Identifining a Therapeutic Window for Intranasal Insulin Administration in a Two-Vessel Rat Model of Forebrain Ischemia and Investigating the Mechanisms of Its Neuroprotective Action

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Cerebral ischemia is a significant medical and social issue, necessitating the development of effective treatment strategies. Due to the complex pathogenesis and prolonged recovery period associated with this condition, drugs with pleiotropic effects, such as intranasally administered insulin (IAI), are of the greatest interest. IAI sprayed in the nasal cavity enters the brain, regulating metabolism through central mechanisms, has neuroprotective and neuro-regulatory effects. It has been proven to be effective in the treatment of neurodegenerative diseases, although data on its effectiveness in cerebral ischemia remain limited. The aim of the work was to search for a “therapeutic window” and evaluate the mechanisms of the neuroprotective effect of IAI when used in rats with cerebral ischemia. Rats with two-vessel forebrain ischemia were administered IAI 2 and 4 hours after an episode of ischemia at a dose of 0.5 IU/rat/day, and then daily for a 7-day period after ischemia. It has been demonstrated that IAI is more effective if animals were treated 2 hours after the ischemic event, compared with administration after 4 hours, despite the subsequent 7-day of IAI treatment. When administered 2 hours after an ischemic event, IAI has been shown to support the expression of components of insulin signaling genes in the hippocampus and normalize the number of cells in the CA1 region. It also stimulates the expression of the anti-apoptotic Bcl-2 gene and reduces the expression of the Gfap and Aif1 genes, which are markers of astrocytes and microglia, and this indicates the anti-inflammatory effect of IAI. In addition, for the first time IAI has been found to stimulate the activity of the thyroid system and prevent the development of post-ischemic hypothyroidism. All these effects were less pronounced or not observed when IAI was administered 4 hours after the ischemic event. Thus, for the first time, we described a “therapeutic window” for the use of IAI in cerebral ischemia and evaluated some of the mechanisms underlying its neuroprotective effects.

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I. Zorina

Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences

编辑信件的主要联系方式.
Email: zorina.inna.spb@gmail.com
俄罗斯联邦, Saint-Petersburg

A. Pechalnova

Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences

Email: zorina.inna.spb@gmail.com
俄罗斯联邦, Saint-Petersburg

E. Chernenko

Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences

Email: zorina.inna.spb@gmail.com
俄罗斯联邦, Saint-Petersburg

D. Avrova

Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences

Email: zorina.inna.spb@gmail.com
俄罗斯联邦, Saint-Petersburg

K. Derkach

Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences

Email: zorina.inna.spb@gmail.com
俄罗斯联邦, Saint-Petersburg

A. Shpakova

Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences

Email: zorina.inna.spb@gmail.com
俄罗斯联邦, Saint-Petersburg

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2. Fig. 1. Effect of intranasal insulin on cell survival in the CA1 region of the hippocampus on day 7 after two-vessel forebrain ischemia/reperfusion. (a) Histogram showing the number of Nissl-positive cells in the CA1 region of the hippocampus. Cells were counted on a 300 μm area, magnification ×20. Data are presented as box-and-whiskers plots (the line in the middle is the median, the box borders are the 25th and 75th percentiles, the tips of the whiskers are the minimum and maximum observed values), n = 6 in all groups, 15–20 images were analyzed for each region. **, ## – differences are significant at p < 0.01; & – differences are significant at p < 0.05. SO – sham-operated rats; IschR – rats that underwent ischemia/reperfusion; IschR-Ins-2 and IschR-Ins-4 – rats that underwent ischemia/reperfusion and received insulin intranasally at a dose of 0.5 IU/rat/day 2 and 4 h after the end of ischemia and then daily for 7 days, respectively. (b) – Photomicrographs of brain sections of the CA1 region of the hippocampus. Nissl staining, magnification ×20, scale bar 100 µm. Designations: 1 – SO; 2 – IschR; 3 – IschR-Ins-2; 4 – IschR-Ins-4.

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3. Fig. 2. Expression of the genes encoding insulin receptor (Insr), insulin receptor substrate-2 IRS2 (Irs2), protein kinase Akt1 (Akt1), and phosphatase PTP1B (Ptpn1) in the hippocampus of rats on day 7 after two-vessel forebrain ischemia/reperfusion and the effect of intranasal insulin on it. Data are presented as M ± SEM (n = 6 in all groups). * and ** – differences are significant with the SO group at p < 0.05 and p < 0.01, respectively; # – differences are significant with IschR at p < 0.05. C – intact rats; SO – sham-operated rats; IschR – rats that underwent ischemia/reperfusion; IschR-Ins-2 and IschR-Ins-4 – rats that underwent ischemia/reperfusion and received insulin intranasally at a dose of 0.5 IU/rat/day 2 and 4 hours after the end of ischemia and then daily for 7 days, respectively.

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4. Fig. 3. Expression of the genes encoding the proapoptotic protein Bax (Bax) and antiapoptotic protein Bcl-2 (Bcl2), as well as their Bax/Bcl2 ratio in the rat hippocampus on day 7 after two-vessel forebrain ischemia/reperfusion, and the effect of intranasal insulin on it. In all groups, n = 6. Normally distributed data are presented as M ± SEM. Data that deviate from normal distribution are presented as box-and-whiskers plots (the line in the middle is the median, the box borders are the 25th and 75th percentiles, and the ends of the whiskers are the minimum and maximum observed values). * and ** – differences are significant with the SO group at p < 0.05 and p < 0.01, respectively; # – differences are significant with IschR at p < 0.05. C – intact rats; SO – sham-operated rats; IschR – rats that underwent ischemia/reperfusion; IschR-Ins-2 and IschR-Ins-4 – rats that underwent ischemia/reperfusion and received insulin intranasally at a dose of 0.5 IU/rat/day 2 and 4 hours after the end of ischemia and then daily for 7 days, respectively.

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5. Fig. 4. Expression of genes encoding brain-derived neurotrophic factor BDNF (Bdnf), glial fibrillary acidic protein GFAP (Gfap), and microglia marker IBA-1 (Aif1) in the hippocampus of rats on day 7 after two-vessel forebrain ischemia/reperfusion, and the effect of intranasal insulin on it. Data are presented as M ± SEM (n = 6 in all groups). * and ** – differences are significant with the SO group at p < 0.05 and p < 0.01, respectively; # and ## – differences are significant with IschR at p < 0.05 and p < 0.01, respectively; & – differences are significant with IschR-Ins-2 at p < 0.05. C – intact rats; SO – sham-operated rats; IschR – rats that underwent ischemia/reperfusion; IschR-Ins-2 and IschR-Ins-4 – rats that underwent ischemia/reperfusion and received insulin intranasally at a dose of 0.5 IU/rat/day 2 and 4 hours after the end of ischemia and then daily for 7 days, respectively.

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6. Fig. 5. Expression of genes encoding cytokines IL1β (Il1b), IL6 (Il6), TNFα (Tnfa) and matrix metalloproteinase MMP9 (Mmp9) in the hippocampus of rats on day 7 after two-vessel forebrain ischemia/reperfusion and the effect of intranasal insulin on it. In all groups, n = 6. Normally distributed data are presented as M ± SEM. Data that do not normally distribute are presented as box-and-whiskers plots (the line in the middle is the median, the box borders are the 25th and 75th percentiles, and the whisker tips are the minimum and maximum observed values). * and ** – differences are significant with the SO group at p < 0.05 and p < 0.01, respectively. C – intact rats; SO – sham-operated rats; IschR – rats that underwent ischemia/reperfusion; IschR-Ins-2 and IschR-Ins-4 – rats that underwent ischemia/reperfusion and received insulin intranasally at a dose of 0.5 IU/rat/day 2 and 4 hours after the end of ischemia and then daily for 7 days, respectively.

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