Intrathecal catheterization has been widely applied in animal experiments, especially those on neuropathic pain. However, the traditional methods still has several limitations. Although some investigators have attempted to improve the traditional methods, the available methods still need to be modified.
Herein, we introduce a modified method for intrathecal catheterization in rats that represents a simple, convenient and a reliable approach for repetitive intrathecal administration of drugs. Prepare a 15 centimeter long PE10 tube. Insert a 20 centimeter long stainless steel wire with two polished ends into the PE10 tube as a support and mark the tube two centimeters from one end.
Cut the sharp tip of a 22G needle and seal the distal end with a pair of forceps. Cut an epidural catheter into one centimeter fragments. Then insert these fragments into the sharptipfree 22G needle and seal the distal end with a pair of heated straight forceps.
This apparatus was called the tube sealing cap. Prepare a 0.3 centimeter times 0.5 centimeter antiallergic band with a pair of scissors. Prepare the instruments for intrathecal catheterization, which was sterilized before surgery.
Included toothed forceps, scissors, a gavage apparatus, a scalpel handle, and number 10 blades. Sterilize the PE10 tube and guide wire with ethanol for approximately two hours. Anesthetize rats with 3%isoflurane at the flow rate of three liters per minute.
Place a rat on the operating table and the hind paw was clipped with a pair of forceps. The absence of hind pole movement in response to stimulation confirmed successful anesthesia. Adequate and analgesia was administrated by intramuscular injection for one milligram per kilogram meloxicam before intrathecal catheterization.
Removes the hair on the lumbar spine region of the back and on the area between two ears with a shaver. Place a centrifuge tube under the abdomen of the rat at the waisthip junction. Sterilize the surgical sites with povidoneiodine solution and ethanol solution three times.
Cover the rat with aseptic dressing and expose the surgical sites. And then wash with normal saline before surgery. Determine the location of the intervertebral space between L5 and L6, by locating the spinal process of L6 at the midpoint between the left and the right bilateral iliac crests.
Fix the skin with the left thumb and the index finger of the operator and then make a three to four centimeters long midline incision just above the spinous process between L4 and S1.Bluntly separate the subcutaneous tissues with a pair of scissors. Locate the intervertebral space between L5 and L6 again. Clamp and lift the L5 dorsal process with a pair of toothed forceps to expand the intervertebral space.
Then bluntly separates the muscles around the vertebral body with a pair of scissors into the top of the L6 dorsal process was completely exposed. When the L5 dorsal process is lifted with a pair of toothed forceps and the intervertebral space is expanded by another pair of forceps, clean the L5 to six intervertebral space with a cotton ball until the inverted V area is completely exposed. Puncture the spine with a 23G needle in the inverted V area, just under the top of the L6 dorsal process.
Carefully insert the PE10 tube containing stainless steel wire into a spinal canal at the puncture site being tilted 30 degree towards the tail. The insertion angle was a adjusted until the PE10 tube could be successfully inserted without any resistance. When the marked area of the PE10 tube reaches the posterior muscle, catheterization is stopped.
Slowly removes the stainless wire from the PE10 tube, then connected the PE10 tube to a syringe, through which 20 microliters of normal saline is injected. After removal of the syringe, saline may have flowed continuously out of the PE10 tube, indicating that the PE10 tube is inserted successfully into the subarachnoid space. Once the PE10 two was confirmed to be unobstructed, suture the muscles on one side of the vertebral body with a four zero suture and make a knot.
Then tie the suture around the PE10 tube and make another knot. Do not cut the suture and the suture the muscles on the other side. Tie the suture around the PE10 tube again.
Make a third knot and cut the suture. Make a 0.5-centimeter long incision at one centimeter just below the midpoint between ears. Bluntly separate the subcutaneous tissues with a pair of scissors.
And insert metal gavage tube through the incision towards the tail. Until the tip is visible in the lumbar incision. Insert the distal end of the PE10 tube into the gavage tube until the PE10 tube exits the other end of the gavage tube.
Then gently withdraws gavage. When the PE10 tube is confirmed to be unobstructed again, suture the remaining muscles around the lumbar incision with a four zero suture. Ties the suture around the PE10 tube and make another knot.
Fix the PE10 tube again. Suture the skin, avoiding damage to the PE10 tube, Then suture the neck skin with a four zero suture. Tie the suture around the PE10 tube and make a knot to fix the PE10 tube.
Once the PE10 tube is confirmed to the unobstructed again, seal the extracorporeal end of the PE10 tube with a sealing cap. The PE10 tube was dried with a piece of tissue and an antiallergic band was tied around the PE10 tube several times to prevent retraction of the PE10 tube during rat movement. After surgery, returned the rat into its cage and closely monitor it during recovery from anesthesia until it regains consciousness.
After the rat is fully awake, remove the sealing cap and inject 20 microliters of 2%lidocaine into the PE10 tube at the rate of 0.2 microliters per second via Hamilton syringe, followed by the injection of 10 microliters of normal saline. Seal PE10 tube with a sealing cap. Place a rat on the table and observe carefully.
Paralysis of the lower limbs after intrathecal injection of lidocaine, indicate successful catheterization. Hind limb paralysis usually lasts approximately 30 minutes. Closely monitor the rat during the recovery period until complete recovery of limb function.
Complete paraplegia of both lower limbs of the rats after intrathecal injunction of lidocaine indicated a successful catheterization. Comparison of the rate of successful catheterization between our modified methods and the previously reported method. The rate of successful intrathecal catheterization for our modified method was 95%which was greater than the 88%rate achieved with the method reported by Hou et al.
Comparison of the long-indwelling catheter rate between our modified method and the previously reported method. The long-indwelling catheter rate at days two, five and seven after intrathecal catheterization was 94%81%and 65%respectively, in a study by Storkson et al. The long-indwelling catheter rate at two, five and seven days after intrathecal catheterization was 95%90%and 85%respectively for our method.
In conclusion, the modified method for intrathecal catheterization may serve as a useful tool for the repetitive intrathecal and administration of drugs and represents a simple, convenient, and a reliable way to shorten the duration of the experiments.
