Abstract: In order to study whether social isolation can cause anxiety in female rodents, adult female rats were separately isolated (single-raised) and four one-cage groups, and behavioral and physiological tests were performed after ten weeks. The behavioral results showed that compared with the group group, the number of uprights in the open field rats decreased, the dark box residence time in the light and dark box test increased, and the number of crossings decreased; indicating that long-term social isolation caused anxiety in female rats. Although there was no difference in the weight of physiological organs associated with stress and reproduction in the two groups, the levels of serum cortisol and progesterone in the monoculture group were lower than those in the group, indicating that long-term social isolation resulted in the hypothalamic salivary-adrenal axis and the lower part of the female. Abnormal regulation of the thalamus-pituitary-gonadal axis may adversely affect the reproductive physiology of females.
Key words: community isolation; female rats; anxiety behavior; blood hormones; physiological indicators
Normal social relations are of great significance to the health of biological individuals. Once destroyed, they will have a negative impact on the body. Long-term social isolation is a survival stress for social animals. This stress model provides a good entry point for the study of human mental illness and is widely used in the research of pathogenesis, new drug development and prevention of human mental disorders.
Social isolation can change the morphology, hormone levels, and neurochemical pathways of the brains of social animals, and the animals exhibit abnormal behavior and physiological characteristics. Previous studies have found that long-term social isolation can lead to hyperactivity in rodents, increased aggressive behavior, lack of normal habitual behavior in the new environment, and loss of prepulse inhibition (PPI). However, the impact of social isolation on rodents is constrained by many factors, depending on the sex age, strain, and duration of isolation of the animals being tested. For example, social isolation can make adult female mice (M. musculus) 'adrenal hypertrophy, shortening the estrous cycle, increasing the ovary and uterus of adult female hamsters (Tscheskia triton), but for male rats (Rattus norvegicus), Male mice (Musmusculus) and mature large hamsters had no effect; 3 weeks old male bong Evan rats were isolated for 12 weeks to increase their preference for alcohol, but this phenomenon did not occur in adulthood. Studies have shown that short-term isolation or isolation for 1 h per day for four weeks will result in a significant increase in serum corticosterone in prairie voles; but long-term segregation has no effect on serum corticosterone
Whether social isolation can cause stress and stress response in rodents is closely related to the lifestyle of rodents under natural conditions. Studies have shown that monoculture has a coercive effect on rats, mice and gerbils such as Merionesunguiculatus, causing an anxiety response and is beneficial to the society of the hamsters (Mesocricetus auratus) and large hamsters living alone. The study of the effects of isolation on stress response in rats is still controversial. For example, some studies have found that social isolation causes elevated corticosteroids (Adreno-('.orticotropic Hormone, ACTH) and corticosterone in rat serum. In some studies, it was reduced, and some studies found that the levels of ACTH and corticosterone were not significantly different between group and isolated animals.
In this study, we will use adult female rats as the research object. Under certain conditions, the stress response of females is significantly higher than that of male rats. However, in the past, there were many male rats used in social isolation research, using elevated cross maze (elevated). Plus maze, EPM) open field and light/dark box Three classic anxiety tests to detect long-term social isolation (isolated for 10 weeks) can cause anxiety in female rats; cortisol, Whether the levels of hormones such as progesterone and estradiol are different from those in the group culture group; whether the weight of organs such as stress, reproduction-related adrenal gland, spleen, uterus, ovary, and foreskin gland has changed. This study will help to further deepen human understanding of rodent social isolation anxiety models.
1 Materials and methods
1.1 Animals
Twenty-four adult female Sprague-Dawley (SD) rats of 2 months old, initial body weight (196.3 ^-242. 6 g), were purchased from Beijing Weitong Tooth China Laboratory Animal Technology Co., Ltd. Considering that female SD rats are variants of Rattus norvegicus, they belong to group animals in the wild. Before the experiment, 4 female rats were kept in cages and fed for 1 week after purchase. The smear of vaginal secretions was observed every day to observe the emotions. For changes in the cycle, rats with normal estrous cycles were selected for subsequent experiments. The light-dark cycle of the room in which the rats were raised was 14I, / lOD, the photoperiod was reversed (lights turned off at 08:00 in the morning, lights up at 22:00), and the room temperature was controlled at (22 ± 2) 0C. Whole reality
During the test, adequate food and water were supplied and the food was standard rat feed. At the time of the experiment, we randomly divided 24 female rats into two groups, of which 12 were housed in the same cage as the group raising group and the other 12 single cages were raised as the social isolation group. Standard rat plastic cages (37 cm X 26 cm X 17 cm) were kept for 10 weeks.
To rule out the effects of estrus on behavioral and hormonal outcomes, we only used data from non-emotional rats for statistical analysis. In order to avoid the influence of human factors on the behavior and hormone levels of rats, we examined the vaginal secretion smears after the behavior test and blood samples in rats to determine their estrus status. The vaginal secretions of estrus rats are all non-nuclear keratinocytes or a small number of epithelial cells.
1. 2 behavior test
All behavioral tests were conducted in a quiet, dark room. The test time was 9:30-16:30 (the dark phase of the rat photoperiod). In the behavior test, the order is measured in the order of the elevated cross maze, the open field and the light and dark box, and each mouse is tested only once a day.
1. 2. 1 elevated cross maze elevated cross maze provided by Shanghai Xinsoft Information Technology Co., Ltd., including two open arms (45 cm X 10 cm), two closed arms (45 cm X 10 cm X40 cm) and 10 cm In the central area of ​​X 10 cm, the closed arm and the open arm intersect in a cross shape, and the entire labyrinth is 74 cm from the ground. The tested rats were acclimated for 30 min in the test room before the test, and the test had normal lighting. The test rats were placed in the central area (head facing the open arm) and allowed to explore freely for 5 min. The activity of each rat in the maze was recorded by the camera, and then the video was observed by the uninformed person, and the number of times the animal entered the open arm and the closed arm (the limbs entered the arm), and was opened separately. The total time spent in the arm and the closed arm.
1.2.2 The market is provided by Shanghai Xinsoft Information Technology Co., Ltd., which is made of plexiglass with a size of 75 cmX 45 cmX and 35 cm (without cover). The floor is divided into 15 15 cm X 15 cm. The grid, the three squares in the middle are called the central grid. At the beginning of the experiment, the mouse was gently placed in the grid at the corner of the field (towards the corner), allowing it to freely detect for 10 min, and the camera recorded all its behavior during this time period. The statistical indicators are as follows: horizontal exercise ability (number of grids crossed), number of uprights (post hind limb standing, front paws vacating or number of times to support the wall), center time (time spent in the center grid), and latency (from Put in the time to leave the corner grid for the first time) and rest time (the limbs stand still).
1.2.3 Light and dark box light and dark box is provided by Shanghai Xinsoft Information Technology Co., Ltd., consisting of two plastic boxes with a size of 35 cm X 25cm X 17 cm, with a tin tube (length 5cm, diameter 5. 5 cm). Connected. One of the boxes was covered with a transparent plexiglass plate, and a 40 W bulb was placed 30 cm above the lid to increase the brightness as a bright box; the other box was sealed with cardboard, and the light was blocked as a black box. During the test, the test mouse is placed in the central area of ​​the bright box, and it is free to explore for 5 minutes in the light box and the black box. The statistical parameters include: the incubation period (the time required from the time of placing to the first time to leave the clear box), the accumulated time in the clear box, the accumulated time in the black box (when all the limbs enter, it is considered to be entered) and between the two boxes. Crossing frequency.
After each animal was tested, the experimenter wiped the labyrinth used (using 75% alcohol first, then water) and dried it with a hair dryer to avoid the residual odor of the previous rat.
The level of anxiety behavior of the rats to be tested.
1. 3 Organ anatomy and determination of blood hormones
After 10 weeks of treatment, at 9:30-10:00 in the morning, the two groups of rats were sacrificed by decapitation, and the trunk blood was collected within 3 minutes. Blood samples were obtained by centrifugation at 4 000 r/min for 4 min at 4 °C. Serum was stored at 200C for determination of cortisol, progesterone and estradiol. Then, the rat's adrenal gland, spleen, ovary, uterus, and foreskin gland were dissected and weighed to the nearest 0. 1 mg. Relative organ weight is calculated by the formula below. Relative organ weight - Absolute organ weight (g) X100 / body weight (g) 0 The hormone content of the serum sample is determined by the method of Iz} 1 radioimmunoassay. The kits used for hormone determination were provided by Beijing Kemei Dongya Biotechnology Co., Ltd. The anti-serum of the human anti-serum is less than 0.0100; the internal and mutual variability is less than 7.4% and 10. 0. %.
1. 4 data analysis
First, a single-sample KS test is used to perform a normality test on the measured data. For normal distribution data, a two-tailed independent sample T test is used; for non-normally distributed data, the Mann Whitney U test is used for statistical analysis. All data were statistically analyzed using SPSS 18. 0, with a significant level of a = 0.05.
2 results
2.1 high-rise labyrinth experiment
Isolated and reared rats did not show significant differences in the behavior of the elevated plus maze test (Table m1)
2. 2 market experiment
Compared with the four caged females, the number of erects in the females was significantly reduced (t=2.543, P=0.023), and there was no significant difference in other behaviors. The residence time of the middle lattice was emotion. The important indicators, although the average value is different, the data is non-normal distribution. The Mann Whitney U test is used for statistical analysis. The results show that there is no significant difference between the group and the monoculture group ( Two = 0.221, P = 0.833) (Table 2)
2. 3 bright box experiment
Compared with the female rats in the group, the time of stay was significantly increased (Ct= 2. 520, the retention time was significantly reduced (Ct-2. 524, the single-raised female rats stopped in the black box P = 0. 047), in the clear box Stop P = 0. 045), the number of crossings between the light and dark boxes is significantly reduced (total crossing frequency: t = 2. 114, P = 0.049), there is no difference in latency (two = 1.629, P = 0.103) (Table 3 ).
2. 4 weight and organ weight changes
There was no significant difference in pre-test initial body weight between monoculture and group female rats ((t=1.290, P=0.210), but isolated feeding
After the first week, the monoculture group showed a significant weight gain compared with the group rats (t=2.570, P=0.023).
The difference continued until the 10th week of isolation (t = 2.900, P = .050) (Figure 1).
However, the relative weights of adrenal gland, spleen, uterus, ovary and foreskin gland between monoculture and group females did not show significant differences. See Table 40.
Our results indicate that long-term social isolation reduces the erect behavior of female rats in the open field. In the light and dark box test, the number of crossings between the light box and the black box is reduced, and the residence time in the black box is increased, indicating that the female rats are Anxiety occurs in long-term social isolation. This is consistent with some of the earlier findings. However, the results of Helleman and Zha et al. showed that social isolation did not cause an anxiety response in rats. The reason for this difference may be due to differences in experimental animal strains, gender differences, and time to perform isolation. For example, Hellemans used male I, Evan rats, and this experiment used female SD strain rats; Hellemans chose Long-evans rats 21 d and 66 d
Isolation was carried out for 3 months. Zhao was isolated in SD rats for 21 days. It was kept in isolation for 8 weeks. This experiment was isolated in adult rats of SD rats. It took 10 weeks. According to previous literature reports, social stress usually reduces the time that animals stay in open arms in an elevated maze. X33 3d} But it is puzzling that in the elevated plus maze test, we did not observe isolated rats and group rats. Differences in behavior. This may be due to the fact that the behavioral parameters in the elevated plus maze are more susceptible to various uncertain test factors, but it may also be more sensitive to the test of the open field and the black and white box in adult SL1 female rats. Need to be further verified.
In terms of body weight, we found that social isolation significantly increased the body weight of female rats in only one week compared to group rats. This may be because isolated rats are eaten more than grouped rats, and studies have shown that even isolated rats that have been fed still show feeding behavior. On the other hand, the increase in body weight (obesity) is also an important indicator for judging social stress in rodents.
In addition to weight gain, changes in stress, reproductive organ weight, and related hormone levels are also relatively straightforward indicators of stress response in animals [?17. However, in our experiments, the relative weights of the adrenal gland, spleen, uterus, ovary, and foreskin of the group and the monoculture did not show differences. This may be because weighing the wet weight of the organ is very susceptible to interference with blood levels and accompanying tissue. Hormone studies have shown that social isolation reduces serum cortisol and progesterone, suggesting that social isolation leads to abnormal regulation of the hypothalamic salivary-adrenal axis and hypothalamic-pituitary Jh} gland axis in females, possibly affecting isolated rats. Physiological functions such as metabolism, reproduction and immunity. Although we found that social isolation caused obvious anxiety behavior in female rats, cortisol decreased significantly. This behavior is inconsistent with hormone levels. It has also appeared in previous studies. For example, high heat stress makes rats anxious. Serum corticosterone levels are decreased. When glucocorticoid levels are normal, rats often have various anxiety behaviors; post-traumatic stress leads to changes in behavior and neurological activity, but cortisol levels in urine Reduce the ratio]. This may be because the results of the anxiety behavior and hormone level tests are highly dependent on the animal's feeding conditions (size of the living space, feed), the test environment, and the chosen test method (the single behavioral method cannot accurately infer the anxiety level of the experimental animals); It may be due to the adaptation of the animal to long-term stress stimuli; in addition, the hormone levels of female rats may be related to the estrous cycle in which they are located.
4 Conclusion
The results of this study indicate that social isolation causes anxiety in female adult rats and causes a decrease in cortisol and progesterone in the blood of isolated rats. The solitary or living lifestyle formed during long-term evolution of animals determines the process of animal domestication. Important factors suitable for breeding conditions, artificial changes in their lifestyle may have an impact on their behavior and physiology. The results of this study will help to further deepen human understanding of rodent social isolation anxiety models. The limitation of this study is that the detection of hormones and behaviors excludes estrus rats, but does not seriously distinguish between other estrus cycles such as estrus, pre-estrus, and late estrus. The impact of social isolation on females in different estrus states needs further study.
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