The overall goal of this procedure is to target multis, segmental intraspinal transplantation of neural precursor cells to the cervical spinal cord, ventral gray matter of both rats and mice. This is accomplished by first preparing the neural precursor cell type of interest for transplantation. Then a laminectomy is performed on the spinal cord at each point of intended cell transplantation.
Next cells are properly injected at the appropriate locations and depths to accurately target cell delivery to the ventral horn of the cervical spinal cord. The final step is to continue to administer immune suppression to the animal until sacrifice to achieve survival of transplanted cells in the spinal cord. Ultimately, results can be obtained for tracking the survival, migration, and differentiation of transplanted neural precursor cells in the adult cervical spinal cord at various time points following transplantation through histological methods such as immunohistochemistry and subsequent microscopy analysis.
Visual demonstration in this method is critical as many of the intricate spinal cord surgical and cell transplantation steps are difficult to learn because they require great care and specific techniques and they're made easier. If these recommended procedures are employed Three days prior to transplantation, immunosuppress the animal by administering daily injections of 10 milligram per kilogram of body weight, cyclosporine A via subcutaneous injection. If rodent derived cells are to be transplanted, or one milligram per kilogram of body weight of both FK 5 0 6 and rapamycin via intraperitoneal injection.
If human derived cells are to be transplanted, also shapes sturdy paperclips to form retractors. Break off part of the paperclip to make a hook and bend the other end of the paperclip into a small loop. Then tie string through the loop of each retractor, autoclave, the retractors, and all instruments to be used in the surgery on the day of the surgery.
Prepare aliquots of glial progenitor cells or other cells to be transplanted according to the procedures outlined in the written protocol. Once the cells are harvested, distribute the cell suspensions between multiple 1.5 milliliter micro centrifuge tubes, and keep on wet ice. Then after administering an appropriate method of anesthesia to the animal, confirm that a suitable anesthetic blame for surgery has been achieved by performing a toe pinch.
Using an electric razor to shave the animal's back, hair from an area slightly rostral to the ears to the middle of the animal's back shave well and remove the cut hair from the skin to prevent hair getting into the incision. Next, soak a piece of gauze with povidone iodine antiseptic solution and apply to the skin over the shaved area. Scrub at least two times with the antiseptic solution and two times with alcohol or diluted disinfectant in a circular manner.
Starting at the incision site going outward toward the hairline. Place a sterile rolled gauze pad on a clean surgery board and tape the pad to the board so that it does not move. Place the animal on top of the rolled gauze in a prune position.
Stretch out both arms of the animal on the pad and then tape the arms to the pad and surgery board to prop up the cervical spinal cord. This will make it easier to work throughout the surgery since the cervical cord is found deep below the surface of the skin. A sterile surgical drape must be used during this procedure, though this is not shown in the video for demonstration purposes.
First, while looking through a surgical microscope set at the lowest magnification, use a sharp sterile scalpel to make a midline incision from the base of the skull to the scapula. Then make an incision through the three muscle layers over the spinal column. Use enough pressure to cut all of the muscle layers with the minimum number of cuts, but do not press too firmly as this may cause damage in deeper tissues with two cotton tipped applicators.
Use a twisting motion to separate the overlying muscle from the paraspinal muscle. Do not cut or tear the muscle as this will cause hemorrhage. Place four of the homemade retractors into position to retract the muscle.
Then pull each of the four retractors to all four corners and tape the strings to the board to secure the retractor. The final exposure should be square or rectangle shaped. Use rat toothed forceps and medium-sized spring scissors to remove the paraspinal muscle from the dorsal surface of the vertebrae.
Take the cuts very close to the bone to better expose the bone surface. Ensure that the cuts are parallel to the surface of the vertebrae, but do not cut down into the cord. Switch the medium-sized spring scissors for a raw share.
Then secure the entire spinal column by grasping the muscle over the C two process with the rat tooth forceps and use the raw she to pull away the muscle at level C four, C five and C six. Then begin a laminectomy at C five. Again, secure the area by holding C two with rat toothed forceps.
Grab the entire lamina with the raw she and position the raw share so that the tool is completely perpendicular to the axis of the spinal column. Slowly and gently crush the lamina without pushing down into the spinal cord. Then gently pull the broken piece of bone upwards.
Next, position the raw ger perpendicular to the spinal column. Then insert the raw ger gently under the bone with a 30 to 60 degree angle relative to the cord surface. Make small cuts to extend the laminectomy to all of the C four to C six lamini.
Do not extend the laminectomy too far laterally as this will cause hemorrhage. Increase the microscope magnification to around 15 x and focus on the cord. Use straight sharp.
Number five forceps to clean off the connective tissue on top of the dura. Then use either mini springing scissors or a micro knife to incise the dura parallel to the axis of the spinal column. As a point just medial to the entry zone of the dorsal rootlets, ensure that the dura is pierced at the site for the injection.
After piercing, the dura cerebral spinal fluid will pour out dry the surface of the cord and the entire exposure with gauze or a cotton tipped applicator. Use forceps to grab and slightly lift the dura away from the spinal cord without pinching or injuring the spinal cord. Extend the incision slightly in the rostral cordal axis using a 10 microliter Hamilton gast type removable needle syringe.
Fitted with a sharp 33 gauge 45 degree beveled metal Hamilton needle. Slowly load the required volume of cell suspension for one injection site. Lower the tip of the needle toward the spinal cord dorsal surface while monitoring its progress through the microscope.
Line up the syringe with the axis of the spinal column to properly target the desired anatomical region, aiming the needle as close to 90 degrees as possible and just medial to the entry zone of the dorsal rootlets. Gently touch the surface of the spinal cord with the tip of the needle, slightly depressing the spinal cord with the needle tip. Then retract the needle until the spinal cord returns to the normal position.
Record this position as Z equals 0.0 using the ruler on the micro manipulator. Next, while visually monitoring progress through the microscope, slightly lower the needle into the spinal cord. Lower the needle to adapt the 1.5 millimeters to target the ventral horn in an adult rat.
Leave the needle in place for two to five minutes before beginning the injection. Ensure that the surgical setup remains undisturbed during this time. Next, inject two microliters of cells over two to five minutes at a constant rate using the pump controller after the injection, leave the needle in place for two to five minutes.
Then carefully withdraw the needle from the spinal cord. Repeat the injection process for other target sites. Once all target sites have been injected, return the microscope to the lowest magnification and suture.
Closed all three of the overlying muscle layers at one time with four zero suture suture muscles at three locations in the rostral cordal axis. Staple the skin closed with nine millimeter wound clips, ensuring that the staples are spaced approximately 0.5 centimeters apart. Tighten the staples with needle holders to prevent the animal from pulling the staples off prior to full wound healing.
Apply povidone IRD antiseptic solution to the incision wound area with soaked gauze and allow the animal to recover from anesthesia on a circulating warm water pad. Continue cyclosporine A or FK 5 0 6 rapamycin injections daily until sacrifice 50, 000 mouse arrived. Glio progenitor cells were transplanted into the ventral horn of spinal cord level C four in an SOD one G 93 a rat.
This image shows that M two positive mouse arrived, transplanted cells survived at one month post transplantation, and that the cells localized to the ventral gray matter, but the injection site medially missed the lateral ventral horn as denoted by the dotted line. As seen here, injection of much higher numbers of cells into this region results in damage at the injection site and along the needle track. Once mastered, this technique can be done in approximately 45 to 60 minutes for six injection sites if it is performed properly.
