Eff ect of platelet automesoconcentrate on the immunological status of experimental animals after liver resection

Objective. To study the indices of cellular and humoral immunity in the blood of rats after liver resection and under conditions of administration of platelet automeso–concentrate. Materials and methods. The study was performed on albino Wistar rats weighing 140 – 245 g. Resection of 2/3 of the liver was performed using the method of aseptic removal of its left and central lobes. During the resection, platelet autologous concentrate at a dose of 1 ml/kg was injected into the liver residue. To characterise the cellular and humoral components of immunity, the levels of leukocytes, lymphocytes, the number of total T– and B–lymphocytes, the level of circulating immune complexes, and the content of immunoglobulins of classes G and M were determined. Results. In the initial stages after liver resection, a marked leukocytosis was observed, accompanied by an increase in the relative number of basophils, eosinophils, rods and monocytes and a decrease in the level of segmented neutrophils and lymphocytes. The level of lymphocytes decreased at the expense of the T–cell population. Against the background of an increase in the number of B–lymphocytes, there was an increase in the levels of immunoglobulins M, G and circulating immune complexes. The injection of platelet autologous concentrate into the liver residue during resection of this organ stimulated the cellular and humoral immunity, as the studied parameters were higher than those of animals that did not receive platelet autologous concentrate. On the 7th day after partial hepatectomy, the studied parameters approached the control values. Conclusions. Components of cellular and humoral immunity can directly or indirectly affect the processes of liver regeneration after liver resection. B–cells after liver resection as antigen–presenting cells can trigger and modulate the immune response, which is enhanced by the introduction of platelet autologous concentrate into the body.

Liver regeneration after liver resection is accompanied by the release of hepatocytes from the G 0 -phase, their activation and several replication cycles [1].As a result of the compensatory hyperplasia process, proliferation processes are intensified and liver volume and weight are restored [2].This requires a large number of genes involved in liver regeneration, which can be divided into three groups: cytokine, growth factor, and metabolic [3].There is a complex interaction between these groups, so a decrease in the expression of one gene rarely leads to a complete blockage of liver regeneration.The immune system plays an important role in the initiation of liver regeneration after liver resection, as new cytokines and receptors have been identified that are involved in the initiation mechanisms [4].
It follows from the above that both the intensity of regenerative processes in the liver and the formation of pathology largely depend on the state of the immune system and its reactivity [5].This is due to the fact that the immune system plays an important role in ensuring the structural and functional homeostasis of the body.Pathological shifts in the immune status of the body can directly or indirectly affect the regenerative processes in the liver after resection of a signifi-cant (65-70%) part of it [6].In addition, the indicators of cellular and humoral immunity can be used to judge the state of the body after partial hepatectomy, the presence of immunotoxicosis syndrome of varying severity, the development of inflammatory processes after resection and the intensity of regenerative processes [7].
Activated platelet-derived factors serve as messengers and regulators of liver regeneration, which are able to influence various interactions between cells and between cells and the extracellular matrix [8].Therefore, it seems promising to use platelet automeso-concentrate (PMC) during liver resection to further stimulate regenerative processes in the liver and reduce the incidence of complications.Platelet AMC contains more than 300 biologically active molecules that are released upon activation from alpha platelets and dense granules and subsequently regulate the process of tissue regeneration [8].The dependence of the regenerative effects of platelet-derived AMP on the immunological status of the organism remains poorly understood.
The a im of the study was to investigate the indices of cellular and humoral immunity in the blood of rats after liver resection and under conditions of platelet-derived AMC administration.

Materi als and methods
The study was conducted on albino Wistar rats weighing 140 -245 g, which were kept on a normal vivarium diet.The animals were kept and all manipulations with them were in accordance with the provisions of the European Convention for the Protection of Vertebrate Animals Used for Research and Scientific Purposes (Strasbourg, 1986) and the recommendations of the VII National Congress on Bioethics "General Ethical Principles of Animal Experiments" (Kyiv, 2019).
At the beginning of the experiment, rats were divided into three groups of 18 in each group: animals undergoing sham surgery without liver manipulation (control -C); animals undergoing resection of 65-70% of the liver (partial hepatectomy -PH); animals in which platelet-derived AMCs at a dose of 1 ml/kg (PH + AMC) were injected into the liver residue during liver resection.
Resection of 2/3 of the liver was performed using the method of aseptic removal of its left and central lobes [9] and anaesthesia by injection of 1% sodium pentobarbital.After liver resection, the animals were brought out of anaesthesia.
Platelet AM C samples injected into animals during liver resection were prepared according to the described method [10] immediately before use.
The rats we re euthanised on days 1, 4 and 7 after liver resection and administration of platelet-derived AMCs using light ether anaesthesia.Fresh blood plasma used in the study was obtained by centrifugation with the anticoagulant sodium citrate for 15 min at 2500 rpm.
To characterise cellular and humoral immunity, the levels of leukocytes, lymphocytes, the number of total T-and B-lymphocytes, the level of circulating immune complexes (CIC), and the content of immunoglobulins (Ig) of classes M and G were determined.
The total numb er of lymphocytes, as well as the number of T-and B-lymphocytes, was determined using light microscopy, and the level of CECs was determined by precipitation of CECs present in the blood with high molecular weight polyethylene glycol.The optical density of the samples was measured in 1 cm thick quartz cuvettes at a wavelength of 450 nm [11].The content of Ig M and Ig G in blood plasma was determined by enzyme-linked immunosorbent assay [12].
The results were statistically analysed using Student's t-test.The results were considered statistically significant at p 0.05.

Results
The development and progression of complications and inflammatory processes after liver resection largely depend on the state of the immune system.In addition, the state of the cellular and humoral immune system can significantly affect the functional capacity of the liver.The intensity of the immune response and its impact on the development of inflammatory processes depend on the functional state of various parts of the immune system.
Determination of the indicators of the cellular immunity showed an increase in the total number of leukocytes in the blood of rats after liver resection during the first week of the experiment, which may indicate the development of inflammatory processes after surgery.The administration of platelet-derived AMCs at a dose of 1 ml/kg did not affect the number of leukocytes on the 1st day after liver resection, since the studied index did not differ from the indexes of animals that were not administered platelet-derived AMCs.On the 4th day, a marked leukocytosis was still observed.However, the level of leukocytes in the blood of rats injected with platelet-derived AMC was 1.3 times lower than the corresponding indicator of rats not injected with platelet-derived AMC.On the 7th day of the experiment, the level of leukocytes in the group of animals injected with platelet-derived AMCs approached the control values, indicating the restoration of metabolic processes in the body (Fig. 1).
An increase in the level of leukocytes may be due to the number of cells of different leukocyte populations.It was found that the leukocyte formula showed a shift in the indices of granulocytes and agranulocytes.Thus, on the 1st day after liver resection, an increase in the relative number of basophils, eosinophils and rod-shaped neutrophils and a decrease in the level of segmented neutrophils in the blood of rats of both experimental groups was detected.Among the agranulocytes in the blood of rats, the number of lymphocytes decreased and the level of monocytes increased (see Table ).
As the period after liver resection increased, the number of basophils, eosinophils and rods of neutrophils decreased, and the number of segmented necrophils increased.However, the approach of the studied parameters to the control values was observed on day 7 of the experiment only in animals that were injected with platelet-derived AMCs into the liver residue during liver resection.As for agranulocytes, as the time of the surgical intervention was removed, the level of lymphocytes increased, and the level of monocytes decreased with the approach to the control values on day 7 of the experiment in rats injected with platelet-derived AMCs.
The decrease in the level of lymphocytes in the short term after liver resection was due to a decrease in the population of UJCS.2023 July/August; 90(4)

The Ukrainian Journal of Clinical Surgery
T-lymphocytes (Fig. 2).Thus, the determination of the state of cellular immunity showed that in animals of both experimental groups on the 1st day after surgery, the content of the T-lymphocyte population decreased to the same extent compared with the control (p 0.05).
A decrease in the number of T lymphocytes can contribute to a decrease in cellular immunity.In addition, the postoperative condition of animals can further suppress the functions of T lymphocytes, which reduces their activity.The decrease in T-lymphocyte content may be due to a decrease in the number of their subpopulation -T-helper and T-suppressors, which indicates a discoordination of the immune system after liver resection [13].
On the 4th day after liver resection, the level of T-lymphocytes in the blood of animals increased, but in rats treated with platelet-derived AMCs, the studied index was at a higher level compared to the index of animals that were not treated with platelet-derived AMCs.On the 7th day after liver resection, the level of T-lymphocytes approached the control values only in the group of rats injected with platelet-derived AMC.
The increase in the level of T lymphocytes is probably due to T helper cells -Th-1 and Th-2, which can produce interleukin (IL)-10, which promotes the conversion of Th-0 to Th-1.The latter regulate the intensity of cellular reactions by synthesising IL-2, IL-12, and tumour necrosis factor-.Th-2 synthesise IL-4, IL-6, IL-10, IL-13, and stimulate humoral reactions by initiating the production of immunoglobulins by B cells.This suggests that the administration of plateletderived AMCs stimulates the T-cell component of immunity, which may be the basis for its clinical efficacy [14].
Changes were detected not only in the T-cell component of immunity, but also in the level of B-lymphocytes.The results of the study showed an increase in the number of B lymphocytes compared to the control on day 1 of the experiment (Fig. 3).Thus, it was found that in animals that did not receive platelet-derived AMCs, the level of B-lymphocytes increased 1.8-fold, and in rats that received platelet-derived AMCs -2.2-fold compared with the control values (p 0.05).On the 4th day after liver resection, the level of B-lymphocytes gradually decreased, but did not reach the control values and was higher in animals injected with platelet-derived AMCs.

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Increasing the number of B cells can help maintain CD169 cells + , which are critical for liver regeneration, as depletion of CD169 cells + leads to a decrease in IL-6 expression after liver resection.This reduces the activation of STAT3 signalling pathways [15].Therefore, the use of platelet-derived AMCs can restore defective liver regeneration by initiating the production of B lymphocytes.On day 7, the number of B-lymphocytes decreased to control levels in both study groups (Fig. 3), which may indicate the completion of liver regeneration processes [15].
The results suggest that the administration of platelet-derived AMCs may contribute to the initiation of an immune response in the body through the population of B lymphocytes that produce immunoglobulins in the body.To test this assumption, we determined the level of Ig M and Ig G in the blood plasma of rats at different times after liver resection.
The analysis of the results showed that along with an increase in the number of B-lymphocytes in the blood plasma, the amount of Ig M and Ig G increased on the 1st day after liver resection, followed by a decrease on the 4th day of the experiment (Fig. 4).An increase in immunoglobulin levels was observed to a greater extent in animals injected with platelet-derived AMC.Thus, while in rats that did not receive platelet-derived AMCs, the level of Ig M increased 2.2 and 1.6-fold compared with the control on days 1 and 4, respectively (Fig. 4, a), in animals that received plateletderived AMCs, the level of Ig M increased 3.5 and 2.8-fold compared with the control on days 1 and 4, respectively (p 0.05).Similar changes were observed in Ig G levels (Fig. 4, b), as their level exceeded the control values by 1.6 and 1.5 times on days 1 and 4, respectively, in animals that did not receive platelet-derived AMC, and by 2.1 and 1.8 times on days 1 and 4, respectively, in animals that received plateletderived AMC (p 0.05).
Increased levels of immunoglobulins in rat blood plasma, on the one hand, may be due to an increase in the concentration of B-lymphocytes that produce antibodies, and on the other hand, the inability of this organ to sequester antigens may be manifested in animals after resection of a significant part of the liver.In addition, decreased hepatic clearance of antigens through the hepatic portal vein system can lead to increased systemic antibody production or impaired antibody excretion by the resected liver [16].
On the 7th day after liver resection, the levels of Ig M and Ig G decreased and approached the control values in both experimental groups.Since the effect of Ig G stimulates the differentiation and proliferation of stellate cells in the liver, a decrease in its level on day 7 of the experiment plays an important role in preventing liver fibrosis.In addition, the approach of the concentration of immunoglobulins in the blood plasma to the baseline level may indicate the cessation of regeneration processes [17].
Along with an increase in the levels of immunoglobulins in the blood plasma, the concentration of CECs increased on days 1 and 4 of the experiment in both experimental groups, with a predominance of this indicator in animals injected with platelet-derived AMCs (Fig. 5).The increase in CIC concentration may be associated with a decrease in liver volume after resection, since these complexes are normally formed in the blood and are phagocytosed and destroyed by phagocytes and liver cells.Approximation of the CIC index to the control values was observed on day 7 of the experiment.

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Thus, components of the cellular and humoral immune system can directly or indirec tly affect the processes of liver regeneration after partial hepatectomy.B cells after liver resection, as antigen-presenting cells, can trigger and modulate the immune response, which is enhanced by the introduction of platelets into the body.In this case, the processes of liver regeneration will be more effective.

Discussion
The immunological status of an organism is determined by various factors, including the functioning of the immune system and the interaction of cells and molecules responsible for immunity.One of the key components of this process is platelets, blood cells known for their role in haemostasis and blood clotting.Recently, researchers have been paying attention to the possible impact of platelet AMP on the immunological status in experimental animals after liver resection.
Liver resection is a serious surgical procedure that can cause a number of changes in the functioning of the immune system.A decrease in liver volume can affect the production of a number of biologically active substances that regulate the immune system.Thus, the study of the effect of platelet AMC is becoming an urgent task to understand the mechanisms underlying these changes.
One of the possible reasons for the use of platelet-derived AMCs is their effect on the regulation of inflammation and immunity.Platelets produce a number of biologically active substances, such as platelet-derived growth factor, which can affect the activity of immune cells.
Experiments on animals after liver resection and the use of platelet-derived AMPK can help determine whether there is an improvement in the immunological status after such an intervention.It is important to keep in mind that the results of such a study may be contextually dependent on the specific conditions of the experiment, the methods used, and the properties of platelet-derived AMPK.
The development and progression of complications and inflammatory processes after liver resection largely depend on the state of the immune system.In addition, the function-al capacity of the liver can significantly affect the state of the cellular and humoral immune system.The intensity of the immune response and its impact on the development of inflammatory processes depend on the functional state of various parts of the immune system.
The study of immune cell parameters confirmed an increase in the total number of leukocytes in the blood of rats during the first week after liver resection, which may indicate the activation of inflammatory processes.The administration of platelet-derived AMCs at a dose of 1 ml/kg did not affect the number of leukocytes on day 1 after surgery, as this indicator did not differ from that of rats that did not receive plateletderived AMCs.On day 4, a pronounced leukocytosis was observed, but the level of leukocytes in the blood of rats injected with platelet-derived AMC was 1.3 times lower than in rats not injected with platelet-derived AMC.On the 7th day of the experiment, the leukocyte count in the animals with plateletderived AMCs approached the values of the control group, indicating the restoration of metabolic processes in the body.

Fig. 1 .
Fig. 1.The level of leukocytes in the blood of rats of experimental groups.Notes.Statistically significant difference: * -compared to the control; # -compared to the animals that underwent liver resection and were not administered platelet-derived AMC (p 0.05).The same in Figs. 2 -5.

Fig. 3 .
Fig. 3. Level of B-lymphocytes in the blood of rats of experimental groups.