Mouse intestinal nerve plexus patch and preparation method thereof
By peeling muscle tissue along the direction of the circular muscle and fixing it with paraformaldehyde and treating it with Triton X-100 solution, the problems of time-consuming and technically demanding enteric plexus detachment were solved, enabling rapid acquisition of complete enteric plexus patches.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SOUTH CHINA AGRICULTURAL UNIVERSITY
- Filing Date
- 2025-12-09
- Publication Date
- 2026-04-21
AI Technical Summary
Existing methods for ripping the enteric nerve plexus are time-consuming and technically demanding, making it difficult to quickly obtain complete enteric nerve plexus tissue.
Muscle tissue was dissected along the direction of the circular muscle, and mouse enteric plexus patches were prepared by combining paraformaldehyde fixation and Triton X-100 solution treatment.
It enables rapid and convenient acquisition of more complete enteric nerve plexus tissue, reducing operation time and technical difficulty.
Smart Images
Figure CN121896149A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of biotechnology, and in particular to a mouse enteric plexus patch and its preparation method. Background Technology
[0002] The enteric nervous system is an independent neural network embedded in the wall of the human digestive tract, containing approximately 500 million neurons—a number approaching the total number of neurons in the spinal cord—and is therefore referred to by the scientific community as the "second brain." This complex system extends from the esophagus to the anus, forming a complete neural regulatory network. The enteric nervous system is mainly composed of two sets of nerve plexuses: the myenteric plexus (Auerbach's plexus, located between the circular and longitudinal muscles of the digestive tract) governs gastrointestinal peristalsis; the submucosal plexus (Meissner's plexus, located in the submucosa) regulates secretion, absorption, and local blood flow. In addition to neurons, the enteric nervous system also includes glial cells (involved in immune defense) and specialized Cajal interstitial cells (ICCs), which act as "intestinal pacemakers" by generating slow-wave potentials to trigger contraction rhythms. The enteric nervous system can independently coordinate gastrointestinal motility (such as migratory complex movements), sphincter opening and closing, digestive enzyme secretion, and mucus secretion without the intervention of the central nervous system (CNS). For example, when food expands the intestines, the enteric nervous system can autonomously trigger local peristaltic reflexes. This autonomy is demonstrated by the fact that even after the vagus nerve is severed, isolated intestinal tissue can still continue to move. The enteric nervous system synthesizes 95% of the body's serotonin (5-HT), 50% of dopamine, and various neuropeptides (substance P, vasoactive intestinal peptide, etc.). These neurotransmitters not only regulate intestinal motility... The gut also influences central mood through the gut-brain axis. Serotonin abnormalities are directly associated with irritable bowel syndrome (IBS), while dopamine is involved in reward mechanisms and motor control. The gut contains 70% of the body's immune cells. The enteric nervous system monitors pathogens, triggers immune responses (such as diarrhea and vomiting), and regulates the balance of gut microbiota. In diseases such as ulcerative colitis (UC), enteric nervous system abnormalities can lead to dysbiosis and mucosal barrier disruption. The vagus nerve is a key pathway between the enteric nervous system and the brain, handling 90% of the signals transmitted from the gut to the brain. This two-way communication explains why psychological stress can lead to elevated glucocorticoid levels, which in turn triggers inflammation of glial cells in the enteric nervous system, creating a vicious cycle of "stress-colitis."
[0003] As a fundamental method for studying neural plexuses, enteric nerve patch technology has irreplaceable value, but current experimental protocols still face challenges such as time-consuming operation and high technical barriers.
[0004] The technical problem of this solution is: how to provide a method for rapidly ripping apart the enteric nerve plexus of mice. Summary of the Invention
[0005] The purpose of this application is to provide a method for rapidly excising the mouse enteric plexus to prepare a mouse enteric plexus patch. Through extensive experiments, this method has been found that when the muscle tissue is peeled off along the direction of the circular muscle during the excising process, it is easier to obtain intestinal tissue carrying a more complete enteric plexus.
[0006] To achieve the above objectives, this application discloses a method for preparing an enteric plexus patch, comprising the following steps:
[0007] Step 1: The intestinal donor is killed, and then intestinal tissue segments from the duodenum, jejunum, ileum or colon are taken, fixed with paraformaldehyde and then dehydrated to obtain dehydrated intestinal tissue segments;
[0008] Step 2: Rinse the dehydrated intestinal tissue segment and remove the mesentery, fat and Paisley's lymph nodes. Then cut the intestinal tissue segment into pieces of about 4-6 mm in length. Place the intestinal pieces flat on a glass slide and add Triton X-100 PBS solution. Then use the smooth side of forceps to fix the intestinal pieces. Then peel the muscle tissue along the direction of the circular muscle to obtain an intestinal slice with the myenteric plexus.
[0009] Step 3: Wash the intestinal slices containing the myenteric plexus, then stained them with fluorescence and mounted them to obtain mouse enteric plexus patches.
[0010] Preferably, the intestinal donor is a mouse, pigeon, pig, or chicken.
[0011] Preferably, the intestinal donor is a mouse, and the method of killing the mouse specifically includes the following steps:
[0012] Step A1: Anesthetize the mouse and cut open its thoracic cavity;
[0013] Step A2: Insert the syringe into the left ventricle at an angle parallel to the horizontal axis of the heart and infuse physiological saline until clear physiological saline flows out of the right atrium. Then, continuously infuse paraformaldehyde into the left ventricle for 5 minutes to obtain lethal mice.
[0014] Preferably, the PBS solution containing Triton X-100 is a PBS solution containing 0.1% Triton X-100 by mass.
[0015] Preferably, step 3 specifically involves: washing the intestinal slices containing the myenteric plexus with Triton X-100 PBS solution, blocking the washed intestinal slices containing the myenteric plexus with blocking solution, incubating with primary antibody and secondary antibody in sequence after blocking, transferring them to a glass slide and fluorescently staining and mounting them to obtain mouse intestinal plexus patches.
[0016] Preferably, the blocking solution is a PBS solution containing 0.3% Triton X-100 and 5% sheep serum by mass.
[0017] During the primary antibody incubation process, the primary antibody incubation solution is a PBS solution containing 0.1% primary antibody, 0.3% Triton X-100, and 5% sheep serum.
[0018] During the secondary antibody incubation process, the incubation solution was a PBS solution containing 0.1% secondary antibody, 0.3% Triton X-100, and 5% sheep serum.
[0019] In addition, this application also discloses a mouse enteric plexus patch, which is prepared by the above-described method for preparing mouse enteric plexus patches.
[0020] The beneficial effects of this application are: the preparation method of the mouse enteric plexus patch provided by this application is convenient and quick. In the process of tearing off the enteric plexus, the muscle is peeled off along the direction of the circular muscle, which makes it easier to obtain intestinal tissue carrying a more complete enteric plexus. Attached Figure Description
[0021] Figure 1 This is a schematic diagram of the cutting procedure before tearing apart the mouse intestine in Example 2;
[0022] Figure 2 This is a schematic diagram of the tearing of the mouse colon in Example 2;
[0023] Figure 3 This is a schematic diagram of the tearing of the small intestine in mice in Example 2;
[0024] Figure 4 This is a schematic diagram of intestinal immunofluorescence in NPY-GFP mice.
[0025] Figure 5 This is a schematic diagram of immunofluorescence staining with mouse intestinal tyrosine hydroxylase antibody (TH).
[0026] Figure 6 This is a schematic diagram of the transection of the colon of a mouse in Comparative Example 1.
[0027] Figure 7 This is a schematic diagram of the intestinal tract of the mouse colon in Comparative Example 2.
[0028] Figure 8 A schematic diagram of tearing apart the small intestine of a pigeon;
[0029] Figure 9 A schematic diagram of the tearing of a pig's small intestine;
[0030] Figure 10This is a diagram illustrating the tearing of a chicken's small intestine. Detailed Implementation
[0031] The present invention will now be clearly and completely described in conjunction with embodiments thereof. It should be noted that, unless specific conditions are specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.
[0032] Example 1: Methods of Lethality in Mice
[0033] 1.1 Anesthesia: Fill the vaporizer with isoflurane, set the oxygen flow rate to 1-2 L / min, and ensure the scavenging system is activated. Place the mouse in a sensing chamber containing 3-5% isoflurane. After the mouse loses its righting reflex, reduce the isoflurane concentration to 1-2% and maintain it.
[0034] 1.2 Surgery: The mouse was placed in a supine position, and a midline abdominal incision was made to expose the thoracic cavity. The thoracic cavity was carefully opened, and the pericardium was completely removed to fully expose the heart.
[0035] 1.3 Perfusion: Insert the needle into the left ventricle near the apex of the heart at an angle roughly parallel to the horizontal axis of the heart. Then turn on the perfusion machine and continuously infuse physiological saline until the liver changes from dark red to almost white, and clear fluid flows out of the right atrium. Quickly replace with paraformaldehyde, continuing for about five minutes. During this process, the mouse's body may wriggle and its tail may be raised. (Note: When the needle penetrates the ventricular wall, you will feel a distinct "loss of resistance". Avoid using excessive force or a steep insertion angle, as this may puncture the interventricular septum or penetrate both ventricles, potentially damaging the heart.)
[0036] Example 2: Acquisition of the enteric nerve plexus
[0037] 2.1 Obtaining intestines
[0038] Fix the mouse in a supine position. Incise the skin, abdominal muscles, and peritoneum along the midline of the abdomen to expose the abdominal cavity. Gently separate the abdominal fat and mesentery, avoiding damage to the colon and adjacent organs (such as the bladder and genitals). The colon is located on the dorsal side of the abdominal cavity, originating in the right iliac fossa (connecting to the cecum) and ending at the level of the third sacral vertebra (connecting to the rectum). Lift the terminal cecum or ileum with forceps, free the colon along the mesentery, and sever its connection to surrounding tissues. The duodenum is located in the right abdominal cavity, is deep red in color, connects to the pylorus of the stomach, and is closely attached to the pancreas posteriorly. The openings of the common bile duct and pancreatic duct are visible on its surface. Using the stomach and cecum as landmarks, the jejunum is located between the duodenum (below the stomach) and the ileum (above the cecum). Usually, the proximal duodenum (5-10 mm from the pylorus) is taken to the beginning of the jejunum (anterior to the ligament of Treitz). Draw paraformaldehyde into a 5 ml syringe, insert the needle into the intestine, and repeatedly flush several times until all intestinal contents are expelled. Immerse in paraformaldehyde and store at room temperature overnight. The next day, replace with 30% sucrose for at least 2 hours to fix.
[0039] 2.2 Tear off the myenteric plexus
[0040] Combination Figure 1-3 After rinsing the selected intestine thoroughly with 0.1% Triton X-100 PBS (a PBS solution containing 0.1% Triton X-100 by weight), carefully remove the mesentery, fat, and Pasch's lymph nodes attached to the jejunum using forceps and scissors. Avoid damaging the intestinal wall. Cut a segment of intestine approximately 5 mm vertically with scissors. Then, under a dissecting microscope, lay the intestinal mass flat on a glass slide and add a drop of 0.1% Triton X-100 PBS solution to protect the mass.
[0041] When dissecting the colon, the smooth surface of forceps is used to fix the intestinal mass, and then the muscle tissue is dissected from the center of the intestinal mass outwards along the direction of the circular muscles. This systematic manipulation exposes the myenteric plexus located between the longitudinal and circular muscles, thus obtaining a complete full-thickness slice specimen. Throughout the process, care should be taken to control the manipulation force appropriately to ensure tissue integrity.
[0042] When separating the duodenum, jejunum, and ileum, first lay the intestinal slice flat on a glass slide, then gently tear off a corner of the slice with forceps (be careful to maintain an appropriate distance between the forceps and the slide to prevent tearing off the intermuscular nerve plexus along with it). When the transparent intermuscular nerve plexus and longitudinal muscle are exposed at one corner of the intestinal slice, fix the transparent part with the back of the forceps, and hold another forceps in your right hand to ensure that the forceps are gripping the outermost serosa or longitudinal muscle, not the deep tissue. The gripping point should be small, and then slowly separate along the direction of the circular muscle.
[0043] Cleaning the myenteric plexus: Soak the torn intestinal pieces in 0.1% Triton X-100 PBS solution and wash on a shaker (80-90 rpm). Repeat the washing three times, 10 minutes each time, replacing the 0.1% Triton X-100 PBS solution each time.
[0044] 2.3 Immunofluorescence chemical tissue staining
[0045] Block for 1 hour with blocking solution. Prepare 600 μL of blocking solution per sample, containing 0.3% Triton X-100 in PBS and 5% normal serum (e.g., sheep serum). Place the samples on a shaker (40-55 rpm) for 1 hour. After blocking, incubate with primary antibody: prepare a solution containing 0.3% Triton X-100 in PBS, 5% normal serum, and a 1:1000 dilution of primary antibody (i.e., primary antibody incubation solution, specifically a PBS solution containing 0.1% primary antibody, 0.3% Triton X-100, and 5% sheep serum). Incubate overnight at 4°C. The next day, the slides were washed again, followed by secondary antibody incubation: the solution contained 0.3% Triton X-100 in PBS, 5% normal serum, and a 1:1000 dilution of secondary antibody (i.e., secondary antibody incubation solution, specifically a PBS solution containing 0.1% secondary antibody, 0.3% Triton X-100, and 5% sheep serum). The samples were placed on a shaker (80-90 rpm) and incubated in the dark for 2 hours. The washing process was repeated. The washed intestinal tissue was then placed on a glass slide, taking care to avoid tissue overlap and air bubbles. After the slide was dry (using a hairdryer to dry the bottom of the slide can accelerate drying), approximately 20 μL of DAPI was pipetted evenly onto the tissue, and then a coverslip was slowly placed on top to complete the mounting.
[0046] Comparative Example 1
[0047] The intestinal nerve plexus of the colon was obtained in the same way as in Example 2, except that the mice were euthanized.
[0048] Comparative Example 2
[0049] The method of killing the mice was the same as in Example 1, except that when tearing apart the myenteric plexus, specifically when separating the colon, the smooth surface of the intestinal block was used to fix the intestinal block, and then the muscle tissue was peeled off from the center of the intestinal block to both sides along the longitudinal muscle direction.
[0050] Results analysis:
[0051] 1. First observe Figure 4-5As can be seen, in the enteric nerve plexus tissue torn off in Example 2, the reticular structure of the duodenum, jejunum, ileum and colon remained intact and did not show obvious damage;
[0052] Furthermore, the fluorescence images of tyrosine hydroxylase antibodies at the four locations mentioned above show that TH-positive neurons are well distributed in the gut and the neuroendocrine network is not damaged.
[0053] 2. Further observation Figure 6-7 It is evident that, during the process of tearing apart the intestinal nerve plexus of euthanized mice, visible ruptures were observed in the intestinal fragments.
[0054] When the muscle is torn vertically, that is, along the longitudinal direction, some muscle tissue shows visible adhesions and fragmentation.
[0055] Furthermore, further observation Figure 8-10 It is evident that when the method for obtaining the colonic nerve plexus described in Example 2 is further applied to pigeons, pigs, and chickens, no visible fragmentation is found in the torn intestinal tissue.
Claims
1. A method for preparing an enteric plexus patch, characterized in that, Includes the following steps: Step 1: The intestinal donor is killed, and then intestinal tissue segments from the duodenum, jejunum, ileum or colon are taken, fixed with paraformaldehyde and then dehydrated to obtain dehydrated intestinal tissue segments; Step 2: Rinse the dehydrated intestinal tissue segment and remove the mesentery, fat and Paisley's lymph nodes. Then cut the intestinal tissue segment into pieces of about 4-6 mm in length. Place the intestinal pieces flat on a glass slide, add Triton X-100 PBS solution, and then use the smooth side of forceps to fix the intestinal pieces. Then peel the muscle tissue along the direction of the circular muscle to obtain an intestinal slice with the myenteric plexus. Step 3: Wash the intestinal slices containing the myenteric plexus, then stained them with fluorescence and mounted them to obtain mouse enteric plexus patches.
2. The method for preparing the enteric plexus patch according to claim 1, characterized in that, The intestinal donor is a mouse, pigeon, pig, or chicken.
3. The method for preparing the mouse enteric plexus patch according to claim 1, characterized in that, The intestinal donor is a mouse, and the method of killing the mouse specifically includes the following steps: Step A1: Anesthetize the mouse and cut open its thoracic cavity; Step A2: Insert the syringe into the left ventricle at an angle parallel to the horizontal axis of the heart and infuse physiological saline until clear physiological saline flows out of the right atrium. Then, continuously infuse paraformaldehyde into the left ventricle for 5 minutes to obtain lethal mice.
4. The method for preparing the mouse enteric plexus patch according to claim 1, characterized in that, The Triton X-100 PBS solution is specifically a PBS solution containing 0.1% Triton X-100 by mass.
5. The method for preparing the mouse enteric plexus patch according to claim 1, characterized in that, Step 3 specifically involves: washing the intestinal slices containing the myenteric plexus with Triton X-100 PBS solution, blocking the washed intestinal slices containing the myenteric plexus with blocking solution, incubating with primary antibody and secondary antibody in sequence after blocking, transferring them to a glass slide and fluorescently staining and mounting them to obtain mouse intestinal plexus patches.
6. The method for preparing the mouse enteric plexus patch according to claim 5, characterized in that, The blocking solution is a PBS solution containing 0.3% Triton X-100 and 5% sheep serum by mass. During the primary antibody incubation process, the primary antibody incubation solution is a PBS solution containing 0.1% primary antibody, 0.3% Triton X-100, and 5% sheep serum. During the secondary antibody incubation process, the incubation solution was a PBS solution containing 0.1% secondary antibody, 0.3% Triton X-100, and 5% sheep serum.
7. A mouse enteric plexus patch, characterized in that, The mouse enteric plexus patch was prepared by any of the methods described in claims 1-6.