PRACA ORYGINALNA

ORIGINAL ARTICLE

MORPHOMETRIC DESCRIPTION OF THE EXCHANGE SEGMENT OF MICROVASCULATURE OF RATS’ SALIVARY GLANDS IN NORMAL CONDITIONS AND CHRONIC ETHANOL INTOXICATION

CHARAKTERYSTYKA MORFOMETRYCZNA SEGMENTU MIKROUNACZYNIENIA GRUCZOŁU ŚLINOWEGO SZCZURA W WARUNKACH PRAWIDŁOWYCH, JAK I W WARUNKACH PRZEWLEKŁEJ INTOKSYKACJI ETANOLEM

Kostyantyn V. Shevchenko, Galyna A. Yeroshenko, Оlena S. Yakushko, Kateryna S. Kazakova, Denys R. Kramarenko

Ukrainian Medical Stomatological Academy, Poltava, Ukraine

Abstract

Introduction: The state of the segments of the microvasculature, especially exchange one, has a significant effect on the organs’ functions. The results of it’s research can be objectified by morphometric method.

The aim: to determine the dynamics of changes in metric indices of the exchange segment of microvasculature of rats’ submandibular glands in normal conditions and in chronic ethanol intoxication.

Materials and methods: 45 albino rats were involved into study. Intact group (n=5) animals were administered with NaCl isotonic solution 4 times a day directly into the stomach. Experimental group (n=40) animals were administered with 40° ethanol 4 times a day directly into the stomach. Animals were sacrificed on 5, 9, 12 and 30 days by overdose of thiopental anesthesia. Lobules of submandibular glands were embedded into epon-812 according to standard procedure.

Results: On day 5 of the experiment the outer diameter of the capillary wall significantly reduced (4,91±0,02 µm), that is 19,5 % less than the values in controls (р<0,05). On day 9 of the experiment it was 15,2 % lower than the value of control group (р>0,05). On day 30 of the experiment the outer capillary diameter was 8 % lower compared to controls (р<0,05).

Conclusions: The early observation showed vasodilatation, confirmed by the constriction of the outer diameter and lumen diameter, accompanied by the thickening of the vascular wall under the influence of chronic ethanol intoxication. The indices did not come to normal values by the 30-th day of the experiment.

Key words: rats, salivary glands, capillaries, ethanol intoxication

Wiad Lek 2019, 72, 3, 323-326

Introduction

The oral cavity has a multi-component system of protection against environmental pathogenic factors. Apparently, the homeostasis of the oral cavity is determined by many factors, but, first of all, by the functional activity of the salivary glands, contributing to both the presence of dental pathology and somatic human health in general [1].

Saliva, which is the product of secretion of the salivary glands, is crucial in keeping the normal state of tissues and organs of the oral cavity. Functions of saliva are diverse [2]. It has been found that quantitative and qualitative changes in saliva greatly determine the resistance of teeth to dental caries [3].

The problem of alcoholism is considered to be relevant worldwide, and is ranking third in the mortality rate after cardiovascular diseases and cancer. The WHO’s statistics reports about 140 million people suffering from chronic alcoholism worldwide, and more than half of them refuse to contact medical professionals for assistance [4, 5].

Salivary glands of almost all patients with alcohol abuse showed marked acinar hyperplasia, dilated ducts, granular and adipose dystrophy of the ductal epithelium. In some cases minor swelling and stromal fibrosis was detected [6].

The state of the segments of the microvasculature, especially exchange one, provided with full gas exchange between the blood, cells and vital substances, has a significant effect on the body.

Some publications report that experimental hyposalivation of both endogenic and exogenic nature shows stable dilatation of the exchange vessels of the microvasculature throughout the observation [7].

Morphometric method is beneficial for objective analysis of the resulting data in the structural elements after the effect of various endogenic and exogenic factors [8].

The aim

The paper was aimed at the determining the dynamics of changes in metric indices of the exchange segment of microvasculature of rats’ submandibular glands in normal conditions and in chronic ethanol intoxication.

Materials and methods

45 outbred albino rats were involved into study. Intact group (n=5) animals were administered with NaCl isotonic solution 4 times a day directly into the stomach. Experimental group (n=40) animals were administered with 12 mg/kg 40° ethanol 4 times a day directly into the stomach [9].

The animals were sacrificed under 25 mg/kg thiopental anesthesia overdose in compliance with the scheduled time periods (on day 5, 9, 12 and 30). Lobules of submandibular glands were embedded into epon-812 according to standard procedure [10]. Semi-thin sections were stained with polychrome stain.

The average values of the outer diameter and diameter of capillary lumen were measured using the microscope with digital Biorex – 3 microphotohead with software, adapted to studies of such type. The thickness of the vascular wall was calculated according to the formula:

Тvw = Do-Dl/2,

where Do is the outer diameter of the capillary wall,

Dl is the inner diameter of the capillary wall,

Тvw is the thickness of the vascular wall of capillaries. Statistical processing of the morphometric data has been carried out using the Microsoft Excel [11].

Animal housing and experiments on them have been carried out in compliance with the “General Ethic Rules for Conducting Experiments on Animals”, adopted by the I National Congress on Bioethics and the requirements of international principles of the “European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes”[12].

Results and Discussion

The morphometric study has established that the average values of the outer diameter of the capillary wall of the lobules of submandibular gland was 6,10±0,05 µm; the inner diameter was 4,16±0,02 µm. The thickness of the vascular wall of capillaries was 0,92 µm (Table I).

On day 5 of the experiment the outer diameter of the capillary wall significantly reduced (4,91±0,02 µm), that is 19,5 % less than the values in controls (р<0,05); similarly, the diameter of the capillary lumen reduced by 12,5 % (Fig.1). Its average values were 3,64±0,02 µm, red blood cells in the lumens were deformed. The thickness of vascular wall was reduced by 30,4 % (0,64 µm) (Table I).

On day 9 of the experiment the outer diameter of capillaries was enlarged by 5,3 % (5,17±0,01 µm) compared to day 5; however, it was 15,2 % lower than the value of control group (р>0,05). The lumen diameter was 3,80±0,02 µm, that was 4,4 % higher the values on day 5 of the experiment but was 8,7 % lower compared to controls (р>0,05). Similarly, the thickness of vascular wall was tending to change. It was 6,3 % higher compared to day 5 of the experiment and 26,1 % (0,68 µm) lower compared to controls (Table I).

On day 12 of the experiment the outer capillary diameter was 5,34±0,03 µm, that was 3,3 % higher the values of the day 9 of the experiment, but was 12,5 % lower compared to controls (р<0,05). The average values of the lumen diameter were 3,87±0,01 µm, that was 1,8 % higher the values of the previous time period of experiment, but was 6,7 % lower the values of controls (р <0,05). The thickness of the vascular wall was 8,8 % higher compared to day 9 of the experiment and 19,6 % lower compared to controls (р <0,05) with the average values of 0,74 µm (Table I).

On day 30 of the experiment the outer capillary diameter of the lobules of submundibular gland was 5,61±0,01 µm, that was 5,1 % higher the values of day 12 of the experiment but 8 % lower compared to controls (р<0,05). The inner capillary diameter was 3,1 % (3,99±0,02 µm) higher the values of the previous time period of experiment, that was 4,1 % lower the values of controls; loose placement of blood corpuscles was detected in the lumens (Fig.2). The thickness of the vascular wall was 9,5 % higher compared to day 12 and significantly 12 % lower compared to controls (р<0,05) with the average values of 0,81 µm (Table I).

The findings of our previous studies have shown the spasm of arterioles in the lobules of the rat’s submandibular glands at the early stages of intoxication with ethanol, which is confirmed by the thinning of the vascular wall by 41.8%. At the ninth day of the experiment, dilatation, manifested by the significant enlargement of the outer diameter and the diameter of the lumen was noted, as well as thinning of the vascular wall. The rates did not come to normal by the thirtieth day of the experiment [13].

It has been established that chronic ethanol intoxication leads to early detection of venous dilatation, which is confirmed by the significant enlargement of the outer diameter and diameter of the lumen along with thinning of the vascular wall. From the twelfth day of the experiment the metric values were tending to restore. The rates did not come to normal by the thirtieth day of the experiment [14]. Thus, the established changes in the exchange segment of the microvasculature are caused by the blood flow decrease from dilated arterioles on the one hand, and hyperhydration of the amorphous component of the intercellular substance of the connective tissue of the stroma of the rats’ submandibular gland lobules on the other hand, at the early stages of the experiment. The findings of our study are in concordance with the findings of other researchers [1, 7], who studied the effect of ethanol and 1% methacrylic ester on the mucous membrane of the masticatory type. However, the authors established a more pronounced reaction of the exchange segment of the microvasculature of the glandular zone of hard palate and gums of rats due to direct exposure of the irritating factor to the surface. The submandibular glands are mediated indirectly by the bloodstream and, to a small extent, retrograde ethanol penetration through the duct system of the glands. The above reduces the manifestations of microcirculation disorders by an average of 10%.

Conclusions

The morphometric study has established that chronic ethanol intoxication affects the exchange segment of the microvasculature of the lobules of submandibular gland.

The early observation showed vasodilatation, confirmed by the constriction of the outer diameter and lumen diameter, accompanied by the thickening of the vascular wall. From day 9 of the experiment the metric values were tending to restore.

The indices did not come to normal values by the 30-th day of the experiment.

References

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2. Débora Alejandra González, Martín Matías Barbieri van Haaster, Emmanuel Quinteros Villarruel. Histamine stimulates secretion of extracellular vesicles with nucleotidase activity in rat submandibular gland. Archives of Oral Biology. 2018;85:201-206.

3. Timofeev AA. Sekretornaya funktsiya slyunnykh zhelez u bolnykh s ostrymi odontogennymi vospalitelnymi zabolevaniyami chelyustey. [Secretory function of the salivary glands in patients with acute odontogenic inflammatory diseases of the jaws]. Sovremennaya stomatologiya. 2011;4:70-74. (in Russian).

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7. Senchakovych YuV, Yeroshenko GA. Morfometrychna kharakterystyka lanok mikrotsyrkuliatornoho rusla pidnebinnykh zaloz shchuriv pry eksperymentalnii hiposalivatsii [Morphometric characteristics of the microcirculatory bed parts of the rat palatal glands at experimental hyposalivation]. Bulletin of Problems Biology and Medicine. 2014;3(112):275-278. (in Ukrainian).

8. Tsukanov DV, Yerochenko GA, Gasyuk NV et al. Morphometric characteristic of microcircular rate of salivary glands after administration of platiphillinum and proserinum. European International Journal of Science and Technology. 2014;3(8):29-34.

9. Ivanochko VM. Uljtrastrukturna orghanizacija reabsorbcijnogho bar’jeru nyrky pry khronichnij alkogholizaciji [Ultrastructural organization of the reabsorption barrier of the kidney at chronic alcoholization]. Tavricheskij mediko-biologicheskij vestnik. 2002;5(3):86-87. (in Ukrainian).

10. Karupu VYa. Jelektronnaja mikroskopija [Electron Microscopy]. Kiev: Vyshchaya shkola; 1984. 207 p. (in Russian).

11. Lapach SN, Chubenko AV, Babich PN. Statisticheskie metody v mediko-biologicheskih issledovanijah s ispol’zovaniem Exel [Statistical methods in biomedical research using Excel]. Kiev: Morion; 2000. 320 p. (in Russian)

12. European convention for the protection of vertebrate animals used for experimental an others scientific purposes/ Strasbourg: Council of Europ, 1986. No 123.52p.

13. Yeroshenko GA, Shevchenko KV, Yakushko OS. Morphometric characteristics of rat salivary glands hemomicrovasculature capacity component under normal conditions and in ethanol chronic intoxication. World of Medicine and Biology. 2018;3 (65):149-152.

14. Silkina YuV, Volkov KS, Shevchenko KV. Morfometrychna kharakterystyka rezystyvnoi lanky hemomikrotsyrkuliatornoho rusla slynnykh zaloz shchuriv pry khronichnii intoksykatsii etanolom [Morphometric characteristics of the resistant link of salivary glands’ hemomicrocircular rate of rats in chronic ethanol intoxication]. Morphologia. 2018;12(1):51-54. (in Ukrainian).

The paper has been written within the research scientific work, entitled “Experimental and morphological study of the effect of transplantants of cryopreserved placenta and other exogenic factors on the morphofunctional state of several internal organs”; State registration number 0113U006185.

Authors’ contributions:

According to the order of the Authorship.

Conflict of interest:

The Authors declare no conflict of interest.

CORRESPONDING AUTHOR

Оlena S. Yakushko

Ukrainian Medical Stomatological Academy

23 Shevchenko str., 36011 Poltava, Ukraine

tel: +380662990448

e-mail: olena.yakushko@gmail.com

Received: 27.11.2018

Accepted: 25.02.2019

Fig. 1. Capillary in the lobule of rat’s submandibular gland on day 5 of the experimental model of chronic ethanol intoxication. Methylene blue stain. 1000 × magnification.

Fig. 2. Capillaries in the periacinar connective tissue of the rats’ submandibular gland on day 30 of the experiment. Methylene blue stain. 1000 × magnification.

Table I. The average values of the capillary diameters of the microvasculature of the lobules of rats’ submandibular gland (µm)

Capillaries

Do

Dl

TVW

Control

6,10±0,05

4,16±0,02

0,92

day 5

4,91±0,02*

3,64±0,02*

0,64

day 9

5,17±0,01*,**

3,80±0,02*,**

0,68

day 12

5,34±0,03*,**

3,87±0,01*,**

0,74

day 30

5,61±0,01*,**

3,99±0,02*,**

0,81

Note: * – р < 0,05 compared with controls; ** – р <0,05 compared with experimental group.