The overall goal of this procedure is to enhance survival and differentiation of neural stem cells transplanted into lesions of severely injured spinal cord in rats. This is accomplished by first making a spinal cord lesion such as a T three transection with most dura intact except a small opening to retain grafted cells. The second step of the procedure is to harvest freshly dissociated neural stem cells from embryonic day 14 rat fetus.
The third step is Resus suspending neural stem cells into each component of fibrin matrices containing growth factor cocktails. The final step is to transplant neural stem cells in fibrin matrices and growth factors into the subacute lesion center, immunofluorescence microscopy of transfected and grafted spinal cords. At seven weeks post grafting can show excellent survival of neural stem cells and their differentiation into mature neurons, which extend remarkable numbers of axons into the host.
The main advantage of this technique over the existing method like graft cell in the cell culture medium or PBS, is that fibrin gel can retain cell into the lesion center and the gross fat cocktail can support their survive. Though this method can provide insight into how neural stem cells grow and function in adult injured spinal cord, it can also be applied to other systems such as traumatic brain injury. Begin the transaction procedure by anesthetizing 150 to 200 gram adult female fisher, 3 44 rats, or 180 to 220 gram athymic nude rats using acceptable methods.
Wait to proceed until the animals are deeply anesthetized and show no responses to tail and paw pinches. Then apply eye ointment, shave the upper thoracic area and clean the skin with Betadine. Start the surgery by cutting the skin and muscle using a number 15 blade exposed a T three vertebra using a surgical retractor, and then using a Perform a laminectomy at the T three vertebra to expose the T three four spinal cord.
Next, make a two millimeter long longitudinal midline incision in the DRA with a number 11 blade. Now place the iridectomy scissors underneath the Dora and cut laterally across the entire right hemicord. Make another cut on the same side, 1.5 millimeters more coly now aspirate the spinal cord tissue between the two cut segments with a blunt 23 gauge needle connected to a moderate intensity vacuum.
Make sure the transection is complete both ventrally and laterally. This completes the lateral HEMI section to extend the lesion to a full width spinal cord. Transection place mirror incisions into the left hemi cord.
Attempt to keep the still intact dura intact, so it will retain a firm graft fibrin matrix. When grafted two weeks later, now suture the muscle, apply antibiotic powder and stable the skin. Immediately post-op.
Inject three milliliters of lactated ringers containing doses of ine and ampicillin. Then maintain rats in a warm incubator until thermal regulation is reestablished. Over the next two weeks, twice a day, empty the bladder and inject the ringers and ampicillin solution.
This can be halted when the reflex of bladder emptying returns. Prepare GFPF 3 44 female rats with an injection of DESI to synchronize embryo collection and make them to mature GFP expressing males on the day of embryo collection and grafting. Have prepared the fibrinogen and thrombin containing growth factor cocktails and store them on ice until they are mixed with the cells.
Collect embryos at day 13.5 to 14.5 Post cotus in cold HBSS buffer. Examine the embryos for GFP expression with a night sea flashlight and filter glasses, or with a fluorescent microscope. Now aseptically, dissect the spinal cord out of each GFP positive fetus.
Remove the overlying meninges and attach spinal ganglia with iridectomy scissors and fine jewelers forceps. Then collect the dissected spinal cords into two milliliters of cold HBSS buffer in a 15 milliliter conical tube. Keep them on ice.
Next, dissociate the spinal cords. Once completed, count the cells with a hemo cytometer and divide them equally into two micro centrifuge tubes. Centrifuge the cells and completely remove the snat with a low vacuum tip over a one to two minute period.
The growth factor cocktails should not be diluted by the remaining supernatant after cell resus. Now suspend each half of the cells in the fibrinogen or thrombin working solution containing the growth factors at a concentration of 250, 000 cells per microliter. Store the cells on ice prior to transplantation.
Begin the transplantation by opening the lesion to T three four spinal cord two weeks after spinal cord transaction. To avoid premature ululation, mix the cells in fibrinogen. Next, using pulled glass pipettes connected to a pico spritzer.
Separately, inject about five microliters fibrinogen cells and five microliters thrombin cells, and to nine points of the lesion site through sealed scar tissue and intact Dora without reopening the lesion site to perform direct cell injection without reopening the lesion site, use a 27 gauge needle to create nine small holes on the surface of the scar tissue and intact Dora one week post lesion, then inject a half volume of the mixed fibrinogen and thrombin solutions into the three middle points of the lesion site and a quarter volume into the three points at the rostral interface and the coddle interface grafted rat NSCS resuspended in PBS lacking a fibrin matrix and growth factors survived poorly in the T three transection site. These cells labeled with GFP only attach to the lesion host margin and leave most of the lesion site empty when cells were co grafted with just fibrin matrix NSC survival improved, but the graft did not fill large lesion cavities. However, embedding rat nscs into fibrin matrices with a growth factor cocktail consistently enhanced their survival, allowed them to fill large lesion cavities and allowed them to differentiate into neurons and glia.
After spinal cord transection 15 to 17, the same result was found with human nscs, which also completely filled the lesion cavity. When assessed seven weeks post grafting a time course study revealed that GFP expressing NSCS survived 24 hours after transplantation, the grafted NSCS gradually became more dense and completely filled the transected lesion cavity by the first week after transplantation, probably due to proliferation. In addition, the grafted NSCS integrated well at the host lesion interface and appear to reduce regions of GFAP Immunoreactivity at the margins of the lesion cavity.
A portion of the grafted NSCS differentiated into new and expressing neurons seven weeks after transplantation. In addition, graft derived spinal cord neurons extended large numbers of axons into the host spinal cord, roly and coddly over long distances. Higher magnification reveal that graft derived axons extended through both white matter and gray matter in the host spinal cord.
Following this procedure, other methods like electrophysiology and behavioral analysis can be used to learn about a functional connectivity of grafted neurons.
