Animal spinal cord total transection injury model and manufacturing method thereof

Abstract

The embodiment of the invention provides an animal spinal cord full transection injury model and a manufacturing method thereof. The method comprises the following steps: providing an experimental spinal cord animal and a self-made trocar needle assembly; determining the position of a T9-T10 vertebral plate of the spinal animal, cutting the skin at the position, and separating muscles and ligaments to expose the gap between the T9-T10 vertebral plate; two opposite sides of the back side of the spinal cord are selected as needle inserting points, the cannula needle head assembly is inserted into the T9-T10 intervertebral plate gap from the needle inserting points, an inner needle core is withdrawn after the dural sac is punctured, and an outer cannula is inserted into the puncture point; repeatedly drawing and inserting the outer sleeve to mash the spinal cord tissue in the dural sac, and sucking out the mashed spinal cord tissue through an external aspirator. According to the embodiment, the vertebral plate does not need to be removed, manufacturing of the injury model is completed on the basis that the integrity of the dura mater is reserved as much as possible, the integrity of blood vessels is reserved to the maximum extent, and the influence of vascular injury on nerve regeneration is eliminated to the maximum extent.

Description

Technical Field

[0001] Embodiments of the present disclosure belong to the technical field of spinal cord injury models, and particularly relate to an animal spinal cord complete transection injury model and a method for making the same. Background Art

[0002] Spinal cord injury (SCI) is a central nervous system injury with a very high disability rate. The incidence rate worldwide is 3.6 - 195.4 cases per million people, and about 78% of the patients are male. In China, the incidence rate of spinal cord injury is 25 - 60 cases per million people. Approximately 50% of the patients are aged 40 - 60 years old, and males are in the majority (2.4 - 5.6:1). Spinal cord injury is mainly caused by traffic accidents, followed by falls and violence. Due to the difficulty of neuron regeneration, it leads to the irreversibility of nerve function loss, seriously reducing the quality of life of SCI patients and generating huge medical costs. Currently, there is no effective treatment plan. Therefore, the research on SCI has always been a hot topic at home and abroad. The establishment of an animal spinal cord injury model provides a scientific research basis for the research on spinal cord injury. Therefore, an ideal animal spinal cord injury model is crucial for the research on spinal cord injury.

[0003] The spinal cord transection injury model can exclude neurons and nerve fibers in the injury area as much as possible, and can effectively observe the repair and regulation effects of exogenous factors on nerve cell regeneration, axon growth, and new synapse formation in the spinal cord injury area. However, the surgical steps of the conventional spinal cord transection model are relatively complex. It is necessary to remove the lamina to fully expose the spinal cord, which causes greater damage to the animal. Moreover, when using a blade to transect the spinal cord, the wound surface is relatively neat, which is quite different from the actual clinical cases. In addition, during the operation of the animal conventional spinal cord transection injury model, while excluding neurons and nerve fibers in the injury area, the blood vessels in the injury area are also removed together, resulting in a large amount of bleeding during the operation, and many complications (such as incision infection, urinary retention, urethral infection, etc.) occur after the operation. The postoperative nursing requirements are higher, and the mortality rate can be as high as 60% - 70%. Summary of the Invention

[0004] Embodiments of the present disclosure aim to at least solve one of the technical problems existing in the prior art, and provide an animal spinal cord complete transection injury model and a method for making the same.

[0005] On the one hand, embodiments of the present disclosure provide a method for making an animal spinal cord complete transection injury model, the method comprising:

[0006] providing an experimental spinal cord animal and a self-made cannula needle assembly; wherein, the cannula needle assembly includes an inner needle core and an outer cannula sleeved outside the inner needle core;

[0007] Determine the position of the T9-T10 laminae of the spinal animal, incise the skin at this position and separate the muscles and ligaments to expose the interlaminar space between T9 and T10;

[0008] Select the opposite two sides on the dorsal side of the spinal cord as the needle insertion points, insert the cannula needle assembly into the interlaminar space between T9 and T10 from the needle insertion points, withdraw the inner needle core after puncturing the dura mater, and insert the outer cannula at the puncture point;

[0009] Insert and withdraw the outer cannula repeatedly to mash the spinal cord tissue in the dura mater, and aspirate the mashed spinal cord tissue through an external suction device to complete the production of the injury model.

[0010] Optionally, before incising the skin at the position of the T9-T10 laminae, the method further includes:

[0011] Anesthetize the spinal animal, and disinfect the skin after shaving the back.

[0012] Optionally, the anesthesia of the spinal animal includes: anesthetizing by intraperitoneal injection of a diluted solution of sodium pentobarbital.

[0013] Optionally, the inner needle core includes a fixedly connected needle core part and a clamping part, and the outer cannula includes a needle-containing tube and a connection cavity that communicate with each other; wherein,

[0014] The needle core part is movably and clearance-fitted with the outer cannula, one end of the needle core part is set to be spiky and extends out of the needle-containing tube, the other end of the needle core part is connected to the clamping part, the clamping part is located outside the connection cavity and is used to control the movement of the needle core part, and the connection cavity is used to connect to the external suction device.

[0015] Optionally, the diameter of the needle core part is 0.4 mm to 0.6 mm, and the height of the inner needle core is 20 mm to 22 mm; the diameter of the needle-containing tube is 0.45 mm to 0.65 mm, and the height of the outer cannula is 19 mm to 21 mm.

[0016] Optionally, the incising the skin at the position of the T9-T10 laminae and separating the muscles and ligaments to expose the interlaminar space between T9 and T10 includes:

[0017] Make a longitudinal skin incision of 0.8 cm to 1.2 cm in the middle of this position, cut open the mucosa, and separate the muscles and ligaments along both sides of the spinous process until the interlaminar space between T9 and T10 is completely exposed.

[0018] Optionally, after aspirating the mashed spinal cord tissue, the method further includes:

[0019] Reset the muscles and ligaments, suture the mucosa and the incised skin; and inject penicillin and glucose diluent through the abdominal cavity.

[0020] Optionally, the injecting penicillin and glucose diluent through the abdominal cavity includes:

[0021] Postoperative care: Place the spinal cord animal on bedding and place it in a room with a temperature of 18 - 22°C; normally provide food and water, inject the penicillin and the glucose diluent into the spinal cord animal through the abdominal cavity every day within the first preset number of days after the operation, manually assist urination a preset number of times every day until the bladder restores the autonomous urination reflex, and change the bedding every second preset number of days;

[0022] The method further includes index evaluation: separately record the operation time, BBB kinematic function score and mortality rate of the spinal cord animal under the injury model and the spinal cord animal under the conventional injury model; wherein, the BBB kinematic function of the two is separately scored by an uninformed observer at different times after the operation.

[0023] Optionally, after sucking out the mashed spinal cord tissue, the method further includes:

[0024] Inject biological materials, and / or stem cells, and / or drugs, and / or nutritional factors into the dural sac through the outer cannula.

[0025] On the other hand, an embodiment of the present disclosure provides an animal spinal cord complete transection injury model, which is made by using the method described above.

[0026] An animal spinal cord complete transection injury model and its manufacturing method according to an embodiment of the present disclosure have the following beneficial effects:

[0027] (1) Using the self-made cannula needle assembly has the advantages of simple operation and good repeatability. Compared with the neat injury ends of the conventional complete transection injury model, the irregular injury ends of this model are closer to the clinical actual situation.

[0028] (2) It has low requirements for experimental equipment, is simple to operate and easy to promote, and has small damage to spinal cord animals, less bleeding, and short wound self-healing time. The spinal cord animals have fewer postoperative complications, high survival rate, and can achieve similar motor function disorders to the conventional complete transection injury model.

[0029] (3) The spinal cord complete transection injury model was created on the basis of preserving the integrity of the dura mater as much as possible, while preserving the integrity of the blood vessels to the maximum extent, making nerve injury a relative single variable and maximally eliminating the effect of vascular injury on nerve regeneration. In addition, biomaterials, stem cells, drugs, and nutritional factors can be injected into the injured area through the outer sheath. The effects of biomaterials, stem cells, drugs, and nutritional factors on the nerve regeneration process and their corresponding molecular mechanisms can be observed and explored. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a schematic flowchart of a method for making an animal spinal cord complete transection injury model according to an embodiment of the present disclosure;

[0031] Figure 2 It is a structural schematic diagram of a trocar needle assembly according to another embodiment of the present disclosure;

[0032] Figure 3 It is a schematic diagram of a specific application of a trocar needle assembly according to another embodiment of the present disclosure;

[0033] Figure 4 This is a schematic diagram of specimen preparation for an animal spinal cord complete transection injury model according to another embodiment of the present disclosure. DETAILED DESCRIPTION

[0034] In order to enable those skilled in the art to better understand the technical solution of the present disclosure, the present disclosure is further described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0035] like Figure 1 As shown, the embodiment of the present disclosure provides a method for preparing an animal spinal cord complete transection injury model. As a specific example, female rats weighing 160 g to 180 g are used as experimental subjects for explanation.

[0036] The method comprises: S110, providing an experimental spinal cord animal and a self-made trocar needle assembly, wherein the trocar needle assembly comprises an inner needle core and an outer sleeve sleeved outside the inner needle core.

[0037] Specifically, in this step, refer to Figure 2, first, provide experimental spinal animals. Select female rats weighing 160 g to 180 g as the experimental subjects. Then, provide a self-made cannula needle assembly 200, which includes an inner needle core 210 and an outer cannula 220. The inner needle core 210 includes a needle core part 211 and a clamping part 212 that are fixedly connected. The outer cannula 220 includes a needle receiving tube 221 and a connecting cavity 222 that communicate with each other. Among them, the needle core part 211 is movably and clearance-fitted in the outer cannula 221. One end of the needle core part 211 is set to be spiky and extends out of the needle receiving tube 221. The other end of the needle core part 211 is connected to the clamping part 212. The clamping part 212 is located outside the connecting cavity 222 and is used to control the movement of the needle core part 211. The connecting cavity 222 is used to connect to the aspirator.

[0038] Further, the diameter of the needle core part can be set to 0.4 mm to 0.6 mm, and the height of the inner needle core is 20 mm to 22 mm. The diameter of the needle receiving tube is 0.45 mm to 0.65 mm, and the height of the outer cannula is 19 mm to 21 mm. Of course, the above components can also be set to other mutually matching sizes, and this embodiment does not make specific restrictions on this.

[0039] Before starting the experiment, the rats also need to be anesthetized. Inject 1% sodium pentobarbital injection intraperitoneally into the rats at a dose of 0.15 ml per 100 g of body weight. After the rats are anesthetized, shave the back and disinfect the skin.

[0040] S120. Determine the position of the T9-T10 laminae of the spinal animal, incise the skin at this position and separate the muscles and ligaments to expose the interlaminar space between T9-T10 laminae.

[0041] Specifically, in this step, determine the position of the T9-T10 laminae of the rats, and make a longitudinal skin incision of 0.8 cm to 1.2 cm in the middle of this position. For example, make a 1 cm skin incision directly above this position, cut open the mucosa, and after seeing the spinous process, separate the muscles and ligaments on both sides of the spinous process. For example, use a mosquito forceps to strip the muscles and ligaments between the T9-T10 laminae until the interlaminar space between T9-T10 laminae is completely exposed.

[0042] S130. Select the two opposite sides on the dorsal side of the spinal cord as the needle insertion points, insert the cannula needle assembly into the interlaminar space between T9-T10 laminae from the needle insertion points, withdraw the inner needle core after piercing the dura mater, and insert the outer cannula at the piercing point.

[0043] Specifically, in this step, refer to Figure 2 and Figure 3, the puncture point is selected on the dorsal side of the spinal cord to avoid damaging the posterior spinal vein on the dorsal side of the spinal cord. Then, one end of the core part 211 of the cannula needle assembly 200 is inserted into the medullary cavity through the interlaminar space between the T9-T10 laminae. After the core part 211 pierces the dura mater, the core part 211 is withdrawn through the clamping part 212, so that the core part 211 is separated from the outer cannula 220. After the core part 211 is separated from the outer cannula, the needle receiving tube 221 is continuously inserted at the puncture point. As an example, one end of the needle receiving tube 221 is set to be flat-headed to facilitate subsequent smashing of spinal cord tissue.

[0044] S140. Repeatedly insert and withdraw the outer cannula to smash the spinal cord tissue in the dura mater, and suck out the smashed spinal cord tissue through an external aspirator to complete the production of the injury model.

[0045] Specifically, in this step, with reference to Figure 2 and Figure 3 , after one end of the needle receiving tube 221 is inserted into the spinal cord of the rat, the rat will shake violently. First, repeatedly insert and withdraw gently to smash the spinal cord tissue, and then connect an external aspirator through the connecting cavity 222 to suck out the smashed spinal cord tissue. So far, the production of the complete transection injury model has been preliminarily completed.

[0046] Furthermore, after sucking out the smashed spinal cord tissue, the method further includes: injecting a biological material, and / or stem cells, and / or a drug, and / or a growth factor into the dura mater through the outer cannula. Interventions such as biological materials, stem cells, drugs, and growth factors through the outer cannula can study the repair effects of different intervention means on nerve regeneration after injury.

[0047] S150. After sucking out the smashed spinal cord tissue, the method further includes: resetting the muscles and ligaments, suturing the mucosa and the incised skin. And injecting penicillin and glucose diluent through intraperitoneal injection.

[0048] Specifically, in this step, after sucking out the smashed spinal cord tissue through the aspirator, reset the muscles and ligaments, and suture the mucosa and the skin. Subsequently, inject penicillin and 5% glucose into it through the abdominal cavity and observe. As a more specific example, when the muscles and ligaments are reset and the mucosa and the skin are also sutured, place the rat on its side on a heating pad and cover it with a tissue paper. After the rat wakes up, let the rat drink water, and then put it into a cage. In the first five days after the operation, inject penicillin and 5% glucose into the rat every day.

[0049] It should be noted that postoperative care is required for the experimental rats: specifically, place the postoperative rats on the bedding and place them in a room with a temperature of 18-22 °C. Normally give food and water. In the first five days after the operation, inject penicillin and 5% glucose into the rats through the abdominal cavity every day. Manually assist in urination 2 times a day until the bladder restores the autonomous micturition reflex, and change the bedding every 3 days.

[0050] Further, it should be noted that the method further includes index evaluation: specifically, the operation time, BBB kinematic function score, and mortality rate of the spinal cord animals under the injury model and the spinal cord animals under the conventional injury model are respectively recorded. Among them, the BBB kinematic function of the two is independently scored by an uninformed observer at different times after the operation.

[0051] Specifically, the operation time, BBB kinematic function score, and mortality rate of the rats under the injury model in this embodiment and the rats under the conventional complete transection injury model are respectively recorded. Among them, the kinematic function scores of the two groups of rats are observed on the 1st, 3rd, 7th, 10th, and 14th days after the injury. To ensure the fairness of the scoring, two uninformed observers are required to independently score the two groups of rats, and then statistical analysis is performed. For example, the data are all expressed in the form of mean ± standard deviation. Tables (1) and (2) below are respectively the observation table of the basic animal conditions and the BBB score table for the determination of kinematic function.

[0052] Table (1)

[0053]

[0054] Table (2)

[0055]

[0056] Through the above analysis, it can be concluded that, compared with the conventional complete transection injury model, the injury model of this embodiment can completely retain the blood vessels of the rat spinal cord (the anterior spinal artery, posterior spinal artery, and posterior spinal vein are completely retained, and there is a certain injury to the central sulcus artery), and the manufacturing method is simple to operate, has little trauma, a low probability of postoperative infection, and few complications in the urinary and digestive systems, so the animal survival rate is high. In addition, the manufacturing method has little injury, high repeatability, and can achieve a similar motor dysfunction to the conventional injury model.

[0057] The method for making a complete transection injury model of an animal spinal cord of the embodiment of the present disclosure, relative to the conventional complete transection injury model, does not require the removal of the lamina in the complete transection injury model of the spinal cord with blood vessels retained, and can use a homemade cannula needle assembly in combination with an aspirator to complete the operation of the complete transection injury, and complete the production of the complete transection injury model of the spinal cord while keeping the dura mater intact as much as possible. At the same time, the method retains the blood vessels of the rat spinal cord as much as possible (the anterior spinal artery, the posterior spinal artery and the posterior spinal vein are completely retained, and the central sulcus artery has certain damage). The production method has less damage, is easy to operate, has high repeatability, and can achieve motor dysfunction similar to the conventional complete transection injury model of the spinal cord, and can study the situation of nerve regeneration while retaining blood supply. At the same time, the interference of biomaterials, stem cells, drugs and nutritional factors can be achieved through the outer cannula, so as to study the repair effect of different intervention methods on nerve regeneration after injury. Since the entire production method is simple to operate, has little trauma, has a low probability of postoperative infection, and has few complications of the urinary and digestive systems, the animal survival rate is high. In addition, the model has good repeatability and has a good prospect of use.

[0058] The method for making an animal spinal cord complete transection injury model of the embodiment of the present disclosure also has the following beneficial effects: (1) The use of a homemade cannula needle assembly has the advantages of simple operation and good repeatability. Compared with the neat injury ends of conventional complete transection injury models, the irregular injury ends of this model are closer to the actual clinical situation.

[0059] (2) The experimental equipment requirements are not high, the operation is simple and easy to promote, and the damage to the spinal cord animals is small, the amount of bleeding is small, and the wound healing time is short. The spinal cord animals have few postoperative complications and a high survival rate, and can achieve motor dysfunction similar to that of the conventional full transection injury model.

[0060] (3) The spinal cord complete transection injury model was created on the basis of preserving the integrity of the dura mater as much as possible, while preserving the integrity of the blood vessels to the maximum extent, making nerve injury a relative single variable and maximally eliminating the effect of vascular injury on nerve regeneration. In addition, biomaterials, stem cells, drugs, and nutritional factors can be injected into the injured area through the outer sheath. The effects of biomaterials, stem cells, drugs, and nutritional factors on the nerve regeneration process and their corresponding molecular mechanisms can be observed and explored.

[0061] Of course, specimens can also be prepared for experimental rats, such as Figure 4 As shown, rats with complete spinal cord transection injury in the vascular preservation group were anesthetized with 1% sodium pentobarbital intraperitoneally and fixed in a prone position after anesthesia. The back skin and muscles were cut open, the vertebral lamina was opened, and the spinal cord tissues of the injured segment and adjacent segments were taken out and observed.

[0062] On the other hand, embodiments of the present disclosure also provide an animal model of complete transverse spinal cord injury, which is prepared by using the method described above. For the specific steps of the method, reference can be made to the relevant descriptions above, and details will not be elaborated here.

[0063] It can be understood that the above embodiments are merely exemplary embodiments adopted to illustrate the principle of the present disclosure. However, the present disclosure is not limited thereto. For those of ordinary skill in the art, various modifications and improvements can be made without departing from the spirit and essence of the present disclosure, and these modifications and improvements are also regarded as the protection scope of the present disclosure.

Claims

1. A method for fabricating an animal model of complete spinal cord transection injury, characterized in that, The method comprises: Provide an experimental spinal cord animal and a homemade trocar needle assembly; wherein the trocar needle assembly comprises an inner needle core and an outer sleeve sleeved outside the inner needle core; Determining the position of the T9-T10 vertebral lamina of the spinal cord animal, cutting the skin at the position and separating the muscles and ligaments to expose the space between the T9-T10 vertebral lamina; Select two opposite sides of the dorsal spinal cord as the needle insertion points, insert the cannula needle assembly into the T9-T10 interlaminar space from the needle insertion points, puncture the dura mater sac and then withdraw the inner needle core, and insert the outer cannula at the puncture point; The outer sleeve is repeatedly inserted and pulled out to crush the spinal cord tissue in the dura mater sac, and the crushed spinal cord tissue is sucked out by an external suction device to complete the preparation of the injury model.

2. The manufacturing method according to claim 1, wherein Before cutting the skin at the T9-T10 lamina position, the method further comprises: The spinal cord animal was anesthetized, and the back skin was prepared and disinfected.

3. The manufacturing method according to claim 2, characterized in that, The anesthetizing of the spinal cord animal comprises: anesthetizing by intraperitoneal injection of sodium pentobarbital dilution.

4. The manufacturing method according to any one of claims 1 to 3, characterized in that, The inner needle core comprises a needle core portion and a clamping portion that are fixedly connected, and the outer sleeve comprises a needle tube and a connecting cavity that are interconnected; wherein, The needle core part is movably fitted in the outer sleeve with a clearance, one end of the needle core part is configured to be in a spike shape and extends out of the needle-receiving tube, the other end of the needle core part is connected to the clamping part, the clamping part is located outside the connecting cavity and is used to control the movement of the needle core part, and the connecting cavity is used to externally connect the suction device.

5. The manufacturing method according to claim 4, characterized in that, The diameter of the needle core is 0.4mm-0.6mm, and the height of the inner needle core is 20mm-22mm; the diameter of the needle tube is 0.45mm-0.65mm, and the height of the outer sleeve is 19mm-21mm.

6. The manufacturing method according to any one of claims 1 to 3, characterized in that, The skin at the T9-T10 vertebral lamina is cut and muscles and ligaments are separated to expose the T9-T10 interlaminar space, including: A 0.8-1.2 cm longitudinal skin incision was made along the middle of the position, the mucosa was cut open, and the muscles and ligaments were separated along both sides of the spinous process until the T9-T10 interlaminar space was completely exposed.

7. The manufacturing method according to claim 6, characterized in that, After aspirating the mashed spinal cord tissue, the method further comprises: The muscles and ligaments are repositioned, the mucosa and the incised skin are sutured; and penicillin and glucose dilution are injected intraperitoneally.

8. The manufacturing method according to claim 7, characterized in that, The intraperitoneal injection of penicillin and glucose dilution includes: Postoperative care: placing the spinal cord animal on a bedding material and placing it in a room with a temperature of 18-22°C; giving food and water normally, injecting the penicillin and the glucose dilution into the spinal cord animal through the abdominal cavity every day within the first preset number of days after surgery, artificially assisting urination for a preset number of times every day until the bladder recovers the autonomous urination reflex, and changing the bedding material every second preset number of days; The method also includes index evaluation: recording the operation time, BBB motor function score and mortality rate of the spinal cord animals under the injury model and the spinal cord animals under the conventional injury model respectively; wherein, the BBB motor function of the two animals is scored independently by blinded observers at different times after surgery.

9. The manufacturing method according to any one of claims 1 to 3, characterized in that, After aspirating the mashed spinal cord tissue, the method further comprises: injecting a biomaterial, and / or stem cells, and / or a drug, and / or a trophic factor into the dural sac through the outer cannula.

10. A complete transection injury model of animal spinal cord, characterized in that, Obtained by using the method according to any one of claims 1 to 9.

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