EtOH Rats Research Articles

Adolescent alcohol binge drinking and withdrawal: behavioural, brain GFAP-positive astrocytes and acute methamphetamine effects in adult female rats

RationaleAlcopop beverages are generally the first alcoholic beverage that young females drink which contain high levels of sugar and alcohol. The over-consumption of these drinks may encourage alcohol co-administration with methamphetamine (METH) impacting on drinking behaviour and glial function.AimsThe aims of this study were to evaluate the effect of adolescent binge alcohol exposure on consumption level, anxiety-like behaviour, cross-sensitization with METH and on astrocyte expression in reward related brain regions.MethodsAdolescent female Sprague-Dawley rats had daily 1-hour oral alcohol consumption of alcopop (ALCP; with sucrose) or ethanol-only (ETOH; without sucrose), transitioned from 5 to 15% (v/v) ethanol content for 34 days. Water and sucrose groups act as controls. During alcohol withdrawal, rats were tested for anxiety on the elevated plus maze (EPM) and locomotor activity following saline or METH (1 mg/kg i.p) treatment. Brains were then collected to assess astrocyte immunofluorescence for glial fibrillary acidic protein (GFAP) in reward-related brain regions.ResultsRats pretreated with 5% ALCP consumed significantly more volume and ethanol intake when compared to 5% EtOH rats. Both ALCP and EtOH groups had a higher preference ratio for 5% than 15% alcohol solutions and ALCP rats had greater ethanol intake at 15% than EtOH rats. Alcohol withdrawal showed no significant differences between groups on anxiety, METH cross-sensitization effects or GFAP intensity in the regions studied.ConclusionsOverall, the addition of sucrose to alcoholic solutions encouraged female rats to consume larger volumes and greater ethanol intake compared to ethanol-only solutions, yet did not have long lasting effects on behaviour and astrocytes.

Microglia Phenotypes Following the Induction of Alcohol Dependence in Adolescent Rats.

Activation of the innate immune system may play a role in the development of alcohol use disorders (AUDs), which often originate with adolescent alcohol abuse. A key player in the innate immune system is microglia, the activation of which occurs along a spectrum from proinflammatory, or M1-like, to anti-inflammatory, or M2-like, phenotypes. Adolescent, male rats were gavaged with ethanol (EtOH) or isocaloric control diet every 8hours for 4days and then sacrificed at 0, 2, 7, and 14days later. Microglia were isolated from the entorhinal cortex and hippocampus by Percoll gradient centrifugation, labeled with surface antigens for activation, and analyzed by flow cytometry. Polarization states of microglia, defined as CD11b+ CD45low cells, were determined by the expression of M1 surface markers, major histocompatibility complex (MHC) II, CD32, and CD86, and M2 surface marker, CD206 (mannose receptor). Cytokine gene expression was measured by reverse transcriptase polymerase chain reaction. Isolated cells were a highly enriched population (>95% pure) of microglia/macrophages according to CD11b immunoreactivity. EtOH rats showed the most dramatic increases in microglia activation markers CD11b and CD45, and M1 (MHC-II) and M2 (CD206) markers at T2, when additional M1 markers CD86 and CD32 were also increased. Surprisingly, proinflammatory gene expression of CCL2, IL-1β, IL-6, and TNF-α generally was decreased at all time points in EtOH rats except for IL-6 which was increased at T0 and TNF-α which was not changed at T0 in either region. Simultaneously, BDNF expression was increased at T2 and T7, while IGF1 and TGF-β gene expression was decreased. Arginase was also increased at T0 in hippocampus, but not changed by alcohol otherwise. These data show that microglia phenotype after alcohol dependence is not a simple M1 or M2 classification, though more indicators of an anti-inflammatory phenotype were observed. Determining microglia phenotype is critical for understanding their role in the development of AUDs.

Impact of intermittent voluntary ethanol consumption during adolescence on the expression of endocannabinoid system and neuroinflammatory mediators

The adolescent brain displays high vulnerability to the deleterious effects of ethanol, including greater risk of developing alcohol use disorder later in life. Here, we characterized the gene expression of the endocannabinoid system (ECS) and relevant signaling systems associated with neuroinflammation and emotional behaviors in the brain of young adult control and ethanol-exposed (EtOH) rats. We measured mRNA levels of candidate genes using quantitative real time PCR in the medial prefrontal cortex (mPFC), amygdala and hippocampus. EtOH rats were generated by maintenance on an intermittent and voluntary ethanol consumption during adolescence using the two-bottle choice paradigm (4 days/week for 4 weeks) followed by 2 week-withdrawal, a time-point of withdrawal with no physical symptoms. Mean differences and effect sizes were calculated using t-test and Cohen's d values. In the mPFC and hippocampus, EtOH rats had significantly higher mRNA expression of endocannabinoid-signaling (mPFC: Ppara, Dagla, Daglb and Napepld; and hippocampus: Cnr2, Dagla and Mgll) and neuroinflammation-associated genes (mPFC: Gfap; and hippocampus: Aif1) than in controls. Moreover, EtOH rats had significantly higher mRNA expression of neuropeptide Y receptor genes (Npy1r, Npy2r and Npy5r) in the hippocampus. Finally, EtOH rats also displayed higher plasma endocannabinoid levels than controls. In conclusion, these results suggest that adolescent ethanol exposure can lead to long-term alterations in the gene expression of the ECS and other signaling systems involved in neuroinflammation and regulation of emotional behaviors in key brain areas for the development of addiction.

Cholinergic rescue of neurocognitive insult following third-trimester equivalent alcohol exposure in rats.

Neonatal ethanol exposure during the third trimester equivalent of human pregnancy in the rat significantly impairs hippocampal and prefrontal neurobehavioral functioning. Postnatal day [PD] 4-9 ethanol exposure in rats disrupts long-term context memory formation, resulting in abolished post-shock and retention test freezing in a variant of contextual fear conditioning called the Context Preexposure Facilitation Effect (CPFE). This behavioral impairment is accompanied by disrupted medial prefrontal, but not dorsal hippocampal expression of the immediate early genes (IEGs) c-Fos, Arc, Egr-1, and Npas4 (Heroux, Robinson-Drummer, Kawan, Rosen, & Stanton, 2019). The current experiment examined if systemic administration of the acetylcholinesterase inhibitor physostigmine (PHY) prior to context learning would rescue prefrontal IEG expression and freezing in the CPFE. From PD4-9, Long-Evans rats received oral intubation of ethanol (EtOH; 5.25 g/kg/day) or sham-intubation (SI). Rats received a systemic injection of saline (SAL) or PHY (0.01 mg/kg) prior to all three phases (Experiment 1) or just context exposure (Experiment 2) in the CPFE from PD31-33. A subset of rats were sacrificed 30 min after context learning to assay changes in IEG expression in the medial prefrontal cortex (mPFC), dorsal hippocampus (dHPC), and ventral hippocampus (vHPC). Administration of PHY prior to all three phases or just context learning rescued both post-shock and retention test freezing in the CPFE in EtOH rats without altering performance in SI rats. EtOH-SAL rats had significantly reduced mPFC but not dHPC expression of c-Fos, Arc, Egr-1, and Npas4. EtOH-PHY treatment rescued mPFC expression of c-Fos in ethanol-exposed rats and increased Arc and Npas4 regardless of dosing condition. While there was no effect of PHY on dHPC or vHPC expression of Arc, Egr-1, or Npas4, this treatment significantly boosted hippocampal expression of c-Fos regardless of ethanol treatment. These findings implicate impaired cholinergic and prefrontal function in cognitive deficits arising from 3rd-trimester equivalent alcohol exposure.

Effects of Isolated or Enriched Housing at Adolescence Upon Ethanol Intake and Anxiety Responses, In Rats Exposed to Prenatal Ethanol

Abstract
 Prenatal alcohol exposure (PAE) is highly prevalent worldwide, and can affect alcohol intake and anxiety responses in the offspring. It is still relatively unknown how PAE interacts with stress exposure at adolescence, and there is a scarcity of treatments to reduce the impact of PAE. The present study assessed alcohol (ethanol) intake and anxiety responses after PAE and tested the modulation of these responses by different housing conditions during early adolescence. Pregnant dams were exposed for 22 hours/day, from gestational day 0, throughout pregnancy, and until postnatal day 7 (PD 7), to a single bottle of 10% ethanol that was mixed in tap water and sucralose (EtOH Group), or to a single bottle of tap water and sucralose (CTRL Group). During PDs 21-42 the offspring was exposed to standard pair housing, isolated housing or enriched housing. Immediately after, they were pair-housed and tested for ethanol intake in 24-hour access, intermittent 2-bottle choice sessions conducted over 4 weeks, and for anxiety responses in the light-dark box test. The EtOH offspring exhibited heightened anxiety (i.e., greater avoidance of the white area of the light-dark box) at PD21, when compared to control (CTRL) rats. Isolated housing induced greater avoidance of the white area, when compared to standard or enriched housing, on PDs 42 and 70. There were no significant EtOH versus CTRL differences in relative (i.e., percent preference vs. water) ethanol intake, yet overall fluid intake and absolute (g/kg) ethanol intake were significantly lower in EtOH versus CTRL rats. Enriched housing during adolescence had a suppressing effect upon absolute ethanol intake during the first week of testing, an effect more noticeable in CTRL than in PAE rats. The present study indicates that PAE or isolated housing is associated with an anxiety-like phenotype. The protracted PAE protocol here employed caused a generalized reduction of intake behaviours. Environmental enrichment was associated with a subtle, yet significant, reduction in absolute ethanol intake.

PROTECTIVE ROLE OF CRUDE EXTRACT OF AMORPHOPHALLUS CAMPANULATUS AGAINST ETHANOL-INDUCED OXIDATIVE RENAL DAMAGE

Objective: The current study investigates the nephroprotective effect of Amorphophallus campanulatus against chronic alcohol-induced oxidative stress and tissue damage.Methods: The rats were simultaneously supplemented with ethanolic extract of A. campanulatus along with ethanol (40% w/v)2 g/kg body weight/day for 30 days to evaluate the nephroprotective effect against alcohol toxicity. Renal antioxidant enzymes, serum urea, creatinine, and pro-inflammatory cytokines were assayed biochemically. Histomorphological and histochemical alterations were detected by Hematoxylin and Eosin, periodic acid Schiff, and Feulgen and Picrosirius stain, respectively. The degree of apoptotic cell death was examined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay technique.Results: Serum urea, creatinine, pro-inflammatory cytokines, tissue TBARS, and activity of glutathione metabolizing enzymes were significantly (p<0.01) elevated, whereas cytosolic and mitochondrial superoxide dismutase, catalase, and levels of reduced glutathione were significantly (p<0.001) decreased in the EtOH group compared to control. However, ethanolic extract of A. campanulatus (ACE) supplementation to the EtOH rats reversed these effects to normal levels. Furthermore, degenerative changes in renal cells with alcohol treatment were minimized to nearness in architecture by ACE supplementation. Glycogen and deoxyribonucleic acid depletion, excess fibrosis due to collagen deposition, and increased apoptotic cell number were also restricted by ACE supplementation, with the higher dose being more promising.Conclusion: Thus ethanol-induced nephrotoxicity was attenuated by ACE treatment by the antioxidative and antiapoptotic property of the extract. Such effects of the extract may be due to the probable presence of different bioactive components in the tuber. Hence, it can be used as a regular nutrient or therapeutic agent to protect the renal cells.

PROTECTIVE ROLE OF CRUDE EXTRACT OF AMORPHOPHALLUS CAMPANULATUS AGAINST ETHANOL-INDUCED OXIDATIVE RENAL DAMAGE

Objective: The current study investigates the nephroprotective effect of Amorphophallus campanulatus against chronic alcohol-induced oxidative stress and tissue damage.Methods: The rats were simultaneously supplemented with ethanolic extract of A. campanulatus along with ethanol (40% w/v)2 g/kg body weight/day for 30 days to evaluate the nephroprotective effect against alcohol toxicity. Renal antioxidant enzymes, serum urea, creatinine, and pro-inflammatory cytokines were assayed biochemically. Histomorphological and histochemical alterations were detected by Hematoxylin and Eosin, periodic acid Schiff, and Feulgen and Picrosirius stain, respectively. The degree of apoptotic cell death was examined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay technique.Results: Serum urea, creatinine, pro-inflammatory cytokines, tissue TBARS, and activity of glutathione metabolizing enzymes were significantly (p<0.01) elevated, whereas cytosolic and mitochondrial superoxide dismutase, catalase, and levels of reduced glutathione were significantly (p<0.001) decreased in the EtOH group compared to control. However, ethanolic extract of A. campanulatus (ACE) supplementation to the EtOH rats reversed these effects to normal levels. Furthermore, degenerative changes in renal cells with alcohol treatment were minimized to nearness in architecture by ACE supplementation. Glycogen and deoxyribonucleic acid depletion, excess fibrosis due to collagen deposition, and increased apoptotic cell number were also restricted by ACE supplementation, with the higher dose being more promising.Conclusion: Thus ethanol-induced nephrotoxicity was attenuated by ACE treatment by the antioxidative and antiapoptotic property of the extract. Such effects of the extract may be due to the probable presence of different bioactive components in the tuber. Hence, it can be used as a regular nutrient or therapeutic agent to protect the renal cells.

Role of Gut-Derived Endotoxin on Type I Collagen Production in the Rat Pancreas After Chronic Alcohol Exposure.

Pancreatic fibrosis is a key pathological feature of alcoholic chronic pancreatitis (ACP). Bacterial endotoxin lipopolysaccharide (LPS) is considered as an important cofactor in the fibrogenesis of ACP. However, there are limitations in the use of exogenous LPS for evaluating the role of endotoxin in ACP pathogenesis. In this study, we determined the relationship between the concentration of LPS in the portal vein and pancreatic type I collagen (Col1) content in chronic alcohol-fed rats. Male Sprague Dawley rats were divided into 2 groups and fed with Lieber-DeCarli isocaloric control (CON) liquid diet or ethanol (EtOH) (15g/kg/d) liquid diet. Eleven CON or EtOH rats were euthanized at the end of week 8, 9, or 10. The plasma LPS from portal vein was determined. Pancreatic inflammatory injury and fibrosis were assessed. Pancreatic stellate cells (PSCs) and macrophages were identified; pancreatic type I collagen alpha 1 (Col1A1) and Toll-like receptor (TLR4) mRNA and protein were examined; pancreatic chemokines and transforming growth factor-beta1 (TGF-β1) were determined. Pancreatic inflammatory scores were increased in 10-week EtOH rats compared with CON rats, but there was no significant difference in collagen deposition between 2 groups. The levels of portal vein LPS and pancreatic TLR4 and Col1A1 mRNA and protein were increased in a time-dependent fashion in EtOH rats, with the highest levels occurring at 10weeks. Additionally, by 8weeks, pancreatic TLR4 and Col1A1 mRNA in EtOH rats were statistically increased as compared to CON rats, whereas portal vein LPS remained unchanged. The number of PSCs and macrophages and expression of chemokines (MCP-1, MIP-1α, and RANTES), TGF-β1, or Col1A1 were significantly increased, each of which was positively correlated with the level of portal vein LPS in 10-week EtOH rats. These results suggest that LPS is associated with alcohol-induced fibrosis in pancreatitis and targeting of bacterial endotoxin may be a promising therapeutic strategy for ACP.

Chronic moderate alcohol drinking alters insulin release without affecting cognitive and emotion-like behaviors in rats.

Because the consumption of alcoholic beverages prevails in society, its effects on diabetes risk is a subject of interest. Extant literature on this issue often disagrees. Here, we probed the effects of chronic moderate ethanol consumption on glucose metabolism in rats. The effect of chronic moderate alcohol drinking on depression- and anxiety-like behaviors and memory was also explored. Adolescent male and female Long-Evans rats consumed saccharin-sweetened 5% (1 week) and 10% ethanol (7 weeks) under a 7.5-h/day (Monday-Friday) access schedule. This exposure was followed by sucrose preference and elevated plus maze (EPM) tests during an intervening week, before a 6-week intermittent-access (Monday, Wednesday, Friday) to 20% unsweetened ethanol in a 2-bottle choice drinking paradigm was implemented (EtOH). A free-feeding control group received water (Water). Our prior work revealed that voluntary ethanol consumption decreases food intake in rats. Hence, a second control group that received water was mildly food-restricted (FR), and their average body weight was matched to that of the EtOH group. During the week following week 6 of intermittent-access to 20% ethanol, rats were submitted to sucrose preference, EPM, and novel object recognition (NOR) tests. Insulin response to a glucose load was subsequently assessed via an oral glucose tolerance test (OGTT). Rats attained and maintained blood ethanol concentrations of ∼55mg/dL that correlated with the dose of sweetened 10% ethanol ingested. Relative to intake by Water controls, EtOH rats consumed less chow. There was no body weight difference between both groups. Neither sex of EtOH rats showed increased depression- and anxiety-like behaviors, as respectively measured by sucrose preference and EPM, nor did they show deficit in object recognition memory during abstinence. Male EtOH rats, however, showed signs of reduced general activity on the EPM. During OGTT, male EtOH rats showed a time-dependent potentiation of insulin release for proper glucose clearance. Such an effect was not observed in females. This landmark study shows that chronic moderate alcohol consumption can have negative metabolic consequences in the absence of overt behavioral deficits, especially in males.

Voluntary ethanol consumption changes anticipatory ultrasonic vocalizations but not novelty response

Novelty and sensation seeking (NSS) and affective disorders are correlated with earlier ethanol (ETOH) consumption, and sustained drinking into adulthood. Understanding the NSS response and affective response before and after voluntary ETOH consumption could elucidate important individual differences promoting sustained ETOH consumption. This study determined that NSS and affective response to rewarding stimulation—measured by ultrasonic vocalizations (USVs)—change after adolescent ETOH voluntary drinking. Rats were tested for their NSS response using the inescapable novelty test. Then rats were tested for their affective response to a natural reward and USVs were measured. The natural reward was experimenter-induced play behavior. Rats were exposed to ETOH for 8 weeks using an intermittent two bottle paradigm. After 8 weeks of voluntary consumption, rats were retested for their response to NSS and affective response to natural reward. Results indicate that voluntary ETOH consumption did not change the response to novelty. Control and ETOH exposed rats decreased their novelty response equally after ETOH consumption, suggesting the decrease was due to age. Importantly, voluntary ETOH consumption changed affective USVs. Compared to water-drinking control rats, ETOH-consuming rats elicited greater anticipatory trill USVs to a natural reward-associated context during a post-drinking probe test. Tickle-induced trill USVs did not change differently between ETOH and control rats. These results provide evidence that voluntary intermittent ETOH exposure increases the anticipation of reward and may represent a form of incentive salience. We postulate these diverging effects could be due to differences in incentive salience or reward processing. Together, these results suggest that voluntary ETOH consumption changes the affective response to conditioned and unconditioned natural rewards and offers a behavioral mechanism for studying affective reward processing after ETOH consumption.

Effects of ceftriaxone on ethanol, nicotine or sucrose intake by alcohol-preferring (P) rats and its association with GLT-1 expression

Increased glutamatergic neurotransmission appears to mediate the reinforcing properties of drugs of abuse, including ethanol (EtOH). We have shown that administration of ceftriaxone (CEF), a β-lactam antibiotic, reduced EtOH intake and increased glutamate transporter 1 (GLT-1) expression in mesocorticolimbic regions of male and female alcohol-preferring (P) rats. In the present study, we tested whether CEF administration would reduce nicotine (NIC) and/or EtOH intake by adult female P rats. P rats were randomly assigned to 4 groups: (a) 5% sucrose (SUC) or 10% SUC [SUC], (b) 5% SUC+0.07mg/ml NIC and 10% SUC+0.14mg/ml NIC [NIC–SUC], 15% EtOH and 30% EtOH [EtOH] and (d) 15% EtOH+0.07mg/ml NIC and 30% EtOH+0.14mg/ml NIC [NIC–EtOH]. After achieving stable intakes (4weeks), the rats were administered 7 consecutive, daily i.p. injections of either saline or 200mg/kg CEF. The effects of CEF on intake were significant but differed across the reinforcers; such that ml/kg/day SUC was reduced by ∼30%, mg/kg/day NIC was reduced by ∼70% in the NIC–SUC group and ∼40% in the EtOH–NIC group, whereas g/kg/day EtOH was reduced by ∼40% in both the EtOH and EtOH–NIC group. The effects of CEF on GLT-1 expression were also studied. We found that CEF significantly increased GLT-1 expression in the prefrontal cortex and the nucleus accumbens of the NIC and NIC–EtOH rats as compared to NIC and NIC–EtOH saline-treated rats. These findings provide further support for GLT-1-associated mechanisms in EtOH and/or NIC abuse. The present results along with previous reports of CEF’s efficacy in reducing cocaine self-administration in rats suggest that modulation of GLT-1 expression and/or activity is an important pharmacological target for treating polysubstance abuse and dependence.

Influence of fat/carbohydrate ratio on progression of fatty liver disease and on development of osteopenia in male rats fed alcohol via total enteral nutrition (TEN)

Alcohol abuse is associated with the development of fatty liver disease and also with significant osteopenia in both genders. In this study, we examined ethanol-induced pathology in response to diets with differing fat/carbohydrate ratios. Male Sprague-Dawley rats were fed intragastrically with isocaloric liquid diets. Dietary fat content was either 5% (high carbohydrate, HC) or 45% (high fat, HF), with or without ethanol (12–13 g/kg/day). After 14, 28, or 65 days, livers were harvested and analyzed. In addition, bone morphology was analyzed after 65 days. HC rats gained more weight and had larger fat pads than HF rats with or without ethanol. Steatosis developed in HC + ethanol (HC + EtOH) compared to HF + ethanol (HF + EtOH) rats, accompanied by increased fatty acid (FA) synthesis and increased nuclear carbohydrate response element binding protein (ChREBP) (p < 0.05), but in the absence of effects on hepatic silent mating type information regulation 2 homolog (SIRT-1) or nuclear sterol regulatory binding element protein (SREBP-1c). Ethanol reduced serum leptin (p < 0.05) but not adiponectin. Over time, HC rats developed fatty liver independent of ethanol. FA degradation was significantly elevated by ethanol in both HC and HF groups (p < 0.05). HF + EtOH rats had increased oxidative stress from 28 days, increased necrosis compared to HF controls and higher expression of cytochromes P450, CYP2E1, and CYP4A1 compared to HC + EtOH rats (p < 0.05). In contrast, HC + EtOH rats had no significant increase in oxidative stress until day 65 with no observed increase in necrosis. Unlike liver pathology, no dietary differences were observed on ethanol-induced osteopenia in HC compared to HF groups. These data demonstrate that interactions between diet composition and alcohol are complex, dependent on the length of exposure, and are an important influence in development of fatty liver injury. Importantly, it appears that diet composition does not affect alcohol-associated skeletal toxicity.

Effects of Chronic Red Wine Consumption on the Expression of Vascular Endothelial Growth Factor, Angiopoietin 1, Angiopoietin 2, and Its Receptors in Rat Erectile Tissue

Long-term consumption of red wine (RW) apparently confers some protection against cardiovascular diseases due to antiatherosclerotic properties of polyphenols and ethanol (EtOH). There is some evidence indicating that they do so by regulating angiogenesis, but the mechanism and the modulator factors involved are largely unknown. The aim of this study was to evaluate the effects of chronic ingestion of RW in vascular structure and in the pattern of expression of vascular growth factors in the rat corpus cavernosum. Male Wistar rats aged 6 mo were treated with RW or an equivalent EtOH solution, as the only liquid source for 6 mo. Expression of vascular endothelial growth factor (VEGF), angiopoietin 1 and angiopoietin 2, and their receptors (VEGFR1, VEGFR2, and Tie2) in cavernous tissue was assayed by immunofluorescence and Western blotting. A reduction of VEGF and VEGFR2 expression, respectively, in smooth muscle and endothelial cells was observed in RW-treated animals, which was balanced by an increase in angiopoietins/Tie2 expression. In EtOH rats, only a decrease in expression of the receptors VEGFR2 and Tie2 was observed. These results, taken together, suggest that antioxidants present in RW activate selected mechanisms for the maintenance of cavernous tissue vascularization. However, functional studies will be necessary to elucidate if RW is of benefit in the prevention of deleterious vascular events associated with ED.

Ethanol-exposed neonatal rats are impaired as adults in classical eyeblink conditioning at multiple unconditioned stimulus intensities

Binge-like exposure to ethanol early in development results in neurotoxic impairments throughout the brain, including the cerebellum and brainstem. Rats exposed to ethanol, during a period of time commensurate with the human third trimester, also show deficits in classical eyeblink conditioning (EBC), a cerebellar-dependent associative learning procedure. The relationship between ethanol-mediated EBC deficits and the intensity of the unconditioned stimulus (US) was explored in the current study. Neonatal rats were intubated and infused with ethanol (EtOH rats), sham-intubated and given no ethanol (SI rats), or reared as unhandled controls (UC rats). As adults, all rats underwent 10 days of 350 ms delay eyeblink conditioning with a tone conditioned stimulus (CS) and one of three co-terminating periorbital shock US. The frequency and topography of the conditioned eyeblink response (CR) were impaired in EtOH rats relative to UC rats. EtOH rats produced fewer CRs, with longer onset latencies, at all US intensities. In contrast, CR amplitude was impaired in EtOH rats at the highest US intensity only. Following conditioning, the unconditioned eyeblink response (UR) was analyzed in subsets of rats from each treatment group at five US intensities. Early ethanol exposure did not impair UR peak amplitude. The deficits in CR production are proposed to result from ethanol-mediated damage within specific regions of the EBC neural circuit.

Adult rats prenatally exposed to ethanol have increased gluconeogenesis and impaired insulin response of hepatic gluconeogenic genes

Rat offspring exposed to ethanol (EtOH rats) during pregnancy are insulin resistant, but it is unknown whether they have increased gluconeogenesis. To address this issue, we determined blood glucose and liver gluconeogenic genes, proteins, and enzyme activities before and after insulin administration in juvenile and adult EtOH rats and submitted adult EtOH rats to a pyruvate challenge. In juvenile rats, basal glucose; peroxisome proliferator-activated receptor-coactivator-1alpha protein and mRNA; and phosphoenolpyruvate carboxykinase enzyme activity, protein, and mRNA were similar between groups. After insulin injection, these parameters failed to decrease in EtOH rats, but glucose decreased by 30% and gluconeogenic enzymes, proteins, and mRNAs decreased by 50-70% in control rats. In adult offspring, basal peroxisome proliferator-activated receptor-coactivator-1alpha protein and mRNA levels were 40-80% higher in EtOH rats than in controls. Similarly, basal phosphoenolpyruvate carboxykinase activity, protein, and mRNA were approximately 1.8-fold greater in EtOH rats than in controls. These parameters decreased by approximately 50% after insulin injection in control rats, but they remained unchanged in EtOH rats. After insulin injection in the adult rats, glucose decreased by 60% in controls but did not decrease significantly in EtOH rats. A subset of adult EtOH rats had fasting hyperglycemia and an exaggerated glycemic response to pyruvate compared with controls. The data indicate that, after prenatal EtOH exposure, the expression of gluconeogenic genes is exaggerated in adult rat offspring and is insulin resistant in both juvenile and adult rats, explaining increased gluconeogenesis. These alterations persist through adulthood and may contribute to the pathogenesis of Type 2 diabetes after exposure to EtOH in utero.

Inhibition of IL-18 reduces myeloperoxidase activity and prevents edema in intestine following alcohol and burn injury

Previous studies have shown that alcohol (EtOH) ingestion before burn injury impaired intestinal barrier and immune function. This study determined whether EtOH and burn injury up-regulate interleukin (IL)-18 and whether IL-18 up-regulation following EtOH and burn injury is a cause for neutrophil recruitment and increased intestinal edema. Rats (250 g) were gavaged with EtOH to achieve a blood EtOH level in the range of 100 mg/dL prior to burn or sham injury (25% total body surface area). A group of rats was treated with Ac-YVAD-CHO (5 mg/kg), an inhibitor of caspase-1 (an enzyme that converts pro-IL-18, an inactive form of IL-18, to mature IL-18), at the time of injury. One day after injury, rats were killed. IL-18 production was determined in circulation and in the supernatants harvested from spleen, mesenteric lymph nodes, and Peyer's patch cell cultures as well as in intestinal tissue homogenates. Neutrophil accumulation in intestine was determined by measuring myeloperoxidase (MPO) activity. We found a significant increase in IL-18 levels in the lymphoid cell supernatants and intestinal tissue homogenates obtained from EtOH and burn-injured rats compared with the rats receiving burn or sham injury. This was accompanied by an increase in intestinal MPO and edema. No demonstrable change in intestinal morphology was observed in any group. Treatment of rats with caspase-1 inhibitor significantly attenuated the increase in IL-18 levels and intestinal MPO activity in EtOH and burn-injured rats. Inhibition of IL-18 also prevented an increase in intestinal tissue water content. As MPO is considered an index of neutrophil infiltration, results presented in this manuscript collectively suggest that IL-18 up-regulation is likely to contribute to the increased neutrophil infiltration and edema in intestinal tissue observed following EtOH and burn injury.

Effects of prenatal alcohol exposure on glucose tolerance in the rat offspring

Low birth weight in humans predisposes to obesity, cardiovascular diseases, and type 2 diabetes in adult life. Alcohol exposure during pregnancy has been associated with fetal growth restriction. We investigated the effects of prenatal exposure to alcohol on glucose metabolism later in the offspring. Female Sprague Dawley rats were given ethanol (ETOH), 4 g/kg/day by gavage throughout pregnancy. Compared with controls, newborn ETOH rats had decreased body size (5.1 ± 0.1 v 6.3 ± 0.1 g, P <.001), plasma insulin (0.44 ± 0.4 v 0.67 ± 0.1 ng/mL, P <.05), and leptin mRNA (P <.05), but they had normal β-cell mass and elevated adipose resistin mRNA and plasma glucose (5.0 ± 0.5 v 3.6 ± 0.3 mmol/L, P <.01). Food intake was decreased in ETOH rats during the fourth week of life, and body weight remained decreased compared with controls until a catch-up growth occurred by 7 weeks of life. At 13 weeks of age, body weight and β-cell mass of ETOH offspring were normal, but plasma glucose and insulin after a glucose challenge were increased compared with controls (P <.05). Adipose leptin and hypothalamic Ob-R mRNA were not different from controls, but resistin was increased (P <.05), and muscle GLUT4 content was decreased (P <.05) in ETOH offspring compared with controls. The data suggest that prenatal alcohol exposure impairs glucose tolerance in the offspring by both inducing insulin resistance and β-cell dysfunction. The prevailing mechanism in 3-month-old rat offspring appears to be insulin resistance, manifested by glucose intolerance and decreased GLUT4 despite hyperinsulinemia. Copyright 2003 Elsevier, Inc. All rights reserved.

Influence of Sex Hormonal Status on Alcohol‐Induced Oxidative Injury in Male and Female Rat Liver

Oxidative stress contributes to the development of liver injury after chronic alcohol intake. Women exhibit greater sensitivity to alcohol-induced liver disease than do men. The aim of the study was to determine the relationship between the sex hormone status of male and female rats and the degree of alcohol-induced oxidative stress in the liver. Male and female rats were pair-fed a liquid diet that contained 36% of their total daily calories as ethanol (EtOH group) or maltose (control group). Blood and liver samples were collected at the end of 8 weeks of diet. Male EtOH rats experienced a reduction in plasma testosterone (T) and an increase in estradiol (E2) levels, with an increase in their calculated E2/T ratio with respect to their controls. Malonaldehyde (MDA) levels, an index of lipid peroxidation, and protein carbonyl content, an index of protein oxidation, in the liver were greater among the EtOH groups in females than in males. In males, an inverse correlation was found between hepatic MDA and circulating T levels, and a direct correlation was disclosed between MDA and estradiol levels. In addition, the hepatic histopathological score correlated inversely with the plasma T levels and directly with the calculated E2/T ratio, an index of feminization. Alcohol-induced oxidative injury, which contributes to hepatic injury in both male and female rats, is enhanced in females compared with males. A role for plasma T levels in protecting male rat liver from ethanol-induced oxidative injury can be hypothesized.

Early Postnatal Ethanol Exposure Has Long‐Term Effects on the Performance of Male Rats in a Delayed Matching‐to‐Place Task in the Morris Water Maze

There is concern that, in the absence of full-blown fetal alcohol syndrome, binge drinking during pregnancy might produce long-term cognitive deficits in offspring. Spatial working memory might be particularly vulnerable in this regard. This is the first study to address this issue in an animal model of binge exposure during the brain growth spurt using a delayed matching-to-place (DMTP) task in the Morris water maze. Infant male rats were gastrostomized and reared artificially from postnatal days (PD) 5 to 18. From PD 6 to 9 they were fed either 6.5 g x kg(-1) x d (-1) ethanol (EtOH) in a binge exposure model (BAC 302 mg/dl) or an isocaloric maltose-dextrin solution (MD). The study included a third suckled control group (SC) that was reared normally. The rats were tested on a series of problems in the DMTP task, first as juveniles (PD 35) and then twice again as adults. Each problem included an initial search trial and a subsequent test trial. The first two phases of testing used delays of either 0 sec or 60 sec between these two trials. The third phase increased this delay to 60 sec and 2 hr. In addition, the rats were tested on a cued task in the water maze. EtOH rats were impaired relative to controls in their ability to relocate the hidden platform on the second trial, which followed the search trial. In Phases 1 and 2, there was no differential effect of ethanol on performance across the 0-sec and 60-sec delay conditions. However, EtOH rats were more affected by the longer 2-hr delay in Phase 3. There were no group differences on the search trial, in swimming speed, or cued-task performance. These findings establish that binge exposure to ethanol during the brain growth spurt results in a long-lasting impairment on the DMTP performance of rats in the water maze.

Fetoprotectivity of the flavanolignan compound siliphos against ethanol-induced toxicity.

Of the three flavanolignans that are found in silymarin (Silybum marianum [L.] Gaertn.), silybin is thought to be the primary therapeutic constituent. To test the capacity of silybin to protect the rat fetus from toxic effects of maternally ingested EtOH we did the following: Adult female rats were assigned to one of four groups; EtOH, EtOH/silybin, pair-fed control, and chow fed control. Silybin was orally administered as Siliphos(R), which is one part silybin to two parts phosphatidylcholine. All groups except the chow-fed control were maintained on a liquid diet throughout pregnancy. On day 21 of pregnancy the rats were killed and the fetuses removed. Gamma glutamyl transpeptidase (GGTP) activity and glutathione (GSH) levels were determined for liver and brain tissue for both the fetuses and the dams. Maternal and fetal GGTP activity in the EtOH rats was significantly higher than that of pair-fed controls, whereas the GGTP activity observed in the Siliphos(R)/EtOH rats was not elevated. Fetal mortality rates in the EtOH rats significantly exceeded those of all three other groups.