The structural base for changes in the interneuronal communication of CA3 neurons in the hippocampus of white rats after severe traumatic brain injury

Цель - изучение пирамидных нейронов поля СА3 гиппокампа белых крыс в динамике после тяжелой черепно-мозговой травмы (ТЧМТ). Методы. ТЧМТ моделировали под наркозом с помощью свободно падающего груза массой 200-250 г с высоты 50 см на теменно-затылочную область. Гиппокамп изучали в контроле (n=5), через 1, 3, 5, 7 и 14 сут после ТЧМТ (n=25). Общую оценку состояния нейронов поля СА3 проводили на препаратах окрашенных гематоксилином-эозином, численную плотность нейронов - при окраске по Нисслю, цитоскелет нейронов изучали с помощью реакции иммунотипирования нейрон-специфического структурного белка (МАР-2), синаптические терминали - иммунотипирования синаптофизина (p38). Для визуализации MAP-2 нейронов и р38 синаптических терминалей использовали мультимерный набор NovolinkTM (DAB) Polymer Detection System (Leica Biosystems Newcastle Ltd, Великобритания). Морфометрический анализ проводили на цветных растровых и бинарных изображениях с использованием плагинов программы ImageJ 1.52s. Определяли относительную площадь зон отека-набухания, численную плотность пирамидных нейронов, количество дистрофически и некробиотически измененных нейронов, общую и относительную площадь синаптических терминалей. Результаты. Через 1 сут после ТЧМТ нарастали явления отека-набухания, увеличивалось количество дистрофически и некробиотически измененных нейронов, уменьшалась общая и относительная площадь терминалей. В течение 14 сут общая плотность нейронов уменьшилась на 31%. Параллельно активировались механизмы нейро- и синаптической пластичности, в результате чего восстанавливался цитоскелет поврежденных нейронов и увеличивалось количество межнейронных синапсов (в 1,32 раза выше контроля). Заключение. Восстановление структур межнейронной коммуникации происходило на фоне уменьшения общей численной плотности пирамидных нейронов. Выявленные изменения рассматриваются как основа перманентной компенсаторно-восстановительной реорганизации межнейронных отношений гиппокампа на фоне вторичной ишемии головного мозга. Aim. To study changes in hippocampal CA3 pyramidal neurons of white rats after severe traumatic brain injury (STBI). Methods. STBI was modeled with a free-falling weight (200-250 g) impact. The hippocampus was studied in control rats (n=5) 1, 3, 5, 7, and 14 days after STBI (n=25). The CA3 field neurons were examined on preparations stained with hematoxylin-eosin and the number of neurons was determined with Nissl staining. The neuronal cytoskeleton was studied by immunotyping of the neuron-specific structural protein MAP-2, and synaptic terminals were studied by immunotyping of synaptophysin (p38). Neuronal MAP-2 and p38 were visualized with a multimeric Novolink™ (DAB) Polymer Detection Systems kit (Leica Biosystems Newcastle Ltd, Great Britain). Morphometric analysis was performed on color raster and binary images using ImageJ 1.52s plugins to determine the relative area of edema and swelling zones, number density of pyramidal neurons, content of dystrophic and necrobiotically altered neurons, and total and relative areas of synaptic terminals. Results. On the next day after STBI, manifestations of edema and swelling and the content of dystrophic and necrobiotically altered neurons were increased whereas the total and relative areas of terminals were decreased. In 14 days, the total density of neurons decreased by 31%, which was in parallel with activation of mechanisms for neuro- and synaptic plasticity. As a result, the cytoskeleton of damaged neurons recovered, and the content of interneuronal synapses increased 1.32 times compared to the control. Conclusion. The structural recovery of interneuronal communication was associated with a decrease in the total number density of pyramidal neurons. These changes were regarded as a base for permanent compensatory and restorative reorganization of hippocampal interneuronal relations in secondary cerebral ischemia.

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Morphofunctional Characteristic of Edema-Swelling of the Cerebral Cortex of White Rats After Severe Traumatic Brain Injury Without the Use of L-Lysine Escinate and Against the Background of Its Use

Russian Sklifosovsky Journal Emergency Medical Care ◽

10.23934/2223-9022-2020-9-2-251-258 ◽

2020 ◽

Vol 9 (2) ◽

pp. 251-258

Author(s):

I. P. Koshman ◽

S. S. Stepanov ◽

A. Y. Shoronova ◽

A. G. Kalinichev ◽

V. A. Akulinin ◽

...

Keyword(s):

Traumatic Brain Injury ◽

Brain Injury ◽

Severe Traumatic Brain Injury ◽

Comparison Group ◽

Main Group ◽

Nerve Tissue ◽

Relative Area ◽

Degree Of Hydration ◽

White Rats ◽

Post Traumatic

Aim of study The study is devoted to a morphometric assessment of the manifestations of edema-swelling of the somatosensory cortex (SSC) of the brain of white rats after severe traumatic brain injury (TBI) without using L-lysine escinate and when using it as a therapeutic effect.Material and methods We stained sections with hematoxylin-eosin and performed morphometric methods. On thin (4 μm) serial frontal sections of SSC, neurons and microvessels in the control (intact animals, n=5) were examined in 1 (n= 5), 3 (n=5), 5 (n=5), 7 (n=5) and 14 (n=5) days after injury without treatment (n=25, comparison group) and with treatment (n=25, main group). In color raster images (lens x100), using the plug-in filter “Find Maxima”, maximum brightness areas (MBA) were determined , which were then analyzed using the “Analyze Particles” program from ImageJ 1.52 s. MBA corresponded to SSC sites with a high degree of hydration of nerve tissue - edema-swelling. Statistical hypotheses were tested using nonparametric criteria.Results and discussion In control animals, a low degree of hydration of SSC tissue was noted (relative area 3-8%). In the comparison group, 1 and 3 days after STBI, foci of edema-swelling covered up to 30% of SSC, in 5 days - up to 15%, in 7 days - up to 20%, in 15 - up to 18%. Significant heteromorphism and heterogeneity of changes in the neuropil around neurons and blood vessels was noted. In the dynamics of the post-traumatic period, the proportion of large foci of edema-swelling (intra- and perineuronal, perivascular) decreased. In the main group, one day after STBI, there was a statistically significantly smaller number of foci of edema-swelling and their total relative area. The values range of these variables significantly decreased. L-lysine escinat affected the water balance most effectively in the acute post-traumatic period (day 1 and 3). The drug “smoothed out” the manifestation peaks (number, focal area) of edema-swelling: the values of the studied morphometric indicators were statistically significantly different. Consequently, morphometric signs of hydropic dystrophy after STBI were detected in both studied groups during the 15 days of observation.Conclusion The degree of SSC nervous tissue hydration increased after STBI. L-lysine escinate statistically significantly reduced manifestations of hydropic dystrophy. The drug significantly affected the degree of hydration of neural tissue observed in the early post-traumatic period.

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Morphofunctional Characteristics of the Hippocampus of White Rats in the Acute Period After Severe Traumatic Brain Injury During the Use of L-lysine Aescinat

Russian Sklifosovsky Journal Emergency Medical Care ◽

10.23934/2223-9022-2020-9-4-529-538 ◽

2021 ◽

Vol 9 (4) ◽

pp. 529-538

Author(s):

I. P. Koshman ◽

A. Yu. Shoronova ◽

S. S. Stepanov ◽

A. G. Kalinichev ◽

V. A. Akulinin ◽

...

Keyword(s):

Traumatic Brain Injury ◽

Brain Injury ◽

Severe Traumatic Brain Injury ◽

Nervous Tissue ◽

Free Water ◽

Maximum Brightness ◽

Acute Period ◽

Degree Of Hydration ◽

White Rats ◽

Post Traumatic

Aim of study. The study is devoted to the effect of L-lysine aescinat on the nervous tissue of the CA1 and CA3 fields of the hippocampus of the brain of white rats in the acute period after severe traumatic brain injury (TBI).Material and methods. TBI was simulated by applying a blow to the parieto-occipital region with a freely falling weight weighing 200-250 grams from a height of 50 cm using a special rail rack. The objectives of the study were: 1) comparative morphometric assessment of the degree of hydration, cyto- and glioarchitectonics of different layers of CA1 and CA3 fields after ischemia without treatment; 2) the effect of L-lysine aescinat on these indicators. We used histological (staining of sections with hematoxylin-eosin and Nissl), immunohistochemical (for NSE, MAP-2 and GFAP) and morphometric methods. On thin (4 μm) serial frontal sections of the hippocampus, neurons, astrocytes, microvessels and neuropiles were studied in control (intact animals, n=5) and 1 and 3 days after injury without treatment (n=10, comparison group) and with treatment ( n = 10, main group). The number density of neurons was determined using the Nissl staining of cells and by the reaction to NSE. The cytoskeleton of neurons was studied by detecting MAP-2, and astroglia by GFAP. On color raster images (staining with hematoxylin and eosin, x100) using the Find Maxima plug-in filter, the zones of maximum brightness were determined, which were then analyzed using Analyze Particles from the ImageJ 1.52s program. Zones of maximum brightness corresponded to areas of the hippocampus with a high degree of hydration of the nervous tissue - edema-swelling. The nature of the distribution, statistical hypotheses, and plotting were checked using Statistica 8.0 software and R environment.Results. In control animals, normochromic neurons without signs of changes in the cytoskeleton prevailed in all layers of fields CA1 and CA3, and a low degree of hydration of the nervous tissue was noted (the relative proportion of zones of maximum brightness was 5–8%). One and 3 days after TBI, there was a statistically significant increase in the focal content of dystrophic and necrobiotically altered neurons (95% confidence interval: 52–78%), manifestations of reactive gliosis were noted, and the proportion of zones of maximum brightness increased to 16%. Statistically significant layer-by-layer differences were revealed between the CA1 and CA3 fields of the hippocampus. The use of L-lysine aescinat had a statistically significant effect on the morphometric parameters of the nervous tissue of the hippocampus.Conclusion. In the early post-traumatic period after TBI, the degree of hydration of the nervous tissue of the hippocampus increased. Heteromorphicity of dystrophic and necrobiotic changes in different layers of CA1 and CA3 fields was noted. L-lysine aescinate had a statistically significant positive effect on these changes. To a greater extent, this is typical for the CA3 field. The revealed changes are considered not only as patho-, but also as sanogenetic structural mechanisms of protection and reorganization of the hippocampus in the acute post-traumatic period.1. In the acute period (1−3 days) after severe traumatic brain injury, the degree of hydration of all components of the hippocampal nervous tissue increased. In the group without treatment, 3 days after injury, the relative volume of edema-swelling zones varied from 10 to 13% in CA1 (control 3-7%) and from 8 to 16% in CA3 (control 5–10%).2. The heteromorphism of hydropic changes in the molecular layer, the layer of pyramidal neurons and the polymorphic layer was established. The maximum increase in the volume of free water (more than twofold) was characteristic of the molecular and polymorphic layer CA1, as well as the polymorphic layer CA3.3. The use of L-lysine aescinat in the acute period significantly changed the manifestations of hydropic dystrophy. One day after injury, the volume of free water increased in comparison with animals without treatment, and then, after 3 days, decreased, but remained higher than in the comparison group. The maximum effect of the drug was noted in field CA3.

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