Modeling method of small animal benign esophageal stenosis model

Abstract

The invention relates to the field of clinical medicine, in particular to a modeling method of a small animal benign esophageal stenosis model, which comprises the following steps: preparing an experimental small animal and endoscope equipment; after a New Zealand rabbit is anesthetized, an animal endoscope is used for entering the esophagus of the New Zealand rabbit, the predicted damaged mucous membrane length (4 cm) is marked at the position 15-20 cm away from the incisor, and a snare is used for conducting electrocoagulation on the whole circumference of the esophagus between the marked points; endoscopic and esophageal radiography examination is carried out 21 days after the modeling operation, the esophagus at the modeling injury part is obviously narrow, and the New Zealand rabbit benign esophageal stenosis model is obtained. According to the modeling method of the small animal benign esophageal stenosis model, clinical esophageal stenosis after an ESD operation can be well simulated, and the blank of small animal models of esophageal stenosis after the ESD operation is filled up. The modeling method using small animals has the characteristics of low cost, convenience in experimental operation and observation and the like, and can be used for exploring related mechanisms and prevention research of ESD postoperative esophageal stenosis.

Description

Technical Field

[0001] The invention relates to the technical field of animal models, and in particular to a modeling method for a small animal benign esophageal stenosis model. Background Art

[0002] Benign esophageal stenosis is often caused by endoscopic submucosal dissection (ESD) for large esophageal lesions, postoperative anastomotic stenosis, chemical corrosion, radiation damage, or esophageal sphincter ring (Schatzki ring). ESD can remove the lesion in one piece and clearly evaluate the histological properties of the lesion and the resection margin of the postoperative specimen, while maintaining the original organ anatomical structure and physiological function. The quality of life after surgery is greatly improved compared with surgery. It has the advantages of less trauma, faster recovery, and good clinical efficacy. It has become the first-line treatment for early esophageal cancer recommended by domestic and foreign guidelines. However, ESD for large-area early esophageal cancer is very likely to be complicated by esophageal stenosis. The incidence of stenosis after ESD for large-area early esophageal cancer is 56% to 76%, while the stenosis rate after full circumferential lesions is as high as 100%. The most common symptom of patients with esophageal stenosis after ESD is dysphagia, often accompanied by reflux, pain behind the sternum, and weight loss, which greatly reduces the quality of life of patients.

[0003] Regarding the prevention of benign esophageal stricture, relevant research at home and abroad still focuses on the stricture after ESD of large esophageal lesions, but there is no unified standard for how to prevent esophageal stricture after ESD. At present, endoscopic balloon dilatation, stent implantation and hormones are the main clinical treatment methods. However, endoscopic balloon dilatation can only temporarily relieve symptoms, and most patients need repeated and frequent dilatation, which may increase the risk of complications such as bleeding and perforation; stent implantation has the risk of granulation tissue hyperplasia and stent displacement; hormones have strong anti-inflammatory effects and are economical, but oral hormones have systemic medication-related side effects, and local injection of hormones has the risk of esophageal perforation, mediastinal abscess, etc., and both oral and local injection of hormones will affect wound healing and are basically ineffective for esophageal stricture after full-circumference ESD. Therefore, further clarifying the molecular mechanism of esophageal stricture after ESD is crucial to finding new therapeutic targets, and animal models are a very important link in testing new treatment options.

[0004] Since esophageal stenosis after ESD is a complication of endoscopic treatment, in order to fully simulate the clinical situation and achieve similar esophageal injury and stenosis formation effects, large animals (pigs, dogs, etc.) are currently used to establish esophageal stenosis models, so as to study the mechanism of esophageal stenosis after ESD and test various methods for preventing and treating esophageal stenosis. However, the use of large animals for experiments has many defects, such as high costs for purchase, feeding, environment, labor, and relative lack of molecular detection methods. Therefore, it is necessary to explore a simpler and more reliable esophageal stenosis model to promote the development of prevention and treatment methods for esophageal stenosis after ESD. In contrast, small animals can overcome the above shortcomings. Summary of the invention

[0005] In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a modeling method for a small animal benign esophageal stenosis model, which is used to solve the problem of the lack of a small animal model of esophageal stenosis after ESD surgery in the prior art. The present invention provides a modeling method for a small animal benign esophageal stenosis model, which can better simulate the formation of esophageal stenosis after ESD surgery in small animals, filling the gap of the current lack of a small animal model of esophageal stenosis after ESD surgery.

[0006] To achieve the above-mentioned object and other related objects, the first aspect of the present invention provides a method for modeling a small animal benign esophageal stenosis model, comprising the following steps:

[0007] Step 1: Fast the New Zealand rabbits for 24 hours, prepare their skin and weigh them on the day of modeling, perform intravenous anesthesia, insert an endoscope through the mouth under general anesthesia, and insert the snare into the esophagus through the endoscope accessory channel;

[0008] Step 2: Mark the estimated length of damaged mucosa of 2 to 8 cm on the esophageal mouth and anus sides 10 to 20 cm away from the incisors of the New Zealand rabbit;

[0009] Step 3: Use the snare to move up and down at the marked position to perform circular cauterization of the entire circumference of the esophagus, with a cauterization length of 2 to 8 cm and a snare power of 20 to 60 W; after the operation is completed, observe whether there is obvious bleeding or perforation in the esophagus, and then inhale and withdraw the endoscope, i.e., the New Zealand rabbit model;

[0010] Step 4: Transfer the modeled New Zealand rabbits to the animal room and use a blood oxygen saturation monitor to observe vital signs until they wake up from anesthesia; fast for 12 to 36 hours after modeling, and inject anti-infection drugs for 1 to 5 consecutive days; continue to feed the rabbits until endoscopic observation shows that the inner wall of the esophagus is intact and narrow, thus obtaining a New Zealand rabbit benign esophageal stricture model.

[0011] In one embodiment of the present invention, the New Zealand rabbits in step 1 weigh 1 to 3 kg, and the New Zealand rabbits are fasted for 15 to 30 hours before the operation;

[0012] In the fourth step, the New Zealand rabbits are fasted for 20 to 30 hours after modeling, and are injected with anti-infection drugs for 2 to 4 consecutive days.

[0013] In one embodiment of the present invention, the New Zealand rabbits in step 1 weigh 1.5 to 2.5 kg, and the New Zealand rabbits are fasted for 22 to 26 hours before the operation;

[0014] In the fourth step, the New Zealand rabbits are fasted for 22 to 26 hours after modeling, and are injected with anti-infection drugs for 2.5 to 3.5 consecutive days.

[0015] In one embodiment of the present invention, the marking position of step 2 is: marking 2 to 6 cm on the esophageal mouth and anus side 15 to 20 cm away from the incisor of the New Zealand rabbit;

[0016] The burning length of step three is 2-6 cm, and the snare power is 30-50 W.

[0017] In one embodiment of the present invention, the marking position of step 2 is: 3 to 5 cm on the esophageal mouth and anus side 15 to 20 cm away from the incisor of the New Zealand rabbit;

[0018] The burning length of step three is 3-5 cm, and the snare power is 35-45 W.

[0019] In one embodiment of the present invention, the marking position of step 2 is: 4 cm is marked on the esophageal mouth and anus side 15 to 20 cm away from the incisor of the New Zealand rabbit;

[0020] The burning length of step three is 4 cm, and the snare power is 40 W.

[0021] In one embodiment of the present invention, the anesthetic drug in step 1 is at least one of urethane, sodium pentobarbital, propofol, sodium thiopental, and valproate;

[0022] The anti-infection drug in step 4 is ceftriaxone sodium or penicillin sodium.

[0023] In one embodiment of the present invention, the anesthetic drug in step one is Zotaxel; and the anti-infective drug in step four is ceftriaxone sodium.

[0024] As described above, the modeling method of a small animal benign esophageal stenosis model of the present invention has the following beneficial effects:

[0025] The postoperative periodic endoscopy, esophageal barium angiography and pathological results of the present invention can illustrate the success of the esophageal stenosis model, which can better simulate the formation of esophageal stenosis after ESD surgery in small animals, filling the gap of the current small animal model of esophageal stenosis after ESD surgery. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The present invention provides steps for the method of modeling a small animal benign esophageal stenosis model.

[0027] Figure 2 Observation of the general condition of the model rabbits.

[0028] Figure 3 Endoscopic manifestations of the esophageal injury and repair process in rabbit models at different time points.

[0029] Figure 4 Pathological manifestations of esophageal injury and repair process in rabbit models at different time points.

[0030] Figure 5 Endoscopic examination and iodized oil contrast examination of the esophagus 3 weeks after surgery showed obvious stenosis of the esophagus at the modeling site.

[0031] Figure 6 HE and Masson staining images of normal esophageal tissue and stricture tissue. DETAILED DESCRIPTION

[0032] The following is a description of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0033] 1. Modeling

[0034] A method for modeling a small animal benign esophageal stricture model, comprising:

[0035] 1. Animal: New Zealand rabbit, about 2 kg;

[0036] 2. Drugs: Shutai, ceftriaxone sodium;

[0037] 3. Modeling steps:

[0038] S1. Purchase 8 New Zealand rabbits weighing about 2 kg and bring them into the room one week in advance to acclimate to the environment;

[0039] S2, 1 day before the surgical model establishment (Day-1), New Zealand rabbits were fasted for 24 hours;

[0040] S3. On the day of modeling (day 0), the rabbits were skinned and weighed, and intravenous anesthesia was performed with Zota (0.1 ml / kg). During anesthesia, the rabbits' respiratory rate and rhythm, corneal reflex and muscle relaxation were observed, and the dose was adjusted according to the anesthesia state. Oxygen was inhaled through a mask to increase the oxygen concentration inhaled by the rabbits.

[0041] S4. Under general anesthesia, the New Zealand rabbits were placed in the left lateral decubitus position, and electrodes were attached to the prepared skin. The oral cavity was opened with a homemade rabbit-specific tooth pad. An experienced digestive endoscopist slowly inserted an ultrafine animal endoscope through the oral cavity. After excluding esophageal lesions that may interfere with the research results, the snare was sent into the middle esophagus (about 15 to 20 cm away from the incisors) through the endoscope accessory channel. The oral and anal sides of the middle esophagus were marked respectively. The estimated length of the damaged mucosa was 4 cm.

[0042] S5. Use the snare to move up and down at the marked position to perform circular cauterization of the entire circumference of the esophagus. The cauterization length is about 3 to 4 cm. The power of the snare is 25 W. After the operation is completed, observe whether there is obvious bleeding or perforation in the esophagus, and then inhale and withdraw the endoscope.

[0043] S6. After the endoscopic operation, transfer the New Zealand rabbit to a quiet and warm animal room and observe its vital signs until it wakes up from anesthesia.

[0044] S7. After modeling, fast for 24 hours, give 20% sugar water to supplement energy, and then transition from liquid diet to normal diet. After modeling, intramuscular injection of ceftriaxone sodium is performed for 3 consecutive days for preventive anti-infection. Continue to feed until the esophageal mucosa is completely healed and stenotic by endoscopy, and the New Zealand rabbit benign esophageal stenosis model is obtained.

[0045] 2. Verification of the effect of animal models

[0046] 1. Evaluation indicators of New Zealand rabbit benign esophageal stricture model

[0047] 1.1 General Observations

[0048] After modeling, the animal's spirit (activity), diet, drinking water, etc. were observed every day, and the body weight was measured and recorded every 3 days. If the animal was found dead, it was quickly dissected, tissue samples were collected, and the cause of death was recorded. If it can be clearly determined that the animal's death was caused by drugs, the abnormal organs were photographed during the dissection and samples were kept and fixed.

[0049] 1.2 Endoscopic examination

[0050] Endoscopic examinations were performed on days 3, 7, 14, and 21 after modeling, and photographs were taken to record the esophageal wound repair and esophageal stenosis.

[0051] 1.3 Esophageal radiography

[0052] Esophageal angiography was performed on the 21st day after modeling to evaluate the esophageal stenosis of the modeling rabbits. After the rabbits were anesthetized with Zota, they were fixed in a supine position on a digital subtraction angiography examination bed, a gastric tube was inserted through the mouth (about 10 cm away from the incisors), and the gastric tube was fixed with tape. Iohexol injection was slowly injected, and the film was exposed at the same time.

[0053] 1.4 Collection of materials

[0054] On the 21st day after modeling, after endoscopic and esophageal angiography examinations, the rabbits were euthanized, the chest organs were exposed, the tissues around the esophagus were observed and the esophagus was separated, the middle and lower esophagus (including the narrow esophagus) was removed, rinsed with saline, placed on a clean towel, the esophagus was cut open along the longitudinal axis, and the esophageal tissue at the lesion was taken for pathological examination.

[0055] 1.5 Esophageal histopathology

[0056] The resected esophageal specimens were fixed in 10% formalin buffer for 24 hours, cut along the long axis and embedded in paraffin, and the paraffin blocks were sliced ​​at a thickness of 4 μm and stained with HE and Masson.

[0057] 2. Verification of the effect of New Zealand rabbit benign esophageal stricture model

[0058] 2.1 Construction of esophageal stenosis model by endoscopic electrocoagulation

[0059] Figure 1 Flow chart of establishing a rabbit esophageal stricture model by endoscopic electrocoagulation. There were 8 New Zealand rabbits in each experimental group and control group, respectively called the modeling group and the control group. All the modeling groups underwent the above-mentioned operation steps, while the control group only underwent endoscopic examination. Figure 1 It can be seen that before the operation, the esophageal mucosa of the New Zealand rabbits was smooth, and the lumen was normal without stenosis; during the operation, a snare was used to perform electrocoagulation and cauterization on the marked segment of the esophagus to damage the mucosa and submucosa, achieving the effect of simulating clinical ESD.

[0060] 2.2 General observation of model rabbits

[0061] After modeling, we observed the general condition of the New Zealand rabbits, including survival rate, body weight, activity (spirit), diet, and drinking water. Since the control group rabbits only underwent endoscopic examination, there was no death before and after the experiment, and their body weight increased in a normal trend, and there were no significant changes in activity, diet, and drinking water. In the first week after esophageal injury, the modeling rabbits were listless, their activity decreased, their diet and drinking water decreased, and their body weight showed a downward trend; in the second week, their spirit and diet improved slightly, but due to the gradual formation of esophageal stenosis, they showed a trend of decreased activity and reduced food intake in the third week. In addition, one animal died of esophageal perforation and mediastinal inflammation on the 5th day after modeling, and two animals died on the 13th and 20th days after modeling, respectively. After autopsy, it was found that the esophagus above the modeling site was significantly dilated, and a large amount of food residues were retained, which was considered to be caused by esophageal stenosis. The overall survival rate of the modeling rabbits was 62.5% (5 / 8). Figure 2 shown.

[0062] 2.3 Endoscopic and pathological manifestations of esophageal injury repair in rabbit models

[0063] The esophageal mucosa of the rabbits in the control group was smooth before and after the experiment, and the lumen was normal without stenosis. The esophageal mucosa of the modeling rabbits was slightly white and edematous after electrocoagulation cauterization; on the third day after modeling, endoscopic review showed the formation of annular ulcers on the esophageal wound surface, and the surface was covered with white fur; on the seventh day after modeling, the white fur at the edge of the wound surface became thinner, and gradually formed granulation tissue with rich blood flow; on the 14th day after modeling, the epithelial tissue at the edge of the wound surface proliferated, and the esophagus was obviously narrowed, and the ultra-fine endoscope could not pass smoothly; on the 21st day after modeling, the mucosal defect was almost covered with uniform epithelial tissue, and esophageal stenosis was formed ( Figure 3 The endoscopic manifestations of the esophageal injury repair process in the above-mentioned rabbit model were basically consistent with the clinical findings.

[0064] On days 0, 3, 7, 14, and 21 after modeling, we collected esophageal tissue from the injured area and performed HE staining to observe the pathological manifestations of the esophageal injury and repair process in the modeled rabbits. On the day of modeling (Day 0), the vascular permeability of the esophageal tissue changed after electrocoagulation and cauterization, and a large number of red blood cells infiltrated; on day 3 after modeling, the submucosal layer was infiltrated by inflammatory cells such as neutrophils, lymphocytes, and macrophages; on day 7 after modeling, the number of inflammatory cells decreased, granulation tissue proliferated, accompanied by a large number of fibroblast proliferation and new blood vessel formation, and regenerated squamous epithelium was seen at the edge, migrating from the edge of the wound to the center; on day 14 after modeling, epithelial cells further regenerated, the inner circular muscle of the muscularis propria was destroyed and replaced by fibrous components; on day 21 after modeling, the wound surface was almost covered with uniform epithelial tissue, the submucosal layer was replaced by collagen fibers, and a large number of fibroblasts were seen arranged in a wavy shape ( Figure 4 The above pathological manifestations of the esophageal injury repair process are basically consistent with the clinical situation.

[0065] 2.4 Successful construction of rabbit esophageal stenosis model

[0066] Endoscopic review and esophageal angiography on the 21st day after modeling showed that the injured segment of the esophagus was significantly narrowed and the esophagus above was significantly dilated ( Figure 5 The esophageal tissues of the control group and the model group were fixed and stained with HE and Masson. The results showed that the normal esophageal tissue structure was observed in the control group, while the esophagus of the model group was significantly thickened, and a large amount of collagen fiber deposition was observed in the submucosal layer ( Figure 6 ), which is highly consistent with the staining results of normal human esophageal tissue and stricture tissue after ESD, further illustrating the reliability of our esophageal stricture model.

[0067] In summary, the postoperative staged pathological results of the present invention can illustrate the success of the esophageal stenosis model, which can better simulate the formation of benign esophageal stenosis after ESD surgery in small animals, filling the gap of the current small animal model of benign esophageal stenosis after ESD surgery. Therefore, the present invention effectively overcomes the various shortcomings in the prior art and has a high industrial utilization value. The above embodiments are only illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with this technology can modify or change the above embodiments without violating the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A method for modeling a small animal benign esophageal stenosis model, characterized in that: The steps include: Step 1: After fasting for 12 to 36 hours, New Zealand rabbits are injected with anesthetic drugs. Under general anesthesia, an endoscope is inserted through the mouth, and the snare is sent into the esophagus through the endoscope accessory channel; Step 2: Mark the estimated length of damaged mucosa of 2 to 8 cm on the esophageal mouth and anus sides 10 to 20 cm away from the incisors of the New Zealand rabbit; Step 3: Use the snare to move up and down at the marked position to perform circular cauterization of the entire circumference of the esophagus, with a cauterization length of 2 to 8 cm and a snare power of 20 to 60 W; after the operation is completed, observe whether there is obvious bleeding or perforation in the esophagus, then inhale and withdraw the scope, thus creating the New Zealand rabbit model; Step 4: Transfer the modeled New Zealand rabbits to the animal room and use a blood oxygen saturation monitor to observe vital signs until they wake up from anesthesia; fast for 12 to 36 hours after modeling, give them sugar water to supplement energy, then transition from liquid diet to normal diet, and inject anti-infection drugs for 1 to 5 consecutive days; continue to feed them until endoscopic observation shows that the inner wall of the esophagus is intact and narrow, thus obtaining a New Zealand rabbit benign esophageal stenosis model.

2. The method for modeling a small animal benign esophageal stenosis model according to claim 1, characterized in that: The New Zealand rabbits in step 1 weigh 1 to 3 kg and are fasted for 15 to 30 hours before the operation; In the fourth step, the New Zealand rabbits are fasted for 20 to 30 hours after modeling, and are injected with anti-infection drugs for 2 to 4 consecutive days.

3. The method for modeling a small animal benign esophageal stenosis model according to claim 1, characterized in that: The New Zealand rabbits in step 1 weigh 1.5 to 2.5 kg and are fasted for 22 to 26 hours before the operation; In the step 4, the New Zealand rabbits are fasted for 22 to 26 hours after modeling, and are injected with anti-infection drugs for 2.5 to 3.5 consecutive days.

4. The method for modeling a small animal benign esophageal stenosis model according to claim 1, characterized in that: The marking position of step 2 is: 2 to 6 cm on the esophageal mouth and anus side 15 to 20 cm away from the incisors of the New Zealand rabbit; The burning length of step three is 2-6 cm, and the snare power is 30-50 W.

5. The method for modeling a small animal benign esophageal stenosis model according to claim 1, characterized in that: The marking position of step 2 is: 3 to 5 cm on the esophageal mouth and anus side 15 to 20 cm away from the New Zealand rabbit incisors; The burning length of step three is 3-5 cm, and the snare power is 35-45 W.

6. The method for modeling a small animal benign esophageal stenosis model according to claim 1, characterized in that: The marking position of step 2 is: 4 cm on the esophageal mouth and anus side 15 to 20 cm away from the New Zealand rabbit incisors; The burning length of step three is 4 cm, and the snare power is 40 W.

7. The method for modeling a small animal benign esophageal stenosis model according to claim 1, characterized in that: The anesthetic drug in step 1 is at least one of urethane, sodium pentobarbital, propofol, sodium thiopental, and Zolpidem; The anti-infection drug in step 4 is ceftriaxone sodium or penicillin sodium.

8. The method for modeling a small animal benign esophageal stenosis model according to claim 1, characterized in that: The anesthetic drug in step one is Shutai; the anti-infective drug in step four is ceftriaxone sodium.

Images