Change in hepatocyte nuclear-cytoplasmic ratio at nontuberculosis mycobacteria infection against the background of immunomodulator action

INTRODUCTION

Currently, the role of non-tuberculosis mycobacteria has significantly increased globally. Mycobacterioses cause notable economic damage to animal production holdings, which is associated with a decreased performance, as well as with early culling of animals and their emergency slaughter [1-3]. High financial costs and lack of effective antiepidemic measures are a threat not only to the holding where mycobacteriosis has been detected, but also to the whole country [4-7]. This issue has not been studied in depth enough and requires increased attention of the scientific and industrial organizations [8-10].

Scientists from Russia and many foreign countries have proved that nontuberculosis mycobacteria have not only sensitizing capacities, but they can also localize inducing specific changes in the animal body and cross-immune reactions to the PPD-tuberculin administration [11-13].

Toxins released by mycobacteria during their vital activity in the macroorganism affect the enzymatic activity of the liver, thus causing pathological changes in the liver tissue, which leads to a decrease in the level of the blood-bile barrier [14-17].

One of the main functions of the liver is transformation of carbohydrates into glycogen, which is the most important energy resource of the body as a whole [18][19]. Moreover, stellate reticulum endotheliocytes have a phagocytic effect that neutralizes the accumulation and transport of toxic substances in the animal’s body [10][20][21].

According to the scientific data of many authors, one of the more effective methods of infectious pathogen control involves the use of immunoprophylaxis drugs [2][22]. Currently, many modern immunomodulators do not demonstrate a sufficiently high ability to stimulate the effectiveness of the immune response to the infectious agents and their toxins in the organism.

Development of modern immunocorrecting agents and their use makes it possible to increase the body’s resistance to the infectious pathogens. An increase in the immune response with the help of a specific immunomodulator enhances the regenerative properties of the cells, tissue and organ as a whole [23-25].

In view of the above material, the goal was set to study the effect of a specific immunomodulator on liver tissue under the laboratory infection with nontuberculosis mycobacteria.

MATERIALS AND METHODS

The research was carried out in a specialized animal keeping facility. Fifteen adult outbred guinea pigs of tortoiseshell color were used in the study. The animals demonstrated negative PPD test results.

A specific complex immunomodulator KIM-M2 was produced by cultivating BCG vaccine strain in a liquid synthetic Sauton medium, then the grown bacterial mass was destructed with an ultrasonic disperser UZDN-1 (Russia) and the resulting suspension was centrifuged at 15,000 rpm. The protein amount was determined in the collected supernatant after its incubation with formalin using bromophenol blue. Hereafter, the protein concentration was brought up to 1 mg/mL with saline solution. The resulting BCG antigen complex was conjugated with polyvinylpyrrolidone (PVP) and polyethylene glycol (PEG) [26].

The designed KIM-M2 series contained 320 mg of PVP and 80 mg of PEG polyions per 1 mg/mL of the protein. A 200 mL sample of the drug was prepared.

The animals were subdivided into three groups of five animals in each. The animals in group 1 (infection control) and group 2 (experimental) were inoculated with Mycobacterium scrofulaceum into the groin area at a dose of 0.001 mg/mL. In 2 weeks, the animals in the experimental group were subcutaneously injected with KIM-M2 immunomodulator into the inner thigh at a dose of 500 mg/mL of the protein. The animals in group 3 (pure control) were injected with a sterile 0.9% saline solution. The microorganisms of pathogenicity groups III–IV were handled in accordance with the sanitary rules and regulations (SanPiN 3.3686-21¹). The specific immunomodulator is based on the antigenic complex of BCG vaccine strain. On day 30 after the start of the experiment, the animals were euthanized under ether anesthesia and bled. Pieces of liver were extracted and fixed in 10% neutral formalin for further work. Further preparation was carried out at STP-120 automatic tissue processor (carousel type; Germany), paraffin blocks were embedded using EC 350 embedding center (Germany). Serial semi-thin sections (5–7 µm) were made using HM-340E microtome (Germany). Histological sections were stained with hematoxylin and eosin according to the generally accepted routine technique.

The work was carried out in compliance with the international principles set out in the Declaration of Helsinki for animals, Directive 2010/63/EU of the European Parliament and of the Council on the protection of animals used for scientific purposes, as well as in accordance with the Guidelines for regulating the use of experimental animals.

Computer morphometry and digital images of micro-preparations were performed in 10 fields using Axio Imager A1 (Zeiss, Germany) light microscope (ocular lens 10×, objective 40×), the area of hepatocytes and their nuclei was measured in micrometers (µm²) using a software package and the AxioVision version 4.8 archiving system.

Statistical processing of the digital data was carried out using Microsoft Office 2010 and involved determination of the arithmetic averages (M) and calculation of the errors of arithmetic averages (m). The significance was determined by the Student’s t-test and differences were considered significant at p < 0.05.

RESULTS AND DISCUSSION

The results of morphological examination demonstrated that in animals of group 1, the liver was visually enlarged and blood-filled, the edges did not converge along the incision, the organ was of loose consistency, dark brown color with large spilled gray lesions, the capsule was edematous and thickened. Beam structure was preserved. The hepatocytes were partially rounded. A large number of non-nuclear hepatocytes were observed. In 50% of the cases, nuclear karyopycnosis was reported, the cell wall was deformed, the cytoplasm was vacuolated and infiltrated into the intercellular space. Kupfer cells were not differentiated due to extensive overgrowth of loose connective tissue. The subendothelial space was expanded.

In KIM-M2 immunized animals in the experimental group, the liver was slightly enlarged, red in color, the capsule was within normal size, the liver lobules were clearly visible, the beam-radial structure was preserved. The centrilobular veins were varicosed. Small single focal hemorrhages were visible on the periphery of the hepatic lobes. Necrotic and dystrophic foci were not detected. There was a moderate connective tissue growth around the triads. The hepatocytes were identified, cell structure was determined, cytoplasm did not infiltrate into the intercellular space, karyopycnotic changes in the nucleus were not detected. Kupfer cells located in the periportal zone of the hepatic lobules were discernible in the sinusoids. No pathological lesions were detected in the intact animals.

The histological test results demonstrated that in the KIM-M2 immunized guinea pigs, the number of mononuclear hepatocytes was 63.5% of the total number of hepatocytes, which was 21.7% higher than in the infected control animals, and 11.0% less than in pure control animals. The proportion of binucleated hepatocytes from the total number of cells was 25.0%, which was 16.7% higher than in case of infection without the use of KIM-M2, and 7.0% higher than in the healthy animals. The number of anucleate hepatocytes was 11.5%, which was 38.4% less compared to infection controls and 4.0% higher compared to pure controls (Table 1).

Based on the data obtained, it can be seen that the specific immunomodulator of microbial origin KIM-M2 enhances the immune response owing to a significant increase in the number of binucleate hepatocytes during intensive mitotic division in animals of the experimental group. The number of mononucleate hepatocytes was lower in comparison with the pure controls, which can be explained by the abundant formation of binucleate hepatocytes. The number of anucleate hepatocytes increased insignificantly.

According to the morphometric study results, the average total area of mononucleate hepatocytes in animals in group 2 was established, which was 2,721.10 µm², which was 53.6% more than in the intact guinea pigs, and 22.8% less than in the infected animals in group 1. The average core area of mononucleated hepatocytes in animals inoculated with KIM-M2 was 289.83 µm², which was 47.6% more than in the intact guinea pigs and 45.0% less than in the animals in the infection control group. In animals in group 1, the average total area of mononucleate hepatocytes was 3,526.94 µm², the average core area of mononucleate hepatocytes was 526.76 µm². In intact animals, the average total area of mononucleate hepatocytes was 1,772.14 µm², the average core area of mononucleate hepatocytes was 196.35 µm² (Table 2).

Based on the results obtained, it can be concluded that the animals in group 2 accumulated potential restorative reserves and polyploidization reserves when exposed to the specific immunomodulator KIM-M2. In guinea pigs from the infection control group, a disturbed hepatocyte mitotic division was observed and the liver destruction process was reported, which slows down the recovery process.

According to the results of calculating the area of liver cells and their nuclei, the intracellular regeneration of the organ was determined by calculating the group nuclear-cytoplasmic ratio, which makes it possible to determine the level of metabolism and compensatory reactions in the body of laboratory animals.

CONCLUSION

Thus, in the animal experiment, it was established that a specific immunomodulator of microbial origin triggers and enhances the process of the liver cellular and intracellular regeneration, which stimulates the body to resist the mycobacterium toxins. In guinea pigs infected with nontuberculous mycobacteria, the destructive processes developed when KIM-M2 was not used, and the mechanism of liver tissue repair was underdeveloped.