A method for constructing an animal model of rabbit cervical insufficiency

Abstract

This invention discloses a method for constructing an animal model of cervical insufficiency, belonging to the field of medical animal model technology, aiming to solve the problem of the lack of a dedicated animal model for cervical insufficiency (CI) in existing research. The method uses healthy female New Zealand rabbits aged 6-8 months and weighing 2.5-3.0 kg in estrus as experimental animals. Individuals in estrus are first selected by vaginal smear method and acclimatized, with fasting for 12 hours before the procedure. Anesthesia is administered via the marginal ear vein with 3% sodium pentobarbital (30 mg / kg). After abdominal opening to expose the cervix, a 12-gauge gavage needle is inserted 1.0-2.0 cm into the cervical canal, and the cervical wall is mechanically scraped 30 times. Then, 0.1 mL of 1 mg / mL lipopolysaccharide solution is injected subserosa at cervical foci at points 4, 8, and 12. The abdomen is closed in layers. Postoperatively, penicillin sodium is injected for anti-infection, and flunixin meglumine for analgesia. The rabbits are then fed as usual. After model establishment, the model was validated through cervical tissue pathological examination (mucosal defects, inflammatory infiltration, smooth muscle rupture), ex vivo cervical tensile testing, and serum inflammatory factor detection (significantly elevated TNF-α and IL-6). This invention employs a combined strategy of "mechanical injury + inflammation induction," along with precise laparotomy and selection of animals in estrus. The model achieves a 100% success rate and a 100% postoperative survival rate, demonstrating excellent stability and reproducibility. It can be used for research on the pathogenesis of cervical infarction (CI) and for evaluating the effectiveness of treatment interventions.

Description

Technical Field

[0001] This invention relates to the field of medical animal model construction technology, specifically to a method for constructing an animal model of cervical insufficiency using New Zealand rabbits in estrus as experimental animals through a combination of mechanical scraping and drug injection. Background Technology

[0002] Cervical insufficiency (CI) is a pathological condition unique to pregnant women. Its core pathological feature is damage to the cervical tissue structure and functional decompensation, manifesting as premature cervical dilation, which can lead to serious adverse pregnancy outcomes such as mid-trimester miscarriage and late-trimester preterm birth, posing a significant threat to maternal and infant health. To deeply understand the pathogenesis of CI and explore effective prevention and treatment methods, constructing a stable, reliable animal model that closely reflects clinical pathophysiological characteristics is a crucial prerequisite.

[0003] Currently, research on cervical insufficiency both domestically and internationally still faces the technical bottleneck of a lack of animal models. Clinical studies have confirmed that mechanical damage to the cervix (such as childbirth trauma, cervical surgical procedures, etc.) is one of the key causes of cervical structural damage and functional weakening, thereby inducing cervical insufficiency (CI). From a pathological correlation perspective, the ultimate harm of cervical insufficiency (miscarriage / premature birth) is consistent with the outcome of animal models of premature birth / miscarriage, and therefore can be used as a model reference. However, existing model construction methods (such as single mechanical damage or simple inflammation induction) have significant limitations: mechanical damage caused solely by cervical dilation or surgical incision cannot simulate the local inflammatory microenvironment often associated with clinical CI; while induction using inflammatory factors such as lipopolysaccharide (LPS) alone is difficult to induce persistent damage to the cervical structure, resulting in poor model stability and low reproducibility. In addition, existing models neglect the influence of the estrous cycle on the physiological state of the cervix in animal selection. The proliferative activity of the cervical mucosa and its sensitivity to damage and inflammation differ significantly in animals at different estrous stages, further exacerbating the variability of experimental results and failing to meet the needs of CI-specific research.

[0004] Based on this, this invention, by referencing the construction ideas of animal models of premature birth or abortion, and combining the core pathological manifestations of cervical insufficiency (cervical structural damage, functional decompensation) and the clear inducing factors (mechanical injury), innovatively proposes a composite modeling strategy of "precise laparotomy + mechanical curettage injury + local lipopolysaccharide induction" on the basis of the modeling logic of animal models of premature birth / abortion. At the same time, it introduces New Zealand rabbits in estrus as experimental animals, simulates clinically relevant cervical injury through mechanical curettage, and introduces inflammatory factors to amplify the cervical pathological response, ultimately achieving a precise simulation of the pathophysiological process of cervical insufficiency, filling the current technical gap of no dedicated animal model for cervical insufficiency, and providing a dedicated experimental tool for cervical insufficiency research. Summary of the Invention

[0005] The purpose of this invention is to provide a method for constructing an animal model of cirrhosis (CI). By combining the synergistic effects of precisely controlled laparotomy, mechanical scraping, and local inflammation induction with the physiological characteristics of New Zealand rabbits in estrus, a highly stable, reproducible animal model that closely resembles the pathological features of clinical CI is constructed, filling the technological gap in dedicated animal models for CI.

[0006] To achieve the above objectives, the present invention adopts the following technical solution, and the parameters of each step have been optimized through pre-experiments to ensure the modeling effect and animal safety: Step 1) Selection of experimental animals: Select healthy female New Zealand rabbits in estrus, aged 6-8 months and weighing 2.5-3.0 kg; Step 2) Grouping: Rabbits were randomly divided into an experimental group and a control group. The experimental group underwent CI model construction, while the control group did not undergo mechanical scraping. The cervix was injected with sterile saline, and the injection volume and location were the same as in the experimental group. Step 3) Preparation of experimental materials and equipment: anesthetic drugs, surgical instruments, syringes, sutures, operating table, iodine, physiological saline, 75% ethanol, gavage injection, lipopolysaccharide solution, gauze, cotton balls, etc. Step 4) Preparation before the experiment: Place the purchased New Zealand rabbits in the above-mentioned standard breeding environment for one week for acclimatization. During this period, they can eat standard rabbit feed and drink water freely. Observe their mental state, food intake and defecation every day, and remove individuals that show abnormalities such as lethargy and loss of appetite. Fasting and water restriction are required before the operation. The skin is prepared and disinfected with iodine.

[0007] Step 5) Anesthesia and Surgical Procedure: Slowly inject 3% sodium pentobarbital into the marginal ear vein. Successful anesthesia is indicated by the disappearance of corneal reflex and the absence of spontaneous limb movement. Maintain the depth of anesthesia using an isoflurane mask. If excessive anesthesia occurs (respiratory rate <20 breaths / min, disappearance of corneal reflex), immediately inject 5 mL of 5% glucose solution intravenously, while simultaneously providing assisted ventilation (30 breaths / min) until vital signs stabilize. Secure the successfully anesthetized rabbit supine on the operating table. Disinfect with iodine solution, drape with a sterile surgical drape, and fully expose the uterus and cervix. Use a gavage needle to mechanically scrape the inner wall of the cervical canal, moving the needle up and down in 0.5-1.0 cm increments. Repeat this 30 times (ensuring damage to the cervical mucosa and superficial muscle layer, but avoiding penetration of the cervical wall). After scraping, select the 4 o'clock, 8 o'clock, and 12 o'clock positions on both sides of the cervix as injection points, injecting 0.1 mL of LPS solution at each point, for a total of 0.3 mL.

[0008] Step 6) Postoperative care: Irrigate the cervix and abdominal operating area, confirm that there are no foreign bodies remaining in the abdominal cavity, suture the surgical incision, disinfect the surgical incision and surrounding area with povidone-iodine after suturing, cover with sterile gauze, and fix with medical tape. Postoperative anti-inflammatory and analgesic treatment is given.

[0009] The present invention is further configured such that, in step 4), the ambient temperature is controlled at 25°C.

[0010] The present invention is further configured such that, in step 5), the dose of sodium pentobarbital is 30 mg / kg, the concentration of isoflurane is maintained at 1%-3%, and the concentration of LPS is 1 mg / mL.

[0011] The present invention is further configured such that: in step 6), 4-0 suture is used to suture the surgical incision.

[0012] The present invention is further configured such that: in steps 4), 5), and 6), the disinfection treatment uses povidone-iodine for comprehensive disinfection and 75% ethanol for deiodination.

[0013] Another objective of this invention is to provide an application of a model constructed using the above-described method for rabbit cervical insufficiency in studying the pathogenesis, pathophysiological changes, and evaluating the effectiveness of related treatment interventions for cervical insufficiency.

[0014] Step 7) Model Validation: Seven days after modeling, the model is subjected to histopathological examination, cervical tension detection and serum inflammatory factor detection. Through multi-dimensional validation, the model is ensured to meet the pathological and functional characteristics of CI.

[0015] This invention employs a combined strategy of "mechanical scraping + local lipopolysaccharide induction," which not only simulates the key etiology of clinical CI (mechanical injury) but also reproduces the local inflammatory microenvironment associated with CI. Furthermore, by combining precise laparotomy procedures, it avoids tissue interference associated with transvaginal procedures. The model is highly consistent with the pathophysiological characteristics of human CI, thus overcoming the shortcomings of existing models that "only simulate the outcome but not the etiology." Excellent stability and repeatability: New Zealand rabbits in estrus were selected as experimental animals to unify the physiological state of the cervix; key parameters such as the number of mechanical curettages (30 times) and the lipopolysaccharide dosage (0.3 mL, 1 mg / mL) were optimized and determined. The success rate of modeling was verified by the examples to be 100%, the postoperative mortality rate of animals was ≤5%, and the experimental results were highly reproducible. Scientific and quantitative detection methods: The innovative use of ex vivo cervical tensile testing (combined with infusion tubing fixation to avoid tissue damage) quantifies cervical functional damage from a biomechanical perspective. Combined with pathological and inflammatory factor detection, a multi-dimensional verification system is formed to ensure the objectivity of model effectiveness assessment. Standardized operation: It specifies experimental materials, operating procedures and parameters in detail, such as anesthetic dosage, incision length, suture specifications, etc. The process is simple and easy to understand, making it easy for experimental personnel to master, reducing the impact of operational differences on experimental results, and is suitable for large-scale experimental research. Beneficial effects

[0016] The model has a strong correlation with clinical practice: This invention uses a combined strategy of "mechanical scraping + local lipopolysaccharide injection" to simultaneously simulate the mechanical damage (such as childbirth trauma and cervical surgery) and local inflammatory microenvironment of clinical CI. Furthermore, it selects animals in estrus (whose cervical mucosal proliferative activity and damage sensitivity are similar to those of women of childbearing age with CI). The pathological characteristics of the model are highly consistent with those of clinical practice.

[0017] High success rate and good stability: After repeated experiments, the modeling success rate of this method reached 100%, and the postoperative survival rate was 100% (no death in the control group). The key parameters (such as the number of scrapings and the lipopolysaccharide dosage) were highly reproducible after optimization, and the model validation indicators of different batches of experiments had small differences (CV value < 10%).

[0018] A robust validation system has been established: breaking through the limitations of traditional single pathological validation, a multi-dimensional validation system of "pathological damage + mechanical properties + inflammatory factors" has been established to comprehensively evaluate the effectiveness of the model and avoid experimental bias caused by misjudgment of a single indicator.

[0019] Highly practical: This model can be used for research on the pathogenesis of cervical infarction (such as the mechanism of cervical smooth muscle cell apoptosis and the regulatory network of inflammatory factors), pathophysiological analysis (such as the degradation pattern of cervical extracellular matrix), and evaluation of the effectiveness of treatment interventions (such as the effectiveness verification of cervical cerclage, anti-inflammatory drugs, and stem cell therapy), covering the needs of basic research and clinical translation. Detailed Implementation

[0020] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the raw materials and reagents used in the following examples can be purchased from conventional biochemical reagent stores or pharmaceutical companies.

[0022] Example: A method for constructing an animal model of rabbit cervical insufficiency, comprising the following steps: Step 1) Selection of experimental animals: Select healthy female New Zealand rabbits, aged 6-8 months, weighing 2.5-3.0 kg. Step 2) Determining estrus: Perform vaginal smears at 9:00 AM daily: Use a sterile cotton swab dipped in physiological saline to insert into the vagina 1-2 cm and rotate to collect the sample. After smearing, stain with Giemsa for 3 minutes. Observe under a microscope. When the proportion of keratinized epithelial cells is ≥80%, white blood cells are ≤3 per high-power field, and the rabbit shows mounting behavior, it is determined to be in estrus. Select rabbits in this state for modeling.

[0023] Step 3) Grouping: Rabbits were randomly divided into an experimental group and a control group. The experimental group underwent CI model construction, while the control group did not undergo mechanical curettage. The cervix was injected with sterile saline, with the injection volume and location the same as the experimental group. All other procedures (anesthesia, disinfection, laparotomy, suturing, etc.) were the same as the experimental group. Step 4) Preparation of experimental materials: Reagents: 3% sodium pentobarbital, isoflurane (for anesthesia), sodium penicillin (for postoperative anti-infection), flunixin meglumine (for postoperative analgesia), 1 mg / mL LPS solution (Escherichia coli O55:B5 strain, prepared with sterile saline), povidone-iodine (for disinfection), sterile saline (for rinsing and tissue protection). Instruments: 12-gauge sterile gavage needle (for cervical curettage), 1mL sterile syringe (for injection), microsurgical instrument kit (including fine forceps, curved scissors, hemostatic forceps), tissue forceps, sterile surgical drape, 4-0 absorbable sutures, electronic balance (for weighing and drug dosage), animal operating table (with limb fixation device), depilatory cream (for skin preparation), sterile cotton balls, electronic universal tensile testing machine (model: WDW-5, accuracy 0.01N, for ex vivo cervical tensile testing); Other: Sterile wet gauze (to protect internal organs during surgery), clean cages (postoperative feeding, temperature 22-25℃, humidity 50-60%).

[0024] Step 5) Pre-experiment preparation: Place the purchased New Zealand rabbits in the above-mentioned standard breeding environment for acclimatization for 1 week. During this period, they can eat standard rabbit feed and drink water freely. Observe their mental state, food intake and defecation every day, and remove individuals that show abnormalities such as lethargy and loss of appetite. 12 hours before the modeling surgery, fast and deprive the rabbits of water. Use an electronic balance to accurately weigh the rabbits and record the data to calculate the dosage of anesthetic drugs. 1 hour before the surgery, use hair removal cream to remove the hair from the lower abdomen to the vulva of the rabbits, and use iodine to preliminarily disinfect the area, followed by 75% ethanol to remove the iodine.

[0025] Step 6) Intraoperative procedures: Anesthesia procedure: Calculate the dosage of 3% sodium pentobarbital based on the rabbit's weight at a dose of 30 mg / kg, and slowly inject it through the marginal ear vein (injection rate 0.5 mL / min); observe the rabbit's corneal reflex and muscle relaxation 5-8 minutes after injection. When the corneal reflex disappears and there is no spontaneous movement of the limbs, the anesthesia is considered successful; maintain the depth of anesthesia with an isoflurane breathing mask.

[0026] Fixation and disinfection: The successfully anesthetized rabbit was fixed supine on the operating table with its limbs secured with restraints and its abdomen facing upward. The lower abdomen to the vulva area was disinfected three times in a progressive manner with iodine solution (from the predetermined incision area outward to the periphery, with a diameter ≥15cm). A sterile surgical drape was then laid, exposing only the midline incision area of ​​the lower abdomen (2-3cm above the pubic symphysis).

[0027] Opening the abdomen to expose the cervix: Make a 5 cm longitudinal incision along the midline of the abdomen, 2-3 cm above the pubic symphysis. First, incise the skin and subcutaneous fat, and bluntly dissect to the anterior sheath of the rectus abdominis muscle using hemostats; cut the anterior sheath along the direction of the rectus abdominis muscle fibers, separate the rectus abdominis muscle bundles, and expose the peritoneum; lift the peritoneum with hemostats and confirm that there are no visceral adhesions, then cut the peritoneum to enter the abdominal cavity. Gently push the small intestine and other visceral organs to the left side of the abdominal cavity with sterile moist gauze to fully expose the uterus and cervix area (the cervix is ​​a light pink cylindrical structure at the junction of the lower end of the uterus and the vagina); gently grasp the connective tissue next to the cervix with tissue forceps (avoid directly grasping the cervical tissue to avoid additional damage), and fix the position of the cervix to facilitate subsequent operations.

[0028] Mechanical curettage of the cervical wall: Holding a 12-gauge sterile gavage needle, slowly insert it into the cervical canal opening to a depth of 1.0-2.0 cm; rotate the gavage needle clockwise at a speed of 10-15 revolutions per minute, while simultaneously moving it up and down along the long axis of the cervical canal (movement range 0.5-1.0 cm), mechanically curetting the inner wall of the cervical canal for a total of 30 times. During the curettage, the force should be controlled by visual observation, aiming for mild congestion of the cervical mucosa without significant bleeding, avoiding excessive damage that could lead to cervical perforation.

[0029] Cervical local lipopolysaccharide injection: After curettage, select one injection point each at the 4, 8, and 12 o'clock positions on the cervix in the subserosal tissue (0.5-1.0 cm from the cervical margin). Use a cotton swab to push aside the surrounding connective tissue. Draw 1 mg / mL lipopolysaccharide solution into a 1 mL syringe and insert it into the injection point at a 45° angle. After confirming no blood return, slowly inject 0.1 mL, for a total of 0.3 mL. After injection, leave the needle in place for 8-10 seconds before withdrawing it. Gently press the injection point with a sterile cotton ball for 10-15 seconds to prevent extravasation or bleeding.

[0030] Closure procedure: Rinse the cervix and abdominal operating area with sterile saline to remove residual medication and tissue debris; reposition internal organs, and after confirming that there are no foreign bodies remaining in the abdominal cavity, suture the peritoneum and anterior rectus abdominis sheath continuously with 4-0 absorbable sutures, and then suture the subcutaneous fat and skin; after suturing, disinfect the surgical incision and surrounding area with povidone-iodine, cover with sterile gauze, and fix with medical tape.

[0031] Step 7) Postoperative care: Anti-infection and analgesia: Immediately after surgery, administer 400,000 U of sodium penicillin per animal via intramuscular injection for 3 consecutive days; simultaneously administer flunixin meglumine 2 mg / kg via intramuscular injection, once on the day of surgery, to relieve pain. Recovery and feeding: Place the rabbit in a warm and quiet cage and closely observe its recovery (usually within 30 minutes after surgery). Before recovery, ensure that the airway is clear. After recovery, resume normal diet and water. Observe the wound healing daily for one week after surgery. If signs of infection such as redness, swelling, or oozing appear, disinfect with iodine solution and administer antibiotics. Remove skin sutures 7 days after surgery.

[0032] Step 8) Model Validation: Seven days after modeling, the model was validated in multiple dimensions to ensure it conformed to the pathological and functional characteristics of CI. Cervical tissue pathological examination: After euthanizing the rabbits, the abdomen was opened through the original surgical incision, and the cervical tissue (including the cervical canal and a small amount of surrounding connective tissue) was completely separated and fixed with 4% paraformaldehyde for 24 hours. After paraffin embedding, 5 μm serial sections were prepared and stained with HE. Microscopic observation was performed to check: ① the integrity of the cervical mucosal epithelium (whether there are defects or shedding); ② inflammatory cell infiltration in the lamina propria (neutrophil count, ≥20 per high-power field is considered positive); ③ smooth muscle fiber arrangement (whether it is disordered or broken). If all three indicators are met, the pathological verification is considered successful.

[0033] Ex vivo cervical tissue tensile testing: Cervical tissue was simultaneously separated, retaining a total length of 2.0 cm. Excess connective tissue was removed, and the tissue was placed in sterile saline at 4℃ (testing to be completed within 30 minutes). The "intermediate thread through infusion tubing" fixation method was used: 2-0 silk thread was passed through the infusion tubing wall in a cross shape (avoiding the cervical tissue), through the cervical canal, and connected to the tensile testing machine clamps. The tensile speed was set to 5 mm / min, and the initial pretension was 0.5 N. The maximum breaking tensile force and elastic modulus were recorded. Model success criteria: Compared with the control group, the maximum force and elastic modulus of the cervical tissue in the model group were significantly reduced, and the difference was statistically significant. P <0.05).

[0034] Serum inflammatory factor detection: 5 mL of peripheral blood was collected via the marginal ear vein before sacrifice, and serum was separated by centrifugation at 3000 rpm for 15 min; serum tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels were detected using ELISA. Model success criteria: Serum TNF-α and IL-6 levels in the model group were significantly higher than those in the normal control group. P < 0.05).

Claims

1. A method for constructing an animal model of cervical incompetence, characterized by, Includes the following steps: (1) Selection of experimental animals: Healthy female New Zealand rabbits in estrus, aged 6-8 months and weighing 2.5-3.0 kg were selected. They were confirmed to be in estrus by vaginal smear test (keratinized epithelial cells account for ≥80%, white blood cells ≤3 per high power field) and had no reproductive system diseases. (2) Preparation of experimental materials: Prepare anesthetic drugs, No. 12 gavage needle, 1mg / mL lipopolysaccharide solution, microsurgical instruments, 4-0 absorbable sutures and tensile testing machine; (3) Preoperative preparation: Animals should be kept in an acclimatized environment for ≥1 week, and fasted and deprived of water for 12 hours before surgery. Skin preparation and disinfection should be performed. (4) Anesthesia and modeling: 3% sodium pentobarbital (30mg / kg) was injected into the ear vein for anesthesia. The abdomen was opened to expose the cervix. The cervical wall was scraped 30 times with a gavage needle. 0.3mL of lipopolysaccharide solution was injected into the 4, 8 and 12 o'clock positions of the cervix. The abdomen was closed in layers (using 4-0 sutures). (5) Postoperative care: intramuscular injection of penicillin sodium for anti-infection, flunixin meglumine for analgesia, and routine feeding; (6) Model validation: The model was validated 7 days after modeling by pathological examination, ex vivo cervical tensile test and serum inflammatory factor test.

2. The method of claim 1, wherein, In step (3), the ambient temperature for adaptive rearing is controlled at 22-25℃ and the humidity at 50-60%.

3. The method of claim 1, wherein, In step (1), the estrus period is determined by vaginal smears: the proportion of keratinized epithelial cells is ≥80%, and the number of white blood cells per high-power field is ≤3. The rabbit shows acceptance of mounting behavior.

4. The method of claim 1, wherein, In step (4), the abdominal incision is a longitudinal incision along the midline of the abdomen, 5 cm long, located 2-3 cm above the pubic symphysis.

5. The method of claim 1, wherein, In step (4), the gavage needle is inserted into the cervical canal to a depth of 1.0-2.0cm, and the rotation speed is 10-15 revolutions per minute during scraping, with an up-and-down movement range of 0.5-1.0cm.

6. The method of claim 1, wherein, In step (4), the concentration of the lipopolysaccharide solution is 1 mg / mL, and it is injected into 3 sides, 0.1 mL on each side, with the injection point 0.5 cm away from the edge of the cervix.

7. The construction method of claim 1, wherein, The pathological examination in step (6) specifically involves taking cervical tissue for HE staining, observing cervical mucosal defects, inflammatory cell infiltration (≥15 inflammatory cells per high-power field), and smooth muscle fiber rupture, and determining that the pathological damage conforms to CI characteristics.

8. The method of claim 1, wherein, In step (6), the tensile test of the ex vivo cervix was performed by fixing it with a thread through the middle of the infusion tube. The test parameters were a tensile speed of 5 mm / min and an initial pretension of 0.5 N. The success criteria for the model were a significant reduction in the maximum force and elastic modulus (P<0.05).

9. The application of the model constructed by the method according to any one of claims 1-8 in the study of the pathogenesis of cervical insufficiency, pathophysiological analysis, and evaluation of the effectiveness of treatment interventions.