Harvesting DRG from Rat Spine: A Procedure to Extract Dorsal Root Ganglion from the Spine of a Rat Model

Protokol

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.

1. 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.
2. 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.
   1. 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.
   2. 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.
   3. 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.
   4. Using forceps, gently detach the spinal cord from the vertebral bones to visualize the DRGs (Figure 1G).
   5. 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.
3. 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.
4. 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.

Sonuçlar

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 exci...

Malzemeler

Name	Company	Catalog Number	Comments
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
Pen/Strep	Gibco	# 15140-122	10,000 Units/mL Penicilin, 10,000 µg/mL Streptomycin
HI FBS	Gibco	# 10082-147	Heat-inactivated Fetal Bovine Serum
DMSO	Fisher Bioreagents	# BP231-100	Dimethyl Sulfoxide