Surgical Transplantation of Tumor Cells into the Spinal Cord of Mice

Summary

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.

Abstract

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.

Introduction

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 a....

Protocol

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

1. Clean and sterilize all surgical instruments thorou.......

Discussion

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.......

Materials

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
Suture needle with thread (medical silk) F303 3-0 75 cm 1/2 5×12 single 19mm black	Shanghai Pudong Jinhuan Medical Products	F303
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