Experimental method for molding rat for treating uterine diverticulum

Abstract

The invention relates to the technical field of biology and new medicine, discloses an experimental method for modeling a rat for treating uterine diverticulosis, and aims to overcome the defects that in the prior art, outer longitudinal muscle fibers and inner annular muscle fibers of uterus are not distinguished, only muscular layers are extensively scraped, and the difference between the degree of inflammation under serosal and the degree of inflammation between the muscular layers after clinical operation is difficult to reproduce. According to the method, 11-12-week-old SPF-grade SD female rats are selected, muscle layer fibers are recognized under the assistance of a microscope, outer 2 / 3 longitudinal muscle fibers are preferentially removed, inner 1 / 3 annular muscle fibers are reserved, lipopolysaccharide layered injection is matched, and the model is constructed in combination with standard postoperative treatment. And a reliable animal model can be provided for meverticulum pathological mechanism research and therapeutic schedule evaluation.

Description

Technical Field

[0001] This invention relates to the fields of biology and new medicine, and in particular to an experimental method for establishing a rat model for treating uterine diverticulum. Background Technology

[0002] Uterine diverticulum is a typical complication following uterine trauma such as cesarean section and myomectomy. Due to poor repair of the myometrial tissue at the surgical incision site, a pouch-like depression structure forms in the uterine wall. Clinically, it often manifests as abnormal uterine bleeding, infertility, and other symptoms, significantly impacting the reproductive health of women of childbearing age. Currently, constructing a rat model with pathological characteristics that closely match clinical findings is a key foundation for conducting research on its pathological mechanisms and evaluating treatment options.

[0003] To meet the need for animal models in pathological research and treatment evaluation of uterine diverticula, several rat uterine diverticulum modeling schemes have been disclosed in the existing technology. For example, the rat uterine scar diverticulum model preparation, identification, and histopathological analysis method (announcement number CN116269896A) involves mechanically damaging the uterus of non-pregnant SD female rats, scraping away the endometrium and part of the myometrium, and injecting LPS to induce inflammation, combined with ultrasound microbubble imaging to achieve non-invasive modeling and identification. Another rat uterine scar diverticulum model preparation, identification, and histopathological analysis method (announcement number CN120284517A) involves constructing a pseudopregnancy model through sequential estrogen and progesterone administration, combined with GBS bacterial infection and suture implantation to achieve pregnancy-related uterine diverticulum modeling.

[0004] However, the existing technology still has two areas that need improvement. First, the myometrial damage lacks structural targeting, failing to distinguish between the longitudinal and circular myometrial fibers outside the uterus. It only roughly scrapes away part of the myometrium with a curette, which cannot accurately simulate the core pathological feature of longitudinal myometrial defects in clinical cesarean section incisions. Second, the inflammation induction is not stratified and controlled. It only achieves inflammation by injecting LPS or implanting infected sutures at a single site, which makes it difficult to reproduce the real pathological state of the difference in the degree of inflammation between the subserosal layer and the myometrium after clinical surgery. Summary of the Invention

[0005] The technical problem to be solved by this invention is that the existing technology has the disadvantages of lacking structural targeting for myometrial damage and lacking stratified control for inflammation induction. To address this, we propose an experimental method for rat modeling in the treatment of uterine diverticulum.

[0006] To achieve the above objectives, this application adopts the following technical solution: an experimental method for establishing a rat model for treating uterine diverticulum, comprising the following steps: S1: Selecting 11-12 week old SPF-grade female SD rats and anesthetizing them by intraperitoneal injection of 45 mg / kg sodium pentobarbital; S2: After shaving and disinfecting the lower abdomen of the rats, making a 1 cm longitudinal incision along the midline, and dissecting layer by layer until the bilateral uteri are exposed; S3: Using ophthalmic scissors, cutting open the entire uterine wall in the center of the uterus, and applying sterile gauze to stop bleeding when oozing occurs; S4: After the uterine wall is laid flat, it is examined under a microscope. Using a 20× objective lens, distinguish between the outer longitudinal and inner circular muscle fibers; use a microscopic scale to confirm the thickness of the myometrium. S5: First, remove the endometrium, then remove the outer longitudinal muscle fibers. If the amount of myometrium removed is insufficient, remove a small amount of the inner circular muscle fibers. S6: Take 10 μL of 1 μg / μL LPS solution and inject it into the subserosal and myometrial junction areas using an ultrafine needle. S7: Suture the uterine wall with 7-0 sutures. After returning the uterus to the abdominal cavity, suture the peritoneum and myometrium with 4-0 sutures, and suture the skin intradermally with 5-0 sutures. S8: Immediately after the procedure, administer an intramuscular injection of penicillin for infection control.

[0007] Preferably, in S4, the outer longitudinal muscle fibers are longitudinal muscle fibers arranged parallel to the long axis of the uterus in the outer 2 / 3 of the total thickness of the myometrium from the serosa side to the endometrial side, and the inner circular muscle fibers are circular muscle fibers arranged around the endometrium in the inner 1 / 3 of the total thickness of the myometrium.

[0008] Preferably, in S5, the removal rate of the outer longitudinal muscle fibers is ≥90%, and the retention rate of the inner circular muscle fibers is ≥70%.

[0009] Preferably, in S6, the ultrafine needle has a diameter of 0.4mm-0.6mm, the subserosal injection depth is controlled at 0.1±0.02mm, and the ultrafine needle injection time is ≥30s.

[0010] Preferably, in S6, the muscle layer junction area is the junction between the outer longitudinal muscle fiber removal area and the inner circular muscle fiber, and the injection time with the ultrafine needle is ≥25s.

[0011] Preferably, in S6, the injection dose ratio of the LPS solution in the subserosa and muscularis junction is 4-6:6-4.

[0012] Preferably, in S7, the uterine wall suturing adopts a combination of first aligning 2-4 stitches, then staggering 2-4 stitches, and finally diagonally suturing 2-4 stitches.

[0013] Preferably, in S5, micro-scissors are used to cut the outer longitudinal muscle fibers at a 30° angle, and the micro-scissor blades advance along the direction of the outer longitudinal muscle fibers.

[0014] Preferably, in step S6, after the ultrafine needle has finished injecting, it is left for 10 seconds before being withdrawn to reduce backflow of the medication.

[0015] Preferably, in S8, penicillin is injected intramuscularly immediately after the operation and injected continuously for 3 days.

[0016] The technical effects and advantages of this invention are as follows:

[0017] In this invention, by identifying the longitudinal muscle fibers in the outer two-thirds and the circular muscle fibers in the inner one-third of the uterus, the outer two-thirds longitudinal muscle fibers are preferentially removed using 30° angle microscissors, while the retention rate of the inner one-third circular muscle fibers is controlled, forming a layered myometrial damage scheme. This replicates the core pathological feature of clinical cesarean section incisions, which are mainly characterized by longitudinal myometrial defects. At the same time, the preservation of the inner circular muscle fibers maintains the structural integrity of the uterus, avoiding the irregular damage and uterine perforation risk caused by the rough scraping of the myometrium in existing technologies. This makes the model's pathological features highly consistent with clinical reality, providing a more reliable structural basis for the evaluation of subsequent treatment plans.

[0018] In this invention, LPS is injected in different locations using an ultra-fine needle, precisely injected into the subserosal region of the uterine wall and the junction of the outer longitudinal muscle fiber removal area and the inner circular muscle fiber area. By controlling the injection depth and injection time, stratified inflammation is induced, replicating the real pathological state of the difference in the degree of inflammation between the subserosal region and the myometrium after clinical surgery. This provides a realistic pathological environment for evaluating the regulatory effect of treatment on inflammation in different areas. Attached Figure Description

[0019] The disclosure of this invention is illustrated with reference to the accompanying drawings. It should be understood that the drawings are for illustrative purposes only and are not intended to limit the scope of protection of this invention. In the drawings, the same reference numerals are used to refer to the same parts:

[0020] Figure 1 This is a schematic flowchart of the experimental method of the present invention;

[0021] Figure 2 This is a pathological section of the uterus of a rat in the normal control group of the present invention;

[0022] Figure 3 A pathological section of the uterus of the rats in the model group of this invention;

[0023] Figure 4 for Figure 3 (c) Enlarged view of the modeling diverticulum area;

[0024] Figure 5 for Figure 3 Enlarged view of the diverticulum area in the middle (d). Detailed Implementation

[0025] It is easy to understand that according to the technical solution of the present invention, without changing the essential spirit of the present invention, those of ordinary skill in the art can propose various interchangeable structural ways and implementation ways. Therefore, the following specific embodiments and drawings are only illustrative descriptions of the technical solution of the present invention, and should not be regarded as the whole of the present invention or regarded as a limitation or restriction on the technical solution of the present invention.

[0026] The present invention provides an experimental method for rat modeling for treating uterine diverticulum. All the instruments used in the experiment are commercially available standardized equipment, and the corresponding models are shown in Table 1, which can meet the requirements of subsequent processing of uterine tissue, pathological morphological observation and quantitative analysis;

[0027]

[0028] Table 1

[0029] The reagents used in the experiment of the present invention and their production units are shown in Table 2. All reagents meet the standards of animal experiments and histopathological examinations, and can exclude the interference of non-reagent factors on the modeling results;

[0030]

[0031] Table 2

[0032] The present invention selects Sprague-Dawley (SD) female rats at the specific pathogen free (SPF) level, which can exclude the interference of microbial infections such as mycoplasma and viruses on uterine pathological changes;

[0033] Ten SD female rats were received before the experiment, 7-8 weeks old. After adaptive observation and health screening, no abnormalities were confirmed;

[0034] In the experimental stage, SD female rats at 11-12 weeks old were actually used. The uterine muscle layer of rats at this age has developed maturely, and the thickness is stable at 0.2-0.3 mm. The pathological repair response to uterine trauma is highly similar to that of the uterus of clinical women of childbearing age;

[0035] It should be noted that all experimental rats were purchased from the Guangdong Provincial Center for Medical Laboratory Animals, with the animal production license number SCXK (Guangdong) 2022-0002 and the animal certificate number 44007200126509, which is convenient for tracing the legality of the animal source and the health status;

[0036] The individual identification of experimental rats adopts a dual numbering method of bromocresol green hair staining combined with cage labels. The individual numbers are distinguished by painting bromocresol green spot combinations on different parts of the rat body surface. At the same time, labels are pasted on the outside of the breeding cages, marking the rat number, the date of entering the cage and the experimental group to avoid confusion;

[0037] All rats were quarantined and observed for 3 days after receipt. Their mental state, food intake, defecation and body surface condition were recorded at 9:00 am every day. Individuals that did not show signs of lethargy, loss of appetite, diarrhea or abnormal body surface were included in subsequent experiments.

[0038] Five rats were housed in each box for the experiment. The animal room environment was kept stable, with the temperature controlled at 22±2℃ and the relative humidity at 50%±5%. A 12h:12h day and night intermittent lighting system was adopted, with light from 8:00 to 20:00 and darkness from 20:00 to 8:00 the next day. Rats were allowed free access to irradiated sterilized standard rat feed.

[0039] Ten rats were used as the normal control group in the experiment. The procedure was as follows:

[0040] C1: Rats were anesthetized by intraperitoneal injection of 45 mg / kg sodium pentobarbital. It was confirmed that the rats' corneal reflex was absent and that they did not respond to pinching pain in their hind limbs.

[0041] C2: The rat was fixed in a supine position on a 37℃ constant temperature operating table. After the hair on the lower abdomen was shaved and disinfected, a 1cm longitudinal incision was made along the midline of the lower abdomen. The skin, subcutaneous fat and anterior sheath of the rectus abdominis muscle were cut layer by layer. The rectus abdominis muscle was bluntly dissected to expose the bilateral uterus.

[0042] C3: Without damaging the uterus, the uterus is directly returned to the abdominal cavity, and the peritoneum and anterior rectus sheath are intermittently sutured with 4-0 non-absorbable sutures, and the skin is sutured intradermally with 5-0 non-absorbable sutures.

[0043] C4: Immediately after surgery, administer penicillin intramuscularly for 3 consecutive days to fight infection. Keep the feeding conditions the same as before surgery.

[0044] Three weeks later, the tissue specimens underwent standard processing. Rats were injected intraperitoneally with 90 mg / kg sodium pentobarbital. Death was confirmed after respiratory and cardiac arrest. The peritoneum was dissected, and intact bilateral uterine tissue was removed. The uterine wall was fixed and trimmed to preserve the entire thickness. The tissue was soaked in distilled water for 24 hours to remove residual fixative, with the water changed every 6 hours. It was then dehydrated three times with 95% ethanol every 30 minutes, followed by two more dehydrations with anhydrous ethanol every 30 minutes. Finally, it was soaked twice with an environmentally friendly clearing agent every 20 minutes. The cleared tissue was then transferred to a 60°C constant temperature environment and infiltrated with histological paraffin for 3 hours, turning the tissue every hour to ensure thorough infiltration. LEICA was used for further processing. ® Paraffin blocks were prepared using the EG1150 embedding mechanism and labeled with experimental groups and rat numbers;

[0045] After the paraffin blocks are prepared, use LEICA ® The RM2235 microtome cut the material into 4μm thick continuous sections, and the sections were placed in a LEICA microtome. ®Flatten the sections on an HI1210 slide spreader, remove wrinkles, and then transfer them to an HI1220 slide dryer for 30 minutes. Afterwards, use a LEICA slide dryer. ® The sections were stained with hematoxylin and eosin (HE) using an AutoStainer-XL staining machine. First, hematoxylin was stained for 5 minutes, followed by rinsing with running water for 1 minute, and then eosin staining for 3 minutes. After staining, the sections were dehydrated with graded ethanol, then cleared with an environmentally friendly clearing agent, and finally mounted with environmentally friendly mounting adhesive. The mounted sections were then placed in an OLYMPUS system. ® The stage of the BX43 microscope is to be observed and images acquired. See the images below. Figure 2 As shown, (a) and (b) are pathological section images of the uterus of rats in the normal control group.

[0046] Example 1

[0047] This embodiment provides an experimental method for establishing a rat model of uterine diverticulum. Thirty SPF-grade female SD rats, aged 11-12 weeks and weighing 280g-320g, were selected. The specific steps are as follows:

[0048] S1: Rats were anesthetized by intraperitoneal injection of 45 mg / kg sodium pentobarbital. It was confirmed that the rats' corneal reflex was absent and that they did not respond to pinching pain in their hind limbs.

[0049] S2: The rat was fixed in a supine position on a 37℃ constant temperature operating table. After shaving and disinfecting the lower abdomen, a 1cm longitudinal incision was made along the midline of the lower abdomen. The skin, subcutaneous fat and anterior sheath of the rectus abdominis muscle were cut layer by layer. The rectus abdominis muscle was bluntly dissected to expose the bilateral uterus.

[0050] S3: Use ophthalmic scissors to cut the uterine wall in the center of the uterus, cutting the entire uterus. If local capillary bleeding occurs during the cutting process, gently press with a sterile gauze for 30 seconds to stop the bleeding, so as to avoid damaging the main uterine blood vessels and affecting subsequent myometrial treatment.

[0051] S4: Lay the cut uterine wall flat on a sterile stage, using OLYMPUS... ® When the BX43 biological microscope is adjusted to the 20× objective lens, the light pink fibers arranged parallel to the long axis of the uterus are the outer 2 / 3 longitudinal muscle fibers, and the light red fibers arranged in a ring around the endometrium are the inner 1 / 3 circular muscle fibers. The total thickness of the myometrium from the serosa side to the endometrial side is confirmed by the microscopic scale.

[0052] S5: First, use microscissors to completely remove the endometrial tissue in the cut area, then hold the LEICA at a 30° angle. ®Microsurgical shears are used to prioritize the removal of the outer two-thirds of the longitudinal muscle fibers. During the shearing process, the shearing is carried out along the fiber direction, and the removal rate of the outer longitudinal muscle fibers is controlled to be ≥90%. If the total amount of muscle layer removed at this time is less than half of the uterine wall muscle layer, a small amount of the inner one-third of the circular muscle fibers is removed to make up the total amount, ensuring that the continuity of the inner one-third of the circular muscle fibers is preserved to be ≥85%.

[0053] S6: Take 10 μL of LPS solution with a concentration of 1 μg / μL, load it into a micropipette equipped with a 0.5 mm ultrafine needle, and slowly insert the needle into the subserosa of the uterine wall. Then, use OLYMPUS... ® Using a BX43 biological microscope, observe the needle depth, controlling it to 0.1±0.02mm, ensuring the needle tip does not penetrate the muscle layer. Slowly inject 5μL of LPS solution over a time of ≥30s to prevent leakage. Then, adjust the needle position and insert it into the junction of the outer longitudinal muscle fiber removal area and the inner 1 / 3 circular muscle fiber area. Slowly inject the remaining 5μL of LPS solution, pausing for 10s after injection before withdrawing the needle to reduce backflow.

[0054] S7: Use 7-0 surgical sutures to suture the uterine wall. First, 2-4 sutures are aligned, then 2-4 sutures are staggered, and finally 2-4 sutures are diagonally sutured. Repeat the operation until the entire uterine segment is sutured. Return the uterus to the abdominal cavity and use 4-0 non-absorbable sutures to intermittently suture the peritoneum and the anterior sheath of the rectus abdominis muscle. Use 5-0 non-absorbable sutures to suture the skin intradermally.

[0055] S8: Immediately after surgery, administer penicillin intramuscularly for 3 consecutive days to fight infection. Keep the feeding conditions the same as before surgery.

[0056] Three weeks later, the specimens underwent euthanasia simultaneously with the normal control group, and subsequent tissue specimens were processed using standard methods. The mounted slides were then placed in OLYMPUS. ® The stage of the BX43 microscope is to be observed and images acquired. See the images below. Figures 3-5 As shown, Figure 3 Images (c) and (d) are pathological sections of the uterus from rats in the model group. Figure 4 (c) An enlarged view of the modeling diverticulum area. Figure 5 Enlarged view of the modeling diverticulum area (d).

[0057] Example 2

[0058] To simulate the pathological characteristics of cesarean section incision layers and further optimize the precision of endometrial and myometrial layer removal, making the ratio of damage to the outer longitudinal layer to preservation of the inner circular layer more consistent with clinical reality, this embodiment provides an experimental method for establishing a rat model for treating uterine diverticulum, which differs from Embodiment 1 in that:

[0059] In S4, via OLYMPUS ®After confirming the total thickness of the muscle layer by observing with a BX43 biological microscope, it was determined that the removal rate of longitudinal muscle fibers in the outer 2 / 3 should be controlled at ≥92%, and the retention rate of circular muscle fibers in the inner 1 / 3 should be controlled at ≥82%.

[0060] In S5, when cutting the outer 2 / 3 of the longitudinal muscle fibers, the speed of advancement along the fiber direction is slightly slower than in Example 1 to ensure a removal rate of 92%, while the amount of inner 1 / 3 of the circular muscle fibers removed is only 80% of that in Example 1.

[0061] The remaining steps are consistent with those in Example 1.

[0062] Example 3

[0063] To simulate the pathological characteristics of cesarean section incision layers and further improve the matching degree between layered myometrial removal and clinically predominantly external longitudinal layer injuries, this embodiment provides an experimental method for rat modeling uterine diverticulum treatment, which differs from Embodiment 1 in that:

[0064] The removal rate of longitudinal muscle fibers in the outer 2 / 3 was controlled at ≥95%, and the retention rate of circular muscle fibers in the inner 1 / 3 was controlled at ≥80%.

[0065] When cutting out the outer 2 / 3 of the longitudinal muscle fibers, gently touch the fibers with the tip of the micro-scissors to confirm their direction and avoid accidentally cutting the inner ring layer. After removing the outer longitudinal muscle fibers, there is no need to cut out the inner ring layer again. Directly confirm the continuity of the inner ring layer ≥80% through a microscope.

[0066] The remaining steps are consistent with those in Example 1.

[0067] Example 4

[0068] To simulate the pathological characteristics of cesarean section incision layers and specifically mimic clinically severe myometrial defects in cesarean section incisions, such as deep incisions leading to almost complete rupture of the outer longitudinal layer and resulting in a diverticulum, this embodiment provides an experimental method for establishing a rat model of uterine diverticulum treatment, which differs from Embodiment 1 in that:

[0069] The removal rate of longitudinal muscle fibers in the outer 2 / 3 was controlled at ≥98%, and the retention rate of circular muscle fibers in the inner 1 / 3 was controlled at ≥70%.

[0070] When removing the outer 2 / 3 of the longitudinal muscle fibers, lay out a microscopic ruler to measure the thickness. After the outer longitudinal fibers are removed, only 1 / 3 of the inner 1 / 3 of the circular muscle fibers are removed, and the inner circular layer is confirmed to be unbroken by microscopy.

[0071] The remaining steps are consistent with those in Example 1.

[0072] Example 5

[0073] To simulate the pathological characteristics of cesarean section incision layers and avoid the backflow of medication during fine-needle injection affecting the inflammatory effect of layering, this embodiment provides an experimental method for establishing a rat model of uterine diverticulum treatment, which differs from Embodiment 1 in that:

[0074] In S6, take 10 μL of LPS solution with a concentration of 1 μg / μL, load it into a micropipette equipped with a 0.4 mm ultrafine injection needle, and control the subserosa injection depth at 0.1 ± 0.02 mm, while extending the injection time to 40 s.

[0075] The remaining steps are consistent with those in Example 1.

[0076] Example 6

[0077] To simulate the layered pathological characteristics of cesarean section incisions, verify the feasibility of LPS layered injection using a combination of large-bore and shallow-depth needles, and ensure that layered inflammation can be achieved with different needle specifications, this embodiment provides an experimental method for establishing a rat model of uterine diverticulum treatment, which differs from Embodiment 1 in that:

[0078] In S6, take 10 μL of LPS solution with a concentration of 1 μg / μL, load it into a micropipette equipped with a 0.6 mm ultrafine injection needle, and control the subserosa injection depth at 0.08 ± 0.02 mm, while extending the injection time to 25 s.

[0079] The remaining steps are consistent with those in Example 1.

[0080] Example 7

[0081] To simulate the layered pathological characteristics of cesarean section incisions, and considering the clinical postoperative pathological features of excessive subserosal exudate and severe inflammation, this embodiment provides an experimental method for establishing a rat model of uterine diverticulum, which differs from Embodiment 1 in that:

[0082] In S6, take 10 μL of LPS solution with a concentration of 1 μg / μL, load it into a micropipette equipped with a 0.5 mm ultrafine injection needle, adjust the subserosal injection dose to 6 μL and the injection time to 35 s, adjust the intermuscular injection dose to 4 μL and the injection time to 25 s, and keep the injection depth at 0.1 ± 0.02 mm.

[0083] The remaining steps are consistent with those in Example 1.

[0084] Example 8

[0085] To simulate the pathological characteristics of cesarean section incision layers, and specifically for diverticula with shallow cesarean section incisions and mild postoperative inflammation, this embodiment provides an experimental method for establishing a rat model of uterine diverticulum treatment, which differs from Embodiment 1 in that:

[0086] The removal rate of longitudinal muscle fibers in the outer 2 / 3 is ≥90%, and the retention rate of circular muscle fibers in the inner 1 / 3 is ≥82%.

[0087] Equipped with a 0.4mm ultrafine injection needle, the subserosal injection dose is 4μL with a bolus injection time of 45s, and the intermuscular injection dose is 6μL with a bolus injection time of 30s;

[0088] The remaining steps are consistent with those in Example 1.

[0089] Example 9

[0090] To simulate the pathological characteristics of cesarean section incision layers, and specifically for cesarean section incisions with thin inner ring layers, such as thin preoperative myometrium, this embodiment provides an experimental method for establishing a rat model of uterine diverticulum, which differs from Embodiment 1 in that:

[0091] The removal rate of longitudinal muscle fibers in the outer 2 / 3 was ≥92%, and the retention rate of circular muscle fibers in the inner 1 / 3 was ≥75%.

[0092] Equipped with a 0.6mm ultrafine injection needle, the subserosa injection depth is 0.08±0.02mm, the dose is 5μL, and the injection time is 25s;

[0093] The remaining steps are consistent with those in Example 1.

[0094] Example 10

[0095] To simulate the pathological characteristics of cesarean section incision layers, and specifically addressing the scenario of diverticulum caused by deep cesarean section incisions and postoperative infection leading to aggravated inflammation, this embodiment provides an experimental method for establishing a rat model of uterine diverticulum treatment, which differs from Embodiment 1 in that:

[0096] The removal rate of longitudinal muscle fibers in the outer 2 / 3 was ≥98%, and the retention rate of circular muscle fibers in the inner 1 / 3 was ≥78%.

[0097] Equipped with a 0.5mm ultrafine injection needle, the subserosal injection depth is 0.12±0.02mm, the dose is 6μL, and the injection time is 35s;

[0098] The remaining steps are consistent with those in Example 1.

[0099] Example 11

[0100] To simulate the pathological features of cesarean section incision layers, reduce the retention rate of the inner 1 / 3 circular muscle fibers, and improve the convenience of actual operation, this embodiment provides an experimental method for establishing a rat model of uterine diverticulum treatment, which differs from Embodiment 1 in that:

[0101] The removal rate of longitudinal muscle fibers in the outer 2 / 3 is ≥95%, and the retention rate of circular muscle fibers in the inner 1 / 3 is ≥72%.

[0102] Equipped with a 0.4mm ultrafine injection needle, the subserosal injection depth is 0.11±0.02mm, the dose is 5μL, and the injection time is 40s;

[0103] The remaining steps are consistent with those in Example 1.

[0104] Comparative Example 1

[0105] This comparative example uses the rat uterine scar diverticulum model preparation method disclosed in CN116269896A. The specific operation is as follows:

[0106] Thirty 11-12 week old SPF-grade non-pregnant female SD rats were selected and anesthetized by intraperitoneal injection of 45 mg / kg sodium pentobarbital. After shaving and disinfection of the lower abdomen, the uterus was exposed by layer-by-layer laparotomy. A 1 cm long incision was made 1 cm from the junction of the two uteri. A 10 mm × 5 mm modeling area was selected on the side near the uterine horn. All endometrial tissue and 0.8-1.2 mm thick myometrial tissue in this area were scraped away with a curette. 0.01 ml of 1 mg / ml LPS was injected into the modeling area and the myometrium. The remaining myometrium and serosa of the uterus were sutured with 6-0 absorbable sutures, and the peritoneum and skin were sutured with 4-0 non-absorbable sutures. Penicillin was injected 3 days after the operation.

[0107] Comparative Example 2

[0108] This comparative example uses the rat uterine scar diverticulum model preparation method disclosed in CN120284517A. The specific operation is as follows:

[0109] Thirty 11-12 week old SPF-grade non-pregnant female SD rats were selected. From days 1 to 16, the rats were subcutaneously injected with 2.5 μg of estradiol and 4 mg of progesterone daily. From days 17 to 23, 50 μg of estradiol was injected subcutaneously daily. Medication was discontinued one day before surgery. Single colonies of Group B Streptococcus standard strains were collected and cultured in Todd-Hewitt broth at 37.5℃ and 5% CO2 for 24 h, then diluted to 1×10⁻⁶. 7 CFU / mL bacterial solution was used to immerse 1 cm long 3-0 sterile silk thread in the bacterial solution for 24 hours for later use. Rats were anesthetized by intraperitoneal injection of 45 mg / kg sodium pentobarbital. After shaving and disinfecting the lower abdomen, the uterus was exposed by layer-by-layer laparotomy. A 2 cm long incision was made 2 cm from the junction of the two uteri. 3-0 silk thread soaked in GBS bacterial solution was inserted into the uterine cavity of one side, and sterile 3-0 silk thread was inserted into the other side. The uterine serosa and myometrium were intermittently sutured with 4-0 absorbable sutures, and the abdomen was closed layer by layer.

[0110] To verify the effectiveness of the rat modeling method for treating uterine diverticulum of the present invention, the consistency between pathological characteristics and clinical findings, and its advantages over existing technologies, the modeling results of Examples 1-11 and Comparative Examples 1-2 were tested. The specific test indicators and results are as follows:

[0111] The rats used in Examples 1-11 and Comparative Examples 1-2 were selected for modeling effectiveness testing, as follows: After dissecting and removing the uterus, the sac-like structures were observed macroscopically, and the percentage of rats that formed diverticula was counted to test the diverticulum formation rate; the percentage of rats that formed diverticula without uterine perforation or abdominal infection was counted to test the modeling success rate; the survival status was recorded daily, and the percentage of rats that survived 3 weeks post-surgery was counted to calculate the post-operative survival rate. The specific test results are shown in Table 3.

[0112]

[0113] Table 3

[0114] According to the data in Table 3, the diverticulum formation rate and modeling success rate of Examples 1-11 of the present invention both reached 85.0%-89.0%, and the postoperative survival rate reached 86.0%-93.0%, which is mainly due to the precise synergy between layered muscle layer removal and layered LPS injection.

[0115] Muscle layer removal procedure using OLYMPUS ® The BX43 biological microscope with a 20× objective lens identifies the outer 2 / 3 longitudinal muscle fibers and the inner 1 / 3 circular muscle fibers, controlling the removal rate of the outer 2 / 3 longitudinal muscle fibers to 90%-98% and the retention rate of the inner 1 / 3 circular muscle fibers to 70%-85%. This not only replicates the pathological characteristics of damage mainly to the outer longitudinal layer in clinical cesarean section, but also maintains the integrity of the uterine structure by preserving the inner circular layer, thus avoiding uterine perforation caused by excessive damage to the myometrium.

[0116] Examples 3 and 11 showed the highest molding efficiency and survival rate, mainly because the combination of parameters ensured sufficient damage to the outer longitudinal layer and adequate support to the inner ring layer, while the LPS layered injection precisely induced inflammation, reducing the risk of perforation.

[0117] The survival rates of Examples 4 and 10 were slightly lower, mainly because the removal rate of the outer longitudinal layer was close to the upper limit of the physiological proportion of the outer longitudinal layer of the rat uterus, which led to a decrease in the mechanical strength of the uterine wall, a prolonged postoperative repair period, and some rats were affected by mild abdominal adhesions.

[0118] Rats modeled in Examples 1-11 and Comparative Examples 1-2 were selected for pathological feature testing, as follows: After HE staining, the rats were observed under a 20× objective lens. The proportion of broken longitudinal fibers was calculated using ImageJ, and the removal rate of the outer 2 / 3 longitudinal muscle fibers and the retention rate of the inner 1 / 3 circular muscle fibers were tested. The number of lymphocytes was counted in 400× fields of view, and the average value of 4 fields of view was taken to test the density of inflammatory cells under the serosal membrane. The test results are shown in Table 4.

[0119]

[0120] Table 4

[0121] According to the data in Table 4, the measured removal rate of longitudinal myofibrous fibers in the outer 2 / 3 was 90.2%-97.2%, mainly due to the accuracy of fiber orientation identification with the 20× objective lens and the 30° angle micro-scissor operation. The retention rate of circular myofibrous fibers in the inner 1 / 3 was 70.5%-84.8%. Even in Examples 4 and 11, the uterine cavity closure was maintained by the continuity of the inner circular fibers, with no risk of perforation.

[0122] The density of inflammatory cells in the subserosal space was 18.5%-28.2 cells / high power field, which was positively correlated with the LPS injection parameters. In Examples 7 and 10, due to the slow diffusion of LPS in the subserosal space and the high local concentration, the density of inflammatory cells reached 28.2 cells / high power field. In contrast, the density of inflammatory cells in Example 8 was 18.5 cells / high power field.

[0123] Comparative Example 1 showed a 70.2% removal rate of longitudinal muscle fibers in the outer 2 / 3 and a 65.3% retention rate of circular muscle fibers in the inner 1 / 3. Due to the complete scraping of the muscle layer without stratification, the muscle layer damage was irregular. In contrast, Comparative Example 2 showed a 68.5% removal rate of longitudinal muscle fibers in the outer 2 / 3. Due to the inhibition of muscle layer repair by pseudoprogesterone and GBS infection, neither of them showed stratified pathological characteristics.

[0124] The model rats of Examples 1-11 and Comparative Examples 1-2 were selected for clinical fit testing, as follows: The proportion of diverticula located 1.5±0.1cm from the uterine horn was counted, corresponding to the lower segment of the human uterus, and the correspondence rate of the injury site was tested; the inflammatory density between the subserosal layer and the myometrium was compared, and a difference of ≥5 diverticula per high-power field was considered to meet the inflammatory stratification; the proportion of shallow diverticula with a diameter of 1.2-2.0mm was counted, and clinically shallow diverticula accounted for 80%; the presence of endometrial epithelial rupture or absence was observed, and the proportion of rats with incomplete epithelial structures was counted. The test results are shown in Table 5.

[0125]

[0126] Table 5

[0127] According to the data in Table 5, the correspondence rate of the injury site was 86.0%-93.0%. Because the modeling operation was performed to locate the central area of ​​the uterus and was precisely controlled at a distance of 1.5±0.1cm from the uterine horn, the correspondence rate of Examples 3 and 11 was the highest, which was highly matched with the anatomical area where clinical diverticulum is common.

[0128] The inflammatory stratification accuracy rate was 80.0%-90.0%. In Example 7, the combination of 6 μL of LPS in the subserosa and 4 μL in the intermuscular layer resulted in a difference in inflammatory cell density between the subserosa and the intermuscular layer of >20 cells / high-power field, demonstrating a significant effect.

[0129] The diverticulum morphology matching rate was 80.0%-86.0%. In Example 3, due to the removal rate of the outer longitudinal segment of 94.8% and the retention rate of the inner ring of 80.3%, the diverticulum diameter was concentrated in the range of 1.2-2.0 mm, and the morphology was consistent with that in clinical practice.

[0130] The incomplete rate of endometrial epithelium was 72.0%-86.0%. In Example 10, due to the removal of 97.1% of the external longitudinal epithelium combined with 6μL of LPS under the serosa, the degree of damage and inflammation was high, the epithelial repair pressure was high, and the incomplete rate was the highest. This is consistent with the pathological rule that the more severe the diverticulum damage, the worse the endometrial integrity.

[0131] The corresponding rate of injury site in Comparative Example 1 was 76.0%, because the incision location was 1 cm away from the uterine horn, which deviated from the clinically common area of ​​diverticulum; the corresponding rate of injury site in Comparative Example 2 was 73.0%, because the uterine position was displaced due to hormones, and both of the corresponding rates were lower than those in the Example.

[0132] The technical scope of this invention is not limited to the content described above. Those skilled in the art can make various modifications and variations to the above embodiments without departing from the technical concept of this invention, and all such modifications and variations should fall within the protection scope of this invention.

Claims

1. An experimental method for modeling a rat for treatment of uterine diverticulum, characterized by, The method comprises the following steps: S1: Select 11-12 week old SPF grade SD female rats, intraperitoneal injection of 45 mg / kg sodium pentobarbital anesthesia; S2: After the lower abdomen of the rat is shaved and disinfected, a 1 cm longitudinal incision is made along the midline, and the bilateral uteruses are separated layer by layer to expose the bilateral uteruses; S3: The uterus is cut in the center with an ophthalmic scissors, and sterile gauze is pressed to stop bleeding when bleeding; S4: After the uterine wall is laid flat, the outer longitudinal and inner circular muscle fibers are distinguished under a microscope with a 20x objective lens, and the muscle layer thickness is confirmed with a micrometer scale; S5: The endometrium is first cut, then the outer longitudinal muscle fibers are cut, and when the muscle layer removal amount is insufficient, a small amount of inner circular muscle fibers are cut; S6: 10 μL of LPS solution with a concentration of 1 μg / μL is injected subserosally and at the junction of the muscle layer with a superfine needle; S7: The uterine wall is sutured with 7-0 suture thread, the uterus is returned to the abdominal cavity, the peritoneum is sutured with 4-0 thread, and the skin is intradermally sutured with 5-0 thread; S8: Immediately after the operation, penicillin is injected intramuscularly to prevent infection.

2. The experimental method for treating uterine diverticulum by rat modeling according to claim 1, characterized in that: In S4, the outer longitudinal muscle fibers are longitudinal muscle fibers arranged in parallel along the long axis of the uterus in the outer 2 / 3 region of the total thickness of the muscle layer from the serosal side to the endometrial side, and the inner circular muscle fibers are circular muscle fibers arranged around the endometrium in the inner 1 / 3 region of the total thickness of the muscle layer.

3. The experimental method for treating uterine diverticulum by rat modeling according to claim 1 or 2, characterized in that: In S5, the removal rate of the outer longitudinal muscle fibers is ≥90%, and the continuity retention rate of the inner circular muscle fibers is ≥70%.

4. The experimental method for treating uterine diverticulum using a rat model according to claim 1, characterized in that: In S6, the superfine needle is 0.4-0.6 mm in size, the subserosal injection depth is controlled to be 0.1±0.02 mm, and the superfine needle injection time is ≥30 s.

5. The experimental method for treating uterine diverticulum by using a rat model according to claim 4, characterized in that: In S6, the junction of the muscle layer is the junction position of the outer longitudinal muscle fiber removal area and the inner circular muscle fiber, and the superfine needle injection time is ≥25 s.

6. The experimental method for treating uterine diverticulum using a rat model according to claim 5, characterized in that: In S6, the injection dose ratio of the LPS solution in the subserosal and muscle layer junction areas is 4-6:6-4.

7. The experimental method for treating uterine diverticulum using a rat model according to claim 1, characterized in that: In S7, the uterine wall is sutured by combining the following steps: aligning and suturing 2-4 stitches, misaligning and suturing 2-4 stitches, and finally diagonally suturing 2-4 stitches.

8. The experimental method for treating uterine diverticulum using a rat model according to claim 1, characterized in that: In S5, the outer longitudinal muscle fibers are cut at an angle of 30° with a microscissors, and the microscissors blade is advanced along the direction of the outer longitudinal muscle fibers.

9. The experimental method for treating uterine diverticulum using a rat model according to claim 1, characterized in that: In S6, after the superfine needle injection is completed, the superfine needle is left for 10 s and then pulled out to reduce the backflow of the drug solution.

10. The experimental method for treating uterine diverticulum using a rat model according to claim 1, characterized in that: In S8, penicillin is injected intramuscularly immediately after the operation, and the injection is continued for 3 days.

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