Method for constructing anal fistula model based on perianal chronic infectious wound surface of rat

Abstract

The invention discloses a method for constructing an anal fistula model based on a rat perianal chronic infectious wound, and belongs to the field of experimental animal modeling, the method comprises the following steps: selecting a 6-8-week male SD rat, adopting a supine position and raising the buttocks to fully expose an operation area, firstly cutting off full skin and subcutaneous tissue at a preset site beside the anus, exposing a superficial fascia layer, and then cutting off a subcutaneous tissue at a preset site beside the anus; and preparing a standardized skin defect wound surface. Then, a quantitative bacterial suspension is implanted into the basal part and the edge subcutaneous part of the wound surface by utilizing a multi-point injection technology, so that pathogenic bacteria form a stable infection focus in the deep layer of soft tissue, and the endogenous infection process evolved from perianal abscess to anal fistula is simulated. The model successfully reproduces the core characteristics of high bacterial load, chronic property, delayed healing and the like of the clinical anal fistula wound surface. The operation process is standard, the success rate is high, the individual difference is small, and a stable and reliable experimental evaluation platform highly simulating the clinical environment is provided for evaluating biological materials with anti-infection, anti-oxidation and healing promoting functions.

Description

Technical Field

[0001] This invention belongs to the field of biomedical experimental animal models, specifically relating to a method for constructing an anal fistula model based on chronic infectious lesions around the anus in rats. Background Technology

[0002] Anal fistula is a highly challenging anorectal surgical condition with a persistently high incidence rate in my country and globally. Epidemiological statistics show that anal fistula is more common in young and middle-aged men, and it is essentially a chronic infectious passage between the rectum or anal canal and the perianal skin. Currently, the standard clinical treatment for anal fistula remains primarily surgical incision, excision, or seton placement, aiming to completely remove the primary infected internal opening and drain necrotic tissue.

[0003] However, surgery is only the beginning of treatment; the postoperative wound healing process is the core factor determining the prognosis. The perianal region has a very unique physiological location, leading to wounds in this area exhibiting significant "difficult-to-heal" characteristics: First, the wound is inevitably exposed to fecal contamination for a long time, and the large number of pathogenic bacteria such as E. coli and Staphylococcus aureus in excrement easily cause recurrent infections; second, the perianal environment is chronically moist and enclosed, lacking sufficient oxygen exchange, which is conducive to the growth of anaerobic bacteria; third, the frequent contraction and relaxation of the anal sphincter generates continuous mechanical stress, which, combined with friction from clothing during walking, causes continuous physical damage to newly formed granulation tissue, creating a vicious cycle. Infected chronic wounds typically exhibit high bacterial load, strong oxidative stress, and persistent inflammation simultaneously. Pathogens and their products can directly damage tissues and trigger a large accumulation of immune cells. Neutrophils and macrophages release reactive oxygen species (ROS) and proteolytic enzymes during bacterial clearance. Excessive ROS can cause lipid peroxidation, cell membrane and mitochondrial damage, affecting fibroblast migration, collagen deposition and angiogenesis, making it difficult for high-quality granulation tissue to form in wounds. Macrophage phenotypic imbalance is also common in chronic wounds; a high proportion of pro-inflammatory phenotypes leads to prolonged inflammation, while insufficient repair-related phenotypes limit tissue regeneration. For chronic infected wounds associated with anal fistulas, antibacterial treatment or single healing-promoting methods are often insufficient; a comprehensive treatment strategy that simultaneously controls infection, reduces ROS, regulates inflammation, and promotes repair is needed.

[0004] In the development of novel biorepair materials (such as responsive hydrogels and drug-loaded fibers), animal experiments are a crucial step in verifying their effectiveness. However, most current research on wound repair still predominantly uses the rat dorsal skin injury model. This model has significant limitations in simulating anal fistula wounds: the rat's dorsal skin is smooth and has a very rich blood supply, and it is far from excretory sources, resulting in a relatively dry and clean environment. This leads to extremely strong self-healing capabilities in dorsal wounds, making it difficult to induce the persistent infection and inflammation commonly seen in clinical practice, and thus failing to truly reflect the drug's ability to control refractory wounds. More importantly, the dorsal model cannot simulate the frequent stretching and friction of the anal area caused by daily activities, nor can it reproduce the continuous challenges posed by the unique bacterial community of the perianal region. Consequently, many materials that perform well in dorsal experiments often fail in clinical applications because they cannot cope with the complex physical environment and severe infections of the perianal region.

[0005] Addressing this technical challenge, establishing a standardized modeling method capable of highly replicating the local environment of anal fistulas is particularly urgent. Especially for advanced "intelligent responsive" biomaterials, the ability to automatically release drugs cannot be realistically verified without a stable evaluation platform that provides an acidic environment, high oxidative stress, and continuous bacterial infection. This invention proposes a rat model of infectious full-thickness perianal skin defects. By establishing standardized wounds at specific anatomical locations and incorporating precisely dosed bacterial implantation, it not only physically simulates the tissue tension and exposure environment of the perianal region but also constructs a chronic inflammatory microenvironment consistent with clinical practice at the molecular level. The establishment of this model effectively fills the gap in the evaluation system for materials specifically designed for anorectal surgery, providing a scientific, stable, and reproducible experimental basis for the screening, efficacy evaluation, and principle research of biomaterials, possessing high academic value and application prospects. Summary of the Invention

[0006] The purpose of this invention is to provide a method for constructing an anal fistula model based on chronic infected perianal wounds in rats, which is simple to operate, has standardized parameters, and highly simulates clinical pathological characteristics. It aims to solve the technical problem that existing back wound models cannot simulate the special anatomical stress and chronic infection microenvironment of the perianal region, and to provide an objective experimental basis for the efficacy evaluation of perianal repair materials.

[0007] A method for constructing an anal fistula model based on chronic infected perianal wounds in rats includes the following steps: (1) Animal fixation and anesthesia: Healthy male SD rats aged 6-8 weeks and weighing 180-240g were used. They were fasted before the operation to reduce fecal contamination. After general anesthesia, they were fixed in a supine position. A hard pad was placed under the tail to raise the buttocks significantly, so as to fully flatten and expose the surgical area centered on the anus. The perianal hair was removed and routine disinfection was performed. (2) Wound preparation: The surgical area is pre-defined in the lateral area 3mm to 10mm away from the anal edge. The full-thickness skin tissue and subcutaneous loose connective tissue are completely removed using surgical instruments until the superficial fascia layer underneath is exposed, and a full-thickness skin defect wound with an area of ​​8mm×8mm to 12mm×12mm is prepared. The anatomical layers are strictly identified during the preparation process to ensure that the instruments only act on the skin and subcutaneous tissue, avoiding and protecting the deep anal sphincter muscle group and main branch blood vessels. (3) Infection induction: In the subcutaneous tissue at the base and edge of the wound described in step (2), a concentration of 0.5 × 10⁻⁶ was injected at multiple points using a microsyringe. 7 CFU / mL up to 1.5 × 10⁻⁶ 7 A suspension of pathogenic bacteria at CFU / mL is injected at a volume of 0.1mL to 0.5mL to allow the pathogens to colonize the deep wound and surrounding tissues. (4) Chronic evolution: The wound is kept open after surgery and is not bandaged, so that the wound is exposed in situ to the microecology of the perianal area rich in bacteria and the mechanical friction environment of daily defecation and activities; after 5 to 8 days of natural evolution, the wound is induced to go through the stages of acute inflammatory exudation, persistent infection and delayed healing, and finally forms a perianal chronic infection wound with the core pathological characteristics of "prolonged inflammatory period and delayed proliferation period", that is, an animal model that simulates the clinical anal fistula microenvironment is successfully constructed.

[0008] Furthermore, in step (1), the rats are specifically selected as 7-week-old male SD rats weighing 200g-220g; the preoperative fasting time is 12 hours; the anesthesia method is specifically to use 1% sodium pentobarbital at a dose of 50mg / kg for intraperitoneal injection, or to combine it with isoflurane inhalation for maintenance anesthesia; the height of the rigid pad is 2-3cm; the routine disinfection is to use 75% alcohol to disinfect three times from the center to the periphery.

[0009] Furthermore, in step (2), the distance between the external incision and the edge of the anus is specifically 5 mm; the instrument is a scalpel or surgical forceps; and the area of ​​the full-thickness skin defect is specifically 10 mm × 10 mm.

[0010] Furthermore, in step (3), the pathogenic bacterium is Staphylococcus aureus; the pathogenic bacterium is introduced by using a microsyringe to inject a concentration of 1.0 × 10⁻⁶ bacteria at 2-3 points in the base of the wound and the surrounding subcutaneous tissue. 7 The total amount of pathogenic bacterial suspension (CFU / mL) injected into a single rat is 0.2 mL.

[0011] 5. The method for constructing an anal fistula model based on chronic perianal infectious wounds in rats according to claim 1, characterized in that, in step (4), the macroscopic characteristics of the chronic evolution and model establishment are that within 48 hours after modeling, the wound shows acute inflammatory exudation without obvious contraction, and on the 5th to 8th day after modeling, the wound transforms into a chronic infection and delayed healing state, which is macroscopically manifested as the formation of purulent secretions or biofilm precursors on the wound surface, increased tension in the surrounding tissues, and the wound area maintained at a higher level than the initial area.

[0012] Furthermore, the rat chronic anal fistula model described in step (4) exhibited the following microscopic histopathological features on day 8 after modeling: HE staining of tissue sections showed a large number of neutrophils and macrophages infiltrating the wound area, and reepithelialization was hindered; Masson trichrome staining showed that the collagen fiber content at the bottom of the wound was low and the arrangement was disordered, and the granulation tissue was immature.

[0013] Furthermore, in the rat chronic anal fistula model described in step (4), after modeling, a suspension was prepared by collecting wound tissue or secretions for bacterial plating culture to verify the presence of a persistent high bacterial load in the wound area and confirm the chronic infection state.

[0014] Furthermore, the model constructed by the method has local infection specificity and systemic biocompatibility; after modeling, HE staining of tissue sections from the heart, liver, spleen, lungs, and kidneys of rats showed that the tissue structure of each organ was intact, the cell morphology was normal, and there were no obvious pathological changes of systemic infection such as inflammatory cell infiltration, tissue necrosis, or congestion.

[0015] Furthermore, the model is used to evaluate the anti-infection performance, wound healing rate, granulation tissue growth, and immune microenvironment regulation of biomedical materials or drugs in a simulated anal fistula environment.

[0016] The beneficial effects of this invention are as follows: (1) Achieving a high degree of consistency between wound anatomical location and clinical environment: The present invention is located in the perianal region, taking into full account the special characteristics of this area, such as active muscle movement and susceptibility to fecal microbial contamination. Compared with the traditional back model, the wound constructed by this model is closer to the clinical anal fistula wound in terms of physiological location and external stress environment, which significantly improves the clinical reference value of experimental conclusions; (2) A stable endogenous chronic infection microenvironment was constructed: By using subcutaneous multi-point precise injection of pathogens, a stable infection focus was successfully established in the deep soft tissue, effectively simulating the pathological evolution after abscess rupture or surgical incision. This "inside-out" infection mode can induce a lasting inflammatory response, avoiding the problems of easy loss of surface bacteria and insufficient infection depth, and ensuring the reliability of the chronic characteristics of the model; (3) It provides a clear and controllable experimental observation window: By limiting the wound size and pathogen concentration, this model can show significant inflammatory characteristics within 24-48 hours after surgery, and due to infection intervention, its natural healing period is significantly longer than that of ordinary injuries. This provides a sufficient time window for observing the effects of different interventions on tissue regeneration, granulation tissue growth and epithelialization process; (4) High standardization of operation and reproducibility of results: This invention standardizes the selection of experimental subjects, fixation of surgical positions, and specific parameters for modeling. Each step is logically clear and has conventional equipment requirements, effectively reducing errors caused by differences in the operation of experimental personnel, and facilitating large-scale application in the field of biomedical research. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the anatomical location and surgical position for rat perianal wound modeling according to the present invention; Figure 2 These are photographs showing the actual process of creating a rat perianal chronic infectious lesion model according to the present invention. Figure 3 Macroscopic morphology of the wound at different time points (D0, D2, D5, D8) after modeling and images processed using ImageJ software; Figure 4 A bar chart showing the wound healing rate at different time points (D2, D5, D8) after modeling; Figure 5 This is a statistical curve showing the change in body weight of experimental rats during the modeling process (D0-D8). Figure 6 HE-stained histopathological images of the major organs (heart, liver, spleen, lung, and kidney) of rats on day 8 after modeling in this embodiment of the invention.

[0018] Figure 7 Figure 1 shows the results of bacterial plasm culture of wound exudate at different time points (D0, D2, D5, D8). Figure 8 HE staining and Masson staining pathological images of wound tissue on day 8 after modeling. Detailed Implementation

[0019] The principles and features of the present invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0020] Example Construction of a rat model of chronic perianal infected wound Animal preparation: Male SD rats aged 6-8 weeks and weighing 200-220g were selected and acclimatized in an SPF environment for 7 days. They were fasted for 12 hours before the operation but given free access to water.

[0021] Anesthesia and positioning: Basic anesthesia was administered via intraperitoneal injection of 1% sodium pentobarbital solution (50 mg / kg), with isoflurane inhalation used to maintain anesthesia as needed during the operation. As shown in Figure 1-2, the rat was fixed in a supine position on a temperature-controlled operating table, with a 2-3 cm high rigid support placed under the sacrum and coccyx to significantly elevate the buttocks, fully flattening and exposing the perianal area.

[0022] Wound preparation: Locate the defect 3-10mm lateral to the anus, remove hair, and disinfect routinely. Using surgical instruments, make incisions layer by layer along the pre-marked lines, completely dissecting the full thickness of skin and subcutaneous loose connective tissue to expose the underlying superficial fascia layer, creating a standardized defect wound with an area of ​​10mm × 10mm. Strictly avoid the deep anal sphincter muscles during the procedure.

[0023] Infection induction: Using a microsyringe, slowly inject multiple points (2-3 points) into the subcutaneous tissue at the base and surrounding edges of the wound, injecting a total of 0.2 mL of Staphylococcus aureus suspension (concentration of 1.0 × 10⁻⁶). 7 (CFU / mL).

[0024] Experimental Example 1: Statistical Analysis of Macroscopic Morphological Changes and Healing Rate in the Model 1. Experimental method: In order to verify the wound model constructed in Example 1, this experiment conducted macroscopic morphological observation and quantitative analysis of the wound at different time points (D0, D2, D5, D8) after modeling.

[0025] (1) Image acquisition: On day 0 (D0) and day 2 (D2), day 5 (D5), and day 8 (D8) after modeling, rats were briefly restrained or lightly anesthetized under the same light source and focal length. A standard millimeter ruler was placed next to the wound as an internal reference, and macroscopic images were taken perpendicular to the wound using a high-definition digital camera. After taking the images, the redness and swelling at the wound edge, the color and characteristics of the exudate, and the growth of scabs and granulation tissue were observed and recorded.

[0026] (2) Area extraction and calculation: Import the collected images into ImageJ image analysis software. First, use the “SetScale” function in conjunction with the standard ruler in the image to calibrate the pixel ratio; then use the “Polygon selections” tool to accurately outline the wound contour along the edge of the unepithelialized granulation tissue, measure and record the actual area of ​​the wound at each time point (denoted as An, and the area at D0 is denoted as A0).

[0027] (3) Healing rate statistics: Calculate the wound healing rate (%) at each time point according to the formula, enter the data into statistical software (such as GraphPad Prism) to draw a bar chart, and conduct intergroup difference analysis.

[0028] 2. Experimental Results: Combined with appendix Figure 3 As shown, in stage D0 (on the day of modeling): the wound shows a full-thickness skin defect with neat edges and a clearly visible superficial fascia layer at the bottom, establishing the initial evaluation criteria.

[0029] Stage D2 (Inflammatory Exudative Phase): 48 hours after modeling, the wound exhibited a significant acute inflammatory response. The base of the wound was covered with a pale yellow inflammatory exudate, and the wound edges showed significant thickening due to inflammatory stimulation. No significant wound contraction was observed at this time, confirming that the subcutaneous implantation of pathogens successfully induced a strong initial inflammatory challenge.

[0030] Stage D5 (Persistent Infection Phase): On the 5th postoperative day, the wound exhibits typical characteristics of chronic infection. A layer of grayish-white or yellowish-brown purulent discharge (characteristics of biofilm precursors) forms on the surface. Although tissue edema has subsided, the inflammatory response remains active. The wound area shrinks slowly, and granulation tissue growth is restricted, showing obvious signs of stunted healing.

[0031] Stage D8 (Delayed Healing Period): On the 8th day after surgery, signs of scab formation begin to appear on the wound, which darkens to a deep brown color. The surrounding tissues are under considerable tension, simulating the slow-healing state caused by frequent mechanical stress after clinical perianal surgery.

[0032] Combined with appendix Figure 4 The wound healing rate was analyzed using ImageJ software. The calculations showed that the healing rate of the model was low in the first 5 days after surgery; by the 8th day, the wound area was still more than 60% of the initial area, and the healing progress was significantly slower than that of conventional sterile skin defects.

[0033] Experimental Example 2: Safety Evaluation of the Model's Systemic Effects on Rats Combined with appendix Figures 5-6This experimental example aims to demonstrate that the bacterial dosage described in this invention only produces local chronic inflammation without causing toxic damage to the vital organ functions and systemic metabolism of rats.

[0034] 1. Experimental Methods: From day 0 before modeling (D0) to day 8 after modeling (D8), rats were weighed and their weight change curves were recorded daily. Their mental state, activity level, and signs of eating and defecating were also monitored. On day 8 after modeling (D8), rats were euthanized using an overdose anesthesia method. The five major organs—heart, liver, spleen, lung, and kidney—were completely removed. After washing with pre-cooled sterile PBS, the organs were fixed in 4% paraformaldehyde for 24-48 hours. Subsequently, the organ tissues underwent gradient dehydration, clearing, and paraffin embedding. Serial sections of 4-5 μm thickness were cut and routinely stained with hematoxylin and eosin (HE). Finally, the sections were examined under an optical microscope (100× and 400×) to identify any systemic infection or toxic pathological changes such as inflammatory cell infiltration, cell degeneration and necrosis, microvascular congestion, or thrombosis.

[0035] 2. Experimental Results: Combined with Appendix Figure 5 Regarding body weight and survival status, the body weight of the model group rats showed a steady upward trend after a slight decrease at D0, with no abnormal decrease observed. Furthermore, the rats maintained good mental state and normal activity during the experiment. (Combined with...) Figure 8 As shown, in terms of pathological evaluation of major organs, HE-stained sections revealed intact tissue structures and normal cell morphology in the heart, liver, spleen, lungs, and kidneys, with no obvious inflammatory cell infiltration, tissue necrosis, or congestion. These results confirm that the modeling protocol used in this invention has excellent biosafety, successfully inducing chronic, non-healing wounds in the perianal region while effectively avoiding systemic toxicity caused by bacterial entry into the bloodstream, ensuring the reliability of this model in subsequent long-term drug evaluations.

[0036] Experimental Example 3: Histopathological Analysis of Wound Tissue The microscopic histological analysis confirmed that the wound was in a chronic, difficult-to-heal state characterized by "prolonged inflammation and delayed proliferation".

[0037] 1. Experimental Methods: Rats were sacrificed on day 8 (D8) after modeling. Full-thickness tissue, including all granulation tissue and normal skin at the periphery, was excised from the wound. After fixation, embedding, and sectioning, the tissue was stained with hematoxylin and eosin (HE) and Masson's trichrome staining, respectively. Under a microscope, the density of inflammatory cell infiltration, the distance of re-epithelialization, the amount of collagen fiber deposition, and the regularity of their arrangement were observed.

[0038] 2. Experimental Results: HE staining: Sections showed extensive infiltration of neutrophils and macrophages in the wound area, with no obvious epidermal cell migration towards the wound center (impaired reepithelialization), confirming that the inflammatory response was still in a highly active phase. Masson staining: Results showed that the wound base had low collagen fiber content and disordered arrangement, sparse distribution of newly formed capillaries, and extremely immature granulation tissue development. Microscopic pathological evidence further confirmed that the model constructed in this invention successfully reproduced the core pathological features of chronically infected, refractory wounds.

[0039] Experimental Example 4: Microbiological Evaluation of Local Bacterial Load in Wounds The presence of a persistent high bacterial load in the wound area after modeling confirmed the formation of a chronic infection microenvironment.

[0040] 1. Experimental Methods: On day 0 (D0) of modeling and on days 2 (D2), 5 (D5), and 8 (D8) after modeling, exudate or central granulation tissue was collected to prepare original-volume bacterial suspensions. After 10-fold serial dilutions, 100 μL of each dilution was evenly spread onto LB agar medium. After incubation at 37°C for 24 hours, photographs were taken and colony-forming units (CFU) were counted.

[0041] 2. Experimental results: such as Figure 7 As shown, throughout the observation period from D0 to D8, a large number of pathogenic bacterial colonies were consistently cultured from the wound site. Although the colony count decreased over time, it remained at a high infection load level on day 8. The subcutaneous multi-point injection method of this invention successfully achieved stable colonization of pathogens in the deep layers of soft tissue, constructing a durable infection microenvironment.

[0042] The above embodiments of the present invention are not intended to limit the scope of protection of the present invention. The implementation of the present invention is not limited thereto. All other modifications, substitutions or alterations made to the above structure of the present invention based on the above content of the present invention, in accordance with ordinary technical knowledge and common practice in the field, without departing from the basic technical idea of ​​the present invention, shall fall within the scope of protection of the present invention.

Claims

1. A method for constructing an anal fistula model based on chronically infected perianal wounds in rats, characterized in that, Includes the following steps: (1) Animal fixation and anesthesia: Healthy male SD rats aged 6-8 weeks and weighing 180-240g were used. They were fasted before the operation to reduce fecal contamination. After general anesthesia, they were fixed in a supine position. A hard pad was placed under the tail to raise the buttocks significantly, so as to fully flatten and expose the surgical area centered on the anus. The perianal hair was removed and routine disinfection was performed. (2) Wound preparation: The surgical area is pre-defined in the lateral area 3mm to 10mm away from the anal edge. The full-thickness skin tissue and subcutaneous loose connective tissue are completely removed using surgical instruments until the superficial fascia layer underneath is exposed, and a full-thickness skin defect wound with an area of ​​8mm×8mm to 12mm×12mm is prepared. The anatomical layers are strictly identified during the preparation process to ensure that the instruments only act on the skin and subcutaneous tissue, avoiding and protecting the deep anal sphincter muscle group and main branch blood vessels. (3) Infection induction: In the subcutaneous tissue at the base and edge of the wound described in step (2), a concentration of 0.5 × 10⁻⁶ was injected at multiple points using a microsyringe. 7 CFU / mL up to 1.5 × 10⁻⁶ 7 A suspension of pathogenic bacteria at CFU / mL is injected at a volume of 0.1mL to 0.5mL to allow the pathogens to colonize the deep wound and surrounding tissues. (4) Chronic evolution: The wound is kept open after surgery and is not bandaged, so that the wound is exposed in situ to the microecology of the perianal area rich in bacteria and the mechanical friction environment of daily defecation and activities; after 5 to 8 days of natural evolution, the wound is induced to go through the stages of acute inflammatory exudation, persistent infection and delayed healing, and finally forms a perianal chronic infection wound with the core pathological characteristics of "prolonged inflammatory period and delayed proliferation period", thus successfully constructing an animal model that simulates the clinical anal fistula microenvironment.

2. The method for constructing an anal fistula model based on chronic perianal infected wounds in rats according to claim 1, characterized in that, In step (1), the rats are specifically selected as 7-week-old male SD rats weighing 200g-220g; the preoperative fasting time is 12 hours; the anesthesia method is specifically to use 1% sodium pentobarbital at a dose of 50mg / kg for intraperitoneal injection, or to combine it with isoflurane inhalation for maintenance anesthesia; the height of the rigid pad is 2-3cm; the routine disinfection is to use 75% alcohol to disinfect three times from the center to the periphery.

3. The method for constructing an anal fistula model based on chronic perianal infected wounds in rats according to claim 1, characterized in that, In step (2), the distance between the external incision and the edge of the anus is specifically 5 mm; the instrument is a scalpel or surgical forceps; the area of ​​the full-thickness skin defect is specifically 10 mm × 10 mm.

4. The method for constructing an anal fistula model based on chronic perianal infected wounds in rats according to claim 1, characterized in that, In step (3), the pathogenic bacterium is Staphylococcus aureus; the pathogenic bacterium is introduced by using a microsyringe to inject a concentration of 1.0 × 10⁻⁶ bacteria at 2-3 points in the base of the wound and surrounding subcutaneous tissue. 7 The total amount of pathogenic bacterial suspension (CFU / mL) injected into a single rat is 0.2 mL.

5. The method for constructing an anal fistula model based on chronic perianal infected wounds in rats according to claim 1, characterized in that, In step (4), the macroscopic characteristics of the chronic evolution and model establishment are that within 48 hours after modeling, the wound shows acute inflammatory exudation without obvious contraction. From the 5th to the 8th day after modeling, the wound transforms into a chronic infection and delayed healing state. Its macroscopic manifestations are the formation of purulent secretions or biofilm precursors on the wound surface, increased tension of surrounding tissues, and the wound area maintained at a higher level than the initial area.

6. The method for constructing an anal fistula model based on chronic perianal infected wounds in rats according to claim 1, characterized in that, The rat chronic anal fistula model described in step (4) exhibited the following microscopic histopathological features on the 8th day after modeling: HE staining of tissue sections showed that there were a large number of neutrophils and macrophages infiltrating the wound area, and reepithelialization was hindered; Masson trichrome staining showed that the collagen fiber content at the bottom of the wound was low and the arrangement was disordered, and the granulation tissue was immature.

7. The method for constructing an anal fistula model based on chronic perianal infected wounds in rats according to claim 1, characterized in that, After the rat chronic anal fistula model described in step (4) is established, a suspension is prepared by collecting wound tissue or secretions for bacterial plating culture to verify the presence of a persistent high bacterial load in the wound area and confirm the chronic infection state.

8. The method for constructing an anal fistula model based on chronic perianal infected wounds in rats according to claim 1, characterized in that, The model constructed by the method has local infection specificity and systemic biocompatibility. After modeling, HE staining of tissue sections from the heart, liver, spleen, lungs, and kidneys of rats showed that the tissue structure of each organ was intact, the cell morphology was normal, and there were no obvious pathological changes of systemic infection such as inflammatory cell infiltration, tissue necrosis, or congestion.

9. The method for constructing an anal fistula model based on chronic perianal infected wounds in rats according to claim 1, characterized in that, The model is used to evaluate the anti-infection performance, wound healing rate, granulation tissue growth, and immune microenvironment regulation of biomedical materials or drugs in a simulated anal fistula environment.

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