Targeted Viral Delivery to Corticospinal Neurons in a Neonatal Rat to Study Spinal Cord Injury

Abstract

Source: Smit, R. D., et al. Targeting the Corticospinal Tract in Neonatal Rats with a Double-Viral Vector using Combined Brain and Spine Surgery. J. Vis. Exp. (2021)

This video demonstrates the targeted delivery of viral vectors to corticospinal neurons in a neonatal rat. The protocol enables precise gene expression using anterograde and retrograde viral injections, facilitating research on neural regeneration and plasticity following spinal cord injury.

Protocol

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

1. Direct injections into the spinal cord targeting axon terminals

1. Using the tip of the glass injection needle, approximate the midline of the spinal cord by identifying the spinal artery. However, if the spinal artery is noticeably off-center or deviates in any way, approximate the midline by using the position of the C2 spinous process and extrapolating this down the length of the spine.
   NOTE: Load the virus into the injector by pipetting ~5 µL onto a piece of parafilm, and position the needle so that the tip rests atop the virus droplet.
2. Once identified, situate the needle just posterior to the C5 lamina at the approximate midline and use this as the reference point. Then, using the micromanipulator, move the needle laterally to the right by 0.3 mm. Lower the needle until it just touches the surface of the spinal cord; from this depth, plunge the needle 0.6 mm into the spinal cord. If necessary, continue to plunge the needle until it punctures the spinal cord; then, retract or lower the needle to the appropriate depth.
3. Inject 1 µL of retro adeno-associated virus2-scCre at 250 nL/min. After the injection is complete, wait 2 min for the virus to diffuse into the spinal cord before slowly withdrawing the needle. Repeat the injections using the same lateral and depth coordinates at two more sites within the spinal window, one at the midpoint and the final just anterior to the T1 lamina.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Flexible Needle Attachment	World Precision Instruments	MF34G-5	For cleaning glass needles and loading red oil into glass needles.
Glass Capillary Tubes	World Precision Instruments	4878	For pulled glass needles - should be designed for nanoliter injectors.
Micromanipulator with a Vernier Scale	Kanetec	N/A	For precise targeting during surgery.
Lab Standard Stereotaxic Instrument	Stoelting	51600	To hold the neonatal sterotaxic holder in place
Lab Standard with Mouse & Neonates Adaptor	51615	For neonatal skull fixation during cranial surgery and spinal injections
Microscope with Light and Vernier Scale Ocular	Leitz Wetzlar	N/A	Used to visualize and measure beveling of pulled glass capillary tubes into functional glass needles.
MicroSyringe Pump Controller	World Precision Instruments	62403	To control the rate of injection.
Nanoliter 2000 Pump Head Injector	World Precision Instruments	500150	To load and inject virus in a controlled fashion.
pAAV-CMV-scCre	Wu lab		Cre plasmid
pAAV-hSyn-DIO-hM3Dq-mCherry (plasmid #44361)	Bryan Roth’s lab through Addgene		DREADD plasmid
PrecisionGlide Needles (25G x 5/8)	Becton, Dickinson and Company	305122	For use with the 1mL and 10 mL syringes to allow injection of the animal model.
Rat Tooth Forceps	Roboz	RS-5152	For griping spinous processes.
Sterile Saline	Baxter Healthcare Corporation	281324	For use in blood clearing, and for replacing fluids post-surgery.
Surgical Gloves	N/A	N/A	For use by the surgeon to maintain sterile field during surgery.
Surgical Heating Pad	N/A	N/A	For maintaining the body temperature of the animal model during surgery.
Surgical Microscope	N/A	N/A	For enhanced visualization of the surgical wound.