Preparation for grafting of a peripheral nerve into a spinal cord injury site begins with pre degeneration of the PNG seven to 10 days prior to grafting. The tibial nerve branch from a donor rat is transected. A cervical level five or C five hemi section is made by aspiration.
The PNG is removed from the donor rat and opposed to the rostral wall of the lesion cavity and secured by suturing aurum to the dura mater. Three weeks later, a dorsal quadrant lesion is made at C seven gel foam saturated with C-H-A-B-C is placed into the cavity for 15 minutes to digest proteoglycans of the glial scar. Two days later, C-H-A-B-C is micro injected adjacent to the lesion site and the distal end of the PNG is opposed to the dorsal quadrant lesion.
Hello, I'm John Hula. I'm the director of the Spinal Cord Injury Research Center at Drexel University College of Medicine. I'm Dr.Veronica Tom from the hula lab and I'm Martha mean.
From the hula lab. Today we will demonstrate a procedure of transplantation of a segment of peripheral nerve into the injured spinal cord of an adult rat. We use this procedure in our laboratory to study axon regeneration and recovery of function after spinal cord injury.
Okay, let's get started. To prepare for microscopic surgery, sanitize the surgical station and autoclave all surgical instruments. Prepare the thermal barrier and the small less than two millimeters cubed pieces of gel foam for micro hemostasis.
Set out cotton swabs, gauze pads, and surgical instruments. Place the anesthetized rat on the preparation table after confirming with a toe pinch that the animal is completely anesthetized. Shave a wide area of fur from the intended surgical field wash surgery site following the aseptic scrub technique described in the accompanying text protocol.
Next place the rat on the thermal barrier cover its nose and mouth with the inhalation cone and reduce the isof fluorine to 3%and flow rate to 0.2 liters per minute. Administer the appropriate dose of antibiotic ampicillin by subcutaneous injection and analgesic buprenorphine by intramuscular injection. The peripheral nerve or PN is transected seven to 10 days prior to its removal to promote degeneration, which provides an environment more suitable for axonal growth than does a fresh nerve preparation.
To begin transection of the pn, lay the animal on its side. Switch to sterile gloves and place a sterile surgical drape around the surgery site. Make a straight line incision from the head of the femur to the knee keeping parallel and approximately three millimeters from the femur cut through the superficial biceps fems muscles along the same line as the skin incision to expose the underlying vastus lateralis muscle.
Once the vastus lateralis muscle is exposed, use scissors to make a small cut in it near the knee and extend this up toward the head of the femur. Gently separate with retractors the vastus laterals until the conjoined tibial and perineal nerve branches of the satic nerve are visualized running parallel to the femur. Next, trace these nerve branches toward the head of the femur until the main trunk of the sciatic nerve is identified.
Just below the bifurcation of the satic nerve, use fine forceps to carefully separate the connective tissue or epineurium surrounding the tibial nerve branch. This is the larger branch. Once the tibial nerve branch is isolated, slip a six oh silk ligature around it and tighten.
Use micro scissors to transect the nerve raw stroll to the ligature. Pass the suture line through the vastus lateralis muscle and tie off in a knot. This will provide a line for easy location of the cut end of the tibial nerve.
When the nerve is harvested. Later, close the vast lateralis muscle with six oh silk sutures. After that, close the biceps PMOs muscle with six oh silk sutures.
Finally, close the skin with Michelle wound clips. Turn off the flow of isof fluorine and remove the inhalation nose cone. Place the animal in a holding cage on a thermal barrier.
Return the animal to its home cage. Once it is alert and active, seven to 10 days later, the PN segment is ready to be harvested. For transplantation to begin place the anesthetized animal on the thermal barrier.
After confirming that the animal is fully anesthetized, remove the wound clips and separate the skin incision. Shaving the area first may not be necessary as the skin should easily pull apart avoiding contamination. By eliminating the need to cut through fur, cut through the suture of superficial muscle to expose the vastus laterals and cut through the suture in this muscle.
Trace the suture line to the cut end of the tibial nerve and remove the suture from the nerve. Use fine forceps to dissect the epineurium from a 15 millimeter length of the tibial nerve And cut through this distal end. Remove the nerve segment and immerse it in sterile hank's balanced salt solution.
We are now ready for the next step, which is to induce a spinal cord injury for transplantation of the PN transplantation may be autologous or heterologous. Place the anesthetized rat on the thermal barrier and administer antibiotics and analgesics as described in the previous section to induce a HEMI section spinal cord injury. First, locate the occipital notch and the prominent T two dorsal vertebral process.
Make an incision the full length between these external landmarks. Cut through the midline of the superficial trapezius muscle and separate the underlying cervical ardis muscles. Insert a spreader to keep the muscles apart.
The deep supraspinal muscles should be cut from the C four C five and C six dorsal vertebral processes. Using R jurors perform a partial laminectomy of C five to expose the entire right side and the medial portion of the left side of the spinal cord. Use the prominent dorsal vein as a landmark for the spinal cord midline.
Next, use a micro scalpel blade to pierce the dura mater and extend the incision over the C five spinal cord. Make a small incision in the underlying subarachnoid and P mater with a pulled glass micro cannula. Begin to aspirate the dorsal right side of the spinal cord using gentle pressure and micro forceps to separate the tissue and facilitate its removal.
Continue through intermediate and ventral portions of the spinal cord to create a two millimeter long cavity that is complete from dorsal to ventral and from midline to the lateral meninges. Place saline soaked gel foam into the HEMI section lesion cavity. To achieve hemostasis, remove the previously harvested segment of peripheral nerve from the Hank solution.
Gently feel the epi from the proximal three millimeters of the nerve segment and oppose this end to the rostral wall of the HEMI section lesion cavity. Secure the segment with 10 oh sutures through the epi uum to the dura mater. Then suture the dura closed over the implanted end of the graft sandwich the graft between two pieces of cel elastic membrane.
Place the graft alongside the C six C seven and T one vertebral processes. Do not oppose the distal nerve end to spinal cord or muscle tissue. Close muscles in layers with four oh sutures and close the skin incision with wound clips.
Finally, place the animal in a holding cage on a thermal barrier and return it to its home cage. Once it is alert and active, regenerated axons that reach the distal end of the PN graft often fail to extend back into the spinal cord. So we use micro injections of chondroitin ACE to degrade the scar tissue surrounding the area of injury.
This procedure is carried out 21 days after the PN transplantation to begin, confirm that the animal is completely anesthetized. Then remove the wound clips and administer antibiotics and analgesics as previously shown. Cut through the superficial sutures to expose the elastic membrane surrounding the nerve graft using fine forceps and spring scissors.
Carefully free the length of nerve from the cel elastic membranes. Reflect the nerve from the surgical field. Perform a partial laminectomy on the right side of the C seven vertebral process.
Pierce the meninges with a micro scalpel blade and by aspiration, prepare a one millimeter cubed dorsal quadrant lesion cavity in the C seven spinal cord. Place saline soaked gel foam into the lesion cavity to achieve hemostasis. After that, replace the gel foam with one soaked with chondroitin nace.
A BC.Treat the lesion site for 15 minutes, providing fresh chondroitin a soaked gel foam every five minutes. Cover the external length of the nerve graft with scholastic membrane. Close the superficial muscles with four oh sutures and the skin incision with wound clips.
Place the animal in a holding cage on a thermal barrier and return it to its home cage once it is alert and active. Two days later, 23 days after PN transplantation, surgery is performed again on the animal to micro inject chondroitin NA into the spinal cord. As before the wound clips are removed from the anesthetized animal and antibiotics and analgesics are administered.
Cut through the superficial sutures to expose the elastic membrane surrounding the nerve graft. Expose the C seven graft site without disturbing the graft. Then use a Hamilton syringe with a pulled glass cannula to micro inject one microliter of chondroitin ace A BC into the right side of the spinal cord, approximately one millimeter distal to the apposition of graft to spinal cord.
Gently peel the EUR from the distal two millimeters of the nerve segment and oppose this end to the C seven lesion cavity. Secure the segment with 10 oh sutures through the epi nearium to the dura mater. Close superficial muscles with four oh sutures and the skin incision with wound clips.
Place the animal in a holding cage on a thermal barrier and return it to its home cage once it is alert and active. When this procedure is optimized, the peripheral nerve segment will closely integrate with the host spinal cord to visualize spinal axons that regenerate into the graft. We diffusion filled the graft with biotinylated dextrin amine or BDA ascending and descending.
Spinal axons will generally enter the graft grow in a relatively straight line parallel to the length of the graft and extend to the distal end of the graft at a rate of approximately one millimeter per day. Peripheral nerve axons will penetrate the adjacent spinal cord. If chondroitin NACE has been used to remove inhibitory proteoglycans from the surrounding scar tissue upon reaching the distal end, very few peripheral nerve axons extend into host tissue after treatment when treated with vehicle.
However, if the distal interface was treated with chondroitin, a significantly more axons emerge from the graft to reinnovate the spinal cord. As shown here in this one representation section out of 15 to 20 sections per animal where axonal outgrowth is observed, this bar graph demonstrates a significant increase in axonal outgrowth after conjoin nace A BC treatment. Immunochemical detection of synaptic markers on spinal cord neurons in response to electrical stimulation of the axons within the graft can be used to assess functional connectivity between transplanted graft and host spinal cord.
This spinal cord region containing the distal apposition site was immuno cyto chemically stained for both biotinylated dextrin amine in green and the synaptic marker synapsin in red arrowheads indicate areas in which there was colocalization of markers indicating that some fibers that regenerated out of the graft formed functional synaptic contacts. Functional connectivity between graft and spinal cord neurons was also demonstrated by assay for CFOs expression. Following synaptic stimulation of host neurons beyond the distal interface, this trans synaptic activation marker can be identified by many CFOs positive neurons in tissue ipsilateral to the graft site, and in higher magnification, very few CFOs positive neurons can be observed in tissue contralateral to the graft.
We have just shown you how to prepare a spinal cord injury site in preparation for transplantation of a peripheral nerve graft to promote regeneration after spinal cord injury. When doing this procedure, it's important to remember to have sufficient nerve length prior to grafting and to have close apposition of the graft to the spinal cord injury site. So that's it.
Thanks for watching and good luck with your experiments.
