Amelioration of ethanol-induced growth retardation by all-trans-retinoic acid and alpha-tocopherol in shell-less culture of the chick embryo

The mechanisms of teratogenic action of ethanol (EtOH) were investigated by testing the hypothesis that all-trans-retinoic acid and/or alpha-tocopherol ameliorates ethanol-induced embryonic growth retardation. Chicken embryos were explanted in shell-less cultures and a single dose of EtOH (15, 30, or 50%) or 50% EtOH with either all-trans-retinoic acid (10(-8)M) or alpha-tocopherol (0.05 M) or a mix of all-trans-retinoic acid (10(-8)M) and alpha-tocopherol (0.05 M) was applied to the center of the blastodisc. EtOH significantly increased the mortality rate and induced growth retardation in a dose-dependent manner. In addition, EtOH increased malondialdehyde (MDA) levels, an indicator of oxidative stress and cell damage, in a dose dependent manner. All-trans-retinoic acid, the active form of Vitamin A, and/or alpha-tocopherol, an antioxidant, co-treatment with EtOH significantly diminished both the EtOH-induced mortality and growth retardation. However, only alpha-tocopherol co-treatment reduced the MDA levels. Thus, the mechanisms of teratogenic action of EtOH appear to involve initiation of oxidative stress as well as perturbation of retinoic acid (RA) signaling. It also appears likely that these mechanisms work independently of each other.     

Acetaldehyde sequestering prevents ethanol-induced stimulation of mesolimbic dopamine transmission

Acetaldehyde (ACD) has been postulated to mediate some of the neurobehavioral effects of ethanol (EtOH). In this study we sought to evaluate whether the stimulatory effects of EtOH on mesolimbic dopamine (DA) transmission are affected by the administration of ACD-sequestering agent D-penicillamine (Dp). To this end we studied the effect of EtOH and ACD in the rat mesoaccumbens pathway by in vivo microdialysis in the nucleus accumbens shell (NAccs), and by single cell extracellular recordings from antidromically identified mesoaccumbens DA neurons in the ventral tegmental area (VTA). Both EtOH (1 g/kg) and ACD (20 mg/kg) administration increased DA levels in the NAccs and increased the activity of mesoaccumbens DA neurons. Pretreatment with Dp (50 mg/kg i.p. 1 h before drug challenge) prevented both EtOH- and ACD-induced stimulation of the DA mesolimbic system without affecting morphine stimulatory actions. These observations add further support to the notion that EtOH-derived ACD stimulates the mesolimbic DA system and is essential in EtOH-induced stimulation of the DA mesoaccumbens system. We conclude that modulation of ACD bioavailability may influence the addictive profile of EtOH by decreasing its psychotropic effects and possibly leading the way to new pharmacological treatments of alcoholism.     

Hepatoprotective potential of 7,8-Dihydroxyflavone against alcohol and high-fat diet induced liver toxicity via attenuation of oxido-nitrosative stress and NF-κB activation

BackgroundFatty liver diseases are the most common and major health concern arises from the modern lifestyle and alcohol (ethanol) abuse. The prevalence of non-alcoholic fatty liver diseases (NAFLD) has been observed prominently in obese and diabetic individuals, while alcoholic liver disease is common in alcoholic persons. Fatty liver disease, such as steatohepatitis, leads to fibrosis, cirrhosis and eventually hepatocellular carcinoma. The present study was designed to investigate the effect of 7,8-Dihydroxyflavone (7,8-DHF) against high-fat diet (HFD) and ethanol (EtOH)-induced hepatotoxicity in rats.MethodsMale Wistar rats (150–200 g) were fed HFD (58% calories from fat) and EtOH (3–15% in drinking water) for 12 weeks. 7,8-DHF was administered intraperitoneally at the dose of 5 mg/kg/day for the last four weeks. After 12 weeks, biochemical, ELISA, RT-PCR, and histological studies have been carried out.ResultsBiochemical analyses revealed the involvement of oxidative stress and inflammation in the liver of HFD and EtOH-fed rats. 7,8-DHF treatment significantly reduced HFD and EtOH-induced oxidative stress as evidenced by the reduction of lipid peroxidation and augmentation of reduced glutathione level. Moreover, IL-1β level was found significantly reduced in 7,8-DHF treated EtOH, HFD and EtOH+HFD groups. The semi-quantitative RT-PCR results indicated down-regulation of Nrf-2 and HO-1 and up-regulation of NF-κB and iNOS mRNA expression level in the liver of HFD and EtOH-fed rats, which was ameliorated by 7,8-DHF treatment.ConclusionThe present study suggested that 7,8-DHF could be an effective pharmacological intervention in combating HFD and EtOH-induced hepatotoxicity.     

Allopregnanolone does not influence ethanol-induced conditioned place preference in DBA/2J mice

Rationale The neurosteroid allopregnanolone (ALLOP; 3-hydroxy-5-pregnan-20-one) produces behavioral and discriminative characteristics similar to that of ethanol (EtOH) and can modulate some of the behavioral and electrophysiological effects of EtOH.Objective The present experiments investigated ALLOP modulation of the effects of EtOH in a place conditioning procedure in male DBA/2J mice.Methods In a series of experiments examining different EtOH doses (1, 2 g/kg) and ALLOP administration times, ALLOP (0, 3.2, 10, 17 mg/kg, IP) was administered four times with EtOH prior to placement on a distinctive floor (CS+). On alternate days, vehicle was administered prior to a saline injection paired with the other floor stimulus (CS–). In a separate experiment, finasteride (0, 50, 100 mg/kg, IP), a 5-reductase inhibitor that blocks ALLOP synthesis, was administered prior to both CS+ and CS– trials. In a final experiment, animals were place conditioned to EtOH alone, and ALLOP (0, 3.2, 10, 17 mg/kg, IP) was administered prior to the preference test only.Results During conditioning, ALLOP increased and finasteride decreased EtOH-stimulated activity compared with vehicle pretreatment. Acquisition of 2 g/kg EtOH-induced conditioned place preference was observed in all mice, regardless of treatment with either ALLOP or finasteride. Similarly, ALLOP did not modulate the expression of EtOH-induced place preference. EtOH increased brain ALLOP levels compared with saline; however, ALLOP administration produced dose-dependent elevations in brain ALLOP levels that were not further augmented by EtOH (2 g/kg) administration.Conclusions These findings indicate that ALLOP does not modulate EtOH-induced place conditioning in male DBA/2J mice.     

Indomethacin does not antagonize the anxiolytic action of ethanol in the elevated plus-maze

The present study was designed to examine whether the prostaglandin (PG) synthesis inhibitor indomethacin (INDO) could antagonize the anxiolytic effects of ethanol (EtOH) in the elevated plus-maze test of anxiety. EtOH (1.6 g/kg) significantly increased the percentage of open arm entries and time spent on the open arms in both inbred C57BL/6J and outbred CD-1 mouse strains. However, this anxiolytic effect of EtOH was not significantly antagonized by pretreatment with INDO (5 and 10 mg/kg) in either strain. EtOH also significantly increased total arm entries in CD-1 mice, but not in the C57BL/6J strain. These data from C57BL/6J mice indicate that the low-dose stimulant properties of EtOH can be dissociated from the anxiolytic action of the drug in the plus-maze task. Finally, although INDO did not antagonize the stimulant effect of EtOH in the plus-maze task (in CD-1 mice), it did attenuate EtOH-induced stimulation of locomotor activity in an open-field arena. Taken together, these results suggest some specificity with regard to the role of PGs in mediating (or modulating) the neurobehavioral actions of EtOH, and further support the notion that the anxiolytic and stimulant effects of EtOH may be mediated by different mechanisms.     

槟榔碱对小鼠酒精急性中枢抑制作用的影响

目的:探讨非选择性毒蕈碱受体(M受体)激动剂—槟榔碱,对小鼠酒精急性中枢抑制作用的影响。方法:测定小鼠的自主活动,观察槟榔碱对酒精诱导的小鼠低活动性的影响。建立酒精诱导小鼠翻正反射消失(loss of therighting reflex,LORR)的模型,观察槟榔碱对LORR潜伏期和持续时间的影响。结果:酒精(1.0、2.0、3.0 g.kg-1)和槟榔碱(0.25、0.51、.0 mg.kg-1)均可剂量依赖性地抑制小鼠的自主活动,但槟榔碱对酒精诱导的小鼠低活动性无影响。槟榔碱(0.25、0.5、1.0 mg.kg-1)对酒精诱导小鼠LORR的潜伏期无影响,但可显著缩短LORR的持续时间。结论:槟榔碱可以拮抗酒精诱导小鼠LORR的药理作用,提示槟榔碱可能具有一定的醒酒作用。     

Behavioral and neurochemical interactions between Group 1 mGluR antagonists and ethanol: potential insight into their anti-addictive properties

Blockade of the mGluR5 subtype of Group 1 metabotropic glutamate receptor (mGluRs) reduces the rewarding effects of ethanol (EtOH), while the effects of mGluR1a blockade remain under-investigated. The present study compared the effects of pretreatment with the mGluR5 antagonist MPEP and the mGluR1a antagonist CPCCPOEt upon behavioral and neurochemical variables associated with EtOH reward in alcohol-preferring C57BL/6J mice. Pretreatment with either antagonist (0-10 mg/kg, IP) dose-dependently reduced measures of EtOH reward in an operant self-administration paradigm and the maximally effective antagonist dose (10 mg/kg) also blocked the expression of EtOH-induced place conditioning, as well as EtOH consumption under 24-h free-access conditions. MPEP pretreatment did not significantly alter the EtOH dose-locomotor response function; however, it prevented EtOH-induced changes in extracellular dopamine, glutamate and GABA in the nucleus accumbens (NAC). In contrast, CPCCOEt shifted the EtOH dose-response function downwards, enhanced the capacity of higher EtOH doses to elevate NAC levels of GABA and lowered extracellular dopamine and glutamate below baseline following EtOH injection. It is suggested that the "anti-alcohol" effects of MPEP may involve an attenuation of the neurochemical signals mediating EtOH reward, whereas those of CPCCOEt may involve an increased sensitivity to the inhibitory effects of EtOH upon brain and behavior.     

The Protective Effects of IGF-1 on Different Subpopulations of DRG Neurons with Neurotoxicity Induced by gp120 and Dideoxycytidine In Vitro

Peripheral neuropathy induced by human immunodeficiency virus (HIV) infection and antiretroviral therapy is not only difficult to distinguish in clinical practice, but also difficult to relieve the pain symptoms by analgesics because of the severity of the disease at the later stage. Hence, to explore the mechanisms of HIV-related neuropathy and find new therapeutic options are particularly important for relieving neuropathic pain symptoms of the patients. In the present study, primary cultured embryonic rat dorsal root ganglion (DRG) neurons were used to determine the neurotoxic effects of HIV-gp120 protein and/or antiretroviral drug dideoxycytidine (ddC) and the therapeutic actions of insulin-like growth factor-1 (IGF-1) on gp120- or ddC-induced neurotoxicity. DRG neurons were exposed to gp120 (500 pmol/L), ddC (50 μmol/L), gp120 (500 pmol/L) plus ddC (50 μmol/L), gp120 (500 pmol/L) plus IGF-1 (20 nmol/L), ddC (50 μmol/L) plus IGF-1 (20 nmol/L), gp120 (500 pmol/L) plus ddC (50 μmol/L) plus IGF-1 (20 nmol/L), respectively, for 72 hours. The results showed that gp120 and/or ddC caused neurotoxicity of primary cultured DRG neurons. Interestingly, the severity of neurotoxicity induced by gp120 and ddC was different in different subpopulation of DRG neurons. gp120 mainly affected large diameter DRG neurons (>25 μm), whereas ddC mainly affected small diameter DRG neurons (≤25 μm). IGF-1 could reverse the neurotoxicity induced by gp120 and/or ddC on small, but not large, DRG neurons. These data provide new insights in elucidating the pathogenesis of HIV infection- or antiretroviral therapy-related peripheral neuropathy and facilitating the development of novel treatment strategies.     

Central ghrelin gastroprotection involves nitric oxide/prostaglandin cross-talk

BACKGROUND AND PURPOSE: Ghrelin, a gut-brain peptide, is considered a gastroprotective factor in gastric mucosa. We investigated the role of prostaglandins (PG) and the possible interplay between PGs and nitric oxide (NO) in ghrelin gastroprotection against ethanol (EtOH)-induced gastric lesions. EXPERIMENTAL APPROACH: We examined the effects of (1) central ghrelin (4 mug per rat) injection on PGE(2) accumulation in normal or EtOH-lesioned gastric mucosa, (2) pretreatment with indomethacin (10 mg kg(-1), p.o.), a non-selective cyclooxygenase (COX) inhibitor, and with a selective COX-1, SC560 (5 mg kg(-1), p.o.) or COX-2 inhibitor, celecoxib (3.5 mg kg(-1), p.o.) on ghrelin gastroprotection against 50% EtOH (1 mL per rat)-induced gastric lesions, (3) the NO synthase inhibitor, L-NAME (70 mg kg(-1), s.c), on gastric PGE(2) content in ghrelin-treated rats and (4) central ghrelin on the expression of constitutive and inducible NOS and COX mRNA and on the localization of the immunoreactivity for COX-2 in the gastric mucosa exposed to EtOH. KEY RESULTS: Ghrelin increased PGE(2) in normal mucosa, whereas, it reversed the EtOH-induced PGE(2) surge. Ghrelin had no effect on mucosal COX-1 expression but reduced the EtOH-induced increase in COX-2 expression and immunoreactivity. Indomethacin and SC560, but not celecoxib, removed ghrelin gastroprotection. L-NAME prevented the PGE(2) surge induced by ghrelin and, like indomethacin, reduced EtOH-induced PGE(2) increase. Ghrelin enhanced eNOS expression and reduced iNOS mRNA. CONCLUSIONS AND IMPLICATIONS: This study shows that COX-1-derived PGs are mainly involved in ghrelin gastroprotection and that the constitutive-derived NO together with PGE(2) are involved in ghrelin gastroprotective activity.     

Comparative effects of FK-506, rapamycin and cyclosporin A, on the in vitro differentiation of dorsal root ganglia explants and septal cholinergic neurons

There is increasing evidence that immunophilins play a role in neural development and differentiation. We have studied the neurotrophic effects of FK-506, rapamycin and cyclosporin A (CsA) on dorsal root ganglia (DRG) taken from different segmental levels (cervical, thoracic and lumbar/sacral), and on rat embryonic septal cholinergic neurons in culture. At a low concentration (1 nM), FK-506 significantly increased (+ 83%) the number of neurites of thoracic DRG expiants. At a higher concentration (100 nM), it also enhanced the neuritogenesis of thoracic (+ 100%) and lumbar/sacral (+ 57%) DRG, but not cervical DRG explants. Rapamycin displayed a converse effect, reducing the development of DRG explants from cervical and thoracic segments. CsA (from 1 to 100 nM) was without effect on DRG neuritogenesis. In contrast to nerve growth factor (NGF), which increased neurite length (+ 116% at 3 ng/ml), neither FK-506 nor rapamycin affected this parameter.     

Effects of exercise on ethanol-induced hypothermia and loss of righting response in C57BL/6J mice.

C57BL/6J mice were given 5 weeks of voluntary wheel running and then studied for ethanol (EtOH) sensitivity as indicated by EtOH-induced hypothermia and loss of righting response (LORR) after 3.8 g/kg EtOH (20% w/v). Mice were assigned to wheel (free access to a running wheel in the home cage) or no wheel conditions, and wheel counts were monitored by a computer at 5-min intervals around the clock. In Experiment 1, duration of EtOH-induced LORR was assessed as amount of time required for the animal to right itself three times in a 30-s period, and body temperature was assessed by rectal probe. Wheel animals showed significantly shorter LORR and significantly less hypothermia at regaining the righting response than no wheel controls. In Experiment 2, temperature was assessed at 45 and 90 min after EtOH challenge. Baseline temperatures for wheel and no wheel animals did not differ, but wheel animals showed dramatic resistance to EtOH-induced hypothermia at both time points. Together with our earlier work, these results provide evidence that prior exercise can offset the effects of EtOH intoxication in several domains of EtOH sensitivity.     

Essential amino acid supplementation decreases liver damage induced by chronic ethanol consumption in rats

The liver sustains the greatest damage from ethanol (EtOH) abuse. EtOH and its metabolites impair hepatocyte metabolism, causing intracellular accumulation of proteins and lipids and increasing radical oxygen species production. These processes are toxic to the mitochondrial respiratory chain and to mitochondrial DNA. We have recently shown that supplementating the diet of rodents with an essential amino acid-enriched mixture (EAAem) significantly increases mitochondrial mass and number in cardiac and skeletal muscles and improves mitochondrial function in aged animals. Thus, in this study we sought to test whether EAAem supplementation could reduce EtOH-induced liver damage. Groups of adult male Wistar rats were fed a standard diet and water ad libitum (the control group), drinking water with 20 percent EtOH (the EtOH group), or drinking water with 20 percent EtOH and EAAem supplementation (1.5 g/kg/day) (the EtOH+EAAem group) for 2 months. The blood EtOH concentration was measured, and markers for fat (Oil-Red-O), mitochondria (Grp75, Cyt-c-ox), endoplasmic reticulum (Grp78), and inflammation (Heme Oxigenase 1, iNOS, and peroxisomes) were analyzed in the liver of animals in the various experimental groups. EAAem supplementation in EtOH-drinking rats ameliorated EtOH-induced changes in liver structure by limiting steatosis, recruiting more mitochondria and peroxisomes mainly to perivenous hepatocytes, stimulating or restoring antioxidant markers, limiting the expression of inflammatory processes, and reducing ER stress. Taken together, these results suggest that EAAem supplementation may represent a promising strategy to prevent and treat EtOH-induced liver damage.     

Cilostazol improves cognitive function in mice by increasing the production of insulin-like growth factor-I in the hippocampus

Insulin-like growth factor I (IGF-I) exerts beneficial effects on cognitive function by inducing angiogenesis and neurogenesis in the hippocampus. We demonstrated that stimulation of sensory neurons in the gastrointestinal tract increased IGF-I production in the hippocampus, and thereby improved cognitive function in mice. Since cAMP plays a critical role in stimulation of sensory neurons, the type III phosphodiesterase (PDE3) inhibitor cilostazol might increase IGF-I production in the hippocampus by stimulating sensory neurons and thus improve cognitive function in mice. We tested this hypothesis in the present study. Cilostazol increased the release of calcitonin gene-related peptide (CGRP) and levels of cAMP in dorsal root ganglion (DRG) neurons isolated from wild-type (WT) mice. Tissue levels of cAMP in the DRG and hippocampus and those of CGRP, IGF-I, and IGF-I mRNA in the hippocampus were increased after 4-week oral administration of cilostazol to WT mice. Levels of expression of c-fos in the spinal dorsal horns, parabrachial nuclei, the solitary tract nucleus, and the hippocampus were also increased in these animals. Significant enhancement of angiogenesis and neurogenesis was observed in the dentate gyrus of the hippocampus after cilostazol administration in WT mice. Significant improvement of spatial learning was also observed in WT mice administered cilostazol. However, none of these effects in WT mice were observed in CGRP-knockout mice. These observations suggest that cilostazol may improve cognitive function in mice by increasing the hippocampal production of IGF-I through stimulation of sensory neurons.     

Corticotropin-releasing factor overexpression decreases ethanol drinking and increases sensitivity to the sedative effects of ethanol

Rationale Corticotropin-releasing factor (CRF) may play a significant role in drug and alcohol abuse.Objective To evaluate the role of CRF in these processes, we examined several ethanol (EtOH) related behaviors in mice that carry a transgene that causes overexpression of CRF.Methods We examined voluntary EtOH drinking, loss of the righting reflex (LORR), EtOH-induced conditioned taste aversion (CTA), and EtOH clearance in littermate transgenic (TG) and non-transgenic (non-TG) mice. In addition, because preliminary results indicated that age exacerbated differences in EtOH consumption between the two genotypes, we performed a cross-sectional and longitudinal evaluation of this trait at two ages (100 and 200 days old).Results We found that TG mice consumed significantly less EtOH and had a lower preference for EtOH-containing solutions compared with their non-TG littermates. We also found that the older drug-naive TG mice drank less EtOH as compared with the younger mice of the same genotype; however, the same relationship did not exist for drug-naive non-TG mice. Prior experience in drinking EtOH when 100 days old led to decreased EtOH drinking when 200 days old in both genotypes. Duration of LORR was longer in the TG mice, EtOH-induced CTA was marginally greater in non-TG mice at the highest dose tested, and there were significant but small differences in EtOH clearance parameters.Conclusions These data show that CRF overexpressing mice voluntarily consume less EtOH. This difference is associated with greater sensitivity to the sedative-hypnotic effects of EtOH, but not with increased sensitivity to the aversive effects of EtOH.     

Ethanol enhances both action potential-dependent and action potential-independent GABAergic transmission onto cerebellar Purkinje cells

Ethanol (EtOH) modulates synaptic efficacy in various brain areas, including the cerebellum, which plays a role in motor coordination. Previous studies have shown that EtOH enhances tonic inhibition of cerebellar granule cells, which is one of the possible reasons for the alcohol-induced motor impairment. However, the effects of EtOH on molecular layer interneurons (MLIs) in the mouse cerebellum have remained unknown. Here we found that MLIs were depolarized by EtOH through enhancement of hyperpolarization-activated cationic currents (I_h). Under physiological conditions, a low EtOH concentration (3-50 mM) caused a small increase in the firing rate of MLIs, whereas, in the presence of blockers for ionotropic glutamate and GABA receptors, EtOH (≥10 mM) robustly enhanced MLI firing, suggesting that synaptic inputs, which seem to serve as the phasic inhibition, could suppress the EtOH-mediated excitation of MLIs and Purkinje cells (PCs). Even in the absence of synaptic blockers, a high EtOH concentration (100 mM) markedly increased the firing rate of MLIs to enhance GABAergic transmission. Furthermore, 100 mM EtOH-facilitated miniature IPSCs via a mechanism that depended on intracellular cyclic AMP, voltage-dependent Ca²⁺ channels, and intracellular Ca²⁺ stores, but was independent of I_h or PKA. The two distinct effects of a high EtOH concentration (≥100 mM), however, failed to attenuate the EtOH-induced strong depolarization of MLIs. These results suggest that acute exposure to a low EtOH concentration (≤50 mM) enhanced GABAergic synaptic transmission, which suppressed the EtOH-evoked excitation of MLIs and PCs, thereby maintaining precise synaptic integration of PCs.     

Plasticity of GABAA receptors after ethanol pre-exposure in cultured hippocampal neurons

Alcohol use causes many physiological changes in brain with behavioral sequelae. We previously observed (J Neurosci 27:12367-12377, 2007) plastic changes in hippocampal slice recordings paralleling behavioral changes in rats treated with a single intoxicating dose of ethanol (EtOH). Here, we were able to reproduce in primary cultured hippocampal neurons many of the effects of in vivo EtOH exposure on GABA(A) receptors (GABA(A)Rs). Cells grown 11 to 15 days in vitro demonstrated GABA(A)R δ subunit expression and sensitivity to enhancement by short-term exposure to EtOH (60 mM) of GABA(A)R-mediated tonic current (I(tonic)) using whole-cell patch-clamp techniques. EtOH gave virtually no enhancement of mIPSCs. Cells pre-exposed to EtOH (60 mM) for 30 min showed, 1 h after EtOH withdrawal, a 50% decrease in basal I(tonic) magnitude and tolerance to short-term EtOH enhancement of I(tonic), followed by reduced basal mIPSC area at 4 h. At 24 h, we saw considerable recovery in mIPSC area and significant potentiation by short-term EtOH; in addition, GABA(A)R currents exhibited reduced enhancement by benzodiazepines. These changes paralleled significant decreases in cell-surface expression of normally extrasynaptic δ and α4 GABA(A)R subunits as early as 20 min after EtOH exposure and reduced α5-containing GABA(A)Rs at 1 h, followed by a larger reduction of normally synaptic α1 subunit at 4 h, and then by increases in α4γ2-containing cell-surface receptors by 24 h. Measuring internalization of biotinylated GABA(A)Rs, we showed for the first time that the EtOH-induced loss of I(tonic) and cell-surface δ/α4 20 min after withdrawal results from increased receptor endocytosis rather than decreased exocytosis.     

Oxidative damage and neurodegeneration in manganese-induced neurotoxicity

Exposure to excessive manganese (Mn) levels results in neurotoxicity to the extrapyramidal system and the development of Parkinson's disease (PD)-like movement disorder, referred to as manganism. Although the mechanisms by which Mn induces neuronal damage are not well defined, its neurotoxicity appears to be regulated by a number of factors, including oxidative injury, mitochondrial dysfunction and neuroinflammation. To investigate the mechanisms underlying Mn neurotoxicity, we studied the effects of Mn on reactive oxygen species (ROS) formation, changes in high-energy phosphates (HEP), neuroinflammation mediators and associated neuronal dysfunctions both in vitro and in vivo. Primary cortical neuronal cultures showed concentration-dependent alterations in biomarkers of oxidative damage, F₂-isoprostanes (F₂-IsoPs) and mitochondrial dysfunction (ATP), as early as 2 h following Mn exposure. Treatment of neurons with 500 μM Mn also resulted in time-dependent increases in the levels of the inflammatory biomarker, prostaglandin E₂ (PGE₂). In vivo analyses corroborated these findings, establishing that either a single or three (100 mg/kg, s.c.) Mn injections (days 1, 4 and 7) induced significant increases in F₂-IsoPs and PGE₂ in adult mouse brain 24 h following the last injection. Quantitative morphometric analyses of Golgi-impregnated striatal sections from mice exposed to single or three Mn injections revealed progressive spine degeneration and dendritic damage of medium spiny neurons (MSNs). These findings suggest that oxidative stress, mitochondrial dysfunction and neuroinflammation are underlying mechanisms in Mn-induced neurodegeneration.     

Diazinon and diazoxon impair the ability of astrocytes to foster neurite outgrowth in primary hippocampal neurons

Evidence from in vivo and epidemiological studies suggests that organophosphorus insecticides (OPs) are developmental neurotoxicants, but possible underlying mechanisms are still unclear. Astrocytes are increasingly recognized for their active role in normal neuronal development. This study sought to investigate whether the widely-used OP diazinon (DZ), and its oxygen metabolite diazoxon (DZO), would affect glial–neuronal interactions as a potential mechanism of developmental neurotoxicity. Specifically, we investigated the effects of DZ and DZO on the ability of astrocytes to foster neurite outgrowth in primary hippocampal neurons. The results show that both DZ and DZO adversely affect astrocyte function, resulting in inhibited neurite outgrowth in hippocampal neurons. This effect appears to be mediated by oxidative stress, as indicated by OP-induced increased reactive oxygen species production in astrocytes and prevention of neurite outgrowth inhibition by antioxidants. The concentrations of OPs were devoid of cytotoxicity, and cause limited acetylcholinesterase inhibition in astrocytes (18 and 25% for DZ and DZO, respectively). Among astrocytic neuritogenic factors, the most important one is the extracellular matrix protein fibronectin. DZ and DZO decreased levels of fibronectin in astrocytes, and this effect was also attenuated by antioxidants. Underscoring the importance of fibronectin in this context, adding exogenous fibronectin to the co-culture system successfully prevented inhibition of neurite outgrowth caused by DZ and DZO. These results indicate that DZ and DZO increase oxidative stress in astrocytes, and this in turn modulates astrocytic fibronectin, leading to impaired neurite outgrowth in hippocampal neurons.