Apoptosis indices in liver cell mitochondria in B16/F10 melanoma growing in presence of chronic neurogenic pain

Введение. Печень по количеству, плотности митохондрий один из самых богатых органов, который также является критическим местом для множества метаболических путей. Цель исследования - изучение показателей апоптоза в митохондриях печени самок мышей линии С57ВL/6 при самостоятельном росте меланомы В16/F10 и на фоне коморбидной патологии - хронической нейрогенной боли. Методика. В эксперименте использовали мышей-самок (n=168) линии С57ВL/6. Группы: интактная (n=21); контрольная (n=21) - создание модели хронической нейрогенной боли (ХНБ), путем двусторонней перевязки седалищных нервов; группа сравнения (n=63) - подкожная трансплантация меланомы B16/F10; основная группа (ХНБ+B16/F10) (n=63) - подкожная трансплантация меланомы В16/F10 через 3 нед после моделировия ХНБ. В митохондриях печени методом ИФА определяли концентрацию: цитохрома С (нг/г белка), каспазы 9 (нг/г белка), Bcl-2 (нг/г белка), AIF (нг/г белка), кальция (Са 2+) (мМоль/г белка). Результаты. В митохондриях клеток печени через 1 нед роста меланомы относительно интактных значений фиксировали нарастание уровней AIF в 2,2 раза, цитохрома С в 1,7 раза (р<0,05) и снижение каспазы 9 в 2,0 раза; через 3 нед - падение кальция в 4,7 раза, AIF в 7,1 раза и цитохрома С в 1,7 раза (р<0,05) и накопление каспазы 9 - 1,6 раза (р<0,05). Развитие опухоли при ХНБ через 1 нед сопровождалось уменьшением концентрации AIF в 29,3 раза и цитохрома С в 2,0 раза по сравнению с контрольными значениями (ХНБ). Через 3 недели роста меланомы на фоне ХНБ фиксировали снижение уровней AIF в 6,6 раза, цитохрома С в 4,7 раза и кальция в 32,8 раза, уровень каспазы 9, напротив, повышался в 1,5 раза (р<0,05). Заключение. Наличие коморбидной патологии - ХНБ при опухолевом процессе способствует раннему возникновению нарушений в электронно-транспортной цепи митохондрий клеток печени. Background. The liver is one of the richest organs in terms of the number and density of mitochondria; it is also a critical site for many metabolic pathways. The aim of the study was to analyze indicators of apoptosis in liver mitochondria in female С57ВL/6 mice with B16/F10 melanoma growing alone and in presence of chronic neurogenic pain. Methods. Female С57ВL/6 mice (n=168) were studied. Animals were divided into groups: intact group (n=21); controls (n=21) with a model of chronic neurogenic pain (CNP) created by bilateral sciatic nerve ligation; comparison group (n=63) with subcutaneous transplantation of B16/F10 melanoma; main group (CNP+B16/F10) (n=63) with subcutaneous transplantation of B16/F10 melanoma 3 wks after modeling CNP. Cytochrome C (ng/g protein), caspase-9 (ng/g protein), Bcl-2 (ng/g protein), AIF (ng/g protein), and calcium (Ca2+) (mmol/g protein) were measured by ELISA in the liver mitochondrial fraction. Results. After 1 wk of melanoma growth, AIF increased by 2.2 times, cytochrome C increased by 1.7 times (p<0.05), and caspase-9 decreased by 2.0 times compared to the intact group values. After 3 wks, calcium decreased by 4.7 times, AIF by 7.1 times, cytochrome C by 1.7 times (p<0.05), and caspase-9 increased by 1.6 times (p<0.05). After 1 wk, tumor development in the presence of CNP was accompanied by decreases in AIF by 29.3 times and cytochrome C by 2.0 times, compared to control CNP values. After 3 wks of melanoma growth in presence of CNP, AIF decreased by 6.6 times, cytochrome C by 4.7 times, and calcium by 32.8 times. Caspase-9, on the contrary, increased by 1.5 times (p<0.05). Conclusions. The presence of CNP comorbidity during the tumor development facilitates earlier occurrence of disorders in the electron transport chain of hepatocyte mitochondria.

Historical aspects of the problem of treatment of trigeminal neuralgia and the role of neurosurgical methods in its solution (literature review)

The trigeminal nerve is a mixed fifth cranial nerve, consisting of motor and sensory components. The sensitive component receives somesthetic information from the skin and mucous membranes of the face into the central nervous system, and the motor component is responsible for the innervation of chewing muscles. One of the manifestations of the pathology of the trigeminal nerve is pain syndrome. Trigeminal neuralgia occupies the main place among neurogenic pain syndrome in the face, is characterized by а severe course and the absence of sufficiently effective methods of treatment. According to the World Health Organization (WHO), the prevalence of trigeminal neuralgia in different countries is 2–5 cases per 100 thousand people per year. Trigeminal neuralgia is classified into 3 etiologic categories. Idiopathic trigeminal neuralgia occurs without apparent cause. Classical trigeminal neuralgia is caused by vascular compression of the trigeminal nerve root. Secondary trigeminal neuralgia is the consequence of a major neurologic disease, e. g., a tumor of the cеrеbеllоpоntine angle or multiple sclerosis. Today, there are many different options for the surgical treatment of trigeminal neuralgia. microvascular decompression of the root, radiosurgical destruction of the Gasser’s node, radiofrequency destruction, glycerol rhizotomy, balloon microcompression are considered the main effective and proven surgical methods for treating trigeminal neuralgia. But the questions of diagnosing the cause of the disease and choosing an adequate surgical method for treating therapeutically resistant trigeminal neuralgia for a particular patient remain open. The development of surgical methods begins from ancient times to the present day. The main stages in the development of neurosurgical treatment methods are presented. The following surgical techniques are described: open method – microvascular decompression, and closed percutaneous destructive methods – radiofrequency destruction, glycerol rhizotomy, balloon compression, radiosurgery, cryodestruction, laser destruction, botulinum toxin injections.

Functional state of cardiomyocyte mitochondria in malignant process in presence of comorbid pathology in experiment

Purpose of the study. An analysis of indices of free radical oxidation and respiration of mitochondria of heart cells in a malignant process in presence of diabetes mellitus and chronic neurogenic pain in experimental animals.Materials and methods. The study included outbred female rats (n=32) and С57ВL/6 female mice (n=84). Experimental groups of rats were: intact group 1 (n=8), control group 1 (n=8) with diabetes mellitus (DM), comparison group 1 (n=8) with standard subcutaneous transplantation of Guerin’s carcinoma, main group 1 (n=8) with Guerin’s carcinoma transplanted after 1 week of persistent hyperglycemia. Experimental groups of mice were: intact group 2 (n=21), control group 2 (n=21) with a model of chronic neurogenic pain (CNP), comparison group 2 (n=21) with standard subcutaneous transplantation of melanoma (B16/F10), main group 2 (n=21) (CNP+B16/F10) with melanoma transplanted 3 weeks after the CNP model creation. Heart mitochondria were isolated by differential centrifugation. Levels of cytochrome C (ng/mg of protein), 8-hydroxy-2'-deoxyguanosine (8-OHdG) (ng/mg of protein), and malondialdehyde (MDA) (μmol/g of protein) were measured in mitochondrial samples by ELISA. Statistical analysis was performed using the Statistica 10.0 program.Results. DM in rats upregulated 8-OHdG by 6.3 times and MDA by 1.9 times (р=0.0000) and downregulated cytochrome C by 1.5 times (р=0.0053) in heart cell mitochondria, compared to intact values. DM+Guerin’s carcinoma in rats increased 8-OHdG by 14.0 times and MDA by 1.7 times (р=0.0000) and decreased cytochrome C by 1.5 times (р=0.0000), compared to intact values. CNP in mice did not affect the studied parameters in mitochondria of the heart. CNP+B16/F10 in mice increased 8-OHdG by 7.1 times and MDA by 1.6 times (р=0.0000) and decreased cytochrome C by 1.6 times (р=0.0008).Conclusions. Comorbidity (diabetes mellitus, chronic neurogenic pain) together with malignant pathology aggravates mitochondrial dysfunction of heart cells with destabilization of the respiratory chain mediated by free radical oxidation processes.

The antinociceptive effects of folic acid using formalin and acetic acid tests in male mice

Background and aims: Antidepressant agents such as imipramine are clinically used to control and treat different types of pain, especially neuropathic pain. Studies have shown the antidepressant-like activity of folic acid (FA). This study aimed to investigate the potential antinociceptive effects of FA using formalin and acetic acid tests in male mice. Methods: Sixty male albino mice (20-30 g) were randomly divided into 10 groups (n=6 in each group) of negative control, positive control (morphine or indomethacin), and FA (10, 15, and 30 mg/kg) groups. In the formalin test, duration of paw licking and biting the right hind paw during acute (0-5 minutes) and chronic (15-60 minutes) pain after intraplantar injection of formalin 2.5% (25 µL) was recorded. In the writhing test, the abdominal constrictions were recorded after the intraperitoneal injection of acetic acid 1%. Results: Only a high dose (30 mg/kg) of FA significantly reduced acute pain (P=0.001) compared with the control group. But all doses of FA significantly decreased chronic pain (P=0.001). In addition, morphine significantly reduced both phases of pain (P=0.020 and P=0.001, respectively). Moreover, indomethacin and all doses of FA decreased the number of abdominal constrictions induced by acetic acid (P=0.001). Conclusion: Compared with acute (neurogenic) pain, FA more potently decreases chronic (inflammatory) pain. Furthermore, FA decreases the parietal pain that could potentially represent antinociceptive effect. However, further studies are needed to elucidate the exact mechanism of FA’s analgesic activity.

INFLUENCE OF CHRONIC NEUROGENIC PAIN ON THE DYNAMICS OF THE NO-SYSTEM FUNCTIONING DURING MELANOMA B16/F10 GROWTH IN MALE MICE

Since B16/F10 melanoma demonstrated gender differences in its growth in the presence of chronic neuropathic pain (cnp) and changes in the system of proangiogenic growth factors, the aim of the study was to analyze levels of components of the no-system in male mice during the growth of transplantable B16/F10 melanoma in the presence of cnp.Material and Methods. 66 male mice С57Вl/6 were used in the experiment. A model of subcutaneous growth of B16/F10 melanoma (during 3 weeks) was created in the cnp presence (sciatic nerve ligation). Concentrations of nos-2, nos-3, l-arginine, citrulline, total nitrite, nitrotyrosine and adma were determined by elisa in intact and tumor tissues.Results. A significant increase in levels of no-synthases was revealed in the skin and tumor tissues in the tumor growth with cnp from week 1, as well as a decrease in the level of total nitrite in the skin, multidirectional dynamics of adma and arginine levels, a steadily increased level of citrulline in the skin and tumor in the dynamics of tumor growth with cnp.Conclusions. Male mice with B16 melanoma growing in the presence of cnp demonstrated a more active functioning of the no-system already from week 1, compared to standard tumor growth, which might result in a greater rate of growth of melanoma with cnp. Significantly higher skin and tumor levels of citrulline in males were a distinctive feature, in contrast to melanoma with standard growth, which could be the result of inhibition of arginine synthesis and formation of a tumor auxotrophic for arginine.

Anti-oedematous and antinociceptive activities assessment of stem bark aqueous extract of Ficus exasperata Vahl. (Moraceae) in rat and mice.

Background: Ficus exasperata Vahl. (Moraceae) is widely used in African traditional medicine for the treatment of various diseases. The present study is undertaken to assess the anti-oedematous and antinociceptive activities of the stem bark aqueous extract of Ficus exasperata in mice and rats. Methods: The anti-oedematous activity was investigated following carrageenan or histamine-induced rat paw oedema models. Antinociceptive activity was evaluated using acetic acid induced writhing test (1 %, 10 ml/kg), capsaicin-induced neurogenic pain (32 µg/mL, 30 µL) and formalin-induced test (1%, 20 µL). Extract was administrated orally at 37.5, 75 and 150 mg/kg. Results: Pre-treatment of rats with Ficus exasperata stem bark aqueous extract exhibited significant inhibition of paw oedema during all the phases of both carrageenan and histamine induced edema in rat. The maximum inhibition percentages were 94.75 % (3 h) and 30.64 % after one hour at the dose of 37.5 mg/kg, respectively, in carrageenan or histamine models. Antinociceptive activity showed that aqueous extract reduced significantly (p < 0.001) the pain induced by acetic acid with an inhibition percentage of 70.8% (150 mg/kg). In the formalin-induced test, the extract also reduced significantly (p < 0.001) licking time during neurogenic phase and inflammatory phase with inhibition percentages of 44.75% and 52.78% respectively at the dose of 75 and 150 mg/kg. In addition, aqueous extract of F. exasperata reduced significantly (p < 0.001) neurogenic pain induced by capsaïcin by 71.28 % at the highest dose (150 mg/kg). Conclusion: This finding suggests that the stem bark aqueous extract of Ficus exasperata possess potent anti-oedematous and antinociceptive activities.

Influence of B16/F10 melanoma growth variant on the level of cytochrome C in mitochondria in various organs of female mice

Introduction. Cytochrome C in mitochondria transfers electrons from complex III to complex IV, and it is a signaling molecule in the apoptosis realization.The objective was to evaluate the level of cytochrome C in cell mitochondria in various organs of female mice with standard and stimulated growth of experimental B16/F10 melanoma.Methods and materials. The experiment was performed on female C57BL/6 mice (n=168). The groups were: intact animals (n=21); controls with a model of chronic neurogenic pain (CNP) (n = 21); group M — standard B16/F10 melanoma transplantation (n=63), group CNP + M — B16/F10 melanoma transplantation 3 weeks after CNP model creation (n=63). The level of cytochrome C (ng / mg protein) were measured by ELISA (Bioscience, Austria). Statistical analysis of results was performed using the «Statistica 10.0» program.Results. After 1 week of standard melanoma growth, an increase in the level of cytochrome C by 2.7 and 1.7 times was detected in mitochondria of the brain and liver; by the 3rd week, it decreased in the liver and skin by 1.7 times. In melanoma mitochondria, the level of cytochrome C was lower than in the skin of intact animals: by 2.5 times after week 1, by 4.5 times after week 2, and by 4.6 times after week 3. After 1 week of stimulated melanoma growth, the level of cytochrome C decreased compared control values: by 2.2 times in the brain, by 1.9 times in the liver, by 1.4 times in the skin; by week 3, it decreased by 4.8 times in mitochondria of the brain, by 4.7 times — in the liver, by 2.3 times — in the heart, by 1.9 times — in the skin. In melanoma mitochondria, the level of cytochrome C was lower than in the skin of intact animals: by 15.3 times after week 1, by 10.3 times after week 2, and by 8.8 times after week 3.Conclusion. Low level of cytochrome C were found in melanoma mitochondria in standard and stimulated tumor growth. The data can be used in the experiment and in clinic for using exogenous cytochrome C as an agent slowing down the malignant process.

Comorbidity: Chronic neurogenic pain affects malignant growth and changes balance of neurosteroids in brain of rats.

e23516 Background: Sex steroids in the brain regulate neurogenesis and the body's response to stress. Chronic neurogenic pain (CNP) and the tumor growth are stress factors that often accompany each other. The purpose of the study was to analyze levels of sex steroid hormones in white matter of the brain of rats with tumor development in presence of CNP. Methods: The study included white outbred male rats (n = 74). In the main groups, a CNP model was created by bilateral sciatic nerve ligation, and after 45 days, M1 sarcoma was transplanted subcutaneously (n = 11) or into the subclavian vein (n = 11). Two comparison groups (each n = 13) included sham operated animals with M1 sarcoma transplanted subcutaneously or into the subclavian vein. Control groups (each n = 13) included animals with CNP or sham operated rats. Levels of testosterone (T), estrone (E1), estradiol (E2), estriol (E3) and progesterone (P4) were measured by ELISA (Cusabio, China) in the brain tissues obtained on day 21 of the tumor growth. Results: Tumors transplanted subcutaneously with and without CNP grew in 100% of animals. Tumor volumes were 1.5 times (p<0.05) greater in animals with CNP, compared with rats without CNP, while the survival in the groups was similar. Levels of all studied hormones, except for E1, in the brain tissue in subcutaneous sarcoma growth were lower in presence of CNP than without it: T and E3–on average by 1.4 times (p<0.05), E2 and P4–by 3.5 times (p<0.05). In rats with intravenous transplantation of M1, tumor nodes in the lungs were registered only in rats with CNP, and the survival of animals was 36 days shorter (p<0.05) than in rats of the corresponding control group. Such specificity of selective neoplastic growth in the pulmonary tissue was combined with lower cerebral T and E3 levels than in the corresponding control–on average by 1.4 times (p<0.05), E2–by 7.2 times, and higher levels of E1–by 1.3 (p<0.05) and P4–by 2.0 times, compared to animals which did not develop the neoplastic process in the lungs without pain. Conclusions: The presence of CNP stimulates the growth of M1 sarcoma in standard subcutaneous inoculation and allows the development of tumors in the lung in intravenous inoculation. The specificity of malignant growth in presence of CNP is accompanied by changes in the brain levels of neurosteroids in rats.

Aminergic status in the brain of urokinase gene-deficient mice with malignant tumors growing in presence of chronic neurogenic pain.

e21582 Background: The fibrinolytic system of the brain is important for its normal functioning and participation in processes that are significant in various stressful influences, including tumor growth and chronic neurogenic pain (CNP). These pathological conditions change the activity of the brain neurotransmitter system. On the other hand, urokinase deficiency is associated with significant inhibition of tumor growth, while CNP – with its stimulation. The purpose of the study was to analyze the effect of CNP on the levels of biogenic amines in the brain of mice with urokinase deficiency (uPA-/-) with transplanted B16/F10 melanoma. Methods: The study included male and female mice: С57ВL/6 (uPA+/+, n = 48) and C57BL/6-Plautm1.1Bug-ThisPlauGFDhu/GFDhu (urokinase gene-knockout - uPA-/-, n = 48). Mouse strains were divided into subgroups (each n = 6): intact; with CNP (bilateral sciatic nerve ligation); 21 days after subcutaneous transplantation of B16/F10 melanoma; 21 days of B16/F10 melanoma growth in presence of CNP (B16/F10+CNP), with tumor transplantation 2 weeks after the sciatic nerve ligation. Levels of adrenaline (A), noradrenaline (NA), dopamine (DA), histamine, serotonin (5HT), and 5-hydroxyindoleacetic acid (5OHIA) were determined in the brain by ELISA (Cusabio, China). Statistical processing - Statistica 10.0. Results: Levels of NA, DA and 5HT in the brain of intact uPA-/- mice were 3.5, 2.1 and 1.9 times higher (p < 0.05), respectively, than in intact uPA+/+ animals, while histamine and 5OHIA were on average 2.0 times lower. The dynamics of cerebral levels of biogenic amines in uPA-/- mice with pathological factors, alone or combined, had practically no gender specificity, with rare exceptions. So, 5HT levels increased up to 4.5 times in uPA-/- mice of both sexes in response to CNP or B16/F10 growth. Melanoma growth in presence of CNP, on the contrary, decreased 5HT by 3-10 times and DA by 1.6 times (p < 0.05) both in males and females, and decreased NA by 1.6 times (p < 0.05) in females. Conclusions: CNP together with melanoma inhibits the initial activation of the HA-, DA- and 5HT-ergic systems in the brain of uPA-/- mice, which may be an important pathogenetic mechanism of the cancellation of genetically determined inhibition of subcutaneous B16/F10 melanoma growth in urokinase deficiency.

Comorbidity affects Вcl-2 levels in mitochondria in C57BL/6 mice with transplantable B16/F10 melanoma.

e21581 Background: Overexpression of the Bcl-2 protein inhibits apoptosis and promotes carcinogenesis. Stress causes signaling leading to cell buffering with Bcl-2 protein above acceptable levels. The purpose of the study was to analyze the influence of comorbidity – chronic neurogenic pain (CNP) – on the Bcl-2 levels in mitochondria of cells of melanoma, the heart, skin and brain in female mice with growing tumors. Methods: Female С57ВL/6 mice were divided into groups: intact group (n = 21); control group with a CNP model – bilateral sciatic nerve ligation (n = 21); group M – B16/F10 melanoma (n = 63); CNP+M group – B16/F10 melanoma was transplanted 3 weeks after the CNP model creation (n = 63). The concentration of Bcl-2 (ng/mg of protein) was determined in mitochondrial samples by ELISA (Thermo Fisher Scientific, Austria). Statictical analysis of results: Statistica 10.0. Results: CNP decreased the Bcl-2 level in heart mitochondria by 1.3 times (p < 0.05), but increased it in skin and brain mitochondria by 5.8 and 1.3 times, respectively. Similar changes were observed in melanoma growth 1 week after its transplantation: Bcl-2 levels decreased in heart mitochondria by 1.3 times, and increased in the skin and brain by 8.9 and 1.3 times, respectively. After 2 weeks of the tumor growth, Bcl-2 in brain mitochondria decreased by 1.7 times, and it started declining in the skin by the 3rd week – by 4 times, compared to intact females. Bcl-2 in tumor mitochondria exceeded the values in the skin by more than 4 times throughout the experiment. Tumor growth in presence of CNP caused a decrease in Bcl-2 in brain mitochondria by 2.4 times after 3 weeks, and in the heart and skin – by 2 and 1.7 times, respectively, after 2 weeks. Bcl-2 in tumor mitochondria in presence of CNP was lower than in the intact skin on average by 1.8 times throughout the experiment. Conclusions: CNP as a comorbidity caused a modulating effect on the mechanisms of survival and apoptosis of cells both in the tumor and in the main organs providing the vital functions of the body - the brain and heart, and also affects the target organ of melanoma - the skin. The results demonstrated the ability of comorbidity to change levels of Bcl-2 in mitochondria depending on the stage of tumor development.