Научная статья на тему 'DISTURBANCES OF MITOCHONDRIAL RESPIRATION OF RAT CEREBRAL NEURONS IN TOTAL AND SUBTOTAL CEREBRAL ISCHEMIA'

DISTURBANCES OF MITOCHONDRIAL RESPIRATION OF RAT CEREBRAL NEURONS IN TOTAL AND SUBTOTAL CEREBRAL ISCHEMIA Текст научной статьи по специальности «Биотехнологии в медицине»

CC BY
0
1
i Надоели баннеры? Вы всегда можете отключить рекламу.
Ключевые слова
MITOCHONDRIAL RESPIRATION / NEURONS / CEREBRAL ISCHEMIA

Аннотация научной статьи по биотехнологиям в медицине, автор научной работы — Bon Elizaveta I., Maksimovich Natalia E., Dremza Iosiph K., Lychkovskaya Maria A.

Objective. To study the parameters of respiration of mitochondria of rat brain homogenates with its total and subtotal ischemia. Methods. The studies used models of total and subtotal cerebral ischemia. Cerebral ischemia was modeled under conditions of intravenous thiopental anesthesia (40-50 mg/kg). Total cerebral ischemia was modeled by decapitation of animals. Subtotal cerebral ischemia was modeled by simultaneous ligation of both common carotid arteries. The sampling of material for the study of tissue respiration of mitochondria was carried out 1 hour and 24 hours after decapitation or ligation.To study mitochondrial respiration, the brain was removed in the cold (0-4°C), dried with filter paper, weighed and homogenized in an isolation medium containing 0.32 M sucrose, 10 mM Tris-HCl, 1 mM EDTA, pH 7.4 (in the ratio 1:10) using a Potter-Evelheim homogenizer with a Teflon pestle according to the modified method. Results. An increase in V1 and V2 and a decrease in the phosphorylation coefficient (ADP/O) indicates proton transfer bypassing the ATP synthase complex. Enzymes of the mitochondrial matrix and cytochrome in this model of cerebral ischemia do not yet have pronounced damage, as evidenced by the high rates of V1 and V2. More pronounced disturbances with the use of succinate than with the use of malate/glutamate indicate a greater damage to the succinate dehydrogenase complex of the electron transport chain, as in the case of total cerebral ischemia. Conclusions. The most pronounced decrease in the respiration indices of the mitochondrial fraction of brain homogenates occurs in total cerebral ischemia due to the complete cessation of the blood supply to the brain neurons. With this method of modeling cerebral ischemia, the appearance of hyperchromic shriveled neurons with pericellular edema is characteristic.

i Надоели баннеры? Вы всегда можете отключить рекламу.

Похожие темы научных работ по биотехнологиям в медицине , автор научной работы — Bon Elizaveta I., Maksimovich Natalia E., Dremza Iosiph K., Lychkovskaya Maria A.

iНе можете найти то, что вам нужно? Попробуйте сервис подбора литературы.
i Надоели баннеры? Вы всегда можете отключить рекламу.

Текст научной работы на тему «DISTURBANCES OF MITOCHONDRIAL RESPIRATION OF RAT CEREBRAL NEURONS IN TOTAL AND SUBTOTAL CEREBRAL ISCHEMIA»

УДК 616.831.31-005.4.-092.913:618.33 3.3.3 Патологическая физиология

DOI: 10.37903/vsgma.2022.2.4 EDN: HANLUR

DISTURBANCES OF MITOCHONDRIAL RESPIRATION OF RAT CEREBRAL NEURONS IN TOTAL AND SUBTOTAL CEREBRAL ISCHEMIA

© Bon E.I., Maksimovich N.Ye., Dremza I.K., Lychkovskaya M.A.

Grodno State Medical University, 80, Gorkogo St., 230009, Grodno, Republic of Belarus

Abstract

Objective. To study the parameters of respiration of mitochondria of rat brain homogenates with its total and subtotal ischemia.

Methods. The studies used models of total and subtotal cerebral ischemia. Cerebral ischemia was modeled under conditions of intravenous thiopental anesthesia (40-50 mg/kg). Total cerebral ischemia was modeled by decapitation of animals. Subtotal cerebral ischemia was modeled by simultaneous ligation of both common carotid arteries. The sampling of material for the study of tissue respiration of mitochondria was carried out 1 hour and 24 hours after decapitation or ligation.To study mitochondrial respiration, the brain was removed in the cold (0-4°C), dried with filter paper, weighed and homogenized in an isolation medium containing 0.32 M sucrose, 10 mM Tris-HCl, 1 mM EDTA, pH 7.4 (in the ratio 1:10) using a Potter-Evelheim homogenizer with a Teflon pestle according to the modified method.

Results. An increase in V1 and V2 and a decrease in the phosphorylation coefficient (ADP/O) indicates proton transfer bypassing the ATP synthase complex. Enzymes of the mitochondrial matrix and cytochrome in this model of cerebral ischemia do not yet have pronounced damage, as evidenced by the high rates of V1 and V2. More pronounced disturbances with the use of succinate than with the use of malate/glutamate indicate a greater damage to the succinate dehydrogenase complex of the electron transport chain, as in the case of total cerebral ischemia.

Conclusions. The most pronounced decrease in the respiration indices of the mitochondrial fraction of brain homogenates occurs in total cerebral ischemia due to the complete cessation of the blood supply to the brain neurons. With this method of modeling cerebral ischemia, the appearance of hyperchromic shriveled neurons with pericellular edema is characteristic.

Keywords: mitochondrial respiration, neurons, cerebral ischemia

НАРУШЕНИЯ МИТОХОНДРИАЛЬНОГО ДЫХАНИЯ НЕЙРОНОВ ГОЛОВНОГО МОЗГА КРЫС ПРИ ТОТАЛЬНОЙ И СУБТОТАЛЬНОЙ ЦЕРЕБРАЛЬНОЙ ИШЕМИИ Бонь Е.И., Максимович Н.Е., Дремза И.К., Лычковская М.А.

Гродненский государственный медицинский университет, Респ. Беларусь, 230009, Гродно, ул. Горького, 80 Резюме

Цель. Изучить показатели дыхания митохондрий гомогенатов головного мозга крыс с его тотальной и субтотальной ишемией.

Методика. В иследованиях использованы модели тотальной и субтотальной ишемии головного мозга. Тотальную ишемию головного мозга моделировали путем декапитации животных. Субтотальную ишемию головного мозга моделировали путем одномоментной перевязки обеих общих сонных артерий. Забор материала для изучения тканевого дыхания митохондрий осуществляли спустя 1 час и 24 часа после декапитации или перевязки. Для исследования митохондриального дыхания головной мозг извлекали на холоде (0-4°С), осушали фильтровальной бумагой, взвешивали и гомогенизировали в среде выделения, содержащей 0,32 М сахарозы, 10 тМ Трис-HCl, 1 mМ ЭДТА, рН 7,4 (в соотношении 1:10), используя гомогенизатор Поттера-Эвельгейма с тефлоновым пестиком согласно модифицированному методу.

Результаты. Увеличение показателей У1 и У2 и уменьшение коэффициента фосфорилирования (АДФ/О) свидетельствует о переносе протонов, минуя АТФ-синтазный комлекс. Ферменты митохондриального матрикса и цитохромы при данной модели ишемии головного мозга еще не имеют ярко выраженных повреждений, о чем свидетельствуют высокие скорости У1 и У2, однако уменьшение коэффициентов акцепторного контроля, дыхательного контроля и фосфорилирования

указывает на разобщениие процессов окисления и фосфорилирования и снижение наработки АТФ при субтотальной ишемии головного мозга. Более выраженные нарушения при использовании сукцината, чем при использовании малата/глутамата свидетельствуют о большем повреждении сукцинатдегидрогеназного комплекса цепи переноса электронов, как и при тотальной ишемии головного мозга.

Заключение. Наиболее выраженное уменьшение показателей дыхания митохондриальной фракции гомогенатов головного мозга происходит при тотальной ишемии головного мозга вследствие полного прекращения кровоснабжения нейронов головного мозга. При данном способе моделирования церебральной ишемии характерно появление гиперхромных сморщенных нейронов с перицеллюлярным отеком.

Ключевые слова: митохондриальное дыхание, нейроны, ишемия головного мозга

Introduction

Energy exchange in the cell is associated with mitochondria, which play an important role in vital processes, participating not only in the formation of ATP, but also in the storage and transmission of hereditary information, apoptosis and plastic processes [3, 11, 12].

Mitochondria are very mobile and plastic organelles that constantly change their shape, have the ability to fusion and subsequent separation. The movement of mitochondria in the cytoplasm is associated with microtubules, which determines their orientation and distribution in the cell. In some cells, mitochondria form long mobile filaments or chains, while in others they are fixed near the places of consumption of ATP [5, 6, 10].

Neurons need a constant supply of ATP for their stability and maintaining the level of potassium ions K + inside the cell, and sodium and calcium ions outside. At rest, the brain consumes up to 20% of the oxygen received by the body. Under normal conditions, effective biological oxidation is the main source of energy-rich phosphate compounds required for the renewal of structures corresponding to the functional activity of cells [5, 9, 10].

Elucidation of the mechanisms of development of energy deficiency in ischemic damage is advisable for detailing the pathogenesis, the ratio of damage and compensation processes in this pathology.

Methods

The experiments were carried out on 88 male outbred white rats weighing 260 ± 20 g in compliance with the Directive of the European Parliament and of the Council No. 2010/63/EU of 22.09.2010 on the protection of animals used for scientific purposes. The studies used models of total (TCI) and subtotal (SCI) cerebral ischemia.

Cerebral ischemia (CI) was modeled under conditions of intravenous thiopental anesthesia (40-50 mg/kg). Total cerebral ischemia was modeled by decapitation of animals. Subtotal cerebral ischemia was modeled by simultaneous ligation of both common carotid arteries (CCA). The sampling of material for the study of tissue respiration of mitochondria was carried out 1 hour and 24 hours after decapitation or ligation of the CCA. The control group consisted of sham-operated rats of the same sex and weight.

To study mitochondrial respiration, the brain was removed in the cold (0-4°C), dried with filter paper, weighed and homogenized in an isolation medium containing 0.32 M sucrose, 10 mM Tris-HCl, 1 mM EDTA, pH 7.4 (in the ratio 1:10) using a Potter-Evelheim homogenizer with a Teflon pestle according to the modified method [1,17].

Mitochondria were isolated by differential centrifugation. The nuclear fraction was separated by centrifugation at 600 g for 10 min (4°C). The resulting supernatant was centrifuged at 8500 g for 10 min (4°C), the mitochondrial pellet was washed twice in the isolation medium and resuspended to a protein concentration of 35-40 mg/ml in the isolation medium and stored in a short tube on ice. Protein concentration was determined by the Lowry method.

To study mitochondrial respiration, a concentrated suspension of mitochondria was introduced into a thermostated sealed polarographic cell with an incubation medium in an amount providing a final protein concentration in the cell of 1 mg/ml. The incubation medium for recording mitochondrial

respiration includes 0.17 M sucrose, 40 mM KCl, 10 mM Tris-HCl, 5 mM KH2PO4, 8 mM KHCO3, 0.1 mM EDTA, pH 7.4.

The principle of operation of a 3.0 ml polarographic cell is based on the registration of oxygen uptake by mitochondria using a built-in Clarke electrode at a temperature of 25°C (Figure 1).

Figure 1. Polarographic cell for studying the respiratory activity of mitochondria. 1 - cell; 2 -thermostatically controlled chamber; 3 - Clarke electrode; 4 - magnetic stirrer; 5 - sealing plug; 6 -channels for dosed anaerobic administration of substrates and ADP; 7 - a sealing ring; 8 - channel for removing air and excess liquid; 9 - fitting for connection to an ultrathermostat.

Registration of changes in oxygen tension (pO2) in the mitochondrial suspension was carried out using an electronic recorder KSP-4. The Clarke electrode was calibrated by successively blowing air (pO2 of air) and gaseous nitrogen (pO2=0 mm Hg) through the cell. After recording the rate of basal (endogenous) respiration in the absence of a substrate (V1), respiration substrates (malate - 2 mM/glutamate - 5 mM or succinate - 5 mM) are alternately introduced into the mitochondrial suspension, and then ADP in an amount of 200 nmol/ml. The obtained polarograms are used to calculate the respiration rate of mitochondria in different metabolic states and the coefficients characterizing the conjugation of oxidation and phosphorylation processes.

The following indicators of mitochondrial respiration were recorded: V1 - the rate of basal respiration, V2 - the rate of substrate-dependent respiration, V3 - the rate of respiration coupled with phosphorylation (after the addition of ADP), V4 - the rate of respiration after the completion of phosphorylation of the added ADP. The indicators characterizing the conjugation of oxidation and phosphorylation processes in mitochondria were determined: the acceptor control coefficient (AK=V3/V2), the respiratory control coefficient (DC=V3/V4) and the phosphorylation coefficient -

The use of solutions of substrates of the complex "malate/glutamate" and succinate makes it possible to assess the degree of functional activity of the electron transfer chain (ETC) in mitochondria in general, in particular - I and II of the ETC complex [7, 8].

To prevent systematic measurement errors, brain samples from the compared control and experimental groups of animals were studied under the same conditions. As a result of research, quantitative continuous data were obtained. Since the experiment used small samples that had an abnormal distribution, the analysis was carried out by methods of nonparametric statistics using the licensed computer program Statistica 10.0 for Windows (StatSoft, Inc., USA). Data are presented as Me (LQ; UQ), where Me is the median, LQ is the value of the lower quartile; UQ is the upper quartile value. Differences between groups were considered significant at p<0.05 (Kruskell-Wallis test with Bonferoni's correction) [2].

Results and their discussion

Compared with the control, at 1-hour TCI in the presence of the substrate "malate/glutamate", which characterizes the state of the first (NADH-dehydrogenase) complex, the electron transport chain V1

H

6

iНе можете найти то, что вам нужно? Попробуйте сервис подбора литературы.

ADP/O.

decreased by 65 (58; 67)%, p<0.05, V2 - by 41 (38; 48)%, p<0.05, V3 - by 25 (22; 38)%, p<0.05, and the phosphorylation coefficient - by 78 (71; 84)%, p<0,05. The rest of the indicators (V4, the coefficient of acceptor control and the coefficient of respiratory control) did not change (p>0.05), table 1.

Table 1. Respiration ischemia when using

indices of the mitochondrial fraction of rat brain homogenates with total cerebral the substrates "malate/glutamate" and "succinate", Me (LQ; UQ)

Groups V1 (ng at O/min x mg protein) V2 (ng at O/min x mg protein) V3 (ng at O/min x mg protein) V4 (ng at O/min x mg protein) (V3/V2) acceptor control coefficient (V3/V4) respiratory control coefficient (ADP/O) phosphorylation coefficient

Malate/glutamate substrate

Control 18 (14;19) 27 (26;27) 51 (48;56) 31 (27;34) 2,0 (1,8,3,0) 1,6 (1,6;1,7) 2,0 (1,9;2,1)

TCI 1 hour 6* (4;9) 16* (11;19) 38* (32;39) 26 (18;30) 2,3 (1,7;3,5) 1,6 (1,5;1,8) 0,5* (0,4;0,6)

TCI 1 day 2* (2;5) 14* (11;19) 28* (18;36) 22 (18;27) 1,8 (1,7;1,9) 1,0*+ (1,0;1,2) 0,0* (0,0;0,0)

Substrate "succinate"

Control 17 (15;17) 34 (28;36) 66 (65;68) 38 (36;40) 2,0 (1,9;2,3) 1,8 (1,7;1,9) 1,9 (1,8;1,9)

TCI 1 hour 9* (6;12) 14* (9;17) 26* (15;32) 26* (15;30) 1,6 (1,2;1,9) 1,0* (1,0;1,0) 0,0* (0,0;0,0)

TCI 1 day 2*+ (1;3) 12* (9;14) 13* (11;16) 13* (11;16) 1,2* (1,1;1,3) 1,0* (1,0;1,0) 0,0* (0,0;0,0)

Note:* - p<0.05 compared with the control group, + - p<0.05 compared with 1 -hour TCI, TCI - total cerebral ischemia

In the presence of succinate substrate, which reflects the work of complex II (succinate dehydrogenase) of the electron transport chain, a decrease in the parameters of energy exchange was established: the rate of basal respiration V1 - by 44 (38; 52)%, p<0.05, the rate of substrate-dependent respiration V2 - by 60 (48; 64)%, p<0.05, respiration rate associated with V3 phosphorylation - by 59 (38; 65)%, p<0.05, respiration rate after completion of phosphorylation of added V4 ADP - by 32 (28; 46)%, p<0.05.

The respiratory control coefficient (V3/V4) decreased by 45 (42; 48)% (p<0.05), the phosphorylation coefficient (ADp/O) at 1-hour CTI was zero. The acceptor control coefficient (V3/V2) did not change (p>0.05). The decrease in the rate of basal respiration Vl was more pronounced when using the substrate succinate (by 21%, p<0.05), which indicates a greater damage to the II complex (succinate dehydrogenase) of the electron transport chain (ETC) during TCI. There were no differences between other indicators (p>0.05).

Under conditions of 1-day TCI in the presence of the substrate "malate/glutamate" at 1-day TCI, Vl decreased by 90 (82; 96)%, p<0.05, V2 - by 46 (42; 55)%, p<0,05, and V3 - by 45 (39; 56)% (p<0.05), the respiratory control coefficient - by 35 (31; 42)% (p<0.05). Indicators V4 and the coefficient of acceptor control did not change (p>0.05). The phosphorylation coefficient (ADP/O) when using both succinate and malate/glutamate with 1-day TCI, as with 1-hour TCI, was equal to zero. Compared to 1hour TCI, the respiratory control coefficient with 1-day TCI was 41 (35; 47)% less (p<0.05). There were no differences in other indicators (V2, V3, V4, acceptor coefficient and phosphorylation coefficient) (p>0.05), table 2.

In the presence of succinate, there was a more significant decrease in V1 than with 1-hour TCI - by 90 (84; 95)%, p<0.05, V2 - by 65 (54; 70)%, p<0.05, V3 - by 78 (54; 87)%, p<0.05, V4 - by 67 (59; 81)%, p<0.05. The respiratory control coefficient decreased by 45 (41; 49)%, p<0.05. Compared with 1-hour TCI, the V1 index was 80 (75; 86)% less (p<0.05) Changes in mitochondrial respiration in relation to the control level when using both substrates were equivalent (p>0.05).

In the SCI group lasting 1 hour, compared with the control group, in the presence of malate/glutamate, V2 increased by 24 (18; 27)%, p<0.05, and the acceptor control coefficient and phosphorylation coefficient decreased by 25 ( 17; 29)%, p<0.05. Other indicators (V1, V3, V4, respiratory control coefficient) did not change (p>0.05). In the presence of the substrate "malate/glutamate" at 1-hour SCI, the mitochondrial respiration indices V1, V2, V3 and V4 were 89 (82; 93)% higher than at 1-hour TCI, p<0.05, 58 (55; 63)%, p<0.05, 24 (21; 29)%, p<0.05 and 32 (27; 37)%, p<0.05, respectively. The respiratory control coefficient did not change (p>0.05), while the acceptor control coefficient was 34 (24; 43)% less, p<0.05,

and the phosphorylation coefficient was 66 (58; 73)% more, p<0.05. The decrease in the phosphorylation coefficient with SCI was less significant - by 53% (p<0.05).

Table 2. Respiration indices of the mitochondrial fraction of rat brain homogenates in subtotal cerebral ischemia when using the substrates "malate/glutamate" and "succinate", Me (LQ; UQ)_

Groups V1 (ng at O/min x mg protein) V2 (ng at O/min x mg protein) V3 (ng at O/min x mg protein) V4 (ng at O/min x mg protein) (V3/V2) acceptor control coefficient (V3/V4) respiratory control coefficient (ADP/O) phosphory lation coefficient

Malate/glutamate substrate

Control 18 27 51 31 2,0 1,6 2,0

(14;19) (26;27) (48;56) (27;34) (1,8,3,0) (1,6;1,7) (1,9;2,1)

SCI 1 18 36* 50 32 1,5* 1,4 1,5*

hour (18;27) (35;38) (48;51) (30;37) (1,3;1,5) (1,3;1,6) (1,4;1,5)

SCI 1 day 6*+ 14*+ 27*+ 21*+ 1,1* 1,4* 1,4*

(1;6) (6;24) (8;42) (7;22) (0,9;1,3) (1,2;1,5) (0,6;1,6)

Substrate "succinate"

Control 17 34 66 38 2,0 1,8 1,9

(15;17) (28;36) (65;68) (36;40) (1,9;2,1) (1,7;1,9) (1,8;1,9)

SCI 1 27* 39* 50* 40 1,3* 1,4* 1,2*

hour (19;27) (37;42) (48;54) (37;41) (1,2;1,3) (1,2;1,4) (1,1;1,2)

SCI 1 day 9*+ (3;14) 13*+ (10;20) 23*+ (20;39) 23 (20;37) 1,3* (1,2;1,3) 1,0* (1,0;1,5) 0,0*+ (0,0;0,2)

Note:* - p<0.05 - in relation to the control level, + - p<0.05 compared to 1 -hour SCI, SCI - subtotal cerebral ischemia

In the presence of succinate substrate, an increase in the rate of basal respiration V1 was noted - by 38 (34; 42)%, p<0.05, the rate of substrate-dependent respiration V2 - by 13 (9; 18)%, p<0.05, rate respiration associated with V3 phosphorylation - by 26 (21; 32)%, p<0.05.

These changes indicate a significant decoupling of oxidation and phosphorylation. The respiration rate after the completion of phosphorylation of the added ADP (V4) did not change (p>0.05). At the same time, the acceptor control coefficient, the respiratory control coefficient and the phosphorylation coefficient decreased by 35 (31; 39)%, p<0.05, 20 (18; 28)%, p<0.05 and by 36 (30; 41)% , p<0.05, respectively, which indicates a decrease in energy production. Compared with 1-hour TCI, with 1-hour SCI in the presence of the substrate "succinate" the rates V1, V2, V3 and V4 were 67 (62; 71)% higher, p<0.05, 64 (58; 68) %, p<0.05, 46 (39; 52)%, p<0.05 and 35 (31; 41)%, p<0.05, respectively. The respiratory control coefficient increased by 30 (24; 36)%, p<0.05. The phosphorylation coefficient at 1hour TCI was zero. When using succinate, the decrease in the respiratory control coefficient was less pronounced with SCI (by 10%, p<0.05).

When using succinate, when using malate/glutamate, in relation to the control level, the phosphorylation coefficient was lower by 11% (p<0.05). Other indicators (V1, V3, V4, respiratory control coefficient, acceptor control coefficient) did not differ (p>0.05). An increase in V1 and V2 and a decrease in the phosphorylation coefficient (ADP/O) indicates proton transfer bypassing the ATP synthase complex. Enzymes of the mitochondrial matrix and cytochrome in this model of CI do not yet have pronounced damage, as evidenced by the high rates of V1 and V2, however, a decrease in the coefficients of acceptor control, respiratory control and phosphorylation indicates the separation of oxidation and phosphorylation processes and a decrease in the production of ATP during SCI. More pronounced disturbances with the use of succinate than with the use of malate/glutamate indicate a greater damage to the succinate dehydrogenase complex of ETC, as in TCI.

In the presence of the substrate "malate/glutamate" at 1-day SCI, compared with 1-day TCI, V1 decreased by 66 (60; 71)%, p<0.05, V2 - by 45 (41; 50)%, p<0.05, V3 - by 47 (39; 52)%, p<0.05, V4 - by 34 (27; 39)%, p<0.05, which is more significant than with 1-hour SCI by 87 (72; 94)%, p<0.05, 61 (58; 73)%, p<0.05 and by 46 (41; 52)%, p<0.05, respectively, except for the value of the V4 indicator, which did not change, p>0.05. The coefficients of acceptor control, respiratory control and phosphorylation decreased by 42 (37; 51)%, p<0.0; by 12 (9; 18)%, p<0.05 and by 25 (21; 32)%, p<0.05, respectively. In the presence of the "malate/glutamate" substrate, the basal respiration rate V1 was 67% higher (p<0.05), the acceptor control coefficient was 39% lower (p<0.05), and the phosphorylation coefficient at 1-day TCI was is zero.

iНе можете найти то, что вам нужно? Попробуйте сервис подбора литературы.

Under the conditions of daily SCI in the presence of succinate substrate, a decrease in V1 was noted - by 47 (39; 51)%, p<0.05, V2 - by 62 (54; 66)%, p<0.05, V3 - by 64 (59 ; 68)%, p<0.05, which is more

pronounced than with 1-hour SCI by 67 (62; 72)%, p<0.05; by 66 (63; 74)%, p<0.05 and by 55 (49; 59)%, p<0.05, respectively. The acceptor control coefficient and the respiratory control coefficient decreased by 35 (29; 41)%, p<0.05 and by 44 (38; 49)%, p<0.05, respectively. The phosphorylation coefficient with 1-day SCl, as well as with 1-day TCI, was equal to zero. Compared with TCl lasting 1 day, with 1-day SCI in the presence of succinate substrate, the V1 rate was 78% higher (p<0.05), and the V3 and V4 rates were higher by 43% (p<0.05). while other parameters did not change (p>0.05).

The use of a mixture of malate with glutamate as a substrate for 1-day SCI showed similar changes in mitochondrial respiration parameters as with succinate, with the exception of a higher phosphorylation coefficient - 1.4 (0.6; 1.6), p<0,05. The decrease in the V1, V2, and V3 indices with 1-day SCI is a consequence of a decrease in the oxygen content for mitochondrial respiration. The suppression of energy processes was more pronounced than with 1-hour SCI, which reflects an extremely low phosphorylation coefficient.

Changes in V1, V2 and V3 indicators at 1-hour SCI and 1-hour TCI were multidirectional. Their increase in SCI is associated with the uncoupling of oxidation and phosphorylation, while a decrease in TCI is associated with a lack of substrates for mitochondrial respiration. The decrease in the rate of basal respiration V1 with 1-day SCI was less pronounced than with TCI: in the presence of succinate - by 43% (p<0.05), and in the presence of malate/glutamate - by 24% (p<0.05).

Conclusions

During cerebral ischemia, damage to the inner mitochondrial membrane occurs due to the activation of free radical oxidation processes [5]. Damage to the inner mitochondrial membrane, in turn, leads to an increase in its permeability and a decrease in the level of the proton gradient due to the transition of protons along the concentration gradient through the formed nonspecific pores into the mitochondrial matrix [13, 19, 20]. As a result, the efficiency of ATP synthesis decreases, and to maintain the intermembrane potential under these conditions, more substrates and oxygen are required [14-16, 18].

Thus, the most pronounced decrease in the respiration indices of the mitochondrial fraction of brain homogenates occurs in total cerebral ischemia due to the complete cessation of the blood supply to the brain neurons. With this method of modeling cerebral ischemia, the appearance of hyperchromic shriveled neurons with pericellular edema is characteristic. In their cytoplasm, the destruction of organelles occurs, the disintegration of neurofibrils and neuropil, which indicates their low functional activity. Simultaneous subtotal ischemia also leads to severe irreversible damage to neurons: at the morphological level, this is manifested by a significant increase in the number of hyperchromic shriveled neurons. Their predominance in the population of neurons in the SCI group corresponds to the inhibition of respiration of the mitochondrial fraction of brain homogenates.

References

1. Барковский Е.В. Современные проблемы биохимии. Методы исследований // Минск: Вышэйшая школа -2013. - 491 c. [Barkovski E.V. Minsk: Vysheyshaya shkola. Minsk: High School. - 2013. - P. 491. (in Russian)]

2. Батин Н. В. Компьютерный статистический анализ данных : учебно-методическое пособие // Минск : Институт подготовки научных кадров НАН Беларуси. - 2008. - 160 с. [Batin N. V. Minsk : Inctitut podgotovki nauchnykh kadrov NAN Belarusi. Minsk: Institute for the Training of Scientific Personnel of the National Academy of Sciences of Belarus. - 2008. - P. 160. (in Russian)]

3. Бонь Е.И. Роль митохондрий в энергетике клетки и характеризующие ее молекулярные маркеры // Оренбургский медицинский вестник. - 2019. - N1. - С. 47-52. [Bon E. I. Orenburgskiy meditsinskiy vestnik. Orenburg medical bulletin. - 2019. - N1. - P. 47-52. (in Russian)]

4. Бонь Е. И. Способы моделирования и морфофункциональные маркеры ишемии головного мозга // Биомедицина. - 2018. - N2. - С. 59-71. [Bon E.I. Biomedicina. Biomedicine. - 2018. - N2. - P. 59-71. (in Russian)]

5. Максимович Н. Е. Головной мозг крысы и его реакция на ишемию: монография // Гродно : ГрГМУ, 2020. - 240 с. [Maksimovich N. E. Grodno: GrSMU. Journal of GrSMU. - 2020. - P. 240. (in Russian)]

6. Baertling F. NDUFA9 point mutations cause a variable mitochondrial complex I assembly defect // Journal of Clinical Genetics. - 2018. - N93. - P. 111-118.

7. Boumans H., Grivell L.A., Berden J.A. The respiratory chain in yeast behaves as a single functional unit // Journal of Biology Chemistry. - 1998. - N273. - P. 4872-4877.

8. Brand M.D., Murphy M.P. Control of electron flux through the respiratory chain in mitochondria and cells // Journal of Biological Reviews. - 1987. - N62. - P. 141-193.

9. Casey R.P. Membrane reconstruction of the energy-conserving enzymes of oxidative phosphorylation // Journal of Biochemistry Acta. - 1984. - N768. - P. 319-347.

10. Chalmers, G. R. Adaptability of the oxidative capacity of motoneurons / G. R. Chalmers, R. R., Roy, V. R. Edgerton // Journal of Brain Reseaarch - 1992. - N570. - P.1-10.

11. DePierre J.W., Ernster L. Enzyme topology of intracellular membranes // Journal of Review Biochemistry. -1988. - N46. - P. 201-261.

12. Hackenbrock C.R. Lateral diffusion and electron transfer in the mitochondrial inner membrane // Journal of Trends Biochemistry. - 1981. - N15. - P. 151-154.

13. Hoffmann C. The effect of differentiation and TGFB on mitochondrial respiration and mitochondrial enzyme abundance in cultured primary human skeletal muscle cells // Journal of Science Report. -2018. - N8. - P. 737740.

14. Holvoet P. Low Cytochrome Oxidase 1 Links Mitochondrial Dysfunction to Atherosclerosis in Mice and Pigs // Journal of PLoS One. - 2017. - N12. - P. 307-312.

15. Klinyerberg M. Principles of carrier catalysis elucidated by comparing two similar membrane translocators from mitochondria, the ADP/ATP carrier and the uncoupling protein // Journal of New York Academic Science. -1985. - N456. - P. 279-288.

16. Leonard K., Haiker H., Weiss H. Three-dimensional structure of NADH: ubiquinone reductase (complex I) from Neurospora mitochondria determined by electron microscopy of membrane crystals // Journal of Molecular Biology. - 1987. - N194. - P. 277-286.

17. Maksimovich N.Ye. Structural and functional features of mitochondria and methods of their study in experiment // MEDICUS. - 2019. - N5. - P. 8-18.

18. Prince R.C. The proton pump of cytochrome oxidase // Journal of Trends Biochemistry Science. -1988. - N13. -P. 159-160.

19. Slater E.C. The Q Cycle, an ubiquitous mechanism of electron transfer // Journal of Trends Biochemistry Science. - 1983. - N8. - P. 239-242.

20. Srere P.A. The structure of the mitochondrial inner membrane-matrix compartment // Journal of Trends Biochemistry Science. - 1982. - N7. - P. 375-378.

Информация об авторах

Бонь Елизавета Игоревна - кандидат биологических наук, доцент, доцент кафедры патологической физиологии им. Д.А. Маслакова УО «Гродненский государственный медицинский университет», Респ. Беларусь. E-mail: asphodela@list.ru

Максимович Наталия Евгеньевна - доктор медицинских наук, профессор, заведующий кафедрой патологической физиологии им. Д.А. Маслакова УО «Гродненский государственный университет», Респ. Беларусь. E-mail: mne@grsmu.by

Дремза Иосиф Карлович - кандитат биологических наук, доцент, доцент кафедры патологической физиологии им. Д.А. Маслакова УО «Гродненский государственный университет», Респ. Беларусь. E-mail: idremza@rambler.ru

Лычковская Мария Александровна - студентка 5 курса педиатрического факультета Гродненского государственного медицинского университета, Респ. Беларусь. E-mail: lychkovskaya.m@gmail.com

Information about the authors

Bon Elizaveta I. - Associate Professor of the Department of Pathological Physiology named D.A. Maslakov Grodno State Medical University, Republic Belarus. E-mail: asphodela@list.ru

Maksimovich Natalia E. - Doctor of Medical Sciences, Professor, Head of the Department of Pathological Physiology named after D.A. Maslakova Grodno State University, Republic of Belarus. E-mail: mne@grsmu.by

Dremza Iosiph K. - Candidate of Biological Sciences, Associate Professor, Associate Professor of the Department of Pathological Physiology. D.A. Maslakov Grodno State Medical University, Rep. Belarus. E-mail: idremza@rambler.ru

Lychkovskaya Maria A. - A fourth-year student of the group number 7 of faculty of Pediatrics Medicine Grodno State Medical University, Rep.Belarus. E-mail: lychkovskaya.m@gmail.com

Конфликт интересов: авторы заявляют об отсутствии конфликта интересов.

i Надоели баннеры? Вы всегда можете отключить рекламу.