This article details a surgical procedure for injecting self-assembling peptides (SAPs) and neural precursor cells (NPCs) into a rat model with a cervical spinal cord injury. The method aims to enhance axonal regrowth and functional recovery by utilizing a scaffold that mimics the extracellular matrix.
Secure an anesthetized rat with a cervical spinal cord injury onto a stereotactic frame.
The lesion site consists of damaged axons and fluid-filled cavities surrounded by infiltrated immune cells, glial cells, and fibroblasts, which hinder axonal regrowth and functional recovery.
Disinfect and incise the skin above the spine. Dissect the muscles to expose the dura, the spinal cord's outer covering.
Excise the scar tissue from the dura to reveal the spinal cord lesion site.
Position a microsyringe above the lesion, containing self-assembling peptides (SAPs), and
inject them into the lesion core on one side of the midline.
Repeat the injection on the opposite side for even distribution.
Next, inject neural precursor cells (NPCs) at sites surrounding the lesion, avoiding the inflamed lesion core to enhance NPC survival.
Once injected, the SAPs self-assemble into a scaffold that mimics the extracellular matrix and bridges the cavities. The scaffold supports NPC survival and differentiation into neural cells for tissue remodeling.
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.
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