A method for constructing a mouse bezoar model

Abstract

The application discloses a method for constructing a mouse bezoar model and particularly relates to the field of animal experiment models. The method comprises the following steps: wrapping the end of the cecum with a special sleeve and then performing ligation, thereby providing an animal model of mechanical bezoar for the first time. The model has small individual differences, short modeling time, uniform modeling degree, higher modeling precision and higher model stability, and can meet the modeling requirements of different bezoar degrees, and is more widely used in various scenes.

Description

Technical Field

[0001] This invention relates to the field of animal model technology, and specifically to a method for constructing a mouse indigestion model. Background Technology

[0002] Food stagnation, also known as food retention or stagnation, is a condition in Traditional Chinese Medicine (TCM) caused by improper diet and impaired spleen and stomach function, leading to food accumulation in the gastrointestinal tract and inability to be properly digested and absorbed. Essentially, food stagnation occurs when food remains in the stomach and intestines beyond their digestive and absorptive capacity, damaging the spleen and stomach and resulting in poor gastrointestinal motility and indigestion. Currently, in TCM research, animal models are often established to simulate the pathological process of food stagnation and evaluate the efficacy of spleen-strengthening and digestion-promoting Chinese herbal medicines.

[0003] Food stagnation is quite similar to functional dyspepsia in modern medicine, and existing dyspepsia models (FD) are mostly constructed using tail-clamping stimulation. However, this method has drawbacks such as long modeling time, large individual variability, atypical pathological features, and difficulty in quantification. In addition, most models are constructed using drug induction, but this method may lead to phenotypic fluctuations due to differences in mouse drug sensitivity, resulting in insufficient model stability. Furthermore, functional dyspepsia models are more suitable for studying the efficacy of drugs (such as prokinetic drugs and digestive enzymes), while non-surgical models are difficult to simulate the mechanical food stagnation state caused by severe gastrointestinal motility disorders. Therefore, there is an urgent need for a highly reproducible, pathologically defined, stable, and reliable animal model construction method for food stagnation to meet the needs of scientific research and drug evaluation. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies by providing a method for constructing a mouse indigestion model.

[0005] On the one hand, the present invention provides a method for constructing a mouse model of food stagnation, which involves surgically ligating the terminal colon of the mouse cecum.

[0006] In some embodiments, the ligation method is as follows: wrapping the colon at the end of the cecum with a cannula, ligating the colon together with the cannula using silk thread, and finally removing the cannula.

[0007] In some embodiments, the sleeve is an openable / closable structure.

[0008] In some embodiments, the opening and closing structure includes, but is not limited to, a split multi-lobed structure or an integral elastic cutout structure.

[0009] The split multi-lobed structure refers to dividing the cannula into several parts, which are mechanically connected or physically adsorbed to achieve detachable fixation. The integrated elastic incision structure is a single cannula body with a single axially extending incision on the side wall. The two edges of the incision have elastic recovery capability, allowing the cannula to be pried open by external force to fit into the intestinal tract, and to automatically close elastically after the external force is removed, forming a wrapping and fixing of the intestinal tract.

[0010] In some embodiments, the opening and closing structure includes, but is not limited to, a magnetic connection structure, a pin-type connection structure, and an adhesive connection structure.

[0011] The magnetic connection structure refers to cutting the sleeve axially and embedding miniature magnets of opposite polarities at the cut. When in use, the sleeve is separated, wrapped around the intestinal tube from both sides of the colon, and then naturally adsorbed and closed by magnetic force.

[0012] The pin-type connection structure refers to cutting the sleeve axially, with a protruding pin on one side of the tube wall and a through-hole on the corresponding position on the other side. The end of the pin has tiny barbs, which can be slightly locked after insertion but are easy to pull out.

[0013] Adhesive connection structure refers to a cannula that is axially cut, but the edges of the cut are sewn with medical-grade nylon Velcro. After the cannula is opened and wrapped around the colon, it is fixed by Velcro.

[0014] During use, the cannula wraps around the colon, compressing it inward. Then, a ligation suture is tied to the cannula. When the cannula is removed, the ligation suture remains on the colon, ensuring that the degree of ligation is consistent for each animal.

[0015] In some embodiments, the sleeve length is ≤5cm.

[0016] In some embodiments, the sleeve length is 1-3 cm.

[0017] In some embodiments, the inner diameter of the sleeve is ≤10mm.

[0018] In some embodiments, the inner diameter of the sleeve is ≤5mm.

[0019] In some embodiments, the inner diameter of the sleeve is ≤3mm.

[0020] In some embodiments, the thread is a surgical suture.

[0021] In some embodiments, a suture is used to puncture and ligate the mesentery without damaging the mesenteric vessels.

[0022] In some embodiments, mice are anesthetized and the surgical site is disinfected before surgery.

[0023] In some embodiments, after ligation, the cecal colon needs to be returned to the abdominal cavity, the wound sutured, and disinfected.

[0024] In some embodiments, the modeling time is ≤30 days.

[0025] In some embodiments, the modeling time is ≤15 days.

[0026] In some embodiments, the modeling time is ≤10 days.

[0027] On the other hand, the present invention provides a mouse model of food accumulation, which is constructed by the above method.

[0028] On the other hand, the mouse indigestion model provided by this invention can be used in screening and / or preparing drugs and health products for the prevention and / or treatment of indigestion or indigestion. Beneficial effects

[0029] By surgically ligating the cecum to directly induce mechanical narrowing, the core pathological link of "physical obstruction of intestinal emptying" is precisely targeted. The terminal colon of the cecum is a key turning point where intestinal contents enter the colon from the ileum. Therefore, obstruction here induces food impaction more directly and is closer to food impaction caused by organic factors in humans. This fills the gap in animal models of "mechanical food impaction" and provides a more precise tool for studying the pathological mechanism of organic food impaction.

[0030] This invention utilizes a specially designed cannula to wrap the colon, ensuring that the degree and location of ligation are the same for each mouse. At the same time, it avoids damage to the mesenteric blood vessels during the modeling process, reduces interference from non-specific pathological factors, and ensures that the modeling mice have small individual differences, uniform modeling degree, more accurate modeling, and higher model stability. In addition, the modeling time is only five days, which is shorter.

[0031] By adjusting the diameter of the sleeve, different degrees of food accumulation models can be induced, meeting the modeling needs of different degrees of food accumulation, and thus expanding the application scenarios. Attached Figure Description

[0032] Figure 1 This is a table recording mouse weight and abdominal circumference.

[0033] Figure 2 These are images of the abdominal states of mice in the sham-operated group (left) and the model group (right).

[0034] Figure 3 These are images of the ileocecal region of mice in the sham-operated group (left) and the model group (right).

[0035] Figure 4 The values ​​represent the levels of MTL, GAS, and VIP in mouse serum.

[0036] Figure 5HE staining images of the duodenum of mice in the sham-operated group (left) and the model group (right). Detailed Implementation

[0037] To facilitate understanding by those skilled in the art, the present invention will be further described below with reference to embodiments. The content mentioned in the embodiments is not intended to limit the present invention. Example 1

[0038] 1. Experimental reagents and consumables 1.1 Laboratory Animals

[0039] 24 SPF-grade male ICR mice, 6 weeks old, weighing 20-30g.

[0040] 1.2 Major Instruments and Consumables

[0041] Small animal anesthesia machine (Reward, R640); pathology slide machine; centrifuge; surgical instruments.

[0042] Motilin (MTL) kit; Gastrin (GAS) kit; Vasoactive intestinal peptide (VIP) kit; 5-0 non-absorbable sutures; 4% paraformaldehyde.

[0043] 2. Experimental Procedure 2.1 Experimental Preparation

[0044] After receiving the experimental mice, they were acclimatized for one week before the formal experiment began. All mice were housed in an SPF-grade animal laboratory, with an ambient temperature of 23±2℃ and a humidity of 40%~60%. The environment was kept in alternating light and dark conditions for 12 hours, and the mice had free access to food and water.

[0045] 2.2 Animal Grouping

[0046] The experimental mice were randomly divided into two groups according to Table 1 below: a sham-operated group of 12 mice and a model group of 12 mice.

[0047] Table 1 Group Molding materials Modeling methods Sham surgery group 5-0 sutures After anesthetizing the mice, the abdominal cavity was opened to expose the cecum and the mesentery near the colon was punctured (to avoid damaging the mesenteric vessels). Model group 5-0 sutures After anesthetizing the mice, the abdominal cavity was opened to expose the cecum. A special cannula was used to wrap the colon, and the terminal colon of the cecum was ligated with sutures (to avoid damaging the mesenteric vessels). The cannula was then removed after the ligation was completed. 2.3 Model Construction 2.3.1 Experimental Preparation

[0048] Preparation of the experimental meal: Weigh 2.5 g of CMC-Na and dissolve it completely in 50 mL of hot water to make a transparent viscous semi-solid. Add 4 g of skim milk powder and 2 g of starch in sequence, stir well, then add 1 g of activated carbon and add water to 62.5 mL to obtain a black semi-solid paste-like experimental meal.

[0049] 2.3.2 Experimental Modeling

[0050] The mice were fasted but allowed free access to water the night before model construction.

[0051] The surgical procedure is as follows: ① The model group mice were anesthetized by inhalation and the experiment began after the mice showed no pinching reflex.

[0052] ② The mouse was placed in a supine position on the operating table, and the skin of the lower left abdomen was prepared and disinfected.

[0053] ③ Make a 5 mm incision in the lower left abdomen of the mouse and remove the cecum and colon from the mouse in sequence.

[0054] ④ Use a specially made cannula (the cannula is an integrated elastic incision structure with an inner diameter of 1.5 mm and a length of 2 cm) to wrap the terminal colon of the mouse cecum.

[0055] ⑤ Use sutures to puncture the mesentery and ligate the cannula along with the colon (to avoid damaging the mesenteric vessels).

[0056] ⑥ After ligation, remove the cannula and return the mouse's cecal colon to the abdominal cavity.

[0057] ⑦ Suture the mouse wound and disinfect it.

[0058] ⑧ Stop anesthetizing the mice and transfer them to a heating pad. Once the mice have recovered, return them to their cages and feed them normally.

[0059] Mice in the sham-operated group underwent the same procedure, but without the use of a cannula or ligation.

[0060] 2.4 Data collection, endpoint sampling, and indicator testing 2.4.1 Process Data Collection

[0061] (1) Mouse body weight observation: Before modeling and before the endpoint sampling, the weight of the mice was weighed and recorded.

[0062] (2) Measurement of mouse abdominal circumference: Before modeling and before the final sampling, the abdominal circumference of the mice was measured and recorded.

[0063] 2.4.2 Sampling at the endpoint

[0064] Five days after mouse modeling, six mice from each group were euthanized, and 0.7 mL of blood was collected from the heart. After standing for 30 min, the blood was centrifuged at 3000 rpm / min for 10 min, and the serum from the supernatant was collected and stored at -80℃. Gastrointestinal tract samples were taken from the mice, washed with physiological saline, and then fixed in 4% paraformaldehyde.

[0065] 2.4.3 Indicator Testing 2.4.3.1 General observation and evaluation of mice

[0066] After modeling, the general appearance indicators of the mice in each group were observed and recorded daily according to Table 2 below.

[0067] Table 2

[0068] 2.4.3.2 Mouse gastric emptying and small intestinal propulsion rate experiment Mice were fasted but allowed free access to water the night before the experiment. The following day, the last 6 mice from each group were selected and each given 10 mL / kg of experimental food (weight denoted as W_actual). They were euthanized by cervical dislocation 20 minutes later. The abdominal cavity of the mice was quickly opened, and the anterior and posterior ends of the stomach were ligated. The stomach was removed and weighed to obtain its total weight (W_total). The entire stomach was cut open along one side of the greater curvature, and the experimental food residue inside was gently washed away with 0.9% sodium chloride solution. After drying, the net weight of the stomach was measured (W_net). The entire small intestine of the rat was taken, laid flat on a specimen measuring plate, and the length from the pylorus to the front of the experimental food (L_anterior) and the length of the entire small intestine (L_total) were measured. The gastric emptying rate and small intestinal propulsion rate were calculated using the formulas.

[0069] Gastric emptying rate = 1 - (W_total - W_net) / W_actual * 100% Small intestine propulsion rate = (Lpre- / Ltotal) * 100% 2.4.3.3 Determination of MTL, GAS and VIP levels in mouse serum

[0070] The levels of MTL, GAS, and VIP in mouse serum were determined by ELISA. The procedure was strictly followed according to the ELISA kit instructions. The specific experimental steps were the same as those for the determination of MTL, GAS, and VIP levels in mouse serum.

[0071] 2.4.3.4 Methods for histological examination 1) Sampling, fixation, dehydration, embedding, and sectioning; a. The gastrointestinal tract of mice was fixed in 4% paraformaldehyde for more than 24 hours; b. Rinse the fixed mouse gastrointestinal tissue with water for 24 hours, and then place it in 70%, 80%, and 95% anhydrous ethanol for 2 hours each for dehydration. c. Clear dewaxing solution, transparent at room temperature for 30 minutes; d. Impregnate overnight at 65°C with paraffin, then embed in paraffin; e. Slice the slices to a thickness of 3-5 μm and dry them in an oven at 60°C.

[0072] 2) Hematoxylin-eosin staining a. Perform transparent dewaxing with xylene dewaxing solution for 15 min, followed by anhydrous ethanol for 1.5 min, 95% ethanol for 1.5 min, 80% ethanol for 1.5 min, and then wash with water for 5 min; b. Stain with hematoxylin for 10 min, then rinse with running water twice, rinsing for 5 min each time; c. Hydrochloric acid-alcohol differentiation for 30 seconds; d. Rinse with water for 5 minutes; e. Stain with eosin for 3-5 minutes; f. Routine dehydration, clearing and mounting: 95% ethanol for 1.5 min, anhydrous ethanol for 1.5 min, and mounting with neutral resin.

[0073] 2.4.4 Data Processing and Result Determination

[0074] The experimental data are expressed as mean ± standard error (Mean ± SEM). All data were processed and statistically analyzed using GraphPad Prism 8.0. The t-test was used to compare the means of two groups, and the ANOVA test was used to compare the means of multiple groups. p < 0.05 was considered statistically significant.

[0075] 3 Results Analysis 3.1 Mouse body weight and abdominal circumference

[0076] as follows Figure 1 As shown, before model construction, there was no significant difference in weight and abdominal circumference between the sham-operated group and the model group. Seven days after model construction, the model mice showed a significant decrease in weight and a significant increase in abdominal circumference compared to the sham-operated group.

[0077] 3.2 Observation and evaluation of the general condition of mice

[0078] Seven days after the model was established, the mice in the sham-operated group exhibited rapid behavior, good mental state, smooth fur, and formed feces, while the mice in the model group exhibited sluggish behavior, lethargy, spiky fur, and loose feces.

[0079] as follows Figure 2 and Figure 3 As shown, 7 days after the model was established, the size of the abdomen and ileocecal region of the mice in the sham-operated group was normal, while the abdomen of the mice in the model group was relatively enlarged and rounded compared to the sham-operated group, and the ileocecal region was also larger than that of the sham-operated group, with a significant increase in chyme in the intestine.

[0080] 3.3 Gastric emptying and small intestinal propulsion rate experiments in mice

[0081] As shown in Tables 3 (mouse gastric emptying experiment) and 4 (mouse small intestinal propulsion rate experiment), 7 days after model construction, the gastric emptying rate of the model group mice was significantly lower than that of the sham-operated group mice, and the small intestinal propulsion rate of the model group mice was significantly lower than that of the sham-operated group mice.

[0082] Table 3

[0083] Table 4

[0084] 3.4 Serum levels of MTL, GAS, and VIP as follows Figure 4 As shown, compared with the sham-operated group mice, the serum levels of MTL and GAS in the model group mice were significantly reduced, while the VIP level was significantly increased by about 50%.

[0085] 3.5 H&E staining of gastrointestinal tract

[0086] as follows Figure 5 As shown, the HE staining of the stomach in the model group mice was largely the same as that in the sham-operated group. The gastric mucosal epithelial structure was intact, with no cell necrosis and a tight arrangement. No necrosis was observed in the glands of the lamina propria, the submucosa, and the muscularis propria. In the sham-operated group, the duodenal microvilli were tightly arranged, and the tight junctions between cells were clearly visible. In the model group, the duodenal mucosa showed many incomplete tight junctions and disrupted continuity. This indicates that the tight junctions of the duodenal mucosa were disrupted, but no organic lesions of the gastrointestinal tissue were observed.

[0087] 4. Experiment Summary

[0088] In this experiment, compared with the sham-operated group, the model group mice showed significantly lower body weight, significantly higher abdominal circumference, and significant differences in general condition and gross anatomical structure of the ileocecal junction. Gastric emptying rate and small intestinal propulsion rate were both significantly reduced, serum MTL and GAS levels were significantly decreased, and VIP levels were significantly increased. Therefore, the mouse model of food stagnation was successfully established.

[0089] In addition, statistical analysis of the data in each group (bar values ​​in each group) showed that the model group had significant differences in all indicators compared with the control group, indicating that the model was successfully constructed. Furthermore, the low dispersion of data within the model group indicated that there were very small differences between mice in the same model, the modeling was uniform and controllable, and the model stability was better.

[0090] Example 2 To investigate whether different inner diameter sleeves can induce models of different degrees, the inner diameter of the sleeve was changed to 2mm or 1mm, while the rest was the same as the experimental modeling section in 2.3.2. It was found that both inner diameter sleeves could successfully induce models. The 1mm sleeve induced a milder model, while the 2mm sleeve induced a more severe model. This indicates that adjusting the sleeve diameter can induce models of different degrees of food accumulation, meeting the modeling requirements for different degrees of food accumulation.

[0091] It should be understood that the above detailed description of the technical solutions of the present invention with reference to preferred embodiments is illustrative and not restrictive. Those skilled in the art can modify the technical solutions described in the embodiments or make equivalent substitutions for some of the technical features based on reading this specification; however, these modifications or substitutions do not cause the essence of the corresponding technical solutions to depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for constructing a mouse model of food stagnation, characterized in that, The construction method involved surgically ligating the terminal colon of the mouse cecum.

2. The method as described in claim 1, characterized in that, The ligation method is as follows: a cannula is used to wrap the colon at the end of the cecum, and then the colon is ligated together with the cannula using silk thread. Finally, the cannula is removed.

3. The method as described in claim 2, characterized in that, The sleeve has an openable and closable structure.

4. The method as described in claim 3, characterized in that, The sleeve length is ≤5cm.

5. The method as described in claim 2, characterized in that, The inner diameter of the sleeve is ≤10mm.

6. The method as described in claim 2, characterized in that, The thread is a surgical suture.

7. The method as described in claim 2, characterized in that, When ligating the colon, the suture punctures the mesentery but does not damage the mesenteric vessels.

8. The method as described in claim 2, characterized in that, The modeling time is ≤30 days.

9. A mouse model of indigestion, characterized in that, It is constructed by the method described in any one of claims 1-8.

10. The use of the mouse indigestion model of claim 9 in screening and / or preparing drugs and health products for the prevention and / or treatment of indigestion or dyspepsia.

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