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An Improved Procedure for Catheterization of the Thoracic Spinal Subarachnoid Space in the Rat

Poon, Y Y. MD*†‡; Chang, Alice Y. W. PhD; Ko, S F. MD§; Chan, Samuel H. H. PhD

doi: 10.1213/01.ANE.0000175506.83900.ce
Technology, Computing, and Simulation: Research Report

Catheterization of the subarachnoid space provides a convenient means to deliver drugs to, or collect cerebrospinal fluid from, the spinal cord in animal experiments, and has been instrumental to our understanding of spinal mechanisms that underlie anesthesia, analgesia, or cardiovascular regulation. Experience gained over the years has revealed several shortcomings of this technique. We report a procedure that encompasses the benefits of direct subarachnoid catheterization of the rat thoracic spinal cord but circumvents the known shortcomings. An intrathecal catheter was fabricated with a small silicon bead at one end of a PE-10 catheter, which was cannulated with a 4/0 suture that served as a guide. Using the L-shape hook of the suture guide as an anchorage, the catheter was advanced into the subarachnoid space until the silicon bead was lodged on a drilled hole (2 × 2 mm) over the lamina proper on the T13 vertebrae. With less surgical trauma, greater precision of placement and firmer anchorage of the catheter, less leakage of cerebrospinal fluid, and minimal mortality or morbidity, our modified procedure for catheterization of the thoracic spinal subarachnoid space in the rat compared favorably to previously reported methods.

IMPLICATIONS: Trauma or improper placement or anchorage of spinal subarachnoid catheterization introduces bias in ensuing experiments. We report a modified procedure for catheterization of the rat thoracic spinal subarachnoid space that compares favorably to previously reported methods and greatly reduces these confounding factors.

*Graduate Institute of Clinical Medicine and Science, Chang Gung University, Kaohsiung; †Center for Neuroscience, National Sun Yat-sen University, Kaohsiung; and Departments of ‡Anesthesiology and §Radiology, Chang Gung Memorial Hospital, Kaohsiung, Taiwan, Republic of China

Supported in part by Chang Gung Memorial Hospital-Kaohsiung (CMRPG8064) to YYP, and the Academic Excellence Program (89-B-FAO8-1-4) from the Ministry of Education to AYWC and SHHC, Taiwan, Republic of China.

Accepted for publication December 23, 2004.

Address correspondence and reprint requests to Samuel H. H. Chan, PhD, Center for Neuroscience, National Sun Yat-sen University, Kaohsiung 80424, Taiwan, Republic of China. Address e-mail to schan@mail.nsysu.edu.tw.

Catheterization of the subarachnoid space provides a convenient means to deliver drugs to (1), or collect cerebrospinal fluid (CSF) from (2), the spinal cord in animal experiments. This method has gained popularity since its original report by Yaksh and Rudy in 1976 (3), and has been instrumental to our understanding of spinal mechanisms that underlie anesthesia, analgesia, or cardiovascular regulation (4–6). Experiences accumulated over the years from research application of spinal subarachnoid catheterization also revealed several shortcomings of this technique. For example, catheterization has been associated with relatively frequent postoperative mortality (7), neurological impairment (8), and failure to gain weight (9). In addition, passage of a long intrathecal catheter inserted via the cisterna magnum (3) to lower spinal levels increases the risk of spinal cord trauma (10). It also causes variations of tip location (11), a crucial determinant of successful subarachnoid spread of administered solution to the intended target (12). Direct thoracic (13) or lumbar (14) subarachnoid catheterization with a shorter catheter has been developed. Although offering obvious benefits, most of the reported procedures involve extensive surgery, including laminectomy, leading to postoperative CSF leakage (15). Other disadvantages include inadvertent epidural catheterization (16) and easy displacement of the catheter from the subarachnoid space during surgery.

Most studies on spinal mechanisms concern sensory or motor function (1,4,5,7–10), and involve subarachnoid catheterization of the lumbar spinal cord. Fewer studies evaluated the spinal mechanisms that underlie cardiovascular regulation, a key component of which is the preganglionic sympathetic neurons located in the intermediolateral column of the thoracic spinal cord. By receiving a tonically active glutamatergic input from the rostral ventrolateral medulla (RVLM), these sympathetic neurons are responsible for maintenance of basal vasomotor tone in the peripheral vasculature (6,17–19). To facilitate evaluation of spinal mechanisms in cardiovascular regulation, the present study was undertaken to develop a procedure that encompasses the benefits of direct subarachnoid catheterization of the thoracic spinal cord whereas circumventing the known shortcomings. We report a method with less surgical trauma, greater precision of placement and firmer anchorage of the intrathecal catheter, less leakage of CSF, and minimal mortality or morbidity. We further validated the application of our thoracic subarachnoid catheterization in functional studies on the tonic descending influence of the RVLM on spinal mechanisms that regulate basal vasomotor tone.

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Methods

All experimental procedures conformed to the guidelines approved by our institutional animal care committee. Adult male Sprague-Dawley rats (300–350 g) purchased from the Experimental Animal Center of the National Science Council, Taiwan, Republic of China, were used. They were housed in an animal room under temperature control (24°–25°C) and 12-h light-dark cycle. Standard laboratory rat chow and tap water were available ad libitum.

Figure 1 illustrates the basic components of our intrathecal catheter. A small bead of silicon glue was placed at one end of a 10-cm-long PE-10 catheter (Clay Adams, Parsippany, NJ), 2 cm from the tip. A 12-cm-long suture (4/0, Miralene; B. Braun, Melsungen, Germany) was inserted into the lumen, with 1 cm protruding from each end of the catheter. A crucial feature is the L-shape hook, formed by bending one end of the suture. The catheter was sterilized in ethylene gas before use.

Figure 1

Figure 1

One-hundred-fifty rats were anesthetized with pentobarbital sodium (50 mg/kg intraperitoneally [IP]). A rectangular area of the skin (3 × 2 cm) above the T8 to L2 vertebrae was shaved and sterilized with povidone iodine. A midline incision was made, and paravertebral muscles attached to the left side of the T11 to L1 vertebrae were reflected from the spinous processes. Under the guidance of a surgical microscope, a hole (2 × 2 mm) was drilled manually with the pointed end of the sharp blade of a periosteal elevator over the middle portion of the left T13 lamina proper until the dura was exposed (Fig. 2a). After application of 0.2 mL of 4% lidocaine to the exposed dura, a slit was made, under higher magnification of the surgical microscope, by traversing the surface of the dura with the tip of a 22-gauge needle, resulting in leakage of clear CSF. The L-shape tip of the suture guide was inserted through the slit and advanced as tangentially as possible into the subarachnoid space (Fig. 2b). Using the hook as an anchorage, the catheter was advanced slowly over the suture guide (Fig. 2c) until the silicon bead was lodged on the drilled hole on the vertebra (Fig. 2d). The suture guide in the catheter was then withdrawn (Fig. 2e) and a small drop of tissue glue (Histoacryl; B. Braun, Tuttingen, Germany) was applied over the silicon bead (Fig. 2f). It should be mentioned that the catheter can be inserted either rostrally or caudally, depending on the spinal segments of interest in subsequent studies. After flushing with 10 μL of sterile artificial CSF (aCSF), the exterior end of the catheter was sealed by heat. The catheter was further secured on the fascia of paravertebral muscle with suture, and its sealed end was buried under the skin. The wound was then closed in layers. Sodium penicillin (10,000 IU; YF Chemical Corporation, Taipei, Taiwan) was given IM to prevent postoperative infection. Animals were returned to the animal room for postoperative recovery in individual cages.

Figure 2

Figure 2

We defined successful subarachnoid catheterization by three criteria. First, animals were observed for motor deficits, leakage of CSF at the wound, bladder dysfunction, or self-mutilation. Second, rats were assessed for normal weight gain during the interim between surgery and experiment, which was 7–14 days. Third, leakage of administered solution after intrathecal application was assessed by injection of Indian ink, given at the end of the experiment, by observing distribution over the targeted segments (T10–12) of the spinal cord.

For comparison, we also performed catheterization of the thoracic subarachnoid space in 58 rats according to the procedures of Yaksh and Rudy (3) and Wang et al. (16). In both instances, a PE-10 catheter (Clay Adams) was introduced either via the exposed atlantooccipital membrane or after exposing the dura after thoracic laminectomy at the T13 spine, respectively. The sealed end of the catheter was buried under the skin after being secured in the neighboring muscle. Animals were returned to individual cages for postoperative recovery in the animal room after receiving sodium penicillin (10,000 IU) IM.

Six implanted rats were anesthetized with pentobarbital sodium (50 mg/kg i.p.). The sealed end of the catheter was retrieved under local anesthesia through a small incision. The sealed end was cut and was inserted with a 30-gauage blunted needle with a hub. The catheter was flushed with 10 μL of sterile aCSF. Four successive intrathecal administrations of contrast medium, given 5 min apart and at 10, 20, 20, and 20 μL each, were delivered at a rate of 10 μL/min by a micro-syringe pump. Radiographs of the animals were taken in the prone position 5 min after each dosing, and the distribution of the contrast medium in the subarachnoid space was recorded.

Thirty implanted rats were anesthetized with pentobarbital sodium (50 mg/kg IP) for preparative surgery, which included cannulation of both left femoral vein and artery. The former was used to provide maintenance of anesthesia by IV infusion of propofol (AstraZenca, Milan, Italy) at a rate of 30 mg · kg−1 · h−1 (20). The arterial cannula was used to measure systemic arterial blood pressure (SAP), which was subject to on-line power spectral analysis (21). During the experiment, animals were placed on a heating pad and allowed to breathe spontaneously with room air. Three equimolar concentrations (75, 150, or 300 nmol) of a non-NMDA (N-methyl-d aspartate) receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (Sigma-Aldrich, St. Louis, MO) or an NMDA receptor antagonist, MK801 (Sigma-Aldrich), were used in 6 separate groups of 5 animals each to study their relative effects on the generation of basal vasomotor tone. The test drug was delivered at a volume of 10 μL via a micro-syringe pump at a rate of 10 μL/min. Changes in the power density of the very low-frequency (VLF) component (0–0.25 Hz) of SAP signals, which represent the basal sympathetic vasomotor outflow (22,23), were recorded for 30 min after each dose of the test drug. Sterile aCSF delivered into the subarachnoid space before the test drug served as vehicle and volume control.

All values are reported as mean ± sem. Changes in the sum total of power density for the VLF component of SAP spectrum over 30 min were compared by one-way analysis of variance to assess group means, followed by the Student-Newman-Keuls multiple-range test for post hoc assessment of individual means. P < 0.05 was considered to be statistically significant.

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Results

As summarized in Table 1, there was a mortality rate of 5% in the 150 rats studied; 4% of the rats exhibited unilateral lower limb paraplegia after surgery, but recovered within a week. Leakage of CSF, either from the wound after surgery or subsequent to intrathecal application of solution during the experiment, was observed in 4% of the rats. All surviving animals had a weight gain of ≥50 g per week. None of them manifested bladder dysfunction or self-mutilation. Based on our stipulated evaluation criteria, we have thus achieved a 95% success rate for subarachnoid catheterization of the thoracic spinal cord using this method. Our results (Table 1) also indicated that our improved procedures compared favorably against catheterization via the atlantooccipital membrane or after thoracic laminectomy.

Table 1

Table 1

We also determined the distribution of solution in the thoracic spinal cord after intrathecal administration in roentgenological experiments. As illustrated in Figure 3, which is representative of results from 6 rats, injection of 10 μL of contrast medium via the tip of our subarachnoid catheter lodged at T13 manifested a restricted distribution between upper lumbar (L1 or L2) and lower thoracic (T11 or T12) spinal segments. Increasing the volume of administered contrast medium to 30 μL (Fig. 3c) or 50 μL (Fig. 3d) extended the distribution to the middle (T5 or T6) or upper (T1 or T2) thoracic spinal cord, respectively. Given at 70 μL (Fig. 3e), the distribution of contrast medium reached the upper cervical spinal cord (C1 to C2). A volume of 10 μL was therefore considered most suitable for restricted distribution of intrathecally administered test drugs.

Figure 3

Figure 3

Figure 4 provides an illustration of the application of thoracic subarachnoid catheterization in functional studies. A tonic descending influence from the RVLM, which is responsible for the maintenance of arterial blood pressure (17), uses glutamatergic neurotransmission in the thoracic spinal cord (18,19). We found that this tonic excitatory influence is also exerted on vasomotor tone. Intrathecal application of equimolar doses (75, 150, or 300 nmol) of either a non-NMDA blocker, CNQX, or a NMDA blocker, MK801, given at a volume of 10 μL, elicited comparable suppression of an experimental index for sympathetic vasomotor outflow (23) that originates from the RVLM (22).

Figure 4

Figure 4

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Discussion

We developed a procedure that encompasses the benefits of direct subarachnoid catheterization of the thoracic spinal cord, and entails less surgical trauma, greater precision of placement and firmer anchorage of the intrathecal catheter, no leakage of CSF, and minimal mortality or morbidity. Roentgenological investigation confirmed proper distribution of solution when administered via the implanted catheter and pharmacological experiments revealed the efficacy of functional blockade of NMDA and non-NMDA receptors in the thoracic spinal cord.

Extensive laminectomy provides a larger plane for the insertion of catheter, but causes greater trauma to the animal and requires a longer recovery period. By drilling a small hole on one side of the lamina proper, our method bypasses completely the process of laminectomy. This, however, has two technical difficulties. The size of the small entry site makes it difficult to identify the dura mater, and the angle of entry into the subarachnoid space is too acute. We overcame these two difficulties by using a guide with appropriate stiffness. Suture (4/0) was selected because its elasticity does not cause spinal injury and its stiffness makes it suitable as a guide. The latter property also allowed us to construct an L-shape hook at the tip of the guide, which turned out to be of crucial importance to the success of our procedure. This hook provided an anchorage in the subarachnoid space and opened a tangential path for the catheter to advance via the guide. Inadvertent epidural catheterization, which causes underestimation of the efficacy of a drug when given intrathecally (24), is thus minimized. We were concerned that drilling a hole on the vertebrae may cause bleeding and produce heat that jeopardizes the spinal cord. We avoided bleeding by drilling on the middle portion of the lamina proper where there are no large vessels. Drilling manually with the pointed end of a periosteal elevator minimized heat accumulation.

We further implemented several measures to minimize injury induced by catheter advancement into the spinal cord, increase precision of placement and anchorage of the intrathecal catheter, and reduce leakage of CSF. First, we selected the shortest possible length of catheter to reach the target site. Shorter passage in the subarachnoid space reduces the risk of spinal injury, decreases variations of tip location in the transverse plane, and avoids excessive drug dilution at the site of action. Second, we used tissue glue to secure the catheter on the bony structure of the spine to prevent inadvertent dislodging of the catheter. Third, the silicon bead served as a marker of catheter length to ensure precision of placement of the catheter in the subarachnoid space. Tissue glue also sealed the space between the silicon bead and the bony entry to prevent postoperative CSF leakage.

In conclusion, by overcoming many of the disadvantages reported for previous methods for subarachnoid catheterization, the procedures reported in this study offer an improved means to deliver drugs to, or collect cerebrospinal fluid from, the spinal cord in our attempts to understand the spinal mechanisms that underlie anesthesia, analgesia, or cardiovascular regulation.

The authors thank Mr. J. H. Chen for preparing Figure 2.

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