Introduction

Cerebrovascular diseases remain a significant problem in modern healthcare. The primary morbidity rate for this group of diseases among the working-age population shows a tendency to increase. Annual non-fatal losses of labor potential due to cerebrovascular diseases range from 61,860 to 118,436 people [1]. In this regard, there is an active search for new methods of treating this pathology.

A key factor in the pathogenesis of vascular brain diseases is impaired perfusion. Normal perfusion of brain tissues is an essential condition for its function. Numerous studies have proven that a decrease in brain tissue perfusion leads to histopathological changes: perivascular and pericellular edema, redistribution of vascular blood filling, changes in the state of neurons and neuroglia, etc. [2,3]. The severity of these changes directly correlates with the duration of hypoperfusion and the volume of the affected vascular bed area [4]. In this connection, the correction of microcirculatory disorders represents an important component of pathogenetic therapy for a wide range of diseases.

It has currently been shown that electromagnetic radiation at frequencies of 150.176...150.664 GHz has a significant impact on processes in the microcirculation system. Previously conducted studies indicate that low-intensity electromagnetic radiation at frequencies of 150.176...150.664 GHz promotes the normalization of microcirculation in the cerebral cortex under conditions of both short-term and prolonged hypoperfusion in white rats [5,6]. Therefore, the aim of this research is to study the morphological changes in brain tissues under the action of low-intensity electromagnetic radiation at frequencies of 150.176...150.664 GHz under conditions of experimental chronic hypoperfusion in white rats.

Objects and Methods

The experimental work was conducted in accordance with the principles of bioethics and the rules of good laboratory practice (GLP). All laboratory animals were kept in a vivarium in compliance with SanPiN 3.3686-21 "Sanitary and Epidemiological Requirements for the Prevention of Infectious Diseases" along with GOST 33215-2014 "Guidelines for the Maintenance and Care of Laboratory Animals. Rules for Equipment of Premises and Organization of Procedures" and "Methodological Recommendations for the Maintenance of Laboratory Animals in Vivariums of Research Institutes and Educational Institutions" (RD-APK 3.10.07.02-09).

Experiments on animals were conducted in accordance with: Directive 2010/63/EU of the European Parliament and of the Council of the European Union of September 22, 2010, on the protection of animals used for scientific purposes, and Federal Law No. 498-FZ of December 27, 2018 (as amended on December 27, 2019) "On the Responsible Treatment of Animals and on Amending Certain Legislative Acts of the Russian Federation."

All surgical manipulations were performed under general anesthesia. For this purpose, intramuscular injection of a solution of tiletamine and zolazepam (Telazol 100 mg®, manufacturer "Zoetis Manufacturing & Research Spain, S.L." Carretera Camprodon s/n – La Riba, 17813-Valle de Bianya, Girona, Spain) at a dose of 10 mg/kg (0.1 ml/kg) body weight and xylazine hydrochloride (Xylanit®, manufacturer LLC "Nita-Farm", Russia) at a dose of 1 mg/kg (0.05 ml/kg) body weight was used.

The experimental study was performed on 34 white male rats weighing 200-220 g, which were divided into three groups. The first group (10 rats) consisted of sham-operated rats (control group). The second group included animals (12 rats) with chronic cerebral hypoperfusion (comparison group). The third group consisted of 12 rats in which a model of chronic cerebral hypoperfusion was reproduced against the background of daily 30-minute exposure to low-intensity electromagnetic radiation at frequencies of 150.176...150.664 GHz.

The model of chronic ischemia was reproduced by ligating the right common carotid artery. For this, after preparing the surgical site, a 0.6 cm incision was made along the anterior edge of the right sternocleidomastoid muscle. The skin, subcutaneous tissue, superficial fascia, and then the anterior wall of the sheath of the right sternocleidomastoid muscle were dissected. The sternocleidomastoid muscle was retracted laterally with a blunt instrument. Next, the right common carotid artery was isolated and separated from the descending branch of the hypoglossal nerve. A ligature was passed under the artery using a Deschamps needle and then tightened. At the end of the operation, the wound was sutured in layers, treated with antiseptic solutions, and an aseptic dressing was applied.

Electromagnetic waves were generated by the "Orbita" apparatus (JSC "Central Research Institute of Measuring Equipment", Saratov, Russia). An area of skin measuring 3 cm² above the xiphoid process of the sternum was irradiated, with the emitter positioned 1.5 cm above the body surface of the animal, at a radiation power of 0.7 mW and a power density of 0.2 mW/cm². Exposure to electromagnetic waves was carried out from the second day after ligation of the right common carotid artery for 6 days. A single irradiation session lasted 30 minutes.

The animals were euthanized on the 7th day after surgery by decapitation. Immediately after decapitation, the animals' brains were gently extracted from the skull, fixed by placing them in a 10% solution of neutral formaldehyde (Biovitrum, Russia), dehydrated in alcohols of increasing concentration, and then embedded in paraffin. Sections 5 µm thick were stained with Mayer's hematoxylin (Biovitrum, Russia) and eosin (Biovitrum, Russia). Bio-Mount (BioOptica, Italy) was used as the mounting medium. The specimens were examined using an AxioImager microscope (Carl Zeiss, Germany). For each group, morphological assessment was performed separately for the right and left hemispheres. Morphological description was carried out according to the accepted algorithm for describing brain microspecimens with meninges: the state of the meninges (structure of the pia mater, blood filling of its vessels, presence of edema, hemorrhages, cellular infiltration, foci of necrosis), the state of the brain parenchyma (blood filling of vessels, presence and severity of edema, presence of hemorrhages, organizing foci, state of neurons).

Results

The conducted studies established that in sham-operated animals, 7 days after the surgical intervention, no pathological changes were detected in the pia mater or the brain parenchyma. No differences in the morphological structure of the tissues of the right and left hemispheres were found (Fig. 1).

Fig. 1 Brain of sham-operated rats. Staining: hematoxylin and eosin. Magnification x63.

In chronic hypoperfusion (comparison group), on the side of the carotid artery ligation (right hemisphere), reduced blood filling of both arterial and venous vessels was noted. In the brain parenchyma of the right hemisphere, severe arterial anemia was also observed – vessels with empty lumens predominated. Most venous vessels were in a collapsed state. In some veins, phenomena of sludge and separation of blood into conglomerates of formed elements and plasma were noted. Pronounced perivascular edema was observed. Also, pericellular edema was visualized throughout the tissue of the right hemisphere. Furthermore, a significant number of dystrophically altered neurons were noted in the right hemisphere. Dystrophic changes in neurons manifested as nuclear swelling, pyknotic changes or karyorrhexis, and chromatolysis. The severity of dystrophic changes in neurons varied in different fields of view from 40 to 70% of neurons (Fig. 2).

Fig. 2 Brain of rats from the comparison group. Staining: hematoxylin and eosin. Magnification x63.

In animals with chronic hypoperfusion, in the pia mater of the contralateral (left) hemisphere, unlike the right hemisphere, arterial hyperemia was observed, and to a lesser extent, venous hyperemia. Diapedetic hemorrhages were noted. In the brain parenchyma, increased blood filling of arteries and veins was also observed. Moreover, unlike the right hemisphere, numerous single erythrocytes were visualized in the lumens of microcirculatory vessels. As in the right hemisphere, perivascular and diffuse pericellular edema were visualized in the contralateral left hemisphere. In some fields of view, single dystrophically altered neurons with signs of karyopyknosis or karyorrhexis were detected (Fig. 3).

Fig. 3. Brain of rats from the experimental group. Staining: hematoxylin and eosin. Magnification x63.

Thus, the obtained data allow us to conclude that chronic hypoperfusion significantly affects the morphology of brain tissue. This is expressed in the restructuring of the vascular bed function. On the affected side (right hemisphere), the number of functioning arterial vessels decreases. On the left side, conversely, activation of blood flow in arterial vessels and capillaries, and to a lesser extent in the venous part of the vascular bed, is noted. Such changes in blood flow in the contralateral hemisphere are likely compensatory in nature and are realized through the activation of collateral blood flow, as well as angiogenesis. In both hemispheres, pronounced diffuse edema of the brain parenchyma is detected, which probably indicates the triggering of mechanisms of ischemic damage and cascades of reactions capable of leading to cell death. This is confirmed by the presence of a significant number of dystrophically altered neurons.

In animals subjected to THF irradiation against the background of prolonged hypoperfusion, in the pia mater of the right hemisphere, unlike the comparison group, uneven blood filling of the vessels is visualized. Both moderately blood-filled arteries and vessels with empty lumens are identified. The veins of the pia mater of the right hemisphere in animals of this group, unlike rats not subjected to THF exposure against the background of prolonged hypoperfusion, are moderately filled with blood. When assessing the blood filling of vessels in the brain parenchyma of this group, unlike the comparison group, uneven blood filling of both arteries and veins was revealed. Some arterial vessels are hyperemic, some vessels are moderately filled with blood, while at the same time, individual arteries with empty lumens are noted. In some arteries, separation of blood into plasma and formed elements is observed. The veins of the right hemisphere in animals of this group predominantly have moderate blood filling; single vessels are in a collapsed state. Unlike the comparison group, in the right hemisphere of animals in this group, individual erythrocytes are visualized in the lumen of microcirculatory vessels. In the right hemisphere of animals in this group, perivascular and pericellular edema are noted, but their severity is less than in the comparison group. In the right hemisphere of the experimental group animals, individual neurons with dystrophic changes in the form of karyopyknosis, karyolysis, and chromatolysis are detected.

In animals subjected to THF irradiation against the background of chronic hypoperfusion, as well as in the comparison group, the arteries of the pia mater of the contralateral (left hemisphere) are hyperemic, and the veins have moderate blood filling. A similar picture of blood vessel filling is observed in the brain parenchyma. At the same time, in the brain parenchyma, as in the comparison group, a large number of single erythrocytes are noted in the lumen of capillaries. Mild perivascular and pericellular edema are observed. Dystrophic changes in neurons in the left hemisphere of rats subjected to THF irradiation against the background of chronic hypoperfusion are not pronounced. Overall, the left hemisphere of the animals irradiated with electromagnetic waves resembles the left hemisphere of animals from the group of rats not subjected to THF exposure against the background of chronic hypoperfusion.

Thus, low-intensity electromagnetic irradiation at frequencies of 150.176...150.664 GHz has a pronounced effect on blood flow in the right hemisphere of the brain (on the affected side). This is manifested by an increase in blood filling of arterial and venous vessels, a reduction in the severity of edema, and dystrophic changes in neurons. It should be noted that in the left hemisphere, as in the animals of the comparison group, signs of activation of collateral blood flow are observed.

Discussion of Results

The obtained data indicate that in animals with ligation of the right carotid artery, a sharp deficit in blood flow occurs in the vessels of the right hemisphere of the brain. The blood flow deficit limits the delivery of oxygen and glucose to the brain tissue. Neuroglial cells, particularly astrocytes, are especially sensitive to the lack of these substances [7]. In response to hypoxia, they release large amounts of neurotransmitters (glutamate, NO, aspartate). These compounds affect neurons, stimulating the opening of calcium channels in them. Calcium ions rush into the neuroplasm along the concentration gradient, triggering apoptosis processes. In modern literature, this chain of events is called the glutamate-calcium cascade [7]. Morphologically, its consequences are reflected in nuclear swelling, pyknotic changes, karyorrhexis, and chromatolysis. Dystrophic changes in neuronal bodies are naturally accompanied by pericellular edema. The described changes correspond to the morphological picture in rats of the comparison group.

Numerous studies have shown that low-intensity electromagnetic radiation at frequencies of 150.176...150.664 GHz promotes the activation of endothelial nitric oxide synthase (eNOS). eNOS enhances the formation of NO from L-arginine [8]. Nitric oxide is a powerful vasoactive compound. Due to its properties, redistribution of cerebral blood flow occurs, which morphologically manifests as uneven blood filling of arteries and veins, ultimately leading to a reduction in perivascular and pericellular edema.

It is also likely that nitric oxide, through feedback systems, inhibits the formation of neuronal nitric oxide synthase (nNOS) in astrocytes. This, in turn, prevents the development of the glutamate-calcium cascade and, accordingly, the development of neuronal apoptosis. Morphologically, this is manifested by a reduction in dystrophic changes such as karyopyknosis, karyolysis, and chromatolysis compared to the comparison group.

Conclusion

Thus, irradiation with low-intensity electromagnetic radiation at frequencies of 150.176...150.664 GHz in animals with modeled chronic hypoperfusion expands the compensatory capabilities of the affected tissue. This creates prerequisites for the development of new methods for the prevention and treatment of chronic cerebral circulatory insufficiency.