Neuroprotective effects of local surface hypothermia during endothelin-1-induced focal ischemia in rat cerebral cortex. I. Electrophysiological analysis

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Abstract

Local therapeutic hypothermia (LTH) is one of the most promising methods for neuroprotection in cerebral ischemia. However, the efficacy of superficial LTH at clinically relevant delays after the onset of ischemic attack remains poorly understood. In the present study, we investigated the neuroprotective effects of LTH in a model of focal ischemia induced by epipial application of the vasoconstrictor Endothelin-1 in the somatosensory cortex region of the rat brain. The neuroprotective effects of LTH were assessed by the level of spontaneous and sensory-evoked electrical activity at different cortical depths using linear electrode arrays. We found that cooling the cortical surface to 28°C using a Peltier element starting from 0, 10, and 60 minutes after Endothelin-1 application caused a significant reduction in the degree of suppression of electrical activity in the ischemic focus formed in the cerebral cortex 3 hours after Endothelin-1 application. The neuroprotective effects of LTH were manifested by a higher level of spontaneous multinit activity, higher power of the local field potential oscillations in theta, alpha and beta bands, as well in a greater amplitude and higher multiunit activity during sensory evoked responses. The neuroprotective effects of LTH were inversely correlated with the delay of LTH onset and were most pronounced with LTH initiated with minimal (0 and 10 minutes) delay after Endothelin-1 application. We also found that only LTH initiated simultaneously with Endothelin-1 application delayed the onsets of spreading depolarization waves and that LTH did not affect the amplitude of negative ultraslow potentials evoked by Endothelin-1. Taken together, the results of the electrophysiological analysis suggest neuroprotective effects of surface LTH, which are particularly pronounced when LTH is minimally delayed from the onset of the ischemic insult in the Endothelin-1-induced focal ischemia model.

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About the authors

G. F. Zakirova

Kazan Federal University

Email: roustem.khazipov@inserm.fr
Russian Federation, Kazan

К. A. Chernova

Kazan Federal University

Email: roustem.khazipov@inserm.fr
Russian Federation, Kazan

R. N. Khazipov

Kazan Federal University; Aix-Marseille University, INMED, IINSERM

Author for correspondence.
Email: roustem.khazipov@inserm.fr
Russian Federation, Kazan; Marseille, France

А. V. Zakharov

Kazan Federal University; Department of Normal Physiology, Kazan State Medical University

Email: roustem.khazipov@inserm.fr
Russian Federation, Kazan; Kazan

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2. Fig. 1. Experimental setup. (a) – layout of metal plate with Peltier element and silicon probe in rat barrel cortex. Arrow shows vibrissa stimulation. Inset on the right – corresponding micrograph (before agar application); (b) – 16-channel linear electrode was inserted vertically into the center of cranial window to record electrical activity of all layers (L1–L6; L – layer) of barrel cortex. Epipial application of ET1 was performed using microinjector. Metal plate with Peltier element was placed near the surface of cerebral cortex and covered with agar; (c) – time plan of experiment in 4 groups of animals.

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3. Fig. 2. Effect of local hypothermia on spontaneous electrical activity of the cerebral cortex in the ischemic focus caused by ET1. (a) – spontaneous electrical activity before ET1 application at a temperature of 39°C (control conditions). Shown are LFP in the frequency range of 0.2–1000 Hz (black lines) and MUA (red vertical segments) at different depths of the cortex; (b) – spontaneous electrical activity 3 h after ET-1 application at a temperature of 39°C. (c, d, e) – examples of electrical activity under conditions of cooling the brain surface to 28°C simultaneously (c), 10 min (d) and 60 min (e) after ET-1 application. NT – normothermia, HT – hypothermia.

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4. Fig. 3. Effect of local hypothermia on the frequency of IVD in the ischemic focus induced by ET1. (a) – IVD (MUA) of layer 5 neurons (individual action potentials – gray lines and averaged – black lines). Examples for all temperature conditions are shown. (b) – frequency of spontaneous IVDs calculated for all electrodes in the cortical column (∑MUA) after 3 h of ET1 action (1 μM) under normothermia conditions (39°C) and upon cooling to a temperature of 28°C, started simultaneously, 10 min and 1 h after ET1 application. Data are normalized to the control level before ET1 application. At the top are shown the probabilities of statistical differences between the groups and the control (from the 100% level) according to the rank sum test for paired samples (p-value), the probabilities of differences between the groups according to the Kruskal–Wallis test (KW p) and significant differences between the groups according to the Dunn test (asterisks); (c) is the dependence of ∑MUA in the ischemic focus on the cooling start time. Normothermic experiments are taken into account with a delay time of 180 min. The black dotted line shows the linear approximation. The box shows the Spearman correlation coefficient and the corresponding significance level; (d) is the level of the IVD frequency relative to the control values ​​in the ischemic focus under different temperature conditions in different layers of the cortical column. The significance level of the difference between the group and the control (from the 100% level, as in panel b) according to the rank sum test for paired samples is shown above the boxes. The boxes show the Spearman correlation coefficients of the parameter on the cooling start time and the corresponding significance levels. NT – normothermia, HT – hypothermia.

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5. Fig. 4. Effect of local hypothermia on sensory-evoked potentials in the ischemic focus induced by ET1. (a) – examples of averaged sensory-evoked potentials (SEPs) at different depths of the cortical column (LFC, black lines) against the background of color-coded SEP maps under control conditions and in ischemic foci induced by ET1 application at different LTG delays; (b) – SEP amplitude calculated for all electrodes in the cortical column (∑SEP) after 3 h of ET1 action under normothermia (39°C) and upon cooling to a temperature of 28°C, started simultaneously, 10 min and 1 h after ET1 application. Data are normalized to the control level before ET1 application. At the top are shown the probabilities of statistical differences between the groups and the control (from the 100% level) according to the rank sum test for paired samples (p-value), the probabilities of differences between the groups according to the Kruskal–Wallis test (KW p) and significant differences between the groups according to the Dunn test (asterisks); (c) is the dependence of ∑SEP in the ischemic focus on the cooling start time. Normothermic experiments are taken into account with a delay time of 180 min. The black dotted line shows the linear approximation. The box shows the Spearman correlation coefficient and the corresponding significance level; (d) is the level of the SEP amplitude relative to the control values ​​in the ischemic focus under different temperature conditions in different layers of the cortical column. The significance level of the difference between the group and the control (from the 100% level, as in panel b) according to the rank sum test for paired samples is shown below the boxes. The boxes show the Spearman correlation coefficients of the parameter on the cooling start time and the corresponding significance levels. NT – normothermia, HT – hypothermia.

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6. Fig. 5. Effect of local hypothermia on sensory-evoked IVDs in the ischemic focus induced by ET1. (a) – examples of sensory-evoked IVD frequency (SE-MUA) graphs at different depths of the cortical column (white lines) against the background of color-coded IVD density maps under control conditions and in ischemic foci induced by ET1 application at different LTG delays; (b) – IVD frequency calculated for all electrodes in the cortical column (∑MUA during SEP), in the time window of 0–30 ms from the onset of SEP, after 3 h of ET1 action under normothermia (39°C) and upon cooling to a temperature of 28°C, started simultaneously, 10 min and 1 h after ET1 application. Data are normalized to the control level before ET1 application. At the top are shown the probabilities of statistical differences between the groups and the control (from the 100% level) according to the rank sum test for paired samples (p-value), the probabilities of differences between the groups according to the Kruskal–Wallis test (KW p) and significant differences between the groups according to the Dunn test (asterisks); (c) – correlation of the degree of recovery of sensory-evoked IVDs from the time of cooling start. Normothermic experiments are taken into account with a delay time of 180 min. The black dotted line shows the linear approximation. The box shows the Spearman correlation coefficient and the corresponding significance level; (d) – the frequency level of sensory-evoked IVDs relative to the control values ​​in the ischemic focus under different temperature conditions in different layers of the cortical column. The significance level of the difference between the group and the control (from the 100% level, as in panel b) according to the rank sum test for paired samples is shown below the boxes. The boxes show the Spearman correlation coefficients of the parameter from the time of cooling start and the corresponding significance levels. NT – normothermia, HT – hypothermia.

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7. Fig. 6. Effect of local hypothermia on the waves of spreading depolarization caused by ET1. (a) – examples of waves of spreading depolarization (SD) at the depth of the 4th layer of the cortex, arising during the first minutes after the application of ET1 for different experimental groups of LTG; (b) – the delay time of the waves of spreading depolarization (SD delay) from the moment of ET1 application depending on the time of LTG onset. The Spearman correlation coefficient and the corresponding level of reliability are shown in the box. NT – normothermia, HT – hypothermia.

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8. Fig. 7. Effect of local hypothermia on negative ultraslow potentials evoked by ET1. (a) – examples of negative ultraslow potentials (NUP) (0–1000 Hz) recorded at the depth of the 4th cortex layer under normothermia and at different LTG delays. Black arrows indicate RD waves; (b) – averaged amplitude of infraslow negative potentials over all cortical layers at different LTG delays. Spearman’s correlation coefficient and the corresponding significance level are shown in the box. NT – normothermia, HT – hypothermia.

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