Application of BD-sourced exosome in construction of affective disorder animal model

Abstract

The invention discloses application of a BD-sourced exosome in construction of an affective disorder animal model. According to the method, the serum exosome derived from a bipolar affective disorder (BD) patient is injected into a mouse body to construct the affective disorder animal model, and the affective disorder animal model comprises a manic animal model constructed by using the serum exosome derived from the BD manic period patient, or a depression animal model constructed by using the serum exosome derived from the BD depression period patient, or a depression animal model constructed by using the serum exosome derived from the BD depression period patient. The method can be used for screening affective disorder drugs, researching and revealing pathogenesis of affective disorder diseases, and has important significance on clinical research of affective disorder diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of BD-derived exosomes in constructing animal models of affective disorders. Background Technology

[0002] Bipolar disorder (BD), formerly known as manic-depressive illness, is a severe, relapsing mental illness characterized by alternating episodes of mania and depression. Manic episodes are characterized by elevated mood, increased energy, and increased activity, while depressive episodes are characterized by depressed mood, decreased energy, and reduced activity. Due to its high prevalence, long course, and severity, BD significantly impacts the daily lives of patients and their families, imposing a substantial economic and psychological burden on families and society. Despite decades of research, the biological mechanisms driving the oscillations between manic and depressive states remain incompletely understood, and current treatments are far from optimal. Lithium, anticonvulsants, and atypical antipsychotics are the cornerstones of BD treatment, but many patients still experience incomplete remission, relapse, or treatment resistance. Therefore, constructing animal models of BD at both common and state-specific levels to elucidate the pathophysiology of BD is crucial for the development of BD biomarkers and novel treatment methods.

[0003] Exosomes are nanoscale extracellular vesicles released by almost all cell types and are considered powerful mediators of intercellular communication. They carry proteins, lipids, metabolites, and nucleic acids, and most importantly, they can cross the blood-brain barrier, influencing neuronal and glial function. In neurology, exosomes are associated with neurodegenerative diseases such as Alzheimer's and Parkinson's, where they propagate misfolded proteins and inflammatory signals. Changes in exosomes have also been reported in schizophrenia and autism spectrum disorders, linking them to central nervous system pathophysiology. However, whether these exosomes functionally participate in the pathophysiology of BD remains uninvestigated. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, the purpose of this invention is to provide the application of BD-derived exosomes in constructing animal models of affective disorders.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of the present invention provides a method for constructing an animal model of affective disorder, the method comprising administering serum exosomes derived from BD patients at different stages to the animals.

[0006] Furthermore, the method includes administering serum exosomes derived from BD patients in the manic phase to animals to obtain an animal model exhibiting manic-like behavior, or administering serum exosomes derived from BD patients in the depressive phase to animals to obtain an animal model exhibiting depressive-like behavior.

[0007] In this invention, the "exosome" refers to nanoscale vesicles secreted by cells, typically with a diameter between 30 and 150 nanometers, and possessing a typical phospholipid bilayer membrane structure. Exosomes encapsulate various bioactive molecules, including proteins, lipids, and nucleic acids (such as DNA, mRNA, and microRNA), whose contents reflect the type and physiological / pathological state of their originating cells. Exosomes play a crucial role in intercellular communication, participating in various physiological and pathological processes such as immune responses, tissue repair, and disease occurrence and development by transmitting biological information. Exosomes are widely present in body fluids, such as blood, saliva, urine, breast milk, and cerebrospinal fluid; almost all cell types can secrete exosomes under normal or pathological conditions. Specifically, the exosomes used in this invention are serum exosomes.

[0008] Furthermore, the manic-like behaviors include excessive exercise, increased interest, high energy, and enhanced social skills.

[0009] Furthermore, the depressive-like behaviors include prolonged immobility, bradykinesia, loss of interest, lethargy, and social withdrawal.

[0010] In this invention, "animal model" refers to a non-human animal that has or displays characteristics of a disease or symptom. Use as an animal model means any use of an animal to study a disease or condition, such as for studying progress or development or responses to new or existing therapies.

[0011] In this invention, animals include non-human vertebrates, more preferably mammals, which refer to all members of the class Mammalia, such as cattle, horses, pigs, sheep, monkeys, pets (e.g., dogs, cats), or rodents. The term "rodent" refers to any and all members of the phylogenetic rodent class (e.g., mice, rats, squirrels, beavers, groundhogs, voles, hamsters, guinea pigs, spiny guinea pigs), including any offspring of all offspring derived therefrom.

[0012] Furthermore, the mammals include mice, rats, rabbits, dogs, pigs, monkeys, sheep, and horses.

[0013] Furthermore, the mammal is selected from mice.

[0014] A second aspect of the present invention provides a method for screening drug candidates for treating mood disorders, the method comprising: administering a drug to be screened to mood disorder animals prepared by the method described in the first aspect of the present invention, analyzing and evaluating the therapeutic effect of the drug to be screened, and selecting a drug that can significantly improve the mood disorder behavior of the animal model.

[0015] In some embodiments, unless otherwise specified, the term "treatment" means reversing or alleviating a disease or condition or one or more of its symptoms used with the term, suppressing the progression of the disease or condition or one or more of its symptoms, or preventing the disease or condition or one or more of its symptoms. The term "treatment" as used in this invention refers to the act of "treatment" as defined above. Therefore, treatment or treatment regimens for diseases in mammals may include one or more of the following: suppressing the growth of the disease, i.e., inhibiting its development; preventing the spread of the disease; alleviating the disease; preventing the recurrence of the disease; and alleviating the symptoms of the disease.

[0016] In some implementations, the improvement refers to a reduction of at least about 10%, at least about 30%, at least about 50%, or at least about 80% in the symptoms of the animal model or the cell phenotype derived from the animal model compared to before drug administration.

[0017] In this invention, the drug candidates can be obtained from a wide variety of sources. For example, they can be obtained by random and directed synthesis of a variety of organic compounds and biomolecules, or libraries of natural compounds in the form of bacterial, fungal, plant or animal extracts, or libraries and compounds of natural or synthetic compounds modified by conventional chemical, physical or biochemical means, or by directed or random chemical modification of known pharmacological agents, such as acylation, alkylation, esterification, amidation, etc., to generate structural analogs.

[0018] Furthermore, the evaluation indicators include animal behavioral tests, neuronal structure, and microglia status.

[0019] Furthermore, animal behavioral tests include the tail suspension test, forced swimming test, sucrose preference test, Y maze test, three-compartment social competence test, water maze test, splash test, open field test, elevated maze test, rotundus test, and chronic restraint. In this invention, the term "tail suspension test," also known as "TST," is a classic and rapid method for evaluating the efficacy of antidepressants, stimulants, and sedatives. Its principle involves mice being suspended by their tails and attempting to escape but failing, thus giving up the struggle and entering a specific state of depressive immobility. This typical "immobility state" reflects a state known as "behavioral despair," a model similar to depression. This model is sensitive to most antidepressants, and its efficacy is significantly correlated with clinical efficacy, thus it is widely used for the initial screening of antidepressants. During the test, the duration of immobility is recorded to reflect the depressive state; antidepressants and stimulants can significantly shorten or alter this immobility state.

[0020] In this invention, the term "forced swimming test," also known as "FST," is a behavioral despair test. It involves placing an animal in a confined environment (such as water) where it struggles desperately to escape but is unable to, creating an inescapable pressure environment. After a period of time, the animal exhibits a typical "immobile state." Observing and recording a series of parameters during the process of the animal developing this despairing immobile state can be used to evaluate the effects of depressants and antidepressants. In the forced swimming test, the movement of the animal's hind limbs is typically used as the criterion for judgment.

[0021] In this invention, the term "saccharin preference test," also known as "SPT," is a behavioral test widely used to assess the emotional state of laboratory animals (such as mice and rats). Its basic principle is to assess an animal's emotional state by measuring its relative preference for saccharin and plain water. It is primarily used to detect levels of happiness and depressive symptoms in animals. This test is based on the observed phenomenon that healthy animals generally prefer sweet foods and drinks, while depressed or melancholic animals may show a reduced preference for sweet substances. SPT can serve as an effective tool for assessing depressive symptoms in laboratory animals. Simultaneously, SPT is also widely used to evaluate the efficacy of antidepressant drugs and observe whether the animal's saccharin preference recovers after drug treatment.

[0022] In this invention, the term "Y-maze test," also known as "YMaze," refers to a behavioral test based on an animal's natural exploratory curiosity. The spontaneous alternation behavior in this test is considered to reflect short-term spatial working memory. It utilizes the animal's innate tendency to explore new environments; during the test, the animal needs to remember previously explored directions each time it changes direction, thus the Y-maze test effectively reflects the animal's spatial working ability. The Y-maze test is generally divided into the Y-maze alternation behavior test and the electrically stimulated Y-maze test. In a specific embodiment of this invention, the Y-maze alternation behavior test is used.

[0023] In this invention, the term "three-compartment social competence test" is a classic animal behavior experiment that assesses a mouse's social competence, social preferences, and social memory by simulating social interactions in its natural environment. The experiment is based on animals' innate social tendencies and their instinct to explore new things, quantifying their social behavioral characteristics by comparing their preference for familiar versus unfamiliar individuals.

[0024] In this invention, the term "water maze test," also known as the "Morris water maze" or "MWM," refers to an experiment in which experimental animals (rats and mice) are forced to swim and learn to find platforms hidden in the water. It is primarily used to test the learning and memory abilities of experimental animals in spatial location and orientation (spatial orientation). It is not only used to evaluate the function of brain regions related to spatial learning and memory, but is also widely applied in scientific research and computer-aided teaching in multiple disciplines, including learning and memory, Alzheimer's disease, hippocampus / outer hippocampus research, intelligence and aging, new drug development / screening / evaluation, pharmacology, toxicology, preventive medicine, neurobiology, animal psychology, and behavioral biology. It has gained widespread recognition worldwide and is the preferred classic test for medical schools to conduct behavioral research, especially research on learning and memory.

[0025] In this invention, the term "splash test" is an animal behavioral experiment that assesses self-grooming behavior (grooming behavior) by simulating dirt on the fur of rodents. This behavior is an innate cleaning habit of rodents, involving complex and stereotyped sequential movements, typically starting from the head and gradually spreading throughout the body to the genitals and tail. Grooming behavior is not only used to maintain the cleanliness of fur and skin but also participates in thermoregulation and emotional expression. In emotional disorder phenotypes, animals may exhibit abnormal self-care abilities, leading to abnormal grooming behavior. The splash test provides a sensitive indicator for assessing the state of emotional disorders in animals by quantifying these behavioral changes.

[0026] In this invention, the term "open field test," also known as the "open box test," is a method for evaluating the autonomous behavior, exploratory behavior, and stress levels of experimental animals in unfamiliar environments. It reflects the animal's autonomous and exploratory behaviors in unfamiliar settings by the frequency and duration of certain behaviors, while the frequency of urination and defecation reflects its stress level. Currently, the open field test is mainly used to observe the animal's autonomous motor abilities, exploratory behavior in unfamiliar environments, stress levels, manic behavior, anxiety, and depressive behavior. Due to its simplicity, feasibility, and accurate data recording, it has become a popular behavioral testing method in the field of animal psychology research.

[0027] In this invention, the term "elevated maze test" includes the "elevated cross maze test" and the "elevated zero maze test," wherein the elevated zero maze test is a modified version of the elevated cross maze, designed to assess the level of depression in rodents by simulating their natural preference for open and enclosed spaces. Rodents are naturally inclined to prefer dark, enclosed spaces (a sense of security), while exhibiting an avoidance tendency towards heights and open spaces (escaping predators). This approach-avoidance conflict is key to assessing depression. The time and number of times an animal spends exploring open areas reflect its level of depression. Animals with high levels of depression will reduce their time spent in open areas and increase their exploration behavior in enclosed areas. In a specific embodiment of this invention, the elevated zero maze test is employed.

[0028] In this invention, the term "rotating bar test" refers to assessing the motor coordination and balance of test animals by forcing them to run on a rotating bar with accelerated acceleration. The test animal is placed on a rotating bar with stable acceleration, and the time it takes to fall off the bar, i.e., the latency period, is recorded. This indicator allows for the detection of the animal's motor learning and coordination abilities, and further analysis of the effects of drugs, diseases, or other interventions on its motor function.

[0029] In this invention, the term "chronic restraint," also known as "CRS" or "chronic restraint stress test," refers to placing experimental animals in an adjustable cylindrical restraint device made of transparent material with ventilation holes. The restraint duration is fixed daily, and food and water are withheld during the daily stress phase. Different durations and stress intensities can be selected based on experimental needs. Duration durations include 1, 2, 3, and 6 hours per day, while stress intensities can be selected for 1, 3, 7, 14, 21, and 28 consecutive days. Due to prolonged restraint in a confined space, animals initially exhibit anxiety, irritability, and attempts to escape. Ultimately, feeling hopeless about escape, they develop symptoms similar to those in humans, such as anhedonia, weight loss, reduced appetite, despair, and fatigue—symptoms that can be improved with antidepressants. Further behavioral evaluations are then conducted.

[0030] Furthermore, the animal behavioral tests were selected from the forced swimming test, the tail suspension test, the open field test, the Y maze test, the splash test, the elevated maze test, and the three-compartment social competence test.

[0031] The third aspect of the present invention provides for any of the following applications: 1) Application of serum exosomes derived from BD patients during the manic episode in the construction of manic animal models.

[0032] 2) Application of serum exosomes derived from BD patients during the onset of depression in the construction of animal models of depression.

[0033] 3) Application of the animal model of affective disorder constructed by the method described in the first aspect of the present invention in screening drug candidates for the treatment of affective disorders.

[0034] Furthermore, the animal model refers to an animal that has or displays characteristics of disease or symptom.

[0035] Furthermore, the animal in question is a mammal.

[0036] Furthermore, the mammals include mice, rats, rabbits, dogs, pigs, monkeys, sheep, and horses.

[0037] Furthermore, the mammal is selected from mice.

[0038] A fourth aspect of the present invention provides a system for constructing an animal model of emotional disorders, the system comprising: The fixing unit is used to fix the animal's head onto the stereo positioning device.

[0039] The first processing unit is used for shaving and disinfection, cutting the scalp along the midline, peeling off the subcutaneous tissue, and exposing the surface of the skull.

[0040] The second processing unit is used to determine the implantation coordinates of the intracranial drug delivery cannula based on the brain atlas, drill a hole at the implantation coordinates of the intracranial drug delivery cannula with a micro drill bit, and vertically insert the intracranial drug delivery cannula into the target area. The implantation coordinates of the intracranial drug delivery cannula are the frontal cortex area.

[0041] The third processing unit is used to suture the scalp and provide postoperative care and recovery.

[0042] The fourth processing unit is used to inject serum exosomes derived from manic BD patients into the brain of the animal through an intracranial administration cannula after the animal has recovered from surgery, in order to obtain a manic animal model, or to inject serum exosomes derived from depressive BD patients into the brain, in order to obtain a depressive animal model.

[0043] Furthermore, the coordinates of the prefrontal cortex region are defined with the anterior fontanelle as the origin, and the coordinates relative to the anterior fontanelle are: AP +2.0 mm, ML ±0.3 mm, DV -1.5 mm.

[0044] Furthermore, the exosomes are injected once every 3 days, for a total of 5 times.

[0045] Furthermore, the number of exosome injection particles is 1×10⁻⁶. 9 Granules / hemispherical.

[0046] In this invention, the term "unit" refers to a software or hardware component that performs a specific function, such as a field-programmable gate array (FPGA), an application-specific integrated circuit (ASIC), an operable medical component, a visualization component, etc. However, the term "unit" is not limited to software or hardware. A "unit" can be configured in an addressable storage medium or can be configured to reproduce one or more processors. Thus, for example, the term "unit" can refer to components such as software components, object-oriented software components, class components, and task components, and can include processes, functions, attributes, procedures, subroutines, program code segments, drivers, firmware, microcode, circuits, data, databases, data structures, tables, arrays, and variables. The functionality provided in components and "units" can be combined into fewer components and "units," or can be further divided into additional components and "units." Furthermore, components and "units" can be implemented to operate one or more central processing units (CPUs) in a device or secure multimedia card.

[0047] A fifth aspect of the present invention provides an apparatus for constructing an animal model of emotional disorders, the apparatus comprising one or more processors and a memory for storing one or more computer programs, which, when executed by the one or more processors, perform the following: Operation 1 is used to fix the animal's head onto the stereo positioning device.

[0048] Step 2, used for shaving and disinfection, involves cutting the scalp along the midline, peeling off the subcutaneous tissue, and exposing the surface of the skull.

[0049] Operation 3 is used to determine the implantation coordinates of the intracranial drug delivery cannula based on the brain atlas. A hole is drilled at the implantation coordinates of the intracranial drug delivery cannula using a micro-drill, and the intracranial drug delivery cannula is vertically inserted into the target area. The implantation coordinates of the intracranial drug delivery cannula are the prefrontal cortex region. The coordinates of the prefrontal cortex region are defined with the anterior fontanelle as the origin, and the coordinates relative to the anterior fontanelle are: AP +2.0 mm, ML ±0.3 mm, DV -1.5 mm.

[0050] Step 4 is used to suture the scalp for postoperative care and recovery.

[0051] Operation 5 is used to inject serum exosomes derived from manic BD patients into the brain of animals through an intracranial drug delivery cannula after the animals have recovered from surgery, in order to obtain a manic animal model, or to inject serum exosomes derived from depressive BD patients into the brain, in order to obtain a depressive animal model.

[0052] Furthermore, the exosomes are injected once every 3 days, for a total of 5 times.

[0053] Furthermore, the number of exosome injection particles is 1×10⁻⁶. 9 Granules / hemispherical.

[0054] The term "device" as used in this invention is not limited to one or a specific number of physical objects. As used herein, a device can be any medical or electronic component having multiple parts that can implement at least some portions of this disclosure. Although the term "device" is used in the following description and examples to describe certain aspects of this disclosure, the term "device" is not limited to a particular configuration, type, or number of objects.

[0055] The terms "processor" or "memory" in this invention include computing devices having one processor or one memory, as well as devices having multiple processors or multiple memories, that can be used to perform some or all of the steps described. "Processor" can include more than one processor, for example, a multi-core design or multiple processors each having a multi-core design.

[0056] Advantages and beneficial effects of the present invention: This invention constructs an animal model of mood disorder by injecting serum exosomes derived from patients with bipolar disorder (BD) into mice. The animal model includes a manic animal model constructed using serum exosomes derived from patients in the manic phase of BD, or a depressive animal model constructed using serum exosomes derived from patients in the depressive phase of BD. This model can be used for screening drugs for mood disorders, studying and revealing the pathogenesis of mood disorders, and is of great significance for clinical research on mood disorders. Attached Figure Description

[0057] Figure 1 The image shows the results of the characterization evaluation of serum exosomes from BD patients. In the image, A represents the Hamilton Depression Rating Scale (HAMD) and Bech-Rafaelsen Mania Rating Scale (BRMS) scores of BD patients; B represents the exosome extraction and separation process; C and D represent the comparable size distribution and concentration of molecular exosomes tracked by nanoparticles; C represents exosomes from manic BD patients; D represents exosomes from depressive BD patients; E represents exosomes from healthy controls; F represents transmission electron microscopy images of exosomes; G represents Western blotting verification of exosomes; H represents the internalization fluorescence image of neurons; and I represents the internalization fluorescence image of microglia.

[0058] Figure 2 The figure shows the effects of exosomes derived from BD patients on mouse behavior. In the figure, A represents the experimental procedure, B represents the total distance traveled in the open field test, C represents the average speed of movement in the open field test, D represents the time at rest in the forced swimming test, E represents the time at rest in the tail suspension test, F represents the time spent in the open area in the elevated zero maze test, G represents the number of times the mouse entered the open area in the elevated zero maze test, H represents the latency period before the mouse began grooming in the splash test, I represents the percentage of spontaneous alternation in the Y maze test, J represents the three-compartment social competence test, K represents social competence, and L represents social novelty.

[0059] Figure 3 The figure shows the effects of exosomes derived from BD patients on mouse neurons. A represents Western blotting images of GFAP and MAP2, B represents the quantification of GFAP in A, C represents the quantification of MAP2 in A, D represents the fluorescence image of MAP2, and E represents the GFAP in mPFC in G. + Cell number, F is the relative fluorescence intensity of GFAP in G, G is the GFAP fluorescence image, H is the relative fluorescence intensity of MAP2 in D, I is the quantification of PSD95 in J, J is the WB image of synapses and neuronal markers, K is the quantification of TH in J, and L is the quantification of SYP in J.

[0060] Figure 4 The images show the effects of exosomes derived from BD patients on mouse microglia. A represents Iba1 PCR results, B represents iNOS Western blotting results, C represents TNFα Western blotting results, D represents CD86 and Arg-1 Western blotting images, E represents the quantification of CD86 in D, F represents the quantification of Arg-1 in D, and G represents Iba1... + Fluorescence image of CD86 in microglia, H represents Iba1 in mPFC. + Total microglia, I represents CD86 in G. + Iba1 + Cell percentage, J is Iba1 + Fluorescence image of Arg-1 in microglia, where K represents Arg-1 in J. + Iba1 + Cell ratio.

[0061] Figure 5 This is a schematic diagram of the system flow for constructing an animal model of emotional disorders provided by the present invention.

[0062] Figure 6 This is a schematic diagram of an apparatus for constructing an animal model of emotional disorders provided by the present invention. Detailed Implementation

[0063] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0064] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are conventional biochemical reagents and are commercially available.

[0065] Example 1: Extraction and Brain Implantation Evaluation of Serum Exosomes from BD Patients I. Experimental Methods 1. Clinical Patient Recruitment: We recruited 10 patients diagnosed with bipolar disorder (BD) from the Third People's Hospital of Foshan City. Diagnostic assessments were conducted by board-certified psychiatrists according to the criteria of the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5). The severity of clinical symptoms was further assessed using the Hamilton Depression Rating Scale (HAMD) and the Bech-Rafaelsen Mania Rating Scale (BRMS). Based on clinical presentation, patients were divided into manic episodes (n = 5) and depressive episodes (n = 5). Five age-matched healthy controls (HC) participants (age = 35 ± 16 years) were recruited as the control group. All participants underwent a structured psychiatric interview conducted by a trained psychiatrist to confirm the diagnosis of BD or exclude any history of mental illness in the HC group. Healthy controls were recruited through hospital advertising and community outreach. Written informed consent was obtained from all participants prior to recruitment.

[0066] 2. Serum Exosome Isolation: Fasting venous blood samples (approximately 10 mL) were collected from each participant. After coagulation at room temperature for 1 hour, the samples were centrifuged at 3000 × g for 10 minutes to separate the serum. The serum was first diluted 1:10 with phosphate-buffered saline (PBS) and then filtered through a 0.2 μm syringe filter to remove cell debris and larger particles. Exosomes were isolated using a qEV size exclusion column (Izon, Oxford, UK) according to the manufacturer's instructions. The exosome-containing fraction was then concentrated at 10000 × g for 60 minutes using a 30 kDa molecular weight cutoff polyethersulfone (PES) centrifuge filter (Vivaspin, Sartorius, Göttingen, Germany). The concentrated exosomes were resuspended in 40 μL of PBS for downstream characterization and functional analysis.

[0067] 3. Exosome Evaluation: Nanoparticle tracking analysis (NTA) was performed using the ZetaView system (Particle Metrix, Germany) to determine particle size distribution and concentration. For morphological evaluation, exosome suspensions were adsorbed onto formvar / carbon-coated copper grids, air-dried, and negatively stained with 2% phosphotungstic acid for 3 minutes. Samples were further dried at 65°C for 5 minutes prior to imaging by transmission electron microscopy (TEM). Exosome protein markers were assessed by Western blot analysis using primary antibodies against CD63 (Santa Cruz, sc-5275), Alix (Abcam, ab186429), and GM130 (Santa Cruz, sc-55590) to confirm exosome purity and exclude contamination from organelles. Purified exosome suspensions were stored in PBS at 20°C until further use.

[0068] 4. Animals: Three-week-old C57BL / 6 mice were used. Under specific pathogen-free (SPF) conditions, animals were housed in groups of five per cage using soft bedding, with controlled temperature (24 ± 2℃), humidity (50% ± 10%), and a 12-hour light / dark cycle. Food and sterile water were readily available.

[0069] 5. Exosome brain entry tracking: Serum exosomes were incubated in sterile PBS containing 5 μm DiI at 37°C for 30 minutes and then fluorescently labeled with the lipophilic dye DiI (Beyotime, China). After labeling, the exosomes were purified through three consecutive filtration steps to remove unbound dye. The purified DiI-labeled exosomes were injected into mice via tail vein. Twenty-four hours after injection, the animals were euthanized, and brain tissue was harvested for immunofluorescence staining to assess the distribution and localization of exosomes.

[0070] II. Experimental Results We first categorized BD patients into those currently experiencing manic episodes and those experiencing depressive episodes based on their Hamilton Depression Rating Scale (HAMD) and Bech-Rafaelsen Mania Rating Scale (BRMS) scores. Figure 1 A). With Figure 1 Procedure B was used for the isolation and identification of serum exosomes. Nanoparticle tracking analysis showed that particles within the expected size range (30-150 nm) had comparable peak diameters in manic, depressed, and healthy controls. Figure 1 CE). Transmission electron microscopy confirmed the characteristic cup-shaped morphology ( Figure 1 F), while Western blotting detected the absence of exosome marker (CD63) and Golgi protein GM130, supporting the purity of the sample. Figure 1 G). To assess their ability to reach the brain, exosomes were labeled with the fluorescent tracer DiI and intravenously injected into mice. Confocal imaging revealed a large number of exosomes aggregated in the mPFC. Labeled vesicles were detected in NeuN-positive neurons and Iba1-positive microglia within this region, confirming that exosomes crossed the blood-brain barrier and were internalized by the neuronal and glial cell populations. Figure 1 HI).

[0071] Example 2: Evaluation of the efficacy of serum exosomes derived from BD patients in constructing an animal model of affective disorder. I. Experimental Methods 1. Animals: Forty 3-week-old C57BL / 6 mice were used. The mice were randomly divided into four experimental groups (n=10 per group, sex balanced): a control group (saline) injected with physiological saline, exosomes from patients with depressive BD (BD-depression-Exo), exosomes from patients with manic BD (BD-mania-Exo), and exosomes from healthy donors (Healthy-Exo).

[0072] 2. Model Construction Method: Mice were anesthetized with isoflurane and fixed in a stereotaxic apparatus (RWD Life Sciences, China). After a midline scalp incision and skull exposure, bilateral guide cannulas (inner diameter 0.34 mm; outer diameter 0.48 mm) were implanted into the frontal cortex (mPFC) using coordinates relative to the anterior fontanelle based on mouse brain atlases: AP +2.0 mm, ML ±0.3 mm, DV -1.5 mm. The cannulas were secured in place with dental cement and stainless steel screws, and animals were allowed a minimum 7-day recovery period before exosome administration. During infusion, a syringe extending 0.5 mm beyond the catheter tip was connected to a microinfusion pump via a polyethylene tube. Exosome suspension was infused bilaterally at a rate of 0.05 μL / min, with a total volume of 0.5 μl (1 × 10⁻⁶) per hemisphere. 9 (Particles / hemispheres). After injection, leave the syringe in place for 10 minutes to promote diffusion and minimize backflow. Infuse every 3 days for a total of 5 times over 2 weeks.

[0073] 3. Behavioral Testing: Systematic behavioral assessments were conducted two days after the last exosome infusion. All behavioral tests were performed by trained researchers who were unaware of the treatment group's condition. To minimize olfactory cues, each instrument was thoroughly cleaned with 75% ethanol between tests. Open field test, elevated zero maze test, three-compartment social competence test, splash test, Y-maze test, forced swimming test, and tail suspension test were performed according to standard procedures in the field.

[0074] 4. Immunofluorescence: After behavioral assessment, mice were deeply anesthetized and perfused with saline via the heart. The brain was dissected and fixed in 4% paraformaldehyde (PFA) at 4°C, followed by cryoprotection in 20% and 30% sucrose solutions prepared in phosphate-buffered saline (PBS) until the tissue precipitated. Coronal sections (35 μm) were prepared using a cryostat and washed with PBS. To minimize nonspecific binding, sections were blocked in rapid blocking buffer (Beyotime, Cat. No. P0260), washed for 10 minutes at room temperature, and then washed three times with PBS. Sections were incubated overnight at 4°C with the corresponding primary antibody. After washing with PBS, sections were incubated with a suitable fluorophore-conjugated secondary antibody at room temperature for 2 hours. Cell nuclei were counterstained with 4',6-diamidinyl-2-phenylindole (DAPI). Fluorescent images were obtained using a Leica TCS SP8 confocal microscope (Leica Microsystems, Germany) for detailed analysis of labeled cells and subcellular structures.

[0075] 5. Antibody information: GFAP (Cell Signaling Technology, #12389), Iba1 (Wako, 019-19741), MAP2 (Millipore, MAB3418), CD86 (Abcam, ab119857), Arg-1 (Abcam, ab91279), PSD95 (Abcam, ab18258), TH (Cell Signaling) Technology, #2792), SYP (Abcam, ab8049), iNOS (BD Biosciences, 610431), TNFα (Abcam, ab6671), β-actin (Sigma-Aldrich, A5441).

[0076] 6. PCR: Total RNA was extracted from mouse brain tissue using TRIzol reagent (Invitrogen, USA). 1 μg of total RNA was reverse transcribed into cDNA using a one-step first-strand cDNA synthesis kit (Genstar Biotech, Beijing, China). Quantitative real-time PCR was performed using a 2×SYBR Green PCR Master Mix (Genstar Biotech) on a real-time PCR detection system. Primer sequences used for amplification are shown in Table 1. All reactions were performed three times, using 2^ The δδCT method was used to calculate relative gene expression, with β-actin as the housekeeping gene for normalization.

[0077] Table 1 Primer Sequences

[0078] II. Experimental Results Animal experimentation procedures are as follows Figure 2 As shown in Figure A. The results showed that mice treated with exosomes derived from manic BD patients exhibited hyperactivity in the open field test. Figure 2 BC), exhibited reduced immobility in forced swimming and tail suspension tests ( Figure 2 DE), showing an increase in exploring with outstretched arms in the elevated zero maze ( Figure 2 FG) exhibited a shorter combing latency in the splash test. Figure 2 H), showed an enhanced and preferred sense of social novelty in the three-room social skills test ( Figure 2 These findings are similar to those observed in patients with manic episodes, characterized by increased activity, reduced hopeless-like behaviors, and enhanced risk-taking and social abilities. In contrast, mice treated with exosomes derived from patients with depressive BD showed the opposite: reduced movement, prolonged immobility, decreased arm-opening exploration, delayed grooming, and diminished social abilities with loss of novelty preference. Figure 2 These phenotypes reflect the core characteristics of depressive episodes, including psychomotor retardation, behavioral hopelessness, loss of interest, and social withdrawal.

[0079] To examine the effects of BD exosomes on glial cell responsiveness and neuronal integrity, we evaluated astrocyte and neuronal markers in the mPFC. Western blot analysis showed that, compared with saline and healthy controls, the exosome-derived exosome treatment groups in manic and depressive BD patients had significantly increased GFAP protein levels, accompanied by significantly decreased MAP2 expression. Figure 3 AC). Immunofluorescence further confirmed the activation of astrocytes, with an increase in the number and fluorescence intensity of GFAP-positive cells. Simultaneously, decreased MAP2 expression accompanied by dendritic tissue disruption was observed in the exosome-treated group. Figure 3 DH).

[0080] We next evaluated synaptic and neuronal proteins. Exosomes derived from BD patients in both manic and depressive phases showed reduced PSD95 expression, indicating postsynaptic structural damage. Figure 3 IJ). Simultaneously, the expression of tyrosine hydroxylase (TH) was also reduced, with a more pronounced decrease after treatment with exosomes derived from manic BD patients. Figure 3 K). Furthermore, synaptophysin (SYP) levels remained unchanged across groups. Figure 3 L).

[0081] In summary, these results indicate that exosomes derived from BD patients induce astrocyte activation and neuronal structural damage in mPFC, characterized by dendritic instability and synaptic protein loss, with more severe effects observed during manic episodes.

[0082] To further characterize the microglial cell response, we assessed activation and polarization markers in each group of mPFCs. In mice receiving exosomes derived from mania or depression, Iba1 mRNA expression was significantly increased, while no changes were observed in healthy exosomes compared to the saline control group. Figure 4 A). At the protein level, iNOS and TNFα levels were elevated in the BD exosome group, and even higher in exosome mice derived from manic BD patients. Figure 4 BC).

[0083] Polarization markers revealed different patterns between the groups. CD86 protein was upregulated in the manic group, while Arg-1 expression was increased in the depressive group. Figure 4 DF). Immunofluorescence results showed that Iba1 + Microglial cell density increased in the manic and depressive exosome groups, but not in the healthy exosome group; CD86 was present during the manic phase. + Iba1 + Arg-1 in cells and during depression + Iba1 + The proportion of cells is larger ( Figure 4 (GK). In summary, exosomes derived from BD patients are associated with microglial activation in mPFCs, manic exosomes are associated with higher levels of M1-related markers, and depressive exosomes are associated with higher levels of M2-related markers.

[0084] The above results collectively demonstrate that exosomes derived from manic BD patients can induce manic-like behavior in mice, and exosomes derived from depressive BD patients can induce depressive-like behavior in mice. Furthermore, exosomes derived from BD patients at different stages can damage neural structures and induce microglia to polarize in different directions.

[0085] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A method of constructing an animal model of affective disorder, characterized by, The method comprises administering serum exosomes derived from BD patients in different stages to animals.

2. The method of claim 1, wherein, The method comprises administering serum exosomes derived from BD patients in the onset stage of mania to animals to obtain an animal model exhibiting mania-like behavior, or administering serum exosomes derived from BD patients in the onset stage of depression to animals to obtain an animal model exhibiting depression-like behavior. Preferably, the mania-like behavior comprises excessive movement, increased interest, high energy, and enhanced social ability. Preferably, the depression-like behavior comprises prolonged immobility, sluggish movement, decreased interest, mental debilitation, and withdrawn social ability.

3. The method of claim 1, wherein, The animal model refers to an animal having or showing characteristics of a disease or condition. Preferably, the animal is a mammal. Preferably, the mammal comprises a mouse, a rat, a rabbit, a dog, a pig, a monkey, a sheep, and a horse. Preferably, the mammal is selected from a mouse.

4. A method of screening for a drug candidate for the treatment of an affective disorder, characterized in that, The method comprises administering a drug to be screened to the animal model of affective disorder prepared by the method of any one of claims 1-3, analyzing the therapeutic effect of the drug to be screened, and evaluating the effect, and selecting a drug that can significantly improve the behavior of the animal model of affective disorder.

5. The method of claim 4, wherein, The evaluation indicators comprise animal behavioral tests, neuronal structure, and microglial cell state. Preferably, the animal behavioral tests comprise a tail suspension test, a forced swimming test, a sucrose preference test, a Y-maze test, a three-chamber social ability test, a water maze test, a splash test, an open field test, an elevated maze test, a rotarod test, and chronic restraint. Preferably, the animal behavioral tests are selected from a forced swimming test, a tail suspension test, an open field test, a Y-maze test, a splash test, and an elevated maze test.

6. Any one of the following applications: 1) Application of serum exosomes derived from BD patients in the onset stage of mania in constructing an animal model of mania; 2) Application of serum exosomes derived from BD patients in the onset stage of depression in constructing an animal model of depression; 3) Application of the animal model of affective disorder constructed by the method of any one of claims 1-3 in screening drug candidates for treating affective disorders.

7. Use according to claim 6, characterized in that, The animal model refers to an animal having or showing characteristics of a disease or condition. Preferably, the animal is a mammal. Preferably, the mammal comprises a mouse, a rat, a rabbit, a dog, a pig, a monkey, a sheep, and a horse. Preferably, the mammal is selected from a mouse.

8. A system for constructing an animal model of mood disorders, characterized by, The construction system comprises: a fixing unit for fixing the head of the animal on a stereotactic instrument; a first processing unit for shaving and disinfecting, incising the scalp along the midline, and stripping the subcutaneous tissue to expose the surface of the skull; a second processing unit for determining an intracranial administration cannula implantation coordinate according to a brain atlas, drilling a hole at the intracranial administration cannula implantation coordinate with a micro drill bit, and vertically inserting an intracranial administration cannula into a target region, wherein the intracranial administration cannula implantation coordinate is a prefrontal cortex region; a third processing unit for suturing the scalp and performing postoperative care and recovery; a fourth processing unit for injecting serum exosomes derived from BD patients in the onset stage of mania into the brain of the animal through the intracranial administration cannula after the animal recovers from the operation to obtain an animal model of mania, or injecting serum exosomes derived from BD patients in the onset stage of depression into the brain of the animal to obtain an animal model of depression.

9. The system of claim 8, wherein, The coordinates of the prefrontal cortex region are defined as follows: the bregma is defined as the origin, and the coordinates relative to the bregma are: AP + 2.0 mm, ML ± 0.3 mm, DV - 1.5 mm; Preferably, the injection cycle of the exosomes is once every 3 days, for a total of 5 times. Preferably, the number of injected particles of said exosome is 1 x 10 9 particles / hemispheres.

10. An apparatus for constructing an animal model of affective disorders, characterized by, The construction device comprises a single or multiple processors, and a memory for storing a single or multiple computer programs that implement the following steps when executed by the single or multiple processors: Operation 1, for fixing the head of the animal on a stereotactic instrument; Operation 2, for shaving and disinfecting, making an incision along the midline of the scalp, stripping the subcutaneous tissue, and exposing the surface of the skull; Operation 3, for determining the intracranial administration cannula implantation coordinates according to the brain atlas, drilling a hole at the intracranial administration cannula implantation coordinates with a micro drill bit, and vertically inserting the intracranial administration cannula into the target region, wherein the intracranial administration cannula implantation coordinates are the prefrontal cortex region; the coordinates of the prefrontal cortex region are defined as follows: the bregma is defined as the origin, and the coordinates relative to the bregma are: AP + 2.0 mm, ML ± 0.3 mm, DV - 1.5 mm; Operation 4, for suturing the scalp, and postoperative care and recovery; Operation 5, for injecting serum exosomes derived from BD patients in the manic phase into the brain of the animal through the intracranial administration cannula after the animal recovers from the operation, to obtain a manic animal model, or injecting serum exosomes derived from BD patients in the depressive phase into the brain of the animal through the intracranial administration cannula after the animal recovers from the operation, to obtain a depressive animal model; Preferably, the injection cycle of the exosomes is once every 3 days, for a total of 5 times. Preferably, the number of injected particles of said exosome is 1 x 10 9 particles / hemispheres.

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