Animal model of trigeminal neuralgia and construction method and application thereof

Abstract

This invention discloses an animal model of trigeminal neuralgia, its construction method, and its applications. This invention successfully constructed an animal model of trigeminal neuralgia and, through behavioral, cellular, and molecular biological experiments, conducted multi-dimensional research to systematically elucidate the association between changes in macrophages and neurons mediated by the NPY-Y2R signaling pathway and trigeminal neuralgia in the animal model. This model can be used for neuroimmunotherapy of trigeminal neuralgia and enables more effective and precise personalized clinical targeted therapy for patients with trigeminal neuralgia.

Description

Technical Field

[0001] This invention relates to the field of trigeminal neuralgia, and more specifically to animal models of trigeminal neuralgia, their construction methods, and applications. Background Technology

[0002] Trigeminal neuralgia (TN) is a refractory neuropathic pain condition of the head and face, characterized by typical neurosensitization. Mild stimulation (trigger points) can elicit paroxysmal, stabbing, and intractable severe pain within the facial distribution area of ​​the trigeminal nerve. Patients often experience negative emotions such as anxiety and depression, placing a significant psychological burden on them. The pathogenesis remains unclear. Current research suggests that the main causes of TN include nerve injury, demyelination, compression of the trigeminal nerve root, and central epileptic discharge-like changes. Some studies on neuropathic pain have also indicated the involvement of immune cells in peripheral and central sensitization processes. However, while most tissues and organs contain resident populations of immune cells, these cells exhibit high heterogeneity due to adaptation to different tissue-specific environments. Furthermore, the mechanism by which immune cells sense neuronal damage and generate a response after trigeminal nerve injury is not fully understood.

[0003] Currently, clinical treatment for trigeminal neuralgia (TN) primarily relies on surgical and pharmacological therapies, including microvascular decompression (MVD) and carbamazepine. While MVD can provide long-term pain relief for TN patients in 70% of cases, its deep surgical location, significant trauma, high risk, and high recurrence rate limit its application in TN treatment. Carbamazepine reduces hyperalgesia by inhibiting sodium ion channels and decreasing neuronal excitability; however, it may cause adverse neurological reactions such as dizziness, fatigue, and drowsiness. Furthermore, as the disease progresses, the remission periods gradually shorten, the pain intensifies, and the response to medication worsens. Therefore, elucidating the pathogenesis of TN and developing more non-invasive and effective treatments is crucial.

[0004] The information in the background section is merely intended to illustrate the general background of the invention and should not be construed as an admission or implication in any way that such information constitutes prior art known to those skilled in the art. Summary of the Invention

[0005] To address at least some of the technical problems in existing technologies, this invention, recognizing the current scarcity of research on the neuroimmune crosstalk mechanisms of trigeminal neuralgia (TN) and the lack of animal models for TN, successfully constructed a novel TN model. Using this model, the invention delves into the crucial role and specific mechanisms of the neuropeptide Y receptor Y2's "neuro-immune" interaction with resident macrophages in the trigeminal ganglion (TG) during TN development, thus providing a new clinical perspective for more effective and precise personalized targeted therapy for TN patients in the future. Specifically, this invention includes the following:

[0006] A first aspect of the present invention provides a method for constructing an animal model of trigeminal neuralgia, comprising the step of administering a neuropeptide Y receptor Y2 agonist to the animal.

[0007] In some embodiments, according to the method for constructing an animal model of trigeminal neuralgia according to the present invention, the agonist comprises a substance capable of promoting the amount and / or activity of neuropeptide Y receptor Y2.

[0008] In some embodiments, according to the method for constructing an animal model of trigeminal neuralgia according to the present invention, the substance includes at least one of small molecule compounds, nucleic acids, polypeptides, proteins, enzymes, antibodies or functional fragments thereof, and gene editing systems.

[0009] In some embodiments, according to the method for constructing an animal model of trigeminal neuralgia according to the present invention, the neuropeptide Y receptor Y2 agonist has the sequence: SKPDNPGEDAPAEDMARYYSALRHYINLITRQRY (SEQ ID No. 1).

[0010] In some embodiments, according to the method for constructing an animal model of trigeminal neuralgia according to the present invention, the method includes:

[0011] (1) The neuropeptide Y receptor Y2 agonist was administered to animals via stereotactic delivery.

[0012] (2) Detect behavioral and / or biochemical indicators of the animals after step (1).

[0013] In some embodiments, the method for constructing an animal model of trigeminal neuralgia according to the present invention includes the following steps:

[0014] (1) Prepare healthy animals;

[0015] (2) Administer a neuropeptide Y receptor Y2 agonist to the trigeminal ganglion of the animal;

[0016] (3) Detect behavioral and / or biochemical indicators of the animal after step (2).

[0017] In some embodiments, according to the method for constructing an animal model of trigeminal neuralgia according to the present invention, wherein,

[0018] The behavioral indicators include at least one of mechanical pain, pain-related emotions, and anxiety-like behaviors.

[0019] The biochemical indicators include the amounts of macrophage markers, neuronal damage markers, and / or inflammatory factors.

[0020] A second aspect of the present invention provides a method for identifying or screening compounds useful for relieving trigeminal neuralgia, comprising the following steps:

[0021] (a) Determine the behavioral and / or biochemical indicators of the animal model to obtain the first measurement data;

[0022] (b) Administer the test compound to the animal model;

[0023] (c) Determine the behavioral and / or biochemical parameters of the animal model after administration of the test compound to obtain second test data; and

[0024] (d) Compare the second test data with the first test data;

[0025] The animal model is obtained using the construction method described in the first aspect.

[0026] In a third aspect, the present invention provides a compound useful for relieving trigeminal neuralgia, which is obtained by the method described in the second aspect.

[0027] In a fourth aspect, the present invention provides a pharmaceutical composition for relieving trigeminal neuralgia, comprising the compounds described in the third aspect.

[0028] This invention demonstrates that by constructing a mouse TN model, administration of either a macrophage scavenger or an NPY2R antagonist can rescue facial hyperalgesia in TN mice. Through behavioral, cellular, and molecular biological experiments, this invention conducts multidimensional research to systematically elucidate the association between NPY2R pathway-mediated macrophage and neuronal changes and trigeminal neuralgia in TN model mice, thus providing a theoretical basis for neuroimmunotherapy strategies for trigeminal neuralgia. Attached Figure Description

[0029] Figure 1This study illustrates the long-term and stable behavioral manifestations of mechanical pain hypersensitivity and cold pain hypersensitivity in mice after TN modeling. (A) Changes in mechanical pain threshold (MPT) 28 days post-surgery. (B) Changes in cold pain threshold 28 days post-surgery. Compared with the control group (Sham), * P <0.05, ** P <0.01, *** P <0.001.

[0030] Figure 2 The following data shows anxiety-like behavior in mice 14 days after TN modeling. (A) Total open field distance. (B) Number of times entering the open field central area. (C) Time spent in the open field central area. Distance of movement in the elevated O-maze open arm (D), number of times entering the O-maze open arm (E), and time spent in the O-maze open arm (F). Distance of movement in the elevated cross open arm (G), number of times entering the cross open arm (H), and time spent in the cross open arm (I). Compared with the Sham group, * P <0.05, ** P <0.01.

[0031] Figure 3 The image shows macrophage infiltration in the TG group 14 days after TN modeling. (A) Heatmap analysis of different immune cell surface markers. The CD68 protein level (B) and mRNA expression (C) in the TG group were significantly increased compared to the Sham group. ** P <0.01. Tnfa (D) and Atf3 (E) mRNA expression was significantly increased, and iNOS and CD206(F) protein expression levels were significantly increased. (G) and (H) protein expression level quantitative analysis graphs. Compared with the Sham group, * P <0.05, ** P <0.01.

[0032] Figure 4 This diagram illustrates how CLO-depleted macrophages rescued hyperalgesia in TN mice and suppressed the inflammatory response in TG. (A) Flowchart of behavioral testing and drug treatment. (B) Changes in facial mechanical pain threshold. (C) Time taken for mice to enter the open arm of the O maze. (D) Distance traveled by mice into the open arm of the O maze. (E) TG Cd68 mRNA expression levels. (F)TG Tnfa mRNA expression level, BL = baseline. Compared with the Veh group,* P <0.05, ** P <0.01.

[0033] Figure 5 The effects of intraTG injection of NPY, NPY1R antagonists, and NPY2R antagonists on the mechanical and cold pain thresholds of the face in mice were shown. The effects of intraTG injection of the control solvent and different concentrations of NPY on the mechanical and cold pain thresholds (A) and (B) of the face in Naïve mice were shown; the effects of intraTG injection of the control agent and three different concentrations of NPY1R antagonists on the mechanical and cold pain thresholds (C) and (D) of the face in TN mice were shown; and the effects of intraTG injection of the control agent and three different concentrations of NPY2R antagonists on the mechanical and cold pain thresholds (E) and (F) of the face in TN mice were shown.

[0034] Figure 6 Knockout was shown Npy2r Effects on the mechanical pain threshold in mice. (A) Npy2r Changes in mechanical pain threshold after TN modeling in KO mice; (B) Npy2r Changes in mechanical pain threshold in KO mice after TG injection of NPY.

[0035] Figure 7 It shows Npy2r Following gene knockout, macrophage infiltration was observed in TG mice 14 days after TN modeling. (A) KO mice showed significantly reduced CD68 protein levels and (B) mRNA expression in TG compared to the WT group.* P <0.05, n=3, (C) Tnfa mRNA expression was significantly reduced compared to the WT group. P <0.05, n=3.

[0036] Figure 8 It was shown that specific activation of Y2R can induce a prolonged decrease in the facial mechanical pain threshold in mice.

[0037] Figure 9 The following data shows anxiety-like behavior in mice 14 days after TG injection of Y2R agonists. (A) Total distance traversed in the open field. (B) Number of times entering the central region of the open field. (C) Time spent in the central region of the open field. (D) Distance traversed in the open arms of the elevated O-maze. (E) Time spent in the open arms of the elevated O-maze. (F) Number of times entering the open arms of the elevated O-maze. (G) Distance traversed in the open arms of the elevated cross maze. (H) Time spent in the open arms of the elevated cross maze. (I) Number of times entering the open arms of the elevated cross maze. Compared with the Sham group, * P <0.05, ** P <0.01, n=6.

[0038] Figure 10 It was shown that specific activation of Y2R can induce macrophage infiltration and upregulation of inflammatory factors in mouse TG.

[0039] Figure 11 The effects of specific activation of Y2R and regulation of macrophages on pain behavior and pain-related negative emotions in mice are shown. (A) Experimental drug administration flowchart. (B) Macrophage depletion rescues facial mechanohypnosis in mice. (C) Macrophage depletion rescues facial cold hypersensitivity in mice. (D) Schematic diagram of the elevated cross maze; after macrophage depletion, the number of times (E) and time (F) of mice entering the open arms of the elevated cross maze significantly increased. (G) Schematic diagram of the elevated O maze; macrophage depletion had no significant effect on the number of times (H) and time (I) of mice entering the open arms of the elevated O maze. Detailed Implementation

[0040] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0041] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0042] Unless otherwise stated, 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. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0043] Those skilled in the art should understand that the step numbers (1), (2), or (a), (b), (c), (d), etc., are only for distinguishing different steps and do not indicate the order of the steps. The order of the above steps is not particularly limited as long as the purpose of the present invention can be achieved. Furthermore, two or more of the above steps can be combined and performed simultaneously. In addition, those skilled in the art should also understand that other steps or operations may be included between steps, such as before and after steps (1)-(2) or (a)-(d), or between any of these steps, for example, to further optimize and / or improve the method described in the present invention.

[0044] Construction of animal models

[0045] A first aspect of the present invention provides a method for constructing an animal model of trigeminal neuralgia, sometimes simply referred to herein as "the method of constructing the present invention", which includes the step of administering a neuropeptide Y receptor Y2 agonist to the animal.

[0046] In the construction method of the present invention, the animals used to construct the animal model are not particularly limited, and are generally non-human mammals, including but not limited to pigs, dogs, cats, mice, etc., with mice being preferred, such as rats and mice.

[0047] In the construction method of the present invention, the administration method of the agonist is not particularly limited. In a preferred embodiment, the administration method is transcranial stereotactic trigeminal ganglion (TG) injection.

[0048] In this invention, the applied drug is a neuropeptide Y receptor Y2 agonist. The terms "agonist," "promoter," and "activator" are used interchangeably. "Receptor agonist" has the meaning commonly understood in the art. In this invention, a neuropeptide Y receptor Y2 agonist includes any substance capable of binding to and activating the neuropeptide Y receptor Y2, thereby producing a biological effect. Through in-depth research, the inventors discovered the key role and specific mechanism of the neuropeptide Y receptor Y2 signaling pathway in the development of neuronal neuropathic hyperalgesia (TN). Specifically, TG injection of NPY can induce transient facial mechanohypnosis. Selective inhibition of NPY1R expression in TG during TN modeling has no significant effect on facial sensation in mice, while selective inhibition of NPY2R expression in TG can transiently rescue facial mechanohypnosis in mice. Therefore, specific activation of the NPY-Y2R signaling pathway can induce pain sensitization in the trigeminal nerve innervation area of ​​mice. This pathway, together with macrophages, mediates the development of pain-related negative emotions and plays a key role in emotional integration. Based on this, without being bound by any theory, any substance that can promote the amount and / or activity of neuropeptide Y receptor Y2 can be used to construct the trigeminal neuralgia animal model of the present invention.

[0049] In this invention, the substance includes at least one of small molecule compounds, nucleic acids, polypeptides, proteins, enzymes, antibodies or their functional fragments, and gene editing systems.

[0050] In a preferred embodiment, the agonist refers to a polypeptide, protein, enzyme, antibody (activating antibody), or functional fragment thereof that targets (or binds to) the neuropeptide Y receptor Y2, thereby activating the neuropeptide Y receptor Y2; or, the agonist may be a nucleic acid encoding the above-mentioned nucleic acid, a carrier containing the nucleic acid, or a cell containing the carrier. In a specific embodiment, the agonist is a polypeptide having the sequence shown in SEQ ID No. 1. The inventors have found that polypeptides having the above-mentioned sequence can achieve at least one of the following compared to existing drugs:

[0051] (1) It causes pain sensitization in the trigeminal nerve innervation area;

[0052] (2) Significantly reduced the facial mechanical pain threshold and prolonged the duration of mechanical pain in animals and exhibited anxiety-like behavior;

[0053] (3) Significantly increases or promotes the amount or expression level of macrophage markers, neuronal damage markers and / or inflammatory factors (including CD68, TNF-α, ATF-3).

[0054] Those skilled in the art will understand that the agonist may also comprise a genetically engineered reagent for overexpression of neuropeptide Y receptor Y2, the genetically engineered reagent comprising a nucleic acid encoding overexpression of neuropeptide Y receptor Y2, a vector containing the nucleic acid, and a cell containing the vector.

[0055] In a preferred embodiment, the construction method of the present invention includes:

[0056] (1) Prepare healthy animals;

[0057] (2) Administer a neuropeptide Y receptor Y2 agonist to the trigeminal ganglion of the animal;

[0058] (3) Detect behavioral and / or biochemical indicators of the animal after step (2).

[0059] In the construction method of the present invention, the behavioral indicators include at least one of mechanical pain, pain-related emotions and anxiety-like behaviors, and the behavioral indicators are preferably mechanical pain; the biochemical indicators include the amount of macrophage markers, neuronal damage markers and / or inflammatory factors, and are preferably the amount of CD68, TNF-α and / or ATF-3.

[0060] In this invention, the amount of neuropeptide Y receptor Y2 agonist is not particularly limited, generally 0.01-1000 nmol / μL, preferably 0.01-500 nmol / μL, or 0.01-400 nmol / μL, or 0.01-300 nmol / μL, or 0.01-200 nmol / μL, or 0.01-150 nmol / μL, or 0.01-100 nmol / μL, or 0.01-50 nmol / μL, or 0.01-40 nmol / μL, or 0.01-30 nmol / μL, most preferably 0.01-20 nmol / μL. Exemplary dosages include 0.01 nmol / μL, 0.05 nmol / μL, 0.1 nmol / μL, 0.2 nmol / μL, 0.3 nmol / μL, 0.4 nmol / μL, 0.5 nmol / μL, 0.75 nmol / μL, 0.95 nmol / μL, 1 nmol / μL, 1.25 nmol / μL, 1.5 nmol / μL, 1.75 nmol / μL, 2 nmol / μL, 2.5 nmol / μL, 2.75 nmol / μL, 3 nmol / μL, 3.25 nmol / μL, 3.5 nmol / μL, 3.75 nmol / μL, 4 nmol / μL, 4.25 nmol / μL, 4.5 nmol / μL, 4.75 nmol / μL, 5 nmol / μL, 5.25nmol / μL, 5.5 nmol / μL, 5.75 nmol / μL, 6 nmol / μL, 6.25 nmol / μL, 6.5 nmol / μL, 6.75nmol / μL, 7 nmol / μL, 7.25 nmol / μL, 7.5 nmol / μL, 7.75 nmol / μL, 8 nmol / μL, 8.25 nmol / μL, 8.5 nmol / μL, 8.75 nmol / μL, 9 nmol / μL, 9.25 nmol / μL, 9.5 nmol / μL, 9.75 nmol / μL, 10nmol / μL, 11 nmol / μL, 12 nmol / μL, 13 nmol / μL, 14 nmol / μL, 15 nmol / μL, 16 nmol / μL, 17nmol / μL, 18 nmol / μL, 19 nmol / μL, 20 The dosage is nmol / μL. The specific dosage can be adjusted according to actual needs. For example, a neuropeptide Y receptor Y2 agonist is administered at 1 nmol / μL.

[0061] In step (3) of the construction method of the present invention, behavioral and / or biochemical indicators of the animal are detected to confirm or optimize the construction conditions. In the present invention, the time for detecting the behavioral and / or biochemical indicators of the animal is not particularly limited. For example, it can be from day 1 to day 20 after drug administration or continuous drug administration, such as day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, day 15, day 16, day 17, day 18, day 19, day 20, etc.

[0062] Methods for identifying compounds useful for alleviating trigeminal neuralgia

[0063] This invention also provides a method for identifying compounds useful for relieving trigeminal neuralgia, sometimes referred to herein simply as "the identification method of this invention," which includes the following steps:

[0064] (a) Determine the behavioral and / or biochemical indicators of the animal model to obtain the first measurement data;

[0065] (b) Administer the test compound to the animal model;

[0066] (c) Determine the behavioral and / or biochemical parameters of the animal model after administration of the test compound to obtain second test data; and

[0067] (d) Compare the second test data with the first test data.

[0068] In the identification method of the present invention, the animal model is obtained by the construction method described in the first aspect of the present invention, and has been administered an appropriate dose of neuropeptide Y receptor Y2 agonist.

[0069] In some implementations, the behavioral indicator is the mechanical pain threshold, and if the mechanical pain threshold of the animal model increases after administration of the test compound, the test compound is identified as a compound useful for relieving trigeminal neuralgia.

[0070] In some implementations, behavioral indicators are the total distance of movement, the number of times the central region is entered and / or the time spent in the central region recorded in the open field test, and / or the distance of movement and / or the time spent in the open arms of the elevated O-maze and elevated cruciate maze. If the total distance of movement, the number of times the central region is entered and / or the time spent in the central region recorded in the open field test of the animal model after administration of the test compound increases, and / or the distance of movement and / or the time spent in the open arms of the elevated O-maze and elevated cruciate maze increases, then the test compound is identified as a compound useful for relieving trigeminal neuralgia.

[0071] In some implementations, the behavioral indicator is anxiety; if the anxiety in the animal model disappears or is relieved after administration of the test compound, the test compound is identified as a compound useful for relieving trigeminal neuralgia.

[0072] In some implementations, the biochemical indicator is the level of macrophage markers. If the level of macrophage markers (CD68 protein or its mRNA level) in the animal model TG decreases after administration of the test compound, the test compound is identified as a compound useful for relieving trigeminal neuralgia.

[0073] In some implementations, the biochemical indicator is a neuronal damage marker. If the level of neuronal damage markers (ATF-3 protein or its mRNA) in the animal model TG decreases after administration of the test compound, the test compound is identified as a compound useful for relieving trigeminal neuralgia.

[0074] In some implementations, the biochemical indicator is an inflammatory factor. If the inflammatory factor (TNF-α) in the TG of the animal model decreases after administration of the test compound, the test compound is identified as a compound useful for relieving trigeminal neuralgia.

[0075] Compounds and pharmaceutical compositions for alleviating trigeminal neuralgia

[0076] The present invention also provides a compound for relieving trigeminal neuralgia, which is a compound obtained by the screening method described above according to the present invention. Preferably, the compound of the present invention is a CSF1R inhibitor (e.g., PLX3397) and / or an NPY2R inhibitor or antagonist (e.g., BIIE0246).

[0077] Those skilled in the art will understand that a pharmaceutical composition may contain the above-mentioned compounds and pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients are not specifically limited, and appropriate excipients may be selected according to actual needs.

[0078] Example 1

[0079] I. Preliminary Experimental Research

[0080] First, a conventional trigeminal neuralgia model was constructed using a partial transection of the infraorbital nerve (pT-ION) model, and its pain behavior and biochemical indicators were detected for preliminary experimental research, as detailed below:

[0081] 1. Experimental Method:

[0082] 1.1 Establishment of the TN animal model

[0083] C57BL / 6J mice were used. In this study, the TN model employed a partial transection of the infraorbital nerve (pT-ION). After general anesthesia with 1.25% avorin, the infraorbital nerve was exposed by making an intraoral approach and incising the junction of the hard palate and the inner surface of the oral mucosa. The infraorbital nerve was freed using a glass needle, then severed, and 1 mm was removed to reduce nerve regeneration. The incision was then sutured with tissue glue and disinfected. The Sham procedure, like the TN procedure, exposed the infraorbital nerve; however, the nerve was not severed in the Sham group.

[0084] 1.2 Pain behavioral testing

[0085] 1.2.1 Mechanical pain

[0086] Mechanical pain threshold testing was conducted daily between 9:00 AM and 4:00 PM. All behavioral tests were performed by an additional experimenter unaware of the animal's grouping. The mechanical pain threshold test was performed top-down. Nine Von Frey fibers of varying strengths (0.023 g, 0.028 g, 0.068 g, 0.166 g, 0.407 g, 0.692 g, 1.202 g, 1.479 g, and 2.042 g) were applied vertically to the V2 region of the mouse's face, bent, and held for 2 seconds. A positive result was defined as the mouse exhibiting head-avoidance, scratching, or body trembling in response to the fiber stimulation; no response was considered negative.

[0087] 1.2.2 Cold pain

[0088] Acetone was instilled onto the face of mice to measure their cold pain threshold. The procedure was as follows: Mice were placed in a test box to acclimatize before the test. Acetone solution was rapidly instilled into the facial skin area innervated by the V3 trigeminal nerve. The time it took for the mouse to rub its face with its forepaws within 1 minute after acetone instillation was recorded. The longer the mouse rubbed its face, the more severe the cold hypersensitivity.

[0089] 1.3 Pain-related emotional and behavioral testing

[0090] 1.3.1 Open Field Experiment

[0091] Mice were placed in an open enclosure (50 cm × 50 cm × 50 cm) and allowed to explore freely for 5 minutes. The central area (25 cm × 25 cm) of the bottom area (50 cm × 50 cm) was considered the central region. The mice's activity trajectory in the open field and the time spent in the central region were recorded by a camera and analyzed using the Smart3.0 system. The following data were recorded: distance traveled in the central region, percentage of distance traveled in the central region, time spent in the central region, percentage of time spent in the central region, number of times the mouse entered the central region, and percentage of times the mouse entered the central region. Less activity in the central region indicated greater anxiety in the mouse.

[0092] 1.3.2 O-shaped elevated maze experiment

[0093] The O-shaped elevated maze consisted of an elevated circle with a diameter of 55 cm, divided into four areas, each separated by a 10 cm high enclosure. Mice were placed within the enclosed arms, their heads facing the junction between the enclosed and open arms, and allowed free movement within the maze for 5 minutes. The entire process was recorded using an infrared camera, and the mouse's movement trajectory was subsequently analyzed using Smart3.0 software. The percentage of distance traveled within the open arms, the percentage of time spent within the open arms, and the number of times the mouse entered the open arms were recorded.

[0094] 1.3.3 Elevated Cross Maze Experiment

[0095] The elevated cross maze consisted of two 35 cm × 5 cm open arms and two 35 cm × 5 cm × 40 cm closed arms. A 5 cm × 5 cm square open area was located in the center of the maze, which was 50 cm above the ground. Mice were placed in the center of the maze with their heads facing the closed arms, and video recordings were started to observe their activity for 10 consecutive minutes. The data was then analyzed using Smart3.0 software. The percentage of distance traveled in the open arms, the percentage of time spent in the open arms, and the number of times the mouse entered the open arms were recorded.

[0096] 1.4 Western Blot

[0097] Mouse triglycerides (TG) were acutely isolated, placed in liquid nitrogen, weighed and recorded, and pre-cooled tissue lysis buffer was added. After protein quantification, the samples were denatured at high temperature. An equal volume of protein samples was used for SDS-PAGE electrophoresis. After transfer to a membrane, the membrane was blocked, followed by incubation with primary antibody, and then with secondary antibody the next day. After cleaning, the chemiluminescent solution was evenly dropped onto the membrane for chemiluminescence.

[0098] 1.5 Real-Time qPCR

[0099] Mouse triglycerides (TG) were acutely isolated and placed in liquid nitrogen. TG RNA was extracted according to the RNA extraction kit instructions, and RNA concentration was detected using Nanodrop 2000. After reverse transcription, RT-qPCR was performed using SYBR Green mix, and data analysis was conducted using 7500 software.

[0100] 1.6 Immunofluorescence staining of tissues

[0101] Mice were perfused with PBS and 4% PFA sequentially. TG was isolated, fixed, graded dehydrated, embedded, and sectioned. Slides containing TG tissue were hydrated with PBS, blocked with BSA, and incubated overnight with primary antibody. The next day, they were incubated with secondary antibody at room temperature and finally mounted with DAPI-containing mounting medium. Images were taken under a laser confocal microscope, and quantitative fluorescence signal analysis was performed for comparison.

[0102] 2. Experimental Results

[0103] 2.1 Within 28 days after establishing the TN model in mice, both the mechanical pain threshold and the cold pain threshold continued to decrease.

[0104] After successfully establishing a mouse TN model, von Frey and acetone were used to detect the mechanical pain threshold and cold pain threshold of the mouse face. Compared with the Sham group, the model group mice had a lower mechanical pain threshold on day 3 post-surgery. Figure 1 A) and cold pain threshold ( Figure 1 Both B) decreased significantly and persisted until 28 days post-surgery. This modeling method yielded a phenotypically stable and long-term TN model.

[0105] 2.2 After establishing the TN model in mice, anxiety-like behaviors were observed.

[0106] Compared to the Sham group, the TN group showed a lower total distance traveled in the open field 14 days after modeling ( Figure 2 A), the number of times entering the central area ( Figure 2 (B) and time spent in the central area ( Figure 2 The C values ​​were significantly reduced in both the elevated O-maze and the elevated cross maze open arm movement distance. Figure 2 (D, G), number of times entering the open arm ( Figure 2 The E and H values ​​and the dwell time in the open arm were both significantly shortened. Figure 2 (F, I). The above experimental results suggest that significant anxiety-like behavior appeared 14 days after the establishment of the TN model in mice.

[0107] 2.3 After establishing the TN model in mice, macrophages infiltrated TG cells, and inflammatory factors were upregulated.

[0108] Fourteen days after modeling, TG tissues from Sham and TN mice were extracted for transcriptome sequencing analysis. The results showed that macrophage markers were upregulated in TG tissues. Figure 3 The expression levels of CD68 protein and mRNA were significantly upregulated (A). Figure 3 (B, C), the expression level of ATF-3 mRNA, a neuronal injury marker, is upregulated ( Figure 3 (E). This embodiment also found that 14 days after TN modeling, pro-inflammatory factors in TG... Tnfa ( Figure 3 The mRNA level of the M1 macrophage group was significantly higher than that of the Sham group. Both the M1 macrophage marker iNOS and the M2 macrophage marker CD206 were significantly upregulated, and the M1 / M2 ratio was significantly increased. Figure 3 These findings suggest that the development of trigeminal ganglion (TN) is accompanied by periganglionic macrophage infiltration, polarization, and activation of inflammatory factors, leading to a local inflammatory response.

[0109] 2.4 Peripheral macrophage scavenger clophosphonate liposomes can salvage TN-induced hyperalgesia by depleting macrophages.

[0110] To further clarify the role of macrophages in trigeminal neuralgia, peripheral macrophages were depleted using clodronate liposomes (CLO). Mice were intraperitoneally injected every 3 days with either clodronate liposomes or a liposome solvent control (Vehicle). Figure 4 A) significantly altered pain behavior in mice. Figure 4 (B). Compared with the Veh group, CLO administration significantly increased the movement time of mice in the open arm of the O maze (B). Figure 4 C) and distance of movement ( Figure 4 (D); Macrophage markers in TG of CLO group mice Cd68 ( Figure 4 E) and Tnfa ( Figure 4 The mRNA expression level of F was significantly reduced.

[0111] 2.5 Inhibiting NPY2R expression can significantly rescue the facial mechanical pain threshold in mice.

[0112] To investigate the effect of NPY2R on pain sensation in mice, NPY was first directly injected into the TG of Naïve mice. The results showed that intra-TG injection of a high concentration of NPY (1.0 nmol) induced transient facial mechanorepression in Naïve mice one day after injection. Figure 5 (A). No significant change was observed in facial cold pain in mice. Figure 5(B). Subsequently, NPY1R antagonist BIBO3304 and NPY2R antagonist BIIE0246 were injected intraTG into TN model mice at 14 days of age, respectively. The results showed that intraTG injection of NPY1R antagonists in TN mice did not significantly alter the mechanical pain threshold (B). Figure 5 C) and cold pain threshold ( Figure 5 (D), injection of NPY2R antagonists can transiently rescue facial mechanorexia in mice. Figure 5 E), has no significant effect on cold pain ( Figure 5 (F).

[0113] 2.6 Knockout Npy2r It can significantly salvage the facial mechanical pain threshold in mice.

[0114] To determine the role of the NPY2R receptor pathway in trigeminal neuralgia, a CRISPR-Cas9 gene knockout strategy was used to construct a [presumably a gene knockout assay]. Npy2r KO mice, wherein the sequence of gRNA1 is ACATAAGTCGATTAACAACTAGG (SEQ ID NO.2), CATAAGTCGATTAACAACTAGGG (SEQ ID NO.3), ACACAGGTGTGAAAGCACATGGG (SEQ ID NO.4) or GACACAGGTGTGAAAGCACATGG (SEQ ID NO.5).

[0115] use Npy2r A TN model was established using KO mice, and the results showed that TN did not cause... Npy2r KO mice exhibited facial mechanical hyperalgesia. Figure 6 A), and NPY injection via TG does not cause a decrease in facial mechanical pain threshold ( Figure 6 (B).

[0116] 2.7 Knockout Npy2r It can reduce TG macrophage infiltration and inflammatory response.

[0117] Npy2r Fourteen days after TN modeling in KO mice, compared with WT mice, neither the HET nor KO mice showed a significant upregulation of the expression levels of the macrophage marker CD68 protein and mRNA in TG. Figure 7 A and B), pro-inflammatory factors Tnfa ( Figure 7 The C) mRNA level in the group was significantly lower than that in the WT group. These results indicate that... Npy2r Knockout significantly reduced TG macrophage infiltration in TN mice and decreased the inflammatory response within TG.

[0118] II. Construction of a new TN mouse model

[0119] Next, this embodiment describes a new method for constructing a TN mouse model, as detailed below.

[0120] 1. Experimental Methods

[0121] 1.1 Stereoscopic Injection

[0122] Transcranial stereotactic trigeminal ganglion (TG) injection was used for drug administration. Specific injection method: C57BL / 6J mice were positioned prone in a stereotactic apparatus, with an opening made in the scalp to expose the anterior and posterior fontanelles. A micro-injection needle was connected to the infusion pump and controller. A group of medium-weight mice were selected, anesthetized with sodium pentobarbital, and their TG coordinates were determined using the stereotactic apparatus with lambda as the zero point. The remaining mice in the same group underwent transcranial TG injection using the same coordinates. A hole was drilled, and the tip of the micro-injection needle was slowly placed on this coordinate, injecting the drug (NPY2R agonist, sequence shown in SEQ ID NO.1, concentration 1 nmol / μL) at a rate of approximately 1 μL / min. To ensure efficient drug diffusion, the needle was left in place for 5 minutes after injection before being slowly withdrawn. Control group mice were injected with saline.

[0123] 1.2 Measurement of behavioral and biochemical indicators

[0124] As shown above.

[0125] 2. Experimental Results

[0126] 2.1 Specific activation of Y2R can induce prolonged mechanical hyperalgesia in mice.

[0127] Injection of a Y2R-specific agonist into naïve mice via TG resulted in a prolonged mechanical hyperalgesia, unlike NPY injection into TG. Figure 8 ).

[0128] 2.2 Specific activation of Y2R can induce anxiety-like behavior in mice.

[0129] Compared to the Saline group, the total distance mice traveled in the open field after TG injection of the agonist ( Figure 9 A), the number of times entering the central area ( Figure 9 (B) and time spent in the central area ( Figure 9 The C values ​​were significantly reduced in both the elevated O-maze and the elevated cross maze open arm movement distance. Figure 9 The D and G values ​​and the dwell time in the open arm were both significantly shortened. Figure 9 (E, H). The above experimental results suggest that mice exhibited significant anxiety-like behavior 14 days after injection of the TG agonist.

[0130] 2.3 Specific activation of Y2R can induce macrophage infiltration and upregulation of inflammatory factors in mouse triglycerides.

[0131] Compared to the Saline group, after injection of the agonist, the expression levels of the macrophage marker CD68 protein and mRNA in TG were significantly upregulated. Figure 10 A and B), neuronal damage markers Atf3 mRNA expression is upregulated ( Figure 10 D); pro-inflammatory factors Tnfa ( Figure 10 The C) mRNA level was significantly higher in the M1 macrophage group than in the Saline group. The M1 macrophage marker iNOS showed an increasing trend, and the M2 macrophage marker CD206 was significantly upregulated. Figure 10 These findings suggest that TN modeling with specific activation of Y2R can induce an inflammatory response within TG, and also indicate that NPY2R receptor-mediated neuroimmune interaction is an important link in the occurrence and maintenance of trigeminal neuralgia.

[0132] Example 2

[0133] Using the mice obtained in Example 1 as a drug screening model, various test compounds were administered. It was found that administration of the CSF1R inhibitor (PLX3397) rescued facial hyperalgesia and improved anxiety-like behavior in the mice. This also confirmed that prolonged hyperalgesia is caused by macrophage activation induced by the NPY2R receptor signaling pathway, thereby amplifying pain signals and leading to peripheral sensitization. Furthermore, after TG injection of the agonist, PLX3397 depleted macrophages in the mice. Figure 11 A) can significantly rescue facial mechanoreathia in mice. Figure 11 B) and cold sensitivity ( Figure 11 (C), and increased the number of times mice entered the elevated cross maze (C). Figure 11 DE) and stay time ( Figure 11 (GI) improves anxiety-like behavior in mice.

[0134] In summary, this invention investigated the regulatory effect of a Y2R selective agonist on trigeminal neuralgia. The results showed that, unlike NPY injection, a single injection of the NPY2R agonist into naïve mice induced a significant decrease in the mechanical pain threshold of the face within 1 day, which persisted until 28 days. Furthermore, changes in macrophages in TG cells 14 days after a single agonist injection were examined, revealing that macrophages in TG cells... Cd68, Tnfa, Atf3The mRNA expression level of the drug was significantly increased. Following TG injection of the agonist, continuous gavage administration of a CSF1R inhibitor for 7 days to deplete macrophages in vivo revealed that 5 days of continuous administration significantly rescued facial mechanical hyperalgesia in mice. Furthermore, anxiety-like behaviors in mice were significantly alleviated one week after drug withdrawal. These results suggest that specific activation of the NPY2R receptor signaling pathway can induce pain sensitization in the trigeminal nerve innervation area of ​​mice. This pathway, together with macrophages, mediates the development of trigeminal neuralgia-related negative emotions and plays a crucial role in emotional integration.

[0135] Although the invention has been described with reference to exemplary embodiments, it should be understood that the invention is not limited to the disclosed exemplary embodiments. Various adjustments or changes may be made to the exemplary embodiments described in this specification without departing from the scope or spirit of the invention. The scope of the claims should be interpreted in the broadest possible sense to cover all modifications and equivalent structures and functions.

Claims

1. A method for constructing an animal model of trigeminal neuralgia, characterized in that, Includes the following steps: (1) Prepare a healthy animal, namely a naïve mouse; (2) Inject 1 nmol / μL of a neuropeptide Y2 receptor agonist into the trigeminal ganglion of the healthy animal, the sequence of which is shown in SEQ ID No.1; (3) After 14 days, the behavioral and biochemical indicators of the animals after step (2) were detected. The behavioral indicators were mechanical pain, pain-related emotions and anxiety-like behaviors. The biochemical indicators were the amounts of macrophage marker CD68 protein or its mRNA, neuronal damage marker ATF-3 protein or its mRNA and inflammatory factor TNF-α. The detection of the upward trend of M1 macrophage marker iNOS and M2 macrophage marker CD206 was further included.

2. The method for constructing an animal model of trigeminal neuralgia according to claim 1, characterized in that, In step (2), the neuropeptide Y2 receptor agonist is injected into the animal using a stereotactic delivery method.

3. A method for screening compounds useful for relieving trigeminal neuralgia, characterized in that, Includes the following steps: (a) Determine the behavioral and biochemical indicators of the animal model to obtain the first measurement data; (b) Administer the test compound to the animal model; (c) Determine the behavioral and biochemical indicators of the animal model after administration of the test compound to obtain the second test data; and (d) Compare the second test data with the first test data; The animal model is obtained by the construction method described in claim 1 or 2; The biochemical indicators are the amounts of macrophage marker CD68 protein or its mRNA, neuronal damage marker ATF-3 protein or its mRNA, and inflammatory factor TNF-α, and further include the M1 macrophage marker iNOS, the M2 macrophage marker CD206, and the increasing trend of the M1 / M2 ratio.

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