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Antinociceptive potency of intrathecal morphine in the rat tail flick test: a comparative study using acute lumbar catheter in rats with or without a chronic atlanto-occipital catheter
Journal of Neuroscience Methods 129 (2003) 33 /39 www.elsevier.com/locate/jneumeth
Antinociceptive potency of intrathecal morphine in the rat tail flick test: a comparative study using acute lumbar catheter in rats with or without a chronic atlanto-occipital catheter Wiliam A. Prado * Department of Pharmacology, Faculty of Medicine of Ribeira˜o Preto, University of Sa˜o Paulo, Av. Bandeirantes 3900, 14049-900, Ribeira˜o Preto, SP, Brazil Received 26 February 2003; received in revised form 17 June 2003; accepted 17 June 2003
Abstract Chronic spinal catheterization via an atlanto-occipital puncture (CAO) has been widely used to study the effects of drugs on spinal nociceptive mechanisms, but this method is associated with spinal cord damage that may change the efficacy of spinally injected analgesics. Using a slight modification of the method of Storkson et al. (J. Neurosci. Methods 65 (1996) 167), the rat spinal cord was acutely catheterized via a lumbar puncture (AL) and the potency of morphine-induced antinociception in the tail flick test was comparatively studied in animals with or without a CAO catheter. The opiate potency via an AL catheter (AD50; 95% confidence limits) was significantly more intense in rats without (0.29 mg; 0.19 /0.47) than in rats with a CAO catheter (1.1 mg; 0.87 / 1.47) and stronger than via a CAO catheter (8.2 mg; 4.6 /14.4). The potency of morphine via a CAO catheter was significantly improved in indomethacin-pretreated rats (1 mg/kg, i.p., twice a day for 5 days), thus indicating that inflammatory changes produced by a CAO catheter are at least in part the reason for the lower efficacy of the opiate. The use of an AL catheter minimizes such spinal changes and permits acute experimental protocols in which more than one spinal injection is necessary. # 2003 Elsevier B.V. All rights reserved. Keywords: Catheterization; Spinal cord; Acute lumbar catheter; Chronic atlanto-occipital catheter; Tail-flick test; Morphine
1. Introduction Chronic intrathecal catheters have been extensively used in rats in behavioral studies on the effects of drugs that interfere with spinal nociceptive mechanisms. The most frequently used method for such purpose consists of catheterization of the subarachnoid space with a polyethylene tubing which is introduced through a puncture in the atlanto-occipital membrane and then advanced along the spinal cord until its tip reaches the lumbar enlargement (Yaksh and Rudy, 1976; LoPachin et al., 1981). Although representing a valuable technique for spinal drug administration, this method has several disadvantages such as spinal compression by the catheter (Li et al., 1985; Gordh et al., 1986; Yaksh et al., 1986;
* Tel.: /55-16-602-3038; fax: /55-16-633-2301. E-mail address: [email protected] (W.A. Prado). 0165-0270/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0165-0270(03)00197-3
Grip et al., 1992), moderate inflammation (Li et al., 1985; Gordh et al., 1986; Yaksh et al., 1986; Svensson et al., 1992) including spinal migration of mononuclear inflammatory cells (Almeida et al., 2000), microglial activation and astrogliosis in the compressed tissue (Jones and Tuszynski, 2001), and demyelination (Yaksh et al., 1986) or neuronal vacuolation (Li et al., 1985). Both tubing size and composition also seem to be important factors affecting the histologic reaction to an indwelling intrathecal catheter (Sakura et al., 1996). The insertion of a catheter between thoracic (Dib, 1984) or lumbar (Martin et al., 1984) vertebrae has been proposed as an alternative to the Yaksh and Rudy’s method, but this does not avoid spinal compression by the catheter and may also induce tissue reactivity. An alternative to injecting drugs spinally using acute needle puncture has been offered (Mestre et al., 1994), but a difficulty remains when more than one spinal injection is required by the experimental protocol.
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Almeida et al. (2000) found the hyperalgesic response to intraplantar carrageenan or PGE2 to be more intense in rats with chronic intrathecal catheters than in naive or sham catheterized rats. They also showed that intraspinal administration of indomethacin to rats with, but not to rats without a chronic catheter caused antinociception in the formalin test. Therefore, the spinal changes produced by the presence of a chronic intrathecal catheter may modify the behavioral response of rodents to the spinal administration of drugs. The present study examines the effect of morphine injected via a chronic atlanto-occipital (CAO) catheter implanted in rats according to the method of Yaksh and Rudy (1976) on the latency for the tail flick reflex to noxious heating of the skin. This effect was then compared with the effects of morphine injected via an acute lumbar (AL) catheter in rats with or without a CAO catheter.
2. Methods 2.1. Subjects and surgery Males Wistar rats (250 /300 g) were used. Animals were housed two to a cage with water and food ad libitum, and kept in temperature controlled rooms (239/ 2 8C) on a 12:12-h light /dark cycle, with the dark cycle beginning at 07:00 h. All tests were held in the morning and conducted according to the IASP guidelines on the use of animals in pain research. Each rat was used only once. Rats were allocated in i.c.v. or i.t. groups. Surgical operations were all performed under anesthesia with tribromoethanol (250 mg/kg, i.p.). For the i.c.v. group, a 10-mm stainless steel guide cannula was implanted as previously described (Miyamoto et al., 1989), using the coordinates (in mm) AP 1.4 from the interaural line, L 1.6 from the sagittal suture and V 3.5 from the skull surface. Soon after the procedures for implant of the guide cannula, some rats in the i.c.v. group had a chronic catheter implanted as described below. For the i.t. group, catheterization of the spinal subarachnoid space was performed. The catheter consisted of a 12 mm length of polyethylene tubing (PE tubing, o.d. /0.4 mm, dead space /10 ml) previously coated with silicone, dried, sterilized by immersion in 70% ethanol, and fully flushed with sterile saline prior to insertion. The CAO catheter was inserted through an incision of the atlanto-occipital membrane up to the lumbar enlargement of the spinal cord (7.5 cm), as described elsewhere (Yaksh and Rudy, 1976). The AL catheter was acutely implanted using a slight modification of a method developed by Storkson et al. (1996). Briefly, a 20-gauge Weiss needle was introduced through the skin into the L5 /L6 intervertebral space. The correct positioning of the needle was assured by a
typical flick of the tail or hind paw. The catheter was then introduced through the needle to protrude 1.5 cm into the subarachnoid space in a cranial direction. The needle was then carefully removed and the tubing anchored to the back skin with a cotton thread suture. Rats with the CAO catheter were used 7 days after surgery. In a separated group of rats with AL catheter we found no significant change in the tail flick latency before or 2 h after surgery (not shown in Section 3). Injections via the AL catheter were then performed 4 h after the catheter implantation to avoid any residual analgesia produced by the anesthetic procedure. In each case, only rats showing no sign of motor impairment were considered for further experimentation. 2.2. Tail-flick test The tail-flick test was conducted as described elsewhere (Roberts and Rees, 1986; Prado et al., 2003). Each animal was placed in a ventilated tube with the tail laid across a wire coil, which was at room temperature (239/2 8C). The coil temperature was then raised by the passage of electric current and the latency for the tail withdrawal reflex was measured. The heating was applied to a portion of the ventral surface of the tail between 4 and 6 cm from the tip. This method is better than the tail immersion in hot water because it minimizes the risk of skin burning when several measurements are done in the same animal (Le Bars et al., 2001). Each trial was terminated after 6 s to minimize the probability of skin damage. Tail flick latency was measured at 5-min intervals until a stable baseline was obtained over three consecutive trials. The latency was measured again within 30 s after drug administration and then at 5-min intervals for up to 40 min. Only rats showing stable baseline latency after up to six trials were used in each experiment. 2.3. Injection procedures Drug or saline was injected intrathecally in a volume of 10 ml over a period of 60 s, followed by 10 ml of sterile saline at the same rate to flush the catheter. Intrathecal injections were monitored by observing the movement of an air bubble along a length of calibrated PE-10 tubing between the intrathecal catheter and the injection syringe. Drug or saline was injected via the CAO catheter (experiment 1A) or via the AL catheter in rats with (experiment 1B) or without (experiment 1C) a CAO catheter. I.c.v. injection of drug or saline was performed using a glass needle (70 /90 mm. o.d.) protected by a system of telescoping steel tubes as described elsewhere (Azami et al., 1980). The assembly was inserted into the guide cannula immediately before the injection and the needle advanced to protrude 0.5 mm beyond the guide cannula
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tip. The volume of injection was 0.5 ml delivered at a constant rate over a period of 3 min, and the needle was removed 20 s after the completion of this procedure. I.c.v. injections were made in rats with (experiment 2A) or without (experiment 2B) a chronic A /O catheter. Experiments 1A and 1C were repeated in groups of rats treated chronically with indomethacin (experiments 3A and 3B, respectively). In these cases, the tail flick latency was measured before the surgical procedures and indomethacin was injected intraperitoneally (1 mg/kg, twice a day) for 5 days. The experiment was conducted 2 days after the last i.p. injection to avoid any residual analgesic effect of indomethacin. 2.4. Examination of catheter position and i.c.v. injection sites At the end of the experiment the correct position of the catheter was indicated by motor paralysis of the hind part of the animal occurring within 15 min after the intrathecal administration of 2% lidocaine (10 ml) followed by saline (10 ml). In order to confirm the correct catheter positioning, 1% methylene blue (10 ml) was injected intrathecally. The rat was then deeply anesthetized with sodium pentobarbital and perfused through the heart with 4% paraformaldehyde in 0.1 M phosphate buffered saline. The spinal cord was cut through the T10 /T11 (rats with the CAO catheter, only) or L4 /L5 (rats with the AL catheter, only) intervertebral disk to look for the catheter tip under a dissecting microscope (4 / magnification). The spinal cords from rats with both CAO and AL catheters were examined at both the T10 /T11 and L4 /L5 planes. Rats showing the catheter tip positioned at sites other than the dorsal spinal cord or dye staining of paravertebral musculature were not considered for data analysis. For examination of i.c.v. injection sites, 1% methylene blue (0.5 ml) was injected through the guide cannula. The rat was anesthetized and perfused through the heart as described above. The brain was then cut with a sharp knife along the coronal plane and the distribution of dye examined under a dissecting microscope (4 / magnification). Only rats showing dye distributed within the ventricular spaces were considered for data analysis. 2.5. Drugs Morphine sulfate and indomethacin were purchased from Sigma-Aldrich Co. (St. Louis, USA), and diluted in sterile saline and 0.1% methyl-cellulose, respectively. 2.6. Statistics The results are reported as mean9/S.E.M. Comparisons between control (saline) and test groups were made by multivariate analysis of variance (MANOVA)
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with repeated measures to compare the groups over all times. The factors analyzed were treatments, time and treatment /time interaction. In the case of treatment / time interaction, one-way analysis of variance followed by the Duncan test was performed for each time. The analysis was performed using the statistical software package SPSS/PC/, version 3.0 and the level of significance was set at P B/0.05. The dose of morphine producing an antinociceptive effect in 50% of the animals in an experimental group at the time of peak effect (AD50), was estimated by the method of Litchfield and Wilcoxon (1949). For calculation, antinociception was arbitrarily considered to occur whenever the latency for the tail flick reflex was equal to or higher than 5 s. Potency ratio (and 95 confidence limits) for the effects of morphine obtained in the two different conditions was also calculated whenever the regression lines obtained did not deviate significantly from parallelism.
3. Results The data in Fig. 1 show a dose-dependent increase in the tail flick latency produced by morphine injected via the CAO catheter (experiment 1A), or via the AL catheter in rats with (experiment 1B) or without (experiment 1C) the CAO catheter (Fig. 1A /C, respectively). The mean baseline latencies obtained in the three experiments were not significantly different. The curves in Fig. 1 were different regarding treatments (F3,29 / 30.8 in A; F3,27 /25.07 in B; F4,35 /34.72 in C; P B/ 0.0001 in all cases) and time /course (F11,286 /38.09 in A; F11,297 /28.45 in B; F11,385 /69.19 in C; P B/0.0001 in all cases), and showed significant treatment /time interactions (F33,286 /4.34 in A; F33,297 /9.35 in B; F44,385 /8.51 in C; P B/0.0001 in all cases). The peak effect of morphine occurred 10 min after drug administration via the CAO catheter and 5 min after administration via the AL catheter in rats without the CAO catheter. The estimated AD50 of morphine was also different in each experimental condition, i.e. 8.2 (95% confidence limits: 4.6 and 14.4), 1.1 (0.87 and 1.47) and 0.29 (0.19 and 0.47) mg for the experiments 1A, 1B and 1C, respectively. The AD50 of morphine under the conditions of experiment C was significantly smaller than under the conditions of experiment 1A (potency ratio /28.27; 95% confidence limits /15.3 and 52) and experiment 1B (3.89; 2.2 and 6.84). The AD50 of morphine under the conditions of experiment 1B was significantly smaller than under the conditions of experiment 1A (7.25; 4.7 and 11.16). A significantly prolonged effect was obtained following morphine (1.6 mg) via AL catheter in rats with CAO catheter (Fig. 1B). The data in Fig. 2 show a dose-dependent increase in the tail flick latency produced by i.c.v. injection of
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Fig. 1. Time /course of the effects of intrathecal saline (10 ml) and various doses of morphine on the rat tail flick test. Injection was made via a CAO (A) or AL catheter in animals with (B) or without (C) a CAO catheter at the time indicated by the arrow. Doses of morphine (in mg/rat) and the number of rats per group (between parentheses) are given in the insert in the upper left corner of each graph. Points are means (9/S.E.M.). (*) Different from saline; (/) different from saline and group of rats treated with morphine 3.33 mg; # different from the remaining groups (P B/0.05).
morphine in rats with (experiment 2A; Fig. 2A) or without (experiment 2B; Fig. 2B) a CAO catheter. The mean baseline latencies obtained for the three experimental groups were not significantly different. The curves in Fig. 2 were different regarding treatments (F3,29 /18.21 in A; F3,26 /26.93 in B; P B/0.0001 in all cases) and time /course (F11,319 /25.4 in A; F11,286 / 21.04 in B; P B/0.0001 in all cases), and showed significant treatment /time interactions (F33,319 /6.23 in A; F33,286 /4.92 in B; P B/0.0001 in all cases). The peak effect of morphine in both experiments occurred 10 min after drug administration. The estimated AD50 of morphine was 1.04 (0.66 and 1.62) and 1.11 (0.78 and 1.57) mg for experiments 2A, and 2B, respectively. The AD50 of morphine under the conditions of experiment 2A was not significantly different from that obtained
under the conditions of experiment 2B (potency ratio / 1.06). The data in Fig. 3 show a dose-dependent increase in the tail flick latency produced by injecting morphine intrathecally via a CAO (experiment 3A; Fig. 3A) or AL (experiment 3B; Fig. 3B) catheter in rats chronically treated with i.p. indomethacin. The mean baseline latencies obtained for the three experimental groups were not significantly different. The curves in Fig. 3 were different regarding treatments (F3,23 /13.87 in A; F3,27 /42.05 in B; P B/0.0001 in all cases) and time / course (F11,253 /48.01 in A; F11,297 /42.53 in B; P B/ 0.0001 in all cases), and showed significant treatment / time interactions (F33,253 /7.0 in A; F33,297 /8.74 in B; P B/0.0001 in all cases). The baseline latencies measured before the chronic treatment with indomethacin were
Fig. 2. Time /course of the effects of intracerebroventricular saline (0.5 ml) and various doses of morphine on the rat tail flick test. Injection was made in rats with (A) or without (B) a CAO catheter at the time indicated by the arrow. Doses of morphine (in mg/rat) and the number of rats per group (between parentheses) are given in the insert in the upper left corner of each graph. Points are means (9/S.E.M). (*) Different from saline; (/) different from saline and group of rats treated with morphine 0.5 mg; # different from the remaining groups (P B/0.05).
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Fig. 3. Time /course of the effects of intrathecal saline (10 ml) and various doses of morphine on the rat tail flick test. Animals were treated with intraperitoneal indomethacin (1 mg/kg, twice a day) for 5 days and the test was conducted 2 days later. Injection of saline or morphine was made via a CAO (A) or AL catheter (B) at the timing indicated by the arrow. Doses of morphine (in mg/rat) and the number of rats per group (between parentheses) are given in the insert in the upper left corner of each graph. Points are means (9/S.E.M.). (*) Different from saline; (/) different from saline and group of rats treated with morphine 1 mg (in A) or 0.125 mg (in B); # different from the remaining groups (P B/0.05).
not included in the Figures, but they did not differ significantly from those obtained on the day of the experiment (baseline latencies of saline-treated rats in Fig. 3). The peak effect of morphine in both experiments occurred 5 min after drug administration. The estimated AD50 of morphine was 2.5 (1.62 and 3.85) and 0.31 (0.19 and 0.49) mg for experiments 3A, and 3B, respectively. The AD50 of morphine under the conditions of experiment 3B was significantly smaller than that obtained under the conditions of experiment 3A (potency ratio / 8.06; 95% confidence limits /4.3 and 14.91). The AD50 of morphine under the conditions of experiment 1C was significantly smaller than that obtained under the conditions of experiment 3A (potency ratio /8.62; 95% confidence limits /4.5 and 16.37), but did not differ significantly from the AD50 obtained under the conditions of experiment 3B (potency ratio /1.06). About 30% of the rats implanted with CAO catheter had motor impairment and, therefore, were not considered for further experimentation. None rat implanted with AL catheter only, experienced any sign of motor impairment, but four rats were discarded due to occlusion of the catheter tip during the injection procedure.
4. Discussion The present experiments confirm that administration of morphine into the subarachnoid space increases the latency for the tail flick reflex of rats to noxious heating of the skin (Yaksh and Rudy, 1977). The potency of morphine, however, differed depending on the method used for spinal catheterization. Morphine administered via the AL catheter was about four times less potent in
rats with than in rats without the CAO catheter and was about 28-fold less potent than when administered via the CAO catheter. Spinal changes produced by the presence of a chronic catheter in the subarachnoid space (see Section 1) may be the reason for the lower potency of morphine when injected via the CAO catheter. However, morphine administered via the AL catheter in rats with the CAO catheter was only about seven times more potent than when injected via the CAO catheter. It is then possible that the spinal diffusion of morphine differs when the drug is injected via the CAO or AL catheter. Restricted diffusion of intrathecally injected dynorphin A due to the development of gliosis and scarring in the vicinity of a CAO catheter has already been proposed (Yaksh and Noueihed, 1985). However, the location of 99mTc-DTPA spreading was comparable following injection via a CAO or AL catheter (Storkson et al., 1996). The AD50 for morphine obtained in this study following injection via the CAO catheter (8.2 mg) was slightly higher than that obtained in earlier studies using similar procedures, such as 2.6 mg (95% confidence limits /0.87 and 7.9) (Schmauss and Yaksh, 1984). Other studies have also found lower AD50 values for morphine such as 2.6 mg (1.6 and 5.3) (Miyamoto et al., 1991) or 1.06 mg (0.87 and 1.28) (Schimoyama et al., 1997) in the tail flick test, or 1.66 mg (1.04 and 2.32) in the tail immersion test (Joo´ et al., 2000), but in these cases the AD50 was calculated as the dose of morphine producing 50% of the maximal effect in the test. For comparison, in our experiments the AD50 (95% confidence limits) for morphine calculated as the dose producing 50% of the maximal effect were 0.26 mg (0.12 and 0.54) and 1.02 mg (0.72 and 1.43) for injection via the AL catheter in rats without or with CAO
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catheter, respectively, and 3.6 mg (2.9 and 4.4) for injection via the CAO catheter. Therefore, whatever the method of calculating the AD50 of morphine, the presence of a CAO catheter reduces the antinociceptive potency of the opiate. Our results agree with a former study showing that dynorphin A (1 /13) is more potent in rats when injected 1 day than 7 days after the implant of a CAO catheter into the subarachnoid space (Long et al., 1988). In contrast, our data differ from a former study showing no change in the antinociceptive potency of morphine regarding the time of permanence of a CAO catheter (Herman and Goldstein, 1985). In this case, however, the method for the calculation of AD50 of morphine injected 1 day after catheter implantation was different from that used for the calculation of AD50 of morphine injected 7/21 days after catheter implantation. The effect of the highest dose of morphine administered via the AL catheter in rats with a CAO catheter had a significantly longer duration than that produced by the remaining doses. We suspect that the presence of both catheters in the same animal somehow reduces the drug distribution throughout the subarachnoid space. The baseline latencies did not differ significantly among the experimental groups, thus indicating that the different ways of catheterizing the subarachnoid space did not change the normal reaction of animals to noxious heating of the skin. This result is in agreement with an earlier report describing no significant difference between naive and CAO cannulated rats in the latency for the animal response in the paw pressure test (Almeida et al., 2000). The AD50 of morphine injected i.c.v. in rats with no catheter (1.11 mg; 95% confidence limits /0.78 and 1.57) did not differ from that obtained from rats with a CAO catheter (1.04 mg; 0.66 and 1.62). Therefore, the changes in the antinociceptive potency of morphine found in our study following different methods of intrathecal catheterization is unlikely to be due to a change in the overall animal responsiveness to noxious stimulation. The AD50 of morphine injected via an AL catheter in animals chronically treated with indomethacin (0.31 mg) did not differ significantly from that obtained in untreated rats (0.29 mg). In contrast, the AD50 of the opiate injected via a CAO catheter was significantly reduced from 8.2 (experiment 1A) to 2.5 mg in indomethacin-treated animals. The baseline latency of indomethacin-treated animals did not differ significantly from that of rats treated with saline. Thus, indomethacin acting synergistically with morphine is unlikely to be the mechanism by which the potency of the opiate increases in indomethacin-treated animals implanted with a CAO catheter. Indomethacin is a standard nonsteroid anti-inflammatory drug, and its efficacy in reducing the AD50 of morphine in chronically but not in acutely cannulated animals points to the involvement
of an inflammatory process that somehow develops in the spinal cord as a local tissue reaction to the chronic permanence of an indwelling catheter. Mononuclear cell infiltration has already been observed in chronically cannulated rats (Almeida et al., 2000) thus indicating that the chronic presence of a catheter in the subarachnoid space induces a local inflammatory process. Inflammation in peripheral tissue increases the antinociceptive potency of various opioid agonists and the spinal levels of dynorphin, and up-regulates the biosynthesis of prodynorphin in nociceptive neurons in dorsal horn neurons, but does not change significantly the opioid receptor density or affinity in the spinal cord (for review, see Przewlocki and Przewlocka, 2001). Elevated levels of spinal dynorphin have also been found following peripheral nerve (Dubner and Ruda, 1992) or spinal cord (Faden et al., 1985; Przewlocki et al., 1988) injury. In contrast, the antinociceptive efficacy of intrathecal morphine decreases in rats with nerve injury (Ossipov et al., 1995). Since chronic pretreatment with indomethacin strongly reduces but does not avoid the loss of antinociceptive potency of morphine in rats with a CAO catheter, it is possible that the indwelling catheter induces an inflammatory reaction that somehow intensifies the spinal injury produced during spinal catheterization. However, further experiments are needed to elucidate the exact mechanism by which a chronic spinal catheter decreases the potency of spinally injected morphine. In agreement to earlier report (Storkson et al., 1996), about 30% of the rats with CAO catheter had motor impairment, whereas no detectable sign of neurological impairment was displayed by rats with AL catheter, only. The implantation of CAO catheter requires several stressful surgical procedures that may interfere with the measurement of nociceptive thresholds based in animal behavioral changes. Thus, a 5 /7-days period is usually respected between surgery and experimental procedures to minimize the interference of the surgical stress on nociceptive threshold measurement. In addition, the implantation of a CAO catheter is known to be accompanied by spinal compression (see Section 1). A morphological examination of eventual spinal compression produced by the AL catheter was not conducted in the present study. In fact, a possibility exists that implantation of an AL catheter can also produce spinal compression, but it would certainly be restricted to a shorter part of the spinal cord. The AD50 of morphine injected via an AL in rats treated chronically with indomethacin did not differ significantly from that obtained in rats with no previous treatment. Thus, any eventual damage to the spinal cord produced during the introduction of an AL catheter does not produce local changes sufficient to modify the potency of morphine. Therefore, the use of an AL catheter apparently minimizes the spinal changes produced by a chronic indwel-
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ling catheter and permits acute experimental protocols in which more than one spinal injection is necessary.
Acknowledgements This study was supported by a grant from FAPESP. I thank Mr P.R. Castania and Mr M.A. Carvalho for skillful technical assistance.
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LoPachin RM, Rudy TA, Yaksh TL. An improved method for chronic catheterization of the rat spinal subarachnoid space. Physiol Behav 1981;27:559 /61. Martin H, Kocher L, Chery-Croze S. Chronic lumbar intrathecal catheterization in the rat with reduced-length spinal compression. Physiol Behav 1984;33:159 /61. Mestre C, Pe´lissier T, Fialip J, Wilcox G, Eschalier A. A method to perform direct transcutaneous intrathecal injection in rats. J Pharmacol Toxicol Methods 1994;32:197 /200. Miyamoto Y, Ozaki M, Yamamoto H. Effect of adrenalectomy on correlation of analgesia with tissue content of morphine. Eur J Pharmacol 1989;167:11 /20. Miyamoto Y, Morita N, Kitabata Y, Yamanishi T, Kishioka S, Ozaki M, Yamamoto H. Antinociceptive synergism between supraspinal and spinal sites after subcutaneous morphine evidenced by CNS morphine content. Brain Res 1991;552:136 /40. Ossipov MH, Lopez Y, Nichols ML, Porreca F. Inhibition by spinal morphine of the tail-flick response is attenuated in rats with nerve ligation injury. Neurosci Lett 1995;199:83 /6. Prado WA, Pelegrini-da-Silva A, Martins AR. Microinjection of renin /angiotensin system peptides in discrete sites within the rat periaqueductal gray matter elicits antinociception. Brain Res 2003;972:207 /15. Przewlocki R, Przewlocka B. Opioids in chronic pain. Eur J Pharmacol 2001;429:79 /91. Przewlocki R, Haarmann I, Nikolarakis K, Herz A, Ho¨llt V. Prodynorphin gene expression in spinal cord is enhanced after traumatic injury in the rat. Mol Brain Res 1988;4:37 /41. Roberts MHT, Rees H. The antinociceptive effects of stimulating the pretectal nucleus of the rat. Pain 1986;25:83 /93. Sakura S, Hashimoto K, Bollen AW, Ciriales R, Drasner K. Intrathecal catheterization in the rat. Improved technique for morphologic analysis of drug-induced injury. Anesthesiology 1996;85:1184 /9. Schimoyama M, Shimoyama N, Inturrisi CE, Elliott KJ. Gabapentin enhances the antinociceptive effects of spinal morphine in the rat tail-flick test. Pain 1997;72:375 /82. Schmauss C, Yaksh TL. In vivo studies on spinal opiate receptor systems mediating antinociception. II. Pharmacological profiles suggesting a differential association of mu, delta and kappa receptors with visceral chemical and cutaneous thermal stimuli in the rat. J Pharmacol Exp Ther 1984;228:1 /12. Storkson RV, Kjorsvik A, Tjolsen A, Hole K. Lumbar catheterization of the spinal subarachnoid space in the rat. J Neurosci Methods 1996;65:167 /72. Svensson BA, Alari L, Post C. Repeated intrathecal injections of dezocine produce antinociception without evidence of neurotoxicity in the rat: a study of morphometric evaluation of spinal cord histology. Anesth Analg 1992;75:392 /9. Yaksh TL, Noueihed R. The physiology and pharmacology of spinal opiates. Ann Rev Pharmacol Toxicol 1985;25:433 /62. Yaksh TL, Rudy TA. Chronic catheterization of the spinal subarachnoid space. Physiol Behav 1976;17:1031 /6. Yaksh TL, Rudy TA. Studies on the direct spinal action of narcotics in the production of analgesia in the rat. J Pharmacol Exp Ther 1977;202:411 /28. Yaksh TL, Noueihed R, Durant P. Studies of the pharmacology and pathology of intrathecally administered 4-anilinopiperidine analogs and morphine in the rat and cat. Anesthesiology 1986;64:54 /66.
Antinociceptive potency of intrathecal morphine in the rat tail flick test: a comparative study using acute lumbar catheter in rats with or without a chronic atlanto-occipital catheter Wiliam A. Prado * Department of Pharmacology, Faculty of Medicine of Ribeira˜o Preto, University of Sa˜o Paulo, Av. Bandeirantes 3900, 14049-900, Ribeira˜o Preto, SP, Brazil Received 26 February 2003; received in revised form 17 June 2003; accepted 17 June 2003
Abstract Chronic spinal catheterization via an atlanto-occipital puncture (CAO) has been widely used to study the effects of drugs on spinal nociceptive mechanisms, but this method is associated with spinal cord damage that may change the efficacy of spinally injected analgesics. Using a slight modification of the method of Storkson et al. (J. Neurosci. Methods 65 (1996) 167), the rat spinal cord was acutely catheterized via a lumbar puncture (AL) and the potency of morphine-induced antinociception in the tail flick test was comparatively studied in animals with or without a CAO catheter. The opiate potency via an AL catheter (AD50; 95% confidence limits) was significantly more intense in rats without (0.29 mg; 0.19 /0.47) than in rats with a CAO catheter (1.1 mg; 0.87 / 1.47) and stronger than via a CAO catheter (8.2 mg; 4.6 /14.4). The potency of morphine via a CAO catheter was significantly improved in indomethacin-pretreated rats (1 mg/kg, i.p., twice a day for 5 days), thus indicating that inflammatory changes produced by a CAO catheter are at least in part the reason for the lower efficacy of the opiate. The use of an AL catheter minimizes such spinal changes and permits acute experimental protocols in which more than one spinal injection is necessary. # 2003 Elsevier B.V. All rights reserved. Keywords: Catheterization; Spinal cord; Acute lumbar catheter; Chronic atlanto-occipital catheter; Tail-flick test; Morphine
1. Introduction Chronic intrathecal catheters have been extensively used in rats in behavioral studies on the effects of drugs that interfere with spinal nociceptive mechanisms. The most frequently used method for such purpose consists of catheterization of the subarachnoid space with a polyethylene tubing which is introduced through a puncture in the atlanto-occipital membrane and then advanced along the spinal cord until its tip reaches the lumbar enlargement (Yaksh and Rudy, 1976; LoPachin et al., 1981). Although representing a valuable technique for spinal drug administration, this method has several disadvantages such as spinal compression by the catheter (Li et al., 1985; Gordh et al., 1986; Yaksh et al., 1986;
* Tel.: /55-16-602-3038; fax: /55-16-633-2301. E-mail address: [email protected] (W.A. Prado). 0165-0270/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0165-0270(03)00197-3
Grip et al., 1992), moderate inflammation (Li et al., 1985; Gordh et al., 1986; Yaksh et al., 1986; Svensson et al., 1992) including spinal migration of mononuclear inflammatory cells (Almeida et al., 2000), microglial activation and astrogliosis in the compressed tissue (Jones and Tuszynski, 2001), and demyelination (Yaksh et al., 1986) or neuronal vacuolation (Li et al., 1985). Both tubing size and composition also seem to be important factors affecting the histologic reaction to an indwelling intrathecal catheter (Sakura et al., 1996). The insertion of a catheter between thoracic (Dib, 1984) or lumbar (Martin et al., 1984) vertebrae has been proposed as an alternative to the Yaksh and Rudy’s method, but this does not avoid spinal compression by the catheter and may also induce tissue reactivity. An alternative to injecting drugs spinally using acute needle puncture has been offered (Mestre et al., 1994), but a difficulty remains when more than one spinal injection is required by the experimental protocol.
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Almeida et al. (2000) found the hyperalgesic response to intraplantar carrageenan or PGE2 to be more intense in rats with chronic intrathecal catheters than in naive or sham catheterized rats. They also showed that intraspinal administration of indomethacin to rats with, but not to rats without a chronic catheter caused antinociception in the formalin test. Therefore, the spinal changes produced by the presence of a chronic intrathecal catheter may modify the behavioral response of rodents to the spinal administration of drugs. The present study examines the effect of morphine injected via a chronic atlanto-occipital (CAO) catheter implanted in rats according to the method of Yaksh and Rudy (1976) on the latency for the tail flick reflex to noxious heating of the skin. This effect was then compared with the effects of morphine injected via an acute lumbar (AL) catheter in rats with or without a CAO catheter.
2. Methods 2.1. Subjects and surgery Males Wistar rats (250 /300 g) were used. Animals were housed two to a cage with water and food ad libitum, and kept in temperature controlled rooms (239/ 2 8C) on a 12:12-h light /dark cycle, with the dark cycle beginning at 07:00 h. All tests were held in the morning and conducted according to the IASP guidelines on the use of animals in pain research. Each rat was used only once. Rats were allocated in i.c.v. or i.t. groups. Surgical operations were all performed under anesthesia with tribromoethanol (250 mg/kg, i.p.). For the i.c.v. group, a 10-mm stainless steel guide cannula was implanted as previously described (Miyamoto et al., 1989), using the coordinates (in mm) AP 1.4 from the interaural line, L 1.6 from the sagittal suture and V 3.5 from the skull surface. Soon after the procedures for implant of the guide cannula, some rats in the i.c.v. group had a chronic catheter implanted as described below. For the i.t. group, catheterization of the spinal subarachnoid space was performed. The catheter consisted of a 12 mm length of polyethylene tubing (PE tubing, o.d. /0.4 mm, dead space /10 ml) previously coated with silicone, dried, sterilized by immersion in 70% ethanol, and fully flushed with sterile saline prior to insertion. The CAO catheter was inserted through an incision of the atlanto-occipital membrane up to the lumbar enlargement of the spinal cord (7.5 cm), as described elsewhere (Yaksh and Rudy, 1976). The AL catheter was acutely implanted using a slight modification of a method developed by Storkson et al. (1996). Briefly, a 20-gauge Weiss needle was introduced through the skin into the L5 /L6 intervertebral space. The correct positioning of the needle was assured by a
typical flick of the tail or hind paw. The catheter was then introduced through the needle to protrude 1.5 cm into the subarachnoid space in a cranial direction. The needle was then carefully removed and the tubing anchored to the back skin with a cotton thread suture. Rats with the CAO catheter were used 7 days after surgery. In a separated group of rats with AL catheter we found no significant change in the tail flick latency before or 2 h after surgery (not shown in Section 3). Injections via the AL catheter were then performed 4 h after the catheter implantation to avoid any residual analgesia produced by the anesthetic procedure. In each case, only rats showing no sign of motor impairment were considered for further experimentation. 2.2. Tail-flick test The tail-flick test was conducted as described elsewhere (Roberts and Rees, 1986; Prado et al., 2003). Each animal was placed in a ventilated tube with the tail laid across a wire coil, which was at room temperature (239/2 8C). The coil temperature was then raised by the passage of electric current and the latency for the tail withdrawal reflex was measured. The heating was applied to a portion of the ventral surface of the tail between 4 and 6 cm from the tip. This method is better than the tail immersion in hot water because it minimizes the risk of skin burning when several measurements are done in the same animal (Le Bars et al., 2001). Each trial was terminated after 6 s to minimize the probability of skin damage. Tail flick latency was measured at 5-min intervals until a stable baseline was obtained over three consecutive trials. The latency was measured again within 30 s after drug administration and then at 5-min intervals for up to 40 min. Only rats showing stable baseline latency after up to six trials were used in each experiment. 2.3. Injection procedures Drug or saline was injected intrathecally in a volume of 10 ml over a period of 60 s, followed by 10 ml of sterile saline at the same rate to flush the catheter. Intrathecal injections were monitored by observing the movement of an air bubble along a length of calibrated PE-10 tubing between the intrathecal catheter and the injection syringe. Drug or saline was injected via the CAO catheter (experiment 1A) or via the AL catheter in rats with (experiment 1B) or without (experiment 1C) a CAO catheter. I.c.v. injection of drug or saline was performed using a glass needle (70 /90 mm. o.d.) protected by a system of telescoping steel tubes as described elsewhere (Azami et al., 1980). The assembly was inserted into the guide cannula immediately before the injection and the needle advanced to protrude 0.5 mm beyond the guide cannula
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tip. The volume of injection was 0.5 ml delivered at a constant rate over a period of 3 min, and the needle was removed 20 s after the completion of this procedure. I.c.v. injections were made in rats with (experiment 2A) or without (experiment 2B) a chronic A /O catheter. Experiments 1A and 1C were repeated in groups of rats treated chronically with indomethacin (experiments 3A and 3B, respectively). In these cases, the tail flick latency was measured before the surgical procedures and indomethacin was injected intraperitoneally (1 mg/kg, twice a day) for 5 days. The experiment was conducted 2 days after the last i.p. injection to avoid any residual analgesic effect of indomethacin. 2.4. Examination of catheter position and i.c.v. injection sites At the end of the experiment the correct position of the catheter was indicated by motor paralysis of the hind part of the animal occurring within 15 min after the intrathecal administration of 2% lidocaine (10 ml) followed by saline (10 ml). In order to confirm the correct catheter positioning, 1% methylene blue (10 ml) was injected intrathecally. The rat was then deeply anesthetized with sodium pentobarbital and perfused through the heart with 4% paraformaldehyde in 0.1 M phosphate buffered saline. The spinal cord was cut through the T10 /T11 (rats with the CAO catheter, only) or L4 /L5 (rats with the AL catheter, only) intervertebral disk to look for the catheter tip under a dissecting microscope (4 / magnification). The spinal cords from rats with both CAO and AL catheters were examined at both the T10 /T11 and L4 /L5 planes. Rats showing the catheter tip positioned at sites other than the dorsal spinal cord or dye staining of paravertebral musculature were not considered for data analysis. For examination of i.c.v. injection sites, 1% methylene blue (0.5 ml) was injected through the guide cannula. The rat was anesthetized and perfused through the heart as described above. The brain was then cut with a sharp knife along the coronal plane and the distribution of dye examined under a dissecting microscope (4 / magnification). Only rats showing dye distributed within the ventricular spaces were considered for data analysis. 2.5. Drugs Morphine sulfate and indomethacin were purchased from Sigma-Aldrich Co. (St. Louis, USA), and diluted in sterile saline and 0.1% methyl-cellulose, respectively. 2.6. Statistics The results are reported as mean9/S.E.M. Comparisons between control (saline) and test groups were made by multivariate analysis of variance (MANOVA)
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with repeated measures to compare the groups over all times. The factors analyzed were treatments, time and treatment /time interaction. In the case of treatment / time interaction, one-way analysis of variance followed by the Duncan test was performed for each time. The analysis was performed using the statistical software package SPSS/PC/, version 3.0 and the level of significance was set at P B/0.05. The dose of morphine producing an antinociceptive effect in 50% of the animals in an experimental group at the time of peak effect (AD50), was estimated by the method of Litchfield and Wilcoxon (1949). For calculation, antinociception was arbitrarily considered to occur whenever the latency for the tail flick reflex was equal to or higher than 5 s. Potency ratio (and 95 confidence limits) for the effects of morphine obtained in the two different conditions was also calculated whenever the regression lines obtained did not deviate significantly from parallelism.
3. Results The data in Fig. 1 show a dose-dependent increase in the tail flick latency produced by morphine injected via the CAO catheter (experiment 1A), or via the AL catheter in rats with (experiment 1B) or without (experiment 1C) the CAO catheter (Fig. 1A /C, respectively). The mean baseline latencies obtained in the three experiments were not significantly different. The curves in Fig. 1 were different regarding treatments (F3,29 / 30.8 in A; F3,27 /25.07 in B; F4,35 /34.72 in C; P B/ 0.0001 in all cases) and time /course (F11,286 /38.09 in A; F11,297 /28.45 in B; F11,385 /69.19 in C; P B/0.0001 in all cases), and showed significant treatment /time interactions (F33,286 /4.34 in A; F33,297 /9.35 in B; F44,385 /8.51 in C; P B/0.0001 in all cases). The peak effect of morphine occurred 10 min after drug administration via the CAO catheter and 5 min after administration via the AL catheter in rats without the CAO catheter. The estimated AD50 of morphine was also different in each experimental condition, i.e. 8.2 (95% confidence limits: 4.6 and 14.4), 1.1 (0.87 and 1.47) and 0.29 (0.19 and 0.47) mg for the experiments 1A, 1B and 1C, respectively. The AD50 of morphine under the conditions of experiment C was significantly smaller than under the conditions of experiment 1A (potency ratio /28.27; 95% confidence limits /15.3 and 52) and experiment 1B (3.89; 2.2 and 6.84). The AD50 of morphine under the conditions of experiment 1B was significantly smaller than under the conditions of experiment 1A (7.25; 4.7 and 11.16). A significantly prolonged effect was obtained following morphine (1.6 mg) via AL catheter in rats with CAO catheter (Fig. 1B). The data in Fig. 2 show a dose-dependent increase in the tail flick latency produced by i.c.v. injection of
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Fig. 1. Time /course of the effects of intrathecal saline (10 ml) and various doses of morphine on the rat tail flick test. Injection was made via a CAO (A) or AL catheter in animals with (B) or without (C) a CAO catheter at the time indicated by the arrow. Doses of morphine (in mg/rat) and the number of rats per group (between parentheses) are given in the insert in the upper left corner of each graph. Points are means (9/S.E.M.). (*) Different from saline; (/) different from saline and group of rats treated with morphine 3.33 mg; # different from the remaining groups (P B/0.05).
morphine in rats with (experiment 2A; Fig. 2A) or without (experiment 2B; Fig. 2B) a CAO catheter. The mean baseline latencies obtained for the three experimental groups were not significantly different. The curves in Fig. 2 were different regarding treatments (F3,29 /18.21 in A; F3,26 /26.93 in B; P B/0.0001 in all cases) and time /course (F11,319 /25.4 in A; F11,286 / 21.04 in B; P B/0.0001 in all cases), and showed significant treatment /time interactions (F33,319 /6.23 in A; F33,286 /4.92 in B; P B/0.0001 in all cases). The peak effect of morphine in both experiments occurred 10 min after drug administration. The estimated AD50 of morphine was 1.04 (0.66 and 1.62) and 1.11 (0.78 and 1.57) mg for experiments 2A, and 2B, respectively. The AD50 of morphine under the conditions of experiment 2A was not significantly different from that obtained
under the conditions of experiment 2B (potency ratio / 1.06). The data in Fig. 3 show a dose-dependent increase in the tail flick latency produced by injecting morphine intrathecally via a CAO (experiment 3A; Fig. 3A) or AL (experiment 3B; Fig. 3B) catheter in rats chronically treated with i.p. indomethacin. The mean baseline latencies obtained for the three experimental groups were not significantly different. The curves in Fig. 3 were different regarding treatments (F3,23 /13.87 in A; F3,27 /42.05 in B; P B/0.0001 in all cases) and time / course (F11,253 /48.01 in A; F11,297 /42.53 in B; P B/ 0.0001 in all cases), and showed significant treatment / time interactions (F33,253 /7.0 in A; F33,297 /8.74 in B; P B/0.0001 in all cases). The baseline latencies measured before the chronic treatment with indomethacin were
Fig. 2. Time /course of the effects of intracerebroventricular saline (0.5 ml) and various doses of morphine on the rat tail flick test. Injection was made in rats with (A) or without (B) a CAO catheter at the time indicated by the arrow. Doses of morphine (in mg/rat) and the number of rats per group (between parentheses) are given in the insert in the upper left corner of each graph. Points are means (9/S.E.M). (*) Different from saline; (/) different from saline and group of rats treated with morphine 0.5 mg; # different from the remaining groups (P B/0.05).
W.A. Prado / Journal of Neuroscience Methods 129 (2003) 33 /39
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Fig. 3. Time /course of the effects of intrathecal saline (10 ml) and various doses of morphine on the rat tail flick test. Animals were treated with intraperitoneal indomethacin (1 mg/kg, twice a day) for 5 days and the test was conducted 2 days later. Injection of saline or morphine was made via a CAO (A) or AL catheter (B) at the timing indicated by the arrow. Doses of morphine (in mg/rat) and the number of rats per group (between parentheses) are given in the insert in the upper left corner of each graph. Points are means (9/S.E.M.). (*) Different from saline; (/) different from saline and group of rats treated with morphine 1 mg (in A) or 0.125 mg (in B); # different from the remaining groups (P B/0.05).
not included in the Figures, but they did not differ significantly from those obtained on the day of the experiment (baseline latencies of saline-treated rats in Fig. 3). The peak effect of morphine in both experiments occurred 5 min after drug administration. The estimated AD50 of morphine was 2.5 (1.62 and 3.85) and 0.31 (0.19 and 0.49) mg for experiments 3A, and 3B, respectively. The AD50 of morphine under the conditions of experiment 3B was significantly smaller than that obtained under the conditions of experiment 3A (potency ratio / 8.06; 95% confidence limits /4.3 and 14.91). The AD50 of morphine under the conditions of experiment 1C was significantly smaller than that obtained under the conditions of experiment 3A (potency ratio /8.62; 95% confidence limits /4.5 and 16.37), but did not differ significantly from the AD50 obtained under the conditions of experiment 3B (potency ratio /1.06). About 30% of the rats implanted with CAO catheter had motor impairment and, therefore, were not considered for further experimentation. None rat implanted with AL catheter only, experienced any sign of motor impairment, but four rats were discarded due to occlusion of the catheter tip during the injection procedure.
4. Discussion The present experiments confirm that administration of morphine into the subarachnoid space increases the latency for the tail flick reflex of rats to noxious heating of the skin (Yaksh and Rudy, 1977). The potency of morphine, however, differed depending on the method used for spinal catheterization. Morphine administered via the AL catheter was about four times less potent in
rats with than in rats without the CAO catheter and was about 28-fold less potent than when administered via the CAO catheter. Spinal changes produced by the presence of a chronic catheter in the subarachnoid space (see Section 1) may be the reason for the lower potency of morphine when injected via the CAO catheter. However, morphine administered via the AL catheter in rats with the CAO catheter was only about seven times more potent than when injected via the CAO catheter. It is then possible that the spinal diffusion of morphine differs when the drug is injected via the CAO or AL catheter. Restricted diffusion of intrathecally injected dynorphin A due to the development of gliosis and scarring in the vicinity of a CAO catheter has already been proposed (Yaksh and Noueihed, 1985). However, the location of 99mTc-DTPA spreading was comparable following injection via a CAO or AL catheter (Storkson et al., 1996). The AD50 for morphine obtained in this study following injection via the CAO catheter (8.2 mg) was slightly higher than that obtained in earlier studies using similar procedures, such as 2.6 mg (95% confidence limits /0.87 and 7.9) (Schmauss and Yaksh, 1984). Other studies have also found lower AD50 values for morphine such as 2.6 mg (1.6 and 5.3) (Miyamoto et al., 1991) or 1.06 mg (0.87 and 1.28) (Schimoyama et al., 1997) in the tail flick test, or 1.66 mg (1.04 and 2.32) in the tail immersion test (Joo´ et al., 2000), but in these cases the AD50 was calculated as the dose of morphine producing 50% of the maximal effect in the test. For comparison, in our experiments the AD50 (95% confidence limits) for morphine calculated as the dose producing 50% of the maximal effect were 0.26 mg (0.12 and 0.54) and 1.02 mg (0.72 and 1.43) for injection via the AL catheter in rats without or with CAO
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catheter, respectively, and 3.6 mg (2.9 and 4.4) for injection via the CAO catheter. Therefore, whatever the method of calculating the AD50 of morphine, the presence of a CAO catheter reduces the antinociceptive potency of the opiate. Our results agree with a former study showing that dynorphin A (1 /13) is more potent in rats when injected 1 day than 7 days after the implant of a CAO catheter into the subarachnoid space (Long et al., 1988). In contrast, our data differ from a former study showing no change in the antinociceptive potency of morphine regarding the time of permanence of a CAO catheter (Herman and Goldstein, 1985). In this case, however, the method for the calculation of AD50 of morphine injected 1 day after catheter implantation was different from that used for the calculation of AD50 of morphine injected 7/21 days after catheter implantation. The effect of the highest dose of morphine administered via the AL catheter in rats with a CAO catheter had a significantly longer duration than that produced by the remaining doses. We suspect that the presence of both catheters in the same animal somehow reduces the drug distribution throughout the subarachnoid space. The baseline latencies did not differ significantly among the experimental groups, thus indicating that the different ways of catheterizing the subarachnoid space did not change the normal reaction of animals to noxious heating of the skin. This result is in agreement with an earlier report describing no significant difference between naive and CAO cannulated rats in the latency for the animal response in the paw pressure test (Almeida et al., 2000). The AD50 of morphine injected i.c.v. in rats with no catheter (1.11 mg; 95% confidence limits /0.78 and 1.57) did not differ from that obtained from rats with a CAO catheter (1.04 mg; 0.66 and 1.62). Therefore, the changes in the antinociceptive potency of morphine found in our study following different methods of intrathecal catheterization is unlikely to be due to a change in the overall animal responsiveness to noxious stimulation. The AD50 of morphine injected via an AL catheter in animals chronically treated with indomethacin (0.31 mg) did not differ significantly from that obtained in untreated rats (0.29 mg). In contrast, the AD50 of the opiate injected via a CAO catheter was significantly reduced from 8.2 (experiment 1A) to 2.5 mg in indomethacin-treated animals. The baseline latency of indomethacin-treated animals did not differ significantly from that of rats treated with saline. Thus, indomethacin acting synergistically with morphine is unlikely to be the mechanism by which the potency of the opiate increases in indomethacin-treated animals implanted with a CAO catheter. Indomethacin is a standard nonsteroid anti-inflammatory drug, and its efficacy in reducing the AD50 of morphine in chronically but not in acutely cannulated animals points to the involvement
of an inflammatory process that somehow develops in the spinal cord as a local tissue reaction to the chronic permanence of an indwelling catheter. Mononuclear cell infiltration has already been observed in chronically cannulated rats (Almeida et al., 2000) thus indicating that the chronic presence of a catheter in the subarachnoid space induces a local inflammatory process. Inflammation in peripheral tissue increases the antinociceptive potency of various opioid agonists and the spinal levels of dynorphin, and up-regulates the biosynthesis of prodynorphin in nociceptive neurons in dorsal horn neurons, but does not change significantly the opioid receptor density or affinity in the spinal cord (for review, see Przewlocki and Przewlocka, 2001). Elevated levels of spinal dynorphin have also been found following peripheral nerve (Dubner and Ruda, 1992) or spinal cord (Faden et al., 1985; Przewlocki et al., 1988) injury. In contrast, the antinociceptive efficacy of intrathecal morphine decreases in rats with nerve injury (Ossipov et al., 1995). Since chronic pretreatment with indomethacin strongly reduces but does not avoid the loss of antinociceptive potency of morphine in rats with a CAO catheter, it is possible that the indwelling catheter induces an inflammatory reaction that somehow intensifies the spinal injury produced during spinal catheterization. However, further experiments are needed to elucidate the exact mechanism by which a chronic spinal catheter decreases the potency of spinally injected morphine. In agreement to earlier report (Storkson et al., 1996), about 30% of the rats with CAO catheter had motor impairment, whereas no detectable sign of neurological impairment was displayed by rats with AL catheter, only. The implantation of CAO catheter requires several stressful surgical procedures that may interfere with the measurement of nociceptive thresholds based in animal behavioral changes. Thus, a 5 /7-days period is usually respected between surgery and experimental procedures to minimize the interference of the surgical stress on nociceptive threshold measurement. In addition, the implantation of a CAO catheter is known to be accompanied by spinal compression (see Section 1). A morphological examination of eventual spinal compression produced by the AL catheter was not conducted in the present study. In fact, a possibility exists that implantation of an AL catheter can also produce spinal compression, but it would certainly be restricted to a shorter part of the spinal cord. The AD50 of morphine injected via an AL in rats treated chronically with indomethacin did not differ significantly from that obtained in rats with no previous treatment. Thus, any eventual damage to the spinal cord produced during the introduction of an AL catheter does not produce local changes sufficient to modify the potency of morphine. Therefore, the use of an AL catheter apparently minimizes the spinal changes produced by a chronic indwel-
W.A. Prado / Journal of Neuroscience Methods 129 (2003) 33 /39
ling catheter and permits acute experimental protocols in which more than one spinal injection is necessary.
Acknowledgements This study was supported by a grant from FAPESP. I thank Mr P.R. Castania and Mr M.A. Carvalho for skillful technical assistance.
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