Long-term optical imaging observation method for mouse spinal cord

Abstract

The invention belongs to the technical field of mouse spinal cord observation, and provides a mouse spinal cord long-term optical imaging observation method which comprises the following steps: step 1, establishing a spinal cord observation cavity; secondly, the cone plate is cut off; step 3, establishing a spinal cord observation window; 4, spinal cord imaging observation; the mouse spinal cord observation device is provided with a connecting rod, a side plate and a stabilizing plate, T12 and L1 outside the mouse spinal cord are conveniently matched, the purpose of observing the mouse spinal cord at the T13 position is achieved, protection is conducted outside the side plate and the stabilizing plate in cooperation with a protection cover, the protection cover can be opened from the side plate and the stabilizing plate in the observation process of the mouse spinal cord, and the mouse spinal cord observation device is convenient to use. And the observation cavity and the observation window are matched with a two-photon fluorescence microscope to achieve the purpose of long-term observation of the T13 mouse spinal cord.

Description

Technical Field

[0001] This invention belongs to the field of mouse spinal cord observation technology, specifically a long-term optical imaging observation method for mouse spinal cord. Background Technology

[0002] Mice and humans share very similar genes, with 99% of mouse genes being homologous to human genes. Although the general structure of the spinal cord in mammals is similar, there are still some differences in neural connections and neural tissue, which makes it essential to study the mouse spinal cord.

[0003] However, the lack of methods for long-term observation of mouse spinal cord leads to difficulties in conducting long-term observations, resulting in unclear observations of the mouse spinal cord.

[0004] To address the problems raised in the background art, those skilled in the art have proposed a long-term optical imaging observation method for mouse spinal cord. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a long-term optical imaging observation method for mouse spinal cord, thereby solving the problem that long-term observation of mouse spinal cord is difficult in the prior art.

[0006] A long-term optical imaging observation assembly for mouse spinal cord includes: a mouse spinal cord, and T12, T13 and L1 located outside the mouse spinal cord; a connecting mechanism fixedly disposed on T12, T13 and L1; and an observation mechanism fixedly disposed on the connecting mechanism for long-term observation of mouse spinal cord.

[0007] Preferably, the connecting mechanism includes two connecting rods fixedly disposed on T12 and L1, and T12 and L14 are respectively fitted with two side plates by the two connecting rods; and the ends of the two side plates are respectively fitted into the depressions on the lower side of the articular processes a of T12, T13 and L1.

[0008] Preferably, the observation mechanism includes a stabilizing plate fixedly mounted on two side plates, and a protective cover fixedly mounted on the two side plates outside the stabilizing plate; a cover glass is fixedly mounted on the T13 and inside the stabilizing plate.

[0009] A long-term optical imaging observation method for mouse spinal cord includes: Step 1, establishing a spinal cord observation cavity; Step 2, removing the cone lamina; Step 3, establishing a spinal cord observation window; Step 4, spinal cord imaging observation.

[0010] Preferably, in step one, a spinal cord observation cavity is established.

[0011] S1. Preoperative preparation: Disinfect tools and implantation components, moisten hemostatic sponge with physiological saline, prepare 0.5% lidocaine solution, weigh mice and prepare analgesics {caspofen, dosage 5.0 mg / kg}, induce anesthesia with {2% isoflurane}, stabilize body temperature and respiratory rate with electric blanket, apply eye ointment, remove hair from waist area, disinfect skin, and administer analgesics;

[0012] S2. Operating procedures

[0013] a. Adjust the animal's position so that the lumbar ridge is aligned with the side plate, and drape it with a towel to enhance aseptic operation;

[0014] b. After confirming the disappearance of the pinch reflex in the mouse's toes, make an incision of about 1.0 cm in the skin along the midline of the lumbar vertebral prominence, and inject lidocaine subcutaneously through the incision at the same time;

[0015] c. Determine the locations of the T12, T13, and L1 vertebrae;

[0016] d. Use sharp microsurgical scissors to penetrate the fascia and paravertebral muscles on both sides of the spinous processes of T12, T13 and L1 vertebrae, and bluntly separate the muscles on the lamina. If there is bleeding, use a hemostatic sponge to stop the bleeding.

[0017] e. Lift and remove the loose paravertebral muscles on both sides of the spinous processes of T12, T13 and L1, taking care not to cut the intervertebral muscles, and to keep the tissue moist, cover the intervertebral muscles with a moistened hemostatic sponge.

[0018] f. Cut the tendons connecting to the T13 articular process. To make these tendons more visible, a spreader can be used to spread the muscles covering the T13 lamina.

[0019] g. Scrape clean the bone surface of all lamina and spinous processes, and remove any remaining muscles attached to the lamina if necessary;

[0020] h. Insert the 2mm thick connecting rod through and leave it in the T12 spinous process, and repeat this operation at the L1 spinous process;

[0021] i. Clamp the side plate onto the special clamp and slide it into the surgical area, then lift the connecting rod so that it is above the side plate;

[0022] j. Gradually and slowly insert the exposed muscles and skin under the side plate, while pushing the side plate towards and against the underside of the spinal articular processes. Before the side plate contacts the vertebrae, adjust the spine by pressing T13 and lower the side plate to make both side plates level, creating a flat foundation for the stable plate to attach.

[0023] k. Remove the soaked hemostatic sponge, dry the surgical area, and cover all intervertebral spaces with Kwik-Sil sealing gel;

[0024] l. Secure the connecting rod to the side plate with instant adhesive;

[0025] m. After the glue dries, suture the skin around the side plate;

[0026] n. Seal the area around the suture with an appropriate amount of quick-drying adhesive;

[0027] o. Loosen the side plate clamps to allow the side plate and connecting rod to be fitted onto the mouse's back;

[0028] P. Apply instant adhesive to the side plate handle, then place the stabilizing plate on top and manually press it to bond it. Reconnect the side plate clamps and further clamp the side plate. When clamping, add instant adhesive to the groove inside the stabilizing plate.

[0029] q. Scrape the exposed vertebral lamina to increase the area for bone cement bonding;

[0030] r. To further prepare the bone for bonding with the bone cement, apply dentin activator gel to the bone surface for 15 seconds, then rinse with saline and blot dry;

[0031] s. Apply a generous amount of flowable cement to cover the prepared bone, and insert the spinous process connecting rod and the edge of the lateral plate;

[0032] t. Remove the cement from the T13 lamina before it hardens to facilitate later laminectomy;

[0033] u. To protect the exposed lamina, the T13 lamina and surrounding cement were covered with Kwik-Si 1 sealant;

[0034] v. Finally, attach the magnetic protective cap to the stabilizing plate to cover the observation chamber;

[0035] S3. Postoperative examination and recovery: Provide the mice with calories, monitor their recovery, administer opioid analgesics, and place them in a non-magnetic cage after they are fully awake.

[0036] Preferably, in step two, the conical plate is removed.

[0037] S1. Preoperative preparation: After the observation cavity is established for at least one week, weigh the mouse and prepare analgesics, induce anesthesia, stabilize body temperature and respiratory rate, and apply eye ointment.

[0038] S2. Operating procedures

[0039] a. Remove the Kwk-Si sealing gel from the T13 lamina, and if necessary, remove the bone cement at the edge of the T13 lamina using a micro-drill;

[0040] b. Make transverse incisions at both the upper and lower ends of the vertebral lamina;

[0041] c. Cut the intervertebral ligaments, separate and remove the lamina, taking care to separate any attachments on the lamina before pulling it apart;

[0042] d. Immediately cover the spinal cord with a moistened hemostatic sponge to maintain moisture and stop the bleeding;

[0043] e. Make a longitudinal incision in the dura mater and pull it laterally to expose the spinal cord. Replace the hemostatic head and, if necessary, expand the laminectomy area to expose the gray dorsal horn.

[0044] f. Cut a piece of fibrosis-inhibiting membrane according to the size of the exposed area of ​​the spinal cord;

[0045] g. Remove the hemostatic sponge and directly cover the spinal cord with the anti-fibrotic membrane;

[0046] h. Placing a moistened hemostatic sponge at the edge of the fibrotic membrane helps limit inward tissue growth;

[0047] i. Remove the hemostatic sponge, apply Kwik-Sil, seal the entire area with sealing glue, fix the fibrosis-inhibiting membrane, and prepare for the next stage;

[0048] i. Finally, attach the magnetic protective cap to the stabilizing plate to cover the observation chamber;

[0049] S3. Postoperative examination and recovery: provide the mice with calories, monitor their recovery, administer opioid analgesics, and proceed directly to step three based on the hemostatic effect. Alternatively, the mice can be placed in a non-magnetic cage after they are fully awake, and the surgery in step three can be performed at a later date as needed.

[0050] Preferably, in step three, a spinal cord observation window is established.

[0051] S1. Preoperative preparation: Weigh the mice and prepare analgesics, induce anesthesia, stabilize body temperature and respiratory rate, and apply eye ointment.

[0052] S2. Operating procedures

[0053] a. Remove the Wik-Si sealing gel and fibrosis-inhibiting membrane from the area to expose the spinal cord;

[0054] b. Immediately place a moistened hemostatic sponge on the spinal cord to maintain moisture and stop bleeding. If necessary, remove the hemostatic sponge placed around the fibrotic membrane in step two to expand the exposed area of ​​the spinal cord.

[0055] c. Cut a piece of anti-fibrotic membrane to the size suitable for exposing the spinal cord, remove the hemostatic sponge, quickly dry it with a cellulose sponge, and immediately place the cut anti-fibrotic membrane directly on the surface of the spinal cord.

[0056] d. Cover the area of ​​the fibrotic membrane and laminectomy site with a small amount of Kwik-Sil sealing gel;

[0057] e. Immediately place a 3.0mm diameter circular coverslip, press for 1 second to squeeze out excess Kwik-si adhesive, and repeat twice;

[0058] f. Let stand for 10 minutes to allow the Kwik-Sil sealant to harden;

[0059] g. Completely cover the edges of the Kwik-Si layer with bone cement to seal and permanently fix the coverslip;

[0060] h. Finally, attach the magnetic protective cap to the stabilizing plate to cover the observation cavity;

[0061] S3. Postoperative recovery: Provide heat, monitor recovery, administer opioid analgesics, and place the mice in a non-magnetic cage after they are fully awake for spinal cord imaging observation.

[0062] Preferably, step four involves spinal cord imaging observation.

[0063] S1. Adaptation training: Before imaging recording, mice need to adapt to the imaging room or imaging equipment for 1 to 3 days.

[0064] S2. Environmental control: The noise level in the imaging room is controlled between 53.0–53.3 dBA {with all instruments off} and 59.7–60.4 dBA {with all instruments on}.

[0065] S3. Imaging while awake.

[0066] a. Remove the mouse from the cage with the observation window, remove the magnetic protective cover, and wipe the imaging window slide clean with an alcohol swab;

[0067] b. Use clamps to hold the stabilizing plate and fix the mouse to the imaging gantry to ensure the mouse remains stable during imaging;

[0068] c. Place the observation window directly below the objective lens (water mirror) of the two-photon fluorescence microscope, and inject an appropriate amount of physiological saline into the imaging cavity;

[0069] d. First, use a 10x scope to make a general observation in order to adjust and determine the observation area;

[0070] e. Then, a 25x or 40x microscope is used for detailed observation and recording of local areas. For photon scanning imaging, a laser wavelength of 920-945nm is selected.

[0071] f. Move the imaging gantry, absorb the water from the imaging chamber, and wipe the imaging window glass again with anhydrous alcohol swabs;

[0072] g. Attach the magnetic protective cap to the stabilizing plate to cover the observation cavity;

[0073] h. Release the clamps from the stabilizing plate and return the mice to their non-magnetic rearing cages.

[0074] Compared with the prior art, the present invention has the following beneficial effects:

[0075] This invention, by incorporating a connecting rod, side plate, and stabilizing plate, facilitates the observation of the mouse spinal cord at the T13 location by using the external T12 and L1 structures. A protective cover is placed outside the side plate and stabilizing plate for protection, allowing the protective cover to be opened from the side plate and stabilizing plate during observation. The observation cavity and window, combined with a two-photon fluorescence microscope, enable long-term observation of the mouse spinal cord at the T13 location. Attached Figure Description

[0076] Figure 1 This is a structural diagram of the mouse spinal cord observed according to the present invention;

[0077] Figure 2 This is a diagram illustrating the operational steps of long-term optical imaging observation of the mouse spinal cord according to the present invention.

[0078] Figure 3 This is a structural diagram of the side plate position of the present invention.

[0079] In the picture:

[0080] 1. Mouse spinal cord; 2. T12; 3. T13; 4. L1; 5. Connecting rod; 6. Lateral plate; 7. Coverslip; 8. Stabilizing plate; 9. Protective cover. Detailed Implementation

[0081] The embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples. The following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention.

[0082] Example

[0083] As attached Figure 1 To be continued Figure 3 As shown:

[0084] The present invention provides a long-term optical imaging observation component for mouse spinal cord, comprising: mouse spinal cord 1, and T122, T133 and L14 located outside mouse spinal cord 1; a connecting mechanism fixedly disposed on T122, T133 and L14; and an observation mechanism fixedly disposed on the connecting mechanism for long-term observation of mouse spinal cord 1.

[0085] refer to Figure 2 and Figure 3 The connecting mechanism includes a connecting rod 5 and a side plate 6;

[0086] By fixing connecting rods 5 to both T122 and T133, T122 and T133 can be connected to two side plates 6 by the two connecting rods 5, and one end of the two side plates 6 is respectively connected to the recess on the lower side of the articular process a of T122, T133 and L14. This makes it easy for the two side plates 6 to be stably engaged with the two connecting rods 5 on both sides of T122, T133 and L14.

[0087] refer to Figure 2 and Figure 3 The observation mechanism includes a coverslip 7, a stabilizing plate 8, and a protective cover 9;

[0088] A stabilizing plate 8 is fixedly connected to the two side plates 6 with screws, allowing the stabilizing plate 8 to be stably installed on the two side plates 6. The stabilizing plate 8 also fixes and reinforces the two side plates 6. A coverslip 7 is fixedly connected to T133 inside the stabilizing plate 8, making it easy to observe the mouse spinal cord 1 through the coverslip 7 at T133. Furthermore, a protective cover 9 is fixedly connected to the two side plates 6 outside the stabilizing plate 8. The protective cover 9 not only protects the stabilizing plate 8 and the coverslip 7, but also allows for easy opening of the protective cover 9 for multiple observations of the mouse spinal cord 1, thus facilitating long-term and convenient observation of the mouse spinal cord 1.

[0089] refer to Figure 1 and Figure 2 A long-term optical imaging observation method for mouse spinal cord also includes the following steps.

[0090] Step 1: Establish observation cavity 1 in mouse spinal cord:

[0091] S1. Preoperative preparation: Disinfect the tools and implantation components {including connecting rod 5, side plate 6, stabilizing plate 8 and protective cover 9}, moisten the hemostatic sponge with physiological saline, prepare 0.5% lidocaine solution, weigh the mouse and prepare the analgesic {caspofen, dosage 5.0 mg / kg}, induce anesthesia with {2% isoflurane}, stabilize body temperature and respiratory rate with an electric blanket, apply eye ointment, remove hair from the waist, disinfect the skin, and administer analgesic.

[0092] S2. Operating procedures

[0093] a. Adjust the animal's position so that the lumbar ridge is aligned with the side plate 6, and place a towel to enhance aseptic operation;

[0094] b. After confirming the disappearance of the pinch reflex in the mouse's toes, make an incision of about 1.0 cm in the skin along the midline of the lumbar vertebral prominence, and inject lidocaine subcutaneously through the incision at the same time;

[0095] c. Determine the locations of the T122, T133, and L14 vertebrae;

[0096] d. Use sharp microsurgical scissors to penetrate the fascia and paravertebral muscles on both sides of the spinous processes of T122, T133 and L14 vertebrae, and bluntly separate the muscles on the lamina. If there is bleeding, use a hemostatic sponge to stop the bleeding.

[0097] e. Lift and remove the loose paravertebral muscles on both sides of the spinous processes of T122, T133 and L14, taking care not to cut the intervertebral muscles, and to keep the tissue moist, cover the intervertebral muscles with a moistened hemostatic sponge.

[0098] f. Cut the tendons connecting the T133 articular processes. To make these tendons more visible, a spreader can be used to spread the muscles covering the T133 lamina.

[0099] g. Scrape clean the bone surface of all lamina and spinous processes, and remove any remaining muscles attached to the lamina if necessary;

[0100] h. Penetrate the 2mm thick connecting rod 5 through and leave it in the T122 spinous process, and repeat this operation at the L14 spinous process;

[0101] i. Clamp the side plate 6 onto the special clamp and slide it into the surgical area, then lift the connecting rod 5 so that it is above the side plate 6;

[0102] j. Gradually and slowly insert the exposed muscles and skin under the side plate 6, while pushing the side plate 6 towards and against the underside of the spinal articular process. Before the side plate 6 contacts the vertebra, adjust the spine by pressing T133 and lower the side plate 6 to make the two side plates 6 level, creating a flat foundation for the attachment of the stabilizing plate 8.

[0103] k. Remove the soaked hemostatic sponge, dry the surgical area, and cover all intervertebral spaces with Kwik-Sil sealing gel;

[0104] l. Fix the connecting rod 5 to the side plate 6 with instant adhesive;

[0105] m. After the glue dries, suture the skin around the side plate 6;

[0106] n. Seal the area around the suture with an appropriate amount of quick-drying adhesive;

[0107] o. Loosen the clamp on the side plate 6 so that the side plate 6 and the connecting rod 5 are fitted onto the back of the mouse;

[0108] P. Apply instant adhesive to the handle of side plate 6, then place the stabilizing plate 8 on top and manually press it to bond it. Reconnect the side plate 6 clamp and further clamp the side plate 6. When clamping, add instant adhesive to the groove inside the stabilizing plate 8.

[0109] q. Scrape the exposed vertebral lamina to increase the area for bone cement bonding;

[0110] r. To further prepare the bone for bonding with the bone cement, apply dentin activator gel to the bone surface for 15 seconds, then rinse with saline and blot dry;

[0111] s. Apply a large amount of flowable cement to cover the prepared bone, and pass it through the edges of the spinous process connecting rod 5 and the lateral plate 6;

[0112] t. Remove the cement from the T133 lamina before it hardens to facilitate later laminectomy;

[0113] u. To protect the exposed lamina, the T133 lamina and surrounding cement were covered with Kwik-Si 1 sealant;

[0114] v. Finally, the magnetic protective cover 9 is attached to the stabilizing plate 8 to cover the observation cavity;

[0115] S3. Postoperative examination and recovery: Provide the mice with calories, monitor their recovery, administer opioid analgesics, and place them in a non-magnetic cage after they are fully awake.

[0116] refer to Figure 1 and Figure 2 Step 2, remove the conical plate:

[0117] S1. Preoperative preparation: After the observation cavity is established for at least one week, weigh the mouse and prepare analgesics, induce anesthesia, stabilize body temperature and respiratory rate, and apply eye ointment.

[0118] S2. Operating procedures

[0119] a. Remove the Kwk-Si sealing gel from the T133 lamina, and if necessary, remove the bone cement at the edge of the T133 lamina using a micro-drill;

[0120] b. Make transverse incisions at both the upper and lower ends of the vertebral lamina;

[0121] c. Cut the intervertebral ligaments, separate and remove the lamina, taking care to separate any attachments on the lamina before pulling it apart;

[0122] d. Immediately cover the mouse spinal cord 1 with a moistened hemostatic sponge to retain moisture and stop bleeding;

[0123] e. Make a longitudinal incision in the dura mater and pull it laterally to expose the mouse spinal cord 1. Replace the hemostatic seam and, if necessary, expand the laminectomy area to expose the gray dorsal horn.

[0124] f. Cut a piece of fibrosis-inhibiting membrane according to the size of the exposed area of ​​the mouse spinal cord 1;

[0125] g. Remove the hemostatic sponge and directly apply the anti-fibrotic membrane to the mouse spinal cord 1;

[0126] h. Placing a moistened hemostatic sponge at the edge of the fibrotic membrane helps limit inward tissue growth;

[0127] i. Remove the hemostatic sponge, apply Kwik-Sil, seal the entire area with sealing glue, fix the fibrosis-inhibiting membrane, and prepare for the next stage;

[0128] i. Finally, the magnetic protective cover 9 is attached to the stabilizing plate 8 to cover the observation cavity;

[0129] S3. Postoperative examination and recovery: provide the mice with calories, monitor their recovery, administer opioid analgesics, and proceed directly to step three based on the hemostatic effect. Alternatively, the mice can be placed in a non-magnetic cage after they are fully awake, and the surgery in step three can be performed at a later date as needed.

[0130] refer to Figure 1 Step 3: Establish an observation window for the mouse spinal cord:

[0131] S1. Preoperative preparation: Weigh the mice and prepare analgesics, induce anesthesia, stabilize body temperature and respiratory rate, and apply eye ointment.

[0132] S2. Operating procedures

[0133] a. Remove the Wik-Si sealing gel and the anti-fibrotic membrane from the area to expose the mouse spinal cord 1;

[0134] b. Immediately place a moistened hemostatic sponge on the mouse spinal cord 1 to maintain moisture and stop bleeding. If necessary, remove the hemostatic sponge placed around the fibrotic membrane in step two to expand the exposed area of ​​the mouse spinal cord 1.

[0135] c. Cut a piece of anti-fibrotic membrane to the size suitable for exposing mouse spinal cord 1, remove the hemostatic sponge, quickly dry it with a cellulose sponge, and immediately place the cut anti-fibrotic membrane directly on the surface of mouse spinal cord 1.

[0136] d. Cover the area of ​​the fibrotic membrane and laminectomy site with a small amount of Kwik-Sil sealing gel;

[0137] e. Immediately place a 3.0mm diameter circular coverslip 7, press for 1 second to squeeze out excess Kwik-si adhesive, and repeat twice;

[0138] f. Let stand for 10 minutes to allow the Kwik-Sil sealant to harden;

[0139] g. Completely cover the edges of the Kwik-Si layer with bone cement to seal it, and permanently fix the coverslip 7;

[0140] h. Finally, the magnetic protective cover 9 is attached to the stabilizing plate 8 to cover the observation cavity;

[0141] S3. Postoperative recovery: Provide heat, monitor recovery, administer opioid analgesics, and after the mice are fully awake, place them in non-magnetic cages for spinal cord imaging observation.

[0142] refer to Figure 1 Step 4: Imaging observation of mouse spinal cord 1:

[0143] S1. Adaptation training: Before imaging recording, mice need to adapt to the imaging room or imaging equipment for 1 to 3 days. This process helps reduce the stress and anxiety of mice during the experiment and ensures the accuracy and reliability of the imaging results.

[0144] S2. Environmental control: The noise level in the imaging room is controlled between 53.0–53.3 dBA {when all instruments are off} and 59.7–60.4 dBA {when all instruments are on}. Noise control helps reduce external interference and ensures that the mice can remain relatively quiet during imaging.

[0145] S3. Imaging while awake.

[0146] a. Remove the mouse with the observation window from the feeding cage, remove the magnetic protective cover 9, and wipe the imaging window slide clean with an alcohol swab;

[0147] b. Use clamps to hold the stabilizing plate 8 and fix the mouse on the imaging gantry to ensure that the mouse remains stable during imaging;

[0148] c. Place the observation window directly below the objective lens (water mirror) of the two-photon fluorescence microscope, and inject an appropriate amount of physiological saline into the imaging cavity;

[0149] d. First, use a 10x scope to make a general observation in order to adjust and determine the observation area;

[0150] e. Then, a 25x or 40x microscope is used for detailed observation and recording of local areas. For photon scanning imaging, a laser wavelength of 920-945nm is selected.

[0151] f. Move the imaging gantry, absorb the water from the imaging chamber, and wipe the imaging window glass again with anhydrous alcohol swabs;

[0152] g. Use a magnetic protective cover 9 to attach to the stabilizing plate 8 to cover the observation cavity;

[0153] h. Release the clamps from the stabilizing plate 8 and return the mice to the non-magnetic rearing cage.

[0154] The embodiments of the present invention are given for the purposes of illustration and description. Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

[0155] Finally, a few points should be noted:

[0156] First, it should be noted in the description of this application that, unless otherwise specified and limited, the terms "installation", "connection" and "linkage" should be interpreted broadly, and can be mechanical or electrical connection, or internal connection between two components, or direct connection. "Up", "down", "left", "right", etc. are only used to indicate relative positional relationship. When the absolute position of the object being described changes, the relative positional relationship may change.

[0157] Secondly: The accompanying drawings of the embodiments disclosed in this invention only involve the structures involved in the embodiments disclosed in this invention. Other structures can refer to the general design. In the absence of conflict, the same embodiment and different embodiments of this invention can be combined with each other.

[0158] Finally: The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0159] The above are all preferred embodiments of this application, and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made in accordance with the structure, shape and principle of this application should be covered within the scope of protection of this application.

Claims

1. A mouse spinal cord long-term optical imaging observation assembly, characterized in that, include: Mouse spinal cord (1), and T12 (2), T13 (3) and L1 (4) located outside the mouse spinal cord (1); The connecting mechanism is fixedly installed on T12(2), T13(3) and L1(4); The observation mechanism is fixedly installed on the connecting mechanism and is used for long-term observation of mouse spinal cord (1).

2. The mouse spinal cord chronic optical imaging observation assembly of claim 1, wherein: The connecting mechanism includes two connecting rods (5) fixedly disposed on T12 (2) and L1 (4), and T12 (2) and L1 (4) are respectively fitted with two side plates (6) by the two connecting rods (5); The ends of the two side plates (6) are respectively engaged with the depressions on the lower side of the articular processes a of T12 (2), T13 (3) and L1 (4).

3. The long-term optical imaging observation component for mouse spinal cord as described in claim 1, characterized in that: The observation mechanism includes a stabilizing plate (8) fixedly mounted on two side plates (6), and a protective cover (9) is fixedly mounted on the two side plates (6) outside the stabilizing plate (8); A cover glass sheet (7) is fixedly installed on the T13 (3) and inside the stabilizing plate (8).

4. A method for long-term optical imaging observation of mouse spinal cord as described in any one of claims 1-3, characterized in that, The following steps are included: Step 1: Establish the observation cavity of the mouse spinal cord (1); Step two, remove the conical plate; Step 3: Establish the mouse spinal cord (1) observation window; Step 4: Imaging observation of mouse spinal cord (1).

5. The long-term optical imaging observation method for mouse spinal cord as described in claim 4, characterized in that: In step one, an observation cavity is established in the mouse spinal cord (1). S1. Preoperative preparation: Disinfect the tools and implantation components {including connecting rod (5), side plate (6), stabilizing plate (8) and protective cap (9)}, moisten the hemostatic sponge with physiological saline, prepare 0.5% lidocaine solution, weigh the mouse and prepare analgesic {caspofen, dosage of 5.0 mg / kg}, induce anesthesia with {2% isoflurane}, stabilize body temperature and respiratory rate with electric blanket, apply eye ointment, remove hair from the waist, disinfect the skin, and administer analgesic; S2. Operating procedures a. Adjust the animal's position so that the lumbar ridge is aligned with the side plate (6), and place a towel to enhance aseptic operation; b. After confirming the disappearance of the pinch reflex in the mouse's toes, make an incision of about 1.0 cm in the skin along the midline of the lumbar vertebral prominence, and inject lidocaine subcutaneously through the incision at the same time; c. Determine the positions of the T12(2), T13(3), and L1(4) vertebrae; d. Use sharp microscissors to penetrate the fascia and paravertebral muscles on both sides of the spinous processes of T12(2), T13(3) and L1(4) vertebrae, and bluntly separate the muscles on the lamina. If there is bleeding, use a hemostatic sponge to stop the bleeding. e. Lift and remove the loose paravertebral muscles on both sides of the spinous processes of T12(2), T13(3) and L1(4), being careful not to cut the intervertebral muscles, and to keep the tissue moist, cover the intervertebral muscles with a moistened hemostatic sponge. f. Cut the tendons connecting the T13(3) articular processes. To make these tendons more visible, a spreader can be used to spread the muscles covering the T13(3) lamina. g. Scrape clean the bone surface of all lamina and spinous processes, and remove any remaining muscles attached to the lamina if necessary; h. Pass the 2mm thick connecting rod (5) through and leave it in the T12 (2) spinous process, and repeat this operation at the L1 (4) spinous process; i. Clamp the side plate (6) onto the special clamp and slide it into the surgical area, then lift the connecting rod (5) so that it is above the side plate (6); j. Gradually and slowly insert the exposed muscles and skin under the side plate (6), while pushing the side plate (6) towards and against the underside of the spinal articular process. Before the side plate (6) contacts the vertebra, adjust the spine by pressing T13 (3) and lower the side plate (6) to make the two side plates (6) level, creating a flat foundation for the attachment of the stabilizing plate (8). k. Remove the soaked hemostatic sponge, dry the surgical area, and cover all intervertebral spaces with Kwik-Sil sealing gel; l. Fix the connecting rod (5) to the side plate (6) with instant adhesive; m. After the glue dries, suture the skin around the side plate (6); n. Seal the area around the suture with an appropriate amount of quick-drying adhesive; o. Loosen the clamps on the side plate (6) so that the side plate (6) and the connecting rod (5) are fitted onto the back of the mouse; P. Apply instant adhesive to the handle of the side plate (6), then place the stabilizing plate (8) on top and manually press it to bond it. Reconnect the side plate (6) clamp and further clamp the side plate (6). When clamping, add instant adhesive to the groove inside the stabilizing plate (8). q. Scrape the exposed vertebral lamina to increase the area for bone cement bonding; r. To further prepare the bone for bonding with the bone cement, apply dentin activator gel to the bone surface for 15 seconds, then rinse with saline and blot dry; s. Apply a large amount of flowable cement to cover the prepared bone, and pass it through the edges of the spinous process connecting rod (5) and the lateral plate (6); t. Before the cement hardens, remove the cement from the T13(3) lamina to facilitate later laminectomy; u. To protect the exposed lamina, the T13(3) lamina and surrounding cement were covered with Kwik-Si 1 sealant; v. Finally, the magnetic protective cover (9) is attached to the stabilizing plate (8) to cover the observation cavity; S3. Postoperative examination and recovery: Provide the mice with calories, monitor their recovery, administer opioid analgesics, and place them in a non-magnetic cage after they are fully awake.

6. The long-term optical imaging observation method for mouse spinal cord (1) as described in claim 5, characterized in that: In step two, the conical plate is removed. S1. Preoperative preparation: After the observation cavity is established for at least one week, weigh the mouse and prepare analgesics, induce anesthesia, stabilize body temperature and respiratory rate, and apply eye ointment. S2. Operating procedures a. Remove the Kwk-Si sealing gel from the T13(3) lamina, and if necessary, remove the bone cement from the edge of the T13(3) lamina with a micro drill; b. Make transverse incisions at both the upper and lower ends of the lamina; c. Cut the intervertebral ligaments, separate and remove the lamina, taking care to separate any attachments on the lamina before pulling it apart; d. Immediately cover the mouse spinal cord (1) with a moistened hemostatic sponge to retain moisture and stop bleeding; e. Make a longitudinal incision in the dura mater and pull it to the side to expose the mouse spinal cord (1), replace the hemostatic seam, and if necessary expand the laminectomy area to expose the gray dorsal horn; f. Cut a piece of fibrosis-inhibiting membrane according to the size of the exposed area of ​​the mouse spinal cord (1); g. Remove the hemostatic sponge and directly apply the anti-fibrotic membrane to the mouse spinal cord (1); h. Placing a moistened hemostatic sponge at the edge of the fibrotic membrane helps limit inward tissue growth; i. Remove the hemostatic sponge, apply Kwik-Sil, seal the entire area with sealing glue, fix the fibrosis-inhibiting membrane, and prepare for the next stage; i. Finally, the magnetic protective cover (9) is attached to the stabilizing plate (8) to cover the observation cavity; S3. Postoperative examination and recovery: provide the mice with calories, monitor their recovery, administer opioid analgesics, and proceed directly to step three based on the hemostatic effect. Alternatively, the mice can be placed in a non-magnetic cage after they are fully awake, and the surgery in step three can be performed at a later date as needed.

7. The long-term optical imaging observation method for mouse spinal cord (1) as described in claim 6, characterized in that: In step three, an observation window is established in the mouse spinal cord (1). S1. Preoperative preparation: Weigh the mice and prepare analgesics, induce anesthesia, stabilize body temperature and respiratory rate, and apply eye ointment. S2. Operating procedures a. Remove the wik-Si sealing gel and the anti-fibrotic membrane in the region to expose the mouse spinal cord (1); b. Immediately place a moistened hemostatic sponge on the mouse spinal cord (1) to maintain moisture and stop bleeding. If necessary, remove the hemostatic sponge placed around the fibrosis-inhibiting membrane in step two to expand the exposed area of ​​the mouse spinal cord (1). c. Cut a piece of anti-fibrotic membrane to the size suitable for exposing the mouse spinal cord (1), remove the hemostatic sponge, quickly wipe it dry with a cellulose sponge, and immediately place the cut anti-fibrotic membrane directly on the surface of the mouse spinal cord (1). d. Cover the area of ​​the fibrotic membrane and laminectomy site with a small amount of Kwik-Sil sealing gel; e. Immediately place a 3.0 mm diameter circular coverslip (7), press for 1 second to squeeze out excess Kwik-si adhesive, and repeat twice; f. Let stand for 10 minutes to allow the Kwik-Sil sealant to harden; g. Completely cover the edges of the Kwik-Si layer with bone cement to seal and permanently fix the coverslip (7); h. Finally, the magnetic protective cover (9) is attached to the stabilizing plate (8) to cover the observation cavity; S3. Postoperative recovery: provide heat, monitor recovery, administer opioid analgesics, and after the mice are fully awake, place them in a non-magnetic cage for spinal cord (1) imaging observation.

8. The long-term optical imaging observation method for mouse spinal cord (1) as described in claim 7, characterized in that: Step four involves imaging and observing the mouse spinal cord (1). S1. Adaptation training: Before imaging recording, mice need to adapt to the imaging room or imaging equipment for 1 to 3 days. S2. Environmental control: The noise level in the imaging room is controlled between 53.0–53.3 dBA {with all instruments off} and 59.7–60.4 dBA {with all instruments on}. S3. Imaging while awake. a. Remove the mouse with the observation window from the feeding cage, remove the magnetic protective cover (9), and wipe the imaging window slide clean with an alcohol swab; b. Use clamps to hold the stabilizing plate (8) and fix the mouse on the imaging gantry to ensure that the mouse remains stable during imaging; c. Place the observation window directly below the objective lens (water mirror) of the two-photon fluorescence microscope, and inject an appropriate amount of physiological saline into the imaging cavity; d. First, use a 10x scope to make a general observation in order to adjust and determine the observation area; e. Then, a 25x or 40x microscope is used for detailed observation and recording of local areas. For photon scanning imaging, a laser wavelength of 920-945nm is selected. f. Move the imaging gantry, absorb the water from the imaging chamber, and wipe the imaging window glass again with anhydrous alcohol swabs; g. Use a magnetic protective cover (9) to attach to the stabilizing plate (8) to cover the observation cavity; h. Release the clamps from the stabilizing plate (8) and return the mice to the non-magnetic rearing cage.

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