Wei-Jung A. Chen

Fetal alcohol syndrome: the vulnerability of the developing brain and possible mechanisms of damage

SummaryFetal alcohol exposure has multiple deleterious effects on brain development, and represents a leading known cause of mental retardation. This review of the effects of alcohol exposure on the developing brain evaluates results from human, animal andin vitro studies, but focuses on key research issues, including possible mechanisms of damage. Factors that affect the risk and severity of fetal alcohol damage also are considered.

Prenatal binge-like alcohol exposure alters neurochemical profiles in fetal rat brain

The majority of studies examining the effects of prenatal exposure to alcohol on neurotransmitter levels have furnished results that are divergent (increase, decrease or no change). The present study assessed six neurochemical compounds [norepinephrine (NE), dopamine (DA), dihydroxyphenylacetic acid (DOPAC), serotonin (5-HT), 5-hydroxyindole acetic acid (5-HIAA), gamma-aminobutyric acid (GABA)] from the same brain tissue. Pregnant Sprague-Dawley rats were given 5.1 g/kg alcohol (by gavage) either daily from embryonic day 1 (E1) through E20 or E20 only. In addition, pairfed/intubated (PF/INT) and ad lib chow (Chow) groups were included as controls. The dams were sacrificed and the fetuses were removed on E20. Binge-like alcohol exposure throughout gestation (E1-E20) produced significantly higher brain to body weight ratios compared with all other groups. Alcohol exposure did not produce changes in NE levels, although the E1-E20 exposure to alcohol reduced the contents of DA and 5-HT compared with the PF/INT and Chow controls. In addition, the E20 alcohol treatment reduced both DA and 5-HT levels compared with the E1-E20 alcohol treatment. DOPAC and 5-HIAA contents were affected by the prenatal treatments insofar as the 5-HIAA levels were decreased in E/1-20 and E20 animals relative to both controls, while the DOPAC levels were decreased in E/1-20, E20 and PF/INT groups compared to the Chow group; however, both metabolites were unaffected by the difference in alcohol treatment duration. Moreover, GABA levels were increased in fetuses exposed to alcohol from E1-E20 compared with all other groups. Collectively, these findings suggest that binge-like alcohol exposure prior to and during neurotransmitter development affects the baseline content of several neurotransmitters.

Prenatal alcohol treatment attenuated postnatal cocaine-induced elevation of dopamine concentration in nucleus accumbens : A preliminary study

Prenatal alcohol exposure has been shown to damage the developing central nervous system (CNS) in a variety of ways, including neuroanatomical anomalies, neurochemical imbalance, and neuropharmacological dysfunction. The present study investigated one of the functional aspects of dopaminergic system in neonatal rats exposed prenatally to a binge-like alcohol paradigm by measuring dopamine concentrations following a single postnatal cocaine challenge. Pregnant Sprague-Dawley rats were given daily intragastric intubations of 5.1 g/kg alcohol solution from embryonic day (E) 1 to 20. Pair-fed and ad lib-fed animals served as controls. On E33 (usually postnatal day 10), offspring from all groups were given injections (IP) of either 0, 20, or 40 mg/kg cocaine. Animals were sacrificed and the substantia nigra/ventral tegmental area (SN/VTA) and nucleus accumbens (NAc) were dissected for the determination of dopamine concentrations using HPLC. Basal dopamine levels (0 mg/kg cocaine group) did not alter as a function of prenatal alcohol treatment in either region. However, acute cocaine injection increased the dopamine content in NAc, but not in SN/VTA, in ad lib-fed animals, and this elevation in dopamine level was significantly attenuated by prenatal alcohol treatment in both female and male animals, and by prenatal pair-fed treatment in male animals. Taken together, these results indicate that there appears to be a regional difference in acute cocaine-induced dopamine elevation, and prenatal binge-like alcohol exposure significantly alters the functional responsiveness of dopaminergic system in NAc. Furthermore, these data suggest that male offspring may be more sensitive to stress-associated or nutritional influences during gestation.

4-Methylpyrazole, an alcohol dehydrogenase inhibitor, exacerbates alcohol-induced microencephaly during the brain growth spurt

Whether alcohol-induced microencephaly occurs as a result of the effect of alcohol or acetaldehyde remains an unanswered, yet important, question. The present study addressed this issue by using an alcohol dehydrogenase (ADH) inhibitor, 4-methylpyrazole (4-MP), that works by blocking the metabolism of alcohol to its primary metabolite acetaldehyde, thereby prolonging the actions of alcohol while minimizing the generation of acetaldehyde. Four groups of artificially reared Sprague-Dawley rat pups were treated with alcohol treatment (3.3 g/kg EtOH or isocalorically matched control formula from postnatal days 4 through 9) and 4-MP administration (IP, 50 mg/kg or saline). A suckle control group was introduced to control the effects of the artificial rearing procedure. On postnatal day 10, all pups were perfused. Alcohol in combination with 4-MP treatment produced a marked microencephaly, as assessed by brain weights or brain to body weight ratios, compared with other artificially reared groups. The peak BACs in the pups that received both alcohol and 4-MP were increased at least twofold compared with those that received alcohol alone. These findings indicate that 4-MP is an effective nontoxic ADH inhibitor and that microencephaly is associated with BAC levels. Most importantly, these results support the hypothesis that alcohol is a causative agent for alcohol-induced microencephaly and implicates the importance of functional ADH activity in attenuating alcohol-induced neuroteratogenicity.

Cocaethylene exposure during the brain growth spurt period: brain growth restrictions and neurochemistry studies.

The concurrent use of alcohol and cocaine has recently attracted attention in the medical research field due to the prevalence of this drug abuse pattern and the exclusive formation of a pharmacologically active substance, cocaethylene (CE). This is the first study to examine the neuroteratogenic effects of cocaethylene exposure during the brain growth spurt (part of the third trimester equivalent) on brain growth restrictions and neurochemical profiles. For the brain growth restrictions study, three groups of artificially reared rat pups were given daily injections of 0, 10 or 20 mg/kg cocaethylene (s.c.) from postnatal days (PDs) 4 through 9. One group of normally reared pups (suckle control) also was used. These pups were perfused on PD 10 and the brains were removed and weighed (forebrain, cerebellum and brainstem). For the neurochemistry study, five groups of artificially reared pups were used and were treated identically to those in the brain growth restrictions study, with the exceptions that animals assigned to acute cocaethylene treatment groups did not receive cocaethylene from PDs 4 through 8 and all animals in this study were sacrificed on PD 9 by decapitation. One suckle control group was included to control the possible artificial rearing effects on the neurochemical measures. Blood and fresh brain tissues (cortex, subcortical structures, cerebellum and brainstem) were collected for blood cocaethylene concentration and neurochemical analyses using GC/MS and HPLC techniques, respectively. The statistical analyses indicated that daily administration of 10 or 20 mg/kg cocaethylene, but not 0 mg/kg cocaethylene, significantly restricted the brain growth (brain weights) in all three brain regions assessed. Furthermore, cocaethylene administration from PDs 4 through 9 produced region-specific alterations in various neurotransmitter concentrations. The changes in neurotransmitter levels were not a function of the responses to the last cocaethylene injection on PD 9, since the outcomes between six days of cocaethylene treatment (PDs 4 to 9) and one day acute treatment (PD 9) were notably different. Furthermore, the artificial rearing procedure appeared to produce significant alterations in various neurotransmitter levels when compared with normally reared (suckle) controls. Collectively, these results suggest that cocaethylene is neuroteratogenic to the developing brain during the third trimester equivalent and the unique formation of cocaethylene resulting from the concurrent use of alcohol and cocaine may represent an increased risk to the developing brain beyond the intrinsic neuroteratogenic effects of cocaine and alcohol individually.

Cocaine exposure during the brain growth spurt failed to produce cerebellar Purkinje cell loss in rat pups

Previous studies in our laboratory indicated that cocaine exposure during the brain growth spurt period, a developmental stage vulnerable to various teratogens, did not produce microencephaly (gross brain weight measures). However, neonatal cocaine exposure has been shown to affect motor coordination and balance, which are both sensitive to cerebellar damage. The purpose of this study was to investigate whether cocaine exposure during the brain growth spurt period could result in the loss of cerebellar Purkinje cells, a neuronal population known to be vulnerable to other teratogenic insults. Sprague-Dawley rat pups were randomly assigned to either cocaine-treated groups (40, 80 mg/kg s.c.) or a gastrostomy control group, and were reared using an artificial-rearing method from postnatal days (PDs) 4 through 9. On PD 10, these animals were perfused and the cerebella were extracted and processed for cell counts. Estimates of Purkinje cell numbers were obtained using a 3-dimensional optical dissector method. The results using this stereological method demonstrated no significant Purkinje cell loss in response to cocaine treatment, even at a dose which has been shown to result in high mortality. The failure of cocaine to produce significant Purkinje cell loss (present finding) or microencephaly (previous finding) odds to the evidence indicating that cocaine is not a potent neuroteratogen.

Cocaine-induced somatic growth deficit during the brain growth spurt is prevented by artificial-rearing

The brain growth spurt is a dynamic period of central nervous system development that has been shown to be particularly vulnerable to a variety of insults. This study was designed to assess the effects of various doses of cocaine during this potentially vulnerable period on survival rate, somatic growth, and brain development, utilizing an artificial-rearing paradigm. Three cocaine-treated groups received SC administration of either 40, 60, or 80 mg/kg/day of cocaine HCl from postnatal days 4 through 9. There were significant differences in survival rates among 60 and 80 mg/kg/day cocaine-treated animals as compared with gastrostomy controls. No statistically significant differences were found with regard to body and brain weights between the various cocaine-treated groups and the gastrostomy control group. Additionally, there was no evidence of cocaines effect on forebrain, cerebellum, or brainstem weights when compared with individually corresponding controls. Taken together with our previous results, these data suggest that cocaine administration during the brain growth spurt period produces a dose-related increase in lethality, and the use of artificial-rearing method seems to lower the lethal threshold of cocaine among these neonates. However, no statistically significant effects of cocaine exposure on brain weight measures were obtained. The most important finding was that the cocaine-induced somatic growth retardation observed previously under normal-rearing conditions was eliminated by using a nutritionally controlled artificial-rearing paradigm, suggesting that cocaine-induced restriction of somatic growth may be a function of interference with normal feeding behavior, and therefore, a secondary effect of cocaine exposure during development.

Alcohol-Induced Inhibition of Cocaine Metabolism and the Formation of Cocaethylene in Neonatal Rats

Due to the significant species differences in the timing of different stages of brain development, to study the effects of drugs during the period equivalent to the third trimester in humans it is necessary to administer the drugs to neonatal rats. rather than in utero. In this study, we investigated the pharmacokinetic interactions between alcohol and cocaine. Such information is critical in understanding the roles of alcohol and cocaine in mediating neuroteratogenesis. Sprague-Dawley rat pups (10 days old) were given IP injections of alcohol (3.3 or 5.0 g/kg) and/or cocaine (40 mg/kg). At 20, 60, or 100 min (60 and 100 min for 3.3 g/kg alcohol only) after injections, 20 microl of tail blood was collected for blood alcohol concentration (BAC) determination. Immediately after tail blood collection, blood was collected and pooled for determinations of blood cocaine (BCC), benzoylecgonine (BBC), and cocaethylene concentration (BCEC). The slopes of the ascending BAC curves were unaffected in the presence of cocaine. BCC levels declined significantly as a function of time after the peak level at 20 min postinjection time. BCC levels were unchanged in pups receiving 3.3 g/kg alcohol, but the levels were significantly higher in 5.0 g/kg pups 20 min after injections. BBC concentrations were reduced to nearly 50% in the presence of alcohol (both doses) 20 min after injections. BCEC was detected at all time points when both alcohol and cocaine were injected. Taken together, these findings indicated that the enzymatic systems involved in converting cocaine to cocaethylene were functional at an early postnatal age, and the metabolism of cocaine was affected by the presence of alcohol in neonatal rats.