Neuroprotective effects of local surface hypothermia during endothelin-1-induced focal ischemia in rat cerebral cortex. II. Morphometric analysis of ischemic lesions

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Abstract

In the present study, we investigated the neuroprotective effects of local therapeutic hypothermia (LTH) in a model of focal ischemia induced by epipial application of Endothelin-1 to the somatosensory cortex of the rat brain by morphometric analysis of ischemic foci formed 3 hours after Endothelin-1 application. The size of ischemic foci was measured in serial coronal brain slices after staining with 2,3,5-triphenyltetrazolium chloride (TTC). It was found that cooling the cortical surface to 28°C using a subdural Peltier element at 0, 10 and 60 minutes delay after Endothelin-1 application, caused a significant reduction in the size of ischemic focus compared to normothermic conditions. The neuroprotective effects of LTH were inversely correlated with the delay of LTH onset from the time of Endothelin-1 application and were most pronounced with LTH initiated with the shortest (0 and 10 minutes) delay after Endothelin-1 application. The size of the ischemic focus was also found to correlate significantly with the degree of electrical activity suppression analyzed in parallel paper. Taken together, the results of morphological and electrophysiological analysis indicate pronounced neuroprotective effects of surface LTH, particularly significant at minimal LTH latency after ischemic onset, in a model of Endothelin-1-induced focal ischemia in the rat cerebral cortex.

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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

G. F. Shaymardanova

Kazan Scientific Centre of RAS

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; Kazan State Medical University

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

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2. Fig. 1. Ischemic foci formed under the action of ET1, under normothermia and LTG. A series of consecutive coronal sections of the brain with an area of ​​ischemic damage formed after 3 hours of ET1 action in animals from 4 experimental groups: under normothermia (39°C) and with local superficial cooling of the cortex to 28°C, started simultaneously, 10 min and 1 hour after ET1 application. The sections are stained with TTS (stained living tissue is red, and unstained areas of ischemic damage are white) and are located in the rostro-caudal direction. The thickness of the sections is 400 μm. * sections in which electrical activity was recorded are indicated (shown at high magnification in Fig. 3).

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3. Fig. 2. Effect of local hypothermia on the volume of ischemic focus caused by ET1. (a) – volume of ischemic foci formed 3 hours after ET1 application (1 μM) under normothermia (39°C) and with superficial local cooling to a temperature of 28°C, started simultaneously, 10 min and 1 h after ET1 application. The probability of statistical differences between the groups according to the Kruskal-Wallis test (KW p) is indicated above the boxes; reliable differences between the groups were revealed according to the Dunn test (asterisk); (b) – correlation of the volumes of ischemic foci from the time of cooling start. Normothermic experiments were taken into account with a delay time of 180 min. The black dotted line shows the linear approximation. The Spearman correlation coefficient and the corresponding level of reliability are shown in the frame. NT – normothermia, HT – hypothermia.

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4. Fig. 3. Examples of ischemic foci caused by ET1 application during normothermia and LTG in the area of ​​electrical activity recording. Sections marked with * in Fig. 1, at high magnification for examples from 4 experimental groups. The boundaries of ischemic foci are outlined with a white dotted line.

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5. Fig. 4. Effect of local hypothermia on the area of ​​the ischemic lesion caused by ET1 in the area of ​​electrical activity recording. (a) – cumulative image of ischemic damage obtained by superimposing the contours of the lesions from individual experiments for the normo- and hypothermia groups. The color scale reflects the lesion probability; (b) – dependence of the lesion area on a slice from the activity recording area on the conditions of normo- and hypothermia. The probability of statistical differences between the groups according to the Kruskal-Wallis test (KW p) is indicated above the boxes; significant differences between the groups were revealed according to the Dunn test (asterisk); (c) – graph of the dependence of the area of ​​ischemic damage in slices from the recording area on the time of LTG onset after ET1 application. Normothermic experiments were taken into account with an LTG delay time of 180 min. The black dotted line shows the linear approximation. The Spearman correlation coefficient and the corresponding reliability level are shown in the frame. NT – normothermia, HT – hypothermia.

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6. Fig. 5. Cluster analysis of morphofunctional metrics of ischemic damage caused by ET1 in different temperature regimes. Graphs of the dependence of the ischemic focus area in slices from the electrical activity recording area on the degree of suppression of various parameters of electrical activity in the ischemic focus: (a) – the total frequency of spontaneous IVDs in all layers of the cortex; (b) – the amplitude of SEPs; (c) – the frequency of sensory-evoked IVDs; (d) – the power of oscillations in the theta (4–7 Hz), alpha (7–15 Hz) and beta (15–30 Hz) frequency ranges. Each point corresponds to one animal. The points are combined into clusters according to the delay of LTG after ET1 application: normothermia (without LTG) – red cluster, LTG with a delay of 0 min – blue cluster, LTG with a delay of 10 min – blue cluster, LTG with a delay of 60 min – green cluster. The columns at the top in the center indicate the values ​​of the Mahalanobis distances between the corresponding clusters and characterize the degree of remoteness of the clusters from each other. The black dotted line shows the linear regression for the data obtained in the ET1 model of ischemia without temperature control [9]. NT – normothermia, HT – hypothermia.

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