CN112293348A - Construction method of mouse model for lumbar intervertebral disc protrusion - Google Patents
Construction method of mouse model for lumbar intervertebral disc protrusion Download PDFInfo
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- 206010050296 Intervertebral disc protrusion Diseases 0.000 title claims abstract description 26
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
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- A61K49/00—Preparations for testing in vivo
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- A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/30—Animals modified by surgical methods
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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Abstract
The invention discloses a method for constructing a mouse model of protrusion of lumbar intervertebral disc, which comprises the following steps: 1) inducing the lumbar intervertebral disc to degenerate by performing lumbar intervertebral disc puncture through a ventral approach; 2) feeding the animal for 4 weeks to obtain degenerated lumbar disc nucleus pulposus tissue; 3) exposing a spinal dural sac and nerve roots through a posterior approach, and placing degenerated intervertebral disc tissues in an intervertebral disc hole area to form nerve root compression to obtain a mouse model of lumbar intervertebral disc protrusion; 4) behavioral testing assessed the appearance and extent of lower limb radiation pain. The invention utilizes the degenerated intervertebral disc tissue to press nerve roots, can induce the severe and lasting lower limb radiating pain symptom, and better simulates the chronic course of clinical lumbar intervertebral disc protrusion; and the mouse is taken as a model animal, and the method has multiple objective and credible pain behavioral measurement methods and abundant transgenic strains, and is favorable for deeply exploring the neurobiological mechanism of lower limb radiating pain caused by lumbar intervertebral disc protrusion.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to a construction method of a mouse model for lumbar disc herniation.
Background
Lumbar intervertebral disc protrusion is one of the main causes of lumbago and lower limb radiation pain, and is quite common clinically. The lumbocrural pain caused by the lumbar intervertebral disc protrusion seriously damages the life quality and the labor capacity of the patients and influences the economic production. At present, the neurobiological mechanism of pain caused by lumbar disc herniation is not completely elucidated. An effective and credible animal model of the lumbar intervertebral disc protrusion is a key scientific research tool for further clarifying the nerve mechanism of lumbocrural pain and developing new clinical drugs.
In early related research, large animals such as pigs, dogs or rabbits are mostly adopted to make lumbar intervertebral disc protrusion models. These large animal models are expensive and relatively convenient to operate, but lack effective behavioral methods to assess pain performance, and can only detect neurological changes by electrophysiology. In recent years, researchers mostly use rats to construct animal models of lumbar intervertebral disc herniation, which can be divided into three types of models of simple inflammation, simple nerve compression and nerve compression + inflammation, and evaluate pain expression by using a limited behavioral method. However, current rat-based models are all made directly with autologous normal disc tissue compressing the nerve roots. The disadvantages of this model are: 1) the normal non-degenerated intervertebral disc is used for pressing nerve roots, the nerve roots are not consistent with the clinical real condition of the lumbar intervertebral disc protrusion, the caused pain symptom is light, and the duration is short (only lasts for about 1-2 weeks); 2) less pain behavioral assessment methods based on rats; 3) the number of rat transgenic strains available for research is small, and the research on the biological mechanism of pain is not favorable for further expanding.
Therefore, a mouse model of protrusion of lumbar intervertebral disc with lasting and reliable pain symptoms, low cost, strong operability and good repeatability is needed at present.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for constructing a mouse model of lumbar intervertebral disc protrusion so as to make up for various defects of the existing related models.
The construction method of the mouse model for treating lumbar intervertebral disc protrusion comprises the following steps: inducing the lumbar intervertebral disc to degenerate by performing lumbar intervertebral disc puncture through a ventral approach; feeding the animal for 4 weeks to obtain degenerated lumbar disc nucleus pulposus tissue; exposing a spinal dural sac and nerve roots through a posterior approach, and placing degenerated intervertebral disc tissues in an intervertebral disc hole area to form nerve root compression to obtain a mouse model of lumbar intervertebral disc protrusion; behavioral testing assessed the appearance and extent of lower limb radiation pain.
Preferably, the model can be constructed by using adult male mice.
Step 1: puncture injury induces lumbar disc degeneration. The adult male mouse after anesthesia is fixed on the operation table top in the supine position, the skin of the abdomen is preserved and disinfected, and a longitudinal incision is made in the middle of the lower abdomen. Exposing the gap between the ilium and lumbar muscles and the abdominal aorta, separating the ilium and lumbar muscles and exposing the milky intervertebral disc; the intervertebral disc is punctured by a needle head with a depth limiting device for 5 times in a total, and the incision is sutured after the puncture is finished.
Step 2: obtaining the degenerated lumbar disc nucleus pulposus tissue. 1 month after mouse intervertebral disc puncture, perfusing with normal saline after anesthesia, rapidly separating and taking out degenerated intervertebral disc after puncture, properly removing peripheral fiber ring and end plate to mainly reserve nucleus pulposus tissue, and soaking and storing with normal saline for modeling of receptor animal.
And step 3: constructing a lumbar intervertebral disc protrusion model. Anaesthetizing, fixing adult male mice in prone position, sterilizing the dorsal skin, centering on L4/5 vertebral body, and taking the median incision. Blunt-separating left paraspinal muscle, exposing vertebral plate, cutting out inner part L4/5 articular process and partial vertebral plate, and exposing L4 nerve root and dural sac; the extracted degenerated nucleus pulposus tissue is filled in the axilla of the junction of the nerve root of the dorsal root ganglion of L4 and the dural sac, and the nerve root is pressed. The incision is sutured layer by layer.
And 4, step 4: behavioral assessment and detection of lower limb radiation pain. Behavioral changes of the lower limb radiating pain of the mice are detected and evaluated by a von Frey test (mechanical pain), a Hargreaves test (thermal pain), an acetone test (cold pain) and the like 1-4 weeks after the operation.
Compared with other existing animal models, the mouse model for lumbar intervertebral disc protrusion, which is constructed by 4 steps, has the following advantages: 1) the nerve root is pressed by the degenerated intervertebral disc tissue, so that the severe and lasting lower limb radiating pain symptom can be induced, and the chronic course of clinical lumbar intervertebral disc protrusion is better simulated; 2) the mouse is taken as a model animal, and the method has various objective and credible pain behavioral measurement methods, and is beneficial to the curative effect evaluation of experimental intervention; 3) the mouse is used as the most common experimental animal and has abundant transgenic strains, thereby conveniently providing a genetic control means, being beneficial to subsequently controlling related molecular targets and deeply exploring the neurobiological mechanism of lower limb radiation pain caused by protrusion of lumbar intervertebral disc.
Drawings
In order to more clearly illustrate the technical solution of the embodiment of the present invention, the method required to be used in the embodiment will be briefly described below by using the attached drawings. It is appreciated that the following drawings depict only some embodiments of the invention and are therefore not to be considered limiting of its scope, for those skilled in the art will readily appreciate that other related drawings may be made from these drawings without the benefit of inventive faculty.
FIG. 1 is a schematic flow chart of a method for constructing a mouse model of prolapse of lumbar intervertebral disc according to the present invention.
Detailed Description
In order to make the technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
All embodiments, implementations and features of the invention can be combined with each other in the invention without contradiction or conflict. In the present invention, conventional devices, apparatuses, components, etc. are either commercially available or self-made according to the present disclosure. In the present invention, some conventional operations and apparatuses, devices, and components are omitted or only described briefly in order to highlight the importance of the present invention.
The existing animal model for prolapse of lumbar intervertebral disc has the following problems: 1) the normal non-degenerated intervertebral disc tissues are used for pressing nerve roots, the nerve roots are not consistent with the clinical real condition of the lumbar intervertebral disc protrusion, the caused pain symptoms are light, and the duration is short (only lasts for about 1-2 weeks); 2) the pain ethology evaluation method aiming at rats and large animals is less, and the popularization and the application of animal models are limited; 3) the transgenic strains of rats and other experimental animals are few, which is not favorable for the deep development of the biological mechanism research of pain. In view of the above, the inventor proposes a novel animal model based on mice, which can not only induce the long-term severe pain symptoms of the mice, but also more accurately simulate the chronic clinical course of lumbar disc herniation; and abundant transgenic mouse strains can conveniently provide a plurality of genetic control means, and are beneficial to the subsequent development of pain experimental intervention research by controlling related molecular targets.
In some examples of the present invention, embodiments provide a method of constructing a mouse model of lumbar disc herniation, the method comprising:
(1) the test mice are subjected to general anesthesia treatment and fixed
In the present example, the test mice were treated with general anesthesia and fixed. In some alternative embodiments, general anesthesia can be performed by administering an appropriate dose of anesthetic depending on the body weight of the mouse.
In this example, the test animal is an adult mouse, and therefore, according to an embodiment of the present invention, the general anesthesia treatment can be performed according to the following steps: adult mice were injected intraperitoneally with pentobarbital sodium at a dose of 0.1 mg/g. Thereby, it is possible to effectively perform general anesthesia on adult mice and maintain an effective predetermined time, wherein pentobarbital sodium is provided and used as an anesthetic.
It is to be noted that the anesthetic may be selected as desired according to the particular needs. Alternatively, as the effectiveness of the anesthetic improves, the sodium pentobarbital may be replaced with other agents that are already available or are in the trial.
(2) Mice were treated for skin preparation.
Skin preparation refers to surgical preparation for shaving hair and cleaning body surface of a corresponding part needing surgical treatment, and is the work of cleaning the surgical area of an animal subjected to surgery before surgery. The skin preparation treatment comprises the following steps: removing body hair, cleaning and disinfecting skin.
(3) Puncture injury induced intervertebral disc degeneration
As a specific implementation scheme, the puncture needle head is optionally 21G, the depth limit is 1mm, and the puncture is carried out for 5 times in a reciprocating mode.
(4) Behavioral testing of mice lower limb radiation pain:
based on the animal model construction method, in the embodiment of the invention, the method for evaluating the lower limb radiation pain caused by the protrusion of the lumbar intervertebral disc is also provided. The evaluation method relates to the behavioral assessment and detection of lower limb radiation pain, and further comprises the following steps: and (5) detecting the behavioral change of the lower limb radiation pain of the mice in 1-4 weeks after the operation. The method is integrated with a plurality of behavioral assessment methods, and specifically comprises the following steps: von Frey test (mechanical pain), Hargreaves test (hot pain), and acetone test (cold pain).
Example 1: injury induced degeneration of lumbar intervertebral disc
(I) animals
Mouse (C57 BL/6); weight: about 25.0 ± 3.0 g; sex: male; age: for 2-3 months.
(II) instruments and materials
Orthopedic surgical instruments, microsurgical surgical instruments, narcotics, puncture needles with depth-limiting devices.
(III) method
1. Anaesthesia
Animals were weighed and anesthetized by intraperitoneal injection with pentobarbital sodium at a dose of 0.1 mg/g.
2. Skin preparation
The supine position is fixed on the operation table, the abdominal skin is removed completely, the iodophor is disinfected locally for 2 times, and the operation part is exposed by laying a sterile operation towel.
3. Puncture operation of lumbar intervertebral disc
(1) Making a 1-1.5cm longitudinal incision in the middle of lower abdomen, cutting skin subcutaneous, retracting small intestine, and exposing gap between ilio-lumbar muscle and abdominal aorta; performing blunt separation to expose milky intervertebral disc; (2) puncturing the intervertebral disc by using a 21G needle with a depth limiting device, limiting the depth to be 1mm, puncturing back and forth for 5 times totally, and suturing an incision after puncturing; (3) 1 month after the puncture, mice subjected to the intervertebral disc puncture operation were irrigated with physiological saline, the degenerated intervertebral discs after the puncture were rapidly separated and removed, and placed in physiological saline to remove the outer part of the annulus fibrosus and end plates, and the nucleus pulposus tissue was retained, so as to compress the nerve root of the recipient animal for modeling.
Example 2: construction of mouse lumbar intervertebral disc protrusion model
(I) animals
Mouse (C57 BL/6); weight: about 25.0 ± 3.0 g; sex: male; age: for 2-3 months.
(II) instruments and materials
Orthopedic surgical instruments, microsurgical surgical instruments, narcotics.
(III) method
1. Anaesthesia
Animals were weighed and anesthetized by intraperitoneal injection with pentobarbital sodium at a dose of 0.1 mg/g.
2. Skin preparation
Fixing the prone position on the operation table, removing skin on the back, sterilizing topical iodophor for 2 times, spreading sterile operation towel, and exposing the operation part.
3. Puncture operation of lumbar intervertebral disc
(1) The median incision was made about 1-1.5cm long, centered on the L4/5 vertebral body. Blunt-separating left paraspinal muscle, exposing vertebral plate, cutting out inner part L4/5 articular process and partial vertebral plate, and exposing L4 nerve root and dural sac; (2) nucleus pulposus tissue from a donor is filled in the axilla at the junction of the dorsal root ganglion nerve root and the dural sac of L4, and the incision is sutured layer by layer.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and various modifications and changes will occur to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (4)
1. A method for constructing a mouse model of protrusion of lumbar intervertebral disc comprises the following steps: 1) inducing the lumbar intervertebral disc to degenerate by performing lumbar intervertebral disc puncture through a ventral approach; 2) feeding the animal for 4 weeks to obtain degenerated lumbar disc nucleus pulposus tissue; 3) exposing a spinal dural sac and nerve roots through a posterior approach, and placing degenerated intervertebral disc tissues in an intervertebral disc hole area to form nerve root compression to obtain a mouse model of lumbar intervertebral disc protrusion; 4) behavioral testing assessed the appearance and extent of lower limb radiation pain.
2. The method for constructing a mouse model of prolapse of lumbar intervertebral disc according to claim 1, wherein the induction of lumbar intervertebral disc degeneration in step 1) is obtained by:
selecting 2-3 month-old male C57BL/6 mouse, weighing 25.0 + -3.0 g, anesthetizing, making a 1-1.5cm longitudinal incision in the middle of lower abdomen, cutting skin, retracting small intestine, and exposing gap between ilio-lumbar muscle and abdominal aorta; performing blunt separation to expose milky intervertebral disc; the 21G needle with the depth limiting device is used for puncturing the intervertebral disc, the depth is limited to be 1mm, the puncture is carried out for 5 times in a reciprocating mode, and the incision is sutured after the puncture is finished.
3. The method for constructing a mouse model of lumbar intervertebral disc protrusion according to claim 1, wherein the mouse model of lumbar intervertebral disc protrusion in the step 3) is obtained by the following steps:
selecting 2-3 month-old male C57BL/6 mouse, weighing 25.0 + -3.0 g, anesthetizing, preparing skin, taking L4/5 vertebral body as center, and making incision at the center of the body to obtain a length of about 1-1.5 cm; blunt-separating left paraspinal muscle, exposing vertebral plate, cutting out inner part L4/5 articular process and partial vertebral plate, and exposing L4 nerve root and dural sac; nucleus pulposus tissue from a donor is filled in the axilla at the junction of the dorsal root ganglion nerve root and the dural sac of L4, and the incision is sutured layer by layer.
4. The method for constructing a mouse model of prolapse of lumbar intervertebral disc according to claim 1, wherein the behavioral assessment and detection method for the lower limb radiation pain of the mouse in the step 4) comprises: von Frey test (mechanical pain), Hargreaves test (hot pain), and acetone test (cold pain).
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| CN113274160A (en) * | 2021-05-07 | 2021-08-20 | 中山大学孙逸仙纪念医院 | Method and system for establishing murine intervertebral disc puncture degeneration model |
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