Journal of Molecular Neuroscience Copyright © 2004 Humana Press Inc. All rights of any nature whatsoever reserved. ISSN0895-8696/04/24:189–199/$25.00
ORIGINAL ARTICLE
A Neuroprotective Peptide Antagonizes Fetal Alcohol Exposure-Compromised Brain Growth Feng C. Zhou,*,1,2 Youssef Sari,1 Teresa A. Powrozek,3 and Catherine Y. Spong 4 Indiana University School of Medicine, 1Department of Anatomy and Cell Biology and 2Program in Medical Neurobiology, and IUPUI, 3Department of Psychology, Indianapolis, IN 46202, 4Section on Developmental and Molecular Pharmacology, Laboratory of Developmental Neurobiology, National Institute of Child Health, Bethesda, MD, 20892 Received December 7, 2003; Accepted December 24, 2003
Abstract We evaluated a 9-amino-acid peptide, SALLRSIPA (SAL), an agonist of activity-dependent neurotrophic factor (ADNF), for its protective properties against fetal alcohol-related brain growth retardation, using an established liquid diet model of alcohol-related neurodevelopmental disorder (ARND) in C57BL/6 mice. Alcohol exposure during neurulation reduced body weight, head size, and specifically brain weight and volume. Major gross brain deficits include underdevelopment of brain areas, cortical thinning, ventricle enlargement, and restricted midline neural tissue growth leading to openings at the roof/floor plate. SALLRSIPA (SAL) treatment increased fetal body weight and restored brain weight, brain volume, and regional brain size. Furthermore, SAL restored cortical thickness, reduced the size and frequency of neural tube openings, and attenuated ventricular enlargement. The ability of SAL to antagonize alcohol-retarded brain growth and development of forebrain and midline neural tube at midgestation suggests its potential use as an antagonist against fetal alcoholrendered microencephaly early in development. Index Entries: Fetal alcohol syndrome; fetal alcohol effect; microencephaly; brain development; neurotrophic factor.
Introduction Maternal alcohol consumption is the leading nongenetic cause of mental retardation in the Western world. The most severe consequences of maternal alcohol abuse are fetal alcohol syndrome (FAS) and alcohol-related neurodevelopmental disorder (ARND) (Abel, 1984; Aase et al., 1995; Stratton et al., 1996), collectively called fetal alcohol spectrum disorder (FASD). FASD is associated with microencephaly, particularly in cortex, limbic system, and
cerebellum. FASD children display substantial cognitive and behavioral deficits (in ARND), and additionally feature craniofacial dysmorphogenesis (in FAS). The features of FASD persisting through childhood cause mental disabilities and psychosocial dysfunctions. Fetal alcohol exposure compromises development at early embryonic stages as seen with respect to small embryonic body, small head and brain size (microencephaly), reduced number of neurons, and neural tube and regional brain dysmorphogenesis
*Author to whom all correspondence and reprint requests should be addressed. E-mail:
[email protected]
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190 (Webster et al., 1983; Sulik et al., 1988; Webster and Ritchie, 1991). The plausible mechanisms of alcoholinduced dysmorphogenesis include excitotoxicity, acetyl alcohol toxicity, apoptosis, glial dysfunction, disruption of retinoic acid signaling, restricted neuronal migration, and so forth (for review, see Goodlett and Horn [2001]). FASD is a preventable disorder. Methods of attenuation include intervention with alcohol drinking and antagonism of alcohol teratogenesis. A vasoactive intestinal peptide (VIP) and its activity-dependent release of molecules, known as activity-dependent neurotrophic factor (ADNF), is a favorable candidate for antagonizing alcoholinduced growth limitations because of its hallmark effect in increasing embryonic growth. ADNF is an extremely potent neurotrophic (Brenneman and Gozes, 1996; Brenneman et al., 1997) or protective agent (Gressens et al., 1997). It additionally has the unique feature of potent efficacy at the femtomolar level (Gozes and Brenneman, 1996), rendering these peptides potentially useful agents to antagonize alcohol-induced ARND. SAL is an agonist of ADNF and has been identified as an extremely potent neurotrophic agent (Brenneman et al., 1998). In a similar family, glia-derived activity-dependent neuroprotective protein (ADNP) and its deduced protein structure, NAPVSIPQ (NAP), have also been identified as a crucial factor for neurodevelopment and as a potent neuroprotectant (Zamostiano et al., 1999; Gozes et al., 2000; Pinhasov et al., 2003). The study on SAL is reported here, and the study on NAP is in progress. In the mouse, prenatal alcohol exposure, depending on dose, pattern, and stage of alcohol exposure, generates a spectrum of teratology and behavioral deficits consistent with clinical FAS (Becker et al., 1996). The C57BL/6 mouse was used in the current study because of its ability to drink large quantities of alcohol and because this mouse has been used for FAS studies with characterized pathological features (Webster et al., 1983; Sulik et al., 1988; Webster and Ritchie, 1991; Olney et al., 2002). We have shown previously that alcohol treatment in liquid diet form, with 20% or 25% ethanol-derived calories (EDCs), in C57BL/6 mice from neurulation through midgestation, achieved a moderate blood alcohol concentration (BAC) similar to those obtained following human consumption. This treatment model also consistently produced characteristic ARND, including reduced embryonic body weight and brain weight in a dose-dependent manner, as well as
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Zhou et al. reduced volume of specific brain regions, cortical thinning, and compromised neural tube at midline (cNTM) tissue (Zhou et al., 2003). In addition, embryos on day 15 (E15) failed to form complete neural tissue at the roof or floor plate at selective regions of the diencephalons, with visible neural tube perforation at the site of future major ventricular regions as alcohol exposure continued. In all cases, the cNTM was accompanied by enlarged ventricles (Zhou et al., 2002; 2003). In this study, we asked, “Can a functional peptide of ADNF protect against alcohol-induced ARND?” Specifically, we investigated the effect of SAL in attenuating the major deficits induced by fetal alcohol exposure, including embryonic body weight and brain weight, head size, regional brain volume, cortical thickness, cNTM, and failure of formation of neural tissue at the roof of the neural tube.
Materials and Methods Animals and Breeding C57BL/6 mice, obtained from Harlan, Inc. (Indianapolis, IN), were acclimated for at least 1 wk and were maintained at the Indiana University Laboratory Animal Research Center in rooms with ad lib chow and water at 22°C room temperature and 30% humidity, on a reverse 12-h light/dark cycle (off at 0700 h). Only nulliparous females were used for mating. In our 2-h breeding paradigm, females were placed with a male for 2 h and checked for a sperm plug immediately after the mating session. Positive sperm plug detection was designated as E0 or hour 0. Within a breeding cohort, females were mated in subgroups separated by 2–3 d. This facilitated contemporaneous pair feeding (below), which required a lag in gestation between the ethanol-consuming dam and its matched pair-fed (PF) dam. Liquid Diets and Alcohol/SAL Administration Pregnant females were weighed daily and, on the day before treatment, assigned pseudorandomly (matched for weight) to one of five treatment groups: free-access ethanol liquid (ALC) diet delivering 25% EDCs as the sole source of nutrients; PF control, yoked individually 2–3 d after to an ALC dam and given matched daily amounts of free-access isocaloric liquid diet, with maltose-dextrin substituted for ethanol; or ad libitum chow and water (Chow) at all times during gestation. The L-form SAL was synthesized in the Molecular Biotechnology Facility, Indiana University School of Medicine, Volume 24, 2004
SAL Attenuates Fetal Alcohol Microencephaly and tested for its efficacy against cultured cells by Dr. D. Brenneman prior to use. The ALC + SAL group was treated with alcohol as in the ALC group and further treated with SAL with a dose adopted from a previous study (20µg/0.2mL). The SAL was freshly prepared in sterile saline and administered once daily ip 30 min before the beginning of the dark cycle. The SAL-only group was the same as the Chow group, but was administered SAL. All treatments were carried from E7 (~168 h postcoitus [PC]) until E15 (~360 h PC), at which time all fetuses were removed for analysis. The fortified liquid Sustacal diet followed the published protocols reported by Middaugh and colleagues (1988, 1995). The diet contains 237 mL of chocolate-flavored Sustacal (Mead Johnson), 1.44 g Vitamin Diet Fortification Mixture (ICN no. 904654), and 1.2 g Salt Mixture XIV (ICN no. 902850). For the ethanol diet, 15.3 mL of 95% ethanol was added to the fortified Sustacal formula, as was water, to make 320 mL of diet with 1 cal/mL (ethanol 4.49% [v/v]). For the isocaloric control diet, 20.2 g Maltose Dextrin (BioServ) was added to the fortified Sustacal formula with water to bring it to 1 cal/mL. One day before treatment, all dams were adapted to the liquid diet by giving them the control diet (without ethanol) as their sole source of calories. Each day between 800 and 900 h, the dams were weighed, the volume of liquid diet consumed during the previous 24 h recorded from 30-mL graduated screw-cap tubes, and freshly prepared diet provided.
Blood Alcohol Levels Maternal blood alcohol levels were tested previously with two sets of five C57BL/6 dams with the 25% EDC diet. In brief, pregnant mice were given the same feeding protocol as the other experimental dams (ethanol diet provided on E7 at 900 h), and two 50-µL tail blood samples were obtained (at 1100 and 1300 h) on E8, E11, and E14. The blood samples were collected in heparinized capillary tubes and centrifuged, and 5-µLplasma samples were analyzed for alcohol concentration using the Analox Alcohol Analyzer, calibrated using a 100 mg/dL ethanol standard. The resulting BACs ranged from 40 to 140 mg/dL from E8 to E14, as reported previously (Zhou et al., 2003). Head Size and Body and Brain Weight The fetuses were removed from the uterus on E15 in phosphate-buffered saline. The head size was Journal of Molecular Neuroscience
191 determined by measuring the widest temporal distance in parallel to the line between the eyes. The brains were removed from the fetuses, by carefully peeling the cartilage (Zhou et al., 2000), and then trimmed between the rostral end of the cortical vesicles to the caudal end of the metencephalic flexure. The brains were then fixed by immersion in 4% paraformaldehyde in phosphate buffer for 24 h before transferring to phosphate buffer alone. Prior to weighing, the body or brain was rolled over a piece of dry absorbent paper to remove excess buffer. Body or brain weights of fetuses from each dam were averaged as one value. Eight dams per group were measured for weight.
Volumetric Measurement and Image Analysis To avoid changes in tissue volume because of shrinkage from tissue processing, both experimental and control groups were embedded in gelatin. All fetal brains were aligned at the same level in gelatin, with fetal brains from ALC with PF, ALC with Chow, or ALC with ALC + SAL, and serial 50-µm coronal sections were cut on a vibrating microtome (Leica). All coronal sections were mounted onto slides, Nissl-stained with methyl green, dehydrated with alcohol, cleared with xylene, and coverslipped for light microscopy and photomicrography. All fetal brain coronal sections were visualized and digitized using a SPOT camera mounted on a Leitz Orthoplan II microscope. Shrinkage among groups following the histological staining processes and dehydration steps was examined. There was no shrinkage in the x/y-axis (area of the sections), but 32% shrinkage was found in the z-axis (thickness) of the sections across the brain regions. There was no differential shrinkage among ALC, ALC + SAL, PF, and Chow. Fresh (prior to dehydration) section thickness was used for volume measurement described below. The area of a particular brain region was determined by drawing a contour around the region and calculating the number of pixels within the contour using an NIH Image System. The thickness of the fresh wet section was determined at ×60 immediately after microtome sectioning. In each fetal brain, 15–20 coronal sections were used for determining the thickness. An average thickness was taken from the sections. Every section was used that contained each brain nucleus. The shape factor in three-dimensional display (differential area between dorsal and ventral view of a section in referring to a specific brain nucleus), if it exists, is minimal. The volume of each fetal brain structure was estimated by multiplying Volume 24, 2004
192 the sum of all measured areas across all sections by the thickness of the fresh wet section. All measurements were done in both hemispheres. Six to ten fetal brains were used per group, with one brain being randomly chosen from each litter. The criteria for selecting the boundaries of a specific region of the fetal brains for measurement were based on neuroanatomical landmarks within the coronal sections. Nissl staining with methyl green was used to visualize the brain profile and borders encountered by the developing neuroepithelium of the selected areas. The ganglionic eminence (g. eminence) was divided into the rostral (including the striatal primordium rostral to pallidal primordium) and the caudal (including the pallidal primordium and the adjacent ventral striatal primordium). The g. eminence was measured rostral from the rostral tip of the g. eminence and caudal to the beginning of the diencephalon. The medial border was set at the lateral ventricle, and the lateral border was designated at the primitive corpus callosum or cortical tissue. The septal nucleus was measured and included the medial/ lateral septum and diagonal band. The characteristic hippocampus primordium was measured between the subicular neuroepithelium and the choroid plexus. The hebenular primordium was measured at the level of the anterior thalamic neuroepithelium. The amygdala was delineated ventral to the piriform cortex or g. eminence and lateral to the internal capsule at the level of the hippocampus. The diencephalon was measured around the third ventricle and included thalamic and hypothalamic areas. The volume of each brain region was obtained by multiplying the area of the measurement (by computer-assisted NIH image analysis) by the average thickness of the fresh section determined under the same microscope.
Cortical Thickness The cortical thickness was measured in the medial frontal cortex and the cingulate cortex. The cortical thickness was measured as the shortest distance between the surface of the cortex and the upper (or medial) border of the primordial corpus callosum. Measurement of cortex was further divided into two major layers: (1) the differentiating layer, including the cortical plate and intermediate zone; and (2) the germinal layer, including the ventricular and subventricular zone, defined by its characteristic cellular profile.
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Zhou et al. Size of Lateral and Third Ventricles Both lateral and third ventricles were delineated quite clearly in the embryo. The lateral ventricle was measured in the telencephalic vesicle, and the third ventricle was measured in diencephalic vesicles with similar criteria to those described in the above section. The volume was again obtained by multiplying the area of the measurement by the average thickness of the fresh section determined under a microscope.
Neural Tube Opening The neural tube opening is defined by incomplete formation of neural tissue (not non-neural membrane) occurring at the floor or dorsal folds. The openings (underneath the wrapping membrane) were observed in whole embryos under a stereomicroscope, followed by microscopic examination of tissue after sectioning, as described previously (Zhou et al., 2003). The brains were embedded in gelatin to avoid tearing of the dorsal or ventral plates. The numbers of brain sections with openings (o) were counted through the entire collection of forebrain sections (n). The ratio of opening (r) along the neural tube axis was determined by the number of sections with openings over the total number of sections measured: r = o/n. The length (l) of the opening along the neural tube axis was determined by multiplying the number of sections with openings (o) by the thickness of the section (average fresh section) (h), as determined by a high-magnification lens: l = o x h (Zhou et al., 2003).
Statistical Analysis For all of the above measurements, one sample was taken from each dam for analysis, with the exception of brain weight, which was an average of brain weights from the whole litter. One-way analysis of variance (ANOVA) was used for analysis among ALC, ALC + SAL, Chow, and PF treatments. After a finding of significance, Fisher’s protected least significant difference (PLSD) was used for pair-wise comparisons. We found that the PF and Chow groups were not different throughout the experimental paradigms. An opening size in an embryo that was greater than 2 standard deviation from the controls was considered significantly compromised. The frequency of cNTM was analyzed in floor and roof closure (neural tissue). An overall frequency of cNTM embryos in each group was also determined.
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Fig. 1. Effect of SAL on fetal alcohol-compromised body and brain weight. Fetal alcohol exposure reduced both body and brain weight at E15. The SAL treatment (20 µg/0.2 mL per dam once daily, ip) throughout the period of alcohol exposure protected the embryos from alcohol-induced reduction with greater effect on brain weight than body weight. Values are expressed as mean ± SEM.; significance of group comparisons used ANOVA and post hoc tests (Fisher’s PLSD). (**, *) p < 0.01 and p < 0.05, respectively. No significant difference was found between Chow and PF groups.
Results Body and Brain Weight and Head Size In agreement with our previous report, alcohol consumption from E7 to E15 reduced body weight and brain weight (Fig. 1) (Zhou et al., 2003), as well as head size (Fig. 2), of the developing embryos. SAL treatment in ALC increased the fetal body weight (p < 0.01) to just below the level of PF and Chow (Fig. 1) and restored the head size (Fig. 2) and brain weight (p < 0.05) (Fig. 1) from that of ALC to levels comparable to PF and Chow. Brain Dimension and Volume of Brain Areas Alcohol, in the current paradigm, also reduced brain dimensions (forebrain volume) from that of PF and Chow, further supporting alcohol-induced brain
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growth retardation. SALLRSIPA(SAL) pretreatment increased the brain dimensions of ALC-treated animals (p < 0.01) to a level comparable to those of PF and Chow (p < 0.05) (Table 1). The alcohol-induced brain area reduction at E15 is brain-wide, with the limbic system being affected most severely (Table 2). Affected regions included the g. eminence, diencephalon, septal nucleus, and to a greater degree, the hippocampus and amygdala (Table 2). SAL increased the size of brain areas in all of the above brain regions (Table 2) to levels similar to those of PF and Chow. No difference was found between PF and Chow in the above parameters.
Neural Tube Opening Fetal alcohol exposure compromised the cNTM, which attenuated timely closure of the neural tube
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Fig. 2. Effect of SAL on fetal alcohol-induced head size reduction. The fetal alcohol exposure in the liquid diet model (with 25% ethanol-derived calories from E7 to E15) retarded the growth of embryos as compared to Chow and PF. The embryos have a smaller head size, with corresponding lower body and brain weight, and smaller brain dimension. The reduction in head size was attenuated by the SAL treatment (ALC + SAL, ip, 20 µg/0.2 mL daily during alcohol treatment). The Chow with SAL treatment (Chow + SAL), however, did not create an abnormally large embryo. Scale bars = 1 mm. Table 1 Effect of Prenatal Alcohol Exposure on Forebrain Dimension Animal groups Forebrain dimension (mm3)
Chow (n = 7)
PF (n = 7)
2.26 ± 0.05
2.26 ± 0.07
ALC (n = 5) 1.77 ± 0.17a–c
ALC + SAL (n = 5) 2.15 ± 0.02
Note: Values are expressed as mean ± SEM; significance of group comparisons used one-way ANOVA and Fishers PLSD. a Significant difference between Chow and ALC groups (p < 0.01). b Significant difference between PF and ALC groups (p < 0.01). c Significant difference between ALC+SAL and ALC groups (p < 0.01).
in the roof and floor plate. The opening was increased in size, as well as in frequency, in the ALC (Fig. 3 and Table 3). This increment is similar to our previous observations (Zhou et al., 2003). SALLRSIPA (SAL) reduced both size and frequency of the opening.
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Ventricle Enlargement Fetal alcohol exposure enlarged ventricular size in the ALC brain as compared with that of PF throughout the lateral and third ventricles. These dilated ventricles were often associated with smaller
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Table 2 Effect of Prenatal Alcohol Exposure on Volumes of Forebrain Structures Fetal brain primordium structures Septal Hippocampal Amygdala Rostral g. eminence Caudal g. eminence Diencephalon Habenular
Chow (n = 7)
PF
ALC
ALC + SAL
(n = 7)
(n = 5)
(n = 5)
0.23 ± 0.01 0.34 ± 0.03 0.27 ± 0.01 0.21 ± 0.01 0.31 ± 0.01 1.14 ± 0.06 0.28 ± 0.02
0.23 ± 0.01 0.34 ± 0.03 0.27 ± 0.01 0.21 ± 0.01 0.30 ± 0.01 1.14 ± 0.09 0.27 ± 0.02
0.17 ± 0.01a,d 0.20 ± 0.04a,c,e 0.20 ± 0.01a,c,f 0.16 ± 0.01 a,c,f 0.23 ± 0.02 a,c,e 0.81 ± 0.05 a,c,e 0.17 ± 0.02 a,c,e
0.20 ± 0.01b.c 0.31 ± 0.02 0.25 ± 0.01 0.20 ± 0.01 0.29 ± 0.01 1.03 ± 0.06 0.25 ± 0.02
Note: Values are expressed as mean ± SEM; significance of group comparisons used one-way ANOVA and Fisher’s PLSD. a,b Significant difference between Chow and ALC, or between Chow and ALC+SAL groups (p < 0.01 and p < 0.05, respectively). c,d Significant difference between PF and ALC , or between PF and ALC+SAL groups (p < 0.01 and p < 0.05, respectively). e,f Significant difference between ALC+SAL and ALC groups (p < 0.01 and p < 0.05, respectively).
Fig. 3. The effect of SAL on fetal alcohol-induced neural tube openings at the roof (A) and floor plate (B) in the third ventricle region. The induction of openings (the same experimental paradigm as in Fig. 2) was antagonized by SAL treatment in all three regions. The alcohol-induced opening in the floor plate was completely attenuated by SAL treatment. The opening was expressed as a ratio of the total axis length examined. For significance of group comparisons, ANOVA and post hoc tests (Fisher’s PLSD) were used. (**, *) p < 0.01 and p < 0.05, respectively. No significant difference was found between Chow and PF groups.
brain size, thus rendering an even smaller brain mass. This was revealed earlier in our brain weight measurements (Fig. 1). SAL attenuated the enlargement of both the lateral and third ventricles (Fig. 4).
in the above cortices, as shown in medial frontal cortex (Fig. 5).
Cortical Thickness Alcohol also reduced the thickness of the medial frontal and cingulate cortices. Alcohol consumption, in our model, caused thinning of cortices, specifically in the differentiating molecular layer and intermediate zone but not in the germinal layer of the ventricular and subventricular zone. SAL treatment increased thickness in the differentiating zone of cortex, but did not further increase the germinal zone
Major Effect on FASD Pregnant C57BL/6 mice exposed to alcohol in a 25% ethanol derived calorie (EDC) liquid diet, produced a characteristic neurodevelopmental disorder featuring microencephaly, hydroencephaly, and cNTM development (Zhou et al., 2003). In this study, we confirmed the previously observed neurodevelopmental disorders with brain growth restriction, including microencephaly (reduced head size, brain
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Discussion
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Zhou et al. Table 3 Frequency of Neural Tube Opening Preneurulation-paradigm alcohol
Area Roof Floor
Chow 0/5 0/5
PF 0/5 0/5
ALC 4/6 1/6
ALC + SAL 2/6 0/6
Fig. 4. In the same treatment paradigm (as in Figs. 2 and 3), SAL protected embryos from alcohol-induced ventricular enlargement. The alcohol-induced ventricle enlargement was attenuated by the SAL treatment in both the third and lateral ventricles. (**) p < 0.01; (*) p < 0.05). No significant difference was found between Chow and PF groups.
weight, and brain volume) and ventricular enlargement. Furthermore, in the alcohol treatment paradigm, we reported a smaller head size and a reduced volume of major brain regions, including limbic areas and cortical thinning. In addition to the above observation, we reported that alcohol compromised neural tissue closure at the midline of the roof and floor plate.
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SAL treatment was found to attenuate alcoholinduced reductions in head size, brain weight, brain dimensions, and regional brain volume. A major feature of SAL protection is that it demonstrated a growth increase in the growth-compromised brain. SAL also attenuates cortical thinning and ventricular enlargement. Because microencephaly is the hallmark deficit in FASD, SAL can be considered as a potential therapeutic agent. SAL treatment also attenuated the incomplete formation of neural tissue covering the roof plate and breach in the floor plate. This has additional significance. It is known that the floor plate/midline tissue receives sonic hedgehog (Shh) from the notochord, as well as producing shh upon its signaling (Ericson et al., 1995.) Shh triggers the production of fibroblast growth factor (FGF) in the floor plate and midline tissue essential for cascaded dorsoventral pattern development and monoamine neuronal restriction (Rubenstein, 1998; Ye et al., 1998). The SAL peptide treatment for developmentalrelated deficits has been tested with an intraperitoneal injection of high doses of alcohol at E8. In that acute alcohol study, pretreatment with an equimolar combination of the SAL and NAP peptides attenuated ethanol-induced fetal death and growth abnormalities, whereas SALalone did not effect fetal survival after heavy alcohol exposure (Spong et al., 2001). Fetal demise has not been a feature of the current moderate ALC model for FASD. We demonstrated further that SAL had a robust effect by increasing brain size and brain mass and amending cNTM tissue in our alcohol liquid diet model. SALLRSIPA(SAL) increases brain growth that would be restricted by moderate alcohol exposure. Thus, SAL demonstrates different levels of protection as a function of alcohol concentration.
Possible Mechanism The mechanism of action by which SAL, ADNF, or ADNP attenuates excitotoxicity is complex and, to date, unclear. Known cellular actions include increased expression of nuclear factor κB (Gozes et al., 1997; Glazner et al., 1999, 2000) and heat shock protein 60 (hsp-60) (Zamostiano et al., 1999; Glazner et al., 2000). These peptides also lead to a reduction in the production of reactive oxygen species and inhibition of oxidative stress (Glazner et al., 1999). These peptides act through a cAMP-independent mechanism, as neither forskolin (an adenylate cyclase activator) nor pituitary adenylate, nor cyclase-
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Fig. 5. In the same treatment paradigm (as in Figs. 2–4), SAL attenuated fetal alcohol-induced cortical thinning, as shown in medial frontal and cingulate cortices (A). The alcohol-induced cortical thinning occurred in the differentiating zone (molecular layer and intermediate zone) but not the proliferating zone (ventricular and subventricular zone), as shown in medial frontal cortex. SALLRSIPA (SAL) increased the thickness of the differentiating layer from the level of the ALC to levels comparable to PF and Chow control groups (B).
activating peptide, provide VIP-like protection (Gressens et al., 1997). The mechanism through which these peptides act therefore seems to require protein kinase C and mitogen-associated protein kinase activation to protect the developing mouse brain against excitotoxicity (Gressens et al., 1999). Cell death and growth abnormalities elicited by alcohol treatment during development are believed to be associated, in part, with severe oxidative damage and generation of reactive oxygen species. NAP and SAL have been shown to exhibit anti-oxidative and anti-
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apoptotic actions in vitro and reduce reactive oxygen species. Again, pretreatment with NAP resulted in a significant decrease in alcohol-associated fetal death. Biochemical assessment of alcohol-treated fetuses indicated that the combination peptide treatment attenuated alcohol-induced decreases in glutathione (Spong et al., 2001). An in vitro study indicated that 100 mM ethanol inhibits cell-cell adhesion mediated by the L1 cell adhesion molecule in L1-expressing NIH/3T3 cells (Wilkemeyer et al., 2002). A study with embryos
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198 cultured for 6 h with 100 mM ethanol showed a marked delay in in vitro development, as seen by a decrease in the number of paired somites. Replacing isoleucine in the amino acid sequence greatly compromised the protective effect of NAP against alcohol toxicity to the cultured embryos (Wilkemeyer et al., 2003), indicating that the peptide has an active component and is not a nutritional supplement source of amino acids. The neurotrophic properties of SAL are yet another possible mechanism by which the peptide attenuates the deficits induced by alcohol. There is a general delay in brain development and maturity in ARND. The neurotrophism of SAL might assist in “catching up” the retarded growth seen during the gestational stage. The increases of general brain growth by SAL are in agreement with this notion. In conclusion, we found that the ADNF agonist, SAL, protected against alcohol-induced deficits in head size, brain weight, brain volume, regional brain size, and cortical thickness during development. SALLRSIPA (SAL) also antagonized the alcoholcompromised midline tissue at the roof and floor plates and attenuated ventricular enlargement. Although the presence of undesired side effects of SAL cannot be completely ruled out at this point, SALtreatment in normal pregnancy does not impose abnormal growth in the above measured paradigms. Our observations, along with current literature, suggest that ADNF and ADNP peptides be considered as candidates for antagonizing alcohol-related dysmorphogenesis because they (1) are extremely potent at femtomolar levels, (2) antagonize fetal demise from intraperitoneal alcohol injection, and (3) attenuate microencephaly and related midline deficits, including compromised neural tube and ventricular enlargement. However, before they can be considered for treatment of alcohol-related developmental dysmorphogenesis, studies will have to be conducted to assess their efficacy, windows of effectiveness at different developmental time points, longterm consequences at postnatal ages, mechanisms and modes of action, and safety at the cellular and organism level, as well as the possible side effects of imbalanced growth or abnormal enhancement.
Acknowledgments This study is supported by U01 AA014829 (C. R. G. and F. C. Z.), R01AA12406 (F. C. Z.), and AA07462. We thank Dr. D. Brenneman for testing
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Zhou et al. the efficacy of SAL prior to our study and Mr. Fang Yung for his assistance in tissue processing.
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SAL Attenuates Fetal Alcohol Microencephaly the developing mouse brain against excitotoxicity. J. Mol. Neurosci. 13, 199–210. Gressens P., Marret S., Hill J. M., Brenneman D. E., Gozes I., Fridkin M., and Evrard P. (1997) Vasoactive intestinal peptide prevents excitotoxic cell death in the murine developing brain. J. Clin. Invest. 100, 390–397. Middaugh L. D., Randall C. L. and Favara J. P. (1988) Prenatal ethanol exposure in C57 mice: effects on pregnancy and offspring development. Neurotoxicol. Teratol. 10, 175–180. Middaugh L. D. and Baggan W. O. (1995) Prenatal maternal ethanol effects on pregnant mice and on offspring viability and growth: influences of exposure time and weaning diet. Alcohol. Clin. Exp. Res. 19(5), 1351–1358. Olney J. W., Tenkova T., Dikranian K., Muglia L. J., Jermakowicz W. J., D’Sa C. and Roth K. A. (2002) Ethanol-induced caspase-3 activation in the in vivo developing mouse brain. Neurobiol. Dis. 9, 205–219. Pinhasov A., Mandel S., Torchinsky A., Giladi E., Pittel Z., Goldsweig A. M., et al. (2003) Activity-dependent neuroprotective protein: a novel gene essential for brain formation. Brain Res. Dev. Brain Res. 144, 83–90. Rubenstein J. L. (1998) Development of serotonergic neurons and their projections. Biol. Psychiatry 44, 145–150. Spong C. Y., Abebe D. T., Gozes I., Brenneman D. E., and Hill J. M. (2001) Prevention of fetal demise and growth restriction in a mouse model of fetal alcohol syndrome. J. Pharmacol. Exp. Ther. 297, 774–779. Stratton K., Howe C., and Battaglia F. (1996) Fetal Alcohol Syndrome: Diagnosis, Epidemiology, Prevention, and Treatment, National Academy Press, Washington, D. C. Sulik K. K., Cook C. S., and Webster W. S. (1988) Teratogens and craniofacial malformations: relationships to cell death. Development 103(Suppl.), 213–231. Webster W. S. and Ritchie H. E. (1991) Teratogenic effects of alcohol and isotretinoin on craniofacial develop-
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