INTRODUCTION
Recently, complex changes in social organization and behavioral restrictions due to the coronavirus disease pandemic have aggravated various types of stress.[1-4] The incidence of diseases caused by psychogenic stress (PS) has increased in adolescents and teenagers. There are concerns regarding its ill-effects on health, growth, and development during growth spurts.[2,4-6] Stress leads to the loss of homeostasis and decline in the functioning of the endocrine system.[7,8] This is particularly evident in the brain and immune cells. It also negatively impacts various organs during the growth stage. Adrenal enlargement, thymic atrophy, and decreased renal weight and volume have also been reported.[9,10] Kidneys synthesize active vitamin D, and hence, they are important regulatory organs for bone metabolism. A decline in renal function promotes bone resorption owing to hypocalcemia and increased parathyroid hormone (PTH) activity.[11-13] Additionally, changes in steroid hormone levels, secreted from the adrenal cortex, affect the hypothalamic-pituitary-adrenal (HPA) axis and the sympathetic nervous system. This suppresses growth hormone levels, leading to a decrease in bone mass.[14,15]
Herrera-Covarrubias et al. [16] and Araujo et al. [17] demonstrated the stress-induced effects of hormones and suggested a link between stress and the endocrine system. In addition, these hormones are affected by oxidative stress, which fluctuates depending on the in vivo conditions such as stress and immune function.[17-19]
In summary, stress induces oxidative stress and hormonal imbalances related to biological functions. Thus, stress can negatively affect normal growth and biological functions. The peak bone mass is closely related to bone disease later in life. Therefore, investigating the effects of PS on the bones and associated organs during the rapid growth stages is necessary.[20,21]
The present study aimed to examine the effects of PS exposure on oxidative stress at the biochemical level during growth. Additionally, this study sought to determine the morphological effects of different frequencies of PS exposure on bone and organ development, with the objective of gaining insight into healthy growth and development.
METHODS
The animal experiments were conducted in accordance with the “Guidelines related to Animal Experimentation at the Aomori University of Health and Welfare (Approval no. 19001).”
1. Experimental animals
Four-week-old male Wistar rats (CLEA Japan, Inc., Tokyo, Japan) were randomly assigned to the control (C, N=6) and PS groups. To further examine the effects of PS frequency, rats were classified into the following three groups: PS once every 4 days (PS-I, N=6), PS once every 2 days (PS-II, N=6), and daily PS (PS-III, N=6). Throughout the rearing period, the room temperature was maintained at 22±2°C, humidity at 55±5%, and darkness maintained from 7 PM to 7 AM. During the rearing period, the experimental animals were fed on CE-2 (CLEA Japan, Inc.), and tap water was freely provided.
2. Protocol of PS exposure
PS was administered using the restraint and water-immersion stress (RWIS) methods, as previously described by Sakamoto et al. [22] and Ohta et al. [23] PS was performed at the same room temperature and humidity conditions as in the usual animal care environment. PS was performed for 4 weeks, once daily for 3 hr. RWIS used small animal fixation adjusters with adjustable lengths to account for individual differences in body size. Rats were placed in the “rat fixation adjuster” that adjusts to two sizes of experimental rats (small, Φ31×20-80 mm; medium, Φ60×60-170 mm). The intake port at the front of the fixation adjuster was moved to the tail to the maximum setting, and the rats were restrained. Afterwards, the restrained rats were transferred to a water tank and submerged in 2 cm of water level.
3. Body weight and body length
Body weights were measured at the same time before the experiment and every week thereafter. On the final day of the experiment, three types of mixed anesthetics were injected intraperitoneally, and body and tail lengths were measured. The body was placed in the dorsal position and measured from the end of the snout to the anterior margin of the anus. The tail length was measured from the posterior margin of the anus to the tip of the tail.
4. Blood collection and anesthesia
Blood sample was collected from the caudal vein of the rats immediately before the start of the experiment and 4 week later after the end of the experiment. The experimental animals were immobilized using a rat fixation adjuster both before and after the experiment, and 1 mL of blood was collected from the tail vein, respectively. Blood was collected from the tail vein both before and after the experiment and centrifuged at 3,000 rpm for 10 min. Anesthesia was prepared with 25 mL of three types of mixed anesthetic agents (Medetomidine Hydrochloride 1.875 mL+Midazolam 2 mL+Butorphanol Tartrate 2.5 mL+ Normal Saline 18.625 mL) and administered intraperitoneally (0.5 mL/BW100 g) after blood collection at the end of the experiment.
5. Biochemistry
The sampled blood was separated into plasma and blood cells. The oxidative stress and antioxidative capacity were then measured using Free Radical Analytical System 4 (FRAS4; H&D srl, Parma, Italy).[24,25] We referred to our previous studies for the explanation and methodologies utilized for the reactive oxygen metabolites-derived compounds (d-ROMs) and biological antioxidant potential (BAP) measurements.[24]
1) Oxidant stress
To measure d-ROMs, 10 μL of thawed plasma was placed into an Eppendorf tube containing acetate buffer with pH 4.8 and mixed by inversion. The mixture was then transferred to a cuvette containing dry chromogen and mixed by inversion. The new mixture was subsequently separated by centrifugation at a constant temperature of 37°C for 60 sec, placed into a photometer, and exposed to light at a wavelength of 505 nm for 5 min to determine the color change.
2) Antioxidant capacity
BAP measurement is a method for evaluating the reduction power of reducing substances in plasma. In total, 50 μL of iron (III) chromogen was mixed into a thiocyanate solution, producing a red color. The solution was then placed in a 505 nm photometer, and the ferric ion concentration was measured for 3 sec. Subsequently, 10 μL of plasma was mixed into this solution, and the color change was measured for 5 min at a constant temperature of 37°C. In addition, oxidation stress index (OSI) (BAP/d-ROMs; relative tolerance to oxidative stress) was calculated using BAP and d-ROMs.
6. Morphology
1) Organs
Subsequently, the chest was opened under anesthesia, and the animals were sacrificed by perfusion with saline solution through the left ventricle. The thymus, heart, spleen, liver, right and left adrenal glands, and kidneys were removed. The removed organs were immersed in 10% neutral buffered formalin solution and stored until analysis. For analysis, they were washed in saline, trimmed, and wiped dry before wet weight was determined. The organs were then oven-dried at 110°C for 24 hr (NDO-420; Tokyo Rikakikai Co., Ltd., Tokyo, Japan), and the dry weight was measured.[21]
2) Bones
The femur and tibia were wet-weighed after removing the muscle tissue and ligaments around bones and the water from bones surface. After measuring the wet weight of the bone, the maximum bone length (50% of the maximum bone length) was measured using an ABS Digimatic Caliper CD-20APX (Mitutoyo Co., Kanagawa, Japan) in 0.1 mm increments. The bone center was then cut vertically, and bone cross-sections were photographed using a digital camera and stored on a computer until further analysis. The photographed bones were oven-dried at 110°C for 24 hr (NDO-420; Tokyo Rikakikai Co., Ltd.), similar to that performed for the organs. The dry weights of bones were determined.[22]
7. Image analysis
The total cross-sectional area, dense bone area, and circumference were measured using WinROOF (Mitani Co., Tokyo, Japan), a general-purpose image-processing package for Windows, to analyze the cross-sectional areas of the central bone.
8. Statistical analyses
The mean and standard deviation were calculated using IBM SPSS Statistics version 27 software (SPSS Inc., Chicago, IL, USA), and relevant t-tests were employed to compare results obtained before and after the experiments for each group. One-way analysis of variance was used for comparisons among the four groups, followed by Scheffe’s test for multiple comparisons. Pearson’s correlation was employed to ascertain the relationship between oxidative stress on organs and bone morphology. The statistical significance level was set at a P value less than 0.05.
RESULTS
1. Body weight and length
Prior to the commencement of the experiment, no significant differences were observed in body weights among the four groups. However, a significant difference was observed from the second week of the experiment (P<0.01). Subsequently, significant differences were observed among the groups at the third week and at the end of the experiment (P<0.001 for each comparison). In comparison to the pre-experimental weight, the final weights of groups C, PS-I, PS-II, and PS-III exhibited a significant increase of 319.9, 262.5, 239.7, and 214.6%, respectively (all P<0.001) (Fig. 1).
Significant differences were observed in the total length and body length among the four groups at the end of the experiment (P<0.001). The total length and body length of group C were significantly lower than those of the other groups (all P<0.001) (Table 1).
2. Organs
1) Thymus
Compared to the wet and dry weights of the thymus gland in group C, those in the PS-III group significantly decreased by 27.8% and 23.5%, respectively (all P<0.05) (Table 2).
2) Spleen
The spleen wet weights were significantly lower in groups PS-II and PS-III than in group C by 27.9% and 35.6%, respectively (all P<0.001). Compared to the spleen wet weight in the PS-I group, those in the PS-II and PS-III groups were significantly decreased by 19.3% (P<0.05) and 27.9% (P<0.01), respectively. In contrast, the spleen dry weight was significantly lower in the PS-II and PS-III groups than in group C by 28.0% and 33.4%, respectively (all P<0.001). It was significantly lower in the PS-III group than in the PS-I group by 24.5% (P<0.01) (Table 2).
3) Adrenal gland
No significant differences were observed in the wet and dry weights of the right and left adrenal glands among the groups (Table 2).
4) Kidney
The wet weight of the right kidney was significantly lower in the PS-I and PS-II groups than in group C by 15.9% and 28.7%, respectively (all P<0.001). The wet weights of the right kidney in the PS-I and PS-II groups were significantly higher than in the PS-III group by 15.2% (all P< 0.01). Dry weights of the right kidney was lower by 15.0% (P<0.01) and 19.5% (P<0.001) in the PS-II and PS-III groups, respectively, than in group C. The PS-III group had a significantly lower dry weight (5.3%; P<0.01) than the PS-I group.
The wet weight of the left kidney was significantly lower in the PS-III group than in group C by 20.8% (P<0.01). In contrast, the dry weights of the left kidney were significantly lower in the PS-II and PS-III groups than in group C by 15.9% and 17.1%, respectively (all P<0.001). It was significantly lower in the PS-III group than in the PS-I group by 5.3% (P<0.05) (Table 2).
5) Heart
The wet weight of the heart was significantly lower by 11.9% in the PS-III group than in group C (P<0.05) (Table 2).
6) Liver
The wet weights of the liver were significantly lower in the PS-I, PS-II, and PS-III groups than in group C by 14.2%, 19.8%, and 25.9%, respectively (all P<0.001). It was significantly lower in the PS-III group than in the PS-I group by 13.7% (P<0.01). In contrast, the dry weights of the liver were significantly lower in the PS-I, PS-II, and PS-III groups than in group C by 18.0%, 22.2%, and 28.0%, respectively (all P<0.001). It was significantly lower in the PS-III group than in the PS-I group by 12.2% (P<0.05) (Table 2).
7) Bones
(1) Weight
The wet weights of the femurs were significantly lower in the PS-I group 7.5% (P<0.05), PS-II group 11.8% (P< 0.001), and PS-III group 17.9% (P<0.001) than in group C. The PS-III group had a significantly lower wet weight (11.2%; P<0.001) than the PS-I group. Moreover, the wet weights of tibias were 11.1% (P<0.01) and 16.5% (P< 0.001) lower in the PS-II and PS-III groups, respectively, than in group C (Fig. 2).
The dry weights of femurs were significantly lower in the PS-I, PS-II, and PS-III groups than in group C by 10.7%, 12.9%, and 18.5%, respectively (P<0.001). The femur dry weight in the PS-III group was significantly lower than that in the PS-I group by 8.6% (P<0.05). In contrast, the tibia dry weight was significantly lower in the PS-1 group by 8.6% (P<0.01), PS-II group by 10.6% (P<0.001), and PS-III group by 15.7% (P<0.001) than in group C. The tibia dry weight was significantly lower in the PS-III group than in the PS-I group (7.8%; P<0.05) (Fig. 2).
(2) Length
Femurs were significantly shorter in the PS-I, PS-II, and PS-III groups than in group C by 4.5%, 5.9%, and 7.0%, respectively (all P<0.001). The femur was significantly shorter in the PS-III group than in the PS-I group by 2.9% (P< 0.01). In addition, the tibias were significantly shorter in the PS-I, PS-II, and PS-III groups than in group C by 2.6% (P< 0.01), 4.3% (P<0.001), and 7.2% (P<0.001), respectively. Tibias were significantly longer in PS-I and PS-II than in PS-III by 4.6% and 3.0%, respectively (all P<0.001) (Fig. 3).
(3) Circumference
The PS-III group had a significantly lower bone circumference of the femur than group C by 8.5% (P<0.01) (Fig. 3).
(4) Cross-sectional area
The total cross-sectional area of the central half of the femur was significantly lower in the PS-III group than in groups C (12.6%) and PS-I (11.1%, P<0.05). The cross-sectional area of the dense bone was significantly lower in the PS-III group than in group C (20.8%, P<0.01) (Fig. 3).
3. Oxidative stress and antioxidant capacity
No significant difference was observed in the pre-experimental d-ROMs, BAP, and OSI among the four groups. A post-experimental comparison among the four groups revealed that d-ROMs were significantly lower in the C Group than in the PS-III Group, (33.7%, P<0.001), PS-I Group (32.1%, P<0.001), and PS-II Group (21.9%, P<0.001). Additionally, BAP was significantly lower (13.3%, P<0.01) in the C Group than in the PS-III Group. However, the post-experimental OSI was significantly lower in group C (24.6%, P<0.05) and group PS-I (28.1%, P<0.01) than in the PS-III (Fig. 4).
4. Correlation between oxidative stress and organ weight
The correlations between the wet weight of organs and the OSI d-ROM were all significantly negative (all P<0.05). The correlation coefficients were as follows: r=−0.517 for the thymus, r=−0.608 for the spleen, r=−0.715 for the right kidney, r=−0.441 for the left kidney, and r=−0.582 for the liver. The right kidney demonstrated a significant negative correlation with the OSI for BAP (r=−0.471), as did the liver (r=−0.567). In contrast, the left adrenal showed a positive correlation (r=0.417) (all P<0.05). In addition, the thymus, spleen, and right kidney demonstrated significant negative correlations with OSI, with r values of −0.431, −0.455, and −0.572, respectively (all P<0.05) (Table 3). Conversely, among the dry weights of the organs, the thymus exhibited a negative correlation of r=−0.480, the spleen r=−0.573, the right kidney r=−0.663, the left kidney r=−0.526, and the liver r=−0.584 for d-ROM (all P<0.05). For BAP, the spleen exhibited a negative correlation of r=−0.478, and the liver exhibited a negative correlation of r=0.533. The left adrenal gland exhibited a positive correlation of r= 0.423 (all P<0.05). Moreover, for OSI, the thymus exhibited a negative correlation of r=−0.424, the spleen r=−0.423, the right kidney r=−0.576, the left kidney r=−0.417, and the liver r=−0.414 (all P<0.05) (Table 3).
5. Correlation between oxidative stress and bone morphology
The femur exhibited a significant negative correlation with d-ROM for wet weight (r=−0.760), dry weight (r= −0.622), bone length (r=−0.611), circumferential length (r= −0.546), bone cross-sectional area (r=−0.567), and dense bone area (r=−0.658). The results were statistically significant at the P value less than 0.01 level for each variable. The correlation coefficients were as follows: −0.458 for wet weight, r=−0.469 for dry weight, r=−0.528 for bone length, r=−0.414 for perimeter, and r=−0.453 for dense bone area. All of these correlations were negative and statistically significant at the P value less than 0.05 level for each variable. For OSI, the correlations between the variables were as follows: wet weight (r=−0.615), dry weight (r=−0.517), bone length (r=−0.448), circumferential length (r=−0.423), transverse bone area (r=−0.468), and dense bone area (r= −0.515). All of these correlations were significant at the 0.05 level. Conversely, significant positive correlations of r= 0.715 to 0.938 were observed between bone morphology items for wet and dry weight of the femur (all P< 0.001) (Table 4).
The tibia exhibited a significant negative correlation of r=−0.720 for wet weight, r=−0.676 for dry weight, and r= −0.763 for bone length for d-ROM (all P<0.001), as well as a significant negative correlation of r=−0.411 for wet weight and r=−0.543 for bone length for BAP (all P<0.05). Furthermore, for OSI, significant negative correlations were observed for wet weight, dry weight, and bone length, with correlation coefficients of r=−0.598, r=−0.559, and r=−0.598, respectively (all P<0.01). Conversely, the bone morphology items demonstrated significant positive correlations between r=0.507 and 0.930 for the wet and dry weight of the tibia (all P<0.05) (Table 4).
DISCUSSION
In this study, we used growing male Wistar rats to investigate the effects of the presence and frequency of PS exposure on oxidative stress. Furthermore, the effects of PS-induced changes in oxidative stress on organ and bone development were examined from a morphological perspective.
The rats showed a rapid increase in body weight and organ development during the early growth stages. However, the growth slows after physical maturation.[26,27] The increase in body weight in the control group observed in this study corroborates the findings of previous studies. However, this increase was not observed in PS-treated groups. Sekine et al. [28] reported that long-term PS in adult rats reduces body weight by 10 to 20 g. Additionally, Flak et al. [29] and Saegusa et al. [30] suggested that this phenomenon is associated with appetite loss due to stress burden. Although the findings of the present study corroborate those of previous studies,[26-30] suppression in body weight increase was greater in our study than in those reported earlier, and it was affected by PS frequency. Thus, the results suggest that the presence or frequency of PS used in this study affects growth in all cases, and that the frequency of PS has a significant effect on the degree of inhibition in development.
Du Ruisseau et al. [31] reported that adult rats subjected to PS for 8 hr per day for 15 days demonstrate thymic atrophy and adrenal enlargement. Therefore, PS induces hormonal changes in various organs. Kapitonova et al. [32] reported that PS causes thymic atrophy during growth. Thymic and hepatic atrophy due to PS was also observed in the present study. This finding corresponds with the results of previous studies;[27,32] however, no significant adrenal enlargement due to PS was observed. Based on the changes observed in body weight and organs (except for the adrenal gland), the present results validate the biological response to PS. The adrenal glands secrete numerous hormones, such as those secreted during growth spurts, that are critical for maintaining biological functions.[33] Hence, the adrenal glands play a critical role in growth by maintaining the homeostasis of the endocrine system. PS-related oxidative stress may affect hepatic function; therefore, consideration of the biological responses to PS is necessary.[9,10,34,35] Rats show rapid growth from the growth stage until maturity, and increase in the weight of various organs manifests as an increase in body weight.[26] The body and organ weights observed in the present study suggest that the presence and frequency of PS are key factors in suppressing development during the growth stage. Therefore, PS may have a greater effect on rats during the growth stage, when the organs and functions are immature, than on rats that have reached maturity.
Foertsch et al. [36] exposed seven-week-old mice to PS for 19 days and observed that PS suppresses bone growth. Cells at the bone growth sites showed an increase in tyrosine hydroxylase expression. This finding suggests that the effects of PS on local adrenaline signal transmission adversely affect the endochondral ossification. Sakai et al. [37] reported that PS can reduce the rate of increase in bone mineral density in six-week-old rats. PS, also, markedly suppressed the growth of the femur and tibia compared to the control group. These findings are comparable to the PS and biological results presented in previous studies.[36,37] In particular, PS caused atrophy of the femur, as demonstrated by measuring bone circumference, cross-sectional area, and compact bone area at a position exactly halfway along the length of the femur. The measurements clearly showed that the PS frequency had a major effect on bone growth during the growth stage. Stress results in a decline in renal function and morphology,[13,34,35] which are associated with bone disease Furthermore, stress-induced PTH overproduction results in aberrant calcium metabolism,[38] and organ growth is highly correlated with bone growth.
In the present study, the PS group showed markedly lower growth rates in all organs compared to the control group. In summary, PS-related growth suppression in various organs influences bone morphology. Bone develops rapidly during growth. However, after attaining peak bone mass in youth, bone mass gradually decreases with age. Thus, bone growth is crucial. The results of the present study on the bones of the subjects were more striking than those of previous studies. The biological responses and physical changes differ between growth and adulthood, with more pronounced changes occurring during the growth phase.[26-30] The definition of the growth phase used in the growth phase experiments is broad, and one-two weeks can result in significant physiological changes in laboratory animals. Furthermore, the type of experimental animals and the intensity of PS were considered as possible reasons for the remarkable results of our experiments. The experimental animals used in this study were “weanling rats,” which are extremely developed during the growth phase. In other words, even during the growth phase, the biological responses and developmental status differ depending on age, which may be reflected in changes in bone mass and different levels of biological responses to PS. Animals showed a decrease in metabolic levels when subjected to stress, as shown by a decrease in the expression levels of enzymes in DNA-synthesizing and energy-generating systems.[39] Therefore, the suppression of all-organ development due to PS exposure in this study was thought to affect bone morphology.
Stress affects the thymus, adrenal glands, and hypothalamus, which are involved in hormone-secretory functions,[40] while hormones affect oxidative stress. Reactive oxygen species (ROS) contribute to immune responses against bacteria and foreign substances that invade the body.[41] However, excessive ROS production and accumulation in vivo leads to increased oxidative stress and oxidative damage to DNA and even normal cells.[41] In vivo oxidative stress is regulated by antioxidants, such as superoxide dismutase and glutathione peroxidase, which are influenced by hormones and other factors.[42] In this study, oxidative stress was significantly increased in the high-frequency PS exposure group compared to the control and low-frequency PS exposure groups, and the response of antioxidant capacity to oxidative stress was significantly increased in the high-frequency PS exposure group. Furthermore, only the left adrenal gland showed a positive correlation with oxidative stress, whereas all other organs showed negative correlations. In addition, all parameters negatively correlated with bone morphology in response to oxidative stress. This suggests that high-frequency PS exposure induces excessive oxidative stress in vivo and has a pronounced effect on the growth of organs, bones, and other organisms.
In this investigation, oxidative stress was significantly higher in the daily high-frequency PS group than in the control and low-frequency PS groups. Antioxidant capacity responded to an increase in oxidative stress. Daily PS exposure induced excessive oxidative stress in vivo, affecting the growth of organs, bones, and other organisms. Bone metabolism, which plays a central role in bone morphology, is constantly regulated by the balance between bone formation and resorption. In particular, bone metabolism is affected by in vivo factors, such as hormones and oxidative stress.[43,44] Stress increases the plasma corticosterone and estradiol levels,[45,46] which are factors that affect bone metabolism. Furthermore, bone-related factors act synergistically or conjugatively in response to hormonal secretion and other internal conditions, forming a complex mechanism for bone metabolism.[47] These findings suggest that, during growth, bones are affected by organ development and oxidative stress conditions, which contribute to bone metabolism. Thus, PS in immature organisms increases oxidative stress, which is reflected in the PTH and HPA system functions during organ development, and a high frequency of PS leads to bone undergrowth and atrophy. These findings suggest that bone is susceptible to organ development and oxidative stress during growth, which affects the body’s internal environment and contributes to its effects on bone metabolism.
In this study, we investigated the morphological changes caused by PS exposure on organ and bone development during growth. We found increased oxidative stress associated with the frequency of PS exposure, suggesting a link between oxidative stress and growth. Thus, the suppression of PS exposure or PS-induced oxidative stress production during growth is important for healthy growth.
Increased oxidative stress with PS in the growth stage has negative effects on organ and bone development, and high frequencies of PS have a greater inhibitory effect on organisms. In other words, removal or suppression of the frequency of PS is important for the healthy development of immature organisms.
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