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* These authors contributed equally
The present protocol describes the development of a reproducible murine model of spinal cord glioma by injecting tumor cells into the intervertebral space, offering a more effective and less invasive approach for research and therapeutic development.
Spinal cord gliomas are commonly malignant tumors of the spinal cord, leading to a high rate of disability. However, uniform treatment guidelines and comprehensive data on spinal cord gliomas remain limited due to the lack of suitable preclinical animal models. Developing a simple and reproducible animal model has become essential for advancing basic and translational research. A murine model is ideal, as the murine spinal cord shares structural similarities with the human spinal cord. This protocol describes the generation of a reproducible murine model of spinal cord glioma by directly injecting tumor cells into the intervertebral space using the spinous process of the seventh cervical vertebra as a guide. Compared to other methods, this approach is more effective and convenient, involving a smaller incision, reduced invasiveness and blood loss, faster recovery, and more stable tumor formation. This model is expected to advance the understanding of disease mechanisms, optimize surgical strategies, and support the development of therapeutic drugs for spinal cord gliomas.
Spinal cord gliomas, including those of the cauda equina, are commonly malignant neoplasms of the spinal cord, with 20%-40% classified as astrocytomas and the remainder as ependymomas1. Based on histological features, spinal cord gliomas are categorized into four grades (I-IV). Grade I and II tumors are considered low-grade gliomas, while grade III and IV tumors are classified as high-grade gliomas. Although spinal cord gliomas can occur at any segment of the spinal cord, they are most frequently found in the cervical region (33% of cases) and are relatively rare in other regions, with 26% of cases in the thoracic region and 24% in the lumbar region2.
Surgery, radiotherapy, and alkylating agents are the primary treatment options for spinal cord gliomas, largely extrapolated from clinical trials on brain gliomas3. However, previous research has demonstrated that, although the histological profiles of spinal cord gliomas resemble those of brain gliomas, the presence of distinct molecular signatures differentiates them from their cerebral counterparts4. In our cohort, spinal cord glioma patients derived no significant benefit from either adjuvant chemotherapy or radiotherapy, underscoring the limited effectiveness of current treatments and the need for new therapeutic strategies5. Therefore, reliable and informative animal models are essential for advancing basic research and preclinical studies.
Currently, several well-established spinal cord glioma models exist, including the method described by Minru et al.6. These models primarily utilize thoracic vertebra removal techniques to expose the spinal cord6,7,8. Although rat models have been employed in the past, they are associated with higher costs, smaller sample sizes, and greater management challenges compared to mouse models. Additionally, more genetically modified experimental mouse models are available than rat models. An immune-competent mouse model is particularly valuable for studying the immune response within the spinal tumor microenvironment and for developing immunotherapeutic strategies for spinal cord gliomas. Furthermore, this method is well-suited for generating patient-derived xenograft models for spinal cord gliomas.
This protocol proposes a safe, technically simple, and rapidly reproducible procedure for creating a spinal cord glioma transplantation model in mice. The model is expected to advance research into the largely unexplored mechanisms underlying glioma progression and facilitate the development of therapeutic drugs for spinal cord gliomas.
This protocol was conducted in compliance with the guidelines approved by the Institutional Committee for the Ethics of Animal Care and Treatment in Biomedical Research at Capital Medical University (AEEI-2021-187). Female C57BL/6 mice, aged 8 weeks and weighing 19-21 g, were used in this study. The reagents and equipment utilized are detailed in the Table of Materials.
1. Pre-surgical preparation
2. Preparation of GL261-luc and B16-F10-luc cells for transplant
NOTE: The GL261-luc GBM cell line was obtained commercially, while the B16-F10-luc melanoma cell line was a gift from Professor Wang Xi. Both cell lines were confirmed to be free of mycoplasma infection through pre-experimental testing.
3. Animal preparation
4. Exposure of cervical spine and determination of insertion point
5. Injection of tumor cells
6. Post-surgical care
7. In vivo bioluminescence imaging
To establish a stable and reliable animal model of spinal glioma, the intervertebral space between the sixth and seventh cervical vertebrae in C57BL/6 mice was identified as the ideal site for inoculation based on literature review and experimental findings10. The seventh cervical vertebra provides a distinct bony landmark, the spinous process (Figure 1G-I), which aids in accurately locating the injection site and stabilizing the inje...
Spinal cord glioma is the most common type of primary malignant tumor in the spinal cord, accounting for over 80% of intramedullary tumors. Pathologically, spinal cord gliomas are primarily classified as ependymomas or astrocytomas, with a particular focus on astrocytomas11. Among astrocytomas, some harbor H3K27M mutations, also known as diffuse midline gliomas (DMGs), which are associated with poor prognoses. A defining feature of spinal cord gliomas is their infiltrative growth pattern, which ma...
No conflicts of interest were declared.
This work was supported by the National Natural Science Foundation of China General Program (Fund No. 8207317). R&D Program of Beijing Municipal Education Commission (Fund No. KZ202210025040). Chinese Institutes for Medical Research, Beijing (Grant No. CX24PY08).
Name | Company | Catalog Number | Comments |
A nutritionally complete food and water gelled diet (Nutra-Gel) | Bio-Serv | N/A | |
Adhesion microscope slides | CITOTEST | 188105 | |
AffiniPure Fab Fragment Goat Anti-Mouse IgG (H+L) | Jacksonimmuno | 115-007-003 | |
B16-F10-luc | Professor Wang Xi's laboratory | N/A | |
Buprenorphine Related Compound A | Sigma-Aldrich | 457071-73-7 | |
CD163 (ABT-CD163) mouse mAb | Immunoway | YM6146 | |
CD86 rabbit pAb | Immunoway | YT7823 | |
Cell counter | Bio-rad | 1450102 | |
Cell Counting Slides | Biorad | 1450011 | |
DAPI/Sealant Dual Solution (Anti-Quenching) | Immunoway | YS0014 | |
Dilator | Jinzhong | D22178 | |
D-Luciferin | PerkinElmer | 122799 | |
DMEM | Gibco | C11995500BT | |
D-PBS | Solarbio | D1040 | |
Fetal Bovine Serum, qualified | Gibco | 10270-106 | |
GL261-luc | Shanghai Zishi Biotechnology | N/A | |
Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 | Invitrogen | A11029 | |
Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 | Life | A21244 | |
Goat Serum | Beyotime | C0265 | |
Hamilton microinjector 10 µL fixed 701N | Hamilton | 80383 | |
In vivo bioluminescent imaging (IVIS Spectrum) | PerkinElmer | N/A | |
Methanol | Fuyu Chemical | 67-56-1 | |
Micro Scissors | Jinzhong | WAA320 | |
Microliter Syringes (10 µL, pointed tip) | Shanghai Gaoge | N/A | |
Microscope cover glass | CITOTEST | 10212440C | |
needle holder 12.5 cm | Jinzhong | JCZ200 | |
Ophthalmic Forceps 10 cm | Jinzhong | JD1060 | |
Ophthalmic Scissors 10 cm | Jinzhong | Y00030 | |
PBS, 10× | Solarbio | P1022 | |
Penicillin-Streptomycin Liquid | Solarbio | P1400 | |
Scalpel Blades | Jinzhong | J0B050 | |
super pap pen | ZSGB-Bio | ZLI-9303 | |
Surgical Knife Handle | Jinzhong | J11010 | |
Surgical scissors 12.5cm straight tip | Jinzhong | J21010 | |
Nylon Surgical Sutures with thread, size 3-0 | UNIFY | N/A | |
Tissue-Tek O.C.T. Compound | SAKURA | 4583 | |
Tribromoethanol | Sigma-Aldrich | T48402 | |
Triton X-100 | Servicebio | GC204003 | |
Trypan Blue Stain Solution, 0.4% | Solarbio | C0040 | |
Trypsin Digestion solutions, 0.25% (without phenol red) | Solarbio | T1350 | |
Tween-20 | Solarbio | T8220 |
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