Modeling method of chronic prostatitis / chronic pelvic pain syndrome mouse model
By combining rat prostate hormone-binding protein PSBP with Freund's adjuvant CFA and using a sheep multivalent inactivated vaccine, a high-efficiency and low-cost CP/CPPS mouse model was established. This solves the problem of imperfect models in existing technologies and meets the needs of hormone level assessment, sexual function testing, and transcriptome research.
Patent Information
- Application Number
- CN202411272877.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-12
- Publication Date
- 2026-01-09
AI Technical Summary
There is a lack of animal models in the current technology that can effectively simulate all the characteristics of human CP/CPPS. Moreover, existing models have problems such as high infection rate, high mortality rate, large influence of hormone levels, and complex operation or high cost, which make it difficult to meet the needs of hormone level assessment, sexual function testing and transcriptome research.
A mouse model was established by combining rat prostate hormone-binding protein PSBP with complete Freund's adjuvant CFA, preparing prostate homogenate and subcutaneously injecting it into mice to simulate the autoimmune response of CP/CPPS, and combining it with sheep multivalent inactivated vaccine to simulate bacterial infection.
It boasts a high modeling success rate and low mortality rate. The model exhibits persistent abnormal phenotypes such as pain sensitization, anxiety, and depression. It is suitable for transcriptomic and proteomic studies, is low-cost and easy to operate, and has a long model maintenance time.
Smart Images
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of bio-device technology for medical research, specifically relating to a mouse model of chronic prostatitis / chronic pelvic pain syndrome (CP / CPPS) in urology and its modeling method. Background Technology
[0002] Chronic prostatitis / chronic pelvic pain syndrome (CP / CPPS) is a common urological disease, accounting for over 90% of chronic prostatitis cases. The estimated incidence of CP / CPPS is 2.2%–13.8%. CP / CPPS presents with a variety of clinical symptoms, including persistent pain in the pelvic and / or genital areas for more than 3 months within the past 6 months without any detectable underlying cause, as well as other manifestations such as lower urinary tract problems and / or sexual dysfunction. Chronic pain often causes emotional and psychological distress such as anxiety and depression, and CP / CPPS is no exception. Therefore, the quality of life of patients with CP / CPPS is significantly reduced.
[0003] Currently, no single model in clinical practice can replicate and reflect all characteristics of human prostate cancer (CP / CPPS), necessitating careful selection of appropriate animal models based on research objectives. Therefore, several animal models have been established using different methods to simulate the pathophysiological progression of CP / CPPS for specific research purposes. The mechanisms of these models are primarily induced by autoimmune responses, sex hormone imbalances, and chemical agents, with rats and mice being the most commonly used experimental animals. However, each model has its unique advantages and limitations. For example, chemical injection methods have led to increased infection and mortality rates in animals. Estrogen-androgen castration models are simple to operate and low in cost, but this modeling method affects hormone levels in the animal and is unsuitable for hormone level assessment, sexual function testing, and transcriptome studies of CP / CPPS. While autoimmune animal models of CP / CPPS exhibit good pathological specificity and similarity to clinical manifestations, making them suitable for studying the pathogenic mechanisms of CP / CPPS, the concentration of prostate protein extracts significantly affects the success rate of model establishment, and the modeling process is time-consuming.
[0004] Based on the current state of the technology, the inventors of this application intend to provide a mouse model of chronic prostatitis / chronic pelvic pain syndrome (CP / CPPS) in urology and a method for establishing the model. Summary of the Invention
[0005] The purpose of this invention is to provide a mouse model of chronic prostatitis / chronic pelvic pain syndrome based on the current state of the technology and the technical problems that need to be solved in clinical practice, for related research, including hormone level assessment of CP / CPPS, sexual function testing requirements, and transcriptome studies.
[0006] Specifically, this application provides a mouse model of chronic prostatitis / chronic pelvic pain syndrome (CP / CPPS) in urology and a method for establishing the model.
[0007] More specifically,
[0008] This invention provides a method for establishing a mouse model of chronic prostatitis / chronic pelvic pain syndrome.
[0009] Prostatic steroid-binding protein (PSBP) is the main secretory protein of the ventral aspect of the rat prostate and has been identified as the major autoantigen in rat EAP models. It can be recognized by the autoimmune system, causing humoral and cellular autoimmune responses in rats. Its characteristics include specific T cell responses and the production of Th1-related isotype autoantibodies. Adoptive transfer of PSBP-specific T cells to their syngeneic receptors also participates in inducing EAP. In addition to lymphocyte infiltration, degranulated mast cells and their released chemical mediators (including histamine, leukotrienes, serotonin, and proteases such as pancreatin) also participate in the inflammatory cascade of prostatitis. The activation and dysregulation of these immune cells, as well as the release of various cytokines and growth factors, including IL-2, IL-4, IL-6, and IL-17, collectively promote prostate lesions. PSBP can induce strong humoral and cellular autoimmune responses, but when mice are injected with prostate extracts plus adjuvants, a more severe disease phenotype can be observed. Therefore, after testing various formulations and dosages of excipients, this invention selected complete Freund's adjuvant (CFA).
[0010] This invention provides a method for establishing, or preparing, a mouse model of chronic prostatitis / chronic pelvic pain syndrome, comprising the following steps:
[0011] (1) Preparation of prostate homogenate: Two 4-month-old rats were sacrificed and their prostate tissue was removed under aseptic conditions. The tissue was ground or homogenized, centrifuged, and the supernatant was added to Freund's complete adjuvant to prepare an injection suspension.
[0012] (2) Preparation of intraperitoneal injection solution for vaccine: At room temperature, under the guidance of the instructions, dissolve the lyophilized powder of inactivated vaccine in aluminum hydroxide gel saline to prepare a prostate homogenate suspension.
[0013] (3) Injection of modeling solution: After anesthetizing the mice, inject prostate homogenate suspension subcutaneously and inject vaccine intraperitoneal injection; repeat the above injection steps until the mouse CP / CPPS model is formed.
[0014] Preferably, the preparation of the prostate homogenate includes: taking 4-month-old Wistar rats, euthanizing them after anesthesia, aseptically dissecting the prostate tissue, washing it, adding physiological saline solution containing 0.5% Triton X-100, homogenizing it with a glass homogenizer on an ice water bath, centrifuging at 4°C and collecting the supernatant; then adjusting the protein concentration to 20-100 mg / mL with 0.1 mol / L PBS buffer (pH 7.2-7.4); before use, thoroughly mixing the prostate homogenate with Freund's complete adjuvant at a volume ratio of 1:(0.5-2), filtering it through a bacterial filter, and preparing an injection suspension.
[0015] This invention incorporates complete Freund's adjuvant into the PSBP at a specific ratio. Preferably, the prostate homogenate and Freund's complete adjuvant are thoroughly mixed at a volume ratio of 1:1 at room temperature.
[0016] Preferably, the preparation of the vaccine intraperitoneal injection solution refers to dissolving 20 doses of lyophilized powder of the multivalent inactivated vaccine for sheep in a sterile aluminum hydroxide gel saline suspension at room temperature.
[0017] Preferably, the injection of the modeling solution includes: taking 6-8 week old mice, anesthetizing them with sodium pentobarbital, and preparing the abdominal skin; taking 4-6 injection sites, subcutaneously injecting a prostate homogenate suspension, and intraperitoneally injecting the vaccine intraperitoneal injection solution; confirming that the mice have recovered from anesthesia and returning them to a clean, sterile cage, and monitoring their activity, food intake, and infection status postoperatively. One month later, the above injection steps are repeated.
[0018] Preferably, the above steps are repeated until the mouse CP / CPPS model is formed after one and a half months.
[0019] Preferably, the modeling method further includes a step of detecting mouse model characteristics, and selecting any one of steps (1)-(3) in claim 1 to further optimize the mouse model based on the mouse model characteristics.
[0020] Preferably, the steps for detecting mouse model characteristics include, but are not limited to, von Frey method for measuring pain threshold, open field test, and pathological examination of prostate specimens from mouse models.
[0021] The von Frey method for measuring pain threshold includes: placing mice in transparent plastic cages with wire mesh bottoms; after acclimatization, measuring the whole-body and local mechanical hypersensitivity of the mice by paw withdrawal threshold and abdomen withdrawal threshold; using von Frey hairs with logarithmically increasing stiffness to test the PWT value of the mice; and determining 50% of the mechanical threshold according to the Dixon up-down method.
[0022] Preferably, the open field test includes measuring the anxiety behavior of mice using an open field test: placing the mouse in the center of the bottom surface of an open field box, with the experimenter away from the open field box, and simultaneously taking a video to record the movement speed, movement distance, rest time, movement distance along the edge or movement distance in the center.
[0023] Preferably, the anxiety behavior of mice is measured using an elevated cross maze. Mice are placed on the central platform of the elevated cross maze, and behavioral indicators such as the time the mice stay in the open arm within 5 minutes and the number of times the mice enter the open arm are recorded.
[0024] Preferably, the depressive behavior of mice is measured using a forced swimming test; mice are placed in a forced swimming pool for 20 minutes every morning at 8:30, and behavioral indicators such as immobility time are recorded; the experimental animals are petted every day before the experiment to eliminate their fear of the experimenter; pathological examination of prostate specimens: prostate tissues taken after euthanizing mice are fixed in 4% paraformaldehyde for 24 hours, then routinely embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), and observed under a light microscope for changes in the prostate gland, ducts, and stroma.
[0025] Accordingly, the present invention provides a mouse model of chronic prostatitis or chronic pelvic pain syndrome. This mouse model was prepared using the modeling method described above.
[0026] The modeling method of the present invention is used to prepare mouse models of chronic prostatitis or chronic pelvic pain syndrome.
[0027] To simulate the role of pathogenic microorganisms in CP / CPPS, this invention uses sheep multivalent inactivated vaccine (trivalent and quadruple protection) as material for intraperitoneal injection in mouse models. Cultures of inactivated and detoxified Clostridium perfringens and Clostridium putrefactivee are used to simulate possible bacterial infection states, so as to more closely approximate the natural CP / CPPS course.
[0028] Compared to existing CP / CPPS models, the advantages of this proposed solution are: high modeling success rate, simple method, low mortality rate, and the presence of a pain sensitization phenotype (chronic pain) and comorbid phenotypes such as anxiety and depression after successful modeling. Furthermore, it achieves this without the use of additional hormones or transcription factors to influence transcriptome expression regulation, laying a foundation for subsequent transcriptome and proteome experiments and possessing broad scientific research applications. The modeling time is approximately 45 days, the modeling materials are readily available, and the phenotype can be maintained for at least 90 days, and up to more than six months. Moreover, the high success rate and low mortality rate per batch contribute to the low overall cost, making this model economically viable. Attached Figure Description
[0029] Figure 1 The image shows paraffin sections of the prostate gland in a mouse CP / CPPS model. (A) Prostate gland of the mouse CP / CPPS model; (B) Prostate gland of a healthy control mouse. HE staining, 200x. Numerous lymphocytes infiltrated the stroma of the mouse CP / CPPS model prostate gland, and edema of the prostate ducts were observed.
[0030] Figure 2 The results of the von Frey method for testing pain thresholds are shown. (A) Schematic diagram of the pain threshold test; (B) Pain threshold at paw withdrawal (p<0.05); (C) Pain threshold at abdominal constriction (p<0.05). The paw represents the systemic pain threshold, and the abdomen represents the local pain threshold. As shown in (B) and (C), compared with the healthy control group, the mouse CP / CPPS model showed a significant decrease in both systemic and lower abdominal local pain thresholds, indicating that the mouse CP / CPPS model is in a state of mechanical pain hypersensitivity, demonstrating that the mouse CP / CPPS model exhibits a phenotype of decreased pain threshold. PWT: Paw-Withdrawal Threshold; ACT: Abdominal Constriction Threshold. Control: Healthy control group; CP / CPPS: Mouse CP / CPPS model group.
[0031] Figure 3 The results of the open field test in the mouse CP / CPPS model are shown. (A) Open field test trajectory recording; (B) 5-minute activity distance (p<0.01); (C) 5-minute central dwell time (p<0.05). Compared with the healthy control group, the total activity distance and central dwell time of the mouse CP / CPPS model were significantly reduced, reflecting a decline in the mice's autonomous movement and exploration abilities, indicating that the mouse CP / CPPS model exhibits an anxiety phenotype. Control: Healthy control group; CP / CPPS: Mouse CP / CPPS model group.
[0032] Figure 4The results of the tail suspension test in the mouse CP / CPPS model are shown. (A) Schematic diagram of the tail suspension test; (B) Curve of immobility time recorded over 5 minutes; (C) Statistical graph of immobility time (p<0.05). The tail suspension test (TST) is similar to forced swimming. It induces "behavioral despair" similar to an immobility period and records the immobility time to reflect the depression level of the mice. The longer the immobility time, the more depressed the mice are. The results showed that the immobility time after tail suspension was significantly abnormal in the mouse CP / CPPS model compared to the healthy control group, indicating that the mouse CP / CPPS model has a depressive phenotype. Control: Healthy control group; CP / CPPS: Mouse CP / CPPS model group. Detailed Implementation
[0033] Example 1
[0034] A mouse model of chronic prostatitis / chronic pelvic pain syndrome is provided, and the specific modeling method is as follows:
[0035] Two 4-month-old Wistar rats were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg / kg) and then sacrificed. Prostate tissue was aseptically harvested, rinsed with physiological saline to remove residual blood, and 2.0 ml of physiological saline solution containing 0.5% Triton X-100 was added. The homogenate was homogenized using a glass homogenizer on an ice-water bath and centrifuged at 4°C for 30 minutes. The supernatant was collected. The protein content of the prostate tissue in the homogenate was determined using a spectrophotometer, and the protein concentration was adjusted to 20 mg / mL with 0.1 mol / L pH 7.4 PBS buffer. The prostate homogenate could be stored in liquid nitrogen or frozen at -80°C. Before use, the prostate homogenate was thoroughly mixed with Freund's complete adjuvant (FCA) at a 1:1 volume ratio and filtered through a 0.22 μm PES bacterial filter to prepare an injectable suspension.
[0036] Preparation of the intraperitoneal injection solution: At room temperature, following the instructions, dissolve 20 doses of lyophilized ovine multivalent inactivated vaccine (trivalent and quadruple protection) powder in 20 mL of the included aluminum hydroxide gel saline suspension to prepare the intraperitoneal injection solution. This mixture should be used immediately and should not be stored overnight.
[0037] Modeling solution injection: C57BL / 6N mice (6-8 weeks old, average weight 20g, SPF grade, routinely fed) were anesthetized with sodium pentobarbital (50mg / kg) via intraperitoneal injection, and the abdomen was prepared. Four to six injection sites were selected, and 0.05mL of prostate homogenate suspension was injected subcutaneously, followed by 0.05mL of vaccine intraperitoneal injection. After confirming the model rats were awake from anesthesia, they were returned to clean, sterile cages. Postoperative monitoring of activity, food intake, and infection was conducted. The above injection steps were repeated after 30 days. By day 45, the mouse CP / CPPS model was established and maintained for at least 90 days.
[0038] Behavioral Experiments: After successful modeling, the pain threshold was measured using the von Frey method. Mice were placed in transparent plastic cages with wire mesh bottoms and allowed to adapt for 30 minutes. The paw withdrawal threshold (PWT) and abdominal withdrawal threshold (ACT) were used to measure the mice's systemic and localized mechanical hypersensitivity to pain. The PWT value was tested using von Frey hairs with logarithmically increasing stiffness. The 50% mechanical threshold was determined using the Dixon up-down method. Anxiety behavior in mice was measured using an open field test. A cubic gray wooden open field box was used. During the experiment, mice were placed in the center of the box's bottom surface, and the experimenter stayed away from the box while recording video for 30 minutes, recording behavioral indicators such as movement speed, movement distance, rest time, distance along the edges, and distance to the center. Anxiety behavior in mice was measured using an elevated cross maze. Mice were placed on the central platform of the elevated cross maze, and behavioral indicators such as the time spent in the open arms and the number of times the mouse entered the open arms were recorded within 5 minutes of movement. Depressive behavior in mice was measured using a forced swimming test. Mice were placed in a forced swimming pool and forced to swim for 20 minutes every morning at 8:30 AM, and behavioral indicators such as immobility time were recorded. For the first 7 days of the experiment, the animals were petted for 5 minutes daily to reduce their fear of the experimenters. The testing room was kept quiet, and the inside of the experimental equipment was cleaned with alcohol after each experiment to avoid affecting the next test. Finally, the video recordings were analyzed using TM-Vision computer software.
[0039] Pathological examination of prostate specimens: Prostate tissue obtained after euthanizing mice was fixed in 4% paraformaldehyde for 24 hours, then routinely embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE). Changes in the prostate gland, ducts, and stroma were observed under a light microscope. Results are as follows: Figure 1 As shown, a large number of lymphocytes infiltrated the prostate stroma and the prostate ducts were edematous in the mouse CP / CPPS model.
[0040] Figure 2The results of the von Frey test for pain threshold are shown, where the foot represents the systemic pain threshold and the abdomen represents the local pain threshold. Compared with the healthy control group, the mouse CP / CPPS model showed a significant decrease in both systemic and lower abdominal local pain thresholds, indicating that the mouse CP / CPPS model is in a state of mechanical pain hypersensitivity, demonstrating that the mouse CP / CPPS model has a phenotype of decreased pain threshold.
[0041] Figure 3 The results of the open field test were shown in the mouse CP / CPPS model. Compared with the healthy control group, the total activity distance and central dwell time of the mouse CP / CPPS model were significantly reduced, which reflects the decline in the mice's voluntary movement ability and exploration ability, indicating that the mouse CP / CPPS model has an anxiety phenotype.
[0042] Figure 4 The results of the tail suspension test in the mouse CP / CPPS model are shown. Similar to forced swimming, the tail suspension test (TST) induces a state of "behavioral despair" similar to immobility, and records the duration of immobility to reflect the mouse's depression level; the longer the immobility, the more depressed the mouse. The results showed that the immobility time after tail suspension was significantly abnormal in the mouse CP / CPPS model compared to the healthy control group, indicating that the mouse CP / CPPS model exhibits a depressive phenotype. Control: Healthy control group; CP / CPPS: Mouse CP / CPPS model group.
[0043] The specific embodiments of the invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and the devices and structures not described in detail should be understood to be implemented in a manner common to the art; those skilled in the art can make various modifications or alterations within the scope of the claims, and make several simple deductions, variations or substitutions, which do not affect the substantive content of the invention.
Claims
1. A method for establishing a mouse model, characterized in that, The mouse model is a mouse model of chronic prostatitis / chronic pelvic pain syndrome, and the modeling method includes the following steps: (1) Preparation of prostate homogenate: Four-month-old rats were sacrificed and their prostate tissue was removed under aseptic conditions. The tissue was ground or homogenized, centrifuged, and the supernatant was added to Freund's complete adjuvant. (2) Preparation of vaccine intraperitoneal injection solution: At room temperature, the inactivated vaccine lyophilized powder is dissolved in aluminum hydroxide gel physiological saline to prepare the vaccine intraperitoneal injection solution; (3) Injection of modeling solution: After anesthetizing the mice, inject prostate homogenate suspension subcutaneously and inject vaccine intraperitoneal injection; repeat the above injection steps until the mouse CP / CPPS model is formed.
2. The method for establishing a mouse model according to claim 1, characterized in that, The preparation of prostate homogenate includes: taking 4-month-old Wistar rats, euthanizing them after anesthesia, removing prostate tissue under sterile conditions, washing it, adding physiological saline solution containing 0.5% Triton X-100, homogenizing it with a glass homogenizer on an ice water bath, centrifuging at 4°C and collecting the supernatant. Then adjust the protein concentration to 20-100 mg / mL with 0.1 mol / L PBS buffer (pH 7.2-7.4). Before use, the prostate homogenate and Freund's complete adjuvant are thoroughly mixed at a volume ratio of 1:(0.5-2), filtered through a bacterial filter, and prepared into an injectable suspension.
3. The method for establishing a mouse model according to claim 2, characterized in that, The prostate homogenate was thoroughly mixed with Freund's complete adjuvant at a volume ratio of 1:1 at room temperature.
4. The method for establishing a mouse model according to claim 1, characterized in that, The preparation of the intraperitoneal injection solution for the vaccine involves dissolving 20 doses of freeze-dried powder of the multivalent inactivated sheep vaccine in a sterile aluminum hydroxide gel saline suspension at room temperature.
5. The method for establishing a mouse model according to claim 1, characterized in that, Injectable modeling solution includes: Six- to eight-week-old mice were anesthetized with sodium pentobarbital and their abdomens were prepared. Choose 4-6 injection sites, subcutaneously inject prostate homogenate suspension, and intraperitoneally inject vaccine intraperitoneal injection solution; After confirming that the mice used for modeling have regained consciousness after anesthesia, they were returned to clean, sterile cages. Postoperative monitoring of activity, feeding, and infection status was conducted. One month later, repeat the above injection steps; The mouse CP / CPPS model was established in one and a half months.
6. The method for establishing a mouse model according to any one of claims 1-5, characterized in that, The modeling method further includes a step of detecting mouse model characteristics, and selecting any one of steps (1)-(3) in claim 1 to further optimize the mouse model based on the mouse model characteristics; The steps for detecting mouse model characteristics include, but are not limited to, von Frey method for measuring pain threshold, open field test, and pathological examination of prostate specimens from mouse models.
7. The method for establishing a mouse model according to claim 6, characterized in that, The von Frey method for measuring pain threshold includes: placing mice in transparent plastic cages with wire mesh bottoms; after acclimatization, measuring the whole-body and local mechanical hypersensitivity of the mice by paw withdrawal threshold and abdomen withdrawal threshold; using von Frey hairs with logarithmically increasing stiffness to test the PWT value of the mice; and determining 50% of the mechanical threshold according to the Dixon up-down method. The open field test includes measuring the anxiety behavior of mice using an open field test: mice are placed in the center of the bottom surface of an open field box, and the experimenter moves away from the open field box while taking pictures to record movement speed, movement distance, rest time, movement distance along the edge or movement distance in the center. Anxiety behavior in mice was measured using an elevated cross maze. Mice were placed on the central platform of the elevated cross maze, and behavioral indicators such as the time the mice stayed in the open arm within 5 minutes and the number of times the mice entered the open arm were recorded. Depressive behavior in mice was measured using a forced swimming test. Mice were placed in a forced swimming pool at 8:30 am every day and forced to swim for 20 minutes. Behavioral indicators such as immobility time were recorded. The experimental animals were petted daily before the experiment to eliminate their fear of the experimenters. Pathological examination of prostate specimens: Prostate tissues taken from euthanized mice were fixed in 4% paraformaldehyde for 24 hours, then routinely embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), and observed under a light microscope for changes in the prostate gland, ducts, and stroma.
8. A mouse model, characterized in that, The mouse model described is a mouse model of chronic prostatitis or chronic pelvic pain syndrome, prepared using any one of the modeling methods described in claims 1-7.
9. The application of the mouse model creation method according to any one of claims 1-7, characterized in that, The modeling method described above is used to prepare mouse models of chronic prostatitis or chronic pelvic pain syndrome.