Synergetic Use of Neural Precursor Cells and Self-assembling Peptides in Experimental Cervical Spinal Cord Injury

The overall goal of this procedure is to introduce a contusion compression clip model of cervical SCI in rats, and a protocol for combined treatment involving SAP injection and NPC transplantation. First, an aneurysm clip is placed around the spinal cord and kept in place for one minute. This results in a bilateral contusion compression injury.

Next SAPs are injected at the injury epicenter NPCs are injected immediately following SAP injection. An osmotic pump with a subdural catheter is inserted for the sustained delivery of growth factors. This procedure results in the delivery of NPCs to the injured spinal cord.

The survival and engraftment of NPCs is supported by combinatorial treatment with SAPs and growth factors. The implications of this technique extend toward a therapy for traumatic spinal cord injury because it provides a clinically relevant model that can be used to study cell-based COMBINATOR therapies that promote spinal cord regeneration. The following experimental protocol was approved by the Animal Care Committee of the University Health Network, Toronto Canada, and is in accordance with the policies established in the guide to the care and use of experimental animals prepared by the Canadian Council of Animal Care.

All surgical procedures must be performed under sterile conditions using sterile equipment and solutions, and according to institution and government guidelines, this procedure uses wist a rats weighing 250 to 270 grams before beginning the procedure. Provide the animals with antibiotics in the drinking water for two days on the day of the procedure. After placing the rat in an anesthesia chamber and inducing anesthesia with 5%ISO fluorine in one to one oxygen and nitrous oxide for one minute, reduce the ISO fluorine to 1.8 to 2.2%Apply off the lambic ointment to the eyes and confirm surgical anesthesia by performing a paw pinch.

Then place the rat onto a heated cushion and fix the head into a stereotaxic frame with a nose cone. After shaving the fur around the cervical spine, disinfect the skin by swabbing with povidone iodine, followed by 70%ethanol after making a midline incision from the region of the C two cervical vertebrae to the prominent processis of the T two thoracic vertebral body. Use surgical scissors to cut through the outer layer of the vertebral muscles directly on the midline in a cranio coddle direction, use surgical scissors to blunt dissect the deeper muscle layers until the processis, osis and laminate are reached.

Insert protractors and hold. Open the muscle layers, then orient the prominent processis posis of the T two vertebra body and identify the targeted levels for laminectomy. Then use a number 15 scalpel to cut through the ligament flay to loosen the lamini and the processis posis.

This next, use a bone clipper to cut through the lamini lateral to the spinal cord and gently remove the lamini without compressing the spinal cord. Now take a moment to identify the emerging nerve roots to spare them from clipping. Then use a hook to loosen the ventral dura from the dorsal side of the vertebral bodies and prepare a corridor for the clip.

Ensure that the ring spring of the clip is set to deliver the desired force. The clip force determines the extent of trauma and compression. Insert the open clip around the spinal cord and let it snap shut to induce a contusion injury.

Clip forces of between 15 and 35 grams are often used with a clipping duration of one minute after the spinal cord injury has been performed. Remove the clip and close the muscles in two layers, followed by closure of the skin. Once the wound is properly closed, stop the anesthesia and observe the rat while it wakes up.

After the animal has regained sufficient consciousness for sternal, recumbent, place the rat in a clean cage. Because of the severity of this type of injury, it is imperative that the following postoperative treatments are followed. First, be sure to administer painkillers such as buprenorphine and meloxicam for three to five days depending on symptoms.

Also prevent dehydration by subcutaneously. Injecting five to 10 milliliters of saline twice a day for three days, and continue to provide antibiotics in the drinking water for five days.Post-surgery. Squeeze the urinary bladder two to three times a day until consistent recovery of bladder function is apparent even after the animals appear recovered.

Be sure to observe the operated animals for neurological deficits and physiological condition. Injection is performed 14 days after spinal cord injury. Before beginning the surgery, prepare the SAPs at a concentration of 1%weight per volume in sterile water, and fill a 100 micrometer glass capillary attached to a Hamilton syringe.

With the solution, attach the Hamilton syringe to the stereotaxic frame. After anesthetizing, the rat affix the head in the stereotaxic frame and disinfect the surgery site with povidone iodine and 70%alcohol. As before, once again, after opening the skin, carefully dissect the pair of vertebral muscles and insert retractors.

Use a number 15 scalpel to remove the scar tissue from the dura and re-expose the lesion site. Then use the tip of a sharp needle to open the dura carefully. Finally, stereotactically, insert the glass capillary to a depth of two millimeters into the traumatized spinal cord and inject the SAPs.

After injecting the SAPs, allow the capillary to remain at the injection site for one minute before removal to allow tissue stretching to accommodate the injected solution. Then repeat the injection at the next site. Here, NPCs, were isolated from the paraventricular zone of an adult DS red mouse and cultivated according to previously published procedures.

Just before micro injection dilute the NPCs in growth medium to a concentration of 50 times 10 to the third live cells per microliter for cell transplantation and load into a glass capillary on a Hamilton syringe. NPCs are injected immediately after injection of SAPs. To do so, insert the glass capillary of the Hamilton syringe at a 0.2 millimeters rostral of the injury site and 1.5 millimeters below the dorsal surface of the spinal cord.

Inject two microliters of the cell suspension at a rate of about 0.5 microliters per minute. After the first injection is complete, allow the capillary to remain in the spinal cord for one minute as before, then retract the capillary, move it to an adjacent site and repeat the injection procedure. After waiting for a minute, as before, retract the capillary and move it two millimeters coddle to the injury site.

Perform two two microliter injections at this location. This image shows all injection sites relative to the spinal cord injury. Implantation of subdural mini osmotic pumps for growth factor support is performed immediately after NPC injections.

First choose an implantation site. Preferred sites are the lateral flanks of the thoracoabdominal region to avoid discomfort caused by the pump itself. After disinfecting the region as before a subcutaneous recess for the pump is prepared.

Next, perform a skip laminectomy of the upper or lower vertebrae adjacent to the spinal cord injury. Here, the spinal cord injury was performed at C seven T one, and so the laminectomy is performed at C five. Now place the pump into the subcutaneous recess, then cut the catheter to the necessary length, and finally use 6.0 sutures to secure it to the pair of vertebral muscles.

Avoiding any movement associated dislocation. Use the tip of a sharp needle to open the dura below the laminectomy. Then introduce the catheter into the subdural space and let it glide in a coddle direction without resistant and without injuring the cord.

Now, check that the catheter runs smoothly and does not have any kinks or folds as shown here. Then close the muscles layer by layer using sutures or clips, and then close the skin. Once the wound is properly closed, stop the anesthesia and observe the animal while it wakes up.

After the animal has regained sufficient consciousness for sternal recumbent, place the rat in a clean cage. Be sure to follow all postoperative treatment procedures, including monitoring of the animal and post-surgical analgesia. Administer immunosuppression treatment two days prior to NPC injection and until seven days post transplantation and until sacrifice respectively.

This fluorescence microscopy image shows a longitudinal section of a traumatized spinal cord of a rat. The staining is DPI fluorescence SAPs labeled with QL six. Fite appear green and have aggregated in the epicenter of the lesion.

Injected DS red positive NPCs have diffused in the rostral and coddle directions indicated by the red fluorescence. The scale bar represents five millimeters. Here a scanning electron microscope image shows nanofiber scaffold formation of assembled SAPs.

The scale bar represents one micrometer. After watching this video, you should have a good understanding of the surgical procedures involved in clip compression, SCI, and how the promising approach of combined treatment with SAPs and NPCs can be studied in this model.