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The Effects of 528 Hz Sound Wave to Reduce Cell Death in Human Astrocyte Primary Cell Culture Treated with Ethanol
Abstract
Objective: Alcohol consumption, because of damaging effects on various tissues including nerves system, causes a major problem in human societies. Nowadays, Usages of the non-pharmacological and non-invasive agents is common. Sound waves which are classified in non-invasive agent, beside on stimulation the auditory cells, also impact on non- auditory cells. It has been reported that the frequency of 528 HZ is related to the note MI and it shown some strange effects such as increasing the ability of DNA repair. Materials and methods: In this paper, with MTT, LDH and ROS assays, it was evaluated the effect of ethanol on astrocytes primary cell culture which exposed to this frequency. Results: The current results show that in IC50 of ethanol, the frequency of 528 Hz increased cells viability about 20% and the level of ROS production has been reduced up to 100%. Conclusion: Therefore, the use of these sound waves can be useful to reduce the toxic effects of ethanol on astrocytes cells culture.
... The study provides evidence that 528 Hz frequency, compared to standard 440 Hz, may have a more pronounced stress-reducing effect, even with a brief exposure of only five minutes. Prior research (Babayi and Riazi 2017) suggests that exposure to a frequency of 528 Hz mitigates the toxic effects of ethanol on human brain cells. The study also reported an increase of human brain cell viability by approximately 20% following exposure to this frequency. ...
... Additionally, the identification of five solfeggio frequencies is intriguing. While the historical and musicological basis of solfeggio frequencies is debated, recent studies suggest potential benefits associated with these frequencies, including stress reduction and cognitive improvement (Babayi and Riazi 2017;Akimoto et al. 2018;Babayi Daylari et al. 2019;Dos Santos et al. 2023). These findings, if replicated with larger sample sizes, could contribute to the understanding of sound frequencies and their potential health impacts with particular reference to chanting Nam-Myoho-Renge-Kyo in the context of Nichiren Shoshu Liturgy. ...
This study investigates the effects of chanting Nam-Myoho-Renge-Kyo on sound frequencies, brain activity, and microbial metabolism. Spectral analysis of chanting revealed peaks at 8 Hz and 116 Hz, corresponding to the first Schumann resonance and a frequency linked to chloride ion movements, respectively. Additionally, five peaks corresponding to solfeggio frequencies were identified. These frequencies are known to exert a positive impact on the human endocrine and autonomous nervous systems; improve survival of human brain cells; decrease anxiety in rats; reverse cognitive and endocrine deficits in zebra fish; reduce total concentration of reactive oxidative species in brain tissue. Brain activity changes were measured using functional near-infrared spectroscopy. Prefrontal cortex activity increased modestly during chanting compared to a pre-chanting baseline, but significantly increased afterwards. This suggests a shift towards focused attention during chanting and enhanced activity afterward. The study also examines the effects of chanting on microbial metabolism. Cultures exposed to chanting, either directly or indirectly, showed increased metabolic activity. This suggests local and non-local effects, similar to previously reported phenomena. Importantly, the study clarifies that the spiritual practice of Nichiren Shoshu Buddhism can't be reduced to mere neurophysiological mechanisms in the brain or any other part of the body. The religious and spiritual experience of practicing Buddhism and chanting Nam-Myoho-Renge-Kyo goes well beyond brain activity, neurotransmitters or quantum phenomena of consciousness. It encompasses a mystic dimension that, although not in contradiction with science, can’t be explained solely by science.
... The 528 Hz sound frequency reduces reactive oxygen species (ROS) by up to 100%, protecting cells from oxidative stress, a key contributor to DNA damage and aging. 4 This helps preserve DNA integrity, supporting cellular longevity. Maintaining and or repairing DNA correlates with longevity and health and is crucial in fields like regenerative medicine. ...
Quantum medicine and regenerative medicine represent transformative frontiers in healthcare. This article explores their possible unification, by emphasizing Tesla waves (scalar waves) and 528 Hz sound frequency as potential bridges to enhance regenerative processes. By integrating quantum principles with regenerative therapies, this innovative approach has the potential to optimize cellular energy, accelerate recovery, and offer non-invasive treatment protocols. Rigorous scientific study is critical to substantiate these approaches and unlock their potential.
... The study provides evidence that 528 Hz frequency, compared to standard 440 Hz, may have a more pronounced stress-reducing effect, even with a brief exposure of only five minutes. Prior research (Babayi and Riazi 2017) suggests that exposure to a frequency of 528 Hz mitigates the toxic effects of ethanol on human brain cells. The study also reported an increase of human brain cell viability by approximately 20% following exposure to this frequency. ...
This study investigates the effects of chanting Nam-Myoho-Renge-Kyo by a long-term Nichiren Shoshu Buddhist practitioner on sound frequencies (A), brain activity (B), and microbial metabolism(C).A. Analysis of sound frequencies: Spectral analysis of chanting revealed peaks at 8 Hz and 116 Hz, corresponding to the first Schumann resonance and a frequency linked to chloride ion movements, respectively. Additionally, five peaks (417, 528, 639, 741, 852 Hz) corresponding to solfeggio frequencies were identified. These frequencies are known to exert a positive impact on the human endocrine and autonomous nervous systems; improve survival of human brain cells; decrease anxiety in rats; reverse cognitive and endocrine deficits in zebra fish; reduce total concentration of reactive oxidative species in brain tissue. B. Study of Brain activity: Brain activity changes were measured using functional near-infrared spectroscopy. Prefrontal cortex activity increased modestly during chanting compared to a pre-chanting baseline, but significantly increased afterwards. This suggests a shift towards focused attention during chanting and enhanced activity afterward. C. Effects on microbial metabolism: Probiotic cultures exposed to chanting, either directly or indirectly, showed increased metabolic activity. This suggests local and non-local effects, similar to previously reported phenomena. Importantly, this study clarifies that the spiritual practice of Nichiren Shoshu Buddhism transcends mere neurophysiological explanations. While brain activity and other bodily processes may be correlated with the practice, the religious and spiritual experience of chanting Nam-Myoho-Renge-Kyoencompasses a mystical dimension that, though not contradicting science, cannot be fully explained by scientific methods alone.
... The pressure waves produced by sounds can affect cells or their structures by determining micro-vibrations, or even generate resonances, i.e., the synchronization of biomolecular oscillatory patterns within cells [50]. Furthermore, acoustic vibrations in the form of a single frequency [51], noise, or music have been shown to influence proliferation, viability, and hormone binding in human cell cultures [52,53] and animal models [54,55]. Acoustic stimuli are therefore of fundamental importance in regulating the spatial interaction between cells, modulating both their individual life and collective behaviors [56,57], and their intracellular and intercellular organization, which are crucial elements that control their function [58]. ...
Vibration and sound are the shaping matrix of the entire universe. Everything in nature is shaped by energy vibrating and communicating through its own sound trail. Every cell within our body vibrates at defined frequencies, generating its peculiar “sound signature”. Mitochondria are dynamic, energy-transforming, biosynthetic, and signaling organelles that actively transduce biological information. Novel research has shown that the mitochondrial function of mammalian cells can be modulated by various energetic stimuli, including sound vibrations. Regarding acoustic vibrations, definite types of music have been reported to produce beneficial impacts on human health. In very recent studies, the effects of different sound stimuli and musical styles on cellular function and mitochondrial activity were evaluated and compared in human cells cultured in vitro, investigating the underlying responsible molecular mechanisms. This narrative review will take a multilevel trip from macro to intracellular microenvironment, discussing the intimate vibrational sound activities shaping living matter, delving deeper into the molecular mechanisms underlying the sound modulation of biological systems, and mainly focusing our discussion on novel evidence showing the competence of mitochondria in acting as energy portals capable of sensing and transducing the subtle informational biofields of sound vibration.
... For another analysis, music with 528Hz has reduced the toxic influence of ethanol, that is the main element of alcoholic beverages. 13 Furthermore, it can induce total reactive oxidative species (ROS) in the brain. 14 Regarding these topics on frequencies, continuous accumulation of evidence would be required. ...
Music has various power for human body and mind. For 12 music tone, two types of scales are Pythagorean Tuning (just intonation) and equal temperament. This minute difference may generate human feeling for music. Music with 528Hz frequency has been in focus as healing music, which is called as “solfeggio frequency music”. Certain frequency music has been known such as 96, 432, 528, 639, 852 Hz, that contributes reducing tension and anxiety. It seems to show beneficial efficacy, but scientific evidence is not enough. Similar to former Mozart effect, further accumulation of evidence and research development will be expected.
... The Solfeggio frequencies associated with chanting, on the other hand, may be responsible for a series of biological effects that may help explaining the increase in psychological resources described by Giannini et al. (2018), andBragazzi et al. (2019). It has been demonstrated that these frequencies have a positive impact on the human endocrine and autonomous nervous system (Akimoto et al. 2018) and improve survival of human brain astrocytes in vitro (Babayi and Riazi 2017). It appears that the positive effects of these frequencies, however, are not limited to humans since it has been demonstrated that they decrease anxiety in rats (Babayi and Riazi 2019) and reverse cognitive and endocrine deficits in zebra fish (Dos Santos et al. 2023), thus indirectly confirming the hypothesis enunciated above that recitation of Nam-Myoho-Renge-Kyo acts not only on human cells. ...
Recent evidence obtained in murine atrial cardiomyocytes suggests that the behavior of tubulin, a constituent of microtubules, is influenced by audible sounds. In the present study, the sequence and the structure of murine and human tubulins were compared with those of human TMC1 (Transmembrane channel-like protein 1), a sound-sensitive protein that is responsible for forming the pore of mechanosensory transduction channels in the hair cells of the inner ear of vertebrates, and with those of bacterial FtsZ (Filamenting temperature-sensitive mutant Z), a bacterial protein homologous to tubulin. The results show that mouse and human alpha-tubulin are 100% identical, whereas sequence homology of human TMC1 and human tubulin alpha 1A chain is scarce, with only 17.31% identity. A higher degree of similarity was observed in the presence of aromatic amino acids as well as in the propensity to form alpha-helices. Based on these results, it is hypothesized that the response of tubulin to audible sounds is mediated by the external alpha-helices as it occurs in TMC1. The sequence homology of human TMC1 and FtsZ from Lactobacillus johnsonii, is moderate with 23.40% identity, however higher than that between TMC1 and tubulin. The overall presence of aromatic amino acids in FtsZ is scarce, but helix propensity is significant. These results are discussed in the context of Orch OR, a theory that postulates that consciousness emerges from quantum computations occurring in microtubules of neurons via information processing mediated by aromatic amino acids of tubulin.
... The Solfeggio frequencies associated with chanting, on the other hand, may be responsible for a series of biological effects that may help explaining the increase in psychological resources described by Giannini et al. (2018), and Bragazzi et al. (2019). It has been demonstrated that these frequencies have a positive impact on the human endocrine and autonomous nervous system (Akimoto et al. 2018) and improve survival of human brain astrocytes in vitro (Babayi and Riazi 2017). It appears that the positive effects of these frequencies, however, is not limited to humans since it has been demonstrated that they decrease anxiety in rats (Babayi and Riazi 2019) and reverse cognitive and endocrine deficits in zebra fish (Dos Santos et al. 2023), thus indirectly confirming the hypothesis enunciated above that recitation of Nam-Myoho-Renge-Kyo acts not only on human cells and systems. ...
Recent evidence obtained in murine atrial cardiomyocytes suggests that the behavior of tubulin, a constituent of microtubules, is influenced by audible sounds. In the present study, the sequence and the structure of murine and human tubulins were compared with those of human TMC1 (Transmembrane channel-like protein 1), a sound-sensitive protein that is responsible for forming the pore of mechanosensory transduction channels in the hair cells of the inner ear of vertebrates, and with those of bacterial FtsZ (Filamenting temperature-sensitive mutant Z), a bacterial protein homologous to tubulin. The results show that mouse and human alpha-tubulin are 100% identical, whereas sequence homology of human TMC1 and human tubulin alpha 1A chain is scarce, with only 17.31% identity. A higher degree of similarity was observed in the presence of aromatic amino acids as well as in the propensity to form alpha-helices. Based on these results, it is hypothesized that the response of tubulin to audible sounds is mediated by the external alpha-helices as it occurs in TMC1. The sequence homology of human TMC1 and FtsZ from Lactobacillus johnsonii, is moderate with 23.40% identity, however higher than that between TMC1 and tubulin. The overall presence of aromatic amino acids in FtsZ is scarce, but helix propensity is significant. These results are discussed in the context of Orch OR, a theory that postulates that consciousness emerges from quantum computations occurring in microtubules of neurons via information processing mediated by aromatic amino acids of tubulin.
... K. Akimoto et al. [24] discovered that music tuned to the frequency of 528 Hz significantly reduced stress after only a few minutes of listening. Another study [27] found that 528 Hz reduced the toxic effects of ethanol, which is the main ingredient in alcoholic beverages. Furthermore, the authors observed that this frequency increased cell life by about 20%. ...
Music is important in everyday life, and music therapy can help treat a variety of health issues. Music listening is a technique used by music therapists in various clinical treatments. As a result, music therapists must have an intelligent system at their disposal to assist and support them in selecting the most appropriate music for each patient. Previous research has not thoroughly addressed the relationship between music features and their effects on patients. The current paper focuses on identifying and predicting whether music has therapeutic benefits. A machine learning model is developed, using a multi-class neural network to classify emotions into four categories and then predict the output. The neural network developed has three layers: (i) an input layer with multiple features; (ii) a deep connected hidden layer; (iii) an output layer. K-Fold Cross Validation was used to assess the estimator. The experiment aims to create a machine-learning model that can predict whether a specific song has therapeutic effects on a specific person. The model considers a person’s musical and emotional characteristics but is also trained to consider solfeggio frequencies. During the training phase, a subset of the Million Dataset is used. The user selects their favorite type of music and their current mood to allow the model to make a prediction. If the selected song is inappropriate, the application, using Machine Learning, recommends another type of music that may be useful for that specific user. An ongoing study is underway to validate the Machine Learning model. The developed system has been tested on many individuals. Because it achieved very good performance indicators, the proposed solution can be used by music therapists or even patients to select the appropriate song for their treatment.
... Moreover, acoustic vibrations in the form of single frequencies [48], noise or music, have been shown to alter proliferation, viability [49], and hormone binding [50] in human cell cultures and in animal models [51,52]. Acoustic stimuli are thus of paramount importance in guiding the spatial interaction between cells, influencing their individual and collective behaviour [53,54], their intra-cellular and inter-cellular organization, which are key elements regulating their function [55]. ...
Mechanical vibrations seem to affect the behaviour of different cell types and the functions of different organs. Pressure waves, including acoustic waves (sounds), could affect cytoskeletal molecules via coherent changes in their spatial organization and mechano-transduction signalling. We analyzed the sounds spectra and their fractal features. Cardiac muscle HL1 cells were exposed to different sounds, were stained for cytoskeletal markers (phalloidin, beta-actin, alpha-tubulin, alpha-actinin-1), and studied with multifractal analysis (using FracLac for ImageJ). A single cell was live-imaged and its dynamic contractility changes in response to each different sound were analysed (using Musclemotion for ImageJ). Different sound stimuli seem to influence the contractility and the spatial organization of HL1 cells, resulting in a different localization and fluorescence emission of cytoskeletal proteins. Since the cellular behaviour seems to correlate with the fractal structure of the sound used, we speculate that it can influence the cells by virtue of the different sound waves’ geometric properties that we have photographed and filmed. A theoretical physical model is proposed to explain our results, based on the coherent molecular dynamics. We stress the role of the systemic view in the understanding of the biological activity.
In recent decades, research on mechanotransduction has advanced considerably, focusing on the effects of audible acoustic waves (AAWs) and low-vibration stimulation (LVS), which has propelled the field of sonobiology forward. Taken together, the current evidence demonstrates the influence of these biosignals on key cellular processes, such as growth, differentiation and migration in mammalian cells, emphasizing the determining role of specific physical parameters during stimulation, such as frequency, sound pressure level/amplitude and exposure time. These mechanical waves interact with various cellular elements, including ion channels, primary cilia, cell–cell adhesion receptors, cell–matrix and extracellular matrix proteins, and focal adhesion complexes. These components connect with the cytoskeletal fibre network, enabling the transmission of mechanical stimuli towards the nucleus. The nucleus, in turn, linked to the cytoskeleton via the linkers of the nucleoskeleton and cytoskeleton complex, acts as a mechanosensitive centre, not only responding to changes in cytoskeletal stiffness and nuclear tension but also regulating gene expression through the transcriptional co-activator YAP/TAZ and interactions between chromatin and the nuclear envelope. This intricate chain of mechanisms highlights the potential of sonobiology in various fields, including dentistry, regenerative medicine, tissue engineering and cancer research. However, progress in these fields requires the establishment of standardized measurement methodologies and biocompatible experimental setups to ensure the reproducibility of results.
Sound healing, based on music therapy, explores the therapeutic potential of specific frequencies for anxiety. The Solfeggio frequency of 528 Hz has gained attention for its ability to resonate with the body's natural frequencies, promoting relaxation, reducing stress, and enhancing emotional well-being. This study explores the potential of pure 528 Hz sound to reduce state anxiety. 48 subjects were randomly assigned to either an experimental group, which received a 3-minute exposure to 528 Hz music, or a control group, which completed a neutral reading task. State anxiety levels were measured using the STAI, pre and post intervention. Data analysis included paired-sample t-tests and a two-way repeated measures ANOVA. Results showed a significant drop in level of anxiety among experimental group (p = 0.022). Additionally, paired-sample t-tests confirmed a decrease in state anxiety scores within the experimental group (p = 0.006). These findings provide empirical evidence suggesting the anxiolytic properties of 528 Hz. This study offers preliminary evidence that 528 Hz sound exposure may effectively reduce state anxiety. Future research should elucidate the mechanisms and examine the effectiveness of this approach in broader contexts. This research contributes to the exploration of sound frequencies as a potential non-invasive intervention for anxiety management.
Testosterone is a nuclear androgen receptor ligand that controls multiple pathways in brain. In addition to the active biosynthesis of steroids in classic steroidogenic organs such as gonads, adrenals and placenta, testosterone also produced in astrocyte cells of brain. Testosterone and its level must be regulated in brain; because, it directly and indirectly affects memory and several key behavioral characteristics. The significance of sound waves on key enzymes that regulate levels of testosterone in brain has not been investigated. The aim of our study was to examine physical stress of such as sound on induction behavioral changes in animal models. According to the current study, sound waves with 528 Hz frequency in 100 dB intensity induce testosterone production in brain by enhancing StAR and SF-1 and reducing P450 aromatase gene expression. Frequency of 528 Hz also reduces total concentration of reactive oxidative species in brain tissue. Prolonged exposure to this sound wave showed reduction of anxiety related behaviors in rats. The results reveal that reduced anxiety is related to increased concentration of testosterone in brain. This study may lead to ascertain a possible therapy in which sounds may be utilized to reduce anxiety in individual.
The present paper is a description of some laboratory experiments carried out in order to investigate the perception and changes in wakefulness occurring during exposure to infrasound.
Perception of infrasound is based on hearing and vibrations in different parts of the body. Threshold of audibility was found to be approximately 110 dB(lin) at 4 Hz and 90 dB(lin) at 20 Hz. Sensations through vibrations were found to occur at about 20 dB above the hearing threshold levels. As far as vibrotactile sensation is concerned no difference was found to exist between deaf and hearing subjects. Hearing sensations could not be registered for neurosensory deafness. 10 deaf and 10 hearing subjects were exposed for 20 minutes at 6 Hz, 115 dB(lin). Reduced wakefulness was noticed among the hearing subjects but not among the deaf subjects.
According to these results, changes in wakefulness of infrasound is based on cochlear stimulation. It is suggested that a reduction in wakefulness that is attributable to infrasound occurs at pressure levels close to the auditory threshold.
Comparison of Ethanol and Acetaldehyde Toxicity in Rat Astrocytes in Primary Culture
This study compared the effects of toxicity of ethanol and its first metabolite acetaldehyde in rat astrocytes through cell viability and cell proliferation. The cells were treated with different concentrations of ethanol in the presence or absence of a catalase inhibitor 2-amino-1,2,4 triazole (AMT) or with different concentrations of acetaldehyde. Cell viability was assessed using the trypan blue test. Cell proliferation was assessed after 24 hours and after seven days of exposure to either ethanol or acetaldehyde.
We showed that both ethanol and acetaldehyde decreased cell viability in a dose-dependent manner. In proliferation studies, after seven days of exposure to either ethanol or acetaldehyde, we observed a significant dose-dependent decrease in cell number. The protein content study showed biphasic dose-response curves, after 24 hours and seven days of exposure to either ethanol or acetaldehyde. Co-incubation in the presence of AMT significantly reduced the inhibitory effect of ethanol on cell proliferation.
We concluded that long-term exposure of astrocytes to ethanol is more toxic than acute exposure. Acetaldehyde is a much more potent toxin than ethanol, and at least a part of ethanol toxicity is due to ethanol's first metabolite acetaldehyde.
Sound waves technology has been applied to different plants. It has been found that sound waves were at different frequencies, sound pressure levels (SPLs), exposure periods, and distances from the source of sound influence plant growth. Experiments have been conducted in the open field and under greenhouse growing conditions with different levels of audible sound frequencies and sound pressure levels. Sound waves at 1 kHz and 100 dB for 1 h within a distance of 0.20 m could significantly promote the division and cell wall fluidity of callus cells and also significantly enhance the activity of protective enzymes and endogenous hormones. Sound waves stimulation could increase the plant plasma-membrane H+-ATPase activity, the contents of soluble sugar, soluble protein, and amylase activity of callus. Moreover, sound waves could increase the content of RNA and the level of transcription. Stress-induced genes could switch on under sound stimulation. Sound waves at 0.1–1 kHz and SPL of (70±5) dB for 3 h from plant acoustic frequency technology (PAFT) generator within a distance ranged from 30 to 60 m every other day significantly increased the yield of sweet pepper, cucumber and tomato by 30.05, 37.1 and 13.2%, respectively. Furthermore, the yield of lettuce, spinach, cotton, rice, and wheat were increased by 19.6, 22.7, 11.4, 5.7, and 17.0%, respectively. Sound waves may also strengthen plant immune systems. It has been proved that spider mite, aphids, gray mold, late blight and virus disease of tomatoes in the greenhouses decreased by 6.0, 8.0, 9.0, 11.0, and 8.0%, respectively, and the sheath blight of rice was reduced by 50%. This paper provides an overview of literature for the effects of sound waves on various growth parameters of plant at different growth stages.
Children prenatally exposed to alcohol can suffer from serious cognitive deficits and behavioral problems as well as from alcohol-related changes in brain structure. Neuropsychological studies have identified deficits in learning and memory as well as in executive functioning both in children with fetal alcohol syndrome and in children with less severe impairments. Both groups of children also exhibit problem behaviors, such as alcohol and drug use, hyperactivity, impulsivity, and poor socialization and communication skills. Brain imaging studies have identified structural changes in various brain regions of these children--including the basal ganglia, corpus callosum, cerebellum, and hippocampus--that may account for the cognitive deficits. Functional brain imaging studies also have detected changes in alcohol-exposed children indicative of deficits in information processing and memory tasks.
The human perception of sound at frequencies below 200 Hz is reviewed. Knowledge about our perception of this frequency range is important, since much of the sound we are exposed to in our everyday environment contains significant energy in this range. Sound at 20-200 Hz is called low-frequency sound, while for sound below 20 Hz the term infrasound is used. The hearing becomes gradually less sensitive for decreasing frequency, but despite the general understanding that infrasound is inaudible, humans can perceive infrasound, if the level is sufficiently high. The ear is the primary organ for sensing infrasound, but at levels somewhat above the hearing threshold it is possible to feel vibrations in various parts of the body. The threshold of hearing is standardized for frequencies down to 20 Hz, but there is a reasonably good agreement between investigations below this frequency. It is not only the sensitivity but also the perceived character of a sound that changes with decreasing frequency. Pure tones become gradually less continuous, the tonal sensation ceases around 20 Hz, and below 10 Hz it is possible to perceive the single cycles of the sound. A sensation of pressure at the eardrums also occurs. The dynamic range of the auditory system decreases with decreasing frequency. This compression can be seen in the equal-loudness-level contours, and it implies that a slight increase in level can change the perceived loudness from barely audible to loud. Combined with the natural spread in thresholds, it may have the effect that a sound, which is inaudible to some people, may be loud to others. Some investigations give evidence of persons with an extraordinary sensitivity in the low and infrasonic frequency range, but further research is needed in order to confirm and explain this phenomenon.
Chapter 3 of this volume (Cserr and Patlak) discussed brain volume regulation principally from the viewpoint of a single organ. This is important both for an understanding of the limits of volume regulation of the brain in situ and, of course, from a clinical viewpoint. In the vertebrate, and especially the highly developed mammalian brain, expansion of the brain will increase the interstitial pressure, P, according to the relationship (Rapoport 1979),
(1)
where k represents the compliance of the brain and is given in units of (mmHg)−1, ΔV is a dimensionless, fractional increase in volume and P is in mm Hg. The compliance of the brain, or its ability to increase volume when the interstitial pressure increases, varies between gray and white matter (Rapoport 1979), and such expansion has to occur at the expense of some other component since the total volume within the rigid skull is fixed.
The effect of maternal alcohol exposure on nerve cell development was investigated in neurons and glial cells cultured from foetal rat brain. Neurons were grown for one week from two-week-old cortical brain cells and glial cells were cultured for four weeks from newborn cortical brain cells
Two types of maternal alcohol treatment were performed; either before and during pregnancy or only until the beginning of pregnancy. In both situations, we found a delayed nerve cell maturation assessed by microscopic observations and determination of enzymatic markers of nerve cell development (non-neuronal and neuron-specific enolase for the neuronal cells, non-neuronal enolase and glutamine synthetase for the glial cells). The results confirmed our previous in vivo experiments pointing out long-lasting effects of maternal alcohol exposure in the offspring.
Ethanol disrupts the physical structure of cell membranes. The most fluid membranes, including those that are low in cholesterol, are the most easily disordered by ethanol. Although the membrane-disordering effect is small, there is pharmacological, temporal, and genetic evidence that it is important. Animals that are resistant to ethanol intoxication because of their genetic background or because of previous exposure to ethanol are found to have brain membranes that are not easily disordered in vitro. An exception is the increased behavioral sensitivity in aging animals, which is not matched by changes in their membranes. When animals are treated chronically with ethanol, their membranes become stiffer, a response that can be regarded as adaptive. Ethanol may favor the uptake of cholesterol or saturated fatty acids into membranes, thus reducing its own effect.
The nervous system is one of the main targets of ethanol toxicity and it has been suggested that astrocytes might play an important role as their integrity is essential for the normal growth and functioning of neurons. Morphological variations of astrocyte cultures were therefore examined after exposure to various doses of ethanol (0.5, 1 and 2%) for different durations (24, 48, 72 and 96 h). The percentage of cell viability and the cell density were calculated and the changes in astrocyte morphology were assessed by an image analysis system (Samba 2005) allowing the characterization of 5 parameters (perimeter, surface, elongation factor, convexity factor and the form factor) of a great number of cells (over 6500). This was necessary because of the high variability in normal cultured astrocyte morphology. A two-way statistical approach (2-factors ANOVA completed by stepwise discriminant analysis) was adopted to emphasize the differences between control and exposed cells. In such conditions, ethanol treated cells became more elongated, less circular and more concave and did not grow like non-exposed cells. The mean pooled values of these parameters tended to be modified as a function of the dose of ethanol. The relationships between parameters clearly separated the groups as a function of the different doses. Finally no significant difference was observed in cell viability and cell density despite lower scores in the groups exposed to the highest dose of ethanol for the longest time. Our results suggest that ethanol might affect astrocytes in two different but probably complementary ways by modifying the cell shape and by altering normal cell development.
Evidence obtained from studies on the mechanisms of action of dopaminergic neurotoxins suggests that glial cells may play an important role in neurodegenerative processes. A possible link between the function of glial cells and nerve cell damage could relate to the ability of astrocytes to convert innocuous compounds into toxic metabolites. Indeed, a mechanism of metabolic activation has been demonstrated in the MPTP (1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine) model of dopaminergic toxicity. The fully oxidized pyridinium metabolite (MPP+) is the ultimate mediator of MPTP neurotoxicity and is apparently able to damage neuronal cells after being formed within and released from astrocytes. Dopaminergic neurons are particularly vulnerable to MPTP toxicity probably because of their ability to accumulate MPP+ and to retain it for a prolonged period of time. Two pathways of MPP+ formation have been identified in astrocytes, one dependent upon the activity of monoamine oxidase (MAO) and the other related to the presence of transition metals. Tetrahydroisoquinolines (TIQs) are neurotoxins similar to MPTP in chemical structure as well as in their requirement for metabolic activation. Original data presented in this study do not support, however, a role of astrocytic MAO in the conversion of N-methyl-1,2,3,4-TIQ to the corresponding quinolinium metabolite.
This is publication number 11130-NP from The Scripps Research Institute. Research was supported by National Institutes of Health grants DA04043, DA04398, and DA08467 from the National Institute on Drug Abuse and AA06420 and AA08459 from the National Institute on Alcohol Abuse and Alcoholism. The authors would like to thank Mike Arends for his valuable assistance with manuscript preparation.
Studies of human addicts and behavioural studies in rodent models of addiction
indicate that key behavioural abnormalities associated with addiction are
extremely long lived. So, chronic drug exposure causes stable changes in the
brain at the molecular and cellular levels that underlie these behavioural
abnormalities. There has been considerable progress in identifying the mechanisms
that contribute to long-lived neural and behavioural plasticity related to
addiction, including drug-induced changes in gene transcription, in RNA and
protein processing, and in synaptic structure. Although the specific changes
identified so far are not sufficiently long lasting to account for the nearly
permanent changes in behaviour associated with addiction, recent work has
pointed to the types of mechanism that could be involved.
Aims:
Ethanol can create progressive neuropathological and functional alterations of neurones. However, the influence of exposure duration is still debated. It is difficult to specify the level of alcohol consumption leading to alcohol-induced brain damage. Moreover, the mechanism of toxicity is assumed to combine direct and metabolically induced effects, although numerous uncertainties remain. Finally, the genotoxic power of ethanol has not fully been investigated in the brain. In the experiment reported herein, primary cultures of neurones were exposed either chronically or acutely to doses of ethanol within the range of blood alcohol levels in intoxicated humans. The impact on the integrity of neurones was assessed by cytotoxicity tests and DNA alterations by single-cell gel electrophoresis (Comet assay) and flow cytometry. Chronic ethanol exposure, even at a low dose, was more harmful to neurones than acute exposure. Both significant reductions in cell viability and DNA alterations were observed in this condition. On the other hand, DNA repair capacities seemed to be preserved as long as the viability measured by specific tests was not affected. Instead, neurones entered a death cell process compatible with apoptosis.
1. Reactive oxygen species and oxidative state are slowly gaining acceptance in having a physiological relevance rather than just being the culprits in pathophysiological processes. The control of the redox environment of the cell provides for additional regulation in relation to signal transduction pathways. Conversely, aberrant regulation of oxidative state manifesting as oxidative stress can predispose a cell to adverse outcome.
2. The phosphatidylinositol 3-kinase/akt pathway is one such pathway that is partially regulated via oxidative state and, in an oxidative stress paradigm such as ischaemic–reperfusion injury, may be inactivated, which can lead to exacerbation of cell death.
3. Activation of nuclear factor (NF)-κB has been associated with oxidative stress. The role of NF-κB in neuronal cell death is widely debated, with major studies highlighting both a pro- and anti-apoptotic role for NF-κB, with the outcome being region, stimulus, dose and duration specific.
4. Oxidative state plays a key role in the regulation and control of numerous signal transduction pathways in the cell. Elucidating the mechanisms behind oxidative stress-mediated neuronal cell death is important in identifying potential putative targets for the treatment of diseases such as stroke.
The gene transcription factor cyclic adenosine monophosphate (cAMP)-responsive element binding (CREB) protein is a nuclear protein that regulates synaptic plasticity via modulating the expression of several (cAMP)-inducible genes. Alcohol addiction is a complex psychiatric disorder and is characterized by a compulsive and uncontrolled pattern of alcohol drinking by an individual in spite of the adverse consequences of its abuse. Ethanol produces both euphoric (reward and reinforcing) and dysphoric (negative withdrawal reactions) effects and these are most likely involved in the initiation and maintenance of alcohol use and abuse. Several neurotransmitter systems in the brain might be involved in the effects of alcohol but the exact molecular mechanisms of both the positive and negative affective states of alcohol abuse are still unclear. Recent research in molecular neurosciences using animal models have identified the role of extended amygdaloid (shell structures of nucleus accumbens [NAc] and central and medial amygdaloid nuclei) CREB signaling in positive and negative affective states of alcohol drinking behaviors. This review article highlights the current findings on the role of nucleus accumbal and amygdaloid CREB signaling in behavioral consequences of alcohol use and abuse.
Ethanol induces oxidative stress in cultured fetal rat cortical neurons and this is followed by apoptotic death, which can be prevented by normalization of cell content of reduced glutathione (GSH). Because astrocytes can play a central role in maintenance of neuron GSH homeostasis, the following experiments utilized cocultures of neonatal rat cortical astrocytes and fetal cortical neurons to determine if astrocytes could protect neurons from ethanol-mediated apoptotic death via this mechanism. In cortical neurons cultured in the absence of astrocytes, ethanol (2.5 and 4 mg/ml; 6-, 12-, and 24-hr exposures) decreased trypan blue exclusion and the MTT viability measures by up to 45% (P < 0.05), increased levels of reactive oxygen species (ROS) by up to 81% (P < 0.05), and decreased GSH within 1 hr of treatment by 49 and 51% for 2.5 and 4 mg/ml, respectively (P < 0.05). This was followed by onset of apoptotic cell death as determined by increased Annexin V binding and DNA fragmentation by 12 hr of ethanol exposure. Coculturing neurons with astrocytes prevented GSH depletion by 2.5 mg/ml ethanol, whereas GSH content was increased over controls in neurons exposed to 4 mg/ml ethanol (by up to 341%; P < 0.05). Ethanol generated increases in neuron ROS and apoptosis; decreases in viability were also prevented by coculture. Astrocytes were largely insensitive to ethanol, using the same measures. Only exposure to 4.0 mg/ml ethanol decreased GSH content in astrocytes, concomitant with a 204% increase in GSH efflux (P < 0.05). These studies illustrate that astrocytes can protect neurons from ethanol-mediated apoptotic death and that this may be related to maintenance of neuron GSH.
Background:
The exact enzymatic mechanisms of ethanol oxidation in the brain are still unclear. The catalase-mediated oxidation of ethanol was demonstrated in rat brain using incubation of brain homogenates with catalase inhibitors. The role of the alcohol dehydrogenase (ADH) or cytochrome P450-dependent system in this process is possible, but has not been confirmed. The objective of the study was to determine the contribution of the different enzymatic pathways to ethanol oxidation in brain homogenates from mice and rats.
Methods:
Three approaches were used to investigate the enzymatic mechanisms of ethanol oxidation in the brain of rats and mice: (1) preincubation of brain homogenates with inhibitors of the ethanol-metabolizing enzymes (catalase, CYP2E1, ADH, and ALDH); (2) utilization of mice with genetic deficiency in ethanol-metabolizing enzymes (catalase, CYP2E1, or both enzymes); and (3) determination of ethanol oxidation in brain subcellular fractions known to have differential activity of ethanol-metabolizing enzymes. The ethanol-derived acetaldehyde (AC) and acetate were determined in brain samples by gas chromatography.
Results:
The catalase inhibitors sodium azide (5 mM) and aminotriazole (5 mM) as well as CYP2E1 inhibitors diallyl sulfide (2 mM) and beta-phenethyl isothiocyanate (0.1 mM) lowered significantly the accumulation of the ethanol-derived AC and acetate in brain homogenates. The ADH inhibitor 4-methyl pyrazole (5 mM) significantly decreased the acetate but not the AC accumulation. Ethanol-derived AC accumulation in brain homogenates of acatalasemic mice was 47% of the control value, 91% in CYP2E1-null mice, and 24% in double mutants (with deficiency of both catalase and CYP2E1). The highest levels of ethanol oxidation were found in microsomal and peroxisomal subcellular brain fractions, where CYP2E1 and catalase are located, respectively.
Conclusions:
Catalase is the key enzyme of ethanol oxidation in the brain of rodents: it may be responsible for about 60% of the process. CYP2E1 plays an important role in ethanol oxidation in the rodent brains. Alcohol dehydrogenase plays a minor role, if any, in this process. Aldehyde dehydrogenase plays the crucial role in the further oxidation of ethanol-derived AC in the brain homogenates.
The role of positive vs negative ethanol reinforcement in ethanol intake of Sardinian alcohol-preferring (sP) rats is unclear.
To test the hypothesis that spontaneous ethanol self-administration of sP rats was sensitive to the opioid receptor antagonist naltrexone, whereas withdrawal-induced, but not spontaneous, ethanol self-administration would be sensitive to corticotropin-releasing factor(1) (CRF(1)) antagonists, implicating differential roles for positive and negative reinforcement, respectively.
Male sP rats operantly (FR1, 30 min/day) self-administered ethanol (10% v/v) until responding stabilized. One group (n=11) was made ethanol dependent through intermittent ethanol vapor exposure. Both nondependent (n = 10) and dependent rats received the CRF(1) antagonist LWH-63 (5, 10, and 20 mg/kg, s.c.). Separate nondependent sP rats (n = 10) received the opioid antagonist naltrexone (16, 50, 150, and 450 microg/kg, s.c.). Finally, CRF(1) antagonists (MJL-1-109-2, LWH-63, and R121919) were studied for their actions on home-cage ethanol drinking in nondependent sP rats (n = 6-8/group) under continuous, limited-access, or stressed conditions.
Naltrexone potently reduced ethanol self-administration in nondependent sP rats. LWH-63 reduced heightened ethanol self-administration of vapor-sensitive, dependent sP rats. CRF(1) antagonists did not reduce ethanol intake in nondependent sP rats. R121919 (10 mg/kg, s.c.) retained antistress activity in sP rats, blunting novelty stress-induced suppression of ethanol intake.
Spontaneous ethanol self-administration of sP rats was opioid dependent with CRF(1) receptors implicated in withdrawal-induced drinking. Opioid and CRF(1) receptors play different roles in ethanol reinforcement and perhaps the ethanol addiction cycle. Such distinctions may apply to subtypes of alcoholic patients who differ in their motivation to drink and ultimately treatment response.
We have employed rat hippocampal astrocytes in culture to investigate the effect of ethanol on reactive oxygen species (ROS) production as well as its effect on [Ca2+]c and GFAP expression. Cells were loaded with the fluorescent probes fura-2 and H2DCFDA for the determination of changes in [Ca2+]c and ROS production respectively, employing spectrofluorimetry. GFAP content was determined by immunocytochemistry and confocal scanning microscopy. Our results show ROS production in response to 50 mM ethanol, that was reduced in Ca2+-free medium (containing 0.5 mM EGTA) and in the presence of the intracellular Ca2+ chelator BAPTA (10 microM). The effect of ethanol on ROS production was significantly reduced in the presence of the alcohol dehydrogenase inhibitor 4-methylpyrazole (1 mM), and the antioxidants resveratrol (100 microM) or catalase (300 U/ml). Preincubation of astrocytes in the presence of 10 microM antimycin plus 10 microM oligomycin to inhibit mitochondria completely blocked ethanol-evoked ROS production. In addition, ethanol led to a sustained increase in [Ca2+]c that reached a constant level over the prestimulation values. Finally, incubation of astrocytes in the presence of ethanol increased the content of GFAP that was significantly reduced in the absence of extracellular Ca2+ and by resveratrol and catalase pretreatment. The data obtained in the present study suggest that astrocytes are able to metabolize ethanol, which induces two effects on intracellular homeostasis: an immediate response (Ca2+ release and ROS generation) and later changes involving GFAP expression. Both effects may underline various signaling pathways which are important for cell proliferation, differentiation and function.
Introductory textbook of psychiatry
- N C Andreasen
- G J Tucker
Andreasen NC, Tucker GJ (1991) Introductory textbook of psychiatry.
Healing codes for the biological apocalypse
- L G Horowitz
Horowitz LG (2000) Healing codes for the biological apocalypse:
Tetrahedron.