Research ArticleAbstract
Objective: Evaluation of changes in the content of ATP synthase in the parietal
cortex and hippocampus of the brain of rats with ischemia of varying severity in a
comparative aspect.
Methods: The experiments were performed on 88 male outbred white rats weighing
260 ± 20 g. Brain ischemia was modeled under conditions of intravenous thiopental
anesthesia (40-50 mg / kg). Total cerebral ischemia was modeled by decapitation of
animals. The brain sampling was carried out 1 hour and 24 hours after decapitation - to
study tissue respiration of mitochondria, as well as 1 hour later to determine the content
of ATP synthase. Subtotal cerebral ischemia was modeled by simultaneous ligation
of both common carotid arteries. The material was taken after 1 hour to determine
the content of ATP synthase. Stepwise subtotal cerebral ischemia was performed by
sequential ligation of both common carotid arteries with an interval of 7 days. The
sampling was carried out 1 hour after ligation of the second common carotid artery in
each of the subgroups. Partial cerebral ischemia was modeled by ligation of one common
carotid artery on the right. The sampling was carried out 1 hour after the operation.
Determination of the content of ATP synthase was carried out by immunohistochemical
method using monoclonal antibodies. For this purpose, after decapitation, the brain was
quickly removed from the rats, pieces of the cerebral cortex were fixed in zinc-ethanolformaldehyde
at + 4 ° C (overnight), then embedded in paraffin.
Results: In the group of stepwise subtotal cerebral ischemia, the smallest decrease
in the content of ATP synthase was observed in the 1st subgroup with an interval
between dressings of 7 days, while the greatest decrease in the content of the enzyme
was noted in the 3rd subgroup with the minimum interval between the dressings of the
common carotid artery (1 day). Modeling of more severe types of ischemic damage led to
pronounced morphological changes in neurons in the parietal cortex and hippocampus
of the rat brain - a decrease in their size, deformation of the perikarya, an increase in the
degree of neuronal chromatophilia with their simultaneous wrinkling and subsequent
death. These disorders were most pronounced in the 3rd subgroup of stepwise subtotal cerebral ischemia with the shortest interval between dressings, which was 1 day, and in
the group of total cerebral ischemia.
Conclusion: Thus, the most pronounced decrease in the content of ATP synthase
was observed in the groups of total cerebral ischemia, subtotal cerebral ischemia and in
the 3rd subgroup of stepwise subtotal cerebral ischemia, with a minimal time interval
between the ligation of the common carotid artery. In stepwise subtotal cerebral
ischemia with an interval between ligation of the common carotid artery of 7 days, the
suppression of the ATP synthase content was not so significant.
Keywords: ATP-Synthase; Cerebral Ischemia; Neurons
Introduction
In cerebral ischemia (CI), a chain of pathogenetic disorders develops in its structures, among which one of the leading is energy deficiency, which leads to the development of cellular pathology [1,2]. The work of enzymes, including sodium-potassium ATPase, is disrupted, leading to an imbalance of ions and cerebral edema [3-6]. A number of molecular markers of the energy activity of mitochondria are known, among which one of the main markers is ATP synthase - an integral protein of the inner mitochondrial membrane that carries out the reaction of ATP formation from ADP [7,8]. Mitochondrial ATP synthase plays an important role in the differentiation of stem cells, promotes the formation of mitochondrial cristae by dimerization and specific regulation [9- 11]. The enzyme belongs to the alpha / beta ATP synthase family. Consists of two structural domains (F1 - extramembrane catalyst and F0 - membrane proton channel), connected by a central rod consisting of γ, δ and ε subunits [12-15], and together with the oligomer of the membrane subunit representing the rotary domain of the enzyme [1,4,7,16,17].Elucidation of the mechanisms of the development of energy deficiency in ischemic damage of varying severity is advisable for detailing the pathogenesis, the ratio of damage and compensation processes in СШ. The aim of the study was to assess changes in the content of ATP synthase in the parietal cortex and hippocampus of the brain of rats with ischemia of varying severity in a comparative aspect. For this purpose, the changes in the content of ATP synthase in rats with partial, subtotal, stepwise subtotal and total СI were studied [3].
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.CI was modeled
under conditions of intravenous thiopental anesthesia (40-50 mg/
kg). Total cerebral ischemia (TCI) was modeled by decapitation of
animals. The brain sampling was carried out 1 hour and 24 hours
after decapitation to study tissue respiration of mitochondria, and
also after 1 hour to determine the content of ATP synthase. Subtotal
cerebral ischemia (SCI) was modeled by simultaneous ligation of
both common carotid arteries (CCA). The material was taken after 1
hour to determine the content of ATP synthase. Graduated subtotal
CI (GSCI) was performed by sequential ligation of both CCA with
an interval of 7 days (subgroup 1), 3 days (subgroup 2), or 1 day
(subgroup 3). The sampling of material was carried out 1 hour after
ligation of the second CCA in each of the subgroups. Partial cerebral
ischemia (PCI) was modeled by ligating one CCA on the right. The
sampling of material was carried out 1 hour after the operation
[3,18].
Determination of the content of ATP synthase was carried out
by immunohistochemical method using monoclonal antibodies.
For this purpose, after decapitation, the brain was quickly removed
from the rats, pieces of the cerebral cortex were fixed in zincethanol-
formaldehyde at + 4°C (overnight), then embedded in
paraffin. Paraffin sections with a thickness of 5 μm were prepared
using a microtome and mounted on glass slides. The preparations
were processed according to the protocol of immunocytochemical
reaction for light microscopy, excluding the procedure of thermal
unmasking of antigens. To determine the immunoreactivity of the
molecular marker of mitochondria ATP synthase (complex V that
forms ATP from ADP), primary monoclonal mouse antibodies (Anti-
ATP5A antibody, Abcam, UK, ab. 14748) were used at a dilution of
1: 2400 at + 4°C, with an exposure of 20 h in a humid chamber.
To detect bound primary antibodies, an EXPOSE Mouse and
Rabbit specific HRP/DAB detection IHC kit Abcam (Great Britain,
ab. 80436) was used. The immunoreactivity of ATP synthase
was studied in the cytoplasm of neurons of the fifth layer of the
parietal cortex and neurons of the CA1 field of the hippocampus
in immunohistochemical preparations based on the optical density
of the chromogen sediment using an Axioscop 2 plus microscope
(Zeiss, Germany), a digital video camera (Leica DFC 320, Germany)
and an image analysis program ImageWarp (Bitflow, USA).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. In connection with the use of small samples with an
abnormal distribution in the experiment, the processing was carried
out by the 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) [19].
Results
In TCI, a decrease in the content of ATP synthase, which characterizes the state of the V-complex of the electron transport chain, was noted in comparison with its level in the control group - by 74 (66; 86) % in the parietal cortex, p<0.05 and by 70 (54; 81) % - in the hippocampus, p <0.05 (Table 1, Figures 1 & 2). At the same time, there were no differences in the content of the enzyme in the parietal cortex and hippocampus (p>0.05).Compared with the value in the “control”, in the SCI group, the content of ATP synthase decreased by 45 (38; 53)% - in the parietal cortex (p <0.05) and by 39 (31; 46)% - in the hippocampus (p<0.05).Compared to the level of the value in the “TCI” group, the content of ATP synthase in rats with SCI was higher in the parietal cortex by 57 (46; 61)%, p<0.05 and by 38 (32; 43)% - in the hippocampus, p<0.05.The decrease in the content of ATP synthase in rats with SIGM was less pronounced than in rats with TCI - by 29% in the parietal cortex (p <0.05) and by 39% in the hippocampus (p <0.05).With PCI, there was a decrease in the content of ATP synthase, compared with the value in the control group - in the parietal cortex by 21 (13; 32)%, p <0.05, in the hippocampus by 8 (4; 13)%, p <0 , 05.
Table 1: The content of ATP synthase in the cytoplasm of neurons in the parietal cortex and field CA1 of the hippocampus of rats with total, subtotal and partial cerebralsischemia,sMes(LQ;sUQ).
Note: * - p <0.05 compared with the “control” group, # - p<0.05 compared with the “TCI” group, + - p<0.05 compared with the “SCI”
group, α - p<0.05 compared with, compared with the 1st subgroup of SCI,
β - p<0.05 in comparison with the 2nd subgroup of GSCI. TCI - total cerebral ischemia, SCI - subtotal cerebral ischemia, PCI - partial
cerebral ischemia, SSCI - stepwise subtotal cerebral ischemia, sg – subgroup
Figure 1: The content of ATP synthase in the cytoplasm of pyramidal neurons in the parietal cortex of rats with total (TCI),
subtotal (SCI), stepwise subtotal (GSCI) and partial cerebral ischemia (PCI). Digital micrograph. Magnification x 40.
Note:
• Subgroup 1 - the interval between CCA dressings 7 days
• Subgroup 2 - the interval between CCA dressings is 3 days
• Subgroup 3 - the interval between CCA dressings 1 day
Figure 2: The content of ATP synthase in the cytoplasm of pyramidal neurons of the CA1 field of the hippocampus of rats with
total (TCI), subtotal (SCI), stepwise subtotal (GSCI) and partial cerebral ischemia (PCI). Digital micrograph. Magnification x 40
Note:
subgroup 1 - the interval between CCA dressings 7 days
subgroup 2 - the interval between CCA dressings is 3 days
subgroup 3 - the interval between CCA dressings 1 day
At the same time, compared with the value in the “TIGM” group,
the content of ATP synthase in the cytoplasm of neurons was 70
(66; 85)% higher in the parietal cortex (p <0.05) and by 58 (41;
76)% - in the hippocampus (p <0.05), and compared with the value
in the “SCI” group - by 28 (19; 38)%, p <0.05 and 35 (27; 42)%, p
<0.05 , respectively. A decrease in the ATP synthase content in the
parietal cortex and hippocampus with PCI is less pronounced than
with SIGM by 24% (p <0.05) and 31% (p <0.05), respectively, and
than with TCI - by 53% (p <0.05) and 62% (p <0.05), respectively.
Compared to the level of ATP synthase in the control group, in the
1st subgroup of SCI (the interval between dressings is 7 days), there
was a decrease in the content of ATP synthase by 23 (12; 37)% in the
parietal cortex (p <0 , 05) and by 7 (3; 12)% - in the hippocampus
(p <0.05), in the 2nd subgroup of GSCI (the interval between
dressings is 3 days), the content of ATP synthase decreased to a
greater extent, amounting to 37 (27; 44)% - in the parietal cortex
(p <0.05) and 26 (20; 34)% - in the hippocampus (p <0.05), and in
the 3rd subgroup (the interval between dressings 1 day) there was
the most significant decrease enzyme content - by 42 (35; 52)%. p
<0.05 and by 33 (27; 42) %, p <0.05, respectively.
In the 3rd subgroup of GSCI (the interval between CCA dressings
was 1 day), the enzyme content was less, compared with the 1st
subgroup, by 27 (22; 32)% - in the parietal cortex (p <0.05) and by
18 (11 ; 23)% - in the hippocampus (p <0.05), and compared with
the 2nd subgroup of GSCI - by 8 (3; 15)% in the hippocampus (p
<0.05), while in the parietal cortex there were no differences was
noted (p> 0.05).Thus, in the “GSCI” group, the smallest decrease in
the content of ATP synthase was observed in the 1st subgroup of
GSCI with an interval between dressings of 7 days, while the greatest
decrease in the enzyme content was noted in the 3rd subgroup with
a minimum interval between CCA dressings (1 day).
Discussion
Compared with the value in the “SCI” group, modeled by onestage
ligation of both CCA, in the 3rd subgroup of SCI in both studied
departments, no differences were found (p> 0.05). The content
of ATP synthase in the 2nd subgroup of SCI was 17% higher than
at SCI (p <0.05) in the hippocampus, while in the parietal cortex
there were no differences (p> 0.05), and in 1 -th subgroup SCI - it
was 30% more in the parietal cortex (p <0.05) and 34% - in the
hippocampus (p <0.05) than in rats with SCI. Compared with the
value in the group “PCI”, in the 1st subgroup of GSCI in both studied
departments there were no differences in the content of ATP
synthase (p> 0.05), in the 2nd subgroup of GSCI it was less by 18%
in the parietal cortex (p <0.05) and by 20% - in the hippocampus
(p <0.05), and in the 3rd subgroup of SIGM the differences from SCI
were more pronounced and amounted to 26% (p <0.05) and 27% (
p <0.05), respectively. The severity of changes in the content of ATPsynthase
in the 2nd and 3rd subgroups of GSCI was greater: by 18%
(p <0.05) - in the hippocampus of the brain of rats of the second
subgroup of SSHM and by 21% - in the parietal cortex (p <0,05)
and 25% in the hippocampus (p <0.05) in the 3rd subgroup of rats
with SCI. The content of ATP-synthase in the 1st subgroup of SSIGM
(the interval between CCA dressings was 7 days) was the closest
to the indicators in the “PCI” group, while in the 3rd subgroup of
GSCI, with a minimum interval between CCA dressings of 1 day, in
to a greater extent was close to the values of the enzyme content
in the “SCI” group, modeled by simultaneous ligation of both CCA,
which indicates a more pronounced damage to the V complex of the
electron transport chain in this model of SCI.
Studies have shown the dependence of the severity of brain
damage in SSHM on the interval between the cessation of blood
flow in both CCA. With a 7-day interval between CCA dressings,
the content of ATP synthase was restored. The results obtained
agree with the previously obtained data. According to which the
smallest morphological changes in neurons were noted in the
“CHIGM” groups and the 1st subgroup “GSCI”, with an interval
between CCA dressings of 7 days. It is obvious that with these
methods of modeling IHM, adaptation processes occur that prevent
the development of pronounced morphological changes and
allow neurons to adapt to conditions of moderate hypoxia. So, for
example, in PCI, the absence of pronounced morphological changes
in rats is explained by the compensation of blood circulation in the
circle of Willis. With GSCI, when the time interval (7 days between
dressings) is sufficient for the development of adaptive processes,
the productivity of mitochondrial respiration increases [5], and,
possibly, the production of nitrogen monoxide and hypoxia-induced
factor are activated [4]. Modeling of more severe types of ischemic
damage leads to pronounced morphological changes in neurons in
the parietal cortex and hippocampus of the rat brain - a decrease
in their size, deformation of perikarya, an increase in the degree
of neuronal chromatophilia with their simultaneous wrinkling and
subsequent death. To the greatest extent, these disorders were
expressed in the 3rd subgroup of SCI with the shortest interval
between dressings, which was 1 day, and in the TCI group [4]. This,
probably, can explain, in comparison with the groups “TCI”, “SCI”
and the 3rd subgroup “SCI”, the higher content of ATP synthase in
the cytoplasm of neurons in the 1st and 2nd subgroups “SCI” and “
PCI “.
Conclusion
Thus, the most pronounced decrease in the content of ATP synthase was observed in the groups “TCI”, “SCI” and in the 3rd subgroup “SCI”, with a minimum time interval between CCA dressings. With GSCI with an interval between CCA dressings of 7 days, the suppression of the content of ATP synthase was not so significant [20,21].
Acknowledgment
• Conflict of interest statement
• The authors declare no conflict of interest.
Funding Sources
State scientific program «To study the processes of damage and adaptation of the brain during its ischemia and the use of correction».
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