GluN1 polypeptide active immunization and sleep deprivation induced anti-NMDAR encephalitis animal model as well as establishment method and application thereof

Abstract

The invention relates to the field of animal models, and discloses an anti-NMDAR encephalitis animal model induced by combination of GluN1 polypeptide active immunization and sleep deprivation as well as an establishment method and application thereof, and the establishment method of the anti-NMDAR encephalitis animal model induced by combination of GluN1 polypeptide active immunization and sleep deprivation comprises the following steps: 1) immunizing an animal with synthesized GluN1359-378 polypeptide according to a primary immunization-enhancement strategy; (2) in the second week after the boosted immunization, sleep deprivation operation is carried out on the animals for 12 hours every day, and the operation lasts for 3 weeks; by adopting the establishment method, the anti-NMDAR encephalitis animal model induced by GluN1 polypeptide active immunization and sleep deprivation can be established; the GluN1 polypeptide active immunization combined sleep deprivation induced anti-NMDAR encephalitis animal model can be applied to research on the morbidity process and mechanism of sleep disorder induced anti-NMDAR encephalitis, research on the morbidity process and mechanism of anti-NMDAR encephalitis, and screening and evaluation of anti-NMDAR encephalitis prevention and treatment drugs.

Description

Technical Field

[0001] The present invention relates to the field of animal models. Specifically, it is about an animal model of anti-NMDAR encephalitis induced by active immunization with GluN1 polypeptide combined with sleep deprivation, a method for establishing the same, and its application. Background Art

[0002] Anti-N-methyl-D-aspartic acid receptor (NMDAR) encephalitis is a severe neuropsychiatric disease mediated by anti-NMDAR antibodies. The global incidence rate is about 1.5 / 1 million, accounting for about 54% - 80% of autoimmune encephalitis in China. The male-to-female incidence ratio is about 1:4, and the median age of onset is 21 years. Most cases present with clinical symptoms such as abnormal mental behavior, speech or movement disorders, epileptic seizures, decreased consciousness level, and autonomic nervous system dysfunction at the onset. There are also some patients whose conditions rapidly deteriorate into severe encephalitis. The average treatment period in the intensive care unit is 1 - 2 months, and the fatality rate is 2.9% - 9.5%. Although early immunotherapy can improve the prognosis, there are still some patients with long-term cognitive impairment or recurrence (the recurrence rate is 12.0% - 31.4%), which brings a heavy burden to patients and their families and poses a challenge to social and economic development.

[0003] The core feature of this disease is that autoantibodies against the extracellular N-terminal domain of the GluN1 subunit of NMDAR are produced in the patient's body. GluN1 antibody (GluN1-Abs) induces the internalization of NMDAR on the neuron surface, resulting in low NMDAR function, thereby triggering cognitive impairment and schizophrenia-like symptoms. Although the pathogenicity of IgG-type GluN1-Abs in anti-NMDAR encephalitis has been confirmed, these antibodies can also be detected in the sera of some healthy individuals, suggesting that other susceptibility factors or triggering factors may be required for the onset of this disease.

[0004] Currently, there is a lack of an effective and stable active immunization animal model for the study of anti-NMDAR encephalitis. The success rate of the conventional animal model of anti-NMDAR encephalitis induced by GluN1 peptide is relatively low, which limits the in-depth study of the mechanism of this disease. Therefore, developing a new technology for inducing an active immunization animal model of anti-NMDAR encephalitis has become an urgent need in current research.

[0005] Sleep is a key process for maintaining physiological balance, which maintains homeostasis by regulating functions such as hormones, metabolism, immune response, tissue repair, and memory consolidation. In recent years, sleep deprivation has become a global problem with wide-ranging effects, including diseases such as cancer, viral infections, metabolic diseases, cardiovascular events, and dementia. The inducements of long-term sleep disorders include work stress, life events, health problems, etc. It is worth noting that insomnia is one of the common early symptoms of anti-NMDAR encephalitis and has a significant impact on the recovery of the disease and quality of life. However, it is currently unclear whether sleep disorders can induce the disease in the presence of GluN1-Abs.

[0006] Therefore, combining the research on sleep deprivation and anti-NMDAR encephalitis and developing new active immunization animal models, especially those that can simulate the impact of sleep disorders on the development of the disease, has become the key to future research. Summary of the Invention

[0007] The purpose of the present invention is to provide an animal model of anti-NMDAR encephalitis induced by active immunization with GluN1 polypeptide combined with sleep deprivation, a method for establishing the same, and its application. This animal model can be used to study the pathogenesis and mechanism of anti-NMDAR encephalitis induced by sleep disorders and for screening and evaluating drugs for the prevention and treatment of anti-NMDAR encephalitis.

[0008] The present invention is achieved through the following technical solutions: A method for establishing an animal model of anti-NMDAR encephalitis induced by active immunization with GluN1 polypeptide combined with sleep deprivation, comprising the following steps:

[0009] 1) Immunize animals with the synthesized GluN1 359-378 polypeptide according to the prime-boost immunization strategies to enable the animals to obtain high-titer and relatively long-lasting serum IgG-type anti-NMDAR antibodies;

[0010] 2) At the 2nd week after the booster immunization, perform sleep deprivation on the animals for 12 hours per day (from 6:00 a.m. to 6:00 p.m.) for 3 weeks; open the blood-brain barrier to promote the entry of peripheral IgG-type anti-NMDAR antibodies into the central nervous system.

[0011] To better implement the method for establishing an animal model of anti-NMDAR encephalitis induced by active immunization with GluN1 polypeptide combined with sleep deprivation of the present invention, the following setting method is particularly adopted: The step 1) specifically includes the following steps:

[0012] 1.1) Preparation process of the GluN1 359-378 polypeptide emulsion: Prepare the GluN1 359-378The polypeptide was diluted with ice-cold sterile normal saline to a 2 mg / ml polypeptide solution, and complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA) was added in an equal volume ratio of 1:1. Two syringes were connected through a three-way valve, and the mixture was slowly aspirated repeatedly until a water-in-oil emulsion was formed to obtain GluN1. 359-378 The polypeptide–CFA emulsion or GluN1 359-378 The polypeptide–IFA emulsion;

[0013] 1.2) Active immunization process: For the first immunization, the GluN1 359-378 polypeptide–CFA emulsion was subcutaneously injected at four points on both sides of the spinal column on the back of the animal, 50 μl per point, for a total of 200 μl per animal; After 2 weeks, a booster immunization was carried out. The GluN1 359-378 polypeptide–IFA emulsion was subcutaneously injected at four points on the back of the animal, 50 μl per point, for a total of 200 μl per animal; After the booster immunization, the mice were intraperitoneally injected with 200 μl of PTX solution (1 ng / μl), and 48 hours later, they were intraperitoneally injected with 200 μl of PTX solution again.

[0014] To further better implement the method for establishing an animal model of anti-NMDAR encephalitis induced by active immunization of GluN1 polypeptide combined with sleep deprivation of the present invention, the following setting method was particularly adopted: The specific content of step 2) is:

[0015] In the second week after the booster immunization, some animals were placed in an automatic sleep deprivation instrument (ZS-SM-II, zslab, China). The gentle mechanical thrust generated by the rotation of the plastic rod on the base was used to prevent the mice from falling asleep. The rotation speed was 5 rpm, and the rotation direction was changed every 15 seconds (alternating between clockwise and counterclockwise); Starting from the time when the light was on (a.m. 6:00, ZT0) until the time when the light was off (p.m. 6:00, ZT12), sleep deprivation was continuously carried out for 12 hours every day for 3 consecutive weeks. The mice were euthanized after the sleep deprivation ended for relevant tests or sent back to the breeding room for continued breeding for future use.

[0016] To further better implement the method for establishing an animal model of anti-NMDAR encephalitis induced by active immunization of GluN1 polypeptide combined with sleep

[0017] deprivation of the present invention, the following setting method was particularly adopted: The specific time period of continuous sleep deprivation for 12 hours every day is: a.m. 6:00~p.m. 6:00.

[0018] To further better implement the method for establishing an animal model of anti-NMDAR encephalitis induced by active immunization of GluN1 polypeptide combined with sleep deprivation of the present invention, the following setting method was particularly adopted: The animals used were 7-week-old, 20±2 g SPF-grade female wild-type C57BL / 6J mice.

[0019] To better implement the method for establishing an animal model of anti-NMDAR encephalitis by active immunization with GluN1 polypeptide combined with sleep deprivation, the following settings are particularly adopted: The synthesized GluN1 359-378 polypeptide is a GluN1 polypeptide containing the extracellular ATD domain (N368 / G369) of the GluN1 subunit, and the sequence is RKLVQVGIYNGTHVIPNDRK, with a purity ≥ 98%. 359-378

[0020] The animal model of anti-NMDAR encephalitis induced by active immunization with GluN1 polypeptide combined with sleep deprivation is characterized in that it is established by using the method for establishing an animal model of anti-NMDAR encephalitis induced by active immunization with the GluN1 polypeptide combined with sleep deprivation.

[0021] Application of the animal model of anti-NMDAR encephalitis induced by active immunization with GluN1 polypeptide combined with sleep deprivation in studying the pathogenesis and mechanism of sleep disorder-induced anti-NMDAR encephalitis.

[0022] Application of the animal model of anti-NMDAR encephalitis induced by active immunization with GluN1 polypeptide combined with sleep deprivation in studying the pathogenesis and mechanism of anti-NMDAR encephalitis.

[0023] Application of the animal model of anti-NMDAR encephalitis induced by active immunization with GluN1 polypeptide combined with sleep deprivation in screening and evaluating drugs for the prevention and treatment of anti-NMDAR encephalitis.

[0024] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0025] (1) The present invention combines active immunization with GluN1 polypeptide and sleep deprivation for the first time, and successfully establishes an animal model of anti-NMDAR encephalitis, providing a new method for studying the pathogenesis of this disease.

[0026] (2) The present invention establishes an animal model with a high success rate through the prime-boost immunization strategy and sleep deprivation operation, solving the problem of the low success rate of existing animal models of anti-NMDAR encephalitis.

[0027] (3) In view of the fact that sleep deprivation can reversibly change the function of the blood-brain barrier, the present invention can prove that the blood-brain barrier plays an important role in the pathogenesis of anti-NMDAR encephalitis.

[0028] (4) The present invention can simulate the key role of sleep disorder in the course of anti-NMDAR encephalitis, enhancing the ability of the model to reproduce the actual clinical pathological process.

[0029] ​(5) The present invention can be used for the research on the pathogenesis of sleep disorder-induced anti-NMDAR encephalitis, the screening and evaluation of potential therapeutic drugs, and has broad application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a technical roadmap of an animal model of anti-NMDAR encephalitis induced by active immunization of GluN1 polypeptide combined with sleep deprivation according to the present invention.

[0031] Figure 2 It is a schematic diagram and result diagram of antibody detection in mice according to the present invention. In the figure, A is a schematic diagram of the grouping and process of antibody detection. B is a representative image of detecting IgG-type GluN1 antibody (GluN1-Ab) by Live cell-based assay (LCBA), and HEK293T cells expressing the extracellular segment of GluN1 subunit are used to detect serum IgG-type GluN1-Abs. C is the result of detecting serum IgG-type GluN1-Ab by ELISA. D is the result of detecting the titer of serum IgG-type GluN1-Ab by LCBA. E is a representative image of co-incubating mouse serum and commercially purchased rabbit anti-GluN1 IgG antibody with normal brain tissue sections to reflect the specificity of serum IgG-type GluN1-Ab.

[0032] Figure 3 It is a result diagram of detecting the permeability of blood-brain barrier (BBB) in the hippocampus of mice according to the present invention. Among them, A-E are the results of BBB detection in GluN1-I group mice, F is a schematic diagram of the process of detecting BBB in sleep deprivation (SD) mice, and G-K are the results of BBB detection in SD mice.

[0033] Figure 4 It is a result diagram of detecting the IgG deposition in the hippocampus of mice according to the present invention. A is a schematic diagram of the detection process, and B and C are the results of IgG detection in the hippocampus of GluN1-I group mice and GluN1-I+SD group mice.

[0034] Figure 5 It is a schematic diagram and result diagram of the cognitive function evaluation process of mice according to the present invention. A is a flow chart of cognitive function detection, B is the result of the Y maze experiment, and C and D are the results of the Morris water maze (MWM) experiment. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0035] The present invention will be further described in detail below in conjunction with embodiments, but the embodiments of the present invention are not limited thereto.

[0036] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the claimed invention, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

[0037] In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of such features. In the description of the present invention, "a plurality" means two or more, unless otherwise specifically defined.

[0038] Example 1:

[0039] The present invention designs a method for establishing an animal model of anti-NMDAR encephalitis induced by active immunization with GluN1 polypeptide combined with sleep deprivation, including the following steps:

[0040] 1) Immunize animals with the synthesized GluN1 359-378 polypeptide according to the prime-boost immunization strategies to enable animals to obtain high-titer and relatively long-lasting serum IgG anti-NMDAR antibodies;

[0041] 2) At the 2nd week after the booster immunization, perform sleep deprivation on the animals for 12 hours per day (from 6:00 a.m. to 6:00 p.m.) for 3 weeks; open the blood-brain barrier to promote the entry of peripheral IgG anti-NMDAR antibodies into the central nervous system.

[0042] Example 2:

[0043] This embodiment is further optimized on the basis of the above embodiment. The same parts as the foregoing technical solutions will not be described herein again. To better implement the method for establishing an animal model of anti-NMDAR encephalitis induced by active immunization with GluN1 polypeptide of the present invention, the following setting method is particularly adopted: The step 1) specifically includes the following steps:

[0044] 1.1) GluN1359-378 Preparation process of polypeptide emulsion: GluN1 359-378 The polypeptide was diluted with ice-cold sterile physiological saline to a 2 mg / ml polypeptide solution, and complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA) was added in equal volume at a ratio of 1:1, and mixed evenly. Two syringes were connected through a three-way valve, and the mixture was aspirated repeatedly and slowly until a water-in-oil emulsion was formed, obtaining GluN1 359-378 polypeptide–CFA emulsion or GluN1 359-378 polypeptide–IFA emulsion;

[0045] 1.2) Active immunization process: For the first immunization, the GluN1 359-378 polypeptide–CFA emulsion was subcutaneously injected at four points on both sides of the spinal column on the back of the animal, 50 μl per point, a total of 200 μl per animal; After 2 weeks, booster immunization was carried out. The GluN1 359-378 polypeptide–IFA emulsion was subcutaneously injected at four points on the back of the animal, 50 μl per point, a total of 200 μl per animal; After the booster immunization, the mice were intraperitoneally injected with 200 μl of PTX solution (1 ng / μl), and 48 hours later, they were intraperitoneally injected with 200 μl of PTX solution again.

[0046] Example 3:

[0047] This example is further optimized on the basis of any of the above examples. The same parts as the previous technical solutions will not be described here again. To better implement the method for establishing an animal model of anti-NMDAR encephalitis by active immunization of GluN1 polypeptide in the present invention, the following setting method is particularly adopted: The specific content of step 2) is:

[0048] In the second week after the booster immunization, some animals were placed in an automatic sleep deprivation instrument (ZS-SM-II, zslab, China). The gentle mechanical thrust generated by the rotation of the plastic rod on the base was used to prevent the mice from falling asleep. The rotation speed was 5 rpm, and the rotation direction was changed every 15 seconds (alternating between clockwise and counterclockwise); Starting from the time when the light was on (a.m. 6:00, ZT0) until the time when the light was off (p.m. 6:00, ZT12), sleep was deprived for 12 hours every day for 3 consecutive weeks. The mice were euthanized or returned to the breeding room for continued breeding after the sleep deprivation (SD) ended.

[0049] Example 4:

[0050] This embodiment is a further optimization based on any of the above embodiments. The same parts as the foregoing technical solutions will not be described herein again. To better implement the method for establishing an animal model of anti-NMDAR encephalitis induced by active immunization with GluN1 polypeptide combined with sleep deprivation, the following specific settings are adopted: The time period of continuous sleep deprivation for 12 hours every day is specifically from 6:00 a.m. to 6:00 p.m.

[0051] Example 5:

[0052] This embodiment is a further optimization based on any of the above embodiments. The same parts as the foregoing technical solutions will not be described herein again. To better implement the method for establishing an animal model of anti-NMDAR encephalitis induced by active immunization with GluN1 polypeptide combined with sleep deprivation, the following specific settings are adopted: The animals used are SPF-grade female wild-type C57BL / 6J mice at 7 weeks old and weighing 20 ± 2 g.

[0053] Example 6:

[0054] This embodiment is a further optimization based on any of the above embodiments. The same parts as the foregoing technical solutions will not be described herein again. To better implement the method for establishing an animal model of anti-NMDAR encephalitis induced by active immunization with GluN1 polypeptide combined with sleep deprivation, the following specific settings are adopted: The synthesized GluN1 359-378 polypeptide is a GluN1 polypeptide containing the extracellular ATD domain (N368 / G369) of the GluN1 subunit, and the sequence is RKLVQVGIYNGTHVIPNDRK, with a purity of ≥ 98%. 359-378 polypeptide, and the sequence is RKLVQVGIYNGTHVIPNDRK, with a purity of ≥ 98%.

[0055] Example 7:

[0056] This embodiment is a further optimization based on any of the above embodiments. The same parts as the foregoing technical solutions will not be described herein again. The animal model of anti-NMDAR encephalitis induced by active immunization with GluN1 polypeptide combined with sleep deprivation is established by using the method for establishing an animal model of anti-NMDAR encephalitis induced by active immunization with GluN1 polypeptide combined with sleep deprivation.

[0057] Example 8:

[0058] This embodiment is a further optimization based on any of Examples 1-7. The same parts as the foregoing technical solutions will not be described herein again. The application of the animal model of anti-NMDAR encephalitis induced by active immunization with GluN1 polypeptide combined with sleep deprivation in studying the pathogenesis and mechanism of sleep disorder-induced anti-NMDAR encephalitis.

[0059] Example 9:

[0060] This example is a further optimization based on any one of Examples 1 to 7. The same parts as the foregoing technical solutions will not be elaborated here. The application of the GluN1 polypeptide active immunization combined with sleep deprivation in inducing an animal model of anti-NMDAR encephalitis in the study of the pathogenesis and mechanism of anti-NMDAR encephalitis.

[0061] Example 10:

[0062] This example is a further optimization based on any one of Examples 1 to 7. The same parts as the foregoing technical solutions will not be elaborated here. The application of the GluN1 polypeptide active immunization combined with sleep deprivation in inducing an animal model of anti-NMDAR encephalitis in the screening and evaluation of drugs for the prevention and treatment of anti-NMDAR encephalitis.

[0063] Example 11:

[0064] This example provides a method for establishing an animal model of anti-NMDAR encephalitis induced by sleep deprivation after GluN1 359-378 polypeptide active immunization.

[0065] The present invention uses the synthesized GluN1 359-378 polypeptide to boost immunize mice. After generating high-titer serum anti-GluN1 antibodies (GluN1-Abs), sleep deprivation (SD) treatment is applied to establish an animal model of anti-NMDAR encephalitis.

[0066] 1. Experimental animals and reagents:

[0067] 1) SPF-grade female wild-type C57BL / 6J mice (7 weeks old, 20 ± 2 g) were purchased from Chengdu Medicilon Inc. and housed in the Experimental Animal Center of West China Hospital, Sichuan University. The selection of female mice was based on clinical observations that the male-to-female incidence ratio of anti-NMDAR encephalitis patients is approximately 1:4. The mice were housed under SPF conditions, with the environmental temperature maintained at 22–25 °C, the humidity at 60%, the light / dark cycle at 12 hours, the light time at 6:00 a.m. (ZT0), and the light-off time at 6:00 p.m. (ZT12). The mice had free access to food and water. All experimental procedures followed the guiding principles of the Animal Care Ethics Committee of West China Hospital, Sichuan University, and necessary measures were taken to reduce the number of animals used and their suffering. At the end of the experiment, the mice were euthanized by intraperitoneal injection of 1% pentobarbital sodium (200 mg / kg).

[0068] 2) Entrust Nanjing Genepeptide Biotech Co., Ltd. to synthesize GluN1 359-378 polypeptide containing the extracellular ATD domain (N368 / G369) of the GluN1 subunit, with the sequence RKLVQVGIYNGTHVIPNDRK, purity ≥ 98%, and stored at -80 °C for later use.

[0069] 3) Complete Freund's adjuvant (CFA, containing Mycobacterium tuberculosis H37Ra, catalog number F5881) and incomplete Freund's adjuvant (IFA, without Mycobacterium tuberculosis H37Ra, catalog number F5506) were both purchased from Sigma-Aldrich, USA. Pertussis toxin (PTX, catalog number 181) was purchased from List Biological, USA.

[0070] 2. Experimental methods:

[0071] 1) Experimental grouping: Mice were randomly assigned during the experiment (i.e., step 1 is not the beginning part of this experimental method, but only to illustrate the grouping situation of mice during the experiment) to the blank control group (Control, CTL), GluN1 359-378 polypeptide immunization group (GluN1-immunized, GluN1-I), blank control + sleep deprivation group (CTL+SD), and GluN1 359-378 polypeptide immunization + sleep deprivation group (GluN1-I+SD).

[0072] 2) Preparation process of GluN1 359-378 polypeptide emulsion: Dilute GluN1 359-378 polypeptide with ice-cold sterile saline to a 2mg / ml polypeptide solution, add CFA or IFA in equal volume by 1:1, mix well, connect two syringes through a three-way valve and aspirate the mixture slowly and repeatedly until a water-in-oil emulsion is formed, obtaining GluN1 359-378 polypeptide–CFA emulsion or GluN1 359-378 polypeptide–IFA emulsion.

[0073] 3) Active immunization process: For the first immunization, inject the GluN1 359-378 polypeptide–CFA emulsion subcutaneously at four points on both sides of the spinal column of the back (before injection, disinfect the skin on both sides of the spinal column of the mouse back with 75% alcohol under isoflurane anesthesia), 50 μl per point, a total of 200 μl per mouse. After 2 weeks, boost immunization was carried out. Inject the GluN1 359-378 polypeptide–IFA emulsion subcutaneously at four points on the back (before injection, disinfect the skin on both sides of the spinal column of the mouse back with 75% alcohol under isoflurane anesthesia), 50 μl per point, a total of 200 μl per mouse, forming GluN1 359-378 polypeptide-immunized mice. Mice in the CTL group were injected with an equal volume of normal saline according to the same procedure. After the boost immunization, all mice were intraperitoneally injected with 200 μl of PTX solution (1 ng / μl). 48 hours later, they were intraperitoneally injected with 200 μl of PTX solution again.

[0074] 4) Sleep deprivation procedure: Some mice in the CTL group or GluN1-I group were placed in an automatic sleep deprivation apparatus (ZS-SM-II, zslab, China). A gentle mechanical thrust generated by the rotation of the plastic rod on the base was used to prevent the mice from falling asleep. The rotation speed was 5 rpm, and the rotation direction was changed every 15 seconds (alternating between clockwise and counterclockwise). Starting from the time when the light came on (a.m. 6:00, ZT0) and ending when the light went out (p.m. 6:00, ZT12), sleep was deprived for 12 hours every day for 3 consecutive weeks. After the sleep deprivation ended, the mice were euthanized for relevant tests or sent back to the breeding room for continued breeding for future use.

[0075] 5) Electrode implantation and sleep recording procedure: Under isoflurane anesthesia, the mice in the CTL+SD group or GluN1-I+SD group were fixed on a stereotaxic apparatus. EEG electrodes were implanted using the following coordinates: positive electrode (AP: -1.0 mm, ML: -1.5 mm), negative electrode (AP: -2.0 mm, ML: 1.5 mm), reference electrode (AP: -4.0 mm, ML: -1.5 mm), and the depth was approximately 0.5 mm. At the same time, two electrodes were implanted in the neck muscles to record electromyogram (EMG). The electrodes were fixed with dental cement and connected to a prefabricated head-mounted fixing device. After the operation, the mice were transferred to a cage and raised individually, and closely observed until they were fully awake. After the electrode implantation, the mice were allowed to recover for 1 week. Subsequently, the mice were placed in the sleep deprivation apparatus for 3 days of adaptation. On the 1st, 7th, 14th, 21st days of the SD stage and the 1st and 7th days after the end of SD, the EEG / EMG signals were recorded using a data acquisition system (Medusa, Bio-Signal Technologies, China), amplified, filtered, and digitized and stored at a sampling rate of 1000 Hz; subsequently, an AI software was used to analyze according to the spectral and topological characteristics of the signals to automatically determine the wakefulness, non-rapid eye movement sleep (NREM), and rapid eye movement sleep (REM) stages. All data were reviewed by experienced sleep researchers and manually corrected if necessary.

[0076] 6) Cognitive function evaluation: The Y maze and MWM experiments were used to evaluate the cognitive function of the mice. To avoid the interference of behavioral tests on the SD effect of the mice, no behavioral tests were performed during the SD period. It was not until the end of SD and the restoration of ad libitum sleep (ALS) that the behavioral training and evaluation of the mice began (as shown in Figure 5 A).

[0077] 3. Detection methods:

[0078] 1) Plasma collection and preparation process for each group of mice: Before cardiac perfusion, collect mouse blood (about 500 μl) by cardiac puncture into a blood collection tube containing EDTA, centrifuge at 1,500×g, 4 °C for 15 minutes, aspirate the supernatant, and store the aliquoted serum samples at -80 °C for later use.

[0079] 2) As shown in Figure 2 Panel B, the process of detecting serum IgG-type GluN1-Ab in each group of mice by Live Cell-Based Assay (LCBA): Seed HEK293T cells in a 96-well plate (Sigma-Aldrich, USA). When the cells cover approximately 50% of the bottom area of the plate, transfect with human GluN1 plasmid (HanBio, China), then return to the cell culture incubator and incubate for 6 hours. Replace the medium and continue to incubate for 24 hours. Dilute the serum samples of each group of mice 1:10 with 10% normal goat serum (NGS) and incubate the transfected HEK293T cells at room temperature for 1 hour. Aspirate the supernatant, add 4% paraformaldehyde to fix for 5 minutes, wash with PBS, permeabilize the membrane with 0.3% TritonX-100 / PBS, add rabbit anti-GluN1 antibody (1:100, ab239949, Abcam, UK) to incubate the cells, and incubate overnight at 4 °C. The next day, after washing the cells with PBS, sequentially add Alexa 594 goat anti-mouse IgG antibody (115-585-003, Jackson ImmunoResearch, USA) and

[0080] 3) As shown in Figure 2 Panel C, the process of detecting serum IgG-type GluN1-Ab in each group of mice by ELISA: GluN1 359-378Polypeptides (1 μg / well) were coated on 96-well plates (Sigma-Aldrich, USA) and incubated overnight at 4°C. The next day, the plates were blocked with 5% bovine serum albumin (BSA) to reduce non-specific binding. Mouse sera from each group were diluted 1:1000 and added to the plates, followed by incubation at room temperature for 2 hours. Then, HRP-conjugated goat anti-mouse IgG antibody (1:10,000, ab205719, Abcam, UK) was added and incubated at room temperature for 1 hour. The plates were thoroughly washed with PBS between the two incubations to remove unbound serum or antibody. 1-Step Ultra TMB ELISA substrate (34028, ThermoFisher Scientific, USA) was added, and the reaction was terminated by adding sulfuric acid after 12 minutes of color development at room temperature. Absorbance was measured at 450 nm using a multi-functional microplate reader (SPARK, TECAN, Switzerland), and corrected with the absorbance at 620 nm. Positive serum antibody determination value was higher than the average measurement value of the CTL group mice plus 3 times the standard deviation (> average measurement value of CTL group mice + 3 × standard deviation).

[0081] 4) Binding Figure 2 As shown in D, the detection process of serum IgG-type GluN1-Ab titers in GluN1-I group mice and GluN1-I+SD group mice: The method was based on LCBA. The initial dilution of serum samples was 1:10. If the result was positive, the samples were successively diluted 1:32, 1:100, 1:320, 1:1000, 1:3200, 1:10,000, and the antibody titer was determined according to the positive result.

[0082] 5) Binding Figure 2 As shown in E, the specificity of serum IgG-type GluN1-Ab was detected using normal mouse brain tissue sections:

[0083] Normal brain tissue sections were blocked with 5% NGS (containing 0.3% Triton X-100) at room temperature for 1 hour, and then co-incubated overnight with sera from CTL group mice, GluN1-I group mice, and commercially purchased rabbit anti-GluN1 IgG antibody (ab109182, Abcam, UK). After washing with PBS, they were incubated with Alexa 594 goat anti-mouse IgG antibody (115-585-003, Jackson ImmunoResearch, USA), 488 goat anti-rabbit IgG antibody (ab96899, Abcam, UK) in the dark for 2 hours. After washing with PBS, the sections were mounted with 50% glycerol / PBS, and images were taken using a fluorescence microscope (Ti2, Nikon, Japan).

[0084] 6) Binding Figure 3In vivo blood-brain barrier (BBB) integrity assessment:

[0085] After anesthesia with isoflurane, mice in the GluN1-I + SD group were injected with fluorescent tracer dyes via the tail vein at the set time points, including FITC-dextran (FITC-dex, 70KD; 46945, Sigma-Aldrich, USA), sodium fluorescein (Na-flu, 0.376KD; F6377, Sigma-Aldrich, USA), and Evans blue (EB; E2129, Sigma-Aldrich, USA), for assessing BBB integrity.

[0086] 7) Binding Figure 4 As shown, the detection process of IgG-type GluN1-Ab deposited in the hippocampal tissue: After anesthesia, perfusion, brain sampling, fixation with paraformaldehyde, dehydration with sucrose, and embedding, mice in the GluN1-I group and the GluN1-I + SD group were made into 10-μm serial coronal brain tissue sections (including the main hippocampal region) and stored at -80 °C for later use. The sections were blocked with 5% NGS (containing 0.3% Triton X-100) at room temperature for 1 hour, then incubated with Alexa 488 goat anti-mouse IgG antibody (115-545-003, Jackson ImmunoResearch, USA) in the dark for 2 hours, stained with DAPI for 10 minutes, washed with PBS, and mounted with 50% glycerol / PBS. Images were taken with a fluorescence microscope (Ti2, Nikon, Japan), and the image data were analyzed using Fiji software (NIH, USA).

[0087] 8) Behavioral tests:

[0088] To avoid interference of behavioral tests with the effect of SD, no behavioral tests were conducted during SD. Behavioral training and evaluation of the mice were only started after the end of SD.

[0089] Morris water maze (MWM) experiment:

[0090] Binding Figure 5 As shown, a circular pool with a diameter of 120 cm was used, filled with an appropriate amount of water mixed with food-grade white pigment, and the water temperature was maintained at 22 - 25 °C. Colorful markers were pasted at the non-water-contact part above the pool according to the directions of east, south, west, and north (E, S, W, N) as visual references. The white platform was placed at the center of a certain quadrant, and its surface was 1 cm below the water surface. Light-shielding curtains were hung around the pool, and indirect lighting was used to minimize external interference.

[0091] Hidden platform experiment: Each mouse was trained twice a day for 5 consecutive days. The experimental mice were randomly placed in one of the four quadrants of the water pool and allowed to search for the hidden platform within 60 seconds. If the mouse failed to find the platform within the specified time, it was guided to swim to the platform and stay for 10 seconds. After the training, the mice were taken out of the water, dried with paper towels and placed in a warm and dry breeding cage. The whole process was recorded by EthoVision XT video tracking software.

[0092] No-platform exploration experiment: 24 hours after the hidden platform experiment, the platform was removed for the spatial exploration test. A water entry point was selected and the mouse was placed in the water. The swimming path within 60 seconds was recorded by EthoVision XT video tracking software.

[0093] Y-maze experiment:

[0094] Combined Figure 5 As shown, the test was carried out in a maze with three equal-length arms, each arm being 50 cm long, 20 cm high and 10 cm wide, arranged at 120°. The mouse was placed in the center of the maze and allowed to freely explore the three arms for 5 minutes. Between each trial, the maze was cleaned with 75% alcohol to remove olfactory cues. The whole process was recorded by EthoVision XT video tracking software. The percentage of spontaneous alternation was calculated according to the following formula: [Number of spontaneous alternations / (Total number of arm entries - 2)] × 100%.

[0095] 4. Experimental results:

[0096] 1) Serum GluN1-Ab detection:

[0097] The experimental protocol and results are as Figure 2 shown in A - D. IgG-type GluN1-Ab was only detected in the sera of mice immunized with GluN1 359-378 polypeptides (GluN1-I group and GluN1-I+SD group), and SD intervention had no significant effect on the titers of IgG-type GluN1-Ab in the sera of these mice. In addition, the binding distribution of IgG-type GluN1-Ab produced by mice in the GluN1-I group in normal mouse brain tissue was basically consistent with the distribution pattern of commercial rabbit anti-GluN1 IgG antibody ( Figure 2 E).

[0098] 2) BBB permeability detection:

[0099] The results are as Figure 3 shown. Compared with the mice in the CTL group, no obvious FITC-dex leakage was observed in the hippocampal region of the mice in the GluN1-I group ( Figure 3(A-E), indicating that the BBB of GluN1-I group mice remained intact at the 2nd week after booster immunization. However, during the SD treatment, a large amount of FITC-dex leakage was observed in the hippocampal region of the mice, indicating that SD intervention significantly increased the BBB permeability. But at 1 week after the end of SD and resumption of ALS, the BBB permeability recovered to the normal level( Figure 3 F-K).

[0100] 3) IgG-type GluN1-Ab deposition in the hippocampal region:

[0101] The protocol and results are as Figure 4 shown. No obvious IgG fluorescence signals were observed in the hippocampal region of GluN1-I group mice at the 2nd, 6th, and 10th weeks after booster immunization. However, at 3 weeks of SD (i.e., the 5th week after booster immunization), a large amount of IgG accumulation was visible in the hippocampal region of GluN1-I+SD group mice. At 1 week of ALS (i.e., the 6th week after booster immunization), the IgG fluorescence intensity in the hippocampal region of GluN1-I+SD group mice decreased to a certain extent compared with that before. At 5 weeks of ALS (i.e., the 10th week after booster immunization), almost no IgG fluorescence signals were detected in the hippocampal region of GluN1-I+SD group mice.

[0102] 4) Behavioral detection:

[0103] The protocol and results of cognitive function assessment are as Figure 5 shown. The cognitive function of GluN1-I group mice did not show significant changes compared with that of CTL group mice at the 2nd, 6th, and 10th weeks after booster immunization, indicating that simple GluN1 polypeptide immunization could not induce encephalitis-like symptoms in mice. The cognitive function of CTL+SD group mice did not show obvious differences compared with that of CTL group mice at 1 week after the end of SD and resumption of ALS (the 6th week after booster immunization). However, the cognitive ability of GluN1-I+SD group mice decreased significantly at the 6th week after booster immunization and recovered to normal at the 10th week after booster immunization. This may be related to the fact that after the end of SD, the BBB repair led to the inability of serum GluN1-Abs to continue to enter the brain (no increase), and at the same time, the antibodies deposited in the brain parenchyma were gradually cleared by phagocytes in the central nervous system.

[0104] Example 12:

[0105] Application of GluN1 polypeptide active immunization combined with sleep deprivation in inducing an animal model of anti-NMDAR encephalitis

[0106] 1. Establish an animal model of anti-NMDAR encephalitis induced by GluN1 polypeptide active immunization combined with sleep deprivation according to the method of Example 11 of the present invention;

[0107] 2. The GluN1 polypeptide active immunization combined with the sleep deprivation-induced anti-NMDAR encephalitis animal model is applied to the study of the pathogenesis of this disease and the screening and evaluation of drugs.

[0108] In summary, the artificially synthesized GluN1 359-378 polypeptide is used to actively immunize female C57 mice according to the primary-booster immunization strategy, which can induce high-titer and long-lasting IgG-type GluN1-Abs. At the 2nd week after booster immunization, the GluN1-I mice were given continuous SD treatment for 3 weeks. A large amount of IgG deposition was observed in the hippocampal region of the GluN1-I+SD group mice. After 1 week of ALS, the IgG in the hippocampal region decreased to a certain extent compared with before, but these animals still showed obvious cognitive impairment, indicating the successful establishment of the GluN1 polypeptide active immunization combined with the sleep deprivation-induced anti-NMDAR encephalitis animal model of the present invention.

[0109] In addition, any combination can be made between various different embodiments of the present invention as long as it does not violate the idea of the present invention, and it should also be regarded as the content disclosed by the present invention.

Claims

1. A method for establishing an animal model of anti-NMDAR encephalitis induced by active immunization with GluN1 polypeptide combined with sleep deprivation, characterized in that: The steps include: 1) The synthesized GluN1 359-378 Peptides are used to immunize animals using a prime-boost strategy; 2) In the second week after booster immunization, the animals were subjected to sleep deprivation for 12 hours per day for 3 weeks.

2. The method for establishing an animal model of anti-NMDAR encephalitis induced by active immunization with GluN1 polypeptide combined with sleep deprivation according to claim 1, characterized in that: The step 1) specifically includes the following steps: 1.1 ) GluN1 359-378 Preparation process of peptide emulsion: GluN1 359-378 The peptide was diluted with ice-cold sterile saline to a 2 mg / ml peptide solution, and complete Freund's adjuvant or incomplete Freund's adjuvant was added in a 1:1 ratio. Two syringes were connected through a three-way valve and the mixture was repeatedly and slowly aspirated until an oil-in-water emulsion was formed to obtain GluN1. 359-378 Peptide – CFA emulsion or GluN1 359-378 Peptide-IFA emulsion; 1.2) Active immunization process: First immunization, GluN1 359-378 The peptide-CFA emulsion was subcutaneously injected at four points on both sides of the dorsal spine of the animals, 50 μl / point, a total of 200 μl / animal; booster immunization was performed 2 weeks later to inject GluN1 359-378 The peptide-IFA emulsion was subcutaneously injected at four points on the back of the animal, 50 μl / point, a total of 200 μl / animal.

3. The method for establishing an animal model of anti-NMDAR encephalitis induced by active immunization with GluN1 polypeptide combined with sleep deprivation according to claim 1, characterized in that: The step 2) is specifically as follows: In the second week after the booster immunization, the animals were placed in an automatic sleep deprivation apparatus. The gentle mechanical thrust generated by the rotation of the plastic rod on the base prevented the mice from falling asleep. The rotation speed was 5 rpm, and the rotation direction changed once every 15 seconds. The sleep deprivation lasted for 12 hours a day, starting from the time the lights were turned on and ending at the time the lights were turned off, for 3 consecutive weeks.

4. The method for establishing an animal model of anti-NMDAR encephalitis induced by active immunization with GluN1 polypeptide combined with sleep deprivation according to claim 3, characterized in that: The specific time period of 12 hours of continuous sleep deprivation every day is: am6:00 ~ pm6:

00.

5. The method for establishing an animal model of anti-NMDAR encephalitis induced by active immunization with GluN1 polypeptide combined with sleep deprivation according to any one of claims 1 to 4, characterized in that: The animals used were 7-week-old, 20±2g SPF-grade female wild-type C57BL / 6J mice.

6. The method for establishing an animal model of anti-NMDAR encephalitis induced by active immunization with GluN1 polypeptide combined with sleep deprivation according to any one of claims 1 to 4, characterized in that: The synthetic GluN1 359-378 The polypeptide is GluN1 containing the extracellular ATD domain of GluN1 subunit 359-378 The polypeptide has the sequence of RKLVQVGIYNGTHVIPNDRK and the purity is ≥98%.

7. Anti-NMDAR encephalitis animal model induced by active immunization with GluN1 polypeptide combined with sleep deprivation, characterized by: The anti-NMDAR encephalitis animal model is established by combining active immunization with GluN1 polypeptide as described in any one of claims 1 to 6 with the method for establishing sleep deprivation-induced anti-NMDAR encephalitis animal model.

8. Application of GluN1 peptide active immunization combined with sleep deprivation-induced anti-NMDAR encephalitis animal model in studying the pathogenesis and mechanism of sleep disorder-induced anti-NMDAR encephalitis.

9. Application of the anti-NMDAR encephalitis animal model induced by active immunization with GluN1 peptide combined with sleep deprivation in studying the pathogenesis and mechanism of anti-NMDAR encephalitis.

10. Application of the animal model of anti-NMDAR encephalitis induced by active immunization with GluN1 peptide combined with sleep deprivation in the screening and evaluation of drugs for the prevention and treatment of anti-NMDAR encephalitis.

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