Persistent stress arousal disorder in rats induced by predation stress

Summary

OBJECTIVE: To investigate whether severe psychological stress can trigger emotional arousal disorder in experimental rats for a long time to establish an ideal animal model of post-traumatic stress disorder.

METHODS: The experiment was performed at the Chengdu General Hospital of the Chengdu Military Region of the People's Liberation Army from 2004-01 to 2005-06. A total of 135 male Wistar rats were randomly divided into a predation-stress group of 75 and a normal control group of 60. The rats were placed in a small cage of a predator stress test chamber, and then a starved cat was placed in the experimental box, so that the cat and the rat had no physiologically harmful contact, resulting in mild panic tremor and Stunning caused by shortness of breath and rapid nasal agitation for 6 to 8 minutes; normal control rats except for no contact with cats, the other treatment procedures are the same. The rats' motor arousal level was measured within 4 months after stress by the motor activity, inquiry behavior, anti-capture reactivity and elevated plus maze test. The spatial learning and memory ability was detected by Morris water maze method. Univariate or two-way analysis of variance was performed using SPSS 9.0 software, as well as SN-K and Tamhane's T2 tests for each group mean.

RESULTS: 135 rats in the experimental rats did not die during the experiment, and all of them entered the results analysis. 1 Compared with the normal control group, the behavior of the crawling field of stress rats was significantly reduced within 1 month after predation stress [(71.5±13.5), (96.8±18.4) points, P < 0.05]; hind limbs at 4 months Sexual standing was still significantly reduced [(11.4 ± 2.1), (17.9 ± 3.3) points, P < 0.05]. 2 Compared with the normal control group, the percentage of the number of open arms and the percentage of staying time of the stressed rats in the elevated plus maze test were significantly lower [(4.8±0.9)%, (20.7±3.7)%, P < 0.01] ;[(0.9±0.2)%, (6.6±1.2)%, P < 0.01]. 3 Compared with the normal control group, the rejection reactivity increased significantly within 2 months after stress [(2.4±0.5), (1.5±0.4) points, P < 0.05]. 4 Compared with the normal control group, the stress on the first day after predation stress in the Morris water maze space training period was significantly extended (32±6), (17±3) s, P <0.05], there was no significant difference in the percentage of time spent on non-target roaming in the four quadrants of the stress rat water maze during the spatial orientation test. There was no significant change in the spatial learning and memory test of rats at 2 weeks after stress.

Conclusion: Simple predation stress successfully induced a variety of emotional arousal obstacles such as decreased sustained motor activity, impaired inquiry behavior, high alertness, anxiety, decreased environmental adaptability, panic escape response, and short-term space. Impaired cognitive ability such as learning and memory, with pathogenic factors and processes similar to post-traumatic stress disorder, can basically simulate its main clinical manifestations.

Key words: stress disorder, post-traumatic; predation stress; emotional awakening; animal model

1 Materials and methods

Design: Randomized controlled animal experiments.

SETTING: Department of Neurology, Chengdu General Hospital of Chengdu Military Command.

MATERIALS: The experiment was completed at the Chengdu General Hospital of the Chengdu Military Region of the People's Liberation Army from 2004-01 to 2005-06. A total of 135 male Wistar rats were selected, and the initial body mass (185±9) g was provided by the Experimental Animal Center of the Institute of Surgery, Daping Hospital, Third Military Medical University of PLA (license number: scxk-(军)2002008). 12 h bright/dark conditions, 22 to 25 °C ambient temperature, separated feeding. The randomized digital table method was divided into 75 predator-stress group and 60 normal control group.

Instrument: According to the literature, the self-prediction stress experiment box (1 m × 1 m × 1 m wire cage, 20 cm × 15 cm × 15 cm small wire cage in the middle, to facilitate the experiment when the rats were placed). According to the literature [5, 6], the self-made behavioral observation box (60 cm × 60 cm × 60 cm no-top wooden box, the bottom of the box is divided into 36 equal squares for the emotional arousal level detection) . An elevated cross maze (wood structure consisting of two 50 cm x 10 cm relative open arms and two 50 cm x 10 cm x 40 cm relative closed arms, open upper part of the closed arm) was prepared according to [7]. There is an open section of 10 cm × 10 cm in the center. The labyrinth is 50 cm from the ground. It is used to evaluate changes in anxiety-like behavior in rats. According to the literature [8], the Morris water maze (1.2 m diameter and 0.5 m high drum) is divided into 4 quadrants (defined as A, B, C, and D in the clockwise direction), and 0.5% to 1.5 after the water is filled. % of the milk powder is added to make the water opaque milky white, the water temperature is controlled at 22 to 25 ° C. The 11 cm diameter platform is fixed in a certain quadrant (in the A quadrant in this experiment), and the platform is 1 cm below the horizontal plane. Learning and memory ability observation).

Design, implementation, and evaluator: Designed as the first author, the data was collected as the first, second, and third authors, and implemented as the first, second, third, and fourth authors, and evaluated as the first and fifth authors. All participants were trained in the relevant professions before the implementation of the experiment.

method:

Model preparation: The rats were placed in a small cage of a predator stress test chamber, and then a starved cat was placed in the experiment box, so that the cat and the rat had no physiologically harmful contact, resulting in slight panic in the rat. The tremors and shortness of breath caused by the shock and rapid nasal agitation for 6-8 minutes to ensure similar stress intensity and stable stress effects in the experimental rats. Rats in the normal control group were treated the same except that they did not touch the cat.

Affective behavior observation: At the 14th, 14th, 1st, and 4th month after predation stress, 12 and 15 rats in the normal control group and the predation stress group were observed respectively. The emotional behavior changes were observed: 1 Rat movement Activity: The rats were gently placed in the small square of the observation box of the open field, and the activity of the animals within 5 minutes was observed. Scoring criteria: Rats 1 /2 or more The body enters the adjacent square from the square in which it is located, that is, 1 point is climbed 1 time, and the total score is used to evaluate the change in exercise activity. 2 Exploring behavior: The method was the same as above, and the hind limb standing score change in rats was observed within 5 min. Scoring criteria: hind limbs standing 1 time 1 point, the total score is used to evaluate the change in inquiry behavior. 3 Repellent reactivity: The rats were lightly grasped with gloves that the animals had never touched, and the reaction was observed. Scoring criteria: 0 easy to grab animals; 1 scream or avoid; 2 scream and avoid; 3 escape; 4 escape and scream; 5 bite or try to bite the glove; 6 active leaping attack; Changes in the characteristics of conflict quality [6]. 4 elevated cross maze experiment: the rat is placed in the middle of the maze, head to the closed arm. The number of times the rats entered the open arms and the closed arms and the retention time of the two arms (which were based on the total limbs or the outriggers of the limbs) were recorded separately. The number of times the rats entered the open arms and the retention time in the open arms were calculated. The percentage of the total number of times (the sum of the number of open arms and closed arms) and the total time (the sum of the staying time of the open arms and the closed arms) was used as an indicator of anxiety.

Spatial learning and memory ability observation: Morris water maze method [8] was used to observe the changes in spatial learning and memory ability on the first day and the second week after predation stress. Days 1 and 2 are space orientation training: 9 days of training on the first day, 2 min / time, record the time from the time when the rats enter the water from different quadrants to find and climb the platform for the training incubation period, and the average of every 3 times as the average training. Latency; 6 training sessions were repeated on day 2; the five average training latency changes were compared to analyze the animal's ability to acquire spatial information. On the third day, the spatial orientation ability test was carried out: the platform was removed first, and then the rats were placed in water for 2 min, and the percentage of the residence time in the four quadrants was recorded to determine the memory storage and retrieval ability of the animals.

MAIN OUTCOME MEASURES: Rat climbing behavior, hindlimb standing, rejection response, elevated cross maze opening and opening time, Morris water maze average training latency and percentage of different quadrant staying time.

Statistical analysis: The single-factor or two-way analysis of variance was performed by SPSS 9.0 software in the Statistics Department of Chengdu General Hospital of Chengdu Military Region, and the S-N-K method of the mean of each group (for rats with homogeneity of variance) Behavior, rejection response, and percentage of open arms) and Tamhane's T2 test (comparison of hindlimb standing, percentage of open arms, and changes in spatial learning and memory ability in rats with irregularities).

2 results

2.1 Analysis of the number of experimental animals The 135 rats involved in the experiment did not die during the experiment, and all of them entered the result analysis.

2.2 Observation of emotional behavior at different times after predation stress in rats

2.2.1 Changes in behavior of the open field and changes in the behavior of the elevated plus maze at different times after predation stress in rats.

Table 1 shows that in the predator-stressed rats, the behavior of the crawling field was significantly reduced within 1 month after stress, indicating that the motor activity decreased, and then gradually returned to normal; while at 4 months, hindlimb standing was still significantly reduced, indicating that it explored The behavior is significantly reduced. Within 4 months after predation stress, the percentage of the number of open arms and the percentage of staying time in stress rats decreased significantly, and recovered slightly at 1 month after stress, but decreased further after 2 months.

2.2.2 Repellent reactivity at different times after predation stress in rats is shown in Table 2. Repellent reactivity in rats was significantly enhanced within 2 months after stress.

3 Discussion

Post-traumatic stress disorder has a variety of clinical manifestations, including repeated recurrence of traumatic experience symptoms, emotional numbness and avoidance symptoms, persistent anxiety, panic escape response, and irritability symptoms. The authors' previous work established two animal models of post-traumatic stress disorder, namely, hippocampal convulsion threshold electrical stimulation and rat-enhanced predation stress. Although both can induce post-traumatic stress disorder-like behavioral abnormalities within 1 month after the modeling of experimental rats, it is difficult to rule out the influence of electrical stimulation and other factors, and the pathogenic factors of post-traumatic emergency disorders are also Large differences; while the latter uses a combination of predation stress and re-environmental stimulation, it is difficult to rule out the effects of two stress effects; at the same time, the two models have limited observation time and the quantitative and qualitative methods of behavioral research used. Insufficient analysis makes it difficult to rule out the influence of subjective factors; therefore, it is necessary to further improve the stress pattern and control the stress intensity to induce stress-related emotional behavior abnormalities in experimental rats for a longer period of time, and quantitatively analyze related emotional arousal The nature of the obstacle.

Given the stress factors and intensity, as well as the extreme helplessness of the victim at the time, it is important in the pathogenesis of post-traumatic stress disorder, using the special predation behavior in the animal food chain to simulate similar pathogenic factors and processes. At the same time, it has been found through trial and error that predation stress for 4 to 9 minutes can cause mild panic tremors in rats, as well as rapid nasal agitation due to shortness of breath caused by shock, and continue this state of shock for 6-8 minutes (ie overall When the stress time is 11 to 17 min), it can ensure the similar stress intensity and stable stress effect of the experimental rats; more interestingly, the stress effect may be optimal at this time, and this is triggered. The experimental rats had obvious emotional behavior disorder during the observation period of 4 months. However, if the stress was insufficient or too strong, the duration was too short or too long, it was difficult to achieve similar effects, but the stress pattern and intensity should be similar. The relationship between the abnormal emotion behaviors is still uncertain and needs to be further explored.

Market behavior observation is an important method to detect the emotional arousal level of experimental animals. Animals entering the strange environment can have obvious protective emotional response, which is of great significance for quantitative analysis of the alertness level, anxiety state and environmental adaptability of experimental animals. The experimental results showed that the behavior of the market was significantly reduced within 1 month after stress in predation-stressed rats, suggesting that the activity of the new environment was significantly reduced; the anti-capture reactivity was significantly enhanced within 2 months after stress. It showed that the degree of shock was serious, the conflict characteristics were enhanced, irritability and struggling behavior were obvious; while hind limb standing was still significantly reduced at 4 months, indicating that the frustration was obvious and the inquiry behavior was significantly suppressed. It can be seen from the experimental results that the predation stress can cause the alarm level of the experimental rats to increase significantly, irritability and adaptability in the new environment, and has been in the panic escape emotional response for a long time after the stress stimulation.

The elevated cross maze test evaluates the anxiety characteristics of experimental animals by using the animal's exploration characteristics of the new environment and the fear of high open arms. The results showed that the percentage of the number of open arms and the percentage of staying time of stress rats decreased significantly within 4 months after predation stress, suggesting that predation stress has a significant long-term anxiety-causing effect on rats; The anxiety of the experimental rats was progressively aggravated within 2 weeks after stress, and was relieved at 1 month, but it was further aggravated after 2 months, indicating that the emotional arousal disorder of experimental rats caused by severe psychological stress. It may be characterized mainly by anxiety-related behaviors, and is very similar to patients with post-traumatic stress disorder. The specific reasons need to be further explored.

The Morris water maze is a spatial learning memory model that is directly related to hippocampal function and can accurately reflect the spatial learning and memory ability of animals. In the space of the Morris water maze, during the training period, the stress rats 1 day after the predation stress spent more time than the normal control group to find the platform, showing that the learning and memory acquisition and consolidation process was affected; During the orientation test period, the spatial orientation ability of stress rats was also significantly impaired, indicating that predation stress can lead to the instability of the memory process of experimental animals, and may also have some influence on the reading or extraction process of stored memory. There was no significant difference in spatial learning and memory between the two groups at 2 weeks after the experiment, suggesting that severe psychological stress only caused short-term learning and memory impairment in experimental animals, and short-lived recognition in patients with post-traumatic stress disorder. The impairment of knowledge is quite similar.