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Introduction. The heart innervation is made up of plexo-ganglionic formation containing sympathetic, parasympathetic, and sensory components. We examined the distribution and neurochemical coding of the ganglia and nerve fibers in the chinchilla’s heart. Material and methods. The heart sections of 10 male and 10 female adult chinchillas were processed in accordance with the thiocholine method for acetylcholine esterase (AChE), and the SPG method for detecting the presence of adrenergic fibers was applied. The routine technique of immunohistochemical (IHC) staining with primary antibodies directed against ChAT, VAChT, DbH, TH, CART, NPY, VIP, GAL and SOM was used. The secondary antibodies were conjugated with Alexa Fluor 488 and Alexa Fluor 555 fluorophores. Results. The epicardium contained ganglia and nerve fibers, the myocardium had a few ganglion neurocytes and nerve fibers, and the endocardium contained only nerve fibers. In the epicardium, AChE-positive fibers were more prevalent than SPG-positive fibers. All the ganglion cells were immunopositive for ChAT and VAChT. Some cells also had a positive reaction to DbH and TH. Fibers containing cholinergic and adrenergic markers were numerous, while many of them were ChAT/DbH- and VAChT/TH-positive. CART/NPY and CART/VIP, as well as CART and GAL, were observed to be colocalized in ganglion neurocytes, as well as in individual cells. The nerve fibers were found to contain all the neurotransmitters we tested for, as well as the following co-occurrences: ChAT/DbH, VAChT/TH, CART/NPY, CART/VIP, CART/GAL, and CART/SOM. Conclusions. Our analysis of the neurochemical profile of the nerve structures in chinchilla’s heart showed that, despite interspecies differences, the general pattern of the distribution of autonomic nervous system structures is similar to that of other mammals’ species, including humans.

Background: The extent of morphological and ultrastructural changes in HeLa cells was assessed by optical, fluorescence and electron microscopy after exposure to various concentrations of physcion, taking into account the biological properties of the test compound. Methods: Cell viability was assessed by MTT assay, while the cell cycle, LC3 expression, apoptosis, change of mitochondrial potential, Bcl-2 protein expression level and the level of reactive oxygen species were analyzed by flow cytometry. Results: As a result of physcion encumbrance, concentration-dependent inhibition of HeLa cell viability and the G0/G1 phase of the cell cycle was observed. Activation of the lysosomal system was also revealed, which was expressed by an increased number of lysosomes, autophage vacuoles and increased expression of the LC3 protein, a marker of the autophagy process. Transmission electron microscopy and fluorescence microscopy showed that physcion induced clear changes in cervical cancer cells, especially in the structure of the nucleus and mitochondria, which correlated with the production of reactive oxygen species by the test compound and indicated the induction of the oxidative process. At the same time, the pro-apoptotic effect of physcion was demonstrated, and this mechanism was dependent on the activation of caspases 3/7 and the reduction in Bcl-2 protein expression. Conclusion: The obtained results indicate an antitumor mechanism of action of physcion, based on the induction of oxidative stress, autophagy and apoptosis.

Background Anthraquinones, including emodin, are compounds with numerous pharmacological properties, including anticancer properties. The aim of this study experiment was to examine the effect of emodin, a natural compound present in the roots and rhizomes of Rheum palmatum, on the induction of mitotic catastrophe in cervical cancer cells. Material and methods HeLa celIs were treated with different emodin concentrations for 48 h, and cell growth was measured with 3-(4-,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolyl. The cell-cycle distribution and the level of apoptosis were determined by means of flow cytometry, using annexin V-fluorescein isothiocyanate staining and propidium iodide. Morphological changes in the mitotic apparatus were evaluated using optical and confocal microscopy techniques. Results Emodin induced an increase in the number of polymorphonuclear cells, giant cells, cells with micronuclei, cells with abnormal mitosis and damaged spindle. The reorganization of F-actin depended on the concentration of emodin. With the increase in emodin concentration, inhibition of mitotic activity was demonstrated, which was manifested by a decrease in the mitotic index, mainly in metaphase of the mitotic process and an increase in the number of cells inhibited in the G2/M phase. At the same time, an increase in the number of apoptotic cells was found. Conclusion Emodin leads to death of cervical cancer cells by induction of a mitotic catastrophe.

Melatonin is a hormone with many different biological activities and therefore seems to be an important factor reducing the harmful effects caused by toxic organophosphorus compounds. In this study, we attempted to evaluate the protective effect of melatonin on liver cells of mice challenged with chemical warfare agent-soman. The study was conducted at the level of ultrastructural and biochemical changes (analysis of the activity of model lysosomal enzymes and assessment of the level of lipid peroxidation). Significant biochemical and ultrastructural changes were found in the studied mouse hepatocytes after administration of soman alone, and soman in combination with melatonin, and the scope of the disclosed changes was dependent on the time of action of the examined factors. Melatonin has shown protective action, shielding liver cells from toxic effects of soman, which may result from its antioxidant properties and stimulation of the lysosomal compartment, the system coordinating the isolation and removal of cell-threatening processes.

Chrysophanol is an anthraquinone with proven antitumor activity against several tumor cell lines. However, its effect on cervical cancer cells is still unknown. Therefore, HeLa cells were exposed to various concentrations of chrysophanol and then subjected to biochemical, ultrastructural, and morphological analysis. It has been shown using flow cytometry and MTT reduction assay that chrysophanol has been shown to inhibit cell viability and arrest cells in the G2/M phase of the cell cycle. Using Annexin V/propidium iodide staining, a significant increase in apoptosis was found after chrysophanol treatment on HeLa cells, and this process was mediated by caspases 3/7 with a clear inactivation of the antiapoptotic Bcl-2 family protein. However, the demonstrated increased number of cells with double-stranded DNA breaks suggests that chrysophanol also causes DNA damage. By means of electron and fluorescence microscopy, a clear effect of chrysophanol on the intensification of degradation processes, on changes in the structure of the nucleus, endoplasmic reticulum and mitochondria was demonstrated. The changes visible in the mitochondria may be related to the increase in the level of free radicals induced by chrysophanol, which induces apoptosis, inter alia, by increasing the permeability of mitochondrial membranes. The range of observed changes depended on the concentration of anthraquinone was tested.

Although vitamin D is included in the group of fat-soluble vitamins, it must be considered as a prohormone. Its active forms including calcitriol have pleiotropic effects and play an important role in the regulation of cell proliferation, differentiation and apoptosis as well as in hormone secretion, and they show anti-cancer properties. Since calcitriol delivery can be beneficial for the organism, and Syrian golden hamsters represent a unique experimental model, we decided to investigate its toxicity in this species. In this study, we injected calcitriol intraperitoneally at doses 0 (control), 0.180±0.009 µg/kg and 0.717± 0.032 µg/kg. Animal behavior was observed for 72 hrs after injection, and afterwards blood and liver and kidney were collected for post-mortem examination, electron microscopy, and hematology analyses. The highest dose of calcitriol induced a change in animal behavior from calm to aggressive, and liver surface showed morphological signs of damage. Following the injection of calcitriol, ultrastructural changes were also observed in the liver and kidneys, e.g. vacuolization and increased number of mitochondria. There was also a trend for increased serum levels of aspartate aminotransferase (AST), but not of alanine aminotransferase (ALT) or GGTP (gamma-glutamyl transpeptidase). There was no change in Ca, Mg and P levels as well as in blood morphology between experimental and control groups. These results indicate that calcitriol at 0.717, but not at 0.18 µg/kg, may induce acute damage of the liver and kidneys, without inducing calcemia. We propose that the hepatotoxic effect of calcitriol in hamster constitutes the primary cause of behavioral changes.

Introduction. Cholinergic and adrenergic innervation of the pancreas in chinchilla (Chinchilla Laniger Molina) was examined in this study. The pancreas is both an exocrine and endocrine gland with autonomic and sensory innervation presented by the numerous nerve fibers and small agglomerations of nerve cells. Material and methods. Investigations were performed on 16 adult chinchillas of both sexes. The material was collected immediately after death of the animals. Histochemical methods: AChE and SPG were used, in addition to routine technique of single and double immunohistochemical (IHC) staining using whole mount specimens and freezing sections with a thickness of 8 to 12 μm. In the immunofluorescence staining, primary antibodies directed against markers used to identify cholinergic — ChAT and VAChT, and adrenergic — DbH and TH neurons. Secondary antibodies were coupled to Alexa Fluor 488 and Alexa Fluor 555 fluorophores. Results. Histochemical studies (AChE) revealed that chinchilla pancreatic cholinergic innervation consisted of ganglionic neurocytes and numerous nerve fibers. These structures are located in the parenchyma of the exocrine part of the organ in close proximity to blood vessels and are present within the walls of the pancreatic ducts and interstitial connective tissue. A delicate fiber network around the Langerhans islets was also observed. The most numerous cholinergic structures were found in the head and tail, and the least numbers were found in the body of the pancreas. The SPG method revealed that adrenergic fibers form a network in the adventitia of blood vessels, and individual fibers run throughout the pancreatic parenchyma. Moreover, adrenergic nerve fibers were observed around the ganglionic neurocytes. This innervation was similar in all parts of the investigated organ. IHC investigations allowed observations of both the cholinergic and adrenergic activities of autonomic nerve structures. Additionally, using ChAT/DbH double staining, colocalization of these substances was observed in the fibers of the pancreatic parenchyma that passed through the cholinergic ganglia. Colocalization of VAChT and TH was found in nerve fibers of the exocrine part, in the walls of blood vessels, and in individual nerve cells. Colocalization of ChAT/DbH and VAChT/TH was observed in the single nerve cells and in the small (2–3 cell) ganglia. ChAT- and DbH-immunopositive nerve fibers were found in the area of the islets of Langerhans. Conclusions. The results indicate a more intense cholinergic innervation of the chinchilla’s pancreas, which is represented by both ganglia and nerve fibers, while adrenergic structures are mainly represented by fibers and only single neurocytes. This arrangement of the investigated structures in this species may imply a major role for hormonal control of exocrine secretion in rodents.

Since the erythrophagocytosis of opsonized erythrocytes is investigated mainly by calculating the phagocytic index using subjective light microscopy evaluation, we present methods for the quantitative and qualitative analysis of human cell erythrophagocytosis. Erythrocytes from two storage periods were used. Using Imaris software, we were able to create a three-dimensional model of erythrophagocytosis. The use of microscopy instead of cytometry revealed a significantly higher number of monocytes and erythrocytes that appeared active in phagocytosis. Spatial reconstruction allowed for detailed analysis of the process by precisely locating erythrocytes in phagocytes. Additionally, a technique of sequential image registration using Nis Elements software allowed for observation of the course of phagocytosis over a range of time intervals. This in vitro research may be helpful for understanding the cellular interactions between monocytes and erythrocytes. The cytometric method-being relatively rapid, sensitive, and specific-can serve as an alternative technique to microscopy in the quantitative analysis of erythrophagocytosis. This allows us to avoid counting the erythrocytes nonspecifically attached to monocytes and gives objective results.

Background and objectives The accumulation of microvesicles in erythrocyte concentrates during storage or irradiation may be responsible for clinical symptoms such as inflammation, coagulation and immunization. Our aim was to determine whether any of the cluster of differentiation (CD) molecules responsible for important functions are present on microvesicles, and if their expression level is dependent on the storage period of erythrocyte concentrates. Material and methods Erythrocyte microvesicles were isolated from 'fresh' (2nd day) and 'old' (42nd day) stored erythrocyte concentrates. Qualitative cytometric analysis of 0·5 µm, erythrocyte-derived, PS-exposing vesicles was performed using the annexin V-FITC, anti-CD235a-PE antibody and calibrated beads. The microvesicles were also visualized under a confocal microscope. The expression of the molecules CD235a, CD44, CD47, CD55, CD59 and of phosphatidylserine (PS) was compared using flow cytometry. Measurements of microvesicle phagocytosis by human monocytes were carried out using a flow cytometer and a confocal microscope. Results The analysis of the microvesicles with calibration beads allowed us to identify these structures with a diameter of about 0·5 µm in the 'fresh' and 'old' samples. At day 2, the microvesicles had elevated expression levels of CD47, reduced expression levels of PS, CD55 and CD59. The phagocytosis index was higher for the microvesicles isolated from the 42-day-old erythrocyte concentrates. Conclusion This research may bring us closer to understanding the factors responsible for erythrocyte ageing and to evaluate the quality of stored red blood concentrates intended for transfusion.

The aim of this study was to evaluate ultrastructural changes in kidney and liver tissue of chicken embryos exposed in ovo to cadmium. Embryonated eggs were injected on the 4th day of incubation with cadmium at the dose of 0, 2, 4 and 8 μg/egg (80 eggs/group). The samples of kidney and liver tissues were collected from embryos at the 14th and 18th day of incubation (E14 and E18) and at hatching day (D1). The tissue structure was evaluated by transmission electron microscopy (Tecnai G2 Spirit). The results indicate that hepatocytes responded to damage caused by toxic cadmium activity with a significant disturbance in the structure of mitochondria and a considerable expansion of the lysososmal system, while glomerular cells additionally reacted with an increased proliferation of peroxisomes. The range of changes observed on the subcellular level was dependent on the dose of cadmium, embryogenesis stage and cell type.

Background Aloe-emodin is an anthraquinone with potential pharmacological properties, including numerous antitumor properties. The purpose of the study was to determine whether aloe-emodin induces mitotic death in cervical cancer cells. Materials and methods Analysis of morphological changes as surrogate mitotic death indicators in HeLa cells was carried out using optical, fluorescence and electron microscopy. Viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide reduction assay. Cell-cycle analysis was performed using flow cytometry. Results Aloe-emodin increased the number of multinucleate cells, giant and micronuclear cells. There was a concentration-dependent decrease in the mitotic index with a predominance of cells in the metaphase of the mitotic process and inhibition of division in the G2/M phase of the cell cycle. The presence of cells with abnormal mitosis and cells with injury to the division spindle was also demonstrated. Conclusion Aloe-emodin induces mitotic catastrophe in cervical cancer cells.

Background: Aloe-emodin belongs to the group of anthraquinones having extremely high biological activity. The aim of this study was to evaluate the range of morphological and biochemical changes in HeLa cells treated with aloe-emodin, especially with regard to the lysosomal compartment. Methods: Marking of lysosomes was performed with neutral red staining for conventional light microscopy and acridine orange staining for confocal microscopy. To evaluate ctivity of lysosomal enzymes and permeability of the lysosomal membrane, spectrophotometric techniques were employed. Results: Aloe-emodin caused increased permeability of lysosomal membranes in HeLa cells, expressed inter alia by extinction of the orange color of acridine orange (lysosomal marker) and in reduction of neutral red uptake by lysosomes. These changes are accompanied by release of cathepsins from the interior of the lysosomes with a simultaneous highly significant increase in their activity in the cytoplasm. Conclusion: The results indicate that aloe-emodin can activate lysosomal pathway-dependent apoptosis in HeLa cells.

BACKGROUND/AIM To search for new antileukemic agents, the chemical structure of phenytoin was modified. A possible cytotoxic activity of three bromoalkyl phenytoin analogs, methyl 2-(1-(3-bromopropyl)-2,4-dioxo-5,5-diphenylimidazolidin-3-yl) propanoate (PH2), 1-(3-bromopropyl)-3-methyl-5,5-diphenylimidazolidine-2,4-dione (PH3) and 1-(4-bromobutyl)-3-methyl-5,5-diphenylimidazolidine-2,4-dione (PH4) on regulated cell death, the cell cycle and cell ultrastructure was assessed. MATERIALS AND METHODS The experiments were performed in vitro on HL-60 and U937 cells, using flow cytometry and electron microscopy methods. RESULTS Application of PH2, PH3, and PH4 resulted in cell surface exposure of phosphatidylserine and plasma membrane impairment, caspase-8, -9, and -3/7 activation, dissipation of mitochondrial membrane potential, DNA breakage, cell-cycle disturbance and cell ultrastructural changes. In general, PH3 appeared to be the most active against the leukemia cells, and all bromoalkyl hydantoins, PH2-PH4, were more active in HL-60 cells than in U937 cells. CONCLUSION The antileukemic activity of the bromoalkyl phenytoin analogs depended on the combination of N-hydantoin substituents and the human cell line used.

Background An example of plant-derived compounds that may be of great importance in oncological therapy is emodin. The aim of this study was to evaluate the range of changes in the lysosomal compartment of HeLa cervical cancer cells treated with emodin. Materials and methods Changes in the lysosomal compartment were evaluated using microscopic techniques. In order to evaluate the activity of lysosomal enzymes and the permeability of the lysosomal membrane, a spectrophotometric technique was employed. Results Emodin induced significant changes in the lysosomal compartment, manifesting as an increase in the number of lysosomes, autophagic vacuoles and the activity of lysosomal hydrolases. Emodin exhibited cytotoxic activity against lysosomes through lysosomal membrane damage with possible leaks of lysosomal hydrolases into the cytoplasm. Conclusion Emodin induces degradation processes and promotes the death of tumor cells through a mechanism that occurs with clear involvement of the lysosomal compartment.

Since the erythrophagocytosis of opsonized erythrocytes is investigated mainly by calculating the phagocytic index using subjective light microscopy evaluation, we present methods for the quantitative and qualitative analysis of human cell erythrophagocytosis. Erythrocytes from two storage periods were used. Using Imaris software, we were able to create a three-dimensional model of erythrophagocytosis. The use of microscopy instead of cytometry revealed a significantly higher number of monocytes and erythrocytes that appeared active in phagocytosis. Spatial reconstruction allowed for detailed analysis of the process by precisely locating erythrocytes in phagocytes. Additionally, a technique of sequential image registration using Nis Elements software allowed for observation of the course of phagocytosis over a range of time intervals. This in vitro research may be helpful for understanding the cellular interactions between monocytes and erythrocytes. The cytometric method-being relatively rapid, sensitive, and specific-can serve as an alternative technique to microscopy in the quantitative analysis of erythrophagocytosis. This allows us to avoid counting the erythrocytes nonspecifically attached to monocytes and gives objective results.