Construction system and device of plateau encephaledema animal model

Abstract

The invention discloses a system and a device for constructing an animal model of plateau encephaledema, the constructed animal model successfully simulates cognitive function impairment and neuroinflammation response related to plateau encephaledema, can effectively reproduce key pathological characteristics of plateau encephaledema, such as oxidative stress, neuroinflammation and neurological function impairment, and can be applied to the field of animal models. The model can be used for researching pathogenesis, early diagnosis and potential treatment strategies of plateau encephaledema. The invention provides a reliable animal model for experimental research and drug screening of plateau encephaledema related diseases.

Description

Technical Field

[0001] The present invention belongs to the field of intelligent medical technology, and in particular, relates to a system and a device for constructing an animal model of high altitude cerebral edema. Background Art

[0002] High Altitude Cerebral Edema (HACE) is a brain disease that occurs when an individual rapidly ascends from low altitude to high altitude and has not adapted to the low oxygen environment. Its main symptoms include headache, vomiting, and abnormal gait. In severe cases, HACE may also lead to cognitive dysfunction, such as difficulty concentrating and memory loss. Given the acute onset of HACE and its potential serious long-term consequences, understanding its mechanisms is crucial for early intervention and improving patient outcomes.

[0003] The animal model of high-altitude cerebral edema is the basis of animal experimental research on HACE, and is of great significance for studying the pathophysiological changes and clinical treatment of HACE. The existing method for constructing an animal model of high-altitude cerebral edema is to place the modeling animal in a low-pressure and low-oxygen chamber for several days, during which time the animal is kept on a normal diet and can be driven away. However, experimental animals have different susceptibility to low pressure and / or hypoxia. For example, rats are not prone to high-altitude cerebral edema or the symptoms of high-altitude cerebral edema are not obvious under low pressure and / or low oxygen conditions; therefore, there is an urgent need for a new method for constructing an animal model of high-altitude cerebral edema to overcome the problem of different susceptibility of animals to low pressure and / or hypoxia. Summary of the invention

[0004] In order to make up for the deficiencies of the prior art, the object of the present invention is to provide a novel system and device for constructing an animal model of high altitude cerebral edema.

[0005] The first aspect of the present invention provides a system for constructing an animal model of high altitude cerebral edema, the system comprising:

[0006] A fixing unit, used to fix the animal's head on the stereotaxic apparatus;

[0007] The first processing unit is used for shaving and disinfection, cutting the scalp along the midline, peeling off the subcutaneous tissue, and exposing the skull surface;

[0008] The second processing unit is used to determine the implantation coordinates of the intracranial drug delivery cannula according to the brain atlas, drill holes at the implantation coordinates of the intracranial drug delivery cannula with a micro drill, and vertically insert the intracranial drug delivery cannula into the target area, wherein the implantation coordinates of the intracranial drug delivery cannula are the bilateral DG areas;

[0009] The third treatment unit is used for scalp suture and postoperative care and recovery;

[0010] The fourth processing unit is used to inject exosomes derived from patients with high-altitude cerebral edema into the animal's brain through an intracranial drug administration cannula after the animal recovers from surgery, so as to obtain an animal model of high-altitude cerebral edema.

[0011] The term "unit" in the present invention refers to a software or hardware component that plays a certain role, 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. The term "unit" can be configured in an addressable storage medium, or can be configured to reproduce one or more processors. Therefore, 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, microcodes, circuits, data, databases, data structures, tables, arrays, and variables. The functions provided in components and "units" can be combined into fewer components and "units", or can be further divided into additional components and "units". In addition, components and "units" can be implemented to operate one or more central processing units (CPUs) in a device or a secure multimedia card.

[0012] Furthermore, the coordinates of the bilateral DG regions are defined as the anterior Bregma point as the origin, and the coordinates relative to Bregma are: AP=-2.1mm, ML= ±1.7mm, DV= -2.1mm.

[0013] Furthermore, an intracranial drug administration cannula fixing unit is also included before the third processing unit, which is used to fix the base of the intracranial drug administration cannula to the skull surface.

[0014] Furthermore, the fixing method includes fixing with medical biological glue or dental cement.

[0015] Furthermore, the diameter of the micro drill is 0.5 mm to 1 mm.

[0016] Furthermore, the depth of the drilling is 0.1-0.2 mm.

[0017] Furthermore, the postoperative care and recovery includes applying antibiotic ointment to the sutured area and / or the area surrounding the sutured area of ​​the animal.

[0018] Furthermore, the postoperative care and recovery also includes administering analgesics to relieve pain after surgery.

[0019] Furthermore, the analgesic is ibuprofen.

[0020] Furthermore, the dosage of ibuprofen is 0.1 mg / kg.

[0021] Furthermore, the postoperative care and recovery also includes monitoring the mouse activity, body weight and / or wound healing.

[0022] Furthermore, the injecting of exosomes derived from patients with high altitude cerebral edema into the animal brain via the intracranial administration cannula comprises connecting the intracranial administration cannula and a microsyringe via a catheter 3 to 7 days after the animal recovers from surgery, and injecting the exosomes derived from patients with high altitude cerebral edema using the microsyringe.

[0023] Furthermore, the injection cycle of the exosomes derived from patients with high altitude cerebral edema is once every 4 days, for a total of 4 injections.

[0024] Furthermore, the number of particles of the exosomes from patients with high altitude cerebral edema per injection was 2.8×10 9 indivual.

[0025] Furthermore, the construction system also includes an anesthesia unit for anesthetizing the animal.

[0026] Furthermore, the anesthesia is inhalation anesthesia.

[0027] Furthermore, the anesthetic drug is isoflurane.

[0028] Furthermore, the concentration of isoflurane is 2% to 3%.

[0029] Furthermore, the animal model is a mouse model.

[0030] Furthermore, the mouse is a C57BL / 6 mouse.

[0031] The term "animal" in the present invention refers to any organism, including mammals, and the steps mentioned in the present invention can be adaptively adjusted according to different animals.

[0032] The second aspect of the present invention provides any of the following applications:

[0033] 1) Application of the animal model constructed by the construction system described in the first aspect of the present invention in screening drugs for preventing or treating high altitude cerebral edema; and / or in the study of the mechanism of high altitude cerebral edema;

[0034] 2) Application of exosomes from patients with HACE in constructing animal models of HACE.

[0035] The third aspect of the present invention provides a device for constructing an animal model of high-altitude cerebral edema, the device comprising: a single or multiple processors, and a memory, the memory being used to store a single or multiple computer programs, and when the single or multiple computer programs are executed by the single or multiple processors, the following are implemented:

[0036] Operation 1 is used to fix the animal's head on the stereotaxic apparatus;

[0037] Operation 2 is used to shave and disinfect, cut the scalp along the midline, peel off the subcutaneous tissue, and expose the skull surface;

[0038] Operation 3 is used to determine the implantation coordinates of the intracranial drug delivery cannula according to the brain atlas, drill a hole at the implantation coordinates of the intracranial drug delivery cannula with a micro drill, and vertically insert the intracranial drug delivery cannula into the target area. The implantation coordinates of the intracranial drug delivery cannula are the bilateral DG areas; the coordinates of the bilateral DG areas are defined as the anterior bregma Bergma point as the origin, and the coordinates are relative to Bregma: AP = -2.1mm, ML = ±1.7mm, DV = -2.1mm;

[0039] Operation 4 is used to suture the scalp and provide postoperative care and recovery;

[0040] Operation 5 is used to inject exosomes derived from patients with high-altitude cerebral edema into the animal's brain through an intracranial drug delivery cannula after the animal recovers from surgery, so as to obtain an animal model of high-altitude cerebral edema.

[0041] Furthermore, the injection cycle of the exosomes derived from patients with high altitude cerebral edema is once every 4 days, for a total of 4 injections.

[0042] Furthermore, the number of particles of the exosomes from patients with high altitude cerebral edema per injection was 2.8×10 9 indivual.

[0043] Furthermore, the construction device also includes: a hair removal unit, a disinfection unit, a cutting unit, a medication unit, and a suturing unit;

[0044] The hair removal parts include: a shaver and surgical scissors;

[0045] The cutting member comprises: a pair of scissors;

[0046] The drug delivery device comprises: a micro-injector;

[0047] The suture piece includes a suture thread.

[0048] The term "device" in the present invention is not limited to one or a specific number of physical objects. As used herein, a device can be any medical or electronic device with multiple components that can implement at least some parts of the present disclosure. Although the following description and examples use the term "device" to describe certain aspects of the present disclosure, the term "device" is not limited to a specific configuration, type, or number of objects.

[0049] The term "processor" or "memory" in the present invention includes a computing device with one processor or one memory and a device with multiple processors or multiple memories that can be used to perform some or all of the steps. A "processor" may include more than one processor, for example, a multi-core design or multiple processors each having a multi-core design.

[0050] Beneficial effects of this application:

[0051] The novel high-altitude cerebral edema animal model construction system disclosed in the present application successfully simulates the cognitive impairment and neuroinflammatory response associated with high-altitude cerebral edema, and can effectively reproduce the key pathological features of high-altitude cerebral edema, such as oxidative stress, neuroinflammation and neurological impairment. This model can be used to study the pathogenesis, early diagnosis methods and potential treatment strategies of high-altitude cerebral edema. The present invention provides a reliable animal model for experimental research and drug screening of high-altitude cerebral edema-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 A schematic diagram of the system flow for constructing an animal model of high altitude cerebral edema;

[0053] Figure 2 A schematic diagram of a device for constructing an animal model of high altitude cerebral edema;

[0054] Figure 3 The experimental results of cognitive dysfunction in mice induced by injection of exosomes from HACE patients are shown in Figure 1. (A): Schematic diagram of exosome injection; (B): Animal experiment flow chart; (C): Open field test results; (D): New object recognition test results; (E): Y-maze test results; (F): New environment feeding inhibition test results; (G): Fear conditioning results;

[0055] Figure 4 The experimental results of oxidative stress response in mice induced by injection of exosomes from HACE patients: (A): expression level of NO in serum of mice in each group; (B): expression level of SOD in serum of mice in each group; (C): expression level of GSH in serum of mice in each group; (D): expression level of MDA in serum of mice in each group; (E): expression level of CAT in serum of mice in each group; (F): expression level of T-AOC in serum of mice in each group; (G): expression level of NO in hippocampus and prefrontal cortex of mice in each group; (H): expression level of SOD in hippocampus and prefrontal cortex of mice in each group; (I): expression level of GSH in hippocampus and prefrontal cortex of mice in each group; (J): expression level of MDA in hippocampus and prefrontal cortex of mice in each group; (K): expression level of CAT in hippocampus and prefrontal cortex of mice in each group; (L): expression level of T-AOC in hippocampus and prefrontal cortex of mice in each group;

[0056] Figure 5 The experimental results of abnormal death of hippocampal neurons in mice caused by injection of exosomes from HACE patients; (A): Schematic diagram of Golgi staining in the DG region; (B): The number of dendritic spines in the DG region of mice in the HACE group was significantly reduced; (C): The number of MAP2-positive cells in the DG region of mice in the HACE group was significantly reduced; (D): Schematic diagram of MAP2 immunofluorescence in the DG region;

[0057] Figure 6 The figures show the experimental results of the proliferation and activation of microglia in the hippocampus and prefrontal cortex of mice induced by injection of exosomes from HACE patients; (A): Schematic diagram of IBA1 immunofluorescence in the DG region; (B): Increased number of microglia in the hippocampus and prefrontal cortex of mice in the HACE group; (CF): Schematic diagram of IBA1 immunofluorescence in the CA1, CA2, CA3 and mPFC regions. DETAILED DESCRIPTION

[0058] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiment of the present invention will be clearly and completely described below in combination with the embodiments of the present invention and the accompanying drawings. It should be clear that the embodiments described in the present invention are only a part of the embodiments that can be implemented in the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention.

[0059] The present invention describes the operation flow in the specification, the drawings and even the claims, which include multiple operation procedures. It should be clearly understood that these operation procedures may not be executed or executed in parallel in the order in which they appear in this article or the drawings. The numbers of the operation steps, such as 101, 102, 103, etc., are only used to distinguish different operations, and the numbers themselves do not represent any execution order. In addition, operations other than the numbers can also be added to the operation procedures of the present invention, and can be executed in sequence or in parallel. In addition, all operations appearing in this article can be omitted in part when necessary. It should be noted that the descriptions such as "first" and "second" mentioned in the present invention are only used to distinguish different operations, devices, modules, messages, etc., and do not limit the order of precedence or the specific type.

[0060] Figure 1 The following is a flow chart of a system for constructing an animal model of high altitude cerebral edema provided by an embodiment of the present invention. Specifically, the system includes:

[0061] A fixing unit 101, used for fixing the animal's head on a stereotaxic apparatus;

[0062] The first processing unit 102 is used for shaving and disinfection, cutting the scalp along the midline, peeling off the subcutaneous tissue, and exposing the skull surface;

[0063] The second processing unit 103 is used to determine the implantation coordinates of the intracranial drug administration cannula according to the brain atlas, drill holes at the implantation coordinates of the intracranial drug administration cannula with a micro drill, and vertically insert the intracranial drug administration cannula into the target area, wherein the implantation coordinates of the intracranial drug administration cannula are the bilateral DG areas;

[0064] The third processing unit 104 is used for suturing the scalp and performing postoperative care and recovery;

[0065] The fourth processing unit 105 is used to inject exosomes from patients with high altitude cerebral edema into the animal's brain through a catheter after the animal recovers from surgery, so as to obtain an animal model of high altitude cerebral edema.

[0066] Furthermore, the coordinates of the bilateral DG regions are defined as the anterior Bregma point as the origin, and the coordinates relative to Bregma are: AP=-2.1mm, ML= ±1.7mm, DV= -2.1mm.

[0067] In some embodiments, each processing unit, fixing unit, and disinfection unit in the system for constructing an animal model of high-altitude cerebral edema may have an independent module for positioning or judgment, and the independent module for positioning or judgment can independently or assist the processing unit, fixing unit, and disinfection unit to complete the procedures to be executed by the construction system.

[0068] In one embodiment, the construction system further comprises an anesthesia unit for anesthetizing the animal.

[0069] In one embodiment, the animal is a mouse. Male C57BL / 6 mice aged 11 weeks and weighing 22±1.5 g were obtained from Beijing Fangyuan Breeding Center, raised under environmental conditions of 23±1°C and 50±1% relative humidity, and placed in an environment of 12 hours of light and dark alternation (lights on from 8:00 am to 8:00 pm every day), and free access to food and water. All animal experiments followed the Guide for the Care and Use of Animals of the National Institutes of Health (NIH Publication No. 80-23) and were approved by the Animal Care and Use Committee of Minzu University of China.

[0070] In a specific embodiment, the present invention adopts the following construction method to construct an animal model of high-altitude cerebral edema: 11-week-old male C57BL / 6 mice (22±1.5g) are selected, and after 1 week of adaptive feeding, catheter implantation is prepared to reduce the pain caused by multiple injections. The specific operation of catheter implantation is: fasting for 12 hours before surgery to ensure safety during surgery, anesthesia is performed with isovolumetric isoflurane (2%~3%), and breathing and heart rate are monitored; then the mouse is fixed on a stereotaxic instrument (RWD 68025), the scalp is shaved, and the surgical area is disinfected with iodine and 75% ethanol; the scalp is incised along the midline (about 1 cm long), and the subcutaneous tissue is gently peeled off with a sterile cotton swab to expose the skull surface; refer to the brain atlas to determine the coordinates of the bilateral DG areas (AP -2.1 mm, ML ±1.7 mm, DV -2.1 The patients were followed up for 3-4 weeks. The patients were followed up for 4-6 weeks. The patients were followed up for 3-6 weeks. The patients were followed up for 4-6 weeks. The patients were followed up for 3-6 weeks. The patients were followed up for 4-6 weeks. The patients were followed up for 3-6 weeks. The patients were followed up for 3-6 weeks. The patients were followed up for 3-6 weeks. The patients were followed up for 4-6 weeks. The patients were followed up for 3-6 weeks. The patients were followed up for 3-6 weeks. The patients were followed up for 3-6 weeks. The patients were followed up for 3-6 weeks. The patients were followed up for 3-6 weeks. The patients were followed up for 3-6 weeks. The patients were followed up for 3-6 weeks. The patients were followed up for 3-6 weeks. The patients were followed up for 3-6 weeks. 8 The exosomes of the particles were injected 4 times to obtain an animal model of high altitude cerebral edema. In one embodiment, the exosomes from patients with high altitude cerebral edema can be injected through a catheter connected to a microinjection pump, preferably at a flow rate of 100 nL per minute.

[0071] In one embodiment, exosomes from HACE patients were obtained from the blood of 11 patients diagnosed with HACE by doctors from the General Hospital of the Tibet Military Region, and 11 healthy volunteers were recruited as a control group (Ctr). All patients signed informed consent. The study protocol was approved by the Ethics Committee of Minzu University of China and followed the relevant guidelines of the Declaration of Helsinki. Figure 3 A shows the schematic diagram of exosome extraction and injection. Figure 3 B outlines the timeline for model development.

[0072] In one embodiment, blood samples from HACE patients and healthy subjects were collected using BD Vacutainer™ serum tubes (Thermo Fisher Scientific, BD367895), which do not contain anticoagulants and allow the blood to coagulate naturally. After collection, the samples were placed at room temperature for 60 minutes to complete the coagulation process, and then centrifuged at 1300 × g for 10 minutes to separate the serum. The separated serum was transferred to a new EP tube and immediately stored at −80°C. Exosomes were extracted using the qEVoriginal70 nm Gen 2 system (IZON, ICO70-13030) according to the manufacturer's instructions and were purified using Vivaspin ® Exosomes were concentrated using 20 ultrafiltration units (Sartorius, 30,000 MWCO PES), and the size distribution of exosomes was determined by nanoparticle tracking analysis using the NanoSight system (NanoSight, London, UK).

[0073] In one embodiment, sera from 2 to 4 patients are combined to extract exosomes, and the extracted exosomes are evenly distributed to the corresponding mouse groups to ensure that the observed effects more accurately reflect the overall group characteristics rather than the variability of individual human samples.

[0074] In one embodiment, the construction system further includes a verification unit for verifying whether the animal model is successfully constructed.

[0075] In one embodiment, the model is successfully constructed by evaluating the cognitive function of the mice. After 4 injections, the cognitive function of the mice is evaluated by a series of behavioral tests.

[0076] In one embodiment, the cognitive function of mice is assessed by an open field test. The open field test (OFT) is a commonly used method to assess the motor activity and exploratory behavior of mice. The experimental apparatus is divided into 16 equal areas, and the mice are initially placed in the central position to adapt to the environment for 2 minutes. This process allows the mice to adapt to the new environment, thereby ensuring their natural response. After the adaptation period, the following three indicators are recorded: the total distance walked, the time spent in the central area, and the number of times the central area is entered, and the recording time is 3 minutes. In order to maintain the objectivity of the analysis, the experiment was performed by an experimenter who was unaware of the grouping of the mice. The results showed that the mice injected with exosomes from HACE patients had a significantly reduced exploration time in the central area in the open field test (OFT) ( Figure 3 C), showing impaired exploratory behavior. In contrast, mice injected with exosomes from healthy individuals or saline showed higher activity levels.

[0077] In one embodiment, the cognitive function of mice is assessed by a novel object recognition test (NOR). The novel object recognition test assesses cognitive function through the natural tendency of mice to explore unfamiliar objects. In the experiment, mice were allowed to explore two identical objects for 10 minutes. After a one-hour rest, one of the objects was replaced with a new object, and the mice entered the experimental area again and explored the new object for 10 minutes. The exploration time and interaction frequency of the mice with new objects and familiar objects were recorded to assess the recognition memory of the mice. The results showed that the time required for mice in the HACE group to recognize new objects was significantly prolonged, indicating that exosomes from HACE patients had a negative impact on learning, memory, and cognitive function ( Figure 3 D).

[0078] In one embodiment, the cognitive function of mice is assessed by a Y-maze test. The Y-maze test measures spatial learning and memory ability by tracking the exploratory behavior of animals in a maze. Mice are placed in the starting arm of the maze and allowed to explore freely. The time spent in each arm and the order of entry into the arm are recorded. As an indicator of working memory, the spontaneous alternation rate is calculated as: [(number of alternations) / (total number of arm entries-2)]×100. The results showed that the number of alternations in the HACE group of mice was less, further reflecting the defects in cognition and memory ( Figure 3 E).

[0079] In one embodiment, the cognitive function of mice was assessed by the novelty-suppressed feeding test (NSF). The novelty-suppressed feeding test assesses the motivation and anxiety levels of mice when exposed to a new environment after a 24-hour fast. In this test, food was placed on a piece of white filter paper and placed in the center of a 50×50×45 cm apparatus. Each mouse was placed individually in a corner of the experimental arena and allowed to explore. The latency to start eating was recorded, revealing the anxiety and motivation of the animals in a new environment. The results showed that the mice in the HACE group spent a longer time approaching the food, further indicating that their exploratory ability was significantly reduced ( Figure 3 F).

[0080] In one embodiment, cognitive function of mice was assessed by fear conditioning. The fear conditioning experiment consisted of two apparatuses: a refrigerated monitoring box (23 × 23 × 30 cm) located inside a larger soundproof box (30 × 30 × 37 cm). The refrigerated monitoring box was equipped with a metal grid for electric shock and recording the vertical and horizontal movements of the animals. Conditioning was performed on the first day, following a specific protocol: the animals remained still for 60 seconds before receiving 12 stimuli. Each stimulus consisted of a conditioned stimulus of 75 dB for 30 seconds, followed by a 30-second interstimulus interval, and finally a 2-second foot shock (30 mA) and 15 seconds of stillness. Short-term memory testing was performed 6 hours later, reducing the number of stimuli to 6. Each trial consisted of a conditioned stimulus and a 30-second follow-up interval, and finally no foot shock. Long-term memory testing was performed on the second day, the day after the short-term test, and the same method was used to assess the quality of long-term memory. The results showed that mice in the HACE group showed less freezing in response to auditory stimuli, while no abnormalities were observed in the other groups ( Figure 3 G).

[0081] In summary, multiple behavioral tests revealed significant cognitive impairment in the HACE group, while mice injected with healthy exosomes showed no such defects. In fact, some cognitive indicators of the healthy exosome group were even better than those of the saline group. This suggests that the exosomes of HACE patients induce mice to exhibit cognitive dysfunction similar to that of patients with HACE, and the HACE mouse model was successfully constructed.

[0082] In one embodiment, the brain tissue and blood of mice were further physiologically evaluated to verify whether the model was successfully constructed. Studies have shown that an imbalance in oxidative stress homeostasis is associated with the pathogenesis of HACE. This imbalance mainly stems from excessive production of reactive oxygen species (ROS) or insufficient antioxidant defense. These factors promote abnormal activation of microglia and damage endothelial tight junctions, ultimately affecting the integrity of the neurovascular unit, which leads to irreversible neuronal death and blood-brain barrier damage, thereby inducing brain edema. In order to evaluate whether HACE exosomes cause abnormalities in oxidative capacity and ROS levels, we measured the expression levels of NO, SOD, GSH-Px, MDA, CAT, and T-AOC in mouse serum, hippocampus (Hip), and medial prefrontal cortex (mPFC) in two brain regions.

[0083] NO is a redox-active molecule that plays a key role in regulating endothelial tension, improving blood flow and oxygen supply, and affecting oxidative stress and inflammatory responses. These functions significantly affect the occurrence and progression of HACE. We measured NO levels in mouse serum, hippocampus, and prefrontal cortex, and the results showed that NO levels in serum and hippocampus of mice injected with HACE exosomes were significantly increased ( Figure 4 A, 4G).

[0084] SOD is an antioxidant enzyme that catalyzes the conversion of superoxide into hydrogen peroxide, thereby affecting the level of oxidative stress. Studies have shown that SOD expression in the brain tissue of experimental mice exposed to high-altitude cerebral edema is significantly increased. We conducted a detailed test on the SOD level of mice injected with HACE exosomes and found that the SOD levels in the hippocampus, prefrontal cortex and serum of mice in the HACE group were abnormally increased ( Figure 4 B, 4H).

[0085] GSH-Px, an antioxidant that can neutralize excess ROS and prevent oxidative damage, was significantly decreased in the hippocampus of HACE group mice ( Figure 4 C, 4I).

[0086] MDA is a byproduct of lipid peroxidation and an indirect marker of oxidative stress. Studies have shown that MDA levels in the hippocampus and cortex of HACE rats are significantly increased. Similarly, we noticed that MDA levels in the blood, prefrontal cortex, and hippocampus of mice in the HACE group were significantly increased ( Figure 4 D, 4J).

[0087] CAT mainly decomposes hydrogen peroxide into water and oxygen, protecting cells from oxidative stress. A decrease in CAT levels can lead to enhanced oxidative stress. In the HACE group mice, we observed a slight increase in CAT expression ( Figure 4 E, 4K).

[0088] T-AOC reflects the overall antioxidant status of various substances and enzymes and is an important indicator of oxidative stress. T-AOC levels in the hippocampus of mice in the HACE group changed significantly. Overall, the HACE group showed active lipid peroxidation and redox imbalance in serum and brain tissue.

[0089] In one embodiment, to evaluate whether exosomes from HACE patients cause neuronal damage in mice, we performed Golgi staining on the DG region of the mouse hippocampus. The results showed that the density of dendritic spines in neurons of mice in the HACE group was significantly reduced ( Figure 5 AB). In addition, immunofluorescence staining of MAP2, which marks neurons in the DG region of the hippocampus, showed that the number of neurons in the HACE group mice was significantly reduced. These findings suggest that exosomes derived from HACE patients severely damaged the hippocampal neurons of mice.

[0090] In one embodiment, in order to further study the number and activation status of microglia in the hippocampus, we labeled microglia with IBA1 and CD68. The results showed that the HACE group mice had a significant decrease in DG ( Figure 6 A) and CA1 ( Figure 6 C), CA2 ( Figure 6D) and CA3 ( Figure 6 E) and mPFC ( Figure 6 F) The number of regional microglia increased, and the number of activated microglia increased significantly ( Figure 6 B). In addition, these microglia displayed abnormal features such as enlarged cell bodies and shortened processes.

[0091] In summary, the above results show that the mice injected with HACE patient-derived exosomes showed cognitive dysfunction and caused redox imbalance in the mouse brain tissue, indicating that injecting HACE patient-derived exosomes into the mouse brain can cause mice to develop symptoms related to high-altitude cerebral edema, indicating that the HACE mouse model was successfully constructed.

[0092] Figure 2 1 is a schematic diagram of a device for constructing an animal model of high altitude cerebral edema provided by an embodiment of the present invention. Specifically, the device comprises: a single or multiple processors, and a memory, wherein the memory is used to store a single or multiple computer programs, and when the single or multiple computer programs are executed by the single or multiple processors, the following are implemented:

[0093] Operation 1 is used to fix the animal's head on the stereotaxic apparatus;

[0094] Operation 2 is used to shave and disinfect, cut the scalp along the midline, peel off the subcutaneous tissue, and expose the skull surface;

[0095] Operation 3 is used to determine the implantation coordinates of the intracranial drug delivery cannula according to the brain atlas, drill a hole at the implantation coordinates of the intracranial drug delivery cannula with a micro drill, and vertically insert the intracranial drug delivery cannula into the target area. The implantation coordinates of the intracranial drug delivery cannula are the bilateral DG areas; the coordinates of the bilateral DG areas are defined as the anterior bregma Bergma point as the origin, and the coordinates are relative to Bregma: AP = -2.1mm, ML = ±1.7mm, DV = -2.1mm;

[0096] Operation 4 is used to suture the scalp and provide postoperative care and recovery;

[0097] Operation 5 is used to inject exosomes derived from patients with high-altitude cerebral edema into the animal's brain through an intracranial drug delivery cannula after the animal recovers from surgery, so as to obtain an animal model of high-altitude cerebral edema.

[0098] In one embodiment, the injection cycle of the exosomes derived from patients with high altitude cerebral edema is once every 4 days, for a total of 4 injections.

[0099] In one embodiment, the number of particles of the exosomes derived from patients with high altitude cerebral edema per injection is 2.8×10 9 indivual.

[0100] Those skilled in the art can clearly understand that, for the convenience and brevity of description, the specific working processes of the systems, devices and units described above can refer to the corresponding processes in the aforementioned method embodiments and will not be repeated here.

[0101] In the several embodiments provided in the present application, it should be understood that the disclosed systems, devices and methods can be implemented in other ways. For example, the device embodiments described above are only schematic. For example, the division of the units is only a logical function division. There may be other division methods in actual implementation, such as multiple units or components can be combined or integrated into another system, or some features can be ignored or not executed. Another point is that the mutual coupling or direct coupling or communication connection shown or discussed can be an indirect coupling or communication connection through some interfaces, devices or units, which can be electrical, mechanical or other forms.

[0102] The units described as separate components may or may not be physically separated, and the components shown as units may or may not be physical units, that is, they may be located in one place or distributed on multiple network units. Some or all of the units may be selected according to actual needs to achieve the purpose of the solution of this embodiment.

[0103] In addition, each functional unit in each embodiment of the present invention may be integrated into one processing unit, or each unit may exist physically separately, or two or more units may be integrated into one unit. The above-mentioned integrated unit may be implemented in the form of hardware or in the form of software functional units.

[0104] A person skilled in the art may understand that all or part of the steps in the various methods of the above embodiments may be completed by instructing related hardware through a program, and the program may be stored in a computer-readable storage medium, and the storage medium may include: a read-only memory (ROM), a random access memory (RAM), a disk or an optical disk, etc.

[0105] A person skilled in the art can understand that all or part of the steps in the above-mentioned embodiment method can be completed by instructing related hardware through a program, and the program can be stored in a computer-readable storage medium. The above-mentioned storage medium can be a read-only memory, a disk or an optical disk, etc.

[0106] The above is a detailed introduction to a computer device provided by the present invention. For a person skilled in the art, according to the concept of the embodiments of the present invention, there may be changes in the specific implementation method and application scope. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A system for constructing an animal model of high altitude cerebral edema, characterized in that: The build system includes: A fixing unit, used to fix the animal's head on the stereotaxic apparatus; The first processing unit is used for shaving and disinfection, cutting the scalp along the midline, peeling off the subcutaneous tissue, and exposing the skull surface; The second processing unit is used to determine the implantation coordinates of the intracranial drug delivery cannula according to the brain atlas, drill holes at the implantation coordinates of the intracranial drug delivery cannula with a micro drill, and vertically insert the intracranial drug delivery cannula into the target area, wherein the implantation coordinates of the intracranial drug delivery cannula are the bilateral DG areas; The third treatment unit is used for scalp suture and postoperative care and recovery; The fourth processing unit is used to inject exosomes derived from patients with high-altitude cerebral edema into the animal's brain through an intracranial drug administration cannula after the animal recovers from surgery, so as to obtain an animal model of high-altitude cerebral edema.

2. The construction system according to claim 1, characterized in that: The coordinates of the bilateral DG regions are defined as the anterior bregma point as the origin, and the coordinates relative to Bregma are: AP = -2.1mm, ML = ±1.7mm, DV = -2.1mm.

3. The construction system according to claim 1, characterized in that: An intracranial drug administration cannula fixing unit is also included before the third processing unit, which is used to fix the base of the intracranial drug administration cannula to the surface of the skull; Preferably, the fixing method comprises fixing with medical biological glue or dental cement; Preferably, the diameter of the micro drill is 0.5 mm to 1 mm; Preferably, the depth of the drilling is 0.1-0.2 mm.

4. The construction system according to claim 1, characterized in that: The postoperative care and recovery includes applying antibiotic ointment to the animal's sutured area and / or the area surrounding the sutured area; Preferably, the postoperative care and recovery also includes administering analgesics to relieve pain after surgery; Preferably, the analgesic is ibuprofen; Preferably, the dosage of ibuprofen is 0.1 mg / kg; Preferably, the postoperative care and recovery also includes monitoring the mouse activity, body weight and / or wound healing.

5. The construction system according to claim 1, characterized in that: The injecting of exosomes from patients with high altitude cerebral edema into the brain of an animal through an intracranial administration cannula comprises connecting the intracranial administration cannula and a micro-injector through a catheter after the animal has recovered for 3 to 7 days after surgery, and injecting the exosomes from patients with high altitude cerebral edema with the micro-injector; Preferably, the injection cycle of the exosomes derived from patients with high altitude cerebral edema is once every 4 days, for a total of 4 injections; Preferably, the number of particles of the exosomes derived from patients with high altitude cerebral edema per injection is 2.8×10 9 indivual.

6. The construction system according to any one of claims 1 to 5, characterized in that: Before the fixing unit, the construction system further includes an anesthesia unit for anesthetizing the animal; Preferably, the anesthesia is inhalation anesthesia; Preferably, the anesthetic drug is isoflurane; More preferably, the concentration of isoflurane is 2% to 3%.

7. The construction system according to any one of claims 1 to 5, characterized in that: The animal model is a mouse model; Preferably, the mouse is a C57BL / 6 mouse.

8. Any of the following applications: 1) Application of the animal model constructed by the construction system according to any one of claims 1 to 7 in screening drugs for preventing or treating high-altitude cerebral edema; and / or in the study of the mechanism of high-altitude cerebral edema; 2) Application of exosomes from patients with HACE in constructing animal models of HACE.

9. A device for constructing an animal model of high altitude cerebral edema, characterized in that: The construction device comprises: a single or multiple processors, and a memory, wherein the memory is used to store a single or multiple computer programs, and when the single or multiple computer programs are executed by the single or multiple processors, the following are implemented: Operation 1 is used to fix the animal's head on the stereotaxic apparatus; Operation 2 is used to shave and disinfect, cut the scalp along the midline, peel off the subcutaneous tissue, and expose the skull surface; Operation 3 is used to determine the implantation coordinates of the intracranial drug delivery cannula according to the brain atlas, drill a hole at the implantation coordinates of the intracranial drug delivery cannula with a micro drill, and vertically insert the intracranial drug delivery cannula into the target area. The implantation coordinates of the intracranial drug delivery cannula are the bilateral DG areas; the coordinates of the bilateral DG areas are defined as the anterior bregma Bergma point as the origin, and the coordinates are relative to Bregma: AP = -2.1mm, ML = ±1.7mm, DV = -2.1mm; Operation 4 is used to suture the scalp and provide postoperative care and recovery; Operation 5 is used to inject exosomes from patients with high altitude cerebral edema into the animal's brain through an intracranial administration cannula after the animal recovers from surgery, so as to obtain an animal model of high altitude cerebral edema; Preferably, the injection cycle of the exosomes derived from patients with high altitude cerebral edema is once every 4 days, for a total of 4 injections; Preferably, the number of particles of the exosomes derived from patients with high altitude cerebral edema per injection is 2.8×10 9 indivual.

10. The construction device according to claim 9, characterized in that The construction device also includes: a hair removal unit, a disinfection unit, a cutting unit, a drug delivery unit, and a suturing unit; The hair removal parts include: a shaver and surgical scissors; The cutting member comprises: a pair of scissors; The drug delivery device comprises: a micro-injector; The suture piece includes: a suture thread.

Images