Construction method and application of herpes zoster neuralgia animal model

Abstract

The invention discloses a construction method and application of a herpes zoster neuralgia animal model. The construction method comprises the following steps: preparing an effective dose of VZV virus protein into immunogen; the immunogen is injected into the body of a non-human mammal, and a continuous pain behavior is induced in the body of the non-human mammal, so that the herpes zoster neuralgia animal model is constructed. The method has the advantages of being high in biological safety, accurate in simulation, stable in model phenotype and good in repeatability.

Description

Technical Field

[0001] This invention relates to methods for constructing animal models in the field of biomedical technology, and particularly to a method for constructing and applying an animal model of herpes zoster neuralgia. Background Technology

[0002] Postherpetic neuralgia (PHN) is the most common chronic complication following varicella-zoster virus (VZV) infection, with an incidence rate of 9%-34%. The typical clinical feature of PHN is severe neuropathic pain lasting for more than 3 months after the skin lesions have healed. Because the pathogenesis of PHN is not fully understood, effective prevention and treatment methods are lacking in clinical practice. Therefore, constructing animal models that can mimic the key features of the disease is crucial for mechanistic research and drug development.

[0003] However, existing mainstream PHN animal models are mainly divided into two categories: VZV live virus models and HSV-1 alternative models. Both models have some limitations that hinder the progress of related research. Specifically, while the VZV live virus inoculation model can induce persistent mechanical hyperalgesia and other pain behaviors in rodents, VZV's strict human tropism prevents effective infection in animals. Furthermore, handling live viruses requires high-level biosafety laboratories, significantly limiting basic research on PHN mechanisms and drug development. On the other hand, while infecting mice with herpes simplex virus type 1 (HSV-1) can cause shingles-like skin lesions and pain behaviors, HSV-1 and VZV are two different viruses. The pathological mechanisms of models established using HSV-1 are fundamentally different from those of PHN induced by human VZV infection, thus limiting the relevance of research findings to clinical translation. Summary of the Invention

[0004] The purpose of this invention is to provide a method for constructing and applying an animal model of herpes zoster neuralgia. This invention has the advantages of high biosafety, accurate simulation, stable model phenotype, and good reproducibility.

[0005] The technical solution of the present invention: a method for constructing an animal model of herpes zoster neuralgia, comprising the following steps: An effective dose of VZV viral protein was prepared into an immunogen; The immunogen was injected into a non-human mammal, inducing persistent pain behavior in the non-human mammal, thereby constructing an animal model of herpes zoster neuralgia.

[0006] The non-human mammal used in the above-mentioned method for constructing an animal model of herpes zoster neuralgia is a mouse.

[0007] In the aforementioned method for constructing an animal model of herpes zoster neuralgia, the VZV viral protein is derived from the culture supernatant of the VZV virus and obtained through purification.

[0008] In the aforementioned method for constructing an animal model of herpes zoster neuralgia, the concentration of the VZV viral protein in the immunogen is from 0.0005 ng / μl to 5 ng / μl.

[0009] In the aforementioned method for constructing an animal model of herpes zoster neuralgia, the single injection dose of the immunogen is 30-50 μl.

[0010] In the aforementioned method for constructing an animal model of herpes zoster neuralgia, the injection is a subcutaneous injection into the sole of the foot.

[0011] The aforementioned method for establishing an animal model of herpes zoster neuralgia involves using behavioral methods to detect pain-related behaviors in animals to confirm the successful establishment of the animal model. The behavioral methods include one or more of mechanical hyperalgesia, thermal hyperalgesia, and cold hyperalgesia.

[0012] An animal model of herpes zoster neuralgia constructed using the aforementioned method.

[0013] The aforementioned animal model of herpes zoster neuralgia is used in screening or evaluating drugs for the prevention or treatment of herpes zoster neuralgia.

[0014] The aforementioned animal model of herpes zoster neuralgia was used in the study of the pathogenesis of herpes zoster neuralgia.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention does not use live VZV virus, thus avoiding uncontrollable biological processes such as virus replication and latency. It does not require operation in a high-level biosafety laboratory, significantly reducing biosafety risks and research costs, and therefore has the advantage of high safety.

[0016] 2. This invention directly uses VZV viral proteins associated with human pathogenicity as pain causative agents to construct a neuroimmune inflammation and pain process driven by VZV antigen specificity, eliminating interference from other viruses such as HSV-1. The model has a higher correlation with human PHN, thus having the advantage of accurate simulation.

[0017] 3. By controlling the dosage and injection site of viral proteins, this invention can induce significant and sustained pain behaviors such as mechanical hyperalgesia, thermal hyperalgesia, and cold hyperalgesia. The model has a high success rate and good reproducibility.

[0018] 4. The model of this invention provides a safe, reliable and effective tool for studying the neuroimmune mechanism of PHN, discovering new drug targets, and screening and evaluating analgesics, and has wide applications. Attached Figure Description

[0019] Figure 1 The image shows the results of a mechanical pain hypersensitivity test in male von Frey experimental rats after a single injection of VZV viral protein into the sole of their paws.

[0020] Figure 2 The image shows the results of thermal pain hypersensitivity detection in male rats after a single injection of VZV viral protein into the sole of their paw using a thermal radiation instrument (Hargreaves method).

[0021] Figure 3 The image shows the results of a single injection of VZV viral protein into the sole of a male rat's foot in an acetone test to detect cold hyperalgesia.

[0022] Figure 4 The image shows the results of a mechanical pain hypersensitivity test after a single injection of VZV viral protein into the sole of the foot of a female rat in the von Frey experiment.

[0023] Figure 5 The image shows the results of thermal pain hypersensitivity detection in female rats after a single injection of VZV viral protein into the sole of their paw using a thermal radiation instrument (Hargreaves method).

[0024] Figure 6 The image shows the results of a single injection of VZV viral protein into the sole of the foot of a female rat in an acetone test, resulting in cold pain hypersensitivity.

[0025] Figure 7 The image shows the changes in the myelin sheath structure of nerve fibers in the main trunk of the sciatic nerve after a single injection of VZV viral protein into the sole of the rat's foot. The upper image is a low-power microscopic observation of the nerve tissue (the scale bar corresponds to a larger field of view), and the lower image is a transmission electron microscopic observation of the nerve fibers (the scale bar is 2 μm, showing the ultrastructure).

[0026] Figure 8 Colocalization of ATF3, a neuronal injury marker, with NF200 neurons in DRG tissue 2 and 4 weeks after a single injection of VZV viral protein into the rat paw.

[0027] Figure 9 Colocalization of ATF3, a neuronal injury marker, with CGRP neurons in DRG tissue 2 and 4 weeks after a single injection of VZV viral protein into the rat paw.

[0028] Figure 10 Colocalization of ATF3 and IB4 neurons, neuronal damage markers, in DRG tissues 2 and 4 weeks after a single injection of VZV viral protein into the rat paw.

[0029] Figure 11 Figure 1 shows the electrophysiological results of neurons in the dorsal root ganglion (DRG). Figure 2 shows the raw current and voltage curves of action potential firing in the control and model groups. Figure 3 shows the minimum stimulation current to generate action potentials. Figure 4 shows the resting membrane potential. Figure 5 shows the amplitude of the action potential. Figure 6 shows the slope of the action potential rise.

[0030] Figure 12 Supplementary results of electrophysiological function testing of dorsal root ganglion (DRG) neurons are shown in Figure f, which shows the action potential decay slope; Figure g shows the after hyperpolarization potential; Figure h shows the number of action potential spikes under different stimulation currents; and Figure i shows the original action potential spike curves under different stimulation currents (100 / 200 / 300 pA). Detailed Implementation

[0031] The present invention will be further described below with reference to the accompanying drawings and embodiments, but this should not be construed as limiting the present invention.

[0032] Example 1: Establishment and experiment of a rat model of herpes zoster neuralgia.

[0033] I. Experimental Materials; 1. Experimental animals: Male and female SD rats, each weighing 180g, were selected and used for experiments after one week of acclimatization. The breeding environment met the standard for routine animal experiments.

[0034] 2. VZV virus-related reagents: VZV virus seed, Vero cells, cell culture grade PBS buffer.

[0035] 3. Experimental instruments: biosafety cabinet, cell culture incubator, 100kD cutoff tangential flow ultrafiltration system, sucrose density gradient centrifuge, ion exchange chromatography equipment, metal bath, microsyringe (specification adapted to 40μl injection volume), von Frey fibers, thermal radiation instrument (specifically for Hargreaves method), transmission electron microscope, laser confocal microscope, whole-cell patch-clamp system.

[0036] II. Experimental Procedure; (a) Preparation of VZV virus proteins; 1. In a biosafety cabinet, the VZV virus seed was inoculated into Vero cells in the logarithmic growth phase using the standard cell inoculation method, and cultured under suitable cell culture conditions for 72 hours.

[0037] 2. After culture, the viral titer in the cell culture medium was determined using standard viral titer detection methods to ensure that the titer reached a TICD50 of 10. 7 about.

[0038] 3. Collect the supernatant of the cell culture medium and concentrate it using a tangential flow ultrafiltration system with a 100kD cutoff to obtain a concentrated virus solution.

[0039] 4. Prepare a sucrose density gradient solution, slowly spread the concentrated virus solution on the top layer of the gradient solution, perform sucrose density gradient centrifugation, and collect samples from the VZV virus protein enrichment intervals based on the sugar content detection results.

[0040] 5. After desaccharification of the collected samples, further purification was carried out by ion exchange chromatography to finally obtain a VZV protein solution with a concentration of 1300 ng / μl, using cell culture grade PBS as the solvent.

[0041] (II) Preparation of VZV virus inactivation protein; Take the VZV virus, place it in a metal bath, set the temperature to 98℃, and heat for 10 minutes to completely remove protein activity, thus preparing VZV virus inactivated protein, and store it at 4℃ for later use.

[0042] (III) Immunogen preparation and animal inoculation; 1. Immunogen preparation: VZV virus protein solutions of different concentration gradients (0.0005 ng / μl, 0.005 ng / μl, 0.05 ng / μl, 0.5 ng / μl, 5 ng / μl) are emulsified to form stable water-in-oil emulsions, thus obtaining immunogens of each concentration gradient.

[0043] 2. Animal Grouping and Inoculation: SD rats were randomly divided into a model group (VP) and a control group (IVP), with half males and half females in each group. In the model group, 40 μl of the corresponding concentration of immunogen was injected once into the sole of one foot using a microsyringe; in the control group, an equal amount of VZV virus inactivating protein was injected once into the sole of one foot. During injection, it was ensured that the drug was injected into the subcutaneous tissue of the foot, avoiding blood vessels and nerves.

[0044] (iv) Model validation and testing; 1. Pain phenotype assessment (behavioral testing): Testing time points: Tests were conducted on days 3, 7, 14, 21, 28, 35, 42, and 56 after injection.

[0045] Mechanical pain hypersensitivity test: The mid-stomach of the injection side of the rat foot was stimulated with von Frey fibers, and the 50% paw withdrawal threshold (PWT) was measured according to the standard test procedure, and the test data were recorded.

[0046] Thermal hyperalgesia test: Using a thermal radiation meter (Hargreaves method), the middle of the sole of the rat's injection side was targeted, and the time from the start of thermal radiation to the rat's withdrawal response was measured, i.e., the withdrawal latency (PWL), and the test data were recorded.

[0047] Cold pain hypersensitivity test: The acetone test method was used. An appropriate amount of acetone was drawn with a microsyringe and quickly dripped into the middle of the sole of the rat's paw on the injection side. The cold pain response of the rat was observed and recorded within 1 minute.

[0048] 2. Evaluation of nerve damage; Transmission electron microscopy: On day 21 after model establishment (3 weeks after modeling), 3 rats were randomly selected from each group. After anesthesia, the main trunk of the sciatic nerve on the injection side was quickly separated. After treatment with 2.5% glutaraldehyde fixation, 1% osmium tetroxide fixation, gradient dehydration, embedding, ultrathin sectioning, staining, etc., the changes in the myelin sheath structure of nerve fibers were observed by transmission electron microscopy.

[0049] Immunofluorescence detection: On day 14 (2 weeks) and day 28 (4 weeks) after modeling, three rats were randomly selected from each group. After anesthesia, tissue from the dorsal root ganglion (DRG) on the injection side was taken, frozen sections were prepared, and after permeabilization and blocking, primary antibodies such as ATF3 antibody, NF200 antibody, IB4 fluorescent probe, and CGRP antibody were added and incubated overnight at 4°C. Then, secondary fluorescent antibody was added and incubated at room temperature. After DAPI staining, the colocalization of ATF3 with NF200, IB4, and CGRP neurons was observed by laser confocal microscopy.

[0050] 3. Electrophysiological testing; On day 7 after the model was established, three rats were randomly selected from each group. After anesthesia, the DRG tissue on the injection side was quickly separated, sections were prepared and placed in the recording chamber. Whole-cell patch-clamp technique was used to detect the electrophysiological characteristics of DRG neurons and record relevant data such as action potential firing frequency.

[0051] (v) Test results; Figure 1 The image shows the results of a mechanical pain hypersensitivity test in male von Frey rats after a single injection of VZV viral protein into the sole of their paws. Figure 1 The 50% withdrawal threshold (PWT) of all groups was close to 15g, indicating that the rats had consistent mechanical pain sensitivity before the experiment. After injection, the PWT of the control group remained at around 12-15g, without obvious hyperalgesia. From 4 hours onwards, the PWT of the model group decreased significantly (most groups dropped below 5g), indicating that VZV virus protein induced mechanical hyperalgesia in male rats.

[0052] Figure 2The image shows the results of thermal pain hypersensitivity detection in male rats after a single injection of VZV viral protein into the sole of their paw using a thermal radiation instrument (Hargreaves method). Figure 2 In the study, the withdrawal latency in all groups was approximately 10–11 seconds, indicating that the rats had consistent thermal pain sensitivity before the experiment. After injection, the withdrawal latency in the control group remained around 10–11 seconds, with no significant thermal pain sensitivity observed. In the model group, starting from 4 hours, the withdrawal latency was significantly shortened (most groups decreased to below 8 seconds, and some even approached 6 seconds), indicating that VZV virus protein induced thermal pain hypersensitivity in male rats.

[0053] Figure 3 The image shows the results of a single injection of VZV viral protein into the sole of a male rat's foot in an acetone test to detect cold hyperalgesia. Figure 3 In the study, the cold pain response scores of all groups were close to 1 point, indicating that the cold pain sensitivity of the rats was consistent before the experiment. After injection, the control group's score remained below 1 point, and no obvious cold pain sensitivity was observed. From 4 hours onwards, the scores of the model group increased significantly (most groups rose to 4-8 points), indicating that VZV virus protein induced cold pain hypersensitivity in male rats.

[0054] Figure 4 The image shows the results of a mechanical pain hypersensitivity test after a single injection of VZV viral protein into the sole of the foot of a female rat in the von Frey experiment. Figure 4 In the study, the PWT of both groups was close to 12g, indicating that the mechanical pain sensitivity of the rats was consistent before the experiment. After injection, the PWT of the control group remained at 10-12g and no pain hypersensitivity was observed. In the model, the PWT dropped significantly to below 5g from 4h and remained at a low level (it did not recover until 56 days), indicating that this concentration of VZV virus protein successfully induced persistent mechanical pain hypersensitivity in female rats.

[0055] Figure 5 The image shows the results of thermal pain hypersensitivity detection in female rats after a single injection of VZV viral protein into the sole of their paw using a thermal radiation instrument (Hargreaves method). Figure 5 In the study, the latency period of foot withdrawal in both groups was close to 11 seconds, and the pain sensitivity was consistent. After injection, the latency period in the control group remained at 10-12 seconds, with no thermal hyperalgesia. The latency period in the model group was significantly shortened to less than 8 seconds from 4 hours onwards and remained at a low level (it did not recover after 56 days), indicating that this concentration of VZV virus protein induced persistent thermal hyperalgesia in female rats.

[0056] Figure 6 The image shows the results of a single injection of VZV viral protein into the sole of the foot of a female rat in an acetone test, resulting in cold pain hypersensitivity. Figure 6At baseline, both groups had cold pain scores close to 1, indicating consistent cold pain sensitivity. After injection, the control group's score remained below 2, with no obvious cold pain sensitivity. The model group's score increased significantly from 4 hours onwards (reaching a maximum of about 7 points) and remained above 4 points (still higher than the control group at 56 days), indicating that this concentration of VZV viral protein induced persistent cold pain hypersensitivity in female rats.

[0057] Figure 7 This image shows the changes in myelin sheath structure of nerve fibers in the main trunk of the sciatic nerve after a single injection of VZV viral protein into the plantar surface of a rat. The upper image is a low-power microscopic observation of the nerve tissue (scale bar corresponds to a larger field of view), and the lower image is a transmission electron microscopic observation of the nerve fibers (scale bar is 2 μm, showing the ultrastructure). Figure 7 As can be seen, in the control group, the cells / fibers in the nerve tissue were regularly arranged and morphologically intact, the myelin sheath of the nerve fibers was continuous and dense, and the axonal morphology was normal, with no obvious damage. In the model group, the nerve tissue showed obvious structural disorder, with loosely arranged cells / fibers; the myelin sheath of the nerve fibers was swollen, broken, and vacuolated (the white area is the myelin sheath damage area), and the axonal structure was destroyed, presenting typical characteristics of nerve injury. This figure verifies the nerve injury basis of the pain model from a morphological perspective: VZV viral protein treatment triggered pathological changes in nerve tissue (especially the myelin sheath), which is the structural reason for hyperalgesia, further supporting the effectiveness of the animal model.

[0058] Figure 8 Two and four weeks after a single injection of VZV viral protein into the plantar surface of rats, the colocalization of neuronal injury markers ATF3 and NF200 in DRG tissues was observed. DAPI (blue) labels cell nuclei, showing cell distribution; ATF3 (red) is a neuronal injury marker (expression increases after neuronal injury); NF200 (green) is a marker of large-diameter myelinated sensory neurons; the merged graph shows the colocalization of ATF3 and NF200. Figure 8In the control group, regardless of whether the model was established for 2 or 4 weeks, ATF3 showed no significant red signal (only a small amount of background was observed), while NF200 showed a uniform distribution of green signal, indicating that the neurons in the control group were not significantly damaged. In the model group, after 2 weeks of modeling, ATF3 showed a significant red signal, and it partially overlapped with the green signal of NF200 in the Merge plot (co-localization). After 4 weeks of modeling, the red signal of ATF3 further increased, and the co-localization ratio with NF200 was even higher. This indicates that after VZV viral protein treatment, myelinated sensory neurons (NF200 positive) in the DRG were damaged, and the damage persisted over time. This figure validates the neural damage in the model at the molecular level: the high expression of ATF3 directly proves neuronal damage, and the damage occurs in myelinated neurons responsible for mechanical or thermal pain sensation, explaining the cellular mechanism of hyperalgesia in previous behavioral tests and improving the effectiveness of the model.

[0059] Figure 9 Two and four weeks after a single injection of VZV viral protein into the plantar surface of rats, the colocalization of neuronal damage markers ATF3 and CGRP in DRG tissues was observed. DAPI (blue) marks cell nuclei, showing cell distribution; ATF3 (red) represents a neuronal damage marker; CGRP (green) is a marker of nociceptive sensory neurons (transmitting pain); and the merged plot shows the colocalization relationship between ATF3 and CGRP. Figure 9 In the control group, ATF3 showed no significant red signal at both 2 and 4 weeks, while CGRP showed a uniform distribution of green signal, indicating that nociceptive neurons in the control group were not damaged. In the model group, ATF3 showed a significant red signal 2 weeks after modeling, and partially overlapped with the green signal of CGRP in the Merge plot (co-localization). Four weeks after modeling, the red signal of ATF3 continued to increase, and the co-localization ratio with CGRP further increased; this indicates that VZV viral protein treatment damaged the nociceptive neurons (CGRP-positive) responsible for transmitting pain in the DRG, and the damage continued to progress over time. This figure explains the cellular mechanism of hyperalgesia in the model from the perspective of pain-transmitting neurons: damage to nociceptive neurons (CGRP-positive) (high ATF3 expression) directly leads to abnormal pain signal transmission.

[0060] Figure 10 Two and four weeks after a single injection of VZV viral protein into the plantar surface of rats, the colocalization of ATF3 and IB4 neurons, markers of nerve injury, was observed in DRG tissue. DAPI (blue) marks cell nuclei; ATF3 (red) is a marker of nerve injury; IB4 (green) is a marker of unmyelinated nociceptive sensory neurons (transmitting pain); the merged graph shows the colocalization relationship between ATF3 and IB4. Figure 10In the control group, ATF3 showed no significant red signal at 2 and 4 weeks of modeling, while IB4 showed a uniform distribution of green signal, indicating that the unmyelinated nociceptive neurons in the control group were undamaged. In the model group, ATF3 showed a significant red signal after 2 weeks of modeling and overlapped with the green signal of IB4 in the Merge plot (co-localization, marked with white arrows). After 4 weeks of modeling, the red signal of ATF3 continued to increase, and the co-localization ratio with IB4 remained at a high level. This indicates that after VZV viral protein treatment, the unmyelinated nociceptive neurons (IB4-positive) responsible for transmitting pain in the DRG were damaged, and the damage persisted. This figure supplements the neuronal damage types in the pain model: not only myelinated neurons (NF200-positive) were damaged, but unmyelinated nociceptive neurons (IB4-positive) were also damaged. The common damage to both types of neurons is the key cellular basis for the various types of hyperalgesia in the model, further enriching the evidence for the pathological mechanism of the model.

[0061] Figure 11 Figure 1 shows the electrophysiological results of neurons in the dorsal root ganglion (DRG). Figure 2 shows the raw current and voltage curves of action potential firing in the control and model groups; Figure 3 shows the minimum stimulation current to generate an action potential; Figure 4 shows the resting membrane potential; Figure 5 shows the action potential amplitude; and Figure 6 shows the action potential rise slope. Figure 11 As can be seen, the control group required 200 pA of stimulation to fire one action potential, while the VZV group only required 100 pA of stimulation to fire two action potentials, indicating that the neurons in the VZV group had higher excitability. The minimum stimulation current for firing action potentials in the model group was significantly lower than that in the control group, proving that neurons were more easily activated (excitability increased) after VZV treatment. However, there were no significant differences in resting membrane potential, action potential amplitude, and rise slope between the two groups, indicating that VZV treatment mainly affected neuronal excitability, rather than basic electrophysiological parameters. This figure verifies the neuronal functional abnormality of the model from an electrophysiological perspective: VZV virus treatment significantly increased the excitability of DRG neurons, which is the core functional mechanism of hyperalgesia (neurons are more likely to fire, and pain signal transmission is enhanced), directly explaining the cause of the hyperalgesic phenotype in behavioral tests.

[0062] Figure 12 Supplementary results of electrophysiological function testing of dorsal root ganglion (DRG) neurons are shown; Figure f shows the action potential decay slope; Figure g shows the after hyperpolarization potential; Figure h shows the number of action potential spikes under different stimulation currents; Figure i shows the original action potential firing curves under different stimulation currents (100 / 200 / 300 pA). Figure 12As can be seen, there was no significant difference (ns) in the action potential decay slope and post-hyperpolarization potential between the two groups, indicating that VZV treatment did not change the basic waveform characteristics of the action potential. In the control group, the number of action potentials only increased slightly with increasing stimulation current. In the model group, the number of action potentials increased significantly with increasing stimulation current (*), and at the same current (e.g., 100 pA), the firing frequency of the VZV group was much higher than that of the control group. This indicates that VZV treatment significantly enhanced the firing capacity of DRG neurons and abnormally increased excitability. This figure further deepens the evidence of neuronal dysfunction at the level of firing frequency: VZV virus treatment not only lowered the activation threshold of neurons (as shown in previous Rheobase results) but also enhanced their sustained firing capacity. This is a direct functional mechanism of excessive pain signal transmission and hyperalgesia, forming a complete causal relationship with previous behavioral and morphological results.

[0063] The behavioral results above showed that, starting from day 3 after injection, the model group rats exhibited significant mechanical hyperalgesia (decreased PWT), thermal hyperalgesia (shortened PWL), and cold hyperalgesia (enhanced cold pain response), and this pain phenotype persisted until day 56 without recovery, showing a significant difference from the control group. Transmission electron microscopy revealed swelling and rupture of the sciatic nerve myelin sheath in the model group rats, while the myelin sheath structure remained intact in the control group. Immunofluorescence assays showed that the colocalization rate of ATF3 neurons with NF200, IB4, and CGRP neurons in the DRG tissue of the model group was significantly higher than that in the control group. Electrophysiological assays showed that the excitability of DRG neurons in the model group was significantly increased, with a statistically significant difference from the control group. Based on the above results, it can be confirmed that this embodiment can construct a rat model of herpes zoster neuralgia.

[0064] Example 2: Application of an animal model of herpes zoster neuralgia in drug screening.

[0065] Using the rat model of herpes zoster neuralgia constructed in Example 1, the efficacy of a candidate analgesic drug (e.g., a novel neuroinflammation inhibitor) was evaluated.

[0066] Rats that successfully developed the model were randomly divided into a model control group and a drug administration group.

[0067] The drug administration group was given the candidate drug by gavage or intraperitoneal injection, while the model control group was given an equal amount of solvent.

[0068] Before and after drug administration, the mechanical pain threshold and thermal pain latency of rats in each group were detected according to the method in Example 1.

[0069] Results analysis: If the pain behaviors (such as increased PWT and prolonged PWL) of rats in the drug administration group are significantly improved compared with those in the model control group, it indicates that the candidate drug has the potential to treat herpes zoster neuralgia.

[0070] Example 3: Study on the pathogenesis of herpes zoster neuralgia Using the herpes zoster neuralgia animal model of Example 1, we explored the key molecular changes and pathophysiological mechanisms related to the nervous and immune systems during the pathogenesis of herpes zoster neuralgia.

[0071] A rat model of herpes zoster neuralgia and age-matched healthy rats (as normal control group) were used.

[0072] At different key time points after modeling, rats were randomly selected from the model group and the normal control group. After anesthesia, dorsal root ganglia, sciatic nerve, spinal cord and related immune tissues were collected. Some samples were used for histological observation and some samples were used for molecular level detection.

[0073] Rats were assessed for pain-related behaviors using standard methods, and phenotypic changes such as hyperalgesia and allergy were recorded.

[0074] The structure of nerve fibers was observed using transmission electron microscopy, and the morphology and distribution of nerve cells and immune cells were analyzed using laser confocal microscopy.

[0075] Western blot and real-time quantitative PCR were used to detect the expression levels and gene transcription of neuroinjury markers, inflammatory factors, key proteins in signaling pathways, and other indicators; immunohistochemistry was used to clarify the tissue localization of target molecules.

[0076] Based on the above detection results, the association between differentially expressed molecules and pain phenotypes and nerve damage was analyzed to preliminarily screen key pathways or molecules involved in the pathogenesis. Their roles can be further verified through in vitro cell experiments. Furthermore, by comparing the detection data of the model group and the normal control group, if the model group shows abnormal expression of specific molecules, damage to neural structures, and changes in pain behavior, and if these three are logically related, the role of that molecule or related pathway in the pathogenesis of herpes zoster neuralgia can be revealed, providing experimental support for elucidating the disease's pathogenesis.

[0077] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for constructing an animal model of herpes zoster neuralgia, characterized in that: Includes the following steps: An effective dose of VZV viral protein was prepared into an immunogen; The immunogen was injected into a non-human mammal, inducing persistent pain behavior in the non-human mammal, thereby constructing an animal model of herpes zoster neuralgia.

2. The method for constructing an animal model of herpes zoster neuralgia according to claim 1, characterized in that: The non-human mammal in question is a rat.

3. The method for constructing an animal model of herpes zoster neuralgia according to claim 1, characterized in that: The VZV virus protein is derived from the culture supernatant of the VZV virus and obtained through purification.

4. The method for constructing an animal model of herpes zoster neuralgia according to claim 1, characterized in that: The concentration of the VZV viral protein in the immunogen is from 0.0005 ng / μl to 5 ng / μl.

5. The method for constructing an animal model of herpes zoster neuralgia according to claim 4, characterized in that: The single injection dose of the immunogen is 30-50 μl.

6. The method for constructing an animal model of herpes zoster neuralgia according to claim 1, characterized in that: The injection was a subcutaneous injection into the sole of the foot.

7. The method for constructing an animal model of herpes zoster neuralgia according to claim 1, characterized in that: The successful establishment of an animal model of herpes zoster neuralgia was confirmed by detecting pain-related behaviors in animals using behavioral methods. The behavioral methods include one or more of mechanical hyperalgesia, thermal hyperalgesia, and cold hyperalgesia.

8. An animal model of herpes zoster neuralgia constructed by the method of any one of claims 1 to 7.

9. The use of the animal model of herpes zoster neuralgia according to claim 8 in screening or evaluating drugs for the prevention or treatment of herpes zoster neuralgia.

10. The application of the animal model of herpes zoster neuralgia according to claim 8 in the study of the pathogenesis of herpes zoster neuralgia.

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