The hypothalamic-pituitary-thyroid axis in critical illness

In severe illness, profound changes occur in the hypothalamic-pituitary-thyroid axis. The observed decrease in serum concentration of both thyroid hormones and thyrotropin (TSH) are not compatible with a negative feedback loop and suggest a major change in setpoint regulation of the hypothalamic-pituitary-thyroid axis. This is supported by post mortem studies showing a decreased expression of thyrotropin-releasing hormone in the hypothalamic paraventricular nucleus of patients with a decreased serum T3 level. In critical illness, serum T3 may even become undetectable without giving rise to an elevated concentration of serum TSH. It is currently not clearly established whether this reflects an adaptation of the organism to illness or instead a potentially harmful condition leading to hypothyroidism at tissue level. There is thus a need for randomized clinical trials in critically ill patients to investigate whether they may benefit from a normalization of thyroid hormone concentration. Recent clinical studies in these patients involving the administration of hypothalamic peptides open up new ways of achieving this.     

Effect of food deprivation and altered thyroid status on the hypothalamic-pituitary-thyroid axis in the rat.

Propylthiouracil (PTU) was administered to rats for different lengths of time with or without food deprivation on the last 2 days. Within 4 days of PTU treatment peripheral 3,5,3'-tri-iodothyronine (T3) fell to low levels and beta-subunit of thyroid-stimulating hormone (beta-TSH) mRNA increased significantly in the anterior pituitary. Pro-thyrotrophin-releasing hormone (pro-TRH) mRNA in the hypothalamic paraventricular nucleus (PVN) increased significantly in the control group of animals by 8 days and in the food-deprived group by day 12; the increment of pro-TRH mRNA in the food-deprived group on day 12 was significantly less than that in the control group. In a second study, animals were treated with intraperitoneal injections of T3 with or without the food deprivation. After 4 days of T3 treatment, peripheral T3 levels were markedly increased and pro-TRH mRNA in the PVN and beta-TSH mRNA in the anterior pituitary were significantly reduced. Food deprivation had no additional suppressive effect. These studies confirm that the predominant effect of food deprivation on the thyroid axis is at the hypothalamic or suprahypothalamic level and that it can, at least in part, overcome the increase in TRH mRNA due to diminished T3 feedback.     

Chronic daily ethanol and withdrawal: 1. Long-term changes in the hypothalamo-pituitary-adrenal axis

BACKGROUND: Hypothalamo-pituitary-adrenal (HPA) function has been demonstrated to be compromised for weeks and even months after alcoholics cease ethanol consumption. Because nonalcoholic subjects with family history-associated increased risk for alcoholism also exhibit compromised HPA function, it is not clear whether defects in the HPA axis of abstinent alcoholics reflect a preexisting condition that may be responsible for increased risk for alcohol abuse versus a persisting adaptational change in response to prolonged alcohol abuse. Consequently, we investigated whether chronic daily ethanol consumption and withdrawal by male Sprague Dawley rats would induce persistent HPA changes consistent with those demonstrated in abstinent alcoholics. METHODS AND RESULTS: In an initial experiment in which ethanol (5%, w/v) was incrementally introduced to liquid diet over a 1 week period followed by 4 weeks of chronic ethanol consumption, not only ethanol-treated rats but also pair-fed control rats exhibited decreased (p < 0.05 vs. ad-libitum-fed controls) anterior pituitary pro-opiomelanocortin (POMC) mRNA concentrations and associated decreases in plasma corticosterone and adrenocorticotropin (ACTH) levels for at least 3 weeks after gradual withdrawal of ethanol from the diet. Pair-feeding-induced decreases (p < 0.05) in thymus and spleen weights suggested that the pair-fed controls were likely stressed in this model, probably in response to the marked and irregular suppression of liquid diet consumption immediately after introduction of ethanol. Consequently, a second model was developed in which ethanol was introduced to the liquid diet much more gradually (i.e., over 3 weeks). In contrast with the rapid ethanol-introduction model, this more prolonged ethanol introduction followed by 4 weeks of chronic daily ethanol consumption increased plasma corticosterone levels (p < 0.05), increased adrenal gland weight (p < 0.05), and decreased thymus and spleen weights (both p < 0.01) without altering any of these parameters in the pair-fed controls. Three weeks after gradual withdrawal of ethanol from the diet, anterior pituitary POMC mRNA concentrations were suppressed (p < 0.05) and thymus and spleen weights were increased (p < 0.05) versus both pair-fed and ad-libitum-fed controls, accompanied by trends for decreased basal plasma corticosterone and adrenal weights. CONCLUSIONS: Chronic daily ethanol treatment induced changes in the HPA axis that persisted for at least 3 weeks after complete cessation of ethanol consumption. These persistent alterations in the HPA axis are similar to the aberrant HPA regulation of abstinent alcoholics, sons of alcoholics, Lewis rats, and individuals who suffer from posttraumatic stress disorder and some types of depression, that is, categories of individuals who all exhibit increased risk for high ethanol consumption. Thus, these chronic daily ethanol-induced persistent changes in the HPA axis may have significant roles in alcohol abstinence syndrome and may increase vulnerability to relapse.     

Chronic ethanol enhances adenosine antiadrenergic actions in the isolated rat heart

BACKGROUND: Chronic alcohol consumption elicits an increase in catecholamine release, which may be detrimental to heart function. Adenosine attenuates adrenergic stimulation via an adenosine receptor-mediated antiadrenergic action. This study investigated the effect of ethanol on adenosine antiadrenergic actions and adenosine release in the rat heart. METHODS: Rats were pair-fed a liquid diet with or without ethanol for 4 weeks or 8 months. Hearts were isolated for determination of contractile function, and coronary effluents were collected for adenosine content. Dose-response relationships for phenylisopropyladenosine (PIA) were determined for hearts adrenergically stimulated by isoproterenol. Experiments were also conducted with normal hearts with or without ethanol (25 mM) administered acutely. The effect of PIA on adenylyl cyclase activities of adrenergic-stimulated crude membrane preparations obtained from alcoholic and nonalcoholic hearts was determined. RESULTS: Acute ethanol reduced basal adenosine release by 39%, but it did not significantly decrease adenosine release during adrenergic stimulation. In hearts chronically treated with ethanol for 4 weeks, adenosine release values before and during adrenergic stimulation were significantly reduced from control values. After 8 months of ethanol, adenosine release was similar with or without adrenergic stimulation. PIA 50% inhibiting concentration (IC50) values for contractile function were reduced from pair-fed control values. Acute ethanol did not significantly change the PIA IC50 value. Chronic ethanol reduced the PIA IC50 for adenylyl cyclase by 96%. CONCLUSIONS: Chronic ethanol treatment increases the antiadrenergic action of adenosine by mechanisms that seem independent of changes in adenosine concentration. Therefore, adenosine-induced cardioprotection against increased catecholamine stimulation is enhanced by ethanol.     

Chronic ethanol consumption reduces delta-and mu-opioid receptor-stimulated G-protein coupling in rat brain

BACKGROUND: Ethanol consumption is thought to enhance the release of endogenous opioids acting at opioid receptors (ORs) in the central nervous system. Prior studies have shown that chronic ethanol consumption in alcohol-preferring rats uncouples mu-ORs from Gi proteins. The purpose of this study was to investigate the potential for uncoupling of the delta- and the mu-OR after chronic ethanol consumption in a nonpreferring rat strain. METHODS: We used radiohistochemical methods to study mu- and delta-OR-stimulated G-protein coupling in brain tissue of rats ingesting liquid diets containing 6.7% ethanol (v/v) for 16 days, as compared with 0% ethanol pair-fed control rats. Sections of brain from pair-fed and ethanol-treated rats were incubated with guanylyl 5'-[gamma-[35S]-thio]-triphosphate ([35S]-GTPgammaS) in the absence and presence of d-Pen2,d-Pen5 enkephalin (DPDPE), a delta-OR agonist, or Tyr-d-Ala-Gly-N(me)Phe-Gly-ol-enkephalin (DAMGO), a mu-OR agonist. RESULTS: DPDPE significantly stimulated [35S]-GTPgammaS binding in the hippocampal dentate gyrus (DG), CA1, cerebellum, and inferior colliculus of untreated pair-fed controls. By contrast, DPDPE-stimulated [35S]-GTPgammaS binding was reduced significantly in those brain regions in the ethanol-consuming group. DAMGO stimulated [35S]-GTPgammaS binding in cortex, caudate, nucleus accumbens, DG, CA1, and superior and inferior colliculi, whereas the DG, CA1, and colliculi showed a significant reduction of binding after chronic ethanol. Basal [35S]-GTPgammaS binding was not different between the two diet groups.CONCLUSIONS These data are the first to demonstrate functional uncoupling of delta-ORs from G proteins after chronic ethanol consumption. Uncoupling may result from modulation of receptors, possibly by internalization or phosphorylation. Alterations in functional coupling of both delta- and mu-ORs and subsequent effects may contribute to continued ethanol consumption.     

Increased ethanol drinking after repeated chronic ethanol exposure and withdrawal experience in C57BL/6 mice

BACKGROUND: The development of dependence may have significant motivational consequences regarding continued use and abuse of ethanol. We have developed a mouse model of ethanol dependence and repeated withdrawals that demonstrates sensitization of seizures and other symptoms of withdrawal. It is unclear whether such experience influences ethanol drinking behavior. The present series of experiments were designed to examine whether repeated cycles of chronic ethanol exposure and withdrawal has an impact on subsequent motivation to voluntarily self-administer ethanol. METHODS: With the use of a modified sucrose-fading procedure, adult male C57BL/6J mice were trained to drink 15% (v/v) ethanol in a limited access procedure (2 hr/day). The animals were not food or water deprived at any time during the experiments. Once stable baseline intake was established, mice were exposed to four cycles of 16 hr of ethanol vapor (or air) in inhalation chambers separated by 8-hr periods of withdrawal. At 32 hr after the last cycle of ethanol exposure, all mice were tested for ethanol intake under limited access conditions for 5 consecutive days. The animals then received a second series of chronic ethanol exposure and withdrawal followed by another 5-day test period for ethanol drinking. RESULTS: Stable daily baseline intake was established in mice that drank 15% ethanol combined with 5% sucrose (experiment 1), 15% ethanol alone (experiment 2), 5% sucrose alone (experiment 3), or 15% ethanol when presented as a choice with water (experiment 4). After repeated cycles of chronic ethanol exposure and withdrawal experience, consumption of ethanol solutions increased over baseline levels and in comparison with control (air-exposed) groups. However, sucrose consumption did not change in mice that were trained to drink 5% sucrose. The increase in ethanol consumption after chronic ethanol exposure and withdrawal experience resulted in a significant increase in resultant blood ethanol levels. CONCLUSIONS: Once the positive reinforcing properties of ethanol were established, chronic ethanol exposure and withdrawal experience resulted in a significant increase in voluntary ethanol drinking that yielded a >2-fold increase in resultant blood ethanol levels. This increase in ethanol intake occurred whether ethanol was presented in combination with sucrose, alone (unadulterated), or as a choice with tap water. Furthermore, this effect seems to be selective for ethanol in that animals that were trained to drink a sucrose solution did not exhibit a change in their intake after similar chronic ethanol exposure. As such, this model may be useful in studying the mechanisms and conditions in which chronic ethanol treatment influences motivation to resume drinking after a period of abstinence (relapse).     

Chronic ethanol exposure impairs neuronal nitric oxide synthase in the rat intestine

BACKGROUND: Nitric oxide (NO), synthesized by neuronal (nNOS), endothelial (eNOS), and inducible (iNOS) nitric oxide synthases, plays an essential role in the physiological functions of the gastrointestinal (GI) tract. Chronic ethanol intake has been shown to interfere with several of these physiological functions, leading to the pathological alterations observed in alcoholic individuals. Our aim therefore was to investigate the effects of chronic ethanol consumption on NOS isoforms in different GI segments. METHODS: Rats received either 20% aqueous ethanol solution or water for 8 weeks. Tissue samples of the duodenum, jejunum, ileum, and colon of the rats were used for measurement of the NOS activity, protein content, and nNOS immunohistochemistry. Anti-HuC/D immunohistochemistry was used to determine the total number of neurons. RESULTS: Measurement of the physiological constitutive NOS (cNOS) activity revealed a 20 times higher activity in the colon than in the small intestine and after chronic ethanol treatment demonstrated a significant decrease in the jejunum, ileum, and colon, while in the duodenum it remained unchanged compared with the control group. The physiological iNOS activity was higher in the ileum and colon than in the duodenum and jejunum, and these levels were not significantly affected by ethanol. Neuronal nitric oxide synthase immunohistochemistry revealed a significant decrease in the numbers of immunostained cells in all investigated intestinal segments, while the total number of myenteric neurons remained constant. The nNOS protein content measured by Western blotting indicated a significant decrease in the colon after ethanol consumption, while in other intestinal segments change was not detectable. CONCLUSIONS: This study has demonstrated for the first time that chronic ethanol consumption has a differential effect on NOS activity, NOS protein content, and the number of nitrergic neurons in different intestinal segments, suggesting that chronic ethanol administration affects the NO pathways in the enteric nervous system.     

Chronic ethanol increases ganglioside sialidase activity in rat leukocytes, erythrocytes, and brain synaptosomes

In view of the chronic alcohol-mediated pathological changes in various sialic acid-deficient glycoconjugates and the potential importance of sialidase in the generation of these glycoconjugates in the blood compartment and in the brain, we have investigated the effects of chronic ethanol feeding for 8 weeks on ganglioside sialidase activities in rat blood and brain. Ganglioside sialidase activity in erythrocytes (whether expressed as units/mg of protein or units/ml of blood) was 1.37- to 1.40-fold higher (p < 0.01) in the ethanol-fed group than in the control group. On the other hand, the same ethanol treatment increased sialidase activity in the leukocyte soluble fraction by 2.50- to 2.60-fold (p < 0.01) and by 1.61- to 1.63-fold (p < 0.01) in the leukocyte particulate fraction, compared with the control group. More importantly, most of the blood compartment sialidase activity was localized in the leukocytes particulate fraction (80 to 86% of total blood activity). Similarly, chronic ethanol treatment increased brain synaptosomal sialidase activity (whether expressed as units/gram of brain or units/mg of protein) 2.16- to 2.43-fold (p < 0.01). In contrast, brain lysosomal sialidase was not significantly altered by ethanol treatment, even though the major proportion of the brain sialidase activity was localized in the lysosomes. The proportion of synaptosomal sialidase activity as the percentage of total brain sialidase activity increased markedly from 13% in the control group to 24% in the ethanol group. Thus, chronic ethanol-mediated increases in sialidase activity in the leukocytes and brain synaptosomes could account for alterations in the ganglioside status of the animal and consequent adverse effects of chronic ethanol on behavioral and pathological changes.     

Chronic ethanol ingestion increases aortic endothelial nitric oxide synthase expression and nitric oxide production in the rat

Background:  Chronic alcohol consumption perturbs cellular function in a variety of organ systems. Previous studies have suggested that moderate alcohol consumption reduces vascular disease, whereas heavier alcohol consumption may worsen it. The mechanisms for these vascular effects of chronic alcohol ingestion continue to be defined and constitute the focus of this study.
Methods:  Male Sprague Dawley rats were fed an isocaloric, Lieber-Decarli liquid diet containing either ethanol (36% calories) or Maltose–Dextrin (substituted for ethanol) for 6 weeks. Telemetric blood pressure measurements were taken before and after ethanol feeding. After the rats were killed, the aortas were analyzed for endothelial nitric oxide (NO) synthase expression and NO production.
Results:  Chronic ethanol ingestion decreased mean arterial pressure and increased aortic NO production as demonstrated by direct ex vivo measurements using iron diethyldithio-carbamic acid as well as analysis of nitrosyl-hemoglobin (NO-Hb) levels. Consistent with these assays of vascular NO production, endothelium-dependent relaxation responses to acetycholine (Ach) were enhanced in ethanol-fed animals. Aortic endothelial nitric oxide synthase expression was also increased by chronic ethanol ingestion.
Conclusions:  These findings demonstrate that a regimen of chronic alcohol ingestion in the rat produced generally salutary effects in the systemic vasculature following a 6-week treatment regimen. These findings extend previous in vitro studies to demonstrate that alcohol has potent effects on vascular endothelial nitric oxide synthase expression, NO production, and vascular function. Consistent with previous reports, these findings confirm that alcohol-induced alterations in the production of reactive nitrogen species play an important role in the pathogenesis of alcohol-mediated tissue effects.     

Development of ethanol withdrawal-related sensitization and relapse drinking in mice selected for high- or low-ethanol preference

Background: Previous studies have shown that high alcohol consumption is associated with low withdrawal susceptibility, while at the same time, other studies have shown that exposure to ethanol vapor increases alcohol drinking in rats and mice. In the present studies, we sought to shed light on this seeming contradiction using mice selectively bred for High‐ (HAP) and Low‐ (LAP) Alcohol Preference, first, assessing these lines for differences in signs of ethanol withdrawal and second, for differences in the efficacy of intermittent alcohol vapor exposure on elevating subsequent ethanol intake. Methods: Experiment 1 examined whether these lines of mice differed in ethanol withdrawal‐induced CNS hyperexcitability and the development of sensitization to this effect following intermittent ethanol vapor exposure. Adult HAP and LAP lines (replicates 1 and 2), and the C3H/HeNcr inbred strain (included as a control genotype for comparison purposes) received intermittent exposure to ethanol vapor and were evaluated for ethanol withdrawal‐induced seizures assessed by scoring handling‐induced convulsions (HIC). Experiment 2 examined the influence of chronic intermittent ethanol exposure on voluntary ethanol drinking. Adult male and female HAP‐2 and LAP‐2 mice, along with male C57BL/6J (included as comparative controls) were trained to drink 10% ethanol using a limited access (2 h/d) 2‐bottle choice paradigm. After stable baseline daily intake was established, mice received chronic intermittent ethanol vapor exposure in inhalation chambers. Ethanol intake sessions resumed 72 hours after final ethanol (or air) exposure for 5 consecutive days. Results: Following chronic ethanol treatment, LAP mice exhibited overall greater withdrawal seizure activity compared with HAP mice. In Experiment 2, chronic ethanol exposure/withdrawal resulted in a significant increase in ethanol intake in male C57BL/6J, and modestly elevated intake in HAP‐2 male mice. Ethanol intake for male control mice did not change from baseline levels of intake. In contrast, HAP‐2 female and LAP‐2 mice of both sexes did not show changes in ethanol intake as a consequence of intermittent ethanol exposure. Conclusions: Overall, these results indicate that the magnitude of ethanol withdrawal‐related seizures is inversely related to inherited ethanol intake preference. Additionally, intermittent ethanol vapor exposure appears more likely to affect high‐drinking mice (C57BL/6J and HAP‐2) than low drinkers, although these animals are less affected by ethanol withdrawal.     

Sex and strain differences in ethanol drinking: effects of gonadectomy

BACKGROUND: Alcohol drinking behavior in rats is known to be sexually dimorphic and strain-dependent. METHODS: To test whether the gonadal steroid milieu exerts activational effects on ethanol intake and can modulate individual sensitivity toward alcohol use and misuse, we examined the effects of gonadectomy on oral self-administration (OSA) of ethanol in male and female rats from different strains. After castration, animals were given continuous free choice between water and ethanol solutions. The ethanol concentration was progressively increased from 2% to 10% and maintained at 6% (the preferred concentration) for 24 days. Ethanol solutions were then withdrawn for 9 days. During the second phase of free-choice drinking, the ethanol concentration was gradually increased every 4 days by the following amounts, in order as listed: 6%, 12%, and 24%. RESULTS: Our results confirm both gender and strain differences in ethanol drinking: females exhibited higher ethanol intake than males, and the WKHA strain drank more than the WKY and SHR strains. However, except for a small decrease in ethanol drinking during the acquisition of ethanol OSA in males after castration, no clear-cut difference was found between gonadectomized and sham-operated animals during the maintenance of ethanol OSA behavior. CONCLUSIONS: These data suggest that gender and strain differences observed are insensitive to gonadal steroids during adulthood, and that different sensitivities to the effect of gonadal steroids do not explain the sex x strain interaction observed in ethanol drinking.     

Chronic ethanol upregulates NMDA and AMPA, but not kainate receptor subunit proteins in rat primary cortical cultures

The present study examined the effects of chronic ethanol exposure on the expression of N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxalone (AMPA) and kainate receptor subunit proteins in rat cortical neuronal cultures grown in media containing 2 mM (high) or 0.1 mM (low) glutamine. Immunoblot analysis of NMDA (NR1, NR2A, NR2B, and NR2D), AMPA (GluR1 and GluR2/3), and kainate (GluR6/7) subunit polypeptides in 3-, 5-, 8-, 10-, and 12 day-old-cultures showed that NMDA receptor subunits NR1, NR2A, and NR2B and AMPA receptor subunits GluR2/3 progressively increased as a function of time, whereas levels of NMDA subunit NR2D were high at day 3 and progressively declined to barely detectable levels by day 12. Levels of AMPA subunit GluR1 and the kainate subunit GluR6/7 remained stable throughout the time course. Replacing the culture media with low glutamine media at culture day 5 did not alter the levels of subunit proteins measured at culture days 9 and 13. However, exposure of low glutamine cultures to 100 mM ethanol for 4 days (starting at culture day 9) significantly increased the levels of NMDA receptor subunits (NR1, NR2A, and NR2B) and AMPA receptor subunits (GluR1 and GluR2/3), but had no effect upon kainate receptor subunits (GluR6/7) or the synapse-associated proteins synapsin I and PSD-95. In contrast, chronic ethanol did not alter the levels of any of these subunit proteins in cells grown in high glutamine. These data demonstrate that under certain experimental conditions, prolonged exposure to ethanol upregulates NMDA and AMPA receptor subunit proteins, but has no effect upon kainate receptor subunit proteins. Because we have previously shown that acute ethanol can inhibit NMDA and AMPA, but not kainate, receptor function in these cultures, the increase in subunit expression likely reflects an adaptive response to the inhibitory effects of ethanol and suggests that both NMDA and AMPA receptors may play an important role in adaptation of the CNS to chronic ethanol.     

Chronic ethanol and iron administration on iron content, neuronal nitric oxide synthase, and superoxide dismutase in rat cerebellum

Excessive chronic ethanol administration to animals has been shown to cause oxidative insults to many body organs, including the liver and brain. In many instances, iron supplementation to the diet may further aggravate ethanol-induced liver damage. However, whether increased dietary iron can enhance the damage in the brain is unknown. In this study, four groups of Sprague-Dawley rats were fed a Lieber-DeCarli liquid diet containing 5% (w/v) ethanol or isocaloric amount of maltase and/or 0.25% (w/v) carbonyl iron for 2 months. At the end of the feeding regimen, iron contents were determined in the plasma, liver, cerebral cortex, and cerebellum. Cerebellar superoxide dismutase (SOD) and nitric oxide synthase (NOS) activities were measured and mRNA levels of MnSOD, CuZnSOD, and nNOS in the cerebellar granule cell layer were quantitated by in situ hybridization. Ethanol treatment alone caused an increase in iron levels in plasma, no change in the liver and cerebral cortex, but a decrease in the cerebellum. Iron supplementation increased liver iron >4-fold but did not alter iron contents in the cerebellum and cortex. All of the mRNA species examined and SOD activity were not affected by either iron or ethanol administration. However, NOS activity in the cerebellum was significantly enhanced by ethanol, whereas iron supplementation had an opposite effect. Our results indicate that iron supplementation to animals consuming ethanol may have tissue-specific effects. Furthermore, ethanol-induced increase in NOS activity in the cerebellum may explain the sensitivity of cerebellar neurons to oxidative insult.     

Chronic intermittent ethanol exposure in early adolescent and adult male rats: effects on tolerance, social behavior, and ethanol intake

Background: Given the prevalence of alcohol use in adolescence, it is important to understand the consequences of chronic ethanol exposure during this critical period in development. The purpose of this study was to assess possible age‐related differences in susceptibility to tolerance development to ethanol‐induced sedation and withdrawal‐related anxiety, as well as voluntary ethanol intake after chronic exposure to relatively high doses of ethanol during adolescence or adulthood. Methods: Juvenile/adolescent and adult male Sprague‐Dawley rats were assigned to one of five 10‐day exposure conditions: chronic ethanol (4 g/kg every 48 hours), chronic saline (equivalent volume every 24 hours), chronic saline/acutely challenged with ethanol (4 g/kg on day 10), nonmanipulated/acutely challenged with ethanol (4 g/kg on day 10), or nonmanipulated. For assessment of tolerance development, duration of the loss of righting reflex (LORR) and blood ethanol concentrations (BECs) upon regaining of righting reflex (RORR) were tested on the first and last ethanol exposure days in the chronic ethanol group, with both saline and nonmanipulated animals likewise challenged on the last exposure day. Withdrawal‐induced anxiety was indexed in a social interaction test 24 hours after the last ethanol exposure, with ethanol‐naïve chronic saline and nonmanipulated animals serving as controls. Voluntary intake was assessed 48 hours after the chronic exposure period in chronic ethanol, chronic saline and nonmanipulated animals using an 8‐day 2 bottle choice, limited‐access ethanol intake procedure. Results: In general, adolescent animals showed shorter durations of LORR and higher BECs upon RORR than adults on the first and last ethanol exposure days, regardless of chronic exposure condition. Adults, but not adolescents, developed chronic tolerance to the sedative effects of ethanol, tolerance that appeared to be metabolic in nature. Social deficits were observed after chronic ethanol in both adolescents and adults. Adolescents drank significantly more ethanol than adults on a gram per kilogram basis, with intake uninfluenced by prior ethanol exposure at both ages. Conclusions: Adolescents and adults may differ in their ability and/or propensity to adapt to chronic ethanol exposure, with adults, but not adolescents, developing chronic metabolic tolerance. However, this chronic exposure regimen was sufficient to disrupt baseline levels of social behavior at both ages. Taken together, these results suggest that, despite the age‐related differences in tolerance development, adolescents are as susceptible as adults to consequences of chronic ethanol exposure, particularly in terms of disruptions in social behavior. Whether these effects would last into adulthood remains to be determined.     

Gender and age at drinking onset affect voluntary alcohol consumption but neither the alcohol deprivation effect nor the response to stress in mice

Background: Epidemiological studies suggest that initiation of alcohol drinking at an early age is associated with an increased risk of developing an alcohol use disorder later in life. Nevertheless, relatively few studies using animal models have investigated the relationship between age of onset of drinking and ethanol drinking patterns in adulthood. Besides age at drinking onset, other factors such as gender could also affect the pattern of development of alcohol consumption. In rodents, many studies have shown that females drink more than males. However, even if it is assumed that hormonal changes occurring at puberty could explain these differences, only one study performed in rats has investigated the emergence of sex‐specific alcohol drinking patterns in adolescence and the transition from adolescence to adulthood. The aim of the present study was to compare the acquisition of voluntary alcohol consumption, relapse‐like drinking (the Alcohol Deprivation Effect—ADE) and stress‐induced alcohol drinking in male and female outbred mice that acquired alcohol consumption during adolescence or adulthood. Methods: Separate groups of naïve female and male WSC‐1 mice aged ± 28 days (adolescents) or ±70 days (adults) were given ad libitum access to water and 6% ethanol solution for 8 weeks (1st to 8th week) before undergoing a 2‐week deprivation phase (9th and 10th week). After the deprivation period, 2‐bottle preference testing (ethanol vs. water) resumed for 3 weeks (11th to 13th). During the 13th week, all animals were subjected to restraint stress for 2 consecutive days. Results: Over the entire time course of the experiment, ethanol intake and preference increased in females (both adults and adolescents). Adolescent animals (both females and males) showed a transient increase in alcohol consumption and preference compared to adults. However, by the end of continuous alcohol exposure (when all mice were adults), ethanol intake was not affected by age at drinking onset. A deprivation phase was followed by a rise in ethanol intake (ADE) that was not affected by sex or age. Finally, stress did not alter alcohol self‐administration either during or after its occurrence. Conclusions: Emergence of greater alcohol consumption in adult females does not seem to be limited to a specific developmental period (i.e., puberty). Age of voluntary drinking onset (adolescence vs. adulthood) does not affect eventual alcohol intake in adult WSC‐1 mice and does not modify the transient increase in ethanol consumption after alcohol deprivation.     

Chronic effects of ethanol under partial inhibition of catalase activity in the rat heart: Light and electron microscopic observations

In order to test the hypothesis that cardiac catalase plays a protective role in the pathogenesis of alcoholic cardiomyopathy, ethanol, comprising 36% of dietary calories, was administered to the rats for 5 weeks under inhibited cardiac catalase activity. Rats were treated with 3-amino-1,2,4-triazole (AT), a specific enzyme inhibitor, or iso-osmotic saline as control. During ethanol feeding, cardiac catalase activity was significantly inhibited at 24 h after AT, 25.6 ± 4.7 IU/mg protein, compared to rats given isoosmotic saline, 51.0 ± 9.1 IU/mg protein (s.d.m.) (P < 0.001). Under light microscopy, observed were intrasarcoplasmic vacuolization and occasional foci of necrosis and fibrosis. Ultrastructural abnormalities included very extensive vesicular changes in the myocardial cells, abnormally increased interstitial fibrosis, dilatation of tubular structures of sarcoplasmic reticulum, increased numbers of lysosomes, dehiscence of intercalated disc and various mitochondrial alterations including amorphous inclusion body. None of the rats given ethanol + saline, pair-fed diet + AT or saline showed comparable changes. Thus, cardiac catalase is considered to play a metabolic and protective role in the rat myocardium chronically exposed to ethanol.     

Chronic effects of ethanol on pharmacokinetics and left ventricular systolic function in rats

BACKGROUND: Observational clinical studies have demonstrated that there is a U-shaped relationship between ethanol consumption and all-cause mortality or risk of ischemic stroke. Although the exact cause of the U-shaped relationship is unclear, the pharmacokinetics of ethanol during the time course of chronic intake of ethanol may be involved, in relation to its cardiotoxic effects. The present study has assessed the cause of the U-shaped relationship between the ethanol consumption and left ventricular (LV) systolic dysfunction in a pharmacokinetic way. METHODS: Male Wistar rats were paired, and either ethanol or control liquid diet was chronically administered for 7 weeks. Then, these rats were subdivided into 3 groups: control liquid-diet-fed rats [EtOH (-)], 3 g/dL ethanol liquid-diet-fed rats (3%EtOH), and 5 g/dL ethanol liquid-diet-fed rats (5%EtOH). Ethanol's cardiotoxicity on LV systolic function was investigated by echocardiography. Ethanol concentration in blood, ethanol pharmacokinetics, and hepatic alcohol dehydrogenase (ADH) activity were evaluated simultaneously. RESULTS: The 5%EtOH group revealed LV systolic dysfunction, associated with a higher ethanol concentration in blood, and lower hepatic ADH activity than the EtOH (-) group; however, the 3%EtOH group did not show LV systolic dysfunction. During the acute ethanol stress, LV systolic dysfunction appeared in both EtOH (-) and 5%EtOH groups, with a higher ethanol concentration in blood and lower hepatic ADH activity than the 3%EtOH group. The 3%EtOH group showed a higher ethanol washout rate, less time-integral of ethanol concentration, and shorter mean residence time of ethanol in blood than the EtOH (-) or 5%EtOH group. CONCLUSIONS: The U-shaped relationship between chronic ethanol consumption and LV systolic dysfunction may be closely related to the pharmacokinetic characteristics of ethanol in blood, which depends on the quantity of chronically drinking alcohol.     

The effects of neuropeptide S on ethanol drinking and other related behaviors in alcohol-preferring and -nonpreferring rats

Background.  Neuropeptide S (NPS) is a 20-amino-acid peptide, identified in the brain and periphery, that is reported to regulate arousal, anxiety, and feeding behavior. Studies were conducted to determine whether this peptide would alter ethanol intake, sucrose intake, anxiety, and general motor activity in alcohol-preferring (P) and -nonpreferring (NP) rats.
Methods.  Experiment 1 : P and NP rats were given 8 weeks of continuous access to ethanol (15% w/v) and water. All rats were implanted with a cannula aimed at either the left or right lateral ventricle and 1 week later were infused with NPS (0.075, 0.3, 1.2 nmol) or artificial cerebrospinal fluid (aCSF) and tested for ethanol, food, and water intake. Experiment 2 : The same doses of NPS were administered to a group of P rats and intake of 2.5% (w/v) sucrose was measured. Experiment 3 : Infusions of NPS (1.2 nmol) or aCSF were administered to P rats prior to a 5-minute test on an elevated plus maze. Experiment 4 : Ethanol naive P and NP rats were infused with NPS (0.075, 0.15, 0.3, 0.6, and 1.2 nmol) or aCSF prior to a 20-minute test in activity monitors.
Results.  NPS reduced ethanol intake in P, but not in NP rats. It did not influence sucrose solution intake in P rats. However, an increase in food intake was seen in both rat lines following lower doses of the peptide. NPS did neither alter anxiety-like behavior in the elevated plus maze test nor was there an effect on general motor activity; however, there was an increase in the amount of time spent in the center of the activity monitors following infusions of 0.6 nmol of NPS in P, but not in NP rats, indicating anxioltyic actions of the peptide.
Conclusions.  These data suggest a role for NPS in the modulation of ethanol drinking and possibly anxiety-like behavior in rats selectively bred for high alcohol drinking.     

Chronic ethanol enhances ectodomain shedding in T cells and monocytes

BACKGROUND: Chronic ethanol (EtOH) has been shown to augment tumor necrosis factor (TNF)-alpha production, and this has been associated with EtOH-induced liver injury. We have recently described a chronic in vitro cell culture model where chronic ethanol exposure results in significantly augmented TNF production in Mono Mac 6 cells, a human monocytic cell line. This enhanced TNF production was redox regulated and associated with increased levels of TNF messenger RNA (mRNA) as well as increased processing of TNF by TNF converting enzyme (TACE), the enzymatic activity of which is regulated by the cellular redox state. We hypothesized that chronic ethanol through oxidative stress activates TACE-mediated ectodomain shedding of the preformed substrates p75 and p55 TNF receptors in Mono Mac 6 cells and L-selectin in Jurkat T cells. METHODS: Mono Mac 6 or Jurkat T cells were treated with EtOH (0, 50, or 100 mM) for 4 to 6 days. Shedding of p75 and p55 TNF receptors (Mono Mac 6 cells) or L-selectin (Jurkat T cells) was induced by stimulation with lipopolysaccharide and phorbol myristate acetate for Mono Mac 6 cells and PMA alone for Jurkat T cells. Shedding was assessed by enzyme-linked immunosorbent assay for shed molecules in the cell supernatant as well as the cell-associated proteins recovered from cell pellets. Steady-state mRNA levels for p75 TNF receptor and L-selectin were determined by ribonuclease protection assay. Cell surface L-selectin and TACE were measured by flow cytometry, and cell associated p55 and p75 TNF receptors were measured by enzyme-linked immunosorbent assay. RESULTS: Chronic EtOH exposure for 6 days resulted in a significant dose-dependent increase in shedding of p75 and p55 TNF receptors from Mono Mac 6 cells and L-selectin from Jurkat T-cells. The enhanced shedding was correlated with an alcohol-induced increase in mRNA levels and cell surface protein levels for these TACE substrates. Although chronic EtOH exposure increased the total amount of p75 and p55 TNF receptor and L-selectin shed into the media, the efficiency of shedding was suppressed by EtOH. In the case of Mono Mac 6 cells, the EtOH exposure increased superoxide production. Inhibition of nicotinamide adenine dinucleotide phosphate (reduced form) oxidase and hydrogen peroxide partially prevented the increased production of p75 TNF receptor in these cells. CONCLUSIONS: These results suggest that chronic EtOH up-regulates p75 and p55 TNF receptors on monocytes and L-selectin on T-cells. However, the TACE-mediated shedding efficiency of these substrates may be inhibited in the presence of EtOH. These results may have implications in monocyte signaling and T-cell trafficking, which may, in part, contribute to immune dysregulation associated with chronic ethanol.