Vitamin E protects against alcohol-induced cell loss and oxidative stress in the neonatal rat hippocampus

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1 Int. J. Devl Neuroscience 22 (2004) Vitamin E protects against alcohol-induced cell loss and oxidative stress in the neonatal rat hippocampus Melissa D. Marino, Michael Y. Aksenov, Sandra J. Kelly Department of Psychology, University of South Carolina, Columbia, SC, USA Received 16 February 2004; received in revised form 15 April 2004; accepted 15 April 2004 Abstract Oxidative stress has been proposed as a possible mechanism underlying nervous system deficits associated with Fetal Alcohol Syndrome (FAS). Current research suggests that antioxidant therapy may afford some level of protection against the teratogenic effects of alcohol. This study examined the effectiveness of antioxidant treatment in alleviating biochemical, neuroanatomical, and behavioral effects of neonatal alcohol exposure. Neonatal rats were administered alcohol (5.25 g/kg) by intragastric intubation on postnatal days 7, 8, and 9. A subset of alcohol-exposed pups were co-administered a high dose of Vitamin E (2 g/kg, or 71.9 IU/g). Controls consisted of a non-treated group, a group given the administration procedure only, and a group given the administration procedure plus the Vitamin E dose. Ethanol-exposed animals showed impaired spatial navigation in the Morris water maze, a decreased number of hippocampal CA1 pyramidal cells, and higher protein carbonyl formation in the hippocampus than controls. Vitamin E treatment alleviated the increase in protein carbonyls and the reduction in CA1 pyramidal cells seen in the ethanol-exposed group. However, the treatment did not improve spatial learning in the ethanol-exposed animals. These results suggest that while oxidative stress-related neurodegeneration may be a contributing factor in FAS, the antioxidant protection against alcohol-induced oxidative stress and neuronal cell loss in the rat hippocampus does not appear to be sufficient to prevent the behavioral impairments associated with FAS. Our findings underscore the complexity of the pathogenesis of behavioral deficits in FAS and suggest that additional mechanisms beyond oxidative damage of hippocampal neurons also contribute to the disorder ISDN. Published by Elsevier Ltd. All rights reserved. Keywords: Alcohol; FAS; Fetal hepatic tissue 1. Introduction The teratogenic effects of alcohol, manifested in the form of craniofacial deformities, low birth weight and growth retardation, and behavioral dysfunction, were first described in the late 1960s (Lemoine et al., 1968) and later termed Fetal Alcohol Syndrome, or FAS (Jones and Smith, 1973). FAS is estimated to affect between 0.33 and 3 in 1000 live births and is now recognized as the leading non-genetic cause of mental retardation in the western world (Abel and Sokol, 1987, 1991). The cost of caring for these children has been estimated at approximately US$ 74.6 million per year, with three quarters of this cost associated with the care of FAS cases with mental retardation (Abel and Sokol, 1991). Therefore, understanding how perinatal alcohol exposure produces behavioral and cognitive deficits is of great medical and economic importance. The determination of alcohol s teratogenic Corresponding author. Tel.: ; fax: address: sandra-kelly@sc.edu (S.J. Kelly). mechanism(s) could conceivably lead to the development of a strategy to alleviate or even prevent these devastating deficits. In vitro analyses and animal models of FAS have provided an effective strategy in the examination of alcohol s neurotoxic effects. This research has uncovered many possible mechanisms that may underlie alcohol neurotoxicity, including the mediation of alcohol s effects through its metabolite acetaldehyde, alcohol-induced fetal hypoxia, alcohol s interaction with and effects on lipid membranes, and alcohol s disruption of the synthesis of proteins and trophic/growth factors (reviewed in Michaelis and Michaelis, 1986). However, these mechanisms seem to converge on the production of free radicals and oxidative stress as an important underlying mechanism of FAS (reviewed in Abel and Hannigan, 1995). Oxidative stress is attractive as a possible mechanism for the alcohol-induced brain damage associated with FAS for many reasons. The brain processes large amounts of O 2 in a relatively small mass, and has a high content of substrates available for oxidation (i.e., polyunsaturated fatty acids /$ ISDN. Published by Elsevier Ltd. All rights reserved. doi: /j.ijdevneu

2 364 M.D. Marino et al. / Int. J. Devl Neuroscience 22 (2004) and catecholamines) in conjunction with low antioxidant activities, making it extremely susceptible to oxidative damage (Halliwell and Gutteridge, 1999). The developing brain, which has only a fraction of the antioxidant enzyme activity of the adult brain, is perhaps even more vulnerable to the neurotoxic effects of oxidative stress than the adult brain (Henderson et al., 1999). In addition, certain regions of the CNS, such as the hippocampus and cerebellum, may be particularly sensitive to oxidative stress because of their low endogenous levels of Vitamin E, an important biochemical antioxidant, relative to other brain regions (Abel and Hannigan, 1995). Such a depressed defense system may be adequate under normal circumstances. However, in pro-oxidative conditions, such as during alcohol exposure, these low antioxidant defenses can predispose the fetal brain to oxidative damage. Several studies have demonstrated that alcohol can cause oxidative damage to lipids, DNA, and proteins in a variety of tissues. Alcohol-induced damage to cellular lipids following acute alcohol exposure has been observed in many tissues (e.g., rat liver homogenates (DiLuzio and Hartman, 1967); cerebellum (Uysal et al., 1986; Rouach et al., 1987; Nordmann, 1987); maternal and fetal hepatic tissue (Henderson et al., 1995; Chen et al., 2000); and fetal brain (Henderson 1et al., 1999)). In addition, a diet high in saturated fats (which are more resistant to peroxidation) was found to alleviate hyperactivity, a common behavioral outcome of fetal alcohol exposure (Abel and Reddy, 1997), suggesting that lipid peroxidation may play an important role in the neuropathology of FAS. Alcohol exposure also induces oxidative damage to nucleic acids, as evidenced by increased levels of 8-OHdG, an oxidatively altered base, in mouse (Weiland and Lauterberg, 1995) and rat (Cahill et al., 1997, 1999) mitochondrial DNA. In addition, DNA fragmentation and nuclear DNA strand breaks characteristics of oxidative DNA damage have also been observed in cultured rat hepatocytes, (Ishii et al., 1996) and in hippocampal and cerebellar tissue from rats administered alcohol chronically (Renis et al., 1996). Finally, oxidative protein damage has been linked with alcohol exposure in a number of studies. Increased protein carbonyl formation one of the most general and commonly used indicators of oxidative protein damage has been observed in the blood of alcoholic patients (Mutlu-Turkoglu et al., 2000; Grattagliano et al., 1996), in the liver of adult male rats exposed to alcohol (Rouach et al., 1997; Bailey et al., 2001; Abraham et al., 2002), and in the intestinal mucosa following alcohol exposure (Altomare et al., 1998). Interestingly, increased protein carbonyl formation in the pancreas and liver of 21-day-old rats that had been exposed to alcohol during development is alleviated by concurrent treatment with folic acid, a vitamin with antioxidant properties, during the period of alcohol exposure (Cano et al., 2001). Finally, an increase in reactive oxidative species has been detected in the cortices of rats exposed to alcohol acutely on either postnatal day 7 or 21 (Heaton et al., 2003). Taken together, the above studies suggest that alcohol exposure results in oxidative stress in a variety of tissues (liver and brain) and under several different alcohol exposure paradigms (fetal and adult; acute and chronic; in vitro and in vivo). These findings imply that oxidative stress is an important factor in the teratogenic effects of alcohol exposure. The role of oxidative stress in alcohol-induced neurotoxicity is also supported by studies showing beneficial effects of antioxidant therapy during alcohol exposure. Pretreatment with the peptide NAPVSIPQ (NAP), which has potent antioxidant properties in vitro, prevents fetal death in a mouse model of FAS (Spong et al., 2001). The treatment also prevents the reduction in glutathione, a common indicator of oxidative stress. Additional studies have demonstrated that the antioxidant vitamins -tocopherol (Vitamin E) and -carotene prevent neurotoxicity in cultured hippocampal neurons exposed to alcohol (Mitchell et al., 1999a,b). These same researchers extended these findings to an in vivo model of FAS, showing that the inclusion of Vitamin E in the diet prevented alcohol-induced Purkinje cell loss in the cerebellum of neonatal rats (Heaton et al., 2000). These studies suggest that antioxidant therapy either before or during alcohol exposure may protect the developing fetus from alcohol s teratogenic effects. However, there has been only one unpublished report that extends these findings to the behavioral deficits associated with FAS and that report was negative (Tran et al., 2003). Further, there has also been a report that melatonin, an antioxidant, does not protect Purkinje cells in the vermis and lobule 1 of the cerebellum from loss after neonatal alcohol exposure (Edwards et al., 2002). Thus, the evidence concerning antioxidant protective effects and alcohol exposure during development is mixed. To investigate whether oxidative stress is a contributing factor in the CNS deficits associated with FAS, an animal model that induces robust deficits in a behavior that has a well-defined neural mechanism must be used. The association between spatial learning and the hippocampus provides such a model. Studies of humans and animals with hippocampal lesions strongly suggest that spatial learning ability is reliant upon an intact hippocampus (Scoville and Milner, 1957; Zola-Morgan et al., 1986; Morris, 1981; Olton and Samuelson, 1976; Morris et al., 1982; Altemus and Almli, 1997). This correlation between hippocampal cell loss and impaired spatial learning ability has been strengthened by similar evidence provided by animal models of FAS. Spatial learning deficits are one of the most commonly reported outcomes in animal models of FAS. These deficits have been observed following both prenatal (Blanchard et al., 1987; Gianoulakis, 1990; Kim et al., 1997) neonatal (Goodlett et al., 1987; Goodlett and Johnson, 1997; Johnson and Goodlett, 2002; Kelly et al., 1988; Pauli et al., 1995) and combined prenatal and neonatal (Cronise et al., 2001) alcohol exposure. The CA1 region of the hippocampus has also proven highly susceptible to cell loss following prenatal (Barnes and Walker, 1981; Miller, 1995), neonatal (West et al., 1986; Bonthius and West, 1990, 1991; Greene et al.,

3 M.D. Marino et al. / Int. J. Devl Neuroscience 22 (2004) ; Miller, 1995; Tran and Kelly, 2003), or combined prenatal and neonatal (Tran and Kelly, 2003) alcohol exposure. Therefore, the association between spatial learning and hippocampal CA1 integrity provides a well-defined neural basis with which to investigate the mechanism of damage that underlies such behavioral deficits. In the present study, we investigated the possible link between oxidative stress, hippocampal damage, and spatial learning deficits induced by alcohol exposure during development. Using an alcohol exposure model shown to produce spatial learning deficits in juvenile rats (Goodlett and Johnson, 1997), we examined the effect of antioxidant treatment on spatial learning in alcohol-exposed animals by measuring performance in the Morris water maze. We performed western blot analyses to measure protein carbonyl formation as an index of oxidative stress in hippocampal tissue. Finally, we assessed hippocampal damage with an unbiased stereological count of CA1 pyramidal cells. We predicted that alcohol-exposed animals would show spatial learning impairments, increased levels of oxidized protein and a reduction in the number of CA1 pyramidal neurons compared to all other groups and that antioxidant treatment would attenuate or alleviate these effects. 2. Experimental procedures 2.1. General methods All animals were housed in the animal colony in the Department of Psychology at the University of South Carolina. The room was maintained at 22 C and on a 12-h light-dark cycle with lights on at h. Late gestation Long-Evans pregnant dams were obtained from Harlan-Sprague Table 1 Treatment of experimental groups Intubation PD6 PD7-9 ET First Milk 5.25 g/kg ethanol Second Milk ET + E First 2 g/kg VE in milk 5.25 g/kg ethanol + 2 g/kg VE in milk Second Milk IC First Intubated only Intubated only Second Intubated only IC + E First 2 g/kg VE in milk 2 g/kg VE in milk Second Intubated only NC First and second ( ) Indicates no intubation was given. VE = Vitamin E. Dawley and were singly housed in polypropylene cages with wood shavings until birth. The day of birth was designated PD1. Litters were left undisturbed until PD4 or 5, at which point the number and sex of pups was determined. Pups from large litters, or from litters with unequal numbers of males and females, were cross-fostered to maintain litters of approximately five males and five females each. The study consisted of two separate cohorts of animals: spatial learning and anatomical experiments (cohort 1), and oxidative stress assay (cohort 2). Table 1 summarizes the treatment groups. Tables 2 and 3 list the number of animals in each group for each set of experiments. Five treatment groups were used in these studies: (1) ET: ethanol only; (2) ET + E: ethanol and Vitamin E co-administration; (3) IC: intubated control; (4) IC + E: intubated with Vitamin E only, and (5) NC: nontreated control. Both cohorts utilized a mixed litter design, and only one animal of each sex from a litter was assigned to a treatment group. Table 2 Body weights and animal numbers for animals used in Morris water maze PD6 PD7 PD8 PD9 PD10 PD21** PD30** ET* M, N = ± ± ± ± ± ± ± 2.3 F, N = ± ± ± ± ± ± ± 1.8 ET + E* M, N = ± ± ± ± ± ± ± 2.7 F, N = ± ± ± ± ± ± ± 2.0 IC M, N = ± ± ± ± ± ± ± 2.6 F, N = ± ± ± ± ± ± ± 1.8 IC + E M, N = ± ± ± ± ± ± ± 2.0 F, N = ± ± ± ± ± ± ± 2.4 NC M, N = ± ± ± ± ± ± ± 2.2 F, N = ± ± ± ± ± ± ± 1.5 Data are reported as group average ± S.E.M. The (*) indicates significantly different from the IC, IC + E and NC groups; the (**) indicates male weights were significantly different from female weights.

4 366 M.D. Marino et al. / Int. J. Devl Neuroscience 22 (2004) Table 3 Body weights and animal numbers for animals used in western blot analysis of protein carbonyls PD6 PD7 PD8 PD9 ET M, N = ± ± ± 0.5* 13.6 ± 0.5** F, N = ± ± ± 0.5* 12.8 ± 0.5** ET + E M, N = ± ± ± ± 0.5 F, N = ± ± ± ± 0.4 IC M, N = ± ± ± ± 0.7 F, N = ± ± ± ± 0.6 IC + E M, N = ± ± ± ± 0.7 F, N = ± ± ± ± 0.5 NC M, N = ± ± ± ± 0.8 F, N = ± ± ± ± 0.5 Data is reported as group averages ± S.E.M.. The (*) indicates significant difference from IC and NC groups only; the (**) indicates significant difference from all other groups. Vitamin E (Sigma, 1000 IU/g) treatment began on PD6. Pups were removed from the cage individually, weighed, and marked with nontoxic colored marker for identification. Then, the pups in the ET + E and IC + E were administered 2.0 g/kg (71.9 IU/g) Vitamin E in 13.9 ml/kg volume of milk to ensure the bioavailability of the Vitamin E. ET pups were intubated with 13.9 ml/kg milk only, and IC pups were intubated without any liquids, because we have previously found that administration of milk to IC pups leads to a significant body weight disparity between the IC and NC groups. Ethanol treatments began on PD7 and continued through PD9. ET pups were administered 5.25 g/kg ethanol in 27.8 ml/kg volume of milk. ET + E pups received the same dose of alcohol supplemented with 2.0 g/kg (71.9 IU/g) Vitamin E. Two hours after the first intubation, ET and ET + E pups were re-intubated with milk only (27.8 ml/kg) to compensate for the decrease in nursing while intoxicated. For the first intubation each day, IC pups were intubated without any liquids and IC + E pups were given 2.0 g/kg (71.9 IU/g) Vitamin E in 13.9 ml/kg milk. The IC and IC+E pups were re-intubated without liquids 2 h following the first intubation. NC pups were weighed and marked daily. There were no differences in survival among treatment groups Blood alcohol concentration procedure Peak blood alcohol concentration was determined as previously described (Dudek and Abbott, 1984; Marino et al., 2002). Blood samples were obtained from ET and ET + E pups of the first cohort 2 h after the first intubation on PD9, a time point which had previously been determined to represent peak BAC following ethanol intubation (Marino et al., 2002) Spatial navigation procedure All animals from each litter of the first cohort were tested in the Morris water maze from PD The apparatus was a white circular tank (122 cm diameter, 62 cm deep) filled with 24 C water at a depth of 30 cm. The water was made opaque by the addition of nontoxic white tempera paint. In the distal cue (invisible platform) condition, a clear Plexiglass platform (15 cm 15 cm) was placed in the center of the northeast quadrant 2 cm below the surface of the water. In the proximal cue (visible platform) condition, an opaque black platform was placed in the center of the southwest quadrant with its surface 1 cm above the surface of the water. The animals were tested for eight sessions in the distal cue condition and for one session in the proximal cue condition. There were four trials per session (1 session per day), each separated by 3 4-min intervals. On each trial, the start point (N, S, E, or W) occurred in a quasi-random manner, with each point occurring only once per session. The experimenter, who stood at the same location during each trial, recorded the latencies to reach the platform with a stopwatch. If the rat located the platform, it was allowed to remain there for 10 s. If the platform was not located, then the rat was directed to the platform by the experimenter and allowed to remain there for 10 s. After the last distal cue trial in session 8, a probe trial was conducted. During the probe trials, the platform was removed and the rat was allowed to swim freely for 45 s. These sessions were recorded by video camera for later scoring. To score the probe trials, a transparent template of the water maze was placed over the television screen during playback of the tapes. On this template, the four different quadrants were marked, as well as the former location of the clear platform. Using this template, the experimenter recorded the amount of time spent in the appropriate quadrant (NE) as well as number of times the animal crossed the previous location of the platform Neuroanatomical procedures Tissue preparation A subset of animals that had completed testing in the water maze was used for stereological analysis of hippocampal CA1 neurons. Animals were deeply anesthetized with 8% chloral hydrate and perfused via the left cardiac ventricle with a Tyrode solution prewash, followed by a modified Karnovsky fixative containing 1.0% (w/v) paraformaldehyde and 1.25% (v/v) glutaraldehyde in 0.2 M phosphate buffer (ph 7.2) for approximately 10 min. The brains were removed and postfixed in 10% buffered formalin until processing (6 12 months) Infiltration and embedding After postfixation, brains were divided at the midline and cut in the coronal plane just rostral to the septum. Either the right or left hemisphere (counterbalanced across groups) was dehydrated through a graded series of alcohols (70,

5 M.D. Marino et al. / Int. J. Devl Neuroscience 22 (2004) , 95%, for 2 h each and 100% for 1 h) prior to infiltration with glycolmethacrylate (GMA) resin (Technovit 7100; Kulzer, Wehrhein, Germany). The hemisphere selection was counterbalanced in order to ensure that variability due to hemisphere was distributed evenly across groups. Tissue was placed in a 50:50 mixture of infiltration solution (100% GMA) and 100% ethanol for 2 h, and then transferred to infiltration solution alone. The tissue was transferred to a new well of infiltration solution each day over the course of 4 days. On the last day, the tissue was embedded in glycolmethacrylate resin using disposable tissue molds Sectioning Sections were cut in the coronal plane with a nonretractable rotary microtome (microtome E, American Optical Corporation) with tungsten-carbide knives (Model 200, Delaware Diamond Knives) using a microtome setting of 30 m. The hippocampus was exhaustively sectioned from the most rostral to the most caudal extent. Every 7th section was collected for sampling, with the first section randomly selected to be within the 1/7th cutting interval. The sections were floated in a cold bath of 20% ethanol, mounted onto clean, uncoated glass slides, and immediately dried at 60 C on a slide warmer prior to staining Staining The sections were stained with a cresyl violet stain modified for use with glycolmethacrylate-embedded tissue. The staining solution contained 100 ml cresyl violet stock, 120 ml 0.1 M glacial acetic acid, and 80 ml 0.1 M sodium acetate solution. The slides were placed in the staining solution for min at room temperature, differentiated in 0.002% acetic acid for 2 min, and rinsed in 96% ethanol (1 dip) and 3 changes of isopropyl alcohol (2 min each). Sections were laid flat to dry, cleared with xylene, and coverslipped using Cytoseal mounting medium and No. 1 glass cover slips from Corning (24 mm 50 mm) Unbiased stereology Unbiased stereological procedures were used to estimate total neuron number in the CA1 layer of the hippocampus. These procedures attempt to maximize precision and efficiency using systematic random sampling and unbiased probes of nuclei Equipment Cell counts were performed using the STEREOLOGER software (version 1.3.2, Systems Planning and Analysis). Video input was captured using a color video camera (Sony Corp., DXC-151A RGB CCD) that was mounted to a Nikon Optiphot-1 light microscope. The digitized image was viewed on a high-resolution computer monitor. The borders of the CA1 layer were outlined using a low-power 5 objective, whereas the neuronal counts were performed under a high-power oil immersion lens (100, N.A. 1.25). A motorized stage controller (MC-XYZ, Applied Scientific Instruments) attached to the microscope encoded and transmitted the X-, Y-, and Z-stage movements to the computer program (resolution of 0.1 m). The Z-axis encoder attached to the fine-focus of the microscope allowed for optical scanning of nuclei Definition of region of interest (CA1) Definition of the reference space (CA1 pyramidal cell layer, or regio superior) was based on the anatomical observations of Blackstad (1956). The neurons of the different regions of the hippocampus are clearly differentiated by size and organization, with the cell bodies and nuclei of CA1 pyramidal cells being small relative to those of the CA3 region. A small transition zone between the CA1 and CA3 (called CA2) contains a layer of pyramidal cells about four to five cells deep, similar in size to CA1 cells but distinctly smaller than CA3 cells. The CA1-subiculum border is defined as the point at which the cells of the CA1 cease to be contiguous. The CA1 region was outlined using the mouse at low power (5 ). The STEREOLOGER software then superimposed a point grid at random over the reference space. Points falling within the reference space were selected; those outside the reference space were deselected. The selected points indicated disector locations at which cells were counted on high magnification (100 ) Cell counting Total number of hippocampal CA1 pyramidal cells was estimated using the optical fractionator method, which has been described previously (Gundersen et al., 1988; West, 1993; West et al., 1991). This method uses a three-dimensional disector probe, or virtual box, to optically section the sample in the Z-axis. These disector probes are placed at random points at fixed intervals within the defined reference space (CA1 region of the hippocampus). Counting parameters that produced an efficient sampling scheme with minimal coefficient of error (CE), one to two neuronal nuclei per disector frame, and total neurons counted per sample, were determined in a pilot study and are described as follows. Counting was performed on approximately sections through the extent of the hippocampus in the coronal plane. The distance between disectors on each section (X Y step size) was 130 m. The area of the square counting frame was 330 m 3 for the CA1 region. Counting was confined to a region 8 m in height (disector height) within the middle of each section. A guard height of 2 m at the top surface of sections, which was not included in the disector height, excluded cells at the cut surface from being included in the count, preventing over-counting. The section thickness was obtained by identifying the top and bottom surfaces of the sections under a 100 oil immersion lens, and the Z-coordinates for both surfaces were recorded with the Z-axis encoder. At least five measurements from each section were used in calculating

6 368 M.D. Marino et al. / Int. J. Devl Neuroscience 22 (2004) the mean section thickness. Total neuron number (N obj ) was estimated by the software using the following equation, where N (total # of neurons) is the sum of neurons counted across all sections; ssf (section sampling fraction) the number of sections sampled / number of sections through the reference space, which was 1/7th for all cases; asf (areal sampling fraction) the area of counting frame/sampling area within each section (asf = area(frame)/area(x y step size)); and tsf (thickness sampling fraction) is the height of the disector/mean section thickness N obj = N 1 ssf 1 asf 1 tsf After defining the region of interest at low power, the oil immersion objective (100 ) was used to perform the actual cell counts. Cell counts were performed by a single experimenter who was blind to the treatment condition of the subjects. At high power, the STEREOLOGER placed the first disector at random and superimposed an unbiased counting frame (Gundersen, 1977) with inclusion and exclusion lines on the image. All subsequent dissectors were placed at a fixed distance (130 m) from each other in both the X- and Y-planes. First, the thickness of the section was determined. The first focal plane of the disector was positioned 2 m below the top of the section (i.e., guard height setting). Counting was then performed through a depth of 8 m (disector height). Neurons were counted if their nuclei first came into focus within the disector height (8 m), were within the disector frame or touching the inclusion lines, and did not touch any exclusion lines. If nuclei were in focus within the guard height setting of 2 m, they were not included in the count. This prevented the over-counting of nuclei that may have been counted in a previous section Western blot for protein carbonyl formation Protein carbonyls were detected using the Oxyblot Protein Oxidation Detection Kit from Serologicals Corp. (Norcross, GA). Samples were treated with 1X 2,4-dinitrophenylhydrazine (DNPH) to derivatize carbonyl groups in protein side chains to 2,4-dinitrophenylhydrazone. The DNP-derivatized protein samples were then separated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Duplicate gels were prepared for each set of samples. After separation, one of the gels was electroblotted to a nitrocellulose membrane and the other was stained with Coomassie Blue to confirm equality of protein load. Membranes were blocked with buffer containing bovine serum albumin (BSA), incubated for 1 h with primary rabbit polyclonal antibody (1:500 dilution) specific to the DNP moiety of the proteins, and then washed three times (5 min per wash) in PBS with 0.01% sodium azide and 0.2% Tween 20. This was followed by 1 h incubation with an alkaline phosphatase-antibody conjugate (goat anti-rabbit IgG, 1:20,000 dilution, Sigma, St. Louis, MO) directed toward the primary antibody. Binding was detected using a substrate for alkaline phosphatase (5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium, SigmaFast tablets, Sigma). Optical density of the staining on the blots was analyzed using the MCID/M7 gel analysis software supplied by Imaging Research Inc. (Ont., Canada). A positive control (supplied with kit) was run with each set of samples. Also, one of the samples in each set was treated with derivatization control solution (which did not contain DNPH) to assess the level of non-specific binding. During image analysis of the blots, the level of binding in the derivatization control sample was subtracted from each DNPH-treated sample to produce a measure of corrected optical density for each sample (Fig. 1). The corrected optical 2.6. Oxidative stress procedures Tissue preparation Rat pups from the second cohort were killed by decapitation 2 h after the final intubation on PD9. The hippocampus and cerebellum were dissected, snap frozen in liquid nitrogen, weighed, and frozen at 80 C until homogenization Homogenization The frozen tissues were homogenized in HEPES buffer containing a protease inhibitor mix (40 mg PMSF and 100 l protease inhibitor cocktail from Sigma) at a ratio of 10 mg tissue:100 l buffer in a glass 175 mm culture tube. The tissue was ground with an Ultra-Turrax T25 homogenizer (Janke and Kunkel). Homogenate was centrifuged at 14,000 rpm for 10 min at 4 C. Supernatant was collected into new centrifuge tube and frozen at 80 C until time of assay. Total protein content of the brain homogenate was determined using the Pierce BCA assay. Fig. 1. Western blot for protein carbonyls in hippocampal tissue extract. Representative Western blot containing a positive oxidized control (+ctl), a sample treated with derivatization control solution (d ctl), and samples from animals in the different groups derivatized with DNPH (lanes 3 12).

7 M.D. Marino et al. / Int. J. Devl Neuroscience 22 (2004) density for each sample was then normalized to protein content by dividing by the ratio of the optical density (OD) of the protein sample to amount protein loaded (in g). 3. Results 3.1. Data analysis Behavioral and physical data were analyzed using analyses of variance (ANOVAs). Significant interactions were analyzed using simple main effects to describe the interaction. Tukey s post hoc analyses were used to examine main effects and simple main effects in behavioral and physical data. The level of significance was P<0.05 except when repeated tests of simple main effects were conducted. In these cases, the level of significance was adjusted by dividing 0.05 by the number of tests conducted. Because ET animals were expected to show a loss in CA1 neurons and increased protein carbonyl formation, the cell counts and western blot data were analyzed using orthogonal a priori comparisons Body weights and BACs Cohort 1: Spatial learning/neuroanatomy animals Body weights for animals used in the water maze and cell counts were analyzed by separate three-way mixed ANOVAs ((Group Sex Day) with Sex and Group as between factors and Day as the within-subjects factor) for PDs 6 9 (during treatment) and PDs 10, 21 and 30 (after treatment). Analysis of PD6 9 body weights showed main effects of Group (F(4, 150) = 7.669, P<0.001) and Day (F(3, 450) = , P<0.001) and a significant interaction of Group Day (F(12, 450) = , P<0.001) (see Table 2). Tukey s post hoc analysis of the main effect of Group revealed that ET and ET + E animals weighed significantly less than IC, IC + E, and NC animals. Simple main effects on the Group Day interaction demonstrated that ET and ET + E animals weighed significantly less than the control groups on PDs 8 and 9 only. Analysis of PD10, 21, and 30 revealed significant main effects of Group (F(4, 87) = 2.886, P = 0.015), Sex (F(1, 87) = 6.184, P = 0.015), and Day (F(2, 174) = , P<0.001) and a significant interaction of Day and Sex (F(2, 174) = , P<0.001). Tukey s post hoc analysis showed that ET animals weighed significantly less than IC + E and NC animals only. Simple main effects on the Sex Day interaction revealed that males weighed significantly more than females on PDs 21 and 30. BACs for these animals were analyzed by a two-way between subjects ANOVA (Group Sex). Mean BACs were mg/dl (±14.84 S.E.M.) for the ET group and mg/dl (±12.90 S.E.M.) for the ET+E group. These values were not significantly different (P = 0.216). Additionally, there was no effect of Sex (P = 0.461) on BAC Cohort 2: Western blot animals Body weights from animals used for protein carbonyl assay were analyzed similarly to the first cohort, except that only body weights from PD6 9 were analyzed. ANOVA revealed a significant main effect of Day (F(3, 210) = , P<0.001) and a significant interaction of Group and Day (F(12, 210) = , P<0.001) (see Table 3). Tukey s post hoc analysis revealed that weights increased significantly each day in all groups. Simple main effects on the Group Day interaction demonstrated that on PD8, ET animals weighed significantly less than IC and NC animals, but did not differ significantly from ET + EorIC+ E animals. On PD9, ET animals weighed significantly less than all other groups. BACs were not measured for this cohort Spatial navigation Distal cue condition A three-way mixed ANOVA (Group Sex Session) with Group and Sex as between-subjects factors and session as a repeated measure revealed significant effects of Group (F(4, 136) = 6.15, P < 0.001)) and Day (F(6, 816) = , P<0.001) on escape latency in the Morris water maze. There was no significant effect of Sex. Tukey s post hoc analyses revealed that ET and ET + E animals had significantly slower latencies than NC, IC and IC + E groups (Fig. 2). In addition, escape latencies decreased significantly each day from the first day of testing until the fourth day of testing. Post hoc analyses on escape latencies for days 5, 6, and 7 revealed no significant differences among groups, indicating that all animals had acquired the task and were performing at asymptote Probe trials The amount of time spent in the probe quadrant during the probe trial was analyzed using a two-way (Group Sex) between-subjects ANOVA. This analysis revealed no significant effects of Group (P = 0.157) or Sex (P = 0.821) and no interactions (group means (seconds) ± S.E.M.: ET = 18.6±1.1, ET +E = 19.6±1.0, IC = 21.0 ±0.9, IC+E = 20.6 ± 0.8, NC = 18.1 ± 1.0). Analysis of the number of probe crossings during the probe trial also demonstrated no significant Sex (P = 0.819) or Group (P = 0.650) differences (group means ± S.E.M.: ET= 4.1 ± 0.4, ET + E = 4.6 ± 0.5, IC = 5.1 ± 0.4, IC + E = 4.7 ± 0.4, NC = 4.4 ± 0.3). These results indicate that, by the end of testing, all animals had learned the correct position of the invisible platform Proximal cue condition Escape latencies to reach the visible (or proximal) platform were analyzed by a two-way (Group Sex) between-subjects ANOVA. There were no significant effects of Group (P = 0.868) or Sex (P = 0.414) and no interactions (group means (seconds) ± S.E.M.: ET = 6.9 ± 0.8,

8 370 M.D. Marino et al. / Int. J. Devl Neuroscience 22 (2004) CA1 pyramidal cell count Fig. 2. Escape latencies in the distal cue condition of the water maze across days of testing (a) and collapsed across days (b). Escape latencies decreased significantly each day from the first day of testing until the fourth day of testing. Latencies on the last 3 days of testing did not significantly differ from each other, indicating that all animals had learned the task (a). Different letters represent significant group differences (b). Error bars represent S.E.M. ET + E = 7.7 ± 0.8, IC = 7.2 ± 0.8, IC + E = 7.2 ± 0.7, NC = 6.8 ± 0.8). This indicates that there were no gross motor or visual deficits that could account for the group differences found in the distal cue condition. The coefficient of error, which indicates the amount of stereological method error between sections within an individual, was computed by the STEREOLOGER program for each subject. Mean coefficient of error (CE), mean section thickness, and mean reference volumes were analyzed by three-way ANOVA (Group Hemisphere Sex). There were no significant effects of Group, Hemisphere, or Sex on any of these measures. The mean CEs of the estimates of neuron number for the CA1 region (group means ± S.E.M.: ET= ± 0.003, ET + E = 0.084±0.002, IC = 0.084±0.002, IC+E = 0.081±0.003) were within the theoretical limits of ideal CE values for an accurate study. Mean section thickness did not differ among groups (group means ( m): ET = 18.6 ± 0.9, ET + E = 18.2 ± 1.7, IC = 18.0 ± 0.3, IC + E = 18.4 ± 0.5), nor did mean reference volumes (group means ( m 3 ) ± S.E.M.: ET = 2.16e 8 ± 1.89e 7,ET+ E = 2.27e 8 ± 1.65e 7,IC= 2.29e 8 ± 1.52e 7,IC+ E = 2.48e 8 ± 1.64e 7 ). Since it was hypothesized that the ET group would be the only group to show a decrease in the number of CA1 cells, CA1 cell counts were analyzed using orthogonal a priori contrasts. Contrast 1 compared the ET group to all other groups; contrast 2 compared the IC and IC + E groups. The overall ANOVA showed no effect of Sex (P = 0.562) or Hemisphere (P = 0.210) on CA1 neuron number. While the overall F values for CA1 neuron number were not significant (F(3, 41) = 2.135, P = 0.099), the planned comparison indicated that ET animals had significantly fewer neurons in this brain region than all other groups (P = 0.032), including ET + E(Fig. 3). This is a medium effect size (ω 2 = 0.086). The planned comparison of IC and IC + E groups was not significant (P = 0.233). NC animals were not included in this study because no significant difference was found between the NC and IC groups in body Fig. 3. Unbiased stereology data for CA1 region of the hippocampus. CA1 pyramidal cell number estimates by unbiased stereology. Unilateral cell number estimates are reported. Groups with different letters were significantly different. Error bars represent S.E.M.

9 M.D. Marino et al. / Int. J. Devl Neuroscience 22 (2004) Fig. 4. Hippocampal protein carbonyls determined by Western blot analysis. OD, which was normalized for each lane by dividing the optical density of the staining on the Western blot by amount of protein loaded, is plotted on the Y-axis. Groups with different letters were significantly different. Error bars represent S.E.M. weight or performance in the water maze, and no significant difference in CA1 neuron number was found between the groups in a previous study (Tran and Kelly, 2003) Protein carbonyl formation Since it was hypothesized that ET animals would have significantly more protein carbonyls compared to all other groups, the same set of orthogonal a priori planned comparisons performed on the CA1 data were performed on this data. A two-way ANOVA (Group Sex) revealed a significant effect of Group (F(3, 61) = 3.44, P = 0.022) on hippocampal protein carbonyls. There was no significant effect of Sex (P = 0.177) and no interaction between Group and Sex (P = 0.862). The planned comparisons indicated that ET animals had significantly higher levels of protein carbonyls (P 0.01) than all other groups (Fig. 4), and there was no significant difference between the IC and IC + E groups (P = 0.89). NC animals were also not included in this study because no significant difference was found between the NC and IC groups in body weight or performance in the water maze. No significant correlation between body weight and protein carbonyl formation was found (R 2 = 0.000). 4. Discussion Alcohol exposure increased protein carbonyl formation in the hippocampus, reduced the number of hippocampal CA1 pyramidal neurons, and produced significant impairments in spatial learning. Some, but not all of these effects were attenuated by antioxidant treatment. Administration of Vitamin E to the ethanol-treated animals resulted in a reduction in protein carbonyls in brain. Also, the antioxidant treatment prevented the alcohol-induced cell loss in the hippocampus but had no significant impact on cell number in control animals. However, the protective effect of the antioxidant treatment was not preserved at the behavioral level. Animals given Vitamin E in addition to alcohol showed similar spatial learning deficits to those exhibited by animals receiving alcohol alone. Thus, it appears that while oxidative stress is occurring in the hippocampus and may contribute to the cell loss in the CA1 region, this effect is probably not sufficient to result in the spatial learning deficits as demonstrated in this model Effects of neonatal alcohol exposure Oxidative protein damage Although a limited number of studies have shown alcohol-induced alterations in some measures of oxidative stress in the brain, such as lipid peroxidation and oxidative DNA damage, this is the first in vivo study to show that alcohol exposure increases protein carbonyl formation in the hippocampus. In fact, this is one of the few studies to show that alcohol induces oxidative stress in the hippocampus, and the only study to our knowledge demonstrating this effect in neonatal hippocampus. A few clinical studies have shown increases in protein carbonyls in blood serum and plasma from human alcoholics (Mutlu-Turkoglu et al., 2000; Grattagliano et al., 1996). Increased protein carbonyls have also been found in the liver of adult rats exposed to alcohol chronically (Abraham et al., 2002; Bailey et al., 2001; Rouach et al., 1997) and in the liver and pancreas of weanling rats exposed to alcohol during gestation and lactation (Cano et al., 2001). However, protein carbonyls

10 372 M.D. Marino et al. / Int. J. Devl Neuroscience 22 (2004) have not previously been measured in brain tissue from rats exposed to alcohol during any period of development (prenatal, postnatal, or both). Therefore, these results support the concept that alcohol induces oxidative stress, in general. They extend previous findings by showing this effect in neonatal tissue, which had not heretofore been examined. While it is possible that the increase in protein carbonyl formation in the hippocampus is the result of a decreased body weight in the ethanol-exposed group, the correlation between these measures is not significant and the difference in body weights is small. This suggests that the contribution of loss of body weight to increased protein carbonyl formation is small. When the reductions in CA1 cell number found in this study are taken into consideration, these results suggest that alcohol-induced oxidative stress in the neonatal hippocampus contributes to the alcohol-induced cell damage associated with FAS CA1 cell number This study is in agreement with previous studies finding that alcohol exposure during the neonatal period results in a reduction in the number of CA1 pyramidal cells (Bonthius and West, 1990, 1991; Diaz-Granados et al., 1993; Greene et al., 1992; Miller, 1995; Tran and Kelly, 2003). However, it is the first study to show this effect in this exposure model (PD7-9), which is a relatively short time period during the brain growth spurt. Other studies have typically used longer exposure periods (PD2 10, Tran and Kelly (2003); PD4 10, Bonthius and West (1990) and Pierce et al. (1989)). However, the results of those studies showed conflicting results. Tran and Kelly (2003) and Bonthius and West (1990, 1991) found significant reductions in CA1 neuron number, while Pierce et al. (1989) did not. In this instance, the method of administration and peak BAC are the most likely culprits for the discrepancy. In the Pierce study (1989), alcohol was administered continuously by artificial rearing, which resulted in relatively low BACs and no significant cell loss in the CA1 region. In the studies that followed (Bonthius and West, 1990, 1991), this regimen was compared to a more condensed mode of administration that resulted in higher peak BACs. Their results showed that only the more condensed administration procedure resulted in CA1 neuron loss. This led them to conclude that peak BAC was the critical factor in neuronal loss. Hence, the cell loss observed in the current study is consistent with those studies that induced high peak BACs. These results suggest that alcohol exposure during the postnatal period alters hippocampal morphology after the cells of that region have finished proliferating, thus implying a neurotoxic mechanism rather than an interference with development Spatial learning The alcohol treatment used in the current study resulted in significant learning impairments classically associated with FAS. Our results are in agreement with previous studies involving brief neonatal alcohol exposure in animal models, which show spatial learning deficits in both adults (Johnson and Goodlett, 2002; Kelly et al., 1988; Pauli et al., 1995; Tomlinson et al., 1998) and juveniles (Goodlett and Johnson, 1997). However, some inconsistencies do exist. In an earlier study, our lab found that alcohol exposure from PD2-10 did not produce spatial learning deficits in juveniles (Cronise et al., 2001). One possible explanation for this discrepancy is the difference in the ethanol doses used (3.0 g/kg in the earlier study versus 5.25 g/kg in the present study). The higher dose used in the current study induced considerably higher BACs (approximately 450 mg/dl) than that used in the previous study (approximately 350 mg/dl). Since peak BAC has been shown to be the critical factor in alcohol-induced learning impairments (Kelly et al., 1988), this may account for the difference between studies. A perhaps more likely explanation of the findings involves the number of trials used in the water maze task. The current study used only 4 trials per day in the water maze task (versus 8 trials per day in the previous study) in order to increase the difficulty of the task and make group differences more apparent. It is possible that the increased number of trials in the earlier study made it easier for the rats to learn the maze, and the subtle effects of alcohol exposure on learning were obscured. Therefore, the differences between the two studies are not surprising. Most importantly, however, our results are consistent with the findings of Goodlett and Johnson (1997), upon which the current administration procedure was based. In their 1997 study, Goodlett and Johnson administered 5.25 g/kg ethanol via 2 daily intragastric intubations instead of the single intubation used in the current study. Although the BACs of animals used in that study ( mg/dl) were lower than those found in the present study ( mg/dl), both studies found similar learning acquisition deficits in juvenile animals and a lack of effect of or interaction with sex. The common finding of learning impairment despite different peak BACs is probably due to the increased difficulty of the water maze task used in Goodlett and Johnson s study. Although they also used 4 trials per day, the number of possible start positions within the maze was 12 in their study, compared to only 4 in the present study. Thus, the maze task in Goodlett and Johnson s study was even more difficult than that in the present study. This likely allowed even more subtle group differences to be observed. In addition, Goodlett and Johnson found that acquisition of learning in a visible cue version of the water maze task was not impaired by the alcohol treatment. Likewise, we found no significant group differences in the visible or proximal cue condition in the present study. This is important since the lack of effects in this version of the task indicate that the longer escape latencies exhibited by the ET and ET + E groups in the invisible or distal cue condition reflect an impairment in spatial localization as opposed to visual or motor deficits.

11 M.D. Marino et al. / Int. J. Devl Neuroscience 22 (2004) Although the ethanol treatment used in this study did result in decreased body weights, this is unlikely to be a factor in the behavioral effects seen. Because the ET + E group showed learning impairments similar to the ET group, but no persistent decreases in body weight, this suggests that the decreased body weight of the ET animals is not responsible for the longer escape latencies in this task. This also suggests that Vitamin E treatment may prevent the decreases in body weight seen in this study. However, it should be noted that in the cohort of animals used for behavioral testing and CA1 cell counts, the body weights of the ET and ET+E did not significantly differ; additionally, the ET + E group did not differ from controls. Therefore, any increases in body weight induced by the Vitamin E treatment are minimal at best and unlikely to affect the behavioral results seen Effects of antioxidant therapy Effects on oxidative stress In the current study, we found that Vitamin E treatment alleviated the increase in protein carbonyl formation associated with alcohol exposure. Although no other studies exist on the effect of antioxidant treatment on alcohol-induced protein carbonyl formation (or alcohol-induced oxidative stress in general), several studies have shown that Vitamin E treatments prevent the production of protein carbonyls in liver (Swierczynski and Mayer, 1998), serum (Zhang and Omaye, 2000), and plasma (Lee et al., 1998) in response to various oxidative stressors. Thus, this is the first study to demonstrate this protective effect of Vitamin E in response to alcohol exposure. Furthermore, our results support the antioxidant role that has been attributed to Vitamin E by previous studies Effects on neuronal loss The protective effect of Vitamin E on neuronal loss is generally consistent with the limited number of studies that have been conducted on this topic. Mitchell et al. (1999a,b) found that both Vitamin E and -carotene increased the survival rate of hippocampal neurons exposed to alcohol in vitro. Both Vitamin E and -carotene completely ameliorated neuronal loss following 400 mg/dl exposure. This concentration, though high, may reflect a physiologically attainable BAC in alcoholic populations and is comparable to the BAC obtained in some binge exposure animal models, including the present study. Thus the cell loss observed in the current study supports the in vitro findings of Mitchell et al. (1999a,b). A later in vivo study from the same laboratory found that acute alcohol exposure during the neonatal period (PD4 5) resulted in a significant loss of cerebellar Purkinje neurons, and that this loss was abrogated by the concurrent administration of Vitamin E (Heaton et al., 2000). Although this study assessed neuron loss in the cerebellum instead of the hippocampus, our finding that Vitamin E protects against neuron loss in vivo is consistent with this general concept. However, it must be noted that recent preliminary findings from Tran et al. (2003) showed that Vitamin E treatment did not prevent the cerebellar cell loss induced by neonatal alcohol exposure. In this study, alcohol was administered to neonatal rats from PD4 9, an exposure paradigm shown previously to reduce the number of cerebellar Purkinje cells (Goodlett et al., 1997). Both the Heaton and Tran studies used comparable doses of Vitamin E (60 IU or 40 mg/kg used by Heaton et al. versus 73 IU or 49 mg/kg used by Tran et al.). Also, the alcohol administration procedures were similar in both studies (PD4 5 via intubation in the Heaton study versus PD4 9 via intubation in the Tran study). However, several differences exist between the two studies. Whereas the Heaton study indirectly estimated cell number from vermal lobule I only, the Tran study used the optical fractionator to directly compute total neuron number from an entire cerebellar hemisphere, including lobules I-HI. In addition, the BACs achieved by Heaton et al. (BACs ranged from 257 to 264 mg/dl) differed somewhat from those achieved in the Tran study (BACs were in the range of 350 to 400 mg/dl). Another important difference between the studies was the amount of time that elapsed between the alcohol exposure and the cell counts. The cell count in Heaton s study was performed at PD9, just a few days after alcohol exposure, whereas Tran and colleagues performed the counts several weeks after alcohol exposure (around PD36). Both found alcohol-induced cell loss, indicating that alcohol exposure induces permanent changes in the cerebellum. However, the lack of effect of Vitamin E in the Tran study might indicate that the protective effects of Vitamin E found in the Heaton study are short-lived, only delaying and not totally preventing, the alcohol-induced cell loss in the cerebellum. Another recent study found no protective effect of the antioxidant melatonin on cerebellar Purkinje cells in the vermis and lobule I (Edwards et al., 2002). Since the timeline of cerebellar development differs from that of the hippocampus, it is difficult to compare these findings with those of the current study. However, our results suggest that the protective effect of Vitamin E on alcohol-induced hippocampal cell loss may be longlasting Effects on behavioral deficits Besides the current study, only one other recent study by has examined the functional effect of antioxidant treatment in an animal model of FAS (Tran et al., 2003). In their study (described in detail above), alcohol exposure produced deficits in eyeblink conditioning in juveniles, but this was not affected by Vitamin E treatment. This is in agreement with our finding that Vitamin E treatment did not prevent alcohol-induced learning deficits. These findings suggest that (1) that CA1 cell loss is not predictive of spatial learning impairments, and (2) that some other processes, outside of oxidative stress, may account for the damage underlying these deficits.