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ABSTRACT: These studies investigated interactions taking place at the mitochondrial membrane in neonatal rat cerebellum following ethanol exposure and focused on interactions between proapoptotic Bax and proteins of the permeability transition pore (PTP), voltage-dependent anion channel (VDAC) and adenine nucleotide translocator (ANT) of the outer and inner mitochondrial membranes, respectively. Cultured cerebellar granule cells were used to assess the role of these interactions in ethanol neurotoxicity. Analyses were made at the age of maximal cerebellar ethanol vulnerability (P4), compared to the later age of relative resistance (P7), to determine whether differential ethanol sensitivity was mirrored by differences in these molecular interactions. We found that, following ethanol exposure, Bax proapoptotic associations with both VDAC and ANT were increased, particularly at the age of greater ethanol sensitivity, and these interactions were sustained at this age for at least 2 h postexposure. Since Bax:VDAC interactions disrupt protective VDAC interactions with mitochondrial hexokinase (HXK), we also assessed VDAC:HXK associations following ethanol treatment and found such interactions were altered by ethanol treatment, but only at 2 h postexposure and only in the P4, ethanol-sensitive cerebellum. Ethanol neurotoxicity in cultured neuronal preparations was abolished by pharmacological inhibition of both VDAC and ANT interactions with Bax but not by a Bax channel blocker. Therefore, we conclude that, at this age, within the constraints of our experimental model, a primary mode of Bax-induced initiation of the apoptosis cascade following ethanol insult involves interactions with proteins of the PTP complex and not channel formation independent of PTP constituents. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2012.
Developmental Neurobiology 07/2012; · 3.55 Impact Factor
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ABSTRACT: These studies investigated ethanol effects on upstream cellular elements and interactions which contribute to Bax-related apoptosis in neonatal rat cerebellum at ages of peak ethanol sensitivity (postnatal day 4 [P4]), compared to later ages of relative resistance (P7). Analyses were made of basal levels of the pro-apoptotic c-jun N-terminal kinase (JNK), Bax, and the 14-3-3 anchoring proteins, as well as the responsiveness of these substances to ethanol at P4 versus P7. Dimerization of Bax with 14-3-3 was also investigated at the two ages following ethanol treatment, a process which sequesters Bax in the cytosol, thus inhibiting its mitochondrial translocation and disruption of the mitochondrial membrane potential. Cultured cerebellar granule cells were used to examine the protective potential of JNK inhibition on ethanol-mediated cell death. Basal levels of JNK were significantly higher at P4 than P7, but no differences in the other proteins were found. Activated JNK, and cytosolic and mitochondrially-translocated Bax were increased in P4 but not P7 animals following ethanol exposure, while protective 14-3-3 proteins were increased only at P7. Ethanol treatment resulted in decreases in Bax:14-3-3 heterodimers at P4, but not at P7. Inhibition of JNK activity in vitro provided partial protection against ethanol neurotoxicity. Thus, differential temporal vulnerability to ethanol in this CNS region correlates with differences in both levels of apoptosis-related substances (e.g., JNK), and differential cellular responsiveness, favoring apoptosis at the most sensitive age and survival at the resistant age. The upstream elements contributing to this vulnerability can be targets for future therapeutic strategies.
Brain research 11/2011; 1432:15-27. · 2.46 Impact Factor
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ABSTRACT: This study investigated ethanol influences on intracellular events that predispose developing neurons toward apoptosis and the capacity of the antioxidant α-tocopherol (vitamin E) and the neurotrophin brain-derived neurotrophic factor (BDNF) to modulate these effects. Assessments were made of the following: (i) ethanol-induced translocation of the pro-apoptotic Bax protein to the mitochondrial membrane, a key upstream event in the initiation of apoptotic cell death; (ii) disruption of the mitochondrial membrane potential (MMP) as a result of ethanol exposure, an important process in triggering the apoptotic cascade; and (iii) generation of damaging reactive oxygen species (ROS) as a function of ethanol exposure.
These interactions were investigated in cultured postnatal day 8 neonatal rat cerebellar granule cells, a population vulnerable to developmental ethanol exposure in vivo and in vitro. Bax mitochondrial translocation was analyzed via subcellular fractionation followed by Western blot, and mitochondrial membrane integrity was determined using the lipophilic dye, JC-1, that exhibits potential-dependent accumulation in the mitochondrial membrane as a function of the MMP.
Brief ethanol exposure in these preparations precipitated Bax translocation, but both vitamin E and BDNF reduced this effect to control levels. Ethanol treatment also resulted in a disturbance of the MMP, and this effect was blunted by the antioxidant and the neurotrophin. ROS generation was enhanced by a short ethanol exposure in these cells, but the production of these harmful free radicals was diminished to control levels by cotreatment with either vitamin E or BDNF.
These results indicate that both antioxidants and neurotrophic factors have the potential to ameliorate ethanol neurotoxicity and suggest possible interventions that could be implemented in preventing or lessening the severity of the damaging effects of ethanol in the developing central nervous system seen in the fetal alcohol syndrome (FAS).
Alcoholism Clinical and Experimental Research 02/2011; 35(6):1122-33. · 3.34 Impact Factor
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ABSTRACT: Prenatal alcohol exposure produces anatomical and behavioral abnormalities associated with fetal alcohol syndrome (FAS). Animal FAS models have demonstrated temporal windows of vulnerability in the developing cerebellum, with substantial ethanol (EtOH)-mediated apoptotic activation during these periods. In rodents, the cerebellum is most sensitive to EtOH on postnatal days 4 to 6 (P4 to P6). At slightly later ages (P7 and later), this region is less vulnerable to EtOH. The present study investigated EtOH effects on mechanisms related to activities of Bad, a proapoptotic member of the Bcl-2 gene family, to further characterize processes underlying these disparate EtOH sensitivities. In healthy cells, Bad is retained in the cytosol by association with 14-3-3, a primarily cytosolic protein. Bad promotes apoptosis by disassociating from 14-3-3 and sequestering Bcl-xL through heterodimerization. This dimerization prevents the neutralizing association of Bcl-xL with Bax, freeing Bax to perform in a prodeath manner. Caspase-dependent cleavage of Bad to a 15-kDa fragment increases its proapoptogenic capacity.
Two hours following EtOH exposure of P4 and P7 animals via inhalation, we determined how exposure affects intracellular localization and proteolytic cleavage of Bad and expression of cerebellar 14-3-3, using subcellular fractionation and Western blot techniques. Ethanol effects on interactions between Bad and 14-3-3 or Bcl-xL at the more vulnerable and less vulnerable ages were determined using an enzyme-linked immunosorbent assay-based technique to detect native protein-protein interactions.
At P4, EtOH increased mitochondrial localization of Bad, expression of a 15-kDa fragment recognized by Bad antibody, and formation of Bad:Bcl-xL complexes. At that more vulnerable age, EtOH also decreased formation of Bad:14-3-3 complexes. At P7, EtOH increased Bad:14-3-3 complexes and reduced Bad:Bcl-xL complexes. Cytosolic 14-3-3 remained unchanged by EtOH at P4 and P7.
Ethanol-induced alterations of Bad-related mechanisms at P4 favor a prodeath response. EtOH does not influence these same mechanisms in a manner that promotes cell death at P7. Divergent Bad-related responses at these 2 developmental ages likely contribute to their differential EtOH vulnerability.
Alcoholism Clinical and Experimental Research 07/2006; 30(6):1031-8. · 3.34 Impact Factor
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ABSTRACT: The developing cerebellum is highly sensitive to ethanol during discrete neonatal periods. This sensitivity has been linked to ethanol-induced alterations in molecules of the Bcl-2 survival-regulatory gene family. Ethanol exposure during peak periods of cerebellar sensitivity, for example, results in increased expression of proapoptotic proteins of this family, while overexpression of the antiapoptotic Bcl-2 protein in the nervous system protects against ethanol neurotoxicity. For the present study, neonatal mice with a targeted deletion of the proapoptotic bax gene were used to determine whether elimination of this protein would mitigate ethanol toxicity. bax knock-out and wild-type mice pups were exposed to ethanol via vapor inhalation during the maximal period of neonatal cerebellar ethanol sensitivity and cerebellar tissue was subsequently assessed for Purkinje and granule cell number and ethanol-mediated generation of reactive oxygen species (ROS). The results revealed that: (1) ethanol exposure during the peak period of cerebellar vulnerability resulted in substantial loss of Purkinje cells in wild-type animals, but not in bax knock-outs; (2) granule cells in the bax gene-deleted animals were not similarly protected from ethanol effects; and (3) levels of ROS following acute ethanol exposure were appreciably enhanced in the wild-type animals but not in the bax knock-outs. These results imply that Bax is important to ethanol-induced Purkinje cell death during critical neonatal periods, but that ethanol effects on granule cells may function at least partially independent of this apoptosis agonist. Amelioration of ethanol-mediated increases in ROS production in the knock-outs may contribute to the observed effects.
Journal of Neurobiology 02/2006; 66(1):95-101. · 3.05 Impact Factor
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ABSTRACT: Exposure of the developing nervous system to ethanol (EtOH) produces neurological aberrations associated with fetal alcohol syndrome. During a well-defined period, cerebellar neurons are highly susceptible to EtOH-induced death, primarily through apoptosis. Neonatal rodent cerebellum is exquisitely sensitive to EtOH on postnatal days 4-6 (P4-6); however, at slightly later developmental ages (P7 and later), EtOH effects are minimal. We have previously shown that EtOH differentially influences expression of apoptosis-related proteins of the Bcl-2 survival-regulatory gene family in P4 and P7 cerebellum. In the present study, the effects of EtOH on multiple functional mechanisms of Bcl-2, Bcl-xL, and Bax were investigated to characterize further the processes underlying these disparate EtOH sensitivities. For these analyses, we addressed the following questions, by using P4 and P7 cerebellar tissue following in vivo exposure: 1) Are there differential patterns of expression of antiapoptotic Bcl-2 or proapoptotic Bax in EtOH-vulnerable Purkinje cells that could contribute to the different degrees of temporal EtOH vulnerability? 2) How does EtOH affect intracellular localization of apoptosis-related proteins? 3) Does cleavage of Bax contribute to EtOH sensitivity? 4) Does EtOH differentially modulate cerebellar protein-protein interactions of Bcl-2, Bcl-xL, and Bax at the vulnerable vs. the resistant ages? Overall, we show that, at P4, the EtOH-mediated effects on Bcl-2, Bcl-xL, and Bax favor a prodeath response, whereas most of the intracellular responses to EtOH exposure at P7 promote survival. Such differential responsiveness likely plays a major role in the disparate ethanol vulnerability at these two postnatal ages.
Journal of Neuroscience Research 10/2005; 81(5):632-43. · 2.74 Impact Factor
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ABSTRACT: Ethanol (EtOH) disrupts the structure and function of the developing nervous system, sometimes leading to birth defects associated with fetal alcohol syndrome (FAS). Animal FAS models indicate that cellular membrane peroxidation, intracellular oxidant accumulation, and suppression of endogenous antioxidant enzymes contribute to the toxic effects of EtOH. Mitochondrially targeted vitamin E (MitoVit E), a chemically engineered form of vitamin E (VE) designed to accumulate in the mitochondria, has been shown to inhibit intracellular oxidant accumulation and cell death more effectively than VE. In previous investigations, we have shown that, in vivo, VE reduces neuronal death in the developing cerebellum of EtOH-exposed animals, and, in vitro, VE prevents apoptotic and necrotic death of EtOH-exposed cerebellar granule cells (CGCs). The present investigation shows that, in a FAS CGC model, 1 nM MitoVit E renders significant neuroprotection against EtOH concentrations as high as 1600 mg/dL. The present study also demonstrates that, in this same model, MitoVit E mitigates EtOH-induced accumulation of intracellular oxidants and counteracts suppression of glutathione peroxidase/glutathione reductase (GSH-Px/GSSG-R) functions, protein expression of gamma-glutamylcysteine synthetase (gamma-GCS), and total cellular glutathione (GSH) levels. In the presence and absence of EtOH, VE amplifies the protein expression levels of gamma-GCS, an enzyme that performs the rate-limiting step for GSH synthesis, and total GSH levels. These results suggest that MitoVit E and VE ameliorate EtOH toxicity through non-oxidant mechanisms-modulations of endogenous cellular proteins-and antioxidant means.
Brain Research 09/2005; 1052(2):202-11. · 2.73 Impact Factor
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ABSTRACT: Ethanol exposure during nervous system development produces a range of abnormalities, and in humans may lead to the fetal alcohol syndrome. Among the mechanisms hypothesized to play roles in ethanol neurotoxicity are altered expression of supportive neurotrophic factors (NTFs), and cellular disturbances in oxidative processes. In this study, ethanol effects on secretion of two NTFs, brain-derived neurotrophic factor, and neurotrophin-3 were analyzed in neonatal rat cerebellar granule cells, and the potential of the antioxidant vitamin E to modulate ethanol effects was investigated. Ethanol exposure in these preparations reduced NTF secretion, but vitamin E appreciably ameliorated the ethanol effects. Possible mechanisms underlying both the ethanol effects on NTF secretion, and the protection of this antioxidant are considered.
Neuroscience Letters 12/2004; 370(1):51-4. · 2.11 Impact Factor
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ABSTRACT: Pycnogenol (PYC), a patented combination of bioflavonoids extracted from the bark of French maritime pine (Pinus maritima), inhibits apoptosis and necrosis of developing neurons exposed acutely to ethanol (EtOH). The present study shows that the protective mechanisms of PYC in EtOH-exposed postnatal day 9 cerebellar granule cells (P9 CGCs) include (1) reduction of reactive oxygen species (ROS) production; (2) counteraction of suppressed copper/zinc superoxide dismutase (Cu/Zn SOD) and glutathione peroxidase/reductase (GSH-Px/GSSG-R) system activities; (3) upregulation of Cu/Zn SOD protein expression; (4) mitigation of the EtOH-mediated exacerbation of catalase (CAT) activity; and, (5) specific binding and inhibition of active caspase-3. These results indicate that the mechanisms by which PYC antagonizes EtOH-induced oxidative stress include oxidant scavenging and modulation of endogenous, cellular proteins. Using findings from the present and previous studies, a model delineating the mechanisms of EtOH effects on the system of antioxidant enzymes in developing CGCs is presented.
Journal of Neurobiology 12/2004; 61(2):267-76. · 3.05 Impact Factor
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ABSTRACT: The detrimental effects of ethanol exposure during nervous system development have been well established. The cellular mechanisms of ethanol neurotoxicity, however, have not been clearly defined. Recent studies suggest that neurotrophin signaling pathways may be involved in ethanol-mediated neuronal death. The present investigation, therefore, was designed to examine ethanol-induced alterations in neurotrophin receptor protein levels in the developing central nervous system (CNS) following chronic ethanol treatment administered during the early neonatal period. For this study, rats were exposed to ethanol via vapor inhalation from postnatal day 4 (P4) to P10. Brains were then dissected on P10 or P21, and Western blots used to quantify expression of neurotrophin receptors TrkA, TrkB, TrkC, and p75. This early postnatal ethanol treatment produced significant alterations in receptor levels in hippocampus, septum, cerebral cortex, and cerebellum. The alterations seen were variable, with decreases generally found in hippocampus and cerebellum, increases noted in septum, and changes in both directions occurring in cortex. These alterations were generally more prevalent in males than in females. While most of the receptor changes observed were transient, sustained or delayed alterations were occasionally seen in hippocampus, cortex, and cerebellum. These results suggest that developmental ethanol exposure modulates expression of these neurotrophin receptors throughout the CNS, alterations which could have wide-ranging effects on functional CNS development. The possible linkage between such changes and abnormalities encountered in the fetal alcohol syndrome are considered.
Journal of Neurobiology 08/2004; 60(1):114-26. · 3.05 Impact Factor
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ABSTRACT: Developmental ethanol exposure produces significant central nervous system (CNS) abnormalities. The cellular mechanisms of ethanol neurotoxicity, however, remain elusive. Recent data implicate altered neurotrophin signaling pathways in ethanol-mediated neuronal death. The present study investigated ethanol-induced alterations in neurotrophin receptor proteins in the rat CNS following chronic ethanol treatment during gestation, via liquid diet to pregnant dams. Brains were dissected on P1 and P10, and Western blots for the neurotrophin receptors TrkA, TrkB, TrkC, and p75 were quantified. Such ethanol treatment produced significant changes in neurotrophin receptor levels in the hippocampus, septum, cerebral cortex, and cerebellum. Receptor levels in hippocampus, septum, and cerebellum, tended to be decreased, while levels in cortex were consistently increased. Males were generally more affected than females. While most of these alterations were transient, sustained or delayed changes were present in P10 septum, cortex, and cerebellum. These results indicate that developmental ethanol exposure produces major changes in the normal physiological levels of the neurotrophin receptors throughout the CNS. These changes in the receptor complement during critical prenatal stages could relate to the anomalous development of the CNS seen in the fetal alcohol syndrome. This relationship is discussed, together with the potential biological effects of such dramatic changes in neurotrophin receptor expression.
Journal of Neurobiology 08/2004; 60(1):101-13. · 3.05 Impact Factor
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ABSTRACT: Developmental ethanol exposure leads to a variety of abnormalities in the central nervous system (CNS). Mechanisms proposed as underlying these effects include alterations of protective antioxidant support, increased generation of harmful free radicals, and altered expression of apoptosis-related proteins. In prior studies, exogenous antioxidant application has been found to reduce ethanol neurotoxicity, but the mechanisms by which this protection is afforded have not been defined. This study was designed to investigate the interactions between ethanol and the antioxidant vitamin E (alpha-tocopherol), with respect to neuronal survival and levels of proteins related to the Bcl-2 survival-regulatory gene family. Neonatal rat cerebellar granule cell cultures were used as a model system. It was found that ethanol significantly impaired neuronal survival in these preparations, and that survival in the presence of ethanol was enhanced by inclusion of vitamin E in the culture medium. This elevated survival was paralleled by increased levels of anti-apoptotic proteins (e.g., Bcl-2, Bcl-xl, activated Akt kinase), and concurrent downregulation of pro-apoptotic proteins (e.g., Bcl-xs). These results suggest that such alterations may represent an important mechanism whereby antioxidants protect against the neurotoxic effects of ethanol in the developing CNS. The possible manner by which these changes are effected are considered.
Developmental Brain Research 07/2004; 150(2):117-24. · 1.78 Impact Factor
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ABSTRACT: The developing central nervous system (CNS) is highly susceptible to ethanol, with acute or chronic exposure producing an array of anomalies and cell loss. Certain periods of vulnerability have been defined for various CNS regions, and are often followed by periods of relative ethanol resistance. In the present study, neonatal rats were acutely exposed to ethanol during a time when peak cell death is found in developing cerebral cortex (postnatal day 7; P7), and during a later neonatal period of ethanol resistance (P21). Comparisons at the two ages were made of basal levels of neurotrophic factors (NTFs), and in addition, ethanol-mediated changes in NTFs, apoptosis-related proteins, antioxidant activities, and generation of reactive oxygen species (ROS) were quantified at 0, 2, and 12 h following termination of exposure. It was found that at P21, basal levels of NTF nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) were considerably higher than at P7, possibly affording protection against ethanol neurotoxicity at this age. Following ethanol treatment at P7, approximately equal numbers of pro-apoptotic and pro-survival changes were produced, although most of the pro-apoptotic alterations occurred rapidly following termination of treatment, a critical period for initiation of apoptosis. At P21, however, the large majority of ethanol-mediated changes were adaptive, favoring survival. We speculate that the capacity of the older CNS to upregulate a number of protective elements within the cellular milieu serves to greatly mitigate ethanol neurotoxicity, while in younger animals, such adjustments are minimal, thus enhancing ethanol vulnerability within this developing region.
Developmental Brain Research 12/2003; 145(2):249-62. · 1.78 Impact Factor
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ABSTRACT: Ethanol produces abnormalities in the developing nervous system, with certain regions being vulnerable during well-defined periods. Neonatal rodent cerebellum is particularly susceptible to ethanol during the early postnatal period [on postnatal days 4-5 (P4-5)], while this region is resistant to ethanol at a slightly later time (P7-9). We assessed basal levels of several substances which may be involved in differential temporal ethanol vulnerability in neonatal cerebellum, and analyzed alterations in these substances after early ethanol exposure.
Assessments were made of neurotrophic factors nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4; apoptosis-related proteins Bcl-2, Bcl-xl, Bax, Bcl-xs, Bad, phosphorylated-Bad, phosphorylated-Akt, and phosphorylated-c-Jun N-terminal kinase; and the antioxidants superoxide dismutase, glutathione reductase, and catalase. These analyses quantified basal levels (in controls), and sequential changes following acute ethanol exposure at the vulnerable and resistant cerebellar periods (P4, P7).
Comparisons of basal levels of the molecules assessed between P4 and P7 revealed higher levels of total proapoptotic Bad at p4, higher levels of the protective pAkt kinase at P7, and lower levels of proapoptotic pJNK at P7. Other basal levels did not differ. While ethanol-mediated alterations were found at both ages favoring both apoptosis and survival, the apoptosis-promoting changes produced on P4 exceeded those on P7, and most occurred within the first 2 hr after exposure, a critical survival/death period. The number of alterations favoring survival were similar at the two ages, but at P7 most occurred within the first 2 hr after exposure, possibly acting in a protective manner.
Differential temporal vulnerability to ethanol in the neonatal cerebellum appears to be paralleled by cellular alterations in neurotrophic factors, apoptosis-regulatory proteins, and/or antioxidant activities which generally favor apoptosis at the most sensitive age and survival at the resistant age.
Alcoholism Clinical and Experimental Research 05/2003; 27(4):657-69. · 3.34 Impact Factor
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ABSTRACT: The developing central nervous system is extremely sensitive to ethanol, with well-defined temporal periods of vulnerability. Many brain regions are particularly susceptible to ethanol during the early neonatal period, corresponding to the human third trimester, which represents a dynamic period of growth and differentiation. For this study, neonatal rats were acutely exposed to ethanol or control conditions at a neonatal age when the developing striatum has been shown to be vulnerable to ethanol (postnatal day 3 [P3]), and at a later age (P14), when this developing region is relatively ethanol-resistant. We then analyzed basal levels of neurotrophic factors (NTFs), and ethanol-mediated changes in NTFs, apoptosis-related proteins, antioxidants, and reactive oxygen species (ROS) generation, which may underlie this differential temporal vulnerability. Sequential analyses were made following ethanol exposure on these two postnatal days, with assessments of NTFs nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4); apoptosis-related proteins Bcl-2, Bcl-xl, Bax, Akt and c-jun N-terminal kinase (JNK); antioxidants superoxide dismutase, glutathione reductase and catalase; and ROS. The results indicated that basal levels of BDNF, and to some degree NGF, were greater at the older age, and that ethanol exposure at the earlier age elicited considerably more pro-apoptotic and fewer pro-survival changes than those produced at the later age. Thus, differential temporal vulnerability to ethanol in this CNS region appears to be related to differences in both differential levels of protective substances (e.g. NTFs), and differential cellular responsiveness which favors apoptosis at the most sensitive age and survival at the resistant age.
Developmental Brain Research 03/2003; 140(2):237-52. · 1.78 Impact Factor
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ABSTRACT: The neonatal cerebellum undergoes an early period of ethanol sensitivity in which profound neuronal loss is seen following acute exposure, while slightly later exposure produces no such loss. This study was designed to determine whether this differential susceptibility is related to differences in ethanol-induced generation of reactive oxygen species (ROS). We found that ethanol treatment on postnatal day 4 (P4), the peak period of cerebellar vulnerability, resulted in ROS increases, but slightly later exposure (on P7) produced no immediate changes in ROS, but reductions were seen at 12 and 24 h following exposure. Exposure on P14 produced consistent decreases in ROS production. Thus, differential responsiveness in oxidative processes may play a major role in the differential temporal ethanol vulnerability of developing cerebellum.
Neuroscience Letters 01/2003; 334(2):83-6. · 2.11 Impact Factor
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ABSTRACT: The sensitivity of the developing central nervous system (CNS) to the deleterious effects of ethanol has been well documented, with exposure leading to a wide array of CNS abnormalities. Certain CNS regions are susceptible to ethanol during well-defined critical periods. In the neonatal rodent cerebellum, a profound loss of Purkinje cells is found when ethanol is administered early in the postnatal period [on postnatal days 4 or 5 (P4-5)], while this neuronal population is much less vulnerable to similar ethanol insult slightly later in the postnatal period (P7-9). Prior studies have shown that neurotrophic factors (NTFs) can be altered by ethanol exposure, and both in vitro and in vivo studies have provided evidence that such substances have the potential to protect against ethanol neurotoxicity. In the present study, it was hypothesized that depletion of an NTF shown to be important to cerebellar development would exacerbate ethanol-related effects within this region, when administration was confined to a normally ethanol-resistant ontogenetic period. For this study, brain-derived neurotrophic factor (BDNF) gene-deleted ("knockout") and wild-type mice were exposed to ethanol via vapor inhalation or to control conditions during the normally ethanol-resistant period (P7 and P8). Two hours after termination of exposure on P8, analyses were made of body weight, crown-rump length, and brain weight. In subsequent investigations, the number and density of Purkinje cells and the volume of cerebellar lobule I were determined, and the expression of anti- and pro-apoptotic proteins and the activities of endogenous antioxidants were assessed. It was found that the BDNF knockouts were significantly smaller than the wild-type animals, with smaller brain weights. Purkinje cell number and density was reduced in ethanol-treated knockout, but not wild-type animals, and the volume of lobule I was significantly decreased in the gene-deleted animals compared to wild-types, but was not further affected by ethanol treatment. The loss of Purkinje cells in the BDNF knockouts was accompanied by decreases in anti-apoptotic Bcl-xl and in phosphorylated (and hence inactivated) pro-apoptotic Bad, and reduced activity of the antioxidant glutathione reductase, while the antioxidant catalase was increased by ethanol treatment in this genotype. In the wild-type animals, anti-apoptotic Bcl-2 was decreased by ethanol treatment, but the pro-apoptotic c-Jun N-terminal kinase (JNK) was markedly diminished by ethanol exposure, while the activity of the protective antioxidant superoxide dismutase (SOD) was significantly enhanced. These results suggest that neurotrophic factors have the capacity to protect against ethanol neurotoxicity, perhaps by regulation of expression of molecules critical to neuronal survival such as elements of the apoptosis cascade and protective antioxidants.
Journal of Neurobiology 06/2002; 51(2):160-76. · 3.05 Impact Factor
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ABSTRACT: Transgenic mice overexpressing NGF in the central nervous system under the control of the glial fibrillary acidic protein (GFAP) promoter were exposed to ethanol via vapor inhalation on postnatal days 4 and 5 (P4–5), the period of maximal cerebellar Purkinje cell sensitivity to ethanol. Wild-type controls were exposed in a similar manner. There were no differences in body weight or size following these procedures, but the transgenic brain weights at this age were significantly greater than wild-type controls. In the wild-type animals, a significant 33.3% ethanol-mediated loss of Purkinje cells in lobule I was detected via unbiased three-dimensional stereological counting on P5. In the GFAP-NGF transgenic animals, however, the 17.6% difference in Purkinje cell number in control and ethanol-exposed animals was not significant. There was a similar difference in Purkinje cell density in both groups, which did reach statistical significance (−32.7% in wild-type ethanol-treated animals, −17% in transgenic ethanol-exposed animals). These results suggest that endogenous overexpression of neurotrophic factors, which have previously been shown to protect against ethanol neurotoxicity in culture, can serve a similar protective function in the intact animal. © 2000 John Wiley & Sons, Inc. J Neurobiol 45: 95–104, 2000
Journal of Neurobiology 11/2000; 45(2):95 - 104. · 3.05 Impact Factor
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ABSTRACT: Background: The cerebellum of the neonatal rat is highly susceptible to ethanol, with profound loss of Purkinje cells resulting from even brief exposure during the first postnatal week. Developmental ethanol exposure previously has been shown to induce free radicals/oxidative stress processes and/or down-regulate protective antioxidants. In an earlier study, we found antioxidants protected against ethanol neurotoxicity in a tissue culture environment. The present study was designed to determine whether similar protection could be manifested in the intact animal.Methods: Neonatal rats were administered a liquid diet via intragastric intubation on postnatal days 4 and 5 (P4-P5), the peak period of ethanol sensitivity in the developing cerebellum. The diet consisted of milk formula with 12% ethanol, the isocaloric substitution of maltose-dextrin for ethanol, or ethanol plus the antioxidant vitamin E. Unbiased three-dimensional counting was utilized to analyze Purkinje cell numbers and density within defined volumes from these animals on P5.Results: These determinations revealed a substantial loss of Purkinje cells in the ethanol-treated animals compared to controls (approximately 30–44%), but this loss was prevented by the inclusion of vitamin E (60IU/100 ml) in the diet. A lower concentration of the antioxidant (30IU/100 ml) was not effective in this regard, however.Conclusions: These results suggest that ethanol-related cerebellar damage during this early postnatal period may be related to oxidative stress processes or the insufficiency of protective antioxidants. Thus, antioxidant treatment may represent a possible therapy for preventing or ameliorating the central nervous system (CNS) damage seen in the fetal alcohol syndrome.
Alcoholism Clinical and Experimental Research 03/2000; 24(4):512 - 518. · 3.34 Impact Factor
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ABSTRACT: Background: The developing cerebellum has been shown to be profoundly affected by exposure to ethanol and to exhibit a temporal pattern of vulnerability. Cerebellar Purkinje cells are particularly susceptible to ethanol on postnatal day 4 or day 5 (P4-5), whereas this population is much less vulnerable to similar ethanol insult slightly later in the postnatal period (P7-9). The purpose of the study was to determine whether differential alterations in neurotrophic factors might be associated with this differential susceptibility.Methods: Neonatal rats were exposed to ethanol via vapor inhalation, and enzyme-linked immunoabsorbent assays were subsequently conducted to assess cerebellar nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3 protein content. These analyses were made after ethanol exposure during the period of maximal cerebellar ethanol sensitivity (postnatal days 4-5 [P4-5]), during a period of much lower sensitivity (P7-8), and during the entire “brain growth spurt” (P4-10).Results: These determinations revealed a significant ethanol-induced decrease in cerebellar nerve growth factor after exposure on P4-5 but not after exposure on P7-8 or P4-10. No significant changes in brain-derived neurotrophic factor or neurotrophin-3 were found with any of the exposure paradigms.Conclusions: These results suggest that alterations in nerve growth factor, which has previously been shown to support cerebellar Purkinje and granule cells, may be a mechanism contributing to the early ethanol susceptibility within these neuronal populations.
Alcoholism Clinical and Experimental Research 09/1999; 23(10):1637 - 1642. · 3.34 Impact Factor
