Experimental anxiety and the reinforcing effects of ethanol in rats

In order to examine the relationship between anxiety and reinforcing effects of alcohol, drug-naive male Wistar rats weighing 250-300 g were classified as "anxious" and "non-anxious" in the elevated plus-maze test. A conditioned place preference test was then used to investigate the reinforcing effects of ethanol (EtOH) on these animals. On 2 alternate days, groups of "anxious", "non-anxious" and "normal" rats received intraperitoneal (ip) injections of EtOH (0.5, 1.0 or 1.5 g/kg) immediately before a 15-min confinement to the white compartment. On the 2 intervening days the same rats received ip injections of saline before confinement to the opposite compartment. On day 5, a 15-min free-choice test was carried out with no injections. Rats classified as "anxious" showed a significant, though not dose-dependent preference for all doses of ethanol compared to saline-treated animals. These data demonstrate that rats regarded as "anxious" are more sensitive to the reinforcing effects of EtOH than "non-anxious" and "normal" Wistar rats and emphasize the relevance of the basal levels of anxiety of rats when trying to detect the reinforcing effects of EtOH.

The decrease in uroporphyrinogen decarboxylase activity induced by ethanol predisposes rats to the development of porphyria and accelerates xenobiotic-triggered porphyria, regardless of hepatic damage

We evaluated the porphyrinogenic ability of ethanol (20% in drinking water) per se, its effect on the development of sporadic porphyria cutanea tarda induced by hexachlorobenzene in female Wistar rats (170-190 g, N = 8/group), and the relationship with hepatic damage. Twenty-five percent of the animals receiving ethanol increased up to 14-, 25-, and 4.5-fold the urinary excretion of delta-aminolevulinate, porphobilinogen, and porphyrins, respectively. Ethanol exacerbated the precursor excretions elicited by hexachlorobenzene. Hepatic porphyrin levels increased by hexachlorobenzene treatment, while this parameter only increased (up to 90-fold) in some of the animals that received ethanol alone. Ethanol reduced the activities of uroporphyrinogen decarboxylase, delta-aminolevulinate dehydrase and ferrochelatase. In the ethanol group, many of the animals showed a 30% decrease in uroporphyrinogen activity; in the ethanol + hexachlorobenzene group, this decrease occurred before the one caused by hexachlorobenzene alone. Ethanol exacerbated the effects of hexachlorobenzene, among others, on the rate-limiting enzyme delta-aminolevulinate synthetase. The plasma activities of enzymes that are markers of hepatic damage were similar in all drug-treated groups. These results indicate that 1) ethanol exacerbates the biochemical manifestation of sporadic hexachlorobenzene-induced porphyria cutanea tarda; 2) ethanol per se affects several enzymatic and excretion parameters of the heme metabolic pathway; 3) since not all the animals were affected to the same extent, ethanol seems to be a porphyrinogenic agent only when there is a predisposition, and 4) hepatic damage showed no correlation with the development of porphyria cutanea tarda.

Low ethanol consumption increases insulin sensitivity in Wistar rats

Several human studies suggest that light-to-moderate alcohol consumption is associated with enhanced insulin sensitivity, but these studies are not free of conflicting results. To determine if ethanol-enhanced insulin sensitivity could be demonstrated in an animal model, male Wistar rats were fed a standard chow diet and received drinking water without (control) or with different ethanol concentrations (0.5, 1.5, 3, 4.5 and 7%, v/v) for 4 weeks ad libitum. Then, an intravenous insulin tolerance test (IVITT) was performed to determine insulin sensitivity. Among the ethanol groups, only the 3% ethanol group showed an increase in insulin sensitivity based on the increase of the plasma glucose disappearance rate in the IVITT (30%, P<0.05). In addition, an intravenous glucose tolerance test (IVGTT) was performed in control and 3% ethanol animals. Insulin sensitivity was confirmed in 3% ethanol rats based on the reduction of insulin secretion in the IVGTT (35%, P<0.05), despite the same glucose profile. Additionally, the 3% ethanol treatment did not impair body weight gain or plasma aspartate aminotransferase and alanine aminotransferase activities. Thus, the present study established that 3% ethanol in the drinking water for 4 weeks in normal rats is a model of increased insulin sensitivity, which can be used for further investigations of the mechanisms involved.

Effect of one-week ethanol treatment on monoamine levels and dopaminergic receptors in rat striatum

We studied the effects of ethanol on the levels of norepinephrine, dopamine, serotonin (5-HT) and their metabolites as well as on D1- and D2-like receptors in the rat striatum. Ethanol (2 or 4 g/kg, po) was administered daily by gavage to male Wistar rats and on the 7th day, 30 min or 48 h after drug administration, the striatum was dissected for biochemical assays. Monoamine and metabolite concentrations were measured by HPLC and D1- and D2-like receptor densities were determined by binding assays. Scatchard analyses showed decreases of 30 and 43%, respectively, in D1- and D2-like receptor densities and no change in dissociation constants (Kd) 48 h after the withdrawal of the dose of 4 g/kg. Ethanol, 2 g/kg, was effective only on the density of D2-like receptors but not on Kd of either receptor. Thirty minutes after the last ethanol injection (4 g/kg), decreases of D2 receptor density (45%) as well as of Kd values (34%) were detected. However, there was no significant effect on D1-like receptor density and a 46% decrease was observed in Kd. An increase in dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC), a decrease in norepinephrine, and no alteration in 5-HT levels were demonstrated after 48-h withdrawal of 4 g/kg ethanol. Similar effects were observed in dopamine and DOPAC levels 30 min after drug administration. No alteration in norepinephrine concentration and a decrease in 5-HT levels were seen 30 min after ethanol (4 g/kg) administration. Our findings indicate the involvement of the monoaminergic system in the responses to ethanol.

Ethanol-induced colitis prevents oral tolerance induction in mice

The gut mucosa is a major site of contact with antigens from food and microbiota. Usually, these daily contacts with natural antigens do not result in inflammatory reactions; instead they result in a state of systemic hyporesponsiveness named oral tolerance. Inflammatory bowel diseases (IBD) are associated with the breakdown of the immunoregulatory mechanisms that maintain oral tolerance. Several animal models of IBD/colitis are available. In mice, these include targeted disruptions of the genes encoding cytokines, T cell subsets or signaling proteins. Colitis can also be induced by intrarectal administration of chemical substances such as 2,4,6-trinitrobenzene sulfonic acid in 50% ethanol. We report here a novel model of colitis induced by intrarectal administration of 50% ethanol alone. Ethanol-treated mice develop an inflammatory reaction in the colon characterized by an intense inflammatory infiltrate in the mucosa and submucosa of the large intestine. They also present up-regulation of both interferon gamma (IFN-gamma) and interleukin-4 (IL-4) production by cecal lymph node and splenic cells. These results suggest a mixed type of inflammation as the substrate of the colitis. Interestingly, cells from mesenteric lymph nodes of ethanol-treated mice present an increase in IFN-gamma production and a decrease in IL-4 production indicating that the cytokine balance is altered throughout the gut mucosa. Moreover, induction of oral tolerance to ovalbumin is abolished in these animals, strongly suggesting that ethanol-induced colitis interferes with immunoregulatory mechanisms in the intestinal mucosa. This novel model of colitis resembles human IBD. It is easy to reproduce and may help us to understand the mechanisms involved in IBD pathogenesis.

Modulation of B lymphocyte function by an aqueous fraction of the ethanol extract of Cissampelos sympodialis Eichl (Menispermaceae)

Cissampelos sympodialis Eichl species are used in folk medicine for the treatment of asthma, arthritis and rheumatism. In the present study, we investigated the immunomodulatory effect of an aqueous fraction of a 70% (v/v) ethanol extract of C. sympodialis leaves on B lymphocyte function. The hydroalcoholic extract inhibited the in vitro proliferative response of resting B cells induced by LPS (IC50 = 17.2 µg/ml), anti-delta-dextran (IC50 = 13.9 µg/ml) and anti-IgM (IC50 = 24.3 µg/ml) but did not affect the anti-MHC class II antibody-stimulated proliferative response of B cell blasts obtained by stimulation with IL-4 and anti-IgM. Incubation with the hydroalcoholic extract used at 50 µg/ml induced a 700% increase in intracellular cAMP levels. IgM secretion by resting B cells (obtained from normal mice) and polyclonally activated B cells (obtained from Trypanosoma cruzi-infected animals) was inhibited by the hydroalcoholic extract. The latter were more sensitive to the hydroalcoholic extract since 6.5 µg/ml induced a 20% inhibition in the response of cells from normal mice while it inhibited the response of B cells from infected animals by 75%. The present data indicate that the alcoholic extract of C. sympodialis inhibited B cell function through an increase in intracellular cAMP levels. The finding that the hydroalcoholic extract inhibited immunoglobulin secretion suggests a therapeutic use for the extract from C. sympodialis in conditions associated with unregulated B cell function and enhanced immunoglobulin secretion. Finally, the inhibitory effect of the hydroalcoholic extract on B cells may indicate an anti-inflammatory effect of this extract.

Can calories from ethanol contribute to body weight preservation by malnourished rats?

Our objective was to compare the use of calories from ethanol by well-nourished and malnourished rats in terms of body weight. Female Wistar rats weighing 170-180 g at the beginning of the study were used. The animals were divided into two groups (N = 12 each): group W received water ad libitum and group E an ethanol solution ad libitum as the only source of liquid throughout the experiment. The concentration of ethanol was increased weekly from 0 to 5, 10, 20 and 40% (v/v). In the well-nourished phase (A), all rats received food ad libitum (AW and AE). Ethanol treatment (AE) was then interrupted and water was offered to both groups. After 2 weeks both AW and AE rats were submitted to food restriction (50% of group AW food consumption), thus initiating the malnutrition phase (M). Liquid was offered as described before to the same W (MW) and E (ME) groups. The weight gain during the 1-week treatment of AE rats was similar to that of AW animals only when AE rats received the 5% (v/v) ethanol solution (9.16 vs 10.47 g). Weight loss was observed after exposure to 10% ethanol (P < 0.05) in spite of maintenance of caloric intake. Malnourished rats presented weight loss, which was attenuated by ethanol intake up to the 20% (v/v) solution and was related to an increased caloric offer. This effect was not observed with the 40% ethanol solution (-9.98 g). These data suggest that calories from ethanol were used to maintain body weight up to the concentration of 10% (v/v) (well-nourished) and 20% (v/v) (malnourished) and that ethanol has a toxic profile which depends on nutritional status.

Comparison of voluntary ethanol intake by two pairs of rat lines used as genetic models of anxiety

The relationship between anxiety-related behaviors and voluntary ethanol intake was examined in two pairs of rat lines by the oral ethanol self-administration procedure. Floripa high (H) and low (L) rats selectively bred for contrasting anxiety responses in the open-field test, and two inbred strains, spontaneously hypertensive rats (SHR) and Lewis rats which are known to differ significantly when submitted to several behavioral tests of anxiety/emotionality, were used (9-10 animals/line/sex). No differences in the choice of ethanol solutions (2%, days 1-4, and 4%, days 5-8, respectively) in a 2-bottle paradigm were detected between Floripa H and L rats (1.94 ± 0.37 vs 1.61 ± 0.37 g/kg for ethanol intake on day 8 by the Floripa H and L rat lines, respectively). Contrary to expectations, the less anxious SHR rats consumed significantly more ethanol than Lewis rats (respective intake of 2.30 ± 0.45 and 0.72 ± 0.33 g/kg on day 8) which are known to be both addiction-prone and highly anxious. Regardless of strain, female rats consumed more ethanol than males (approximately 46%). The results showed no relationship between high anxiety and voluntary intake of ethanol for Floripa H and L rats. A negative association between these two variables, however, was found for SHR and Lewis rat strains. Data from the literature regarding the association between anxiety and alcohol intake in animal models are not conclusive, but the present results indicate that factors other than increased inborn anxiety probably lead to the individual differences in ethanol drinking behavior.

Effects of chronic ethanol treatment on monoamine levels in rat hippocampus and striatum

We studied the effects of ethanol on concentrations of noradrenaline (NE), dopamine (DA) and serotonin (5-HT) and their metabolites in rat hippocampus and striatum. Ethanol (2 or 4 g/kg, po, from a 20% aqueous solution) was administered daily to male Wistar rats (4-13 per group) for 30 days and animals were sacrificed 30 min or 48 h after the last administration. Monoamines were measured by HPLC and considered significant at P < 0.05. A 47% increase in 5-HT levels was observed in the hippocampus with 4 g/kg ethanol in the 30-min protocol. Ethanol (2 and 4 g/kg) decreased DA (2114.5 ± 126.4 and 1785.1 ± 234.2 ng/g wet tissue, respectively) and 3,4-dihydroxyphenylacetic acid (DOPAC, 1477.6 ± 132.1 and 1218.8 ± 271.7 ng/g wet tissue, respectively) levels, while the higher dose also decreased NE (159.8 ± 13.5), 5-HT (228.0 ± 46.8) and 5-hydroxy-3-indoleacetic acid (5-HIAA, 304.4 ± 37.2 ng/g wet tissue), in the striatum after a 48-h withdrawal as compared to controls (DA: 3063.9 ± 321.3; DOPAC: 2379.6 ± 256.0; NE: 292.8 ± 50.2; 5-HT: 412.4 ± 36.2; 5-HIAA: 703.9 ± 61.4 ng/g wet tissue). In the 30-min protocol, ethanol (2 or 4 g/kg) decreased striatal NE (66 and 70%) and DA (50 and 36%) levels. On the other hand, increases were seen in 5-HIAA (146 and 153%) and 5-HT (59 and 86%) levels. Ethanol (2 g/kg, po) increased the homovanillic acid (HVA)/DA ratio (129%) in the striatum in the 30-min protocol, while at the higher dose it increased the HVA/DA ratio in the 48-h protocol (61%). These results indicate alterations in monoamines, mainly in the striatum, after chronic ethanol, which are influenced by dose and by the length of time after the last drug administration.

Long-term ethanol intoxication reduces inflammatory responses in rats

The anti-inflammatory effects of long-term ethanol intoxication were determined during ethanol treatment and withdrawal on the basis of neutrophil and eosinophil migration, hind paw edema and mast cell degranulation. Male Wistar rats (180-200 g, around 2 months of age) were exposed to increasing concentrations of ethanol vapor over a 10-day period. One group was evaluated immediately after exposure (treated group - intoxicated), and another was studied 7 h later (withdrawal group). Ethanol inhalation treatment significantly inhibited carrageenan- (62% for the intoxicated group, N = 5, and 35% for the withdrawal group, N = 6) and dextran-induced paw edema (32% for intoxicated rats and 26% for withdrawal rats, N = 5 per group). Ethanol inhalation significantly reduced carrageenan-induced neutrophil migration (95% for intoxicated rats and 41% for withdrawn rats, N = 6 per group) into a subcutaneous 6-day-old air pouch, and Sephadex-induced eosinophil migration to the rat peritoneal cavity (100% for intoxicated rats and 64% for withdrawn rats, N = 6 per group). A significant decrease of mast cell degranulation was also demonstrated (control, 82%; intoxicated, 49%; withdrawn, 51%, N = 6, 6 and 8, respectively). Total leukocyte and neutrophil counts in venous blood increased significantly during the 10 days of ethanol inhalation (leukocytes, 13, 27 and 40%; neutrophils, 42, 238 and 252%, respectively, on days 5, 9 and 10, N = 7, 6 and 6). The cell counts decreased during withdrawal, but were still significantly elevated (leukocytes, 10%; neutrophils, 246%, N = 6). These findings indicate that both the cellular and vascular components of the inflammatory response are compromised by long-term ethanol intoxication and remain reduced during the withdrawal period.

The time course of ethanol tolerance: associative learning

The effect of different contextual stimuli on different ethanol-induced internal states was investigated during the time course of both the hypothermic effect of the drug and of drug tolerance. Minimitters were surgically implanted in 16 Wistar rats to assess changes in their body temperature under the effect of ethanol. Rat groups were submitted to ethanol or saline trials every other day. The animals were divided into two groups, one receiving a constant dose (CD) of ethanol injected intraperitoneally, and the other receiving increasing doses (ID) during the 10 training sessions. During the ethanol training sessions, conditioned stimuli A (tone) and B (buzzer) were presented at "state +" (35 min after drug injection) and "state -" (170 min after drug injection), respectively. Conditioned stimuli C (bip) and D (white noise) were presented at moments equivalent to stimuli A and B, respectively, but during the saline training sessions. All stimuli lasted 15 min. The CD group, but not the ID group, developed tolerance to the hypothermic effect of ethanol. Stimulus A (associated with drug "state +") induced hyperthermia with saline injection in the ID group. Stimulus B (associated with drug "state -") reduced ethanol tolerance in the CD group and modulated the hypothermic effect of the drug in the ID group. These results indicate that contextual stimuli acquire modulatory conditioned properties that are associated with the time course of both the action of the drug and the development of drug tolerance.

The combination of atorvastatin and ethanol is not more hepatotoxic to rats than the administration of each drug alone

Animal studies and premarketing clinical trials have revealed hepatotoxicity of statins, primarily minor elevations in serum alanine aminotransferase levels. The combined chronic use of medicines and eventual ethanol abuse are common and may present a synergistic action regarding liver injury. Our objective was to study the effect of the chronic use of atorvastatin associated with acute ethanol administration on the liver in a rat model. One group of rats was treated daily for 5 days a week for 2 months with 0.8 mg/kg atorvastatin by gavage. At the end of the treatment the livers were perfused with 72 mM ethanol for 60 min. Control groups (at least 4 animals in each group) consisted of a group of 2-month-old male Wistar EPM-1 rats exposed to 10% ethanol (v/v) ad libitum replacing water for 2 months, followed by perfusion of the liver with 61 nM atorvastatin for 60 min, and a group of animals without chronic ethanol treatment whose livers were perfused with atorvastatin and/or ethanol. The combination of atorvastatin with ethanol did not increase the release of injury marker enzymes (alanine aminotransferase, aspartate aminotransferase, and lactic dehydrogenase) from the liver and no change in liver function markers (bromosulfophthalein clearance, and oxygen consumption) was observed. Our results suggest that the combination of atorvastatin with ethanol is not more hepatotoxic than the separate use of each substance.

Participation of the cholinergic system in the ethanol-induced suppression of paradoxical sleep in rats

Sleep disturbance is among the many consequences of ethanol abuse in both humans and rodents. Ethanol consumption can reduce REM or paradoxical sleep (PS) in humans and rats, respectively. The first aim of this study was to develop an animal model of ethanol-induced PS suppression. This model administered intragastrically (by gavage) to male Wistar rats (3 months old, 200-250 g) 0.5 to 3.5 g/kg ethanol. The 3.5 g/kg dose of ethanol suppressed the PS stage compared with the vehicle group (distilled water) during the first 2-h interval (0-2 h; 1.3 vs 10.2; P < 0.001). The second aim of this study was to investigate the mechanisms by which ethanol suppresses PS. We examined the effects of cholinergic drug pretreatment. The cholinergic system was chosen because of the involvement of cholinergic neurotransmitters in regulating the sleep-wake cycle. A second set of animals was pretreated with 2.5, 5.0, and 10 mg/kg pilocarpine (cholinergic agonist) or atropine (cholinergic antagonist). These drugs were administered 1 h prior to ethanol (3.5 g/kg) or vehicle. Treatment with atropine prior to vehicle or ethanol produced a statistically significant decrease in PS, whereas pilocarpine had no effect on minutes of PS. Although the mechanism by which ethanol induces PS suppression is not fully understood, these data suggest that the cholinergic system is not the only system involved in this interaction.

Effect of chronic ethanol consumption in female rats subjected to experimental sepsis

The objective of this research was to evaluate the interference of ethanol consumption by female rats with cytokines involved in the sepsis process and its correlation with mortality, the main outcome of sepsis. Female Wistar rats in estrus phase were evaluated in three experiments. Experiment 1 (n=40) was performed to determine survival rates. Experiment 2 (n=69) was designed for biochemical analysis, measurement of cytokine and estrogen levels before and after sepsis, and experiment 3 (n=10) was performed to evaluate bacterial growth by colony counts of peritoneal fluid. In all experiments, treated animals were exposed to a 10% ethanol/water solution (v/v) as the single drinking source, while untreated animals were given tap water. After 4 weeks, sepsis was induced in the rats by ip injection of feces. In experiment 1, mortality in ethanol-exposed animals was delayed compared with those that drank water (48 h; P=0.0001). Experiment 2 showed increased tumor necrosis factor alpha (TNF-α) and decreased interleukin-6 (IL-6) and macrophage migration inhibitory factor in septic animals exposed to ethanol compared to septic animals not exposed. Sepsis also increased TNF-α and IL-6 levels in both ethanol- and water-exposed groups. Biochemical analysis showed higher creatinine, alanine aminotransferase and aspartate aminotransferase and decreased glucose levels in septic animals that were exposed to ethanol. In experiment 3, septic animals exposed to ethanol showed decreased numbers of colony-forming units than septic animals exposed to water. These results suggest that ethanol consumption delays the mortality of female rats in estrus phase after sepsis induction. Female characteristics, most probably sex hormones, may be involved in cytokine expression.

Concomitant stress potentiates the preference for, and consumption of, ethanol induced by chronic pre-exposure to ethanol

Ethanol abuse is linked to several acute and chronic injuries that can lead to health problems. Ethanol addiction is one of the most severe diseases linked to the abuse of this drug. Symptoms of ethanol addiction include compulsive substance intake and withdrawal syndrome. Stress exposure has an important role in addictive behavior for many drugs of abuse (including ethanol), but the consequences of stress and ethanol in the organism when these factors are concomitant results in a complex interaction. We investigated the effects of concomitant, chronic administration of ethanol and stress exposure on the withdrawal and consumption of, as well as the preference for, ethanol in mice. Male Swiss mice (30–35 g, 8-10 per group) were exposed to an ethanol liquid diet as the only source of food for 15 days. In the final 5 days, they were exposed to forced swimming stress. Twelve hours after removal of the ethanol liquid diet, animals were evaluated for ethanol withdrawal by measuring anxiety-related behaviors and locomotor activity. Twenty-four hours after evaluation of ethanol withdrawal, they were evaluated for voluntary consumption of ethanol in a “three-bottle choice” paradigm. Mice exposed to chronic consumption of ethanol had decreased locomotor activity during withdrawal. Contrary to our expectations, a concomitant forced swimming stress did not aggravate ethanol withdrawal. Nevertheless, simultaneous ethanol administration and stress exposure increased voluntary consumption of ethanol, mainly solutions containing high concentrations of ethanol. These results showed that stressful situations during ethanol intake may aggravate specific addiction-related behaviors.