Method for constructing non-organic (psychological) erectile dysfunction rat model

Abstract

The invention discloses a manufacturing method of a non-organic erectile dysfunction rat model, which comprises the following steps: firstly, applying chronic mild stress to male rats; and then, through a sexual behavior test, a sucrose water uptake experiment and an apomorphine experiment, screening out rats with erectile dysfunction (manifestation as decrease in insertion ratio and negative in apomorphine experiment) and depression-like manifestation (manifestation as decrease in sucrose water uptake) as non-organic erectile dysfunction model rats. The model rat manufactured by the invention is similar to human patients in etiology and behaviors (erectile dysfunction, depression, sexual arousal and sexual driving force inhibition), and has better surface validity. Brain function changes similar to those of a human patient can be found through functional magnetic resonance scanning of the model rat manufactured through the method, and it is prompted that the model rat has pathogenesis similar to those of the human patient. The model can replace human patients to be used for central molecular mechanism research.

Description

Technical Field

[0001] The present invention relates to a method for preparing a rat model of non-organic erectile dysfunction, in particular to an animal model which is similar to human patients with non-organic erectile dysfunction in etiology and behavior and has good surface validity, and belongs to the field of andrology. Background Art

[0002] Non-organic erectile dysfunction refers to erectile dysfunction caused by interpersonal relationships or psychological states (such as depression or anxiety) as the main or only factor. At present, some functional magnetic resonance imaging studies have found that patients have abnormal brain function and structure, but the central molecular mechanism behind it is still unclear [Jin, C., M. Guan, M. Dong, J. Wu, Z. He, X. Chen, D. Shi, J. Ren, G. Shi, and X. Zhang, Aberrant baseline brainactivity in psychogenic erectile dysfunction patients: a resting state fMRI study [J]. Brain Imaging & Behavior, 2018, 12(5): 1393-404.]. Due to ethical restrictions, molecular mechanism research cannot be conducted directly on the human brain, and the central molecular mechanism research of non-organic erectile dysfunction still requires the establishment of animal models.

[0003] At present, the method for constructing animal models of non-organic erectile dysfunction is mainly to expose animals to various stress conditions, including immobilization method [COLLU R, GIBB W, DUCHARME J R. Role of catecholamines in the inhibitory effect of immobilization stress on testosterone secretion in rats [J]. Biology of Reproduction, 1984, 30(2): 416-22.], foot shock method [Ishikawa, M., C. Hara, S. Ohdo, and N. Ogawa, Plasma corticosterone response of rats with sociopsychological stress in the communication box [J]. Physiology & Behavior, 1992, 52(3): 475-80.], cold water immersion method [Bidzinska, B., F.Petraglia, S. Angioni, AD Genazzani, M. Criscuolo, G. Ficarra, A.Gallinelli, GP Trentini, and AR Genazzani, Effect of different chronic intermittent stressors and acetyl-l-carnitine on hypothalamic beta-endorphinand GnRH and on plasma testosterone levels in male rats [J]. Neuroendocrinology, 1993, 57(6): 985-90.], exercise, food restriction, noise, fasting, crowding and social group stress [Retana-Márquez, S., H. Bonilla-Jaime, G. Vázquez-Palacios, R. Martínez-García, and J.Velázquez-Moctezuma, Changes in masculine sexual behavior, corticosterone and testosterone in response to acute and chronic stress in male rats [J]. Hormones and Behavior, 2003, 44(4): 327-37.]. In these models, the sexual behavior of male rats was inhibited, as shown by prolonged mounting latency, prolonged insertion latency, and decreased insertion rate. Therefore, these models have certain similarities with human patients. However, in these models, the stress conditions are mostly severe and of short duration (no more than 20 days). In human patients, non-organic erectile dysfunction is mostly related to long-term chronic mental illness or long-term interpersonal relationship problems. Therefore, there are still differences in etiology between the existing models and human patients. Finally, there is a lack of research on the verification and comparison of animal models and human patients in terms of magnetic resonance imaging. . Summary of the invention

[0004] Purpose of the invention: In order to overcome the shortcomings of existing models, the present invention provides a method for preparing a non-organic erectile dysfunction rat model based on a depression model.

[0005] Technical solution: To achieve the above purpose, the present invention first applies chronic mild stress to male rats, and then screens out rats with erectile dysfunction (manifested by decreased insertion ratio and negative apomorphine test) and depression-like performance (manifested by decreased sucrose water intake) as non-organic erectile dysfunction model rats through sexual behavior test, sucrose water intake experiment, and apomorphine experiment. The model rats prepared by the present invention are similar to human patients in etiology and behavior (erectile dysfunction, depression, inhibition of sexual arousal and sexual drive), and have good face validity. The intracavernous pressure test of the model rats prepared by the present invention is normal, which can exclude organic erectile dysfunction of the penis. Functional magnetic resonance imaging of the model rats prepared by the present invention can reveal brain function changes similar to those of human patients, further verifying the similarity between the model and human patients.

[0006] The specific steps of making the non-organic erectile dysfunction rat model of the present invention are as follows: Wistar male (6-7 weeks old, weighing 180-220 g) and female (6-7 weeks old, weighing 160-180 g) rats were given free access to food and water. During the first week of adaptation, rats were housed in cages of 5 each, then male rats were housed in individual cages, while female rats were still housed in cages of 5 each until the end of the experiment.

[0007] Starting from the second week, all male rats underwent a sucrose water intake experiment.

[0008] At the 4th week, the female mice underwent bilateral ovariectomy, and after recovery from surgery, hormones were injected subcutaneously to induce artificial estrous cycles for sexual behavior testing.

[0009] At week 4, male mice began to undergo sexual behavior testing.

[0010] In the 5th and 6th weeks, the average of the last three sucrose water intake test results was taken as the baseline value of each rat. According to the baseline value, the rats were divided into two groups: control group and model group.

[0011] Starting from the 7th week, male rats were first subjected to three apomorphine experiments. Rats that showed erections in all three apomorphine experiments (100 μg / kg apomorphine was injected subcutaneously in the neck, dissolved in 0.5 mg / kg ascorbic acid in saline solution) continued the experiment, and the model group began to implement 6 weeks of chronic mild stimulation.

[0012] At the end of the 12th week, chronic mild stress ended.

[0013] At week 13, sexual behavior tests and three apomorphine experiments were performed.

[0014] Among the rats in the model group, the rats that met the following three conditions at the same time were non-organic erectile dysfunction model rats: a. The results of the sucrose water intake experiment at the end of the 12th week were both reduced by more than 30% compared with the average value and baseline value of the control group; b. The insertion ratio in the sexual behavior test (the ratio of the number of insertions to the number of insertions plus the number of ridings in the 30-minute sexual behavior test) was lower than 0.43; c. No erection was observed in the three apomorphine experiments at the 13th week.

[0015] The experimental rats can be Wistar or SD rats, either ordinary or SPF grade. The chronic mild stress includes the following: intermittent lighting: the lighting is turned on / off every 2 hours; cage tilt: the cage is tilted 45 degrees; stroboscopic irradiation: the rats are irradiated with stroboscopic light at a frequency of 5 Hz; white noise: white noise is played continuously; two-by-two cages: two male rats are placed in a cage, and each time they are replaced with a different stranger rat from the last time; wetting the bedding: the bedding is wetted with water, and new dry bedding is replaced after the process is completed; no operation. Each chronic mild stress lasts for 12 hours, and a new stress method is replaced every 12 hours.

[0016] The chronic mild stress may last for 4-8 weeks. If it is too short, the success rate of the model may be too low, while if it is too long, there may be too many rats without sexual behavior, making it impossible to determine the insertion ratio.

[0017] The method for preparing a non-organic erectile dysfunction rat model according to claim 1 is characterized in that the frequency of the sucrose water intake experiment can be from 3 times a week to once every 2 weeks, and can be adjusted as needed during the experiment.

[0018] The method for preparing a non-organic erectile dysfunction rat model according to claim 1 is characterized in that the frequency of sexual behavior testing can be from 3 times a week to once every 3 weeks, and can be adjusted as needed during the experiment.

[0019] The non-organic erectile dysfunction rat model prepared by the method according to any one of claims 1 to 6 is characterized in that the model rats are similar to human patients in etiology and behavior (erectile dysfunction, depression, sexual arousal and inhibition of sexual drive), and have good face validity. The model rats prepared by the present invention were subjected to intracavernous pressure test, and the results showed no statistical difference compared with the blank control group rats, indicating that the penile erectile function was normal and there was no organic erectile dysfunction. Functional magnetic resonance imaging of the model rats prepared by the present invention revealed similar brain function changes as those of human patients, indicating that they have similar pathogenesis as human patients.

[0020] Beneficial effects: Compared with the prior art, the method for preparing a rat model of non-organic erectile dysfunction of the present invention has the following advantages: The rat model prepared by the present invention has good stability, and the rat model continues to show erectile dysfunction for at least 1 week after the end of chronic mild stress.

[0021] The rat model prepared by the present invention has a high modeling rate. Taking chronic mild stress for 6 weeks as an example, the modeling rate can reach more than 80%.

[0022] The rat model prepared by the present invention has undergone milder and longer-lasting stress conditions, has depression-like manifestations, and exhibits erectile dysfunction, sexual arousal inhibition and sexual drive inhibition in sexual behavior, which is similar to human patients in etiology and behavior and has good surface validity.

[0023] Functional magnetic resonance imaging (FMRI) scans of the model rats prepared by the present invention reveal similar changes in brain function as those of human patients, suggesting that the model rats have similar pathogenesis as those of human patients and can replace the human body for the study of central nervous system mechanisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The manufacturing flow chart of the present invention in Example 1; Figure 2 The chronic mild stress process of the present invention in Example 1; Figure 3 The insertion ratio of the control group and the model group in the present invention at the 13th week of Example 1; Figure 4The present invention performs functional magnetic resonance imaging in Example 1, and the comparison results of ALFF values ​​between the model group and the control group (marking the voxels with significant differences). Voxels with significant differences are marked with different colors according to different t values. From A to K are coronal magnetic resonance images marked with pseudo-color according to t values ​​in the order from head to tail. A to J are images of increased ALFF values ​​in the model group compared to the control group. K is an image of decreased ALFF values ​​in the model group compared to the control group. The color legend is in the lower right corner. Hip: hippocampus; VCx: visual cortex; AMY: amygdala; Pir: piriform cortex; Hyp: hypothalamus; Thal: thalamus; SCx: sensory cortex; CPu: caudate-putamen; Cg: cingulate gyrus; MCx: motor cortex; ICx: insular cortex; Figure 5 The chronic mild stress process of Example 4 of the present invention; Implementation

[0025] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0026] The present invention first applies chronic mild stress to male rats, and then screens out rats with erectile dysfunction (manifested by decreased insertion ratio and negative apomorphine test) and depression-like performance (manifested by decreased sucrose water intake) as non-organic erectile dysfunction model rats through sexual behavior tests, sucrose water intake experiments, and apomorphine experiments. The model rats prepared by the present invention are similar to human patients in etiology and behavior (erectile dysfunction, depression, inhibition of sexual arousal and sexual drive), and have good face validity. Functional magnetic resonance imaging of the model rats prepared by the present invention can reveal brain function changes similar to those of human patients, further verifying the similarity between the model and human patients.

[0027] The specific implementation methods of the present invention are as follows: Example 1

[0028] (1) Male Wistar rats (6-7 weeks old, weighing 180-220 g) and female Wistar rats (6-7 weeks old, weighing 160-180 g) were given free access to food and water. During the first week of adaptation, rats were housed in cages of five each. Subsequently, male rats were housed in individual cages, while female rats were still housed in cages of five each until the end of the experiment.

[0029] (2) Starting from the second week, all male rats underwent a sucrose water intake experiment (twice a week from 2 to 7 weeks and once a week from 8 to 12 weeks).

[0030] (3) At the 4th week, the female mice underwent bilateral ovariectomy. After recovery from surgery, hormones were injected subcutaneously to induce artificial estrous cycles for sexual behavior testing.

[0031] (4) At the 4th week, male mice began to undergo sexual behavior testing (twice a week).

[0032] (5) In the 5th and 6th weeks, the average of the last three sucrose water intake test results was taken as the baseline value for each rat. The rats were divided into two groups according to the baseline value: control group and model group.

[0033] (6) Starting from the 7th week, male rats were first subjected to three apomorphine experiments. Rats that showed erections in all three apomorphine experiments (100 μg / kg apomorphine dissolved in 0.5 mg / kg ascorbic acid in saline solution injected subcutaneously in the neck) continued the experiment. The model group began to implement 6 weeks of chronic mild stimulation (see the procedure). Figure 2 ).

[0034] (7) At the end of the 12th week, chronic mild stress ends.

[0035] (8) At week 13, sexual behavior tests and three apomorphine experiments were conducted.

[0036] (9) Among the model group rats, rats that simultaneously met the following three conditions were considered to be non-organic erectile dysfunction model rats: a. the results of the sucrose water intake experiment at the end of the 12th week were both more than 30% lower than the average value and baseline value of the control group; b. the insertion ratio in the sexual behavior test (the ratio of the number of insertions to the number of insertions plus the number of ridings in the 30-minute sexual behavior test) was lower than 0.43; c. no erection was observed in the three apomorphine experiments at the 13th week.

[0037] The final mold success rate is about 83%. Example 2

[0038] The operation procedure is similar to that of Example 1, except that the duration of chronic mild stress is 4 weeks.

[0039] The final moldability rate is about 55%. Example 3

[0040] The operation procedure is similar to that of Example 1, except that the duration of chronic mild stress is 8 weeks.

[0041] The final mold success rate is about 86%. Example 4

[0042] The operation process is similar to that of Example 1, except that the chronic mild stress process is shown in Figure 5 .

[0043] The final mold success rate is about 83%. Example 5

[0044] The operation process is similar to that of Example 1, except that the frequency of the sucrose water intake experiment is once every 2 weeks, and the frequency of the sexual behavior test is once every 3 weeks.

[0045] The final mold success rate is about 83%.

[0046] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a rat model of non-organic (psychological) erectile dysfunction, the characteristics of which include Follow these steps: (1) Wistar male (6-7 weeks old, 180-220 g) and female (6-7 weeks old, 160-180 g) rats were given free access to food and water. During the first week of adaptation, rats were housed in cages of 5, then male rats were housed in individual cages, while female rats were still housed in cages of 5 until the end of the experiment. (2) Starting from the second week, all male rats underwent sucrose water intake experiments. (3) At the fourth week, female rats underwent bilateral ovariectomy. After recovery from surgery, hormones were injected subcutaneously to induce artificial estrus cycles for sexual behavior tests. (4) At the fourth week, male rats began to undergo sexual behavior tests. (5) At the fifth and sixth weeks, the average of the last three sucrose water intake test results was taken as the baseline value for each rat. Rats were divided into two groups according to the baseline value: control group and model group. (6) From the 7th week onwards, male rats were first subjected to three apomorphine experiments. Rats that showed erections in all three apomorphine experiments (100 μg / kg apomorphine dissolved in 0.5 mg / kg ascorbic acid saline solution injected subcutaneously in the neck) continued the experiment, and the model group began to implement 6 weeks of chronic mild stimulation. (7) At the end of the 12th week, chronic mild stress ended. (8) At the 13th week, sexual behavior tests and three apomorphine experiments were performed. (9) Among the rats in the model group, rats that met the following three conditions at the same time were non-organic erectile dysfunction model rats: a. The results of the sucrose water intake experiment at the end of the 12th week were both reduced by more than 30% compared with the average value and baseline value of the control group; b. The insertion ratio in the sexual behavior test (the ratio of the number of insertions to the number of insertions plus the number of ridings in the 30-minute sexual behavior test) was less than 0.43; c. No erections were observed in the three apomorphine experiments in the 13th week.

2. The method for preparing the non-organic erectile dysfunction rat model according to claim 1, characterized in that: The experimental rats selected can be Wistar or SD rats, either ordinary grade or SPF grade.

3. The method for preparing the non-organic erectile dysfunction rat model according to claim 1, characterized in that: Chronic mild stress included the following: intermittent lighting: the light was turned on / off every 2 hours; cage tilt: the cage was tilted 45 degrees; stroboscopic irradiation: the rats were irradiated with stroboscopic light at a frequency of 5 Hz; white noise: white noise was played continuously; two-by-two co-cage: two male rats were placed in a cage, and each time they were replaced with a different stranger rat; wetting the bedding: the bedding was wetted with water, and new dry bedding was replaced after the process was completed. No operation. Each chronic mild stress lasted for 12 hours, and a new stress method was replaced every 12 hours.

4. The method for preparing the non-organic erectile dysfunction rat model according to claim 1, characterized in that: Chronic mild stress can last for 4-8 weeks. If it is too short, the model success rate may be too low. If it is too long, there may be too many rats without sexual behavior, making it impossible to determine the insertion rate.

5. The method for preparing the non-organic erectile dysfunction rat model according to claim 1, characterized in that: The frequency of the sucrose water intake experiment can be from 3 times a week to once every 2 weeks, and can be adjusted as needed during the experiment.

6. The method for preparing the non-organic erectile dysfunction rat model according to claim 1, characterized in that: The frequency of sexual behavior testing can range from 3 times a week to once every 3 weeks, and can be adjusted as needed during the experiment.

7. A non-organic erectile dysfunction rat model prepared according to the method of any one of claims 1 to 6, characterized in that: The model rats are similar to human patients in etiology and behavior (erectile dysfunction, depression, sexual arousal and inhibition of sexual drive), and have good face validity. The intracavernous pressure test of the model rats prepared by the present invention is normal, and organic erectile dysfunction of the penis can be excluded. Functional magnetic resonance imaging of the model rats prepared by the present invention can reveal similar brain function changes as those of human patients, suggesting that they have similar pathogenesis as human patients.

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