94 Проблеми ендокринної патології № 3, 2023 ISSN 2518-1432 (Online); ISSN 2227-4782 (Print) https://doi.org/10.21856/j-PEP.2023.3.12 ULTRASTRUCTURAL CHANGES OF THE PINEAL GLAND IN EPILEPSY* T. A. Litovchenko¹, A. E. Dubenko2, S. O. Sazonov2, V. A. Florikian¹, O. P. Zavalna¹, O. Y. Sukhonosova¹ ¹ Kharkiv National Medical University, Kharkiv, Ukraine; 2 SI «Institute of Neurology, Psychiatry and Narcology of the National Academy of Medical Sciences», Kharkiv, Ukraine t.litovchenko@yahoo.com Epilepsy as a global health problem affects about 1 % of the population. The occurrence of epilepsy appears to be due to the presence of various factors, such as neurological, percep- tual, psychological, and social, that can either cause or aggravate the disease [1]. There is no doubt about the pathogenetic role of neurotransmitter systems' disorders in the pathogenesis of epilepsy. Monoaminergic systems deserve special attention in the con- text of the problem of formation of epileptic activity. The prerequisite for this is that monoam- inergic neural systems form pathways to the neocortical parts, limbic system, cerebellum, hypothalamus; have an activating or inhibitory effect on the structures of the intermediate, an- terior and neocortical parts of the brain; have not only neurotransmitter, but also neuromod- ulatory properties; participate in the regula- tion of brain homeostasis; are determined in structures with low convulsive thresholds; in the process of metabolism in the brain, they form a large number of products characterized by neurotropic activity. The circadian rhythm of epileptic seizures was described more than 2,000 years ago, and the first modern scientific research was con- ducted in the late nineteenth century. In these early studies, approximately two-thirds of peo- ple with epilepsy showed circadian patterns of epileptic seizures (day, night). Daytime seizures are known to cluster during wakefulness or late afternoon, while nighttime seizures oc- cur frequently before falling asleep, sleeping and early in the morning before waking up [2]. This circadian pattern of seizures is usu- ally well preserved. The circadian rhythm of epileptic seizures has a huge impact on sleep (and vice versa), and the link between epilepsy and the sleep-wake cycle is being actively stu- died. Clinical and experimental observations have shown that sleep deprivation can trigger seizures [3, 4]. * The study was conducted on the authors' own initiative as part of the scientific theme 0102U002. This publication was prepared without any external source of funding’ should appear. The authors assume responsibility for the published work. The authors guarantee absence of competing interests and their own financial interest when carrying out the re- search and writing the article. The manuscript was received by the editorial staff 26.02.2023. 95Проблеми ендокринної патології № 3, 2023 Експериментальні дослідження A lot of studies have shown that epilepsy is closely related to circadian rhythms [5]. For example, Quigg (2000) reported an associa- tion with the circadian rhythm of some types of epileptic seizures in both humans and ex- periments [6]. In another study, Durazzo et al. (2008) found that most epileptic seizures that occur in the occipital and temporal lobes oc- cur during the day, while seizures that occur in the frontal and parietal lobes usually occur at night during sleep. Thus, endogenous circa- dian rhythm differently affects the manifesta- tions of epilepsy in different areas of the brain [7, 8]. Temporal-partial seizures are characte- rized mainly by the spread of adhesions during the phase of slow sleep [9]. Temporal lobe epi- lepsy is often pharmacologically resistant and the most common type of symptomatic epilepsy involving the hippocampus, entorhinal cortex, and amygdala [10]. Circadian rhythms are endogenously con- trolled, which are almost 24-hour cycles of behavioral and physiological processes, such as the sleep-wake cycle, hormone production, regulation of body temperature and blood pres- sure, and a number of other physiological indi- cators [11]. The excitability of neurons is homeostati- cally regulated by excitatory and inhibitory influences in the nervous system. Hype rex- citability, which is caused by a violation of this delicate balance, causes hypersynchronous electrical discharges of brain neurons, which can clinically manifest as epileptic seizures. It was shown that neurotransmitter receptors and ion channels are rhythmic expression and activity in the circadian regulation. Analysis of the binding of radioactive ligand by several neurotransmitter receptors in the rat brain showed that the cerebral cortex has the highest variations and the cerebellum the lowest. The hippocampus has circadian patterns of ligand- binding activity of α1-adrenergic and benzodi- azepine receptors. Changes in the rhythm of membrane excitability and electrical activity of neurons are provided mainly by changes in potassium conductivity [12, 13]. The expression of pyridoxalkinase, an enzyme involved in the metabolism of pyridoxal phosphate and neu- rotransmitters (e.g., serotonin and dopamine), is regulated by circadian transcription factors bZIP PAR [14]. Thus, circadian mechanisms modulate neuronal excitability at several le- vels, and their destruction can cause excessive uncontrolled excitability. Further studies have confirmed that the main circadian rhythm genes, BMAL1 and CLOCK, affect both the seizure threshold and excitability [15]. Melatonin is an amine hormone synthe si- zed and secreted by the pineal gland that regu- lates circadian rhythm and seasonal responses. Melatonin is the main hormone secreted by the pineal gland, which is synthesized from tryp- tophan and serotonin. Previous studies have shown that epilepsy is also associated with melatonin levels. For example, administration of exogenous melatonin can reduce the number of seizures, extend the latent period before the seizure, and reduce the severity of epilepsy [16, 17]. In a study by Mosi´nska et al. (2018) sug- gested that melatonin may control the frequen- cy and threshold of epileptic seizures, which regulates the expression of circadian rhythm genes. However, the exact mechanism underly- ing this protective effect and whether melato- nin can restore circadian rhythm gene expres- sion in epilepsy has not been fully studied. Circadian rhythm is associated with epi- lepsy through two main mechanisms. First, it is known that circadian rhythm genes (BMAL1 and CLOCK) and their complex of transcription factors, BMAL1-CLOCK, affect the expression of other genes causally involved in epilepsy (for example, PAR DBP, TEF, and HLF). It was re- ported that disabling CLOCK in excitatory py- ramidal neurons in rats led to a decrease in the seizure threshold and an increase in the num- ber of attacks during sleep. This was combined with a decrease in the formation of «dendritic spines» and changes in the electrophysiological characteristics of neural networks containing excitatory pyramidal cells. The same phenom- enon was observed in mice with BMAL1 dis- abled [18, 19]. The second mechanism is related to the mTOR pathway, which is known to be regulat- ed by circadian rhythm factors [20]. It has been shown that mutations in the genes of mTOR in- hibitors, TSC1 and TSC2, and other regulatory proteins can lead to disinhibition of the mTOR pathway, which leads to the development of sei- zures [21, 22]. 96 Проблеми ендокринної патології № 3, 2023 Експериментальні дослідження Despite a significant body of research on the role of melatonin in the development of epileptic seizures, the cause of changes in the level of this hormone in patients with epilepsy remains unknown, as well as whether these changes are persistent or a dynamic condition associated with epileptic seizures as such. Given that almost all melatonin synthe- sis occurs in the pineal gland and structural changes in this gland are an indicator of per- sistent (possibly irreversible) violation of its sec retory function, the aim of the study was to identify ultrastructural changes in the pineal gland in an experimental model of epilepsy. MATERIALS AND METHODS Histological examinations were performed on Wistar rats, aged 5–6 months and weighing an average of 200 g. The animals were kept in standard vivarium conditions on a normal diet. The 36 rats were used in the experiment, the control group consisted of 10 similar animals. Epileptic seizures were simulated by re- peated action of a sinusoidal amplitude-mod- ulated electric current with a carrier frequen- cy of 5000 Hz, a modulation frequency of 150 Hz, a modulation depth of 100 %, a strength of 50–60 mA, exposure 3c through cutaneous electrodes superimposed on the rat neck. For each animal, the threshold current strength was determined — the current strength that causes an initial epileptic reaction. Stimulation was started with a current of 35 mA, gradu- ally increasing by 10 mA until the appearance of tonic extension of the hind limbs. The rats developed spontaneous generalized seizures af- ter 9–14 stimulations. Material for histological examinations was taken after stable formation (for > 1 month) of repeated seizures. Ultrathin sections, after contrast with Reynolds lead cit- rate, were studied under an electron microscope at an accelerating voltage of 75 kV. The increase was selected adequate to the study and ranged from 20,000 to 60,000 times. The control tissue Fig. 1. Ultrastructure of pinealocytes (pinealocyte nuclei) of the epiphysis of rats with experimental epilepsy. The increase is 31000. Contrasted with lead citrate. 97Проблеми ендокринної патології № 3, 2023 Експериментальні дослідження Fig. 2. Ultrastructure of pinealocytes (invagination of the nuclear envelope) of the pineal gland of rats with experimental epilepsy. The increase is 30,000. Contrasted with lead citrate. was taken from intact animals under similar conditions. Electron microscopic examination of the pineal gland of rats with experimental epilepsy in comparison with the control group of animals revealed dystrophic and destructive changes of organelles and membranes. Experiments on animals in this research are approved by authors’ affiliations institu- tions. When working with animals, we followed international and Ukrainian recommenda- tions for working with experimental animals (Counsil for International Organizations of Medical Sciences (CIOMS) (1985); Guide for the care and use of laboratory animals 1996) [23, 24]. RESULTS AND THEIR DISCUSSION The nuclei of pinealocytes (Fig. 1) were large with evenly distributed chromatin granu- les. They contain rather large nucleoli. The matrix of nuclei was significantly enlightened. However, the nuclear membrane was clearly outlined and had small areas of destruction. It formed shallow intussusception. In the peri- nuclear zone of the cytoplasm, the number of organelles decreased, and this zone acquired electronic transparency. A significant number of pinealocytes contained nuclei, the nuclear shells of which formed very deep intussuscep- tion (Fig. 2). Chromatin in such nuclei was lo- cated in the form of accumulations of granules along the nuclear membrane. The nuclear mem- brane was not subject to significant changes. The cytoplasm of individual pinealocytes, the number of which is small, had a significant dilution (Fig. 3). The perinuclear zone of the cytoplasm had practically no organelles. The number of mitochondria and cisterns of the en- doplasmic reticulum is significantly reduced. Free ribosomes and polysomes are absent. Hyaloplasm had a low electron density. Few mitochondria contained a small number of cry- stals. The mitochondrial matrix had a coarse 98 Проблеми ендокринної патології № 3, 2023 Експериментальні дослідження fibre structure. Focal lysis of membrane struc- tures was very often observed. A significant number of pinealocytes con- tained a large number of mitochondria, with a fine-grained matrix, the size of which varied widely (Fig. 4). In general, mitochondria had a rounded shape, but there were also rod-shaped. At the same time, sometimes, electronically transpar- ent zones were found in the matrix. Some mito- chondria had almost no cristae. The agranular endoplasmic reticulum was moderately vacuo- lated, and its tanks contained a substance of very low electron density. The reduction in the number of ribosomes and polysomes in the cy- toplasm is noteworthy. Sometimes there are pinealocytes that have mitochondria of various shapes and sizes, the main feature of which is the location of crysts along the axis of the organelle, as well as the appearance of electron-transparent vacuoles in the mitochondrial matrix (Fig. 5). The lamellar cytoplasmic Golgi complex (Fig. 6) was significantly hypertrophied, the parallel arrangement of its smooth membranes is broken. The vacuoles of the Golgi complex be- came very large. Autophagosomes were almost always present in the area of localization of the latter. The cytoplasmic membrane of pinealocytes was very loose and lost the typical structure of the elementary membrane. There is a signifi- cant increase in its osmophilicity. In addition, individual apoptotically altered pinealocytes were found in the epiphysis tis- sue in experimental rats with epilepsy (Fig. 7). The cytoplasm of such pinealocytes has a fair- ly high electron density. The nuclei contain electron-dense chromatin and are significant- ly reduced in size. Numerous ribosomes and polysomes are present in the cytoplasm. It is characterized by a significant polymorphism in the size and shape of numerous mitochon- dria, which contain a large number of clearly Fig. 3. Ultrastructure of pinealocytes (cytoplasmic dilution) of the epiphysis of rats with experimental epilepsy. The increase is 26000. Contrasted with lead citrate. 99Проблеми ендокринної патології № 3, 2023 Експериментальні дослідження contoured crystals and have a well-contoured outer membrane. There is a gradual absorp- tion of cellular organelles by a large number of autophagosomes. The cytoplasmic membrane retains its integrity, but significantly changes its configuration and has deep intussusception. Thus, the cell in the apoptosis stage retains functionally active organelles, which ensure the process of utilization of cytoplasmic struc- tures. Thus, under the conditions of modeling epi- lepsy in experimental rats, dystrophic changes in pinealocytes were detected, which are struc- turally manifested in sharp mitochondrial ede- ma with a decrease in the number of crysts, partial destruction of external membranes and crysts of mitochondria, vacuolization of cis- terns of the agranular endoplasmic reticulum, a decrease in the number of ribosomes, as well as enlightenment of the matrix of the nucle- us. These changes indicate a violation of the synthetic function of pinealocytes and energy processes. Violation of the protein-synthesizing function of pinealocytes is confirmed by the detected reduction of the lamellar cytoplasmic Golgi complex. As a result, catabolic processes develop at the intracellular level, the structural manifestation of which is the presence of au- tophagosym and lipid inclusions in the cyto- plasm. A small number of cells had apoptotic changes with intact membranes and function- ally active organelles that provided the process- es of utilization of cytoplasmic structures. The study of the control group's material did not reveal such changes. Experimental studies have confirmed the presence of persistent morphological changes in the pineal gland in epilepsy, which is the rea- son for the decrease in its functional activity. In recent decades, a number of systemic functions of melatonin have been established, largely determining the vital functions of the body. These aspects of melatonin are usually well known [25]. Despite sufficient work on the role of mela- tonin in epileptogenesis, the results are often Fig. 4. Ultrastructure of pinealocytes (mitochondria) of the epiphysis of rats with experimental epilepsy. The increase is 32000. Contrasted with lead citrate. 100 Проблеми ендокринної патології № 3, 2023 Експериментальні дослідження Fig. 5. Ultrastructure of pinealocytes (structure of mitochondria) of the pineal gland of rats with experimental epilepsy. The increase is 38000. Contrasted with lead citrate. divergent, obtained on different clinical and experimental material and are not subject to systematic analysis and comparison, although the participation of melatonin in the formation of epileptic activity is not in doubt. Our experi- mental data on the morphological changes of the pineal gland confirm the conclusion about a stable, possibly irreversible, decrease in gland function in epilepsy due to damage and disrup- tion of the structure of its cells. Opinions about the anticonvulsant proper- ties of melatonin are contradictory. Guo et al. (2009) reported that serum melatonin levels were reduced in children with epilepsy and febrile seizures and recommended the use of melatonin as adjunctive therapy [26]. Another study presented data from patients with re- sistant temporal lobe epilepsy, which showed a decrease in melatonin in the interictal period and its increase by 3 times after seizures. The authors believe that melatonin has anticonvul- sant properties [27] and such an increase is a compensatory response. There is evidence of decreased melatonin levels in patients with nocturnal and diurnal focal seizures. Molina- Carballo et al. in their study showed that the level of melatonin in the serum increased du- ring seizures and returned to normal values after 1 hour and concluded that such an in- crease in melatonin secretion may represent the body's response to seizures and aimed at achieving homeostasis [28]. Schapel et al. (1995) concluded that mela- tonin production increases in untreated pa- tients with active epilepsy and has a circadian model with phase differences compared to con- trols [29]. But there is the opinion of a number of authors who deny the significant role of chan- ges in melatonin levels in the development and relief of epileptic seizures [30]. Other studies have shown sufficient efficacy of exogenous melatonin as adjunctive therapy in incurable epilepsy [31, 32]. 101Проблеми ендокринної патології № 3, 2023 Експериментальні дослідження Fig. 6. Ultrastructure of pinealocytes (Golgi complex) of the epiphysis of rats with experimental epilepsy. The increase is 37000. Contrasted with lead citrate. Our studies of the ultrastructure of the pineal gland confirm a steady decrease in its functional activity in epilepsy. Most likely, this is due to the depletion of compensatory mecha- nisms in the conditions of epileptic brain. This opinion is indirectly confirmed by the role of melatonin in the physiology of mitochondria as a protector of neuronal mitochondrial oxi- dative phosphorylation under toxic effects and under conditions of hypoxia-reoxygenation [33]. In addition, melatonin is a unique regulator of apoptosis and neuroplasticity [34]. This is espe- cially important given the widespread presence of melatonin receptors in the CNS and, espe- cially, in the hippocampus (CA3 field, tradition- ally plays a leading role in epileptogenesis) [35]. Melatonin plays a role in Ca2+ homeosta- sis, and it inhibits the increase in Ca2+ caused by acidification and prevents the increase in Ca2+ caused by glutamate in the cerebral cortex of rats and acts as an antiexatotoxic and an- ti-inflammatory molecule in neurons [36, 37]. Melatonin also increases the neuroplasticity of the hippocampus [38]. CONCLUSIONS The established morphological changes of the pineal gland indicate stable and possibly irreversible changes in the gland, which is ac- companied by a significant decrease in mela- tonin production. Given the polyfunctionality of this hormone, its deficiency can be one of the causes of a number of metabolic disor- ders, changes in neural networks, impaired permeability of synaptic membranes, and as a consequence, increased epileptic activity of neurons. Further activation of free radical pro- cesses, energy deficiency, hemodynamic disor- ders in epilepsy contribute to an increase in melatonin deficiency, depletion of the function- al reserves of the pineal gland and the death of melanocytes. 102 Проблеми ендокринної патології № 3, 2023 Експериментальні дослідження Fig. 7. Ultrastructure of pineal pinealocyte rats with experimental epilepsy. The increase is 29000. Contrasted with lead citrate. The epileptic seizures, from a neurophysio- logical point of view, can be considered as a vio- lation of the physiological bioelectrical activity of neurons, their rhythm. 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Life Sci 2014;100(2): 97-109. doi: 10.1016/j.lfs.2014.01.085. 37. Hardeland R. Int J Molec Sci 2019;20(5): 1223. doi: 10.3390/ijms20051223. 38. Domínguez-Alonso A, Valdés-Tovar M, Solís-Chagoyán H, Benítez-King G. Int J Molec Sci 2015;16(1): 1907- 1927. doi: 10.3390/ijms16011907. ULTRASTRUCTURAL CHANGES OF THE PINEAL GLAND IN EPILEPSY T. A. Litovchenko¹, A. E. Dubenko2, S. O. Sazonov2, V. A. Florikian¹, O. P. Zavalna¹, O. Y. Sukhonosova¹ ¹ Kharkiv National Medical University, Kharkiv, Ukraine; 2 SI «Institute of Neurology, Psychiatry and Narcology of the National Academy of Medical Sciences», Kharkiv, Ukraine t.litovchenko@yahoo.com Circadian mechanisms modulate neuronal excitability at several levels, and their destruction can cause excessive uncontrolled excitability. Previous studies have shown that epilepsy is associated with melatonin levels. Melatonin is a powerful chronobiotic secreted from the pineal gland; helps to maintain normal circadian rhythms and is used to treat some neurological and psychiatric disorders. Circadian rhythms and sleep/wake mechanisms play role in the epilepsy. Melatonin deficiency can decrease seizure threshold, and hence could in- crease seizure activity. The aim of the study was to identify ultrastructural changes in the pineal gland in an experimental model of epilepsy. Materials and methods. The experiment was carried out on Wistar rats, 5–6 months of age, weight of about 200 g, 36 experimental and 10 control animals. Electrically provoked experimental epileptic seizures model in rats was reproduced. The rats developed spontaneous generalized seizures after 9–14 stimulations. Material for histological examinations was taken after stable formation (for > 1 month) of repeated seizures. 104 Проблеми ендокринної патології № 3, 2023 Експериментальні дослідження Ultrathin sections, after contrast with Reynolds lead citrate, were studied under an electron microscope at an accelerating voltage of 75 kV. The increase was selected adequate to the study and ranged from 20,000 to 60,000 times. Results. Experimental studies have confirmed the presence of stable dystrophic, up to apoptosis of some cells, morphological changes of the pineal gland in the experimental model of epilepsy in rats, which is the rea- son for the decrease in its functional activity. Conclusions. Our experimental data on the morphological changes of the pineal gland confirm the conclu- sion about a stable, possibly irreversible, decrease in gland function in epilepsy due to damage and disruption of the structure of its cells. Given the polyfunctionality of melatonin, its deficiency can be one of the causes of changes in neural networks, impaired permeability of synaptic membranes, and as a consequence, increased epileptic activity of neurons. K ey wor d s : epilepsy, experimental epileptic seizures, pineal gland, melatonin. УЛЬТРАСТРУКТУРН² ЗМ²НИ ШИШКОПОД²БНО¯ ЗАЛОЗИ ПРИ ЕП²ЛЕПС²¯ Літовченко Т. А.¹, Дубенко А. Є.2, Сазонов С. О.2, Флорікян В. А.¹, Завальна О. П.¹, Сухоносова О. Ю.¹ ¹ Харківський національний медичний університет, м. Харків, Україна; 2 ДУ «Інститут неврології, психіатрії та наркології Національної академії медичних наук України», м. Харків, Україна t.litovchenko@yahoo.com Циркадні механізми модулюють нейрональну збудливість на кількох рівнях, їх руйнування може викликати надмірну неконтрольовану збудливість. Попередні дослідження показали, що епілепсія пов'язана з рівнем мелатоніну. Мелатонін є потужним хронобіотиком, що виділяється шишкоподібною залозою; допомагає підтримувати нормальний циркадний ритм і використовується для лікування деяких неврологічних і психіатричних розладів. Циркадні ритми та механізми сну/неспання відігра- ють роль у розвитку епілепсії. Дефіцит мелатоніну може знизити судомний поріг і, отже, збільшити судомну активність. Метою дослідження було виявлення ультраструктурних змін епіфіза на експери- ментальній моделі епілепсії. Матеріали та методи. Експеримент проводили на щурах лінії Вістар віком 5–6 місяців, масою близько 200 г, 36 піддослідних і 10 контрольних тварин. Було відтворено модель експерименталь- них епілептичних нападів, спровокованих електричним струмом, на щурах. Після 9–14 стимуляцій у щурів розвивалися спонтанні генералізовані напади. Матеріал для гістологічного дослідження брали після стійкого формування (протягом > 1 місяця) повторних нападів. Ультратонкі зрізи після кон трастування цитратом свинцю за Рейнольдсом досліджували під електронним мікроскопом при прискорювальній напрузі 75 кВ. Збільшення було обрано адекватним дослідженню і коливалося від 20 000 до 60 000 разів. Результати. Експериментальними дослідженнями підтверджено наявність стійких дистрофіч- них, аж до апоптозу деяких клітин, морфологічних змін епіфіза при експериментальній моделі епі- лепсії у щурів, що є причиною зниження його функціональної активності. Висновки. Отримані експериментальні дані про морфологічні зміни шишкоподібної залози під- тверджують висновок про стійке, можливо незворотне, зниження функції залози при епілепсії вна- слідок пошкодження і порушення структури її клітин. Враховуючи поліфункціональність мелатоніну, його дефіцит може бути однією з причин змін нейронних мереж, порушення проникності синаптичних мембран і, як наслідок, підвищення епілептичної активності нейронів. К л ю ч о в і с л о в а : епілепсія, експериментальні епілептичні напади, шишкоподібна залоза, мелатонін.