The average body weight of ethanol-fed mice was significantly higher (p<0.02; N=8) than that of pair-fed controls or mice in the alcohol + ALC group. Conversely, the mean weight of whole-brain tissue of alcohol-fed mice was lower than that of the control or alcohol + ALC group after 8 weeks of liquid diet administration (see ). The average amount of intake per animal was about 17.0 ml during the control liquid diet acclimation period. The pattern of food intake during pair-feeding was about 12, 15, 16, and 18 ml/mouse, respectively, at week 1, 2, 3, and 4–8. Pair-feeding of all other conditions was based on the volume of EtOH liquid diet intake in the alcohol condition.
Body and brain weights and blood alcohol levels in alcohol-fed mice
Alcohol-fed animals exhibited behavioral changes during the ethanol period. We observed that these animals were physically less active and were less alert compared with the pair-fed control or the alcohol-diet plus ALC animals. We speculated that the change in behavior was related to impairment of synaptic transmission resulting from alcohol-induced oxidative damage and mitochondrial energy depletion, leading to loss of neuronal activity. Thus, we analyzed the low-frequency stimulus (LFS) and LTP synaptic transmission of neuronal activity in ex vivo frontal cortical brain tissue slices. These analyses revealed a significant reduction in LTP and LFS synaptic transmission in the CA1 region in brain slices from alcohol-fed mice compared with pair-fed controls (). As anticipated, ALC prevented the alcohol-elicited loss of LTP and LFS synaptic transmission in the frontal cortex, suggesting that ALC blocked the loss of neuronal activity, perhaps by stabilizing mitochondrial function. These data are consistent with our recent findings that, in culture, ALC exposure protects primary human neurons from EtOH/Acetaldehyde (Ach) insult [17
Fig. 1 ALC prevented alcohol-induced reduction in LTP synaptic transmission in frontal cortex tissue slice. LTP synaptic responses were induced by a constant current stimulation (twin pulses, 150–300 μA, 40 μs, 0.05 Hz) of Schafer collateral (more ...)
To evaluate the neuroprotective effects of ALC in our animal model, we first examined the changes in the expression of neuronal marker protein neurofilament (NF) in frontal cortex coronal sections by immunofluorescence, using antibody to neurofilament protein. The data indicated that ethanol administration diminished NF fluorescence in cortical sections. However, daily coadministration of ALC with ethanol liquid diet prevented the loss of NF from cortical neurons in these animals (). The mitochondrial-specific antioxidant CoQ10 afforded very minimal neuroprotection from chronic alcohol administration (data not shown), similar to our unpublished data in cell culture studies, which was attributed to the impermeability of exogenous CoQ10 across the cellular and mitochondrial membranes. Thus, we focused here on the protective effects of ALC, rather than that of CoQ10, from alcohol insult. Because the integrity of the neurofilaments was affected by alcohol ingestion, we determined the susceptibility of specific types of neurons to chronic alcohol in take. Here, we identified the loss of dopaminergic neurons by loss of fluorescence in TH protein and cholinergic neurons by the loss of fluorescence in ChAT in coronal sections of the brain. Our findings revealed that dopaminergic neurons, mostly localized in the substantia nigra region, exhibited degeneration after ethanol administration (). Similarly, cholinergic neurons detected in the outer region of the brain striatum in ethanol-fed mice were less distinct compared with control and ALC + EtOH-fed mice (). Coadministration of ALC seemed to protect both dopaminergic and cholinergic neurons from alcohol insults in this region.
Fig. 2 ALC administration protected frontal cortical neurons from depletion of NF from chronic alcohol feeding. (A–C) Immunohistochemical staining for marker protein (neurofilaments, NF) on 5-μm tissue sections from (A) control, (B) EtOH, and (more ...)
In agreement with our recent in vitro findings, we observed a twofold increase in the intensity of the 135-kDa iNOS protein in the frontal cortex after chronic alcohol ingestion compared with controls (). Immunohistochemistry and confocal microscopy studies showed distinctive staining of neurofilaments around iNOS in the cytoplasm of neurons, particularly in brain tissues from alcohol ingestion (). Coadministration of ALC with ethanol for 8 weeks significantly decreased alcohol-elicited iNOS protein induction in this brain region (). Because the staining for neurofilaments and iNOS was localized differentially within the same cell body, we further confirmed the localization of iNOS in neurons by colocalizing with NeuN protein. Antibody to NeuN is specific to the nucleus and cytoplasm of neurons only, and our results clearly provide the evidence that iNOS was indeed perfectly colocalized with NeuN, as shown in . Interestingly, we observed fewer neurons in brain tissues from the EtOH condition compared with the control or the ALC + EtOH condition ().
Fig. 3 Alcohol-induced iNOS protein colocalized with neurofilaments in brain frontal cortex. Protein extracts derived from the frontal cortex were analyzed for alterations in iNOS protein content, whereas coronal tissue sections were analyzed for iNOS subcellular (more ...)
Fig. 4 Induction of iNOS in the brain perfectly colocalizes with NeuN in neuronal cell bodies. Immunofluorescence histochemical stainings of iNOS (red), NeuN (green), and DAPI nuclei (blue) in brain tissue sections. (A–C) Control brain tissue, (D–F) (more ...)
We then examined the levels of 3-nitrotyrosine protein adducts (3-NT immunoreactivity is a signature of peroxynitrite binding to proteins). We examined their subcellular localization in frontal cortex. Antibody to 3-NT detected three immunoreactive proteins with band sizes of 25, 55, and 160 kDa. Because the immunoreactive bands for 25 and 160 kDa were very weak (bands were not accurately quantifiable after minimizing background), we present the data obtained from the 55-kDa immunoreactive protein band. We found that ethanol administration caused a 2.3-fold increase in the 3-NT protein level, which paralleled a significant induction of 3-NT protein staining that was, again, distinctly colocalized with the neuronal marker NeuN, suggesting that 3-nitrotyrosine protein adducts were formed because of iNOS induction (). Unlike CoQ10, administration of ALC significantly blocked the ethanol-elicited up-regulation of 3-NT protein to levels that approached those of controls. These results confirm our recent findings that EtOH/Ach caused a 45% induction of iNOS protein level in primary human neurons [17
]. As a positive control for 3-NT formation, brain tissue slices from control animals were incubated (1 h in CO2
incubator) with an authentic peroxynitrite-generating compound, 3-morpholinosydnonimine hydrochloride (SIN-1; 300 μM), in the absence and presence of equimolar uric acid, a peroxynitrite scavenger. For the presence of uric acid condition, tissue slices were exposed to uric acid for 20 min in a CO2
incubator before the addition of SIN-1 and uric acid was present at the time of SIN-1 treatment. As anticipated, the increased level of 3-NT protein expression after SIN-1 treatment was prevented by coadministration of uric acid ().
Fig. 5 Induction of iNOS correlates with increased 3-nitrotyrosine protein adduct levels in frontal cortical neurons. Protein extracts were examined for changes in 3-NT protein content, whereas tissue sections were used for 3-NT subcellular localization. (A) (more ...)
Because iNOS induction and 3-NT formation led to neuroinflammation, we also studied the localization of 3-NT protein in astrocytes and microglia in brain tissue from ethanol-fed, their pair-fed controls, and alcohol + ALC-fed mice. Interestingly, 3-NT was weakly colocalized with the astrocytic marker protein GFAP (), but was not detected in microglia (data not shown), suggesting that alcohol intake up-regulated iNOS mostly in neurons. However, numbers of distinctly stained microglia were higher in brain tissue from alcohol-fed animals (46 cells/μm2) than in pair-fed control mice (10 cells/μm2) in the same brain region (), suggesting the development of an inflammatory process as a result of microgliosis in ethanol-fed mice.
Fig. 6 Detection of nitrotyrosine immunoreactivity in frontal cortical astrocytes. (A–D) Brain frontal tissue sections were stained for 3-NT protein adducts (red) and merged with the astrocyte-specific protein marker GFAP (green). (A) 3-NT in control (more ...)
Because iNOS induction did not seem to be a prominent source of oxidative stress in astrocytes and microglia, we examined the localization of NOX1 protein with the astrocyte marker protein GFAP and the microglia marker protein Iba1 in the frontal cortex. Immunohistochemical detection revealed an association between NOX1 activation and astroglial activation in alcohol-fed mice as evidenced by the colocalization of NOX1 protein with GFAP () and Iba1 (). Again, ALC treatment partially neutralized the effects of alcohol on NOX1 protein expression in astrocytes and microglia.
Fig. 7 NOX1 protein colocalized with astrocytes (GFAP) in the frontal cortex. Double immunohistochemistry of GFAP (green) with NOX1 (red) protein (A, B) in control brain tissue, (C, D) in alcohol brain tissue, and (E, F) in coadministration of alcohol and ALC (more ...)
Fig. 8 Brain frontal cortex tissue sections from chronic studies were stained for colocalization of NOX1 (red) protein with microglia (Iba1; green). (A) Control, (C) EtOH, and (E) EtOH + ALC; NOX1 alone. (B) Control, (D) EtOH, and (F) EtOH + ALC, colocalization (more ...)
To assess the association between NOX1 activation and oxidative damage in the brain, we then evaluated the changes in NOX1 protein level and formation of the ROS-mediated lipid peroxidation product 4-HNE. Antibody to NOX1 detected multiple immunoreactive bands with a prominent band size of approximately 65 kDa, which was the expected molecular weight of NOX1 protein. Ethanol administration caused a 2.5-fold increase in the intensity of the 65-kDa NOX1-immunoreactive band in brain tissues compared with brains from pair-fed controls (). Elevation in NOX1 protein level was accompanied by a 2.8-fold increase in 4-HNE protein adduct content in alcohol-fed mice compared with controls (). Coadministration of ALC (but not malonyl coenzyme A or CoQ10) almost completely abrogated the up-regulation by alcohol of NOX1 and 4-HNE protein in the brain. To verify the formation of 4-HNE (a signature of ROS oxidatively damaged proteins), we measured ROS production by a direct electron paramagnetic resonance method in this brain region. These analyses confirmed our findings with 4-HNE that chronic alcohol ingestion significantly enhanced the production of ROS, and, interestingly, ALC diminished ROS generation (). Thus, our findings indicate that ALC neutralizes oxidative damage in the brain caused by chronic ethanol administration.
Fig. 9 Detection of NOX1, 4-HNE, and ROS in brain tissue samples. Brain homogenate protein extracts from chronic ethanol liquid diet-fed or pair-fed control mice were analyzed for changes in NOX1 protein and 4-HNE protein. (A) Immunoreactive bands of NOX1 (65 (more ...)