In this video, we demonstrate the extraction of dorsal root ganglion or DRG from a harvested rat spine for downstream applications.
Protocol
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Harvest and Preparation of DRGs (estimated timing: 2 h, day 8)
NOTE: Experiments with mice and rats require approval from the IACUC. In some countries, the use of chicken eggs also requires approval.
Get six to seven week-old Sprague Dawley rats (~200 g in weight) to extract DRGs. NOTE: One rat should yield ~40 cervical and thoracic DRGs. Mouse DRG also integrates in the CAM; however, the conditions for this species need to be optimized independently.
In a laminar flow cabinet, harvest DRGs from cervical and thoracic regions following the protocol for mouse DRG extraction published elsewhere. Follow Figure 1 for orientation on how to harvest DRGs.
Euthanize the rat by cardiac puncture after administration of Ketamine/ Xylazine injected intraperitoneally. Clean the rat skin with 70% ethanol and remove the rat spine using a scissor. Do not perform cervical dislocation because this would damage the cervical DRGs.
Separate the cervical, thoracic, and lumbar regions with the same scissor, following the schematic anatomic representation and gross images provided in the Figure 1A-D. Place the spine sections in a 10 cm culture dish with 1X PBS to keep tissues wet.
With a delicate bone scissor, open the vertebral bones in the dorsal and ventral aspects, separating the spine in two lateral halves (Figure 1E-F). Place the tissue sections in a clean 10 cm dish with fresh 1X PBS.
Using forceps, gently detach the spinal cord from the vertebral bones to visualize the DRGs (Figure 1G).
With fine forceps held underneath each DRG, grasp it and pull it out from the bone cavity in which it is lodged. Do not hold the DRG directly because this will cause tissue damage. Do not trim the axon bundles from the DRG (Figure 1H). NOTE: Avoid using lumbar DRGs since these have reduced integration in the CAM. For DRG region location, follow the schematic illustration and gross anatomic images in Figure 1A-D.
Immediately after harvesting, place each DRG into DMEM culture medium supplemented with 2% Penicillin/Streptomycin (Pen/Strep) and 10% heat-inactivated Fetal Bovine Serum (FBS) to help prevent bacterial contamination of the DRGs. Group all DRGs in the same 6 cm culture dish with 4 mL of culture medium.
After harvesting all DRGs, transfer them to a new culture dish with DMEM culture medium supplemented with 2% Pen/Strep plus 10% FBS and containing 1.25 µg/mL of red fluorescent dye. Incubate for 1 h in the cell culture incubator.
Representative Results
Figure 1: DRG extraction on day 8. A. Rat schematic illustrating the anatomical location of the spine. B. Diagram of the rat vertebrae configuration showing different body regions; green for cervical, dark blue for thoracic, orange for lumbar and light blue for sacral vertebrae. C-D. Ventral aspect of the rat spine after surgical excision; separation of the regions as illustrated in B. E. Dissection of the vertebrae to open the spinal cord canal, separating the vertebral bodies into two lateral sections containing the DRGs. Section should cut through the dorsal and ventral aspect of each vertebral bone at the midline. F. Gross aspect of the opened thoracic spine. G. After the spinal cord is displaced, DRGs are easily visible in the vertebral canals (arrow heads pointing 3 DRGs). H. Stereomicroscopic image of one DRG (arrow) with the corresponding axon bundles (arrowhead). Scale bars: C, D, F, and G, 1 cm; H, 1 mm.
Declarações
The authors have nothing to disclose.
Materials
PBS (1x) pH 7.4
Gibco
# 10010-023
Phosphate Buffered Saline
Sprague Dawley rats (females)
Charles River laboratories
Strain code: 001
6-7 weeks old (190-210g in weight)
Dumont # 5 fine forceps
Fine Science Tools (FST)
# 11254-20
Used to harvest DRG
Extra fine Graefe forceps, curved
Fine Science Tools (FST)
# 11151-10
Used to graft DRG onto the CAM on day 8 and to harvest CAM tissue on day 17
Extra fine Graefe forceps, straight
Fine Science Tools (FST)
# 11150-10
Used to graft DRG onto the CAM on day 8 and to harvest CAM tissue on day 17
DMEM (1x)
Gibco
# 11965-092
Dulbeecco`s Modified Eagle Medium
CellTracker Red CMTPX fluorescent dye
Life Technologies
# C34552
Reconstitute 50µg in 40µL of DMSO and stock at -20oC. Use 1µL of stock solution/mL of culture medium
Harvesting DRG from Rat Spine: A Procedure to Extract Dorsal Root Ganglion from the Spine of a Rat Model. J. Vis. Exp. (Pending Publication), e20476, doi: (2023).