A tail suspension test apparatus and method for assessing behavioral despair in mice after stroke

Abstract

The application discloses a tail suspension test device and method for evaluating the behavioral despair of mice after stroke, wherein a slope plate is arranged below the hanging piece for hanging the mice in the hanging chamber, so as to prevent the mice from struggling due to movement coordination disorders, to help the suspended mice keep balance, and to prevent the mice from shaking even if the movement ability of the mice is impaired, and the application is particularly suitable for evaluating the despair of the mice with impaired movement coordination after stroke. The application supports part of the body weight of the mice through the slope plate, the pain caused during the test is slight, and the application can also be used for rats once the size of the device is adapted to the rats. The application is a modified detection method (mTST for short) of TST, and is used for evaluating the behavioral despair of the mice after stroke. The application can be applied to the evaluation of the curative effect of antidepressant drugs on the model mice of chronic mild stress (CMS) and post-stroke depression (PSD), and is favorable to the basic research of diseases such as PSD and the clinical transformation of drugs.

Description

Technical Field

[0001] This invention relates to a tail suspension test device and method for assessing behavioral despair in mice after stroke, belonging to the technical field of experimental research on behavioral despair in mice after stroke. Background Technology

[0002] Stroke is the third leading cause of death and disability worldwide. Patients recovering from stroke often exhibit signs of mood and affective disturbances, particularly depression. This post-stroke depressive state is defined as post-stroke depression (PSD). Most affected individuals develop mild to moderate depression shortly after a stroke, but depression can appear months or years later. The incidence of depression among stroke survivors is approximately one-third within one year. Patients with PSD are more likely to experience cognitive impairment, suicidal tendencies, and long-term disability. The development of PSD can be influenced by a variety of factors and is similar to, but not identical to, major depressive disorder (MDD). However, several recent multicenter, double-blind, randomized, placebo-controlled studies do not support the use of selective serotonin reuptake inhibitors (SSRIs), the first-line antidepressants, after stroke, leaving a gap in the clinical treatment of PSD. Further research is needed to identify the causes and interventions for the prevention and treatment of PSD.

[0003] Depressive mood and anhedonia are two fundamental symptoms of major depressive disorder (MDD), and at least one must be present along with other key symptoms for a diagnosis of MDD. Clinically, an individual's self-report provides crucial information about depression. However, in the laboratory, it is difficult to determine whether a mouse is “depressed.” The inability of humans and mice to communicate verbally is partly to this end. Fortunately, several behavioral tests have been established and widely used to assess depressive-like states in rodents by briefly observing species-specific behaviors that resemble certain aspects of human depression. Examples include the suspended tail test (TST) and forced swimming test (FST) for despair, the sucrose preference test (SPT) for anhedonia, and the novelty-inhibiting feeding test (NSFT), open field test (OFT), and elevated cross maze test (EPMT) for assessing anxiety symptoms in depressive-like states in mice. While these tests have also been used by some researchers to assess depressive-like behaviors in rodent PSD models, new problems have arisen in their application, particularly when performing FST or TST in mice after a stroke. Midbrain artery occlusion (MCAO) alone, or in combination with chronic mild stress, is commonly used to induce PSD in mice. However, the MCAO model is associated with large infarcts in the cerebral cortex and striatum, resulting in significant motor impairments. These can interfere with the assessment of depression. For example, mice may choke on water due to lack of coordination during FST or shake violently during TST. Classical FST or TST is not suitable for assessing behavioral despair in rodents following severe stroke.

[0004] The suspended tail test (TST) is a classic method for assessing behavioral despair in mice and is commonly used in basic and preclinical research on depression. However, the TST itself depends on motor function, and the results may be misleading when applied to mice with severely impaired motor coordination, such as mice with post-stroke depression models. In general, CMS simultaneously induces behavioral despair (increased resting time compared to controls in both the FST and TST) and anhedonia (decreased sucrose preference compared to controls in the SPT), such as... Figure 1 As shown in the diagram. However, we found that ischemic mice showed reduced sucrose preference in the SPT compared to control mice, but similar immobility times in the FST and TST. Figure 1 ad). Motor function impairment caused by MCAO (adj.) Figure 1 e,f) can explain this difference, while MCAO does not affect motor activity ( Figure 1 g). Impaired motor function makes coordination difficult for mice when hanging or swimming, so they instinctively struggle for balance rather than for survival, resulting in reduced immobility time. Furthermore, label-free pose estimation of TST videos was performed using the deep learning framework DeepLabCut35. Figure 1 h) revealed characteristic features of the hind paw trajectories in MCAO mice. The two hind paw trajectories of the control group and CMS mice were relatively separate, but the trajectories of the MCAO mice largely overlapped, further supporting the unbalanced movement of MCAO mice in TST. Figure 1 i). In summary, the classic TST and FST provide misleading information when used to assess behavioral despair in mice after stroke. Summary of the Invention

[0005] The purpose of this invention is to provide a tail suspension test device and method for assessing behavioral despair in mice after stroke. It is a modified method based on the classic TST, which avoids interference from impaired motor function and can be used to assess behavioral despair in mice after stroke. This solves the technical problem that the existing FST or TST is not suitable for assessing behavioral despair in rodents after severe stroke.

[0006] The present invention provides a tail suspension test device for assessing behavioral despair in mice after stroke, comprising a suspension chamber, the front side of which is a transparent plate, a camera located at the front of the suspension chamber, a suspension device for suspending mice installed at the top of the suspension chamber, a pressure sensor connected to the suspension device, and an inclined plate assembly inside the suspension chamber, the inclined plate assembly comprising an inclined plate arranged at an angle and a sliding support member, the sliding support member being located at the bottom of the inclined plate to support the inclined plate, the inclined plate being inclined upward from front to back and located below the suspension device, the angle between the inclined plate and the horizontal plane being 40°-80°.

[0007] The bottom of the inclined plate is connected to a base plate. The sliding support includes a base plate and guide rails respectively arranged on the left and right sides of the base plate. The base plate is fixedly connected to the bottom of the inclined plate. The two guide rails are arranged in parallel and extend in the front-back direction. The inner sides of the two guide rails that are close to each other are provided with guide rail grooves. The two sides of the base plate are slidably installed in the guide rail grooves respectively. Each guide rail groove is provided with a locking member for locking the base plate.

[0008] The locking component includes a U-shaped component with its opening facing the corresponding guide rail groove and being locked onto the guide rail groove. The lower side of the U-shaped component is pressed against the base plate, and the upper side of the U-shaped component is located above the guide rail. The upper side of the U-shaped component is provided with a threaded hole, and a bolt for tightening the guide rail is installed in the threaded hole.

[0009] The base plate has a handle at its front end for adjusting the position of the ramp.

[0010] The left and right sides and the top of the suspension chamber are opaque panels, while the rear side and the bottom of the suspension chamber are white luminous back panels. The hanging component is a hook.

[0011] The present invention provides a tail suspension test method for assessing behavioral despair in mice after stroke, which adopts the following technical solution: A tail suspension test method for assessing behavioral despair in mice after stroke, which uses the above-mentioned tail suspension test device, includes the following steps: (1) cut a piece of tape, which is used to fix the tape to the mouse tail; (2) place the mouse on the test table and let it move freely, mark a distance of 3 cm from the end of the mouse tail, straighten the mouse tail, fix one end of the tape cut in step (1) to the ventral side of the mouse tail, and fix the other end to the dorsal side of the mouse tail, with the two ends of the tape attached to the mouse tail having equal lengths, both being the length from the end of the mouse tail to the 3 cm section, and then glue the two ends of the tape together. (2) Ensure the mouse's tail is tightly wrapped with tape, forming a loop in the middle of the tape; (3) Weigh the mouse with the tape attached and record its weight as X grams; (4) Hang the littermate mouse with the tape attached in the hanging chamber, hang the tape loop on the hanger, keep the mouse's back facing the camera, move the ramp until the mouse's front paws just touch the surface of the ramp, and when the pressure sensor's digital display flashes between X-1 and X grams, the ramp is successfully positioned. Lock the ramp in this fixed position; (5) Repeat step (4) with the same mouse until the other hanging chambers are debugged. Remove the mouse from the hanging chamber, remove the tape, stroke the mouse and put it back in its original cage. (6) Open the preview window of the computer behavior recording software connected to the camera, adjust the camera parameters to obtain a clear video, and record an 11-second background video of the hanging chamber without mice; (7) Repeat the operation process of steps (1)-(3), and then suspend the experimental test mice in the hanging chamber, keeping the back of the mice facing the camera, ensuring that the suspended mice can only touch the surface of the inclined plate with their front paws, and the pressure sensor readings are between X-1 and X grams; (8) Record the mice for a fixed duration, avoiding noise, light and significant environmental changes during the test; After the video recording is completed, remove the mice from the hanging chamber and remove the tape from the mice's tails. Touch the mouse and put it back in its original cage; (9) Repeat the test procedure, that is, repeat the above steps (7)-(8) until all mice to be tested have completed the test; (10) Select the video taken in step (8) for analysis, open the video recorded in the experiment in the behavior analysis software, and set the analysis box. The analysis box is the area covered by the software analysis. Ensure that the analysis box can accommodate the mouse's body at any point in time in the video recorded in step (8); perform automatic behavior analysis using the behavior analysis software; (11) Obtain the behavior analysis results of the mouse in step (10). The analysis results include the mouse's stillness, movement and other times. Export the behavior analysis results and compare the stillness time of different mice.

[0012] There are 4 hanging chambers, and the 4 hanging chambers are used simultaneously for the tail suspension test of mouse behavioral despair; in step (7), each mouse tail is attached with an adhesive strip, and the 4 mice are hung in the 4 hanging chambers in turn, keeping the mouse's back facing the camera each time; the hanging standard of each mouse should be consistent with that in step (4), and the 4 mice tested in the same session should be hung in turn as quickly as possible, and the hanging of the 4 mice should be completed within 10 seconds.

[0013] The video recording duration in step (8) is 6 minutes; the video recording duration in step (6) is 11 seconds.

[0014] During each test, the backlight of the suspension chamber was turned on and all other lights in the test chamber were turned off. Throughout the test, the backlight of the suspension chamber was kept as the only light source. Before each test, the suspension chamber was cleaned with odorless detergent and the inclined plate was thoroughly wiped with 70% ethanol to dispel the odor left by the mice that had been tested previously.

[0015] In step (10), the analysis box is larger than the mouse body, and a space of one body width is left on the left and right sides of the mouse body in the analysis box.

[0016] The beneficial effects of this invention are as follows: This invention is a modified TST (mTST) detection method used to assess behavioral despair in stroke-affected mice. An inclined plate is added below the suspension device suspending the mouse in the suspension chamber to prevent the mouse from struggling due to motor coordination impairment, thus helping the suspended mouse maintain balance. Even with impaired motor function, it prevents the mouse from swaying, making it particularly suitable for assessing despair in stroke-affected mice with impaired motor coordination. This invention supports part of the mouse's weight via the inclined plate, resulting in less pain during testing. Furthermore, once the device size is adapted for rats, it can also be used in rats. This invention can be applied to detect the efficacy of antidepressants in mouse models of chronic mild stress (CMS) and post-stroke depression (PSD), which is beneficial for basic research on PSD and the clinical translation of drugs.

[0017] The present invention provides a simple testing procedure for assessing behavioral despair in mice. During the test, the immobile state of the mice is objective and easily identifiable. This invention possesses all the advantages of classic TST (Traumatic Stimulation Test). Compared to FST (Free-Side Stimulation Test), it does not cause hypothermia in mice. Once released from the experiment, the mice immediately resume normal spontaneous activity without special post-experimental treatment. Furthermore, FST is more suitable for rats than mice because rats are better swimmers. Using mice in TST is significantly less costly than using rats in FST, and TST can also be used for research on transgenic animals. More importantly, the present invention's mTST procedure overcomes the misinterpretation of experimental results due to impaired motor coordination in classic TST and can be used to assess behavioral despair in mice after stroke. The use of an inclined board in this invention also prevents the suspended mice from twisting or rotating their bodies and keeps their backs facing the experimenter or camera. Therefore, during the mTST test of this invention, the movement of the mouse's limbs is easily observed, and the mice prefer to place their front paws on the inclined board rather than climb up with their tails, solving the pole-climbing problem in TST. Attached Figure Description

[0018] Figure 1 This is a classic experimental diagram showing that TST and FST are not suitable for assessing behavioral despair in mice after stroke.

[0019] Figure 2 This is an experimental graph showing the effect of the inclined plate angle on the mTST stationary time;

[0020] Figure 3 These are mTST's equipment design drawings;

[0021] Figure 4 This is a schematic diagram of mouse suspension in mTST;

[0022] Figure 5 This is an experimental diagram of mTST testing the behavior of despair in mice exposed to MCAO or CMS;

[0023] Figure 6 This is an experimental diagram of behavioral despair in mice exposed to PSD, as detected by mTST.

[0024] Figure 7 This is an overall schematic diagram of a tail suspension test device for assessing behavioral despair in mice after stroke, according to the present invention.

[0025] Figure 8 yes Figure 7 A schematic diagram of a medium-pressure sensor;

[0026] Figure 9 yes Figure 7 Schematic diagram of the sliding support component;

[0027] Figure 10 yes Figure 7Schematic diagram of the inclined plate and the bottom plate;

[0028] In the diagram: 1-Suspension chamber, 2-Guide rail, 3-Locking component, 4-Slope plate, 5-Handle, 6-Hook, 7-Pressure sensor, 8-Conversion module, 9-Conversion module main control board, 10-LCD display screen, 11-Zero adjustment button. Detailed Implementation

[0029] The embodiments of the present invention will be described in detail below, but the present invention can be implemented in many different ways as defined and covered by the claims.

[0030] like Figures 7 to 10 As shown, an embodiment of the present invention discloses a tail suspension test device for assessing behavioral despair in mice after stroke. The device includes a suspension chamber 1, with a transparent front panel, opaque left and right sides and top surface, and a white luminescent back panel on the rear and bottom surfaces. A camera is positioned at the front of the suspension chamber. A hook 6 is mounted on the top of the suspension chamber 1 for suspending the mouse. A pressure sensor 7 is connected to the hook 6. An inclined plate assembly is located inside the suspension chamber 1, comprising an inclined plate 4 and a sliding support. The sliding support is positioned at the bottom of the inclined plate to support the inclined plate 4. The inclined plate 4 is inclined upwards from front to back and is located below the hook 6. The angle between the inclined plate 4 and the horizontal plane is 40°-80°. In this preferred embodiment, the angle between the inclined plate 4 and the horizontal plane is 60°. The inclined plate 4 is transparent.

[0031] The bottom of the inclined plate 4 is connected to a base plate. The sliding support includes guide rails 2 respectively disposed on the left and right sides of the base plate. The base plate is fixedly connected to the bottom of the inclined plate 4. The two guide rails 2 are arranged in parallel and extend in the front-back direction. The inner sides of the two guide rails 2 that are close to each other are provided with guide rail grooves. The two sides of the base plate are slidably installed in the guide rail grooves. Each guide rail groove is provided with a locking member 3 for locking the base plate. The locking member 3 includes a U-shaped member. The opening of the U-shaped member faces the corresponding guide rail groove and is locked in the guide rail groove. The lower side of the U-shaped member is pressed against the base plate, and the upper side of the U-shaped member is located above the guide rail. The upper side of the U-shaped member is provided with a threaded hole, and a bolt for tightening the guide rail is installed in the threaded hole.

[0032] The base plate has a handle 5 at its front end for adjusting the position of the inclined plate.

[0033] The requirements for each component in this embodiment are as follows:

[0034] Guide rails: Material requirements: smooth and supportive. Such as stainless steel or thickened acrylic sheet.

[0035] Locking components: Material requirements, ease of operation, and prevent slippage after locking. Examples include adjusting nuts.

[0036] Inclined panels: Material requirements: transparent and smooth, supportive, and stable. Acrylic sheets are an example.

[0037] Handle: Material requirements: transparent and silky smooth, such as acrylic sheet.

[0038] Hooks: Material requirements: smooth, strong and inelastic, such as stainless steel.

[0039] Pressure sensor: Can be assembled with a hook, recommended accuracy is 0.1g, suitable range for mice is 0~100g, suitable range for rats is 0~500g. High sensitivity, real-time LCD display of readings, and the reading can be zeroed. Minimum requirement is an accuracy of not less than 1.0g. Pressure sensor 7 is connected to conversion module 8, conversion module main control board 9, LCD display 10, and zeroing button 11 via circuitry.

[0040] Placing an inclined board under the suspended mouse can provide support for the mouse. Figure 2 a) to prevent MCAO (middle cerebral artery occlusion) mice from struggling to maintain balance. When the board was tilted to 60°, the immobility time in the sham-operated group was approximately 95 seconds, while that in the MCAO group was approximately 194 seconds, showing a significant difference between the two groups. Figure 2 b). When the ramp was tilted to 40° or 80°, the immobility time of MCAO mice and sham-operated mice increased to 200 seconds or more. Figure 2 b). Mice can only touch an 80° inclined board with their forepaws, but they can stand on a 40° board and rely on the 80° board with both forepaws and hind paws. Figure 2 a). Therefore, a 40° or 80° incline provides relatively comfortable support for the mice, greatly reducing the despair caused by suspension. A preferred embodiment of the invention uses a 60° incline.

[0041] An embodiment of the present invention provides a tail suspension test method for assessing behavioral despair in mice after stroke, which is performed using the suspension chamber described above and includes the following steps:

[0042] (1) Cut a piece of tape to be used to fix the mouse tail;

[0043] (2) Place the mouse on the test table and let it move freely. Mark a distance of 3 cm from the end of the mouse's tail. Straighten the mouse's tail. Fix one end of the tape cut in step (1) to the ventral side of the mouse's tail and the other end to the dorsal side of the mouse's tail. The lengths of the two ends of the tape attached to the mouse's tail are equal, both being the length from the end of the mouse's tail to the 3 cm section. Adhere the two ends of the tape together and ensure that the mouse's tail is tightly wrapped by the tape. A loop is formed in the middle of the tape.

[0044] (3) Weigh the mouse with the tape attached and record the weight of the mouse as X grams;

[0045] (4) Hang the mouse with tape attached in the hanging chamber, hang the tape loop on the hanging device, keep the mouse's back facing the camera, move the inclined board until the mouse's front paws just touch the surface of the inclined board, when the digital display of the pressure sensor flashes between X-1 and X grams, the inclined board is successfully positioned, and lock the inclined board in this fixed position.

[0046] (5) Repeat step (4) using the same mouse until the other hanging chambers are properly set up. Remove the mouse from the hanging chamber, remove the tape, stroke the mouse, and return it to its original cage;

[0047] (6) Open the preview window of the behavior recording software connected to the camera, adjust the camera parameters to obtain a clear video, and record an 11-second background video of the mouse-free hanging room.

[0048] (7) Repeat the operation process of steps (1)-(3), and then suspend the experimental test mouse in the suspension chamber, keeping the mouse's back facing the camera, ensuring that the suspended mouse can only touch the surface of the inclined plate with its front paws, and the pressure sensor displays a number between X-1 and X grams.

[0049] (8) Record videos of mice for a fixed duration, avoiding noise, light and significant environmental changes during the test; after the video recording is completed, remove the mice from the hanging chamber, remove the tape from the mice's tails, stroke the mice and put them back in their original cages.

[0050] (9) Repeat the test procedure, that is, repeat the above steps (7)-(8) until all mice to be tested have completed the test;

[0051] (10) Select the video taken in step (8) for analysis. Open the video recorded in the experiment in the behavior analysis software and set the analysis box. The analysis box is the area covered by the software analysis. Ensure that the analysis box can accommodate the mouse's body at any point in time in the video recorded in step (8). Perform automatic behavior analysis using the behavior analysis software.

[0052] (11) Step (10) obtains the behavioral analysis results of the mice. The analysis results include the time the mice are still, move, and other times. The behavioral analysis results are exported and the still time of different mice is compared.

[0053] Preferably, in this embodiment there are 4 suspension chambers, and the angle between the inclined plate and the horizontal plane in each suspension chamber is 60°. The 4 suspension chambers are used simultaneously for the tail suspension test of mouse behavioral despair. In step (7), each mouse tail is attached with an adhesive tape ring, and the 4 mice are hung in the 4 suspension chambers in turn, keeping the mouse's back facing the camera each time. The suspension standard for each mouse should be consistent with that in step (4). The 4 mice tested in the same session are suspended in turn as quickly as possible, and the suspension of the 4 mice is completed within 10 seconds.

[0054] During each test, the backlight of the suspension chamber was turned on and all other lights in the test chamber were turned off. Throughout the test, the backlight of the suspension chamber was kept as the only light source. Before each test, the suspension chamber was cleaned with odorless detergent and the inclined plate was thoroughly wiped with 70% ethanol to dispel the odor left by the mice that had been tested previously.

[0055] The invention will be described below with reference to specific experimental procedures:

[0056] I. Experimental Materials

[0057] Animals: Adult male C57BL / 6 mice.

[0058] Key point: The age and sex of mice can affect immobility, therefore mice used in each independent experiment should be of the same sex and similar age (e.g., 9 to 10 weeks old). Furthermore, age-similar mice also tend to be similar in size and shape, eliminating the need for experimenters to adjust the position of the ramp between tests on different mice.

[0059] Animal husbandry and handling: Three to four mice are housed per cage, with free access to food and water. The housing should be kept at a constant temperature (21 ± 2°C), humidity (55 ± 10%), and a standard 12-hour light-dark cycle. Mice should be touched at least daily for one week prior to initiating the mTST program of this invention to acclimate them to the experimenter. If mice are on the CMS program, touch each mouse for 2 minutes after daily stress. If mice are on the MCAO program, daily treatment should be suspended on the first and second days after surgery. When touching mice, experimenters should not wear strong perfumes or other strong scents, as this may cause aversion or interference with behavioral assessment.

[0060] Reagents: 70% ethanol, odorless cleaning agent, medical tape.

[0061] Reagent Preparation: Medical tape should be selected to be strong enough to withstand the weight of the suspended mice, yet soft enough to avoid injury; therefore, cotton tape is recommended. Prepare the tape segment before each test. Cut a 16 cm long (2.0 cm wide) segment of tape, fold the 16 cm segment in half along its long axis, and glue the middle 10 cm together (see...). Figure 4 a) The 3 cm section at each end will be used to attach the tape to the mouse's tail. Do not prepare all the tape segments too early, as the exposed 3 cm ends of the tape segments will lose their stickiness over time.

[0062] II. Operating Procedures for the mTST Detection Method of the Invention (Steps 1-21):

[0063] (a) Animal adaptation: Time 1 hour:

[0064] 1. Ensure a constant temperature (21 ± 2°C) and humidity (55 ± 10%) in the laboratory.

[0065] 2. One hour before the test begins, place the mice in a corner of the testing room to allow them to acclimatize.

[0066] Important Notes: Regarding circadian rhythms, it is recommended that all mice tested in independent experiments be tested in the same morning (9:00-12:00) or the same afternoon (14:00-17:00). On a device with four hanging chambers, approximately 48 mice can be tested within 3 hours. If the number of mice in an independent experiment exceeds 48, and testing is required in both the morning and afternoon, then mice assigned to the morning test should be acclimatized between 8:00-9:00, and mice assigned to the afternoon test should be acclimatized between 13:00-14:00.

[0067] (ii) Equipment debugging: 0.5 hours.

[0068] While adapting the mice (step 2), the experimenter can simultaneously adjust the equipment (steps 3-7).

[0069] 3. Turn on the background light in the suspension chamber and turn off all other lights in the testing room. Throughout the entire test, keep the background light in the suspension chamber as the sole light source.

[0070] 4. Wrap the mouse's tail with 16 cm of tape and weigh the mouse according to the procedure described in steps 9-11 (record the mouse's weight as X grams). Hang the mouse on a hook and pull it through the tape attached to its tail ( Figure 4 (ad) Keep the mouse's back facing the camera. Pull or push the ramp along the track until the mouse's front paws just touch the surface of the ramp. Figure 4 f). Simultaneously, the digital display of the pressure sensor will flash between X and X-1 grams, indicating successful positioning of the ramp. Once the ramp is in place, the locking mechanism on the guide rail is engaged.

[0071] Important Note: The test mouse used in this step should not be included in the actual experiment. In other words, an additional mouse is needed for adjusting the inclined plane. This mouse should be a littermate of the mouse included in the actual experiment to ensure that the inclined plane position determined using this mouse is suitable for the mice in the actual experiment.

[0072] Key Step: The placement of the inclined plane is crucial for the success of mTST. Generally, since mice of the same age have similar body sizes, the position of the inclined plane is generally applicable for independent experiments. However, if there are significant differences in mouse weight or the inclined plane position is unsuitable, it needs to be adjusted promptly. The standard for adjustment is the pressure sensor reading. The experimenter should determine the appropriate position by carefully observing the distance between the inclined plane and the mouse. If the inclined plane is not in contact with the mouse (…), the experimenter should proceed with caution. Figure 4 If e), the suspended mouse is not supported, and the digital display of the pressure sensor will flash between X and X+1 grams. If the ramp is too close to the mouse ( Figure 4 (g) The mouse tilts and rests against the board, and the digital display of the pressure sensor flashes between X-1 and X-2 grams. The pressure changes detected by the pressure sensor will help the experimenter determine the proper position of the inclined plate.

[0073] 5. Open the preview window of the computer behavior recording software. Adjust the camera parameters to obtain clear video of the mouse.

[0074] 6. Remove the mouse from the hook and record an 11-second video of the mouse-free hanging chamber background.

[0075] Warning: Do not remove the tape from the mouse before preparing the last hanging chamber. Gently stroke the mouse and return it to its original cage.

[0076] Important Note: This video will be used to analyze background noise during mouse inactivity (Step 20). Record background videos for each suspension chamber of the multi-channel setup. It is recommended to record background videos before each independent experiment and not to share the same background video between different experiments.

[0077] 7. For the preparation of other hanging chambers, please repeat steps 4-6.

[0078] (iii) Tail suspension test: The test time varies depending on the number of test animals; 15 minutes per session (4 mice per session).

[0079] 8. Before each test, clean the hanging chamber with unscented dish soap and thoroughly wipe the inclined plate with 70% ethanol to remove the odor left by the mice that were previously tested.

[0080] Key step: Thorough cleaning will remove all olfactory traces that may attract or frighten the mice in the test, avoiding any negative impact on the mice's behavior.

[0081] 9. Cut a 16 cm long strip of tape (2.0 cm wide). Fold the 16 cm long tape in half along its long axis. Glue the middle 10 cm together, leaving 3 cm at each end to secure the tape to the tail. Figure 4 a).

[0082] Key point: Note that the length of the tape segment should be determined by the distance between the suspension hook and the floor of the chamber. Reference: When the height of the suspension chamber is 38cm, we choose a length of 16cm so that the mice can be suspended at a height of about 30cm above the floor of the chamber.

[0083] 10. Gently stroke the mouse for 10 seconds, then place it on the testing table and allow it to move freely. Mark a distance of 3 cm from the end of the mouse's tail. Gently straighten the mouse's tail, fold a 16 cm long strip of tape in half, and fix one end (3 cm) of the tape to the ventral side of the mouse's tail and the other end (3 cm) to the dorsal side of the mouse's tail. Then, glue the two ends of the tape together. Figure 3 b), so that the 16 cm long tape forms a loop ( Figure 3 c). Gently pinch the two ends of the adhesive tape with your fingers to ensure that the mouse's tail is tightly wrapped with tape.

[0084] Key step: The mouse's tail should be directly wrapped in the tape, ensuring the mouse is suspended with a straight tail. Suspending by a bent tail is painful for the mouse and should be avoided.

[0085] Key point: The tape should be applied 3 cm from the tip of the mouse's tail. Suspending through the shorter end of the tail (e.g., 1 cm) is painful for the mouse and should be avoided. Although suspending through the base of the tail may be more comfortable for the mouse, the likelihood of the tail climbing will increase significantly.

[0086] 11. Weigh the tape and the mouse together, then return the mouse to its cage. The weight will be used as a reference for step 14.

[0087] 12. Repeat steps 9-11 to test the other 3 mice in the same test. Number them 1 to 4. Clearly record the group and number of each mouse in each test.

[0088] Key: Each group of mice in each independent experiment should be tested simultaneously in each mTST experiment. Mice in the same group should be tested according to a randomization protocol generated by a computer program. The positions of mice in the four chambers should be rotated so that mice from the same group are placed in different hanging chambers in each experiment.

[0089] Key point: Do not proceed to step 13 until all mice being tested in the same field are ready to be suspended.

[0090] 13. Hang the first mouse in the first suspension chamber, with the tape loop attached to its tail. Figure 4 (cd) Keep the mouse's back facing the camera. Then hang the second mouse in the second hanging chamber, the third mouse in the third hanging chamber, and finally hang the fourth mouse in the fourth hanging chamber.

[0091] Key Step: The suspension criteria for each mouse should be consistent with those in step 4; otherwise, the inclined plane position determined in step 4 will not be applicable to mice tested subsequently. For example, the tape loop should be placed at the bottom of the hook. It is recommended that steps 4 and 14 be performed by the same experimenter.

[0092] Key point: The four mice being tested in the same session should be suspended sequentially as quickly as possible. Using tape loops, we can typically suspend four mice within 10 seconds.

[0093] Key point: It is recommended to hang the mice in sequence so that the recorded videos can be easily matched with the corresponding mice.

[0094] 14. Check the position of the inclined plane for each mouse in the suspension chamber. Ensure that the suspended mouse can only touch the surface of the inclined plane with its front paws. Figure 4 f). Meanwhile, the digital display of the pressure sensor should indicate that the pressure is approximately equal to the weight of step 11 minus 1 gram.

[0095] Key Step: The correct placement of the inclined plate is crucial for the modified tail suspension test (mTST). The plate position is adjusted according to step 4. If the inclined plate does not contact the mouse ( Figure 4 e), then no support will be provided to the suspended mouse. If the inclined board is too close to the mouse ( Figure 4 g), the mouse will lean against and rest on the board. Only when the inclined board is accurately positioned ( Figure 4 f) is necessary to ensure the mice maintain their balance and helplessness during the test.

[0096] Key point: This step should be completed as quickly as possible. In most cases, the ramp does not need to be adjusted again after positioning in step 4. If adjustment is necessary, the ramp position should be adjusted within 10 seconds. Unlock the locking mechanism and pull or push the ramp until the mouse's front paws just touch the ramp surface. Note that adjustments will extend the suspension time before the formal test. Once the mouse is suspended, recording should begin as soon as possible. It is recommended that any additional adjustments should not exceed 1 minute.

[0097] 15. Open the preview window of the behavior recording software (Depression software, Clever Sys, Inc.) to ensure that each mouse's movement is clearly captured. Set the recording duration to 6 minutes and start recording.

[0098] Key steps: Avoid noise, light, and significant environmental changes during testing. The four mice being tested simultaneously should be isolated both auditorily and visually.

[0099] 16. After the video recording is finished, remove the mice from the hooks, gently remove the tape from their tails, and put them back into their respective cages.

[0100] 17. Save each video to the same folder using a filename that identifies the group and the mouse number.

[0101] 18. Repeat the testing procedure (steps 9-18) until all mice have completed the test.

[0102] IV. Data Analysis (Time may vary depending on the number of videos):

[0103] 19. Select the background captured in step 6 for analysis. Open the video recorded in the experiment in the behavior analysis software and set the analysis frame. The analysis frame is the area covered by the software analysis. Ensure that the analysis frame can accommodate the mouse's body at any point in the 6-minute video.

[0104] Important note: Information outside the analysis frame will not be read during analysis. Therefore, the defined analysis frame should be larger than the mouse's body, leaving a space equal to the width of the mouse's body on both the left and right sides. No instances of mice struggling outside this analysis frame were observed during testing.

[0105] Important note: Do not share analysis boxes and background settings between different experiments.

[0106] 20. Select the folder for the experiment and start the automatic behavior analysis using the parameters set in step 19.

[0107] 21. Export the results of the behavioral analysis (immobility, movement, and others) to Excel, and calculate the immobility time of the mice within 5 minutes after the end of the testing phase as an indicator of behavioral despair.

[0108] The experimental schedule is as follows: (1) Steps 1-2, animal adaptation: 1 hour; (2) Steps 3-7, equipment debugging: 0.5 hours (can be done simultaneously with Steps 1-2); (3) Steps 8-18, tail suspension test: depending on the number of mice tested; 15 minutes per session (4 mice per session); (4) Steps 19-21, data analysis: depending on the number of videos, each video analysis takes about 3 minutes.

[0109] III. Experimental Results

[0110] In the classic TST, escape behavior in mice can be categorized into three types: running, body contortion, and body jerking. In the modified mTST, due to the support of the inclined plane, body contortion and body jerking are rarely observed. The movement of the mice is almost entirely running, even uncoordinated running. Because the right hemisphere is exposed to ischemia during the preparation of the MCAO model, significant motor dysfunction was observed in the left hind limb when the mice were suspended in the mTST. If ischemia occurs in the left hemisphere, then the right hind limb is primarily affected. Although the movement characteristics of ischemic mice are unique, they are closely related to the impaired motor function. As in the classic TST, the immobility time during the mTST test in this invention is a key indicator of behavioral despair and can be easily read from the video using behavioral analysis software. It is recommended to calculate the immobility time for the last 5 minutes of the 6-minute mTST test. Immobility latency is not reported in the classic TST and is not recommended to be reported in the mTST either.

[0111] To validate the effectiveness of the mTST assay in assessing behavioral despair in mice after stroke, 9- to 10-week-old C57BL / 6 mice were exposed to MCAO or sham surgery, and mTST was performed one week later. Figure 5 a). The results showed that, compared with the sham surgery group, the immobility time in the MCAO group was significantly increased ( Figure 5 b). Furthermore, a single ketamine treatment reduced immobility time in MCAO mice, indicating a rapid antidepressant effect. Figure 5 b). Therefore, the mTST detection method is effective in measuring behavioral despair in mice after stroke and is sensitive to antidepressant treatment. Label-free pose estimation of the video using the deep learning framework DeepLabCut35 revealed the trajectory set of each paw during mTST ( Figure 5 c), which differs from the dispersed trajectories in the classic TST ( Figure 1 i). This indicates that the mice move more regularly and symmetrically during mTST. Even in MCAO mice, the overlap of the trajectories of the two hind paws due to movement incoordination is greatly reduced during mTST. Figure 5 c). Then mTST was repeated on days 7, 14, and 28 after MCAO ( Figure 5 d). The results showed that MCAO mice had longer immobility times on days 7 and 14 than sham-operated mice. Figure 5 e), demonstrating the feasibility of weekly interval repetition measurements. However, on day 28, there was no significant difference in immobility time between the sham-operated group and the MCAO group. A possible reason is the spontaneous recovery of depressive-like behavior in the MCAO mice, as sucrose preference in the SPT was similar between the sham-operated and MCAO groups on day 28. Figure 5f). However, we cannot rule out the possibility that repeated measures reduced inter-group differences, therefore we recommend not repeating mTST more than three times in the same batch of mice. We also demonstrated the utility of the mTST protocol in assessing behavioral despair in mice exposed to CMS (a classic model). Figure 5 As shown in gh, both the increase in immobility time induced by CMS and the decrease in immobility time induced by ketamine treatment were revealed by both mTST and classic TST. Notably, the immobility time in each group in mTST was slightly longer than that in classic TST. This is likely a reasonable result due to the support of the sloping plate in mTST. Finally, the applicability of the mTST protocol to the PSD mouse model was examined. Figure 6 As shown in figure a, PSD was induced in 9- to 10-week-old C57BL / 6 mice via MCAO (day 0) and CMS (days 7 to 36), followed by chronic fluoxetine treatment from day 7 to 36. Grid walking task and rotarod test demonstrated impaired motor function in PSD mice at 2 and 5 weeks post-MCAO. Figure 6 b, c). Using the mTST assay, we successfully identified behavioral despair differences between the sham surgery group and the PSD group at two time points. Figure 6 d, e). Furthermore, after 4 weeks of treatment instead of 1 week, the antidepressant effect of fluoxetine was clearly observed, manifested as a reduction in immobility time (d, e). Figure 6 Therefore, the mTST assay can also be used to measure behavioral despair in PSD model mice and to evaluate the effectiveness of chronic antidepressant treatment.

[0112] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A tail suspension test device for assessing behavioral despair in mice after stroke, comprising a suspension chamber, the front side of which is a transparent plate, a camera positioned at the front of the suspension chamber, and a suspension device for suspending the mouse mounted on the top of the suspension chamber, the suspension device being connected to a pressure sensor, characterized in that: The left and right sides and top of the suspension chamber are opaque panels, and the rear side of the suspension chamber is a white luminescent back panel. The suspension chamber is equipped with an inclined plate assembly, which includes an inclined plate and a sliding support. The sliding support is located at the bottom of the inclined plate to support it. The inclined plate is inclined upward from front to back and is located below the hanging device. The angle between the inclined plate and the horizontal plane is 40°-80°. When in use, keep the mouse's back facing the camera and the mouse's front paws just touching the surface of the inclined plate.

2. The tail suspension test device for assessing behavioral despair in mice after stroke, as described in claim 1, is characterized in that: The bottom of the inclined plate is connected to a base plate. The sliding support includes a base plate and guide rails respectively arranged on the left and right sides of the base plate. The base plate is fixedly connected to the bottom of the inclined plate. The two guide rails are arranged in parallel and extend in the front-back direction. The inner sides of the two guide rails that are close to each other are provided with guide rail grooves. The two sides of the base plate are slidably installed in the guide rail grooves respectively. Each guide rail groove is provided with a locking member for locking the base plate.

3. The tail suspension test device for assessing behavioral despair in mice after stroke, as described in claim 2, is characterized in that: The locking component includes a U-shaped component with its opening facing the corresponding guide rail groove and being locked onto the guide rail groove. The lower side of the U-shaped component is pressed against the base plate, and the upper side of the U-shaped component is located above the guide rail. The upper side of the U-shaped component is provided with a threaded hole, and a bolt for tightening the guide rail is installed in the threaded hole.

4. The tail suspension test device for assessing behavioral despair in mice after stroke, as described in claim 2, is characterized in that: The base plate has a handle at its front end for adjusting the position of the ramp.

5. The tail suspension test device for assessing behavioral despair in mice after stroke, as described in claim 1, is characterized in that: The bottom surface of the suspension chamber is a white luminous back panel, and the hanging component is a hook; the angle between the inclined plate and the horizontal plane is 60°.

6. A tail suspension test method for assessing behavioral despair in mice after stroke, comprising using the tail suspension test apparatus as described in claim 1, characterized in that, Includes the following steps: (1) Cut a piece of tape to be used to fix the mouse tail; (2) Place the mouse on the test table and let it move freely. Mark a distance of 3 cm from the end of the mouse's tail. Straighten the mouse's tail. Fix one end of the tape cut in step (1) to the ventral side of the mouse's tail and the other end to the dorsal side of the mouse's tail. The lengths of the two ends of the tape attached to the mouse's tail are equal, both being the length from the end of the mouse's tail to the 3 cm section. Adhere the two ends of the tape together and ensure that the mouse's tail is tightly wrapped by the tape. A loop is formed in the middle of the tape. (3) Weigh the mouse with the tape attached and record the weight of the mouse as X grams; (4) Hang the littermate mice with tape attached in the hanging chamber, hang the tape loop on the hanger, keep the mouse's back facing the camera, move the ramp until the mouse's front paws just touch the surface of the ramp. When the digital display of the pressure sensor flashes between X-1 and X grams, the ramp is successfully positioned. Lock the ramp in this fixed position. (5) Repeat step (4) with the same mouse until the other hanging chambers are calibrated. Remove the mouse from the hanging chamber, remove the tape, stroke the mouse and put it back in its original cage. (6) Open the preview window of the behavior recording software connected to the camera, adjust the camera parameters to obtain a clear video, and record an 11-second background video of a mouse-free hanging room; (7) Repeat the operation process of steps (1)-(3), and then suspend the experimental test mouse in the suspension chamber, keeping the mouse's back facing the camera, ensuring that the suspended mouse can only touch the surface of the inclined plate with its front paws, and the pressure sensor displays a number between X-1 and X grams. (8) Record videos of mice for a fixed duration, avoiding noise, light and significant environmental changes during the test; after the video recording is completed, remove the mice from the hanging chamber, remove the tape from the mice's tails, stroke the mice and put them back in their original cages. (9) Repeat the test procedure, that is, repeat the above steps (7)-(8) until all mice to be tested have completed the test; (10) Select the video taken in step (8) for analysis, open the video recorded in the experiment in the behavior analysis software, and set the analysis box. The analysis box is the area covered by the software analysis. Ensure that the analysis box can accommodate the mouse's body at any point in time in the video recorded in step (8); use the behavior analysis software to perform automatic behavior analysis; (11) Step (10) obtains the behavioral analysis results of the mice. The analysis results include the time the mice are still, move, and other times. The behavioral analysis results are exported and the still time of different mice is compared.

7. The tail suspension test method for assessing behavioral despair in mice after stroke, as described in claim 6, is characterized in that: There are 4 hanging chambers, and the 4 hanging chambers are used simultaneously for the tail suspension test of mouse behavioral despair; in step (5), each mouse tail is attached with an adhesive tape ring, and the 4 mice are hung in the 4 hanging chambers in turn, keeping the mouse's back facing the camera each time; the hanging standard of each mouse should be consistent with that in step (4), and the 4 mice tested in the same session should be hung in turn as quickly as possible, and the hanging of the 4 mice should be completed within 10 seconds.

8. The tail suspension test method for assessing behavioral despair in mice after stroke, as described in claim 6, is characterized in that: The video recording duration in step (8) is 6 minutes; the video recording duration in step (6) is 11 seconds.

9. The tail suspension test method for assessing behavioral despair in mice after stroke, as described in claim 6, is characterized in that: During each test, the backlight of the suspension chamber was turned on and all other lights in the test chamber were turned off. Throughout the test, the backlight of the suspension chamber was kept as the only light source. Before each test, the suspension chamber was cleaned with odorless detergent and the inclined plate was thoroughly wiped with 70% ethanol to dispel the odor left by the mice that had been tested previously.

10. The tail suspension test method for assessing behavioral despair in mice after stroke, as described in claim 6, is characterized in that: In step (10), the analysis box is larger than the mouse body, and a space of one body width is left on the left and right sides of the mouse body in the analysis box.

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