RU2290859C1 - Method for predicting experimental hypothyroidism in laboratory animals - Google Patents

Abstract

FIELD: medicine, diagnostics.

SUBSTANCE: it is necessary to measure a patient's body temperature followed by detecting functional thyroid activity. Additionally, at periodicity of once weekly, not rarely, one should investigate daily thermoregulation rhythms due to measuring body temperature values at 7 a.m., 11 a.m., 3 p.m., 7 p.m.,11 p.m. and 7 a.m. sharp. At achieving the sign of resistant decrease of body temperature values in experimental animals during all rhythm phases against analogous values in control group one should put the diagnosis to be "an experimental hypothyroidism". The innovation enables to obtain a reliable model of hypothyroidism more fully depicting the nature of pathophysiological shifts.

EFFECT: higher accuracy and efficiency of diagnostics.

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Description

The invention relates to medicine, in particular experimental pathophysiology, and can be used in the functional diagnosis of experimental hypothyroidism in laboratory animals.

There are many ways to obtain an experimental model of hypothyroidism in laboratory rats, which differ from each other both in the average daily dose and the duration of administration of thyreostatics, in particular mercazolil.

So, some authors use small average daily doses of mercazolil, for example 0.3 mg / 100 g body weight (1). Others, on the contrary, are high - up to 20 mg / 100 g of the mass of the animal (2). At the same time, the duration of thyreostatic administration varies from 21 to 25 days (1, 2). Under these conditions, when reproducing the above methods, it is difficult for the experimenter to determine the time for obtaining a reliable model of hypothyroidism, which in turn depends on the average daily dose of thyreostatic and the duration of its administration. An affirmative diagnosis of “Experimental mercazolyl hypothyroidism” can be made only after decapitation of the animals, followed by determination of the level of thyroid hormones. If hypothyroidism is not confirmed, the experimenter loses all the biological material, that is, the entire group of animals.

A known method for the diagnosis of experimental hypothyroidism, including the study of symptoms such as lethargy, drowsiness, sluggishness, lowering body temperature, hypercholesterolemia, leukopenia, anemia (3).

The disadvantage of this method is that all these symptoms, except for determining the level of thyroid hormones, are not reliable signs of hypothyroidism. And the level of thyroid hormones in small animals requires their preliminary killing.

Closest to the claimed diagnosis of experimental hypothyroidism in laboratory animals is the classical model of experimental hypothyroidism, reproduced by oral administration of thyrostatics mercazolilum in laboratory rats at the rate of 20 mg / 100 g of body weight daily for 3 weeks (2).

The disadvantage of this method is that in this case there is no objective monitoring of the state of the thyroid gland at the intermediate stages of the experiment and diagnostic criteria that can reliably establish the occurrence of hypothyroidism in animals. Against this background, side effects and a distortion of the model of pathophysiological disorders in thyroid pathology are not ruled out. All this makes it difficult to decide on the feasibility of stopping the further administration of thyreostatics.

The objective of the invention is the development of a method for the diagnosis of experimental hypothyroidism in laboratory animals, reflecting the receipt of the full clinical characteristics of the hypothyroid state.

A method for diagnosing experimental hypothyroidism makes it possible to obtain a new technical effect - obtaining a reliable model of hypothyroidism that most fully reflects the essence of pathophysiological shifts in the experiment, as well as increasing the efficiency of research due to the saving of medication, laboratory diagnostic tools and biological material.

The indicated technical effect is achieved by the fact that a method is proposed for diagnosing experimental hypothyroidism in laboratory animals caused by the oral administration of thyreostatics, including measuring body temperature and then determining the functional activity of the thyroid gland, characterized in that the daily rhythm of thermoregulation is further investigated by measuring body temperature at 07 ⁰⁰ , 11 ⁰⁰ , 15 ⁰⁰ , 19 ⁰⁰ , 23 ⁰⁰ and 07 ⁰⁰ h with a frequency of, for example, at least once a week. When experimental animals achieve a sign of a steady decrease in body temperature in all phases of the rhythm, the diagnosis of “experimental hypothyroidism” is established with respect to similar indicators of the control group, which in turn allows you to proceed to the next stages of the study.

It is known that hypothermia is regarded as a diagnostic sign of hypothyroidism. In the currently known methods for diagnosing hypothyroidism, they resort to a single measurement of body temperature values during the day, which reduces the accuracy of the diagnosis. For example, the values of body temperature in experimental animals, in particular in rats, undergo significant fluctuations during the day. Moreover, the magnitude of these fluctuations in healthy animals of the control group can reach 3 ° C, that is, in the range from 35.9 ° C to 38.9 ° C - Fig.1.

As can be seen from figure 1, the body temperature in a healthy rat in some cases can be calculated as a sign of hypothyroidism (35.9 ° C when measured at 07 ⁰⁰ h), and in others as a sign of hyperthyroidism (38.7 and 38.9 ° C when measured at 19 ⁰⁰ h and 23 ⁰⁰ h, respectively).

The option of a single measurement of body temperature at a strictly defined time of the day is also unacceptable in the diagnostic plan, since acrophases and rhythm bathyphases can shift during the day.

In turn, the application of the principle of chronobiological studies in the claimed method improves the accuracy of diagnosis and allows you to:

- firstly, to make a timely decision on the abolition of the further administration of thyreostatics and, therefore, to prevent overdose and related side effects;

- secondly, decide on the need to continue the introduction of thyreostatics to obtain a complete classical picture and laboratory data confirming the completion of the current stage of the experiment;

- thirdly, to guarantee the receipt for scientific purposes of additional laboratory data confirming the reliability of the experimental model of hypothyroidism;

- fourthly, to prevent at the initial stage of the experiment the development of complications leading to culling and death of laboratory animals;

- fifthly, in general, to increase the effectiveness of scientific research due to the saving of medical, laboratory diagnostic tools and the rational use of biological material.

The method is as follows. Regardless of the experimenter’s chosen method of introducing hypothyroidism, it is known that they differ in the average daily dosage and duration of the administration of thyreostatics, additionally, with a frequency of, for example, at least once a week, daily rhythm of thermoregulation is studied by measuring body temperature at 07 ⁰⁰ , 11 ⁰⁰ , 15 ⁰⁰ , 19 ⁰⁰ , 23 ⁰⁰ and 07 ⁰⁰ hours. Moreover, a steady decrease in body temperature values by 0.3-1.0 ° C in all phases of the rhythm in rats of the experimental group in comparison with the control, as established, is an additional diagnostic criterion for obtaining a reliable model of hypothyroidism.

The conducted biorhythmological studies allow us to conclude that the first stage of the experiment is completed - obtaining a model of hypothyroidism and, in turn, proceed to the next one after partial slaughter of animals to obtain a set of clinical and laboratory data - general analysis and blood biochemistry, measurement of weight and height indicators, determination the level of thyroid hormones in the blood serum - TSH, T ₄ , T ₃ , pathomorphology of organs and tissues of slaughtered animals.

And, on the contrary, when obtaining other ratios of temperature curves, which can be expressed either as a continuous or partial excess of the analyzed parameters of the experimental group over the control, they come to the conclusion that the first stage is incomplete and the need to continue the course of administration of thyreostatics.

The invention is characterized by the following examples of the method for the diagnosis of experimental hypothyroidism.

Example 1 - the diagnostic significance of chronobiological studies in establishing the sign of inaccuracy of the experimental model of hypothyroidism at low daily doses of thyreostatic. Simulation of hypothyroidism in adult male rats weighing 180-220 g by intragastric administration of mercazolil in small doses at the rate of 1 mg / 100 g of animal body weight. All animals are divided into 2 groups of 12 rats each: 1st — control, 2nd — experimental. Since the 8th day of the experiment, examined the circadian rhythms of thermoregulation by measuring body temperature at 07 ^00, 11 ^00, 15 ^00, 19 ^00, 23 ⁰⁰ and 07 ⁰⁰ hr - Table 1, Figure 2. The research results were processed by the method of variation statistics using the indicator of the accuracy of the experience, R.

Table 1
The body temperature of rats on the 8th day with the introduction of merkazolil in an average daily dosage of 1 mg / 100 g; (M ± m), n = 12 Time of day (hour) 7 ⁰⁰ 11 ⁰⁰ 15 ⁰⁰ 19 ⁰⁰ 23 ⁰⁰ 7 ⁰⁰ Body temperature, ° С (control group) 38.4 ± 0.07 36.9 ± 0.11 37.8 ± 0.15 37.0 ± 0.09 38.0 ± 0.09 38.0 ± 0.07 The accuracy indicator of experience, P,% 0.19 0.30 0.40 0.25 0.25 0.19 Body temperature, ° С (experimental group) 37.4 ± 0.09 36.5 ± 0.14 37.0 ± 0.16 37.0 ± 0.10 36.9 ± 0.22 37.0 ± 0.18 The accuracy indicator of experience, P,% 0.25 0.39 0.45 0.45 0.59 0.49

As can be seen from figure 2, in this case there is no "sign of a steady decline in the control group." So, the values of the indices of the control and experimental groups at 19 ⁰⁰ h coincide and amount to 37.0 ° С.

Based on the foregoing, it was concluded that it is impossible to diagnose “experimental hypothyroidism” and, therefore, the incompleteness of the current stage of the experiment. It was decided to continue the introduction of merkazolil.

Repeated studies of diurnal rhythms of thermoregulation were carried out after a week, i.e. on the 15th day from the beginning of the experiment - the data are presented in table 2 and figure 3.

As can be seen from figure 3, and in these terms was not achieved "a sign of a steady decrease in body temperature in all phases of the rhythm in relation to the same indicators of the control group", which also did not allow the diagnosis: "experimental hypothyroidism."

table 2
The body temperature of rats on the 15th day with the introduction of merkazolil in an average daily dosage of 1 mg / 100 g; (M ± m), n = 12 Time of day, h 7 ⁰⁰ 11 ⁰⁰ 15 ⁰⁰ 19 ⁰⁰ 23 ⁰⁰ 7 ⁰⁰ Body temperature, ° С (control group) 37.1 ± 0.11 38.0 ± 0.07 37.3 ± 0.15 38.9 ± 0.10 36.9 ± 0.09 37.4 ± 0.14 The accuracy indicator of experience, P,% 0.31 0.20 0.41 0.28 0.24 0.37 Body temperature, ° С (experimental group) 36.1 ± 0.08 36.1 ± 0.12 37.3 ± 08 37.7 ± 0.13 35.8 ± 0.17 35.9 ± 0.14 The accuracy indicator of experience, P,% 0.23 0.33 0.22 0.36 0.48 0.41

Table 3
The content of T ₃ , cT ₄ and TSH in the blood serum of laboratory rats with experimental hypothyroidism (M ± m) Names of animal groups The number of animals in the group The duration of the experiment, day T ₃ (n / mol / l) C T ₄ (p / mol / l) TSH μMU / ml Control 12 - 1.88 ± 0.13 16.68 ± 0.50 0.124 ± 0.01 Experimental No. 4 - merkazolil 1 mg / 100 g 12 - * 2.32 ± 0.07 * 8.19 ± 0.08 * 0.216 ± 0.01 * difference with control is statistically significant (P <0.05)

In order to confirm the diagnostic value of chronobiological studies on the 16th day of the experiment, animals were decapitated, followed by determination of the levels of thyroid hormones - 3,5,3 '- triiodothyronine (T ₃ ), free thyroxine (cT ₄ ) and thyroid stimulating hormone (TSH). The data are presented in table 3.

As one would expect, the results of an enzyme-linked immunosorbent assay of blood serum confirmed the forecast made on the basis of biorhythmological studies to obtain an unreliable model of hypothyroidism. Thus, the concentration of 3,5,3 '- triiodothyronine (T ₃ ) in the blood serum of rats of the experimental group was 2.32 ± 0.07 n / mol / l versus 1.88 ± 0.13 n / mol / l in control animals , which no longer allows to establish a reliable diagnosis of "experimental hypothyroidism."

Example 2 - the diagnostic significance of chronobiological studies in establishing the sign of inaccuracy of the experimental model of hypothyroidism at high daily doses of thyreostatic. Simulation of hypothyroidism in adult male rats weighing 180-220 g also by intragastric administration of mercazolil in already high daily doses at the rate of 20 mg / 100 g of animal body weight. All animals are divided into 2 groups of 12 in each: 1st — control; 2nd - experimental.

Starting from the 8th day from the start of the experiment, daily rhythm of thermoregulation according to the scheme was studied by measuring body temperature at 07 ⁰⁰ , 11 ⁰⁰ , 15 ⁰⁰ , 19 ⁰⁰ , 23 ⁰⁰ and 07 ⁰⁰ h. The data are presented in Fig. 4 and in table 4 .

As can be seen from figure 4, in this version of the experiment there is no "sign of a steady decrease in body temperature in all phases of the rhythm in relation to the same indicators of the control group" and, moreover, there is a sign of rhythm desynchronosis: at 11 ⁰⁰ hours the body temperature of experimental animals (37.5 ° С) significantly exceeds the same indicator in the control group (37 ° С); bathyphase (the lowest value of the studied parameter) in experimental animals is shifted by 23 ⁰⁰ h, i.e. the structure of daily rhythms of thermoregulation is broken.

Table 4
The body temperature of rats on the 8th day with the introduction of merkazolil in an average daily dosage of 20 mg / 100 g; (M ± m), n = 12 Time of day (hour) 7 ⁰⁰ 11 ⁰⁰ 15 ⁰⁰ 19 ⁰⁰ 23 ⁰⁰ 7 ⁰⁰ Body temperature, ° С (control group) 38.0 ± 0.07 37.0 ± 0.06 36.9 ± 0.15 37.6 ± 0.11 37.2 ± 0.08 37.2 ± 0.14 The accuracy indicator of experience, P,% 0.19 0.17 0.42 0.30 0.24 0.39 Body temperature, ° С (experimental group) 36.9 ± 0.39 37.5 ± 0.16 36.7 ± 0.11 36.8 ± 0.14 36.3 ± 0.13 36.9 ± 0.13 The accuracy indicator of experience, P,% 1.06 0.44 0.31 0.38 0.36 0.35

The data of biorhythmic studies testified to significant pathophysiological changes in the animal organism, which does not allow us to talk about obtaining a reliable model of experimental hypothyroidism in this embodiment. In this group, a lethal outcome was observed in 3 rats, in addition, complications such as alopecia, nosebleeds, development of a shock condition, tooth loss, in particular lower incisors, were observed in almost all animals.

Indeed, with the use of such high doses of merazolyl, the level of thyroid hormones decreases (T ₃ , cT ₄ ), the concentration of TSH increases, but from the point of view of experimental pathophysiology in this case it is impossible to talk about obtaining a reliable model of hypothyroidism due to the numerous complications that result from the breakdown protective and adaptive mechanisms in animals - table 5.

Table 5
The content of T ₃ , cT ₄ and TSH in the blood serum of laboratory rats with experimental hypothyroidism (M ± m) Names of animal groups The number of animals in the group The duration of the experiment in days T ₃ (n / mol / l) ct ₄ (p / mol / l) TSH, μme / ml Control 12 - 1.88 ± 0.13 16.68 ± 0.50 0.124 ± 0.01 Experimental No. 1 - merkazolil 20 mg / 100 g * * * 9 - 1.44 ± 0.10 6.0 ± 0.23 0.196 ± 0.02 * difference with control is statistically significant (P <0.05)

Thus, the identification of violations in the daily rhythms of thermoregulation made it possible to diagnose the receipt of an unreliable hypothyroidism model.

Example 3 - the diagnostic significance of chronobiological studies in establishing the sign of reliability of the experimental model of hypothyroidism at optimal doses of thyreostatics. Modeling of hypothyroidism in adult male rats weighing 180 ... 220 g by intragastric administration of mercazolil in optimal doses at the rate of 5 mg / 100 g of animal body weight. All animals were divided into 2 groups of 12 in each: 1st — control, 2nd — experimental. Since the 8th day of the experiment, examined the circadian rhythms of thermoregulation by measuring body temperature at 07 ^00, 11 ^00, 15 ^00, 19 ^00, 23 ⁰⁰ and 07 ⁰⁰ hours. The research results are presented in figure 5 and in table 6.

Table 6
The body temperature of rats on the 8th day with the introduction of merkazolil in an average daily dosage of 5 mg / 100 g; (M ± m), n = 12 Time of day (hour) 7 ⁰⁰ 11 ⁰⁰ 15 ⁰⁰ 19 ⁰⁰ 23 ⁰⁰ 7 ⁰⁰ Body temperature (° C) (control group) 38.4 ± 0.07 36.9 ± 0.11 37.8 ± 0.15 37.1 ± 0.09 38.0 ± 0.09 38.0 ± 0.07 The accuracy indicator of experience (P) in% 0.19 0.30 0.40 0.25 0.25 0.19 Body temperature (° C) (experimental group) 36.7 ± 0.12 35.9 ± 0.12 36.0 ± 0.13 37.3 ± 0.17 36.2 ± 0.11 36.1 ± 0.13 The accuracy indicator of experience (P) in% 0.33 0.34 0.36 0.46 0.31 0.30

As can be seen from figure 5, on the 8th day of the experiment it is not yet possible to establish the diagnosis of “experimental hypothyroidism” and go to the next stage of the study, since the experimental animals did not achieve a sign of a steady decrease in body temperature in all phases of the rhythm with respect to to similar indicators of the control group

In connection with the foregoing, a decision was made about the need to continue the course of administration of thyreostatics for another seven days.

As the analysis of the correlation of temperature curves shows, and within these periods it is not possible to establish the diagnosis of “experimental hypothyroidism”.

A week later, that is, on the 15th day from the start of the experiment, a repeated study of the daily rhythms of thermoregulation was conducted. The data are presented in Fig.6 and in table 7.

Table 7
The body temperature of rats on the 15th day with the introduction of merkazolil in an average daily dosage of 5 mg / 100 g; (M ± m), n = 12 Time of day, h 7 ⁰⁰ 11 ⁰⁰ 15 ⁰⁰ 19 ⁰⁰ 23 ⁰⁰ 7 ⁰⁰ Body temperature, ° С (control group) 37.4 ± 0.12 37.3 ± 0.0 36.9 ± 0.11 37.9 ± 0.1 37.5 ± 0.10 37.1 ± 0.11 The accuracy indicator of experience, P,% 0.34 0.20 0.30 0.38 0.28 0.31 Body temperature, ° С (experimental group) 35.3 ± 0.11 36.8 ± 0.12 36.9 ± 0.08 37.0 ± 0.07 35.2 ± 0.15 34.7 ± 0.06 The accuracy indicator of experience, P,% 0.32 0.34 0.23 0.21 0.42 0.18

Therefore, the administration of thyreostatic was continued. Exactly a week later, that is, on the 22nd day from the start of the experiment, the study of diurnal rhythms of thermoregulation was repeated.

Table 8
The body temperature of rats on the 22nd day with the introduction of merkazolil in an average daily dosage of 5 mg / 100 g; (M ± m), n = 12 Time of day, h 23 ⁰⁰ 7 ⁰⁰ 11 ⁰⁰ 15 ⁰⁰ 19 ⁰⁰ 23 ⁰⁰ 7 ⁰⁰ Body temperature, ° С (control group) 37.2 ± 0.08 37.9 ± 0.13 36.8 ± 0.10 38.1 ± 0.10 38.3 ± 0.05 37.4 ± 0.12 37.0 ± 0.16 The accuracy indicator of experience, P,% 0.23 0.34 0.29 0.26 0.15 0.33 0.45 Body temperature, ° С (experimental group) 35.9 ± 0.07 35.9 ± 0.11 35.8 ± 0.09 37.2 ± 0.09 37.2 ± 0.07 35.9 ± 0.09 35.5 ± 0.07 The accuracy indicator of experience, P,% 0.21 0.33 0.27 0.24 0.20 0.27 0.19

According to the analysis of the data presented in Fig.7 and in table 8, the diagnosis of "experimental hypothyroidism" was made and, therefore, a conclusion was made about the completion of the current stage of the experiment and the possibility of moving to the next. In this variant of the experiment, a sign was obtained of a steady decrease in body temperature in all phases of the rhythm in relation to the same indicators in the control group.

The results of an enzyme-linked immunosorbent assay for the content of thyroid hormones unambiguously confirmed (table 9) the validity of the conclusion made on obtaining a reliable model of hypothyroidism.

As can be seen from table 9, the level of thyroid hormones (T ₃ and T ₄ ) in experimental animals was reduced in comparison with the same indices of the control group and vice versa, the level of thyroid stimulating hormone (TSH) was increased, which objectively confirms the previously diagnosed diagnosis.

Table 9
The content of T ₃ , with T ₄ and TSH in the blood serum of laboratory rats with experimental hypothyroidism (M ± m) Names of animal groups The number of animals in the group The duration of the experiment, day T ₃ (n / mol / l) c T ₄ (p / mol / l) TSH μMU / ml Control 12 - 1.88 ± 0.13 16.68 ± 0.50 0.124 ± 0.01 Experimental No. 3, mercazolil 5 mg / 100 g * * * 12 - 1.6 ± 0.02 3.4 ± 0.12 0.164 ± 0.01 * difference with control is statistically significant (P <0.05)

Literature taken into account during the examination:

1. Augustinovich M.S., Detyuk E.S. Morpho-cytochemical features of the progeny gonads with decreased thyroid function of the female // Transactions of Crimean honey. in-ta - 1986.- T.109. - S.119-121.

2. Kuzmak N.I. The effect of thyreostatic drugs and thyroidectomy on the content of sialic acids in blood serum and liver in rats // Questions honey. chemistry. - 1978.- T.24, v. 1. - S. 52-56.

3. Potemkin VV Endocrinology. - M .: Medicine, 1987. - S.137-153.

Claims (1)

A method for diagnosing experimental hypothyroidism in laboratory animals, including measuring body temperature with subsequent determination of the functional activity of the thyroid gland, characterized in that in addition, with a frequency of, for example, at least once a week, daily rhythms of thermoregulation are examined by measuring body temperature at 07 ⁰⁰ , 11 ^00, 15 ^00, 19 ^00, 23 ⁰⁰ and 07 ⁰⁰ hours, and when the experimental animals of feature sustained reduction in body temperature rhythm indicators in all phases with respect to the analogous m the control group is diagnosed "experimental hypothyroidism."

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