Modeling Sensory Axon Regeneration in a Rat Using Dorsal Root Ganglion Injection and Dorsal Root Crush Injury

Samenvatting

Source: Cheah, M., et al. Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration. J. Vis. Exp. (2017).

This video demonstrates a step-by-step procedure for injecting a viral suspension into the dorsal root ganglia (DRGs) and inducing a controlled axonal injury via a dorsal root crush in an anesthetized rat. The protocol involves exposing the DRGs, delivering a growth-promoting viral vector to facilitate axonal regeneration, and severing axonal connections to the spinal cord to model sensory nerve injury and regeneration.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Injecting DRG and Performing a Dorsal Root Crush Injury

NOTE: This is an extremely delicate surgery. It is advisable to practice on a few dead animals first to familiarize with the anatomy before advancing to live animal surgery.

1. Locate the prominent C2 and T2 spinous processes over the skin. Make a skin incision between the C2 and T2 spinous processes with a no.10 scalpel blade (once the skin is opened, a white, fibrous-tissue midline should be visible on the first layer of muscle; the first layer of muscle has a "jelly-like" texture).
2. Make a similar-sized incision on the first layer of muscle along the white midline using a scalpel blade. Do not go beyond the prominent T2 spinous process, as there is a major arterial branch from the descending aorta located near there.
   NOTE: Heavy bleeding can occur anywhere from this step onwards. Always stop the bleeding and remove the blood by using sterilized cotton buds or surgical absorbable sponges to allow clear visualization at all times. Proceeding with the procedure "blindly" may result in unwanted damage to the spinal cord, DRG, or dorsal root, leading to unexpected adverse effects in the animal later on.
3. Retract the first layer of muscle using two retractors, one placed rostrally and one caudally; the second layer of muscle, with a striated appearance, should be visible.
4. Locate the midline of the second layer of muscle (where two longitudinal muscles can be observed connected by a thin, membranous tissue). Dissect the membranous tissue using a pair of microscissors to separate the two longitudinal muscles. Avoid using a scalpel blade if possible, as this may result in unnecessary muscle damage and bleeding.
5. Adjust the retractors accordingly to expose the third layer of thin muscle covering the spine. The spinous processes can be felt by lightly touching with a pair of forceps over the third layer of muscle.
6. Make a small incision on the third layer of muscle using microscissors and gently scrape off the muscle from the bone using a curette or scalpel in a sideway manner to clearly expose the vertebrae. If required, cut some of the muscle or tendon away to make the laminectomy easier.
7. To expose the left C5 - C8 DRGs, perform a left hemi-laminectomy on the C4 - T1 vertebrae by carefully removing part of the lamina and pedicle using a pair of fine rongeurs. The DRG is located near the transverse foramen of the vertebrae.
   NOTE: C3 - C7 vertebrae do not have a prominent spinous process. The C5 DRG is located between the C4 and C5 vertebrae, the C6 DRG is between the C5 and C6 vertebrae, and so on, while the C8 DRG is between the C7 and T1 vertebrae. There is no C8 vertebra, despite the presence of a C8 DRG and a C8 spinal cord segment.
8. Once enough of the DRG has been exposed for injection, prepare the syringe by placing the virus-filled microliter syringe fitted with a custom-made, 33-gauge blunt needle onto the stereotaxic syringe holder. Before injection, use a 30-gauge beveled needle to make a small superficial opening on each of the targeted DRG to assist with the insertion of the injection needle.
9. Insert the 33-gauge needle into the center of the DRG by turning the knobs gently to adjust the stereotaxic coordinates. Do not over-insert the needle, as this may cause fluid to leak out from the ventral side of the DRG. Should leakage occur, adjust the position of the needle immediately.
   NOTE: The numeric stereotaxic coordinates are not used; however, it is helpful to utilize the frame to hold the needle for injection.
10. Inject 1 µL of the virus into each DRG at 0.2 µL/min using an infusion syringe pump. Wait for an additional three minutes before withdrawing the needle. During the injection, the DRG will slowly change color if the virus solution contains a colored dye.
11. To perform a concurrent C5 - C8 dorsal root crush injury, crush each root three times for 10 s each using a pair of fine-tipped forceps (Bonn Micro), opposing the ends of the forceps completely; a white line in the tissue should appear at the crush site. Do not go deeper than required with the forceps, as this may cause damage to the ventral root.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Fast Green FCF dye	Sigma-Aldrich	F7258	For visualizing colorless solution. Recommended concentration: 1%
Olsen-Hegar Needle Holder	Fine Science Tools	FST 12502-12
Friedman Pearson Rongeur Curved 0.7mm Cup	Fine Science Tools	FST 16121-14
Bonn Micro Forceps	Fine Science Tools	FST 11083-07	For performing dorsal root crush injury
Tissue Separating Scissors	Fine Science Tools	FST 14072-10
Fine Scissors	Fine Science Tools	FST 14058-11
Micro-Adson Forceps	Fine Science Tools	FST 11018-12
Goldstein Retractor	Fine Science Tools	FST 17003-03
Vannas Spring Scissors (straight)	Fine Science Tools	FST 15018-10
SURGIFOAM Absorbable Gelatin Sponge	Ethicon	1972	For bleeding control
Microliter Syringe RN701 (10 μl)	Hamilton	80330
Custom-made Removable Needle (for DRG injection)	Hamilton	7803-05	33 gauge, 38 mm, point style 3
UltraMicroPump with SYS-Micro4 Controller	World Precision Instruments	UMP3-1