Morphological features of histogenic differon cells in connective tissue of guinea pigs’ lungs after sensitization with ovalbumin

Results. We have established the regularity of morphological changes dynamics in the cellular elements of pulmonary connective tissue. Experimental sensitization and inhalated allergization with ovalbumin leads to a statistically significant increase in the average number of fibroblasts and fibrocytes throughout the observation period in all experimental groups. It has been proved that the dynamics of cells has a multidirectional character, demonstrated by indicators of the fibroblast/fibrocyte coefficient, which shows the disproportion in the fibroblast/fibrocyte ratio and proves the tendency to the development of fibrosis in guinea pigs’ pulmonary connective tissue in case of experimental sensitization with ovalbumin.

Актуальные вопросы фармацевтической и медицинской науки и практики. 2021. Т. 14, № 2(36). С. 220-225 The variety of connective tissue functions, such as morphogenetic, reparative, biomechanical, metabolic reflects a wide range of its morphological adaptive changes in response to various factors, including allergenic ones [1,2]. Airway hyperreactivity develops as a result of allergic airway inflammation in case of experimental sensitization with ovalbumin [3]. Asthma pathogenesis is represented as a multiple process in which nonspecific and specific immune and neuroendocrine points are involved. Interactions between them lead to the development of asthma hallmarksinflammatory process and as its result -airway remodeling [4][5][6]. A specific sign of connective tissue reaction in response to the action of an allergen as a result of the development of allergic inflammation is subepithelial fibrosis in airways [7]. The thickened basal lamina of the airway epithelium and the connective tissue of their respiratory mucosa contain an increased number of collagens types I, III and V, fibronectin and tenascin, which is a pathognomonic point of allergic inflammation and is not observed in case of other inflammatory diseases of the respiratory system [7,8]. The appearance and accumulation of deposits from glycosaminoglycans and collagen in the ground substance of extracellular matrix of connective tissue is associated with fibroblasts' or myofibroblasts' secretory function. The latter originate from resident cells of pulmonary connective tissue, such as fibroblasts and myocytes [8,9]. The mechanisms of differentiation of fibroblasts and myocytes into myofibroblasts, actively synthesized collagen types I, III, V in case of allergic inflammation, are currently being actively investigated by scientists [10,11]. However, morphological changes in the cellular component of the connective tissue of airways and lungs, experimentally sensitized, have not been sufficiently studied in the chronobiological aspect.

Aim
To estimate morphological features of histogenic differon cells in connective tissue of guinea pigs' lungs after sensitization with ovalbumin.

Materials and research methods
The experiment was performed on 48 male guinea pigs weighing 450-600 g, which were housed under standard environmental conditions in the animal facility of Zaporizhzhia State Medical University. All experimental procedures and the animal care were carried out according to ethical guidelines (Strasbourg, 1986;Kyiv, 2001).
Experimental model of allergic airway disease. Allergic airway inflammation was induced as previously described [12]. Guinea pigs received three subcutaneous injections and eight inhalations with ovalbumin (OVA) (Sigma Aldrich, USA). We use alum (10 mg/mL in saline) as an adjuvant (AlumVax Hydroxide vaccine adjuvant, OZ Biosciences France) with OVA injections to counteract tolerance.
Experimental design. We use six groups of guinea pigs in the experimental protocol (8 guinea pigs each). Animals of the first four groups with asthma model were euthanized with an overdose of thiopental (50 mg/kg) on the 23 rd , 30 th , 36 th and 44 th days of the experiment respectively; control guinea pigs of group 5 were administered with equivalent amount of saline; group 6 was intact. We subdivided the experiment to the early (23 rd , 30 th days of the experiment) and late (36 th and 44 th days of the experiment) periods of the pulmonary allergic inflammatory process. Paraffin sections of guinea pigs' lungs were stained by the method of Mason to estimate the number and distribution of collagen fibers, alcyan blue with a critical concentration of MgCl 2 0.2 M to determine the dynamic of glycosaminoglycans distribution and morphometric evaluation of histogenic differon cells of connective tissue [13]. The sections were examined by light microscope (Primo Star, Zeiss, Germany). We counted the number of fibrocytes and fibroblasts per 5000 μm 2 in 10 fields of lung strip in each animal and their fibroblast/fibrocyte ratio coefficient. Fibroblasts and fibrocytes were identified and differentiated by their morphological points at the lightoptical level. Fibroblasts are large polygonal cells up to 50 μm in size with rounded or oval euchromatic nucleus, have processes. Fibrocytes are spindle-shaped cells with the large, identical-shaped, dense nucleus, little volume of cytoplasm and long processes.
Statistical analysis was performed using Microsoft Excel and Statistica for Windows 13 (StatSoft Inc., № JPZ804I382130ARCN10-J). Data were evaluated by Shapiro-Wilk test and the Kolmogorov-Smirnov test. Values are expressed as means M ± m. Multiple comparisons were made using the parametric Student's t-test (P*) and the nonparametric U-Whitney-Mann test (P**). The obtained data were compared between the median and interquartile range Me (Q 1 ; Q 3 ). P value of <0.05 was considered significant.

Results
Morphological evaluation of guinea pigs' lungs after experimental sensitization with ovalbumin reveals reactive morphological changes in the connective tissue of airways and pulmonary interstitium. There is edema and disorganization of collagen fibers and changes in the tinctorial properties of the ground substance, such as more intensive metachromatic staining, compared to the control group. Along with this, we observe a reaction from the fibroblastic elements of the connective tissue of lung. Some of the fibroblasts increase in size, the cytoplasm acquires a more pronounced basophilia. Some cells divide mitotically. In addition, in case of ovalbumin sensitization, intense diffuse and focal perivascular and peribronchial infiltration by lymphocytes is observed (Fig 1).
Morphometric assessment of diffusely located histogenic differon cells of connective tissue in lungs of intact group guinea pigs has shown that their average number was as follows: fibrocytes 6.50 ± 0.24, fibroblasts 7.50 ± 0.16, fibroblast/fibrocyte coefficient 1.46 ± 0.06. We have not found statistically significant difference in the number of cells between control and intact groups (P*/** > 0.05), indicated that the procedure has not influenced changes in data content from connective tissue cells. Therefore, we compare results of the experimental and control groups in further observation.
The number of fibrocytes and fibroblasts in the connective tissue of guinea pigs' lungs changes after sensitization and inhalation with ovalbumin. Analysis of the average number of fibrocytes in the early period of allergic inflammatory process in the lungs reveals that their number increases statistically significantly compared to the control group since the 23 rd day after the experiment and is 9.5 ± 0.09 in the field of view, which is 1.7 times more than in the control group (Fig. 2). We have declared a tendency to an increase in the number of fibrocytes on the 30 th day of the experiment in the 2 nd experimental group. There is also a statistically significant difference between the average number of fibrocytes in animals of the control group and after sensitization with ovalbumin, observed in the late period of the experiment (36 th and 44 th days) in animals of 3 rd and 4 th experimental groups. Thus, on the 36 th day after the start of the experiment, the average number of fibrocytes is 12.88 ± 0.15 in the field of view, the coefficient of increase is 2, compared to the control group. On the 44 th day of observation, the maximum value of the average number of fibrocytes reaches 13.38 ± 0.14 in the field of view. The maximum coefficient of increase (2.3) is observed in the 4 th experimental group, compared to the control group (Fig. 4).
Analyzing the number of connective tissue fibroblasts in guinea pigs' lungs in the early period of allergic inflammation, we have found that their number increases statistically significantly (P*/** < 0.05) from the 23 rd day of the experiment (12.75 ± 0.21 in the field of view), compared to the control group (Fig. 3). The maximum coefficient of increase in the average number of fibroblasts compared to the control group is 1.8 and is observed on the 23 rd day of the experiment in the 1 st experimental group. Fibroblast/ fibrocyte coefficient is 1.37 ± 0.03 in the 1 st experimental group, which is slightly less than the same indicator in the control group. We have reported the tendency to increase of the number of fibroblasts on the 30 th day of the experiment in the 2 nd experimental group. The fibroblast-fibrocyte coefficient in the 2 nd experimental group is 1.30 ± 0.04 (Fig. 4).
The statistically significant difference in the average number of fibroblasts compared to similar indicators in animals of the control group is also observed in the late period of allergic inflammatory process in lung (36 th and 44 th day after the start of the experiment) in animals of the 3 rd and 4 th experimental groups. On the 36 th day after the start of the experiment, the average number of fibroblasts ss 10.38 ± 0.27 in the field of view, the magnification factor is 1.5, compared to the control group. The tendency to gradually significant decrease of the number of fibroblasts is shown with increasing experiment time. Fibroblast/fibrocyte coefficient decreases statistically significantly in 1.8 times in the late period of development of allergic inflammatory process in lung, compared to the similar indicator in the control group.

Discussion
Thus, our study determines the pattern of dynamics of morphological changes in the cellular elements of the respiratory connective tissue. Experimental sensitization and challenging with ovalbumin leads to a statistically significant increase in the average number of fibroblasts and fibrocytes during all observation periods in all experimental groups. The multidirectional nature of their dynamics attracts attention. The maximum number of fibroblasts occurs during the first period of pulmonary allergic inflammation on the 23 rd day of observation, after which there is a tendency to gradually reduce their content to the 44 th day of the experiment. In contrast to the dynamics of fibroblasts, the average content of fibrocytes gradually increases during the experiment, with the maximum coefficient of increase on the 44 th day. This multidirectional nature of cell dynamics reflects the fibroblast/fibrocyte coefficient, during the experiment, indicated a disproportion in the fibroblast -fibrocyte ratio, especially in the late period of allergic inflammation. The latter fact is a confirmation of the tendency to the development of fibrosis and sclerotic processes in connective tissue of guinea pigs' lungs during experimental sensitization with ovalbumin. Our morphological observation of guinea pigs' lung connective tissue cells in general confirmed with previous studies. Previously reported persistently elevated number of fibrocytes in bronchial mucosa is observed in chronically undertreated or corticosteroid-resistant asthma and are associated with persistent airway inflammation and connective tissue remodeling of the bronchial wall [10]. The asthmatic bronchial epithelium is predominant source of fibrocyte chemoattractants in asthma and contributes with T helper type 2 lymphocytes and eosinophils to promote the proliferation and remodeling function of recruited fibrocytes. The presence of elevated numbers of fibrocytes in the bronchial mucosa of allergic patients may also increase the risk of acute exacerbations because these cells can amplify T helper type 2 inflammation to the clinically relevant allergen and can promote further inflammation [4,14,15]. Several studies have revealed subepithelial fibrosis, primarily mediated by submucosal resident fibroblasts proliferated and differentiated into myofibroblasts [9,10]. However, authors have shown that oncostatin M-induced products of connective tissue cells, such as monocyte chemoattractant protein 1 (MCP-1), IL-6, and PGE2 can modulate macrophage function, including the expression of oncostatin M produced by macrophages and neutrophils, indicating feedback loops characterized macrophage and connective tissue cell interaction in extracellular matrix remodeling and allergic inflammation [16]. Oncostatin M receptor (OSMRβ chains) is expressed by pulmonary connective tissue fibroblasts and bronchial smooth muscle cells. Scientists have associated lung extracellular matrix hyperproduction with defective epithelial -connective tissue crosstalk during allergic inflammation, suggesting the aberrant activation of the asthmatic epithelial-mesenchymal trophic unit (EMTU) may lead to disease pathogenesis [14]. A similar trend has been recently showed in studies of other scientists [9,17].
In conclusion, our results may become a part of the foundation for the further investigation of morphological basis of mechanisms of epithelial neuroendocrine -immune interaction in the animal models of widespread diseases.

Conclusions
1. There is a gradual increase in the average number of fibrocytes (respectively 9.50 ± 0.09 in the 1 st experimental group to 13.38 ± 0.14 in the 4 th experimental group) and a decrease in the average number of fibroblasts (12.75 ± 0.21 in the 1 st experimental group to 10.37 ± 0.27 in the 4 th experimental group) from the 23 rd to the 44 th day after the start of experimental sensitization with ovalbumin in guinea pigs' lungs.
2. A decrease of the fibroblast/fibrocyte coefficient from 1.37 ± 0.03 in the early period to 0.82 ± 0.03 reflects a divergent nature in the dynamics of the number of connective tissue cells in the late period of allergic inflammation, indicated a tendency of development of fibrosis in the connective tissue of guinea pigs' lungs.   Prospects for further research. We are planning to study the components of ground substance of extracellular matrix and ultramicroscopic changes in the connective tissue cells of guinea pigs' lung in case of experimental sensitization with ovalbumin.

Funding
This research is a part of the research work of Zaporizhzhia State Medical University "Immunomorphological characteristics of internal organs under the influence of endo-and exogenous factors on the body", state registration No. 0118U004250.