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Abstract

This study explores the impact of the extract from the bursa of Fabricius on the bone marrow of mice with CPA-induced immunodeficiency. Myelogram analysis revealed that CPA administration resulted in lymphopenia and suppression of lymphopoiesis, while granulocyte sprouts are activated, indicating a non-specific immune response. The treatment of mice with CPA-induced immunodeficiency with the extract from the bursa of Fabricius stimulated the lymphoid lineage in the bone marrow, as evidenced by an increase in lymphoblasts and mature lymphocytes. However, there was no significant restoration of lymphocyte numbers in peripheral blood, possibly due to the lack of antigen stimulation. The extract also led to an increase in neutrophilic and erythroid cell content. Additionally, administration of the extract from the bursa of Fabricius stimulated cell mitotic activity and increased the total number of myelokaryocytes. These findings suggest a significant immunotropic effect of the extract on bone marrow under conditions of immunodeficiency, resulting in the activation of lymphoid and erythroid sprouts, as well as an increased leukocyte and granulocyte content. The demonstrated ability of the extract to counteract the cytostatic effect of CPA highlights the potential for further research into isolating and identifying active peptides with immunostimulatory properties‎.

Keywords: Bone marrow, Cell cycle, Immune modulation, Secondary immunodeficiencies, Myelogram

Introduction  

Secondary immunodeficiencies are acquired pathological conditions where there is a disruption in the immune response mechanisms, leading to a reduced overall immune system activity and its ability to fight against infectious agents effectively. Immunodeficiency can occur independently or develop secondarily due to various external and internal factors. Factors contributing to weakened immune defenses include excessive physical activity, cancer, inadequate protein intake, dysfunction of regulatory systems, exposure to toxins, and prolonged use of drugs. Immunodeficiency states are relatively common and are characterized by an increased susceptibility to viral and bacterial pathogens [1-20].

Secondary immunodeficiencies, which involve impaired immune responses and increased susceptibility to pathogens, can develop under the influence of factors such as cyclophosphamide (CPA), a commonly used agent to induce immunodeficiencies in laboratory animals. With the increasing need for innovative immunotropic drugs, bioorganic materials derived from animals, such as bursa extract, show promise. Bursa extract has low toxicity and specific effects, making it appealing for developing immunomodulatory treatments. Despite its known immunostimulatory properties, the impact of bursa extract on central immune organs like the bone marrow remains understudied, highlighting the importance of researching its effects on the bone marrow of mice with experimental immunodeficiency [5-8, 21-36].

Materials and Methods

C57BL/6 mice were utilized for the experiments due to their small size, low maintenance requirements, and ability to adapt well to vivarium conditions [37-39]. However, limitations arise from the specific parameters associated with these animals, complicating result interpretation and their applicability to humans. The experimental portion of the study involved three-month-old male C57BL/6 mice raised and housed in the vivarium in strict adherence to the sanitary, hygienic, and veterinary standards for laboratory animals [6, 40-48]

All manipulations and interventions in the experimental protocol adhered to Directive 2010/63/EU of the European Parliament and the Council regarding the protection of animals used for scientific purposes. The study received official approval from the Ethics Committee of the Institute of Immunology and Physiology of the Ural Branch of the Russian Academy of Sciences, following the established procedure.

The standard conditions in the vivarium included a constant air temperature of +20–22° C, free access to food (Delta Feeds, a mixed food for laboratory mice and mice - LbK 120 C-19, RF) and water. The experimental animals were randomly divided into three groups with the same number of six mice in each group.

Three experimental groups were formed:

1. Control group of mice (n = 6),
2. Animals (n = 6) in which immunodeficiency was modelled (CPA)
3. The animals (n = 6) were injected intraperitoneally with a bursa extract at a dose of 0.1 ml/20 g against the background of immunodeficiency modelling.

The experimental procedures were terminated, and the animals were excluded from the study on the seventh day after the creation of the experimental state of immunodeficiency. Animals in all experimental groups were premedicated 15-20 minutes prior to planned euthanasia: a 2% solution of xylazine was administered intramuscularly at a rate of 1 ml per 1 kg body weight, as was the drug Zoletil-100 at a dose of 0.3 ml/kg. All procedures were carried out in accordance with the approved protocols and ethical standards for the treatment of laboratory animals.

To model the secondary immunodeficiency state in laboratory animals, a single injection of the cytotoxic drug cyclophosphamide (Endoxan®, Baxter Oncology GmbH, Germany) was administered into the peritoneal cavity. The dosage was calculated individually for each mouse based on body weight and was 200 mg/kg. The solution was prepared immediately before administration by dissolving the drug in a sterile 0.9% sodium chloride solution so that the concentration of the active component was 20 mg/ml. The animals in the control group received an equivalent volume of isotonic 0.9% sodium chloride solution. Before the manipulations were carried out, the body weight of all animals was recorded to ensure accurate dosing.

The mechanism of the cytostatic effect of CPA is based on DNA alkylation. CPA arrests the cell cycle in stages G0/G1 and S through the formation of cross-links and DNA damage.

Cyclophosphamide has a cytotoxic effect that induces cell apoptosis. As an antitumor agent, it inhibits the processes and proliferation of bone marrow cells, resulting in a decrease in the cellular elements in the hematopoietic tissue and a reduction in its functional activity.

To establish the immunodeficiency model, laboratory mice were administered a single intraperitoneal injection of the cytostatic drug, cyclophosphamide, at a dose of 200 mg/kg body weight. The drug was dissolved in a sterile 0.9% sodium chloride solution to achieve a final concentration of 20 mg/ml. The control group of animals received an injection of an isotonic solution without the active substance added.

To quantify the total number of myelokaryocytes extracted from the femoral marrow, a method involving mechanical disruption of the tissue with a rubber bulb into a test tube containing a 3% acetic acid solution with methylene blue as a coloring agent was used. The cell suspension was homogenised by pumping it through a needle attached to a syringe and then collected in a leucocyte pipette. The sample was diluted 20-fold with the same acetic acid solution.

The Goryaev chamber was cleaned with acetic acid solution before use, and the contents of the pipette were added carefully to the chamber. The cells were counted in 20 large squares of the chamber.

The total number of myelokaryocytes was calculated using the following formula:

K = n × 20 × 250 × 4000 / 20

(1)

Where:

• TMC – total myelokaryocyte count;
• n — the number of cells counted;
• 20 — the dilution coefficient;
• 250 is — the conversion factor for the volume of one large square (1/250);
• 4000 — the multiplier for recalculating the concentration from 1 µl to a total volume of 4000 µl;
• 20 – the number of large squares taken into account.

In order to evaluate not only the total number but also the distribution of myelokaryocytes in the bone marrow smear, they were morphologically differentiated. The starting material was obtained by flushing bone marrow tissue into a petri dish with a drop of isotonic solution. The tissue fragments were carefully dispersed with sterile forceps until a homogeneous cell suspension was achieved. The resulting suspension was applied to a clean glass slide to create a smear. Once the preparation had naturally dried, it was fixed in ethyl alcohol. Staining was performed using the Romanovsky-Giemsa method with a 15-minute exposure time. After staining was completed, the slides were washed under running water. The morphological analysis of the cell elements was conducted at a magnification of ×100 using an immersion system on a Leica DM 2500 microscope (Germany).

The analysis revealed data on the absolute number of myelokaryocytes (in millions of cells per femur), categorized by their morphological subtypes, and the percentage distribution of different cell forms was calculated.

Results and Discussion

When examining the qualitative and quantitative composition of bone marrow cells, known as myelogram, in laboratory mice of the C57BL/6 line after a single injection of cyclophosphamide (CPA), a decrease in the total number of lymphoid cells was observed compared to intact animals. This decrease affected both mature lymphocytes and their precursors, lymphoblasts (Table 1).

 

 

 

The likely cause of induction is the direct stimulating effect of bursal peptides, as well as an indirect effect through the activation of morphogenesis factors produced by the liver for hematopoietic cells involved in erythro- and myelopoiesis processes. This observation suggests that bone marrow progenitor cells may be in G-1 phase with a transition to the S phase of the cell cycle (Figure 1).

 

Figure 1. Bone marrow myelogram parameters in C57BL/6 mice after administration of CPA and the extract from the bursa of Fabricius (x 106 cells/femur)

The consistent number of immature erythrocyte precursors in mice after CPA administration confirmed the hematological analysis data, indicating the development of an anemic condition. This was also evidenced by a decrease in the level of young forms of erythrocytes in the bone marrow.

It is important to note that the total number of myelokaryocytes increased by 10% compared to the control group, which is associated with the rapid growth of neutrophilic cell lines. In animals with experimental immunodeficiency, the use of extract from the bursa of Fabricius led to the activation of lymphoid growth in the bone marrow. This effect was achieved by increasing the content of lymphoblasts and mature lymphocytes, as well as increasing the total number of lymphoid line cells compared to intact individuals and groups without treatment.

However, no significant lymphocyte restoration was observed in the peripheral blood. This is likely due to the absence of an antigenic stimulus necessary for the migration of lymphocytes from the bone marrow into systemic circulation. Other studies have shown that stimulation of lymphocyte production with bursal peptides becomes more pronounced only after prior sensitization of the body with viruses or other antigens. Thus, in this case, lymphoid cells increased the reserve population, which can be activated when the antigen load increases due to viral or bacterial pathogens.

In C57BL/6 mice, treatment with the extract from the bursa of Fabricius increased the content of neutrophilic cell lines and their precursors compared to the control sequence, as well as to the model of immunodeficiency without therapy. An increase in the number of erythroid elements in all experimental groups after extract administration was also detected (Table 2), indicating a multifactorial effect of the drug on hematopoietic processes.

The observed increase in the proliferation of leukocytes and erythroid precursors following the introduction of the extract from the bursa of Fabricius was evidenced by a significant increase in the total number of nuclear cells in the bone marrow, which was approximately 1.5 times higher than that in the control group. Additionally, an increase in mitotic activity was observed, indicating a higher frequency of cell divisions.

Conclusion

Thus, it has been established that the extract from the bursa of Fabricius has a pronounced immunotropic effect on the bone marrow when modeling immunodeficiencies. It stimulates the mitotic activity of cells, increases the total number of myelokaryocytes, and simultaneously activates lymphoid and erythroid sprouts. These changes were accompanied by an increase in the absolute leukocyte and granulocyte content in the peripheral blood, as well as a tendency to normalize the erythrocyte indices.

The ability of the extract to compensate for the cytostatic effect of CPA opens up prospects for further research aimed at isolating and identifying active peptides with immunostimulating activity. The obtained data allow us to consider the extract from the bursa of Fabricius as a potential basis for creating drugs intended to correct immunodeficiency processes.

Acknowledgments: The authors thank the administration of the Ural State University of Economics for the opportunity to research its basis.

Conflict of interest: None

Financial support: None

Ethics statement: The study was conducted according to the guidelines of the Declaration of Helsinki.

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