An immune emulsion, its preparation method and application in establishing an animal model of demyelinating Guillain-Barré syndrome

Abstract

The present invention provides an immune emulsion, a preparation method thereof, and an application in constructing an animal model of demyelinating Guillain-Barré syndrome, belonging to the technical field of animal model construction. The immune emulsion provided by the present invention comprises GalC at 0.8-1.2 mg / mL, hemocyanin at 0.8-1.2 mg / mL, complete Freund's adjuvant at 0.8-1.2 mg / mL, and a solvent. Immunizing Japanese white rabbits with the immune emulsion provided by the present invention can successfully establish an animal model of demyelinating GBS. Moreover, the characteristics of the demyelinating GBS animal model constructed by the present invention in terms of clinical manifestations, electrophysiology, and molecular pathology (especially macrophage infiltration) are highly consistent with those of human AIDP patients, providing an important tool for studying the pathogenesis and treatment regimens of demyelinating neuropathies such as GBD / AIDP, and can be used for screening and testing specific drugs, having profound scientific research significance and important application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of animal model construction, and particularly relates to an immune emulsion, a preparation method thereof, and an application thereof in constructing an animal model of demyelinating Guillain-Barré syndrome. Background Art

[0002] Guillain-Barré syndrome (GBS) is the most common and severely disabling autoimmune disease in the department of neurology. The pathogenesis of GBS is still unclear. Generally, it is believed that exogenous pathogens induce the human body to produce pathogenic antibodies through the molecular mimicry mechanism, and the latter attack the myelin sheath or axon of the peripheral nerve, and then activate the complement and lead to the formation of the membrane attack complex, which is the basic mechanism of GBS onset. At present, the main clinical treatment methods for GBS are plasma exchange and intravenous immunoglobulin (IVIG), but clinical data show that only 65% of patients are effective for the above methods, about 20% of patients still have severe disabilities one year after treatment, and 5% of patients die due to poor treatment effects, causing a great burden to society, families and individuals.

[0003] According to different pathological characteristics, GBS can be divided into two types: demyelinating type and axonal type. Among them, acute inflammatory demyelinating polyneuropathy (AIDP) is the most classic and common demyelinating type of GBS. Its pathogenic antibody is the IgG antibody of galactocerebroside (GalC). The main lesion is the segmental demyelination of the motor and sensory nerve roots and peripheral nerves. The clinical manifestation is acute symmetrical flaccid limb paralysis, with or without paresthesia. Since AIDP is an autoimmune disease, the autoimmune anti-ganglioside IgG antibody can bind to the ganglioside on the peripheral nerve myelin sheath and then trigger the complement to bind to its constant region, forming a membrane attack complex and damaging the peripheral nerve. Animal models are important tools for exploring the pathogenesis of GBS / AIDP and finding new treatment methods. At present, there is still no generally recognized animal model for the study of AIDP. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide an immune emulsion that can be used to construct an animal model of demyelinating Guillain-Barré syndrome, so that the characteristics of the constructed model are highly consistent with those of human AIDP patients in terms of clinical manifestations, electrophysiology, and molecular pathology.

[0005] In order to achieve the above invention purpose, the present invention provides the following technical solutions:

[0006] The present invention provides an immune emulsion, comprising GalC, hemocyanin, complete Freund's adjuvant and a solvent, wherein the concentration of GalC is 0.8 - 1.2 mg / mL, the concentration of hemocyanin is 0.8 - 1.2 mg / mL, and the concentration of complete Freund's adjuvant is 0.8 - 1.2 mg / mL.

[0007] Preferably, the solvent comprises physiological saline.

[0008] The present invention also provides a method for preparing the above immune emulsion, comprising the following steps: dissolving GalC in an ethanol solution with a volume fraction of 95%, mixing it with physiological saline to obtain a dissolution solution, mixing the dissolution solution and hemocyanin, and then mixing it with complete Freund's adjuvant to form an emulsion, so that the final concentrations of GalC, hemocyanin and complete Freund's adjuvant are independently 0.8 - 1.2 mg / mL.

[0009] Preferably, the volume-mass ratio of the ethanol solution with a volume fraction of 95% to GalC is 0.1 mL:1 mg, and the volume ratio of the ethanol solution with a volume fraction of 95% to physiological saline is 1:4.

[0010] The present invention also provides an application of the above immune emulsion or the immune emulsion prepared by the above preparation method in constructing an animal model of demyelinating Guillain - Barré syndrome.

[0011] The present invention also provides a method for constructing an animal model of demyelinating Guillain - Barré syndrome, comprising the following steps: immunizing Japanese white rabbits with the above immune emulsion or the immune emulsion prepared by the above preparation method until the rabbits become ill.

[0012] Preferably, when immunizing Japanese white rabbits with the immune emulsion, the injection immunization is carried out at a dose of 0.8 - 1.2 mg GalC / rabbit.

[0013] Preferably, the injection is multi-point subcutaneous injection on the back.

[0014] Preferably, the injection interval is 3 weeks / time.

[0015] The present invention also provides an application of the model constructed by the above method in screening drugs for preventing and / or treating Guillain - Barré syndrome.

[0016] The beneficial effects of the present invention:

[0017] The present invention for the first time provides an immune emulsion that can be used to construct a demyelinating GBS animal model. Immunizing big ear white rabbits with the immune emulsion provided by the present invention, the established demyelinating GBS animal model is highly consistent with human AIDP patients in terms of clinical manifestations, electrophysiology, and molecular pathology (especially macrophage infiltration), providing an important tool for studying the pathogenesis and treatment regimens of demyelinating neuropathies such as GBD / AIDP, and can be used for the screening and testing of specific drugs, having profound scientific research significance and important application prospects.

[0018] The present invention for the first time clarifies the clinical characteristics, electrophysiology, and neuropathological characteristics of AIDP. Specifically, the clinical score of the AIDP model rabbits is significantly increased, mainly manifested in abnormal postures, inability to walk upright, neck weakness, slipping weakness of the front and hind limbs, and absence of the righting reflex on the left and right sides; it is clarified that the serum anti-GalC IgG antibody of the AIDP model rabbits is positive; it is clarified that the amplitudes of the compound muscle action potentials at the medial malleolus, popliteal fossa, and sciatic notch of the AIDP model rabbits decrease, the waveforms are discrete, the latencies increase, the durations increase, and the conduction velocities decrease, and the shortest latency of the F wave at the medial malleolus increases; it is clarified that pathogenic antibodies are generated on the nerve root fibers of the AIDP model rabbits, triggering complement activation and the formation of the membrane attack complex, attacking the myelin sheath and the Ranvier node region of the nerve fibers, causing myelin sheath damage and sodium channel destruction, resulting in demyelinating lesions of the nerve fibers and macrophage infiltration. Description of the Drawings

[0019] Figure 1 Results of the electrophysiological study of the rabbit models in Example 1 and Comparative Example 1, where A is the compound muscle action potential at the medial malleolus, popliteal fossa, and sciatic notch of the rabbit model, and B is the F wave at the medial malleolus of the rabbit model;

[0020] Figure 2 Results of the toluidine blue staining of the demyelinating lesions of the anterior and posterior nerve roots in the lumbosacral part of the spinal cord of the rabbit model, where Normal is the group in Comparative Example 1 (normal rabbits), AIDP is the group in Example 1, the arrows indicate the demyelinated sites, Ventral root: anterior spinal root; Dorsal root: posterior spinal root;

[0021] Figure 3Electron microscopy showed demyelinating lesions of the rabbit model's nerve roots. Among them, A is the cross-section of the nerve root fibers of the rabbit model in Comparative Example 1, and B - H are the cross-sections of the nerve root fibers of the rabbit model in Example 1 (AIDP). In Figure B, the arrow indicates that the myelin sheath of the nerve root fibers in the AIDP rabbit model becomes thinner, and the asterisk indicates macrophage infiltration in the nerve fiber space; Figure C shows that the myelin sheath of the nerve fiber is stripped, and the high-resolution image in the square is shown in Figure D; the arrow in Figure D indicates the position where the myelin sheath is being stripped; Figure E shows the late stage of nerve fiber demyelination, where the myelin sheath becomes extremely thin and macrophages are phagocytosing myelin debris. The magnified areas of the white and black frames in Figure E are shown in Figures F and G respectively; the arrow in Figure F indicates the position where the myelin sheath is being stripped, the myelin sheath is being stripped and has become very thin, and # indicates the axon; Figure G shows macrophages phagocytosing myelin debris, where * indicates macrophages and the arrow indicates the phagocytosed myelin debris; Figure H shows the appearance of axons with complete demyelination (i.e., naked axons) caused by the stripping of the myelin sheath, surrounded by macrophages containing a large amount of myelin debris. In Figure H, # indicates the naked axon and * indicates macrophages;

[0022] Figure 4 IgG antibody deposition on the nerve fibers of the rabbit model. Compared with Comparative Example 1 (Normal), the cross-section of the nerve fibers of the rabbit model in Example 1 (AIDP) showed obvious IgG antibody deposition, indicating that pathogenic antibodies were produced in the AIDP rabbit's nerve fibers and damaged the myelin sheath of the nerve fibers (dotted area). MBP: Myelin basic protein, Tubulin: Tubulin, Rabbit IgG: Rabbit IgG antibody;

[0023] Figure 5 Complement activation on the nerve fibers of the rabbit model. Compared with Comparative Example 1 (Normal), the longitudinal section of the nerve fibers of the rabbit model in Example 1 (AIDP) showed obvious IgG antibody deposition and complement activation (C3c: Complement C3 complex). The dotted area indicates the position of complement activation, which led to myelin sheath damage and the production of myelin debris (arrow within the dotted line);

[0024] Figure 6 IgG antibody deposition and myelin sheath reduction on the nerve fibers of the rabbit model. Compared with Comparative Example 1 (Normal), the cross-section of the nerve fibers of the rabbit model in Example 1 (AIDP) showed obvious rabbit IgG antibody deposition (p < 0.0001) and a decrease in MBP expression (p < 0.0001), indicating that its myelin sheath was damaged;

[0025] Figure 7In the rabbit model, the number of sodium channels on the nerve fibers decreased. Compared with Comparative Example 1 (Normal), the cross-section of the nerve fibers of the rabbit model in Example 1 (AIDP) showed a significant decrease in the number of sodium channels (p<0.001), indicating that the sodium channels, an important structural protein in the Ranvier nodes of the nerve fibers, were damaged. The arrows indicate the sodium channels, NaV: sodium channel;

[0026] Figure 8 In the rabbit model, Caspr1 on the nerve fibers did not change significantly. Compared with Comparative Example 1 (Normal), the cross-section of the nerve fibers of the rabbit model in Example 1 (AIDP) showed that the number of Caspr1 did not change significantly (p = 0.178), indicating that Caspr1, a structural protein in the paranodal region of the nerve fibers, was not significantly damaged. The arrows indicate Caspr1, Caspr1: Contactin-associated Protein 1, contactin-associated protein 1;

[0027] Figure 9 In the rabbit model, macrophages infiltrated and phagocytosed myelin debris. Compared with Comparative Example 1 (Normal), the cross-section of the nerve fibers of the rabbit model in Example 1 (AIDP) showed that a large number of macrophages infiltrated after myelin injury and phagocytosed myelin debris. The arrows in the left figure indicate myelin debris, the arrows in the middle figure indicate macrophages, and the arrows in the right figure indicate the co-localization of macrophages and myelin debris, indicating that macrophages phagocytosed myelin debris. Detailed implementation mode

[0028] The present invention provides an immune emulsion, which includes GalC, hemocyanin, complete Freund's adjuvant, and a solvent. The concentration of GalC is 0.8 - 1.2 mg / mL, the concentration of hemocyanin is 0.8 - 1.2 mg / mL, and the concentration of complete Freund's adjuvant is 0.8 - 1.2 mg / mL.

[0029] The present invention has no special limitation on the specific sources of GalC, hemocyanin, and complete Freund's adjuvant. In the present invention, the concentration of GalC in the immune emulsion is preferably 0.9 - 1.0 mg / mL, the concentration of hemocyanin is preferably 0.9 - 1.0 mg / mL, and the concentration of complete Freund's adjuvant is preferably 0.9 - 1.0 mg / mL. The solvent preferably includes physiological saline, and the physiological saline is preferably physiological saline with a mass-volume fraction of 0.9%. The present invention has no special limitation on the specific source of the physiological saline.

[0030] The present invention also provides a method for preparing the above-mentioned immune emulsion, which comprises the following steps: dissolving GalC in an ethanol solution with a volume fraction of 95%, mixing it with physiological saline to obtain a solution, mixing the solution with hemocyanin, and then mixing it with complete Freund's adjuvant until an emulsion is formed, so that the final concentrations of GalC and hemocyanin are independently 0.8-1.2 mg / mL.

[0031] In the present invention, the volume-mass ratio of the ethanol solution with a volume fraction of 95% to GalC is preferably 0.1 mL:1 mg. The volume-mass ratio of the 95% ethanol solution defined in the present invention to GalC can, on the one hand, completely dissolve Galc, and on the other hand, can minimize the toxicity of ethanol. If the ratio is too low, Galc cannot be completely dissolved, and if it is too high, the toxicity of ethanol will increase. The volume ratio of the ethanol solution with a volume fraction of 95% to physiological saline is preferably 1:4.

[0032] In the present invention, after dissolving GalC and mixing it with physiological saline, it is preferably ultrasonicated with an ultrasonic crusher until the solution becomes clear. The power of the ultrasonic wave is preferably 50-80 Hz, and the time of the ultrasonic wave is preferably 3-5 min to obtain a solution. Then the solution is mixed with an equal mass of hemocyanin so that the concentrations of GalC and hemocyanin are both 1.6-2.4 mg / mL.

[0033] In the present invention, it is preferred to use a needle-type injection mixer to mix the solution containing GalC and hemocyanin with an equal volume of complete Freund's adjuvant until an emulsion is formed. The specific operation of the mixing is as follows: suck the solution containing GalC and hemocyanin and complete Freund's adjuvant with equal volume into two syringes respectively, connect the two syringes with a double-headed needle, drain the air, first quickly push the solution containing GalC and hemocyanin into the syringe containing complete Freund's adjuvant, and then alternately push the syringe until a viscous emulsion (water-in-oil) is formed, so that the final concentration of GalC is 0.8-1.2 mg / mL. In the present invention, after obtaining the emulsion, it is preferably identified. The preferred method for the identification is: drop the emulsion into cold water. If it remains intact and does not disperse, floats on the water surface in a droplet shape, it means that the emulsification is complete and it is a qualified water-in-oil agent; if the emulsion disperses when dropped into cold water, it is an unqualified water-in-oil agent.

[0034] The present invention also provides an application of the above-mentioned immune emulsion or the immune emulsion prepared by the above-mentioned preparation method in constructing an animal model of demyelinating Guillain-Barré syndrome.

[0035] The present invention also provides a method for constructing an animal model of demyelinating Guillain-Barré syndrome, which comprises the following steps: immunizing a large-ear white rabbit with the above-mentioned immune emulsion or the immune emulsion prepared by the above-mentioned preparation method until it gets sick.

[0036] The specific source of the Japanese white rabbits with big ears in the present invention is not particularly limited. Preferably, male Japanese white rabbits about 3 months old are used. The specific feeding method of the Japanese white rabbits with big ears in the present invention is not particularly limited. In a specific embodiment, the feeding amount is 80 - 120 g per day, and sufficient drinking water is provided.

[0037] In the present invention, when immunizing Japanese white rabbits with big ears with the immune emulsion, it is preferably injected and immunized at a dose of 0.8 - 1.2 mg GalC per rabbit, and more preferably at a dose of 0.9 - 1.0 mg GalC per rabbit. The injection is preferably subcutaneous multi-point injection on the back each time, and the interval between each injection is preferably 3 weeks per time. In the present invention, the number of injections is preferably 3 - 10 times to obtain a demyelinating Guillain - Barré syndrome animal model.

[0038] The present invention also provides an application of the model constructed by the above method in screening drugs for preventing and / or treating Guillain - Barré syndrome.

[0039] The technical solutions provided by the present invention are described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0040] Example 1

[0041] (1) Select male Japanese white rabbits about 3 months old, with a body weight of 2.0 - 2.5 Kg, a daily feeding amount of 100 g, and sufficient drinking water.

[0042] (2) Prepare the immune emulsion: Dissolve GalC with ethanol with a volume fraction of 95% (add 0.1 mL of 95% ethanol per 1 mg), add physiological saline (95% ethanol: physiological saline = 1:4), then use an ultrasonic crusher for ultrasonic treatment, with a power of 50 Hz and a time of 3 min until the solution becomes clear. Then dissolve an equal mass of hemocyanin with the obtained solution so that the concentrations of GalC and hemocyanin are both 1.6 mg / mL. Use a needle - type injection mixer to mix the solution containing GalC and hemocyanin with an equal volume of complete Freund's adjuvant into an emulsion. The specific operation is as follows: Aspirate the solution containing GalC and hemocyanin and complete Freund's adjuvant with equal volumes into two syringes respectively, connect the two syringes with a two - way needle, drain the air. First, quickly push the solution containing GalC and hemocyanin into the syringe containing complete Freund's adjuvant, and then alternately push the syringe barrels until a viscous emulsion (water - in - oil) is formed. Make the final concentration of GalC 0.8 mg / mL. Drop the emulsion into cold water. If it remains intact and does not disperse, floating on the water surface in a droplet shape, it means the emulsification is complete, and it is a qualified water - in - oil agent. Sub - pack it with a 1 mL syringe at 1 mL per vial for subsequent experiments. If the emulsion disperses when dropped into cold water, it is an unqualified water - in - oil agent, and it needs to be prepared again.

[0043] (3) Immunize the rabbits subcutaneously at multiple points on the back with the immune emulsion at a dose of 1.2 mg of GalC per rabbit every 3 weeks for 3 times to obtain an animal model of demyelinating Guillain-Barré syndrome.

[0044] Example 2

[0045] (1) Select male Japanese white rabbits about 3 months old, weighing 2.0 - 2.5 Kg, with a daily feeding amount of 80 g and sufficient drinking water.

[0046] (2) Prepare the immune emulsion: Dissolve GalC in ethanol with a volume fraction of 95% (add 0.1 mL of 95% ethanol per 1 mg), add normal saline (95% ethanol:normal saline = 1:4), then use an ultrasonic crusher for ultrasonic treatment at a power of 80 Hz for 4 min until the solution becomes clear. Then dissolve an equal mass of hemocyanin with the obtained solution so that the concentrations of both GalC and hemocyanin are 2.4 mg / mL. Mix the solution containing GalC and hemocyanin with an equal volume of complete Freund's adjuvant into an emulsion using a syringe-type injection mixer. The specific operation is as follows: Draw the solution containing GalC and hemocyanin and complete Freund's adjuvant in equal volumes into two syringes respectively, connect the two syringes with a double-headed needle, exhaust the air, first quickly push the solution containing GalC and hemocyanin into the syringe containing complete Freund's adjuvant, and then alternately push the syringe barrels until a viscous emulsion (water-in-oil) is formed. Make the final concentration of GalC 1.2 mg / mL. Drop the emulsion into cold water. If it remains intact and does not disperse, floating on the water surface in droplets, it means the emulsification is complete, and it is a qualified water-in-oil agent. Aliquot it into 1 mL syringes at 1 mL per syringe for subsequent experiments.

[0047] (3) Immunize the rabbits subcutaneously at multiple points on the back with the immune emulsion at a dose of 0.8 mg of GalC per rabbit every 3 weeks for 10 times to obtain an animal model of demyelinating Guillain-Barré syndrome.

[0048] Example 3

[0049] (1) Select male Japanese white rabbits about 3 months old, weighing 2.0 - 2.5 Kg, with a daily feeding amount of 120 g and sufficient drinking water.

[0050] (2) Preparation of immune emulsion: Dissolve GalC in 95% ethanol (add 0.1 mL 95% ethanol to 1 mg), add physiological saline (95% ethanol: physiological saline = 1:4), and then use an ultrasonic disruptor to sonicate at 60 Hz for 5 min until the solution becomes clear, and then use the resulting solution to dissolve an equal amount of hemocyanin so that the concentrations of GalC and hemocyanin are both 2.0 mg / mL. Use a needle injection mixer to mix the solution containing GalC and hemocyanin with an equal volume of complete Freund's adjuvant to form an emulsion. The specific operation is as follows: Aspirate an equal volume of the solution containing GalC and hemocyanin and complete Freund's adjuvant into two syringes respectively, connect the two syringes with a two-way needle, exhaust the air, first quickly push the solution containing GalC and hemocyanin into the syringe containing complete Freund's adjuvant, and then push the needles alternately until a viscous emulsion (oil-in-water) is formed. Make the final concentration of GalC 1.0 mg / mL, drop the emulsion into cold water, if it remains intact and does not disperse, and floats on the water surface in droplets, it is completely emulsified and is a qualified oil-in-water preparation, use a 1mL syringe to dispense 1mL / vial for subsequent experiments.

[0051] (3) The immune emulsion was injected subcutaneously at multiple points on the back of each rabbit at a dose of 1.0 mg GalC every 3 weeks. After 6 injections, a demyelinating Guillain-Barré syndrome animal model was obtained.

[0052] Comparative Example 1

[0053] The difference from Example 1 is that step (2) is omitted, 0.8 mg of normal saline is injected in step (3), and the rest is the same as Example 1.

[0054] Example 4

[0055] After the third immunization, the models obtained in Example 1 and Comparative Example 1 were weighed and clinically scored daily.

[0056] Clinical scoring was performed according to the scoring criteria, and the onset of the disease was defined as a clinical score of 10 points or higher. The results are shown in Table 1. The rabbits in Example 1 (AIDP rabbits) scored 15 points on the first day of the onset of the disease, mainly manifested as abnormal posture, abnormal walking, neck weakness, weak front and rear limb sliding, and loss of righting reflex of left and right side turning. The rabbits in Comparative Example 1 (control group) scored between 0 and 2 points all the time, with no obvious abnormalities. This shows that the method of the present invention successfully constructed an animal model of demyelinating Guillain-Barré syndrome.

[0057] Table 1 Clinical scoring results of the models obtained in Example 1 and Comparative Example 1

[0058]

[0059]

[0060] Example 5

[0061] Collect the auricular marginal venous blood of the models obtained in Example 1 and Comparative Example 1, and determine the plasma anti-GalC IgG antibody titer by enzyme-linked immunosorbent assay. The results show that the anti-GalC IgG antibody of the rabbits in Example 1 was positive (OD = 0.11, +) during the onset of the disease, and the anti-GalC IgG antibody of the rabbits in Comparative Example 1 was negative (-).

[0062] Example 6

[0063] Anesthetize the rabbits in Example 1 and Comparative Example 1 with 3% sodium pentobarbital at a dose of 1 mL / kg respectively, and conduct sciatic nerve motor conduction studies using electrophysiological instruments. The recording electrode was inserted into the medial gastrocnemius muscle belly of the hind limb, the reference electrode was inserted into the plantar tendon 0.5 cm distal to it, the ground electrode was inserted subcutaneously in the upper limb, and the stimulating electrodes stimulated the sciatic nerve at the medial malleolus, popliteal fossa, and sciatic notch respectively. Record the amplitude, latency, and duration of the compound muscle action potential at the medial malleolus, popliteal fossa, and sciatic notch respectively and calculate the conduction velocity; record the F wave at the medial malleolus and record the minimum value among the 10-15 latencies of the elicited F waves. The results are as Figure 1 and Table 2 show.

[0064] Table 2 Electrophysiological research results of the AIDP rabbit model

[0065]

[0066] The results show that compared with the rabbits in the Comparative Example 1 group, the amplitude of the compound muscle action potential at the medial malleolus, popliteal fossa, and sciatic notch of the rabbits in the Example 1 group decreased during the onset of the disease, the waveform was discrete, the latency increased, the duration increased, and the conduction velocity decreased ( Figure 1 A, Table 2), and the shortest latency of the F wave at the medial malleolus increased ( Figure 1 B, Table 2).

[0067] Example 7

[0068] After deeply anesthetizing the model rabbits in the Example 1 and Comparative Example 1 groups with 3% sodium pentobarbital respectively, first perfuse 1 L of normal saline through the heart, and then perfuse 1 L of 2% paraformaldehyde. After perfusion, take the anterior and posterior nerve roots of the lumbosacral spinal cord.

[0069] a. Toluidine blue staining experiment: Place the taken anterior and posterior nerve roots of the lumbosacral spinal cord in a strong fixing solution containing 0.01 M PBS, 1% paraformaldehyde, and 3% glutaraldehyde and soak overnight at 4°C, then fix at room temperature in 1% osmium tetroxide solution for 80 minutes. After gradient dehydration, perform paraffin embedding, and then section with a paraffin microtome and conduct toluidine blue staining. The results are as Figure 2 shown. Obvious demyelinating lesions occurred in the nerve fibers of the rabbits in the Example 1 group after the onset of the disease.

[0070] b. Electron microscopy experiment: The extracted anterior and posterior nerve roots of the lumbosacral spinal cord were immersed in a fixing solution containing 0.2 M PB, 4% paraformaldehyde, and 50% glutaraldehyde at 4°C overnight, and then fixed in 1% osmium tetroxide at 4°C in the dark for 120 minutes. After gradient dehydration, resin embedding was performed, and then sectioning was carried out using an ultramicrotome. Uranyl acetate and lead citrate staining were performed, and the myelin sheath damage and macrophage infiltration on the cross-section of nerve fibers were observed under the electron microscope. The results are as Figure 3 shown. Demyelinating lesions were shown on the nerve fibers. Macrophage infiltration was observed in the spaces between nerve fibers and phagocytosis of damaged myelin sheath fragments, indicating that macrophages play a role in clearing myelin sheath fragments.

[0071] c. Immunofluorescence experiment: The extracted anterior and posterior nerve roots of the lumbosacral spinal cord were immersed in 4% paraformaldehyde fixing solution at 4°C overnight, and then dehydrated in 30% sucrose solution at 4°C for more than 24 hours. OCT embedding medium was used for embedding, and then longitudinal and cross sections of the nerve roots were made using a cryostat. The sections were immersed in absolute ethanol at -20°C for 20 minutes and then washed with PBS to remove excess embedding medium. A blocking solution containing 10% goat serum and 0.3% Triton X-100 was added, and the sections were blocked at room temperature in a wet box for 2 hours. Subsequently, the primary antibodies were incubated overnight at 4°C, the secondary antibodies were incubated for 2 hours at room temperature, and an anti-fluorescence quencher was added for mounting. Images were taken using a Zeiss Imager Z2 fluorescence microscope from Germany. The primary antibodies and their ratios are as follows: goat anti-C3c (Abcam, #ab64684, 1:200), mouse anti-MBP (Abcam, #ab62631, 1:300), mouse anti-β-3 Tubulin (Thermo Fisher, #MA1-118, 1:500), rabbit anti-NaV (Abcam, #ab65166, 1:200), rabbit anti-Caspr (Abcam, #ab34151, 1:200), mouse anti-RAM11 (Dako, #M0633, 1:100). The secondary antibodies and their ratios are as follows: donkey anti-rabbit IgG AF647 (Abcam, #ab150075, 1:500), goat anti-mouse IgG2b AF555 (Thermo Fisher, #A21147, 1:1000), goat anti-mouse IgG AF488 (Thermo Fisher, #A32723, 1:1000), donkey anti-rabbit IgG AF488 (Abcam, #ab150073, 1:500). The results are as Figures 4 - 9 shown.

[0072] There was obvious IgG antibody deposition and complement activation on the nerve fibers of the AIDP rabbit model (Example 1 group), while there was no antibody deposition on the nerve fibers of normal rabbits (Control group 1) Figure 4) and complement activation( Figure 5 );Myelin sheath reduction on nerve fibers in the AIDP rabbit model (p < 0.0001)( Figure 6 ), significantly decreased sodium channel number (p < 0.001)( Figure 7 ), no significant difference in Caspr number( Figure 8 );There was obvious macrophage infiltration in the AIDP rabbit model, and phagocytosis of the shed myelin fragments( Figure 9 ).

[0073] In summary, the experimental results show that immunizing Japanese white rabbits with an emulsifier prepared by mixing GalC, hemocyanin and complete Freund's adjuvant can produce pathogenic antibodies and trigger complement activation and the formation of membrane attack complexes, attacking the myelin sheath of nerve fibers and the nodes of Ranvier, resulting in demyelinating lesions of nerve fibers and macrophage infiltration.

[0074] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. Use of an immune emulsion in the preparation of a reagent for constructing an animal model of demyelinating Guillain-Barré syndrome, Characterized in that, The immune emulsion includes GalC, hemocyanin, complete Freund's adjuvant and a solvent, wherein the concentration of GalC is 0.8 - 1.2 mg / mL, the concentration of hemocyanin is 0.8 - 1.2 mg / mL, and the concentration of complete Freund's adjuvant is 0.8 - 1.2 mg / mL.

2. The use according to claim 1, Characterized in that, The solvent includes physiological saline.

3. The use according to claim 1, Characterized in that, The preparation method of the immune emulsion comprises the following steps: dissolve GalC with an ethanol solution with a volume fraction of 95%, mix it with physiological saline to obtain a solution, mix the solution and hemocyanin, and then mix it with complete Freund's adjuvant until it becomes an emulsion, so that the final concentrations of GalC, hemocyanin and complete Freund's adjuvant are independently 0.8 - 1.2 mg / mL.

4. The use according to claim 3, Characterized in that, The volume-mass ratio of the ethanol solution with a volume fraction of 95% to GalC is 0.1 mL:1 mg, and the volume ratio of the ethanol solution with a volume fraction of 95% to physiological saline is 1:4.

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