Method for experimental modeling of drug-induced rhinitis in immature rats by intranasal administration of alpha-adrenergic receptor agonists

Abstract

FIELD: experimental medicine.SUBSTANCE: invention relates to physiology, otolaryngology, pediatrics and pharmacology. Immature rats are administered an alpha-adrenergic receptor agonist intranasally three times a day at a dose of 3 μg / 100 g of animal weight. The period of administration of the drug is 28 days.EFFECT: method allows modeling changes in the nasal mucosa corresponding to drug-induced rhinitis in immature Wistar rats.1 cl, 3 dwg, 2 tbl, 1 ex

Description

[0001] The invention relates to experimental medicine, namely to the modeling of drug-induced rhinitis and can be used in the field of physiology, otolaryngology, pediatrics and pharmacology.

[0002] Currently, rhinitis medicamentosa is a pressing problem. It is known that with long-term topical application, intranasal adrenergic agonists act directly on α1- and α2-adrenergic receptors in the nasal cavity, causing vasoconstriction and symptoms of nasal obstruction (rhinitis medicamentosa) [1, 6, 7, 8, 9].

[0003] Adequate modeling of the processes under the action of intranasal adrenergic agonists on the nasal mucosa in adolescents is ethically not reproducible and difficult to study.

[0004] A method for modeling drug-induced rhinitis in adult rats has been demonstrated, which has been confirmed by histological examination. The following morphological changes have been described: almost complete loss of cilia across the entire epithelial layer in all layers of the nasal mucosa, desquamation of epithelial cells, and, in some cases, necrosis of the epithelial lining. The lamina propria exhibited pronounced lymphohistiocytic infiltration, perivascular, and interstitial edema [5].

[0005] The limitation of the method is that the study was conducted on mature rats, and only local changes in the nasal mucosa were studied.

[0006] A method for modeling paranasal sinusitis in rats is known [3], which consists in the fact that Staphylococcus aureus, phage type 80 / 81, is injected directly into the mucous membrane of the nasal cavity of the experimental animal in a volume of 0.2 cm 3containing 2 billion microbial bodies. According to the authors, this method reduces trauma and simplifies the technology for producing the experimental model. However, this method does not allow for studying changes in the nasal mucosa caused by prolonged exposure to intranasal adrenergic agonists.

[0007] A method for modeling inflammation in the paranasal sinuses during chronic tobacco smoking is described [4], which consists in the fact that, under experimental conditions, rats are placed in an inhalation chamber into which tobacco smoke containing 98 mg / m3 is supplied 3 Carbon monoxide emissions from cigarettes containing 11 mg of tar and 0.8 mg of nicotine were measured at a rate of 1 cigarette per 8-10 minutes, with smoke delivered daily for 1.5 hours over a 6-month period. However, this method also does not allow for studying changes in the nasal mucosa caused by long-term exposure to intranasal adrenergic agonists.

[0008] The objective of the present invention is to develop a method for experimental modeling of drug-induced rhinitis in immature Wistar rats.

[0009] The technical result of the claimed invention is changes in the nasal mucosa in a model of drug-induced rhinitis in immature Wistar rats.

[0010] The technical result of the claimed invention is achieved due to the fact that immature Wistar rats were administered alpha-adrenergic receptor agonists intranasally for 28 days.

[0011] The details, features, and advantages of the present invention follow from the following description of the implementation of the claimed technical solution using drawings, which show:

[0012] Fig. 1 - Normal histological structure of the mucous membrane in immature Wistar rats. Hematoxylin and eosin staining x 400.

[0013] Fig. 2 - Surface of the nasal mucosa. Focal desquamation of the epithelium. Hematoxylin and eosin staining x 200.

[0014] Fig. 3 - Necrosis of the epithelial lining. Hematoxylin and eosin staining x 200.

[0015] The method is as follows.

[0016] The drug-induced rhinitis model was performed in immature 40-day-old Wistar rats. Their weight at the start of the experiment ranged from 100 to 150 grams. The rats in the drug-induced rhinitis model were 70 days old at the time of the study.

[0017] The animals were randomized into two groups: the main group (n=12) and the control group (n=12). Rats in the main group were administered nasivin drops intranasally for 28 days at a dose of 3 μg / 100 g of animal weight three times a day (daily dose 9 μg / 100 g). The control group received a similar volume of saline solution intranasally and at the same time. The dosage for immature rats was calculated based on weight (Table 1).

[0018] Table 1.

[0019] Calculation of Nazivin dosage for modeling drug-induced rhinitis in immature rats

[0020] Parameters Dosage Age: From 40 to 70 days 0.05% solution of Nazivin, 1 drop 3 times a day for 28 days Weight from 100-150 g to 250 g Single dose 0.003 mg / 100 g of body weight Daily dose 0.009 mg / 100 g of body weight

[0021] The animal study protocol is presented in Table 2.

[0022] Table 2

[0023] Animal Study Protocol

[0024] Day number Procedure Comments 1 Examination of the animal Animal condition assessment, weighing 1-28 Examination of the animal Taking the study drug 29 Examination of the animal Animal condition assessment 30 Anesthesia and tissue collection Taking histological material for examination

[0025] Study duration: 30 days.

[0026] Comments: Animal examinations are aimed at promptly detecting any changes in their condition. All procedures are carried out to ensure the welfare of the animals and obtain reliable scientific data. Tissue collection is performed after anesthesia to minimize stress on the animals.

[0027] Procedure Description:

[0028] Day 1: At the beginning of the study, each animal undergoes an initial examination to assess its general condition. They are weighed to determine the drug dosage. The data is recorded in a log.

[0029] Days 1-28: During this period, animals receive the study drug daily. During this period, animals are regularly examined to monitor their condition and response to the drug.

[0030] Day 29: The animals are re-examined to assess their current condition.

[0031] Day 30: The animals are weighed and the data is recorded in a log. The anesthetic dosage is calculated based on the animals' weight. Nasal mucosal tissue is collected and preserved. The resulting material is then sent for histological examination.

[0032] On the 30th day, histological material was collected. After anesthesia, the nasal turbinate was isolated from the animal with a 25% Urethane solution (Sigma) at a dose of 800 mg / kg intraperitoneally. The nasal mucosa tissue was prepared for the study: the nasal mucosa tissue was placed in 10% formalin for 24-48 hours. Then the tissue was embedded in paraffin [2]. Paraffin sections with a thickness of 3-4 μm, prepared on a microtome (MicromHM 340E, Germany) from paraffin blocks, were fixed on glass slides. The sections were deparaffinized and dehydrated by sequential incubation in toluene for 2 changes of 5 minutes, absolute ethanol for 2 changes of 5 minutes, and 95% ethanol for 2 changes of 3 minutes. The slides with sections were rinsed in distilled water and transferred into distilled water.Hematoxylin was applied to the sections for 5 minutes, then rinsed under running water. Eosin was used to stain the nuclei. Eosin was applied to the histological sections, left for 20-30 seconds, and then rinsed under distilled water. The specimens were then inoculated with Canada balsam. The hematoxylin-eosin-stained sections were analyzed to determine morphological changes in the nasal mucosa.

[0033] Research results.

[0034] The mucous membrane of the nasal cavity is normal.

[0035] The epithelial lining is represented by a single-layer, multi-row, columnar, ciliated epithelium containing goblet cells (Fig. 1). The epithelium is located on a basement membrane, which separates it from the lamina propria of the mucosa. The latter is represented by loose, fibrous, irregular connective tissue with numerous blood vessels and the presence of mucous and serous glands. Lymphocytes, plasma cells, as well as isolated leukocytes and mast cells, are found here. Neutral mucopolysaccharides are detected in the lamina propria of the mucosa and, to a lesser extent, in the epithelial lining. Acidic mucopolysaccharides are absent in the epithelium and are detected in some connective tissue fibers and the walls of individual blood vessels of the lamina propria of the mucosa. Intra- and extravascular fibrin is usually not detected in normal nasal mucosa (Fig. 1).

[0036] Nasal mucosa in the drug-induced rhinitis model after 28 days of administration of Nazivin:

[0037] Morphological changes were detected in all layers of the mucosa. In all cases, the normal structure of the epithelium was disrupted. Most notable was the almost complete loss of cilia over the entire surface of the epithelial layer. Desquamation of epithelial cells was observed in some areas (Fig. 2), and in some cases, necrosis of the epithelial lining (Fig. 3). Uneven perfusion of the blood vessels was noted, with some vessels either empty or, conversely, dilated and engorged. Plasma impregnation was observed in the walls of some vessels. Mucoid swelling affected not only the connective tissue but also the vascular walls. Young and mature fibrin were detected in the lumens of some vessels.

[0038] Conclusion:

[0039] Modeling of drug-induced rhinitis with intranasal administration of Nazivin in immature rats is characterized by morphological changes in all layers of the mucous membrane: almost complete loss of cilia on the entire surface of the epithelial layer, desquamation of epithelial cells, and in some cases, necrosis of the epithelial lining; in the lamina propria there was pronounced lymphohistiocytic infiltration, perivascular and interstitial edema.

[0040] The claimed method makes it possible to create a model of drug-induced rhinitis in immature rats by intranasal administration of alpha-adrenergic receptor agonists, close to the conditions of development of this process in adolescents, which will make it possible to study the pathogenesis of the disease and physiological processes in the tissues of the nose.

[0041] Sources of information

[0042] 1. Ovchinnikov A.Yu., Miroshnichenko N.A., Ryabinin V.A., Nikolaeva Yu.O. Long-acting topical decongestants. Medical Council. 2020; (16): 134-138. https: / / doi.org / 10.21518 / 2079-701X-2020-16-134-138.

[0043] 2. Sarkisov D.S., Perov Yu.L. Microscopic technique. M.: Medicine, 1996.- 544 p.

[0044] 3. Method for simulating paranasal sinusitis. Russian patent of 1999 RU 2129736 C1. Invention according to IPC G09B23 / 28. Patent RU No. 2129736; cl. G09B 23 / 28, 1999.

[0045] 4. Method for experimental modeling of inflammation in the paranasal sinuses during chronic tobacco smoking in rats. Russian patent 2014 RU 2522954 C2. Invention according to IPC G09B23 / 28 .RU 2522954 C2, 20.07.2014.

[0046] 5. Tursunov RM, Zarechnova HH Study of the effect of a decongestant (naphthyzine) on the morphology of the nasal mucosa of rats. Bulletin of KRSU. 2012; 12 (2): 162–165.

[0047] 6. Akhsanuddin S., Povolotsky R., Tayab R., et al. Adverse events associated with intranasal sprays: an analysis of the database of the Food and Drug Administration and a literature review. Ann Otol Rinol Laryngol. November 2021; 130(11):1292-1301. DOI: 10.1177 / 00034894211007222.

[0048] 7. Alromaih S., Alsagaf L., Aloraini N., et al. Drug-induced rhinitis: a narrative review. Ear Nose Throat J. 2022 November 15:1455613221141214. DOI: 10.1177 / 01455613221141214.

[0049] 8. Liva G, Karatzanis A, Prokopakis E. Review of rhinitis: Classification, types, pathophysiology. J Clin Med. 2021; 10(14): 3183.

[0050] 9. Massink A., Amelia T., Karamychev A., Izerman A. Allosteric modulation of receptors conjugated with G-protein, amiloride and its derivatives. Prospects for drug discovery? Med Res Rev. 2020 March; 40(2):683-708. DOI: 10.1002 / MED.21633.

Claims

A method for experimental modeling of drug-induced rhinitis in immature rats, including intranasal administration of a drug, characterized in that an alpha-adrenergic receptor agonist is administered into the nose three times a day at a dose of 3 μg / 100 g of animal weight, for a period of 28 days.

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