Analgesic Mechanisms of Ketamine in the Presence and Absence of Peripheral Inflammation

The protocol for this study was approved by the Sapporo Medical University Animal Care and Use Committee. Experiments were conducted in male Sprague-Dawley rats (weighing 250–300 g, Japan SLC, Hamamatsu, Japan), which were housed individually in a temperature-controlled (21 ± 1°C) room with a 12-h light–dark cycle and given free access to food and water.

All surgical procedures were performed during general anesthesia (isoflurane 3% in oxygen). In some rats, a polyethylene intrathecal catheter (PE-10; Becton Dickinson, Clay Adams, NJ) was inserted 15 mm cephalad into the lumbar subarachnoid space at the L4–L5 intervertebrae with the tip of the catheter located near the lumbar enlargement of the spinal cord to administer the drugs intrathecally. 21The catheter was tunneled subcutaneously and externalized through the skin in the neck region. The volume of dead space of the intrathecal catheter was 15 μl. At least 6 days of postsurgical recovery was allowed before animals were used in experiments. Each animal was used in only one experiment. Only animals that showed normal behavior and motor function and that had shown complete paralysis of the tail and bilateral hind legs after administration of 2% lidocaine 10 μl through the intrathecal catheter were used.

In other rats, to measure noradrenaline and 5-hydroxytryptamine (serotonin) concentrations in the lumbar cerebrospinal fluid (CSF), a dialysis probe was inserted 30 mm cephalad into the lumbar subarachnoid space at the L4–L5 intervertebrae, positioning the dialysis area of the probe located at the lumbar enlargement of the spinal cord. The dialysis probe was constructed according to the method described in our previous report. 22A 6-cm cupraphan hollow fiber with an ID of 200 μm, an OD of 220 μm, and 50-kd molecular weight cutoff (DM-22, Eicom Co., Kyoto, Japan) was coated with a thin layer of epoxy glue (Devcon Co., Danvers, MA) along the whole length, except for a 2-cm region in the middle. To make the fiber firm enough for implication, a Nichrome-Formvar wire with a 78-μm ID (A-M systems, Inc., Everret, WA), was passed through the fiber. Each end of the fiber was attached to 2-cm polyethylene catheters (PE-10, Clay Adams), and each end of polyethylene catheters was then attached to an 8-cm Teflon tube with an ID of 100 μm and an OD of 400 μm (JT-10, Eicom). A U-shaped loop was formed by gently bending the whole fiber in the middle, and the two pieces were bound together at the fiber–polyethylene catheter connection using epoxy glue. The dead space volume of the dialysis probe was 7 μl. The two distal ends of the probe were tunneled subcutaneously and externalized through the skin in the neck region. The experiments were performed 5 days after the implantation of the dialysis probe. In the experiments, we used only animals that showed normal behavior. The rats were used only once for the experiments. After each experiment, rats were killed with an overdose of pentobarbital, and the position of the dialysis probe was then confirmed by gross examination of the spinal cord.

All drugs were purchased from Sigma Chemical Co. (St. Louis, MO). Intrathecal drug administration was accomplished using a microinjection syringe (Hamilton, Remo, NV) connected to the intrathecal catheter in awake, briefly restrained rats. Yohimbine hydrochloride, methysergide hydrochloride, and ketamine hydrochloride were dissolved in physiological saline. Intrathecal drug administration was performed manually over a 10-s period in a single injection volume of 10 μl followed by a flush of 15 μl physiological saline. Systemic drug administration was intraperitoneally performed in a single injection volume of 1 ml.

Thermal nociceptive testing was conducted using an analgesimeter (Plantar Test 7370; Ugo Basile, Cometio Varese, Italy). Radiant heat was applied on the plantar surface of hind paws. The thermal nociceptive threshold was evaluated as paw withdrawal latency (PWL) from the heat source. Bulb intensity was adjusted so that the baseline PWL was 10–12 s. To avoid tissue damage, cutoff time was 20 s.

To determine the antinociceptive effects of systemic or intrathecal ketamine, rats received intrathecal (10, 50, 100, or 500 μg) or intraperitoneal ketamine (10, 25, or 50 mg/kg) in a random fashion after determination of basal PWL. PWLs were measured at 5, 10, 15, 20, 30, 45, and 60 min after the injections. In addition, some rats received intrathecal saline, 10 μg yohimbine, or 15 μg methysergide 10 min before intraperitoneal administration of ketamine. These doses of the antagonists have no effect on the thermal threshold in normal rats. 16,23 

Unilateral inflammation was induced by injection of λ-carrageenan (2 mg) into the plantar surface of the left hind paw during 1% isoflurane anesthesia. λ-Carrageenan was suspended in normal saline by sonication and administered in a 0.1-ml injection volume. After the animal had recovered from isoflurane anesthesia, it was then placed in a Plexiglass box that permitted observation.

The experiments were designed to investigate the effects of ketamine on PWLs after thermal hyperalgesia was established by carrageenan injection. After basal PWL was obtained, carrageenan was injected subcutaneously on the left hind paw. Consistent with previous reports, 24our preliminary studies revealed that maximum shortening of the PWL was sustained for 3–6 h after carrageenan injection. Accordingly, 3 h after the carrageenan injection, rats received intraperitoneal or intrathecal ketamine. After ketamine administration, PWLs were measured at 5, 10, 15, 20, 30, 45, and 60 min. In addition, some rats received intrathecal saline, 10 μg yohimbine, or 15 μg methysergide 10 min before intraperitoneal administration of ketamine.

The dialysis probe was perfused with artificial CSF (ACSF; 140 mm NaCl, 4.0 mm KCl, 1.26 mm CaCl², 1.15 mm MgCl², 2.0 mm Na²HPO⁴, 0.5 mm NaH²PO⁴, and pH 7.4) at a constant flow rate of 3 μl/min. The samples were collected as 20-min fractions and divided into two samples: 30 μl of dialysate collected for analysis of noradrenaline and 30 μl for 5-hydroxytryptamine. The samples were frozen at −80°C until analysis.

The rats were placed in a plastic cage with dimensions of 30 × 30 × 35 cm. The animals freely moved in the cage during the dialysis experiments. Three consecutive samples were collected for determination of basal levels after an initial washout period of 120 min, and then rats received intraplantar saline (0.1 ml) or a carrageenan (2 mg, 0.1 ml) injection. Three hours after intraplantar injection, 50 mg/kg ketamine or saline was intraperitoneally administered.

Noradrenaline and 5-hydroxytryptamine in the dialysate were analyzed using high-performance liquid chromatography with electrochemical detection (Degasser; DG-100, liquid chromatograph; EP-100, electrochemical detector; ECD-100, Eicom). The chromatographic condition were as follows: column, Eicompak (MA-5ODS 4.6 × 150 mm, Eicom); mobile phase: 0.1 m phosphate buffer (pH 6.0) containing 5.0% methanol, 0.02 mm EDTA, and 0.54 mm sodium 1-octanesulfonate for the analysis of noradrenaline, 0.1 m phosphate buffer (pH 6.0) containing 20.0% methanol, 0.02 mm EDTA, and 0.72 mm sodium 1-octanesulfonate for the analysis of 5-hydroxytryptamine; working electrode: glassy carbon (WE-3G, Eicom); flow rate, 0.23 ml/min. Detector voltage was set at 0.45 V. Detector temperature was set at 25.0°C. Retention time for noradrenaline was 10.75 min, and that for 5-hydroxytryptamine was 14.85 min.

To determine the in vitro  recovery of noradrenaline and 5-hydroxytryptamine across the dialysis probe used in the present study, a dialysis probe was put into aliquots containing a 10-μm concentration of noradrenaline and 5-hydroxytryptamine, and perfused with ACSF at a constant flow rate of 3 μl/min at room temperature. The in vitro  recovery was estimated based on the levels of noradrenaline and 5-hydroxytryptamine in 20-min dialysis sample.

The PWLs were represented as mean ± SD. The noradrenaline and 5-hydroxytryptamine concentrations were represented as mean ± SD of the percentage of basal levels. Changes in PWLs after intraplantar carrageenan were compared with basal PWLs using a paired t  test. To assess the effects of drugs, changes in PWLs were analyzed using a two-way analysis of variance followed by multiple between-group comparisons using the Bonferroni correction, and PWLs after ketamine administration were compared with those before ketamine using a one-way analysis of variance followed by Bonferroni correction within a single group. With regard to noradrenaline and 5-hydroxytryptamine concentrations, the statistical significance was determined by a two-way analysis of variance followed by multiple between-group comparisons using the Bonferroni correction. A P  value < 0.05 was considered to be statistically significant.

Intraperitoneal ketamine 25 and 50 mg/kg showed several characteristic behavioral effects in addition to analgesic properties. After intraperitoneal injection of ketamine, the rats showed head weaving and circling behavior that persisted for 15 and 40 min at the 25- and 50-mg/kg doses of ketamine, respectively. Neither 10 mg/kg intraperitoneal ketamine nor intrathecal ketamine resulted in any abnormal behavior.

The basal PWLs were 10.37 ± 0.22 s. Intraperitoneal ketamine produced dose- and time-dependent antinociceptive effects, with the peak effect at 10 or 15 min after administration (fig. 1). Neither intrathecal 10 μg yohimbine nor 15 μg methysergide alone affected basal PWLs (data not shown). Intrathecal pretreatment with yohimbine or methysergide, but not saline, significantly inhibited the antinociceptive effects of 50 mg/kg ketamine administered intraperitoneally (P < 0.01 or 0.001, respectively;fig. 2). Pretreatment did not improve motor effects associated with injection of 50 mg/kg ketamine. Intrathecal ketamine at the doses (10–500 μg) used did not produce any antinociceptive effects (fig. 3).

Carrageenan injection significantly reduced the PWLs of the injected paw (from 11.4 ± 0.6 s to 3.5 ± 0.2 s;P < 0.001), but not of the contralateral side (from 11.6 ± 0.2 s to 11.2 ± 0.5 s), 3 h after carrageenan administration.

At doses of 25 and 50 mg/kg, intraperitoneal ketamine provoked head weaving and circling behavior that persisted for 15 and 40 min, respectively. Figure 4Ashows the antihyperalgesic effect of 50 mg/kg systemic ketamine in carrageenan-treated rats, which was apparent at 5 min and persisted for 45 min. The peak effects of ketamine were observed at the time point of 10 min after intraperitoneal injection. As shown fig. 4B, intraperitoneal ketamine reversed the shortened PWL on the ipsilateral side in a dose-dependent manner but had no effects on the contralateral side. Figure 5Ashows the antihyperalgesic effect of 500 μg intrathecal ketamine. The significant increases in PWLs of the injected paw were observed for 30 min. The peak effects of intrathecal ketamine were observed at the time point of 10 min after injection. As shown in fig. 5B, intrathecal ketamine reversed the shortened PWL on the ipsilateral side in a dose-dependent manner but had no effects on the contralateral side. Intrathecal ketamine did not evoke any motor effects. Pretreatment with intrathecal yohimbine 10 μg or methysergide 15 μg did not alter the antihyperalgesic effects of intrathecal and intraperitoneal ketamine on the shortened PWLs in the carrageenan-treated rats (table 1).

The basal levels of noradrenaline and 5-hydroxytryptamine were 2.2 ± 0.47 and 0.94 ± 0.21 pg/20 μl, respectively. The in vitro  recovery of noradrenaline and 5-hydroxytryptamine were estimated to be 28.49 ± 0.01% and 29.37 ± 0.02%, respectively (n = 4), across the dialysis probe.

There were not significant differences between the efflux of noradrenaline and 5-hydroxytryptamine before ketamine administration and the basal levels (fig. 6). In saline-treated rats, 50 mg/kg intraperitoneal ketamine significantly increased the efflux of noradrenaline at 20, 40, 60, 80, and 100 min after injection of ketamine compared with the basal levels (P < 0.01). In carrageenan-treated rats, intraperitoneal ketamine significantly increased the efflux of noradrenaline at 20, 40, and 60 min after injection of ketamine compared with the basal levels (P < 0.01); however, the magnitude of increase in noradrenaline efflux was significantly smaller than that in saline-treated rats (P < 0.01;fig. 6). Intraperitoneal ketamine 50 mg/kg significantly increased the efflux of 5-hydroxytryptamine at 20, 40, 60, 80, and 100 min after injection of ketamine, compared with basal levels, in saline-treated rats (P < 0.01). In carrageenan-treated rats, the efflux of 5-hydroxytryptamine increased at 20, 40, and 60 min after injection of ketamine compared with basal levels (P < 0.01); however, the magnitude of increase in 5-hydroxytryptamine efflux was significantly smaller than that in saline-treated rats (P < 0.01;fig. 6).

In the present study, systemic ketamine produced antinociceptive effects, with increases in the concentrations of noradrenaline and 5-hydroxytryptamine in the lumbar CSF. We also observed that pretreatment with intrathecal yohimbine and methysergide inhibited the antinociceptive effects of systemic ketamine. On the other hand, intrathecal ketamine did not produce any antinociceptive effects. Additionally, our previous study demonstrated that the transection of the lower thoracic spinal cord abolished the antinociceptive effects of ketamine as assessed by the tail flick latency. 16Taken together, our results suggest that ketamine activates the monoaminergic descending inhibitory pathway at the supraspinal sites but not at the spinal level, resulting in antinociceptive effects. An electrophysiologic study suggested the activation of the supraspinal pain inhibition system by ketamine, 17consistent with our findings. A well-documented descending antinociceptive system is arising from the periaqueductal gray matter and nucleus raphe dorsalis and relaying through the rostral ventromedial medulla oblongata. 25These neurons project to the dorsal horn of the spinal cord and release monoamines such as 5-hydroxytryptamine and noradrenaline, resulting in the inhibition of the nociceptive transmission within the spinal cord. 25It has been indicated that the activation of the monoaminergic descending inhibitory system increases the concentrations of noradrenaline and 5-hydroxytryptamine in the lumbar CSF. 26–28In the present study, we showed increases in monoamine concentration in CSF after systemic ketamine. The time courses of the change in concentrations of monoamines in the CSF were not identical to those of antinociceptive effects after ketamine administration. We speculate that this discrepancy may be a result of the time lag between the changes in the monoamine concentration in CSF and in synaptic cleft, or the accumulation of noradrenaline and 5-hydroxytryptamine efflux into CSF. The precise mechanisms underlying the interaction of ketamine with the monoaminergic descending inhibitory pathway remain unclear. Although it has been reported that ketamine activates the monoaminergic descending inhibitory system through the opioid receptors, 15Hustveit et al.  29suggested that agonistic actions of ketamine on opioid receptors play a minor role in its analgesic effects.

We did not find any antinociceptive effects of intrathecal ketamine in the present study. Klimscha et al.  11also demonstrated that intrathecal ketamine did not have any antinociceptive effects using the tail flick test. On the other hand, Crisp et al.  20suggested that intrathecal ketamine produced the antinociceptive effects through spinal opioidergic, noradrenergic, and serotonergic system. Tung and Yaksh 13showed the weak antinociceptive effects of intrathecal ketamine through serotonergic but not opioidergic or noradrenergic systems. The reason for the difference in the effects of intrathecal ketamine is unclear. However, Klimscha et al.  11indicated that high dose of intrathecal ketamine (500 μg), which was smaller than the dose used in the study by Crisp et al. , 20produced supraspinal effects. In addition, Tung and Yaksh 13suggested that a local anesthetic effect by intrathecal ketamine could not be ruled out.

In the present study, 25 and 50 mg/kg systemic ketamine impaired motor function. Therefore, there is a possibility that the ketamine-induced motor impairment affected the withdrawal latency to the noxious heat. In the present study, however, intrathecal pretreatment with yohimbine and methysergide inhibited the antinociceptive effect of ketamine but not ketamine-induced motor impairment. Additionally, systemic ketamine produced motor impairment in the rat with peripheral inflammation similar to the untreated rats, and ketamine reversed the hyperalgesia to the ipsilateral side without any effects on the contralateral side. These results indicate that ketamine-induced prolongation of the withdrawal latency observed in the present study is a result of antinociceptive effects but not motor impairment.

The activation of NMDA receptors in the spinal cord is believed to play a critical role in the development and maintenance of the pathologic pain states, such as hyperalgesia and allodynia, as observed after nerve and tissue injuries. It is well known that the activation of spinal NMDA receptors is involved in carrageenan-induced hyperalgesia. 7Several lines of evidence have indicated that ketamine has the antagonistic property for the NMDA receptors, in a noncompetitive manner. 30In the present study, both intrathecal and systemic ketamine reversed the hyperalgesia on the ipsilateral side without any effects on the contralateral side. Pretreatment with intrathecal yohimbine or methysergide did not inhibit these antihyperalgesic effects of ketamine in carrageenan-treated animals. In addition, systemic ketamine significantly increased noradrenaline and 5-hydroxytryptamine in the saline-treated rats as compared with the carrageenan-treated rats. These results suggested the inhibition of the NMDA receptor activation by ketamine, rather than the activation of monoaminergic descending inhibitory system, in the rats with peripheral inflammation. Consistent with our results, intrathecal ketamine reversed the thermal hyperalgesia without any effects on the contralateral side to the inflammation in the rat model of the carrageenan-induced unilateral peripheral inflammation. 7,11In a rat model of peripheral neuropathy, in which the NMDA receptors were also thought to be activated, the antihyperalgesic effects of both intrathecal and systemic ketamine were specific to the neuropathic hind limb, whereas no consistent change in behavior was observed on the contralateral side. 8–10Recently, it has been reported that the activation of the NMDA receptors not only at the spinal cord but also at the supraspinal site, including the thalamus and rostral ventromedial medulla oblongata, was related to the maintenance of hyperalgesia associated with inflammation. 31,32This would indicate that systemic ketamine inhibited the activation of NMDA receptors at the supraspinal sites as well as the spinal site. It is unclear why the analgesic mechanism of ketamine in the presence of inflammation is different from that in the absence of inflammation. Recent evidence has indicated that the central sensitization of the wide dynamic range spinothalamic tract neurons attenuates the periaqueductal gray matter–evoked inhibition, indicating disinhibition. 33Therefore, in the stage of carrageenan-induced peripheral inflammation that evokes the sensitization of wide dynamic range neurons in the spinal cord, 34it might be that ketamine does not activate the supraspinal inhibitory systems because of disinhibition.

In conclusion, the present study demonstrated that ketamine produced antinociceptive effects through the activation of monoaminergic descending inhibitory system, whereas, in the unilateral peripheral inflammation-induced hyperalgesic state, the inhibition of NMDA receptor activation rather than the activation of the monoaminergic system contributes to the antihyperalgesic effects of ketamine. The mechanisms of the antinociceptive and antihyperalgesic properties of ketamine are different.