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0EVALUATION OF MORPHOFUNCTIONAL FEATURES OF THE NUCLEAR APPARATUS OF HEPATOCYTES DURING
LIVER RESECTION
1Isayeva N.Z., 2Mansurova D.A., 3Khusanova M.A.
1,2,3Alfraganus University https://doi.org/10.5281/zenodo.13884839
Abstract. Subject of 25% liver resection in the early stages of the experiment (3-7 days) showed that in the post-resection period, both the mitotic division figures and the yield of nucleoli were very insignificant (3-5%). However, on the 15th day of the post-resection period, the number of binucleated hepatocytes and nucleoli migration were increased. The studied data showed that a number of binucleated hepatocytes not only increases, but also participates in the cellular renewal of the liver, and, consequently, in the processes of organ regeneration.
Keywords: liver, resection, binuclear hepatocytes, nucleolus, regeneration, migration.
The analysis of morphological picture of animals subjected to 25% liver resection in the early terms of the experiment (3-7 days) showed that in the postresection period both mitotic division figures and nuclei output were very insignificant (3-5%). However, on the 15th day of the postresection period, the number of binucleated hepatocytes and migrating nuclei increased markedly. The studied data showed that the number of bi-nucleated hepatocytes not only increased, but also participated in cell renewal of the liver, and, consequently, in the regeneration processes of the organ.
The liver is a unique organ that maintains homeostasis. It performs transformation of exogenous amino acids, carbohydrates, detoxification and excretion of endogenous metabolic products and xenobiotics into bile. Among the elements of the nuclear apparatus of hepatocytes, a very small cell, the nucleus, is of great importance. Our studies have shown that when it is necessary to synthesize a large amount of protein for the needs of the liver itself or the body, the nucleus of rabbit hepatocytes can migrate into the cytoplasm and participate in synthetic processes [1, 2, 8].
In order to establish whether this phenomenon of nucleus exit exists not only in rabbits but also in other mammals, we selected widely used laboratory animals - white mongrel rats, and as an experiment - 25% liver resection (left lateral lobe of the liver). Sexually mature animals weighing 180-200g under mild ethominal anesthesia were subjected to laparotomy and liver resection was performed according to the generally accepted technique. Then the animals were slaughtered at 3, 7, 15 and 30 days after the operation.
Liver pieces were processed by the above methods, and histological preparations from both control-intact and operated experimental animals were analyzed under a microscope.
The first scientific study of the process of liver regeneration was conducted in 1931 by E. M. Hiqins and R. M. Anderson. To study the mechanism of regeneration, various experimental approaches were used, which were based on the evaluation of liver regeneration, after its chemical damage, surgical resection in vivo, as well as studies of hepatocyte proliferation in primary culture in the presence of risk factors in vitra.
The most widely studied example of liver regeneration is the model of its regrowth after resection. It has been noted that after resection of liver tissue, compensatory regeneration occurs with the formation of new cellular elements by mitotic division of the remaining ones. Thus, liver regeneration is a compensatory growth process in which the resected lobe does not grow, but the mass of the remaining lobes increases as a consequence of cell proliferation [5;6;12].
The level of proliferative activity of hepatocytes depends to some extent on the volume of resection: the larger the portion of the liver removed during surgery, the higher the proliferative pool of cells. Removal of less than 40% of tissue is accompanied by low DNA synthesis, which makes us suggest that the triggering effect of removing a small amount of tissue is insufficient (perhaps, under these circumstances, functional deficiency does not develop). It is known that after resection of 60--70% of liver tissue, the remaining part of the liver experiences a significant energy deficit that persists for 1 day. Subsequently, the energy status increases and approaches the initial values after 5-7 days.
Purpose of the study. To study the effect of resection on nucleus migration and to investigate the role of nucleus migration on the regenerative processes of the liver in rats.
Materials and Methods. The liver of sexually mature rats of both sexes served as the material. The experiment was conducted on 25 mongrel white rats weighing 180-200 g in compliance with the principles of humane treatment of animals as set forth in the Helsinki Declaration and the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes [2]. The animals were kept on a standard vivarium diet with free access to food and water.
Animals were subjected to 25% liver resection and were decapitated on 3, 5, 7, 15 and 30 days after resection, under ethominal anesthesia (40 mg/kg of animal body weight). For morphometric and light-optical studies (LEICA GALEN III microscope) histological preparations were fixed in 10% buffered aqueous formaldehyde solution and examined according to the generally accepted technique. Morphometric study of liver preparations was performed at 1000x magnification on 5 p,m thick sections stained with hematoxylin-eosin using the method of point morphometric grids overlaying [1].
The number of mononuclear and dinuclear hepatocytes, hepatocyte diameter, nuclei were determined [10]. Statistical processing of the study results was performed by the method of variation statistics using the software package "Statistica 10" with the use of the parametric Student's t-criterion [3]. Differences of the compared values were considered statistically significant at p < 0.05. All experimental work was performed in compliance with the rules of bioethics approved by the European Convention for the Protection of Vertebrate Animals Used for Laboratory or Other Purposes.
Sections for histologic studies were stained with hematoxylin-eosin, and preparations were photographed using a microscope model N-800M, under an immersion objective (x100). The experimental group consisted of 26 rats. The material of the study was liver of sexually mature mongrel rats.
Results and Discussion. The results of the conducted studies showed that the most important data were obtained when analyzing the morphological picture of animals subjected to 25% liver resection. In the early terms of the experiment (3-7 days), both figures of mitotic division and nuclei exit were insignificant (3-5%). However, the number of exiting nuclei markedly
increased on the 15th day about 8-10 times (preliminary data), compared to the control, in a small area of the field of view slightly prominent nuclei were detected (Fig. 1).
Hepatocytes are polygonal in shape, their cytoplasm is oxyphilically colored, nuclei contain 2- 3 small nuclei or sometimes one large nucleus. In Fig. 1 a binuclear hepatocyte is in the center, each nucleus of which contains one nucleus, and next to it another hepatocyte is, in which it is clearly visible how the nucleus bulges towards the cytoplasm, obviously, it is at the stage of penetration of the nuclear envelope.
By 30 days of the postoperative period the number of nuclei output (1.75±0.88) significantly decreased, approaching the norm. Thus, at liver resection, along with the increase in the number of mitoses on the 3-5 days of the experiment, the output of nuclei (1,31±0,21**) in hepatic cells with the formation of many nuclear and young cells markedly increases. A characteristic feature of mitotic activity of hepatocytes in small resections is that it proceeds without reaching peak values, i.e. there is no pronounced synchronization of mitotic cycles of hepatocytes. This fact was the basis for some authors to consider the recovery of liver mass after such resection (10-30% of liver parenchyma) as a peculiar model of regeneration impairment [Mitchell C. et al., 2005]. It is believed that such dynamics of proliferation is explained by a later onset of the peak activity of the main growth factors and cyclins D1, A, E [Mitchell C. et al., 2005]. Based on this, some authors consider the proliferation of hepatocytes after small resections (1030% of liver parenchyma) as a kind of a model of violation of the mitotic cycle of hepatocytes, in which it is impossible to synchronize cell proliferation [Lambotte L. et al., 1997, Li J. et al., 2009]. The obtained data testify not only to the increase of dinuclear hepatocytes, but also to their participation in cell renewal in the liver, hence, in the processes of organ regeneration. One of the indicators of the intensification of regenerative processes in the liver is an increase in the content of dinuclear hepatocytes (T.A. Zaletaeva, 1963; B.P. Solopaev 1980; L.K. Romanova, 1984; I.V. Stupin et al., 1995, etc.). The mechanism of their origin is not quite clear. Most researchers currently deny amitotic division of nuclei in any tissue. At the same time, there appeared indirect evidence that a significant part of mitoses in the liver is acytokinetic, which leads to the formation of binuclear hepatocytes [2;3;4].
At 3 days after resection, the liver of experimental animals contained a large number of mitotically dividing hepatocytes. A large number of dividing hepatocytes was detected up to 7 days after surgery, then they became less. However, single figures of mitosis were found even 10 days after the operation. At the final stages of regeneration among hepatocytes binuclear cells were more frequently found (8,89±2,1**).
The diameter of nuclei of mononuclear hepatocytes increases on the 5th day (2.59±0.44**) and then steadily decreases. On the 30th day (2,59±0,44**) it is close to control, diameter of nuclei of binuclear cells sharply increases on the 5th day (1,97±0,31**) and begins to decrease till the 15th day (1,41±0,74*), then there follows a jump-like increase on the 30th day (1,71±0,88**) deviated from control. The number of nuclei of mononuclear cells slightly increases on the 5th day (1,90±1,05), then it steadily decreases till the 30th day. On the 30th day the index is slightly less than the control one (1,75±0,88). The number of nuclei of bi-nuclear cells sharply decreases on the 5th day (1,31±0,21**). Up to day 10 it does not change (1,31±0,21**). After the 15th day it starts to grow (1,51±0,84**) and by the 30th day the index is close to the control (1,68±0,95).
USWL, V * r v mm ' ^ ¡j
Fig. 1. Rat liver with partial resection. Hematoxylin and eosin staining. The cytoplasm of the liver cell is stained oxyphilic, and the large basophilic nucleus often contains one large nucleolus. In a mononuclear hepatocyte, the large nucleolus is at the stage of exiting through the nuclear membrane. Zoom x100. Fig. 2. Rat liver after partial resection. Hematoxylin and eosin staining. A. In the center of the figure is a figure of mitotic division of a hepatocyte. Zoom in x 40
Morphometric indices of rat liver in normal and after 25% resection In rat liver parenchyma after 25% resection, the number of both mononuclear and dinuclear hepatocytes increased on the 3rd day compared to control, the diameter of peripheral hepatocytes was slightly increased, the diameter of hepatocytes around the lobule was significantly increased 0, 9 times (16,7+0,6), the diameter of nucleus is increased in comparison with control data (7,3+0,3), the diameter of nuclei is also increased on the 3rd day p=0,019 (1,97+0,1), the number of nuclei is markedly decreased p=0,0046 (1,3+0,1)in comparison with the previous and subsequent days. On the 5th day in dynamics the number of uninuclear p<0.0000 (12,8+0,4) and binuclear p<0.0000 (8,9+0,2) hepatocytes increased in comparison with the previous days, the diameter of hepatocytes of the periphery (17+0,5) and around the lobules P=0.0002 (17,9+0,2) were markedly increased. The number of nuclei were markedly decreased compared with the control. On the 7th day, the number of uninucleated 1.8 times P<0.0000 (16.9+0.4) and dinuclear cells 1.8 times P<0.0000 (8.9+0.2) were sharply increased. Diameter of nuclei of bi-nuclear cells decreases p=0,031 (5,3+0,1), diameter of hepatocytes around lobules increased 1,14 times p=0,0004 (18,1+0,3), number of nuclei of bi-nuclear hepatocytes starts to increase p=0,044 (1,5+0,1). On the 15th day, the number of mononuclear 2.0 times p<0.0000 (18,4+0.3) and dinuclear cells 2.0 times p<0.0000 (9,2+0.3). Diameter of nuclei of binuclear cells without
changes, diameter of hepatocytes around lobules increased 1,14 times p=0,012 (17,9+0,6), number of nuclei of binuclear hepatocytes starts to increase p=0,044 (1,5+0,1). The rest of the indices without obvious changes. Thus, the diameter of peripheral hepatocytes slightly increases by 5 days, on the 7th day it decreases, and on the 15th day a sharp increase is observed. The diameter of hepatocytes around the central vein stably increases after resection and remains until the 15th day. The largest diameter is reached on the 7th day. The diameter of nuclei of mononuclear hepatocytes increases on the 3rd day and then steadily decreases. On the 7th day it is close to the control, the diameter of nuclei of dinuclear cells sharply increases on the 3rd day and begins to decrease up to the 7th day, then a jump-like increase follows, on the 15th day it deviates from the control.
The number of nuclei of mononuclear cells slightly increases on the 3rd day, then it steadily decreases until the 15th day. On the 15th day the index is slightly less than the control one. The number of nuclei of dinuclear cells sharply decreases on the 3rd day. Up to the 5th day it does not change. After the 7th day it starts to grow and by the 15th day the index is close to the control.
At liver resection, nuclei of hepatocytes participate in regenerative processes along with mitosis (3-5 days), their output increases on the 7th day of the experiment, hence, nuclei necessarily participate in the restoration of the lost mass of the organ.
At liver resection, nuclei of hepatocytes participate in regenerative processes along with mitosis (3-5 days), their output increases on the 15th day of the experiment, therefore, nuclei necessarily participate in the restoration of the lost mass of the organ.
Conclusions. Thus, we can conclude that at liver resection, along with mitotic division of hepatic cells there is also migration of hepatocyte nuclei, moreover, this process is stimulated, especially on the 7th day after the surgical period, which may indicate the regenerative value of the nucleus exit into the cytoplasm of the cell. The increase in the number of multinucleated hepatocytes, according to literature data, indicates the activation of protein-synthetic function of the cell. One way or another, the increase in the number of dinuclear hepatocytes, together with hypertrophy of hepatic cells, can be regarded as a single compensatory-adaptive mechanism aimed at normalization of the structure and function of the organ.
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