’s protection
could be more closely associated with its antioxidant prop-
erties than with edema reduction. However, we emphasize
that no confirmatory brain edema assessments were done in
the abovementioned study. For example, if binge alcohol–
induced brain edema had been found to be unaffected in vivo
by L-644, 711 or BUM for blood-brain barrier, metabolic, or
other reasons, the Hamelink et al. (2005) conclusions would,
to prevent edema in HEC slices binge-exposed to alcohol
(Fig. 5C). In addition, if a mechanism other than edema de-
terrence (such as free radical trapping) was the principal
neuroprotective one employed by furosemide, the diuretic
would have been expected to significantly reduce binge
alcohol–induced neurodamage in all affected regions, but it
failed to do so in the olfactory bulb glomeruli (Collins et al.,
1998), a region not examined by Hamelink et al. (2005).
Table 1 also summarizes that, despite lacking antioxidant
capability, ATZ diuretic, the primary focus of these current
experiments, prevented binge alcohol–induced tissue water
accumulation and neurodegeneration both in HEC slice cul-
tures and in vivo. A carbonic anhydrase inhibitor and cere-
brovascular dilator stimulus (Settakis et al., 2003), ATZ has
been reported to reduce ischemic brain edema in rats (Czer-
nicki et al., 1994), but there apparently is no further infor-
mation linking the diuretic to possible neuroprotection.
Furthermore, and particularly germane to our alcohol exper-
iments, ATZ and several arylsulfonamide isomers have been
shown to potently inhibit the AQP4 water channel (Huber
et al., 2007); the possible importance of this effect is discussed
Torasemide, a sulfonylurea-based compound that is con-
sidered a more potent loop diuretic than furosemide but, ac-
cording to the ORAC results, possesses no antioxidant ability,
has had some success as a neuroprotective agent in stro-
ke=edema models (Plangger, 1992; Staub et al., 1994). It also
selectively inhibits Cl?transport-related glial swelling, at-
evoked cell volume increases (Staub et al., 1993). In our HEC
slice cultures, torasemide largely blocked the LDH release
evoked by binge alcohol exposure=withdrawal. Although
torasemide might protect through other molecular mecha-
nisms such as blocking of angiotensin=angiotensin receptor
pathways(Fortuno etal.,1999;Muniz etal.,2001),the resultis
consistent with the view that brain tissue overhydration and
its downstream effects are potentially important factors in
alcohol’s neurotoxic mechanism.
The failure of L-644, 711 to protect against binge alcohol–
induced neurotoxicity in the HEC slice cultures (Fig. 6) and in
vivo (Hamelink et al., 2005) indicates that the compound,
which primarily inhibits Cl?=HCO3?exchange, may not ef-
fectively counter the binge alcohol–induced brain water in-
creases. Although lacking sufficient sample for in vivo binge
alcohol=edema studies, we note that in a stroke model in rats,
1991). With respect to BUM, in harmony with the in vivo re-
sults of Hamelink et al. (2005), this diuretic did not protect
against binge alcohol neurodamage in the HEC slice culture
model. We further showed that, unlike ATZ and furosemide,
HEC slice edema induced by binge alcohol was not prevented
by this diuretic, possibly explaining its lack of neuroprotec-
tion. Some considerations that might underlie this are that
BUM is considerably less potent than furosemide in terms of
inhibition of the Cl?-extruding KCC2 transporter, but is a
500-fold stronger inhibitor of the electroneutral Naþ-Kþ-2Cl?
(NKCC1) co-transporter (Payne et al., 2003). This difference in
potencies is thought to underlie BUM’s reported absence of
Table 1. Effects of Diuretics, Antioxidants, and L-644-711 on Binge Alcohol-Induced Brain Edema
and=or Neurotoxicity in Hippocampal-Entorhinocortical (HEC) Slice Cultures and In Vivo:
Comparison with In Vitro Antioxidant Capability
in HEC slices
in HEC slices
neurotoxicity in vivo
aHamelink et al. (2005).
bCrews et al. (2006).
cYes in Alkamuls solubilizer (Hamelink et al., 2005); no in corn oil (Crews et al., 2006).
n.d., not determined; BHT, butylated hydroxytoluene.
ALCOHOL NEURODAMAGE AND BRAIN EDEMA269
anti-epileptic effects as compared to furosemide’s inhibition
of Kþ-induced epileptiform activity in hippocampal slices
(Margineanu and Klitgaard, 2006). It is therefore possible that
this divergence between BUM and furosemide is also im-
portant in the context of inhibition=prevention of alcohol-
induced brain edema and neurodegeneration.
Revisiting the issue of furosemide and its effectiveness,
the diuretic’s neuroprotective effect against binge alcohol–
induced damage thus might arise from a combination of ac-
tions not available to BUM or L-644, 711. Its prevention of
alcohol-induced brain edema (Collins et al., 1998) could first
derive from the diuretic’s potent inhibition of the brain-
specific KCC2 co-transporter as mentioned, with resulting
restoration of cellular ionic strength and volume. Of interest
is that apoptotic events induced by etoposide in fibroblasts—
the translocation ofcytosolic BAXprotein to the mitochondria
and the consequent release of mitochondrial cytochrome c—
were suppressed by furosemide (Karpinich et al., 2002). The
explanation was that BAX translocation is the outcome of a
conformational alteration in BAX resulting from ionic and
pH changes in the cytosolic milieu, changes that furosemide
might counter via inhibition of Cl?extrusion. Parenthetically,
whether classically apoptotic events such as cytochrome c
release contribute significantly to alcohol-induced neurode-
rats using a terminal indicator of apoptosis, TUNEL staining,
translocation and cytochrome c release should be examined
in the binge alcohol models, since these events can arise in-
dependent of classical apoptosis mechanisms.
We do not question, however, that additional mode of fu-
possibly the reduction of brain edema) could well be anti-
oxidative. Oxidative stress has been postulated by us and
several others as fundamental to alcohol’s neurotoxic mech-
anism; indeed, as shown in Table 1, administration of selected
anti-oxidants (notably cannabidiol, vitamin E, and butylated
hydroxytoluene) provided significant neuroprotection in
binge alcohol–intoxicated rats (Hamelink et al., 2005; Crews
et al., 2006), but the sources of reactive oxygen species (ROS)
are imprecisely understood. Other potential actions of furo-
semide appear to be less relevant to the alcohol models. The
diuretic is reported to be a GABA-A receptor antagonist at
concentrations similar to those used in HEC slice cultures, but
antagonism is manifested primarily in the cerebellum and not
in the hippocampus and cortex (Korpi and Luddens, 1997).
A key associated point is that our preliminary finding of
increased AQP4 during binge alcohol–induced edema and
neurodegeneration in HEC slices could be of emerging sig-
nificance in terms of alcohol’s edema-based mechanism. The
aquaporin water channel family consists of several gene
products, with AQP4 the primary form in brain (Gunnarson
et al., 2004). While expressed primarily in astroglia (Amiry-
Moghaddam et al., 2003), it is also expressed by microglia
activated by inflammatory stimuli (Tomas-Camardiel et al.,
2004). Increasing evidence indicates that AQP4 activity plays
aninstigating partin cellular (cytotoxic)glial edemain animal
models of trauma, stroke and ischemia (Taniguchi et al., 2000;
Badaut et al., 2007; Neal et al., 2007). Although the precise
nature (i.e., cytotoxic versus vasogenic) of the binge alcohol–
dependent edema is still uncertain, we suspect that glial
edema is an important component, and AQP4 could thus
have an early neuropathological role. However, it is still
possible that AQP4 elevation is a cellular survival response to
the edema and associated neuroinflammatory responses ra-
ther than (or in addition to) a causative step. For example,
upregulation of a number of cell death=survival genes, in-
cluding AQP4, was associated with ischemic neuroprotection
due to inflammatory (endotoxin) brain preconditioning
(Mallard and Hagberg, 2007). Studies with knockdown or
knockout models are needed to answer this question.
Our current view is that, by whatever molecular process it
is initiated, brain edema (in part cytotoxic) and associated cell
stress deformation due to repetitive high alcohol exposure
and withdrawal promotes pro-inflammatory processes en-
compassing activation of PLA2 and excessive mobilization of
AA, with increased oxidative stress as one downstream out-
come (Lehtonen and Kinnunen, 1995; Basavappa et al., 1998).
The work of Crews et al. (2004) suggests that increased pro-
inflammatory cytokines (e.g., TNFa), may also be involved.
While intracellular ROS elevations due to the alcohol=alcohol
withdrawal stress and cellular edema can result from a
xanthine oxidase, ribonucleotide reductase, NADPH oxidase,
and mitochondrial leakage), AA is sometimes the major
contributor in a neurodegenerative ROS process (Bobba et al.,
2008). AA could produce oxidative stress enzymatically and
indirectly via NADPH oxidase induction (Dana et al., 1998); it
also could aggravate the edema (Chan et al., 1983; Winkler
et al., 2000). In addition, ROS could be positive feedback sig-
nals, further activating PLA2isoforms (Martinez andMoreno,
2001). Possibly distinct from ROS generation, AA could fuel
cell death processes by stimulating mitochondrial perme-
ability transition (Scorrano et al., 2001). Glutamate, released
by astrocytic swelling as well as by AA (Freeman et al., 1990;
Kimelberg and Mongin, 1998), can exacerbate oxidative stress
via a non-excitotoxic pathway involving inhibition of gluta-
thione biosynthesis (oxidative glutamate toxicity (Tan et al.,
2001). Of note, our current inhibitor studies with HEC slice
cultures indicate that blockade of PLA2 activity is neuropro-
tective against binge alcohol treatment (Brown et al., 2008).
However, a further mechanistic possibility is the potential
integrative role in alcohol-dependent neurodamage for nu-
transcription factor which is reported to be upregulated by
polyunsaturated fatty acids like AA (Maziere et al., 1999) as
well as by alcohol in cultures and binge alcohol intoxication in
vivo (Zima and Kalousova, 2005; Crews et al., 2006; Zou and
In summary, since two diuretics that lack antioxidant po-
tency, ATZ and torasemide, prevent both brain edema and
binge alcohol–induced neurodegeneration, we argue that
brain edema is likely to be a critical factor leading to neuro-
degeneration, with its suppression by these diuretics, as well
as by furosemide (but not by BUM or possibly L-644, 711)
providing significant neuroprotection. Nevertheless, addi-
tional protective mechanisms could exist for each of the
effective agents. Also, AQP4 water channels, evidently in-
or maintenance of the observed edema. Further studies are
needed to understand the mechanisms by which repetitive
alcohol intoxication and withdrawal promote brain edema,
and how=whether AQP4 is centrally involved.
270SRIPATHIRATHAN ET AL.
We gratefully acknowledge N. Achille for assistance in
HEC slice cultures, Dr. H.L. Kimelberg for L-644, 711, and the
Loyola Medical Center Alcohol Research Program. The re-
search was supported in part by NIH (grants T32 AA13527,
R21 AA011543) and a Potts award.
Author Disclosure Statement
No competing financial interests exist.
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