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EXPERIMENTAL OUTBRED RATS MODELING OF PULMONARY FIBROSIS
Elvina M. Sharipova; Vazira Z. Jalalova
Bukhara State Medical Institute
Resume:
The impact of COVID-19 on lung function is the most important in the post-
coronavirus period. The effects of experimentally induced inflammation of the respiratory
tract have not been thoroughly studied. The pathogenesis of the development of fibrosis in
the lungs is local and systemic production of proinflammatory cytokines, followed by
narrowing of the respiratory tract. In the post-COVID years, there are many studies
describing the signs of pulmonary fibrosis, such as increased fibroblast proliferation and
deposition of extracellular matrix in the late stages of acute lung inflammation. This article
describes the modeling of fibrosis in an experiment induced by daily exposure to nitrogen
dioxide in the upper respiratory tract of experimental animals.
Thus, it should be noted that the modeling we have chosen seems possible and effective,
which allows us to study the mechanisms of pulmonary fibrosis development and ways of
early diagnosis, timely treatment and prevention.
Key words:
experimental model, pulmonary fibrosis, outbred rats, modeling of pulmonary
fibrosis in rats.
INTRODUCTION
The incidence of idiopathic pulmonary fibrosis (IPF) has been reported in several studies
worldwide and appears to be increasing, but different case ascertainment methodologies and
classification systems have prevented reliable comparisons between studies. Several review
articles from different countries on incidence and prevalence have been reviewed, but
current evidence suggests that there is no statistical analysis of overall incidence and
mortality data worldwide [ Hutchinson J. (2015)].
Work has been conducted on the mediastinal lymph nodes of patients with IPF, revealing
differential expression profiles than those of patients with lung cancer, indicating distinct
immune-mediated pathways regulating fibrogenesis and carcinogenesis. PD-1 expression in
mediastinal lymph nodes is consistent with that in lung tissue. Low-dose pembrolizumab
may have an antifibrotic effect. Clinical trials aimed at endotyping patients based on
mediastinal lymph node profiling and, accordingly, the introduction of targeted therapies
such as PD-1 inhibitors are highly anticipated. With the gradual understanding of the
pathogenesis of IPF, new progress has been made in the treatment of IPF [ Karampitsakos T.
(2023)].
However, there is room for progress before satisfactory efficacy can be achieved. Recent
experiments have confirmed that the PD-1/PD-L1 pathway can interact with various cell
types and pathways and is involved in promoting fibrosis and immune regulation in IPF.
Simultaneously, animal experiments have shown that the use of PD-1/PD-L1 inhibitors
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reduces the symptoms of pulmonary fibrosis. In this brief review, we present the effects of
PD-1/PD-L1 in IPF; ongoing studies suggest that this may offer a new direction for future
IPF therapy [ Tan J. (2024)].
Enlarged mediastinal lymph nodes (LNs) are common in idiopathic pulmonary fibrosis (IPF)
and are known to be associated with the severity of pulmonary fibrosis. However, the
relationship between mediastinal LNs and IPF prognosis is not entirely clear to date [ Sin S.
(2017)].
Another study was performed on a population-based study of 152 patients with IPF, of
whom 135 (89%) received antifibrotic treatment for IPF during the study follow-up. Among
the 62 patients with LNE who underwent follow-up chest CT and received antifibrotic
treatment, 57 (92%) had persistent evidence of mediastinal LNE over time [ Sgalla G.
(2020)]. The results showed that diffuse mediastinal lymph node involvement predicts
clinically significant functional deterioration in patients with IPF [ de Souza Xavier Costa N,
(2017)].
Currently existing treatments for IPF have limited efficacy and the prognosis of the disease
remains disappointing. The latest advances in understanding several interrelated
pathogenetic mechanisms of IPF is the identification of various molecular phenotypes
resulting from complex interactions between genetic, epigenetic, transcriptional, post-
transcriptional, metabolic and environmental factors. For accurate and early diagnosis of IPF
and improvement of the prognosis of the disease, it is necessary to develop and validate
diagnostic and prognostic biomarkers [ Namakanova O.A. (2024)]. Antifibrotic therapy with
potentially new drugs requires the use of biomarkers that make it possible to prognosticate
the course of the disease and assess the effectiveness of drug therapy, which in turn
contributes to a wider use of personalized therapy [Anaev E.Kh. (2017)].
In this work, we attempted to create a model of pulmonary fibrosis on white mongrel rats,
which will show how a fibrosis-like condition is formed in the lungs in an experiment,
which makes it possible to study the relationship of this pathology with other organs and
systems, provoked by the introduction of nitrogen dioxide into the respiratory tract.
MATERIALS AND METHODS
In the experiment, sexually mature white mongrel rats weighing 180-220 g were used to
model pulmonary fibrosis. All ethical requirements were met and approved by the protocol
of the Ethics Committee of the Ministry of Health of the Republic of Uzbekistan ( (No.
6/16-1699 dated September 27, 2022 ) [ Barnoev A.I., 2023 ] .
Pulmonary fibrosis itself was provoked by passive introduction of nitrogen dioxide into the
respiratory tract of experimental animals. For this purpose, experimental rats were placed in a
special box and exposed to NO
2
30-40 mg/m
3
using a specially adapted fan with an outlet
pipe through which toxic substances are released into the open atmospheric air. On the
opposite side there is a vent that is tightly closed during poisoning of experimental animals.
The role of the toxic substance was performed by nitrogen dioxide obtained by the chemical
reaction of nitric acid with zinc metal. To create a concentration of the toxic substance in the
range exceeding the MAC (single concentration in the air of 0.4 mg/m3), namely 30-40
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mg/m3 (the model was proposed by L.N. Danilov et al., 2009), the reaction of nitric acid with
zinc inside the box was used.
The reaction used concentrated nitric acid and Zn 0.08 mg, respectively. The concentration
of vapors of the toxic substance in the air was derived using chemical and mathematical
equations. As a result of the reaction of nitric acid with metal, vapors of the toxic substance
nitrogen dioxide are released, which, with prolonged poisoning for 90 days, daily cause
inflammatory connective tissue changes in the lung parenchyma. A fan was mounted inside
the chamber to create the same concentration of NO
2
throughout the entire volume of the
box. The animals were exposed to the toxic substance daily for 30 minutes three times a day
with an interval of 15 minutes between poisonings. The ventilation intervals of the chamber
were made to remove the accumulated carbon dioxide released during the breathing of the
animals. The box was ventilated by exhaust through an exhaust pipe with an open vent in the
chamber. After each ventilation of the chamber, the toxic substance was re-injected.
As a result, it was found that after 60 days, the surface of the visceral membrane in the lungs,
the drainage function of the lymph nodes, sharply decreases. As a result, varying degrees of
narrowing of the lymphatic vessels that have reached a morphofunctional passive state lead
to varying degrees of tissue fluid retention in the interstitial tissue of the lung tissue, chronic
obstructive and inflammatory diseases of the lungs. At later stages from 60 to 90 days,
significant changes in the lung tissue were formed, such as vascular fibrosis and perivascular
fibrosis, as well as interstitial and interalveolar fibrosis. With these clinical and
morphological changes in the bronchopulmonary lymph nodes, functional changes of
varying degrees occur.
The experimental animals were divided into 2 groups by age, 6- and 9-month-old, and
correspondingly control groups.
The removed lung tissue samples were fixed in 10% formalin, embedded in paraffin, and
after hardening, standard 6-7 μm thick sections were prepared. The sections were stained
with hematoxylin and eosin and histochemically with alcian blue. The prepared histological
preparations were studied under an NLCD-307B binocular microscope (Novel, China). The
study materials were subjected to statistical processing using parametric and nonparametric
analysis methods. Accumulation, adjustment, systematization of the initial information and
visualization of the obtained results were carried out in Microsoft Office Excel 2010
spreadsheets. Statistical analysis was performed using the IBM SPSS Statistics v.23 program
(developer - IBM Corporation).
RESULTS AND DISCUSSION
The model we proposed was used to determine and study morphofunctional and structural
changes in the lung tissue and regional lymph nodes, in particular axillary and
bronchopulmonary. Under experimental conditions, pulmonary fibrosis is manifested by a
continuous change of inflammatory processes, pathological processes and reparative
regeneration processes. A significant part of the experiment was modeled to study changes
in the lymph nodes, while long-term changes in lung tissue were studied superficially.
In our study, we used a model of pulmonary fibrosis induced by passive introduction of a
toxic substance into the respiratory tract of a rat. The proposed model allows us to evaluate
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stepwise changes in the acute inflammatory period, chronic obstructive stage and chronic
inflammation stage, as well as the later period of fibrosis formation.
The simulation lasted 90 days, with an intermediate study of lung tissue and regional lymph
nodes on the 30th and 60th days. The results obtained from white outbred rats in the group
with the simulation of pulmonary fibrosis, but not subjected to correction, were studied.
Secondary follicles developing in the germinal center are revealed. As a result, foci of
diffuse hyperplasia are revealed in the lymphoid follicles of the cortex. In the secondary
lymphoid follicles, lightly stained cells consist of the reproductive center (germinative) and
a cluster of lymphocytes with a dark-colored crown around it. Intensively growing B-
lymphocytes, lymphoblasts, macrophages, dendritic cells and lymphocytes are found in the
germinal center of the lymphoid follicles. The following results were obtained in a study of
the bronchopulmonary lymph nodes of white outbred rats with experimentally induced
pulmonary pneumosclerosis.
It was found that at the initial stage of modeling (day 30) a small amount of collagen was
deposited, which indicates only the formation of fibrosis. Accordingly, a longer period will
be required for the accumulation of a larger amount of collagen in the lung tissue, taking this
conclusion into account, we continued the modeling up to 90 days. The same conclusion
was made regarding changes in the bronchopulmonary lymph nodes.
In conclusion, it was found that long-term exposure of the respiratory tract of
experimental animals to nitrogen dioxide leads to the expected changes, namely, typical
signs of pulmonary fibrosis with the deposition of collagen fibers in the lung tissue, and also
gave informative results in the study of regional lymph nodes. All of the above leads us to a
clear understanding of the problem of pulmonary fibrosis, which in turn may help us
completely rid humanity of this destructive disease in the future.
CONCLUSION
The presented modeling is noninvasive , reproducible and quite feasible, which was carried
out by introducing nitrogen dioxide into the respiratory tract of rats. A more thorough study
of the pathogenetic processes of pulmonary fibrosis reveals an understanding of this
pathology in general and possible complications, and also makes it possible to find new
ways in early diagnosis, therapy and prevention, which is the main advantage of our study.
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