Mouse spinal cord calcium signal monitoring system based on nociceptive stimulation

Abstract

The invention relates to the technical field of biomedical experimental equipment, in particular to a mouse spinal cord calcium signal monitoring system based on nociceptive stimulation, which comprises a spine windowing covering device used for covering a window in mouse spine windowing, a spine fixing device used for avoiding mouse body movement, and a thermal stimulation device used for stimulating mouse pain, the spine windowing covering device comprises a covering main plate, and an observation window is arranged on the covering main plate. The device can effectively protect a mouse spine windowing part, can stably fix mice of different body types, prevents the mice from body movement when the mice are subjected to nociceptive stimulation, guarantees the stability of an experiment process and the accuracy of data, can accurately control the temperature of a heating plate through a temperature control plate of the thermal stimulation device, and improves the experiment efficiency. The device can accurately apply noxious thermal stimulation to a mouse, simulate a real noxious stimulation environment, and provide reliable experimental conditions for researching a pain mechanism.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical experimental equipment, and in particular to a mouse spinal cord calcium signal monitoring system based on noxious stimulation. Background Art

[0002] Calcium imaging refers to a method that uses calcium ion indicators to monitor calcium ion concentrations within tissues. In neurological research, calcium imaging is widely used to monitor neuronal activity. Specifically, the technique requires pre-injection of relevant mouse tissue. For example, in this patent, a virus containing a calcium ion indicator is injected into the mouse spinal cord tissue, which infects the target neurons in the spinal cord. Later, by monitoring the strength of calcium ion signals in the virus-infected neurons, neuronal activity in vivo is indirectly reflected. However, this process requires a high-end microscope and the ability to observe neurons as clearly as possible under microscopy in vivo. This requires surgically opening the spinal cord to expose the spinal cord tissue. When monitoring spinal cord calcium signals in mice, effective protection and observation of the fenestration site are difficult. Furthermore, the mouse cannot be stably fixed, resulting in movement during noxious stimulation, which can interfere with experimental results and affect the accuracy of the monitoring data. Therefore, a system for monitoring spinal cord calcium signals in mice that can address these issues is urgently needed. Summary of the Invention

[0003] The purpose of the present invention is to provide a mouse spinal cord calcium signal monitoring system based on noxious stimulation. The system can effectively protect the mouse spinal window, stably fix the mouse, accurately apply noxious thermal stimulation, and accurately monitor the mouse spinal cord calcium signal, thereby providing reliable experimental data for pain-related research.

[0004] In order to solve the above technical problems, the present invention adopts the following technical solutions: The present invention provides a mouse spinal cord calcium signal monitoring system based on noxious stimulation, comprising: a spinal fenestration covering device for covering a window during spinal fenestration surgery on mice; A spinal fixation device to prevent mouse movement; a thermal stimulation device for painful stimulation of mice; The spinal column window covering device comprises a covering main board, on which an observation window is provided.

[0005] Furthermore, the spinal window covering device also includes a covering sub-plate, which is made of transparent material. The covering main plate is provided with a recessed portion and a clamping portion. The observation window is arranged in the recessed portion. The covering sub-plate covers the observation window in the recessed portion, and the clamping portions are arranged at both ends of the covering main plate.

[0006] Furthermore, the spinal fixation device includes: a device base, a device vertical plate, a vertical slide plate, a loading platform, and a supporting component. The device vertical plate is vertically arranged at one end of the device base, the vertical slide plate can slide up and down on the device vertical plate, the loading platform is connected to the vertical slide plate, and the supporting component is arranged on the loading platform.

[0007] Furthermore, a first strip hole is provided on the loading platform, and the supporting assembly includes a clamping structure, a horizontal fixed rod, a vertical sliding rod, a sliding block, a first limiting bolt, a first limiting bolt, and a third limiting bolt. Two through holes are provided on the sliding block, and the clamping structure is arranged on the horizontal fixed rod. One end of the horizontal fixed rod passes through a through hole on the sliding block and is limited by the first limiting bolt. One end of the vertical sliding rod passes through another through hole on the sliding block and is limited by the second limiting bolt. The other end of the vertical sliding rod passes through the first strip hole and is fixed to the loading platform by the third limiting bolt.

[0008] Furthermore, the clamp structure includes upper jaws, lower jaws, an elastic member, and a fastening bolt. The upper jaws and the lower jaws are mirror-slidably arranged on a horizontal fixed rod. The elastic member is arranged on the horizontal fixed rod and is located between the upper jaws and the lower jaws. The fastening bolt is arranged on the end of the horizontal fixed rod. Rotating the fastening bolt can make the upper jaws and the lower jaws engage with each other.

[0009] Furthermore, a limiting rod is provided on the upper jaw, and a limiting hole matching the limiting rod is provided on the lower jaw. The limiting rod is inserted into the limiting hole, and the limiting rod can slide freely in the limiting hole when the fastening bolt is rotated.

[0010] Furthermore, an extension plate is provided on the loading platform, and the first strip-shaped hole is provided on the extension plate.

[0011] Furthermore, the spinal fixation device includes: a device base, a device vertical plate, a vertical sliding plate, a loading platform, a supporting assembly, and a fourth limiting bolt. The device vertical plate is provided with a second strip hole, the device vertical plate is vertically arranged at one end of the device base, the fourth limiting bolt passes through the second strip hole to set the vertical sliding plate on the device vertical plate, the loading platform is connected to the vertical sliding plate, and the supporting assembly is arranged on the loading platform.

[0012] Furthermore, the thermal stimulation device includes a temperature control plate, a heating plate, and a heat dissipation block. The heating plate is arranged on the heat dissipation block, and the temperature control plate is electrically connected to the heating plate.

[0013] Furthermore, two supporting components are provided and are mirror-imaged on the horizontal fixing rod.

[0014] The spinal window covering device of the present invention can effectively protect the spinal window part of the mouse through the design of the covering main board, observation window, covering sub-board and groove structures. At the same time, the transparent covering sub-board makes it convenient for the experimenter to clearly observe the spinal cord part through the observation window. At the same time, water medium can be added to the covering sub-board to form a water mirror, providing good observation conditions for calcium signal monitoring. The spinal window covering device is covered and fixed on the window part of the mouse, and the supporting component of the spinal fixation device is clamped on the clamping part of the covering main board. The position of the stage is adjusted. The cooperation between the stage and the supporting component can stably fix mice of different sizes, prevent the mice from moving when subjected to harmful stimulation, and ensure the stability of the experimental process and the accuracy of the data. The temperature control plate of the thermal stimulation device can accurately control the temperature of the heating plate, and can accurately apply harmful thermal stimulation to the mouse, simulating a real harmful stimulation environment, and providing reliable experimental conditions for studying pain mechanisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Schematic diagram of the spinal fenestration and covering device structure of the mouse spinal cord calcium signal monitoring system under noxious stimulation; Figure 2 Schematic diagram of the spinal fixation device structure for the mouse spinal cord calcium signal monitoring system under noxious stimulation Figure 1 .

[0016] Figure 3 Schematic diagram of the spinal fixation device structure for the mouse spinal cord calcium signal monitoring system under noxious stimulation Figure 2 .

[0017] Figure 4 Schematic diagram of the stage structure of the mouse spinal cord calcium signal monitoring system under noxious stimulation.

[0018] Figure 5 Schematic diagram of the vertical structure of the mouse spinal cord calcium signal monitoring system based on noxious stimulation.

[0019] Figure 6 Schematic diagram of the thermal stimulation device structure of the mouse spinal cord calcium signal monitoring system under noxious stimulation.

[0020] Among them, the spinal window cover device-1, cover main plate-11, observation window-101, recessed portion-102, clamping portion-103, cover sub-plate-12, spinal fixation device-2, device base-21, device vertical plate-22, second strip hole-221, vertical slide plate-23, loading platform-24, first strip hole-241, extension plate-242, support component-25, clamp structure-251, upper clamp teeth-2511 , lower jaw-2512, elastic part-2513, fastening bolt-2514, limiting rod-2515, limiting hole-2516, horizontal fixing rod-252, vertical sliding rod-253, sliding block-254, first limiting bolt-255, first limiting bolt-256, third limiting bolt-257, fourth limiting bolt-26, thermal stimulation device-3, temperature control plate-31, heating plate-32, heat dissipation block-33. DETAILED DESCRIPTION

[0021] In order to make the objects and advantages of the present invention more clearly understood, the present invention is further described below in conjunction with embodiments; it should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0022] The preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood by those skilled in the art that these embodiments are only used to explain the technical principles of the present invention and are not intended to limit the scope of protection of the present invention.

[0023] It should be noted that, in the description of the present invention, terms such as "up", "down", "left", "right", "inside", and "outside" indicating directions or positional relationships are based on the directions or positional relationships shown in the accompanying drawings. This is only for the convenience of description and does not indicate or imply that the device or element must have a specific orientation, be constructed and operated in a specific orientation. Therefore, it cannot be understood as a limitation on the present invention.

[0024] Furthermore, it should be noted that, in the description of the present invention, unless otherwise expressly specified or limited, the terms "mounted," "connected," and "connected" should be understood in a broad sense. For example, they may refer to fixed connections, detachable connections, or integral connections; mechanical connections or electrical connections; direct connections or indirect connections through an intermediate medium; and internal communication between two components. Those skilled in the art will understand the specific meanings of the above terms in the present invention based on specific circumstances.

[0025] The present invention is further described in detail below with reference to the accompanying drawings.

[0026] See Figure 1The spinal fenestration covering device includes a main cover plate 11 and a sub-cover plate 12. The main cover plate 11 is provided with an observation window 101. The sub-cover plate 12 is made of a transparent material and has a recessed portion 102 and a clamping portion 103. The observation window 101 is located in the recessed portion 102. The sub-cover plate 12 covers the observation window 101 in the recessed portion 102. The clamping portions 103 are located at both ends of the main cover plate 11.

[0027] See Figure 2-Figure 5 The spinal fixation device 2 includes: a device base 21, a device vertical plate 22, a vertical sliding plate 23, a loading platform 24, a supporting assembly 25, and a fourth limiting bolt 26. A second strip hole 221 is provided on the device vertical plate 22. The device vertical plate 22 is vertically arranged at one end of the device base 21. The fourth limiting bolt 26 passes through the second strip hole 221 to set the vertical sliding plate 23 on the device vertical plate 22, and limits the position of the vertical sliding plate 23 on the device vertical plate 22. The loading platform 24 is threadedly connected to the vertical sliding plate 23. There are two supporting assemblies 25, which are mirror-imaged on the horizontal fixing rod 252. A downward arc-shaped recessed portion is provided on the top of the loading platform 24. An extension plate 242 is provided on the workbench 24, and a first strip hole 241 is set on the extension plate 242. The supporting assembly 25 includes a clamping structure 251, a horizontal fixed rod 252, a vertical sliding rod 253, a sliding block 254, a first limiting bolt 255, a first limiting bolt 256, and a third limiting bolt 257. Two through holes are provided on the sliding block 254. The clamping structure 251 is set on the horizontal fixed rod 252. One end of the horizontal fixed rod 252 passes through a through hole on the sliding block 254 and is limited by the first limiting bolt 255. One end of the vertical sliding rod 253 passes through another through hole on the sliding block 254 and is limited by the second limiting bolt 256. The other end of the vertical sliding rod 253 passes through the first strip hole 241 and is fixed to the workbench 24 by the third limiting bolt 257. The clamp structure 251 includes an upper jaw 2511, a lower jaw 2512, an elastic member 2513, and a fastening bolt 2514. The upper jaw 2511 and the lower jaw 2512 are mirror-imaged and slidingly arranged on the horizontal fixed rod 252. The elastic member 2513 is arranged on the horizontal fixed rod 252 and is located between the upper jaw 2511 and the lower jaw 2512. The fastening bolt 2514 is arranged on the end of the horizontal fixed rod 252. Rotating the fastening bolt 2514 can cause the upper jaw 2511 and the lower jaw 2512 to engage with each other. The upper jaw 2511 is provided with a limiting rod 2515, and the lower jaw 2512 is provided with a limiting hole 2516 that cooperates with the limiting rod 2515. The limiting rod 2515 is inserted into the limiting hole 2516. When the fastening bolt 2514 is rotated, the limiting rod 2515 can slide freely in the limiting hole 2516.

[0028] See Figure 6The thermal stimulation device 3 includes a temperature control plate 31 , a heating plate 32 , and a heat sink 33 . The heating plate 32 is disposed on the heat sink 33 . The temperature control plate 31 and the heating plate 32 are electrically connected.

[0029] When using the mouse spinal cord calcium signal monitoring system based on noxious stimulation of the present invention, a spinal fenestration surgery is first performed on the mouse: that is, 40 mg / kg sodium pentobarbital is injected intraperitoneally to anesthetize the mouse, and the spinal cord fenestration surgery is started after the mouse has no response to the pain stimulus. The mouse's back is shaved and the skin is prepared, disinfected with iodine tincture, and deiodinated with alcohol. A longitudinal incision is made along the mouse's back, and the left back muscles are bluntly separated with pointed tweezers. After exposing the T13 and L1 spinous processes, the vertebral bodies are cut along the pedicles on both sides and the T13 and L1 vertebral bodies are removed. Under a stereomicroscope, sufficient hemostasis is performed, the surgical field is cleaned, 1% agarose solution is added, the spinal fenestration covering device is covered, and the spinal fenestration covering device is fixed with dental cement. Next, place the mouse on the stage, adjust the position of the clamp structure in the holding assembly, and rotate the fastening bolts 2514 so that the upper and lower jaws 2511 and 2512 engage the clamping portion 103 covering the ends of the main board 11. Adjust the position of the vertical slide plate on the device's vertical plate to position the mouse at the appropriate height. Specifically, loosen the first, second, and third limit bolts. Adjust the clamp structure to the appropriate position by sliding the horizontal fixed rod and vertical slide rod on the slide block and moving the vertical slide rod within the first strip hole. Tighten the limit bolts. Then, rotate the clamp structure's fastening bolts so that the upper and lower jaws engage the clamping portion, firmly securing the mouse. Place the spinal cord window close to the microscope lens. Add sterile water to the lens and begin imaging. Imaging parameters are set as follows: scan speed: 30 Hz / frame, pixels: 512×512, laser excitation wavelength: 920 nm. During the imaging process, the central blood vessels of the spinal cord were used as a landmark to distinguish the left and right sides, and the imaging depth was about 250 μm below the surface of the spinal cord. Finally, the mouse's left foot was secured to a heating plate with the palm resting against it. Before stimulation began, the temperature was set to 25°C and maintained for at least 10 minutes. The thermal stimulation temperature was set to 45°C and the duration was set to 10 seconds. This precise noxious thermal stimulation was applied to the mouse, while spinal cord calcium signals were monitored and recorded using relevant monitoring equipment.

[0030] The present invention is not limited to the above-mentioned specific implementation methods. Various changes made by ordinary technicians in this field based on the above-mentioned concept without creative work are all within the scope of protection of the present invention.

Claims

1. A mouse spinal cord calcium signal monitoring system based on noxious stimulation, characterized in that: The system comprises: a spinal fenestration covering device (1) for covering a window during spinal fenestration surgery in mice; Spinal fixation device to prevent mouse movement (2); Thermal stimulation device for painful stimulation of mice (3); The spinal column fenestration covering device (1) comprises a covering main board (11), and an observation window (101) is provided on the covering main board (11).

2. The mouse spinal cord calcium signal monitoring system based on noxious stimulation according to claim 1, characterized in that: The spinal window covering device (1) further includes a covering sub-plate (12), the covering sub-plate (12) being made of a transparent material, the covering main plate (11) being provided with a recessed portion (102) and a clamping portion (103), the observation window (101) being arranged in the recessed portion (102), the covering sub-plate (12) covering the observation window (101) in the recessed portion (102), and the clamping portion (103) being arranged at both ends of the covering main plate (11).

3. The mouse spinal cord calcium signal monitoring system based on noxious stimulation according to claim 1, characterized in that: The spinal fixation device (2) comprises: a device base (21), a device vertical plate (22), a vertical slide plate (23), a loading platform (24), and a supporting component (25). The device vertical plate (22) is vertically arranged at one end of the device base (21), the vertical slide plate (23) can slide up and down on the device vertical plate (22), the loading platform (24) is connected to the vertical slide plate (23), and the supporting component (25) is arranged on the loading platform (24).

4. The mouse spinal cord calcium signal monitoring system based on noxious stimulation according to claim 3, characterized in that: The loading platform (24) is provided with a first strip hole (241), and the supporting assembly (25) includes a clamping structure (251), a horizontal fixing rod (252), a vertical sliding rod (253), a sliding block (254), a first limiting bolt (255), a first limiting bolt (256), and a third limiting bolt (257). The sliding block (254) is provided with two through holes. The clamping structure (251) is set on the horizontal fixing rod (252), one end of the horizontal fixing rod (252) passes through a through hole on the sliding block (254) and is limited by the first limiting bolt (255), one end of the vertical sliding rod (253) passes through another through hole on the sliding block (254) and is limited by the second limiting bolt (256), and the other end of the vertical sliding rod (253) passes through the first strip hole (241) and is fixed to the loading platform (24) by the third limiting bolt (257).

5. The mouse spinal cord calcium signal monitoring system based on noxious stimulation according to claim 4, characterized in that: The clamp structure (251) comprises an upper clamp tooth (2511), a lower clamp tooth (2512), an elastic member (2513), and a fastening bolt (2514); the upper clamp tooth (2511) and the lower clamp tooth (2512) are arranged on a horizontal fixing rod (252) in a mirror-image sliding manner; the elastic member (2513) is arranged on the horizontal fixing rod (252) and is located between the upper clamp tooth (2511) and the lower clamp tooth (2512); the fastening bolt (2514) is arranged on the end of the horizontal fixing rod (252); and rotating the fastening bolt (2514) can cause the upper clamp tooth (2511) and the lower clamp tooth (2512) to engage with each other.

6. The mouse spinal cord calcium signal monitoring system based on noxious stimulation according to claim 5, characterized in that: The upper jaw (2511) is provided with a limiting rod (2515), and the lower jaw (2512) is provided with a limiting hole (2516) that matches the limiting rod (2515). The limiting rod (2515) is inserted into the limiting hole (2516), and when the fastening bolt (2514) is rotated, the limiting rod (2515) can slide freely in the limiting hole (2516).

7. The mouse spinal cord calcium signal monitoring system based on noxious stimulation according to claim 4, characterized in that: An extension plate (242) is provided on the loading platform (24), and the first strip-shaped hole (241) is provided on the extension plate (242).

8. The mouse spinal cord calcium signal monitoring system based on noxious stimulation according to claim 1, characterized in that: The spinal fixation device (2) comprises: a device base (21), a device vertical plate (22), a vertical sliding plate (23), a loading platform (24), a supporting assembly (25), and a fourth limiting bolt (26). The device vertical plate (22) is provided with a second strip hole (221). The device vertical plate (22) is vertically arranged at one end of the device base (21). The fourth limiting bolt (26) passes through the second strip hole (221) to set the vertical sliding plate (23) on the device vertical plate (22). The loading platform (24) is connected to the vertical sliding plate (23). The supporting assembly (25) is arranged on the loading platform (24).

9. The mouse spinal cord calcium signal monitoring system based on noxious stimulation according to claim 4, characterized in that: The thermal stimulation device (3) comprises a temperature control plate (31), a heating plate (32), and a heat dissipation block (33). The heating plate (32) is arranged on the heat dissipation block (33). The temperature control plate (31) is electrically connected to the heating plate (32).

10. The mouse spinal cord calcium signal monitoring system based on noxious stimulation according to any one of claims 1 to 9, characterized in that: The supporting components (25) are provided with two, and are mirror-imaged and arranged on the horizontal fixing rod (252).

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