Intrathecal Delivery of Antisense Oligonucleotides in the Rat Central Nervous System

This protocol allows the direct delivery of therapeutics into the rat's central nervous system to evaluate the pharmacokinetics, pharmacodynamics, and efficacy of novel therapeutics in rat disease models. This procedure is safe, effective, and does not require expensive equipment or surgical tools. Demonstrating the procedure will be Yi Chen and Yi Luo, research scientists from my laboratory.

To prepare the special guide cannulas, use a rotary tool with a cut off wheel to cut off both ends of a 19 gauge needle to obtain an approximately one and a half to two centimeter long guide cannula. Then use the grinding wheel of the rotary tool to smooth both ends. To prepare the catheter wire assembly, cut one eight centimeter long piece of 011 inch diameter PE10 tubing to serve as the intrathecal catheter for each animal.

Use an ethanol-resistant marker pen to make a mark two centimeters from one end of each piece of tubing. For each animal, insert an 11 centimeter long stylet wire cut from polytetrafluoroethylene coated stainless steel wire into the lumen of the eight centimeter PE10 catheter. To prepare the delivery catheter assembly, cut a piece of five to 10 centimeter 023 inch diameter PE50 catheter and insert a 23 gauge tubing adapter into one end of the catheter.

Then insert a 30 gauge needle with a hub cut off into an approximately 0.5 centimeter piece of PE10 tubing and connect the PE10 tubing to the other end of the catheter. To prepare a guide cannula-needle assembly, place a guide cannula over the end of a 23 gauge needle. Before beginning the surgery, place a heating pad on the surgery table and cover the pad with a sterile drape sheet.

Place a 50mL conical centrifuge tube onto the sheet and confirm a lack of response to toe pinch in the anesthetized experimental rat. After weighing the animal, shave the back from the tail to the caudal thoracic spine and place the shaved rat onto the sterile sheet in the prone position. Position the 50mL tube under the abdomen to flex the spine in the lumbar region, and apply ophthalmic ointment to the eyes.

Next, subcutaneously inject one milligram per kilogram of sustained release Buprenorphine and clean the exposed skin with sequential povidone and alcohol scrubs. Drape the animal with a fenestrated sterile transparent sheet. For placement of the catheter and injection of the compound, identify the two natural pits between the muscles above the shaved pelvis, and with one hand holding the pits, use the other hand to gently press and feel the spine from the caudal to rostral direction to locate the first major indentation between the vertebrae.

After identifying the intervertebral space between the S1 and L6 vertebrae, move slightly rostrally to identify the intervertebral space between the L5 and L6 vertebrae. Use a scalpel to make a no more than two centimeter long incision in skin and muscle capsule at this site along the midline from the rostral to caudal direction so that the injection site will be at the center of the incision. Use dissection scissors to dissect away the connective tissue until the muscle layer can be visualized.

Make a one centimeter incision in the muscle capsule immediately lateral to the dorsal spinal process of the L6 lumbar vertebra. Position the guide cannula needle assembly near the anterior aspect of the sixth lumbar vertebra and push the assembly into the intervertebral space along the anterior aspect of the sixth vertebra so that the end of the needle penetrates the spinal canal. Use blunt forceps to locate the dorsal spinal process of the L6 lumbar vertebra then push the needle along the anterior aspect of the sixth vertebra into the invertebrata space.

Push the guide cannula in place along the needle, remove the needle, and insert the catheter wire assembly into the guide cannula. Angling the catheter at an approximately 45 degree angle to the spinal canal, force the end of the catheter approximately 0.3 centimeters into the canal. Remove the stylet wire approximately 2.5 centimeters from the intrathecal tip of the catheter and advance the catheter into the spinal canal until the two centimeter marking on the catheter is just visible below the muscle.

When the catheter is in place, remove the guide cannula to leave the catheter and stylet wire in place. Advance the catheter into the spinal canal until the two centimeter mark is at the entrance of the canal and completely withdraw the stylet wire. Cerebral spinal fluid may be visualized entering the implanted catheter.

Then connect the delivery catheter assembly to the distal end of the implanted catheter via the 30 gauge needle end. Next, load 60 microliters of sterile saline into a 100 microliter syringe and load a bolus of 30 microliters of the test compound into a second syringe. Connect the saline syringe to the tubing adapter end of the delivery catheter assembly and flush 20 microliters of sterile saline into the intrathecal space.

When all of the saline has been flushed, replace the saline syringe with the bolus loaded syringe and inject 30 microliters of the test compound into the intrathecal space over a period of 30 seconds. When all of the bolus has been delivered, replace the bolus loaded syringe with another saline loaded syringe and flush the catheter with an additional 40 microliters of saline. When the second volume of saline has been delivered, detach the delivery catheter assembly from the implanted catheter and use a pair of very hot heat-sterilized dissection forceps to clamp down on the tubing to aseptically cut and heat-seal the implanted catheter.

Use absorbable monofilament sutures to secure the heat-sealed catheter to the connective tissue and then use nonabsorbable sutures to close the skin. Then use gauze and saline to wash any blood from the skin and place the rat in a heated incubator with monitoring until full recompency. In this representative experiment, very good knockdown was obtained in all of the regions collected after single bolus antisense oligonucleotide delivery as demonstrated.

However, some degree of regional variability was measured with the spinal cord exhibiting the highest percentage of knockdown. After a successful injection of the antisense oligonucleotide, or other therapeutics, into an appropriate model, one can evaluate the pharmacokinetics, pharmacodynamics, and efficacy of the therapeutics.