A method for preparing an esophageal visceral perception abnormal rat model

Abstract

The present application belongs to the field of medicine and biotechnology, and particularly relates to a preparation method of an esophageal visceral perception abnormal rat model. The preparation method is to give regular esophageal acid stimulation to young rats, adjust the speed, total amount, etc., and through repeated experiments, a modeling method is found which can maintain the integrity of the esophageal mucosa, not cause damage to the esophageal mucosa, and induce long-term changes in the central system management of the visceral perception area. After the rats grow up, the esophageal chemical stimulation is given, and through the changes in the behavior pattern of the rats, the changes in the key pathway NMDA pathway receptor of the central management of the visceral perception area, etc., the establishment of the esophageal visceral perception abnormal rat model is verified. The esophageal visceral perception abnormal rat model prepared by the method has the advantages of simple preparation method, low modeling cost, and therefore has high practical value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medical biotechnology, and particularly relates to a preparation method of an esophageal visceral perception abnormal rat model. BACKGROUND

[0002] Esophageal visceral perception abnormality is an important pathogenesis of many refractory functional esophageal diseases. People or animals with visceral hypersensitivity have a more sensitive reaction than normal healthy people or animals when the esophagus receives a certain degree of stimulation. At present, since the mechanism of visceral perception abnormality has not been elucidated, effective measures have not been found, and therefore many patients with related diseases cannot be effectively treated. Therefore, preparing an animal model that can be used for experiments has very important significance for elucidating the role of visceral perception abnormality in refractory functional esophageal diseases and for future targeted drug development.

[0003] One of the most difficult problems in the preparation of a visceral perception abnormal animal model is that experimental animals cannot express their feelings in language. In the past, in the esophageal hypersensitivity model, it was often only possible to indirectly reflect whether there was visceral hypersensitivity by calculating the levels of pain-related substances in the neural center after modeling. Another problem is that excessive stimulation in the esophagus often leads to significant damage to the esophageal mucosa, resulting in esophageal erosion, ulceration, and even perforation.

[0004] For example, Yang Min's team developed a visceral hypersensitivity rat model prepared by esophageal acid perfusion. Specifically, on day 14 of the experiment, adult healthy Sprague-Dwley (SD) rats were subjected to esophageal acid perfusion. The animals were fasted for 12-16 h, and anesthetized by intraperitoneal injection of 2.5% sodium pentobarbital (1 mg / kg). The anesthetized animals were fixed in a supine position with the head elevated at 20-30 degrees. The abdominal wall and stomach wall were incised, and a drainage tube was placed at the cardia to collect the liquid dripped from the esophagus. A single-lumen perfusion tube (inner diameter 0.50 mm, outer diameter 0.80 mm) was placed in the esophagus through the mouth, with the opening located 2-3 cm above the junction of the esophagus and the stomach. The catheter was fixed, and the other end was connected to a continuous perfusion pump. 0.1 mol / L hydrochloric acid was used for perfusion. The perfusion liquid was maintained at 37°C, and the speed was 10 ml / h for a total of 50 min (see, for example, Yang Min et al., "Establishment and evaluation of an animal model of esophageal visceral hypersensitivity", Journal of Practical Surgery, 2008, Vol. 17, No. 2, pp. 77-79; Yang Min et al., "Effect of esophageal acid perfusion on Fos expression in the central nervous system of a visceral hypersensitivity model rat", Chinese Journal of Digestion, July 2006, Vol. 26, No. 7, pp. 448-452). However, this model did not evaluate whether the esophageal mucosa was intact after modeling, and if there was esophageal erosion after acid perfusion, it would be difficult to identify it as a visceral hypersensitivity model. In addition, this study only used the pain-related substance c-fos to indirectly evaluate whether the modeling was successful, and did not evaluate the reaction of the model animals when their viscera were stimulated after modeling.

[0005] For another example, the team of Chen Ying developed an esophageal visceral sensitivity animal model by continuously stimulating the esophagus of a rabbit, specifically: first, a self-made dilation balloon (ERCP catheter with a balloon at the front end) and a PTCA pressure pump are needed. The young rabbits (rabbit age 45 ± 2d) are fixed in a supine position on a rabbit table, and the mouth is opened with an oral speculum. The lower esophagus is dilated twice a day, with a diameter of 0.9 cm, each time lasting 30 s, with an interval of half an hour, for a total of 14 days, and then the catheter is pulled out after decompression. The animals are fasted for more than 8 hours before the experiment starts every day, and free water is provided (see, for example, Chen Ying et al., "Preparation of an esophageal visceral sensitivity animal model", Journal of Tongji University (Medical Edition), Vol. 29, No. 2, April 2008, pp. 15-18). However, although the research results show that continuous mechanical stimulation increases esophageal visceral sensitivity without causing histological changes in the esophageal mucosal epithelium and early ultrastructural changes in the esophageal tissue, this model requires the use of rabbits as experimental animals, the use of self-made dilation balloons, and a long experimental duration, all of which result in a more complex modeling procedure and higher modeling costs, and mechanical stimulation and acid stimulation, which are more commonly studied, are two different types of esophageal stimulation, and their mechanisms for causing esophageal visceral sensitivity may not be the same. Therefore, it cannot be determined whether the method of replacing acid stimulation with mechanical stimulation is more advantageous.

[0006] It can be seen that the animal models used in previous esophageal visceral hypersensitivity studies are not ideal and may have more or less defects. Previous models often only prove that the substances related to pain perception in the neural central region of the animal change after modeling, and fail to prove through in vivo experiments that the response of the model rat esophagus to re-stimulation is different from that of the non-model rat, and do not provide evidence that the esophageal mucosa of the model animal has no visible damage, or the model preparation process is complex and the modeling cost is high.

[0007] Based on previous exploration of the pathogenesis of functional diseases, repeated stimulation of peripheral organs at an early stage of life may cause the occurrence of abnormal visceral perception through brain-gut interaction mechanisms. Based on the above background, the present application induces abnormal visceral perception in rats by repeatedly perfusing acid into the esophagus of young rats, and then verifies the existence of persistent abnormal visceral perception by giving the adult rats chemical stimulation of acid in the esophagus, through the characteristics of the rats, such as the existence of behavioral patterns different from ordinary rats and abnormal expression of NMDA receptor subunits in the brain.

[0008] The success of the esophageal visceral perception abnormality rat model is evaluated as follows: after the esophageal stimulation modeling process, the esophageal mucosa has no visible damage; when the esophagus is stimulated, the response of the model group animals is significantly different from that of non-visceral hypersensitivity animals; the NMDA pathway receptor level in the brain region involved in the regulation of visceral sensation changes. SUMMARY

[0009] The present application aims to overcome the deficiencies of the prior art and provide a preparation method of a new esophageal visceral perception abnormal rat model.

[0010] Specifically, the present application is realized through the following technical solutions:

[0011] The present application provides a preparation method of an esophageal visceral perception abnormal rat model, which comprises the following steps:

[0012] (1) from 7 days old, all young rats are reared in a same litter with their parent female rats under suitable conditions;

[0013] (2) when the young rats are 7-14 days old, the young rats are given esophageal acid perfusion every day, and the perfusion is continuously performed for 8 days;

[0014] (3) then the esophageal stimulation is stopped, and the normal feeding is performed until the young rats are 8 weeks old, and thus the esophageal visceral perception abnormal rat model is obtained.

[0015] As an optional mode, in the above preparation method, in step (1), the rats are SPF level SD rats, and the suitable rearing conditions are suitable environmental temperature and humidity, 12h / 12h light and dark alternating environment, and free feeding and drinking.

[0016] As an optional mode, in the above preparation method, in step (2), the intubation depth of the esophageal acid perfusion is 2.5-3.0 cm.

[0017] Preferably, the intubation depth of the esophageal acid perfusion is 2.8 cm.

[0018] As an optional mode, in the above preparation method, in step (2), the acid used for the esophageal acid perfusion is 0.1N HCl.

[0019] As an optional mode, in the above preparation method, in step (2), the perfusion amount of the esophageal acid perfusion is 20 μl, and the perfusion time is 1 min.

[0020] As an optional mode, in the above preparation method, in step (2), the catheter used for the perfusion is a small animal esophageal mucosa continuous micro-perfusion catheter.

[0021] Preferably, the catheter used for the perfusion is a small animal esophageal mucosa continuous micro-perfusion catheter which is self-made by the present inventors.

[0022] More preferably, the perfusion used catheter is the catheter described in CN112914782A or CN214805675U.

[0023] As an optional mode, in the above preparation method, in step (3), at 8 weeks of age, the mucosa of the lower esophagus and the cardiac part of the rat is smooth, and neither erosion nor ulceration is observed.

[0024] As an optional mode, in the above preparation method, in step (3),

[0025] At 8 weeks of age, in the case of intact esophageal mucosa, after receiving acute intragastric acid perfusion stimulation again, the rat can still exhibit relatively more active behavior compared with the normal control rat.

[0026] As an optional mode, in the above preparation method, in step (3), at 8 weeks of age, in the case of intact esophageal mucosa, after receiving acute intragastric acid perfusion stimulation again, the expression of the dorsal NMDA receptor NR1 subunit in the dorsal hippocampus of the rat brain is significantly up-regulated compared with the normal control rat, indicating that the model rat has a synchronous change in the NMDA receptor pathway, a key pathway for central visceral perception.

[0027] It should be understood that, within the scope of the present application, each of the above technical features of the present application and each of the technical features specifically described below (such as the examples) can be combined with each other to form a new or preferred technical solution. Due to the limited space, they will not be repeated one by one here.

[0028] Compared with the prior art, the present application has the following beneficial effects:

[0029] The present application adjusts the speed, total amount, etc. by giving regular esophageal acid stimulation to young rats, and through repeated experiments, a modeling method is found which can not only maintain the integrity of the esophageal mucosa without causing damage to the esophageal mucosa, but also induce long-term changes in the central system management of visceral perception area. After the rats grow up, by giving intragastric chemical stimulation, through the change of the rat behavior pattern and the change of the key pathway NMDA pathway receptor in the central management of visceral perception area, the establishment of the esophageal visceral perception abnormal rat model is verified. The esophageal visceral perception abnormal rat model prepared by the method of the present application has the advantages of simple preparation method and low modeling cost, and therefore has high practical value. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 Comparison of esophageal gross morphology of the control group (A) and the experimental group (B) after growing up.

[0031] Figure 2 Comparison of body weight growth rate between the experimental model group and the control group.

[0032] Figure 3: The total distance of spontaneous movement of the model group and the control group rats after adulthood. Among them, NS, young rats of esophageal saline infusion group; NH, young rats of esophageal acid infusion group.

[0033] Figure 4 : The total distance of spontaneous movement of the model group and the control group rats after adulthood. Among them, NS, young rats of esophageal saline infusion group; NH, young rats of esophageal acid infusion group.

[0034] Figure 5 : The expression change of the dorsal NMDA receptor NR1 subunit of the dorsal hippocampus of the model group and the control group rats after receiving esophageal acid stimulation again. DETAILED DESCRIPTION

[0035] The present application will be further described below with reference to specific examples. It should be understood that the specific examples described herein are intended to explain the present application, and are not intended to limit the scope of the present application.

[0036] The specific techniques or conditions not mentioned in the examples are carried out according to the techniques or conditions described in the literature in the art, or according to the product instructions. The reagents or instruments not mentioned by the manufacturer are all conventional products that can be purchased through regular channels.

[0037] The experimental methods in the following examples are all conventional methods, unless otherwise specified. The test materials used in the following examples are all commercially available products, unless otherwise specified.

[0038] EXAMPLE

[0039] 1. Preparation method of the esophageal visceral perception abnormal rat model of the present application

[0040] SPF level SD rats (provided by the Experimental Animal Science Department of the Medical Department of Peking University, a total of 32, 4 died after adulthood due to intubation and other factors, finally 6-8 in each of the four experimental groups), from 7 days old, all young rats were reared in a suitable environment with their parent female rats, the environmental temperature and humidity were suitable, and the light:dark environment alternated for 12h:12h, with free water and food. When the young rats were 7-14 days old, the rats were divided into two groups, and the rats were given esophageal acid infusion (NH group) or physiological saline infusion (NS group) every day.

[0041] (1) Through the dissection of the esophagus of the suckling rats, the length of the esophagus of the 7-day-old suckling rats was determined, and the intubation depth was determined to be 2.8 cm;

[0042] (2) Insert the self-made catheter (see CN112914782A, CN214805675U) into the tube;

[0043] (3) Establish the esophageal acid perfusion rate of suckling rats, 0.1N HCl, 20μL, a total of 1min perfusion, 8 days in a row. The control group was perfused with normal saline at the same speed and total volume. After that, stop the esophageal stimulation and feed normally for 8 weeks.

[0044] 2. Model verification method

[0045] (1) At 8 weeks of age, the rats were sacrificed and the esophagus was removed, the esophagus was cut along the longitudinal axis to the cardia part, and then flattened to observe the general morphology of the esophageal mucosa.

[0046] (2) During the feeding stage of young rats, the body weight of the two groups of rats was measured on the same day every week, and the body weight data was recorded.

[0047] (3) The animal spontaneous activity video analysis system (JLBehv-LR4, Jiliang Software Technology Co., Ltd., Shanghai, China) was used to record the spontaneous activity of rats. The spontaneous activity of rats was recorded after the end of esophageal acid perfusion in young rats, before the second intervention in adulthood, and 3 hours after acute esophageal acid perfusion in adulthood.

[0048] (4) After the rats were decapitated, the brain tissue was quickly removed, weighed, embedded, frozen at -80℃, sectioned, and the brain regions were determined and sampled. Western blot method was used to detect the level of NMDA receptor NR1 subunit.

[0049] 3. Model verification results

[0050] (1) At 8 weeks of age, the esophageal morphology of the model group (NH group) and the control group (NS group) rats was compared, and the mucosa of the lower esophagus and the cardia part of the two groups was smooth, and no erosion, ulceration or other lesions were found. Figure 1 )。

[0051] (2) The experimental model group and the control group were fed to adulthood (8 weeks of age), and after 2 weeks, there was no significant difference in body weight gain rate Figure 2 )。

[0052] (3) After the model group and the control group rats reached adulthood (8 weeks of age), there was a significant difference in spontaneous movement between the two groups of rats. The experimental results showed that the total distance of the model group rats was significantly higher than that of the control group rats (47380±4120mm vs 33580±3087mm) Figure 3 )。

[0053] (4) Model group and control group rats were given acute esophageal acid perfusion stimulation again after adulthood, and the animal spontaneous activity video analysis system recorded the behavior of rats. Due to the stimulation, the activities of rats in both groups were inhibited, but the total mileage of the spontaneous activity of the model group rats was still higher than that of the control group rats, and the mileage reduction rate of the model group rats (57.98%) was lower than that of the control group (79.68%) Figure 4 ). It is suggested that in the case of intact esophageal mucosa, the rats can still exhibit relatively more active behavior after receiving esophageal chemical stimulation again.

[0054] (5) The expression of dorsal NMDA receptor NR1 subunit in the dorsal hippocampus of model group and control group rats was significantly up-regulated after receiving esophageal acid stimulation again. Adult rats can cause a significant increase in dorsal NR1 level, and the increase is higher than that of the control group. It is suggested that the central visceral perception key pathway NMDA receptor pathway in model rats changes synchronously Figure 5 ).

[0055] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalent technologies, the present application also intends to include these modifications and variations.

Claims

1. A method for preparing a rat model of esophageal visceral sensory abnormalities, characterized in that: The method induces long-term changes in the central nervous system's visceral sensory regions of rats, and the preparation method includes the following steps: (1) Starting from 7 days old, all young rats were housed together with their parent female rats under suitable conditions; (2) When the young rats are 7-14 days old, they are given esophageal acid perfusion daily for 8 consecutive days. The insertion depth of the esophageal acid perfusion cannula is 2.5-3.0 cm, the acid used for esophageal acid perfusion is 0.1N HCl, the perfusion volume is 20 μL, and the perfusion time is 1 min. (3) After that, the esophageal stimulation was stopped and the rats were fed normally until they were 8 weeks old, thus obtaining a rat model of esophageal visceral perception abnormality. At 8 weeks old, with the esophageal mucosa intact, after receiving acute esophageal acid perfusion stimulation again, the rats were still able to exhibit relatively more active behavior compared with normal control rats. The expression level of NMDA receptor NR1 subunit in the dorsal hippocampus of the rats was significantly upregulated compared with normal control rats.

2. The preparation method according to claim 1, characterized in that: In step (1), the rats are SPF-grade SD rats, and the suitable feeding conditions are suitable ambient temperature and humidity, 12 h / 12 ​​h light and dark alternation environment, and free access to food and water.

3. The preparation method according to claim 1, characterized in that: In step (2), the catheter used for perfusion is a small animal esophageal mucosa continuous micro-perfusion catheter.

4. The preparation method according to claim 1, characterized in that: In step (3), at 8 weeks of age, the mucosa of the lower esophagus and cardia of the rats was smooth and no erosion or ulceration was observed.

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