Mouse multi-dimensional behavior linkage monitoring device and method based on von Frey stimulation

Abstract

The invention relates to a von Frey stimulation-based mouse multi-dimensional behavior linkage monitoring device and a von Frey stimulation-based mouse multi-dimensional behavior linkage monitoring method. The device comprises a fixing assembly, a plurality of acquisition modules and a data processing module. In the fixing assembly, fixing devices restrain the trunk in a mouse containing hole through movable clamping pieces, the head of a mouse moves freely, and a bearing base is mechanically connected with the multiple fixing devices. The stimulation parameter acquisition module records the von Frey stimulation intensity, the stimulation position and the stimulation duration time applied to the head and face of the mouse in real time; the facial action acquisition module is used for acquiring a head and face specific reaction caused by von Frey stimulation; the expression response acquisition module captures facial feature expression changes of the mouse based on the image; the trunk linkage behavior acquisition module acquires associated activities of the trunk of the mouse after the mouse is stimulated by von Frey; the tail action acquisition module is used for acquiring mouse tail specific reaction caused by von Frey stimulation; and the data processing module carries out time calibration on the various data and calculates multi-dimensional behavior linkage monitoring data of the mouse based on the various data.

Description

Technical Field

[0001] This invention relates to the field of mouse fixation technology, and in particular to a multidimensional behavioral linkage monitoring device and method for mice based on von Frey stimulation. Background Technology

[0002] Trigeminal neuralgia (TN) is a neuropathic pain disorder characterized by paroxysmal, severe pain in the head and face. Its pathogenesis is complex and not fully understood. Establishing stable and reliable animal models and conducting mechanosensitivity assessments are crucial for elucidating the disease mechanism and developing treatment options. In the mechanosensitivity assessment system for neuropathic pain, the von Frey test is widely recognized as the gold standard for assessing the mechanosensitivity threshold in animals due to its clear principle and relatively simple operation. This test involves applying nylon threads (von Frey threads) of varying stiffness to specific areas of the animal's body surface to induce nociceptive reflexes such as foot withdrawal, head retraction, and scratching. The 50% reflex threshold is calculated using algorithms such as Up-Down to quantify the animal's mechanosensitivity level. However, when applied to the mechanosensitivity assessment of the TN model, the inherent technical limitations of the von Frey test are significantly amplified because the pain location in the TN model is concentrated in the head and face, and the nature of the pain is highly specific. This creates a prominent contradiction with the need for accurate model assessment, resulting in several shortcomings.

[0003] Currently, the von Frey test suffers from a misalignment between reflex thresholds and pain experience. The core indicator of the von Frey test is the nociceptive reflex threshold of an animal to mechanical stimuli, but this threshold differs fundamentally from the animal's actual pain experience. Currently, the reflex threshold recorded by the test only characterizes the sensitivity of peripheral nociceptors and cannot comprehensively quantify the core characteristics of pain intensity, duration, and emotional association caused by trigeminal nerve injury in mice, leading to a deviation between the assessment results and the pathological nature of pain in the model.

[0004] Furthermore, technical limitations in the operational process led to high variability and low repeatability of the test data. The TN model's evaluation targets are concentrated in a small area of ​​the mouse's head and face, such as the cheek area innervated by the trigeminal nerve and the beard area. The anatomical structures of these areas are very delicate, which makes the von Frey test even more demanding on operational techniques. In routine operation, ensuring the consistency of stimulation parameters is not easy: on the one hand, the experimenter needs to manually apply the von Frey wire vertically to the target area. It is almost impossible to ensure that the application angle, loading speed, and duration are completely consistent across different test cycles and different operators. Even a small parameter deviation can have a significant impact. On the other hand, the target area of ​​the head and face is itself very small. Even a slight shift in the stimulation position, from the cheek skin to the hair follicle area at the root of the beard, will cause significant fluctuations in the response rate due to the different distribution density of nerve endings. These uncontrollable factors at the operational level, coupled with research indicating that the traditional up-down algorithm may have estimation errors, ultimately make it difficult to compare test data from different operators in the same laboratory or between different laboratories, thus greatly reducing the reliability of the results.

[0005] The data interpretation is clearly deceptive, stemming from the subjectivity and information loss inherent in the positive response criteria. The von Frey test's "50% retraction threshold" (e.g., 0.4g), calculated using an up-down algorithm, possesses a numerical appearance and is easily mistaken for an objective quantitative indicator. However, the generation of this indicator is significantly subjective. Research indicates that traditional up-down algorithms may suffer from estimation errors when parameters are improperly set, further impacting the objectivity of the results. In TN model assessments, the determination of a positive response relies entirely on the experimenter's subjective observation, with significant differences in the criteria for defining response intensity among different researchers. For example, slight head deflection and hesitant head retraction in mice, compared to violent head retraction accompanied by defensive behavior such as full-body struggling, are all routinely recorded as positive responses, but the pain intensity they reflect differs by orders of magnitude. Positive criteria can vary between different laboratories, and even among different researchers within the same laboratory, leading to extremely high inter-investigator variability. This black-and-white recording method loses a significant amount of information about the intensity and nature of pain. This black-and-white binary classification method not only leads to high variability among researchers, but also fails to capture the dynamic changes in pain (such as response latency and duration) and the differences in nature (such as the distinction between sharp and dull pain), making the numerical threshold results unable to truly represent the severity of pain in the model.

[0006] The singularity of assessment dimensions leads to an imbalance in the assessment of local reflexes and systemic responses. Trigeminal neuralgia (TN) is a local manifestation of a systemic disease. In addition to increased mechanosensitivity in the head and face, TN model mice often exhibit a series of systemic pain-related behaviors, such as reduced activity, abnormal grooming behavior, and social avoidance. However, the conventional von Frey test focuses solely on reflex behaviors in the target area of ​​the head and face, using the presence of head withdrawal / scratching as the sole assessment criterion, completely ignoring compensatory responses in other areas such as the trunk and limbs, as well as systemic behavioral changes. This localized assessment model results in an assessment system that only covers the local reflex dimension of pain response, lacking crucial information from the integrated dimension of systemic behavior. This fails to comprehensively and systematically characterize the pathophysiological state of the TN model, potentially leading to biased assessments of drug efficacy or intervention measures.

[0007] CN219089745U discloses an adjustable mouse restraint device, including a mouse restraint device cylinder and a restraint base. The adjustable mouse restraint device also includes an adjustable support rod, a restraint collar, a mouse adjustable ventilation cage, and a mouse tail-fixing cloth. The mouse tail-fixing cloth is fixed to the tail of the mouse restraint device cylinder. The adjustable support rod allows for height adjustment according to the operating environment, and the restraint collar allows for tightness adjustment. This improves upon existing mouse restraint devices, which are mostly fixed cylinders, requiring the mouse tail to be twisted or the operator to inject from the side during tail vein injection, thus reducing the success rate of tail vein injection. This utility model uses a mouse tail-fixing cloth for tail restraint, avoiding damage to the mouse tail, ensuring animal welfare, and improving the success rate of tail vein injection. However, this technical solution cannot specifically restrain the mouse's limbs and allow head movement, nor can it monitor multi-dimensional indicators after stimulation, or distinguish between pain and stimulus reflex behavior.

[0008] As described above, this invention proposes a multi-dimensional behavioral linkage monitoring device for mice based on von Frey stimulation. For von Frey mechanical stimulation experiments on the head and face of mice (such as whisker area, muzzle, cheek, etc.), it simultaneously realizes precise application of stimulation, acquisition of behavioral signals of the head, face and trunk, and linkage analysis of data, solving the problems of difficulty in matching stimulation intensity with behavioral response and inaccurate capture of subtle movements in traditional experiments.

[0009] Furthermore, on the one hand, there are differences in understanding among those skilled in the art; on the other hand, the applicant studied a large number of documents and patents when making this invention, but due to space limitations, not all details and contents were listed in detail. However, this does not mean that the present invention does not possess the features of these prior art. On the contrary, the present invention already possesses all the features of the prior art, and the applicant reserves the right to add relevant prior art to the background art. Summary of the Invention

[0010] To address the shortcomings of existing technologies, this invention provides a mouse multidimensional behavioral linkage monitoring device based on von Frey stimulation from a first aspect. The device includes a fixation component, a stimulation parameter acquisition module, a facial movement acquisition module, an expression response acquisition module, a trunk linkage behavior acquisition module, a tail movement acquisition module, and a data processing module.

[0011] The restraint system includes restraints and a support base. The restraints secure the mouse by confining its torso within a mouse hole via movable snap-fit ​​connectors, while allowing free head movement. The support base is mechanically connected to several restraints, which are arranged to isolate the visual field of adjacent mice. A stimulus parameter acquisition module records in real-time the intensity, location, and duration of the von Frey stimulus applied to the mouse's head and face, using these parameters as baseline parameters for behavioral response analysis. For head- and face-specific responses induced by von Frey stimulation, a facial motion acquisition module acquires the mouse's head rotation angle, whisker wagging amplitude, and the amplitude of muscle contraction vibrations induced by von Frey stimulation. An expression response acquisition module captures characteristic facial expression changes in mice based on images, including image recognition of eye socket tightening, nose and cheek puffing, and ear backward movement amplitude. The trunk linkage behavior acquisition module collects the associated trunk movements of mice after von Frey stimulation. Specifically, it collects the deformation of the trunk fixation support to determine the trunk torsion angle and the pressure changes on the mouse trunk against the support to determine the trunk stress response, thus aiding in the assessment of pain / tactile response intensity. The tail movement acquisition module targets the mouse's tail-specific response to von Frey stimulation, collecting the number and amplitude of tail twitches and the amplitude of tail wagging. Tail tension is collected based on the pressure caused by tail muscle tension. The data processing module performs time calibration on the various data transmitted from the stimulation parameter acquisition module, facial movement acquisition module, facial expression response acquisition module, trunk linkage behavior acquisition module, and tail movement acquisition module, and calculates multi-dimensional behavioral linkage monitoring data of the mice based on these data.

[0012] This technological solution, based on a von Frey stimulation-based multidimensional behavioral monitoring device for mice, has achieved several significant breakthroughs. By integrating a stimulation parameter acquisition module, the device can record in real-time the intensity, location, and duration of mechanical stimulation applied to the mouse's head and face. This overcomes the subjectivity and instability inherent in traditional experiments that rely on manual manipulation, making the stimulation process more standardized and quantifiable. Simultaneously, the device goes beyond simply observing behaviors like withdrawal. Through facial movement acquisition, expression response acquisition, trunk-related behavior acquisition, and tail movement acquisition modules, it simultaneously captures various behavioral signals, including head rotation angle, whisker swaying amplitude, eye socket tightening, nose and cheek puffing, ear retraction, trunk twisting, limb pressure changes, and tail twitching, swaying, and muscle tension. This enables multidimensional data acquisition, ranging from local responses to systemic stress. This information allows for assessments of mouse pain or tactile responses that go beyond a simple presence or absence, reflecting the intensity, dynamic changes, and behavioral patterns of the response, significantly improving the comprehensiveness and accuracy of the assessment.

[0013] In terms of data processing, this invention uses a data processing module to perform time calibration on data from different sensors, achieving millisecond-level time synchronization with the stimulus initiation point as a unified benchmark. This linked analysis method can clearly present the temporal relationship between stimuli and various behavioral responses, helping to identify the latency and duration characteristics of different responses, thus enabling a more scientific assessment of the mouse's true feelings. Furthermore, the design of the fixation components fully considers the physiological characteristics and behavioral needs of experimental animals. The fixator restrains the torso with movable clips but allows free head movement. Combined with a silicone support and ventilation structure, this ensures test stability while reducing additional stress from restraint. The support base can connect multiple fixators simultaneously and isolates adjacent mice with a field-of-view baffle, supporting batch testing to improve efficiency while avoiding mutual interference between animals. Overall, this device, through systematic structural design and multi-source data fusion, effectively overcomes the problems of inconsistent stimulus parameters, limited behavioral information, and strong subjectivity in traditional von Frey tests, providing more objective, refined, and reliable technical support for pain mechanism research and drug efficacy evaluation.

[0014] According to a preferred embodiment, the stimulation parameter acquisition module includes a stimulation intensity sensor, a stimulation location locator, and a stimulation timer. The stimulation intensity sensor is located at the end of the von Frey handle and determines the mechanical stimulation intensity in real time by acquiring the tensile force generated by the bending of the fibers when the fibers on the handle contact the mouse skin. The stimulation location locator is located on the surface of a silicone support for supporting the head and face, and identifies the stimulation location based on preset network coordinates. The stimulation timer starts recording the stimulation duration when the tensile force value acquired by the stimulation intensity sensor exceeds a tensile force threshold.

[0015] This technique integrates a stimulus intensity sensor, a position locator, and a timer to achieve full quantification and precise control of the von Frey stimulation process. The stimulus intensity sensor collects fiber bending tension in real time, ensuring objective and accurate mechanical stimulus intensity data; the position locator uses the grid coordinates on the surface of the silicone support to precisely locate the stimulation site, avoiding human error; and the timer is triggered by a tension threshold to accurately record the duration of the stimulus. The three components work together to effectively eliminate errors caused by subjective judgment in traditional operations, improving the repeatability and data reliability of the experiment, and providing accurate benchmark parameters for subsequent behavioral response analysis.

[0016] According to a preferred embodiment, the facial motion acquisition module includes a head rotation angle sensor, a whisker sway sensor, and a muscle activity sensor. The head rotation angle sensor is positioned at the temporal region of the mouse to acquire the head rotation angle in real time in both horizontal and vertical directions; the whisker sway sensor is positioned near the head of the restraint device, and its infrared illumination range covers the whisker swaying area, acquiring changes in infrared light flux caused by whisker swaying to determine the whisker swaying amplitude; the muscle activity sensor acquires the amplitude of muscle contraction vibrations at the muzzle and / or cheek induced by von Frey stimulation.

[0017] This technology utilizes a multi-sensor synergy to precisely quantify subtle head and facial behaviors in mice. A head rotation angle sensor captures real-time horizontal and vertical head movements, objectively recording avoidance or withdrawal responses; a whisker sway sensor detects minute whisker swaying using changes in infrared light flux, sensitively reflecting changes in sensory sensitivity; and a muscle activity sensor captures the vibration amplitude of the muzzle and cheek muscles, identifying pain-related actions such as biting and twitching. This approach overcomes the limitations of traditional manual observation, comprehensively and dynamically reflecting the intensity and behavioral characteristics of mice's local responses to stimuli, thus improving the objectivity and resolution of the data.

[0018] According to a preferred embodiment, the facial expression acquisition module includes an eye socket tightening action acquisition unit, a nose and cheek action acquisition unit, and an ear action acquisition unit. The eye socket tightening action acquisition unit is positioned on a support base corresponding to the top of the mouse's head and captures the outline of the mouse's eye socket using an imaging method capable of penetrating the mouse's eye hairs. This allows the data processing module to identify eye socket tightening actions based on a first distance change between preset eye socket feature points. The nose and cheek action acquisition unit is positioned at the mouse's nose and snout and both cheeks and acquires pressure values. This allows the data processing module to determine nose puffing actions and / or cheek puffing actions based on changes in pressure values ​​at the nose and snout and both cheeks. The ear action acquisition unit is positioned on a support base at a specified distance from the outer side of the mouse's ear and records a second distance change between the ear edge and the ear action acquisition unit by vertically illuminating the ear edge with a laser beam. This allows the data processing module to determine whether the ear has moved backward based on the second distance change.

[0019] This data acquisition method automates and quantifies the recognition of facial expression changes in mice. The eye socket contraction action acquisition unit uses hair-penetrating imaging to accurately capture changes in the eye socket contour; the nose and cheek action acquisition unit uses pressure sensors to monitor the bulging movements of the nose and cheeks in real time; and the ear action acquisition unit uses laser displacement technology to sensitively detect ear displacement. This allows for objective and continuous recording of pain-related facial features such as eye socket contraction, nose and cheek bulging, and ear displacement, avoiding the subjectivity of manual scoring. This method improves the sensitivity and reliability of pain assessment in mice, and is particularly suitable for scenarios where the head is restricted and full-body behavior cannot be observed, providing stable data support for non-invasive pain intensity assessment.

[0020] According to a preferred embodiment, the trunk-linked behavior acquisition module includes a trunk torsion sensor and a limb movement sensor. The trunk torsion sensor is a strain gauge installed in the trunk fixation bracket of the restraint device. When the mouse's trunk twists, the data processing module determines the trunk torsion angle based on the deformation degree of the strain gauge. The limb movement sensor is a pressure sensor installed in the trunk fixation bracket corresponding to the forelimbs and hindlimbs respectively in the restraint device. It collects pressure change data caused by the movements of the forelimbs and hindlimbs, allowing the data processing module to determine the trunk stress response based on the pressure change data.

[0021] This invention achieves precise quantification of trunk stress responses in mice through trunk torsion sensors and limb movement sensors. Strain gauges, embedded in a trunk fixation device, sensitively detect deformation during trunk torsion, thereby accurately calculating the torsion angle and reflecting the intensity of avoidance or defensive actions. Pressure sensors, corresponding to the forelimbs and hindlimbs respectively, collect real-time pressure changes caused by limb movement, capturing limb struggle or tension behaviors induced by stress. This setup allows the invention to objectively record trunk-related responses induced by von Frey stimulation, overcoming the limitations of assessment relying solely on facial behavior.

[0022] According to a preferred embodiment, the tail movement acquisition module includes a tail twitching acquisition unit, a tail wagging behavior acquisition unit, and a tail tension detection sensor. The tail twitching acquisition unit is located at the mid-distal end of the mouse tail and acquires the acceleration caused by tail vibration, allowing the data processing module to determine the tail twitching behavior based on the acceleration and its duration. The tail wagging behavior acquisition unit is located on the sidewall of the tail movement area within the restraint and forms an infrared light curtain perpendicular to the tail; when the mouse tail wags, it acquires the frequency of infrared light blocking to calculate the wagging amplitude. The tail tension detection sensor is located at the proximal end of the mouse tail and detects the pressure when the tail muscles are tense.

[0023] This invention achieves multi-dimensional and precise capture of mouse tail behavior using this technology. The tail twitching acquisition unit sensitively detects the intensity and duration of tail vibrations using an accelerometer, accurately identifying twitching behavior; the tail wagging behavior acquisition unit uses an infrared light curtain to record the frequency of tail wagging for calculating the wagging amplitude, achieving non-contact dynamic monitoring; and the tail tension detection sensor senses real-time pressure changes in the proximal muscles of the tail, reflecting the degree of muscle tension under stress or tension. The combination of these three technologies comprehensively quantifies the tail's movement characteristics and muscle activity, effectively supplementing facial and trunk behavioral information and improving the completeness of overall stress response assessment.

[0024] According to a preferred embodiment, the restraint includes a first fixing frame and a second fixing frame rotatably connected thereto; when the first fixing frame and the second fixing frame are assembled, a mouse-holding hole is formed inside the restraint; a movable locking member for locking the mouse below the neck is provided in the mouse-holding hole at the first end of the restraint corresponding to the mouse's head, so that the mouse can move its head; the movable locking member adjusts the locking position by a screw at the first end; a silicone support for supporting the head and face is provided at the first end of the restraint; a torso fixing support for fixing the mouse's torso is provided on the inner wall of the mouse-holding hole; a tail sealing plate and a tail sealing plate screw are provided at the second end of the restraint close to the mouse's tail, the tail sealing plate screw being used to adjust the position of the tail sealing plate to prevent the mouse from curling up.

[0025] This restraint device forms a mouse-holding hole through a rotatably connected first and second fixing frame, achieving a balance between stable confinement and localized freedom of movement for the mouse. Movable latches, combined with screw adjustments, precisely secure the area below the neck, allowing free head movement and facilitating the acquisition of head and facial behaviors in a natural state. A silicone support provides flexible support, reducing restraint stress. A trunk fixation support ensures trunk stability, facilitating accurate detection of behaviors such as twisting. The tail cap, adjusted by a screw, prevents tail curling from interfering with behavioral responses while preserving tail movement freedom. The overall structure balances restraint and physiological naturalness, improving the accuracy and repeatability of stimulus response acquisition, and is suitable for simultaneous multimodal behavioral monitoring.

[0026] According to a preferred embodiment, the first and second fixing frames of the fixture are provided with a plurality of ventilation holes to maintain ventilation conditions.

[0027] By incorporating ventilation holes in the first and second restraint frames of the restraint system, a well-ventilated environment was effectively maintained in the mice's space, reducing heat and stress caused by prolonged restraint and improving their comfort. Good ventilation helps stabilize physiological states, reduces interference from non-experimental factors, and improves the reliability of behavioral data and the reproducibility of the experiment.

[0028] According to a preferred embodiment, the support base and the fixator are connected by a plug to fix the position of the fixator; the support base is provided with a field of vision baffle between adjacent fixators to isolate the field of vision of adjacent mice; the support base is provided with a vent corresponding to the position of the vent hole of the fixator.

[0029] This invention enables rapid assembly and disassembly of the restraints and precise positioning through the insertion rod connection, improving experimental efficiency and position repeatability; the field-of-view baffles set between adjacent restraints effectively block visual contact between mice, avoid social interference, and ensure the independence of the behavioral responses of each mouse; the ventilation holes on the support base correspond to the ventilation holes of the restraints, ensuring air circulation and maintaining a good microenvironment.

[0030] This invention provides, from a second aspect, a method for multidimensional behavioral linkage monitoring of mice based on von Frey stimulation. The method includes: restraining the mouse's trunk within the mouse-holding hole of a restraint device using movable clips, while allowing free head movement; mechanically connecting several restraint devices to a support base, arranging the restraint devices to isolate the visual field of adjacent mice; a stimulation parameter acquisition module that records in real-time the intensity, location, and duration of the von Frey stimulation applied to the mouse's head and face; using the von Frey stimulation intensity, location, and duration as benchmark parameters for behavioral response analysis; and, for head-face specific responses induced by von Frey stimulation, a facial movement acquisition module that acquires the mouse's head rotation angle, whisker swaying amplitude, and muscle contraction vibration amplitude induced by von Frey stimulation; an expression response acquisition module that captures characteristic facial expression changes in mice based on images, including image recognition of eye socket tightening, nose and cheek puffing, and ear backward movement amplitude; and a trunk linkage behavior acquisition module that acquires data on the mouse's response to von Frey stimulation. The study included the acquisition of associated trunk movements following Frey stimulation. Specifically, the deformation of the trunk fixation device was collected to determine the trunk torsion angle, and changes in pressure on the mouse trunk against the fixation device were collected to determine the trunk stress response, aiding in the assessment of pain / tactile response intensity. A tail movement acquisition module was used to collect the number and amplitude of tail twitches and the amplitude of tail wagging, based on the pressure caused by tail muscle tension. Stimulation parameter acquisition, facial movement acquisition, facial expression response acquisition, trunk-related behavior acquisition, and tail movement acquisition modules transmitted various data to a data processing module. The data processing module performed time calibration on the data and calculated multi-dimensional behavioral linkage monitoring data of the mouse based on these data.

[0031] The method of this invention achieves simultaneous multi-dimensional behavioral monitoring of mice under von Frey stimulation through systematic design. This method overcomes the limitations of traditional von Frey tests that rely solely on the presence or absence of withdrawal, enabling a refined and objective assessment from single reflexes to coordinated behaviors across multiple body parts, significantly improving the accuracy and repeatability of pain sensitivity detection. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the logic module of the mouse multidimensional behavior linkage monitoring device based on von Frey stimulation provided by the present invention; Figure 2 This is a flowchart illustrating the multidimensional behavioral linkage monitoring method for mice based on von Frey stimulation provided by the present invention. Figure 3 This is a schematic diagram of the overall structure of the fixator provided by the present invention; Figure 4 This is a schematic diagram of the top structure of the fixator provided by the present invention; Figure 5 This is a schematic diagram of the bottom structure of the fixator provided by the present invention; Figure 6 This is a schematic diagram of the structure of the first end of the fixator provided by the present invention; Figure 7 This is a schematic diagram of the structure of the fixture and the bearing base in a combined state provided by the present invention; Figure 8 This is a schematic diagram of the structure of various sensors at the first end of the fixator provided by the present invention; Figure 9 This is a schematic diagram of the fixture provided by the present invention in the open state; Figure 10 This is a schematic diagram of two placement methods of the bearing base provided by the present invention; Figure 11 This is a scene diagram of a mouse with various sensors installed, as provided by the present invention, being fixed in place; Figure 12 This is a schematic diagram of the arrangement of several fixers provided by the present invention.

[0033] List of reference numerals 100: Fixing component; 101: Fixer; 102: First fixing frame; 103: Second fixing frame; 104: Movable snap-fit ​​connector; 105: Mouse hole; 106: First end screw; 107: Second end screw; 108: Tail sealing plate screw; 109: Vent hole; 110: Rotating shaft; 111: Insert rod; 112: Locking component; 113: Elastic shaft; 114: Bearing base; 115: Ventilation opening; 116: Field of view baffle; 117: Locking hole; 118: Fixer in open state; 119: Silicone support; 120: Torso fixing support; 121: Tail sealing plate; 200: Stimulation parameter acquisition module; 210: Stimulation intensity sensor; 220: Stimulation position Positioner; 230: Stimulation timer; 240: Memory; 300: Facial motion acquisition module; 310: Head rotation angle sensor; 320: Whisker swaying sensor; 330: Muscle activity sensor; 400: Facial expression acquisition module; 410: Eye socket tightening motion acquisition unit; 420: Nose and cheek motion acquisition unit; 430: Ear motion acquisition unit; 500: Trunk linkage behavior acquisition module; 510: Trunk torsion sensor; 520: Limb activity sensor; 600: Tail motion acquisition module; 610: Tail twitching acquisition unit; 620: Tail wagging behavior acquisition unit; 630: Tail tension detection sensor; 700: Data processing module. Detailed Implementation

[0034] The following is a detailed explanation with reference to the accompanying drawings.

[0035] The von Frey test is a behavioral experimental method used to assess the sensitivity of animals (especially rodents such as mice and rats) to mechanical stimuli. It is widely used in the field of pain research, particularly in the detection of mechanical allodynia and tactile hyperalgesia in chronic pain models.

[0036] The von Frey test uses a standardized set of single filaments (called von Frey filaments), each with a specific bending force (usually measured in grams). During the experiment, these filaments are applied vertically to the animal's skin surface (such as the sole of the foot, face, or back) from below, with pressure gradually increased until the filaments bend. The animal is observed to exhibit avoidance responses such as limb withdrawal, foot lifting, or paw licking. By using a series of stimuli with increasing or decreasing intensities, combined with the Up-Down method (or Dixon method), the threshold for a 50% positive response is calculated as a quantitative indicator of mechanosensitivity. Due to its ease of operation, lack of complex equipment, and relatively quantifiable results, the von Frey test is widely recognized as one of the gold standards for assessing peripheral nerve injury, inflammatory pain, and mechanical abnormal pain within neuropathic pain.

[0037] Although the von Frey test is widely regarded as one of the gold standards for assessing mechanosensitivity, its inherent limitations are significantly amplified when applied to animal models of trigeminal neuralgia (TN), exposing a range of scientific and methodological problems.

[0038] The von Frey test essentially measures the threshold of nociceptive reflexes, rather than directly reflecting an animal's pain experience. The head withdrawal or scratching behavior exhibited by mice after stimulation is a rapid spinal reflex arc response. While this reflexive behavior can serve as an indirect indicator of pain-related sensitivity, it cannot be equated with subjective pain perception, thus raising fundamental questions about the validity of the test.

[0039] Furthermore, the technical challenges of the procedure lead to high variability and low reproducibility in experimental results. Because von Frey threads must be applied precisely to tiny areas of the face at a stable angle, speed, and duration, even minor deviations in the application can trigger drastically different behavioral responses. This process is highly dependent on the experimenter's subjective judgment and skill, further exacerbating data instability. Traditional analytical methods' criteria for positive responses are inherently subjective and prone to simplification. While the final output may seem precise, such as a 50% retraction threshold of 0.4g, this standard often includes behavioral responses of vastly different degrees (e.g., slight hesitation in retraction versus violent, full-body struggle for escape) as positive, ignoring differences in response intensity and quality, leading to misjudgments of pain levels. Judgment criteria may also fluctuate between different researchers and even within the same experimenter at different times, resulting in significant between- and within-researcher variability.

[0040] Traditional methods focus solely on positive responses in the head and face, neglecting potential behavioral changes in other parts of the body. This limits the comprehensive assessment of overall pain response patterns. Therefore, there is a pressing need to establish an improved experimental system for von Frey stimulation of specific areas of the mouse head and face (such as the whisker area, muzzle, and cheeks). This system would allow for precise control of the stimulation process, simultaneous acquisition of behavioral signals from the head, face, and trunk, and the use of data linkage analysis techniques to address the challenges of accurately matching stimulus parameters with behavioral responses and accurately capturing subtle movements in traditional methods. This would improve the objectivity, sensitivity, and repeatability of mechanorepain assessment.

[0041] Therefore, this invention improves the structure of a mouse restraint device and proposes a device and method for monitoring multidimensional behavioral linkages in mice based on von Frey stimulation. This invention also proposes a mouse restraint device. Furthermore, this invention can provide a processor, server, or cloud server for calculating multidimensional behavioral linkage monitoring data in mice.

[0042] Example 1 Restraining mice in a cylindrical restraint device is a common experimental paradigm, especially when precise stimulation localization or electrophysiological recording is required. In this setup, the mouse's range of motion is severely restricted, causing some key pain indicators (such as scratching the face with forepaws) to disappear. However, many other reliable behaviors remain as indicators of pain intensity. The assessment focus shifts entirely to head, neck, body, and tail movements. The following are key behaviors that can serve as indicators of pain intensity in mice under such restraint: head / neck movements (rapid head retraction, violent head shaking, head turning to avoidance), facial expression changes (eye socket tightening, nose / cheek puffing, ear retraction or flattening), body struggles (body twisting, limb withdrawal, abdominal tightening), and tail wagging (rapid twitching, lateral swaying, tail taut and upward curling).

[0043] Among these, head / neck movements are the most critical indicators. Even when the body is fixed, the head and neck can still move, making them the most important and reliable indicators of pain: rapid head retraction is a core indicator. When the von Frey filament touches the painful area, the mouse will exhibit a very rapid and violent head retraction movement backward or to the side. The amplitude and speed of this movement are positively correlated with the degree of pain. After retraction, the mouse may violently and repeatedly shake its head, i.e., violently shake its head. This is a clear signal of trying to get rid of the painful stimulus, and the number and duration of head shaking can be recorded. In addition, the mouse may continuously turn its head away from the stimulus and maintain this posture, i.e., head turning avoidance, which is an active avoidance behavior.

[0044] In addition to head / neck movements, facial expressions, body struggles, and tail wagging can also serve as supplementary indicators for assessing pain levels. Facial expressions include eye socket tightening, nose or cheek puffing, and ears moving back or flattening. Body struggles are mainly manifested as body twisting, limb retraction, and abdominal tightening. Tail wagging is characterized by rapid twitching, swaying from side to side, or a taut and upturned tail. These behaviors, combined with head / neck movements, can be used to comprehensively assess the mouse's pain status.

[0045] Facial expression changes are an increasingly important indicator for assessing pain status in mice. This indicator is assessed using the Mouse Grimace Scale (MGS), which has the significant advantage of accurately reflecting pain status even with only slight head movements, as subtle changes in facial features can accurately reflect the mouse's pain state.

[0046] Key features of pain-related facial expressions in mice can be categorized into three types: first, eye socket tightening, manifested as squinting or even closing the eyes; second, nose / cheek bulging, specifically manifested as tension and bulging of the muscles in the nose and cheeks; and third, ear displacement or flattening, i.e., the ears fold backward and downward instead of the normal upright position.

[0047] The specific application of key facial expression features is as follows: before and after applying stimulation to mice, their facial state is recorded by taking photos or recording videos. Then, scores are made based on the above facial features. The scores are positively correlated with the degree of pain of the mice, that is, the higher the score, the more severe the pain of the mice.

[0048] Body struggling is a key indicator of pain in mice restrained in a cylindrical cage 101. Although the mice cannot extend their limbs, they can still twist and struggle within the cylinder. This struggling is distinctly different from minor positional adjustments, manifesting as a generalized, sudden, and forceful writhing motion. From an indicator perspective, this behavior is a unique manifestation of the mouse's escape response within a confined space; therefore, the intensity and duration of the struggling can serve as effective indicators for quantifying the intensity of pain in mice.

[0049] Tail movements, including tail twitching or wagging, are typical behaviors in mice when they are excited, stressed, or anxious, and are also of significant reference value in pain assessment. When mice are stimulated by pain, their tails will twitch rapidly and tensely, sometimes exhibiting rapid tail rattling similar to a rattlesnake, and occasionally striking the inner wall of the restraints. This specific tail movement is a strong indicator of pain status and is a good auxiliary indicator for assessing the degree of pain in mice.

[0050] Based on the above key indicator collection requirements, this embodiment provides a mouse multi-dimensional behavioral linkage monitoring device based on von Frey stimulation. The device includes a fixation component 100, a stimulation parameter acquisition module 200, a facial movement acquisition module 300, an expression response acquisition module 400, a trunk linkage behavior acquisition module 500, a tail movement acquisition module 600, and a data processing module 700. Preferably, as shown... Figure 1 As shown, the stimulus parameter acquisition module 200, facial motion acquisition module 300, facial expression response acquisition module 400, trunk linkage behavior acquisition module 500, and tail motion acquisition module 600 are all connected to the data processing module 700 via wired or wireless means to transmit data.

[0051] In this invention, such as Figure 7As shown, the fixation assembly 100 includes a fixator 101 and a support base 114. The fixator 101 fixes the mouse in a manner that restrains the torso within the mouse hole 105 by means of a movable snap-fit ​​104 while allowing the head to move freely. The support base 114 is mechanically connected to a plurality of fixators 101 and the fixators 101 are arranged in a manner that isolates the field of vision of adjacent mice.

[0052] In this invention, the stimulation parameter acquisition module 200 records in real time the intensity, location, and duration of the von Frey stimulus applied to the head and face of the mouse, and sends the von Frey stimulus intensity, location, and duration as benchmark parameters for behavioral response analysis to the data processing module 700.

[0053] In this invention, targeting the head-face specific response induced by von Frey stimulation, the facial motion acquisition module 300 acquires the mouse's head rotation angle, whisker swaying amplitude, and muscle contraction vibration amplitude induced by von Frey stimulation, such as... Figure 1 As shown. The facial expression acquisition module 400 captures characteristic facial expression changes in mice based on images. Specifically, it recognizes the amplitude of eye socket tightening, nose and cheek puffing, and ear retraction movements based on image recognition. Figure 1 As shown.

[0054] In this invention, the trunk linkage behavior acquisition module 500 acquires the associated trunk movements of mice after being stimulated by von Frey. The trunk linkage behavior acquisition module 500 also acquires the degree of deformation of the trunk fixation support 120 to determine the trunk torsion angle. Furthermore, the trunk linkage behavior acquisition module 500 acquires the pressure changes of the mouse's trunk on the trunk fixation support 120 to determine the trunk stress response. The trunk torsion angle and trunk stress response are used to assist the data processing module 700 in determining the intensity of the mouse's pain / tactile response.

[0055] The trunk-linked behavior acquisition module 500 is equipped with a micro data processor to perform the corresponding data conversion steps. If the trunk-linked behavior acquisition module 500 is not equipped with a micro data processor, the raw data can be sent to the data processing module 700, which will then perform the corresponding data conversion steps to obtain data on trunk torsional angle and trunk stress response.

[0056] In this invention, the tail movement acquisition module 600 collects the number and amplitude of tail twitches in response to von Frey stimulation in mice, as well as the amplitude of tail wagging. It also collects tail tension based on the pressure caused by the tension of the tail muscles. Figure 1 As shown.

[0057] In this invention, the data processing module 700 performs time calibration on various types of data transmitted by the stimulus parameter acquisition module 200, facial movement acquisition module 300, facial expression response acquisition module 400, trunk linkage behavior acquisition module 500, and tail movement acquisition module 600, and calculates multi-dimensional behavioral linkage monitoring data of mice based on various types of data.

[0058] The present invention provides a detailed description of the fixed component 100, the stimulus parameter acquisition module 200, the facial movement acquisition module 300, the facial expression response acquisition module 400, the trunk linkage behavior acquisition module 500, the tail movement acquisition module 600, and the data processing module 700.

[0059] According to a preferred embodiment, the comprehensive Figures 3 to 6 As shown, the fixture 101 includes a first fixture 102 and a second fixture 103 rotatably connected thereto. Preferably, the rotatably connected element is a rotating shaft 110. A locking element 112, such as a snap-lock, is also provided on the non-connecting side of the first fixture 102 and the second fixture 103. Figure 7 The fastener 118 is shown in the open position.

[0060] comprehensive Figures 3 to 6 As shown, after the first fixing frame 102 and the second fixing frame 103 are assembled, a mouse-holding hole 105 is formed inside the fixing device 101. Preferably, the mouse-holding hole 105 is a cylindrical hole. Preferably, a movable latching member 104 for engaging the mouse's neck and below is provided in the mouse-holding hole 105 at the first end of the fixing device 101 corresponding to the mouse's head, so as to restrain the mouse's torso within the mouse-holding hole 105. The movable latching member 104 does not fix the head, allowing the mouse's head to move freely.

[0061] Preferably, comprehensive Figures 3 to 6 As shown, the movable latching member 104 includes at least three arc-shaped pieces. The ends of the three arc-shaped pieces are connected in sequence by an elastic shaft 113 equipped with a coil spring, so that the movable latching member 104 has an outward expansion tendency when forming an approximately semi-circular structure.

[0062] comprehensive Figures 3 to 6 As shown, the movable snap-fit ​​connector 104 adjusts its snap-fit ​​position via the first end screw 106. Specifically, the first end screw 106 connects to the movable snap-fit ​​connector 104 located inside the first fixed frame 102 via a through hole on the first fixed frame 102. Figure 6As shown, when the first end screw 106 screws into the mouse hole 105, it pushes the movable locking member 104 towards the second fixing frame 103. The two wings of the movable locking member 104 retract along the inner wall of the mouse hole 105, thereby adaptively fixing mice of different sizes. Preferably, the movable locking member 104 also adjusts its locking position via the second end screw 107. The combined movement of the two screws on the movable locking member 104 allows it to more stably fix the mouse below the neck.

[0063] comprehensive Figures 3 to 6 As shown, the first end of the fixator 101 is provided with a silicone support 119 for supporting the head and face. Preferably, as shown... Figure 8 and Figure 11 As shown, the silicone support 119 lifts the mouse starting from its neck. Preferably, as... Figure 11 As shown, the silicone support 119 should be an arc-shaped support structure that conforms to the physiological curve of the mouse's head and face, and a locally flat section should be designed with the whisker area as the core area.

[0064] Preferably, such as Figure 11 As shown, the arc-shaped support structure closely conforms to the contours of the mouse's head and face, achieving stable fixation to prevent head displacement during experiments and thus reducing pressure stress on the mouse through the flexibility of silicone. An XY-axis grid coordinate system is established with the whisker area as the origin. The precision of the XY-axis grid can be set as needed. Locally flat sections are designed in the whisker area and surrounding core stimulation areas to ensure the accuracy of the grid coordinate system and avoid coordinate shifts caused by the curvature of the surface. The flat surface also facilitates the standardized attachment of RFID tags (ensuring a stable recognition distance between the tag and the RFID reader at the handle end, improving the accuracy of position recognition in standardized experiments). Here, the whisker area and surrounding core stimulation area are the extensions of the left and right whisker areas.

[0065] The inner wall of the mouse placement hole 105 is provided with a torso fixing bracket 120 for fixing the mouse's torso. Preferably, the torso fixing brackets 120 can be arranged in groups of 2 to 4 in a circle to fix the mouse's torso in the same circle. Preferably, at least two groups of torso fixing brackets 120 can be provided on the inner wall of the mouse placement hole 105. Preferably, the torso fixing brackets 120 can be evenly distributed circumferentially. In the radial direction, there are intervals between different groups of torso fixing brackets 120. The intervals can be equal or unequal.

[0066] like Figure 4 As shown, the second end of the restraint 101, near the mouse's tail, is equipped with a tail sealing plate 121 and a tail sealing plate screw 108. The tail sealing plate screw 108 is used to adjust the position of the tail sealing plate 121 to prevent the mouse from curling up. Figure 4As shown, the tail cover 121 is provided with a hole that allows the mouse's tail to pass through, so that the tail can be laid out comfortably.

[0067] According to a preferred embodiment, the comprehensive Figures 3 to 6 As shown, the first fixing frame 102 and the second fixing frame 103 of the fixture 101 are provided with a number of ventilation holes 109 to maintain ventilation conditions.

[0068] According to a preferred embodiment, such as Figure 5 As shown, the bottom of the retainer 101 is provided with a plug 111. The number of plugs 111 can be two (see...). Figure 5 (There can be more than two.) (Comprehensive) Figures 6 to 10 As shown, the support base 114 and the retainer 101 are connected by insert rods 111 to fix the position of the retainer 101. To increase the stability of the retainer 101, the number of insert rods 111 can be set to three. The support base 114 is provided with a locking hole 117. A spring clamping component is provided in the locking hole 117. When the insert rod 111 is inserted into the locking hole 117, the spring clamping component locks the insert rod 111.

[0069] comprehensive Figures 7 to 12 As shown, the support base 114 has a field of vision baffle 116 between adjacent fixation devices 101 to isolate the field of vision of adjacent mice and prevent visual communication between the mice.

[0070] comprehensive Figures 7 to 12 As shown, the support base 114 is provided with a vent 115 corresponding to the position of the vent 109 of the fixation device 101. When the fixation device 101 is installed on the support base 114, the vent 115 and the vent 109 form a ventilation channel, so that the temperature inside the mouse hole 105 is the same as the external environment, avoiding the mouse from suffering from environmental pain.

[0071] Preferably, comprehensive Figures 7 to 12 As shown, the extension direction of the vent 115 is perpendicular to the axis of the fixture 101, so that the fixture 101 in both the horizontal and vertical directions can achieve ventilation.

[0072] Preferably, such as Figure 10 As shown, the support base 114 can be placed in a way that makes the fixture 101 vertical or horizontal, to adapt to different experimental needs.

[0073] According to a preferred embodiment, such as Figure 1 As shown, the stimulation parameter acquisition module 200 includes a stimulation intensity sensor 210, a stimulation location locator 220, and a stimulation timer 230.

[0074] A stimulation intensity sensor 210 is disposed at the end of the von Frey handle. When the fibers on the handle contact the mouse skin, the mechanical stimulation intensity is determined in real time by collecting the tensile force generated by the bending of the fibers. Preferably, the stimulation intensity sensor 210 is a miniature tensile sensor. When the fibers contact the mouse skin, the miniature tensile sensor collects the tensile force generated by the bending of the fibers in real time, converts the tensile force value into an electrical signal, and sends it to the data processing module 700. Preferably, the tensile force value is correlated with the mechanical stimulation intensity.

[0075] Specifically, a miniature tension sensor is selected, and the size of the miniature tension sensor is checked to ensure it is compatible with the installation space at the end of the handle.

[0076] A mounting groove and threading hole are machined at the end of the von Frey handle to embed a miniature tensile sensor, which is then fixed and cured with biocompatible epoxy resin. The fiber is rigidly connected to the sensor's sensing surface via a medical-grade stainless steel clip, and pre-bending is used to verify the sensor's effectiveness.

[0077] Then perform calibration and debugging. Store the calibration curve to the data processing module 700, and recalibrate periodically (e.g., weekly) or when anomalies are detected, and record the information.

[0078] The von Frey handle is fixed and connected to a miniature tension sensor and a data processing module 700. When the fiber comes into contact with the mouse skin, the miniature tension sensor collects the bending tension value in real time and converts it into an electrical signal, which is then transmitted to the data processing module 700.

[0079] like Figure 3 and Figure 8 As shown, the stimulation location locator 220 is disposed on the surface of the silicone support 119 used to support the head and face, and identifies the stimulation location based on a preset grid coordinate system. The surface of the silicone support 119 is divided into a grid and marked according to the preset grid coordinate system. For example, the preset grid coordinate system is marked as follows: an XY axis coordinate system is established with the whisker area as the origin. Preferably, the experimenter manually inputs the stimulation location through a matching touch screen, or attaches an RFID tag to the corresponding area of ​​the silicone support 119, and the location is automatically identified by an RFID reader at the handle end.

[0080] Specifically, the silicone tray 119 is pre-treated. For example, a grid is laser-engraved on the surface of the tray, establishing an XY-axis coordinate system with the whisker area as the origin, resulting in clear and wear-resistant coordinate scales. The stimulation position locator 220 is preferably an RFID tag. The RFID tag is fixed to a pre-set groove on the surface of the silicone tray 119, ensuring precise alignment with the grid coordinates, and then connected to the positioning system and the data processing module 700.

[0081] In manual mode, researchers input the target location via a companion touchscreen to achieve automatic positioning. For example, the target location could be a position at a certain distance from the left tentacle area.

[0082] The standardized experiment can be performed in an automatic mode. An RFID tag is attached to the target area of ​​the silicone tray 119. When the RFID reader at the handle end approaches, it automatically identifies the location and sends the feedback to the data processing module 700.

[0083] Before use, the stimulus location locator 220 is verified for positioning. It is tested using standard coordinate points, and calibration is checked before each batch of experiments.

[0084] The stimulation timer 230 starts recording the stimulation duration when the tension value collected by the stimulation intensity sensor 210 is greater than the tension threshold.

[0085] Preferably, the stimulation timer 230 is connected to the stimulation intensity sensor 210 and receives the tension value. When the detected tension value is greater than a threshold (which corresponds to the trigger state of fiber contact with skin), the stimulation timer 230 automatically starts timing and records the duration of stimulation in real time; when the detected tension value returns to zero, it indicates that the stimulation has ended, the stimulation timer 230 automatically stops timing, and generates complete timestamp data including the stimulation start time and stimulation end time.

[0086] Preferably, the stimulus intensity sensor 210 can also be connected to the memory 240. The above parameters are transmitted to the memory 240 via low-noise wires and are synchronously cached with the behavioral response signal on the same time axis.

[0087] According to a preferred embodiment, such as Figure 1 As shown, the facial motion acquisition module 300 includes a head rotation angle sensor 310, a whisker swaying sensor 320, and a muscle activity sensor 330. For specific head and face responses induced by von Frey stimulation (such as head avoidance rotation, rapid retraction, violent head shaking, whisker swaying, biting movements, etc.), the facial motion acquisition module 300 achieves quantitative acquisition through high-sensitivity sensors.

[0088] like Figure 3 and Figure 8 As shown, the head rotation angle sensor 310 is set at the temporal region of the mouse to collect the head rotation angle in the horizontal and vertical directions in real time.

[0089] Preferably, the head rotation angle sensor 310 includes multiple miniature tilt sensors, preferably two, but more can be added depending on actual monitoring needs. The miniature tilt sensors are embedded in the silicone support 119 and precisely correspond to the position of the mouse's temporal region.

[0090] Specifically, two miniature tilt sensors are embedded in the silicone support 119 at the temporal region of the mouse. These two sensors correspond to the X and Y axes, respectively, and are used to monitor in real time the rotation angles of the mouse's head during horizontal lateral avoidance movements and vertical head-up / head-down movements. When the detected rate of angle change exceeds a preset threshold, the data processing module 700 determines that the mouse has exhibited an active avoidance response.

[0091] like Figure 3 , Figure 4 , Figure 8 and Figure 11 As shown, the tentacle swing sensor 320 is located near the head position of the fixture 101, and its infrared light illumination range covers the tentacle swing area. It collects the change in infrared light flux caused by the tentacle swing to determine the tentacle swing amplitude.

[0092] Specifically, such as Figure 8 As shown, the whisker oscillation sensor 320 is preferably an array-type infrared pair sensor. The array-type infrared pair sensor is positioned on both sides of the mouse's head, and its infrared light illumination range covers the whisker oscillation area, with the infrared optical axis perpendicular to the natural downward direction of the whiskers. When the mouse's whiskers oscillate, the whiskers block the infrared light, and the array-type infrared pair sensor collects the change in luminous flux and sends it to the data processing module 700. The data processing module 700 calculates the whisker oscillation amplitude based on the change in luminous flux.

[0093] An array-type infrared photodiode sensor is essentially a form of optical sensor (photoelectric sensor). Its core working principle is to detect changes in light signals (light intensity, luminous flux, etc.) and convert them into electrical signals, thereby monitoring the state of a target. The array-type infrared photodiode sensor consists of an infrared emitting tube and an infrared receiving tube. By emitting infrared light of a specific wavelength, when the tendrils swing and block the infrared optical axis, the luminous flux detected by the infrared receiving tube changes. The array-type infrared photodiode sensor converts this change in light signal into an electrical signal and sends it to the data processing module 700, which ultimately calculates parameters such as the tendril swing amplitude and frequency.

[0094] More preferably, an array of infrared photodiode sensors is installed at a distance from the tentacles on the outer side of the tentacle area. Several pairs of these array-type infrared photodiode sensors can be configured to achieve full coverage of the left and right tentacles. The infrared optical axis is perpendicular to the natural downward direction of the tentacles. When the tentacles swing, they block the infrared light. The array-type infrared photodiode sensors detect this change in light flux and can calculate the swing amplitude and frequency of the tentacles in real time. The array-type infrared photodiode sensors then transmit the swing amplitude and frequency to the data processing module 700.

[0095] When the amplitude of the tentacle's swing is less than the minimum swing threshold, the data processing module 700 determines it as a slight tremor. When the amplitude of the swing is greater than the maximum swing threshold, the data processing module 700 determines it as a violent swing.

[0096] The muscle activity sensor 330 collects the amplitude of muscle contraction vibrations in the muzzle and / or cheek induced by von Frey stimulation.

[0097] like Figure 3 and Figure 8 As shown, preferably, the muscle activity sensor 330 is configured as a miniature piezoelectric film sensor. The miniature piezoelectric film sensor is positioned on the silicone support 119 at locations corresponding to the front of the mouse's snout and cheek skin.

[0098] When von Frey stimulation induces muscle contraction vibrations in mice, a miniature piezoelectric film sensor collects changes in the electrical signals caused by these vibrations and sends them to a data processing module 700. The data processing module 700 calculates the muscle vibration amplitude based on the electrical signals and quantifies it as muscle contraction intensity. Based on the muscle contraction intensity, the data processing module 700 distinguishes different response types: vibration amplitude less than the minimum amplitude is considered no response; vibration amplitude between the minimum and maximum amplitudes is considered a slight twitching; and vibration amplitude greater than the maximum amplitude and duration greater than a preset duration is considered a biting action. Muscle contraction vibrations include, for example, the high-frequency vibrations generated by the jaw muscles during biting behavior.

[0099] According to a preferred embodiment, such as Figure 1 As shown, the facial expression acquisition module 400 includes an eye socket tightening action acquisition unit 410, a nose and cheek action acquisition unit 420, and an ear action acquisition unit 430. This facial expression acquisition module 400 is used to capture characteristic facial expression changes in mice induced by von Frey stimulation (eye socket tightening, nose and cheek bulging, and ear retraction). Through a combination of high-precision imaging and sensing, it enables the visual recording and quantitative analysis of subtle facial expressions, providing a more intuitive assessment basis for pain / tactile responses.

[0100] like Figure 8 , Figure 9 and Figure 11 As shown, the orbital tightening action acquisition unit 410 is set on the support base 114 corresponding to the position of the mouse's head, and captures the outline of the mouse's orbital cavity in an imaging manner that can penetrate the hair of the mouse's eye, so that the data processing module 700 can identify the orbital tightening action based on the first distance change between preset orbital feature points.

[0101] For example, such as Figure 8As shown, two high-definition infrared cameras are installed on the top of the support base 114 (preferably at the position directly in front of the mouse's eyes). These cameras can penetrate the mouse's eye hairs and clearly capture the outline of the eye socket, avoiding the stimulation of the mouse by visible light.

[0102] Image recognition algorithm: By using preset orbital feature points (such as the inner corner of the eye, the outer corner of the eye, the midpoint of the upper eyelid, and the midpoint of the lower eyelid), the algorithm calculates the first distance change between feature points in real time.

[0103] like Figure 3 and Figure 8 As shown, the nose and cheek movement acquisition unit 420 is set at the position of the mouse's nose and snout and both cheeks and collects pressure values, so that the data processing module 700 determines the nose puffing movement and / or cheek puffing movement based on the pressure value changes of the nose and snout and both cheeks.

[0104] For example, four flexible pressure sensors are embedded in the silicone support 119 at the locations corresponding to the mouse's snout (from the tip of the nose to around the nostrils) and both cheeks. The flexible pressure sensors fit the skin but do not compress it. A sudden increase in pressure value of the sensor at the snout corresponds to a nose puffing-out action; an increase in pressure value of the sensor on one or both cheeks corresponds to a cheek puffing-out action.

[0105] like Figure 8 and Figure 11 As shown, the ear motion acquisition unit 430 is set on a support base 114 at a specified distance from the outside of the mouse ear. The second distance change between the ear edge and the ear motion acquisition unit 430 is recorded by vertically irradiating the edge of the auricle with a laser beam, so that the data processing module 700 can determine whether the ear has moved backward based on the second distance change.

[0106] For example, such as Figure 8 and Figure 11 As shown, a miniature laser displacement sensor is installed on each side of the support base 114 (corresponding to the outer side of the mouse ear). The laser beam is perpendicularly irradiated onto the edge of the ear (a preset reference position in the resting state). The change in the second distance between the edge of the ear and the miniature laser displacement sensor is recorded in real time. When the second distance increases by a preset second distance threshold (i.e., the ear moves backward and away from the miniature laser displacement sensor), it is determined as an ear movement backward.

[0107] According to a preferred embodiment, such as Figure 1 As shown, the trunk linkage behavior acquisition module 500 includes a trunk torsion sensor 510 and a limb movement sensor 520. The trunk linkage behavior acquisition module 500 acquires the associated trunk movements of mice after being stimulated by von Frey (such as body torsion, limb withdrawal, abdominal tension, etc.) to help determine the intensity of pain / tactile response (such as strong stimulation may cause whole-body withdrawal).

[0108] like Figure 3 and Figure 8 As shown, the trunk torsion sensor 510 is a strain gauge disposed within the trunk fixation bracket 120 of the fixator 101. When the mouse's trunk twists, the data processing module 700 determines the trunk torsion angle based on the degree of deformation of the strain gauge. The trunk fixation bracket 120 can be made of flexible silicone material.

[0109] For example, two flexible strain gauges (arranged symmetrically along the long axis of the torso) are embedded in the torso fixation bracket 120 of the fixation device 101. When the mouse torso is twisted, the strain gauges deform and the torsional angle is calculated by the change in resistance.

[0110] like Figure 3 and Figure 8 As shown, the limb movement sensor 520 is a pressure sensor installed in the trunk fixation bracket 120 corresponding to the forelimb and hindlimb respectively in the fixation device 101. It collects pressure change data caused by the movement of the forelimb and hindlimb, so that the data processing module 700 can determine the trunk stress response based on the pressure change data.

[0111] For example, a thin-film pressure sensor is installed at both the front (corresponding to the forelimb position) and rear (corresponding to the hindlimb position) of the trunk fixation device 120 to detect pressure changes during limb pushing or retraction. A sudden increase in pressure (such as in a forelimb pushing device) or a sudden decrease in pressure (such as in limb curling) can be considered a trunk stress response. The proximal end of the mouse tail refers to the position close to the mouse's trunk or the root of its appendages. On the tail, the part closest to the tail root (where it connects to the trunk) is the proximal end. The distal end of the mouse tail refers to the position further away from the mouse's trunk or the root of its appendages, i.e., closer to the tip. For example, the tip of the tail is the distal end. The mid-distal end of the mouse tail refers to the area between the middle and distal ends, emphasizing that it is neither the base nor the very tip, but rather the middle part towards the outer / terminus.

[0112] According to a preferred embodiment, such as Figure 1 As shown, the tail motion acquisition module 600 includes a tail twitching acquisition unit 610, a tail wagging behavior acquisition unit 620, and a tail tension detection sensor 630.

[0113] In response to von Frey stimulation-induced tail-specific responses in mice (such as rapid twitching, swaying from side to side, and tail tightening and lifting), the tail motion acquisition module 600 uses high-precision sensing and dynamic capture technology to quantify and record subtle tail movements, supplementing evidence of whole-body pain / tactile responses in addition to head and face reactions.

[0114] like Figure 11 As shown, the tail twitching acquisition unit 610 is located at the mid-distal end of the mouse tail to acquire the acceleration caused by the vibration of the mouse tail, so that the data processing module 700 can determine the tail twitching behavior based on the acceleration and the duration of acceleration.

[0115] For example, a miniature triaxial accelerometer is fixed to the distal end of the mouse's tail using medical tape to avoid affecting tail movement. The miniature triaxial accelerometer is used to capture subtle vibrations in the distal end of the tail. When the rate of change of acceleration exceeds a preset acceleration change threshold (preferably perpendicular to the tail axis) and the duration is less than a preset time threshold, it is determined to be a twitch. The data processing module 700 automatically counts the number of twitches and the amplitude of each twitch.

[0116] like Figure 4 and Figure 9 As shown, the tail-wagging behavior acquisition unit 620 is disposed on the side wall of the tail movement area within the restraint 101 and forms an infrared light curtain perpendicular to the tail. When the mouse's tail wags, the frequency of infrared light blocking is acquired to calculate the wagging amplitude.

[0117] For example, the infrared light curtain is also an array of infrared photocell sensors. At least one set of arrayed infrared photocell sensors is arranged on both sides of the tail's active area, forming an infrared light curtain perpendicular to the tail. When the tail swings left and right, it blocks one or both sides of the infrared receiver. The data processing module 700 calculates the tail swing amplitude based on the number and frequency of the blocked infrared receivers. The arrayed infrared photocell sensors are arranged along the length of the tail and are spaced apart from each other.

[0118] The tail tension sensor 630 is placed near the end of the mouse tail to detect the pressure when the tail muscles are tensed.

[0119] For example, a miniature pressure sensor was embedded inside a silicone ring fixed near the proximal end of a mouse's tail to detect the pressure exerted on the silicone ring when the tail muscles tightened. When the pressure value increased compared to the resting state, it was determined to be a tail muscle tightening action, and the recorded twitching / tail wagging behavior at this time was more likely to be related to pain.

[0120] Preferably, the stimulation intensity sensor 210 is located at the end of the von Frey handle. When the von Frey fiber comes into contact with the mouse skin, the stimulation intensity sensor 210 determines the mechanical stimulation intensity in real time by collecting the tensile force generated by the fiber bending, and converts the tensile force value into an electrical signal and sends it to the data processing module 700. The data processing module 700 triggers a synchronization signal (TTL level), setting this moment as the stimulation start point. All behavioral response sensors (tilt, infrared, piezoelectric, strain gauge, etc.) record data based on this timestamp, ensuring millisecond-level alignment of stimulation intensity, reaction time, and reaction intensity.

[0121] The data processing module 700 has a built-in clock chip to avoid time drift during long-term experiments and ensure that response data from multiple stimuli can be compared laterally.

[0122] When mice are restrained within the cylindrical restraint 101, although the important assessment indicator of forelimb scratching is difficult to obtain, the focus of assessment can be shifted to behaviors such as head retraction, head shaking, body struggling, and tail movements. Although the Up-Down threshold method remains the gold standard in this field, this invention, by redefining the criteria for positive response and combining behavioral scoring with facial expression analysis (MGS), can provide a comprehensive and reliable assessment of the degree of pain in mice.

[0123] Example 2 This embodiment is a further improvement on embodiment 1, and repeated content will not be described again.

[0124] Habituation training is necessary. Mice must undergo this training before formal experiments. For several consecutive days, the mice are placed in the hysteresis apparatus, with the daily placement time gradually increasing, for example, from 15 minutes to 1 hour, until the mice no longer exhibit tension or struggling behavior due to the restraint itself. Otherwise, it will be difficult to distinguish whether the reaction is triggered by restraint stress or by the pain response itself.

[0125] Baseline establishment: Before conducting model establishment or drug intervention, baseline tests were performed on mice that had adapted to the restraints, and their normal response data to von Frey silk were recorded.

[0126] This embodiment provides a method for monitoring multidimensional behavioral linkages in mice based on von Frey stimulation, such as... Figure 2 As shown, the method is as follows.

[0127] S100: Fixed mice.

[0128] The mouse's torso is restrained within the mouse-holding hole 105 of the restraint 101 by the movable clip 104, while the head moves freely. Several restraints 101 are mechanically connected to the support base 114, and the restraints 101 are arranged in a way that isolates the field of vision of adjacent mice.

[0129] Specifically, first, the fixation device 101 is inserted into the support base 114, then the first fixation frame 102 is opened, the mouse is placed in the second fixation frame 103, the first fixation frame 102 is closed, the tail sealing plate 121 is adjusted, and the two movable locking pieces 104 at the front and rear are adjusted to hold the mouse securely.

[0130] S200: Record the intensity, location, and duration of von Frey stimulation applied to the head and face of mice.

[0131] The stimulation parameter acquisition module 200 records in real time the intensity, location, and duration of the von Frey stimulus applied to the head and face of mice, and uses the intensity, location, and duration of the von Frey stimulus as the baseline parameters for behavioral response analysis.

[0132] S300: Collects the mouse's head rotation angle, whisker swaying amplitude, and muscle contraction vibration amplitude induced by von Frey stimulation.

[0133] In response to the head and face-specific responses induced by von Frey stimulation, the facial motion acquisition module 300 acquires the head rotation angle, whisker swaying amplitude, and muscle contraction vibration amplitude induced by von Frey stimulation in mice.

[0134] S400: Collect data related to the amplitude of eye socket contraction, nose and cheek puffing, and ear posterior movement in mice.

[0135] The facial expression acquisition module 400 captures characteristic facial expression changes of mice based on images, including the eye socket tightening action, nose and cheek puffing action, and ear backward movement amplitude based on image recognition.

[0136] S500: Collects data on trunk torsional angle and related trunk stress response.

[0137] The trunk linkage behavior acquisition module 500 acquires the associated activities of the mouse trunk after being stimulated by von Frey. The trunk torsion angle is determined based on the degree of deformation of the trunk fixation support 120, and the trunk stress response is determined based on the pressure change of the mouse trunk on the trunk fixation support 120, so as to help judge the intensity of pain / tactile response.

[0138] S600: Collects the number and amplitude of tail twitches, and the amplitude of tail wagging behavior.

[0139] The tail movement acquisition module 600 targets the mouse tail-specific response induced by von Frey stimulation, acquiring the number and amplitude of tail twitches, the amplitude of tail wagging behavior, and tail tension based on the pressure caused by the tension of the tail muscles.

[0140] S700: Time-calibrates various types of data and calculates multi-dimensional behavioral linkage monitoring data of mice based on various types of data.

[0141] The stimulus parameter acquisition module 200, facial movement acquisition module 300, facial expression response acquisition module 400, trunk linkage behavior acquisition module 500, and tail movement acquisition module 600 transmit various types of data to the data processing module 700. The data processing module 700 performs time calibration on various types of data and calculates multi-dimensional behavioral linkage monitoring data of mice based on various types of data.

[0142] It should be noted that the specific embodiments described above are exemplary. Those skilled in the art can devise various solutions inspired by the disclosure of this invention, and these solutions all fall within the scope of this invention and its protection. Those skilled in the art should understand that this specification and its accompanying drawings are illustrative and not intended to limit the scope of the claims. The scope of protection of this invention is defined by the claims and their equivalents. This specification contains multiple inventive concepts; phrases such as "preferredly" or "according to a preferred embodiment" indicate that the corresponding paragraph discloses an independent concept. The applicant reserves the right to file divisional applications based on each inventive concept.

Claims

1. A multi-dimensional behavioral linkage monitoring device for mice based on von Frey stimulation, characterized in that, The device includes: The fixation assembly (100) includes a fixator (101) and a support base (114). The fixator (101) fixes the mouse in such a way that the torso is restrained within a mouse hole (105) by a movable snap-fit ​​(104) while the head moves freely. The support base (114) is mechanically connected to a plurality of the fixators (101) and the fixators (101) are arranged in such a way that they isolate the field of vision of adjacent mice. The stimulation parameter acquisition module (200) records the intensity, location and duration of von Frey stimulation applied to the head and face of mice in real time. The facial motion acquisition module (300) acquires the mouse's head rotation angle, whisker swaying amplitude, and muscle contraction vibration amplitude induced by von Frey stimulation. The facial expression response acquisition module (400) is based on image recognition of the mouse's eye socket tightening action, nose and cheek puffing action and ear back movement amplitude; The trunk linkage behavior acquisition module (500) acquires the degree of deformation of the trunk fixation support (120) to determine the trunk torsion angle, and acquires the pressure change of the mouse trunk on the trunk fixation support (120) to determine the trunk stress response, so as to help judge the intensity of pain / touch response. The tail movement acquisition module (600) collects the number and amplitude of tail twitches and the amplitude of tail wagging behavior in response to von Frey stimulation in mice, and collects tail tension based on the pressure caused by the tension of the tail muscles. The data processing module (700) performs time calibration on various types of data transmitted by the stimulus parameter acquisition module (200), facial movement acquisition module (300), facial expression response acquisition module (400), trunk linkage behavior acquisition module (500) and tail movement acquisition module (600), and calculates multi-dimensional behavioral linkage monitoring data of mice based on various types of data.

2. The mouse multidimensional behavior linkage monitoring device according to claim 1, characterized in that, The stimulus parameter acquisition module (200) includes: The stimulation intensity sensor (210) is set at the end of the von Frey handle to determine the mechanical stimulation intensity in real time by collecting the tensile force generated by the bending of the fibers when the fibers on the handle come into contact with the mouse skin. Stimulation position locator (220) is set on the surface of silicone support (119) for supporting the head and face, and identifies the stimulation position based on preset network coordinates; The stimulation timer (230) starts recording the stimulation duration when the tension value collected by the stimulation intensity sensor (210) is greater than the tension threshold.

3. The mouse multidimensional behavior linkage monitoring device according to claim 1 or 2, characterized in that, The facial motion acquisition module (300) includes: A head rotation angle sensor (310) is set at the temporal region of the mouse to collect the head rotation angle in the horizontal and vertical directions in real time. The tentacle swing sensor (320) is located near the head position of the fixture (101), and its infrared light illumination range covers the tentacle swing area. It collects the change in infrared light flux caused by the tentacle swing to determine the tentacle swing amplitude. A muscle activity sensor (330) acquires the amplitude of muscle contraction vibrations at the snout and / or cheeks induced by von Frey stimulation.

4. The mouse multi-dimensional behavior linkage monitoring device according to any one of claims 1 to 3, characterized in that, The facial expression response acquisition module (400) includes: The orbital tightening action acquisition unit (410) is set on the support base (114) corresponding to the top of the mouse's head. It captures the outline of the mouse's orbital area in an imaging manner that can penetrate the hair around the mouse's eyes, so that the data processing module (700) can identify the orbital tightening action based on the first distance change between preset orbital feature points. The nose and cheek movement acquisition unit (420) is set at the position of the mouse's nose and snout and both cheeks and collects pressure values, so that the data processing module (700) determines the nose puffing movement and / or cheek puffing movement based on the pressure value changes of the nose and snout and both cheeks. The ear motion acquisition unit (430) is set on a support base (114) at a specified distance from the outside of the mouse ear. The second distance change between the ear edge and the ear motion acquisition unit (430) is recorded by vertically irradiating the edge of the auricle with a laser beam, so that the data processing module (700) can determine whether the ear has moved backward based on the second distance change.

5. The mouse multi-dimensional behavior linkage monitoring device according to any one of claims 1 to 4, characterized in that, The trunk linkage behavior acquisition module (500) includes: The trunk torsion sensor (510) is a strain gauge installed in the trunk fixation bracket (120) of the fixation device (101). When the mouse's trunk is torsioned, the data processing module (700) determines the trunk torsion angle based on the degree of deformation of the strain gauge. The limb movement sensor (520) is a pressure sensor installed in the trunk fixation bracket (120) corresponding to the forelimb and hindlimb respectively in the fixation device (101). It collects pressure change data caused by the movement of the forelimb and hindlimb, so that the data processing module (700) determines the trunk stress response based on the pressure change data.

6. The mouse multi-dimensional behavioral linkage monitoring device according to any one of claims 1 to 5, characterized in that, The tail motion acquisition module (600) includes: A tail twitching acquisition unit (610) is set at the mid-distal end of the mouse tail to acquire the acceleration caused by the vibration of the mouse tail, so that the data processing module (700) can determine the tail twitching behavior based on the acceleration and the duration of the acceleration. The tail-wagging behavior acquisition unit (620) is set on the side wall of the tail activity area inside the fixation device (101) and forms an infrared light curtain perpendicular to the tail; when the mouse tail wags, the blocking frequency of infrared light is collected to calculate the wagging amplitude. A tail tension sensor (630) is placed near the end of the mouse tail to detect the pressure when the tail muscles are tensed.

7. The mouse multidimensional behavior linkage monitoring device according to any one of claims 1 to 6, characterized in that, The fixture (101) includes a first fixture (102) and a second fixture (103) rotatably connected thereto. When the first fixing bracket (102) and the second fixing bracket (103) are assembled, a mouse hole (105) is formed inside the fixing device (101). The mouse holder (101) corresponding to the mouse head has a movable locking member (104) in the mouse hole (105) at the first end for locking the mouse below the neck, so that the mouse can move its head; the movable locking member (104) adjusts the locking position by the first end screw (106). The first end of the fixator (101) is provided with a silicone support (119) for supporting the head and face. The inner wall of the mouse placement hole (105) is provided with a torso fixing bracket (120) for fixing the mouse torso. The second end of the restraint (101) near the mouse's tail is provided with a tail sealing plate (121) and a tail sealing plate screw (108), the tail sealing plate screw (108) being used to adjust the position of the tail sealing plate (121) to prevent the mouse from curling up.

8. The mouse multidimensional behavior linkage monitoring device according to any one of claims 1 to 7, characterized in that, The first fixing frame (102) and the second fixing frame (103) of the fixture (101) are provided with a number of ventilation holes (109) to maintain ventilation conditions.

9. The mouse multi-dimensional behavioral linkage monitoring device according to any one of claims 1 to 8, characterized in that, The bearing base (114) and the fixture (101) are connected by a plug (111) to fix the position of the fixture (101); The support base (114) has a field of vision baffle (116) between adjacent fixation devices (101) to isolate the field of vision of adjacent mice; The support base (114) is provided with a vent (115) corresponding to the position of the vent (109) of the fixture (101).

10. A method for monitoring multidimensional behavioral linkages in mice based on von Frey stimulation, characterized in that, The method includes: The mouse's torso is restrained within the mouse hole (105) of the restraint (101) by the movable snap fastener (104), while the head moves freely. Several of the restraints (101) are mechanically connected to the support base (114) and arranged in a manner that isolates the field of vision of adjacent mice. The stimulation parameter acquisition module (200) records in real time the intensity, location and duration of the von Frey stimulus applied to the head and face of the mouse. The facial motion acquisition module (300) acquires the mouse's head rotation angle, whisker swaying amplitude, and muscle contraction vibration amplitude induced by von Frey stimulation. The facial expression acquisition module (400) is based on image recognition of the mouse's eye socket tightening action, nose and cheek puffing action and ear back movement amplitude; The trunk linkage behavior acquisition module (500) acquires the degree of deformation of the trunk fixation support (120) to determine the trunk torsion angle, and acquires the pressure change of the mouse trunk on the trunk fixation support (120) to determine the trunk stress response, so as to help judge the intensity of pain / touch response. The tail movement acquisition module (600) collects the number and amplitude of tail twitches in response to von Frey stimulation, the amplitude of tail wagging behavior, and tail tension based on the pressure caused by the tension of the tail muscles. The stimulus parameter acquisition module (200), facial movement acquisition module (300), facial expression response acquisition module (400), trunk linkage behavior acquisition module (500) and tail movement acquisition module (600) transmit various types of data to the data processing module (700). The data processing module (700) performs time calibration on various types of data and calculates multi-dimensional behavioral linkage monitoring data of mice based on various types of data.

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