Ildiko Dobos

The Effects of Ketamine and Its Enantiomers on the Morphine- or Dexmedetomidine-induced Antinociception after Intrathecal Administration in Rats

Background The spinal administration of some N-methyl-d-aspartate receptor antagonists results in antinociception and potentiates the effects of opioids and &agr;2-adrenoceptor agonists, but ketamine and its enantiomers have not been examined. The present study investigated the interactions of racemic ketamine, R (−)-ketamine and S (+)-ketamine with morphine and with dexmedetomidine. Methods Intrathecal catheters were implanted into male Wistar rats. Three days later, the acute nociceptive sensitivity was assessed using the tail-flick test. Analgesic latencies were converted to the percentage maximum possible effect. The dose that yielded 50% of the maximum possible effect (ED50) and dose–response and time–course curves were determined for the ketamines (30–300 &mgr;g), morphine (0.1–3.0 &mgr;g), dexmedetomidine (0.3–10.0 &mgr;g), and mixtures of two doses of ketamines (30 or 100 &mgr;g) with different doses of morphine or dexmedetomidine for fixed-dose analysis. Results Neither racemic ketamine nor its enantiomers alone had a significant effect on the tail-flick test, with the exception of the highest dose of racemic ketamine, which caused motor impairment. Morphine and dexmedetomidine each produced dose-dependent antinociception, with ED50 of 1.7 &mgr;g (95% confidence interval: 1.04–2.32) and 4.85 &mgr;g (3.96–5.79), respectively. A low dose (30 &mgr;g) of racemic ketamine or its enantiomers did not influence the ED50 of morphine significantly. Coadministration of 100 &mgr;g racemic ketamine or S (+)-ketamine, but not R (−)-ketamine, significantly enhanced and prolonged the antinociceptive effect of morphine. Both doses of racemic ketamine or its isomers significantly decreased the ED50 value for dexmedetomidine, although the higher dose of racemic or S (+)-ketamine had the highest potency. One-hundred micrograms of racemic ketamine or S (+)-ketamine also prolonged the effects of dexmedetomidine. Conclusions These data indicate that racemic ketamine and S (+)-ketamine, but not R (−)-ketamine, exhibit similar effectiveness in potentiating the antinociceptive effects of both morphine and dexmedetomidine.

The synergistic antinociceptive interactions of endomorphin-1 with dexmedetomidine and/or S(+)-ketamine in rats.

Spinal administration of the endogenous &mgr;-opioid agonist peptide, endomorphin-1, results in antinociception in rodents, but there are few data about its interaction with other antinociceptive drugs. We investigated the antinociceptive interactions at the spinal level of endomorphin-1 with the N-methyl-d-aspartate antagonist S(+)-ketamine, the &agr;2-adrenoceptor agonist dexmedetomidine, or both in awake rats. Nociception was assessed by the tail-flick test. Dose-response curves were determined for endomorphin-1 (0.6–50 &mgr;g), for dexmedetomidine (0.1–10 &mgr;g), for mixtures of S(+)-ketamine (30 or 100 &mgr;g) with endomorphin-1 (2–18 &mgr;g) or of endomorphin-1 with dexmedetomidine in a fixed ratio (4:1), and for the triple combination of the three drugs after intrathecal administration. Endomorphin-1 and dexmedetomidine both produced dose-dependent antinociception. The coadministration of 100 &mgr;g S(+)-ketamine significantly enhanced the antinociceptive effect of 6 &mgr;g endomorphin-1. Isobolographic analysis of the combinations of endomorphin-1 and dexmedetomidine revealed a synergistic interaction between these drugs. The 80% effective dose for the triple combination was significantly less than that for either binary combination. These data indicate that S(+)-ketamine and dexmedetomidine, acting via different receptors, produce synergistic antinociceptive interaction with endomorphin-1 at the spinal level. Furthermore, the triple combination of an opioid agonist, an &agr;2-adrenoceptor agonist, and an N-methyl-d-aspartate receptor antagonist shows potent antinociceptive activity.

Antinociceptive effect of the S(+)-enantiomer of ketamine on carrageenan hyperalgesia after intrathecal administration in rats

Ketamine exerts antinociceptive effects in many pain tests.We investigated the antinociceptive effect of intrathecally administered racemic ketamine and its S(+)- and R(-)-enantiomer on carrageenan-induced thermal hyperalgesia with a paw withdrawal test and acute pain (hot plate and tailflick) tests. Rats were prepared with a chronic lumbar intrathecal catheter to receive either saline or ketamine enantiomers in cumulative doses. None of the ketamines (10, 50, or 100 [micro sign]g) had any effect on the withdrawal latency of the contralateral, noninjected paw. In the injected paw, intrathecal saline did not alter carrageenan-induced thermal hyperalgesia, whereas intrathecally applied S(+)-, R(-)-, and racemic ketamine decreased thermal hyperalgesia. However, compared with saline, racemic ketamine had a higher efficacy than S(+)-ketamine, whereas R(-)-ketamine did not achieve statistical significance. Neither S(+)- nor R(-)-ketamine had a significant effect in the tailflick test (10, 100, or 500 [micro sign]g). In the hot plate test, only the largest dose of ketamine (500 [micro sign]g) caused a non-stereospecific, significant increase in hot plate latency; this dose caused supraspinal effects as well. The results demonstrate that the behavioral hyperalgesia associated with carrageenan-induced hindpaw inflammation in rats is attenuated by the intrathecal administration of racemic and S(+)-ketamine, but not R(-)-ketamine, which only displayed an insignificant trend toward a dose-response relationship. This finding warrants further studies to investigate a possible clinical advantage of preservative-free S(+)-ketamine over the currently used preservative containing racemic mixture. Implications: In rats, intrathecal S(+)-ketamine was effective for treating inflammatory pain. Although racemic ketamine has a greater efficacy, S(+)-ketamine is available as a preservative-free drug and might be of clinical interest for future neuraxial administration in different pain states. (Anesth Analg 1998;86:561-5)

Effect of intrathecal agmatine on inflammation-induced thermal hyperalgesia in rats

Agmatine, an endogenous ligand, interacts both with the alpha2-adrenoceptors and with the imidazoline binding sites. The effect of intrathecally administered agmatine on carrageenan-induced thermal hyperalgesia was investigated by means of a paw-withdrawal test in rats. The effect of agmatine on morphine-induced anti-hyperalgesia was also studied. Intrathecal agmatine in doses larger than 250 microg caused a decrease in the pain threshold, with vocalization and agitation lasting for several hours in all animals. Agmatine alone at 1-100 microg did not give rise to any change in the thermal withdrawal threshold in the contralateral non-inflamed paw. Agmatine pretreatment was found to dose-dependently attenuate the thermal hyperalgesia induced by intraplantar carrageenan. The effect of 100 microg agmatine was completely lost by 60 min, whereas the effect of 50 microg was of similar magnitude but exhibited a longer duration. Agmatine posttreatment had a slighter effect. Agmatine pretreatment (100 microg) together with 1 microg morphine (subeffective dose) has significantly higher anti-hyperalgesic effect then the individual compounds by themselves. These are the first data demonstrating the behavioral and anti-hyperalgesic effects of intrathecal agmatine. The results reveal important interactions between intrathecal agmatine and opioids in thermal hyperalgesia.

The antinociceptive effect of intrathecal kynurenic acid and its interaction with endomorphin-1 in rats

Kynurenic acid as an endogenous ligand antagonizes all types of ionotropic glutamate receptors, with preferential affinity for the glycine-binding site of the N-methyl-D-aspartate (NMDA) receptor. The purpose of the present study was to investigate the antinociceptive potency of continuously administered kynurenic acid on carrageenan-induced thermal hyperalgesia by means of a paw withdrawal test in awake rats. The possible interaction between kynurenic acid and the endogenous mu-opioid receptor agonist peptide, endomorphin-1, was examined in the same set-up. Kynurenic acid at the higher doses (1-4 microg/min) significantly decreased the thermal hyperalgesia and increased the paw withdrawal latencies on the non-inflamed side. These doses were also associated with motor impairment on both sides. Low doses of kynurenic acid (0.01-0.1 microg/min) potentiated, but did not prolong, the antinociceptive effect of endomorphin-1 (0.1-1 microg/min) on the inflamed side. There was no sign of motor impairment during the combined treatment. These findings demonstrate that the combination of low doses of these two endogenous ligands provides effective and well-controlled antinociception without side effects.

The significance of intrathecal catheter location in rats

Although chronic intrathecal catheterization is a widely used method in rats, few calibration experiments have been performed. In this study, we investigated the correlation between the side position of the catheter tip and the side differences observed in the motor and sensory disturbances after intrathecal administration of lidocaine to a large number of rats. The existence of a sensory block was determined by the paw withdrawal test. The motor impairment was assessed by observing the complete clubbing of the hindpaw and measuring the hindpaw grip strength. After experimental use, we established the position of the catheter tip. The catheter tips were variously located in all directions of the transverse plane in the rat spinal subarachnoid space. Lidocaine administration (100 or 500 microg/5 microL; n = 264 and 112, respectively) led to dose-dependent motor and sensory disturbances. The effect of 100 microg of lidocaine exhibited side differences; i.e., the extents of both motor (r = 0.77) and sensory (r = 0.60 and r = 0.67 for the right and the left side, respectively) disturbances correlated significantly with the location of the catheter tip. Our data have shown that detection of the paralytic and/or antinociceptive effect of small-dose lidocaine before planned experiments is a simple and reliable method for prediction of the location of the catheter tip. We suggest that the position of the catheter might cause side differences in the drug effect, especially if small doses of drugs are administered and their effects are investigated on both sides.

Antinociceptive effect of continuous intrathecal administration of endomorphin-1

&NA; Endomorphin‐1 is a novel endogenous opioid peptide with high affinity and selectivity for the &mgr;‐opioid receptor. Earlier results have shown that it causes antinociception in different pain tests, but its effect is short‐lasting. The purpose of the present study was to investigate the antinociceptive potency of continuously administered endomorphin‐1 on carrageenan‐induced thermal hyperalgesia by means of a paw withdrawal test in awake rats. The possible interaction between endomorphin‐1 and the C‐terminal octapeptide of the novel endogenous peptide nocistatin (bPNP‐3‐8P) was examined in the same experimental set‐up. Continuous administration of endomorphin‐1 (0.1, 0.3, 1 or 2 &mgr;g/min for 60 min) did not influence the paw withdrawal latencies of the normal paws. On the inflamed side, endomorphin‐1 dose‐dependently decreased the thermal hyperalgesia during continuous administration. The cessation of administration resulted in a gradual decrease in the antinociceptive effect of endomorphin‐1. bPNP‐3‐8P (0.003–30 &mgr;g, administered cumulatively) significantly decreased the heat hyperalgesia at higher doses (3 and 30 &mgr;g). Continuous administration of bPNP‐3‐8P (0.03, 0.1 and 1 &mgr;g/min) did not potentiate the antinociceptive effect of endomorphin‐1; instead, it even shortened the duration of its effect. The results demonstrate that continuous administration of endomorphin‐1 is an effective method of inhibiting thermal hyperalgesia in rats. Furthermore, the fragment bPNP‐3‐8P itself has low antinociceptive potency and does not potentiate the antinociceptive effect of endomorphin‐1 under these circumstances.

Evaluation of endomorphin-1 on the activity of Na+,K+-ATPase using in vitro and in vivo studies

The goal of this study was to investigate the effects of endomorphin-1 on Na(+),K(+)-ATPase activity in mouse brain synaptosome in vitro, and its antinociceptive interaction with the Na(+),K(+)-ATPase inhibitor ouabain. Endomorphin-1 (0.1 nM-10 microM) produced a concentration-dependent (EC(50): 43.19 nM, CI: 23.38-65.71 nM, E(max): 25.86%, CI: 24.53-27.20%), naloxone-reversible increase of the synaptosomal Na(+),K(+)-ATPase activity. The intrathecally (i.t.) administered endomorphin-1 (2-20 microg) produced a dose-dependent short-lasting increase in the tail-flick latency. Ouabain itself (1-1000 ng, i.t.) did not cause antinociception. Treatment with 10 ng ouabain significantly decreased the antinociceptive effect of 2 microg endomorphin-1, but none of the other combinations did significantly differ from the endomorhin-1-treated groups. These data indicate that endomorphin-1 increases the activity of Na(+),K(+)-ATPase in vitro but this effect may play a weak role in the antinociception induced by intrathecal endomorphin-1.

Inhibition of glucosylceramide synthase reversibly decreases the capsaicin-induced activation and TRPV1 expression of cultured dorsal root ganglion neurons.

&NA; Recent studies have demonstrated significant changes in the neuronal ganglioside status associated with altered functional states of nociceptive primary sensory neurons. In the present study, therefore, the effects of the inhibition of glucosylceramide synthase, the key enzyme of ganglioside synthesis, were studied on chemically defined populations and on the activation of TRPV1 of cultured adult rat sensory ganglion neurons. In control cultures, capsaicin resulted in the activation of TRPV1 in 29.7 ± 2.5% of the neurons, as assessed with the cobalt uptake assay. Pretreatment of the cultures for 4 days with an inhibitor of glucosylceramide synthase, d‐threo‐1‐phenyl‐2‐decanoylamino‐3‐morpholino‐1‐propanol (d‐PDMP), significantly decreased the proportion of capsaicin‐activated neurons to 11.6 ± 1.2%. Immunohistochemistry demonstrated that, in control cultures, 37.5 ± 1.4% of the neurons displayed TRPV1 immunoreactivity, whereas in d‐PDMP‐treated cultures the proportion of TRPV1‐immunoreactive neurons was diminished to 18.2 ± 2.1%. Further experiments disclosed that these effects of d‐PDMP were reversible. The capsaicin‐, but not the high potassium‐induced release of CGRP, was also significantly reduced after d‐PDMP treatment, as measured with ELISA. The proportions of IB4‐ and CGRP‐positive neurons were not significantly affected by d‐PDMP. The present observations demonstrate that inhibition of neuronal ganglioside synthesis profoundly modulates the expression of the TRPV1 receptor, apparently leaving other markers of nociceptive neurons, such as CGRP and IB4, unaffected. The findings indicate that as yet unidentified ganglioside(s) synthesized by the glucosylceramide synthase pathway may be essential for nociception through mechanisms which may implicate membrane lipid raft function and/or altered nerve growth factor signaling, which are essential for the TRPV1 receptor function.

Long-term changes in the antinociceptive potency of morphine or dexmedetomidine after a single treatment.

Acute tolerance develops after a single administration of opiate or &agr;2-adrenergic agonists, but the characteristics of the delayed type of acute tolerance have not been analyzed in acute and inflammatory thermal pain tests. We investigated the long-term changes in the antinociceptive potency of morphine (10 mg/kg) injected intraperitoneally and the &agr;2-adrenoceptor agonist dexmedetomidine (150 &mgr;g/kg intraperitoneally) on acute heat pain (tail-flick test) sensitivity and on carrageenan-induced inflammatory thermal hyperalgesia (paw withdrawal test) after a second injection 7 days later. The first treatment did not influence the baseline values on Day 8 in either test. In the tail-flick test, the antinociceptive potency of morphine, but not that of dexmedetomidine, was significantly decreased after repeated administration, suggesting a delayed type of acute tolerance to morphine. In contrast, the antihyperalgesic effect of morphine in the paw withdrawal test did not change after repeated injection, whereas the potency of dexmedetomidine was increased on Day 8. There were significant differences between the inflamed and noninflamed sides on Day 1 but not on Day 8, revealing an increased potency of the drugs on the inflamed side. There was no sign of cross-tolerance between the two drugs in either pain test. These data indicate long-term changes in the antinociceptive potency of morphine or dexmedetomidine after single treatment in different heat pain tests.