Plastic brain toxicity detection method

Abstract

The invention relates to the technical field of plastic brain toxicity detection, and discloses a plastic brain toxicity detection method, which comprises the following steps: giving a preset number of food particles and water to mice in a target environment, and randomly dividing the mice into groups; after the mice are weighed, MNP with a preset concentration is administered to the experimental group regularly every day, normal saline is administered to the control group, 3D behavioral recording is conducted on the mice after several days of continuous administration, and behavioral analysis is conducted according to the recording result; and after behavioral analysis, performing injection anesthesia on the mice in each group, sampling brain tissues of the mice in each group, and performing brain cell analysis based on the brain tissues of the mice so as to detect the plastic brain toxicity. The toxicity of micro-plastic or nano-plastic can be efficiently and accurately detected through systematic experiment steps.

Description

Technical Field

[0001] This invention relates to the field of plastic brain toxicity detection technology, and in particular to a method for detecting plastic brain toxicity. Background Technology

[0002] Microplastics / nanoplastics (MNPs) are small in size, with nanoplastic particles having diameters of only a few or tens of nanometers. However, due to technological limitations, the minimum visibility of existing fluorescence microscopes is only 200 nm, so it is impossible to see whether nanoplastic particles have entered the brain under a fluorescence microscope.

[0003] Determining the neurotoxicity of nanoplastic microparticles has become an urgent problem to be solved. Summary of the Invention

[0004] Therefore, to address the poor performance of existing technologies in detecting the neurotoxicity of nanoplastic particles, a new method for detecting the neurotoxicity of plastics is proposed. The method includes:

[0005] In the target environment, mice were given a preset number of food particles and water. The mice were randomly divided into groups. The environment in which the mice were placed maintained a 12:12 hour light-dark cycle. Each group included experimental groups that were treated with intranasal drops of suspensions of microplastics or nanoplastics with fluorescent labels of different sizes, and control groups that were treated with intranasal drops of physiological saline.

[0006] After weighing the mice, the experimental group was given a preset concentration of MNP at regular intervals every day, while the control group was given physiological saline. After several days of continuous administration, the mice were recorded in 3D, and the behavioral analysis was performed based on the recording results.

[0007] Following behavioral analysis, mice in each group were anesthetized by injection, and brain tissue samples were collected from each group. Brain cell analysis was then performed based on the mouse brain tissue to detect plastic brain toxicity.

[0008] In one embodiment, the experimental groups include a 26nm MNP group, a 1μm MNP group, a 20μm MNP group, and a 120μm MNP group. The 26nm MNP group refers to a mouse group treated with intranasal drops of fluorescently labeled plastic microparticle suspension with a size of 26nm MNP, the 1μm MNP group refers to a mouse group treated with intranasal drops of fluorescently labeled plastic microparticle suspension with a size of 1μm MNP, the 20μm MNP group refers to a mouse group treated with intranasal drops of fluorescently labeled plastic microparticle suspension with a size of 20μm MNP, and the 120μm MNP group refers to a mouse group treated with intranasal drops of fluorescently labeled plastic microparticle suspension with a size of 120μm MNP.

[0009] In one embodiment, after weighing the mice, the experimental group is administered a preset concentration of MNP at regular intervals daily, while the control group is administered physiological saline. After several days of continuous administration, 3D behavioral recordings are performed on the mice, and behavioral analysis is conducted based on the recording results. The steps include:

[0010] According to different groups, the mice in each group were placed in an open field consisting of a transparent box and a base plate to walk freely. The open field was placed inside a support frame, which was lined with opaque material. A light source was set on the top of the support frame, and four cameras were installed around the support frame to record images of the mice’s behavior. Behavioral analysis was performed based on the behavioral images and behavioral recognition models.

[0011] In one embodiment, the step of anesthetizing mice in each group after behavioral analysis, sampling brain tissue from each group of mice, and performing brain cell analysis based on the mouse brain tissue to detect plastic brain toxicity includes:

[0012] After deep anesthesia, mice were perfused with paraformaldehyde and phosphate-buffered saline (PBS). Brain tissue samples were collected, fixed, dehydrated, and embedded. The tissues were then sliced ​​into 30 μm thick sections using a cryostat.

[0013] Tissues were stained with c-Fos antibody in single-well floating culture dishes. After staining, the tissues were mounted, dried, and then scanned using a virtual microscope slide scanning system. Cell counting was used to quantitatively analyze the number of neurons exhibiting c-Fos signals, thereby analyzing the activation status of brain cells and completing brain cell analysis to detect plastic brain toxicity.

[0014] The proposed method for detecting plastic brain toxicity involves administering a predetermined number of food particles and water to mice in a target environment. The mice are randomly divided into groups, with the environment maintaining a 12:12-hour light-dark cycle. Each group includes experimental groups treated with intranasal drops of fluorescently labeled microplastics or nanoplastics of varying sizes, and a control group treated with intranasal drops of physiological saline. After weighing the mice, the experimental groups are administered a predetermined concentration of microplastics (MNPs) daily, while the control group is administered physiological saline. After several days of continuous administration, 3D behavioral recordings of the mice are performed, and behavioral analysis is conducted based on the recordings. Following the behavioral analysis, the mice in each group are anesthetized, and brain tissue samples are collected. Brain cell analysis is then performed based on the brain tissue to detect plastic brain toxicity. This method enables efficient and accurate detection of the toxicity of microplastics or nanoplastics through a systematic experimental procedure. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] in:

[0017] Figure 1 This is a flowchart of a plastic brain toxicity detection method in one embodiment;

[0018] Figure 2 This is a multi-view video capture device for a plastic brain toxicity detection method in one embodiment;

[0019] Figure 3 This is a graph showing the behavioral analysis results of a plastic brain toxicity detection method in one embodiment;

[0020] Figure 4 This is a graph showing experimental data from a plastic brain toxicity detection method in one embodiment;

[0021] Figure 5 This is a diagram showing the distribution of MNPs in the brain in one embodiment of the plastic brain toxicity detection method.

[0022] Figure 6 This is a diagram showing the activation of c-Fos in the brain in one embodiment of the plastic brain toxicity detection method. Detailed Implementation

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the terminology used herein in the specification of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application; the terms "comprising" and "having," and any variations thereof, in the specification, claims, and foregoing drawings of this application, are intended to cover non-exclusive inclusion. The terms "first," "second," etc., in the specification, claims, or foregoing drawings of this application are used to distinguish different objects, not to describe a particular order.

[0024] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Please see Figure 1 As shown, Figure 1 A schematic flowchart of a plastic brain toxicity detection method according to an embodiment of the present invention includes the following steps:

[0027] Step S101: In the target environment, give the mice a preset number of food particles and water, and randomly divide the mice into groups. The environment in which the mice are located maintains a 12:12 hour light-dark cycle. Each group includes experimental groups that are treated with suspensions of microplastics or nanoplastics with fluorescent labels of different sizes by nasal drops, and control groups that are treated with physiological saline by nasal drops.

[0028] In one embodiment, the experimental groups include a 26nm MNP group, a 1μm MNP group, a 20μm MNP group, and a 120μm MNP group. The 26nm MNP group refers to a mouse group treated with intranasal drops of fluorescently labeled plastic microparticle suspension with a size of 26nm MNP, the 1μm MNP group refers to a mouse group treated with intranasal drops of fluorescently labeled plastic microparticle suspension with a size of 1μm MNP, the 20μm MNP group refers to a mouse group treated with intranasal drops of fluorescently labeled plastic microparticle suspension with a size of 20μm MNP, and the 120μm MNP group refers to a mouse group treated with intranasal drops of fluorescently labeled plastic microparticle suspension with a size of 120μm MNP.

[0029] As an example, male C57BL / 6J mice (6 weeks old) were given food pellets and water, and kept in the target environment for C57BL / 6J mice under a 12:12 hour light-dark cycle. Mice were randomly divided into 5 groups of 5–10 mice each. Fluorescently labeled microplastics or nanoplastics were used, with sizes of 26 nm MNP, 1 μm MNP, 20 μm MNP, and 120 μm MNP, respectively. The control group received saline. Suspensions of the above-mentioned microplastics or saline were administered to mice via nasal drops. After weighing, mice were administered 10 mg / kg MNP or saline, respectively, at 14:00 daily for 7 consecutive days. On day 8, mice underwent 3D behavioral recording or an open field test (OFT). Then, on day 9, mice were anesthetized by intraperitoneal injection of 1% pentobarbital, and brain tissue was sampled.

[0030] Step S102: After weighing the mice, administer the experimental group with a preset concentration of MNP at regular intervals every day, and administer the control group with physiological saline. After several days of continuous administration, record the mice’s behavior in 3D and perform behavioral analysis based on the recording results.

[0031] In one embodiment, mice in different groups are placed in an open area consisting of a transparent box and a base plate to walk freely. The open area is placed inside a support frame, which is covered with an opaque material. A light source is installed on the top of the support frame. Four cameras are installed around the support frame to record images of mouse behavior. Behavioral analysis is performed based on the behavioral images and a behavioral recognition model.

[0032] As an example, a multi-view video capture device such as Figure 2 As shown. The target environment for the mouse refers to placing the mouse in a multi-view video capture device. This device includes a circular open area consisting of a transparent acrylic box (cylinder) and a white plastic base, a support frame (cube), cameras, computer equipment, and a light source. The white plastic base can have a checkerboard pattern. The circular open area is located at the bottom center of the support frame. Cameras are installed on the four sides of the support frame and are connected to the computer equipment. The light source is located at the top of the support frame and is used to adjust the brightness. The support frame is surrounded by an opaque material to prevent the light source from scattering. For example, the mouse walks freely in the circular open area consisting of the transparent acrylic box and the white plastic base. The base of the area has a diameter of 50cm and a height of 50cm. The circular open area is placed in the center of a 90×90×75cm³ movable stainless steel support frame. The support frame is surrounded by a thick, rough, black, opaque rubber pad to prevent light reflection. Four Intel RealSense D435 cameras are installed around the support frame. The activity of mice in a circular clearing was simultaneously recorded at 30 frames per second using a PCI-E USB-3.0 data acquisition card and the Python sense2 camera interface package. A light source providing uniform and stable white background light was located at the top of the supporting frame. The camera and display were connected to a high-performance computer (i7-9700K, 16GB RAM), equipped with a 1TB SSD and a 12TB HDD as the software and hardware platform required for image acquisition. Animal activity was recorded for 15 minutes. Behavioral parameters of the animals were analyzed using Pr sim 8.0 (GraphPad Software).

[0033] It should be noted that the reference Figure 3Through 3D behavioral recording and analysis, it was concluded that MNPs caused spontaneous behavioral disorder in animals after entering the brain. Significant differences were found between the #5, #7, #20, and #23 fine behavior experimental groups and the control group. Specifically, #5 represented walking with head down to explore; #7 represented staying in one place and sniffing; #20 represented running quickly with head down to explore; and #23 represented walking relatively briskly and exploring.

[0034] As yet another example, see reference Figure 4The experimental data, while showing no significant effect of 26nm MNPs and 1μm MNPs treatment on mouse movement from the total distance and average velocity analysis in 3D behavioral studies, revealed that 1μm MNPs treatment induced anxiety-like behavior in mice, as parameters such as the time spent in the center, the distance traveled in the center, and the number of times the mice entered the center showed a decreasing trend; most importantly, 1μm MNPs treatment significantly reduced the distance traveled in the center / distance traveled at the edge. The experimental data can be obtained through the following steps: 1. Camera position calibration: During calibration, 185 pairs of calibration images with 11×8 checkerboard patterns were captured within the field of view of the main camera and three secondary cameras, and calibration parameters were calculated. Then, the calibration parameters of these three camera pairs were calculated using Capture software. This process ensured the accurate calibration of the multi-camera system, providing precise spatial location data for subsequent 3D behavioral analysis. (This step is only necessary when camera calibration parameters are unknown or the cameras have been moved.) 2. Data collection: After determining the camera position parameters, pre-experimental preparations were performed, including adjusting the lighting and setting up the open field. Mice were placed in a circular enclosure 50 cm in diameter and 50 cm high, consisting of a transparent acrylic box and a white plastic floor, positioned as determined in step 1 to ensure complete recording of the mice's activity range. Each group of animals was allowed 30 minutes to acclimatize to the laboratory environment before entering the experiment. Subsequently, behavioral data was simultaneously collected from four perspectives using BehaviorAtlasCapture (DC01) software at a frame rate of 30 frames per second and a resolution of 848*480, with each mouse recorded for 15 minutes. Next, BehaviorAtlas 3D behavioral data analysis is performed, specifically including: 1. 3D reconstruction: The videos from the four perspectives of all samples are loaded into BehaviorAtlas Analyzer software. For each perspective, 16 keypoints are tracked (the 16 keypoints include: nose, left ear, right ear, neck, left front matrix, right front matrix, left hip, matrix, right hip matrix, left front claw, right front claw, left hip claw, right hip claw, back, root position, middle position, tip position), generating corresponding 2D coordinate data. Then, using the camera parameters obtained in step 1, 3D reconstruction is performed, generating an N-frame × 48 matrix, where each row represents one frame of data, and each column represents the x, y, or z coordinates of a body point.2. Behavior Clustering and Segmentation: After obtaining the 3D coordinate data, the BehaviorAtlas algorithm (Huang et al., Nature Communications, 2021) is used to decompose the data into multiple action sequences, with a default of 40 categories. These actions represent different motion patterns or postures. After clustering, the software cuts the video from viewpoint 1 into independent action segments based on the start and end frames of each action segment, and categorizes them into folders with corresponding numbers (1 to 40). This step also yields 39 kinematic parameters, including velocity, motion intensity, and body shape parameters. 3. Action Segment Annotation: For the 40 action categories obtained from unsupervised clustering, manual annotation is performed on the video clips for each category.

[0035] Step S103: After behavioral analysis, mice in each group were anesthetized by injection, brain tissue of mice in each group was sampled, and brain cell analysis was performed based on the mouse brain tissue to detect plastic brain toxicity.

[0036] In one embodiment, mice under deep anesthesia were perfused with paraformaldehyde and phosphate-buffered saline (PBS) to collect brain tissue samples. After fixation, dehydration, and embedding, the tissue was sectioned into 30 μm thick slices using a cryostat. The tissue was stained with c-Fos antibody in single-well floating culture dishes. After mounting and drying, the stained sample slices were scanned using a virtual microscope slide scanning system. Cell counting was used to quantitatively analyze the number of neurons exhibiting c-Fos signals, thereby analyzing the activation status of brain cells and completing the brain cell analysis. As an example, a VS120 virtual microscope slide scanning system can be used to scan the stained sample slices.

[0037] As an example, through fluorescent brain slice images, it was observed that only the 1μm MP group entered the paraventricular nucleus (PVN), lateral septal nucleus (LS), bed nucleus of stria terminalis (BST), and olfactory bulb (OB) regions of the hypothalamus in the 26nm, 1μm, 20μm, and 120μm MNP groups. Figure 5As shown. The specific experimental steps include: Ⅰ. Deeply anesthetized mice, perfused with 4% paraformaldehyde (PFA) and phosphate-buffered saline (PBS); Ⅱ. Brain harvested and fixed with 4% PFA; Ⅲ. Fixed brain dehydrated with 30% sucrose; Ⅳ. Embedding; Ⅴ. Leica cryostat sectioning the tissue into 30μm thick slices; Ⅵ. Antibody staining of the tissue in single-well floating culture dishes: 1. Wash the embedding agent (OCT), rinse three times with PBS solution, and place on a shaker to mix [room temperature (RT); speed: 35-45 rpm; time: 10 min]; 2. Blocking: Block in blocking solution [10% NGS in 0.3% PBST] for one hour [RT, 18-20 rpm, 1 hour (the solution preparation method for this step is: first prepare 0.3% PBST with PBS and Triton, such as 498.5 mL PBS plus 1.5 mL Triton). Then add normal goat serum (NGS) at a ratio of 1:9; 3. Stain with DAPI (4',6-diamidino-2-phenylindole, commonly used to observe the morphology and number of cell nuclei), dilute the DAPI stock solution (2 mg / ml) with PBS to prepare a 1:5000 DAPI dilution, and incubate at room temperature for 5-10 min [RT, 18 rpm, 5-10 min]; 4. Wash with DAPI, rinse three times with PBS, 10 min each time, 3× [RT, 35-45 rpm, 10 min]; 5. Mount the slide and air dry; 6. Mount the slide with mounting medium and store it in a slide box. Store the slide box in a refrigerator at 4℃; 7. Scan the stained sample slides with a VS120 virtual microscope slide scanning system and collect the results.

[0038] As yet another example, see reference Figure 6Through fluorescent brain slice images, it can be seen that there is strong c-Fos activation in the PVN, LS, BST and OB brain regions, which are closely related to emotions. The experiment was conducted using the following steps: 1. Mice were deeply anesthetized and perfused with 4% paraformaldehyde (PFA) and phosphate-buffered saline (PBS); 2. After brain harvesting, the brain was fixed with 4% PFA; 3. The fixed brain was dehydrated with 30% sucrose; 4. Embedding was performed; 5. The tissue was sectioned into 30 μm thick slices using a Leica cryostat; 6. The embedding medium (OCT) was washed, rinsed three times with PBS solution, and shaken on a shaker [room temperature (RT); speed: 35-45 rpm; time: 10 min]; blocking was performed in blocking solution [10% NGS in 0.3% PBST] for one hour [RT, 18-20 rpm, 1 h] (the solution preparation method for this step is as follows: first prepare 0.3% PBST with PBS and Triton, such as 498.5 mL PBS plus 1.5 mL Triton. Then add normal goat serum (NGS) at a ratio of 1:9).

[0039] Incubate primary antibody: Dilute primary antibody with 0.1% PBST. Primary antibody is prepared using Rabbit Antibiotic-Fos (1:500; 2250, Cell Signaing Technology, USA), diluted 1:500. Aspirate the blocking solution completely, do not wash, and incubate with the primary antibody. Recommended incubation conditions are [4℃, 18-20 rpm, 12-14 h]; incubation can also be completed at room temperature for one hour. Wash the primary antibody with PBS: rinse three times, 10 min each time, 3× [RT, 35-45 rpm, 10 min]. Incubate with the secondary antibody: dilute the fluorescent secondary antibody with PBS. The secondary antibody is ALEXA FLUOR@488-conjugated affi n ipure fab fragment goat ant i-rabbit (1:400; 111-547-003; Jackson Immunological Research, USA). Incubate at a 1:400 ratio [RT, 18-20 rpm, 1-3 h]. Incubation must be performed in the dark. Stain with DAPI: after secondary antibody incubation, dilute the DAPI stock solution (2 mg / ml) with PBS to prepare a 1:5000 DAPI solution. Incubate with diluted buffer (I) at room temperature for 5-10 min [RT, 18 rpm, 5-10 min]; wash with secondary antibody and DAPI, rinse three times with PBS, 10 min each time, 3× [RT, 35-45 rpm, 10 min]; mount the slides and air dry; mount with mounting medium or 60% glycerol, and store in a slide box. Store the slide box at 4℃; scan the stained slides using a VS120 virtual microscope slide scanning system to collect results.

[0040] It should also be noted that the conclusion that microplastics / nanoplastics have neurotoxicity can be demonstrated by the following aspects: ① microplastics / nanoplastics can enter the brain; ② microplastics / nanoplastics entering the brain can cause spontaneous behavioral disturbances in experimental animals; ③ microplastics / nanoplastics entering the brain can cause anxiety-like behaviors in animals; ④ microplastics / nanoplastics entering the brain can activate brain regions related to anxiety. Methodologically, firstly, this invention uses nasal ingestion as the animal administration method, which makes it easier for microplastics / nanoplastics to enter the nervous system and can reduce side effects; secondly, this invention uses a low-cost and efficient multi-view 3D animal motion capture system to characterize the 3D kinematics of animals when studying behavioral phenomena; finally, this invention uses animal brain slices and immunohistochemistry to analyze the activity of various regions of the animal brain, thereby determining the affected areas and forms of the brain. (1) Regarding the method of drug delivery: When choosing a method of drug delivery to treat brain diseases, it is necessary to consider how to allow the drug to cross the BBB and be quickly utilized by the target tissue. Intranasal delivery of drugs accumulates under the nasal mucosa and directly and rapidly enters the central nervous system (CNS) and quickly migrates to the lesion site through chemotaxis. It has the characteristics of being non-invasive, low infection, rapid absorption, easy to control the dosage, convenient for repeated administration, bypassing the BBB, and reducing systemic side effects. Compared with systemic doses, intranasal drug delivery shows better targeting and less drug accumulation in peripheral organs, which has a significant advantage in the treatment of CNS diseases. (2) Regarding behavioral experiments: First, the 3D motion learning framework solves the problem of multi-timescale animal behavior. Animal behavior is self-organized into a multi-scale hierarchical structure from bottom to top, including posture, action, and behavior spectrum. By mapping the "letter-word-sentence" of human language to the "posture-action-behavior spectrum" in animal behavior, posture and action are low- to mid-level elements, while the high-level behavior spectrum is a stereotyped pattern of actions that follow inherent transmission rules in certain semantic environments. The focus of two-stage pose and motion decomposition is to extract non-locomotor movement (NM) features from the first two layers. Secondly, the 3D motion learning framework emphasizes the dynamics and temporal variability of behavior. The most critical aspect of unsupervised methods is to define an appropriate metric to quantify the relationship between samples, to measure the similarity between NM segments, so that the model can automatically search for repeatable NM sequences. (3) Fluorescent microplastics / nanoplastics are used for easy observation.

[0041] The above-described embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be included within the protection scope of the present invention.

Claims

1. A method for detecting the brain toxicity of plastics, characterized in that, The method for detecting the neurotoxicity of plastics includes: In the target environment, mice were given a preset number of food particles and water. The mice were randomly divided into groups. The environment in which the mice were placed maintained a 12:12 hour light-dark cycle. Each group included experimental groups that were treated with intranasal drops of suspensions of microplastics or nanoplastics with fluorescent labels of different sizes, and control groups that were treated with intranasal drops of physiological saline. After weighing the mice, the experimental group was given a preset concentration of MNP at regular intervals every day, while the control group was given physiological saline. After several days of continuous administration, the mice were recorded in 3D, and the behavioral analysis was performed based on the recording results. Following behavioral analysis, mice in each group were anesthetized by injection, and brain tissue samples were collected from each group. Brain cell analysis was then performed based on the mouse brain tissue to detect plastic brain toxicity.

2. The method for detecting the brain toxicity of plastics according to claim 1, characterized in that, The experimental groups included a 26nm MNP group, a 1μm MNP group, a 20μm MNP group, and a 120μm MNP group. Specifically, the 26nm MNP group was a mouse group treated with intranasal drops of fluorescently labeled plastic microparticle suspension with a size of 26nm MNP, the 1μm MNP group was a mouse group treated with intranasal drops of fluorescently labeled plastic microparticle suspension with a size of 1μm MNP, the 20μm MNP group was a mouse group treated with intranasal drops of fluorescently labeled plastic microparticle suspension with a size of 20μm MNP, and the 120μm MNP group was a mouse group treated with intranasal drops of fluorescently labeled plastic microparticle suspension with a size of 120μm MNP.

3. The method for detecting the brain toxicity of plastics according to claim 2, characterized in that, After weighing the mice, the experimental group was administered a pre-set concentration of MNP daily at regular intervals, while the control group was administered physiological saline. After several days of continuous administration, 3D behavioral recordings were performed on the mice, and behavioral analysis was conducted based on the recording results. The steps included: According to different groups, the mice in each group were placed in an open field consisting of a transparent box and a base plate to walk freely. The open field was placed inside a support frame, which was lined with opaque material. A light source was set on the top of the support frame, and four cameras were installed around the support frame to record images of the mice’s behavior. Behavioral analysis was performed based on the behavioral images and behavioral recognition models.

4. The method for detecting the brain toxicity of plastics according to claim 1, characterized in that, The steps following behavioral analysis, including anesthetizing mice in each group by injection, sampling brain tissue from each group of mice, and performing brain cell analysis based on the mouse brain tissue to detect plastic brain toxicity, include: After deep anesthesia, mice were perfused with paraformaldehyde and phosphate-buffered saline (PBS). Brain tissue samples were collected, fixed, dehydrated, and embedded. The tissues were then sliced ​​into 30 μm thick sections using a cryostat. Tissues were stained with c-Fos antibody in single-well floating culture dishes. After staining, the tissues were mounted, dried, and then scanned using a virtual microscope slide scanning system. Cell counting was used to quantitatively analyze the number of neurons exhibiting c-Fos signals, thereby analyzing the activation status of brain cells and completing brain cell analysis to detect plastic brain toxicity.

Images