Assessment of the level of interleukin-1β expression in the cerebral cortex of mice in a model of post-traumatic stress disorder: methodological recommendations

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Abstract

The purpose of this study was to determine the influence of transcardial perfusion, as well as social hierarchy in male mice, on the level of gene expression in the cerebral cortex of mice using the example of the proinflammatory cytokine IL-1β. Material and methods: the study was carried out on non-linear male mice aged 3 months, 10 of which were subjected to stress according to a single prolonged stress protocol. The development of a stress disorder was confirmed by behavioral tests in the “Open Field” and “Elevated Plus Maze” mazes. The control group consisted of 10 mice that were not exposed to any effect. Then, from each group, 5 mice underwent transcardial perfusion, the rest were anesthetized and killed without perfusion. To determine expression levels, mRNA was isolated from the cerebral cortex, cDNA was synthesized, followed by real-time PCR.

Results: Mice stressed according to the single prolonged stress protocol demonstrated a rigid social hierarchy, while the behavior of dominant males in the Open Field and Elevated Plus Mazes was significantly different from the behavior of subordinates. As a result of stress in mice, the level of IL-1β expression in the cerebral cortex significantly increases compared to control animals, both in the case of transcardial perfusion and without it. In the control mice group, there was a trend between perfused and nonperfused animals toward lower levels of IL-1β expression in perfused animals, but there was no statistical significance. In the stress group, the expression level of IL-1β was significantly higher in non-perfused animals compared to perfused animals.

Conclusion: Our study showed that stress in male mice leads to increased conflicts against the backdrop of a rigid social hierarchy with a clear distinction between dominant and subordinate males. At the same time, the behavior of dominant males in the “Open Field” and “Elevated Plus Maze” mazes differs significantly from the behavior of subordinates, which is reflected in the study statistics. Also, when assessing the expression levels of interleukins in the brain, transcardial perfusion is recommended to remove blood cells from the brain vessels, since the level of expression differs in perfused and non-perfused animals.

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About the authors

Georgii Mikhailovich Dzhobadze

Federal State Autonomous Institution of Higher Education “IKBFU named after Immanuel Kant", Educational and ScientificCluster "Institute of Medicine and Life Sciences (MEDBIO)"

Author for correspondence.
Email: aoagovs@gmail.com
ORCID iD: 0009-0005-0065-2126

Student of High School of Life Sciences

Russian Federation, st. Universitetskaya, 2, Kaliningrad, 236041

Anastasia A. Bruzgina

Federal State Autonomous Institution of Higher Education “IKBFU named after Immanuel Kant", Educational and ScientificCluster "Institute of Medicine and Life Sciences (MEDBIO)"

Email: anastasiabruz@gmail.com
ORCID iD: 0009-0000-8492-9578

Student of High School of Life Sciences

Russian Federation, st. Universitetskaya, 2, Kaliningrad, 236041

Ekaterina A. Kurilova

Federal State Autonomous Institution of Higher Education “IKBFU named after Immanuel Kant", Educational and ScientificCluster "Institute of Medicine and Life Sciences (MEDBIO)"

Email: ekaterinakuuurilova@gmail.com
ORCID iD: 0000-0003-0031-116X

PhD Student of High School of Life Sciences

Russian Federation, st. Universitetskaya, 2, Kaliningrad, 236041

Oksana P. Tuchina

Federal State Autonomous Institution of Higher Education “IKBFU named after Immanuel Kant", Educational and ScientificCluster "Institute of Medicine and Life Sciences (MEDBIO)"

Email: otuchina@kantiana.ru
ORCID iD: 0000-0003-1480-1311

Head of Laboratory for Synthetic Biology, PhD, Docent

Russian Federation, st. Universitetskaya, 2, Kaliningrad, 236041

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2. Fig. 1. Determination of dominant (a)and subordinate (б, в) male mice in the stress group by tail injury

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3. Fig. 2. Behavioral phenotyping of mice in the “Open Field” maze. a – photograph of the setup and representative tracks of a control and stressed survivor, б – time spent by control (empty bars) and stressed (gray bars) animals in different parts of the maze, as well as a comparison of dominant males (gray bars) with the rest (gray bars with shading) in the stress. Data are presented as mean ± SEM. ** – p≤0.01, *** – p≤0.001 (t-test)

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4. Fig. 3. Behavioral phenotyping of mice in the elevated plus maze. a – photograph of the setup and representative tracks of a controland stressed survivor, б – time spent by control (empty bars) and stressed (gray bars) animals in different parts of the maze, as wellas a comparison of dominant males (gray bars) with the rest (gray bars with shading) in the stress. Data are presented as mean ± SEM.* – p≤0.05, ** – p≤0.01 (t-test)

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5. Fig. 4. Expression levels of the pro-inflammatory interleukin IL-1β in the cerebral cortex of mice: a – comparison of animals from the control group after transcardial perfusion (empty bars with shading) and without perfusion (empty bars); б – comparison of animals from the stressed group after transcardial perfusion (gray bars with shading) and without; в – comparison of animals from the control (empty bars) and stressed (gray bars) groups without perfusion; г – comparison of animals from the control (empty bars) and stressed (gray bars) groups after transcardial perfusion. Data are presented as mean ± SEM. * – p≤0.05 (t-test)

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