PRACA ORYGINALNA

ORIGINAL ARTICLE

MORPHOFUNCTIONAL STATUS OF CARDIO-VASCULAR SYSTEM OF RATS WITH CONGENITAL HYPOTHYREOSIS

STATUS MORFOLOGICZNO-CZYNNOŚCIOWY UKŁADU SERCOWO-NACZYNIOWEGO SZCZURÓW Z WRODZONĄ NIEDOCZYNNOŚCIĄ TARCZYCY

Svitlana M. Chuhray1, Viktoria E. Lavrynenko3, Rostyslav F. Kaminsky2, Iryna V. Dzevulska2, Olexandr V. Malikov2, Olexandr I. Kovalchuk2,3, Liudmyla M. Sokurenko1,3

1Department of Histology and Embryology, Bogomolets National Medical University, Kyiv, Ukraine

2Department of Human anatomy, Bogomolets National Medical University, Kyiv, Ukraine

3Department of Fundamental medicine, Taras Shevchenko National University of Kyiv, Kyiv, Ukraine

Abstract

Introduction: There is an increasing number of cases of congenital hypothyroidism. One of the most common complications of hypothyroidism is damage to the cardiovascular system, which in 30-50% of patients leads to the development of arterial hypertension.

The aim: studying the features of the ultrastructure of myocardial capillaries in mature rats with hypothyroidism.

Materials and methods: Experiments were conducted on 40Wistar line rats with congenital hypothyroidism: juvenile young (45-day) and sexually mature (100-day) rats, as well as intact animals of the corresponding age. While extracted from the experiment rats of all experimental groups had their arterial pressure measured using a plethysmograph while the development of hypothyroidism was controlled by the immune-enzymatic method. Electron microscopic examination of the left ventricular myocardium and morphometric study of volumetric and quantitative densities, cross-section area, and form factor of micropinocytotic vesicles were conducted.

Results: In the sexually mature rats with congenital hypothyroidism the quantitative density of the capillaries in the myocardium decreases. Activation of transcytosis is accompanied by significant violations of vesiculation. Some of the endothelial cells of experimental animals contain a moderate amount of transport vesicles, while others are overfilled with these structures and desquamate into the lumen. In older rats with congenital hypothyroidism there is a further dilution of capillaries, the development of hypoxic state in them, mucinous edema of interstitial space, decrease of biosynthetic and transport processes activity.

Conclusions: In young (45-day) rats with congenital hypothyroidism the direction and expressiveness of compensatory processes is to enhance the transcytosis processes. Dystrophic-destructive changes are manifested by apoptosis in some endothelial cells, decrease in the number of biosynthetic organelles, lysis and edema of their cytoplasm. In sexually mature (100-day) rats with congenital hypothyroidism destructively-dystrophic processes in the blood capillaries of the myocardium are approximately balanced with compensatory-adaptive.

Key words: myocardium, blood capillaries, rat, congenital hypothyroidism, electron microscopy

Wiad Lek 2019, 72, 2, 229-233

INTRODUCTION

In recent decades more and more attention of researchers is being attracted by prevalence of hypothyroidism – a disease caused by decrease in the biological effect of thyroid hormones on the tissue level or their deficiency in the body. The number of cases of congenital hypothyroidism (CHT) is increasing. One of the most common complications of hypothyroidism is a violation of the cardiovascular system, which in 30-50% of patients leads to the development of arterial hypertension [1-3]. However, although in people of different ages clinical manifestations and the course of hypothyroidism differ significantly [4, 5], data on morphofunctional changes in organs and tissues in the age aspect has not been identified in the literature.

THE AIM

In this regard, the purpose of the research was to study the features of the ultrastructure of myocardial blood capillaries in mature rats with hypothyroidism, that is, structures where metabolism occurs and which are largely responsible for tissue damage in the heart.

MATERIALS AND METHODS

Experiments were performed on 40 white rats of the Wistar line, which were under standard vivarium conditions of the Bogomolets National Medical University. The juvenile (45-day) and sexually-grown (100-day) Wistar rats with CHT were studied. Control was provided by intact rats of the Wistar line of the same age. Congenital hypothyroidism was modeled by inhibition of thyroid gland by thyreostatic – Mercazolil. Starting from the 14th day of perinatal development pregnant females were administered with the drug. After birth, the animal received it with the mother’s milk, and in the future – while self-feeding. The drug was administered throughout the experiment daily at a dose of 20 mg / kg of body weight. The solvent was water. The development of hypothyroidism was controlled by the immune-enzymatic method on the Sunrise RC device, TEKAN in both control and hypothyroidic rats when they were removed from the experiment. Experiments with animals were carried out in accordance with the Law of Ukraine “On protection of animals from cruelty” (2006), “General ethical principles of animal experiments”, adopted by the First National Congress on Bioethics (Kiev, 2001).

While extracted from the experiment rats of all experimental groups had their AP (arterial pressure) measured using a plethysmograph, while the development of hypothyroidism was controlled by the immune-enzymatic method on the Sunrise RC device, TEKAN.

Electron microscopic examination of the left ventricular myocardium of experimental rats was conducted. The material was processed in accordance with the generally accepted techniques of electron microscopic examination [6]. Ultra-thin sections were made with ultratome Reihart (Austria) and examined using an electron microscope PEM-125K.

Morphometric studies were carried out on a semi-automatic device for graphic research processing using the program “Organelle”. Volumetric and quantitative densities, cross-section area, form factor of micropinocytotic vesicles (MPV) were studied. Statistical processing was carried out using the Student parametric criterion and the nonparametric Kolmogorov-Smirnov criterion according to the principle of variation statistics.

RESULTS

Changes in functional parameters in experimental rats

At 45-day rats with CHT AP does not reach meaningful quantities yet. At 100 days after birth AP increases compared with control animals. In this case, about half of animals with CHT showed increase of this parameter (Table I).

The existence of persistent hypothyroidism is confirmed by a significant decrease in the concentration of free thyroxine in blood plasma during the whole experiment, starting with the early stages of the disease and becoming the most pronounced in the final stages (Table I), compared with control.

Ultrastructure of blood capillaries of myocardium in rats

In the myocardium of 45-day rats with congenital hypothyroidism, capillaries with both narrowed and enlarged lumen (Fig.1A, B) as well as flattened microvessels can be observed. This may indicate, on one hand, the decrease in the intensity of the capillaries formation, and, on the other, the involvement of a greater number of capillaries in the microcirculation, which leads to the decrease in the number of reserve, non-functioning microvessels. Most capillaries have a flattened lumen surface, which contains single microvilli and invaginations (Fig. 1B).

However, there are capillaries with different magnitude of cervical protrusions at different stages of separation from the cell. Such microclazmatosis outgrowths can further lose connection with endothelial cells and get into the bloodstream.

Endothelial cells of moderate electron density with well-preserved cytoplasm are prevalent. They contain large nuclei of oval or somewhat irregular shape. Mitochondria in a small number are located mainly in the near-nuclear zone. They are usually oval, with an unevenly enlightened matrix and irregularly located crystae (Fig.1.A). The channels of the endoplasmic reticulum are mainly short, contain ribosomes on their membranes. The latter, in the form of discrete units and polyribosomal complexes unevenly fill the nuclear-containing and peripheral regions of the endothelial cells. Lysosomes, multivesicular corpuscles, secretory vesicles of the Golgi complex, secretory granules are not expressed. There are also rare elements of the cytoskeleton – microfilaments and microtubules.

In 100-day rats with congenital hypothyroidism ultrastructural changes become more expressed and more prevalent in the blood capillaries of the myocardium. Part of the capillaries have dilated lumen and thinned nuclear-containing and peripheral areas. Endothelial cells that form these capillaries have a preserved ultrastructure, but they also show damage to organelles. Some cells have only isolated tubules and ribosomes even within the nuclear-containing regions. Channels of the granular endoplasmic reticulum are often fragmented, degranulated, dilated. Dictiosomes of the Golgi complex are flattened and have blurred membranes that give them a look of a homogeneous mass of moderate electron density.

The number of mitochondria is negligible. They show electron-dense matrix, destruction of external mitochondrial membranes, fragmented crystae. In some mitochondria, the outer membrane is spirally twisted, thus resembling myelin sheaths. That is, the formation of myelin-like structures, which is a sign of a violation of lipid peroxide oxidation.

Attention is drawn to increasing electron density of inter-endothelial junctions, enlargement of the obliteration spots and storage of finely dispersed material, which may worsen the paracellular route of substances transport. Basal lamina is sometimes thinned, destructured, sometimes thickened, and loose.

Another feature that further increases in 100-day rats with congenital hypothyroidism is the prevalence of blood capillaries at different stages of destruction. Endothelial cells with increased electron density of the cytoplasm, picnotically altered nuclei, a large number of microclazmatosis outgrowths on the luminal and in some cases on basal surfaces are observed. In the myocardium, areas of perivascular edema are common.

Micropinocytotic vesicles in endothelial cells of myocardial blood capillaries

Micropinocytotic vesicles in endothelial cells of myocardial circulatory capillaries in 45-day rats with congenital hypothyroidism may be attached to the basal or lumen surfaces of the cells or have free forms unequally, even within one endothelial cell locating in the cytoplasm (Fig.1.A). Morphometric analysis has shown that in this period of study in 1 μm3 of the cytoplasm of endothelial cells of the myocardium of hypothyroid rats there are 400,7 + 28.9 micropinocytotic vesicles, which significantly differs from this index in control (Table II).

In 100-day rats with congenital hypothyroidism, the micropinocytotic vesicles undergo major qualitative and quantitative changes. Cells without vesicles are observed. But most endothelial cells are filled with these structures. They are located along the basal or luminal surface densely one next to another, forming complexes of various shapes. In some cases, when too many vesicles are gathered, their excess is isolated in the form of microclazmatosis outgrowths and excreted out of endothelial cells.

As for morphometric analysis, it has shown that the vesicle density in 100-day hypothyroid rats is 443.7 + 25.9 / μm3, which is significantly higher than the control index (Table II). In intact animals the number of endothelial cells with a small amount of micropinocytotic vesicles decreases with age, but they still prevail in the population.

DISCUSSION

So, in 45-day rats with CHT, the ultrastructure of the myocardium capillaries is heteromorphic. This heterogeneity of the structure is due to changes that, on one hand, are compensatory, and on the other destructively-dystrophic. The processes of edema of endothelial cells in animals with СHT are widespread and expressive. Swollen endothelial cells commonly form sequestrums of the cytoplasm with fluid of edema, which are separated and observed both in the capillary lumen and perivascularly. This may be due to the fact that persistent deficiency of thyroid hormones observed in 45-day rats leads to violations in the synthesis of Na + / K + -ATPase and ion channel proteins [7, 8]. Changes in the Na + / K + exchange function, free Na + and K + channels of plasmalemma lead to an increase in its permeability for Na + ions, and as a result, development of significant edema in the endothelial cells.

In rats with CHT, there is a decrease in the number of biosynthetic organelles. It should be noted that hypoplastic processes are typical for hypothyroidism, as thyroid hormones are necessary for the metabolism of all cells. Due to their deficiency, synthesis and neogenesis of both organelles and their compounds are violated.

At the same time, in the capillaries of 45-day rats with CHT, processes of transendothelial transfer of substances are intensified, as evidenced by an increase in the quantitative density of MPV. Intensification of the processes of substances transfer is a manifestation of adaptive processes that develop in response to the influence of the pathological factor. Another symptom of these processes in the capillaries is the local thinning of peripheral parts of the endothelial cells to the size of MPV. All these changes are due to changes in the physical and chemical composition of the plasma membranes. Thyroid hormones, regulating Na + / Ca2 + metabolism through the expression and phosphorylation of ion channels, affect the content of membrane-connected Ca2 + [9, 10] That is, the cause of increased transcytosis may be, on one hand, increased fluidity of the plasma membrane, and on the other hand, destabilization and changes in its conformation, that lead to increase in the processes associated with the formation of invaginations and vesicles.

Another feature of the ultrastructure of myocardium capillaries in young rats with CHT is the presence of electron-dense endothelial cells. This condition is characteristic for apoptotic cells, and the receptor pathway plays a primary role in triggering of endothelial cell apoptosis with the participation of the nuclear and cytoplasmic receptors of thyroid hormones.

With increasing of age of rats with CHT up to 100 days both compensatory-adaptive and destructively-dystrophic changes become more expressive. The prevalence of endothelial cells with signs of edema is negligible compared with the previous observation period. Perhaps this is a consequence of the death of edematous endothelial cells, on one hand, and on the other, capillaries of the myocardium in both groups of observations undergo changes in the rigidity of the plasma membrane, which do not allow the development of expressed edema. The prevalence of capillaries with well-preserved ultrastructure can be explained by the manifestations of compensatory processes, while the decrease in the number of organelles taking part in biosynthesis – to destructive ones. The largest changes refer to the number of MPV. The activation of substances transfer is aimed, obviously, at the compensation of lowered transcytosis in myocardium due to destruction of the part of the capillaries accompanied by their insufficient formation due to the lack of hormones of thyroid gland. However, endothelial cells are overfilled with MPV forming conglomerates, which, as a rule, desquamate into the lumen of the capillaries as microclazmatosis outgrowths. Such ultrastructural changes can be dystrophic manifestations, which are the result of significant destabilization and conformation of the plasma membranes in the endothelial cells. In places where inter-endothelial junctions were disconnected “windows” were formed. Active transcytosis through them contributes to increase of perivascular edema, worsening the trophism of cardiomyocytes and is a manifestation of dystrophic processes.

CONCLUSIONS

1. In young (45-day) rats with CHT the direction and expressiveness of compensatory processes, is to enhance the transcytosis. Dystrophic-destructive changes are manifested by apoptosis of some endothelial cells, a decrease in the number of biosynthetic organelles, lysis and edema of their cytoplasm.

2. In blood capillaries of the myocardium in sexually mature (100-day) rats with CHT destructive-dystrophic processes are approximately balanced with compensatory and adaptive ones. In the myocardium, the quantitative density of the capillaries decreases. Activation of transcytosis occurs against significant violations of vesiculation. Some of the endothelial cells of experimental animals contain a moderate amount of transport vesicles, while others are overfilled with these structures and desquamate into the lumen.

REFERENCES

1. Pertsova, T.O., Kulikova, OM Stan Renin-Angiotensin-Aldosterone system in patients with hypothyroidism and arterial hypertension. Endocrinology. 2004; 9 (1): 97-100.

2. Katerhenchuk VI, Beregova O.P. Cardiovascular hypothyroidism masks. Internal medicine. 2007; 3 (3): 50-55.

3. Fletcher AK., Weetman AH. Hypertension and hypothyroidism. J. Hum Hypertens. 1998; 12: 79-82.

4. Selivanova GB Cardiovascular manifestations of hypothyroidism: the role and place of hypertension. Cardiologist. 2006; 4: 64-69.

5. Anderson G., Biakeman N., Streeten D. The effect of age on prevalence of secondary form of hypertension in 4429 consecutively referred patients. J. Hypertens. 1994; 12(5): 609-615.

6. Karupu V.Ya. Electron microscopy. K.: Vishcha School, 1984: 208 p.

7. Strukov AI, Serov V.V. Pathological Anatomy. M .: Medicine, 1995: 688p.

8. Kahaly G.J., Dillmann W.H. Thyroid hormone action in the heart. Endocrine Rev. 2005; 26: 704–728.

9. Gick G.G., Melikian J., Ismail-Beigi F. Thyroidal enhancement of rat myocardial Na,K-ATPase: preferential expression of alpha 2 activity and mRNA abundance. J.Membr. Biol. 1990; 115: 273-282.

10. Ojamaa K., Sabet A., Kenessey A., Shenoy R., I. Klein Regulation of rat cardiac Kv1.5 gene expression by thyroid hormone is rapid and chamber specific. Endocrinology. 1999; 140: 3170 – 76.

Acknowledgements: The authors would like to thank the employees of the Institute of Pathologists Problems Bogomolets National Medical University prof. L.O. Stechenko and T. P. Kuftyreva.

Authors’ contributions:

According to the order of the Authorship.

Conflict of interest:

The Authors declare no conflict of interest.

CORRESPONDING AUTHOR

Liudmyla Sokurenko

Department Histology and Embryology

of National О.О. Bogomolets Medical University

tel: +380675010815

e-mail: l-sokurenko@i.ua

Received: 03.10.2018

Accepted: 18.01.2019

Table I. Changes in the content of free thyroxine in blood plasma and AT figures

45 days

100 days

Control

CHT

Control

CHT

Arterial pressure, mm Hg

98,7±6,1

112,7±7,3

109,4±5,3

131,6±5,2*,**

Thyroxine, pmol/l

7,63±0,99

3,96±0,46*

8,58±0,72

2,77±0,76*

Note: * – р<0, 05 in comparison with the data of the Control; ** – р<0, 05 in comparison with the data of the other term group.

Fig. 1. Blood capillaries of the left ventricular myocardium in 45-day rat with CHT. Electron microscopic photo. Magnification: А-22000; B -19000: Lumen (1) of the capillary. Nucleus (2), micropinocytotic vesicles (), interendothelial junctions() in endothelial cells. Basal lamina ().

Fig. 2. Blood capillaries of the left ventricle myocardium of 100-day rats with CHT. Electron microscopic photo. Magnification: А-40000; B-60000. Interendothelial junctions (),micropinocytotic vesicles () in endothelial cells. Basal lamina ().

Table II. The morphometric indexes of MPV in endothelial cells of myocardial circulatory capillaries

Age of animals

Groups

Volumetric density, %

Quantitative density,

1/ μm3

Average area,

10-2 μm2

Form factor

45 days

Control

18,09+1,04

282,7+15,9

0,57+0,01

0,84+0,05

CHT

17,15+0,83

400,7+28,*

0,46+0,01*

0,86+0,04

100 days

Control

21,95+0,85

332,5+18,6

0,57+0,01

0,85+0,07

CHT

23,00+1,49

443,7+25,9*

0,55+0,01

0,84+0,06

Note: * – р<0, 05 in comparison with the data of the Control;