Analytic grade reagents were purchased from Sigma Aldrich (St. Louis, MO), Cell Signaling Technology (Danvers, MA), Santa Cruz Biotechnology (Santa Cruz, CA), and Mitosciences (Eugene, OR). Diet ingredients were obtained from Research Organics (Cleveland, OH) or MP Biomedicals (Irvine, CA). HT22 cells, a murine hippocampal cell line, were the generous gift of Dr. David Schubert (Salk Institute, San Diego, CA). All reagents for mitochondrial respiration assay were purchased from Seahorse Bioscience (North Billerica, MA).

Male Sprague-Dawley rats (Charles River, Wilmington, MA) were 3 months old at the beginning of this study. They were housed individually at controlled temperature (22˚C - 25˚C) and humidity (55%), with ad libitum access to water and a 12-hour-light/dark cycle. All animal experimentation was conducted in accordance with the Guide to the Care and Use of Laboratory Animals [DHHS Publication No. (NIH) 85 - 23, revised 1996, Office of Science and Health Reports, DRR/NIH, Bethesda, MD] and was approved by the University of North Texas Health Science Center Animal Care and Use Committee.

The induction of ethanol dependence was accomplished by a method that has been routinely employed in our laboratory [21]. Rats were divided into 4 groups (5 - 7 rats/group) according to ethanol-diet and MB treatment. The four groups were control dextrin-diet groups with or without MB treatment and ethanol-diet groups with or without MB treatment. Rats in the ethanol-diet groups received a liquid diet containing 7.5% (wt./vol.) ethanol for 5 weeks. Control-diet rats received a liquid diet with dextrin isocalorically substituted for ethanol. MB (0.5 mg/kg, once/day, I.P.) was injected for last 5 days of ethanol-diet and on the first day of EW. This regimen of MB administration is based on the rationale that most of alcoholic patients who seek for treatments receive medication near and/or during a detoxification phase. The liquid diets are nutritionally balanced containing multiple ingredients including pulverized casein, vitamin and mineral mixtures, sucrose, and cellulose [22]. One hundred ml of fresh diet was placed in each cage daily for 5 weeks and then abruptly removed to initiate EW stress. Thereafter, animals were fed conventional chow pellets. The experience of EW stress in these rats was ensured according to a standard method of monitoring EW signs such as tremor, rigidity, and hyperactivity [21].

Rats were humanely sacrificed under anesthesia [xylazine (20 mg/kg, I.P.) and ketamine (100 mg/kg, I.P.)] 24 hours after the removal of ethanol-diet. Immediately thereafter, cerebellar mitochondria were isolated by conventional differential centrifugation with slight modifications [23]. We chose cerebellum because this brain area is one of the most vulnerable brain areas to ethanol or EW. Briefly, cerebellum was dissected, rinsed, and rapidly transferred to a homogenizer containing ice-cold isolation buffer (320 mM sucrose, 1 mM dipotassium ethylene diamine tetraacetic acid, 10 mM Tris-Hcl). The homogenate was centrifuged at 1330 g for 5 minutes at 4˚C and the supernatant was saved. The pellet was resuspended in 0.5 volume of the original isolation buffer and centrifuged again under the same conditions. The two supernatants were combined and centrifuged further at 21,200 g for 10 minutes. The resulting pellet was resuspended in 12% Percoll solution (GE Healthcare BioSciences AB, Uppsala, Sweden) and centrifuged at 6900 g for 10 minutes. The resulting soft pellet was washed once with mitochondrial isolation buffer and centrifuged at 6900 g for 10 minutes. The pellet containing mitochondria was used in this study. Bradford assay was conducted to analyze mitochondrial protein concentrations according to manufacturer’s instructions (Bio-Rad, Hercules, CA, USA) and a previous method [24].

Mitochondrial respiration was assessed by measuring mitochondrial O₂ consumption rate (pmoles/minutes) according to a method provided by the XF respirometer manufacturer (Seahorse Bioscience). XF sensor cartridge was hydrated overnight in XF calibration buffer (at 37˚C, no CO₂). Isolated mitochondria were diluted with mitochondrial assay solution (Seahorse Bioscience) to yield a final concentration of 200 µg/ml. Diluted mitochondria (50 µl) were transferred into each well of XF microplate and spun down at 4˚C for 10 - 20 minutes at 2000 - 3600 g. A consistent monolayer of mitochondrial adhesion to the bottom of the wells was visually ensured. A volume of 450 µl of succinate (5.5 mM) and rotenone (2.2 µM) was then added to each well. The XF microplate was warmed at 37˚C (no CO₂) for 8 - 10 minutes and placed in XF respirometer. Real-time (data are obtained while mitochondria respire) mitochondrial respiration was subsequently recorded every 5 - 7 minutes.

Rats were deeply anesthetized with the mixture of ketamine (60 mg/kg, I.P.) and xylazine (10 mg/kg) at 24 hours of EW and perfused intracardially with saline, followed by 4% paraformaldehyde in phosphate buffer. Cerebellar areas containing vermis were divided into halves by a midsagittal cut and this sample was used for the immunohistochemistry. Briefly, the fixed tissues were rinsed in 70% ethanol overnight, dehydrated using different ethanol concentrations, xylene, and mixture of xylene/ paraffin, and then embedded in paraffin. For consistency from animal to animal, multi-tissue blocks from the same groups were embedded together. The sections started approximately 1.5 mm parasagital from the midline of the cerebellar vermis and ended after 250 sections at 2.5 mm. For each rat, the 9th section from the midline was used for the immunohistochemistry analysis [21]. The slides were deparaffinized in xylene, rehydrated through decreasing ethanol concentrations, and washed with phosphate-buffered solution (PBS). The slices were then moisturized at 95˚C for antigen retrieval. A primary antibody against COX subunit IV (Cell signaling, Danvers, MA) was diluted (1:1000) in blocking buffer (10% normal goat serum). COX is composed of 13 subunits. We chose subunit IV because this subunit is particularly vulnerable to EW [25]. The slices were rinsed in PBS and then incubated with broad spectrum poly-horseradish-peroxidase-conjugate for 40 minutes. The antigen-antibody bindings were visualized with a diaminobenzidine color reaction and examined with an inverted Carl Zeiss microscope and a digital camera. All photographs were taken of the cerebellar cortex containing Purkinje layers. Slides stained with non-immunoreactive serum used for a negative control had negligent diaminobenzidine staining.

The HT22 cell line was originally selected from HT4 cells that were immortalized from mouse primary hippocampal neurons using a temperature-sensitive small virus- 40 T antigen. HT22 cells were maintained as described previously [26]. Briefly, HT22 cells were grown in DMEM, supplemented with 10% charcoal-stripped fetal bovine serum (HyClone, Logan, UT) and 1% penicillin/ streptomycin at 37˚C in an atmosphere containing 5% CO₂ and 95% air. HT22 cells (10⁶ cells) were plated into Petri dishes (Greiner Bio-One, Monroe, NC). The following day, the cells were exposed to vehicle solution (ethanol-free DMEM medium) or 100 mM ethanol for 3 days. At the end of the 3 day-ethanol exposure, ethanolcontaining solution was abruptly replaced with ethanolfree solution (DMEM medium) to create EW stress and then incubated for 4 hours before assays began.

HT22 cells (1000 cells/well) were seeded into XF microplates and exposed to ethanol (100 mM) for 3 days, and abruptly withdrawn from ethanol to create EW stress [18, 27]. The cell plate was processed according to a manufacture’s (Seahorse Bioscience) instruction, placed on an O₂ sensor cartridge, and inserted into the XF respirometer. This step was done immediately after 4 hours of EW. MB’s protection against EW-induced mitochondrial respiratory suppression was tested by treating cells with MB (250 nM) during the 4-hour-EW period. The role of COX in mitochondrial respiration was tested by treating cells with a COX inhibitor (NaN₃, 0.1 µM). NaN₃ was preloaded in the reagent delivery chambers of the O₂ sensor cartridge and injected into the wells while the XF respirometer was reading O₂ consumption rate. The effects of antioxidants on mitochondrial respiration were tested by preloading [50 µM of CoQ10 (Coenzyme Q10) or 1 µM of BHT (2,6-di-tert-butyl-4-methylphenol)] in this way. In addition, these antioxidants were chronically administered to a separate set of cells during the 4 hours of EW. Control cells were exposed to only dimethyl sulfoxide. O₂ consumption rates (pmoles/minutes) were obtained approximately every 5 - 7 minutes.

Since glutamate is known to mediate hyperexcitatory EW stress, we tested the effect of glutamate in the presence or absence of MB on COX protein content in HT22 cells. HT22 cells were treated with 3 mM glutamate for 24 hours with or without MB treatment (250 nM) for last 4 hours. After cells were allowed to grow overnight, a protease inhibitor and 1 ml of radioimmunoprecipitation assay buffer were added to lyse cells. Cells were then quickly collected and placed on ice. Bradford protein assay was conducted to assess total protein amount. Samples containing 30 µg of protein were resolved by SDS-PAGE on 10% cross-linked gels and then electrophoretically transferred onto polyvinylidene fluoride membranes. Nonspecific binding was blocked with 5% fat-free milk. Blots were washed in PBS containing 0.05% Tween 20 and then probed overnight with mouse monoclonal antibodies (Mitosciences, Eugene, OR) against COX subunit IV (1:1000 dilution). Blots were then incubated with horseradish peroxidase-conjugated secondary antibodies (1:10,000 dilution) for one hour at room temperature. Bands were detected using immunoblotting luminescence (UVP, Upland, CA) and quantified by an image densitometer. Immunoblottings for β-actin were carried out as a positive and a loading control.

The effect of MB on cell death induced by EW and/or glutamate was tested using Calcein assay. HT22 cells were exposed to ethanol (100 mM) for 3 days and withdrawn for 4 hours as described earlier. MB (250 nM) and/or glutamate (3 mM) was treated during the 4-hour of EW period. A separate set of ethanol-free cells was exposed to glutamate (3 mM) for 24 hours with or without MB treatment for last 4 hours to test MB’s protection against glutamate cytotoxicity. The role of COX in the effect of MB or glutamate was tested by NaN₃ (0.1 µM) cotreatment during EW. Following the removal of the medium from the 96-well plates, the cells were incubated with fluorogenic Calcein-AM (2.5 µM, Molecular Probes, Eugene, OR) in PBS. Twenty minutes later, fluorescence intensity was determined using a Bio-Tek FL600 microplate reader (Winooski, VT) with an excitation/emission filter set at 485/530 nm. Cell culture wells treated with methanol served as a blank condition.

All numerical data are expressed as means ± standard error of mean (SEM) and were analyzed by a singlefactor-ANOVA (EW with or without drug treatment) or two-factor-ANOVA (EW × drug treatment). When ANOVA detected statistically significant treatment effects, post hoc Tukey’s tests were performed to identify specific differences between groups. p values of < 0.05 were taken to indicate statistical significance.

We have previously demonstrated that mitochondria from ethanol withdrawn cells are less able to respire than mitochondria from healthy cells [3]. Using brain tissues obtained from ethanol withdrawn rats, the current study extended this phenomenon to MB’s protection. Figure 1 illustrates that brain mitochondria from ethanol withdrawn rats show a smaller degree of respiration than those from control-diet rats or MB-treated ethanol withdrawn rats. MB-treatment to control-diet rats did not significantly alter mitochondrial respiration (data not shown). These data indicate that EW suppresses mitochondrial respiration in a manner that is protected by MB.

Our previous study has shown that EW suppresses COX expression in rats. We tested whether MB attenuates such effect of EW on COX. Ethanol withdrawn rats show COX positive immunostaining (green fluorescence) to a lesser degree than control-diet rats (Figure 2). MB-treated ethanol withdrawn rats show much more COX positive staining than vehicle-treated ethanol withdrawn rats. These results indicate that MB treatment preserves COX expression upon EW stress.

We next investigated how MB improves mitochondrial respiration compromised by EW stress (Figure 3). We hypothesized that MB’s up-regulation of COX mediates protection against EW-induced mitochondrial respiratory suppression. To test this hypothesis, we coadministered MB with a COX inhibitor (NaN₃) during the 4 hours of EW in cells. Separately, MB followed by NaN₃ was acutely administered while XF respirometer recorded basal mitochondrial respiration. Both chronic and acute treatments of MB attenuated the inhibiting effect of EW on mitochondrial respiration. However, this effect of MB was diminished in the presence of NaN₃. These results indicate that COX mediates the MB’s protection against EW-induced mitochondrial respiratory suppression.

MB has been reported to exert an antioxidant activity such that it minimizes electron leakage from the electron transfer complexes and the formation of reactive O₂ species (ROS). In contrast, EW increases ROS produc-

tion and protein oxidation [17,28]. If MB’s antioxidant activity mediates its mitochondrial respiratory protection, treating cells directly with antioxidants would increase mitochondrial respiration upon EW stress. We administered antioxidants BHT and CoQ10 during the 4 hours of EW (Figure 4(a)) or acutely injected (Figure 4(b)) them into cells while basal respiration was recorded. However, neither compound was able to increase mitochondrial respiration of ethanol withdrawn or control-cells. The failure of these antioxidants to increase mitochondrial respiration of ethanol withdrawn cells suggests that an antioxidant activity may not mediate or not directly mediate MB’s protection against EW-induced mitochondrial respiratory suppression.

Considering that mitochondria provide cellular energy for cell survival, it is reasonable to speculate that mitoprotective MB protects cell viability. We have previously demonstrated that EW provokes cell death [27] and neuronal loss [21]. Since glutamate is known to mediate hyperexcitatory EW stress [29,30], we hypothesized that MB protects cell survival from EW through glutamate inhibition. Figure 5 illustrates that EW decreases cell viability and this effect of EW is substantially attenuated

by MB treatment. However, MB was less cytoprotective after glutamate cotreatment (Figure 5(a)). Reciprocally, glutamate treatment acted like EW stimulus by inhibiting cell viability and this effect of glutamate was attenuated by MB treatment (Figure 5(b)). These results indicate that there is a counteracting relationship between MB and glutamate at the level of cell viability and that MB protects cells from EW stress by inhibiting glutamate insults.

We investigated whether MB protects cell survival from EW or glutamate through COX upregulation. If this was the case, a COX inhibitor treatment would blunt the MB’s protection. We indeed observed that MB was less protective against EW (Figure 7(a)) or glutamate (Figure 7(b)) cytotoxicity in the presence of NaN₃. NaN₃ treatment alone (without MB) during EW exacerbated EW-induced cell death. In addition, glutamate at the dose that inhibited cell viability decreased the protein content of COX and this effect of glutamate was attenuated by MB treatment (Figure 7(c)). These results support the idea that COX mediates MB’s protection against EWor glutamate-induced cytotoxicity.

If MB’s protection against glutamate cytotoxicity is

mediated through its antioxidant activity, a prooxidant drug (H₂O₂) would blunt the MB’s protection. H₂O₂ alone inhibited cell viability and this effect was blunted in the presence of MB, indicating MB’s antioxidant protection for cell survival (Figure 6(a)). However, the cell viability of glutamate-exposed cells did not differ after treatment with MB alone or in combination with H₂O₂ (MB + H₂O₂) (Figure 6(b)). These data indicate that a prooxidant stimulus does not alter MB’s protection against glutamate cytotoxicity.