Ethane-beta-sultam Modifies the Activation of the Innate Immune System Induced by Intermittent Ethanol Administration in Female Adolescent Rats Alcoholism & Drug Dependence

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Introduction
'Binge drinking' is becoming an increasing medical and social problem, particularly amongst adolescents. Intermittent alcohol abuse or 'binge drinking', is deined as a period of excessive drinking, (5 or more alcoholic drinks consecutively over a 4-6 h period), which is then followed by a period of abstinence. he corticolimbic brain regions appear to be susceptible to binge-induced degeneration and induced relearning deicits, [1], particularly during adolescence when neurogenesis is occurring. Over the past few years some of the underlying biochemical and neurochemical processes involved in the cognitive [2] and electrophysiological abnormalities [3] have been identiied in various animal models of binge drinking.
Clinical studies have clearly shown that chronic alcohol abuse is associated with increases in infections [4,5], which are caused by alcohol-induced changes in the innate and adaptive immune systems. here is an inability of white cells to migrate to the site of infection or inlammation, as well as functional changes in lymphocytes, [6] natural killer cells, monocytes and macrophages [7] . In addition, alcohol will increase the permeability of the gut; such that gut derived endotoxins will be transported to the liver via the portal vein to stimulate tolllike receptors to induce inlammation and the release of damaging pro-inlammatory cytokines [8]. he release of pro-inlammatory cytokines will activate phagocytic cells, such as macrophages and microglia and induce inlammation in both the liver [9] and speciic brain regions [10]. It is noteworthy that as the excessive alcohol intake continues, there is adaptation by these cells, particularly in the brain, to reduce the inlammatory response by altering the gene expression of various transcription factors, e.g. NFkappaB, [11] which is referred to as neuro-adaptation. Over the period of chronic alcohol abuse, >10 years, there will be progressive loss of behavioural control, caused by decreased frontal cortical regulation of attention and cognitive lexibility, combined with increased limbic negative feelings [12]. In addition the hippocampus will be adversely afected by chronic ethanol abuse, and shows decreased hippocampal volume as well as deicits in hippocampal-dependent learning and memory [13].
In contrast, binge drinking will rapidly induce inlammation in the periphery [14] and hippocampus and prefrontal cortex brain regions in a rat model of binge drinking [14,15], as well as altering the ratio of pro-inlammatory cytokines to anti-inlammatory cytokines in the blood of University binge drinkers [16]. he explanation for such vulnerability in young binge drinkers is related to the fact that there is ine-tuning of speciic neuronal connections, via synaptic pruning, during this adolescent period [17]. Two brain regions show particularly marked ontogenetic alterations during adolescence, the prefrontal cortex, where considerable remodeling occurs within regions which form an interconnecting network of circuitry and the hippocampus, where hippocampal stem cells are present in the subgranular zone, inside the dentate gyrus granule cell layer. hese neural stem cells are linked to hippocampal function, which include learning, memory and mood [18]. It is in these two regions where binge drinking has its most profound neurotoxic efect, inducing adverse changes in structural integrity which could result in a variety of cognitive deicits [3]. Furthermore such vulnerability occurs within a relatively short time, e.g. approximately 2 years, of commencing a binge drinking regime in susceptible adolescents.
he excitatory amino acid glutamate plays an important role in alcoholism. Glutamate mediates approximately 70% of synaptic transmission, reaching concentration in the low millimolar range. Once released into the synaptic clet it can bind to one of the three types of ionotrophic glutamate receptors, the N-methyl-D-aspartate receptor, NMDA, the α-amino-3-hydroxy-5-methylisoxazole-4propionic acid receptor and the kainite receptor. In addition glutamate can bind to metabotrophic glutamate receptors in the perisynaptic regions or on the presynaptic terminal. Glutamate is cleared from the extracellular environment by sodium dependent excitatory amino acids, EAAT, which include GLT1, EAAT2 and EAAT5. In addition, EAAT1 and EAAT2 are expressed in glial cells and can remove excess glutamate [19]. Chronic alcohol abuse will inhibit neuronal NMDA receptor function, the NR2B containing NMDA receptors being particular sensitive to inhibition by ethanol. Microdialysis studies have shown that there are no changes in extracellular glutamate in various brain regions in an animal model of chronic alcohol abuse, although during detoxiication there is a rapid increase in glutamate release [20]. In contrast, microdialysis studies in a rat model of a binge drinking revealed signiicantly increased extracellular glutamate in the hippocampus ater only 3 weeks of a binge drinking regime [14]. he precise mechanisms as to how ethanol alters extracellular glutamate are unknown, although it may be due to ethanol-induced changes in glutamate uptake by glial cells [21][22][23].
he sulphonated amino acid taurine is widely distributed in human tissue, being present at high concentrations, 50mM in leucocytes, microglia and macrophages, where it plays an important anti-inlammatory role [24]. In our recent studies [25], we clearly showed that the anti-inlammatory property of taurine was mediated via stabilisation of IkaBa, thus preventing activation of NFkappaB. Although taurine can be synthesised intracellularly from cysteine and methionine, the diet is the main source for human nutrition. Taurine uptake from the plasma is tightly controlled by the taurine transporter, TauT, such that supplementation with taurine will only transiently increase taurine levels within the liver and to a lesser extent in the brain [25].
Since TauT activity is decreased by inlammation [26][27][28], this might indicate that the ability of such cells to protect themselves from inlammation will be decreased. herefore taurine analogues which are able to traverse cellular membranes independently of TauT may enhance intracellular taurine levels and promote anti-inlammatory pathways Beta sultam is an analogue of β-lactams, a group of compounds which are able to inactivate serine enzymes, i,e, elastase, which is released in response to inlammatory stimuli and plays a major role in protein digestion following phagocytosis [29]. he parent substituted β -sultam does not inhibit serine enzymes but is slowly hydrolysed to taurine. It therefore has the potential to difuse across cellular membranes independent of TauT and increase intracellular taurine content. In addition, it has also been shown that cetriaxone, an FDA approved β -lactam antibiotic, reduced ethanol consumption in alcohol preferring rats [30] which in part is due to the up regulation of glutamate transporter 1 [31]. herefore ethane-β-sultam may also diminish extracellular glutamate levels by a comparable mechanism, and diminish the neurotoxicity of binge drinking.
Since we had shown the anti-inlammatory action of ethane-βsultam in vitro in macrophages and microglia in our earlier cell culture studies [25], it was of interest to ascertain whether it would have an anti-inlammatory action in an animal model of binge drinking. In addition, since β-lactams antibiotics alter glutamate transporter 1, it was of interest to ascertain whether extracellular glutamate content might be inluenced by ethane-β-sultam. Lastly, since proinlammatory cytokines play an important role in the modulation of learning, memory, neural plasticity and neurogenesis [32], the binge drinking rats were assessed in a water maze trial to investigate whether their learning, possibly impaired by binge drinking, could be rectiied by ethane-β-sultam administration.

Animals
Adolescent Wistar female rats (Harlan-Nossan, Milan, Italy) at puberty (6 weeks of age), with average body weights of approximately 125-155 g, were housed under controlled humidity and temperature with 12h dark/light cycles and a free supply of food and water within a polypropylene cage. All animals were treated in accordance with the Italian Guidelines for Animal Care (D. L. 116/92) and European Communities Council Directives (86/609/ECC).

Animal treatment and binge drinking regime
he rats were randomly assigned to various binge treatment groups, each with n=4, repeating the binge treatment on at least two diferent occasions (total number of rats in each experimental group=8). A minimum number of 3 rats in each group completed the various analysis/tests at the end of each binge treatment. Animals were administered by gavage 1 g/kg ethanol or 2 g/kg ethanol +/ethane-β -sultam. Ethanol doses (20%) were administered 3x /day with 3 h intervals on 2 consecutive days by gavage, followed by 5 days abstinence. his was repeated for a total of three weeks. he control rats were administered either ethane-β-sultam or water alone at the same time points as the binge drinking ethanol rats ( Figure 1). he synthesis of ethane-β-sultam has been previously described [25]. Ethane-β-sultam was freshly prepared before each administration (2.86 mg/ml) and given by gavage at a dose of 28 mg/ kg. he administration of ethane-β-sultam was initiated one week before commencing the binge drinking regime, this compound being administered by gavage, each morning at 0900 h. It was then continued daily for the subsequent 3 weeks of the binge drinking regime, at 09.00 h or 30 min before the irst daily dose of ethanol.

Microdialysis
he rats underwent surgical procedures at the end of the second week of the binge drinking regime, ive days before the administration of the last ethanol dose, as previously described [14]. Rats were anaesthetised with chloral hydrate (400 mg/kg i.p), mounted onto a stereotaxic frame (Stellar, Stoelting Co., Wood Dale, IL, USA) and a guide cannula (concentric design, CMA Microdialysis AB, Stockholm, Sweden) was implanted vertically into the right ventral hippocampus, using the following coordinates, relative to Bregma and skull surface: AP -4.8, L -5.2, V-4.0 at the ventral extent of the guide cannula. he rats were allowed to recover for 5 days ater the surgery, ater which time microdialysis was commenced on the last day of the three weeks binge drinking regime, when a vertical microdialysis probe (2 mm exposed surface, CMA 12, CMA Microdialysis AB, Stockholm, Sweden) was inserted into the guide cannula. he inlet of the probe was connected to a microdialysis pump (CMA/100, CMA Microdialysis AB, Stockholm, Sweden) and the ventral hippocampus perfused with artiicial cerebrospinal luid (aCSF) consisting of, 3.0 mM KCl, 1.0 mM MgCl 2 , 140 mM NaCl, 1.2 mM CaCl 2 , 0.27 mM NaH 2 PO 4 , 7.2 mM glucose and 1.2 mM Na 2 HPO4 at pH 7.4 at a rate of 2µl/min [14]. Ater a stabilisation period of 1 h, the perfusion fractions were collected every 30 minutes. he fractions collected at -90, -60 and -30 min were taken as representative of the basal extracellular concentrations for glutamate and taurine. Ater the collection of the -30 min microdialysis fraction, rats were administered ethanol or water, i.e. during the irst 30 min collection of the stimulated period, which lasted a further 5 h duration (10 fractions of 30 min. he correct positioning of the probe was conirmed at a later time (see below).
To measure the extracellular concentrations of glutamate and taurine, the microdialysis samples were treated with OPA-reagent for pre-column derivatisation, which consisted of mercaptoethanol and O-phthalaldehyde (OPA). he amino acid derivatives were separated with 5µm reverse-phase Nucleosil C18 column (250 × 4 mm; Machery-Nagel, Duren, Germany), maintained at room temperature. he mobile phase consisted of 0.1 M potassium acetate (pH adjusted to 5.48 with glacial acetic acid) and methanol with a 3 linear step gradient from 25% to 90% methanol (low rate of 1.0 ml/min). Column eluent was analysed using a High-Performance Liquid Chromatography (HPLC) reverse-phase Shimadzu spectroluorimeter system (Shimadzu Italia S.r.l., Milan, Italy) set to an excitation wavelength of 340nm and emission wavelength of 455nm (controlled by Class-VPTM 7.2.1 SP1 Client/Server Chromatography Data System) [14].

Alveolar macrophage isolation and blood collection
Alveolar macrophages were isolated from rats within 24 h of completing microdialysis. Rats were anaesthetised with Nembutal, prior to a small incision in the trachea, to allow a small tube to be inserted into the lungs. A phosphate bufer solution (pH 7.4), approximately 40 ml, was used to lavage the alveolar macrophages from the lungs, which were recovered ater centrifugation at 1,500 rpm for 10 min. Cells at densities of 1 × 10 5 or 2 × 10 5 were pipetted into wells (Corning Inc. USA) containing culture medium Dulbecco media, 10% foetal calf serum, streptomycin (100 µg/ml) and penicillin (100 µg/ ml). he alveolar macrophages were let for 24 h to adhere to the wells. he supernatant was then removed and the cells resuspended in culture medium and stimulated with LPS (1 µg/ml) for 24 h. he supernatants were removed and stored at -20 o C prior to further analysis for NO and the two cytokines, IL6 and TNFα. he rats were then decapitated, the brains removed, and blood collected. he blood was spun at 3000 rpm for 15 minutes to collect serum, which was then stored at -20°C prior to taurine analysis.
In a separate experiment, blood was removed from the tail vein of adolescent binge drinking rats +/-ethane-β-sultam rats, n=8, at timed intervals ater the irst, second and third ethanol administration. he blood alcohol concentrations were estimated by an enzymatic method, where the conversion of NAD + to NADH results in an increase in absorbance at 340 nm that is proportional to the ethanol concentration

Nitrite analysis
he levels of nitrites in the cell supernatants were evaluated by combining 100 µl aliquots with an equal volume of Greiss reagent (2.5% phosphoric acid, 1% sulphanilamide and 0.1% naphthalene diamine dihydrochloride). he mixture was incubated for 10 min at room temperature and optical density measured at 540 nm. Standards were prepared in the range 1-50 µM.

Cytokine analyses
IL6 and TNFα were assayed in the supernatants by ELISA (R & D System, Inc. UK)

Serum taurine analysis
Trichloroacetic acid (2%) was added to the serum to precipitate proteins and the supernatant recovered ater centrifugation at 3000 r.p.m. for 15 minutes. he supernatant was diluted 1:500, ater which its taurine content was assayed by HPLC with luorescence detection of the o-phthalaldehyde derivative.

Brain preparation for histological investigation
he brains were removed from each rat at the completion of the pulmonary lavage. he brains were initially preserved in 4% formaledehyde in bufer solution and then cryopreserved in 30% sucrose solution prior to being frozen in isopentane at -80 o C prior to analysis. For cutting, the frozen brains were mounted in the cryostat (Bright Instruments, UK) and coronal sections, 20 microns, cut

Cresyl fast violet staining (CFV)
CFV staining was used to stain the neurons in the hippocampus, 8-10 sections for each rat. he neurons are substantially larger than microglia cells and are clearly distinguishable from these phagocytic cells by their morphology. he correct positioning of the probe in the CA1 hippocampal region was conirmed by this stain. Immunohistochemisty OX-6 and iNOS immunohistochemical staining: Every 7th slide was stained for presence of MHC-II [14] and iNOS, which constituted approximately 8-10 brain sections for each rat. Slides were rehydrated in changes of ethanol, circled with a pap pen (Daido Sangyo Co. Ltd., Tokyo, Japan) and then let in phosphate bufer saline (PBS; 16 g NaCl, 2.3 g Na 2 PO 4 , 0.4 g KH 2 PO 4 , in 400 ml adjusted to pH 7.4). Endogenous peroxide activity was blocked by 1% H 2 O 2 in 100% methanol (45 min). Slides were washed and incubated (1 h) irst in 5% normal horse serum (Vector Laboratories, UK) with PBS/Triton X (PBS, 0.1% Triton X-100; Sigma-Aldrich, UK) then in the same solution with the OX-6 antibody (Serotec Ltd., Oxford, UK), 1:500 dilution or the iNOS antibody 1:200 dilution, and refrigerated for 20 h. he slides were washed in PBS/TX prior to incubation with 5% normal horse serum and 0.5% anti-mouse IgG (2nd antibody) in PBS/TX (90min). he slides were washed and the ABC mix applied (Vector stain Elite Kit, Vector Laboratories, UK) and slides covered (1 h). ABC mix was washed of with PBS and the chromogen, 3,3'-diaminobenzidine (DAB; 5 ml H 2 O, 2 drops bufer, 4 drops DAB, 2 drops H 2 O 2 ; Vector Laboratories, UK) added and let (5-15 min) until the brain sections had turned pale brown. Slides were then dehydrated and mounted, as described earlier.

Stereological cell quantiication
Neuronal cell counts were made on the CFV stained slides (approximately 8 slides per brain) from within the dentate gyrus regions region from -4.3mm to -4.52mm bregma (Figure 2a). he hippocampal regions were maintained in the same position in both hemispheres, within the "fork" of the hippocampus, encompassing the polymorph layer of the dentate gyrus and CA1 neurons. Microglia counts encompassed the entire region of the CA1 hippocampal region from -4.30mm to -4.5mm bregma (number of rats =8) (Figure 2a). A computer based stereology sotware system (Image Pro, Media Cybernetics, PA, USA) attached to a Nikon Eclipse E8-microscope (Nikon Instruments, Surrey, UK) and JVC (London, UK) 3CCD camera was used. Briely, for each section, an area of interest was delineated manually with relation to previously published boundaries, to create an Area of Interest (AOI) (Figure 2b). he sotware system then created counting frames (100 × 60µm) which fell within the AOI using the uniform random sampling method. he total area of the counting frames relative to the area of the AOI gives the Area Sampling Fraction (ASF). he height of the optical dissector, which was measured by taking an average of 3 random points across the section using a Heidenhain microcator (Hedenhain, Traunreut, Germany), relative to the section thickness gives the Height Sampling Fraction (HSF). he Section Sampling Fraction (SSF) was 1/7 as every 7th section in either the dendate gyrus or total hippocampus was analysed. To avoid edge efects, when counting microglia or neurons, within the counting frames, "acceptance" and "forbidden" lines were used ( Figure   2c). Total cell estimates were calculated as follows, where n equal the number of cells counted:

Cognitive function
In order to minimise stress, the rats were handled daily throughout their period in the animal house and during the treatment phase preceding the water maze experiment. Twenty four hours ater the conclusion of the binge drinking regime +/-ethane-β-sultam, the rats commenced training in the spatial version of the Morris water maze test. he water maze apparatus consisted of a circular pool (169 cm width, and 50 cm depth) made of white plastic. he pool was illed to a depth of 30-35 cm with water maintained at room temperature (20 + 1 o C) and made opaque by the addition of a non-toxic black ink. A hidden platform (30 cm high, 12 cm diameter) was placed under the water in the centre of one quadrant of the pool. Extra-maze visual cues (i.e. coloured paper in diferent forms was placed on the edge of the wall) and two lights as well as an auditory cue, (a radio) remained in ixed positions throughout the experiments. Training in the Morris Water Maze Task consisted of 4 trials/day for 4 days during which the rat learned to navigate to a submerged platform located in a constant spatial position. he rat was released into the water in one quadrant of the tank and the latency (time in seconds) to climb onto the platform was recorded. he starting point for each trial was in a diferent quadrant for three trial runs while the fourth trial run reverted to the original quadrant. If the rat had not found the platform ater 60 seconds it was placed on the platform by the experimenter, and let there for 20 seconds to collect visual spatial information.
Five hours ater the last training test, a probe test was carried out in which each rat was placed in the water in the 1 st quadrant, and given 30  seconds to ind the position of the platform in the pool, which had been removed. he time that the rat spent in the 1 st quadrant, (dial entry), was recorded as well as the time in the quadrant where the platform had been, (target dial). Ater 30 seconds, the rat was removed from the tank.

Statistical evaluation
he data are presented as mean ± standard error, SEM or mean ± standard deviation, SD. Statistical analysis was carried out using one-way or two-way ANOVA as appropriate, followed by the post hoc

Body weight of rats
he body weight of all rats, measured at the beginning and the end of the binge drinking regime, indicated comparable weight gains in all of the treatment groups, when administered water or ethanol +/ethane-β-sultam ( Figure 3).

Blood ethanol levels
Blood ethanol levels increased during the period of the binge drinking regime. Ater the 2 g/kg dose, administered 3x during one day at 3 hourly intervals, the peak blood ethanol level was 0.5 g/l at 30 minutes ater the irst binge, while the blood ethanol peak concentration was at 1h, 0.88 g/l and 1.32 g/l ater the second and third dose, respectively. However the rate of clearance was similar for each dose. he dose of 1 g/kg ethanol induced blood ethanol levels 50% lower. he administration of ethane-β-sultam did not signiicantly alter either the blood ethanol concentrations or the ethanol clearance rates.

Serum and macrophage taurine levels
Serum taurine levels assayed in each treatment group are shown in (Figure 4) Ethanol treatment induced a statistically signiicant decrease in the concentration of serum taurine in the rats administered 2 g/kg EtOH (two way Anova, post hoc test: p<0.01 versus control), but not at the lower dose of 1 g/kg. When ethanol treatment was associated with ethane-β-sultam, the serum concentration of taurine showed a statistically signiicant increase, in both, 1 and 2 mg/kg ethanoladministered groups, as compared to those not administered ethaneβ-sultam (p<0.05, 1 and 2 g/kg EtOH + ethane-β-sultam, versus EtOH alone). he treatment with ethane-β-sultam alone, however, did not modify the serum levels of taurine in the control group.
he mean concentration of taurine assayed in the macrophages isolated from the ethanol-treated rats +/-ethane-β-sultam supplementation did not show any statistically signiicant diference.

Pro-inlammatory markers released from alveolar macrophages before and ater stimulation with lipopolysaccharide, LPS
Parallel release of each of these pro-inlammatory markers from the alveolar macrophages in diferent treatment conditions can be observed in (Figure 5). he LPS-induced release of TNFα is shown in (Figure 5a) and the release of NO and IL-6, before and 24 h ater LPS stimulation, in (Figure 5b) and (Figure 5c), respectively. Following LPS stimulation, there were statistically signiicant increases in each of these markers in the binge drinking rats administered either 1 g/ kg or 2 g/kg, by comparison to the control group administered water alone (p<0.05 or p<0.01 by two way Anova followed by post hoc test, see ( Figure 5) legend for details). In both 'binge drinking' EtOH groups, where ethane-β-sultam was co-administered during the binge drinking regime, there were signiicant decreases in the release of the pro-inlammatory markers from the alveolar macrophages, to almost control values.

Hippocampal taurine and glutamate microdialysate content ater binge drinking + /-ethane-β-sultam supplementation
he extracellular concentrations of taurine and glutamate were measured by microdialysis in the ventral hippocampus, under basal conditions and following the last EtOH dose of the binge drinking  regime. he basal extracellular levels of taurine were not afected by ethanol treatment, either 1 g/kg or 2 g/kg. Overall, ethane-β-sultam administration induced a statistically signiicant increase in the basal extracellular concentration of taurine in the hippocampal microdialysate, as compared to the non-supplemented rats (two way ANOVA, +/-ethane-β-sultam F 1.37 =7.29, p<0.01) (Figure 6a). In the post hoc group comparison, however, presence versus absence of ethane-β-sultam was statistically signiicant only for the control group. he basal extracellular concentration of glutamate showed a statistically signiicant increase in the rats administered 2 g/kg ethanol as compared to the control group (two way ANOVA post hoc comparison p<0.05). Such increases were abolished in rats administered 2 g/kg ethanol +ethane-β-sultam, (two way Anova post hoc comparison versus 2 g/kg, p<0.05), (Figure 6b). No further signiicant changes in taurine or glutamate extracellular concentrations were evident, ater the last ethanol dose, during the 5 h of microdialysis, in any of the animal groups.

Activation of microglia in the hippocampus ater binge drinking +/-ethane-β-sultam
Representative microphotographs of the region within the hippocampus where OX-6-immunopositive microglia were present, in binge drinking +/-ethane-β-sultam supplemented rats, are shown in the two upper panels of ( Figure 7a). Stereological cell counts of the activated microglia in the CA1 region of the hippocampus, showed    that, contrary to the absence of OX-6 positive cells in the control group +/-ethane-β-sultam, there was a highly signiicant increase in these phagocytic cells in the binge drinking rats which received either 1 g/kg or 2 g/kg ethanol (Two way ANOVA, post hoc p<0.0001 versus controls). Supplementation with ethane-β-sultam signiicantly reduced the number of activated microglia in the 1g/kg ethanol administered rats, by approximately 50% (Two way ANOVA, post hoc p>0.01 versus control), but did not reduce the numbers of these inlammatory cells in the 2 g/kg ethanol administered rats (Figure 7b).
Further immunohistochemical studies with then iNOS antibody conirmed that there was co-localisation of iNOS with the activated microglia, (Figure 7a), thereby conirming that such microglia were releasing pro-inlammatory cytokines as well as NO.

Cell counts of neurons in the dendate gyrus brain region ater binge drinking +/-ethane-β-sultam
Stereological cell counts of the neurons in the dendate gyrus region identiied statistically signiicant losses of neurons in the areas where the activated microglia had been observed, (Figure 8), in both 1 and 2 g/kg EtOH-treated groups (two way ANOVA, post hoc group comparisons versus control, p<0.01 and p<0.05, respectively) which were not signiicantly diferent from each other. Ethane-β-sultam restored the neuronal number to almost that of the control group in the binge drinking rats which had been administered 1 g/kg ethanol (post hoc group comparison, p<0.01). However, no protection against neuronal loss was evident in the rats administered 2 g/kg + ethane-βsultam.

Morris water maze studies
Spatial learning and memory was assessed in the rats at the conclusion of the binge drinking regime. It was of interest to note that the rats administered ethanol showed no fear of the water and immediately started to swim when placed in the pool. In contrast, control rats as well as the rats, which had received ethanol + ethaneβ-sultam were timid and slow to commence the task. Task acquisition along the 4 days is shown in Figure 9, where mean values of the escape latency time (seconds) are plotted against number of days. Global statistical analysis (Figure legend for details) was performed using the General Linear Model (GLM) for repeated measures, which indicated a diference of the escape latency for the factor binge treatment only at the end of the acquisition period, day 4. hus for multiple comparisons, a two way ANOVA applied to escape latency values obtained on day 4, showed that the ethanol-fed adolescent female rats, both 1 and 2 g/kg, had performed with signiicantly higher latencies to ind the hidden platform than the control group (post hoc comparison versus control, p<0.05), (Figure 9). In the 1 g/kg rats administered ethane-β-sultam, there were signiicantly lower latencies (post hoc comparison versus ethanol alone, p<0.05), similar to those of the controls, (Figure 9). In contrast, the 2 g/kg rats administered the pro-taurine drug showed identical higher latencies as compared to those administered ethanol   alone. here were no signiicant diferences between the controls or the binge drinking rats +/-ethane-β-sultam in the dial entry or probe trials.

Discussion
In these present studies the activation of the innate immune system by intermittent alcohol administration was modiied in both the periphery and the brain hippocampal region ater the administration of ethane-β-sultam, as exempliied by decreases in both the activation of phagocytic cells, (macrophages and microglia) and the associated release of pro-inlammatory markers. It was predicted that ethaneβ-sultam would increase cellular levels of taurine, as a result of its slow hydrolysis to taurine; however only marginal changes in plasma and macrophage taurine levels were analysed ater ethane-βsultam in these present studies. Increasing taurine status (by taurine supplementation) inluences ethanol metabolism [25], as exempliied by increased ethanol clearance from the blood, which was associated with altered brain activities of aldehyde dehydrogenase and catalase. Beta-lactam antibiotics, such as cetriaxone also inluence ethanol elimination rates, [33] as a result of inhibition of liver ALDH activity by the N-methyltetrazolethiomethyl group on the 3-position of the cephem nucleus [33]. However since ethane-β-sultam has no methyltetrazolethiomethyl group on the 3-position of the ethane-βsultam molecule it would not be expected to alter enzymes involved in ethanol metabolism. No changes in ethanol metabolism occurred in these present studies, which might have been a factor in the induction of a pro-inlammatory state in the brain.
he pro-inlammatory state induced by a binge drinking regime has been previously reported by many investigators, signiicant damage being reported in limbic association regions, including the cortex and the hippocampus, in particular the ventral dentate gyrus. he extent of such inlammatory changes is possibly associated with the ethanol dose, duration of ethanol intoxication and the number of periods of ethanol cessation. he biochemical and neurochemical changes induced remain unclear. It is reported that binge drinking does not induce changes in N-methyl-D-aspartate (NMDA) sensitivity or in the brain density of voltage-gated calcium channels [1]. Furthermore the levels of blood ethanol achieved in these present studies ater 3 successive ethanol administration was not excessively high. he increased glutamate concentrations may therefore be an important factor in the neurotoxicity observed in the binge drinking model. he binge drinking regime induced a signiicant increase in hippocampal glutamate ater 1 g/kg and 2 g/kg ethanol, which could be due to the multiple withdrawal episodes occurring between the ethanol drinking periods [20]. In our previous studies of chronic alcohol loading in experimental animals, there was a cumulative efect of several withdrawal episodes where basal glutamate content increased in speciic brain regions [20]. Glutamate can stimulate glial cells towards an inlammatory phenotype. Supplementation with ethane-β-sultam reduced hippocampal glutamate levels, which returned towards control values. he mode of action involved in this diminution is unclear although it is reported that β-lactam antibiotics are potent stimulators of GLT1 expression and protein content in the hippocampal CA1 astrocytes and mixed neuron/glial cortical cultures [34]. Up regulation of these speciic glutamate transporters may decrease extracellular glutamate content. Interestingly, when administered to animal models of motor neuron degeneration, there was a delayed loss of neurons and muscle cells as well as a reduction of hypercellular gliosis, [34] which was associated with decreased extracellular glutamate levels [31,35]. he pathway involved in the activation of the promoter region of the GLT1 gene is unknown. It was of note that there was a reduction in extracellular glutamate content in the hippocampus region ater ethane-β-sultam supplementation, in the controls as well as the binge drinking rats possibly indicating an increased GLT1 expression. Clearly this would need to be analysed in future studies. he release of cytokines by peripheral cells, as identiied in the alveolar macrophages, may readily compromise the endothelial function and permeability of the blood brain barrier [36], thereby facilitating the migration of inlammatory cells into the brain to further promote neuro-inlammation [37]. In addition it is of interest that the hippocampus highly expresses the pro-inlammatory cytokine receptors, e.g TNFα receptors, which may account for its vulnerability to systemic proinlammatory cytokines [38]. Glial cells play important roles in the nurturing of the neurons, as well as important roles in the immune and inlammatory response. In preliminary studies, acute ethanol doses (20mM-100mM) were shown to induce limited activation of an immortilised cell line, N9, (Unpublished data Ward and Nayak). Similarly, primary microglia, incubated between 7 and 24h with ethanol, 50mM, also showed only a marginal activation with increased NO release [39]. However in another study of murine macrophages, low to moderate levels of ethanol, (10-50mM) did stimulate TLR4, which triggered MAPKs pathways, translocation of NFkB to the nucleus and the release of pro-inlammatory cytokines and NO. However higher doses, 100mM were inhibitory [39]. An ethanolinduced priming stimulus of the microglia may certainly be an initial event in binge drinkers, when high circulating levels of ethanol may be achieved. Other factors may further potentiate the pro-inlammatory phenotype.
In these present studies OX-6 was used to stain and measure activated microglia in the dentate gyrus region of the hippocampus  ater 3 weeks, which induced the release of pro-inlammatory cytokines as well as NO, the latter being conirmed by immunohistochemical staining. Higher ethanol doses, 5 g/kg, administered in a single 4-day binge study, elicited a more widespread microglia iniltration in all regions of the hippocampus [40], which were associated with a range of central mediators of inlammation, e.g. pro-inlammatory cytokines, as well as COX2 and iNOS. However, this model of binge drinking had no prolonged period of abstinence from ethanol. In another study when 5 g/kg, was administered intra-gastrically for a shorter time period, every 8 h for 4 days, [41] there was no evidence of increased cytokine release in various brain regions. However there are no reported studies of the efect of β-lactam antibiotics on pro-and anti-inlammatory cytokines in the brain. A decrease in the number of inlammatory microglia was evident ater ethane-β-sultam supplementation, as well as a decrease in NO expression (as assayed by immunohistochemical techniques), which might be related to the decrease in glutamate content or some other unknown biochemical efect of β-lactams.
he neuro-inlammatory changes induced by binge drinking in this present study were paralleled by hippocampal neuronal loss. Neuronal loss has also been reported in other studies [1,42] although the ethanol concentrations used were much higher, 4-9 g/kg. he dentate gyrus region is particularly vulnerable since it contains neural progenitor cells, which will proliferate throughout life, but particularly during adolescence, to form neurons, astrocytes and oligodendrocytes. High doses of ethanol were shown to decrease the survival of these neural progenitor cells [43]. In another study of marque monkeys, where a binge type regime was administered for varying time periods, there were signiicantly decreased numbers of actively dividing type 1, 2a, and 2b cell types without signiicantly altering the early neuronal type 3 cells. Such results, as concluded by the authors, were caused by alcohol interfering with the division and migration of hippocampal pre-neuronal progenitors [44].
Pharmaceutical agents may prevent the neurotoxicity of binge drinking. For example, the anti-oxidant butylated hydroxytoluene reversed binge induced brain damage, possibly via NFκb inhibition, and blocked ethanol inhibition of neurogenesis in several brain regions ater the administration of very high doses of ethanol (8-12g/kg/day) 3 x /day for 4 days with no abstinence period [45]. Administration of indomethacin to adolescent rats, exposed to ethanol 3g/kg for 2 consecutive days at 48h intervals, abolished both COX-2 and iNOS expression, as well as cell death and behavioural deicits [46].
Another approach would be to prevent the activation of transcription factors, which mediate inlammation, i.e. NFκB. Taurine a sulphonated amino acid will prevent NFκB activation by stabilising IκBα and preventing its phosphorylation [25]. Although ethaneβ-sultam diminished the activation of the innate immune system in both the alveolar macrophages in the periphery and the glial cells in the hippocampus, its exact mode of action awaits detailed investigation since its administration was not associated with signiicant increases in taurine cellular levels.
he immune system plays an important role in both brain function and behavioural processes [32]. Peripheral inlammation can profoundly afect the functioning of the brain with respect to memory and cognition. In early studies, ethanol was shown to disrupt acquisition of a spatial task in adolescent rats in the Morris Water Maze [47] although Rajendran and Spear [48] indicated that this was a stressful technique which was not substantiated in the less stressful test-the sand box maze. Chronic binge-type ethanol exposure, 5 g/ kg every 48 h for 20 days, showed evidence of tolerance to ethanolinduced spatial deicits, when tested immediately. In mice, which were chronically alcoholised by administration of 10% alcohol for 5 months followed by withdrawal, there were glia cell activations in frontal cortex and striatum, which were associated with cognitive and anxietyrelated behavioural impairment [49]. In our present study it was shown that spatial learning and memory was impaired in rats administered intermittent alcohol for 3 days, which was corrected by the pro-drug in the 1g/kg ethanol administered rats.
hese present studies have shown that administration of ethaneβ-sultam signiicantly reduced the inlammatory response both in the periphery and in the brain [50,51]. Although increased taurine levels were not discernible in some of the tissues, possibly due to homeostatic controls, a reduction in neuro-inlammation and neuronal cell loss occurred as well as an improvement in ethanol-associated cognitive impairment. hese studies have identiied the importance of the innate immune system in the toxicity of binge drinking.