Ultrastructural reorganization of the constituent components of the rat periodontium under conditions of chronic opioid exposure

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Introduction
Opioid addiction has acquired the characteristics of an epidemic in recent years and is a serious problem in modern medicine [6,18,19]. Chronic use of narcotic substances leads to addiction, when there is a need for higher doses, after all, the constant and long-term use of opioids in 23 % of people leads to the development of opioid addiction [1,19]. In view of this, experimental models for the purpose of studying the action of opioids, namely, the study of their analgesic potential, side effects, behavioral reactions, as well as their effects on various organs and systems, are of increasing interest recently [7,8,12,17].
It should be noted that among users of narcotic substances, a significant prevalence of diseases of the oral cavity is noted, in comparison with the general population [9,11,20]. Taking into account the fact that the constituent components of the periodontium react quite distinctly to the action of various factors and homeostasis disturbances, the study of pathological changes in the oral cavity of addicted persons is relevant [21,23]. The researchers note that comprehensive knowledge about the condition of the organs and tissues of the oral cavity in opioid-dependent persons can ensure the effectiveness of diagnostic and therapeutic measures [5,10,22].
Analysis of modern scientific data shows that the problem of the pathogenesis of inflammatory and dystrophic periodontal diseases remains unsolved [4,14,15]. The reflection of the pathomorphological picture in the periodontal tissues is a rather complex process, and changes occur after a certain period of time with the involvement of various mechanisms of pathogenesis [2,3,13,16]. In this regard, the study of pathomorphological changes at the ultrastructural level in the organs of the oral cavity, periodontal tissues, which develop against the background of the longterm effect of opioid agents, is undoubtedly an urgent problem today.
The aim of the study was to find out the peculiarities of the submicroscopic organization of the tissues of the periodontal complex at the end of the fourteenth week of the experimental action of the opioid analgesic nalbuphine.
All animals were kept in a vivarium and work on keeping, care, labeling and all the other manipulations were carried out in compliance with the provisions of the European Convention for the Protection of Vertebrate Animals used for experimental and other scientific purposes (Strasbourg, 1985). The Commission on Bioethics of Danylo Halytskyi Lviv National Medical University has stated that the study meets ethical requirements in accordance with the order of the Ministry of Health of Ukraine No. 231 of 01.11.2000 (Protocol No. 10 of 24.05.2021).
For electron microscopic examination, pieces of periodontal soft tissue in the area of the gingival margin of the jaws were used, which were immediately immersed in a large drop of 2.5 % glutaraldehyde solution diluted in 0.1 M phosphate buffer (pH 7.36) with sucrose. Pieces were cut from the strips, tissue blocks were transferred to tubes with a fixing solution of the same composition. Subsequently, tissue blocks were fixed with a 2 % solution of osmium tetroxide in 0.1 M phosphate buffer (pH 7.36) with the addition of sucrose. For dehydration and preparation for impregnation with water-insoluble resins, fabric blocks washed from the remains of fixatives were passed through alcohols of increasing concentration. Fabric blocks were immersed in epon-araldite by passing through solutions of increasing resin concentration. Ultrathin sections were prepared on a УМТП-3M ultramicrotome using glass knives. For the study, sections of silver or gently lemon color were selected and contrasted in a 2 % solution of uranyl acetate, and then lead citrate. Studying and photographing the material was carried out with the help of an electronic microscope ПЕМ-100-01 (Ukraine) at magnifications of 1500x -20000x on the microscope screen.

Results
Electron microscopic examination of rats of the I (control) group revealed layer-by-layer arrangement of gingival epithelium cells, clear and even contours of nuclei, and a low density of organelles in the cytoplasm were established. Ordered bundles of collagen fibers, intercellular substance and cells of the fibroblastic series are visualized in the periodontium. In the hemomicrovessels, a clear organization of endothelial cells, intercellular contacts, and small perivascular spaces containing constituent components of connective tissue has been established.
At the ultrastructural level, after 14 weeks of opioid exposure, significant changes were found in the studied periodontal components of laboratory animals. In the epitheliocytes of the basal layer of the free part of the gums, uneven contours of the nuclei are determined, the karyolemma forms rather deep intussusceptions, and karyorrhexis phenomena are observed in some places. The perinuclear space is of uneven width, in some places significantly expanded. Nucleoli are not detected in the electron light karyoplasm. In some areas, the contours of the plasmalemma are vaguely traced. Violation of the integrity of intercellular contacts, their significant reduction in individual areas was revealed. In places, intercellular contacts are visualized in the form of osmiophilic clusters. The intercellular spaces are mostly expanded and uneven, which is caused by edematous phenomena due to excessive fluid accumulation. In the karyolemma of the spinous layer of cells of the epithelium of the free part of the gums, deep intussusceptions were found, in the karyoplasm there are segregated components of nucleoli (Fig. 1). Vacuole-like formations are observed in structureless illuminated areas of the cytoplasm, which is a sign of the development of partial focal cell necrosis. Organelles in the cytoplasm are mostly damaged, destroyed cristae and a lighted matrix are found in swollen mitochondria. Bundles of tonofilaments have vague contours, mostly thickened. Electron-dense inclusions of keratohyalin of various sizes are visualized in the cytoplasm of the granular layer of epitheliocytes of the free part of the gums.
The contours of the nuclei of the epithelium of the gingival sulcus are uneven, the karyolemma forms multiple intussusceptions. Nucleoli are not detected in the illuminated karyoplasm. Organelles in the cytoplasm of epitheliocytes of the basal layer are destructively changed. In most of the mitochondria, lightening of their matrix and damaged cristae were found, vacuolization of mitochondria was observed in some places (Fig. 2). Destructive changes of tonofilaments are determined, where signs of their fragmentation are revealed, in some places they are lysed. In significant areas, intercellular contacts are not clearly visualized, most are reduced, which indicates the phenomenon of acantholysis. Intercellular spaces are mostly uneven and significantly expanded. In the cytoplasm of cells of the spinous layer, osmiophilic inclusions are observed, which is caused by increased keratinization of the epithelial plate in the area of the gingival sulcus of white rats.
In the epithelial cells of the attached part of the gums, apoptotic phenomena are observed, as well as structureless, illuminated areas of the cytoplasm, lysosomes, and osmiophilic inclusions (Fig. 3). The development of necrotic changes is caused by significant damage and destruction of organelles, as well as fragmentation or lysis of bundles of tonofilaments. Small pyknotic nuclei with uneven contours and heterochromatin in the karyoplasm are found in some of the epitheliocytes of the attached part of the gums. The karyolemma is mainly osmiophilic, the phenomena of karyorrhexis, in some places -karyolysis, as well as signs of destruction of karyoplasm are observed. Violation of intercellular contacts, their indistinctness and uneven expansion of intercellular spaces were revealed.
Ultrastructural studies demonstrated pronounced changes in periodontal components. Disruption of the orderly arrangement of collagen fibers and destructive changes, in particular, their fragmentation, destruction of fibers in places, were found in the bundles. The illumination of the amorphous component of the intercellular substance of the connective tissue is determined (Fig. 4).
Submicroscopic organization of fibroblasts and fibrocytes changes significantly, macrophages are activated. In fibroblasts, altered nuclei and damaged organelles in the cytoplasm are identified. Signs of karyopyknosis in fibrocytes, karyorrhexis phenomena in places. There is an expansion of the perinuclear space, indistinct contours of     Heterochromatin is determined on a significant area of osmiophilic karyoplasm. In the cytoplasm, organelles are mostly destructively changed, lysosomes, vacuole-like formations, expanded and fragmented tubules of the granular endoplasmic reticulum, and unevenly thickened cisterns of the Golgi complex are observed. Swollen mitochondria with destroyed cristae and lightened matrix were found (see Fig. 4).
Submicroscopically, significant changes were found in the links of the hemomicrocirculatory channel of the mucous membrane of the gums. In the wide lumens of the hemocapillaries, the formed elements of the blood, mostly erythrocytes, are determined. The phenomenon of "sludge phenomenon" is observed. Endotheliocyte nuclei are destructively altered, with signs of karyopyknosis and karyorrhexis. Numerous deep intussusceptions of the karyolemma and dense accumulations of heterochromatin in the karyoplasm were revealed. Destroyed organelles and few pinocytotic vesicles can be found in the cytoplasm. In some areas, vacuole-like structures are visualized. The basal membrane is vaguely contoured, in some places it is significantly thickened. Perivascular spaces are expanded, where thickened bundles of collagen fibers are determined, which is caused by the development and progression of sclerotic changes (Fig. 5).
In the venules, the signs of dilatation, hemoptysis and "sludge phenomenon" predominate. n the lumen of the venules, erythrocytes are identified, places where platelets accumulate. In the nuclei of endotheliocytes, osmiophilic karyoplasm with accumulation of heterochromatin, karyopyknosis and uneven contours of karyolemma are visualized (Fig. 6). The perinuclear space is expanded. Brightened areas, electron-dense inclusions, vacuoles, as well as damaged organelles and vacuolated mitochondria are observed in the cytoplasm, which is a sign of cytolysis. The adluminal surface of endotheliocytes is focally exfoliated in some areas and protrudes into the lumen of venules. The basal membrane thickens significantly, its contours are indistinct in places. Collagen fibers were found in significantly expanded perivascular spaces.

Discussion
The results obtained by us of the electron microscopic study of the constituent components of the periodontium against the background of chronic opioid exposure are compared with the data of the ultrastructural study in patients with periodontopathies. In particular, with generalized periodontitis, researchers submicroscopically detected destroyed contacts between epitheliocytes and signs of spongiosis in the layers of the gingival epithelium, the formation of which was caused by the penetration of serous fluid into the intercellular spaces, which had a significant volume [2,13,16]. The destruction of mitochondrial cristae and vacuolization of their matrix, the phenomena of apoptosis, cell nuclei acquired an irregular shape and lost nucleoli and chromatin were also noted [3]. In individual cells of the basal layer of the gingival epithelium, the researchers identified foci of destruction of the cell membrane [2,16].
In our studies, we also observed fragmented or lysed bundles of tonofilaments, significant damage and destruction of organelles, phenomena of karyorrhexis and vacuole-like formations in illuminated areas of the cytoplasm, which is a sign of the development of partial focal necrosis. According to the researchers, in patients with generalized periodontitis, the lumen of hemocapillaries was filled with erythrocytes and platelets. The basal membrane is loose, in the cytoplasm of endotheliocytes, areas of lightening, mitochondria with disorganized cristae were determined. Nuclei of irregularly shaped endotheliocytes with karyolemma intussusception, destructive nucleoli were present, which indicated damage to the hemomicrocirculatory channel of the periodontium, causing ischemia of the gingival mucosa [3].  In patients with generalized periodontitis with accompanying cardiovascular pathology, obturation of the lumen of vessels of the gingival mucosa, desquamation of the endothelium, apoptosis of endotheliocytes, and obturation of blood vessels with formed blood elements were noted, which indicated the development of local endothelial dysfunction [13]. The given data in patients with periodontitis are compared with the results obtained by us of the study of microvessels under the experimental action of opioid, where submicroscopically in the lumen of the hemocapillaries of animals, the phenomenon of "sludge phenomenon", destructively changed nuclei of endotheliocytes, with signs of karyopyknosis, and damaged organelles and vacuole-like structures were found in the cytoplasm, which indicated the progression of hypoxia. 2. With the long-term effect of an opioid analgesic, signs of karyorrhexis, segregation of nucleoli, spongiosis, acantholysis, apoptosis, as well as vacuole-like formations, which are a manifestation of focal necrosis of epitheliocytes, were revealed. Fragmentation and destruction of periodontal collagen fibers, destructive changes in fibrocytes and fibroblasts were found in the periodontium.
3. Ultrastructural reorganization of the cellular components of the periodontium is caused by vascular disorders of a systemic nature, which are manifested by the phenomena of dilatation, hemoptysis, sludge phenomenon, dystrophic changes in the endotheliocytes of hemocapillaries and venules, and the progression of sclerotic changes, which significantly disrupts the trophism of rat periodontal tissues.