Indian Journal of Animal Research

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Research Article

Indian Journal of Animal Research, volume 54 issue 2 (february 2020) : 155-159

Investigation of Therapeutic Effects of Thermal Waters of Afyonkarahisar Region in Stress-Induced Mice

Bülent Elitok1,*
1Afyon Kocatepe University, Faculty of Veterinary Medicine, Department of Internal Medicine-03200, Afyonkarahisar/Türkiye.
Cite article:- Elitok Bülent (2020). Investigation of Therapeutic Effects of Thermal Waters of Afyonkarahisar Region in Stress-Induced Mice . Indian Journal of Animal Research. 54(2): 155-159. doi: 10.18805/ijar.B-1187.

ABSTRACT

In this study, 20 Albino mice of the same daily age were used. All the  animals were restrained in well-ventilated 50 ml tubes (plus 3 or 4 cm long  middle tubes), which were slipped over the tail to restrict movement even more and left undisturbed under an opaque duration of the stress (30 min). After stress procedure was applied in all animals, 20 mice which have same alive weight average and constitute the study material were divided into two groups as control group (CG) (n=10) and study groups (SG) (n=10) for a 21-day treatment period equally and randomly. Normal tap water was added to the CG mice’ drinkers, while the hot spring water that was brought from the source each day as fresh added to SG mice’ drinkers and they were allowed to reach ad libitum. Clinical, hematological and blood biochemical parameters were measures on 1, 7, 14 and 21 days after treatment  in all the animals. The results of this study showed that cortisol and glucose levels decreased in stressed mice treated with Süreyya I Hot Spring Water, while ALB, TP, T3 and T4 levels increased and these changes were statistically significant (p<0.06) when compared to the control group. 

INTRODUCTION

Stress - often called the “disease of the century”- is a topic of interest among researchers to evaluate the participation of the central nervous system and the entire body (Quelhas Martins et al., 2015). Endocrine response to stress exposure is characterized by increased ACTH and cortisol secretion (Magiaku 2006; Whitworth 2005; Folkman 2013). Cortisol is a glucocorticoid hormone released by adrenal glands in response to different stimuli and the levels of salivary cortisol reflect the concentration of free cortisol in the blood (Dunbar et al., 2010; Ranabir and Reetu 2011; Sarkar et al., 2013; Elitok and Kilic, 2019).
       
Balneotherapy traditionally refers to using thermal mineral waters (also mud/peloid) in different combinations (Fioravanti et al., 2017; Karagülle et al., 2018). This therapy method is a clinically effective complementary approach in the treatment of stress-related pathologies, because it is leading to antiinflammatory, analgesic, antioxidant, chondroprotective and anabolic effects together with neuroendocrineimmune regulation (Kerdes et al., 2017; Galvez et al., 2018).
       
The aim of this experimental study was to determined whether mineral rich-Süreyya I Hot Spring Water effectivness on the treatment of stress.

MATERIALS AND METHODS

In this study, 20 Albino mice of the same daily age were used. The animals were kept in plastic cages in a stable environment with equal humidity and heat conditions for 12 hours night and 12 hours day at Afyon Kocatepe University Experimental Animals Application and Research Center. During the study, animals were allowed to receive ad libitum mouse feed. Mice were given Tetracosactide (Synacthen, Ciba-Geigy) in saline 2.8 μg/day/5 days and stress effect was induced (Denton et al., 1999.). Moreover, all the  animals were restrained in well-ventilated 50 ml tubes (with holes of 3-4 cm intervals on it for the animal to easily breathe, Fig 1) and left undisturbed under an opaque box 30 minutes duration of the stress (Kim and Han, 2006; Zimprich et al., 2014). After stress procedure, 20 mice were divided into two groups as control group (CG) (n=10) and study groups (SG) (n=10) for a 21-day treatment period equally and randomly. Normal tap water was added to the CG mice’ drinkers, while the hot spring water that was brought from the source each day as fresh added to SG mice’ drinkers and they were allowed to reach ad libitum. CG mice were bathed in the 35±2°C tap water as same hour every day, while SG mice were bathed in the same temperature with fresh hot spring water for 15 minutes. Blood samples were collected from the cephalocele vein via the method described by Hem et al., (1998)  in all of the CG and SG group animals.
       

Fig 1: Stress tubes for mouse.


 
Experimental part of this study was made in Experimental Animals Application and Research Center of Afyon Kocatepe University and conducted in accordance with Afyon Kocatepe University Experimental Animals Ethics Committee Instructions (AKUEAECI) under the report with reference number 42-18 and was supported by Afyon Kocatepe University Scientific Research Projects Committee (SRPC) under the number 18.KARIYER.238.
 
Characteristics of Süreyya I hot spring
 
In this study, the possible therapeutic effects of hot spring water on DM were obtained from Süreyya I spring in center of Ýscehisar District of Afyonkarahisar Province. Süreyya I Hot Spring Water which is volcanic spring and has the property of being the only carbon dioxide water of the region, has been reported as sodium bicarbonate, carbon dioxide, fluoride and silicon thermomineral water class by Ýstanbul University Çapa Medical Faculty Department of Medical Ecology and Hydroclimatology and mineralization in total has been reported as 4046.8 g/L by Ýzmir Community Health Laboratory. Although not specified in this report, it is also accepted in calcium water class since its calcium content is> 150 mg/L (Quattrini et al., 2017).
 
Clinical examination
 
The body weight (T), mobilization, feed and water consumption, whether they developed lesions of mice were examined and body temperatures, heart (P) and respiratory frequencies (R) of the animals were measured at the determined measurement times.
 
Hematological examinations
 
Hematological parameters such as Erythrocyte (RBC), total leukocytes (WBC), hematocrit (HCT), hemoglobin (HB), mean corpusculer volume (MCV), mean hemoglobin concentratiion (MCH) and mean corpusculer hemoglobin concentration (MCHC) were determined using by commercial test kits and Chemray Brand blood counting device.
 
Blood biochemical examinations
 
Cortisol (CORT), triiodothyronine (T3) and thyroxine (T4) measurements in blood biochemical examinations were done  by ELISA reader device (ChemWell Chromate 4300 Elisa Reader, Awareness Technology, Inc. Martin Hwy. Palm City, USA) by using Elisa kits (Sunred Biological Technology Company Co., Shangai/China) Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Total Protein (TP), Albumin (ALB), Glucose (GLU) levels were measured on the Cobas Integra 400 Plus Roche Brand (Roche Diagnostics GmbH, Germany) Analyzer.
 
Statistical analysis
 
Statistical analyzes of the groups were made according to ANOVA method. Duncan test was used to determine the importance of intragroup differences in the study group. Statistical analyzes were performed using Windows compatible SPSS 18.1 (Inc., Chicago, II, USA) software. Data were expressed as mean ± standard error and p <0.05 was considered significant.

RESULTS AND DISCUSSION

In this study, sex was not considered a factor in the statistical analysis of the data.
 
Clinical findings
 
Clinical findings of animals are shown in Table 1. There was no statistically significant difference in terms of T in the measured time periods (p>0.05), when compared with the pre-study, it was found that P and R frequencies showed significant (p<0.05) differences in terms of average after stress formation and it was found that these averages were significantly higher in SG animals (p<0.05) compared to CG animals during the treatment process.
 

Table 1: Statistical comparison of body temperature, pulse and respiratory in the animals.


 
Hematological examination
 
It was observed that WBC, MCV, MCH and MCHC levels increased significantly after stress formation (p<0.05), whereas RBC, HG and HCT levels were significantly (p<0.05) decreased. In the post-treatment comparisons, WBC, MCV, MCH and MCHC averages in SG animals were lower (p<0.05) and RBC, HG and HCT levels were higher (p<0.05) Table 2.
 

Table 2: Hematological findings of the animals.


 
Blood Biochemical Examination
 
The averages of blood biochemical analysis findings are shown in Table 3. It was observed that AST, ALT, GLU and CORT levels increased significantly (p<0.05) after stress induction and TP, ALB, T3 and T4 levels decreased significantly (p<0.05). During treatment period, it was observed that these changes reversed, in terms of increasing and decreasing parameters and these changes were found statistically more important (p<0.05)  in SG animals compare to the CG animals.
 

Table 3: Blood biochemical findings of the animals.

  
 
Findings  obtained lower WBC levels in the CG animals were consistent as observed by  Terui et al., (1980), which reported increased glucocorticoid secretion suppresses WBC levels. It was also reported that the animals treated with ACTH had high RBC levels and as a result of that HCT and HB levels increased by activation of erythropoiesis due to decreased oxygen. This adaptive response relates to  numbers of erythrocytes and higher levels of circulating HB and HCT for higher metabolism (Costa et al., 1985; Thompson et al., 1987). RBC, HB and HCT levels measured in our study were also higher in SG after treatment and our those findings were consistent with the findings of the aforementioned researchers (Costa et al., 1985; Thompson et al., 1987).
       
When compared to the SG animals, higher ALT and AST levels in the CG animal were detected in all the time periods after treatment. Findings are consistent , corroborate with the findings of other researchers, who reported that the transaminase levels increased more than two-fold in the animals under stress (Cwynar et al., 2014).
       
Cortisol can be considered as a sensitive and reliable stress biomarker, even though patient-specific characteristics such as age, gender and individual differences in the daily pattern of secretion of this hormone should be taken into account to better assess the effects of stressful stimuli (Van Cauter et al.,1996; Hellhammer et al., 2009; Antonelli and Donelli, 2018). Corticosterone concentrations in pregnant lizards increased more than seven-fold over basal levels following injection of ACTH. (Preest et al., 2005). Formun Altý Tetracosactide is a synthetic peptide analogue which consists of the active N-terminal amino acids 1–24 of the ACTH molecule (Waller and Sampson, 2018). These researchers claim that cortisol levels increased 9-fold in animals with tetracostacid. Our these findings were consistent with the results of the studies (Calamari et al., 2007; Reddy et al., 2018) reported that hypercalamo-pituitar-adrenal axis stimulation in the cases under physical and physiological stress resulted in hypercortisolemia causing an increase in glucose production in the liver and increased blood glucose levels. After stress f, there it was decrese in the levels of TP and ALB in all the animals. Some researchers (Hassan and Roussel, 1975) attributed the reason related to  breaking down of proteins by triggering gluconeogenesis and leads to a decrease in blood TP and ALB levels. CG animals had lower levels of TP and ALB compared to the animals in SG, as data support.
       
In our study, the most lower levels of T3 and T4 were determinerdfollowing stress in all the animals. When groups seperated and begun to treatment with differnet agents, it was clearly shown that T3 and T4 levels continue to rise in both groups, but the most higher levels were determined in the animals treated with Sureyya I Hot Spring Water.  Stress inhibits the secretion of thyroid stimulating hormone (TSH) (Goldstein, 1987; Helmreich et al., 2005) and thyroid hormone by the action of glucocorticoids on the central nervous system (Ranabir and Retu, 2011; Gupta et al., 2019). Our findings obtained in this study deal with T3 and T4 were consistent with the findings of the study (Colavlta et al., 1983) on the goat breeds.
       
The findings obtained at the end of this study were evaluated as a whole; it was observed that cortisol and glucose levels decreased in stressed mice treated with Süreyya I Hot Spring Water, while ALB, TP, T3 and T4 levels increased, and these changes were statistically significant (p<0.06) when compared to the control group.

CONCLUSION

It has been concluded that the Süreyya I Hot Spring Water reduce stress in experimental animals.

ACKNOWLEDGEMENT

Special thanks to Mr. Suayp Demirel for his supporting during study.

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