Elizaveta I. Bon *, Maksimovich N.E.,, Zimatkin S.M.,, Kokhan N.V.,
Abstract
Target. Analysis of changes in the morphological characteristics of neurons in such phylogenetically different parts of the rat cerebral cortex in different periods after total cerebral ischemia.
Methodology. Experiments were performed on 42 male outbred white rats with an initial weight of 240±20 g. Total cerebral ischemia in outbred white rats was modeled by decapitation. The material was taken at the 1st, 5th, 15th, 30th, and 60th minutes, as well as 5 and 24 hours after decapitation.
Results. With total cerebral ischemia, a significant decrease in the size of neurons and deformation of the perikarya were observed. Normochromic neurons completely disappeared at the 60th minute. The number of hyperchromic neurons increased and then progressively decreased. Shriveled neurons accounted for the majority of cells in the studied areas of the cortex at 30-60 minutes, and then, after 5 and 24 hours, cells with pericellular edema predominated in the neuron population.
Conclusion. The obtained data on histological changes in neurons of phylogenetically different parts of the cerebral cortex in the dynamics of total cerebral ischemia provide the basis for further detailed study of post-mortem changes in the brain, determining the time of death, creating a fundamental basis for studying the properties of neurons, including their transition from one functional state to other.
Introduction
Currently, in medicine, the concept of death is based on evidence of a permanent absence of brain function. A number of methods are used to diagnose the functioning of the brain: electroencephalography, assessment of cranial nerve reflexes, and studies of cerebral blood flow. On histopathological examination, post-mortem changes include edema, hemorrhages, infarcts, necrosis, ischemic softening, wrinkling and deformation of neurons, and pycnosis of their nuclei. In the hemispheres of the cerebellum, swelling and venous plethora are often found, in the subthalamic region and the optic tubercle – an area of spotted lysis. The most typical histological change in brain death is edema of its tissues followed by rupture of blood vessels [2-12]. Previous studies on the study of morphological changes in neurons of the parietal and cortex and hippocampus in subtotal cerebral ischemia of the brain showed a decrease in the size of perikarya and an increase in the number of hyperchromic and hyperchromic shriveled neurons [6,5]. At the same time, it is of interest to quantitatively study changes in the size, shape, and degree of neuron cytoplasm chromatophilia in different periods after total experimental cerebral ischemia.
The aim was to analyze changes in the morphological characteristics of neurons in such phylogenetically different parts of the cerebral cortex (parietal cortex and hippocampus) in rats in different periods after total cerebral ischemia.
Methodology
The experiments were performed on 42 male outbred white rats with an initial weight of 240 ± 20 g in compliance with the requirements of the Directive of the European Parliament and of the Council No. 2010/63/EU of September 22, 2010 on the protection of animals used for scientific purposes. Animals were kept in an air-conditioned room (22°C) under mixed lighting on a standard vivarium diet and free access to food and water, in groups of no more than 5 individuals in a vivarium cage.
The use of rats as experimental animals is due to the similarity of angioarchitectonics and morphology of the cerebral cortex in rats and humans. Total cerebral ischemia in white outbred rats was modeled by decapitation. The material was taken at the 1st, 5th, 15th, 30th, and 60th minutes, as well as 5 and 24 hours after decapitation. After decapitation, the brain was quickly removed, pieces of the anterior part of the cerebral cortex were fixed in Carnoy's fluid. Serial paraffin sections were stained with 0.1% toluidine blue by the Nissl method and for the detection of ribonucleoproteins by Einarson.
The study of histological preparations, their microphotography, morphometry and densitometry of the chromogen sediment in histological preparations were performed using an Axioscop 2 plus microscope (Zeiss, Germany), a digital video camera (LeicaDFC 320, Germany) and ImageWarp image analysis program (Bitflow, USA). The localization of the parietal cortex and hippocampus cortex in histological preparations of the rat brain was determined using a stereotaxic atlas [12]. At least 30 neurons of the fifth layer of the parietal cortex and the pyramidal layer of the field CA1 of the hippocampus were evaluated in each animal, which provided a sufficient sample size for subsequent analysis. The number of large pyramidal neurons per unit area of sections of the cerebral cortex was determined on paraffin sections. Cells were isolated from the total number of cells according to the intensity of cytoplasm staining (chromatophilia). There were several types: normochromic – moderately colored; hyperchromic – dark; hyperchromic – very dark, with deformed perikarya; hypochromic – light colored; shadow cells are almost transparent. The number of each cell type was counted.
After a preliminary check for the normality of the distribution of indicators, the obtained data were analyzed by the methods of nonparametric statistics using the Statistica 10.0 program for Windows (StatSoft, Inc., USA). The results are presented as Me (LQ; UQ), where Me is the median, LQ is the value of the lower quartile; UQ is the value of the upper quartile. Differences between the indicators of the control and experimental groups were considered significant at p<0>
Research results
In rats with total cerebral ischemia (TCI), changes in the size (S) and shape (form factor, elongation factor) of neuronal perikarya and the degree of chromatophilia of their cytoplasm were studied at certain time intervals (5 minutes, 15 minutes, 30 minutes, 1 hour, 5 hours, 1 day) in the parietal cortex (PC) and hippocampus (Hp). After 5 minutes of TIGM, there were no changes in the size, shape, and ratio of neurons in terms of the degree of cytoplasmic chromatophilia in both studied sections of the cerebral cortex, compared with those in the control group.
At the 15th minute of TCI, there was a decrease in the size of the PC and Hp neurons – by 47% (p<0>
At the same time, compared with changes at 15 minutes of TCI, after 30 minutes of the ischemic period, S neurons decreased in PC by 51% (p<0>
By the 60th minute of TCI, in comparison with the indicators in the control group, S of PC neurons decreased by 74% (p<0>
By 5 hours of TCI, the area of perikaryons of PC neurons in experimental rats was only 1/6 part (p<0>
By 24 hours of TCI, changes in the size of neurons in both studied structures, compared with manifestations of brain ischemia, was not observed (P> 0.05).
The shape of the neurons changed already by the 15th minute of TCI. Neurons became more elongated in both studied areas – the elongation factor (elongation index) increased by 25% (p<0>0.05), and the elongation factor increased by 50
