Thyrotoxicosis and colloid goitre simulation method

FIELD: medicine.

SUBSTANCE: invention refers to experimental medicine, namely to thyrotoxicosis simulation ensured by endogastric introduction of Thyreotom in weekly graded doses 25 mkg/kg to 200 mkg/kg within 6 weeks followed by evaluation of functional activity of thyroid gland.

EFFECT: method provides creation of such thyroid gland pathology ensuring a clinical presentation of hyperfunctioning thyroid gland in blood and peripheral organs, and simulating colloid goitre pathology in thyroid parenchyma.

2 tbl

 

The invention relates to medicine, namely to endocrinology.

One of the necessary conditions for the successful solution of tasks in experimental pharmacology is the use of accurate models of the studied diseases are able to fully and objectively reflect the essence of pathophysiological changes in the organism of experimental animals.

Currently known methods of simulation of experimental hyperthyroidism are exogenous introduction experimental animal preparations of one of the thyroid hormones (for example, triiodothyronine). This model of hyperthyroidism can reproduce the clinical picture of hyperthyroidism and to study the effect of excess thyroid hormones in the blood to the peripheral organs and body systems (cardiovascular, nervous system, liver, skeletal muscles, the level of sex hormones).

However, it should be noted that in patients with severe functional hyperthyroidism in the blood increased the content of total T4that correlates with the level of total T3. It is known that both thyroid hormone can cause a number of pharmacological effects, since they have their own receptors in some target cells. Therefore, to reproduce the most complete clinical picture of the experimental is hyperthyreosis necessary to the hormonal status was enhanced by both thyroid hormones.

Furthermore, it should be noted that the method of modeling of hyperthyroidism using exogenous introduction thyroid hormone feedback mechanism in thyroid parenchyma develops the clinical picture is not typical for the simulated pathology in other tissues of the body. When modeling the experimental hyperthyroidism (thyrotoxicosis) the organism is saturated with exogenous thyroid hormones, which leads to inhibition of thyroid-stimulating activity of the pituitary gland, and thyroid gland loses factor, stimulating its secretory activity. The consequence of this is the development of morphological patterns of hypothyroidism in the tissues of the thyroid gland, while in the peripheral organs can be observed functional hyperthyroidism.

Modeling structural thyroid diseases is also of considerable interest, because the thyroid gland is always accompanied by a characteristic change in its structure. Since the most common form of structural pathology of the thyroid gland is a colloid goiter, the study of the effect of drugs on the recovery processes of the morphofunctional state zonesmarines thyroid as the drive is very informative and allows you to determine the nature of thyrotropic action of the investigational drug.

There are many ways of obtaining experimental model of hyperthyroidism on laboratory rats, differing as used thyroid hormone, and average daily dose, duration, and method of its introduction. Most often for this purpose use drugs of one of the synthetic thyroid hormones. Much less is currently used in organic preparations of thyroid hormones (thyroidin).

There is a method of modeling of thyrotoxicosis, including intragastric administration of thyroidin experimental animal within 7 days and twice a day subcutaneously izadrina dose of 0.04 g/kg (AV. St. USSR №1735892). On the 8th day determine body weight, record the electrocardiogram, define rectal temperature and oxygen consumption. The comparison of obtained results with the original data shows a decrease in the body weight of 26.5%, increase in heart rate of 98 beats / min, the increase in rectal temperature of 2.8°C and oxygen consumption on 0,019 l/100 g of body weight for 1 hour, which indicates the development of thyrotoxicosis in experimental animals.

The disadvantage of this method is the development of ORGANOARSENIC effects of thyroidin containing untreated thyroid hormones. For this reason, the thyroidin currently has extremely is of limited use in experimental, and clinical pharmacology.

Also there is a method of modeling of hyperthyroidism using thyroxine in a dose of 50 mg/100 g in 10 days, /1/, the development of hyperfunctional state the authors judged on the content in serum T4, T3 and TSH.

The effect of experimental hyperthyroidism on the activity of acetylcholinesterase and Na(+), K (+) - and Mg(2+)-ATPase used intragastric administration of thyroxine in a dose of 25 mg/100 g for 14 days /3/.

In the study of the antioxidant system in rats with experimental hyperthyroidism, used to model the pathology of triiodothyronine in the dose of 20 mg/100 g for 1, 3 and 5 days /5/. The disadvantage of these methods is the inferiority of the clinical picture of hyperthyroidism, because the increased levels of hormones occurs only at the expense of the injected hormone.

When studying the enzyme activity of the heart muscle in experimental thyrotoxicosis to simulate pathology used thyroxine, which rats were treated at a concentration of 12 mg/l in drinking water /2/. The disadvantage of this method is the absence of a precise input of thyroid hormone and monitor individual animal drug.

The closest technical solution is the simulation method of thyrotoxicosis and colloides the goiter, including intragastric administration of an experimental animal thyroid hormones, in particular teratoma /6/. In the study of thyroid-stimulating activity of tincture of bacon for modeling experimental hyperthyroidism has used the drug tireotom containing a combination of both thyroid hormones, which was administered to the experimental animals at a dose of 20 mg/100 g for 8 weeks. The clinical pathology in this way of modeling was characteristic functional of hyperthyroidism. The disadvantage of this method is the danger of animals (especially young animals) with a long introduction large doses of thyroid hormones.

The objective of the invention is the development of simulation method of hyperthyroidism in laboratory animals, reflecting the receipt of a complete clinical characteristics hyperthyroid condition (hyperthyroidism or thyrotoxicosis), obtaining a reliable model of colloid goiter, as well as improving the efficiency of research by saving laboratory diagnostics and biological material.

The aim was the creation within a single experiment of this model, the pathology of the thyroid gland, which produces in the blood and peripheral organs clinical picture of hyperthyroidism and thyroid parenchyma will create the pathology of colloid goiter.

This technical effect is achieved by the use of the simulation method of thyrotoxicosis and colloid goiter, including intragastric administration of an experimental animal, teratoma with subsequent determination of the functional activity of the thyroid gland. Tireotom injected in increasing weekly doses ranging from 25 μg/kg 200 μg/kg, for 6 weeks.

Experimental studies were conducted on 60 white nonlinear rats male weight 200-270, All animals were kept in standard vivarium conditions VILAR on a balanced diet with full content of iodine (1.00 mg/kg feed).

Model experimental thyrotoxicosis was obtained by daily oral administration of the drug Tireotom (he became-Chemie, Germany), increasing weekly doses of 25, 50, 100, 150, 200 μg/kg of the Drug was administered in the form of water mist for 6 weeks. Then, to confirm the diagnosis of hyperthyroidism, 10 rats were slaughtered by decapitation, blood samples are taken to determine the level of thyroid hormones in serum and secretion of the thyroid glands to determine their mass and morphological analysis of tissue.

After a four-week break 40 animals with experimental thyrotoxicosis used to further study the effects of investigational drugs on Voss is the resolution of the thyroid gland.

Control normotensive thyroid served 10 intact rats that received distilled water.

To assess the functional state of the thyroid gland of rats used methods, with the greatest diagnostic significance: radioimmunological determination of thyroid hormones in serum and morphological analysis of the tissues of the thyroid gland.

Radioimmunological assay (RIA) free triiodothyronine (T3) and thyroxine (T4) in the serum of rats was performed using sets Total T4RIA KIT and Total T3RIA KIT company Immunotech, Bekman (Czech Republic). The concentration of hormones considered automatically by the calibration curve in the γ-counter company LKB (Sweden). For morphological analysis of both lobes of the thyroid gland of rats immediately after separation were weighed on a torsion balance to determine the absolute mass (in mg) with a precision of 0.01, and were fixed in 10% solution of neutral formalin. Histological processing was performed by standard methods (Merkulov, G. A. Course histopathological techniques / Haircolour. - M.: Medicine, 1969. - 423). Paraffin sections with a thickness of 5-7 μm were prepared on a Reichert microtome (Austria). The color of the samples was carried out according to traditional methods, hematoxylin-eosin (O.V. Volkov Basis of histology with histological technique / Obvalov, Ukeles the s - M.: Medicine, 1971. - 271 (C).

For an objective assessment of the functional status of the thyroid gland used quantitative methods of analysis of tissue components, including those developed for endocrine organs (Ochmanski, 2002; Wllicb, 1975; Kraska, 1980; Thu, 1990). Stereoscopic method point account using a computer program on histological sections were determined relative area of the tissue components of the thyroid gland: follicular epithelium and colloid, interfollicular Islands, desquamated epithelium, connective tissue, lymphatic infiltration and vascular bed.

With the aid of an eyepiece micrometer was determined parameters of thyroid follicles: the average height of the follicular epithelium (h), the average size of the cores of thyrocytes (d), the average diameter of follicles (D). Produced by counting the proportion (%) of follicles of different size diameters in the overall sample. In a variation range of diameters was taken as a small follicles up to 50 µm), medium (up to 100 μm and larger than 100 μm. Conduct at least 100 measurements 40 follicles each sample, only held not less than 400 measurements for each indicator.

Statistical analysis was performed using the software package statistical analysis Statistica 6,0 (StatSoft, USA). We used the criterion of Mann-Whitney the La comparison of independent samples, the Wilcoxon test to compare related samples. To assess the significance of differences between samples from the distribution, approaching normal, used t-student test.

In tables 1 and 2 present data on thyroid status, change of body weight and thyroid gland, as well as morphological and functional characteristics of the tissue of the thyroid gland in rats in the simulation of experimental hyperthyroidism.

According to the results of radioimmunoassay analysis, the simulation method of hyperthyroidism using increasing doses of teratoma, the levels of both thyroid hormones in the serum of experimental animals was increased. So, the content of thyroxine and triiodothyronine were respectively 117% and 122% of the average values of these parameters in the blood serum of intact rats, and the value of the index deyodirovania was close to normal. At the same time was also a significant reduction in body weight of animals, as well as the reduction and the increase in the relative weight of the thyroid gland as compared with intact animals (p<0,05) (table 1).

Results morphological analysis of the tissues of the thyroid gland of rats treated with tireotom showed the presence polymerforschung colloid goiter. In the structure of the thyroid gland of rats was dominated by medium follicles (56,8) and small (26,6%) size and the maximum number of large follicles (16,6%) compared with the levels in all other groups studied animals. The variability in size of follicles was in the range from 20,84 to 259,37. While on the periphery of the shares were detected significantly increased follicles engaged aggregated colloid and tendency to the formation of thereareplaces, and in the center of the body follicles smaller size.

In most histological preparations of the rat thyroid gland was observed low follicular epithelium, the average height was 6.2±3,68 μm. Core thyrocytes were flattened form, with an average area of 11.1±3,39 μm2that is less than the average value of this indicator in the group of intact rats (p<0,05). It is recognized that reducing the height of the thyroid epithelium and reduction of the area of the nuclei of thyrocytes indicates a decrease in the functional activity of the thyroid gland, which in this case is connected with a long introduction thyroid hormones and blocking your own hormone production of the thyroid gland.

These results are also confirmed by morphometric analysis of tissue components of the thyroid gland of rats treated with tireotom. So, it is established that the introduction of teratoma in rats more than half of the gland volume was occupied by colloid 54,1±16,89% (intact and 39.9±13,26%), while the mean diameter of follicles was 74.3±38,02 that is above the average value of this indicator in the intact animals (p<0,05).

About the reduction in the functional activity of the thyroid gland shows a decline in the relative volume of follicular and interfollicular thyroid epithelium, which had respectively 20,8±11,27% and 4.1±5,47% of the volume of the gland, the values of these indicators in intact rats reached 33.7±9,11% and 8.1±9.27 per cent respectively.

It should be noted the presence of some necrotic manifestations observed in the parenchyma of the thyroid gland of rats treated with tireotom in increasing the dose. Thus, the observed increase in the relative volume of the endothelial cells of 5.8±11,31% (intact 1,3±2,60%), which was inversely correlated with the volume of follicular thyrocytes (r= - 0.54) and to a lesser extent with the volume interfollicular thyroid cells (r=-0,28). Apparently, an excess of thyroid hormones lead to destructive processes in the follicles, affecting not only midorikawa walls, but the walls of the follicles, ultimately leading to their partial fragmentation. As a result, in some parts of the body were observed foci of lymphocytic and macrophage infiltration, the relative volume of which was 0.9±2,82%, higher values of this indicator in the intact animals (0,1±0,43%). On the destructive processes in the stroma of the organ also indicate hemorrhage associated most likely with the destruction of the walls of blood vessels. The study of the vascular bed showed the presence of foci of hemorrhage, while the relative size of the vascular bed was 5.1±2,48%, which is considerably lower than in the group and the stroke animals (11,8±4,80%). Also noted is the increase in the relative volume of the connective tissue, in this group of animals 8,9±10,42% against 5,0±4,07% in intact rats (table 2). However, it should be noted that this increase has a direct correlation with the number of endothelial thyrocytes (r=0.43), and according to the morphological picture of stroma accompanied not by processes sclerosing thyroid gland and the presence of inflammation, edema and necrotic process of connective tissue, it is also a small number of fibroblastic cells number in the stroma.

It can be concluded that the morphological picture of the tissues of the thyroid gland of rats treated with increasing doses of thyroid hormones characteristic of severe pathology of hypothyroidism with polymorphismin type structure colloid goiter.

Thus, the developed experimental model of thyrotoxicosis in rats, which reflects the clinical picture of functional hyperthyroidism with increased content of both thyroid hormones T3T4in the serum of experimental animals, while in the thyroid parenchyma obtained morphological picture of severe thyroid hypofunction with polymorphismin type structure colloid goiter.

The advantages of the proposed method simulation of hyperthyroidism are:

1) who is very useful to reproduce the most characteristic picture of hormonal changes, observed in most cases of hyperthyroidism, which allows to use this model to study the effect of excess thyroid hormones in the blood by organs and systems in the experimental animal (cardiovascular, nervous system, liver, muscle tissue, and so on);

2) the use of increasing doses of teratoma allows to obtain the morphological picture of colloid goiter in rats, which can be used to study therapeutic effect of drugs on the recovery processes of the functional activity of the thyroid gland in the conditions of goiter pathology with Hypo-functional colloid type structure of the gland;

3) the use of increasing doses of teratoma is safe and reduces the loss of animals during the experiment.

Sources of information

1. Aragao CN, Souza LL, Cabanelas A, Oliveira KJ, Pazos-Moura CC. Effect of experimental hypo - and hyperthyroidism on serum adiponectin.: Metabolism. 2007 Jan; 56(1): 6-11.

2. Araujo AS, Ribeiro MF, Enzveiler A, Schenkel P, Fernandes TR, Partata WA, Irigoyen MC, Llesuy S, Bello-Klein, A. Myocardial antioxidant enzyme activities and concentration and glutathione metabolism in experimental hyperthyroidism // Mol Cell Endocrinol. 2006 Apr 25; 249(1-2): 133-9.

3. Carageorgiou H, Pantos C, Zarros A, Stolakis V, Mourouzis I, Cokkinos D, Tsakiris S. Effects of hyper - and hypothyroidism on acetylcholinesterase, (Na(+), K (+))- and Mg (2+)-ATPase activities of adult rat hypothalamus and cerebellum. Metab Brain Dis. 2007 Mar; 22(1): 31-8.

4. Ozdemir S, Yucel R, Dariyerli N, Toplan S, Akyolcu MC, Yigit G, Hatemi H. The effects of experimental hyperthyroidism on hemorheology and plasma fibrinogen cncentration // Endocrine. 2006 Oct; 30(2): 203-5.

5. Sahoo DK, Roy A, Bhanja S, Chainy GB. Experimental hyperthyroidism-induced oxidative stress and his or her of antioxidant defence system in rat testis // Indian J Exp Biol. 2005 Nov; 43(11): 1058-67.

6. The effect of tincture of bacon on thyroid function in experimental Hypo - and hyperthyroidism in rats / Laslau, Vgenerally, Essmann, A.a.semenov // Chemical and pharmaceutical journal: scientific-technical and production journal. - 2005. Volume 39, N5. - P.23-25.

Table 1.
Indicators of functional activity of the thyroid gland of rats in conditions of experimental hyperthyroidism and normal.
IndicatorsEd. dimensionthe intacttireotom
T4nmol/l39,95 [27,63; 49,84]46,80 [25,41; 55,05]
T30,73 [0,60; 0,77]0,89 [0,58; 1,30]
Index deyodirovaniaT3/T40,022±0,0141 0,023±0,0068
The body weight of the animalg366,7±30,31of 174.4±26,97
the mass of the thyroid glandmg21,6±3,7515,4±3,02*
The relative weight of the thyroid glandmg/kg6,2±0,898,8±2,31*
* - marked, statistically significant differences between groups (t-student test).
Table 2.
Morphological and functional changes in the thyroid gland of rats in conditions of introduction of teratoma.
indicatorsEd. dimensionthe intacttireotom
Follicular thyrocytes%33,7±9,1120,9±11,27
colloid%39,9±13,2654,1±16,89
stroma%5,1±4,078,9±10,42
Interfollicular thyrocytes%8,1±9,274,1±5,47
Endothelial thyrocytes%1,3±2,605,9±11,30
vessels%11,8±4,85,2±2,48
leukocytes%0,1±0,430,9±2,82
The diameter of the folliclemcm59,0±24,8374,2±38,02
The height of the epithelium mcm8,2±2,696,1±1,89
The area of the nuclei of thyrocytesμm214,1±3,4511,1±3,39

The simulation method of thyrotoxicosis and colloid goiter, including intragastric administration of an experimental animal, teratoma with subsequent determination of the functional activity of the thyroid gland, characterized in that tireotom injected in increasing weekly doses ranging from 25 to 200 mcg/kg for 6 weeks.



 

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