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Research Article | DOI: https://doi.org/10.31579/2766-2314/152
*Corresponding Author: E. I. Bon, Grodno State Medical University, Grodno, Belarus.
Citation: V. Lelevich, E. I. Bon, T. I. Ilyuchik, E. A.Yankovskaya, (2025), State of Tissue Respiration of Brain Regions in Chronically Alcoholic Rats Exposed to Ethanol and Succinate in in Vitro, J, Biotechnology and Bioprocessing, 6(2); DOI:10.31579/2766-2314/152
Copyright: © 2025, E. I. Bon. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Received: 05 March 2025 | Accepted: 10 March 2025 | Published: 16 March 2025
Keywords: chronic alcohol intoxication; alcohol withdrawal; rats; tissue respiration; brain homogenates
The problem of the development of alcohol dependence remains relevant due to insufficient research on the processes occurring in the brain during prolonged exposure to ethanol. The aim of the study is to assess the rate of oxygen consumption by homogenates of the cerebral cortex and cerebellum of rats under conditions of chronic alcoholization, ethanol withdrawal, as well as the influence of ethanol and succinate in vitro on it. The rate of oxygen consumption by homogenates of the cerebral cortex and cerebellum of rats receiving ethanol for 8 months was studied, as well as during the period of ethanol withdrawal on endogenous substrates, during incubation with a solution of ethanol and succinate. An increase in the rate of oxygen consumption by brain homogenates on endogenous substrates was found during chronic alcohol intoxication of rats, a decrease during the withdrawal of ethanol on the 1st and 3rd days, a stimulating effect of ethanol in the cerebral cortex on the 3rd day of abstinence, as well as a stimulating effect of succinate in the groups of control animals and with chronic alcohol intoxication. Thus, chronic alcoholization of rats leads to the development of dependence of tissue respiration on the presence of ethanol in the cell. The absence of a stimulating effect of succinate in the groups with ethanol withdrawal indicates significant activation of the succinate dehydrogenase pathway in these animals.
Alcohol has an adverse effect not only on the health of an individual, but also on social and demographic processes in society [1]. Prolonged intake of ethanol in the body leads to the formation of physical dependence, to which there is a hereditary predisposition [2]. Up to half of people with long-term alcohol use experience a withdrawal state where consumption is significantly reduced or stopped. In the most severe form, it can be life-threatening [3]. However, its pathogenesis requires further investigation [4].
According to some authors, the manifestation of clinical signs of alcohol withdrawal syndrome is based on disorders of metabolic processes in the mitochondria [5]. Chronic alcohol consumption in experimental animals causes hypoxia in various organs [6,7]. It has been shown that brief cyclical episodes of moderate hypoxia and reoxygenation in experimental animals for several days or weeks cause adaptation that protects the brain from glutamate excitotoxicity caused by ethanol withdrawal, mitochondrial damage, reduced ATP synthesis, oxidative stress, accumulation of beta-amyloid [8], and protects rat cerebellar mitochondrial cytochrome c oxidase from the stress associated with ethanol withdrawal [9], reduces alcohol consumption and withdrawal symptoms [10]. In the pathogenesis of withdrawal syndrome, the functional and biochemical aspects of this condition are the least studied. There is little literature evidence that both acute alcoholism and chronic alcoholism are accompanied by metabolic shifts in the mitochondria of nerve cells, which leads to a decrease in the intensity of energy-producing processes.
Thus, the processes that occur in brain cells during chronic alcohol intoxication (CAI) and abstinence require further study, the question remains unclear: does ethanol itself affect the cells or is it the effect of more toxic acetaldehyde?
The aim of the study was to evaluate the rate of oxygen consumption by homogenates of the cerebral cortex and cerebellum of rats under conditions of chronic alcoholism, ethanol withdrawal, as well as the effect of ethanol and succinate in on it in vitro.
Experiments were performed on 28 white outbred male rats. The experiments were conducted in accordance with the principles of experimental and clinical bioethics. The weight of the animals was 160-230 g.
CAI was modeled by the method of incomplete water deprivation [11]. The experimental group of rats consumed an ethanol solution as the only source of liquid for 8 months. The concentration of the ethanol solution during the first 2 weeks was 5%, the next 2 weeks-10%, and then until the end of the experiment – 15%. The animals were kept on dry food. The control group was kept in similar conditions and consumed water.
Alcohol withdrawal syndrome was modeled in chronically alcoholic rats (8 months) by replacing the ethanol solution with water for periods of time equal to the 1st and 3rd days.
The following experimental groups were formed: 1st-control (n=7), 2nd-CAI (n=7), 3rd – 1st day of ethanol withdrawal (n=7), 4th-3rd day of ethanol withdrawal (n=7).
After decapitation, the animals ' brains were quickly removed from them, cleaned of blood, and холодеthe cerebral cortex and cerebellum were isolated in the cold. Homogenates were prepared.
Tissue respiration was determined by the rate of oxygen consumption (SPC) by rat brain homogenates. The procedure was performed in п polarographicclosed temperature-controlled cell with a volume of 1.25 ml using a Clark electrode [12]. During the experiment, after recording the initial oxygen consumption (respiration on endogenous substrates), an ethanol solution in the final concentration of 50 mmol/l was added to the medium and changes in O2 consumption were recorded2. After that, 5 mmol/l of sodium succinate was added to the cell and the oxygen consumption stimulated by it was recorded.
The results were expressed as median (Me) and scattering (25, 75 percentiles). Nonparametric criteria and Kruskal-Wallis, U-Mann-Whitney, and TWilcoxon T-test were used to compare the values Вилкоксона. The differences were considered statistically significant at p<0>
In the homogenates of the cerebral cortex of rats with CAI, an increase in SPC was observed in comparison with the control group by 83.3%, p=0.02 (Table 1).
| Endogenous | Ethanol (50 mol/l) | Succinate (5 mmol / l) | |
Control (n=8) | 0,006 (0,0046; 0,009) | 0,0059 (0,0026; 0,0078) | 0,024 > (0,02; 0,027) |
KHAI (n=7) | 0,011 * (0,0087; 0,014) | 0,014 (0,007; 0,021) | 0,029 > (0,025; 0,036) |
1 day of ethanol withdrawal (n=7) | 0,0036 + (0,0013; 0,0065) | 0,011 (0,0087; 0,016) | 0,018 (0,005; 0,026) |
3 days of ethanol withdrawal (n=6) | 0,009+ (0,003; 0,009) | 0,013# (0,0096; 0,014) | 0,011 (0,0076; 0,012) |
Table 1: Rate of oxygen consumption by homogenates of the cerebral cortex of chronically alcoholic rats duringin in vitro incubation with ethanol solution and succinate, Iu (25 %; 75 %), ml O2 x min / g of tissue
Table 1. The rate of oxygen consumption by homogenates of the cerebral cortex of chronically alcoholized rats during in vitro incubation with ethanol solution and succinate, Me (25%; 75%), ml O2 x min / g tissue
Notes:
1 - * - statistically significant differences with the control group,
2 - + - with the KHAI group,
3 - # – with the same group, respiration on endogenous substrates,
4 - > – with the same group, incubation with ethanol solution
Withdrawal of ethanol on day 1 causes a decrease in SPC in rats compared to CAI by 67.3%, p=0.0027, and withdrawal of ethanol on day 3-by 18.2%, p=0.045.
To study the direct effects of ethanol, experiments were conducted with its addition in in vitro. It was found that ethanol increases SPC in the group of rats with ethanol withdrawal on day 3 by 44.4%, p=0.04, compared with endogenous respiration in the same group of rats. After incubation of homogenates with ethanol, stimulation of respiration with succinate leads to an increase in SPC by homogenates of the cerebral cortex of rats in control animals by 306.8% (p=0.01) and in the group with CAI – by 107.1% (p=0.01). No stimulating effect of succinate was found in the groups of rats with ethanol withdrawal.
Similar results were obtained when studying tissue respiration in cerebellar homogenates. So, in CAI, there is an increase in SEC compared to the control group by 225.0 %, p=0.002 (Table 2).
| Endogenous | Ethanol (50 mol/l) | Succinate (5 mmol / l) | |
Control (n=8) | 0,004 (0,003; 0,0057) | 0,0052 0,0052(0,004; 0,0069) | 0,021> (0,014; 0,028) |
KHAI (n=7) | 0,013* (0,011; 0,018) | 0,017 (0,0076; 0,024) | 0,031> (0,024; 0,044) |
1 day of ethanol withdrawal (n=7) | 0,0028+ (0,0015; 0,0047) | 0,007 (0,0032; 0,015) | 0,021 (0,02; 0,025) |
3 days of ethanol withdrawal (n=6) | 0,0018*+ (0,0014; 0,0023) | 0,013 (0,0061; 0,024) | 0,021 (0,015; 0,023) |
Table 2: Rate of oxygen consumption гомогенатамиby cerebellar homogenates of the brain of chronically alcoholized rats duringin in vitro incubation with ethanol solution and succinate, Iu (25 %; 75 %), ml O2 x min / g of tissue
Table 2. The rate of oxygen consumption by cerebellar homogenates of the brain of chronically alcoholized rats during in vitro incubation with ethanol solution and succinate, Me (25%; 75%), ml O2 x min / g tissue
Notes:
1 - * - statistically significant differences with the control group,
2 - + - with the KHAI group,
3 - > – with the same group, incubation with ethanol solution
Withdrawal of ethanol in rats on day 1 causes a decrease in SPC compared to CAI by 78.5%, p=0.001, on day 3 of withdrawal-by 86.2%, p=0.006. On day 3 of ethanol withdrawal, SPC decreases by 55.0% compared to the control group, p=0.049.
However, the addition of ethanol to the cerebellar homogenates did not lead to changes in the studied indicator.
When cerebellar homogenates were stimulated to breathe with succinate, an increase in SPC was also observed only in the control group by 303.8%, p=0.02, and in the group of rats with CAI by 82.4%, p=0.01. In both groups of rats with ethanol withdrawal, the stimulating effect of succinate was absent.
It can be assumed that in CAI, an increase in SPC reflects an increased need for oxygen in the nervous tissue, which indicates the existence of adaptive mechanisms that arise in response to prolonged alcohol intake in the body. This adaptive mechanism allows efficient functioning of the processes of tissue respiration, ensuring the oxygen demand of the brain in alcoholic rats. The decrease in the rate of oxygen consumption after ethanol withdrawal may be due to a decrease in the activity of tissue respiration enzymes, which is confirmed by literature data indicating a decrease in the activity of cytochrome oxidase in the cerebral cortex at 6 months of alcoholization of animals and a decrease in the activity of succinate dehydrogenase at the 12th month of alcoholization [13]. Our previous studies showed a decrease in the intensity of NADH-dependent oxidation during abstinence in rats [14].
An increase in SPC with the addition of ethanol in гомогенатахrat cortical homogenates with the elimination of ethanol on day 3 indicates that ethanol itself is a factor in increasing SPC. According to Komissarova I. A., it is the drop in the concentration of acetaldehyde after ethanol withdrawal in long-term alcoholized rats that is the main factor leading to a decrease in the activity of NADH22-dependent dehydrogenases, changes in metabolic processes in general, and leads to the development of alcohol withdrawal syndrome [15]. However, our data suggest that ethanol itself can directly affect the rate of oxygen consumption гомогенатамиby rat brain homogenates and, most likely, activate tissue enzymes. Apparently, ethanol was an activator of tissue respiration when added to гомогенатамbrain homogenates of rats with alcohol withdrawal.
Inhibition of the respiration rate after ethanol withdrawal and its pronounced increase during incubation of homogenates with ethanol indicate the need for the presence of ethanol in an increased concentration for the processes of tissue respiration in alcoholic rats. The absence of a stimulating effect of succinate in groups of animals with ethanol withdrawal and literature data indicate an increase in the activity of succinate dehydrogenase with alcohol withdrawal in rats.
During chronic alcohol intoxication in rats, an increase in the respiratory activity of homogenates of the cerebral cortex on endogenous substrates occurs.
During the period of ethanol withdrawal, oxygen utilization by rat brain homogenates is disrupted, and the rate of oxygen consumption in the cerebral cortex and cerebellum decreases on the 1st and 3rd days of abstinence.
Ethanol in in vitro does not change the rate of oxygen consumption by rat brain homogenates during chronic alcohol intoxication. In alcohol withdrawal, ethanol stimulates the rate of oxygen consumption in the cerebral cortex on the 3rd day of ethanol withdrawal.
The addition of succinate to brain homogenates increases the rate of oxygen consumption in groups of control animals and those with chronic alcohol intoxication. The absence of a stimulating effect of succinate in the groups with ethanol withdrawal indicates a significant activation of the succinate dehydrogenase pathway in these animals.
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My article, titled 'No Way Out of the Smartphone Epidemic Without Considering the Insights of Brain Research,' has been republished in the International Journal of Clinical Case Reports and Reviews. The review process was seamless and professional, with the editors being both friendly and supportive. I am deeply grateful for their efforts.
To Dear Erin Aust – Editorial Coordinator of Journal of General Medicine and Clinical Practice! I declare that I am absolutely satisfied with your work carried out with great competence in following the manuscript during the various stages from its receipt, during the revision process to the final acceptance for publication. Thank Prof. Elvira Farina
Dear Jessica, and the super professional team of the ‘Clinical Cardiology and Cardiovascular Interventions’ I am sincerely grateful to the coordinated work of the journal team for the no problem with the submission of my manuscript: “Cardiometabolic Disorders in A Pregnant Woman with Severe Preeclampsia on the Background of Morbid Obesity (Case Report).” The review process by 5 experts was fast, and the comments were professional, which made it more specific and academic, and the process of publication and presentation of the article was excellent. I recommend that my colleagues publish articles in this journal, and I am interested in further scientific cooperation. Sincerely and best wishes, Dr. Oleg Golyanovskiy.
