Recording changes in biochemical parameters in vivo in the ischemic stroke model
- Authors: Khramova Y.V.1,2, Kotova D.A.2, Ivanova A.D.2, Pochechuev M.S.1, Kelmanson I.V.2, Trifonova A.P.2,3, Sudoplatov M.A.2,4, Katrukha V.A.1,2, Sergeeva A.D.1,2, Raevskii R.I.2, Solotenkov M.A.1, Fedotov I.V.1,5, Fedotov A.B.1,5,6, Belousov V.V.2,4,7,8, Bilan D.S.2,4
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Affiliations:
- Lomonosov Moscow State University
- Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences
- Moscow Institute of Physics and Technology (National Research University)
- Pirogov Russian National Research Medical University
- Russian Quantum Center “Skolkovo”
- National University of Science and Technology “MISiS”
- Federal Center of Brain Research and Neurotechnologies, Federal Medical Biological Agency
- Institute for Cardiovascular Physiology, University Medical Center Göttingen, Georg-August University
- Issue: Vol 18, No 4 (2023)
- Pages: 487-490
- Section: Conference proceedings
- URL: https://genescells.ru/2313-1829/article/view/623461
- DOI: https://doi.org/10.17816/gc623461
- ID: 623461
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Abstract
Stroke is a significant and socially insidious disease that ranks second among fatal diseases according to the World Health Organization [1]. Understanding the molecular mechanisms behind the pathogenesis of this ailment will enable the development of more effective preventative measures and treatment strategies to minimize the negative consequences of stroke. Despite the abundance of experimental data, most of which were acquired indirectly, the study of the dynamics of biochemical parameters in brain tissue in real time during the acute phase of ischemic stroke is difficult. The use of genetically-encoded sensors creates novel possibilities for monitoring alterations in different biochemical and metabolic parameters in vivo tissues.
In this study, we evaluated pH changes, hydrogen peroxide production (an important type of biologically active ROS), and polysulfide synthesis in various types of brain tissue cells of SHR rats during the development of ischemic stroke in real time using sensors such as SypHer3s (for pH detection), HyPer7 (for H2O2 detection), and PersIc (for polysulfide detection). Middle cerebral artery occlusion was used to simulate an ischemic stroke. The in vivo sensor signals were registered with a fiber optic setup that was created in the laboratory of spectroscopy and nonlinear optics at Moscow State University.
The studies revealed that in the acute phase of stroke, acidosis occurred in the cytoplasm of neurons in the caudate nucleus, the epicenter of ischemia. The pH mutated from 7.25±0.08 to 6.7±0.15 within the first few seconds after arterial occlusion initiation. A gradual increase in pH was observed after the initial drop, which persisted throughout reperfusion but did not return to the original value in all animals. In the penumbra zone, a wave-like shift in sensor signal was detected, whereas no change in sensor signal was noted in the healthy hemisphere. Investigation of the dynamics of H2O2 formation in the mitochondrial matrix of caudate neurons revealed minimal sensor oxidation during ischemia/reperfusion in the acute phase of stroke, indicating low ROS production. Nevertheless, a substantial increase in the sensor signal was detected after 24 hours following the surgery. Thus, the confirmation of oxidative stress development in the affected hemisphere differed from the commonly accepted view in terms of its dynamics. Previously, it was believed that excessive production of H2O2 leading to oxidative stress and related brain cell death occurred primarily in the acute phase. However, a comparison of hydrogen peroxide production dynamics in neurons and astrocytes revealed differences between these cell populations. It was discovered that as early as 12 hours after middle cerebral artery occlusion, the sensor signal in astrocytes increased more intensely than in neurons. This trend persisted until the end of the measurements, 40 hours after surgery. The observed distinctions may stem from glial cells’ protective function in counteracting the harmful consequences of hydrogen peroxide on neurons, along with their contribution to maintaining the myelin structure in the brain. Additionally, the role of astrocytes in neuroinflammation development is noteworthy. Reactive sulfur species, in addition to reactive oxygen species, appear to be significant contributors to the development of pathological processes. The PersIc sensor signal measurement did not show any disparities between the caudate nucleus of the healthy hemisphere and the hemisphere affected by stroke development in terms of polysulfide and persulfide appearance detection. However, the area surrounding the core infarction is noteworthy due to the observed bouts of acidosis using the SypHer3s sensor. Our findings suggest a potential association between these bouts, spreading depolarization, changes in calcium concentration, and the development of neuroinflammation. These reactions may ultimately lead to the synthesis of polysulfides, known modulators of inflammatory reactions.
Thus, our data provides valuable additions to the existing knowledge on metabolic changes that take place during the progression of ischemic brain injury.
Full Text
Stroke is a significant and socially insidious disease that ranks second among fatal diseases according to the World Health Organization [1]. Understanding the molecular mechanisms behind the pathogenesis of this ailment will enable the development of more effective preventative measures and treatment strategies to minimize the negative consequences of stroke. Despite the abundance of experimental data, most of which were acquired indirectly, the study of the dynamics of biochemical parameters in brain tissue in real time during the acute phase of ischemic stroke is difficult. The use of genetically-encoded sensors creates novel possibilities for monitoring alterations in different biochemical and metabolic parameters in vivo tissues.
In this study, we evaluated pH changes, hydrogen peroxide production (an important type of biologically active ROS), and polysulfide synthesis in various types of brain tissue cells of SHR rats during the development of ischemic stroke in real time using sensors such as SypHer3s (for pH detection), HyPer7 (for H2O2 detection), and PersIc (for polysulfide detection). Middle cerebral artery occlusion was used to simulate an ischemic stroke. The in vivo sensor signals were registered with a fiber optic setup that was created in the laboratory of spectroscopy and nonlinear optics at Moscow State University.
The studies revealed that in the acute phase of stroke, acidosis occurred in the cytoplasm of neurons in the caudate nucleus, the epicenter of ischemia. The pH mutated from 7.25±0.08 to 6.7±0.15 within the first few seconds after arterial occlusion initiation. A gradual increase in pH was observed after the initial drop, which persisted throughout reperfusion but did not return to the original value in all animals. In the penumbra zone, a wave-like shift in sensor signal was detected, whereas no change in sensor signal was noted in the healthy hemisphere. Investigation of the dynamics of H2O2 formation in the mitochondrial matrix of caudate neurons revealed minimal sensor oxidation during ischemia/reperfusion in the acute phase of stroke, indicating low ROS production. Nevertheless, a substantial increase in the sensor signal was detected after 24 hours following the surgery. Thus, the confirmation of oxidative stress development in the affected hemisphere differed from the commonly accepted view in terms of its dynamics. Previously, it was believed that excessive production of H2O2 leading to oxidative stress and related brain cell death occurred primarily in the acute phase. However, a comparison of hydrogen peroxide production dynamics in neurons and astrocytes revealed differences between these cell populations. It was discovered that as early as 12 hours after middle cerebral artery occlusion, the sensor signal in astrocytes increased more intensely than in neurons. This trend persisted until the end of the measurements, 40 hours after surgery. The observed distinctions may stem from glial cells’ protective function in counteracting the harmful consequences of hydrogen peroxide on neurons, along with their contribution to maintaining the myelin structure in the brain. Additionally, the role of astrocytes in neuroinflammation development is noteworthy. Reactive sulfur species, in addition to reactive oxygen species, appear to be significant contributors to the development of pathological processes. The PersIc sensor signal measurement did not show any disparities between the caudate nucleus of the healthy hemisphere and the hemisphere affected by stroke development in terms of polysulfide and persulfide appearance detection. However, the area surrounding the core infarction is noteworthy due to the observed bouts of acidosis using the SypHer3s sensor. Our findings suggest a potential association between these bouts, spreading depolarization, changes in calcium concentration, and the development of neuroinflammation. These reactions may ultimately lead to the synthesis of polysulfides, known modulators of inflammatory reactions.
Thus, our data provides valuable additions to the existing knowledge on metabolic changes that take place during the progression of ischemic brain injury.
ADDITIONAL INFORMATION
Funding sources. The study was supported by a grant from the Russian Science Foundation No. 22-15-00299.
Authors' contribution. All authors made a substantial contribution to the conception of the work, acquisition, analysis, interpretation of data for the work, drafting and revising the work, and final approval of the version to be published and agree to be accountable for all aspects of the work.
Competing interests. The authors declare that they have no competing interests.
About the authors
Y. V. Khramova
Lomonosov Moscow State University; Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences
Author for correspondence.
Email: yul.khramova@gmail.com
Russian Federation, Moscow; Moscow
D. A. Kotova
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences
Email: yul.khramova@gmail.com
Russian Federation, Moscow
A. D. Ivanova
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences
Email: yul.khramova@gmail.com
Russian Federation, Moscow
M. S. Pochechuev
Lomonosov Moscow State University
Email: yul.khramova@gmail.com
Russian Federation, Moscow
I. V. Kelmanson
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences
Email: yul.khramova@gmail.com
Russian Federation, Moscow
A. P. Trifonova
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences; Moscow Institute of Physics and Technology (National Research University)
Email: yul.khramova@gmail.com
Russian Federation, Moscow; Dolgoprudny
M. A. Sudoplatov
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences; Pirogov Russian National Research Medical University
Email: yul.khramova@gmail.com
Russian Federation, Moscow; Moscow
V. A. Katrukha
Lomonosov Moscow State University; Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences
Email: yul.khramova@gmail.com
Russian Federation, Moscow; Moscow
A. D. Sergeeva
Lomonosov Moscow State University; Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences
Email: yul.khramova@gmail.com
Russian Federation, Moscow; Moscow
R. I. Raevskii
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences
Email: yul.khramova@gmail.com
Russian Federation, Moscow
M. A. Solotenkov
Lomonosov Moscow State University
Email: yul.khramova@gmail.com
Russian Federation, Moscow
I. V. Fedotov
Lomonosov Moscow State University; Russian Quantum Center “Skolkovo”
Email: yul.khramova@gmail.com
Russian Federation, Moscow; Moscow
A. B. Fedotov
Lomonosov Moscow State University; Russian Quantum Center “Skolkovo”; National University of Science and Technology “MISiS”
Email: yul.khramova@gmail.com
Russian Federation, Moscow; Moscow; Moscow
V. V. Belousov
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences; Pirogov Russian National Research Medical University; Federal Center of Brain Research and Neurotechnologies, Federal Medical Biological Agency; Institute for Cardiovascular Physiology, University Medical Center Göttingen, Georg-August University
Email: yul.khramova@gmail.com
Russian Federation, Moscow; Moscow; Moscow; Göttingen, Germany
D. S. Bilan
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences; Pirogov Russian National Research Medical University
Email: yul.khramova@gmail.com
Russian Federation, Moscow; Moscow
References
- Pega F, Nafradi B, Momen NC, et al. Global, regional, and national burdens of ischemic heart disease and stroke attributable to exposure to long working hours for 194 countries, 2000–2016: A systematic analysis from the WHO/ILO Joint Estimates of the Work-related Burden of Disease and Injury. Environment International. 2021;154:106595. doi: 10.1016/j.envint.2021.106595
Supplementary files
