Method for constructing zebrafish epilepsy model through intramuscular injection of PTZ

Abstract

The invention relates to a construction method of a zebrafish epilepsy model for intramuscular injection of PTZ, and belongs to the technical field of medical molecular biology. The method comprises the following steps: preparing a microsyringe with a set volume; preparing a PTZ solution, and injecting the PTZ solution with set solubility into the microsyringe prepared in the step S1; treating the zebra fish to reduce the activity of the zebra fish; and injecting the PTZ solution into the back muscle of the zebra fish through a microsyringe. The PTZ solution can quickly enter blood circulation after being injected into back muscle, so that the drug effect is quickly shown, and an epileptic seizure model is established in a short time. According to the method for rapidly inducing epilepsy, the model has high reproducibility and is suitable for large-scale experiments and preclinical research.

Description

Technical Field

[0001] The invention relates to the technical field of medical molecular biology, and in particular to a method for constructing a zebrafish epilepsy model by intramuscular injection of PTZ. Background Art

[0002] Current research methods for zebrafish epileptic seizures all have drawbacks that are difficult to overcome, making it difficult to achieve controllable epileptic seizures. It is difficult to accurately control the dosage of drugs that trigger epileptic seizures, making it impossible to effectively control the severity and frequency of epileptic seizures. Injections can easily cause injuries, and unstable operation methods not only affect the physiological state of the experimental subjects, but also make epileptic seizures difficult to predict and control due to individual injury differences, further increasing the difficulty of achieving controllable epileptic seizures. Summary of the invention

[0003] Based on this, it is necessary to provide a method for constructing a zebrafish epilepsy model by intramuscular injection of PTZ to address the problem of poor controllability in epilepsy model construction.

[0004] A method for constructing a zebrafish epilepsy model by intramuscular injection of PTZ, the method comprising the following steps:

[0005] S1. Prepare a microsyringe with a set volume;

[0006] S2, preparing a PTZ solution, and injecting the PTZ solution of a set solubility into the micro-syringe prepared in step S1;

[0007] S3, treating zebrafish to reduce the activity of zebrafish;

[0008] S4. Inject PTZ solution into the dorsal muscle of zebrafish using a microsyringe.

[0009] The present application discloses a method for constructing a zebrafish epilepsy model by intramuscular injection of PTZ. The use of a micro-syringe ensures the precise dosage of the PTZ solution, avoids the effects of excessive or insufficient injection volume on zebrafish, and ensures the consistency and repeatability of the model. By setting the volume of the micro-syringe, researchers can accurately adjust the injection volume of PTZ, thereby inducing consistent epileptic seizure reactions in zebrafish and ensuring the reliability of the experimental results. Compared with the traditional injection method, the micro-syringe can reduce the dosage fluctuation caused by improper operation, thereby improving the accuracy and controllability of the experiment. PTZ (γ-aminobutyric acid transaminase inhibitor) is a drug used to induce epilepsy in animal models, and the accurate configuration of the solution is the key to ensuring the biological activity of the PTZ solution. By accurately configuring the concentration of the PTZ solution, the effectiveness of PTZ is ensured, thereby enhancing its ability to induce epilepsy. By adjusting the solubility, the frequency, intensity and duration of epileptic seizures can be effectively optimized, providing consistent results for subsequent experiments. By reducing the activity of zebrafish, the stress response before the injection of PTZ is reduced, making it more relaxed and better able to accept the injection. This operation helps to avoid experimental interference caused by excessive activity or stress and improves the consistency of the model. Reducing the activity of zebrafish helps its muscles to be in a more stable state, thereby optimizing the absorption efficiency of PTZ solution and ensuring that the biological effect of the drug can be maximized. Accurately injecting PTZ solution into the back muscles of zebrafish with a microsyringe can ensure the target area of ​​drug action. This injection method can avoid uneven distribution of drugs in the body and ensure that PTZ can reach sufficient concentration in the back area of ​​zebrafish to induce epileptic seizures. Intramuscular injection of PTZ solution can simulate the mechanism of naturally occurring epileptic seizures. PTZ quickly induces epileptic reactions by acting on specific areas of the nervous system (such as cerebral cortex, brainstem, etc.), which is convenient for studying the pathophysiological process of epilepsy. Through this step, zebrafish can quickly enter an epileptic state, and researchers can observe the symptoms and manifestations of epileptic seizures (such as muscle tremors, loss of balance, etc.) in real time, providing a powerful model for the study of the mechanism of epilepsy. After injection into the back muscles, PTZ solution can quickly enter the blood circulation, so that the drug effect can be quickly manifested, thereby establishing an epileptic seizure model in a short time. This rapid method of inducing epilepsy makes the model highly reproducible and suitable for large-scale experiments and preclinical studies.

[0010] In one embodiment, the specific steps of injecting the PTZ solution into the back muscle of zebrafish by microinjection are as follows:

[0011] S41, scanning the zebrafish, identifying the back region of the zebrafish, and obtaining back region information;

[0012] S42, determining the injection point, injection depth and injection volume according to the age, weight and dorsal area information of the zebrafish;

[0013] S43. Inject PTZ solution through a microsyringe, and control the injection speed through feedback information from zebrafish during the injection process.

[0014] Precise positioning of the dorsal region is key to ensuring the effectiveness of injected drugs. By scanning the zebrafish and identifying the dorsal region, it is possible to ensure that the PTZ solution is injected into the correct location, avoiding uneven or incomplete drug effects due to improper injection. Dorsal muscles usually have a higher blood supply and can absorb drugs more effectively. By scanning and identifying the dorsal region, it is possible to ensure that the drug is injected into this efficient absorption area, thereby improving the bioavailability of the PTZ solution and ensuring that the drug effect is maximized. Customizing the injection protocol according to the age and weight of the zebrafish can ensure that the injection depth and injection volume of each zebrafish are adapted to its physiological characteristics. For example, young zebrafish and zebrafish with lower body weight usually require smaller injection volumes and shallow injection depths, while adult zebrafish can withstand higher drug doses and deeper injection depths. In this way, drugs can be injected at the optimal dose based on physiological differences, reducing the risk of overdose or underdose. Adjusting the injection volume and injection depth according to the physiological state of the zebrafish (such as age and weight) can avoid damage to the zebrafish caused by excessive injection volume or improper injection depth or adverse reactions caused by drug overdose. More importantly, this helps ensure uniform distribution and rapid absorption of the drug in the body, avoiding adverse consequences caused by drug deposition in local tissues. Determining the injection point and depth not only makes the injection operation more precise, but also ensures that the drug acts on the correct physiological area, avoiding incomplete drug absorption or local toxic reactions caused by the wrong injection location. In this way, the injection operation becomes more controllable and precise, which helps to improve the accuracy and consistency of experimental results. Feedback information controls the injection speed so that the injection process can be adjusted in real time according to the physiological response of zebrafish. For example, zebrafish may show different stress responses or physiological changes (such as accelerated heart rate, muscle contraction, etc.) when receiving PTZ solution injection. These feedback signals can instantly reflect the physiological state of zebrafish, thereby adjusting the injection speed. This method can prevent the injection from being too fast or too slow, ensure a smooth and uniform drug injection process, and reduce the potential harm of drugs to zebrafish. The injection speed of PTZ solution is crucial to the construction of zebrafish epilepsy model. By adjusting the injection speed through feedback information, the injection speed of the drug concentration can be accurately adjusted according to the feedback signals of the zebrafish (such as physiological indicators, movement status, etc.), so that the drug can act on the zebrafish nervous system to the greatest extent, thereby inducing the expected epileptic seizure response. Dynamic adjustments during drug injection can help researchers simulate the process of epileptic seizures more accurately and improve the biological relevance of experimental results. By monitoring the real-time feedback information of zebrafish and adjusting the injection speed, the experimental conditions can be optimized in real time to ensure the stability of the injection process. This not only helps to control the effect of PTZ-induced epilepsy, but also effectively avoids uneven distribution of drugs in the body or excessive or weak drug reactions, ensuring that stable and consistent results are obtained in each experiment.

[0015] In one embodiment, the specific steps of scanning the zebrafish, identifying the back region of the zebrafish, and obtaining the back region information are as follows:

[0016] S411, scanning the zebrafish, and dividing the zebrafish into a head region, a dorsal region, a lateral region, an abdominal region, and a tail region;

[0017] S412, identifying the morphology of the dorsal region of the zebrafish;

[0018] S413, performing grid processing on the back area to obtain grid information of the back area;

[0019] S414, scanning the thickness of the back region, and matching the thickness information with the grid information of the back region.

[0020] By scanning zebrafish and dividing them into different regions (such as the head, back, side, abdomen, and tail), researchers were able to analyze each physiological region of the zebrafish independently. This regional division helps to analyze and understand the physiological structure of zebrafish more accurately, ensuring that experimental operations (such as injection, measurement, etc.) can focus on specific areas and avoid unnecessary physiological interference. The dorsal region is usually one of the main sites for drug injection in zebrafish. By accurately identifying the dorsal morphology, it can ensure that the injection point selection is more scientific and reasonable. Especially when performing drug injection or other interventions, accurate identification of the dorsal morphology helps to determine the appropriate injection depth and injection location to avoid unnecessary effects on other regions (such as the abdomen, tail, etc.). Gridding makes the morphology of the dorsal region of zebrafish more detailed and visualized, and each small area of ​​the back can be analyzed independently. Gridding not only provides geometric information of the dorsal region, but also allows accurate measurements from local areas, such as the thickness of the dorsal muscles, the length of the dorsal fin, the curvature of the spine, etc., thereby providing more accurate spatial information for subsequent operations. Gridding information can provide spatial distribution data of the dorsal region, which is of great value for subsequent drug injection and treatment. By analyzing the gridding information, we can understand the differences in drug absorption, distribution, and metabolism in different areas of the back region, thereby providing data support for the selection of drug injection volume and injection location. Scanning the thickness of the back region can provide accurate depth data, so that the injection depth can be adjusted according to the actual thickness of the back. By matching the thickness information of the back region with the gridding information, it can be ensured that the drug is injected to the appropriate depth to avoid shallow injections that result in insignificant drug effects, or too deep injections that result in local damage. The matching of the thickness of the back region with the gridding information can not only provide detailed spatial guidance for injections, but also perform multi-dimensional analysis by fusing different biological information. This helps researchers gain a deeper understanding of the role of the back region in drug absorption, neural responses, tissue damage, etc., and provides strong data support for subsequent experimental design.

[0021] In one embodiment, the specific steps of determining the injection point, injection depth and injection volume according to the age, weight and dorsal area information of the zebrafish are as follows:

[0022] S421, matching the age and weight of the zebrafish with the grid information of the dorsal region of the zebrafish;

[0023] S422, determining the injection point, injection depth and injection volume of the zebrafish according to the age, weight and grid information of the back region of the zebrafish;

[0024] S423. Adjust the injection depth and injection volume based on the feedback information from the zebrafish.

[0025] By matching the age and weight of zebrafish with the gridded information of their dorsal region, the researchers were able to create a biometric database that accurately reflects the physiological structure and developmental status of zebrafish. The gridded information of the dorsal region of each zebrafish provides more detailed geometric data, which helps to accurately measure and analyze the thickness, morphology and relationship between the dorsal region and other physiological characteristics (such as age and weight). The age and weight of zebrafish are matched with the gridded information of the dorsal region to form a data-based decision-making framework. This approach can support more scientific and accurate experimental decisions through quantitative biological information, avoid relying on intuition or imprecise speculation, and make the entire experimental process more controllable and efficient. By comprehensively considering the age, weight and gridded information of the dorsal region of zebrafish, the injection point and injection depth can be determined more accurately. The thickness of the dorsal region of zebrafish of different ages is different, and there are also differences in the muscle layer and blood supply of the back, so the accuracy of the injection depth and injection point is crucial. Combined with the gridded information, incomplete drug injection or local damage caused by improper injection position or too shallow / deep depth can be avoided. The thickness and muscle state of the dorsal region directly affect the absorption efficiency of the drug. By combining the gridded data of the dorsal region, researchers can better understand the structural characteristics of the dorsal region of zebrafish, determine the best location for drug injection, and ensure that the drug can be quickly absorbed and exert the maximum biological effect. This helps to improve the bioavailability of the drug and ensure that the establishment of the epilepsy model is more stable and reliable. By obtaining feedback information from zebrafish in real time (such as physiological reactions, behavioral changes, etc.), the injection depth and injection volume can be adjusted dynamically. For example, if the zebrafish shows a strong stress response or discomfort, the researchers can reduce the injection depth or slow down the injection speed in real time to reduce damage to the zebrafish. If the zebrafish does not respond adequately to a certain dose, the injection volume can be appropriately increased. This flexible adjustment makes the entire injection process more in line with the physiological needs of each zebrafish and improves the accuracy and safety of the injection.

[0026] In one embodiment, the specific steps of matching the age and weight of the zebrafish with the grid information of the back region of the zebrafish are as follows:

[0027] S4211. Combine data from different sensors to obtain comprehensive information about the dorsal region of zebrafish;

[0028] S4212, data alignment: spatially align data of different modalities to ensure that data of different modalities at the same physical location have consistent coordinates;

[0029] S4213, Denoising and Standardization: Denoise the data of different modalities and ensure that the data of each modality has a uniform scale through standardization;

[0030] S4214, extracting features from the back region of zebrafish;

[0031] S4215. Dynamic matching between zebrafish age, weight and dorsal region characteristics is achieved through a deep reinforcement learning model.

[0032] The data provided by different sensors can complement each other and generate more accurate and rich information about the back region. For example, visual sensors perform very well on the surface texture and muscle distribution of zebrafish skin, while ultrasonic or infrared sensors can provide more in-depth information. Through this comprehensive information acquisition, the physiological condition and structural characteristics of zebrafish can be better analyzed, providing an accurate basis for the formulation of injection plans. Data alignment ensures that data from different sensors can be compared and fused in the same spatial framework. This step is the basis of data analysis and can ensure that the data collected by all sensors have a consistent coordinate system in physical space. Through this alignment, the spatial differences caused by different data sources can be eliminated and the comparability between different data modalities can be improved. Through denoising and standardization, data from different modalities can be fused at the same scale, avoiding the difference in fusion effect caused by data of different scales. This helps to improve the quality of data fused from different sensors, thereby obtaining more accurate features of the zebrafish back region and promoting the progress of subsequent analysis. Feature extraction can transform complex data into interpretable and quantifiable biological features through algorithms, making it easier for researchers to understand the changing laws of the zebrafish back region. This interpretability makes the experimental results more operational and valuable, and provides a basis for further optimizing the drug injection scheme. Through continuous feedback learning, the deep reinforcement learning model makes the matching of the physiological characteristics of zebrafish with the characteristics of the dorsal region have strong adaptive capabilities. This means that even if the physiological state of the zebrafish changes during the experiment, the model can respond quickly and adjust the matching strategy to ensure accuracy during the injection process.

[0033] In one embodiment, the specific steps of combining data from different sensors to obtain comprehensive information about the back region of the zebrafish are as follows:

[0034] S42111, Infrared imaging: Through infrared imaging technology, images of heat distribution in zebrafish are obtained to reveal the physiological state of the fish and the blood circulation in the dorsal area;

[0035] S42112. Ultrasound imaging: Obtain three-dimensional structural data of the back area through ultrasound, and detect the thickness and density of the muscle layer through sound wave reflection.

[0036] S42113. Optical imaging: Obtain detailed information of the fish body through optical imaging, which is used to obtain the outer contour of the back.

[0037] Infrared imaging technology can capture the thermal distribution in zebrafish in real time. These thermal distributions reflect the physiological activities in zebrafish, including metabolism, blood circulation, organ function, etc. The temperature changes in the dorsal area can reveal the local blood flow and muscle activity, which is crucial for understanding the health status and response of zebrafish. Ultrasound imaging technology can penetrate into the dorsal area of ​​zebrafish and obtain three-dimensional structural data. Through the principle of sound wave reflection, the thickness and density of the muscle layer, fat layer and other tissues in the dorsal area can be detected. This structural information is crucial for determining the injection depth, dosage and injection point selection. Optical imaging can provide high-resolution images, which helps to clearly capture the appearance details of zebrafish. By obtaining the outline of the back, the location of the injection point can be accurately calibrated to avoid errors. This is especially important for drug injection in zebrafish, because the accurate positioning of the dorsal area directly affects the effectiveness and accuracy of the injection.

[0038] In one embodiment, the specific steps of determining the injection point, injection depth and injection volume of the zebrafish by using the grid information of the age, weight and back region of the zebrafish are as follows:

[0039] S4221, inputting the weight, age, and geometric features of the back region of the zebrafish into a regression model, and training the model to obtain a mapping relationship;

[0040] S4222. Simulate the needle penetration process in the back of zebrafish, optimize the predicted injection point based on the information of different ages, weights and back thickness, and ensure that the needle can accurately penetrate in the predetermined area without damaging other organs;

[0041] S4223, constructing a regression model between thickness and injection depth, and establishing a depth prediction model through regression analysis;

[0042] S4224. The following nonlinear regression model is used to calculate the injection volume. The expression of the injection volume is as follows:

[0043]

[0044] Among them, V inj is the injection volume, W is the weight of zebrafish, D inj is the injection depth, and α, β, γ are constants obtained by fitting the experimental data.

[0045] By inputting the weight, age and geometric characteristics of the dorsal region of zebrafish into the regression model, the mathematical relationship between the physiological characteristics of zebrafish and injection-related parameters (such as injection point, injection depth, and injection volume) can be constructed. The model can accurately predict the optimal injection strategy for different individuals, thereby greatly improving the accuracy of experimental design. By simulating the process of needle penetration, the model can predict the movement trajectory of the needle under different conditions, ensuring that the needle can enter the dorsal region of the zebrafish according to the expected path during the injection process without damaging other important organs. This precise control greatly reduces the risk of accidental injury and improves the survival rate of zebrafish and the success rate of the experiment. By establishing a prediction model between thickness and injection depth through regression analysis, the optimal injection depth under different dorsal thickness conditions can be accurately predicted. This depth prediction can help researchers adjust the injection strategy according to the specific body shape of zebrafish, thereby ensuring that the drug is accurately injected into the target tissue. By calculating the injection volume through a nonlinear regression model, the drug dose required for each zebrafish can be accurately predicted based on the weight, injection depth and model fitting constants (α, β, γ) of the zebrafish. The weights of factors such as weight and depth in the injection volume prediction are comprehensively considered, avoiding the overdose or underdose that may be caused by simple quantitative predictions.

[0046] In one embodiment, the specific steps of adjusting the injection depth and injection volume according to the feedback information of the zebrafish are as follows:

[0047] S4231, collecting the electrical activity of zebrafish back muscles through an electromyography sensor to monitor the degree and strength of muscle contraction;

[0048] S4232, obtaining heart rate and respiratory rate in real time through electrocardiogram and respiratory sensors;

[0049] S4233, filtering the collected feedback signals to remove noise signals and extract effective features;

[0050] S4234. Establish a feedback-based depth adjustment model and fit it through a nonlinear regression model:

[0051]

[0052] Where M(t) is the muscle reaction intensity at time t, D inj is the injection depth, F feedback The feedback information is extracted;

[0053] S4235. According to the real-time feedback information, an incremental adjustment method is used to optimize the injection depth. After each feedback information is input, an adjustment amount is obtained, and the injection depth is adjusted according to the adjustment amount.

[0054] By monitoring the electrical activity of the muscles, the immediate response of zebrafish to injection stimulation can be captured, especially in the early stages of drug injection. Such real-time data helps researchers quickly evaluate the effects of drugs and make timely adjustments to avoid adverse reactions caused by overdose. Fluctuations in heart rate and respiratory rate are sensitive indicators of drug side effects, especially after the injection of neurological drugs (such as PTZ), which may cause abnormalities in the cardiovascular and respiratory systems. Real-time monitoring of these indicators can help researchers identify potential adverse reactions in advance and take timely adjustment measures to ensure the safety of the experiment. After filtering, key features related to the physiological response of zebrafish (such as muscle contraction intensity, heart rate fluctuations, etc.) can be extracted. These features are the basis for subsequent modeling and dynamic adjustment of injection parameters. The data after feature extraction has higher analytical value and can provide more accurate input for nonlinear regression models. The feedback-based depth adjustment model can dynamically adjust the injection depth according to real-time physiological feedback (electromyography, electrocardiography and other signals). This nonlinear regression model can accurately fit the complex relationship between physiological signals and injection depth, so as to intelligently adjust the injection strategy according to feedback information and improve the accuracy of injection. The incremental adjustment method can gradually transform feedback information into actual operational adjustments, making the injection depth adjustment process smoother and more flexible. Compared with large adjustments, incremental adjustments can avoid the instability caused by excessive or insufficient adjustments and improve the controllability of the experiment.

[0055] In one of the embodiments, the specific steps for preparing a microsyringe of a set volume are as follows: a glass microelectrode tip is pulled by a needle puller, and the tip pulled by the needle puller is a glass microelectrode tip with a diameter of 0.2μm-0.4μm; the glass microelectrode tip is connected to the syringe with a hot melt glue gun. The diameter of the glass microelectrode tip pulled by the needle puller ranges from 0.2μm to 0.4μm, which meets the requirements of microscopic operations. This precise microelectrode tip can ensure better penetration and less damage during the injection process, which is especially important for neuroelectrophysiological measurements or drug injections that require delicate operations in experiments. Using a hot melt glue gun to connect the glass microelectrode tip to the syringe can quickly complete the assembly and provide a firm fixation effect. Hot melt glue has strong adhesion and can fix the microelectrode tip in a short time to prevent the electrode from falling off or shifting due to vibration or operational errors during the injection process.

[0056] In one embodiment, the specific steps of treating the zebrafish to reduce the activity of the zebrafish are: selecting 3-month-old zebrafish with an average weight of 300 mg, and placing the zebrafish prone in the cracks of a moist cotton pad after a 0°C ice water bath. The activity of the zebrafish can be effectively reduced by treating 3-month-old zebrafish weighing about 300 mg in a 0°C ice water bath. The low-temperature ice water bath will inhibit the metabolism and movement ability of the zebrafish, causing it to enter a temporary paralysis state. In this process, the movement ability of the zebrafish is greatly reduced, thereby reducing its reactivity and behavioral changes, which is convenient for subsequent drug injection and operation.

[0057] In one embodiment, the specific steps of preparing the PTZ solution are: weighing a predetermined amount of PTZ powder with a micro-spoon, dissolving the PTZ powder in physiological saline, and the concentration of the PTZ solution is 350 mg / kg PTZ solution. Accurately weighing a predetermined amount of PTZ powder with a micro-spoon and dissolving it in physiological saline can ensure the accurate concentration of the PTZ solution. Setting the concentration of PTZ solution to 350 mg / kg ensures that each zebrafish receives a consistent amount of drug, thereby facilitating the standardization and repeatability of the experimental results.

[0058] In one embodiment, the maximum injection volume of the PTZ solution is less than 10 microliters. An injection volume less than 10 microliters can reduce the physical damage of drug injection to zebrafish tissues. In microinjection, a small volume of injection can accurately deliver drugs without causing excessive mechanical pressure or trauma to the back muscles or other tissues of the zebrafish. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 Flow chart of the method for constructing a zebrafish epilepsy model by intramuscular injection of PTZ;

[0060] Figure 2 Flow chart of the specific steps for injecting PTZ solution into the dorsal muscle of zebrafish via a microsyringe;

[0061] Figure 3 A flowchart of the specific steps for scanning zebrafish, identifying the dorsal region of zebrafish, and obtaining dorsal region information;

[0062] Figure 4 Flow chart of the specific steps for determining the injection point, injection depth, and injection volume based on the age, weight, and dorsal region information of zebrafish;

[0063] Figure 5 Flowchart of the specific steps for matching the age and weight of zebrafish with the gridded information of the dorsal region of zebrafish;

[0064] Figure 6Flowchart of the specific steps to combine data from different sensors to obtain comprehensive information about the zebrafish dorsal region;

[0065] Figure 7 Flow chart of the specific steps for determining the injection point, injection depth and injection volume of zebrafish based on the grid information of zebrafish age, weight and dorsal region;

[0066] Figure 8 Flow chart of the detailed steps for adjusting injection depth and injection volume using feedback information from zebrafish. DETAILED DESCRIPTION

[0067] In order to more clearly understand the above-mentioned purpose, features and advantages of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be noted that the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

[0068] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Therefore, the protection scope of the present invention is not limited to the specific embodiments disclosed below.

[0069] The following describes the method for constructing a zebrafish epilepsy model by intramuscular injection of PTZ according to some embodiments of the present invention with reference to the accompanying drawings.

[0070] like Figure 1 As shown, this embodiment discloses a method for constructing a zebrafish epilepsy model by intramuscular injection of PTZ. The method for constructing a zebrafish epilepsy model by intramuscular injection of PTZ comprises the following steps:

[0071] S1. Prepare a microsyringe with a set volume;

[0072] S2, preparing a PTZ solution, and injecting the PTZ solution of a set solubility into the micro-syringe prepared in step S1;

[0073] S3, treating zebrafish to reduce the activity of zebrafish;

[0074] S4. Inject PTZ solution into the dorsal muscle of zebrafish using a microsyringe.

[0075] The present application discloses a method for constructing a zebrafish epilepsy model by intramuscular injection of PTZ. The use of a micro-syringe ensures the precise dosage of the PTZ solution, avoids the effects of excessive or insufficient injection volume on zebrafish, and ensures the consistency and repeatability of the model. By setting the volume of the micro-syringe, researchers can accurately adjust the injection volume of PTZ, thereby inducing consistent epileptic seizure reactions in zebrafish and ensuring the reliability of the experimental results. Compared with the traditional injection method, the micro-syringe can reduce the dosage fluctuation caused by improper operation, thereby improving the accuracy and controllability of the experiment. PTZ (γ-aminobutyric acid transaminase inhibitor) is a drug used to induce epilepsy in animal models, and the accurate configuration of the solution is the key to ensuring the biological activity of the PTZ solution. By accurately configuring the concentration of the PTZ solution, the effectiveness of PTZ is ensured, thereby enhancing its ability to induce epilepsy. By adjusting the solubility, the frequency, intensity and duration of epileptic seizures can be effectively optimized, providing consistent results for subsequent experiments. By reducing the activity of zebrafish, the stress response before the injection of PTZ is reduced, making it more relaxed and better able to accept the injection. This operation helps to avoid experimental interference caused by excessive activity or stress and improves the consistency of the model. Reducing the activity of zebrafish helps its muscles to be in a more stable state, thereby optimizing the absorption efficiency of PTZ solution and ensuring that the biological effect of the drug can be maximized. Accurately injecting PTZ solution into the back muscles of zebrafish with a microsyringe can ensure the target area of ​​drug action. This injection method can avoid uneven distribution of drugs in the body and ensure that PTZ can reach sufficient concentration in the back area of ​​zebrafish to induce epileptic seizures. Intramuscular injection of PTZ solution can simulate the mechanism of naturally occurring epileptic seizures. PTZ quickly induces epileptic reactions by acting on specific areas of the nervous system (such as cerebral cortex, brainstem, etc.), which is convenient for studying the pathophysiological process of epilepsy. Through this step, zebrafish can quickly enter an epileptic state, and researchers can observe the symptoms and manifestations of epileptic seizures (such as muscle tremors, loss of balance, etc.) in real time, providing a powerful model for the study of the mechanism of epilepsy. After injection into the back muscles, PTZ solution can quickly enter the blood circulation, so that the drug effect can be quickly manifested, thereby establishing an epileptic seizure model in a short time. This rapid method of inducing epilepsy makes the model highly reproducible and suitable for large-scale experiments and preclinical studies.

[0076] like Figure 2 As shown, in addition to the features of the above embodiments, this embodiment further defines:

[0077] The specific steps for injecting PTZ solution into the dorsal muscle of zebrafish via a microsyringe are as follows:

[0078] S41, scanning the zebrafish, identifying the back region of the zebrafish, and obtaining back region information;

[0079] S42, determining the injection point, injection depth and injection volume according to the age, weight and dorsal area information of the zebrafish;

[0080] S43. Inject PTZ solution through a microsyringe, and control the injection speed through feedback information from zebrafish during the injection process.

[0081] Precise positioning of the dorsal region is key to ensuring the effectiveness of injected drugs. By scanning the zebrafish and identifying the dorsal region, it is possible to ensure that the PTZ solution is injected into the correct location, avoiding uneven or incomplete drug effects due to improper injection. Dorsal muscles usually have a higher blood supply and can absorb drugs more effectively. By scanning and identifying the dorsal region, it is possible to ensure that the drug is injected into this efficient absorption area, thereby improving the bioavailability of the PTZ solution and ensuring that the drug effect is maximized. Customizing the injection protocol according to the age and weight of the zebrafish can ensure that the injection depth and injection volume of each zebrafish are adapted to its physiological characteristics. For example, young zebrafish and zebrafish with lower body weight usually require smaller injection volumes and shallow injection depths, while adult zebrafish can withstand higher drug doses and deeper injection depths. In this way, drugs can be injected at the optimal dose based on physiological differences, reducing the risk of overdose or underdose. Adjusting the injection volume and injection depth according to the physiological state of the zebrafish (such as age and weight) can avoid damage to the zebrafish caused by excessive injection volume or improper injection depth or adverse reactions caused by drug overdose. More importantly, this helps ensure uniform distribution and rapid absorption of the drug in the body, avoiding adverse consequences caused by drug deposition in local tissues. Determining the injection point and depth not only makes the injection operation more precise, but also ensures that the drug acts on the correct physiological area, avoiding incomplete drug absorption or local toxic reactions caused by the wrong injection location. In this way, the injection operation becomes more controllable and precise, which helps to improve the accuracy and consistency of experimental results. Feedback information controls the injection speed so that the injection process can be adjusted in real time according to the physiological response of zebrafish. For example, zebrafish may show different stress responses or physiological changes (such as accelerated heart rate, muscle contraction, etc.) when receiving PTZ solution injection. These feedback signals can instantly reflect the physiological state of zebrafish, thereby adjusting the injection speed. This method can prevent the injection from being too fast or too slow, ensure a smooth and uniform drug injection process, and reduce the potential harm of drugs to zebrafish. The injection speed of PTZ solution is crucial to the construction of zebrafish epilepsy model. By adjusting the injection speed through feedback information, the injection speed of the drug concentration can be accurately adjusted according to the feedback signals of the zebrafish (such as physiological indicators, movement status, etc.), so that the drug can act on the zebrafish nervous system to the greatest extent, thereby inducing the expected epileptic seizure response. Dynamic adjustments during drug injection can help researchers simulate the process of epileptic seizures more accurately and improve the biological relevance of experimental results. By monitoring the real-time feedback information of zebrafish and adjusting the injection speed, the experimental conditions can be optimized in real time to ensure the stability of the injection process. This not only helps to control the effect of PTZ-induced epilepsy, but also effectively avoids uneven distribution of drugs in the body or excessive or weak drug reactions, ensuring that stable and consistent results are obtained in each experiment.

[0082] like Figure 3As shown, in addition to the features of the above embodiments, this embodiment further defines:

[0083] The specific steps for scanning zebrafish, identifying the dorsal region of zebrafish, and obtaining dorsal region information are as follows:

[0084] S411, scanning the zebrafish, and dividing the zebrafish into a head region, a dorsal region, a lateral region, an abdominal region, and a tail region;

[0085] S412, identifying the morphology of the dorsal region of the zebrafish;

[0086] S413, performing grid processing on the back area to obtain grid information of the back area;

[0087] S414, scanning the thickness of the back region, and matching the thickness information with the grid information of the back region.

[0088] By scanning zebrafish and dividing them into different regions (such as the head, back, side, abdomen, and tail), researchers were able to analyze each physiological region of the zebrafish independently. This regional division helps to analyze and understand the physiological structure of zebrafish more accurately, ensuring that experimental operations (such as injection, measurement, etc.) can focus on specific areas and avoid unnecessary physiological interference. The dorsal region is usually one of the main sites for drug injection in zebrafish. By accurately identifying the dorsal morphology, it can ensure that the injection point selection is more scientific and reasonable. Especially when performing drug injection or other interventions, accurate identification of the dorsal morphology helps to determine the appropriate injection depth and injection location to avoid unnecessary effects on other regions (such as the abdomen, tail, etc.). Gridding makes the morphology of the dorsal region of zebrafish more detailed and visualized, and each small area of ​​the back can be analyzed independently. Gridding not only provides geometric information of the dorsal region, but also allows accurate measurements from local areas, such as the thickness of the dorsal muscles, the length of the dorsal fin, the curvature of the spine, etc., thereby providing more accurate spatial information for subsequent operations. Gridding information can provide spatial distribution data of the dorsal region, which is of great value for subsequent drug injection and treatment. By analyzing the gridding information, we can understand the differences in drug absorption, distribution, and metabolism in different areas of the back region, thereby providing data support for the selection of drug injection volume and injection location. Scanning the thickness of the back region can provide accurate depth data, so that the injection depth can be adjusted according to the actual thickness of the back. By matching the thickness information of the back region with the gridding information, it can be ensured that the drug is injected to the appropriate depth to avoid shallow injections that result in insignificant drug effects, or too deep injections that result in local damage. The matching of the thickness of the back region with the gridding information can not only provide detailed spatial guidance for injections, but also perform multi-dimensional analysis by fusing different biological information. This helps researchers gain a deeper understanding of the role of the back region in drug absorption, neural responses, tissue damage, etc., and provides strong data support for subsequent experimental design.

[0089] like Figure 4 As shown, in addition to the features of the above embodiments, this embodiment further defines:

[0090] The specific steps to determine the injection point, injection depth, and injection volume based on the zebrafish age, weight, and dorsal region information are as follows:

[0091] S421, matching the age and weight of the zebrafish with the grid information of the dorsal region of the zebrafish;

[0092] S422, determining the injection point, injection depth and injection volume of the zebrafish according to the age, weight and grid information of the back region of the zebrafish;

[0093] S423. Adjust the injection depth and injection volume based on the feedback information from the zebrafish.

[0094] By matching the age and weight of zebrafish with the gridded information of their dorsal region, the researchers were able to create a biometric database that accurately reflects the physiological structure and developmental status of zebrafish. The gridded information of the dorsal region of each zebrafish provides more detailed geometric data, which helps to accurately measure and analyze the thickness, morphology and relationship between the dorsal region and other physiological characteristics (such as age and weight). The age and weight of zebrafish are matched with the gridded information of the dorsal region to form a data-based decision-making framework. This approach can support more scientific and accurate experimental decisions through quantitative biological information, avoid relying on intuition or imprecise speculation, and make the entire experimental process more controllable and efficient. By comprehensively considering the age, weight and gridded information of the dorsal region of zebrafish, the injection point and injection depth can be determined more accurately. The thickness of the dorsal region of zebrafish of different ages is different, and there are also differences in the muscle layer and blood supply of the back, so the accuracy of the injection depth and injection point is crucial. Combined with the gridded information, incomplete drug injection or local damage caused by improper injection position or too shallow / deep depth can be avoided. The thickness and muscle state of the dorsal region directly affect the absorption efficiency of the drug. By combining the gridded data of the dorsal region, researchers can better understand the structural characteristics of the dorsal region of zebrafish, determine the best location for drug injection, and ensure that the drug can be quickly absorbed and exert the maximum biological effect. This helps to improve the bioavailability of the drug and ensure that the establishment of the epilepsy model is more stable and reliable. By obtaining feedback information from zebrafish in real time (such as physiological reactions, behavioral changes, etc.), the injection depth and injection volume can be adjusted dynamically. For example, if the zebrafish shows a strong stress response or discomfort, the researchers can reduce the injection depth or slow down the injection speed in real time to reduce damage to the zebrafish. If the zebrafish does not respond adequately to a certain dose, the injection volume can be appropriately increased. This flexible adjustment makes the entire injection process more in line with the physiological needs of each zebrafish and improves the accuracy and safety of the injection.

[0095] like Figure 5 As shown, in addition to the features of the above embodiments, this embodiment further defines:

[0096] The specific steps to match the age and weight of zebrafish with the gridded information of the dorsal region of zebrafish are as follows:

[0097] S4211. Combine data from different sensors to obtain comprehensive information about the dorsal region of zebrafish;

[0098] S4212, data alignment: spatially align data of different modalities to ensure that data of different modalities at the same physical location have consistent coordinates;

[0099] S4213, Denoising and Standardization: Denoise the data of different modalities and ensure that the data of each modality has a uniform scale through standardization;

[0100] S4214, extracting features from the back region of zebrafish;

[0101] S4215. Dynamic matching between zebrafish age, weight and dorsal region characteristics is achieved through a deep reinforcement learning model.

[0102] The data provided by different sensors can complement each other and generate more accurate and rich information about the back region. For example, visual sensors perform very well on the surface texture and muscle distribution of zebrafish skin, while ultrasonic or infrared sensors can provide more in-depth information. Through this comprehensive information acquisition, the physiological condition and structural characteristics of zebrafish can be better analyzed, providing an accurate basis for the formulation of injection plans. Data alignment ensures that data from different sensors can be compared and fused in the same spatial framework. This step is the basis of data analysis and can ensure that the data collected by all sensors have a consistent coordinate system in physical space. Through this alignment, the spatial differences caused by different data sources can be eliminated and the comparability between different data modalities can be improved. Through denoising and standardization, data from different modalities can be fused at the same scale, avoiding the difference in fusion effect caused by data of different scales. This helps to improve the quality of data fused from different sensors, thereby obtaining more accurate features of the zebrafish back region and promoting the progress of subsequent analysis. Feature extraction can transform complex data into interpretable and quantifiable biological features through algorithms, making it easier for researchers to understand the changing laws of the zebrafish back region. This interpretability makes the experimental results more operational and valuable, and provides a basis for further optimizing the drug injection scheme. Through continuous feedback learning, the deep reinforcement learning model makes the matching of the physiological characteristics of zebrafish with the characteristics of the dorsal region have strong adaptive capabilities. This means that even if the physiological state of the zebrafish changes during the experiment, the model can respond quickly and adjust the matching strategy to ensure accuracy during the injection process.

[0103] like Figure 6 As shown, in addition to the features of the above embodiments, this embodiment further defines:

[0104] The specific steps to combine the data from different sensors to obtain comprehensive information about the zebrafish dorsal region are as follows:

[0105] S42111, Infrared imaging: Through infrared imaging technology, images of heat distribution in zebrafish are obtained to reveal the physiological state of the fish and the blood circulation in the dorsal area;

[0106] S42112. Ultrasound imaging: Obtain three-dimensional structural data of the back area through ultrasound, and detect the thickness and density of the muscle layer through sound wave reflection.

[0107] S42113. Optical imaging: Obtain detailed information of the fish body through optical imaging, which is used to obtain the outer contour of the back.

[0108] Infrared imaging technology can capture the thermal distribution in zebrafish in real time. These thermal distributions reflect the physiological activities in zebrafish, including metabolism, blood circulation, organ function, etc. The temperature changes in the dorsal area can reveal the local blood flow and muscle activity, which is crucial for understanding the health status and response of zebrafish. Ultrasound imaging technology can penetrate into the dorsal area of ​​zebrafish and obtain three-dimensional structural data. Through the principle of sound wave reflection, the thickness and density of the muscle layer, fat layer and other tissues in the dorsal area can be detected. This structural information is crucial for determining the injection depth, dosage and injection point selection. Optical imaging can provide high-resolution images, which helps to clearly capture the appearance details of zebrafish. By obtaining the outline of the back, the location of the injection point can be accurately calibrated to avoid errors. This is especially important for drug injection in zebrafish, because the accurate positioning of the dorsal area directly affects the effectiveness and accuracy of the injection.

[0109] like Figure 7 As shown, in addition to the features of the above embodiments, this embodiment further defines:

[0110] The specific steps for determining the injection point, injection depth, and injection volume of zebrafish based on the age, weight, and grid information of the dorsal region of the zebrafish are as follows:

[0111] S4221, inputting the weight, age, and geometric features of the back region of the zebrafish into a regression model, and training the model to obtain a mapping relationship;

[0112] S4222. Simulate the needle penetration process in the back of zebrafish, optimize the predicted injection point based on the information of different ages, weights and back thickness, and ensure that the needle can accurately penetrate in the predetermined area without damaging other organs;

[0113] S4223, constructing a regression model between thickness and injection depth, and establishing a depth prediction model through regression analysis;

[0114] S4224. The following nonlinear regression model is used to calculate the injection volume. The expression of the injection volume is as follows:

[0115]

[0116] Among them, V inj is the injection volume, W is the weight of zebrafish, D injis the injection depth, and α, β, γ are constants obtained by fitting the experimental data.

[0117] By inputting the weight, age and geometric characteristics of the dorsal region of zebrafish into the regression model, the mathematical relationship between the physiological characteristics of zebrafish and injection-related parameters (such as injection point, injection depth, and injection volume) can be constructed. The model can accurately predict the optimal injection strategy for different individuals, thereby greatly improving the accuracy of experimental design. By simulating the process of needle penetration, the model can predict the movement trajectory of the needle under different conditions, ensuring that the needle can enter the dorsal region of the zebrafish according to the expected path during the injection process without damaging other important organs. This precise control greatly reduces the risk of accidental injury and improves the survival rate of zebrafish and the success rate of the experiment. By establishing a prediction model between thickness and injection depth through regression analysis, the optimal injection depth under different dorsal thickness conditions can be accurately predicted. This depth prediction can help researchers adjust the injection strategy according to the specific body shape of zebrafish, thereby ensuring that the drug is accurately injected into the target tissue. By calculating the injection volume through a nonlinear regression model, the drug dose required for each zebrafish can be accurately predicted based on the weight, injection depth and model fitting constants (α, β, γ) of the zebrafish. The weights of factors such as weight and depth in the injection volume prediction are comprehensively considered, avoiding the overdose or underdose that may be caused by simple quantitative predictions.

[0118] like Figure 8 As shown, in addition to the features of the above embodiments, this embodiment further defines:

[0119] The specific steps for adjusting the injection depth and injection volume using the feedback information from the zebrafish are as follows:

[0120] S4231, collecting the electrical activity of zebrafish back muscles through an electromyography sensor to monitor the degree and strength of muscle contraction;

[0121] S4232, obtaining heart rate and respiratory rate in real time through electrocardiogram and respiratory sensors;

[0122] S4233, filtering the collected feedback signals to remove noise signals and extract effective features;

[0123] S4234. Establish a feedback-based depth adjustment model and fit it through a nonlinear regression model:

[0124]

[0125] Where M(t) is the muscle reaction intensity at time t, D inj is the injection depth, F feedback The feedback information is extracted;

[0126] S4235. According to the real-time feedback information, an incremental adjustment method is used to optimize the injection depth. After each feedback information is input, an adjustment amount is obtained, and the injection depth is adjusted according to the adjustment amount.

[0127] By monitoring the electrical activity of the muscles, the immediate response of zebrafish to injection stimulation can be captured, especially in the early stages of drug injection. Such real-time data helps researchers quickly evaluate the effects of drugs and make timely adjustments to avoid adverse reactions caused by overdose. Fluctuations in heart rate and respiratory rate are sensitive indicators of drug side effects, especially after the injection of neurological drugs (such as PTZ), which may cause abnormalities in the cardiovascular and respiratory systems. Real-time monitoring of these indicators can help researchers identify potential adverse reactions in advance and take timely adjustment measures to ensure the safety of the experiment. After filtering, key features related to the physiological response of zebrafish (such as muscle contraction intensity, heart rate fluctuations, etc.) can be extracted. These features are the basis for subsequent modeling and dynamic adjustment of injection parameters. The data after feature extraction has higher analytical value and can provide more accurate input for nonlinear regression models. The feedback-based depth adjustment model can dynamically adjust the injection depth according to real-time physiological feedback (electromyography, electrocardiography and other signals). This nonlinear regression model can accurately fit the complex relationship between physiological signals and injection depth, so as to intelligently adjust the injection strategy according to feedback information and improve the accuracy of injection. The incremental adjustment method can gradually transform feedback information into actual operational adjustments, making the injection depth adjustment process smoother and more flexible. Compared with large adjustments, incremental adjustments can avoid the instability caused by excessive or insufficient adjustments and improve the controllability of the experiment.

[0128] In addition to the features of the above-mentioned embodiments, the present embodiment further defines: the specific steps for preparing a microsyringe of a set volume are as follows: a glass microelectrode tip is pulled by a needle puller, and the tip pulled by the needle puller is a glass microelectrode tip with a diameter of 0.2μm-0.4μm; the glass microelectrode tip is connected to the syringe with a hot melt glue gun. The diameter of the glass microelectrode tip pulled by the needle puller ranges from 0.2μm to 0.4μm, which meets the requirements of microscopic operations. This precise microelectrode tip can ensure better penetration and less damage during the injection process, which is especially important for neuroelectrophysiological measurements or drug injections that require delicate operations in experiments. Using a hot melt glue gun to connect the glass microelectrode tip to the syringe can quickly complete the assembly and provide a firm fixation effect. Hot melt glue has strong adhesion and can fix the microelectrode tip in a short time to prevent the electrode from falling off or shifting due to vibration or operational errors during the injection process.

[0129] In addition to the features of the above embodiment, this embodiment further defines: the specific steps of treating zebrafish and reducing the activity of zebrafish are: selecting 3-month-old zebrafish with an average weight of 300 mg, and placing the zebrafish prone in the cracks of a moist cotton pad after a 0°C ice water bath. The activity of zebrafish can be effectively reduced by treating 3-month-old zebrafish weighing about 300 mg with a 0°C ice water bath. Low-temperature ice water baths inhibit the metabolism and motor ability of zebrafish, causing them to enter a temporary paralysis state. In this process, the motor ability of zebrafish is greatly reduced, thereby reducing its reactivity and behavioral changes, which is convenient for subsequent drug injection and operation.

[0130] In addition to the features of the above embodiments, this embodiment further defines that the specific steps of preparing the PTZ solution are: weighing a predetermined amount of PTZ powder with a micro-spoon, dissolving the PTZ powder in physiological saline, and the concentration of the PTZ solution is 350 mg / kg PTZ solution. Accurately weighing a predetermined amount of PTZ powder with a micro-spoon and dissolving it in physiological saline can ensure the accurate concentration of the PTZ solution. Setting the concentration of PTZ solution to 350 mg / kg ensures that each zebrafish receives a consistent amount of drug, which helps standardize and repeatable experimental results.

[0131] In addition to the features of the above embodiments, this embodiment further defines that the maximum injection volume of the PTZ solution is less than 10 microliters. An injection volume less than 10 microliters can reduce the physical damage to zebrafish tissues caused by drug injection. In microinjection, a small volume of injection solution can accurately deliver drugs without causing excessive mechanical pressure or trauma to the back muscles or other tissues of the zebrafish.

[0132] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0133] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for constructing a zebrafish epilepsy model by intramuscular injection of PTZ, characterized in that: The method for constructing a zebrafish epilepsy model by intramuscular injection of PTZ comprises the following steps: S1. Prepare a microsyringe with a set volume; S2, preparing a PTZ solution, and injecting the PTZ solution of a set solubility into the micro-syringe prepared in step S1; S3, treating zebrafish to reduce the activity of zebrafish; S4. Inject PTZ solution into the dorsal muscle of zebrafish using a microsyringe.

2. The method for constructing a zebrafish epilepsy model by intramuscular injection of PTZ according to claim 1, characterized in that: The specific steps of injecting PTZ solution into the back muscle of zebrafish by microinjection are as follows: S41, scanning the zebrafish, identifying the back region of the zebrafish, and obtaining back region information; S42, determining the injection point, injection depth and injection volume according to the age, weight and dorsal area information of the zebrafish; S43. Inject PTZ solution through a microsyringe, and control the injection speed through feedback information from zebrafish during the injection process.

3. The method for constructing a zebrafish epilepsy model by intramuscular injection of PTZ according to claim 2, characterized in that: The specific steps of scanning the zebrafish, identifying the back region of the zebrafish, and obtaining the back region information are as follows: S411, scanning the zebrafish, and dividing the zebrafish into a head region, a dorsal region, a lateral region, an abdominal region, and a tail region; S412, identifying the morphology of the dorsal region of the zebrafish; S413, performing grid processing on the back area to obtain grid information of the back area; S414, scanning the thickness of the back region, and matching the thickness information with the grid information of the back region.

4. The method for constructing a zebrafish epilepsy model by intramuscular injection of PTZ according to claim 3, characterized in that: The specific steps of determining the injection point, injection depth and injection volume according to the age, weight and dorsal area information of the zebrafish are as follows: S421, matching the age and weight of the zebrafish with the grid information of the dorsal region of the zebrafish; S422, determining the injection point, injection depth and injection volume of the zebrafish according to the age, weight and grid information of the back region of the zebrafish; S423. Adjust the injection depth and injection volume based on the feedback information from the zebrafish.

5. The method for constructing a zebrafish epilepsy model by intramuscular injection of PTZ according to claim 4, characterized in that: The specific steps of matching the age and weight of the zebrafish with the grid information of the back region of the zebrafish are as follows: S4211. Combine data from different sensors to obtain comprehensive information about the dorsal region of zebrafish; S4212, data alignment: spatially align data of different modalities to ensure that data of different modalities at the same physical location have consistent coordinates; S4213, Denoising and Standardization: Denoise the data of different modalities and ensure that the data of each modality has a uniform scale through standardization; S4214, extracting features from the back region of zebrafish; S4215. Dynamic matching between zebrafish age, weight and dorsal region characteristics is achieved through a deep reinforcement learning model.

6. The method for constructing a zebrafish epilepsy model by intramuscular injection of PTZ according to claim 5, characterized in that: The specific steps of combining data from different sensors to obtain comprehensive information about the zebrafish back region are as follows: S42111, Infrared imaging: Through infrared imaging technology, images of heat distribution in zebrafish are obtained to reveal the physiological state of the fish and the blood circulation in the dorsal area; S42112. Ultrasound imaging: Obtain three-dimensional structural data of the back area through ultrasound, and detect the thickness and density of the muscle layer through sound wave reflection. S42113. Optical imaging: Obtain detailed information of the fish body through optical imaging, which is used to obtain the outer contour of the back.

7. The method for constructing a zebrafish epilepsy model by intramuscular injection of PTZ according to claim 4, characterized in that: The specific steps of determining the injection point, injection depth and injection volume of the zebrafish according to the grid information of the age, weight and back region of the zebrafish are as follows: S4221, inputting the weight, age, and geometric features of the back region of the zebrafish into a regression model, and training the model to obtain a mapping relationship; S4222. Simulate the needle penetration process in the back of zebrafish, optimize the predicted injection point based on the information of different ages, weights and back thickness, and ensure that the needle can accurately penetrate in the predetermined area without damaging other organs; S4223, constructing a regression model between thickness and injection depth, and establishing a depth prediction model through regression analysis; S4224. The following nonlinear regression model is used to calculate the injection volume. The expression of the injection volume is as follows: Among them, V inj is the injection volume, W is the weight of zebrafish, D inj is the injection depth, and α, β, γ are constants obtained by fitting the experimental data.

8. The method for constructing a zebrafish epilepsy model by intramuscular injection of PTZ according to claim 4, characterized in that: The specific steps of adjusting the injection depth and injection volume by feedback information from zebrafish are as follows: S4231, collecting the electrical activity of zebrafish back muscles through an electromyography sensor to monitor the degree and strength of muscle contraction; S4232, obtaining heart rate and respiratory rate in real time through electrocardiogram and respiratory sensors; S4233, filtering the collected feedback signals to remove noise signals and extract effective features; S4234. Establish a feedback-based depth adjustment model and fit it through a nonlinear regression model: Where M(t) is the muscle reaction intensity at time t, D inj is the injection depth, F feedback The feedback information is extracted; S4235. According to the real-time feedback information, an incremental adjustment method is used to optimize the injection depth. After each feedback information is input, an adjustment amount is obtained, and the injection depth is adjusted according to the adjustment amount.

9. The method for constructing a zebrafish epilepsy model by intramuscular injection of PTZ according to claim 1, characterized in that: The specific steps of preparing the microsyringe of set volume are as follows: The needle puller pulls the glass microelectrode tip, and the tip pulled by the needle puller is a glass microelectrode tip with a diameter of 0.2μm-0.4μm; Attach the glass microelectrode tip to the syringe using a hot glue gun.

10. The method for constructing a zebrafish epilepsy model by intramuscular injection of PTZ according to claim 1, characterized in that: The specific steps of treating the zebrafish to reduce the activity of the zebrafish are: selecting 3-month-old zebrafish with an average weight of 300 mg, placing the zebrafish prone in the crack of a wet cotton pad after a 0°C ice water bath; And / or the specific steps of preparing the PTZ solution are: weighing a predetermined amount of PTZ powder with a micro-medicine spoon, dissolving the PTZ powder in physiological saline, wherein the concentration of the PTZ solution is 350 mg / kg PTZ solution; And / or the maximum injection volume of the PTZ solution is less than 10 μl.

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