Cytoarchitectonic features of the neocortex, archicortex and amygdala of white rats after a 20-minute occlusion of the common carotid arteries

Purpose. The study is focused on glioarchitectonics of the neocortex, archicortex and amygdala of Wistar white rats in normal state and after a 20-minute occlusion of the common carotid arteries. Materials and methods. Light (stained with hematoxylin and eosin) microscopy, immunohistochemistry (GFAP), and morphometry were used to study the distribution, shape, and area of GFAP-immunopositive brain cells in the normal range ( n = 5) and at days 1, 3, 7, 14, 30 ( n = 25) after acute ischemia. Results. Focal changes were found in the density of the glial network: decrease and increase in the local content of GFAP-positive material. Reactive, dystrophic and necrobiotic changes in neurons after acute ischemia were accompanied by reorganization of neuroglia and increase in the neuroglia index in certain zones by 1.2–1.5 times. The surface area of the particles in GFAP-positive astrocytes in sections of the neocortex in the control was 8.4–18.1, but after 3 days after ischemia this rate in some parts of the neocortex rose to 45.0–59.3%. In the hippocampus this rate was 8.1% and 16.2%, and in the amygdala it was 12.6% and 21.2%. Hypertrophy of mature astrocytes was manifested by the increase in the diameter, degree of branching and length of their processes. Conclusion. The obtained data are considered as a phenomenon of ischemic preconditioning and activation of protective processes in neuro-glio-vascular microcomplexes.

Заключение. Полученные данные рассматриваются в аспекте феномена ишемического прекондиционирования и активации защитных процессов в нейро-глио-сосудистых микрокомплексах. __________________________ godendrogliacytes and microgliacytes are regarded as an integrated cellular system providing the protection, restoration and restructuring of damaged neural tissue [4,5]. Earlier studies of neuroglia have shown that its cells provide metabolic support to neurons and play a key role in the regulation of blood flow in the areas of active brain neurons (release of a neurotransmitter). Astrocyte networks, being an analogue of the lymphatic system, perform draining and detoxifying functions in the central nervous system. These functions of astrocytes are of great importance in the acute postischemic period, when the maximum manifestations of cell swelling are revealed [5].
The effect of ischemia on brain neuroglia of experimental animals was also studied [5,6]. However, the purpose, objectives, experimental models, research methods, brain areas, and the duration of the observation period in those studies were different from those in our work. No data on comparison of the neocortex, archicortex and amygdala glioarchitectonics within 30 days after reperfusion are present. Mainly, gliocytes in the necrotic focus and perinecrotic zone were studied during long-term carotid artery ligation, i.e. irreversible changes in neurons. Thus, the details of the reorganization of glioarchitectonics in the neocortex, archicortex and amygdala have not been compared. Therefore, the existing morphological studies are insufficient for understanding the impact of global ischemia on the glioarchitectonics of these brain areas.
The purpose of this study was to carry out a comparative analysis of the structural basis of the glial cell reorganization in the neocortex, different regions of the hippocampus and nuclei of the amygdala after a 20-minute occlusion of the common carotid arteries.

MATERIAL AND МETHODS
The work was carried out at Omsk State Medical University and was approved by the local Ethics Acute 20-minute cerebral ischemia induced by common carotid artery occlusion ((CCAO), two-vessel model of incomplete global ischemia without hypotension) was modeled with premedication (atropine sulfate 0.1 mg/kg, subcutaneously) and general anesthesia (Zoletil 100, 10 mg/kg). The general condition of animals in the postoperative period was estimated in points taking into account the principles of investigating the brain physiology and pathophysiology [7].
Biopsy of the material was performed at days 1 (n = 5), 3 (n = 5), 7 (n = 5), 14 (n = 5) и 30 (n = 5) after ischemia. False-operated (without arterial occlusion) animals of the same age served as a control (n = 5). The brain was fixed by perfusion of 4% paraform solution on 0.1 M phosphate buffer (pH 7.4) through the ascending part of the aortic arch. Serial frontal sections (2-4 µm) at the level of sensorimotor cortex, hippocampus and amygdala (MT) [8] were stained with hematoxylin and eosin, and with the immunohistochemical method for glial fibrillary acidic protein (GFAP, Leica Biosystems Newcastle Ltd., UK). Verification was performed according to the recommendations of the reagent manufacturer, finishing staining was done with hematoxylin. The Leica DM 1000 microscope was used for digital microphotography (200 fields of view from each studied area for a period).
Morphometric analysis was performed on binary images using ImageJ 1.46 software. The relative area of GFAP-positive material in the field of view was determined. The nature of the distribution of the specified value was evaluated using the Kolmogorov-Smirnov criterion. Statistical hypotheses were tested using the χ2 test (StatSoft Statistica 8.0) [9]. The results were presented as a percentage (95% confidence interval -95% CI, for 200 fields of view for each reagion for a period of time, StatSoft Statistica 8.0; MedCalc 11.6.1.0). In the course of statistical analysis, the null hypothesis was rejected at p ≤ 0.05.

RESULTS
In the brain of control animals, the GFAP-positive material was localized in the bodies and processes of astrocytes between normochromic neurons (Fig. 1).
Due to local features of cytoarchitectonics and relative density of cells in the nervous tissue, the studied brain areas differed in the proportion of GFAP-positive material per one neuron. This was due to the fact that in the neuron area (layers of pyramidal cells) the minimum relative area of GFAP-positive material (cytoplasm and processes of astrocytes) per unit area of the slice was in the neocortex and CA1, the maximum -in CA4 and amygdala (MT). Statistically significant differences in paired comparison are shown in the table (Table 1).  П р и м е ч а н и е . СМК -сенсомоторная кора, ÇФ -зубчатая фасция, СА -сонная артерия, МТ -миндалевидное тело, CI -доверительный интервал. * различия статистически значимы при парном сравнении с СА1 гиппокампа и СМК (критерий χ2).
Thus, despite the uniform distribution of glial cells in normal conditions, neuroglia relations in the neocortex, hippocampus and amygdala had features associated with the density of neurons.
It was found that after 20 minutes of CCAO, diffuse reversible microfocal ischemic changes of the neurons and the corresponding local response of neuroglia in the neocortex, hippocampus and amygdala dominated. In the studied brain parts, areas with low and high density of processes were identified, that is not typical of the normal state (Fig. 2).
That is, reactive, dystrophic and necrobiotic changes in neurons after acute ischemia were accompanied by reorganization of neuroglia and increase in the neuroglia index in certain zones by 1.2-1.5 times (χ2 > 8.2; p < 0.01).
Hypertrophy of mature astrocytes was manifested by an increase in the diameter, branching degree and length of their processes (Fig. 2-4).
At the same time, the average relative area of GFAP-positive material (taking into account high and low density zones) calculated on 200 random Only in the neocortex, at days 1 and 3 after ischemia, a statistically significant difference by the Min-Max difference was revealed. This was explained by focal changes (Tables 2, 3).
Thus, using the immunohistochemical study of the GFAP distribution, common regularities and features of the glioarchitectonics of the neocortex, hippocampus and amygdala in normal conditions and N o t e . CI -95% confidence interval. * in comparison with the control, the differences are statistically significant at p ≤ 0.05 (c2 test).