Indicators of the cell cycle in the thyroid gland in rats when using infusion of 0.9% NaCl solution on the background of thermal skin burns

Systemic damage of the organs, including the thyroid gland, is one of the key factors in the pathogenesis of burn disease due to thermal skin burns. The aim of this study was to investigate the indices of the cell cycle and DNA fragmentation of thyroid gland cells in rats with the use of infusion of 0.9% NaCl solution against the background of thermal skin burns. Experimental studies were conducted on 60 white male rats weighing 160180 g, which was subjected to thermal burns of the skin of 2-3 degrees with a total area of 21-23% of the body surface. The first 7 days rats were infused with 0.9% NaCl solution into the inferior vena cava. Animals were removed from the experiment by decapitation (after 1, 3, 7, 14, 21, and 30 days). DNA content in the nuclei of the cells of the thyroid gland of rats was determined by flow cytometry. The statistical processing of the obtained results was carried out in the license package "STATISTICA 6.1" using nonparametric estimation methods. After 1 day after thermal skin damage and using 0.9% NaCl solution, lower (p<0.05) values of the S-phase index (0.234±0.094) were found compared to the control group without burn (0.652±0.134). The maximum decrease (p<0.01) of S-phase indicators (0.622±0.110 and 0.214±0.105, respectively) and a significant increase (p<0.01) of the SUB-G0G1 interval (5.288±0.840) compared to similar control group values (2.594±0.628) is observed after 3 days. The S-phase against the background of the introduction of 0.9% NaCl solution and thermal skin burn remained significantly lower than those of the similar control groups at 7 (p<0.01), 14 (p<0.05) and 21 days (p<0.05). At 14 days after thermal skin injury, the SUB-G0G1 interval (p<0.05) was lower than in the control group of rats. After 30 days, the G0G1 phase parameters were significantly lower (p<0.01), and the G2+M phase values were significantly (p<0.01) higher than those in the control group at the same time. Thus, it was found that 0.9% NaCl solution was not effective enough to correct cell division disorders during the entire observation period after skin burns.


Introduction
The urgency of the problem of therapy of thermal burns of the skin and burn disease (BD) is caused by the increase in the number of burn injuries in modern society, the lack of efficiency of existing methods of therapy, the high frequency of development of complications of a systemic nature [14]. The inefficiency of the proposed methods of treatment is not least due to the complex pathogenesis of this damage, the numerous factors responsible for the cascade of pathological processes in thermal burns [13]. For this reason, the worldwide study of the mechanisms and pathogenetic factors of BD at the tissue, cell, subcellular and molecular levels perform, which deepens knowledge about this process and identifies potential targets of therapy [3].
One of the key factors in the pathogenesis of burn disease due to thermal burns of the skin, many researchers [6,10,18] are considering systemic damage to the organs of the endocrine system, which manifests itself both functionally and at the cellular and subcellular level. A special role in the pathogenesis of BD according to modern data [18] is given to the interaction of the triangle pituitary gland -adrenal cortex, pituitary gland -thyroid gland.
It is known [8] that patients with severe thermal burns of the skin have decreased plasma triiodothyronine concentrations, low thyroxine and normal range, or slightly decreased thyrotropic hormone concentrations. This ensemble of change is collectively known as the "nonthyroid disease" syndrome [1]. The degree of manifestation of this disease is associated with the prognosis of the disease, but there is no evidence for the causality of this association. It is assumed [18] that the development of this condition is a consequence of the acute phase response to systemic irritation and microelement constraints. Thyroid injury pathogenetically is associated with the level of endogenous intoxication and the development of general inflammatory response, established a clear link between changes in thyroid hormone metabolism and activation of various proinflammatory cytokines [15].
Today, it is believed [7] that the problem of correction of thyroid gland damage remains an open question in the treatment of BD, and a problem that requires an urgent solution, given the important role of the functioning of this gland in the metabolism of the skin and other vital tissues (bone, connective tissue) that provide the restoration of homeostasis in the body. However, the most accurate method for assessing cell division is the DNA cytometry method, which is nowadays defined as a reference for the establishment of apoptosis markers, and such that allows dividing the cell phases into separate components [2]. We did not find any data on studies of thyroid cell division by DNA cytometry against the background of burn skin damage.
The aim of the study was to investigate the indices of the cell cycle and DNA fragmentation of the thyroid gland in rats using infusion of 0.9% NaCl solution against the background of thermal skin burns.

Materials and methods
Experimental studies were conducted on 60 white male rats weighing 160-180 g, conducted at the Research Thermal burns of the skin of 2-3 degrees were carried out by applying to the pre-shaved lateral surfaces of the trunk of rats four copper plates (each with a surface area of 13.86 cm2) for 10 seconds, which were pre-heated for 6 minutes in water with a temperature of 100?C [9]. The total area of skin lesions was 21-23%. The first 7 days of rats were infused with 0.9% NaCl solution into the inferior vena cava. Animals were removed from the experiment by decapitation (after 1, 3, 7, 14, 21, and 30 days). Shaving, catheterization of veins, staging of skin burns, and decapitation of rats were performed under the conditions of intravenous propofol anesthesia (calculated at 60 mg/ kg animal weight).
Within the framework of the agreement on scientific cooperation between the Research Center of National Pirogov Memorial Medical University, Vinnytsya and the Department of Histology, Cytology and Embryology of the Odessa National Medical University, DNA content in the nuclei of thyroid cells of rats was determined by flow DNA cytometry on a multifunction flow cytometer "Partec PAS" (Partec, Germany) [17]. Determined: G0G1 is the percentage of G0G1 phase cells to all cells in the cell cycle (DNA content = 2c); S is the percentage of the phase of DNA synthesis to all cells of the cell cycle (DNA content > 2c and < 4c); G2+M is the percentage ratio of the G2+M phase to all cells in the cell cycle (DNA = 4c). Determination of DNA fragmentation (SUB-G0G1, apoptosis) was performed by isolating the RN2 regions on DNA histograms before the G0G1 peak, indicating nuclei of cells with a DNA content < 2c.
The statistical processing of the obtained results was carried out in the license package "STATISTICA 6.1" using nonparametric estimation methods. The significance of the difference in values between the independent quantitative values was determined using the Mann-Whitney U test.

Results
It is established that the background of the introduction of 0.9% NaCl solution 1 day after thermal burns of the skin marked changes in the cell cycle indices of the thyroid gland -statistically significant decrease in the number of cells in phase S (p<0.01), which indicates insufficient restoration of the damaged cell population. Other indicators of the cell cycle have no significant or trending differences with those of the group without skin burn (Table 1). No significant or trending differences for G0G1, G2+M, and SUB-G0G1 were identified (see Table 1).
On the presented DNA-histogram ( Fig. 1) of thyroid cells 1 day after skin burn on the background of the introduction of 0.9% NaCl solution, the level of DNA fragmentation in the interval SUB-G0G1 was 3.32%, and phase S -0.19%, indicating expressed inhibition of DNA synthesis.
The maximum reduction in the percentage of thyroid cells in phase S (p<0.01) and, at the same time, a peak increase in the average level of SUB-G0G1 interval (p<0.01) was established after 3 days of observation from the start of thermal skin burn on the background of 0.9% NaCl solution introduction (see Table 1). No significant or trending differences were found for G0G1 and G2+M indicators (see Table 1).
In the presented DNA histogram (Fig. 2) of nuclear suspension of cells of the thyroid gland of rats 3 days after skin burn on the background of the introduction of 0.9% NaCl solution, SUB-G0G1 (RN2, DNA fragmentation) was 5.33%, indicating the presence of a significant group of cells who are in the state of apoptosis activation. After 7 days after thermal skin burns and 0.9% NaCl solution, a significantly lower (S<0.01) value of S-phase was observed and a slight tendency (p=0.076) to greater values of SUB-G0G1 interval compared to the group without skin burn (see Table 1). No significant or trending differences for G0G1 and G2+M indicators were detected (see Table 1).
After 14 days after thermal skin burn, significantly lower (p<0.05) values of the number of cells in the S-phase and higher (p<0.05) values of the level of the DNA fragmentation index in the interval SUB-G0G1 against the background of the introduction of the first seven days of 0.9% NaCl solution and compared to similar indicators in animals without burns (see Table 1). No significant or trending differences were found for G0G1 and G2+M.
At day 21 after thermal skin burn, significantly lower (p<0.05) cell counts in the S-phase were observed against the background of administration of the first seven days of 0.9% NaCl solution and compared with similar indices in animals without burns (see Table 1). No significant or trending differences for G0G1, G2+M, and SUB-G0G1 were identified (see Table 1). 30 days after skin burns, against the background of the introduction of the first seven days 0.9% NaCl solution tended (p=0.060) to lower values of the number of cells in the Sphase, significantly (p<0.01) less than the value of the G0G1 phase and higher (p<0.01) value of the G2+M phase    compared with similar values in animals without burns (see Table 1). No significant or trending differences were observed for the SUB-G0G1 interval (see Table 1).
In the presented DNA histogram (Fig. 3)

Discussion
Analyzing the obtained data of thyroid cell cycle indexes against the background of thermal burns of the skin and infusion of 0.9% NaCl solution and comparing the results with the data of other similar studies [12,16] we can make some generalizations. The most pronounced cell cycle abnormalities were observed 3 days after thermal skin damage, although the first signs of these disorders in the form of a significant decrease in DNA synthesis (p<0.01) were observed after 1 day. However, established changes after 1 day are only the initiation of further damage to the thyroid gland, as changes at the subcellular level precede changes at the tissue and cellular levels, with subsequent disruption of the functioning of the organ. There are several factors that can be explained by the increase in the negative effect of burn injury due to increased toxicity of products, the stress of organ depletion and the potential activation of protective mechanisms, which is known to be often observed in burn skin damage [1,11]. The thyroid gland in this case is included in the systemic damage of the organs of the endocrine system against the background of burn injury, which is manifested both functionally and at the cellular and subcellular levels and has been established in many studies [16,18].
Perhaps the changes we recorded in the first days of observation appear due to the complex effects of burn toxins, the imbalance of hormonal regulation at the level of the hypothalamic-pituitary system, and the launch of a protective mechanism to inhibit damage, which are wellknown factors of protection in critical states [5,16]. However, our further observations in the following terms indicate insufficient protective effect and deepening of the violation of the cell cycle of the thyroid cells using 0.9% NaCl solution.
As is known [10], changes at the subcellular level precede changes at the cellular and histological levels, which corresponds to the hypofunctional state of the organ. Therefore, we can consider our indicators more sensitive marker of damage to the thyroid gland than morphological studies, which can potentially indicate the further development of deep morpho-functional damage to the gland, which is characteristic when using DNA cytometry [2,12]. It should also be noted that these disorders were observed even in the long term of our study, which can affect the regeneration processes not only in the gland itself, but also in skin cells, the regeneration of which is controlled by the level of thyroid hormones [4,15]. That is why damage to the thyroid gland against the background of burn injury of the skin is an important element of pathogenesis and requires effective correction [8,14]. We can note the lack of effectiveness of using 0.9% NaCl solution to correct cell division disorders throughout the observation period.
The prospect of further research is to study the effect of other infusion solutions on thyroid cell cycle indices against the background of thermal skin burns.

Conclusions
1. 1 day after thermal skin damage and use of 0.9% NaCl solution, lower (p<0.05) values of the S-phase indicator were found compared to the control group of rats (0.9% NaCl solution without burn), reflecting a significant disturbance of the thyroid cell cycle.
2. The maximum decrease (p<0.01) of S-phase indicators and a significant increase (p<0.01) of the SUB-G0G1 interval compared to the same control group was observed after 3 days. The S-phase indicators against the background of the introduction of 0.9% NaCl solution and thermal skin burn remained significantly lower than those of the similar control groups at 7 (p<0.01), 14 (p<0.05) and 21 days (p<0.05). At 14 days after thermal skin injury, the SUB-G0G1 interval (p<0.05) was lower than in the control group of rats.
3. After 30 days, the G0G1 phase indicators were significantly lower (p<0.01) and the G2+M phase indicators were significantly (p<0.01) higher than those established in the control group at the same time.