In vivo study of the role of hydrogen peroxide in the development of ishemic stroke in a model of streptozotocin-induced type I diabetes in rats using genetically encoded biosensor HyPer7

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Abstract

Diabetes mellitus is a significant risk factor for the development of complications following ischemic stroke. However, the precise mechanisms through which elevated glycemic status impacts neuronal metabolism during ischemia remain unclear. One probable reason for the exacerbation of post-ischemic consequences under hyperglycemia is oxidative stress, the indicator of which may be H2O2. In this research, we used the genetically encoded fluorescent biosensor HyPer7 to exhibit the hydrogen peroxide dynamics in the matrix of neuronal mitochondria during ischemic stroke under hyperglycemic and normal glucose conditions.

The research was conducted on SHR rats with normal and high blood glucose levels. Type I diabetes mellitus was induced by injecting streptozotocin, which is toxic to pancreatic β-cells. For the expression of the fluorescent biosensor HyPer7-mito in the neuronal mitochondria of the caudate nucleus region, a suspension of adeno-associated virus particles carrying the sensor gene under the neuronal promoter was administered under stereotactic control. After the rats were injected, optical fibers with a ceramic adapter were implanted directly into their brain striatum, enabling us to register the H2O2 indicator signal using a highly sensitive fluorescence excitation and detection system developed by the Institute of Photonics and Nonlinear Spectroscopy at Moscow State University. The biosensor signal recording was continuously performed in real-time from the moment the animal was anesthetized. While recording, anesthetized rats underwent middle cerebral artery occlusion, which supplies corpus striatum.

Using HyPer7 in the ischemic stroke model described, we observed that the H2O2 concentration dynamics in the affected hemisphere of rats with normal and elevated blood glucose levels were similar during the acute phase of stroke and one day after occlusion. Oxidation of the biosensor was observed in both animal groups during both ischemia and reperfusion. However, HyPer7 exhibited the most marked response one day after the occlusion of the middle cerebral artery, indicating a significant rise in hydrogen peroxide concentration.

Furthermore, the metabolic activity of brain tissue was evaluated through staining slices obtained 24 hours after occlusion with 2,3,5-triphenyltetrazolium chloride. Rats with diabetes exhibited 2.6 times larger brain damage, indicating that hyperglycemia exacerbates the consequences of ischemic stroke. Furthermore, there was a higher mortality rate in the hyperglycemic group following ischemic stroke. This indicates that the consequences of ischemic stroke were more severe under hyperglycemia, with 25% of the animals in the hyperglycemic group dying before the experiment ended, whereas none of the control group animals died.

Our study found that an elevated glycemic status does not impact the creation of H2O2 during the acute phase of stroke or one day after occlusion. However, it significantly intensifies damage to brain tissue and raises mortality rates.

Full Text

Diabetes mellitus is a significant risk factor for the development of complications following ischemic stroke. However, the precise mechanisms through which elevated glycemic status impacts neuronal metabolism during ischemia remain unclear. One probable reason for the exacerbation of post-ischemic consequences under hyperglycemia is oxidative stress, the indicator of which may be H2O2. In this research, we used the genetically encoded fluorescent biosensor HyPer7 to exhibit the hydrogen peroxide dynamics in the matrix of neuronal mitochondria during ischemic stroke under hyperglycemic and normal glucose conditions.

The research was conducted on SHR rats with normal and high blood glucose levels. Type I diabetes mellitus was induced by injecting streptozotocin, which is toxic to pancreatic β-cells. For the expression of the fluorescent biosensor HyPer7-mito in the neuronal mitochondria of the caudate nucleus region, a suspension of adeno-associated virus particles carrying the sensor gene under the neuronal promoter was administered under stereotactic control. After the rats were injected, optical fibers with a ceramic adapter were implanted directly into their brain striatum, enabling us to register the H2O2 indicator signal using a highly sensitive fluorescence excitation and detection system developed by the Institute of Photonics and Nonlinear Spectroscopy at Moscow State University. The biosensor signal recording was continuously performed in real-time from the moment the animal was anesthetized. While recording, anesthetized rats underwent middle cerebral artery occlusion, which supplies corpus striatum.

Using HyPer7 in the ischemic stroke model described, we observed that the H2O2 concentration dynamics in the affected hemisphere of rats with normal and elevated blood glucose levels were similar during the acute phase of stroke and one day after occlusion. Oxidation of the biosensor was observed in both animal groups during both ischemia and reperfusion. However, HyPer7 exhibited the most marked response one day after the occlusion of the middle cerebral artery, indicating a significant rise in hydrogen peroxide concentration.

Furthermore, the metabolic activity of brain tissue was evaluated through staining slices obtained 24 hours after occlusion with 2,3,5-triphenyltetrazolium chloride. Rats with diabetes exhibited 2.6 times larger brain damage, indicating that hyperglycemia exacerbates the consequences of ischemic stroke. Furthermore, there was a higher mortality rate in the hyperglycemic group following ischemic stroke. This indicates that the consequences of ischemic stroke were more severe under hyperglycemia, with 25% of the animals in the hyperglycemic group dying before the experiment ended, whereas none of the control group animals died.

Our study found that an elevated glycemic status does not impact the creation of H2O2 during the acute phase of stroke or one day after occlusion. However, it significantly intensifies damage to brain tissue and raises mortality rates.

ADDITIONAL INFORMATION

Funding sources. The study was supported by the Russian Science Foundation, grant No. 22-15-00299.

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About the authors

A. P. Trifonova

Moscow Institute of Physics and Technology (National Research University); Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences

Author for correspondence.
Email: trifonova.ap@phystech.du
Russian Federation, Dolgoprudny; Moscow

D. A. Kotova

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences

Email: trifonova.ap@phystech.du
Russian Federation, Moscow

A. D. Ivanova

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences

Email: trifonova.ap@phystech.du
Russian Federation, Moscow

M. S. Pochechuev

Moscow State University

Email: trifonova.ap@phystech.du
Russian Federation, Moscow

Yu. V. Khramova

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences; Moscow State University

Email: trifonova.ap@phystech.du
Russian Federation, Moscow; Moscow

M. A. Sudoplatov

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences; Pirogov Russian National Research Medical University

Email: trifonova.ap@phystech.du
Russian Federation, Moscow; Moscow

V. A. Katrukha

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences; Moscow State University

Email: trifonova.ap@phystech.du
Russian Federation, Moscow; Moscow

A. D. Sergeeva

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences; Moscow State University

Email: trifonova.ap@phystech.du
Russian Federation, Moscow; Moscow

R. I. Raevskii

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences

Email: trifonova.ap@phystech.du
Russian Federation, Moscow

M. A. Solotenkov

Moscow State University

Email: trifonova.ap@phystech.du
Russian Federation, Moscow

I. V. Fedotov

Moscow State University; Russian Quantum Center “Skolkovo”; Texas A&M University

Email: trifonova.ap@phystech.du
Russian Federation, Moscow; Moscow; Texas, USA

A. B. Fedotov

Moscow State University; Russian Quantum Center “Skolkovo”; National University of Science and Technology “MISiS”

Email: trifonova.ap@phystech.du
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: trifonova.ap@phystech.du
Russian Federation, Moscow; Moscow; Moscow; Göttingen, Germany

A. M. Zheltikov

Texas A&M University

Email: trifonova.ap@phystech.du
United States, Texas

D. S. Bilan

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences; Pirogov Russian National Research Medical University

Email: trifonova.ap@phystech.du
Russian Federation, Moscow; Moscow

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