Migraine headache animal model and construction method thereof

Abstract

The invention discloses a migraine animal model and a construction method thereof. The construction method comprises the steps that SD rats are placed in a clean-level environment to be adaptively bred for at least one week, and the rats freely ingest and drink water; and at least continuously adding a calcitonin gene related peptide solution into the nasal cavity of the rat once a day for at least 7 days. According to the animal model construction method, the CGRP closely related to migraine pathogenesis is taken as a target, and the CGRP is continuously dropped into the nasal cavity, so that rats generate migraine-like behaviors such as increased mechanical pain threshold, increased spontaneous pain behavior and increased photophobia degree, and the operation method is simple. The animal model construction method can be realized only by dripping the CGRP solution into the nasal cavity of the rat, is non-invasive and non-interventional in operation, and is simple and high in applicability. The migraine rat model is stable in phenotype, can be used for researching the pathogenesis of migraine and evaluating a new treatment method, and is expected to provide more reliable technical support and research thought for research on related aspects such as the pathogenesis of migraine and treatment targets.

Description

Technical Field

[0001] The invention belongs to the technical field of constructing experimental animal models in livestock breeding, and in particular relates to a migraine animal model and a construction method thereof. Background Art

[0002] Migraine is a common chronic neurovascular disease with complex pathophysiological mechanisms and unknown etiology. At present, animal models of migraine, such as epidural inflammatory soup stimulation model, intraperitoneal injection of nitroglycerin model, and cortical spreading depression (CSD), have been established, which can partially simulate migraine-like symptoms. However, since the invention of the above animal models is mostly based on phenotypes, there are certain limitations. A single model cannot fully simulate the symptoms of migraine, especially the characteristics of recurrent and unilateral attacks, and can often only reflect part of the pathophysiological mechanism of migraine.

[0003] Calcitonin gene-related peptide (CGRP) is a neuropeptide that plays a central role in the pathophysiology of migraine. Serum levels increase during migraine attacks, and intravenous infusion of CGRP can induce migraine-like attacks in patients with migraine. CGRP is a potent microvascular dilator that exists in two forms: α-CGRP and β-CGRP. During acute migraine attacks, the release of CGRP leads to vasodilation of the pericranial vessels and acts on CGRP receptors on pain fibers to transmit pain information to the central nervous system, leading to central sensitization.

[0004] Therefore, there is an urgent need to design an animal model based on the pathogenesis of migraine and which is easier to operate, so that researchers can effectively understand and study the occurrence mechanism, development patterns and intervention measures of migraine, in order to provide reliable technical support and research ideas for related research on the pathogenesis of migraine, therapeutic targets, etc. Summary of the invention

[0005] In view of the above technical problems, the present invention provides a migraine animal model and a method for constructing the same.

[0006] The technical solution provided by the present invention is as follows: In a first aspect, the present invention provides a method for constructing a migraine animal model, comprising the following steps: SD rats were placed in a clean environment for at least one week for adaptive breeding, with free access to food and water; After the adaptation, the calcitonin gene-related peptide solution was dripped into the nasal cavity of the rats once a day for at least 7 consecutive days.

[0007] In a possible implementation, the once a day is once a day at a fixed time period.

[0008] In a possible implementation manner, the CGRP solution is prepared by dissolving the CGRP in physiological saline or phosphate buffered saline (PBS) solvent.

[0009] Furthermore, the concentration of the calcitonin gene-related peptide solution is 2-6 μg / mL.

[0010] Furthermore, the concentration of the calcitonin gene-related peptide solution is 3-5 μg / mL.

[0011] Preferably, the concentration of the calcitonin gene-related peptide solution is 5 μg / mL.

[0012] In a possible implementation, the dripping amount of the calcitonin gene-related peptide solution is 30 μL / time.

[0013] In a second aspect, the present invention provides a migraine animal model constructed using the method described in the first aspect.

[0014] In a third aspect, the present invention provides a method for evaluating migraine-like behavior in the migraine animal model according to the second aspect, comprising the following steps: Rats were tested for mechanical hyperalgesia to obtain the mechanical pain threshold; Rats were subjected to cold stimulation test to obtain the cold pain threshold; The rats were subjected to a photophobia test to assess photophobia behavior; The rats were subjected to immunofluorescence test to detect the expression levels of c-Fos and CGRP.

[0015] In one possible implementation, photophobia testing, a method for assessing photophobia behavior, includes: The rats were placed in the center of the light-dark box and allowed to freely explore to acclimate to the environment; After acclimation, the test was conducted to detect the running trajectory of the rats, and the time spent in the light and dark chambers and the number of times they moved between the two chambers were recorded; Assess photophobic behavior.

[0016] In a possible implementation, the method of performing immunofluorescence test on rats to detect the expression levels of c-Fos and CGRP comprises the following steps: Sample pretreatment: rat cells were treated with a fixative, a permeabilizing agent, and a blocking solution in sequence to obtain a first sample; Primary antibody incubation: Incubate the first sample with a solution of specific c-Fos and CGRP antibodies to allow the antibodies to fully bind to the antigens, thereby obtaining a second sample; Washing: washing the second sample to remove unbound primary antibody and obtain the third sample; Secondary antibody incubation: incubating the third sample with a secondary antibody corresponding to the host species of the primary antibody to obtain a fourth sample; the secondary antibody is used to bind to the fluorophore; Washing: washing the fourth sample to remove unbound secondary antibody to obtain the fifth sample; Nuclear staining and sealing: staining and sealing the cell nuclei of the fifth sample to obtain the sixth sample; Detection: Detect the fluorescence signal of the sixth sample.

[0017] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects: 1. The animal model construction method described in the present invention is based on the CGRP target and can simulate the main symptoms of migraine, including hyperalgesia and photophobia.

[0018] 2. The animal model construction method of the present invention can be achieved by simply dripping CGRP solution into the rat's nasal cavity. It is non-invasive and non-invasive, and the method is simple and has strong applicability.

[0019] 3. The animal model obtained by the animal model construction method of the present invention has a stable phenotype and good repeatability.

[0020] 4. The animal model described in the present invention can be used to study the pathogenesis of migraine and evaluate new treatment methods, in order to provide more reliable technical support and research ideas for the study of the pathogenesis of migraine, therapeutic targets and other related aspects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the experimental process; Figure 2 This is a graph showing the trend of changes in the periorbital pain threshold of rats during the modeling period of nasal instillation of CGRP solution; Figure 3 This is a graph showing the changing trend of cold pain threshold scores in rats during the modeling period of nasal instillation of CGRP solution; Figure 4 The trend graph of the time spent by rats in the bright compartment in the light-dark box test during the modeling period of nasal instillation of CGRP solution; Figure 5 The trend chart of the number of times rats moved from one compartment to another in the light-dark box test during the modeling period of nasal instillation of CGRP solution; Figure 6 shows immunofluorescence staining of some brain regions; Fig. 6A , c-Fos expression in TNC site before and after nasal instillation of CGRP; Figure 6B , Expression of CGRP in TNC before and after nasal instillation of CGRP. DETAILED DESCRIPTION

[0022] The content of the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but the content of the present invention is not limited thereto.

[0023] Unified explanation of the English abbreviations that appear below: 1. CGRP: calcitonin gene-related peptide; 2. TNC: caudate of the spinal trigeminal nucleus; 3. c-Fos: fos proto-oncogene; 4. SPF: specific pathogen free; 5. SD: Sprague-Dawley; 6. PBS: phosphate buffered saline; 7. BSA: bovine serum albumin; 8. DAPI: 4',6-diamidino-2-phenylindole.

[0024] In the following examples, the rats were all SPF grade (specific pathogen free grade) 6-8 week old male SD (Sprague-Dawley) rats purchased from Hunan Slake Jingda Biotechnology Co., Ltd. and raised in the SPF environment of the Animal Experiment Center of Renmin Hospital of Wuhan University.

[0025] Example 1 See also Figure 1 , a method for constructing a migraine animal model, the steps are as follows: 3-5 rats were kept in each cage, and food and water were freely available. Rats lived in a 12-hour light / 12-hour dark environment (8:00-20:00 a.m. daily), the temperature in the rat room was maintained at 23-24°C, and the humidity was maintained at 55±5%. Each rat was randomly assigned to the experimental group and the control group using a random number table; Before the start of the experiment, the rats were acclimatized for at least one week; After the adaptation, the calcitonin gene-related peptide solution was dripped into the nasal cavity of the rats once a day for 7 consecutive days.

[0026] In a possible implementation, the once a day is once a day at a fixed time period.

[0027] In one possible implementation, the method of dripping the calcitonin gene-related peptide solution into the rat's nasal cavity is: grab the rat and fix its head so that the rat is facing up, and drip the calcitonin gene-related peptide solution into the rat's nasal cavity drop by drop multiple times through a microsyringe. After the solution is dripped, it is suspended for at least 1 minute to ensure that the rat inhales all the solution.

[0028] It should be noted that whether the CGRP solution is completely inhaled by the rats can be observed with the naked eye.

[0029] In a possible implementation, the CGRP solution is prepared by dissolving the CGRP in physiological saline or phosphate buffered saline (PBS).

[0030] Furthermore, the concentration of the calcitonin gene-related peptide solution is 2-6 μg / mL.

[0031] Furthermore, the concentration of the calcitonin gene-related peptide solution is 3-5 μg / mL.

[0032] Furthermore, the dripping amount of the calcitonin gene-related peptide solution is 30 μL / time.

[0033] In this example, 10 healthy adult SD rats weighing 250-300 g were selected as experimental subjects. 30 μL of 5 μg / mL CGRP solution was dripped into the nasal cavity of each rat at 8-10 am every day for 7 consecutive days.

[0034] Example 2 The method for evaluating the migraine-like behavior of the migraine animal model described in Example 1 comprises the following steps: 1. Perform mechanical hyperalgesia test on rats to obtain the mechanical pain threshold.

[0035] Specifically, mechanical hyperalgesia was assessed using von Frey filaments. A series of standardized von Frey filaments were applied continuously to the orbital region of rats, starting with a 0.008 g filament, with an interval of 1-2 minutes between consecutive stimulations. A positive response was considered when the mouse stroked the face with the ipsilateral forepaw and quickly retracted the head from the stimulation or vocalized. If the response was negative, the mechanical stimulation was gradually increased until a positive response was obtained and then decreased until a negative result was observed. The minimum force that produced a positive response was recorded as the threshold force. Each mouse was tested at least three times, and the average threshold was measured. The results are shown in Figure 2 shown.

[0036] 2. Perform cold stimulation test on rats to obtain the cold pain threshold.

[0037] Specifically, after the rats have adapted to the test environment, they are fixed with a rat fixator or other device, and cold stimulation is generated by the test equipment, such as using a cold plate or dripping acetone of a certain concentration on the hind limbs of the rats. The rats' reactions to the cold stimulation, such as lifting their feet, licking their paws, or struggling, are observed. The rats' reaction time to the cold stimulation is recorded, and the cold pain threshold of the rats is evaluated. The results are as follows: Figure 3 shown.

[0038] 3. Perform photophobia test on rats to evaluate photophobia behavior.

[0039] Specifically, rats were placed in the center of a light-dark box and allowed to freely explore to acclimate to the environment; After acclimation, the test was conducted to detect the running trajectory of the rats, and the time spent in the light and dark chambers and the number of times they moved between the two chambers were recorded; Assess photophobic behavior.

[0040] It should be noted that the light-dark box consists of a light chamber and a dark chamber with a passage between the two parts (each part has a size of 30×30×20 cm). The box is placed in a dedicated room where mice can adapt before testing.

[0041] 4. Perform immunofluorescence test on rats to detect the expression levels of c-Fos and CGRP.

[0042] Specifically, the steps are as follows: (1) Sample preparation: Rats were anesthetized with isoflurane and fixed by perfusion. Brain tissue was obtained and embedded and fixed to prepare brain tissue sections.

[0043] (2) Fixation: Fix the sections with a fixative (such as 4wt% paraformaldehyde or cold methanol) to maintain tissue morphology and antigen integrity.

[0044] (3) Permeabilization: Treat the sample with a permeabilization agent (such as 0.1 wt%-0.25 wt% Triton X-100) so that the antibody can access the antigen.

[0045] (4) Blocking: Use blocking solution (such as 1wt% BSA or 10wt% serum) to block nonspecific binding sites.

[0046] (5) Primary antibody incubation: Specific c-Fos and CGRP antibodies are diluted in solvent A and incubated on the sample to allow the antibody to fully bind to the antigen. The solvent A is made of 1 wt% BSA (bovine serum albumin) dissolved in PBS (phosphate buffered saline).

[0047] (6) Washing: Wash the samples with PBS or PBST (a mixture of 99.9wt% PBS and 0.1wt% Tween 20) to remove unbound primary antibody.

[0048] (7) Secondary antibody incubation: Use the indirect method to incubate with the secondary antibody corresponding to rat. The secondary antibody is usually conjugated with a fluorophore (such as FITC, TRITC, Cy3, etc.). The secondary antibody includes Cy3-conjugated goat anti-rabbit secondary antibody, FITC-conjugated goat anti-mouse secondary antibody, etc.

[0049] (8) Washing: Wash the sample again to remove unbound secondary antibody.

[0050] (9) Nuclear staining: Use DAPI (4',6-diamidino-2-phenylindole) or similar reagents to stain the cell nuclei.

[0051] (10) Sealing: Use anti-fluorescence fading sealing agent to seal the slides.

[0052] (11) Detection: Observe and record the fluorescence signal using a fluorescence microscope.

[0053] It should be noted that FITC is fluorescent isothiocyanate, TRITC is tetrabromofluorescein, and Cy3 is cyanine dye.

[0054] Test results: Figure 2 This is a graph showing the change trend of periorbital pain threshold in rats during the modeling period of nasal instillation of CGRP solution, where CON represents the control group rats with nasal instillation of saline, and CGRP represents the model group rats with nasal instillation of CGRP. Figure 2 It can be seen that after nasal dripping of CGRP, the rats' periorbital pain threshold continued to decrease, and after 7 days the threshold was basically below 1 gram; while after nasal dripping of normal saline, the rats' periorbital pain threshold remained basically unchanged, maintained at around 8 grams. The results show that nasal dripping of CGRP reduces the mechanical pain threshold of rats.

[0055] Figure 3 This is a graph showing the trend of cold pain threshold scores in rats during the modeling period of nasal instillation of CGRP solution, where CON represents the control group rats with nasal instillation of saline, and CGRP represents the model group rats with nasal instillation of CGRP. Figure 3 It can be seen that after nasal dripping of CGRP, the cold pain threshold score of rats continued to increase, and the threshold was basically around 8 points after 7 days; while after nasal dripping of normal saline, the cold pain threshold score of rats remained basically unchanged, maintained at around 2 points. This shows that nasal dripping of CGRP lowered the cold pain threshold of rats.

[0056] Figure 4 This is a graph showing the time that rats stayed in the bright compartment during the light-dark box test during the nasal instillation of CGRP solution modeling. CON represents the control group of rats that received nasal instillation of saline, and CGRP represents the model group of rats that received nasal instillation of CGRP. Figure 4 It can be seen that after nasal dripping of CGRP, the time that rats stayed in the bright compartment continued to decrease, and after 7 days, the proportion of time spent in the bright compartment in the total test time dropped to less than 20%; while after nasal dripping of normal saline, the time that rats stayed in the bright compartment remained basically unchanged, maintaining at about 45%. This shows that nasal dripping of CGRP increased the photophobia of rats, and photophobia is one of the important precursor symptoms of migraine.

[0057] Figure 5 The trend chart of the number of times rats moved from one compartment to another in the light-dark box test during the modeling period of nasal instillation of CGRP solution, where CON represents the control group rats with nasal instillation of saline solution, and CGRP represents the control group rats with nasal instillation of saline solution. Figure 5 It can be seen that after nasal dripping of CGRP, the number of holes pierced by the rats continued to decrease, and after 7 days, the number of holes pierced dropped to about 1 time; while after nasal dripping of normal saline, the number of holes pierced by the rats remained basically unchanged, maintained at about 5 times. This shows that nasal dripping of CGRP increased the photophobia of rats.

[0058] FIG6 is immunofluorescence staining of some brain regions; CON represents rats in the control group that received nasal drops of normal saline, and CGRP represents rats in the control group that received nasal drops of normal saline. Fig. 6A , c-Fos expression in TNC site after nasal instillation of CGRP and saline; Figure 6B , Expression of CGRP in TNC after nasal instillation of CGRP and saline. Fig. 6A It can be seen that compared with nasal saline, the immunofluorescence intensity of c-Fos in the rat TNC increased after nasal CGRP instillation, which indicates that nasal CGRP instillation activated the trigeminovascular system of rats. Figure 6B It can be seen that compared with nasal drops of normal saline, the immunofluorescence intensity of CGRP in the TNC site of rats increased after nasal drops of CGRP, which indicates that nasal drops of CGRP can increase the expression level of CGRP protein in the TNC site of rats.

[0059] Combining the data and analysis results, it can be seen that the method of constructing a migraine rat model by nasal instillation of CGRP in this embodiment is consistent with the characteristics of migraine. The migraine rat model provided in this embodiment has a stable phenotype, which helps researchers to understand the pathogenesis, development rules and intervention measures of migraine more conveniently and effectively.

[0060] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for constructing a migraine animal model, characterized in that: The following steps are involved: SD rats were placed in a clean environment for at least one week for adaptive breeding, with free access to food and water; After the adaptation, the calcitonin gene-related peptide solution was dripped into the nasal cavity of the rats once a day for at least 7 consecutive days.

2. The method for constructing a migraine animal model according to claim 1, characterized in that: The once a day means once a day at a fixed time period.

3. The method for constructing a migraine animal model according to claim 1, characterized in that: The calcitonin gene-related peptide solution is prepared by dissolving the calcitonin gene-related peptide in physiological saline or phosphate buffer.

4. The method for constructing a migraine animal model according to claim 3, characterized in that: The concentration of the calcitonin gene-related peptide solution is 2-6 μg / mL.

5. The method for constructing a migraine animal model according to claim 4, characterized in that: The concentration of the calcitonin gene-related peptide solution is 3-5 μg / mL.

6. The method for constructing a migraine animal model according to claim 1, characterized in that: The dripping amount of the calcitonin gene-related peptide solution is 30 μL / time.

7. A migraine animal model, characterized in that: The method according to any one of claims 1 to 6 is used to construct.

8. The method for evaluating migraine-like behavior in a migraine animal model according to claim 7, characterized in that: The following steps are involved: Rats were tested for mechanical hyperalgesia to obtain the mechanical pain threshold; Rats were subjected to cold stimulation test to obtain the cold pain threshold; The rats were subjected to a photophobia test to assess photophobia behavior; The rats were subjected to immunofluorescence test to detect the expression levels of c-Fos and CGRP.

9. The method for evaluating migraine-like behavior in a migraine animal model according to claim 8, characterized in that: Photophobia testing. Methods for assessing photophobia behavior include: The rats were placed in the center of the light-dark box and allowed to freely explore to acclimate to the environment; After acclimation, the test was conducted to detect the running trajectory of the rats, and the time spent in the light and dark chambers and the number of times they moved between the two chambers were recorded; Assess photophobic behavior.

10. The method for evaluating migraine-like behavior in a migraine animal model according to claim 8, characterized in that: The method for performing immunofluorescence test on rats to detect the expression levels of c-Fos and CGRP comprises the following steps: Sample pretreatment: Rat brain tissue sections were treated with a fixative, a permeabilizing agent, and a blocking solution in sequence to obtain a first sample; Primary antibody incubation: Incubate the first sample with a solution of specific c-Fos and CGRP antibodies to allow the antibodies to fully bind to the antigens, thereby obtaining a second sample; Washing: washing the second sample to remove unbound primary antibody and obtain the third sample; Secondary antibody incubation: incubating the third sample with a secondary antibody corresponding to the host species of the primary antibody to obtain a fourth sample; the secondary antibody is used to bind to the fluorophore; Washing: washing the fourth sample to remove unbound secondary antibody to obtain the fifth sample; Nuclear staining and sealing: staining and sealing the cell nuclei of the fifth sample to obtain the sixth sample; Detection: Detect the fluorescence signal of the sixth sample.

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