Suspension tail testing device and method for evaluating behavioral despair of mice after stroke

Abstract

The invention discloses a tail suspension testing device and method for evaluating behavioral despair of a stroke mouse, an inclined plate is added below a suspension piece for suspending the mouse in a suspension chamber, the mouse is prevented from struggling due to dyskinesia so as to help the suspended mouse keep balance, even if the athletic ability of the mouse is damaged, the mouse can be prevented from shaking, and the mouse is prevented from being damaged. The method is especially suitable for evaluating despair feelings of mice with impaired motion coordination after stroke. Parts of weight of the mouse are supported by the inclined plate, pain caused during testing is light, and the device can be used for the mouse once the size of the device adapts to the mouse. The invention relates to an improved detection method for TST (mTST for short), which is used for evaluating the behavioral despair of mice after stroke, can be applied to the aspect of detecting the curative effect of antidepressant drugs in chronic mild stress (CMS) and post-stroke depression (PSD) model mice, and is beneficial to the fundamental research of diseases such as PSD and the like and the clinical transformation of drugs.

Description

Technical Field

[0001] The invention relates to a tail suspension test device and method for evaluating behavioral despair in post-stroke mice, and belongs to the technical field of behavioral despair experimental research in post-stroke mice. Background Art

[0002] Stroke is the third leading cause of death and disability worldwide. Patients recovering from stroke often show signs of mood and affective disorders, especially depression. The depressive state after stroke is defined as poststroke depression (PSD). Most affected individuals develop mild to moderate depression soon after the stroke, but depression may develop months or years later. The incidence of depression in stroke survivors is approximately one-third at one year. Patients with PSD are more likely to experience cognitive deficits, suicidal tendencies, and long-term disability. The onset of PSD may be influenced by multiple factors and is similar 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-choice antidepressant, after stroke6-8, leaving a gap in the clinical treatment of PSD. Further research is needed to identify causes and interventions to prevent and treat PSD.

[0003] Depressed mood and anhedonia are two basic symptoms of major depressive disorder (MDD), and at least one of them must be present along with the other key symptoms to be diagnosed with MDD. In the clinic, individual self-reports provide key information about depression. However, in the laboratory, it is difficult to determine whether a mouse is “depressed” or not. This is partly due to the fact that humans and mice cannot communicate through language. Fortunately, several behavioral tests have been established and widely used to assess depression-like states in rodents by briefly observing species-specific typical behaviors that resemble some aspects of human depression, such as the tail suspension test (TST) and forced swim test (FST) for despair, the sucrose preference test (SPT) for anhedonia, and the novelty suppressed feeding test (NSFT), open field test (OFT), and elevated plus maze test (EPMT) for assessing anxiety symptoms in depression-like states in mice. Although these tests have also been used by some researchers to assess depression-like behaviors in rodent PSD models, new problems have arisen in their application, especially when FST or TST is performed after a stroke in mice. Middle cerebral 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 choked on water due to lack of coordination in the FST or shook violently in the TST. The classic FST or TST are not suitable for assessing behavioral despair in rodents after severe stroke.

[0004] The tail suspension test (TST) is a classic method for assessing behavioral despair in mice and is commonly used in basic and preclinical studies of depression. However, the TST itself relies on motor function, and the test results may be misleading when applied to mice with severe motor coordination impairment, such as post-stroke depression model mice. In general, CMS simultaneously induces behavioral despair (increased immobility time compared with controls in FST and TST) and anhedonia (decreased sucrose preference compared with controls in SPT) in mice, as shown in Figure 2. Figure 1 However, we found that ischemic mice had reduced sucrose preference in the SPT but similar immobility times in the FST and TST compared with control mice ( Figure 1 ad). MCAO-induced motor impairment ( Figure 1 e,f) could explain this difference, while MCAO did not affect motor activity ( Figure 1 g). Impaired motor function makes it difficult for mice to coordinate when hanging or swimming, so mice instinctively struggle for balance rather than survival, resulting in reduced immobility time. In addition, the deep learning framework DeepLabCut35 was used to perform markerless pose estimation on TST videos ( Figure 1 h) The characteristics of the hind paw trajectories of MCAO mice were found. The two hind paw trajectories of the control and CMS mice were relatively separate, but the trajectories of MCAO mice overlapped greatly, further supporting the unbalanced movements of MCAO mice in TST ( Figure 1 i). In conclusion, the classical TST and FST provide misleading information when used to assess behavioral despair in post-stroke mice. Summary of the invention

[0005] The purpose of the present invention is to provide a tail suspension test device and method for evaluating behavioral despair in mice after stroke. The device and method are a modified method based on the classic TST, which avoids the interference of impaired motor function and can be used to evaluate behavioral despair in mice after stroke, so as to solve the technical problem that the FST or TST in the prior art is not suitable for evaluating the behavioral despair of rodents after severe stroke.

[0006] The present invention discloses a tail suspension test device for evaluating behavioral despair of mice after stroke, which adopts the following technical scheme: a tail suspension test device for evaluating behavioral despair of mice after stroke, comprising a hanging chamber, the front side of the hanging chamber being a transparent plate, a camera being arranged in front of the outside of the hanging chamber, a hanging piece for suspending mice being installed on the top of the hanging chamber, a pressure sensor being connected to the hanging piece, an inclined plate assembly being arranged inside the hanging chamber, the inclined plate assembly comprising an inclined plate and a sliding support member arranged in an oblique direction, the sliding support member being arranged at the bottom of the inclined plate to support the inclined plate, the inclined plate being inclined upward from front to back and being located below the hanging piece, and the angle between the inclined plate and the horizontal plane being 40°-80°.

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

[0008] The locking piece includes a U-shaped piece, the opening of the U-shaped piece faces the corresponding guide rail groove and is stuck on the guide rail groove, the lower side of the U-shaped piece is pressed against the bottom plate, the upper side of the U-shaped piece is located above the guide rail, and a threaded hole is provided on the upper side of the U-shaped piece, in which a bolt for tightening the guide rail is installed.

[0009] The front end of the bottom plate is provided with a handle for adjusting the position of the inclined plate.

[0010] The left and right side surfaces and the top surface of the hanging chamber are all opaque plates, the rear side surface and the bottom surface of the hanging chamber are all white luminous back panels, and the hanging piece is a hook.

[0011] The tail suspension test method for evaluating the behavioral despair of mice after stroke of the present invention adopts the following technical scheme: A tail suspension test method for evaluating the behavioral despair of mice after stroke, which adopts the above-mentioned tail suspension test device, comprises the following steps: (1) cutting a piece of tape, which is used to fix on the mouse tail; (2) placing the mouse on the test table, allowing it to move freely, marking a distance of 3 cm from the end of the mouse tail, straightening the mouse tail, fixing one end of the tape cut in step (1) on the ventral side of the mouse tail, and fixing the other end on the dorsal side of the mouse tail, the length of the two ends of the tape pasted on the mouse tail is equal, both are the length of the end of the mouse tail to the 3 cm section, and gluing the two ends of the tape together. (3) Weigh the mouse with the tape and record its weight as X grams; (4) Hang the same litter of mice with the tape in the hanging chamber, hang the tape loop on the hanging fixture, keep the mouse's back facing the camera, move the inclined board until the mouse's front paws can 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. Lock the inclined board in this fixed position; (5) Repeat step (4) with the same mouse until the other hanging chambers are debugged, take the mouse out of the hanging chamber, remove the tape, touch the mouse and put it back to the 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 hang the experimental test mouse in the hanging chamber, keep the mouse's back facing the camera head, ensure that the suspended mouse can only touch the surface of the inclined board with its front paws, and the number displayed by the pressure sensor is between X-1 and X grams; (8) Record the video of the mouse for a fixed length of time, avoiding noise, light and significant environmental changes during the test; after the video recording is completed, remove the mouse from the hanging chamber and remove the tape on the mouse's tail. Touch the mouse and return it to its original cage; (9) Repeat the test procedure, that is, repeat the above steps (7)-(8) until all the 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 frame. The analysis frame is the area covered by the software analysis. Make sure that the analysis frame can accommodate the body of the mouse at any time point in the video recorded in step (8); use the behavior analysis software to perform automatic behavior analysis; (11) Step (10) obtains the behavior analysis results of the mouse, the analysis results include the mouse's immobility, movement and other times, export the behavior analysis results, and compare the immobility time of different mice.

[0012] There are 4 hanging chambers, which are used for the tail suspension test of mouse behavior despair at the same time; in step (7), a tape ring is attached to the tail of each mouse, and 4 mice are hung in the 4 hanging chambers in turn, with the backs of the mice 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 duration of the video recorded in step (8) is 6 minutes; in step (6), the duration of the video recorded is 11 seconds.

[0014] During each test, turn on the illuminated back panel of the hanging chamber and turn off other lights in the test room. Keep the illuminated back panel of the hanging chamber as the only light source throughout the test. Before each test, clean the hanging chamber with odorless detergent and wipe the inclined plate thoroughly with 70% ethanol to dispel the odor left by the mice tested previously.

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

[0016] The beneficial effects of the present invention are as follows: the present invention is a modified TST detection method (mTST for short), which is used to evaluate the behavioral despair of mice after stroke. An inclined board is added under the hanging piece for hanging mice in the hanging chamber to prevent mice from struggling due to motor coordination disorders, so as to help the hanging mice maintain balance, and even if their motor ability is impaired, the mice can be prevented from shaking. The present invention is particularly suitable for evaluating the despair of mice with impaired motor coordination after stroke. The present invention supports part of the weight of the mice through the inclined board, and the pain caused during the test is relatively light, and once the size of the device is adapted to rats, it can also be used for rats. The present invention can be used in detecting the efficacy of antidepressant drugs in chronic mild stress (CMS) and post-stroke depression (PSD) model mice, which is beneficial to the basic research of diseases such as PSD and the clinical transformation of drugs.

[0017] The test procedure for evaluating the behavioral despair of mice in the present invention is simple, and the behavioral immobility of mice during the test is objective and easy to identify. The present invention has all the advantages of the classic TST. Compared with the FST, it does not cause hypothermia in mice. Once the mice are released from the experiment, they will immediately resume normal spontaneous activity without special post-experimental treatment. In addition, the FST is more suitable for rats than mice because rats are better at swimming than mice. The cost of using mice in the TST is much lower than using rats in the FST, and the TST can also be used for the study of transgenic animals. More importantly, the mTST procedure of the present invention overcomes the influence of impaired motor coordination on the misjudgment of experimental results in the classic TST, and can be used to evaluate the behavioral despair of mice after stroke. The use of the inclined board in the present invention also prevents the hanging mice from twisting or rotating their bodies and keeps the back of the mice facing the experimenter or the camera. Therefore, during the mTST test of the present invention, the movement of the mouse limbs is easy to observe, and the mouse prefers to put its front paws on the inclined inclined board instead of climbing up with its tail, solving the climbing pole problem in the TST. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is an experimental diagram that the classic TST and FST are not suitable for evaluating behavioral despair in mice after stroke; Figure 2 This is an experimental diagram of the effect of the inclined board angle on the immobility time of mTST; Figure 3 It is the equipment design drawing of mTST; Figure 4 is a schematic diagram of mouse suspension in mTST; Figure 5 This is a diagram of the mTST experiment to detect behavioral despair in mice exposed to MCAO or CMS; Figure 6 This is a diagram of the mTST experiment to detect behavioral despair in mice exposed to PSD; Figure 7 is an overall schematic diagram of a tail suspension test device for evaluating behavioral despair in mice after stroke according to the present invention; Figure 8 yes Figure 7 Schematic diagram of the pressure sensor; Fig. 9 yes Figure 7 Schematic diagram of the sliding support; Fig.10 yes Figure 7 Schematic diagram of the middle inclined plate and the bottom plate; In the figure: 1-suspension chamber, 2-guide rail, 3-locking piece, 4-inclined plate, 5-handle, 6-hook, 7-pressure sensor, 8-conversion module, 9-conversion module main control board, 10-liquid crystal display, 11-zero adjustment button. DETAILED DESCRIPTION

[0019] The following is a detailed description of embodiments of the present invention, but the present invention can be implemented in many different ways as defined and covered by the claims.

[0020] like Figures 7 to 10 As shown, a tail suspension test device for evaluating the behavioral despair of mice after stroke according to an embodiment of the present invention comprises a hanging chamber 1, the front side of the hanging chamber 1 is a transparent plate, the left and right side surfaces and the top surface of the hanging chamber 1 are opaque plates, the rear side surface and the bottom surface of the hanging chamber 1 are white luminous back plates, a camera is arranged in front of the hanging chamber, and a hanging piece for hanging mice is installed on the top of the hanging chamber 1, and the hanging piece is a hook 6. A pressure sensor 7 is connected to the hanging piece, and an inclined plate assembly is arranged inside the hanging chamber 1, and the inclined plate assembly comprises an inclined plate 4 and a sliding support member arranged in an oblique direction, and the sliding support member is arranged at the bottom of the inclined plate to support the inclined plate 4, the inclined plate 4 is inclined upward from front to back and the inclined plate 4 is located below the hanging piece, and the angle between the inclined plate 4 and the horizontal plane is 40°-80°, and the angle between the inclined plate 4 and the horizontal plane is 60° as a preferred embodiment, and the inclined plate 4 is a transparent plate.

[0021] The bottom of the inclined plate 4 is connected to a bottom plate, and the sliding support member includes guide rails 2 respectively arranged on the left and right sides of the bottom plate, the bottom 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, and the inner sides of the two guide rails 2 close to each other are provided with guide rail grooves, and the two sides of the bottom plate are respectively slidably installed in the guide rail grooves, and each guide rail groove is respectively provided with a locking member 3 for locking the bottom 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 stuck on the guide rail groove, the lower side of the U-shaped member is pressed against the bottom plate, the upper side of the U-shaped member is located above the guide rail, and 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.

[0022] A handle 5 is provided at the front end of the bottom plate for adjusting the position of the inclined plate.

[0023] The requirements for each component in this embodiment are as follows: Guide rail: Material requirements: smooth and supportive, such as stainless steel or thickened acrylic plate.

[0024] Locking parts: material requirements, easy operation, no sliding after limit fixing, such as adjusting nuts.

[0025] Inclined board: Material requirements: transparent and smooth, supportive, and not shaky. Such as acrylic board.

[0026] Handle: Material requirements, transparent and silky, such as acrylic sheet.

[0027] Hook: Material requirements: smooth, strong and non-elastic, such as stainless steel.

[0028] Pressure sensor: can be assembled with the hook, with a recommended accuracy of 0.1g, a mouse adaptation range of 0~100g, and a rat adaptation range of 0~500g. High sensitivity, LCD real-time display reading, reading can be reset to zero. The minimum requirement is an accuracy of not less than 1.0g. The pressure sensor 7 is connected to the conversion module 8, the conversion module main control board 9, the LCD display 10, and the zero adjustment button 11 through the circuit.

[0029] Placing a slanted board under the hanging mouse can provide support for the mouse ( Figure 2 a) Prevent MCAO (middle cerebral artery occlusion) mice from struggling to maintain balance. When the board was tilted to 60°, the immobility time of the sham group was about 95 seconds, while that of the MCAO group was about 194 seconds, showing a significant difference between the two groups ( Figure 2 b). When the inclined board 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 the 80° inclined board with their front paws, but they can stand on the 40° board and lean on the 80° board with their front and hind paws ( Figure 2 a). Therefore, the inclined board at 40° or 80° provides a relatively comfortable support for the mouse, greatly alleviating the despair caused by hanging. The preferred embodiment of the present invention adopts the inclined board at 60°.

[0030] A tail suspension test method for evaluating behavioral despair in mice after stroke according to an embodiment of the present invention is performed using the suspension chamber in the above test, and comprises the following steps: (1) Cut a piece of tape to fix it on the mouse's tail; (2) Place the mouse on the test bench and allow it to move freely. Mark a distance of 3 cm from the end of the mouse's tail, straighten the mouse's tail, and 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 pasted on the mouse's tail are equal, both of which are the length from the end of the mouse's tail to the 3 cm section. Glue the two ends of the tape together and ensure that the mouse's tail is tightly wrapped by the tape, and a loop is formed in the middle of the tape. (3) Weigh the mouse with the tape attached and record its weight as X grams; (4) Hang the mouse with tape in the hanging chamber, hang the tape ring on the hanging fixture, keep the mouse's back facing the camera, move the inclined board until the mouse's front paws can 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 the inclined board is locked in this fixed position; (5) Repeat step (4) with the same mouse until the other hanging chambers are debugged. Take the mouse out of the hanging chamber, remove the tape, touch the mouse and return it to its original cage; (6) Open the preview window of the behavior recording software on the computer connected to the camera, adjust the camera parameters to obtain a clear video, and record an 11-second video of the mouse-free hanging chamber background; (7) Repeat steps (1) to (3), and then hang the experimental test mouse in the hanging chamber, keeping the mouse's back facing the camera head, ensuring that the suspended mouse can only touch the surface of the inclined board with its front paws, and the number displayed by the pressure sensor is between X-1 and X grams; (8) Perform video recording of the mice for a fixed length of time, 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 on the tail of the mice, touch the mice, and put them back to their original cages; (9) Repeat the test procedure, i.e., repeat the above steps (7) to (8) until all mice to be tested have completed the test; (10) Select the video captured in step (8) for analysis, open the video recorded in the experiment in the behavior analysis software, and set the analysis frame, which is the area covered by the software analysis. Make sure that the analysis frame can accommodate the body of the mouse at any time point 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, which include the immobility, movement and other times of the mice. The behavioral analysis results are exported and the immobility times of different mice are compared.

[0031] Preferably, in this embodiment, there are four hanging chambers, and the angle between the inclined plate and the horizontal plane in each hanging chamber is 60°. The four hanging chambers are used for the tail suspension test of mouse behavior despair at the same time; in step (7), a tape ring is attached to the tail of each mouse, and four mice are hung in four hanging chambers in turn, with the back of the mouse facing the camera each time; the hanging standard of each mouse should be consistent with that in step (4), and the four mice tested in the same session should be hung in turn as quickly as possible, and the hanging of the four mice should be completed within 10 seconds.

[0032] During each test, turn on the illuminated back panel of the hanging chamber and turn off other lights in the test room. Keep the illuminated back panel of the hanging chamber as the only light source throughout the test. Before each test, clean the hanging chamber with odorless detergent and wipe the inclined plate thoroughly with 70% ethanol to dispel the odor left by the mice tested previously.

[0033] The present invention is described below in conjunction with specific experimental processes: 1. Experimental Materials Animals: Adult male C57BL / 6 mice.

[0034] CRITICAL: The age and sex of mice may affect immobility, so mice used in each independent experiment should be of the same sex and of similar age (e.g., 9 to 10 weeks old). In addition, mice of similar age are of similar shape and size, which allows the experimenter to avoid adjusting the position of the inclined board between tests of different mice.

[0035] Animal husbandry and handling: Three to four mice are housed per cage with free access to food and water. The housing room should maintain a constant temperature (21 ± 2°C), humidity (55 ± 10%) and a standard 12-hour light-dark cycle. Mice should be handled at least daily for one week before starting the mTST procedure of the present invention to acclimate them to the experimenter. If mice undergo the CMS procedure, each mouse is handled for 2 minutes every day after daily stress. If mice undergo the MCAO procedure, daily handling should be suspended on the first and second days after surgery. When handling mice, experimenters should not wear strong odors such as strong perfumes, which may cause disgust or interfere with behavioral assessments.

[0036] Reagents: 70% ethanol, odorless detergent, medical tape.

[0037] Reagent preparation: Medical tape should be strong enough to bear the weight of the hanging mouse, but soft enough to avoid hurting the mouse. Cotton tape is recommended. Prepare tape segments before each test. Cut a 16 cm long (2.0 cm wide) tape segment, fold the 16 cm tape in half along the long axis, and tape the middle 10 cm together (see Figure 4 a). The 3 cm portion of each end will be used to attach the tape to the mouse's tail. Do not prepare all of the tape segments too early in advance, as the exposed 3 cm end of the tape segments will lose stickiness over time.

[0038] 2. mTST detection method of the present invention - operating procedures (steps 1 to 21 below): 1. Animal adaptation: time: 1 hour: 1. Ensure constant temperature (21 ± 2°C) and humidity (55 ± 10%) in the laboratory.

[0039] 2. One hour before the test, place the mice in a corner of the testing room for acclimatization.

[0040] Note: Regarding circadian rhythm, it is recommended to test all mice in independent experiments in the same morning (9:00-12:00) or the same afternoon (14:00-17:00). On a device with 4 hanging chambers, approximately 48 mice can be tested in 3 hours. If the number of mice in independent experiments exceeds 48 and the experimenter needs to test in both the morning and afternoon, the mice assigned to the morning test should be acclimated at 8:00-9:00 and the mice assigned to the afternoon test should be acclimated at 13:00-14:00.

[0041] (ii) Equipment debugging: time 0.5 hours.

[0042] While acclimating the mouse (step 2), the experimenter can simultaneously commission the equipment (steps 3-7).

[0043] 3. Turn on the background light of the hanging room and turn off other lights in the test room. During the entire test process, keep the background light of the hanging room as the only light source.

[0044] 4. Wrap the mouse's tail with 16 cm of tape and weigh the mouse according to the procedure described in steps 9-11 (the mouse weight is recorded as X grams). Hang the mouse on the hook and attach the tape to its tail ( Figure 4 ad), keep the mouse's back facing the camera. Pull or push the inclined board along the track until the mouse's front paws just touch the surface of the inclined board ( Figure 4 f). At the same time, the digital display of the pressure sensor will flash between X and X-1 grams, indicating the successful positioning of the ramp. Once the ramp is in place, lock the locking piece on the guide rail.

[0045] Note: The trial mouse used in this step should not be included in the formal experiment. In other words, an additional mouse is needed for the adjustment of the inclined board. This mouse should be a littermate of the mice included in the formal experiment to ensure that the inclined board position determined with this mouse is appropriate for the mice in the formal experiment.

[0046] Critical Steps: The positioning of the inclined board is critical to the success of the mTST. Usually, the position of the inclined board is universal for independent experiments because mice of the same age are similar in size. If there is a large deviation in the weight of the mice, the inclined board position is not appropriate and needs to be adjusted in time. The adjustment standard is the pressure sensor reading. The experimenter should determine the appropriate position by carefully observing the distance between the inclined board and the mouse. If the inclined board does not touch the mouse ( Figure 4 e), the suspended mouse is not supported and the digital display of the pressure sensor will flash between X and X + 1 g. If the inclined board is too close to the mouse ( Figure 4g), the mouse will lean and rest on the board, and the digital display of the pressure sensor will flash between X-1 and X-2 grams. The pressure changes detected by the pressure sensor will help the experimenter determine the appropriate position of the tilt board.

[0047] 5. Open the preview window of the computer behavior recording software and adjust the camera parameters to obtain a clear video of the mouse.

[0048] 6. Remove the mouse from the hook and record an 11-second video of the mouse-free hanging chamber background.

[0049] WARNING: Do not remove the tape on the mouse until after preparing the last hanging chamber. Pet the mouse and return it to its original cage.

[0050] IMPORTANT: This video will be used to analyze the noise background during the immobility period (step 20). Take a background video for each hanging chamber of the multichannel apparatus. It is recommended to take a background video before testing in each independent experiment and not to share the same background video between different experiments.

[0051] 7. For the preparation of other hanging chambers, repeat steps 4-6.

[0052] (iii) Tail suspension test: The test time varies according to the number of animals tested; each session lasts 15 min (4 mice per session).

[0053] 8. Before each test, clean the hanging chamber with unscented dishwashing liquid and wipe the inclined plate thoroughly with 70% ethanol to dissipate the odor left by previously tested mice.

[0054] Critical Step: Thorough cleaning will remove all olfactory traces that may attract or scare the mice being tested and avoid negatively affecting mouse behavior.

[0055] 9. Cut a 16 cm long tape (2.0 cm wide), fold the 16 cm long tape in half along the long axis, glue the middle 10 cm together, and leave 3 cm at each end to fix the tape on the tail ( Figure 4 a).

[0056] KEY: Note that the length of the tape piece should be determined by the distance between the hanging hook and the chamber floor. Reference: When the height of the hanging chamber is 38 cm, we choose a length of 16 cm so that the mouse can be suspended at a height of approximately 30 cm above the chamber floor.

[0057] 10. Touch the mouse for 10 seconds, place it on the test bench, 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 the 16 cm long tape in half, fix one end of the tape (3 cm) on the ventral side of the mouse's tail, and the other end (3 cm) on the dorsal side of the mouse's tail, and then glue the two ends of the tape together ( Figure 3 b) Make a loop with the 16 cm long tape ( Figure 3 c). Gently pinch the two ends of the adhesive tape with your fingers to ensure that the mouse tail is tightly wrapped by the tape.

[0058] CRITICAL STEP: The mouse tail should be wrapped straight in tape, ensuring the mouse is suspended by a straight tail. Suspension by a bent tail is painful for the mouse and should be avoided.

[0059] CRITICAL: The tape should be applied 3 cm from the end of the mouse's tail. Hanging by a shorter end of the tail (e.g., 1 cm) is painful for mice and should be avoided. Although hanging by the base of the tail is more comfortable for mice, the likelihood of tail climbing increases significantly.

[0060] 11. Weigh the tape and mouse and place the mouse back in the cage. The weight will be used as a reference in step 14.

[0061] 12. Repeat steps 9-11 to test 3 more mice in the same test. Number them 1 to 4. Clearly record the group and number of each mouse in each test.

[0062] CRITICAL: Each group of mice in each independent experiment should be tested simultaneously in each mTST experiment. Mice from the same group should be tested according to the randomization scheme generated by the computer program. The position of mice in the 4 chambers should be rotated so that mice from the same group are placed in different hanging chambers in each experiment.

[0063] CRITICAL: Do not proceed to step 13 until all mice to be tested in the same field are ready to be hung.

[0064] 13. Hang the first mouse in the first hanging chamber with the tape loop attached to its tail ( Figure 4 cd), with 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 the fourth mouse in the fourth hanging chamber.

[0065] Critical Step: The hanging criteria for each mouse should be consistent with that in step 4, otherwise the position of the inclined board determined in step 4 will not be appropriate for the mice tested subsequently. For example, the tape loop should be placed at the bottom of the hook. It is recommended that the same experimenter perform steps 4 and 14.

[0066] CRITICAL: Four mice tested in the same session should be suspended in sequence as quickly as possible. Using the tape loop, we usually complete the suspension of 4 mice within 10 seconds.

[0067] CRITICAL: It is recommended to hang the mice in order so that the recorded videos are easy to match with the corresponding mice.

[0068] 14. Check the position of the inclined board for each mouse in the hanging chamber. Make sure that the hanging mouse can only touch the surface of the inclined board with its front paws ( Figure 4 f). At the same time, the digital display of the pressure sensor should show a pressure approximately equal to the weight in step 11 minus 1 gram.

[0069] Critical Step: The correct position of the inclined board is critical for the modified tail suspension test (mTST). The position of the inclined board is adjusted according to step 4. If the inclined board does not touch the mouse ( Figure 4 e), the hanging mouse will not be supported. If the inclined board is too close to the mouse ( Figure 4 g), the mouse will lean and rest on the board. Figure 4 f) to ensure the body balance and helpless state of the mice during the test.

[0070] CRITICAL: This step should be completed as quickly as possible. In most cases, the inclined board does not need to be adjusted again after it is positioned in step 4. If adjustment is necessary, the position of the inclined board should be adjusted within 10 seconds. Unlock the locking pieces and pull or push the inclined board until the mouse's front paws just touch the inclined board surface. Note that adjustments will extend the suspension time before the actual test. Once the mouse is suspended, recording should be started as soon as possible. It is recommended that additional adjustments should not exceed 1 minute.

[0071] 15. Open the preview window of the behavior recording software Depression software (Clever Sys, Inc.) to ensure that every mouse movement is clearly captured. Set the recording time to 6 minutes and start recording.

[0072] Critical Steps: Avoid noise, light, and significant environmental changes during testing. Four mice tested simultaneously should be auditorily and visually isolated.

[0073] 16. When the video recording is finished, remove the mice from the hooks, gently remove the tape from their tails, and place them back into their corresponding cages.

[0074] 17. Save each video in the same folder with a file name that identifies the group and mouse number.

[0075] 18. Repeat the testing procedure (steps 9-18) until all mice have completed the test.

[0076] 4. Data analysis (depending on the number of videos, the time will vary): 19. Select the background captured in step 6 for analysis. Open the video recorded in the experiment in the behavioral analysis software and set the analysis frame, which is the area covered by the software analysis, to ensure that the analysis frame can accommodate the body of the mouse at any time point in the 6-minute video.

[0077] Important: Information outside the analysis box will not be read during analysis, so the analysis box should be defined larger than the mouse body, leaving a body width of space on both sides of the mouse body. No mice have been found to struggle outside of such an analysis box.

[0078] Important: Do not share analysis frames and background settings between different experiments.

[0079] 20. Select the folder for the experiment and start the automated behavior analysis using the parameters set in step 19.

[0080] 21. Export the results of behavioral analysis (immobility, movement, and others) to Excel and calculate the immobility time of mice within 5 minutes after the test phase as an indicator of behavioral despair.

[0081] The experimental time is arranged as follows: (1) Steps 1-2, animal adaptation: 1 hour; (2) Steps 3-7, equipment debugging: 0.5 hours (can be performed simultaneously with steps 1-2); (3) Steps 8-18, tail suspension test: depending on the number of mice tested; each session is 15 minutes (4 mice per session); (4) Steps 19-21, data analysis: depending on the number of videos, each video analysis takes about 3 minutes.

[0082] 3. Experimental Results In the classic TST, the escape behavior of mice can be divided into three types: running, body twisting, and body twitching. In the modified mTST, due to the support of the inclined board, body twisting and body twitching of mice are rarely observed. The movement type of mice is almost entirely running, or even uncoordinated running of the limbs. Because the right hemisphere is exposed to ischemia when the MCAO model is prepared, the left hind limbs show obvious motor dysfunction when the mice are suspended in the mTST. If ischemia occurs in the left hemisphere, the right hind limbs will be mainly damaged. Although the movement characteristics of ischemic mice are unique, they are closely related to impaired motor function. Just like in the classic TST, the immobility time during the mTST test in the present invention is a key indicator of behavioral despair, which can be easily read from the video by behavioral analysis software. It is recommended to calculate the immobility time of the last 5 minutes of the 6-minute mTST test. The immobility latency is not reported in the classic TST, and it is not recommended to report it in the mTST.

[0083] To verify 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 subjected to mTST one week later ( Figure 5 a). The results showed that the immobility time in the MCAO group was significantly increased compared with the sham operation group ( Figure 5b). In addition, a single ketamine treatment reduced the immobility time in MCAO mice, indicating a rapid antidepressant effect ( Figure 5 b). Thus, the mTST assay is effective in measuring behavioral despair in post-stroke mice and is sensitive to antidepressant treatment. Markerless pose estimation from videos using the deep learning framework DeepLabCut35 revealed that the trajectory of each paw during the mTST was concentrated ( Figure 5 c), which is different from the scattered trajectory in the classical TST ( Figure 1 i). This indicates that the mice moved more regularly and symmetrically during the mTST. Even for MCAO mice, the overlap of the two hind paw trajectories caused by uncoordinated movements was greatly reduced during the mTST ( Figure 5 c). mTST was then measured repeatedly on days 7, 14, and 28 after MCAO ( Figure 5 d). The results showed that the immobility time of MCAO mice was longer than that of sham-operated mice on days 7 and 14 ( Figure 5 e), indicating the feasibility of repeated measurements at weekly intervals. However, on day 28, there was no significant difference in the immobility time between the sham and MCAO groups. The possible reason is the spontaneous recovery of the depressive-like behavior of MCAO mice, because the sucrose preference of the SPT on day 28 was similar between the sham and MCAO groups ( Figure 5 f). However, we cannot exclude the possibility that repeated measurements reduced intergroup differences and therefore recommend not repeating the mTST more than three times in the same batch of mice. We also demonstrated the utility of the mTST protocol for assessing behavioral despair in mice exposed to CMS, a classic model. Figure 5 As shown in Figure 3, the increase in immobility time caused by CMS and the decrease in immobility time caused by ketamine treatment can be revealed by mTST as well as the classical TST. It is worth noting that the immobility time of each group in the mTST was slightly greater than that in the classical TST. This may be a reasonable result due to the support of the inclined board in the mTST. Finally, whether the mTST protocol is applicable to the PSD mouse model was examined. Figure 6 a, PSD was induced in 9- to 10-week-old C57BL / 6 mice by MCAO (day 0) and CMS (days 7 to 36), and chronic fluoxetine treatment was given from day 7 to 36. The grid walking task and rotarod test demonstrated impaired motor function in PSD mice 2 and 5 weeks after MCAO ( Figure 6 b, c). Using the mTST detection method, we successfully found differences in behavioral despair between the sham operation group and the PSD group at two time points ( Figure 6 d,e). In addition, the antidepressant effect of fluoxetine was clearly observed after 4 weeks of treatment, but not after 1 week of treatment, as evidenced by a reduction in immobility time ( Figure 6d,e). Therefore, the mTST assay can also be used to measure behavioral despair in PSD model mice and evaluate the effects of chronic treatment with antidepressants.

[0084] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A tail suspension test device for evaluating the behavioral despair of mice after stroke, comprising a suspension chamber, the front side of the suspension chamber being a transparent plate, a camera being arranged in front of the outside of the suspension chamber, a suspension piece for suspending mice being installed on the top of the suspension chamber, and a pressure sensor being connected to the suspension piece, characterized in that: An inclined plate assembly is provided inside the suspension chamber, and the inclined plate assembly includes an inclined plate and a sliding support member arranged at an angle. The sliding support member is arranged at the bottom of the inclined plate to support the inclined plate. The inclined plate is inclined upward from front to back and is located below the hanger. The angle between the inclined plate and the horizontal plane is 40°-80°.

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

3. The tail suspension test device for evaluating behavioral despair in post-stroke mice according to claim 2, characterized in that: The locking piece includes a U-shaped piece, the opening of the U-shaped piece faces the corresponding guide rail groove and is stuck on the guide rail groove, the lower side of the U-shaped piece is pressed against the bottom plate, the upper side of the U-shaped piece is located above the guide rail, and a threaded hole is provided on the upper side of the U-shaped piece, in which a bolt for tightening the guide rail is installed.

4. The tail suspension test device for evaluating behavioral despair in post-stroke mice according to claim 2, characterized in that: The front end of the bottom plate is provided with a handle for adjusting the position of the inclined plate.

5. The tail suspension test device for evaluating behavioral despair in post-stroke mice according to claim 1, characterized in that: The left and right side surfaces and the top surface of the hanging chamber are all opaque panels, the rear side surface and the bottom surface of the hanging chamber are all white luminous back panels, the hanging piece is a hook; the angle between the inclined plate and the horizontal plane is 60°.

6. A tail suspension test method for evaluating behavioral despair in mice after stroke, which uses the tail suspension test device described in claim 1, characterized in that: The following steps are involved: (1) Cut a piece of tape to fix it on the mouse's tail; (2) Place the mouse on the test bench and allow it to move freely. Mark a distance of 3 cm from the end of the mouse's tail, straighten the mouse's tail, and 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 pasted on the mouse's tail are equal, both of which are the length from the end of the mouse's tail to the 3 cm section. Glue the two ends of the tape together and ensure that the mouse's tail is tightly wrapped by the tape, and a loop is formed in the middle of the tape. (3) Weigh the mouse with the tape attached and record its weight as X grams; (4) Hang the same litter of mice with tape in the hanging chamber, hang the tape loop on the hanging fixture, keep the mouse's back facing the camera, move the inclined board until the mouse's front paws can 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; (5) Repeat step (4) with the same mouse until the debugging of other hanging chambers is completed, take the mouse out of the hanging chamber, remove the tape, touch the mouse and put it back to the original cage; (6) Open the preview window of the behavior recording software on the computer 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 a mouse; (7) Repeat steps (1) to (3), and then hang the experimental test mouse in the hanging chamber, keeping the mouse's back facing the camera head, ensuring that the suspended mouse can only touch the surface of the inclined board with its front paws, and the number displayed by the pressure sensor is between X-1 and X grams; (8) Perform video recording of the mice for a fixed length of time, 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 on the tail of the mice, touch the mice, and put them back to their original cages; (9) Repeat the test procedure, i.e., 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 frame, which is the area covered by the software analysis, to ensure that the analysis frame can accommodate the body of the mouse at any time point in the video recorded in step (8); perform automatic behavior analysis using the behavior analysis software; (11) Step (10) obtains the behavioral analysis results of the mice, which include the immobility, movement and other times of the mice. The behavioral analysis results are exported and the immobility times of different mice are compared.

7. The tail suspension test method for evaluating behavioral despair in mice after stroke according to claim 6, characterized in that: There are 4 hanging chambers, and the 4 hanging chambers are used for the mouse behavioral despair tail suspension test at the same time; in step (5), a tape ring is attached to the tail of each mouse, and 4 mice are hung in the 4 hanging chambers in turn, with the backs of the mice 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 evaluating behavioral despair in mice after stroke according to claim 6, characterized in that: The duration of the video recorded in step (6) is 6 minutes; in step (9), the duration of the video recorded is 11 seconds.

9. The tail suspension test method for evaluating behavioral despair in mice after stroke according to claim 6, characterized in that: During each test, turn on the illuminated back panel of the hanging chamber and turn off other lights in the test room. Keep the illuminated back panel of the hanging chamber as the only light source throughout the test. Before each test, clean the hanging chamber with odorless detergent and wipe the inclined plate thoroughly with 70% ethanol to dispel the odor left by the mice tested previously.

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

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