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Morphological characteristics of the rats brain under hypergravity

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 Research Article

Morphological characteristics of the rats brain under

hypergravity
Aleksei V. Tverskoi*, Vitaliy N. Morozov, Anastasia A. Petrenko, Tatyana S. Mukhina, Aleksandr S.
Parichuk

ABSTRACT

Aim: The objective of the research was to study the morphological changes in the most sensitive areas of the brain under
hypergravity, using standard general histological and histochemical, as well as immunohistochemical methods with antibodies to
glial fibrillary acid protein, neurofilament protein, Ki67 proliferation markers, and p53 apoptosis. Materials and Methods: The
experiment was performed on 40 white Wistar rats divided into 4 groups: 1st - control, 2nd - rats exposed to hypergravity for 8 min
at a centrifuge rotation rate of 5,000 rpm, 3rd - rats exposed to hypergravity 3 times during the 1st day for 5 min and at a rotation
rate of 4,000 rpm, and 4th - rats exposed to hypergravity 3 times for 5 min and a rotation speed of 4,000 rpm for 3 days.After the
end of the experiment, morphological changes in the brain were examined. Results: It was established that the animals of the first
group died from compression and dislocation of the brain resulted from overloads. Rats from the second group had clinical signs,
such as weakness, one- and two-sided paresis, which completely passed within an hour. The histological picture of the brain had
no specific features. In the third and fourth groups, the clinical symptoms were more pronounced, remaining until the end of the
1st day. Morphological picture of the immunohistochemical examination had no features but showed severe microcirculation
disorders when stained with hematoxylin and eosin. Conclusion: Thus, the hypergravitational model of cerebral ischemia allows
obtaining circulatory hypoxia of the brain, which is confirmed by clinical data and general histological methods.

KEY WORDS: Cerebral ischemia, Hippocampus, Hypergravity, Morphological changes, Rats

INTRODUCTION of staining, followed by morphometric measurement

Acute and chronic disorders of cerebral circulation
lead to the development of a number of diseases, such
as strokes and dyscirculatory encephalopathies. This
determines the importance of searching for new drugs,
innovative molecules, and compounds,[1,2] methods of
their administration, schemes of application, for which
the experimental pharmacology should conduct studies
on pharmacological targets,[3,4] and in vivo models.[5,6]
Currently, a large number of models of focal and total
brain ischemia are used. In this regard, the definition and
selection of the experimental model are a key step in the
implementation of the study. The next important point
is the choice of a method to evaluate the effectiveness
of the experimental model. To do this, a huge range of
clinical and morphological methods is used, such as the
assessment of behavioral, neurological, and cognitive
disorders, histological and histochemical methods
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of neurons in the most sensitive regions to hypoxia,
determination of the concentration of S-100 protein,
and neuron-specific enolase (NSE) in the blood by
the method of immunoelectrochemiluminescence
and electroencephalography. Morphological methods
include the study of brain tissue using a survey
hematoxylin and eosin staining, Nisslthionin staining,
immunohistochemical staining with antibodies to
protein S-100, glial fibrillary acid protein (GFAP),
NSE, as well as immunofluorescence method using
FluoroJade reagent.[7]
To create total cerebral ischemia in experimental
models, two- or four-vessel models are used
with ligation of two common carotid arteries or
a combination of a common carotid ligation with
occlusion of two vertebral arteries, respectively.
This model gives severe brain ischemia in 90% of
experimental animals. This group also includes the
model of gravitational ischemia. Models of focal brain
ischemia are divided into thromboembolic - a model
with occlusion of common carotid arteries with the

Department of Human Anatomy and Histology, Belgorod State University 308015, Belgorod, Russia

*Corresponding author: Aleksei V. Tverskoi, Department of Human Anatomy and Histology, Belgorod State University
308015, Belgorod, Pobeda Street 85, Russia. E-mail: tverskoy@bsu.edu.ru

Received on: 19-10-2017; Revised on: 27-11-2017; Accepted on: 20-12-2017

30 Drug Invention Today | Vol 9 • Issue 3 • 2017

 Aleksei V. Tverskoi, et al.
use of endothelin and a model of thrombosis caused
by photochemistry.
Objective
The objective of our research was to study the
acute morphological changes in the rats’ brain in a
gravitational model of cerebral ischemia.
MATERIALS AND METHODS
The material of the study was 40 white male Wistar
rats, which were divided into four experimental
groups. The first control group included 10 animals
not exposed to gravitational ischemia. The second
group consisted of 10 animals exposed to gravitational
ischemia once for 8 min, with a centrifuge rotation
rate of 5,000 rpm. The third group included 10
animals, which were exposed 3 times for 5 min at a
rotation speed of 4,000 rpm during the 1st day with
an interval of 1 h. The fourth group consisted of 10
animals exposed 3 times for 3 days with parameters
similar to experimental group 3. To carry out the
experiment, a centrifuge was used with the ability to
adjust the speed from 500 to 8,000 per minute and
the rotation arm length of 1 m, at the end of which
2 containers were installed to fix animals. Outside
the experiment, the animals were kept in standard
vivarium conditions with free access to water and
food. After the exposure, the neurologic symptoms
were assessed using the McGraw grading scale in the
modification by Gannushkina;[8,9] the activity of rats
was studied in an elevated cross labyrinth by Panlab
Harvard Apparatus LE 846 and using an infrared
activity monitor by Panlab Harvard Apparatus
LE 8825. The animals were withdrawn from the
experiment after the 3rd day, by overdose of anesthesia.
For histological examination, a brain was taken and
fixed in a 10% solution of buffered neutral formalin
for 24 h, after which the specimen was subjected to
standard processing on Leica TP 1020 apparatus,
then embedded into paraffin, cut into slices of 4–5
µm thick, and stained with hematoxylin and eosin
using standard protocols and techniques on Leica
EG 1150 H, Leica RM 2245, and Leica autostainer
XL. To stain neurons of the brain, thionine was used
according to Nissl method. Immunohistochemical
reactions were performed with GFAP antibodies
(clone EP672Y, CellMarque), NF (clone SP6,
CellMarque), p53 (clone DO7, CellMarque), and Ki
67 (clone NE14, Biogenex). The streptavidin-biotin
method (LSAB Kit) was used; chromogen was used
as diaminobenzidine. Epitope retrieval was performed
by heating in citrate buffer (pH 6.0) for 40 min at a
temperature of 93–95°C. The immunohistochemical
study was performed in the immunohistochemistry
department of the Belgorod regional pathoanatomical
bureau (T.S. Mukhin bureau). Photoprotection and
morphometric analysis were performed using Mirax
Drug Invention Today | Vol 9 • Issue 3 • 2017
Desk (Carl Zeiss Microimaging GMbH, Germany)
and Statistica 10.0 software (USA).
Main Part
For the study, all animals were divided into four
groups. The control group included 10 animals kept
under normal vivarium conditions and withdrawn
from the experiment on the 3rd day. Morphological
examination of the brain did not reveal any findings,
and the microscopic picture corresponded to the
typical histological structure of the brain of healthy
animals [Figure 1a].
The second group of animals consisted of 10
individuals hypergravitated for 8 min at a rotation
rate of 5,000 rpm. A macro-microscopic examination
of the brain revealed a picture of the compression
and dislocation of the brain with signs of trunk
herniation. Microscopic study revealed an uneven
plethora of the choroid and vascular plexuses of the
3rd and 4th ventricles, focal plethora of capillaries of
white and gray matter, some of which with diapause
hemorrhages and perivascular edema. However, most
capillaries are anemic, collapsed, with single red
blood cells in the lumen [Figure 1b].
The third group of animals included 10 individuals
exposed 3 times to hypergravity for 5 min at a rotation
rate of 4,000 rpm. The animals were withdrawn from
the experiment immediately after the third exposure to
hypergravity. Microscopic examination revealed signs
of focal transient brain ischemia, more pronounced in
its areas with the greatest sensitivity to hypoxia - in
the hippocampus, the cortex of the frontal lobe, and
cerebellum. In the hippocampus, mainly in the regions
CA1 and CA3, signs of disorganization of the layers of
neurons and their partial cell depletion were revealed.
Changes in the nuclei of neurons were recorded in
the form of swelling and fuzziness of the contours.
Polymorphic changes of the chromatophilic substance in
the form of focal chromatography, lumpy condensation,
and fuzziness of the structure of apical dendrites of
a b
Figure 1: :(a) Normal histological structure of CA1 area
of the rat’s hippocampus (control group), (b) hemorrhage
in the ventricle and perivascular edema. Hematoxylin and
eosin a, b×400
31
 Aleksei V. Tverskoi, et al.
a b
Figure 2: Ki67 (a) and p53 (b)-negative reaction in 4th
experimental group; ×400
a b
Figure 3: Glial fibrillary acidic protein-positive reaction in
the 1st (control) group (a) and 4th experimental group (b);
×400
a 6. Shakhno EA, Savitskaya TA, Pokrovskaya TG, Yakushev VI,
b
Figure 4: Neurofilament-positive reaction in the 1st (control)
group (a) and 4th experimental group (b),×400
pyramidal neurons were also revealed. These changes
in neurons are of typical, non-specific nature and are
observed both in acute and chronic stressful effects and
in different models of cerebral ischemia.[10,11]
The immunohistochemical study was performed on brain
specimens of rats of group 4 exposed to hypergravity
3 times for 3 days. Mitotic activity and signs of
proliferation both in staining with hematoxylin and eosin
and with the use of Ki67 were not revealed [Figure 2a].
Identification of the neurons in the frontal lobe and the
hippocampus revealed no p53-positive cells [Figure 2b].
32
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The reaction with GFAP, which is an intermediate
filament, results in a bright positive staining of
astrocytes and glial cells, satellite cells, Schwann cells
covering the axons of the hippocampal neurons, and
the frontal lobe [Figure 3a and b].
A bright positive staining with the neurofilament
protein, related to the cytoskeleton, was detected both
in the control and in the experimental group [Figure 4].
CONCLUSION
1. The obtained data testify that, under the experimental
hypergravity and resulting total brain ischemia,
microcirculatory disorders such as perivascular,
pericellular edema, diapedesis hemorrhages,
stasis in the vessels, and cerebral hemorrhages
predominate on the day 1–3.
2. No changes in the proliferative activity of neurons
most sensitive to hypoxia of brain regions are
observed, as well as qualitative disorders in the
synthesis of cytoskeleton proteins.
3. A hypergravitational model of cerebral ischemia
can be used to create total circulatory hypoxia.
REFERENCES
1. Kravchenko DV, Beskhmelnitsyna EA, Korokin MV,
Avtina TV, Sernov LN, Tishin AN, et al. Molecular screening
of prospective candidates for TRPA1 ion channel selective
antagonists. Res Result 2016;2:63-6.
2. Bogus SK, Dukhanin AS, Kucheryavenko AF,
Vinakov DV, Suzdalev KF, Galenko-Yaroshevsky PA.
Pleyotropicantiaggregant effects of an innovative antiarrhythmic
of class III SS-68, an indole derivative. Res Result 2017;3:3-13.
3. Ragulina VA, Kostina DA, Dovgan AP, Burda YE,
Nadezhdin SV. Nuclear factor kappa B as a potential target for
pharmacological correction endothelium-associated pathology.
Res Result 2017;3:114-24.
4. Danilenko LM, Klochkova GN, Kizilova IV, Korokin MV.
Metabolic cardioprotection: New concepts in implementation of
cardioprotective effects of meldonium. Res Result 2016;2:95-100.
5. Molchanova OV, Pokrovskaya TG, Povetkin SV, Reznikov KM.
Endothelioprotective property of the combination of the thioctic
acid and rosuvastatin shown in the endothelial dysfunction
models. Res Result 2016;1:9-15.
Pokrovskii MV, Grinshpan DD. Use of L-arginine immobilised
on activated carbon for pharmacological correction of
endothelial disfunction. Res Result 2016;1:30-5.
7. Wright NT, Prosser BL, Varney KM, Zimmer DB,
Schneider MF, Weber DJ. S100A1 and calmodulin compete
for the same binding site on ryanodine receptor. J BiolChem
2008;283:26676-83.
8. Gannushkina IV. Cerebral circulation in different types of
circulatory hypoxia of the brain. Vestnik of Russian Academy
of Medical Sciences 2000;9:22-7.
9. McGraw CP, Pashayan AG, Wendel OT. Cerebral infarction
in the Mongolian gerbil exacerbated by phenoxybenzamine
treatment. Stroke 1976;7:485-8.
10. Dolzhikov AA, Tverskoi AV, Bobyntsev II, Kriukov AA,
Belykh AE. Morphometric study of hippocampal neurons in
chronic immobilization stress. Res Result 2015;4:62-5.
Source of support: Nil; Conflict of interest: None Declared
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