Research method for influence on TBI rear impulse behavior

Abstract

The invention discloses a research method for influence on a post-TBI impulse behavior, and relates to the technical field of traumatic brain injury treatment. The method comprises the following steps: S1, preparing experimental animals, instruments and equipment, reagents and solutions, and grouping the experimental animals; s2, establishing a mouse TBI model by adopting a controllable cortex attack (CCI) mode, and judging the injury severity of the mouse TBI model through nerve function defect scoring (NSS); according to the method, by adopting an immune western blot method, immunofluorescent staining and real-time fluorescent quantitative PCR, the change of lipid droplet metabolism and neuronal functions after TBI can be deeply analyzed from protein and gene levels, multi-level data support is provided, transcranial direct current stimulation and adenosine A2a receptor regulation are used as intervention means, and the method has the advantages that the method is simple and convenient to operate, and the application prospect is wide. The improvement effect of the tDCS on the impulse behavior after the TBI is explored, and particularly, a mechanism that the tDCS regulates A2AR and Plin2 to inhibit lipid droplet accumulation provides a new thought for TBI treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of traumatic brain injury treatment, and in particular to a method for studying the influence of impulsive behavior after TBI. Background Art

[0002] Traumatic brain injury (TBI) commonly occurs in various accidental situations, such as traffic accidents, falls, military operations, and attacks. After treatment and rehabilitation, most patients often have behavioral sequelae on the basis of the physical damage of the primary disease. Impulsive behavior is a behavioral change that occurs under normal physiological conditions but is prone to occur in diseases such as traumatic brain injury. Most patients have increased irritability, verbal or physical aggression, temper tantrum, impatience, and poor decision-making or judgment. The emergence of impulsive behavior is related to the brain's cortex, hippocampus, amygdala and other brain areas, but the specific neural mechanism of impulsive behavior is not clear, which also limits the application of treatment methods;

[0003] Lipids are essential for cellular function, especially in the brain and nervous system, where they are involved in neuronal function, membrane integrity, and myelination. Dysregulation of lipid metabolism is associated with neurological diseases, and traumatic brain injury (TBI) can lead to significant changes in lipid metabolism. Lipid droplets are the main storage form of intracellular lipids and play an important role in lipid metabolism, protein degradation, membrane synthesis, and cell protection. However, excessive accumulation of lipid droplets can be harmful, and imbalance in their regulation may lead to lipid toxicity. Our study found that lipid droplets increased significantly in the TBI-damaged area and were highly overlapped with neurons, which may be the cause of impaired cortical neuronal function. Therefore, regulating the abnormal increase in cortical lipid droplets after TBI may be a target for treating increased impulsive behavior, and transcranial direct current stimulation (tDCS) is a non-invasive technology that regulates motor and cognitive functions. It has been found that it can inhibit impulsive behavior after TBI. Summary of the invention

[0004] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a research method for the influence of impulsive behavior after TBI.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A method for studying the effects of impulsive behavior after TBI, comprising the following steps:

[0007] S1: Prepare experimental animals, instruments, reagents and solutions, and group the experimental animals;

[0008] S2: The mouse TBI model was established by controlled cortical impact (CCI), and the severity of injury in the mouse TBI model was determined by the neurological deficit score (NSS);

[0009] S3: Transcranial direct current stimulation (tDCS) and in vivo drug administration in mice;

[0010] S4: The mice were behaviorally tested using the 5-choice serial reaction time task (5-CSRTT);

[0011] S5: Western blot analysis of mouse brain;

[0012] S6: Immunofluorescence staining (IF) of mouse brain;

[0013] S7: Real-time fluorescence quantitative PCR (qPCR) was performed on mouse brain tissue to detect genes related to lipid droplet formation;

[0014] S8: Use statistical software to perform statistical analysis on the results obtained in the above steps.

[0015] Preferably: in step S1, the experimental animals are preferably C57BL / 6J male mice weighing 22-26 grams, and the mice are randomly divided into six groups: sham operation group (Sham), traumatic brain injury group (TBI), sham operation + transcranial direct current intervention group (Sham+tDCS), traumatic brain injury + transcranial direct current intervention group (TBI+tDCS), traumatic brain injury + adenosine A2a receptor antagonist group (TBI+SCH58261), traumatic brain injury + adenosine A2a receptor agonist group (TBI+CGS21680).

[0016] Further: In step S3, the implementation method of tDCS is: the anode is placed in the catheter with normal saline, and the cathode chest and abdominal electrode cream is fixed, 0.3mA×15min / day×7 days.

[0017] Further: in step S3, the in vivo administration includes slowly pushing the CGS21680 solution and the SCH58261 solution into the brain tissue at a speed of 0.5 ul / min.

[0018] As a preferred solution of the present invention: in step S4, the 5-CSRTT behavior training includes a habituation training phase, a training phase and a test phase, and the test phase includes a long ITI (LITI) test and a variable ITI (VITI) test.

[0019] As a further solution of the present invention: in step S5, the antibody solution used for Western blot identification is rabbit anti-ADRP / Perilipin2 and mouse anti-GAPD.

[0020] As a further solution of the present invention: in step S6, the primary antibody solution for immunofluorescence staining is rabbit anti-ADRP / Perilipin2, rabbit anti-Iba-1, mouse anti-NeuN, goat anti-A2AR and mouse anti-GFAP.

[0021] On the basis of the above scheme: in step S7, real-time fluorescence quantitative PCR (qPCR) detects genes related to lipid droplet formation including Elov15, Fasn, Gdpd3, Plin2, PPARA, PRKAA1, mogat1, Sphk1, etc.

[0022] On the basis of the above scheme: In step S8, the results were statistically analyzed using prism8 statistical software, all results were presented as mean ± SEM, and the differences between different groups were tested using one-way analysis of variance (ANOVA) and Dunnett's multiple comparisons.

[0023] The beneficial effects of the present invention are:

[0024] 1. A research method for the effect of impulsive behavior after TBI. By using immunoglobulin, immunofluorescence staining and real-time fluorescence quantitative PCR, it can deeply analyze the changes in lipid droplet metabolism and neuronal function after TBI at the protein and gene levels, providing multi-level data support, and using transcranial direct current stimulation and adenosine A2a receptor regulation as intervention methods to explore its improvement effect on impulsive behavior after TBI, especially the mechanism of tDCS inhibiting lipid droplet accumulation by regulating A2AR and Plin2, which provides a new idea for the treatment of TBI.

[0025] 2. A research method for the effect of impulsive behavior after TBI, which determined that tDCS may affect the occurrence of impulsive behavior by affecting the accumulation of lipid droplets in the cortical lesion after TBI, and this regulatory effect of tDCS is through A 2A This is achieved by R affecting Plin2, a key molecule in lipid droplet metabolism. Plin2 may be a key target for treating increased impulsive behavior after TBI.

[0026] 3. A research method for the effect of impulsive behavior after TBI, which quantitatively evaluates the impulsive behavior of mice through the 5-choice serial reaction time task (5-CSRTT), can accurately reflect the changes in cognitive function and impulsive behavior of mice after TBI, and combined with the tDCS intervention effect, provides a scientific basis for behavioral regulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a flow chart of a research method for the effect of impulsive behavior after TBI proposed by the present invention;

[0028] Figure 2 This is a statistical diagram of the reduction of impulsive behavior in TBI mice after tDCS treatment, according to a research method for the effect of impulsive behavior after TBI proposed by the present invention;

[0029] Figure 3 It is a schematic diagram of tDCS inhibiting the expression of Plin2 in the cortex of TBI mice in a research method for the effect of impulsive behavior after TBI proposed by the present invention;

[0030] Figure 4 A is a research method for the effect of impulsive behavior after TBI proposed by the present invention. 2A Schematic diagram of R regulating the expression of Plin2. DETAILED DESCRIPTION

[0031] The technical solution of this patent is further described in detail below in conjunction with specific implementation methods.

[0032] The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.

[0033] Embodiment 1:

[0034] A method for studying the effects of impulsive behavior after TBI, such as Figure 1-4 As shown, the following steps are included:

[0035] S1: Prepare experimental animals, instruments, reagents and solutions, and group the experimental animals;

[0036] The preferred experimental animals are C57BL / 6J male mice weighing 22-26 grams, and the mice are randomly divided into: (1) sham operation group (Sham), (2) traumatic brain injury group (TBI), (3) sham operation + transcranial direct current intervention group (Sham + tDCS), (4) traumatic brain injury + transcranial direct current intervention group (TBI + tDCS), (5) traumatic brain injury + adenosine A2a receptor antagonist group (TBI + SCH58261), (6) traumatic brain injury + adenosine A2a receptor agonist group (TBI + CGS21680);

[0037] The instruments and equipment used include: craniocerebral injury instrument, micropipette, surgical instruments, microplate reader, fully automatic gel imaging instrument, protein electrophoresis instrument, electrotransfer instrument, -80℃ refrigerator, low-temperature centrifuge, cryostat, confocal microscope, brain stereotaxic instrument, microinjection pump, constant temperature oven;

[0038] The reagents used include: protease phosphatase inhibitor cocktail, RIPA lysis buffer (strong), QuickBlockWestern primary antibody diluent, BCA protein concentration assay kit, sodium dodecyl sulfate (SDS), goat blocking serum, antibody diluent, paraformaldehyde, anhydrous ethanol, TritonX-100, sucrose, Tween-20, TBST buffer, DAPI solution (ready-to-use), anti-fluorescence attenuation mounting medium, sodium alginate, bovine serum albumin BSA (full components), PAGE gel rapid preparation kit (10%), two-color prestained protein Maker, SDS-PAGE protein loading buffer, ECL chemiluminescent substrate kit, glycine, tris(hydroxymethylaminomethane), methanol, sodium chloride, Anti-GFAP antibody, ADRP / Perilipin2Polyclonalantibody, BODIPY TM 493 / 503, DMSO, Anti-GFAP antibody, Anti-A2A adenosine receptor, recombinant Anti-Iba1 antibody, Anti-NeuN antibody, CY3-labeled goat anti-mouse antibody, 488-labeled goat anti-mouse antibody, CY3-labeled goat anti-rabbit antibody, CY3-labeled donkey anti-goat antibody, 488-labeled donkey anti-rabbit antibody, 647-labeled donkey anti-goat antibody, HRP-labeled goat anti-rabbit antibody, HRP-labeled goat anti-mouse antibody, Anti-GAPDH antibody, Stripping Buffer, PVDF membrane, protein-free rapid blocking solution;

[0039] S2: The mouse TBI model was established by controlled cortical impact (CCI) and the severity of injury in the mouse TBI model was determined by NSS score;

[0040] The mice were anesthetized by intraperitoneal injection of 2.5% tribromoethanol (10 ml / kg), the hair on the head of the mice was shaved, and then the skin of the head was cut along the midline of the scalp. The meninges of the mice were rubbed with hydrogen peroxide to expose the skull. Then, a craniotomy with a diameter of 4-5 mm was performed in the left parietal cortex using an electric drill. The center of the craniotomy was located between the anterior fontanelle and the lambdoid suture, and then the bone pieces were removed. The mice were fixed on the fixed bracket of the craniocerebral injury instrument, and the model was made using an aerodynamic impact device, the tip of which was a metal cylinder with a diameter of 3 mm, and the cortex was struck by controlled impact. The specific impact parameters were depth (2 mm below the dura mater), impact speed (3.5 m / s), and impact dwell time (500 ms). After the impact, the bone pieces left over from the craniotomy were reset to the impact site, the wound was sutured, and the mice were kept warm using an electric blanket until they were fully awake and could move freely and were put back into the cage.

[0041] One day after TBI modeling, the severity of injury in the mouse TBI model was judged by NSS score (neurological deficit score). The scoring items and scoring criteria are shown in the following table. The total score is 20 points. The higher the score, the more severe the injury:

[0042] Neurological deficit score

[0043]

[0044]

[0045] S3: Transcranial direct current stimulation and in vivo drug administration to the mouse brain region;

[0046] Electrode implantation was performed on the third day after TBI modeling in mice: the mice were anesthetized with 2.5% tribromoethanol (10 ml / kg) and fixed in a stereotaxic apparatus. After exposing the skull, three screws were implanted at AP = -2 mm and ML = 2 mm to form a triangular bracket. The central positioning implant had an inner diameter of 1 mm. 3 The polycarbonate catheter was fixed with dental cement to keep the tube free of obstruction. On the 7th day after surgery, transcranial direct current stimulation (tDCS group: the anode was placed in the catheter saline, the cathode was fixed with chest and abdominal electrode paste, 0.3 mA × 15 min / day × 7 days) was started. The TBI group was given stimulation, while the sham group only omitted the current output, and the rest of the operations were completely the same.

[0047] Use a scalpel to cut the skin to expose the skull and mark the location for installing the injection needle (the location is the injury range of traumatic brain injury in mice, AP = -2mm, ML = -2.1mm, DV = -1mm). Install three bone screws around the injection needle and use a locator to attach the injection needle to the marked location.

[0048] When administering drugs in vivo to the mouse brain region, first remove the catheter cap of the injection needle tube, and connect the injection needle tube to the microinjection pump; slowly push the CGS21680 solution and SCH58261 solution (50nM) into the brain tissue at a speed of 0.5ul / min (dissolved in dimethyl sulfoxide, and then prepared with normal saline, ready for use), and wait for 5 minutes after the solution completely enters the brain; after the end, remove the connected catheter, install the catheter cap, and put the mouse back into the mouse cage for continued feeding; the same experimenter administers drugs to the mouse brain region at the same time every day for 3 consecutive days;

[0049] S4: 5-choice serial reaction time task (5-CSRTT);

[0050] After the mice adapted to the environment, they were placed in a 5-CSRTT training room for 30 minutes a day to get used to the training room and receive sugar water or pellet food rewards by touching their noses. After 3 days, the stimulation lights of the five holes were turned on and the mice could get rewards by touching their noses in any hole. Before entering the training, the mice had to receive more than 50 rewards in a maximum of 10 training sessions for two consecutive days.

[0051] Training phase: divided into 6 phases, training once a day, 5 days a week, must meet specific criteria (Table 1); at the beginning of the experiment, the lights in the training bin and reward bin are on, and the mice need to put their noses into the food reward bin to get the reward; then, after the ITI, the stimulus light of a random hole is on, and the mice need to put their noses into the hole within the specified time to get the reward; errors, omissions or premature responses will be punished by turning off the light for 5 seconds; each experiment lasts 30 minutes or 100 tests;

[0052] Testing phase: After mice achieved stable performance in stage 6 of training for at least 7 consecutive days, they were tested with a long ITI (LITI) for 45 min, an ITI of 7 s, and a stimulus duration of 1 s. One week later, they were retested under constant ITI (7s) conditions, with saline or d-amphetamine injected intraperitoneally 30 min before the test. Subsequently, the test was repeated using a randomly assigned variable ITI (VITI) of 2, 5, 10, or 15 s. During the testing period, mice were tested daily in stage 6 to maintain a stable baseline.

[0053] The standards for each stage of CSRTT behavior training are shown in the following table:

[0054]

[0055]

[0056] Behavioral scoring: Total number of tests to complete the training phase: The total number of tests required to complete the training phase from phase 1 to phase 6;

[0057] Total number of trials per test = correct responses + incorrect responses + missed responses that occurred during LITI or VITI;

[0058] Latency to correct response: the time delay from nose poking into the light cue hole after stimulus onset;

[0059] Persistent behavior score: the total number of repeated nose touches to the same orifice that occurred between a correct response and the consumption of the reward;

[0060] %Accuracy = [correct responses / (correct + incorrect responses)] × 100%

[0061] % Omission = [Omission / (Correct Response + Incorrect Response + Omission Response)] × 100%

[0062] % Early response = [early response / (correct response + incorrect response + missed response + early response)] × 100%

[0063] S5: Western blot identification of mouse brain: anesthetize the mouse, decapitate it, separate the skull, take out the brain tissue and put it on ice; weigh the brain tissue, and prepare lysis solution according to RIPA: protease inhibitor: phosphatase inhibitor = 50:1:1 (100ul for every 10mg tissue); add lysis solution and sterilized steel beads to a centrifuge tube, put it in a -20℃ precooled grinding box, set the grinding program (60Hz, 15s / 5s interval, 5 times); after grinding, transfer to a new tube, let it stand for 30min, and put it on ice; precool the centrifuge, centrifuge (12000 rpm, 20min, 4℃), carefully transfer the supernatant and record the volume;

[0064] Then, the protein concentration was determined using the BCA method. After the determination was completed, 5x Loading Buffer was added to the calculated sample, oscillated and mixed, and centrifuged at low speed. The sample was placed in a 95°C metal bath and boiled for 10 minutes. The sample was divided into 200ul EP tubes according to the sample loading amount and marked.

[0065] Store in a -80℃ refrigerator;

[0066] Western blot was performed on the mouse brain samples for identification. The antibody solutions used in the primary antibody incubation of Western blot were rabbit anti-ADRP / Perilipin2 (1:3000) and mouse anti-GAPDH (1:5000); the secondary antibody solutions used in the secondary antibody incubation were goat anti-mouse (1:5000) and goat anti-rabbit (1:5000);

[0067] S6: Immunofluorescence staining (IF) of mouse brain;

[0068] The mice were anesthetized by intraperitoneal injection and fixed. The chest cavity was cut open to expose the heart. Normal saline was perfused from the left ventricle until the liver turned white, and then 4% paraformaldehyde was perfused until the mice became rigid. The brain tissue was removed, fixed with 4% paraformaldehyde and washed with PBS, dehydrated in 30% sucrose solution, and finally frozen in a -80℃ refrigerator;

[0069] The brain tissue was then taken out from the -80°C refrigerator for trimming, embedded in OCT and placed in a freezing microtome for slicing. The slice thickness was 25 μm. The sliced ​​brain slices were placed in a 24-well plate containing 0.02% sodium azide and stored in a 4°C refrigerator. The brain slices were then taken out for immunofluorescence staining and BODIPY staining.

[0070] The primary antibody solutions for immunofluorescence staining were: rabbit anti-ADRP / Perilipin2 (1:200), rabbit anti-Iba-1 (1:200), mouse anti-NeuN (1:200), goat anti-A 2A R (1:200), mouse anti-GFAP (1:400);

[0071] The results of immunofluorescence staining showed that the expression of lipid droplets in the cortical region of TBI mice could be significantly reduced after tDCS treatment. It was found that BODIPY was co-expressed with NeuN in large quantities, but not with GFAP and Iba-1, indicating that the lipid droplets produced after TBI were mainly present in neurons;

[0072] A 2A R plays a role in neuroregulation and anti-inflammatory effects. The results of Western blot showed that compared with the Sham group, the expression of Plin2 in the TBI group was increased. After intervention with adenosine A2a receptor agonist (CGS21680), the expression of Plin2 showed a further upward trend, while after intervention with adenosine A2a receptor antagonist (SCH58261), the expression of Plin2 decreased. In the double immunofluorescence staining test, A 2A R can regulate the changes of Plin2, and the changes of Plin2 are related to A 2A R is highly consistent and overlaps;

[0073] S7: Real-time fluorescence quantitative PCR (qPCR) was performed on mouse brain tissue. The 96-well plate of real-time fluorescence quantitative PCR contained detection of genes related to lipid droplet formation, such as Elov15, Fasn, Gdpd3, Plin2, PPARA, PRKAA1, mogat1, Sphk1, etc., including a negative control for genomic DNA contamination, a primer set with high sensitivity and specificity for detecting untranscribed and repetitive genomic DNA, and three reverse transcription control (RTC) wells, which can be used to verify the efficiency of RT reaction by qPCR detection. The qPCR specific detection is a template synthesized by the built-in external RNA control of the first-strand synthesis kit;

[0074] Comparing the changes of Sphk1, Elov15, Fasn, Gdpd3, mogat1, Plin2, PRARA, and PRKAA1 between the Sham group and the TBI group, Fasn, Gdpd3, mogat1, Plin2, PRARA, and PRKAA1 in the TBI group showed significant differences, and the change of Plin2 was the most obvious. It was further found that Plin2 was significantly increased in the cortical area of ​​the TBI group, and tDCS could inhibit the increase of Plin2 in the cortical area after TBI. Immunofluorescence co-staining of Plin2 and NeuN in the cortical area showed that compared with the control group, the expression of Plin2 in the TBI group was more obvious, tDCS could effectively inhibit the expression of Plin2 after TBI, and the expression of Plin2 was highly overlapped with the neuronal marker NeuN. Further immunofluorescence staining of Plin2 and BODIPY was performed, and the results once again confirmed that tDCS can inhibit the expression of Plin2 after TBI, and the expression trend of BODIPY is consistent with and highly overlaps with Plin2.

[0075] At the same time, the immunofluorescence results showed that compared with the Sham group, BODIPY and A 2A After tDCS and adenosine A2a receptor antagonist intervention, BODIPY and A 2A R expression was significantly decreased, and NeuN and A 2A R highly overlaps, proving that tDCS can inhibit cortical neurons A 2A R expression while inhibiting the abnormal accumulation of lipid droplets;

[0076] S8: Statistical analysis of the results obtained in the above steps was performed using prism8, and all results are presented as mean ± SEM. Data with one variable and multiple conditions were analyzed using one-way analysis of variance (ANOVA), and Dunnett's multiple comparisons were used to test differences between different groups;

[0077] In this research method, mice were trained using a pre-constructed impulse behavior detection device. After all mice reached the required standards for completing all 5-CSRTT training, TBI modeling and transcranial direct current stimulation were performed and the impulsive behavior of each group of mice was detected. There was no difference in accuracy between the TBI group and the sham mice, and tDCS did not affect the accuracy of the TBI group and the sham group.

[0078] In terms of the rate of ignoring stimulation signals, TBI will cause a significant increase in the proportion of mice ignoring signals, and tDCS can reduce the rate of ignoring signals after TBI; in terms of premature occurrence rate, we found that the impulsive behavior of mice increased significantly after TBI, and tDCS can effectively reduce the occurrence of impulsive behavior in TBI mice.

[0079] It was determined that tDCS may affect the occurrence of impulsive behavior by affecting the accumulation of lipid droplets in the cortical lesion after TBI, and this regulatory effect of tDCS is through A 2A This is achieved by R affecting Plin2, a key molecule in lipid droplet metabolism. Plin2 may be a key target for treating increased impulsive behavior after TBI.

[0080] The above is a preferred specific implementation manner of the present invention, and the protection scope of the present invention is not limited thereto. Any modification, equivalent substitution and improvement made by any technician familiar with the field within the technical scope disclosed by the present invention in combination with the prior art or public common sense, within the spirit and principle of the present invention, shall be covered by the protection scope of the present invention.

Claims

1. A method for studying the effects of impulsive behavior after TBI, characterized in that: The steps include: S1: Prepare experimental animals, instruments, reagents and solutions, and group the experimental animals; S2: The mouse TBI model was established by controlled cortical impact (CCI), and the severity of injury in the mouse TBI model was determined by the neurological deficit score (NSS); S3: Transcranial direct current stimulation (tDCS) and in vivo drug administration in mice; S4: The mice were behaviorally tested using the 5-choice serial reaction time task (5-CSRTT); S5: Western blot analysis of mouse brain; S6: Immunofluorescence staining (IF) of mouse brain; S7: Real-time fluorescence quantitative PCR (qPCR) was performed on mouse brain tissue to detect genes related to lipid droplet formation; S8: Use statistical software to perform statistical analysis on the results obtained in the above steps.

2. A method for studying the effects of impulsive behavior after TBI according to claim 1, characterized in that: In step S1, the experimental animals are preferably C57BL / 6J male mice weighing 22-26 grams, and the mice are randomly divided into six groups: sham operation group (Sham), traumatic brain injury group (TBI), sham operation + transcranial direct current intervention group (Sham+tDCS), traumatic brain injury + transcranial direct current intervention group (TBI+tDCS), traumatic brain injury + adenosine A2a receptor antagonist group (TBI+SCH58261), and traumatic brain injury + adenosine A2a receptor agonist group (TBI+CGS21680).

3. A method for studying the effects of impulsive behavior after TBI according to claim 2, characterized in that: In step S3, tDCS is implemented as follows: the anode is placed in the catheter in normal saline, and the cathode chest and abdominal electrodes are fixed with cream, 0.3 mA × 15 min / day × 7 days.

4. A method for studying the effects of impulsive behavior after TBI according to claim 3, characterized in that: In step S3, the in vivo administration includes slowly pushing the CGS21680 solution and the SCH58261 solution into the brain tissue at a speed of 0.5 ul / min.

5. A method for studying the effects of impulsive behavior after TBI according to claim 4, characterized in that: In step S4, the 5-CSRTT behavior training includes a habituation training phase, a training phase, and a test phase, and the test phase includes a long ITI (LITI) test and a variable ITI (VITI) test.

6. A method for studying the effects of impulsive behavior after TBI according to claim 5, characterized in that: In step S5, the antibody solutions used for Western blot identification are rabbit anti-ADRP / Perilipin2 and mouse anti-GAPDH.

7. A method for studying the effects of impulsive behavior after TBI according to claim 6, characterized in that: In step S6, the primary antibody solution for immunofluorescence staining is rabbit anti-ADRP / Perilipin2, rabbit anti-Iba-1, mouse anti-NeuN, goat anti-A2AR and mouse anti-GFAP.

8. A method for studying the effects of impulsive behavior after TBI according to claim 7, characterized in that: In step S7, real-time fluorescence quantitative PCR (qPCR) detects genes related to lipid droplet formation, including Elov15, Fasn, Gdpd3, Plin2, PPARA, PRKAA1, mogat1, Sphk1, etc.

9. A method for studying the effects of impulsive behavior after TBI according to claim 8, characterized in that: In step S8, the results were statistically analyzed using prism8 statistical software, and all results were presented as mean ± SEM. One-way analysis of variance (ANOVA) and Dunnett's multiple comparisons were used to test the differences between different groups.

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