The overall goal of this procedure is to achieve spatially restricted gene expression in the mouse spinal cord by stereotactically injecting a viral vector into the lumbar dorsal horn. The first step of the procedure is to locate and expose the lumbar vertebra. The second step is to remove the dorsal portion of the vertebra in order to reveal the spinal cord.
A procedure termed laminectomy. Next, a glass pipette connected to a micro syringe pump is positioned over the dorsal horn and lowered into the spinal cord. The final step is to slowly inject the vector suspension.
Two weeks later, transfection of dorsal horn neurons is demonstrated by the expression of green fluorescent protein, which allows tracking the virus's distribution. This method helps elucidate the function of neurons in the some of the sensory pathway. We use it in studies that focus specifically on the development of inflammatory or neuropathic pain.
Visual demonstration is critical for learning how to position the mouse into the stereotactic frame. It's, it is essential for the success of the procedure that the spine is fixed properly because the laminectomy is such a delicate technique. Martin Mole, a graduate student from my laboratory, will be demonstrating the procedure.
John Wang, a research assistant, will be helping him One or two days prior to the procedure. Pul glass pipettes to obtain a tip diameter of 40 micrometers and bevel the tip at an angle of 20 degrees on the day of the surgery. Begin the procedure by disinfecting the surgical equipment and preparing the sterile surgical tools to set up the stereotaxic frame.
Mount the micro syringe injector onto the manipulator and plug the device in. Attach one of the glass pipettes to the micro syringe using the compression fitting. Now remove the plunger from the micro syringe.
Fill the syringe with the mineral oil, then reinsert the plunger and push it all the way to the tip. Carefully Avoid creating any air bubbles. Then insert the micro syringe into the holder on the injector.
Next, remove the virus from the freezer and prepare the virus particle suspension just before use by diluting it with sterile phosphate buffered saline to the desired particle concentration. Pipette five microliters of the virus suspension onto a small piece of param. Then lower the glass pipette tip into the drop and pull the plunger to fill the micro syringe.
At the end. Create a small air bubble at the pipette tip to prevent it from clogging. In this step, prepare the surgery area by wiping the bench in the heating pad with disinfectant.
Then anesthetize the mouse by isof fluorine inhalation. Next, apply lubricant on each eye to protect them from drying during the operation Afterward, shave the fur from the lower back to the neck of the mouse. Disinfect the skin with alternating wipes of a topical antiseptic, such as chlorhexidine or povidone iodine, and 70%ethanol.
Then infiltrate the incision site with 0.25%bupivacaine diluted one to 10 with physiological saline. Subsequently incised the skin at the coddle end of the rib cage, two to three centimeters along the midline and separate the fascia covering the spine. Identify vertebra L one, which is located coddled to the vertebra, holding the last pair of ribs and expose it by removing the small spinal muscles and ligaments attached to its dorsal surface.
Then slightly lift and hold vertebra L one with a pair of adds and forceps. Next, use a dedicated laminectomy forceps to remove the dorsal portion of the vertebra, including the spinous process and the lamina to expose the spinal cord. Avoid damaging the dura mater or the spinal cord during this procedure.
Then transfer the mouse onto a heating plate in the stereotaxic frame and monitor the temperature of the mouse during the subsequent operation. Now use the v notch spikes to stabilize the spine so that the vertebra does not move during breathing. Next, bring the micro syringe closer so that the pipette tip is above the laminectomy site.
Lower the plunger until you see the virus suspension exiting the pipette. Remove the droplet with sterile gauze. Then position the pipette tip at the rostral most portion of the exposed spinal cord.
Center the pipette above the posterior median sulcus and move the tip 500 micrometers laterally. After that, lower the tip to the surface of the cord and puncture the dura mater. Subsequently, lower the tip of the glass, pipette 300 micrometers into the spinal cord.
Inject one microliter of virus suspension at a rate of 200 nanoliters per minute. At the end of the injection, wait at least two minutes before slowly retracting the pipette. Repeat the injection procedure at the coddle mosts portion of the exposed spinal cord to achieve complete distribution of the viral vector in spinal segment L four.
The two injection sites are located rostral and coddle to the L four segment. To avoid tissue damage in the target region after the injection release, vertebra L one from the V notch clamps and remove the mouse from the stereotaxic frame. Next, suture the fascia with five oh Vicryl.
The closed fascia provides cover for the laminectomy site. Then close the skin with nylon sutures or surgical staples. Provide post-surgical analgesia for 72 hours with daily subcutaneous injections of carprofen at five milligrams per kilogram.
Transfer the mouse to a recovery cage with soft, non particular bedding. Place the mouse on the side for comfortable breathing. Then monitor the animal until it is fully alert.
Ambulance and starts drinking. Monitor the post-surgical recovery by daily inspection for the first three days. Then every other day or three days per week until the experiment is complete.
Seven to 10 days after the operation when wound healing is complete. Remove the skin sutures or staples. This figure shows the expression of enhanced green fluorescent reporter protein or EGFP in the left dorsal horn of the L four spinal cord.
Two weeks after the stereotaxic injection of R-A-A-V-E-G-F-P neurons were immunostain for neuronal nuclei Protein shown in red Arrowhead. Point to scattered transfected glial cells in the dorsal column. Approximately 80%of neurons in the medial dorsal horn were infected With practice.
The procedure can be completed in 30 to 40 minutes, and please remember to maintain sterile conditions during the procedure. After watching this video, you should have a good understanding of how to perform intra pine chimal injections into the dose ho of the mouse spinal cord. We are using the technique for gene manipulation, but you may adapt it for other purposes such as intraspinal drug application.
