The overall goal of this procedure is to promote remyelination and clinical improvement in animals with established demyelination via transplantation of neural stem cells into the spinal cord of JHMV infected mice. This is accomplished by first preparing the animal. The second step of the procedure is to perform a laminectomy at thoracic vertebrae.
10 cells are then injected into the exposed spinal cord. After a successful injection, the wound is closed. Ultimately, presence of mouse neural stem cells in the spinal cord.
Parenchyma can be visualized by methods such as fluorescent microscopy, Neuro stem cell transplantation into the spinal cord of JHMB. Infected mice can help answer key questions into the field of cell replacement therapies for the treatment of demyelinating diseases, such as how the cells migrate, proliferate, and differentiate also how the cells affect the central nervous system. And if we can modify the cells or the spinal cord environment they're transplanted into to enhance their therapeutic benefits.
Although this method can provide insight into cell replacement therapy in a viral model of demyelination, it can also be applied to other models of demyelination and injury, such as experimental autoimmune encephalomyelitis, EAE, and spinal cord injury. Before preparing cells for injection, prepare all necessary solutions. Next, clean and sterilize all equipment.
Prepare the surgical area and set up the micro manipulator to prepare the cells for transplantation trypsin eyes, and resuspend them in a 50 milliliter conical vial. Wash the cells to be transplanted three times in HBSS. Count the cells before the final spin.
After the final, spin down decant HBSS and leave the vial in an upside down position to prevent droplets from reaching the pellet. Dry the inside walls with a UV irradiated sterile Kim wipe. Do not allow the pellet to dry.
Holding the tube upright slowly and very delicately. Re suspend the cells in half the final desired volume of HBSS. Ultimately, the cells should be at a concentration of 100, 000 cells per microliter.
Measure the total volume by pipetting the suspension into a pipette tip, and then adjusting the dial on the pipetter until the entire suspension is in the pipette tip. This will tell you how much more HBSS is needed for the desired concentration. Bring up the cells to the desired volume.
By adding HBSS. You may transfer the cells to an eph tube. Place them back on ice.
Check the viability of the cells if they must be on ice for longer than two hours. To begin the surgical procedure, confirm the proper level of anesthesia through the toe pinch reflex. Use electric clippers to shave the dorsal area of the mouse from the lower back to the neck.
Once the area is shaved, apply a thin layer of hair removal cream to remove any remaining hair. After one to two minutes, wipe the area clean with dry gauze and then with cause lightly soaked in soapy water. The surgical area should now be free of hair.
Lastly, sterilize the prepared area with Betadine solution and apply ointment to the eyes. To avoid dryness, position the mouse dorsal side up. Make an incision over the laminectomy site spanning from about thoracic vertebrae.
T eight to T 11. Exaggerate the curvature of the spine and locate T 10. It is the first vertebrae with a flat process and is generally two vertebrae rostral to the apex of the curvature of the spine.
With the gray forceps, firmly secure the spinal column. At T nine, use a scalpel to score the space between the two spiny protrusions of T 10 and T 11. Next, scrape the muscle layer away to expose the bone underneath, making sure that the curvature of the scissors is pointing away from the cord.
Slowly insert one blade of the scissors between T 10 and T 11. Slide the blade between the cord and the pedicle of T 10 and snip the pedicle Repeat on the other side. Lift the lamina to expose the cord underneath and carefully snip it off.
Be sure not to leave any free or jagged bone fragments behind. Once all the bone fragments are cleared, wipe any blood away with sterile cotton swabs. First, prepare the Hamilton syringe by attaching the needle with the needle nut.
Insert the plunger and flush several times with water. 70%ethanol and HBSS once cleaned. Remove the plunger and loosen the needle nut.
Pull the needle away from the syringe to prevent back pressure. When loading the cells load 15 microliters of cells into the syringe by pressing the tip of the pipette tightly into the back of the syringe. Insert the plunger about five millimeters and then tighten the needle nut.
Depress the plunger until some of the cell suspension is seen exiting the needle. Make sure there are no bubbles in the syringe, and lay the syringe down horizontally to prevent the cells from making a gradient by gravity. Returning back to the animal, use hemostats to grip the S spinous doci muscle.
Connecting the spines of T eight and T nine. Clamp the hemostat to the left micro manipulator arm so that the mouse's front paws are in the air and its rear paws. Lightly touch a platform of sterile paper towels.
Attach the syringe to the right micro manipulator arm at a 70 degree angle and slide the syringe to the lowest possible position before clamping. Stabilize the mouse by pinning its tail against the paper towels. Slowly lower the needle towards the cord.
Insert the tip one millimeter into the opposite hemisphere through the dorsal midline. The tip of the needle should be in the gray mat or close to, but not in the central canal. Slowly inject 2.5 microliters of cells at a rate of one microliter per five seconds.
After injecting the cells, wait 10 seconds and retract the needle. A 10th of a turn. Retract at this pace every 10 seconds until the needle is out of the cord.
Injecting the cells too fast or removing the needle too quickly may cause flx of cell suspension. If this occurs, discard the mouse from the study. Once removed from the cord, quickly retract the needle and detach the syringe from the micro manipulator arm.
Again, laying it down horizontally. Release the mouse and transfer it to a suturing table. Repeat this procedure for each mouse until the syringe is emptied.
Discard the cells and reload if clumping is visible, flushing and cleaning the syringe between loads. After the injection, the animal is transferred to a separate location To be sutured. Pass the suture needle through the superficial fascia on both sides of the incision.
Pull the suture through to bring the fascia together and to cover the exposed spinal cord at the site of the removed lamina. Use a needle holder in forceps to tie three knots and trim away as much threat as possible. One suture is usually sufficient while pulling away the skin from the mouse.
To avoid stapling the underlying muscle, close the incision with two to three staples. Once the wound is closed, inject 0.5 milliliters of lactated ringer solution subcutaneously into the lower back, but away from the incision. Place the mouse on its side in its cage, making sure the surgery site does not contact the cage bedding.
Monitor the animals until fully recovered. This image shows successfully transplanted GFP labeled mouse neural stem cells in a coronal section of the spinal cord. 21 days post-transplant.
After watching this video, you should have a good idea of how to transplant neuro stem cells into the spinal cord of JHMV infected mice Once mastered. It is possible to perform this technique on 40 mice in about three hours.
