CYTOLOGICAL PICTURE OF APOPTOSIS IN LIVER CELLS

ABSTRACT

In liver cells, there is a certain life cycle, after which the cells undergo natural death. Sufficient material has been accumulated on the mechanisms of apoptotic death, and they are often performed in vitro in cell cultures or in vivo in living tissues or organs of the body. However, the most accessible and simplest method for revealing the morphological features of apoptotic cells is light microscopy. In this paper, the dynamics of the cytological picture of apoptosis in human and some animal liver cells is presented in a comparative aspect.

АННОТАЦИЯ

В клетках печени существует определенный жизненный цикл, по истечению которого клетки подвергаются естественной гибели. О механизмах апоптотической гибели накоплен достаточный материал и зачастую они выполнены in vitro- в культурах клеток или же in vivo- в живых тканях или органах организма. Однако наиболее доступным и простым методом выявления морфологических особенностей апоптотических клеток является световая микроскопия. В данной работе в сравнительном аспекте приводятся динамика цитологической картины апоптоза в клетках печени человека и некоторых животных.

Keywords: apoptosis, natural death, morphology, cytology, liver.

Ключевые слова: апоптоз, естественная гибель, морфология, цитология, печень.

Introduction. Apoptosis is a programmed cell death process that plays an important role in maintaining tissue homeostasis. In the liver, there is also a certain life cycle, after which the cells undergo natural death. To date, significant material has been accumulated on the mechanisms of apoptotic death, in most cases they are performed on cell cultures - in vitro, or in living tissues, organs of the body - in vivo, which is not always identified with the true course of apoptosis in living tissue. In this regard, the most accessible and simple method for detecting apoptotic cells in order to determine their morphological features is light microscopy of histological preparations. Previously, we studied the features of the morphological course of apoptosis in the liver cells of rabbits and white rats using light-optical and electron microscope methods. In this paper, the dynamics of the cytological picture of apoptosis in human liver cells and some other mammals is presented in a comparative aspect.

Material and methods. The material was the liver of mature outbred rabbits, white rats, domestic pigs and humans. Rabbits and rats were kept under normal vivarium conditions; domestic pigs were selected from farms of one offspring without signs of any pathology. The liver of patients was obtained from the mortuary of the Tashkent Dental Medical Institute, who died from various diseases. Animals were slaughtered in compliance with ethical standards, after opening the abdominal cavity, the liver was removed and processed by conventional histological methods. Sections 5-7 µm thick were stained with hematoxylin-eosin or van Gieson and viewed under immersion (x 100) of a DN-300 M microscope with a digital camera.

Research results. When stained with hematoxylin-eosin in the liver of a rat, hepatocytes are clearly distinguished, having a polygonal shape and oxyphilic cytoplasm with one or two basophilic stained nuclei. Morphological analysis of the liver parenchyma of various animals and humans showed that in most cases apoptotic cells are extremely rare, and in isolated cases more often, about 20 fields of view account for one apoptosis. Moreover, the initial signs of apoptosis in all animals and humans are approximately identical and are characterized by the appearance of single vacuoles in the hepatocyte cytoplasm (Fig. 1a). Then the number of vacuoles in the cytoplasm of the hepatocyte increases from 4-5 to 8-10, and at this stage apoptosis can be clearly identified (Fig. 1b), since there are no such vacuoles in the surrounding cells.

Figure 1. Apoptosis in hepatocytes. Explanations in the text

Then, large vacuoles in the cytoplasm of the hepatocyte disintegrate into smaller ones, more precisely vesicles, the cell acquires a spherical shape, and the cell nucleus shrinks (Fig. 1c). It should be mentioned that, as a rule, single cells undergo natural death, however, we detected apoptosis of 3 adjacent hepatocytes (Fig. 1d). At the final stage of apoptosis, the vesicular state of the cytoplasm is replaced by a delicate mesh, the cell acquires a strictly spherical shape, the cytoplasm looks light, and the nucleus becomes even more pycnotized and the cell swells towards the sinusoid. Probably, after the rupture of the cell membrane, the contents of the cell are washed out into the lumen of the vessel.

In contrast to the rat liver, in the liver of rabbits, according to morphological features, we have identified two types of hepatocyte apoptosis. The first type corresponds to the apoptosis of white rats described above. Large vacuoles also appear in the cytoplasm of the cell, which gradually fill the cytoplasm, the nucleus becomes pycnotized and the cell acquires a spherical shape, and therefore differ sharply from its neighbors. (Fig. 2a). In the liver of rabbits, the second type of apoptosis is also observed, when the cell cytoplasm is homogenized and stained oxyphilically, and the nucleus is fragmented into chromatin clumps. In this case, the cell also acquires a rounded shape, but does not contain vacuoles; however, the cell border is also clearly distinguished (Fig. 2b). The fact that two types of apoptosis do occur in the liver of rabbits is evidenced by the figure (Fig. 2c), where both types of apoptosis are found in a small area of the liver parenchyma. Cytological signs of both types differ from each other. In the first type of apoptosis, the cell has a spherical shape, the cytoplasm is strongly clarified and is represented by small vesicular elements, and the nucleus is pycnotized (the lower part of the figure). In the second type of apoptosis, the cytoplasm of the hepatocyte is stained oxyphilically, contains neither vacuoles nor vesicles, and the nucleus is fragmented into large clumps (upper part of the figure). At the final stage of both the first and second types of apoptosis, swollen cells protrude towards the sinusoid, and sometimes are eliminated into the lumen of the vessel (Fig. 2d).

Figure 2. Apoptosis in hepatocytes. Explanations in the text

Pig liver when stained according to van Gieson, lobules are clearly distinguished, the boundaries of which are presented in the form of straight lines, represented by thin layers of connective tissue. The hepatic triads are located in the region of the corners of the lobules. The hepatic plates and sinusoidal capillaries are oriented toward the center of the lobule, where the central vein is exposed. In the porcine liver, similarly to the liver of rabbits and rats, apoptosis occurs in single cells and, as a rule, they are found in the perivenous and occasionally in the intermediate zones of the lobule. Apoptotically altered porcine liver hepatocytes are characterized by uneven contours and the appearance of 2-3 large vacuoles in their cytoplasm and slight condensation of chromatin under the nuclear membrane (Fig. 3a). Then, the number of vacuoles in the cytoplasm increases to 5-8, as a result, the volume of the cell increases significantly, and the concentration of nuclear chromatin increases under the nuclear envelope, and thread-like structures appear. At the next stage, large vacuoles break up into smaller ones, therefore, sometimes along with 3-4 medium-sized vacuoles, numerous small vesicular elements appear in the cytoplasm (Fig. 3b, c, d). In rare cases, within the lobule, several cells at different stages of apoptosis are detected at once. In any case, in an apoptotic cell, large vacuoles break up into small vesicles, and the cell itself becomes rounded and the cytoplasm becomes brightly light, and such a cell is easy to detect in the liver parenchyma. Usually, not only mononuclear, but also binuclear hepatocytes undergo apoptosis. In such cases, the cytoplasm and nuclei of hepatocytes also change, moreover, in this case, the transition of different-sized vacuoles (Fig. 3c) into smaller vesicular structures (Fig. 3d) is clearly visible, which fill the cytoplasm with small uniform vesicles, probably corresponding to apoptotic bodies.

Figure 3. Apoptosis in hepatocytes. Explanations in the text

In the final stage of apoptosis in pig liver cells, apoptotic cells most clearly acquire a spherical shape, the volume of the cell increases significantly compared to the surrounding ones, and vesicular structures give them a bright, light appearance, which is why we designated them as “boiling cytoplasm” (Fig. 3d). At the end of the process, the apoptotic cell disintegrates into small bodies and, together with the pycnotized nucleus, are probably eliminated into the lumen of the sinusoid. The appearance of macrophages and the capture of apoptotic vacuoles by neighboring hepatocytes were not detected in our studies.

Discussion of the obtained results. In the adult organism, cell death in the liver is balanced with cell neoplasm, and apoptosis plays an important role in this process. As our studies have shown, mitotic division is extremely rare in the liver (only in rats, one mitosis was found per 2000 cells), obviously this is due to the peculiarity of the organ, where, along with cell division, polyploidy and mononuclearity occur. However, compared with mitosis, apoptosis was more common; moreover, it had a similar course in all the studied patients. As usual, single cells undergo apoptosis, occasionally a group of cells, and in the main, in the perivenous, sometimes in the intermediate zones of the hepatic lobule. According to the cytological course and depending on the predominant change in the cytoplasm or nucleus, we have identified two types of it. The first type is characterized by clarification of the cytoplasm and pycnosis of the nucleus, and the second type, by acidophilic staining of the cytoplasm and clumpy disintegration of the nucleus, respectively, designated as "boiling cytoplasm" and/or "nuclear catastrophe". Although both types of apoptosis are transient processes, certain stages can be distinguished during them. In the first type of apoptosis: vacuolization and an increase in cell volume; vesiculation and rounding; formation of apoptotic bodies and pycnosis of the nucleus; rejection and elimination of cellular decay into the lumen of the sinusoid. The second type of apoptosis is characterized by a sharp acidophilia of the cytoplasm and clumpy disintegration of the nuclear chromatin, which develop simultaneously and it is not possible to single out any stages. As our studies show, all stages of the first type of apoptosis are well detected on histological preparations, with staining with hematoxylin-eosin and according to van Gieson. Apoptosis of the first type begins with the appearance of 2-3 vacuoles, the volume of each is larger than the cell nucleus, and then their number reaches 7-8 and the cell increases sharply in volume. The appearance of vacuoles in the hepatocyte cytoplasm is referred to in some works as cell membrane vesiculation.

At the next stage, the vacuoles break up into small vesicles and the cells become rounded, acquire a spherical shape, the cytoplasm of the cell looks like a “boiling cytoplasm”, and the nucleus undergoes pycnosis. As a result of these changes, the cytoplasm is clarified, clearly distinguished from other cells, and the cell swells towards the lumen of the sinusoid. The second type of apoptosis was more common in rabbits and rare in rats. The second type of apoptosis is characterized by acidophilic staining of the hepatocyte cytoplasm and fragmentation of the nucleus into clumps of chromatin. Obviously, the second type of apoptosis is a transient process, since it is rarely detected in the liver parenchyma, and therefore it cannot be divided into separate stages. The second type of apoptosis is characterized by cell breakdown into granular structures, fragmentation of the nucleus into chromatin clumps, as well as wedging of erythrocytes between the hepatocyte cytoplasm. However, cell rounding, separating them from the hepatic lamina and washing out apoptotic bodies into the lumen of the sinusoid are identical with the first type of apoptosis. Some studies indicate that apoptotic bodies are taken up by neighboring cells or macrophages. Biochemical studies have shown that the process of apoptosis consists of 4 separate stages: initial, effector, degradation and absorption. Obviously, morphological studies - vacuolization, vesiculation correspond to the effector stage, and the formation of apoptotic bodies - the stage of degradation, the stage of elamination - absorption. Probably, the effector phase is associated with reorganization of intracellular actin filaments, namely, peripheral cortical annular bundles, as a result of contractions, the cell membrane forms swellings, and vacuoles appear in the cell. At the next stage, microtubules of the cytoskeleton are depolymerized, as a result of which the cell acquires a rounded shape, and the cell takes on a foamy structure, the cell “boils”. As a result, the cell is fragmented into small apoptotic bodies and eliminated into the vessel lumen.

Apoptosis plays an important role in morphogenesis and is a mechanism for constant control of the size of organs. Apoptosis is well detected both with hematoxylin staining and with van Gieson. And it comes to light as in the form of light or sharply acidophilic cells with a dense fragment of nuclear material. In the analysis of the liver parenchyma of humans and some animals, apoptosis was more common than mitotic division. After the fundamental work of Ker, apoptosis is assigned as a fundamental physiological process that maintains a certain balance of cells in the organ. Observations show that apoptosis occurs according to a certain morphological stereotype. Apoptotic cells were found predominantly in the central and intermediate zones of the hepatic lobule. However, in contrast to animals, in humans, apoptotic cells were detected in groups around the central vein. The study of the dynamics of the cytological picture of apoptosis in humans and animals makes it possible to distinguish the following stages: vacuolization, vesiculation - "boiling" of the cytoplasm, spherization, wrinkling of the nucleus and extrusion - rejection of the cell with the leaching of the cell contents into the lumen of the sinusoid (in the first type of apoptosis). Acidophilic staining of the cytoplasm with fragmentation of the nucleus and elamination of decay products into the lumen of the sinusoid (with apoptosis of the second type), the latter was mainly detected in rabbits and white rats. In this regard, we have identified two types of apoptosis "boiling cytoplasm" and nuclear pycnosis (first type of apoptosis) and acidophilic cytoplasm and nuclear decay (second type of apoptosis). In the liver of humans and pigs, we found only the first type of apoptosis, accompanied by vacuolization of the cytoplasm and pycnosis of the nucleus. At the final stage, pycnosis of the nucleus occurs and the cell eliminates into the lumen of the sinusoid. The last stages of apoptosis, separation of the cell from the liver plate, rupture of the cell membrane and leaching of the contents of the cytoplasm were clearly detected in the liver of rabbits and rats. Apoptotic bodies are engulfed by neighboring cells or macrophages. Therefore, we found only the first type of apoptosis in the pig's liver, from which it follows that only the first type of apoptosis is characteristic of the pig. In the literature, a detailed picture of the morphological course of apoptosis is quite rare, referring to the fact that apoptosis is a rare phenomenon, a fleeting process, morphologically difficult to identify and very laborious. Other studies indicate the possible verification of apoptosis, when stained with hematoxylin-eosin. Indeed, the process of detecting apoptosis is tedious and time consuming. However, it remains the only way to identify apoptosis in tissues.

References:

1. Altieri D.C. Survivin and IAP proteins in cell-death mechanisms // Biochem J. - 2010. - Vol. 430. - No. 2. - R. 199-205.
2. Cagnol S., Chambard J.C. ERK and cell death: mechanisms of ERK-induced cell death-apoptosis, autophagy and senescence // FEBS J. - 2010. - Vol. 277. - No. 1. - P. 2-21.
3. Galitsky, V. A. The emergence of eukaryotic cells and the origin of apoptosis / V. A. Galitsky // Cytol. - 2005. - T. 47, No. 2. - S. 103-120.
4. Titov S.A., Krutko V.N. Modern ideas about the mechanisms of aging (Review) // Human Physiology. - 1996. - T. 22. - No. 2. - S. 118-123.