This article describes a method for intrathecal delivery of test compounds in anesthetized rats. The procedure involves catheter placement in the lumbar spinal canal, allowing for the administration of therapeutic agents directly into the cerebrospinal fluid.
Start with an anesthetized rat with an implanted guide cannula in the lumbar spinal canal.
Insert a catheter-wire assembly through the guide cannula at an angle into the intrathecal space, where cerebrospinal fluid or CSF circulates.
Remove the guide cannula and slowly advance the catheter deeper.
Then, remove the wire entirely and connect the catheter to a delivery system.
Inject sterile saline into the spinal canal, ensuring clear passage.
Slowly administer a test compound solution into the CSF, followed by a flush with sterile saline.
Detach the delivery system, cut the catheter, and heat-seal its end.
Suture the heat-sealed catheter to the connective tissue and then close the skin over the catheter. Clean the site and allow the rat to recover.
The test compound spreads through the CSF to the spinal cord and brain, which comprise the central nervous system or CNS.
Within the CNS, it enters the neurons and offers therapeutic potential.
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 no more than 2-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 1-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 processes of the L6 lumbar vertebra. Then push the needle along the anterior aspect of the sixth vertebra into the intervertebral 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 2 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 2 centimeter mark is at the entrance of the canal, and completely withdraw the stylet wire. Cerebrospinal 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, bead 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 non-absorbable 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 recumbency.
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