Norepinephrine transporter: a candidate gene for initial ethanol sensitivity in inbred long-sleep and short-sleep mice

BACKGROUND: Altered noradrenergic neurotransmission is associated with depression and may contribute to drug abuse and alcoholism. Differential initial sensitivity to ethanol is an important predictor of risk for future alcoholism, making the inbred long-sleep (ILS) and inbred short-sleep (ISS) mice a useful model for identifying genes that may contribute to alcoholism. METHODS: In this study, molecular biological, neurochemical, and behavioral approaches were used to test the hypothesis that the norepinephrine transporter (NET) contributes to the differences in ethanol-induced loss of righting reflex (LORR) in ILS and ISS mice. RESULTS: We used these mice to investigate the NET as a candidate gene contributing to this phenotype. The ILS and ISS mice carry different DNA haplotypes for NET, showing eight silent differences between allelic coding regions. Only the ILS haplotype is found in other mouse strains thus far sequenced. Brain regional analyses revealed that ILS mice have 30 to 50% lower [3H]NE uptake, NET binding, and NET mRNA levels than ISS mice. Maximal [3H]NE uptake and NET number were reduced, with no change in affinity, in the ILS mice. These neurobiological changes were associated with significant influences on the behavioral phenotype of these mice, as demonstrated by (1) a differential response in the duration of ethanol-induced LORR in ILS and ISS mice pretreated with a NET inhibitor and (2) increased ethanol-induced LORR in LXS recombinant inbred (RI) strains, homozygous for ILS in the NET chromosomal region (44-47 cM), compared with ISS homozygous strains. CONCLUSIONS: This is the first report to suggest that the NET gene is one of many possible genetic factors influencing ethanol sensitivity in ILS, ISS, and LXS RI mouse strains.     

Interaction between S-propranolol and ethanol in mice selectively bred for ethanol sensitivity: the inbred short- and long-sleep mice

BACKGROUND: We have studied the effect of a beta-adrenergic blocking agent, S-propranolol, on the response of mice to anesthetic doses of ethanol. We used the selectively bred short and long sleep (ISS and ILS) mice. These mice were selected for their differential sensitivity to anesthetic doses of ethanol and then inbred. The study was prompted by the finding that the effect of ethanol on the firing rate of cerebellar Purkinje cells is modulated by beta-adrenergic input. In addition, this firing rate depression by ethanol is highly correlated with the anesthetic potency of ethanol. We were attempting to find a behavioral correlate of this effect of beta-adrenergic agents in the ISS and ILS mice. METHODS: We studied the effect of S-propranolol plus ethanol on the sleep time and blood ethanol at awakening in the inbred ILS and ISS mice. We administered anesthetic doses of ethanol with and without S-propranolol. We conducted studies of the rate of disappearance of ethanol in the presence of S-propranolol and carried out sleep time and metabolic studies with mice in an incubator held at 32 to 33 degrees C. RESULTS: We found that S-propranolol caused a prolonged anesthetic time brought about by ethanol but only in ISS mice. There was no significant difference in the blood ethanol levels at awakening with or without S-propranolol, indicating that S-propranolol had no effect on the brain sensitivity. Subsequently, we showed that this was due to a profound hypothermia caused by a combination of S-propranolol and ethanol. This was greater in the ISS mice because a larger dose of ethanol was required for the anesthetic effect of ethanol. The effect on ethanol disappearance rate, temperature drop, and anesthesia time all were largely reversed by placing the animals in an incubator at 32 to 33 degrees C. CONCLUSIONS: Profound hypothermia lowers the ethanol disappearance rate when both S-propranolol and ethanol are given. The effect of S-propranolol is likely due to the blockade of beta-adrenergic receptors that prevents thermogenic responses to the hypothermia brought about by ethanol. The results indicated that there might be a genetic effect controlling the hypothermic response to the combination of S-propranolol and ethanol. Further experiments to investigate this are reported in a subsequent article. We could find no evidence of a central nervous system effect of S-propranolol on the hypnotic actions of ethanol in these strains of mice.     

MK-801-induced locomotor activity in long-sleep x short-sleep recombinant inbred mouse strains: correlational analysis with low-dose ethanol and provisional quantitative trait loci

BACKGROUND: Low doses of the N-methyl-D-aspartate receptor (NMDAR) antagonist MK-801 (dizocilpine) or ethanol increase locomotor activity to a lesser extent in long-sleep (LS), than in short-sleep (SS), mice. LS mice also have fewer brain [3H]MK-801 binding sites than SS mice. In this study, LSXSS recombinant inbred (RI) mice were used to investigate whether different NMDAR densities contribute to differential MK-801 activation and whether common genes are involved in initial sensitivity to MK-801-and ethanol-induced activation. METHODS: Locomotor activity was measured for 90 min after saline or MK-801 injection. Quantitative autoradiographic analysis of [3H]MK-801 binding was used to measure densities of NMDARs in seven brain regions. The ethanol (1-2 g/kg) activation scores from Erwin and colleagues (1997) were used for correlational analysis, as was their method for quantitative trait loci (QTL) analysis. RESULTS: Both saline and MK-801 (0.3 mg/kg, given intraperitoneally) induced a continuum of locomotor responses across the LSXSS RI strains. There was a 4-fold range of MK-801 difference scores (MK-801 score-saline baseline), with the RI 9 and RI 4 strains representing low and high responders, respectively. Dose-response experiments with these two strains confirmed that 0.3 mg/kg MK-801 produced significant activation, similar to previous results with LS and SS mice. However, unlike previous LS/SS results, lower densities of NMDARs were not observed in the RI 9 than in the RI 4 mouse brains. No significant genetic correlations were observed between MK-801-induced and ethanol-induced responses in the LSXSS RI mice. Two provisional MK-801 activation QTLs were identified (p < 0.01) on chromosomes 11 and 19, neither in common with those mapped for ethanol activation. CONCLUSIONS: Different densities of brain NMDARs are unlikely to account for the differential activation of LSXSS RI mice by MK-801. Additionally, in the RI mice either separate sets of genes regulate low dose MK-801- and ethanol-induced locomotor responses or the overlapping subset of genes controlling these two behaviors is small (< or =10%).     

N-methyl-D-aspartate receptor responses are differentially modulated by noncompetitive receptor antagonists and ethanol in inbred long-sleep and short-sleep mice: behavior and electrophysiology

BACKGROUND: Short-sleep (SS) mice exhibit higher locomotor activity than do long-sleep (LS) mice when injected with low doses of ethanol or the noncompetitive N-methyl-D-aspartate receptor (NMDAR) antagonist MK-801 (dizocilpine). SS mice also have higher densities of brain NMDARs. However, two strains of LS X SS recombinant inbred (RI) mice also show differential activation to ethanol and MK-801, but have similar numbers of NMDARs. Here we used inbred LS (ILS) and SS (ISS) mice to investigate further the relationship between NMDARs and sensitivity to the stimulant effects of low doses of ethanol. METHODS: Open field activity and spontaneous alternations were measured after saline or drug injection. [3H]MK-801 binding parameters were determined in hippocampus, cortex, striatum, and nucleus accumbens. Extracellular field excitatory postsynaptic potentials (fEPSPs) were recorded in the CA1 region of hippocampal slices. RESULTS: Systemic injection of either ethanol or MK-801 increased locomotor activity to a greater extent in ISS mice than in ILS mice. The competitive NMDAR antagonist 2-carboxypiperazin-4-yl-propyl-1-lphosphonic acid (+/- CPP) depressed activity of ILS, but not ISS, mice. No strain differences were observed in spontaneous alternations or in the number or affinity of NMDARs in the brain regions examined. Likewise, the magnitudes of hippocampal NMDAR-mediated fEPSPs were similar in ILS and ISS mice and were inhibited to the same extent by a competitive NMDAR antagonist. However, both ethanol and the NMDAR NR2B receptor antagonist ifenprodil inhibited the late component of hippocampal NMDAR fEPSPs to a greater extent in ISS, than in ILS, mice. CONCLUSIONS: Differential ethanol- and MK-801-induced behavioral activation in ILS and ISS mice was not associated with differences in NMDAR number. Nonetheless, pharmacological differences in hippocampal NMDAR responsiveness suggest that ISS mice express NMDARs that have a greater sensitivity to noncompetitive, but not competitive, NMDAR antagonists. These differences, which may reflect differences in NMDAR subunit composition, could underlie the differential responsiveness to low doses of ethanol in ILS and ISS mice.     

Locomotor activity induced by noncompetitive NMDA receptor antagonists versus dopamine transporter inhibitors: opposite strain differences in inbred long-sleep and short-sleep mice

Background: The actions of ethanol in the brain involve multiple neuroreceptor systems, including glutamatergic N‐methyl‐D‐aspartate receptor (NMDAR) channels. In a novel environment, both ethanol and the noncompetitive NMDAR antagonist MK‐801 stimulate locomotor activity to a lesser extent in inbred long‐sleep (ILS) mice compared with inbred short‐sleep (ISS) mice. The behaviorally activating effects of noncompetitive NMDAR antagonists are thought to involve increased monoamine neurotransmission. Thus, in this study, we sought to determine whether: (1) habituation to the behavioral environment alters the differential locomotor‐stimulant effects of noncompetitive NMDAR antagonists in ILS and ISS mice and (2) the differential behavioral sensitivity of ILS and ISS mice to noncompetitive NMDAR antagonists is mediated through direct inhibition of the dopamine transporter (DAT). Methods: Open field locomotor activity was measured following acute systemic injection of saline or drug. [³H]DA uptake parameters were determined in striatal synaptosomes prepared from drug‐naïve mice. Results: Habituation to the testing environment abolished the strain differences in saline‐induced locomotor activity. However, ethanol‐ as well as MK‐801‐treated ILS mice still exhibited reduced locomotor activity compared with ISS mice, suggesting that a drug‐environment interaction is not the primary explanation for the strain differences. The noncompetitive NMDAR antagonists phencyclidine and ketamine also induced significantly lower locomotor activity in ILS than in ISS mice. In contrast, the DAT inhibitors cocaine and GBR 12909 and the DA releaser amphetamine induced greater locomotor activity in ILS than in ISS mice, a strain difference opposite that of the noncompetitive NMDAR antagonists. Furthermore, the differential behavioral effect found with DAT inhibitors was not mediated by differences in the affinity nor number of striatal DATs between ILS and ISS mice. Conclusions: Our results support the conclusion that the differential locomotor‐stimulant effects of ethanol and noncompetitive NMDAR antagonists in ILS and ISS mice are not mediated through direct inhibition of DAT.     

Differential effects of ethanol on gamma-aminobutyric acid-A receptor-mediated synaptic currents in congenic strains of inbred long and short-sleep mice

BACKGROUND: Ethanol enhances gamma-aminobutyric acid (GABA)A receptor-mediated responses in the brain, and this enhancement is greater in a mouse line behaviorally more sensitive to ethanol (long sleep) than in a line (short sleep) behaviorally less ethanol sensitive (assayed by loss of righting; sleep time). Quantitative trait locus (QTL) analysis of inbred long sleep (ILS) and inbred short sleep (ISS) phenotypes revealed four chromosomal regions (Lore1, Lore2, Lore4, and Lore5) that together account for approximately 50% of ethanol-induced sleep-time variance. Congenic strains were generated, each of which is homozygous for one of four ISS Lore QTLs on the ILS background. These congenic mouse strains are ideally suited for asking which QTL regions might correlate with other phenotypes that differ between ILS and ISS mice. Here we used the congenics to investigate altered GABAA responses to ethanol. METHODS: Evoked GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs) were measured by whole-cell voltage-clamp recording procedures in CA1 pyramidal neurons in hippocampal brain slices. RESULTS: GABAA IPSC responses in hippocampal brain slices from ILS mice were significantly enhanced by 80 mM ethanol, whereas those from ISS mice were not affected. ILS.Lore2 and ILS.Lore5 congenic strains were significantly enhanced by 80 mM ethanol, similar to the background (control) ILS mice. However, ethanol had no significant effect on GABAA responses in ILS.Lore1 and ILS.Lore4 congenic mice, similar to the ISS mice, thus reflecting the influence of ISS alleles on the ILS phenotype. CONCLUSIONS: Our results suggest that alleles located in the Lore1 and Lore4 QTL regions confer ethanol sensitivity of GABAA receptor-mediated IPSCs. Thus, for these QTLs, GABAA IPSCs may represent an endophenotype of sedative/hypnotic sensitivity to ethanol. Although the Lore2 and Lore5 QTL regions have a significant effect on sleep time, they do not play a significant role in the differential ethanol enhancement of GABAA IPSCs between ILS and ISS mice.     

Ethanol-induced malfunction of neutrophils respiratory burst on patients suffering from alcohol dependence

Background: Polymorphonuclear, neutrophil granulocytes (PMN) play a major role in the control of infections, and people who abuse alcohol are susceptible to infections. Resistance against infections ensues intracellularly following initial phagocytosis of microorganisms with the oxygen‐dependent respiratory burst, the key enzyme of which is the respiratory burst oxidase, whereby oxygen radicals are produced for microbial destruction. To date there is insufficient information available in connection with the process of impaired defence against infection in patients suffering from alcohol dependence. Therefore, our investigation was carried out to determine the influence of alcohol exposition on the formation of oxygen radicals and the respiratory burst. Methods: 4.5 ml of whole blood was taken from 10 healthy adults and 10 patients suffering from alcohol dependence. An additional 3.5 ml of whole blood was taken from the alcoholic patients for determination of the blood alcohol concentration. The respiratory burst of PMN was tested using the Four‐Colour‐Continuous Flow Cytometer. Each experimental procedure consisted of 4 test samples [negative controls, Escherichia coli, FMLP‐supplement (N‐formyl‐l‐methionyl‐l‐leucyl‐l‐phenylalanin), PMA‐supplement (phorbol‐12‐myristate‐13‐acetate)]. Differing concentrations of ethanol were also introduced to each of the tests performed (0.20 to 4.00 g/l). Results: Ethanol revealed a marked decrease of burst activity in those patients suffering from alcoholism with increased alcohol concentration. A dependence between the burst activity and the ethanol concentration was seen to be statistically significant. This effect was only evident after stimulation with E. coli and FMLP in those patients with alcohol dependence. Conclusion: The results presented in this study show an impairment in the function of PMN in those patients addicted to alcohol due to the decrease in burst activity. In view of the results of the different stimuli, the second‐messenger effects were not evident. A clarification of this phenomenon could well be assumed as an allosteric receptor effect on the burst oxidase, namely, a direct effect on the phagocytosis interaction between circulating granulocytes and causative organisms.     

Increased ethanol drinking after repeated chronic ethanol exposure and withdrawal experience in C57BL/6 mice

BACKGROUND: The development of dependence may have significant motivational consequences regarding continued use and abuse of ethanol. We have developed a mouse model of ethanol dependence and repeated withdrawals that demonstrates sensitization of seizures and other symptoms of withdrawal. It is unclear whether such experience influences ethanol drinking behavior. The present series of experiments were designed to examine whether repeated cycles of chronic ethanol exposure and withdrawal has an impact on subsequent motivation to voluntarily self-administer ethanol. METHODS: With the use of a modified sucrose-fading procedure, adult male C57BL/6J mice were trained to drink 15% (v/v) ethanol in a limited access procedure (2 hr/day). The animals were not food or water deprived at any time during the experiments. Once stable baseline intake was established, mice were exposed to four cycles of 16 hr of ethanol vapor (or air) in inhalation chambers separated by 8-hr periods of withdrawal. At 32 hr after the last cycle of ethanol exposure, all mice were tested for ethanol intake under limited access conditions for 5 consecutive days. The animals then received a second series of chronic ethanol exposure and withdrawal followed by another 5-day test period for ethanol drinking. RESULTS: Stable daily baseline intake was established in mice that drank 15% ethanol combined with 5% sucrose (experiment 1), 15% ethanol alone (experiment 2), 5% sucrose alone (experiment 3), or 15% ethanol when presented as a choice with water (experiment 4). After repeated cycles of chronic ethanol exposure and withdrawal experience, consumption of ethanol solutions increased over baseline levels and in comparison with control (air-exposed) groups. However, sucrose consumption did not change in mice that were trained to drink 5% sucrose. The increase in ethanol consumption after chronic ethanol exposure and withdrawal experience resulted in a significant increase in resultant blood ethanol levels. CONCLUSIONS: Once the positive reinforcing properties of ethanol were established, chronic ethanol exposure and withdrawal experience resulted in a significant increase in voluntary ethanol drinking that yielded a >2-fold increase in resultant blood ethanol levels. This increase in ethanol intake occurred whether ethanol was presented in combination with sucrose, alone (unadulterated), or as a choice with tap water. Furthermore, this effect seems to be selective for ethanol in that animals that were trained to drink a sucrose solution did not exhibit a change in their intake after similar chronic ethanol exposure. As such, this model may be useful in studying the mechanisms and conditions in which chronic ethanol treatment influences motivation to resume drinking after a period of abstinence (relapse).     

Longitudinal Observations of Monoamine Oxidase B in Alcoholics: Differentiation of Marker Characteristics

The marker characteristics of monoamine oxidase B (MAO B) in human platelets were investigated in a clinical study of 59 alcoholics (diagnosed according to the criteria of ICD-10) observed over a period of 6 months. Demographic and family history were obtained by a structured interview, including the substance abuse section of CIDI (Composite International Diagnostic Interview). The patient's personality was assessed by Cloninger's Tridimensional Personality Questionnaire (TPQ). Blood samples were first drawn during chronic intoxication (day of admission to the hospital for detoxication), after short-term abstinence (8 days later), medium-term (3 months later), and long-term abstinence (6 months later). A group of 22 matched healthy nonalcoholics served as controls studied under sober conditions and during acute intoxication (4 hr after ingestion of 1 g ethanol/kg body weight). All platelet samples were investigated with 6 kynuramine concentrations as substrate (fluorometric assay) in the absence and presence of 200 itim ethanol (ETOH) in vitro. MAO B activity was significantly reduced in alcoholics during chronic intoxication (V_max: 2.70 ± 0.15 nmol/min/mg protein) compared with sober (V_max: 3.25 ± 0.23 nmol/min/mg protein) and acutely intoxicated controls that turned to normal during abstinence. However, MAO B activity obtained during medium- and long-term abstinence was significantly lowered in patients with high novelty-seeking and impulsiveness scores in the TPQ, a history of suicide attempts, or an alcoholic mother. The affinity of MAO B (K_m values) was unchanged in alcoholics at any time investigated. Addition of ETOH in vitro reduced the affinity. This effect was less pronounced when the blood had been obtained during chronic intoxication and after short-term abstinence, suggesting tolerance toward ETOH. It is demonstrated that reduced MAO B values may serve as state and trait markers of alcoholism and that they can be disentangled in a longitudinally designed study.     

Brain GABA-Transaminase and Monoamine Oxidase After Chronic Ethanol Treatment in Rats

The activities of γ-aminobutyrate aminotransferase (GABA-T) and monoamine oxidase (MAO-A and -B) were estimated in various brain regions of rats exposed to ethanol for 90 weeks. During the first period (weeks 1–58), the rats had access to both ethanol (10% w/v) and water during a 24-hr interval at the end of each week. At this point, the animals were given either a saline injection (intraperito-neally, group 1) or an ethanol injection (2.0 g/kg ip, group 2). During the second period (weeks 59–90), the rats in groups 1 and 2 had continuous access to both ethanol and water. The third group was composed of untreated control rats. Compared with controls, there was an increase of 20–45% in the mean brain GABA-T activity in both groups of ethanol-treated rats. However, analysis of the data for the individual ethanol-treated rats revealed a considerable difference in brain GABA-T activity. Thus, -30% of the ethanol-treated rats showed approximately twice the activity of rats in the exposed groups and in the control group. There was no connection between ethanol intake, water intake, or body weight and GABA-T activity in any of the brain regions examined. There was no effect of ethanol in vitro on the activity of GABA-T in the brain cortex in concentrations of 20–100 mki, whereas acetaldehyde inhibited the activity by 15% at these concentrations. The present results suggest that there is a bimodal distribution with respect to the effect of ethanol on rat brain GABA-T activity. With regard to MAO, the activities (both forms) in the brain remained unaffected after 90 weeks of ethanol treatment.     

鸡胚低分子提取物对D-半乳糖拟衰老小鼠线粒体DNA缺失突变的影响

目的 探讨鸡胚低分子提取物对D-半乳糖诱导的衰老小鼠线粒体DNA(mtDNA)缺失突变的影响.方法 采用D-半乳糖诱导制备衰老小鼠模型并用鸡胚低分子提取物处理.用聚合酶链反应技术和琼脂糖凝胶电泳检测mtDNA缺失片段,密度扫描技术对扩增片段进行相对定量.缺失片段构建质粒后,直接测序进行鉴定.结果 全部小鼠的肝、大脑皮质与海马组织内均存在4239 bp的mtDNA缺失片段.衰老模型鼠不同组织mtDNA缺失的相对百分含量均明显高于正常对照组(均为P%0.01),而鸡胚低分子提取物可明显降低模型鼠肝、大脑皮质与海马组织内mtDNA缺失的比例(均为P＜0.05).肝组织中mtDNA缺失的比例较大脑皮质和海马组织更高.结论 4239 bp的mtDNA缺失片段普遍存在于小鼠中,鸡胚的低分子提取物可以明显减少D-半乳糖拟衰老小鼠4239 bp的mtDNA缺失的发生.     

催老小鼠重要脏器氧化水平和抗氧化能力的变化

目的　研究D 半乳糖催老小鼠心、肝、脑、肺、肾等重要脏器氧化水平和抗氧化能力的改变 ,并对其进行比较。　 方法 　 2 0只 2月龄雄性小鼠随机分为催老组和正常对照组 ;催老组每日皮下注射D 半乳糖 10 0mg·kg-1,正常对照组每日皮下注射等量生理盐水 ,持续 42d。以丙二醛 (MDA)含量反映氧化水平 ,超氧化物歧化酶(SOD)活性反映抗氧化能力。　 结果 　与正常对照组相比 ,D 半乳糖催老组小鼠心、肝、脑、肺、肾等重要脏器MDA含量明显增加 (P <0 0 1) ;肝、脑、肺和肾的SOD活性显著降低 (P <0 0 5 ) ,而心脏SOD活性无明显变化。　 结论　D 半乳糖催老小鼠心、肝、脑、肺、肾等重要器官氧化水平增加 ;肝、脑、肺、肾等器官抗氧化水平下降 ;各器官在改变程度上存在一定差异     

Formation of malondialdehyde adducts in livers of rats exposed to ethanol: role in ethanol-mediated inhibition of cytochrome c oxidase

BACKGROUND: Previous studies in our laboratory demonstrated that short-term ethanol consumption by maternal rats increased the hepatic levels of 4-hydroxynonenal (HNE) in both the adult and the fetus. Additionally, HNE inhibited cytochrome c oxidase (COX) by forming adducts with the enzyme subunits. The present study examined modification of COX by another major aldehydic lipid peroxidation product, malondialdehyde (MDA), and its role in COX inhibition by ethanol. METHODS AND RESULTS: It is demonstrated in vitro that MDA inhibits the activity of purified COX while forming adducts with the enzyme. Compared with HNE, MDA is a more potent inhibitor of COX. Overnight incubation at room temperature caused an 80% decrease in COX activity by MDA versus a 67% decrease by HNE. MDA produced marked inhibition of COX activity at physiologically relevant concentrations, e.g., 43% inhibition at 10 microM. Although our previous studies documented that HNE formed adducts primarily with subunit IV of COX via histidine residues, the current report showed that MDA forms adducts with both subunit IV and subunit V via lysine residues. Furthermore, both aldehydes induce carbonyl formation in subunit IV. The in vivo role of MDA in the impairment of COX by ethanol is assessed in both adult and fetal liver after maternal ethanol consumption. CONCLUSIONS: The results showed that: (1) there are significant increases in MDA levels in liver homogenate as well as mitochondria in both adult and fetal livers after ethanol exposure; (2) these MDA levels are in the nanomole/mg protein range, in contrast to picomole/mg protein range of HNE in identical setting; and (3) ethanol-induced production of MDA is accompanied by enhanced formation of MDA adducts with COX. These findings suggest that MDA may play at least as equally an important role as HNE in ethanol-induced inhibition of COX.     

千斤拔醇提物抗小鼠运动疲劳的作用

目的 探讨千斤拔醇提物的抗疲劳作用及其机制,为其在保健食品的开发提供实验依据.方法 健康昆明小鼠32只,随机分为4组,每组8只,即正常对照组,3个不同剂量组(25,50,100 mg/kg),连续灌胃10 d后,分别测定负重游泳时间、肝糖原(HG)含量、血清尿素氮(BUN)和血乳酸脱氢酶(LDH)活性,血清SOD(SOD)活性、丙二醛(MDA)含量、谷胱甘肽-过氧化物酶(GPX)活性.结果 低、中、高剂量千斤拔能显著增加小鼠负重游泳时间,增加HG含量,减少血清BUN的含量,降低LDH活性.结论 高剂量千斤拔并能显著增加血清SOD活性,降低MDA含量,增强GPX活性,说明千斤拔具有明显的抗疲劳作用.     

Maternal ethanol exposure is associated with decreased plasma zinc and increased fetal abnormalities in normal but not metallothionein-null mice

BACKGROUND: Ethanol profoundly affects fetal development, and this is proposed to be due primarily to a transient fetal zinc (Zn) deficiency that arises from the binding of Zn by metallothionein (MT) in the maternal liver. Zn homeostasis and fetal outcome were investigated in normal (MT+/+) and metallothionein-null (MT-/-) mice in response to ethanol exposure. METHODS/RESULTS: Mice were treated with saline or ethanol (0.015 m/g intraperitoneally at 0 and 4 hr) on day 8 of gestation (Gd8), and the degree of fetal dysmorphology was assessed on Gd18. The incidence of external abnormalities was significantly increased in offspring from MT+/+ dams exposed to ethanol, where 27.4% of fetuses were affected. MT-/- ethanol-, MT+/+ saline-, and MT-/- saline-treated dams had fetuses in which the frequencies of abnormalities were 2.2, 6.4, and 6.9%, respectively. To investigate Zn homeostasis, nonpregnant mice were killed at intervals over 16 hr after ethanol injection. Liver MT concentrations in MT+/+ mice were increased 20-fold by 16 hr, with a significant elevation evident by 4 hr, whereas liver Zn levels were also significantly increased by 2 hr and maintained for 16 hr. In parallel with these changes, plasma Zn concentrations in MT+/+ mice decreased by 65%, with minimum levels of 4.5+/-0.3 micromol/liter at 8 hr. Conversely, MT-/- mice exhibited increased plasma Zn concentrations, with peak values of 20.8+/-0.3 observed at 4 hr. CONCLUSION: These findings link the teratogenic effect of ethanol to the induction of maternal MT and the limitation of fetal Zn supply from the plasma.     

Effects of gangliosides on ethanol-induced neurodegeneration in the developing mouse brain

Background: Ethanol exposure induces apoptotic neurodegeneration in the developing rodent brain during synaptogenesis. This process has been studied as a model for fetal alcohol syndrome. Previously, we have shown that gangliosides and LIGA20 (a semisynthetic derivative of GM1 ganglioside) attenuate ethanol‐induced apoptosis in cultured neurons. In the present study, the effects of GM1 and LIGA20 on ethanol‐induced apoptotic neurodegeneration were examined using an in vivo neonatal mouse model. Methods: Seven‐day‐old C57BL/6By (B6By) mice were pretreated twice with intraperitoneal administration of GM1 (30 mg/kg), LIGA20 (2.5 mg/kg), or saline, followed by subcutaneous injection of either saline or ethanol (2.5 g/kg) twice with a 2 hours interval. Then the brains were: (1) perfusion‐fixed 24 hours after the first ethanol injection, and the extent of neurodegeneration was assessed by cupric silver staining of the brain sections, or (2) perfusion‐fixed 8 hours after the first ethanol injection, and the sections were immunostained with anti‐cleaved (activated) caspase‐3 antibody to evaluate caspase‐3 activation. Results: The comparison of cupric silver stained coronal sections indicates that ethanol‐induced widespread neurodegeneration in the forebrains of B6By mice was reduced overall by GM1 and LIGA20 pretreatments. The extent of neurodegeneration detected by silver impregnation and activated caspase‐3 immunostaining was quantified in the cingulate and retrosplenial cortices, which were the regions most severely affected by ethanol. The results indicate that GM1 and LIGA20 pretreatments induced statistically significant reductions—approximately 50% of the ethanol‐treated samples—in silver impregnation and activated caspase‐3 immunostaining. No significant differences were observed between saline controls and samples treated with GM1 or LIGA20 alone. Conclusions: These results indicate that GM1 and LIGA20, which have been shown to be neuroprotective against insults caused by various agents, partially attenuate ethanol‐induced apoptotic neurodegeneration in the developing mouse brain.     

Relationship of brain ethanol metabolism to the hypnotic effect of ethanol. II: Studies in selectively bred rats and mice

BACKGROUND: To clarify the role of brain acetaldehyde in the hypnotic effect of ethanol, we compared the ethanol-oxidizing capacity (rate of acetaldehyde accumulation) and catalase and aldehyde dehydrogenase activity in the brains of animals genetically selected for different sensitivities to the hypnotic effect of ethanol. METHODS: We used high, low, or control alcohol-sensitive rats (HAS, LAS, and CAS) and short- and long-sleep mice (SS and LS), as well as SS x LS recombinant inbred mice with known strain differences in mean duration of ethanol-induced sleep. We studied the rate of accumulation of acetaldehyde from ethanol in brain homogenates of these animals and correlated those values with their hypnotic sensitivity to ethanol. RESULTS: Acetaldehyde accumulation from ethanol was significantly higher in the brain homogenates from HAS rats and LS mice with high sensitivity to the hypnotic effect of ethanol in vivo, compared with LAS rats and SS mice with low sensitivity to ethanol. A correlation was found between the duration of ethanol-induced sleep and the in vitro rate of accumulation of ethanol-derived acetaldehyde in the brains of recombinant SS x LS mice strains. There was no correlation of sleep time with brain catalase levels. There were no line differences in brain catalase or aldehyde dehydrogenase or in alcohol or aldehyde dehydrogenase activity in livers of LAS, CAS, and HAS rats or in SS and LS mice. CONCLUSIONS: A correlation between the brain acetaldehyde accumulation, but not catalase levels, and the central effect of ethanol was demonstrated in animals genetically differing in initial sensitivity to the hypnotic effect of ethanol.     

APP17肽对D-半乳糖脑老化模型小鼠神经元凋亡的影响

目的观察D-半乳糖脑老化小鼠(DGAM)海马内凋亡相关蛋白Fas、Fas配体(Fas-L)、核因子κB(NFκB)、C-Fos、C-Jun的表达及其脑神经元是否发生凋亡,并观察APP17肽的作用. 方法用D-半乳糖(D-gal)制造脑老化小鼠模型,给模型小鼠皮下注射APP17肽进行预防或治疗.D-gal皮下注射8周后,小鼠灌注取脑,行脑切片,一部分切片做免疫组织化学染色,观察各组小鼠海马内凋亡相关蛋白Fas、Fas-L、NFκB、C-Fos、C-Jun的表达;另一部分切片用凋亡试剂盒检测神经元是否发生凋亡. 结果对照组小鼠海马内Fas、Fas-L、C-Fos、C-Jun阳性染色神经元的数目分别为20.60±4.60、20.00±4.24、9.93±3.79、13.44±4.18,DGAM组小鼠上述各种抗体的阳性染色神经元数目分别为42.80±15.83、38.53±8.13、23.25±5.18、23.47±8.49,DGAM组比对照组明显增加(P＜0.05).DGAM组海马内NFκB的阳性染色神经元数目为13.93±5.31,比对照组小鼠的23.93±3.69明显减少(P＜0.05).用APP17肽预防或治疗后小鼠上述各种蛋白质的表达恢复到接近对照组小鼠的水平.对照组小鼠未发现凋亡的神经元;DGAM组小鼠脑神经元出现大量凋亡,用APP17肽预防或治疗后,神经元凋亡的数目与对照组小鼠接近. 结论 DGAM海马内凋亡相关蛋白表达异常,脑神经元发生凋亡.APP17肽可恢复各种凋亡相关蛋白的表达,阻止神经元凋亡,维持神经元的正常功能.     

Alcoholic cardiomyopathy in mice. Myocardial glycogen, lipids and certain enzymes.

Chronic ethanol feeding to C57B1 mice for 25 weeks caused an increase in heart size, both absolutely and relative to body weight. When ethanol was fed with a diet having a methionine imbalance, the activities of acetyl CoA synthetase were depressed. Ethanol also caused functional changes in the heart mitochondria measured as a decrease in $\text{P}\text{O}$ ratios; thus it appears that alcohol per se can affect the heart. The correlation of the biochemical abnormalities in mitochondria with the ultrastructural pathology observed suggest that the C57B1 mouse may serve as a useful experimental animal model for the study of alcoholic cardiomyopathy.     

Effects of early postnatal exposure to ethanol on retinal ganglion cell morphology and numbers of neurons in the dorsolateral geniculate in mice

Background: The adverse effects of fetal and early postnatal ethanol intoxication on peripheral organs and the central nervous system are well documented. Ocular defects have also been reported in about 90% of children with fetal alcohol syndrome, including microphthalmia, loss of neurons in the retinal ganglion cell (RGC) layer, optic nerve hypoplasia, and dysmyelination. However, little is known about perinatal ethanol effects on retinal cell morphology. Examination of the potential toxic effects of alcohol on the neuron architecture is important because the changes in dendritic geometry and synapse distribution directly affect the organization and functions of neural circuits. Thus, in the present study, estimations of the numbers of neurons in the ganglion cell layer and dorsolateral geniculate nucleus (dLGN), and a detailed analysis of RGC morphology were carried out in transgenic mice exposed to ethanol during the early postnatal period. Methods: The study was carried out in male and female transgenic mice expressing yellow fluorescent protein (YFP) controlled by a Thy‐1 (thymus cell antigen 1) regulator on a C57 background. Ethanol (3 g/kg/d) was administered to mouse pups by intragastric intubation throughout postnatal days (PDs) 3 to 20. Intubation control (IC) and untreated control (C) groups were included. Blood alcohol concentration was measured in separate groups of pups on PDs 3, 10, and 20 at 4 different time points, 1, 1.5, 2, and 3 hours after the second intubation. Numbers of neurons in the ganglion cell layer and in the dLGN were quantified on PD20 using unbiased stereological procedures. RGC morphology was imaged by confocal microscopy and analyzed using Neurolucida software. Results: Binge‐like ethanol exposure in mice during the early postnatal period from PDs 3 to 20 altered RGC morphology and resulted in a significant decrease in the numbers of neurons in the ganglion cell layer and in the dLGN. In the alcohol exposure group, out of 13 morphological parameters examined in RGCs, soma area was significantly reduced and dendritic tortuosity significantly increased. After neonatal exposure to ethanol, a decrease in total dendritic field area and an increase in the mean branch angle were also observed. Interestingly, RGC dendrite elongation and a decrease in the spine density were observed in the IC group, as compared to both ethanol‐exposed and pure control subjects. There were no significant effects of alcohol exposure on total retinal area. Conclusions: Early postnatal ethanol exposure affects development of the visual system, reducing the numbers of neurons in the ganglion cell layer and in the dLGN, and altering RGCs’ morphology.