Surgical Transplantation of Mouse Neural Stem Cells into the Spinal Cords of Mice Infected with Neurotropic Mouse Hepatitis Virus

Carbajal, Kevin S.; Weinger, Jason G.; Whitman, Lucia M.; Schaumburg, Chris S.; Lane, Thomas E.

1. Attach the needle with needle nut to the Hamilton syringe and clean them by flushing several times with water, then 70% ethanol, and finally HBSS. Insert plunger after each fill with water, 70% ethanol, or HBSS. 2. Prepare the Hamilton syringe for cell loading by removing the plunger and loosening the needle nut and pulling the needle away from the syringe to prevent backpressure. Ensure that careful handling of the needle and nut are done with sterile gloves. 3. Load 15μl of cells into pipette tip and press the tip tightly into the back end of the syringe to load the cells into the syringe. 4 . Insert the plunger about 5mm and then tighten the needle nut. 5. Depress plunger until some of the cell suspension is seen exiting the needle. 6. Make sure there are no bubbles in the syringe and lay the syringe down in the horizontal position to prevent the cells from making a gradient by gravity. 7. Grab hold of the laminectomized mouse by the spinalis dorsi muscle connecting the spines of T8 and T9 (Figure 2B , C). 8. Clamp the hemostat to the left (vertical) micromanipulator arm so that the mouse's front paws are in the air and its rear paws lightly touching a platform of sterile paper towels as in Figures 2A and 2B . 9. Attach the syringe to the right micromanipulator arm (at a 70° angle) and slide the syringe to the lowest position possible before clamping. 10. Stabilize the mouse by pinning its tail against the paper towels and slowly lower the syringe ( Figure 2B ). 11. Lower the needle towards the cord and insert the needle 1mm into the opposite hemisphere through the dorsal midline ( Figure 2C ). The tip of the needle should be in the gray matter close to the central canal. 12. Slowly inject 2.5μl of cells. Inject at a rate of 1μl / 5 seconds. After injecting the cells, wait a 10 seconds and retract the needle a tenth of a turn at a time every 10 seconds until the needle is out of the cord. Pay attention to possible efflux of cell suspension. 13. Quickly retract the syringe and detach it from the micromanipulator arm. Lay the syringe down horizontally. 14. Release the mouse and transfer to suturing table. 15. Repeat steps 5.7-5.14 for each mouse until the syringe is emptied. Sterilize tools (in the sterilizer) and the needle (by wiping with ethanol) between animals. Discard cells and reload if clumping is visible. 16. Clean the syringe as in step 5.1 between loads.