SD rat neuropathic pain model dorsal root ganglion cell extraction method
By optimizing the construction and cell culture technology of neuropathic pain model in SD rats, the complex problems of dorsal root ganglion cell extraction and culture process were solved, efficient and stable cell extraction and culture were achieved, and the accuracy and reliability of neuropathic pain research was improved.
Patent Information
- Application Number
- CN202510001310.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-02
- Publication Date
- 2025-04-11
AI Technical Summary
In the prior art, the extraction and culture process of dorsal root ganglion cells is complex, with low cell survival rate, low purity and poor culture effect, which limits the accuracy and reliability of neuropathic pain research.
By optimizing the construction of neuropathic pain models of SD rats, behavioral testing, culture medium preparation, cell culture and immunofluorescence verification, the extraction efficiency and culture quality of dorsal root ganglion cells are improved. Specific methods include selecting SD rats for selective ligation of sciatic nerve branches, preparing suitable culture media, using polylysine-coated culture plates, and verifying the characteristics of cells by immunofluorescence technology.
Efficient extraction and stable culture of dorsal root ganglion cells was achieved, the adherence rate and survival rate of cells were improved, the subsequent experiments were ensured smoothly, and reliable model and data support was provided for neuropathic pain research.
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Figure CN119799637A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of dorsal root ganglion cell extraction, in particular to a method for extracting dorsal root ganglion cells of a SD rat neuropathic pain model. Background Art
[0002] In the prior art, neuropathic pain is a chronic pain state caused by damage or dysfunction of the nervous system. In order to deeply study the pathogenesis of neuropathic pain and explore effective treatment methods, it is crucial to establish a stable and reliable animal model. SD rats have become commonly used experimental animals in the field of neuroscience research because of their physiological characteristics similar to those of humans, strong fertility, and easy breeding and management. In the construction of neuropathic pain models, selective ligation of sciatic nerve branches (SNI) is a classic and effective modeling method. This method simulates common clinical nerve damage by ligating specific branches of the sciatic nerve (such as the tibial nerve and the common peroneal nerve) while leaving other branches (such as the sural nerve) intact. Dorsal root ganglia (DRG), as a key component of the sensory nervous system, play an important role in pain signal processing. DRG neurons can sense and transmit pain information from the periphery. Abnormalities in their structure and function are closely related to the occurrence and development of neuropathic pain.
[0003] However, the extraction and culture process of dorsal root ganglion cells is relatively complicated, requiring delicate operation and suitable culture conditions. Traditional extraction methods often have problems such as low cell survival rate, low purity, and unsatisfactory culture effect, which limits the accuracy and reliability of subsequent experimental research. Therefore, developing an efficient, stable and reliable method for extracting dorsal root ganglion cells in a SD rat neuropathic pain model is of great significance for promoting the in-depth development of neuropathic pain research. Based on this, the present invention proposes an improved method for extracting dorsal root ganglion cells in a SD rat neuropathic pain model, which improves the extraction efficiency and culture quality of dorsal root ganglion cells by optimizing animal model construction, behavioral testing, culture medium preparation, cell culture and immunofluorescence verification. Summary of the invention
[0004] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a method for extracting dorsal root ganglion cells from a SD rat neuropathic pain model.
[0005] In order to achieve the above object, the present invention adopts the following technical solutions:
[0006] A method for extracting dorsal root ganglion cells from a SD rat neuropathic pain model comprises the following steps:
[0007] S1: SD rats were selected as experimental animals and subjected to selective ligation of sciatic nerve branches (SNI). The tibial nerve and common peroneal nerve in the sciatic nerve were ligated, and the sural nerve was kept intact. After surgery, the changes in tactile sensitivity of the lateral area of the rat paw were observed to verify the success of the model construction.
[0008] S2: The tactile sensitivity test was performed using a VONfrey ciliary mechanical stimulation needle to evaluate the changes in tactile threshold in the lateral area of the paw of rats after SNI surgery;
[0009] S3: Prepare DMEM / F12 complete medium and neural cell-specific medium for the growth and maintenance of dorsal root ganglion cells;
[0010] S4: Poly-lysine-coated culture plates are used to improve cell adhesion and survival rates;
[0011] S5: Dorsal root ganglia were extracted from SD rats after SNI surgery under sterile conditions. After digestion, centrifugation, and resuspension, they were inoculated into six-well plates for culture. During the culture process, the culture medium was replaced regularly to observe the cell growth.
[0012] S6: Immunofluorescence technology was used to verify the expression of sodium ion channel 1.7 in dorsal root ganglion cells extracted from the SNI model.
[0013] Preferably: the preparation method of the DMEM / F12 complete medium in S3 comprises: taking 44 mL of DMEM / F12 basal culture medium into a sterile tube in a clean bench, adding 5 mL of fetal bovine serum, 500 μL of penicillin-streptomycin mixture (100X) and 500 μL of glutamine, filtering with a 0.22 um filter and transferring to a new tube, marking the preparation date and wrapping the sealing film tightly, and storing at 4°C for later use.
[0014] Further: The preparation method of the special culture medium for neural cells in the S3 includes: taking 48 mL of Neurobasal basal culture base into a sterile bottle in a clean bench, adding 1 mL of SM1, 500 μL of penicillin-streptomycin mixture (100X) and 500 μL of glutamine, filtering with a 0.22 um filter and transferring to a new bottle, marking the preparation date and wrapping the sealing film tightly, and storing at 4°C for future use.
[0015] Further: the method for coating the culture plate with poly-lysine in S4 includes: coating the culture plate with 0.1 mg / mL poly-lysine the day before, washing it three times with PBS buffer solution before use and then drying it for later use.
[0016] As a preferred solution of the present invention: the method for extracting and culturing dorsal root ganglion cells in S5 comprises: placing SNI postoperative SD rats in a supine position under sterile conditions, decapitating and opening the thoracic and abdominal cavities after anesthesia, inserting microscissors from the opening of the caudal vertebral canal to cut the vertebral canal and remove the exposed spinal cord, picking up dorsal root ganglia, rinsing with HBSS buffer solution and temporarily storing in DMEM / F12 complete culture medium, and inoculating into six-well plates for culture after digestion, centrifugation and resuspension.
[0017] As a further scheme of the present invention: the specific steps of S5 include: removing the DMEM / F12 complete medium, washing three times with PBS buffer solution, adding 1 mL of 0.25% trypsin and 1 mL of type I collagenase to digest at 37°C for 1 hour, shaking clockwise several times every 10 minutes, and then adding 2 mL of DMEM / F12 complete medium to terminate digestion, and pipetting until there are no obvious tissue blocks.
[0018] As a further solution of the present invention: the immunofluorescence verification step in S6 includes: cell fixation, permeabilization, blocking, primary antibody incubation, secondary antibody incubation, sealing and observation.
[0019] On the basis of the above scheme: the cell fixation step includes: after washing the sample with PBS, fixing the cells in the culture plate with a freshly prepared 4% paraformaldehyde solution for 30 minutes, and then washing the sample with PBS.
[0020] On the basis of the above scheme: the primary antibody incubation step includes: adding primary antibody (NaV1.7, 1:200), incubating overnight at 4°C in the dark, and then washing the sample with PBS.
[0021] Based on the above scheme: the secondary antibody incubation step includes: adding secondary antibody FITC, incubating at room temperature for 45 minutes, and then washing the sample with PBS.
[0022] The beneficial effects of the present invention are:
[0023] 1. A method for extracting dorsal root ganglion cells from a SD rat neuropathic pain model. By selecting SD rats and performing selective ligation of the sciatic nerve branches (SNI), a neuropathic pain model can be stably constructed. The model is highly repeatable and reliable, providing a basis for subsequent dorsal root ganglion cell extraction and research.
[0024] 2. A method for extracting dorsal root ganglion cells from a SD rat neuropathic pain model, which effectively improves the extraction efficiency of dorsal root ganglion cells through surgical operations under sterile conditions, the rational use of digestive enzymes, and cell processing steps such as centrifugation and resuspension. This not only saves experimental time and cost, but also ensures the smooth progress of subsequent experiments.
[0025] 3. A method for extracting dorsal root ganglion cells from a SD rat neuropathic pain model, which significantly improves the cell adhesion rate and survival rate by preparing DMEM / F12 complete culture medium and nerve cell-specific culture medium suitable for the growth and maintenance of dorsal root ganglion cells, and using poly-lysine-coated culture plates.
[0026] 4. A method for extracting dorsal root ganglion cells from a SD rat neuropathic pain model was developed. Immunofluorescence technology was used to verify the expression of sodium channel 1.7 in dorsal root ganglion cells extracted from the SNI model. This not only further confirmed that the cells met the characteristics of the neuropathic pain model, but also provided a scientific basis for subsequent drug screening and treatment research. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 This is a fluorescence statistical result diagram of a method for extracting dorsal root ganglion cells in a SD rat neuropathic pain model proposed by the present invention;
[0028] Figure 2 The present invention provides a flow chart of a method for extracting dorsal root ganglion cells from a SD rat neuropathic pain model. DETAILED DESCRIPTION
[0029] The technical solution of this patent is further described in detail below in conjunction with specific implementation methods.
[0030] The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.
[0031] Embodiment 1:
[0032] A method for extracting dorsal root ganglion cells from a SD rat neuropathic pain model comprises the following steps:
[0033] S1: Animal preparation and model construction:
[0034] SD rats were selected as experimental animals and subjected to selective ligation of sciatic nerve branches (SNI); specifically, the tibial nerve and common peroneal nerve in the sciatic nerve were ligated, while the sural nerve was kept intact; changes in tactile sensitivity in the lateral area of the rat paw were observed after surgery to verify the successful construction of the model, in which the lesion caused obvious hypersensitivity in the lateral area of the paw, which was innervated by the retained sural nerve;
[0035] S2: Behavioral testing:
[0036] The tactile sensitivity test was performed using VONfrey (ciliary mechanical stimulation needle) to evaluate the changes in tactile threshold in the lateral area of the paw of rats after SNI surgery;
[0037] S3: Culture medium preparation:
[0038] DMEM / F12 complete medium and neural cell-specific medium are prepared for the growth and maintenance of dorsal root ganglion cells. The culture method comprises the following steps:
[0039] (1) Place 44 mL of DMEM / F12 basal medium in a sterile, clean tube in a clean bench;
[0040] (2) Add 5 mL of fetal bovine serum, 500 μL of penicillin-streptomycin mixture (100X), and 500 μL of glutamine;
[0041] (3) Filter through a 0.22 μm filter and transfer to a new tube. Label the culture medium name and preparation date on the lid, wrap it tightly with sealing film, and store at 4°C for future use.
[0042] The method for culturing a special culture medium for neural cells comprises the following steps:
[0043] (1) Take 48 mL of Neurobasal culture medium and place it in a sterile, clean bottle in the clean bench;
[0044] (2) Add 1 mL SM1, 500 μL penicillin-streptomycin mixture (100X), and 500 μL glutamine;
[0045] (3) After filtering with a 0.22 μm filter, transfer the culture medium to a new bottle tube, label the culture medium name and preparation date on the cap, wrap the cap tightly with sealing film, and store at 4°C for future use;
[0046] S4: Poly-lysine coated culture plate:
[0047] A poly-lysine coated culture plate is used to improve the cell adhesion rate and survival rate. The culture method includes: using 0.1 mg / mL poly-lysine to coat the culture plate the day before, washing it three times with PBS buffer solution before use and then drying it for later use;
[0048] S5: Dorsal root ganglion cell culture:
[0049] Dorsal root ganglia are extracted from SD rats after SNI surgery under sterile conditions, and after digestion, centrifugation, resuspension and other steps, they are inoculated into six-well plates for culture; during the culture process, the culture medium is regularly replaced and the cell growth is observed. The culture method includes the following steps:
[0050] (1) SNI postoperative SD rats were placed in a supine position under sterile conditions, anesthetized with sodium pentobarbital, and then decapitated and the thoracic and abdominal cavities were opened;
[0051] (2) Insert microscissors from the opening of the coccygeal canal, cut the canal open along the dorsal midline of the spine, remove and expose the spinal cord. The dorsal root ganglia can be seen along both sides of the spine next to the intervertebral foramen. Carefully pick them out with a microscope, rinse them with HBSS buffer solution, and temporarily store them in DMEM / F12 complete culture medium.
[0052] (3) Remove the DMEM / F12 complete medium, wash three times with PBS buffer solution, add 1 mL of 0.25% trypsin and 1 mL of type I collagenase, and digest at 37°C for 1 hour. Shake clockwise several times every 10 minutes during the digestion. Then add 2 mL of DMEM / F12 complete medium to terminate the digestion. Pipette 20 times until there are no obvious tissue blocks.
[0053] (4) Inoculate cells into a six-well plate, with 105 cells per well, and culture in a 37°C, 5% carbon dioxide incubator. After 4 h of culture, the cells begin to adhere to the wall, and the Neurobasal medium is replaced with a full medium, and then the Neurobasal medium is replaced with a half medium every day;
[0054] S6: Immunofluorescence validation:
[0055] Immunofluorescence technology was used to verify the expression of sodium channel 1.7 in dorsal root ganglion cells extracted from the SNI model to further confirm that the cells met the characteristics of the neuropathic pain model. The verification process included the following steps:
[0056] (1) Cell fixation: Wash the sample with PBS. Fix the cells in the culture plate with freshly prepared 4% paraformaldehyde solution for 30 minutes, then wash the sample with PBS;
[0057] (2) Permeabilization: 0.5% triton X-100 (prepared in PBS) was used to permeabilize the cells for 10 min, and then the samples were washed with PBS;
[0058] (3) Blocking: Block with 2% BSA for 30 minutes, then wash the samples with PBS;
[0059] (4) Primary antibody incubation: add primary antibody (NaV1.7, 1:200) in dark conditions at 4°C overnight, then wash the samples with PBS;
[0060] (5) Secondary antibody incubation: Add secondary antibody FITC, incubate at room temperature for 45 min, and then wash the samples with PBS;
[0061] (6) Sealing and observation: Add DAPI for staining for 10 minutes, wash the sample with PBS, add 20 μL of mounting medium for sealing, and observe under a fluorescence microscope.
[0062] Embodiment 2:
[0063] A method for extracting dorsal root ganglion cells from a SD rat neuropathic pain model. After experiments and identification, the purity of rat DRG neurons was greater than 90% during neuron identification. The fluorescence intensity of sodium channel 1.7 in the neurons was significantly increased compared to that of unmodeled mice. Figure 1 It is the fluorescence statistical result graph.
[0064] The above is a preferred specific implementation manner of the present invention, and the protection scope of the present invention is not limited thereto. Any modification, equivalent substitution and improvement made by any technician familiar with the field within the technical scope disclosed by the present invention in combination with the prior art or public common sense, within the spirit and principle of the present invention, shall be covered by the protection scope of the present invention.
Claims
1. A method for extracting dorsal root ganglion cells from a SD rat neuropathic pain model, characterized in that: The following steps are involved: S1: SD rats were selected as experimental animals and subjected to selective ligation of sciatic nerve branches (SNI). The tibial nerve and common peroneal nerve in the sciatic nerve were ligated, and the sural nerve was kept intact. After surgery, the changes in tactile sensitivity of the lateral area of the rat paw were observed to verify the success of the model construction. S2: The tactile sensitivity test was performed using a VONfrey ciliary mechanical stimulation needle to evaluate the changes in tactile threshold in the lateral area of the paw of rats after SNI surgery; S3: Prepare DMEM / F12 complete medium and neural cell-specific medium for the growth and maintenance of dorsal root ganglion cells; S4: Poly-lysine-coated culture plates are used to improve cell adhesion and survival rates; S5: Dorsal root ganglia were extracted from SD rats after SNI surgery under sterile conditions. After digestion, centrifugation, and resuspension, they were inoculated into six-well plates for culture. During the culture process, the culture medium was replaced regularly to observe the cell growth. S6: Immunofluorescence technology was used to verify the expression of sodium ion channel 1.7 in dorsal root ganglion cells extracted from the SNI model.
2. The method for extracting dorsal root ganglion cells from a SD rat neuropathic pain model according to claim 1, characterized in that: The preparation method of the DMEM / F12 complete medium in S3 includes: taking 44 mL of DMEM / F12 basal culture medium into a sterile tube in a clean bench, adding 5 mL of fetal bovine serum, 500 μL of penicillin-streptomycin mixture (100X) and 500 μL of glutamine, filtering with a 0.22 um filter and transferring to a new tube, marking the preparation date and wrapping the sealing film tightly, and storing at 4°C for future use.
3. The method for extracting dorsal root ganglion cells from a SD rat neuropathic pain model according to claim 2, characterized in that: The preparation method of the S3 medium for neural cells comprises: taking 48 mL of Neurobasal basal culture medium into a sterile bottle in a clean bench, adding 1 mL of SM1, 500 μL of penicillin-streptomycin mixture (100X) and 500 μL of glutamine, filtering with a 0.22 um filter and transferring to a new bottle, marking the preparation date and wrapping the sealing film tightly, and storing at 4° C. for future use.
4. The method for extracting dorsal root ganglion cells from a SD rat neuropathic pain model according to claim 3, characterized in that: The method for coating the culture plate with poly-lysine in S4 includes: coating the culture plate with 0.1 mg / mL poly-lysine the day before, washing the plate with PBS buffer solution three times before use, and then drying the plate for later use.
5. The method for extracting dorsal root ganglion cells from a SD rat neuropathic pain model according to claim 4, characterized in that: The extraction and culture method of dorsal root ganglion cells in S5 comprises: placing the SNI postoperative SD rat in a supine position under sterile conditions, decapitating the rat after anesthesia to open the thoracic and abdominal cavities, inserting microscissors from the opening of the caudal vertebral canal to cut the vertebral canal to remove the exposed spinal cord, picking the dorsal root ganglia, rinsing them with HBSS buffer solution and temporarily storing them in DMEM / F12 complete culture medium, and then inoculating them into a six-well plate for culture after digestion, centrifugation and resuspension.
6. The method for extracting dorsal root ganglion cells from a SD rat neuropathic pain model according to claim 5, characterized in that: The specific steps of S5 include: removing DMEM / F12 complete medium, washing three times with PBS buffer solution, adding 1 mL of 0.25% trypsin and 1 mL of type I collagenase to digest at 37°C for 1 hour, shaking clockwise several times every 10 minutes, and then adding 2 mL of DMEM / F12 complete medium to terminate digestion, and pipetting until there are no obvious tissue blocks.
7. The method for extracting dorsal root ganglion cells from a SD rat neuropathic pain model according to claim 6, characterized in that: The immunofluorescence verification steps in S6 include: cell fixation, permeabilization, blocking, primary antibody incubation, secondary antibody incubation, sealing and observation.
8. The method for extracting dorsal root ganglion cells from a SD rat neuropathic pain model according to claim 7, characterized in that: The cell fixation step comprises: after washing the sample with PBS, fixing the cells in the culture plate with a freshly prepared 4% paraformaldehyde solution for 30 minutes, and then washing the sample with PBS.
9. The method for extracting dorsal root ganglion cells from a SD rat neuropathic pain model according to claim 8, characterized in that: The primary antibody incubation step includes: adding primary antibody (NaV1.7, 1:200), incubating overnight at 4°C in the dark, and then washing the sample with PBS.
10. The method for extracting dorsal root ganglion cells from a SD rat neuropathic pain model according to claim 9, characterized in that: The secondary antibody incubation step includes: adding secondary antibody FITC, incubating at room temperature for 45 minutes, and then washing the sample with PBS.