Vol 159, No 1 (2021)

Original Study Articles

Autophagy as a life support marker of isolated hepatocytes

Bgatova N.P., Dossymbekova R.S., Taskaeva J.S., Miroshnichenko S.M., Knyazev R.A., Solovieva A.O., Sharipov K.O., Tungushbaeva Z.B.


AIM: The work aimed to reveal structural signs of autophagy in the cytoplasm of isolated hepatocytes in the dynamics of their cultivation.

MATERIALS AND METHODS: The cultivated hepatocyte culture cell cycle was studied by flow cytofluorometry. The cells were cultured for 1, 24, and 48 hours. Morphometric analysis was performed using of the computer program Image J. The diameters of the nuclei and cytoplasm of hepatocytes, the volumes of nuclei and cytoplasm, and the nuclear-cytoplasmic ratio were determined. The concentration of intracellular organelles and autophagy was evaluated with magnification by 30000 times.

RESULTS: The cell cycle arrest in the G0/G1 stage after 24 hours of hepatocyte cultivation and the preservation of their viability by hour 48 of the experiment without increase in the percentage of cells in the apoptosis stage were revealed. The decrease in the absolute count of cells was registered, as well as an increase in the nuclear-cytoplasmic ratio indicating a decrease in the proportion of hepatocyte cytoplasm in the course of cultivation. After 24 hours of cultivation, autophagosomes with fragments of cytoplasm, glycogen rosettes, and autolysosomes with partially degraded material were revealed in the cell cytoplasm. By hour 48 of the study, a significant decrease in the volume density of glycogen and mitochondria was noted, as well as an increase in basal autophagy in hepatocytes, with a prevalence of glycophagy and mitophagy.

CONCLUSIONS: Autophagy maintains cellular homeostasis of isolated hepatocytes under standard culture conditions, as evidenced by a decrease in the volume density of glycogen and mitochondria, and an increase in basal autophagy in the hepatocyte cytoplasm. The findings indicate the contribution of autophagy to the survival of the primary culture of hepatocytes and can be used as an indicator of the adequacy of culturing conditions.

Morphology. 2021;159(1):5-12
pages 5-12 views

Monoamine oxidase B in developing histaminergic neurons of the rat brain

Zimatkin S.M., Zaerko A.V.


BACKGROUND: Histaminergic neurons of the brain play an important role in the regulation of many functions, systems, and reactions of the body, as well as in the pathogenesis of many pathological conditions and diseases. In the brain, histamine acts as a neurotransmitter and is localized mainly in histaminergic neurons. All histaminergic neurons of the hypothalamus, unlike other types of neurons, have high activity of monoamine oxidase type B (MAO B) which is a key enzyme of histamine metabolism in the brain.

AIM: The work aimed to perform parallel assessment of MAO B activity and immunoreactivity in rat hypothalamus histaminergic neurons in the process of postnatal ontogenesis.

MATERIALS AND METHODS: The study was conducted on samples of the hypothalamus of 5, 10, 20, 45, and 90 days old offspring of outbred white rats (45 rats), conforming to the “Guidelines for the Use of Animals in Research”. Sections of the hypothalamus were processed histochemically to detect MAO B activity and immunohistochemically using antibodies to MAO B.

RESULTS: It was revealed that the activity and immunoreactivity of the enzyme of histamine oxidative deamination and the marker enzyme of the hypothalamic histaminergic neurons, monoamine oxidase type B were not detected in cytoplasm of histaminergic neurons on the day 5 after birth. Then these indicators were simultaneously increasing from the day 10 to the day 90 of postnatal ontogenesis.

CONCLUSIONS: The synchronism of the postnatal development of MAO B activity and immunoreactivity in histaminergic neurons of the brain indicates a parallel accumulation of MAO B protein and its enzymatic activity, reflecting the formation of their specific, mediator metabolism.

Morphology. 2021;159(1):13-19
pages 13-19 views

Immunophenotypic characteristics of inducible NO synthase expression in dentate gyrus of mature rats in modeling depression and its pharmacological correction

Smirnov A.V., Ekova M.R., Tyurenkov I.N., Volotova E.V.


AIM: The work aimed to investigate inducible NO synthase (iNOS) expression in dentate gyrus in mature rats when modeling depression, as well as the establish the pharmacological correction possibility of detected changes with Phenibut and compounds under laboratory codes of RSPU-189, RSPU-135.

MATERIALS AND METHODS: Depressive-like behavior in animals was modeled by combining stressful stimuli such as loud sound, pulsating bright light, and vibration simultaneous with constant restriction of mobility and fluctuations in temperature of environment for 7 days (daily for 30 minutes). Changes in level of iNOS expression in dentate gyrus were assessed by calculating relative area of immunoreactive material (IRM) and staining intensity in points from 0 to 3.

RESULTS: Compared with the control group, rats with experimental depression showed an increase in expression of iNOS-IRM in cytoplasm of neuronal perikarya in granular layer of dentate gyrus, as well as an increase in relative area of iNOS-IRM in neuropil and nerve cells. The use of the compound RSPU-189 (salifen) demonstrated to a greater extent the corrective effect, since in the cytoplasm of neuronal perikarya in granular layer of dentate gyrus of rats, there was a decrease in the expression of iNOS-IRM, as well as a decrease in the relative area of iNOS-IRM in neuropil and nerve cells, which corresponded to values of these parameters in the control group of animals.

CONCLUSIONS: An experimental modeling of depression in dentate gyrus of mature rats revealed an increase of iNOS-IRM expression, the decrease of which was noted in its pharmacological correction with the compound RSPU-189 (salifen), which may indicate the predominant neuroprotective effect of this compound on GABAergic neurotransmission mechanisms.

Morphology. 2021;159(1):21-28
pages 21-28 views

Fixation with zinc-formalin as an adequate substitution of Zenker-formol for the histochemical staining of the pancreatic islet cells

Manskikh V.N.


AIM: The work aimed to determine the possibility of using zinc-formalin fixation instead of Zenker-formol to identify selectively all the main types of cells (A, B, D) of the pancreatic Langerhans islets by staining using classical histochemical methods.

MATERIALS AND METHODS: Pancreatic samples of the naked mole rat (Heterocephalus glaber, Rüppell, 1842) were fixed for 24 hours in zinc-formalin fixative containing 300 ml of 37% formaldehyde, 50 g of zinc chloride, 1.9 ml of glacial acetic acid, and 2 L of distilled water. After fixation, the standard procedures for paraffin embedding and staining with Heidenhain’s azan and a combination of azan and Gomori’s paraldehyde-fuchsin were performed according to routine protocols.

RESULTS: It was revealed that a distinctive differential staining of all three major types of islet cells can be obtained if pancreatic samples are fixed in a zinc-formalin fixative for 24 hours. At the same time, as after fixation with Zenker-formol (but not with formalin solution or Bouin fixative), not only A and B, but also D-cells are clearly identified. Comparison of the immunohistochemical data on the distribution pattern of various cells in the pancreatic islet in a naked mole rat with that detected by histochemical stains showed their complete coincidence.

CONCLUSIONS: Zinc-formalin can be used as a more convenient and safer fixative for detecting Langerhans islet cells (including D cells) by staining with Heidenhain’s azan and Gomori’s paraldehyde-fuchsin instead of mercury salt-containing Zenker-formol.

Morphology. 2021;159(1):29-32
pages 29-32 views

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