Neuroscience
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Direct Injection of a Lentiviral Vector Highlights Multiple Motor Pathways in the Rat Spinal Cord
Chapters
Summary March 15th, 2019
This protocol demonstrates injection of a retrogradely transportable viral vector into rat spinal cord tissue. The vector is taken up at the synapse and transported to the cell body of target neurons. This model is suitable for retrograde tracing of important spinal pathways or targeting cells for gene therapy applications.
Transcript
Viral vectors can introduce genetic material into cells to compensate for defective genes, to up-regulate important growth proteins, or to manufacture markers that can trace neural circuits. Introducing viral vectors into the nervous system is challenging due to innate biological barriers. Direct injection into spinal cord tissue bypasses these barriers, allowing for access to cell bodies or synapses.
Correctly targeting the L1 to L4 spinal levels can be difficult. It's important to use landmarks to verify the target and remember that these segments line to the T11 to T13 vertebrae. On the day of the procedure, plug the injector into the micro-pump and place the micro-pump into a micromanipulator with a vernier scale.
Use a syringe with a flexible needle to carefully load a colored dye into one of the glass needles and insert the glass needle into the injector. Then confirm that the needle is seeded correctly into the washers, and that the injector cap is screwed on tight, and that the steel injector needle is extended approximately three-quarters of the length of the glass needle. Before beginning the procedure, thaw at least one microliter, plus a small volume of extra virus per injection from freezer storage, and confirm a lack of response of toe pinch in an anesthetized rat.
Shave along the dorsal midline from the hips to the inferior angle of the scapulae of the animal, pulling the skin taut for an easier and more precise shave, and disinfect the exposed skin with sequential 5%iodine and 70%ethanol scrubs. Apply ophthalmic ointment to both eyes. Then place the animal on a sterile cloth and place gauze underneath the bladder to collect any urine.
To identify the site of the skin incision, press the fingers gently at the last rib to locate the L1 vertebra. Using this vertebra as a landmark and holding the skin taut by gentle spreading while pressing firmly with the scalpel, use a number 10 surgical blade to make a three to four-centimeter skinned incision ending just inferior to L1 to expose the muscle. Use forceps and scissors to feel for the spinous processes.
Make small rostral cuts to allow room to grab securely onto an upper process with rat tooth forceps. And make two long, deep cuts as close to the processes as possible. After securing the lateral muscles in place, clear the muscle around the processes to determine the shape of their heads.
Then visualize the shapes of the heads of the spinous processes in order to identify the laminectomy site. The T11 and the joining T12 and T13 processes will be the site of the laminectomy. Once the target area has been correctly identified, gently spread the vertebrae to reveal the intervertebral ligaments, and use rongeurs to take small bites of the spinous processes and the dorsal aspect of the vertebrae.
Clear the bone away from the midline so that the midline blood vessel can be observed, leaving a window that clearly reveals the spinal cord tissue and is free of debris. Then fasten stabilizing forceps to the spinous processes rostral and caudal to the laminectomy window to secure the animal in a spinal holder. And raise the abdomen of the animal to negate the effect of breathing movements.
To load the virus into the injector, pipette approximately five microliters of the thawed stock onto a piece of Parafilm, and position the glass needles so that the tip is inside the drop. Use the micro-pump to withdraw up to four microliters of virus at a rate of 20 to 100 nanoliters per second, and set the controller to inject before releasing a small amount of virus from the needle tip to ensure that it is not blocked. Wipe off any excess virus with a laboratory wipe and position the micromanipulators so that the vernier scale is visible.
Place the needle at the midline of the spinal cord, and use the vernier scale on the micromanipulator to direct the needle laterally 0.8 millimeters. Then lower the needle to the spinal chord until it is indenting, but not puncturing, the dura, and use a quick twisting motion to puncture the dura with the needle until the needle tip has sunk to a depth of 1.5 millimeters. Once the needle is in place, program the injector to deliver the virus at a rate of 400 nanoliters per minute, and confirm that virus is entering the spinal chord by observing the progress of the dye front.
Once the injection is finished, allow the needle to rest in the spinal cord for two to five minutes to facilitate the diffusion of the virus, before slowly withdrawing the needle for positioning at the next injection site. After the last injection, remove the animal from the spinal holder to allow the removal of any devices used to spread the lateral muscle. Then close the muscle with a 4.0 chromic catgut suture and staple the skin with nine-millimeter wound clips.
Four weeks after a successful injection, significant GFP expression is observed in the neurons within the gray matter of the thoracic spinal cord on the side, ipsilateral to the injection. In the white matter, GFP expression is observed in the axons in the ipsilateral cord, especially in areas typical to the propriospinal axons. A higher magnification of the gray matter neurons reveals an example of a typical signal expression in neuronal cell bodies and dendrites.
GFP expression in neurons can also be observed in brainstem nuclei, such as within the pontine reticular formation. The key factors for a successful spinal cord injection procedure are correct vertebral targeting and ensuring that virus is entering the tissue If a rostral spinal cord injury is performed prior to this procedure, direct injection can be used to trace neurons that reestablish connections around the injury.
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