Badanie histologiczne i mikroskopia elektronowa przyusznych gruczołów ślinowych u odwodnionych szczurów w różnym wieku

Denys P. Biletskyy, Oleg A. Ustiansky, Olena S. Maksymova, Pavel A. Moskalenko, Alexey A. Tymoshenko,
Anna S. Degtyarenko, Viktoriia Yu. Harbuzova , Yevhen I. Dubovyk, Andrii
P. Voznyi, Gennadii F. Tkach

SUMY STATE UNIVERSITY, SUMY, UKRAINE

ABSTRACT

Introduction: Water-salt metabolism disorders is one of the main factor of salivary gland pathology development.

The aim: To study the morphological structure of the parotid salivary gland of young, mature and old rats at micro- and ultrastructural levels under water deprivation.

Materials and methods: The experiment was carried out on thirty six laboratory male rats of different ages (young, mature and old). The rats of the control group received normal volume of drinking water. The rats of the experimental group were deprived of water for 6 days. Light microscope “OLYMPUS” and transmission electron microscope JEM-1230, (JEOL, Japan) were used for structural analysis.

Results: Obtained results revealed increasing numbers of vacuoles in the serous cells, the enlarged cisterns of endoplasmic reticulum and Golgi apparatus tubules, the condensed chromatin and the nuclei with significant invaginations in parotid gland of the rats of all age groups. The area of the acinuses more changed in young rats, the decrease was 34.61 % (P = 0.007). The internal diameter of capillaries most decreased in the dehydrated old rats by 23.76 % (P = ٠.٠٠9) in comparison with all study groups.

Conclusions: Water deprivation brings about the structure changes of the parotid gland at microand ultrastructural levels the intensity of which depends on the age of animals. The most dramatic changes have occurred in young and old rats.

Wiad Lek 2018, 71, 2 cz. II, -313

 

INTRODUCTION

Water determines all life processes in organs and tissues, and it is an integral part of metabolic processes. The water deficiency brings about the dysfunction or suspension of the synthetic, excretory, detoxification cell functions and the deterioration of functions in the organism [1]. The organism provides a constant water homeostasis by regulating water intake and losses of fluid under normal conditions. However, there are several factors that may break the normal water-electrolyte balance [2].

Water loss as little as 2 % of the body weight leads to a disorder of the organism function, especially older individuals have a higher risk of developing dehydration independent of their health state [3]. Several factors cause the dehydration in the elderly. With the aging process, the kidneys lose one-third of their nephrons that significantly reduces their ability to reabsorb the nutrients and to concentrate water [4]. Also, the loss in muscle mass usually leads to the total body water decrease [5]. The sensitivity of osmoreceptors was decreased that led to the diminution of the thirst perception [6]. Therefore, even healthy elderly have a tendency of developing the moderate dehydration which they cannot compensate by the water intake as compared with younger people [7].

On the contrary, children are very sensitive even to the smallest water deficit. A child’s organism contains more water than an adult organism. Moreover, they have high metabolic rate and imperfection of excretory and neurohumoral regulation mechanisms of the water and salt balance [8, 9]. Adults are also prone to the dehydration that could be caused by a long-term lack of water intake in the extreme situations, working under the high temperature conditions, diseases of the gastrointestinal tract, hyperthermia, dyspnea, caffeine abuse, alcohol abuse, uncontrolled drug administration [10].

The water deprivation plays a crucial role in the pathogenesis of many diseases of the gastrointestinal tract, the urinary system and the endocrine organs. The organs of the oral cavity are also closely related to the water-salt metabolism and provide a normal vital function of the organism [11].

The parotid gland is one of the major salivary glands which performs many functions. The gland produces the largest amount of serous secretion compared with other large salivary glands. [12, 13]. The serous secretion consists of α-amylase (ptyalin), lysozyme, lactoferrin, lipase, peroxydase, DNase, RNase, opsonins, leukins, histatins, secretory sIg [14, 15], thromboplastin, antiheparin substance, prothrombin, activators and inhibitors of fibrinolysis, epidermal growth factor [16], phosphatase, hyaluronidase and kallikrein, mineral elements (calcium, phosphorus, fluorides), the proline-rich proteins and statherins [17]. The violation of water-salt metabolism is one of the main factors that cause the development of salivary gland pathology [18].

Based on the general tendency for the growth of population ageing, climate changes, health deterioration of young people, the salivary gland dysfunction can become a common problem in the future [19].

THE AIM

The aim of research is to study the morphological structure of the parotid gland of young, mature and old rats at micro- and ultrastructural levels under water deprivation.

MATERIALS AND METHODS

The experiment was carried out on thirty six laboratory male rats ranging in the age of 4–6 months (young rats), 7–9 months (mature rats) and 20–22 months (old age rats). The animals were divided into two groups: experimental and control (18 rats). The rats of the control group received normal volume of drinking water. The rats of the experimental group were deprived of water for 6 days. Following six days from the beginning of the experiment, all rats were euthanized by the intramuscular injection of ketamine and xylazine. The parotid glands were taken out to perform the histological and ultrastructural analysis.

Animals were kept in vivarium conditions (the vivarium of Medical Institute of Sumy State University). The animal care and experiments were carried in accordance with the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (Strasburg, 1986); Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the Protection of Animals Used for Scientific Purposes; and The General Ethical Principles for Experiments on Animals, which were accepted by the First Bioethics National Congress (Kyiv, 2001). The study was approved by the Ethic Committee of Medical Institute of Sumy State University (№ 7/07.03.14).

For histology, the parotid gland was fixed in a 10 % formalin buffered solution. After fixation, the specimens were dried in an ascended alcohol series, cleared in two changes of xylene and embedded in molten paraffin. The 5-μm-thick slices were sectioned with a rotary microtome. To study the general structural features of the parotid gland, the samples were stained with hematoxylin and eosin (H&E). The specimens were analyzed using a light microscope “OLYMPUS” with a digital camera (Baumer optronic Typ: CX 05 with lenses 4, 10 × 40 and binocular 10).

For ultramicroscopic study, the 1 mm3 pieces of parotid gland were fixed in 2.5 % solution of glutaraldehyde and the postfixation was carried out in 1 % osmium tetroxide solution, and then the samples were dehydrated in ascended alcohol series. After immersion in propylene oxide, the specimens were embedded in epoxy resins mixture. The ultrathin slices (40–60 nm) were sectioned and stained with 1 % toluidine blue, and then the sections were examined by the light microscope. The ultrathin sections (40–60 nm) were stained with uranyl acetate and lead citrate according to Reynolds method and studied with a transmission electron microscope (JEM-1230, JEOL, Japan).

We had conducted the study with the following parameters: area of acini, internal diameter of intercalated ducts and internal diameter of capillaries. Determining the reliability of differences was performed using Student’s t-test (t). The value of P < 0.05 was considered as significant. All statistical analyses were performed using the Statistical Package for Social Science Program (SPSS for Windows, version 15.0, SPSS Inc., Chicago, IL, USA).

RESULTS AND DISCUSSION

The micro- and ultrastructure of the parotid gland had been studied previously in control rats (Fig. 1a, b, с) [20]. Therefore, only the changes caused by the influence of dehydration will be discussed within the scope of the present article. It should be noted only that the excessive growth of the connective tissue around the intercalated and striated ducts was observed in the parotid gland of old rats of the control group and at the same time their lumens were slightly narrowed (Fig. 1c). At the electron microscopic study of the parotid gland in young rats there was found a greater amount of electron-transparent granules (Fig. 2a) compared with mature rats (Fig. 2b). In the case of the old rats, the serous cells of parotid gland had the lipid inclusions and larger secretory granules (Fig. 2c).

In the case of the dehydrated young rats, the microscopic study of the parotid gland has revealed the serous cells having the enlightened cytoplasm and numerous vacuoles. The inter-acinar septums were almost not visualized. The epithelial cells of intercalated ducts were flattened and their lumens were narrowed. The local hemorrhages were observed around capillaries and venules (Fig. 1d).

The thinned and well-visualized interlobular connective-tissue septums were observed in the parotid gland of mature rats. The epithelial cell nuclei of the striated ducts were elongated and placed closer to the apical surface of the cell in different directions. The lumens of the striated ducts were irregularly shaped and extended. The myoepithelial cells were spindle-shaped and converged towards each other (Fig. 1e).

The histological study of the parotid gland of the elderly rats revealed a large number of vacuolated serous cells of the acini. Their nuclei have taken on various forms and the nuclei of other cells were pyknotic. The glandular cells without nuclei with a significantly enlightened cytoplasm were encountered. The numerous microcystic changes and lipid inclusions were observed in lieu of destroyed glandulocytes. Occasionally, the lumens of intercalated ducts were not visualized. The capillary lumens were emptied and their walls collapsed (Fig. 1f).

Morphometric parameters after experiment had the following changes. The area of acinuses decreased by 34.61 % (P = 0.0065), 23.49 % (P = 0.0175) and 14.25 % (P = 0.1592) in dehydrated young rats, mature rats and old rats respectively. The internal diameter of intercalated ducts decreased by 26.67 % (P = 0.0494), 14.08 % (P = 0.4097) and 11.94 % (P = 0.0022) in dehydrated young rats, mature rats and old rats respectively. The internal diameter of capillaries decreased by 21.53 % (P = 0.0001), 16.27 % (P = 0.0415) and 23.76 % (P = 0.009) in dehydrated young rats, mature rats and old rats respectively in comparison with the control group (Table I).

At the ultrastructural level, the nuclei of serous cells had the thinned karyolemma and the small invaginations in the parotid gland of the dehydrated young rats. The nucleoli were fluffed up and enlightened. The chromatin was condensed by large lumps at the periphery of the nucleus. The nuclei have been destroyed in some cells and their sites were occupied by the osmiophilic accumulations of the nuclear material. The perinuclear space was enlarged. The cytoplasm of secretory cells contained more electron-transparent secretory granules and numerous vacuoles in comparison with the control group. The cisterns of the granular endoplasmic reticulum were extended and emptied, and had the appearance of vesicles of different sizes and shapes. The Golgi apparatus tubules were also extended and there were a large number of dilated vesicles with homogeneous contents around them. The intercellular canaliculi contained the residues of fine granular material (Fig. 2d).

The cytoplasm serous cells of the parotid gland of dehydrated mature rats contained the secretory granules which tended to fusion with each other. The vacuolation of the granular endoplasmic reticulum cisterns was observed. The capillary lumens were narrowed and emptied. The nuclei of their endothelial cells had the irregular shape, the karyolemma formed deep invaginations, and the chromatin had the condensed state and concentrated along the nuclear membrane (Fig. 2e).

The elongated and irregularly shaped nuclei of serous cells were found on the ultrathin sections of the parotid gland of the dehydrated rats. The karyolemma formed invaginations and had enlarged pores. The perinuclear spaces were locally expanded. The cytoplasm contained the large lipid inclusions. The mitochondria were dilated and had the disoriented cristae and an enlightened matrix. The cisterns of the granular endoplasmic reticulum were significantly expanded and sometimes fragmented. The Golgi apparatus tubules were disorganized and randomly oriented. They were surrounded by small electron-transparent vesicles. The cytoplasm contained the large number of vacuoles of different calibers and contained few mature small secretory granules in comparison with the control group (Fig. 2f).

The body fluid level reduction leads to the development of hypovolemia and the redistribution of extra- and intracellular fluid. The increasing hypovolemia is accompanied by the compensatory mechanisms depletion, and the volume of the blood circulating decreases. The violation of the microcirculation occurs in organs and tissues. The concentration of electrolytes is increased in the cells as well as the hydrated films structure of protein molecules is transformed; the proteins solubility and the active surface of cell membranes are reduced, and their intercellular interactions, the regulatory signals perception, the active and passive transport are violated. The abovementioned changes contribute to the cytoplasm pH reduction, the oxidative stress and the chemical reactions which usually don’t proceed in a fully hydrated cell [21]. Water enters the acinar cells from the blood plasma and ensures all its internal processes. Dehydration causes the hypotension of the parotid gland [22] and leads to its dysfunction [19].

The previous work [23] demonstrated the narrowed capillaries with the swollen endothelial cells and thickened basement membrane in the supraoptic nucleus of mature rats under water deprivation. In our study, we have observed the vessels lumen narrowing of microcirculatory bloodstream, the local hemorrhages around capillaries and venules, the emptied capillaries and the collapse of their walls. Furthermore, the most dramatic changes were in young and old rats. The scientists argue that the system of adaptive and compensatory mechanisms is not quite perfect at young ages [8] and in the case of the old age, their disruption occurs as well the involutive and dystrophic processes are intensified [24].

In the studies of the effect of water deprivation on the cells of a subcommissural organ, J. Leonieni, and G. J. Gilbert revealed the formation of numerous and deep invaginations of the nuclear membrane, the filling of the cytoplasm by empty and extended vacuoles of the endoplasmic reticulum and by vesicles with a homogeneous or fine-grained material of the low electron density. The membrane systems of the Golgi apparatus were expanded and the quantity of Golgi apparatus’s vesicles and vacuoles was increased. The authors also noted that the mitochondrial structure had not changed and the lumens of capillaries were narrowed, and their endothelial cells had a swollen appearance [25, 26].

In the study of transitional epithelium of the urinary tract of rats under effect of water deprivation, B. Monis and D. Zambrano found the increase of sizes and number of cytoplasmic vesicles in cells compared to the control animals. It was also shown that the vesicles had formed clusters in dehydrated rats; the number of lysosomes which contained densely packed round and polyhedral osmiophilic structures and fragments of membranes was increasing. The endothelial cells cytoplasm of capillaries had a large number of pinocytic vesicles but the authors were not sure that their quantity was significantly different from the normal cells [27].

In our research, we have observed the extended tubules of the endoplasmic reticulum and Golgi complex in the serous cells of parotid salivary glands in the dehydrated rats of all age groups that can indicate about the similar mechanism of action of the dehydration on the cells of different organs in comparison with the previous studies. Furthermore, J. Leonieni and L. Rechardt say that the tubules expansion of the endoplasmic reticulum and the Golgi complex serve as the evidence of the secretory cells functioning enhancement [25]. However, taking into consideration that these tubules are mostly empty, it can be suggested that their secretory function is unproductive. During the study of supraoptic nucleus cells of the mature rats under water deprivation, the dilated cisterns of the endoplasmic reticulum and the numerous flattened vesicles of the Golgi apparatus have been found [23, 28].

We have found the enhanced vacuolization of serous cells cytoplasm in the young and old rats. In their studies, B. Monis and D. Zambrano allege that these are the water containing pinocytic vesicles the amount of which increased under dehydration [27]. These changes can reflect the compensatory mechanisms at the cellular level, which consist thereby in the water conservation in the cells. Although from the viewpoint of other authors, the observed spindle vesicles in the cytoplasm of serous cells of dehydrated young rats are a special barrier which transfers the fluid from the cytoplasm on the surface of the cell membrane [29]. Thus, the cell can transfer the water to the interstitial space and the capillaries to maintain the blood volume. In the study of adrenal glands of the dehydrated guinea pigs, H. A. Kader and co-authors found that the cell cytoplasm of the zona fasiculata and zona reticularis was pale and vacuolated compared to control animals [30]. The authors suggested that these changes were the symptom of hyperactivity of these zones.

The general tendency for all age groups was the accumulation of chromatin into the globs under karyolemma and the formation of the invaginations of the nuclear membrane. The intensity of these changes also depended on age. Thus, they had more pronounced effects in young rats. In addition to the mentioned changes, the osmiophilic accumulations of the nuclear material were observed in lieu of the destroyed nuclei. B. Monis and D. Zambrano demonstrated that the nuclei of dehydrated rats had not changed. In the research dedicated to the examination of supraoptic nucleus of dehydrated rat cells [27], it was shown that the cell nuclei had become round and had lost the invaginations of the karyolemma [23].

The dilated mitochondria with disoriented cristae and enlightened matrix were observed in the study of ultrathin sections of the parotid gland of elderly rats. The changes of mitochondria structure were not found in the rats of mature and young age. B. Monis and D. Zambrano also noted that the mitochondria in dehydrated mature rats were unchanged [27]. Following water deprivation, the shape, size and number of mitochondria had not changed that was found during the study of supraoptic cell nucleus of mature rats [23].

CONCLUSIONS

Water deprivation brings about the structure changes of a parotid gland at micro- and ultrastructural levels the intensity of which depends on the age of animals. Thus, the area of the acinuses more changed in young rats, the decrease was 34.61 % (P = 0.007). The area of the acinuses had the smallest changes in the dehydrated old rats, the area decreased by 14.25 % (P = 0.159). The changes of internal diameters of intercalated ducts had the same tendency. The internal diameter of capillaries most decreased in the dehydrated old rats by 23.76 % (P = 0.009) in comparison with all study groups.

REFERENCES

1. Popkin BM, D’Anci KM, Rosenberg IH. Water, hydration, and health. Nutrition Reviews. 2010;68(8):439–458. doi: 10.1111/j.1753-4887.2010.00304.x

2. Faes MC, Spigt MG, Rikkert O. Dehydration in geriatrics. Medscape. 2007;10(9):590–596.

3. Begum MN, Johnson CS. A review of the literature on dehydration in the institutionalized elderly. Journal of Clinical Nutrition and Metabolism. 2010;5(1):47–53. http://dx.doi.org/10.1016/j.eclnm.2009.10.007

4. Kositzke JA. A question of balance-dehydration in the elderly. Journal of Gerontological Nursing. 1990;16(5):4–9. https://doi.org/10.3928/0098-9134-19900501-04

5. Davis KM, Minaker KL. Disorders of fluid balance: dehydration and hyponatremia. Principles of geriatric medicine and gerontology. 1994;3:1182–1190.

6. Rolls BJ, Wood RJ, Rolls ET, Lind H, Lind W, Ledingham JG. Thirst following water deprivation in humans. Am J Physiol Regulatory Integrative Comp Physiol. 1980;239(5):476–482. http://ajpregu.physiology.org/content/239/5/R476

7. Phillips PA, Rolls BJ, Ledingham JG, Forsling ML, Morton JJ, Crowe MJ, Wollner L. Reduced thirst after water deprivation in healthy elderly men. N Engl J Med. 1984;311 (12):753–759. DOI: 10.1056/NEJM198409203111202

8. Bizzarri C, Pedicelli S, Cappa M, Cianfarani S. Water balance and “salt wasting” in the first year of life: the role of aldosterone-signaling defects. Hormone Research in Paediatrics. 2016;86:143–153. DOI: 10.1159/000449057

9. Manz F. Hydration in children. J Am Coll Nutr. 2007;26:562–569.

10. Bossingham MJ, Carnell NS, Campbell WW. Water balance, hydration status, and fat-free mass hydration in younger and older adults. Am J Clin Nutr. 2005;81:1342–1350. http://ajcn.nutrition.org/content/81/6/1342.long

11. Kleiner SM. Water: an essential but overlooked nutrient. J Am Diet Assoc. 1999;99:200–206. DOI: 10.1016/S0002-8223(99)00048-6

12. Edgar M, Dawes C, O’Mullane D. Saliva and oral health. Rev Assoc Med Bras. 2014;56:1–9.

13. Humphrey SP, Williamson RT. A review of saliva: normal composition, flow, and function. Journal of Prosthetic Dentistry. 2001;85:162–169. DOI: 10.1067/mpr.2001.113778

14. Edgar WM. Saliva: its secretion, composition and functions. Br Dent J. 1992;172:305–312.

15. Schenkels LC, Veerman EC, Nieuw Amerongen AV. Biochemical composition of human saliva in relation to other mucosal fluids. Crit Rev Oral Biol Med. 1995;6:161–175.

16. Singh M, Singhal U, Bhasin GK, Panday R, Aggarwal SK. Oral fluid: Biochemical composition and functions: A review. J Pharm Biomed Sci. 2013;37:1932–1941.

17. Amerongen AV, Veerman EC. Saliva: the defender of the oral cavity. Oral Dis. 2002;8:12–22.

18. Carlson ER, Webb DE. The diagnosis and management of parotid disease. Oral and Maxillofacial Surgery Clinics of North America. 2013;25:31–48. DOI: 10.1016/j.coms.2012.10.001

19. Ship JA, Fischer DJ. The relationship between dehydration and parotid salivary gland function in young and older healthy adults. J Gerontol A Biol Sci Med Sci. 1997;52:310–319.

20. Pinkstaff CA. The cytology of salivary glands. International review of cytology. 1980;63:141–261. https://doi.org/10.1016/S0074-7696(08)61759-3

21. França MB, Panek AD, Eleutherio ECA. Oxidative stress and its effects during dehydration. Comparative Biochemistry and Physiology. 2007;146:621–631. https://doi.org/10.1016/j.cbpa.2006.02.030

22. Young JA, van Lennep EW. Transport in salivary and salt glands. Membrane transport in biology. 1979;4:563–674.

23. I. Ultrastructure of the supraoptic nucleus of normal and dehydrated rats. Acta Physiologica. 1969;77:9–29.

24. Schmitt R, Melk A. Molecular mechanisms of renal aging. Kidney International. 2017;92:569–579. DOI: 10.1016/j.kint.2017.02.036

25. Leonieni J, Rechardt L. The effect of dehydration on the uhrastructure and cholinesterase activity of the subcommissural organ in the rat. Z Zellforsch Mikrosk Anat. 1972;133:377–387.

26. Gilbert GJ. Subcommissural organ secretion in the dehydrated rat. Anat Rec. 1958;132:563–567.

27. Monis B, Zambrano D. Transitional epithelium of urinary tract in normal and dehydrated rats. Zeitschrift fiir Zellforschung. 1968;85:165–182.

28. Tweedle CD, Hatton GI. Ultrastructural comparisons of neurons of supraoptic and circularis nuclei in normal and dehydrated rats. Brain Research Bulletin. 1976;1:103–121.

29. Walker BE. Electron microscopic observations on transitional epithelium of the mouse urinory blodder. J. Ultrastructure research. 1960;3:345–361. https://doi.org/10.1016/S0022-5320(60)90014-9

30. Kader HA, Fawzy S, Metwally S, Khaalawii SEl, Zagloui SS. Effect of dehydration and rehydration on some parenchymatous organs of the guinea pig. Sci. Med. J. Cai. Med. Synd. 1990;2:177–202.

This study is the part of the scientific research work of the Ministry of Education and Science of Ukraine “The morphofunctional monitoring of the state of organs and the organism systems under conditions of the disorder of homeostasis” (state registration number 0109U008714).

ADDRESS FOR CORRESPONDENCE

Yevhen Dubovyk

Prokofieva str., 48A, ap. 63, Sumy, Ukraine

tel: +380 0509731924

e-mail: janitor@ukr.net

Received: 20.11.2017

Accepted: 09.03.2018

Figure 1. Light micrographs of parotid salivary gland of the young (a), mature (b) and old (c) rats of the control group and young (d), mature (e) and old (f) rats of the experimental group. (a, b) (control group) shows normal architecture of parotid gland: the intercalated ducts (ID), the lumen of striated duct (SD), acinus (А), interlobular connective-tissue septums (IS). (c) the narrowed lumens of intercalated ducts (ID), the overgrowth of connective-tissue around striated ducts (SD) (intercalated and striated ducts). (d) (experimental group) the numerous vacuoles in the cytoplasm of serous cells (V), the local hemorrhages around capillaries and venules (H). (e) the extended lumen of striated duct (SD), the thinned interlobular connective-tissue septums (IS), the interlobular septum of connective tissue. (f) the microcystic changes (Mc) and lipid inclusions (L), the narrowed lumen of the capillary (C). H&E staining.

Figure 2. Transmission electron micrographs of parotid gland of young (a), mature (b), and old (c) rats of the control group and young (d), mature (e) and old (f) rats of the experimental group. (a, b) (control group) the nuclei of oval serous cells (N), (dG) the secretory granules with high electron density and the immature secretory granules (iG), the granular endoplasmic reticulum (rER) the Golgi apparatus (G), the mitochondria (M). (c) the lipid inclusions (L) and the electron-dense secretory granules (dG). (d) (experimental group) the osmiophilic clusters of nuclear material (OC), the immature secretory granules (iG), the vacuoles (V). (e) the fusion of secretory granules (iG), the narrowed lumen of the capillary (C), the deep invaginations of endotheliocytes’ karyolemma (E). (f) the large lipid inclusions (L), the disoriented cristae of mitochondria (M), the cisterns of the granular endoplasmic reticulum (rER), the disorganized Golgi apparatus tubules (G), the vacuoles (V), the diminished nuclei with condensed chromatin (N)

Table I. Comparison of the morphometric data between the studied groups (Mean ± SEM).

Parameters

Young (control)

Young (exp)

Mature (control)

Mature (exp)

Old
(control)

Old (exp)

AA, µm2

230.62 ± 19.11

150.81 ± 13.36

291.12 ± 22.85

222.75 ±
7.53

182.53 ± 13.79

156.52 ± 10.11

IDID, µm

15.79 ± 0.89

11.58 ± 1.66

14.31 ± 2.32

12.29 ± 0.36

12.11 ± ٠.٢٨

10.66 ± 0.22

IDC , µm

8.92 ± 0.25

7.01 ± 0.19

9.86 ± 0.53

8.25 ± 0.44

9.59 ± 0.56

7.31 ± 0.43

Note: AA – area of acini, IDID – internal diameter of intercalated ducts,

IDC – internal diameter of capillaries, exp – experimental group.