Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

The overall goal of this procedure is to describe in detail a contusion model of spinal cord injury in mice, followed by a transplantation of neural stem cells. This is accomplished by first labeling postmortem, neural precursor cells or PM NPCs with the vital tracer PKH 26. Next, a dorsal incision is made on the mouse and the spinal cord is exposed.

Then an infinite horizon or IH impactor device is used to create a lesion on the spinal cord. Finally, the PKH 26 labeled PM and PCs are injected into the tail vein of the mouse. Ultimately, the fate of engrafted PKH 20 6:00 PM and PCs at the lesion site is followed.

The injury model replicates the spinal cord injury man. The primary mechanical injury is followed by progressive secondary degeneration where cellular and pharmacological treatment are studied. The described applications may provide a methodological basis to obtain further insights into faith.

Induction of stem cells transplanted into a spinal cord while degeneration and inflammatory events are taking place. Demonstrating the lesion will be Dr.Tony Laja Longo PhD student in the laboratory. While the tail injection will be performed by Dr.Ache, that is a postdoctoral fellow in the lab, Beginning with neural stem cells that meet the criteria as described in the text protocol.

Resus suspend the cells to a concentration of one times 10 to the six cells per 150 microliters. Prepare at least 1.2 times 10 to the six cells per mouse to have enough cells for loading the pipette. With neural stem cell medium, wash the cells three times in a 10 milliliter conical vial, spinning at 500 times G for five minutes at room temperature to pellet the cells.

After each wash, before the final spin count, the cells then after spinning, aspirate the supernatant, leaving about 25 microliters of liquid to prevent aspirating any cells. Next, prepare a two X suspension by adding one milliliter of diluent C to the cell palate and use gentle pipetting to resuspend it immediately. Before staining, prepare a two x dye solution in diluent C by adding four microliters of the PKH 26 ethanol D solution to one milliliter of diluent C in a tube and mix well.

To disperse quickly add the one milliliter of two x cell suspension to one milliliter of two XD solution and immediately mix the sample by pipetting. After incubating the cell die suspension for one to five minutes, stop the staining by adding an equal volume of 1%BSA solution in HBSS and incubate for one minute. Then centrifuge the cells at 500 times G for 10 minutes and carefully remove the supernatant.

Use 10 milliliters of complete medium to resuspend the cell pellet for assessment of cell recovery, cell viability and fluorescence intensity. Centrifuge the cells discard the supernatant and add 10 milliliters of HBSS to the cell pellet. Wash twice with HBSS and resuspend the cells in a sterile physiological solution.

After preparing the surgical area and a mouse according to the text protocol, place the animal dorsal side up on a slide warmer to avoid hypothermia during the surgery with a scalpel, make a vertical incision over the region of interest from T seven to T 12 using forceps. Hold the skin and superficial fat pad found between the space between the fifth and sixth thoracic processes under a stereo microscope. Count the process under the blood vessel as T six, then move to T seven.

Then place a little bearing under the ventral side of the mouse to increase the curvature of the spine and use grof forceps to block the spine to immobilize it. Next, use a scalpel to cut the para of vertebral muscles bilaterally from T seven to T 10 at the vertebral level until the dorsal surface of the lamina contacts the scalpel tip. Then use a scalpel to tick off the junction from T seven to T 10 at the vertebral level until the dorsal surface of the lamina contacts the scalpel tip stop in the space between the T eight and T nine tiny protrusions.

Then use micro scissors to cut the tissue between T eight and T nine and T nine and T 10. Now use the rongeur to remove the T nine process. Expose the junction by using micro scissors to carefully scrape away the muscle layer.

Continue until the bone is exposed. Use forceps to remove the muscles from the lamina and open a small space between the vertebrae. Then gently insert the micro scissors under the bone.

Cut the lamina on both sides and use the forceps to remove it, exposing the cord. Use the ron jour to remove any free or jagged bone fragments that are left behind. Then remove the top half of the T nine dorsal process.

After following the IH impactor device protocol, according to the guidelines in the text, use four oh absorbable suture to close the incision by first covering the exposed spinal cord at the site of the removed lamina. Taking care not to pinch off the underlying muscles. Use two or three reflex clips to close the skin.

Inject two milliliters of saline in the lower back subcutaneously and place the mouse in a prewarm cage on a heating pad to recover. To carry out a tail vein injection of cells. Resuspend the cells in the test tube and load 75 microliters into a 0.3 milliliter syringe, ensuring that no bubbles are present in the cell suspension.

To avoid cell sedimentation, keep the syringe in a horizontal position. Place the mouse underneath the heat lamp to dilate the tail veins. Then gently pull the mouse into the restrainer to visualize the lateral tail vein, which appears as a narrow blue line.

Use an alcohol swab to clean the tail, and once the vein is visualized, grab the tail vein between the middle finger and thumb of the left hand. The T nine contusion demonstrated in this video caused the transient loss of hind limb function three times 10 to the fifth cells or PBS were injected into the tail vein in three injections carried out 30 minutes, six hours, and 18 hours after injury. As shown here, within two to three weeks, PBS treated injured, mice improved and hind limb function reached three points of basal mouse scale or BMS mice treated with PM.NPCs, however, showed a higher recovery and reached 4.5 points of BMS as seen here, most engrafted pm NPCs labeled with PKH 26, accumulated at the edges of the lesion forming clusters.

Soon after administration, the transplanted cells migrated along the lesion edges and in a more diffused fashion where they differentiated into neurons. At 30 days after lesion and transplantation, the cell body of pm NPCs increased in size and in most cells, dendritic like processes were obvious and fully immuno stain by the specific antibodies to map to While obtaining this pro procedure, it is important to remember that higher forces make may increase mortality in the animal due to a more severe injuries. Thus, we suggest to use our paradigm of lesioning paradigm that is followed by a more reproducible process of recovery.

After watching this video, you should have a good understanding of how to obtain a reproducible experimental model of traumatic spinal cord injury in the mouse by using the infinite horizon impactor.