The overall goal of this procedure is to safely deliver and accurately target biologic therapeutics to specific intraparenchymal sites of interest within the spinal cord. This is accomplished by first preparing the animal for surgery. Next, the laminectomy is carried out and the spinal derrick is assembled.
Then the dura is opened and the intraspinal injections are performed. Finally, the dura musculature, fascia, and skin are closed. Ultimately, results can be obtained that show safety and targeting outcomes through the collection of behavioral and targeting data.
The technique portrayed in the present manuscript is that of the use of a stabilizing platform, as well as a flexible cannula for the delivery of a biological payload into the spinal cord. The advantages of this approach are that it allows for accuracy in delivery, replacing standard stereotactic techniques, which are hard to apply in the spinal cord due to its mobility. In addition to accuracy, the technique provides a margin of safety, such that movement of the patient.
In this case, a pig does not result in damage to the spinal cord because of the immobilizing platform and the movement of the spinal cord within the canal that occurs during respiration and during the heartbeat doesn't further damage the spinal cord due to the flexibility of the cannula. After fasting the mini pig for 12 hours and sedating it using an intramuscular injection of ketamine, ace, promazine, and atropine, intubate the animal and maintain it on oxygen and one to 3%isof fluorine. Monitor the depth of anesthesia as outlined in the written protocol.
Clip the hair on the back and head of the animal. Transport the sedated animal to the operating room and place it in a prone position on a custom frame to mimic positioning on a Jackson spinal surgical table. The frame utilizes adjustable slings that are placed under the chest and pelvis of the animal, allowing the abdomen to hang free, and therefore minimizing pressure on the abdomen and chest and consequent epidural venous bleeding.
The frame also provides external immobilization of the spine. For the procedure, place the animal on a heated recirculating pad to maintain body temperature. Next, insert a marginal ear vein catheter for fluid administration and drug delivery during surgery.
Then prepare the surgical field with alcohol and chlorhexidine or Betadine solution and place surgical drapes on the surgical field. Make a 10 to 15 centimeter skin incision and use monopolar electrocautery to bilaterally dissect the paraspinal musculature lare. Next, begin a dorsal multi-level laminectomy using Ron jewels and a surgical drill to remove the spinous processes and perform a laminotomy of the vertebrae overlying the C3 to C five or L two to L four segments.
The spinal Derrick is a device designed to deliver gene and cell therapies into the spinal cord to secure the device to the patient, make one centimeter skin incisions above and below the primary incision. Place percutaneous posts through the incisions and mount them to the lamina to expose the required area of spinal cord and complete the laminectomy. Insert the integrated retractor blades and attach them to the derrick.
Retract the tissue while pulling upward on the spinous processes. Cut away the interspinous ligaments and ligamentum flam and complete the laminectomy. The cannula is comprised of a 30 gauge needle attached to 30 gauge astic tubing with two metal sheaths, one outer mobile sheath and one inner immobile sheath.
Using a Hamilton lure lock attached to a micro injector pump fit the distal end and with a 24 gauge outer cannula that sits on the proximal end of the injection needle flange and sheath, the proximal end to expose the spinal cord. First, administer a bolus of methyl prednisone. Then carefully place attack to lift the dura off of the cord.
Use a Woodson dental tool and an 11 blade to make a 2.5 centimeter incision through the dura. Then use four aught nulon suture to reflect the dura away from the peel layer and secure it to the deep paraspinal musculature. Place surgical patties around the cord.
Then attach the platform rail system by top first, loading the gondola onto the two bars and mounting the Z drive on the universal joint. Next, place the loaded cannula on the micro drive and use the universal joint on the micro injection platform to adjust the coronal and sagittal angles, which will ensure a trajectory orthogonal to the surface of the spinal injections, follows the placement of the cannula. Then position the needle medial to the dorsal root entry zone or dres, and pull up the rigid metal outer sleeve, leaving the flexible tubing exposed.
Using a pre calibrated micro injector pump inject into the target site. Then leave the needle in place for an additional one minute to prevent cell reflux up the cannula injection tracted. After removing the needle, relocate the stereotaxic apparatus to the next target site, two to four millimeters away along the rostral coddle axis to avoid visible blood vessels on the dorsal surface of the spinal cord.
After gently removing the spinal Derrick used four aught neural on to close the dura in a watertight fashion fashion. Once the deep muscular layer and fascia layers are closed with Vicryl suture, use three aught nylon suture to close the skin following anesthesia recovery extubate and monitor the animal for two hours. For postoperative analgesia, staple a transdermal fentanyl patch to the back of the animal for three days.
Additionally, administer buprenorphine for up to three days postoperatively if necessary. Then transfer the animal to an individual cage and monitor it at least once daily. For food consumption, defecation and micronutrition behavioral data is collected to assess neurological morbidity.
As previously described, the safety of the procedure is determined by the ability of an animal to return to preoperative baseline. Transient neurological deficits should mostly resolve between postoperative days one and seven with some variations depending on breeds and procedure. Permanent morbidity is defined by lasting neurological deficits, which do not resolve by the time the animals reach iacuc default endpoint Following this procedure.
Other methods such as immunochemistry can be used to ascertain important data such as the viability of the cells, migration of the cells, and the accuracy of the injection.
