Anil K. Rattan

Role of spinal opioid receptors in the antinociceptive interactions between intrathecal morphine and bupivacaine.

In studies on the clinical management of pain, a combination of morphine and bupivacaine is more effective than either of them alone in producing analgesia. The present study was designed to examine the effect of bupivacaine on morphine-induced antinociception as measured by the tail-flick test in the rat. To understand the basis of this interaction, the effect of bupivacaine on the binding of opioid ligands to their spinal opioid receptors in the rat also was investigated. Intrathecal administration of 5, 20, or 50 micrograms bupivacaine significantly potentiated the antinociception produced by intrathecal administration of 10 micrograms morphine. There was more than a 10-fold increase in the area under the curve (AUC0-60 min) for morphine-induced antinociception in the presence of bupivacaine. At higher doses of morphine (20 micrograms), bupivacaine was not very effective, increased AUC0-60 min for antinociception by only about 25%, and in fact significantly decreased the total duration of morphine-induced antinociception. Radioreceptor assays done with rat spinal cord membrane preparations revealed that bupivacaine (0.1-10 nM) inhibited the binding of specific ligands to mu-receptors but increased the binding to delta- and kappa-receptors. The authors conclude that the facilitation of morphine-induced antinociception by bupivacaine may be associated with a conformational change in the spinal opioid receptors induced by bupivacaine. Although increasing the binding of morphine to kappa-opioid receptors is the most prominent effect, the binding of opioid ligands to all spinal receptors is inhibited at high doses of bupivacaine.

Inhibition of Morphine-induced Tolerance and Dependence by a Benzodiazepine Receptor Agonist Midazolam in the Rat

We investigated whether midazolam administration influenced morphine-induced antinociception and tolerance and dependence in the rat. Antinociception was assessed by the tail-flick (TF) and the hot-plate test (HP 52°C). Morphine tolerance developed after daily single injections of morphine for 11 days. The effect of midazolam on morphine-induced antinociception and tolerance was assessed by giving daily injections of various doses of midazolam for 11 days. The first injection of saline or midazolam was given intraperitoneally and 30 min later morphine (10 mg/kg body weight) was administered subcutaneously. Antinociception was monitored by measuring TF and HP latencies 60 min after the second injection. Midazolam was injected at four different concentrations: 0.03, 0.1, 0.3, and 3 mg/kg body weight. Chronic administration of morphine resulted in the development of tolerance to antinociception in both TF and HP tests, with rats exhibiting baseline antinociception on Day 9. Animals treated with midazolam alone showed little antinociception on Days 3–9. However, midazolam administration in morphine-treated animals attenuated morphine-induced tolerance to antinociception on Days 1–11 as measured by the tail-flick test. Midazolam also decreased the jumping behavior following naloxone injections in morphine-dependent rats. These results suggest that midazolam may prolong the effects of morphine by delaying morphine-induced development of tolerance to antinociception. Midazolam also attenuated a decrease in weight gain induced by chronic injections of morphine.

The effect of morphine tolerance dependence and abstinence on immunoreactive dynorphin (1–13) levels in discrete brain regions, spinal cord, pituitary gland and peripheral tissues of the rat

The effect of morphine tolerance dependence and protracted abstinence on the levels of dynorphin (1-13) in discrete brain regions, spinal cord, pituitary gland and peripheral tissues was determined in male Sprague-Dawley rats. Of all the tissues examined, the highest level of dynorphin (1-13) was found to be in the pituitary gland. Among the brain regions and spinal cord examined, the levels of dynorphin (1-13) in descending order were: hypothalamus, spinal cord, midbrain, pons and medulla, hippocampus, cortex, amygdala and striatum. The descending order for the levels of dynorphin (1-13) in peripheral tissues was: adrenals, heart and kidneys. In morphine tolerant rats, the levels of dynorphin (1-13) increased in amygdala but were decreased in pons and medulla. In morphine abstinent rats, the levels of dynorphin (1-13) were increased in amygdala, hypothalamus and hippocampus. The levels of dynorphin (1-13) were increased in pituitary but decreased in spinal cord and remained so even during protracted abstinence. The levels of dynorphin (1-13) in the peripheral tissues of morphine tolerant rats were unaffected. However, in the heart and kidneys of morphine abstinent rats, the levels of dynorphin (1-13) were increased significantly. It is concluded that both morphine tolerance and abstinence modify the levels of dynorphin (1-13) in pituitary, central and peripheral tissues. Morphine abstinence differed from non-abstinence process in that there were additional changes (increases) in the levels of dynorphin (1-13) in brain regions (hypothalamus and hippocampus) and peripheral tissues (heart and kidneys) and may contribute to the symptoms of the morphine abstinence syndrome.(ABSTRACT TRUNCATED AT 250 WORDS)

DIFFERENTIAL EFFECTS OF INTRATHECAL MIDAZOLAM ON MORPHINE-INDUCED ANTINOCICEPTION IN THE RAT : ROLE OF SPINAL OPIOID RECEPTORS

The antinociceptive effects of an intrathecally administered benzodiazepine agonist midazolam, alone and in combination with morphine, were examined in the rat by using the tail-flick test. The duration of antinociceptive effect produced by midazolam was significantly less (P less than 0.05) than that produced by morphine. Low doses of midazolam (10 micrograms) and morphine (10 micrograms) produced a synergistic effect in prolonging antinociceptive effect. However, at higher doses (20 or 30 micrograms), these drugs reduced the extent of antinociception produced by each other. Naloxone administration prevented antinociception produced by these drugs, indicating interactions between midazolam and opioid receptors. Midazolam had dual effects on the binding of opioid ligands to the spinal opioid receptors. At low dose, it potentiated the displacement of [3H]naloxone by morphine. At higher doses, midazolam inhibited the binding of opioid ligands to their spinal receptors in the following order: kappa greater than delta greater than mu. These results indicate that differential antinociceptive effects of midazolam on morphine-induced antinociception involve interaction of this benzodiazepine with spinal opioid receptors.

Effect of Chronic Treatment with Morphine, Midazolam, and Both Together on β-Endorphin Levels in the Rat

We have recently reported that a short-acting anesthetic and analgesic drug midazolam can produce analgesia and decrease morphine tolerance and dependence in the rat by interacting with the opioid system. This study was designed to investigate the effect of midazolam, morphine, and both together on beta-endorphin levels in the rat. Male Sprague-Dawley rats were divided into four groups: (1) saline-saline; (2) saline-morphine; (3) midazolam-saline, and (4) midazolam-morphine groups. First, saline or midazolam injection was given IP and after 30 min a second injection of saline or morphine was given subcutaneously once daily for 11 days. Animals were sacrificed on 11th day 60 min after the last injection, to measure beta-endorphin by radioimmunoassay. Saline-morphine-treated animals showed a significant increase in beta-endorphin levels in the cortex, pons, medulla, lumbar spinal cord, adrenals, and spleen, and a decrease only in its level in pituitary. Midazolam-saline-treated animals showed a significant increase in beta-endorphin levels only in the medulla, and a decrease in its levels in hippocampus, striatum, and adrenals. Saline-morphine-treated animals did not show any changes in plasma beta-endorphin, but animals treated with midazolam-saline had a significant decrease in plasma beta-endorphin. In rats treated with morphine and midazolam together, beta-endorphin levels in cortex, lumbar spinal cord, and spleen decreased to the similar levels observed in rats treated with saline-saline; in pons and cervical spinal cord the levels were even lower than that found in saline-saline group. The decrease in pituitary beta-endorphin in morphine-midazolam-treated rats was due to morphines own activity, whereas the decrease in plasma beta-endorphin in hippocampus in the morphine-midazolam group was a synergistic effect of morphine and midazolam. The beta-endorphin level in adrenal glands in the morphine-midazolam-treated animals was not different from that found in rats treated with morphine alone but was still higher than that in the saline-saline group. In general, it appears that chronic treatment with morphine stimulates the beta-endorphinergic system. A concomitant treatment with midazolam abolishes the stimulatory effect of morphine on the beta-endorphinergic system. These results may help us in understanding the intrinsic mechanisms involved in narcotic tolerance and dependence.

Effect of chronic treatment with morphine, midazolam and both together on dynorphin(1–13) levels in the rat

We have recently reported that midazolam, a benzodiazepine receptor agonist that is also a short acting anesthetic and analgesic drug, can produce analgesia and decrease morphine tolerance and dependence in the rat by interacting with the opioidergic system. This study was designed to investigate the chronic effect of midazolam and/or morphine on the levels of dynorphin(1-13) in the pituitary gland, different brain regions, spinal cord and peripheral tissues of the rat. Four sets of animals were used: (I) saline-saline; (II) midazolam (0.03, 0.3 or 3.0 mg/kg, body wt., i.p.)-saline; (III) saline-morphine (10.0 mg/kg, body wt., s.c.); and (IV) midazolam-morphine (0.03, 0.3 or 3.0 mg/kg midazolam + 10.0 mg/kg morphine) groups. The first saline or midazolam injection was given i.p. and after 30 min, the second injection of saline or morphine was given s.c. daily for 11 days. Animals were sacrificed on the 11th day, 60 min after the last injection and dynorphin(1-13) was measured in indicated tissues by radioimmunoassay method. The midazolam treated animals showed a significant decrease in dynorphin(1-13) levels in the cortex, cerebellum, cervical region of spinal cord, heart and adrenals, and a significant increase in the hypothalamus, striatum and lumbar region of the spinal cord. The morphine treated animals showed a significant decrease in dynorphin(1-13) levels in the pituitary gland, hypothalamus, hippocampus, striatum, cerebellum, pons, medulla, kidneys, adrenals and spleen, and a significant increase only in the lumbar region of the spinal cord. When both drugs were injected together there was no effect on pituitary gland, kidneys and spleen. These drugs antagonize each others effect on dynorphin(1-13) in the hypothalamus, striatum, cerebellum, pons, medulla and heart. However, the midazolam-morphine combination significantly increases dynorphin(1-13) levels in the hippocampus, cortex, midbrain, cervical and lumbar regions of the spinal cord, and adrenals. These results suggest the involvement of dynorphin(1-13) in the inhibition of morphine-induced tolerance and dependence by midazolam in the rat. These results may also help us in understanding the intrinsic mechanisms involved in narcotic tolerance and dependence.

Met-enkephalin alteration in the rat during chronic injection of morphine and/or midazolam

We have recently reported that the short-acting anesthetic and analgesic drug midazolam can produce analgesia and decrease morphine tolerance and dependence in the rat by interacting with the opioid system. This study was designed to investigate the effect of midazolam, morphine, and both together on met-enkephalin levels in the rat. Male Sprague-Dawley rats were divided into four groups: (1) saline-saline; (2) saline-morphine; (3) midazolam-saline, and (4) midazolam-morphine groups. First, a saline or midazolam injection was given intraperitoneally and after 30 min a second injection of saline or morphine was given subcutaneously once daily for 11 days. Animals were sacrificed on the 11th day 60 min after the last injection to measure met-enkephalin by radioimmunoassay. Morphine tolerant animals showed a significant increase in met-enkephalin levels in the cortex (137%) and midbrain (89%), and a significant decrease in met-enkephalin levels in the pituitary (74%), cerebellum (34%) and medulla (72%). Midazolam treated animals showed a significant decrease in met-enkephalin levels in the pituitary (63%), cortex (39%), medulla (58%), kidneys (36%), heart (36%) and adrenals (43%), and a significant increase in met-enkephalin levels in the striatum (54%) and pons (51%). When morphine and midazolam were injected together, midazolam antagonized the increase in met-enkephalin levels in cortex and midbrain region and the decrease in met-enkephalin level in the medulla region observed in morphine tolerant animals. These results indicate that morphine tolerance and dependence is associated with changes in the concentration of met-enkephalin in the brain. Midazolam may inhibit morphine tolerance and dependence by reversing some of the changes induced in met-enkephalin levels in brain by morphine in morphine tolerant and dependent animals.

Effect on morphine-induced catalepsy, lethality, and analgesia by a benzodiazepine receptor agonist midazolam in the rat

Previously we have shown that intrathecal administration of midazolam can increase or decrease morphine-induced antinociception, depending upon relative concentration of these drugs by modulating spinal opioid receptors, and it also can inhibit morphine-induced tolerance and dependence in the rat. Now we report that midazolam also influences catalepsy, lethality, and analgesia induced by morphine in the rat. In the acute treatment, animals were first treated with saline or midazolam (0.03 to 30.0 mg/kg, b.wt., IP), and 30 min later with a second injection of saline or morphine (1.0 to 100.0 mg/kg, b.wt., SC). The catalepsy was measured 60 min after the second injection and lethality was checked after 24 h. Midazolam injection increased the morphine-induced catalepsy and lethality. In the chronic treatment, animals were injected with two injections daily for 11 days. The first injection consisted of saline or midazolam (0.03 to 3.0 mg/kg, b.wt., IP), and 30 min later with a second injection of saline or morphine (10.0 mg/kg, b.wt., IP) was given. Lethality, antinociception, and body weight were measured. Chronic morphine treatment also increased lethality in a dose-dependent manner. Chronic treatment with midazolam and morphine increased the antinociception on day 11, as measured in the tail-flick and hot-plate tests. Midazolam administration also prevented the morphine-induced weight loss. These results suggest a strong interaction between midazolam and morphine in altering catalepsy, lethality, and analgesia in rat.

Antagonism of antinociception produced by intrathecal clonidine by ketorolac in the rat : The role of the opioid system

The management of severe pain may require “balanced analgesia,” involving the use of analgesics with different modes of action. Clonidine, an &agr;2-adrenoreceptor agonist produces analgesia by itself as well as when given with morphine and local anesthetics. Ketorolac is indicated for the management of moderately severe acute pain and causes analgesia equivalent to morphine. This study was designed to investigate whether the addition of ketorolac promotes antinociception produced by intrathecal administration of clonidine in male Sprague-Dawley rats. Intrathecal injection of clonidine (1–30 &mgr;g) induced a dose-dependent increase in antinociception as measured by the tail flick (TF) and hot plate tests. Ketorolac alone (150–600 &mgr;g) increased the antinociception by 50%–60% only in the TF test. Ketorolac (10 &mgr;g) decreased clonidine (10 &mgr;g)-induced antinociception from 69.1% ± 7.8% to 23.5% ± 1.6% (P < 0.05) in the TF test and 35.7% ± 4.7% to 4.5% ± 0.1% (P < 0.05) maximum possible effect in the hot plate test. Ketorolac also antagonized the effect of 30 &mgr;g of clonidine. The opioid receptor antagonist naloxone antagonized the antinociceptive effect of clonidine and ketorolac, indicating the involvement of the opioid system in the antinociception produced by clonidine or ketorolac. However, neither clonidine nor ketorolac (10−8 to 10−3 M) inhibited the binding of specific ligands to &mgr;-, &dgr;-, and &kgr;-opioid receptors, indicating a lack of direct interaction of clonidine and ketorolac with opioid receptors. These results suggest that intrathecal injection of ketorolac antagonizes the antinociception produced by clonidine. Implications Clonidine and ketorolac are two important drugs used to give pain relief to patients. We observed that ketorolac inhibits clonidine-induced analgesia in the rat. We recommend that this drug interaction should be taken into account when both clonidine and ketorolac are used together to alleviate pain in patients.

The neurotoxic actions of ibotenic acid on cholinergic and opioid peptidergic systems in the central nervous system of the rat

The neurotoxic effects produced by ibotenic acid (IA) induced chemical lesions of the central nervous system (CNS) cholinergic system were examined on the opioid peptidergic system in adult rats. Forebrain cholinergic systems were bilaterally lesioned by the infusion of IA (1 or 5 micrograms/site) into the nucleus basalis magnocellularis (NBM). One week after the injections, the animals were sacrificed, and activities of acetylcholinesterase (AChE), choline acetyltransferase (ChAT) and concentrations of beta-endorphin (beta-End) and Met-enkephalin (Met-Enk) were measured in different brain regions. Animals treated with IA showed a decrease in the activity of ChAT (-24%), AChE (-36%) and beta-End level (-33%) in the frontoparietal cortex (FC). For the first time we report that these changes were associated with a compensatory increase in the activity of ChAT (+27%), AChE (+25%), beta-End level (+66%) in the remaining part of the cortex, i.e. cortex devoid of frontal cortex (C-FC). Met-enkephalin level increased by 59% in the frontoparietal cortex and did not change in the cortex devoid of frontal cortex upon IA treatment. These results suggest that IA treatment results in changes in the activity of cortical ChAT and AChE, and beta-End level in the same direction. Injection of IA in the NBM did not cause a change in the activity of ChAT or AChE in other brain regions such as hippocampus, striatum or midbrain. In addition to cortex devoid of frontal cortex, midbrain also showed a significant increase in the beta-End level in the IA treated animals. However, pituitary beta-End decreased in the neurotoxin treated animals.(ABSTRACT TRUNCATED AT 250 WORDS)