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Keywords:

  • corticosterone;
  • ethanol;
  • FJ-B;
  • hippocampus;
  • mifepristone;
  • neurodegeneration

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. FINANCIAL DISCLOSURES
  9. Authors Contribution
  10. References

Excessive ethanol (EtOH) use leads to impaired memory and cognition. Using a rat model of binge-like intoxication, we tested whether elevated corticosterone (Cort) levels contribute to the neurotoxic consequences of EtOH exposure. Rats were adrenalectomized (Adx) and implanted with cholesterol pellets, or cholesterol pellets containing Cort in order to achieve basal, medium, or high blood concentrations of Cort. Intragastric EtOH or an isocaloric control solution was given three times daily for 4 days to achieve blood alcohol levels ranging between 200 and 350 mg/dl. Mean 24-hour plasma levels of Cort were ∼110 and ∼40 ng/ml in intact EtOH-treated and intact control animals, respectively. Basal Cort replacement concentrations in EtOH-treated Adx animals did not exacerbate alcohol-induced neurodegeneration in the hippocampal dentate gyrus (DG) or the entorhinal cortex (EC) as observed by amino-cupric silver staining. In contrast, Cort replacement pellets resulting in plasma Cort levels twofold higher (medium) than normal, or greater than twofold higher (high) in Adx-Cort-EtOH animals increased neurodegeneration. In separate experiments, pharmacological blockade of the Type II glucocorticoid (GC) receptor was initiated with mifepristone (RU38486; 0, 5, 15 mg/kg/day, i.p.). At the higher dose, mifepristone decreased the number of degenerating hippocampal DG cells in binge-EtOH–treated intact animals, whereas, only a trend for reduction was observed in 15 mg/kg/day mifepristone-treated animals in the EC, as determined by fluoro-jade B staining. These results suggest that elevated circulating Cort in part mediates EtOH-induced neurotoxicity in the brain through activation of Type II GC receptors.


INTRODUCTION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. FINANCIAL DISCLOSURES
  9. Authors Contribution
  10. References

Glucocorticoids (GCs) are secreted by the adrenal glands in response to a variety of physical, emotional, and chemical stressors. Release of natural GCs [cortisol in primates, corticosterone (Cort) in rats and mice] is normally adaptive and results in energy mobilization and redistribution from tissues such as immune and reproductive systems to organ systems that utilize oxygen and glucose to meet short-term homeostatic challenges, such as cardiovascular and pulmonary tissues. Prolonged chronic stress or elevated GCs promote insidious tissue pathologies, including neuronal brain damage. Neurotoxicity and neuronal death represent deleterious consequences of GC over-secretion (Sapolsky, Krey & McEwen 1986). In rodents, stress and elevated GC concentrations have been shown to suppress adult neurogenesis (Gould & Tanapat 1999), cause marked loss of pyramidal cells from the CA3region of the hippocampus (Sapolsky & Pulsinelli 1985; Watanabe, Gould & McEwen 1992) and hippocampal atrophy in patients with high and persistent GC levels because of Cushing's syndrome (Starkman et al. 1992). One structure particularly vulnerable to GC-mediated neurodegeneration is the hippocampus (Sapolsky & Pulsinelli 1985), which contains the highest density of the central nervous system (CNS) GC receptors (McEwen, Dekloet & Rostene 1986). GCs are thus thought to ‘endanger’ hippocampal neurons, reducing their likelihood of survival under a variety of neurotoxic insults such as excitotoxicity (Stein-Behrens et al. 1992), transient global ischemia (Sapolsky & Pulsinelli 1985), hypoglycemia or exposure to antimetabolites (Sapolsky 1985), cholinergic or serotonergic neurotoxins (Johnson, Moore & Morin 1989; Hortnagl et al. 1993) and oxygen radical generators (McIntosh & Sapolsky 1996). Accordingly, adrenalectomy (Adx) did protect hippocampal pyramidal cells from age-related loss (Landfield, Baskin & Pitler 1981) and hippocampectomy led to increased basal plasma Cort concentrations (Magarinos, Somoza & De Nicola 1987), as a result of disrupting the negative feedback loop in which the hippocampus has been persistently implicated. Although to a lesser extent, GCs have also been shown to enhance neurotoxicity in the striatum and cortex (Sapolsky & Pulsinelli 1985).

Chronic alcohol consumption is known to ultimately lead to structural pathologies of the brain followed by learning and memory impairment, and other reduced cognitive functions in humans and animals (Oscar-Berman, Hutner & Bonner 1992; Bowden & Mccarter 1993; Jarrard 1993; Obernier et al. 2002b). Animal studies, using a binge-like alcohol exposure model mimicking a single cycle of binge intoxication in human alcoholics (Majchrowicz 1975), have shown that neurodegeneration can occur after large doses of alcohol administered over 3–4 days. Such a model reliably produces neuronal damage in corticolimbic areas including hippocampal structures such as the dentate gyrus (DG), and the entorhinal cortex (EC) (Collins, Corso & Neafsey 1996; Zou et al. 1996; Corso et al. 1998; Crews et al. 2000; Hamelink et al. 2005; Cippitelli et al. 2010a,b). Importantly, alcohol is known to activate the hypothalamic-pituitary-adrenal (HPA) axis and to elevate circulating GCs (Rivier, Bruhn & Vale 1984; Thiagarajan, Mefford & Eskay 1989), suggesting that alcohol-mediated CNS neurotoxicity could be due in part to chronically elevated GCs. Furthermore, in vitro studies have recently shown that Cort treatment exacerbates damage associated with excitotoxicity as well as ethanol (EtOH) withdrawal in rat hippocampal slices (Mulholland et al. 2005, 2006). Endogenous GCs exert their effects by binding to two distinct intracellular receptor subtypes: the GC receptor Type II (GR) and the mineralocorticoid receptor (MR) Type I. Paradoxically, the GR has a lower affinity for GCs than MRs and only becomes highly occupied at high GC concentrations, such as those seen following an alcohol insult (Rivier et al. 1984). In contrast, MRs are rapidly saturated at low GC concentrations (Joels & Dekloet 1994). This would give rise to the possibility that GRs may play a role in mediating the effects of GCs in alcohol-induced neurotoxicity. In order to better understand EtOH-induced neurotoxicity in the rodent binge model, animals were subjected to binge-like intoxication, characterized in part by elevated Cort levels, with manipulation of circulating Cort levels or pharmacological blockade of the GR with the selective GR antagonist mifepristone (RU38486). Mifepristone is also a progesterone antagonist abortifacient; therefore, it is possible that some of the observed neurodegenerative/neuroprotective effects attributed to GR events are mediated in part through progesterone receptors, which was not addressed in this study.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. FINANCIAL DISCLOSURES
  9. Authors Contribution
  10. References

Animals

Male Sprague Dawley rats (Taconic Farms, Rockville, MD, USA) weighing approximately 250 g were maintained in a temperature and humidity-controlled vivarium on a 12-hour light/dark cycle with water and food available ad libitum. Upon arrival, all animals were group housed, but individually housed after implantation of chronic indwelling gastric cannulae and Adx. All surgical procedures were performed in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Rats were randomly assigned to either liquid-diet control groups with or without EtOH, Adx, or Cort pellet treatment, and were subjected to implantation of chronic indwelling gastric cannula, and Adx or sham operated followed by Cort pellet replacement. Plasma Cort and EtOH levels were monitored by tail bleeds in all groups. Forty-µm frozen brain sections were obtained from each animal after infusion and fixation and stained for dead or dying neurons with the amino cupric silver stain, as previously described and performed by Neuroscience Associates (Knoxville, TN, USA, 37934). In a separate study, groups of intact rats were intubated by gavage with diet alone or with EtOH and given 0, 5 or 15 mg/kg mifepristone with or without EtOH. Frozen brain sections were obtained for analysis with fluoro-jade B (FJ-B), which stains for dead or dying neurons. Plasma from tail bleeds was assayed by radioimmunoassay (RIA) for Cort levels and by Sigma diagnostic kits or gas chromatography for EtOH in appropriate treatment groups.

Gastric cannula implantation and Adx

All surgical procedures were performed under ketamine hydrochloride/xylazine (80:10 mg/kg i.p.) anesthesia. Intragastric catheterization was carried out as previously described (Hamelink et al. 2005). Bilateral Adx was performed by making a single 3-cm dorsal incision, immediately caudal to the rib cage, followed by bilateral 1.5-cm incisions through the dorsal abdominal musculature. The adrenal glands were surgically removed and the incisions sutured. The gastric cannula, Adx, and Cort-cholesterol surgeries were performed during the same surgical session. Cort-cholesterol pellets were implanted subcutaneously between the scapulae in the basal, medium, or high Cort level groups with 15% Cort 100 mg pellets, 15% Cort 250 mg pellets, or 60% Cort 250 mg pellets, respectively. The efficacy of pellet implantation and the maintenance of plasma Cort concentrations were evaluated by measuring plasma Cort levels by RIA. Intact animals underwent sham surgery. Following surgeries each animal received an i.p. injection of gentamicin (8.5 mg/kg) and ampicillin (70 mg/kg) and was placed on a heating pad until they became ambulatory, at which time they were returned to their cages and fed ad lib.

Binge alcohol treatment

Identical to what was reported previously (Hamelink et al. 2005), 7 days after cannula implantation, all rats were given ad lib access to alcohol-free liquid diet for 3 days formulated to provide 16.9% of calories as protein, 59.2% carbohydrate, and 23.9% fat (Research Diets Inc., Allentown, NJ, USA). The morning of the next day, alcohol administration was begun (day 1 of the 4-day binge). Beginning at lights on (day 1), all rats were given a priming dose of 5 g EtOH followed by 12 ml of liquid diet via gastric cannula every 8 hours. In the EtOH-treated animals, the 12 ml of liquid diet was modified to contain 10–12% less calories from carbohydrates, which was replaced with an equal number of EtOH calories. Rats were rated for their level of behavioral intoxication at the time of each EtOH feeding and given the appropriate EtOH dose, as reported by (Majchrowicz 1975) for 4 days. On the morning of the fifth day, animals were deeply anesthetized with an i.p. injection of ketamine hydrochloride and xylazine (80:10 mg/kg) and transcardially perfused via gravity flow. Blood was cleared from the animal's vasculature with 200 ml of wash solution (0.8% sodium chloride, 0.4% dextrose, 0.8% sucrose, 0.5% sodium nitrite, 0.023% calcium chloride, and 0.034% sodium cacodylate) followed by 250 ml of fixative solution (4% sucrose, 4% paraformaldehyde, and 1.43% cacodylic acid). Animals destined to be stained with FJ-B were perfused with 200 ml of normal saline followed by 250 ml of 4% paraformaldehyde in phosphate-buffered saline (PBS).

In a follow-up study to the Adx-Cort-replacement or Mifepristone experiment, rats were subjected to a 4-day binge intoxication procedure as previously described (Hamelink et al. 2005; Cippitelli et al. 2010a,b). However, liquid diet of a slightly different composition from the Cort studies, namely Nestle Good Start Infant Formula (Glendate, CA, USA), was used. In brief, alcohol (20% v/v) with diet or diet alone was administered via 18-gauge gavage needles. Alcohol-treated animals were given an initial priming dose of 5 g/kg. Additional alcohol was administered every 8 hours for four consecutive days at 7:00 am, 3:00, and 11:00 pm based on the animal's behavioral intoxication level, as determined using a 6-point behavioral intoxication scale (Majchrowicz 1975). Controls received equal volumes of the vehicle (water with addition of 6% sucrose and 14.7% Nestle Good Start Formula powder). Rats received from 8.5–10.5 ml of diet alone or diet plus Etoh at each dosing. Rats received a daily injection of either mifepristone (5 or 15 mg/kg) or vehicle 60-min prior to the 3:00 pm gavage.

Blood EtOH and plasma Cort levels

In the intragastric studies, blood was obtained daily by tail bleed approximately 2 hours after the initial daily EtOH administration for analysis of plasma EtOH and Cort levels. In addition, blood was obtained multiple (four) times per day from some animals in order to determine mean 24-hour Cort levels. Blood alcohol levels were determined using a standard alcohol dehydrogenase-based diagnostic kit (Sigma Chemical Co., St Louis, MO, USA) or gas chromatography with head-space sampling using a flame ionization detector. Plasma Cort was determined by 125I RIA (ICN Biomedicals kit, Costa Mesa, CA, USA). The sensitivity of the assay was ∼1.56 ng Cort per ml plasma.

Amino-cupric silver staining

Neurodegeneration was assessed using the amino-cupric-silver technique of De Olmos as performed by Switzer's Neuroscience Associates group (De Olmos, Beltramino & de Olmos de 1994; Switzer 2000). In brief, brains were removed from the skulls 24 hours after perfusion and immersed in a solution containing 20% glycerol and 2% dimethyl sulfoxide to prevent freeze artifacts and then embedded in a gelatin matrix in groups of 16. The brain matrices were then sectioned in the coronal plane by sliding microtome (40 µm in thickness). Every eighth section was then stained.

Sections were rinsed three times in deionized water and then placed for 4 days in an aqueous mixture of silver and copper nitrate, pyridine, and EtOH. Sections were then transferred through acetone, a diamine-silver solution, reduced in a weak formaldehyde solution, and bleached in a preparation of potassium ferricyanide and sodium borate (to remove unreduced silver). Sections were then mounted, dried, and counter-stained with neutral red. Coronal sections from the ventral hippocampus, both left and right side (two each side), were analyzed for degeneration counts at 6.00 and 6.32 mm posterior to bregma (Paxinos & Watson 1986). Degenerating cells were defined as dark, argyrophilic neurons with dendrites clearly visible. Dark objects not clearly identified as neurons were not counted. The number of counts was determined from two consecutive circular microscope fields at 20× magnification for each tissue section. Data for hippocampal degeneration are presented as counts per square millimeter by dividing total number of degenerating cells counted in eight microscope fields by the calculated area of the fields counted (1-mm diameter counting field). Two sections from both the left and right side of the EC were also counted at 7.00 and 7.32 mm posterior to bregma, in one microscope field at 20× magnification.

FJ-B staining

FJ-B was purchased from Histochem, Inc. (Jefferson, AR, USA) and used as a marker of degenerating neurons as previously described (Schmued & Hopkins 2000). FJ-B staining was used because it is equally sensitive but methodologically simpler than the previously described classical amino-cupric silver staining. It has been established that the results obtained by these two methods are highly correlated (Obernier, Bouldin & Crews 2002a) with our own personal observation. In brief, 3 hours after the last alcohol gavage, rats were anesthetized and perfused first with normal saline followed immediately with 4% paraformaldehyde in PBS. Horizontal 20-µm cryosections were obtained, allowing visualization in the same section of both EC and ventral hippocampus containing the DG. Sections were mounted directly on gelatin-coated slides, and stained for FJ-B according to the manufacturer's protocol. Dried slides were cleared in xylene and cover-slipped with Cytoseal (Richard-Allan Scientific, Kalamazoo, MI, USA). Alternate sets of sections were stained with cresyl violet in order to verify basic histology. Cell density analysis was on a Leica DMLB microscope using an FITC filter set, and BioQuant imaging software (R&M Biometrics, Nashville, TN, USA). Six horizontal sections containing the hippocampus and the EC, both left and right side, were analyzed for degenerating cells between 5.82 and 6.10 mm ventral from bregma (Paxinos & Watson 1998). Results for EC degeneration are depicted as counts per square mm by dividing the total number of degenerating cells found in 48 examined microscope fields, equivalent to 16.8 mm2 (single field area was 0.35 mm2 × 4 fields per side × 2 sides per section × 6 sections per animal, for a total of 16.8 mm2) with a 20× microscope objective. Degenerating granule cells of the entire DG were measured using a semiautomatic stereology system (Bioquant). Data for EC and DG degenerating cells are presented as number per square millimeter.

Drug administration

Mifepristone (RU38486, Cayman Chemical Company, Ann Arbor, MI, USA) was dissolved in propylene glycol by ultrasonication and injected intraperitoneally (i.p.) at the volume of 1 ml/kg. Animals received 0, 5, or 15 mg/kg of Mifepristone because previous work demonstrated that the antagonist exerts functional effects at these dose levels (Koenig & Olive 2004).

Statistical analysis

Data for argyrophilic or FJ-B positive cells or Cort levels are reported as mean values ± standard error. The data from each experiment was analyzed by one-way analysis of variance (ANOVA) and when a statistical significant threshold was reached, ANOVA was followed by Newman–Keuls post hoc test for pairwise comparisons. For all statistical analysis, differences between control and experimental groups were considered significant if P < 0.05.

RESULTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. FINANCIAL DISCLOSURES
  9. Authors Contribution
  10. References

Confirmation of plasma Cort levels in intact and treated animals

Plasma Cort levels depicted in Fig. 1a represent mean 24-hour Cort levels obtained from four samples per day (7:00 am, 1:00 pm, 7:00 pm, and 1:00 am) for 4 days in intact EtOH-treated or controls (Fig. 1a; main effect: F(1, 9) = 9.39, P < 0.02). These results serve as a 24-hour index of Cort levels for comparison with the pellet animals (Fig. 1b), because the Adx-pellet animals are devoid of a diurnal Cort rhythm. Figure 1b Cort levels are the result of samples taken 2 hours after the first daily dosing over the 4-day binge exposure. Cort levels in intact non-EtOH–treated animals (Fig 1a) were not significantly different from the Adx-basal Cort groups with or without EtOH treatment (Fig 1b). Cort levels in the Adx, EtOH-treated medium or high Cort pellet groups (Fig 1b) were not significantly different from the intact EtOH-treated rats (Fig 1a); however, Cort-levels in the medium or high Cort-Adx group (Fig. 1b) differed significantly from the basal-Adx Cort group with or without EtOH (Fig. 1b; main effect: F(3,42) = 79.77, P < 0.001).

image

Figure 1. (a) Mean 24-hour plasma cortcosterone (Cort) levels in intact ethanol (EtOH)-treated and controls. *P < 0.05 difference versus intact controls (n = 6 animals per group or 24 samples). (b) Plasma Cort levels in adrenalectomized (Adx) animals with basal, medium, or high Cort pellet replacement and EtOH treatment. Data are given as the mean ± standard error of the mean of ng Cort per ml of plasma. ***P < 0.001 Adx-medium Cort + EtOH and Adx-High Cort + EtOH versus Adx-Basal Cort + Control (Results represent data obtained from 11 to 12 animals per group or 44 to 48 Cort samples). For further details see Results and Materials and Methods

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Elevated Cort increases alcohol-induced neurodegeneration in the DG and EC

Degenerating neurons were not observed in the hippocampus or EC of intact or Adx-Cort replaced animals, unless exposed to EtOH. Degenerating cells were evident in the olfactory glomerular layer, the piriform cortex, the DG, and the EC. The most intense damage was found in the granule cells of the DG and the layer III pyramidal cells of the lateral EC (Figs 2 & 3). Both Adx-Cort replaced medium and high pellet animals treated with EtOH illustrated greater neurodegeneration than the Adx-basal Cort + Control-treated animals both in the hippocampal DG (Fig. 2; main effect: F(3,29) = 6.19, P < 0.003) and the EC (Fig. 3; main effect: F(3,29) = 7.35, P < 0.001). Post hoc analysis of EtOH treatment plus basal Cort versus EtOH plus medium or high Cort levels revealed a statistically significant increase in the EC (P < 0.05) whereas, only a marginally significant change was seen in the DG.

image

Figure 2. Elevated corticosterone (Cort) exacerbates binge ethanol (EtOH)-induced neurotoxicity in the dentate gyrus (DG) of the hippocampus of adrenalectomized (Adx) rats. Sections were stained by amino-cupric silver staining to visualize neurodegeneration. Panels (a), (b), and (c) show photomicrographs of degeneration in the dentate gyrus (DG) of rats without EtOH (a), with EtOH (b) and EtOH-Cort groups (c). (d) Graphical representation of argyrophilic cell quantitation in the DG. Data represent mean values of argyrophilic positive cells/mm2 ± standard error of the mean. Significant difference from control *P < 0.05, **P < 0.01 (results represent data obtained from 7 to 11 animals). For further details see Results and Materials and Methods

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image

Figure 3. Elevated corticosterone (Cort) exacerbates binge ethanol (EtOH)-induced neurotoxicity in the entorhinal cortex (EC) of adrenalectomized (Adx) rats. Sections were stained by amino-cupric silver staining to visualize neurodegeneration. Panels (a), (b), and (c) show photomicrographs of degeneration in the EC of rats without EtOH (a), with EtOH (b), and EtOH-high Cort groups (c). (d) Graphical representation of argyrophilic cell quantitation in the entorhinal cortex. Data represent mean values of argyrophilic positive cells/mm2 ± standard error of the mean. Significant difference from control *P < 0.05, **P < 0.01 (results represent data obtained from 7 to 11 animals). For further details, see Results and Materials and Methods

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Mifepristone treatment reduces alcohol-induced neurodegeneration

Alcohol treatment induced substantial neuronal cell death in the DG as well as the EC. In the DG, neurotoxicity was reduced by daily treatment with mifepristone (Fig. 4; main effect: F(3,29) = 3.73, P < 0.03). Post hoc analysis showed that animals treated with the 15 mg/kg dose of mifepristone compared with alcohol only significantly reduced the level of neurodegeneration (P < 0.05). Alcohol administration caused a similar neurotoxicity in the EC, in particular, in layer III pyramidal cells. However, even though there was a main effect of alcohol similar to that seen in the DG, there was no statistically significant effect of mifepristone (15 mg/kg/day) treatment (P = 0.078) on EC neurotoxicity (Fig. 5; main EtOH effect: F(3,29) = 6.11, P < 0.003). In areas other than the hippocampal DG granule cells and the EC Layer III pyramidal cells, FJ-B positive cells were too few to be reliably quantified; therefore, it was not possible to observe any neuroprotective effects of mifepristone in other areas of the rat brain. In summary, the FJ-B results indicate that binge-like alcohol exposure produced neuronal cell death both in the DG and EC, and treatment with mifepristone once daily throughout the binge procedure resulted in a neuroprotective effect in the DG, whereas, just a trend to decrease the number of neurodegenerating cells in the EC was observed. It is of interest to note that the observed neuroprotective or blocking effect of mifepristone was apparent even though mifepristone caused a dose-related enhancement of Cort levels on top of the already EtOH elevated levels (Fig. 6).

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Figure 4. Binge ethanol (EtOH)-induced neurotoxicity in intact animals in the dentate gyrus (DG) of the hippocampus, and its reduction by the glucocorticoid type II receptor antagonist mifepristone (Mif). Sections were stained by fluoro-jade B (FJ-B) to visualize neurodegeneration. Panels (a), (b), and (c) show representative sections visualizing labeled neurons in animals without EtOH (a), with EtOH (b), and EtOH +15 mg Mif/day (c). Panel (d) shows quantitation of histological data, demonstrating EtOH-induced neurodegeneration and its reduction by Mif at 15 mg/kg/day. Data are given as the mean number of FJ-B positive cells/mm2 ± standard error of the mean. *P < 0.01, EtOH + 15 mg/kg Mif versus EtOH + 0 mg/kg Mif (results represent data obtained from 7 to 9 animals). For further details see Results and Materials and Methods

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image

Figure 5. Binge alcohol-induced neurotoxicity in the entorhinal cortex (EC), demonstrates only a neuroprotective trend in the presence of the glucocorticoid type II receptor antagonist mifepristone (Mif). Sections were stained by fluoro-jade B (FJ-B) to visualize neurodegeneration. Panels (a), (b), and (c) show representative sections visualizing labeled neurons in animals without EtOH (a), with EtOH (b), and EtOH +15 mg Mif/day groups (c). Panel (d) shows quantitation of histological data, demonstrating EtOH-induced neurodegeneration. Data are given as the mean number of FJ-B positive cells/mm2 ± standard error of the mean; #P < 0.07 EtOH + 15 mg/kg Mif versus EtOH + 0 mg/kg Mif (results represent data obtained from 7 to 9 animals). For further details see Results and Materials and Methods

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Figure 6. Activation of the hypothalamic-pituitary-adrenal axis by binge-like EtOH treatment and enhancement by the glucocorticoid type II receptor antagonist mifepristone (Mif). Data are given as the mean ± standard error of the mean of ng of corticosterone per ml of plasma. Significant difference from control **P < 0.01, ***P < 0.001 (results represent data obtained from 8 to 9 animals or 32 to 36 Cort samples). For further details see Results and Materials and Methods

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Mifepristone enhances EtOH-induced activation of the HPA axis

A robust activation of the HPA axis occurred in response to binge alcohol exposure. Cort levels were determinated from daily plasma samples obtained 2 hours after lights off. ANOVA showed a highly significant main effect of the EtOH treatment [F(3,28) = 8.20, P < 0.001]. Mifepristone treatment did not induce a statistically significant increase of Cort levels over EtOH treatment alone (post hoc analysis, P > 0.05), although a trend toward increased Cort response was clearly observed following treatment with mifepristone at the 15 mg/kg dose (Fig. 6).

DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. FINANCIAL DISCLOSURES
  9. Authors Contribution
  10. References

Excessive alcohol exposure in both laboratory animals and clinical studies leads to activation of the HPA axis (stress axis) and site-specific neurodegeneration in the central nervous system. Memory, learning and other cognitive abilities are compromised in alcoholics and EtOH exposed animals. Even after protracted periods of abstinence, certain deficits remain, providing evidence for persistent damage. Morphological and structural studies reveal the hippocampus and cerebellum to be preferentially damaged by excessive alcohol consumption. The availability of a binge-like alcohol intoxication model that produces region-specific damage over a 4-day time frame has enabled a practical analysis of the events responsible for the observed alcohol-induced neurodegeneration.

In earlier non-EtOH studies of brain cytotoxicity, elevated GCs have been shown to energetically endanger or weaken hippocampal neurons, so that in the presence of coincident insults, the observed damage is worsened (Sapolsky et al. 1986). It is envisioned in intoxicating, EtOH-exposure studies that the endangering or weakening effects of EtOH act in concert with elevated stress axis GCs. EtOH alone contributes to cytotoxicity, but the indirect or EtOH-induced secondary effects on humoral (elevated GCs) and nutritional (energy availability and utilization) events likely act as coincident insults. In light of the clear evidence that the binge-type alcohol model produces ongoing elevated Cort levels throughout each binge treatment, it was germane to determine the relative contribution of the elevated Cort levels versus that of the direct effect of EtOH exposure on cytotoxicity.

We carried out a series of endocrine organ ablation and hormone replacement studies, aimed at defining the intrinsic regulatory importance of normal to elevated Cort levels concident with EtOH intoxication. We first evaluated the possibility that elevated plasma levels of Cort contribute to the neurotoxic effects of a binge-like alcohol exposure, as assessed in EtOH-challenged animals subjected to narrowly maintained plasma Cort concentrations in Adx animals with implanted Cort pellets. Neuronal cell death was assessed by counting the number of argyophilic positive hippocampal DG granule cells and entorhinal cortical cells, two brain areas previously shown to be sensitive to alcohol-induced neurotoxicity (Collins et al. 1996; Crews et al. 2000; Hamelink et al. 2005). Because we found that supra-normal Cort replacement in the Etoh-treated animals was associated with enhanced EtOH-induced neurotoxicity, we then examined the causal role of EtOH-induced Cort elevations for neurotoxicity in the binge-like intoxication model by examining whether GR blockade with the specific antagonist mifepristone (RU38486) would result in reduced numbers of dead or dying DG and EC cells as detected by FJ-B staining. Several groups of animals were Adxed and given replacement Cort level therapy designed to achieve circulating Cort levels from non-stressed intact animals with basal 24-hour mean Cort levels of approximately 40 ng/ml or less up to a 24-hour mean stressed level of 150 ng/ml, while being challenged with intoxicating doses of EtOH in the 4-day binge model (Fig. 1a & b). The goal was to achieve a range of plasma Cort levels that was close to the mean 24-hour basal level in one group (non-stressed) through a range of Cort concentrations in other groups that would approximate binge-type alcohol-induced levels (stressed). Given that implanted Cort pellets achieve a constant 24-hour circulating concentration of Cort without a diurnal rhythm, it seemed logical to use mean daily Cort levels as a yardstick of daily Cort exposure. It was not possible to superimpose binge-EtOH treatment on Adx animals without a minimal circulating or permissive level of Cort, because in the absence of minimal Cort replacement, animals do not survive the binge EtOH challenges. In preliminary studies, non-EtOH–treated, Adx animals with mean Cort levels maintained from ∼40 to ∼150 ng/ml produced no visible CNS neurodegeneration. In contrast, binge-like EtOH exposure resulted in neurotoxity in all treatment groups. Furthermore, EtOH-induced neurodegeneration was enhanced at Cort levels ∼2-fold higher (medium Cort) than 24-hour mean basal levels, but did not increase further with an additional doubling of plasma Cort levels (high Cort). Both the alcohol-treated medium and high Cort pellet groups had higher argyrophilic cell counts in the EC and DG granule cells than the intact EtOH-treated basal Cort level group.

Our neurodegeneration results are consistent with the notion that a narrow range of circulating Cort is required to optimize each Cort-dependent effect or system (Akana et al. 1985). It would appear that mean 24-hour Cort plasma levels of around 75 ng/ml or higher are above permissive or optimal replacement concentrations for the brain regions examined, because these Cort levels exacerbated alcohol-induced neurotoxicity in the binge model. In order to directly examine the hypothesis that elevated GCs act as a coincident insult with EtOH to enhance neurotoxicity, a second study was performed in which the important regulatory GR was blocked with increasing concentrations of mifepristone, a specific GR blocker. Daily treatment with mifepristone in the rodent EtOH binge model resulted in a dose-related decrease in the number of degenerating neurons in both the EC and DG granule cells of the hippocampus, although this reduction was statistically significant only in the hippocampus. Together, our data obtained using adrenal gland ablation, Cort level replacement and GR blockade provides consistent and compelling evidence that elevated Cort levels in the rodent in the presence of intoxicating levels of EtOH enhances EtOH-induced neurodegeneration. Furthermore, we have recently shown that preventing EtOH-induced neurotoxicity leads to a rescue of the cognitive impairment that is otherwise associated with (Cippitelli et al. 2010a,b) binge EtOH treatment in the rodent model. Therefore, from a pharmacotherapeutic standpoint, it is likely that keeping GC levels at permissive or low levels, or partially blocking brain GRs might reduce EtOH-induced neurotoxicity and slow the insidious, cognitive decline in binge-type alcoholics or EtOH-dependent patients.

Although the findings of these studies are consistent and informative within themselves, they do not lend themselves to a concise understanding of the cellular and molecular mechanisms through which chronic EtOH exposure directly or indirectly causes brain damage. Borrowing from a number of previous studies and recent neurobiological reviews (Crews, Zou & Qin 2011; Frank, Watkins & Maier 2011; Kelley & Dantzer 2011; Yakovleva et al. 2011) that focus on EtOH-stress axis activation with concomitant, manifold neurobiological effects, it is clear that multiple cascades that define excitotoxicity, proinflammation, generation of reactive oxygen species, and oxidative stress are pivotal elements in EtOH-induced neurotoxicity. In summary, stressors (e.g. EtOH, GCs) prime the host neuroinflammatory response and chronic exposure to EtOH in vivo activates microglia and astrocytes in the brain, which increases proinflammatory cytokines. Stress, EtOH and hormonal signals activate the oxidation sensitive transcription factor NF-kB, which binds to DNA and increases the transcription of many genes including chemokines, cytokines, oxidases, and proteases. Through the milieu of these events, chronic EtOH exposure in concert with elevated GCs lead to the observed neurodegeneration in our rodent episodic or binge model of neurodegeneration.

Acknowledgements

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. FINANCIAL DISCLOSURES
  9. Authors Contribution
  10. References

This work was supported by the National Institute on Alcohol Abuse and Alcoholism (NIAAA)-Intramural Research Program (IRP). We thank Dr. Melanie Schwandt and Karen Smith for careful revision of the paper.

FINANCIAL DISCLOSURES

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. FINANCIAL DISCLOSURES
  9. Authors Contribution
  10. References

The authors declare no biomedical financial interests or potential conflicts of interest.

Authors Contribution

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. FINANCIAL DISCLOSURES
  9. Authors Contribution
  10. References

AC and RE were responsible for conceiving the idea and writing the manuscript. All other authors provided critical reviews for final version of manuscript.

References

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. FINANCIAL DISCLOSURES
  9. Authors Contribution
  10. References
  • Akana SF, Cascio CS, Shinsako J, Dallman MF (1985) Corticosterone: narrow range required for normal body and thymus weight and ACTH. Am J Physiol 249:R527R532.
  • Bowden SC, Mccarter RJ (1993) Spatial memory in alcohol-dependent subjects—using a push-button maze to test the principle of equiavailability. Brain Cogn 22:5162.
  • Cippitelli A, Damadzic R, Frankola K, Goldstein A, Thorsell A, Singley E, Eskay RL, Heilig M (2010a) Alcohol-induced neurodegeneration, suppression of transforming growth factor-beta, and cognitive impairment in rats: prevention by Group II metabotropic glutamate receptor activation. Biol Psychiatry 67:823830.
  • Cippitelli A, Zook M, Bell L, Damadzic R, Eskay RL, Schwandt M, Heilig M (2010b) Reversibility of object recognition but not spatial memory impairment following binge-like alcohol exposure in rats. Neurobiol Learn Mem 94:538546.
  • Collins MA, Corso T, Neafsey EJ (1996) Neuronal degeneration in rat cerebrocortical and olfactory regions during subchronic ‘Binge’ intoxication with ethanol: possible explanation for olfactory deficits in alcoholics. Alcohol Clin Exp Res 20:284292.
  • Corso TD, Mostafa HM, Collins MA, Neafsey EJ (1998) Brain neuronal degeneration caused by episodic alcohol intoxication in rats: effects of nimodipine, 6,7-dinitro-quinoxaline-2,3-dione, and MK-801. Alcohol Clin Exp Res 22:217224.
  • Crews FT, Braun CJ, Hoplight B, Switzer RC, Knapp DJ (2000) Binge ethanol consumption causes differential brain damage in young adolescent rats compared with adult rats. Alcohol Clin Exp Res 24:17121723.
  • Crews FT, Zou J, Qin L (2011) Induction of innate immune genes in brain create the neurobiology of addiction. Brain Behav Immun 25:S4S12.
  • De Olmos JS, Beltramino CA, de Olmos de LS (1994) Use of an amino-cupric-silver technique for the detection of early and semiacute neuronal degeneration caused by neurotoxicants, hypoxia, and physical trauma. Neurotoxicol Teratol 16:545561.
  • Frank MG, Watkins LR, Maier SF (2011) Stress and glucocorticoid-induced priming of neuro inflammatory responses: potential mechanisms of stress-induced vulnerability to drugs of abuse. 25:S21S28.
  • Gould E, Tanapat P (1999) Stress and hippocampal neurogenesis. Biol Psychiatry 46:14721479.
  • Hamelink C, Hampson A, Wink DA, Eiden LE, Eskay RL (2005) Comparison of cannabidiol, antioxidants, and diuretics in reversing binge ethanol-induced neurotoxicity. J Pharmacol Exp Ther 314:780788.
  • Hortnagl H, Berger ML, Havelec L, Hornykiewicz O (1993) Role of glucocorticoids in the cholinergic degeneration in rat hippocampus induced by ethylcholine aziridinium (AF64A). J Neurosci 13:29392945.
  • Jarrard LE (1993) On the role of the hippocampus in learning and memory in the rat. Behav Neural Biol 60:926.
  • Joels M, Dekloet ER (1994) Mineralocorticoid and glucocorticoid receptors in the brain—implications for ion permeability and transmitter systems. Prog Neurobiol 43:136.
  • Johnson RF, Moore RY, Morin LP (1989) Lateral geniculate lesions alter circadian activity rhythms in the hamster. Brain Res Bull 22:411422.
  • Kelley KW, Dantzer R (2011) Alcoholism and inflammation: neuroimmunology of behavioral and mood disorders. Brain Behav Immun 25:S13S20.
  • Koenig HN, Olive MF (2004) The glucocorticoid receptor antagonist mifepristone reduces ethanol intake in rats under limited access conditions. Psychoneuroendocrinology 29:9991003.
  • Landfield PW, Baskin RK, Pitler TA (1981) Brain aging correlates—retardation by hormonal-pharmacological treatments. Science 214:581584.
  • Magarinos AM, Somoza G, De Nicola AF (1987) Glucocorticoid negative feedback and glucocorticoid receptors after hippocampectomy in rats. Horm Metab Res 19:105109.
  • Majchrowicz E (1975) Induction of physical dependence upon ethanol and the associated behavioral changes in rats. Psychopharmacologia 43:245254.
  • McEwen BS, Dekloet ER, Rostene W (1986) Adrenal-steroid receptors and actions in the nervous-system. Physiol Rev 66:11211188.
  • McIntosh LJ, Sapolsky RM (1996) Glucocorticoids may enhance oxygen radical-mediated neurotoxicity. Neurotoxicology 17:873882.
  • Mulholland PJ, Self RL, Hensley AK, Little HJ, Littleton JM, Prendergast MA (2006) A 24 hours corticosterone exposure exacerbates excitotoxic insult in rat hippocampal slice cultures independently of glucocorticoid receptor activation or protein synthesis. Brain Res 1082:165172.
  • Mulholland PJ, Stepanyan TD, Self RL, Hensley AK, Harris BR, Kowalski A, Littleton JM, Prendergast MA (2005) Corticosterone and dexamethasone potentiate cytotoxicity associated with oxygen-glucose deprivation in organotypic cerebellar slice cultures. Neuroscience 136:259267.
  • Obernier JA, Bouldin TW, Crews FT (2002a) Binge ethanol exposure in adult rats causes necrotic cell death. Alcohol Clin Exp Res 26:547557.
  • Obernier JA, White AM, Swartzwelder HS, Crews FT (2002b) Cognitive deficits and CNS damage after a 4-day binge ethanol exposure in rats. Pharmacol Biochem Behav 72:521532.
  • Oscar-Berman M, Hutner N, Bonner RT (1992) Visual and auditory spatial and nonspatial delayed-response performance by Korsakoff and non-Korsakoff alcoholic and aging individuals. Behav Neurosci 106:613622.
  • Paxinos G, Watson C (1986) The Rat Brain in Stereotaxic Coordinates. San Diego: Academic Press.
  • Paxinos G, Watson C (1998) The Rat Brain in Stereotaxic Coordinates. San Diego: Academic Press.
  • Rivier C, Bruhn T, Vale W (1984) Effect of ethanol on the hypothalamic-pituitary-adrenal axis in the rat—role of corticotropin-releasing factor (Crf). J Pharmacol Exp Ther 229:127131.
  • Sapolsky RM (1985) Glucocorticoid toxicity in the hippocampus: temporal aspects of neuronal vulnerability. Brain Res 359:300305.
  • Sapolsky RM, Krey LC, McEwen BS (1986) The neuroendocrinology of stress and aging—the glucocorticoid cascade hypothesis. Endocr Rev 7:284301.
  • Sapolsky RM, Pulsinelli WA (1985) Glucocorticoids potentiate ischemic injury to neurons: therapeutic implications. Science 229:13971400.
  • Schmued LC, Hopkins KJ (2000) Fluoro-Jade B: a high affinity fluorescent marker for the localization of neuronal degeneration. Brain Res 874:123130.
  • Starkman MN, Gebarski SS, Berent S, Schteingart DE (1992) Hippocampal formation volume, memory dysfunction, and cortisol levels in patients with Cushing's syndrome. Biol Psychiatry 32:756765.
  • Stein-Behrens BA, Elliott EM, Miller CA, Schilling JW, Newcombe R, Sapolsky RM (1992) Glucocorticoids exacerbate kainic acid-induced extracellular accumulation of excitatory amino acids in the rat hippocampus. J Neurochem 58:17301735.
  • Switzer RC, III (2000) Application of silver degeneration stains for neurotoxicity testing. Toxicol Pathol 28:7083.
  • Thiagarajan AB, Mefford IN, Eskay RL (1989) Single-dose ethanol administration activates the hypothalamic-pituitary-adrenal axis: exploration of the mechanism of action. Neuroendocrinology 50:427432.
  • Watanabe Y, Gould E, McEwen BS (1992) Stress induces atrophy of apical dendrites of hippocampal CA3 pyramidal neurons. Brain Res 588:341345.
  • Yakovleva T, Bazov I, Watanabe H, Hauser KF, Bakalkin G (2011) Trannscriptional control of maladaptive and protective responses in alcoholics: a role of the NF-kB system.
  • Zou JY, Martinez DB, Neafsey EJ, Collins MA (1996) Binge ethanol-induced brain damage in rats: effect of inhibitors of nitric oxide synthase. Alcohol Clin Exp Res 20:14061411.