Opioid and nonopioid mechanisms may contribute to dynorphin's pathophysiological actions in spinal cord injury

It has been suggested that the opioid dynorphin, an endogenous agonist for kappa-opiate receptors, contributes to secondary tissue damage after spinal cord injury. To evaluate this hypothesis further, effects of intrathecally administered dynorphin (Dyn) A-(1-17), dynorphin antiserum, or the kappa-selective opiate antagonist nor-binaltorphimine (nor-BNI) were studied in rats subjected to standardized impact trauma to the thoracic spinal cord. Effects of intrathecal Dyn A-(1-17) were also compared to those of Dyn A-(2-17), which is inactive at opiate receptors, in uninjured and injured animals. Both Dyn A-(1-17) and Dyn A-(2-17) produced motor dysfunction in uninjured rats, but Dyn A-(1-17) was approximately 2.5 times more potent. At lower doses of Dyn A-(1-17), paraparesis was markedly attenuated by nor-BNI; nor-BNI was less effective at higher doses of Dyn A-(1-17) and did not modify the motor dysfunction produced by Dyn A-(2-17). Treatment with dynorphin antiserum significantly improved outcome after trauma as compared to control treatment with normal rabbit serum or leucine-enkephalin antiserum. Dyn A-(1-17), but not Dyn A-(2-17) at similar doses, exacerbated neurological dysfunction after spinal cord injury. Pretreatment with nor-BNI attenuated neurological dysfunction after traumatic spinal cord injury to a similar degree in rats administered saline or Dyn A-(1-17). These observations support the hypothesis that dynorphin contributes to certain pathophysiological changes after traumatic spinal cord injury through both opiate-receptor (kappa-receptor)-mediated and nonopioid mechanisms.     

Mixed opioid/nonopioid effects of dynorphin and dynorphin related peptides after their intrathecal injection in rats

Dynorphin dose-dependently increased the tail flick latency of rats to radiant heat following its intrathecal injection. This effect was accompanied by an alteration in motor function that was characterized by a flaccid extension of the hindlimbs and flaccidity of the tail. Naloxone (10 but not 1 mg/kg) blocked the antinociceptive effect and motor disturbance produced by dynorphin. The non-opioid analogue des-Tyr1-dynorphin(1-13) also increased tail flick latency and produced paralysis. Dynorphin(1-8) significantly elevated tail flick latency without affecting motor function. Furthermore, the effect of dynorphin(1-8) was blocked by 1 mg/kg naloxone. These data suggest a possible physiological role of dynorphin in influencing motor function in the spinal cord and a role of dynorphin(1-8) in modulating pain transmission. Another finding of the present study was that dynorphin was approximately ten times more potent in producing its effects when injected one day after surgery compared to when it when it was injected one week or more after surgery.     

Structure-activity analysis of dynorphin A toxicity in spinal cord neurons: intrinsic neurotoxicity of dynorphin A and its carboxyl-terminal, nonopioid metabolites

Dynorphin A [dynorphin A (1-17)] is an endogenous opioid peptide that is antinociceptive at physiological concentrations. Levels of dynorphin A increase markedly following spinal cord trauma and may contribute to secondary neurodegeneration. Both kappa opioid and N-methyl-d-aspartate (NMDA) receptor antagonists can modulate the effects of dynorphin, suggesting that dynorphin is acting through kappa opioid and/or NMDA receptor types. Despite these findings, few studies have critically examined the mechanisms of dynorphin A neurotoxicity at the cellular level. To better understand how dynorphin affects cell viability, structure-activity studies were performed examining the effects of dynorphin A and dynorphin A-derived peptide fragments on the survival of mouse spinal cord neurons coexpressing kappa opioid and NMDA receptors in vitro. Time-lapse photography was used to repeatedly follow the same neurons before and during experimental treatments. Dynorphin A caused significant neuronal losses that were dependent on concentration (> or = 1 microM) and duration of exposure. Moreover, exposure to an equimolar concentration of dynorphin A fragments (100 microM) also caused a significant loss of neurons. The rank order of toxicity was dynorphin A (1-17) > dynorphin A (1-13) congruent with dynorphin A (2-13) congruent with dynorphin A (13-17) (least toxic) > dynorphin A (1-5) ([Leu(5)]-enkephalin) or dynorphin A (1-11). Dynorphin A (1-5) or dynorphin A (1-11) did not cause neuronal losses even following 96 h of continuous exposure, while dynorphin A (3-13), dynorphin A (6-17), and dynorphin A (13-17) were neurotoxic. The NMDA receptor antagonist MK-801 (dizocilpine) (10 microM) significantly attenuated the neurotoxic effects of dynorphin A and/or dynorphin-derived fragments except dynorphin A (13-17), suggesting that the neurotoxic effects of dynorphin were largely mediated by NMDA receptors. Thus, toxicity resides in the carboxyl-terminal portion of dynorphin A and this minimally includes dynorphin A (3-13) and (13-17). Our findings suggest that dynorphin A and/or its metabolites may contribute significantly to neurodegeneration during spinal cord injury and that alterations in dynorphin A biosynthesis, metabolism, and/or degradation may be important in determining injury outcome.     

Prostaglandin E2 release evoked by intrathecal dynorphin is dependent on spinal p38 mitogen activated protein kinase

Spinal dynorphin has been hypothesized to play a pivotal role in spinal sensitization. Although the mechanism of this action is not clear, several lines of evidence suggest that spinal dynorphin-induced hyperalgesia is mediated through an increase in spinal cyclooxygenase products via an enhanced N-methyl-D-aspartate (NMDA) receptor function. Spinal NMDA-evoked prostaglandin release and nociception has been linked to the activation of p38 mitogen activated protein kinase (p38). In the present work, we show that intrathecal delivery of an N-truncated fragment of dynorphin A, dynorphin A 2-17 (dyn2-17), which has no activity at opioid receptors, induced a 8-10-fold increase in phosphorylation of p38 in the spinal cord. The increase in phosphorylated p38 was detected in laminae I-IV of the dorsal horn. Moreover, confocal microscopy showed that the activation of p38 occurred in microglia, but not in neurons or astrocytes. In awake rats, prepared with chronically placed intrathecal loop dialysis catheters, the concentration of prostaglandin E2 in lumbar cerebrospinal fluid was increased 5-fold by intrathecal administration of dyn2-17. Injection of SD-282, a selective p38 inhibitor, but not PD98059, an ERK1/2 inhibitor, attenuated the prostaglanin E2 release. These data, taken together, support the hypothesis that dynorphin, independent of effects mediated by opioid receptors, has properties that can induce spinal sensitization and indicates that dyn2-17 effects may be mediated through activation of the p38 pathway. These studies provide an important downstream linkage where by dynorphin may act through a non-neuronal link to induce a facilitation of spinal nociceptive processing.     

Intrathecal dynorphin A administration causes pressor responses in rats associated with an increased resistance to spinal cord blood flow

Hemodynamic responses (blood pressure, as well as cardiac output (CO), peripheral and CNS blood flow changes measured via radioactive microspheres) were analyzed in anesthetized rats 2 min following intrathecal (IT) administration (10 microliters) of either 5-ion control solution or 20 nmol of dynorphin A(1-13) into the lower thoracic space (T10-T12). Mean arterial pressure (MAP) significantly increased within 2 min following IT dynorphin A(1-13) due to rise in total peripheral resistance, whereas CO significantly declined. Two minutes post-IT-dynorphin A(1-13) administration spinal cord blood flow also significantly decreased for 2 cm anterior and 1 cm posterior from the tip of the spinal catheter, which reflected a significant elevation in tissue flow resistance of spinal cord vessels in spite of the reduction of CO. As well, tissue blood flow resistance was also increased at this time in the kidneys and adrenal glands. The results indicate that within 2 min after intrathecal dynorphin A(1-13) administration an acute increase in blood flow resistance of spinal cord vessels around the tip of the spinal catheter occurs, at a time when the animal is also hypertensive. It is suggested that the associated pressor response may, in part, be caused by dynorphin A evoking localized ischemia.     

Effect of calcium ion on analgesia of opioid peptides

The analgesic effect produced by subcutaneous injection (SC) of morphine was antagonized by intracerebroventricular (ICV) but not intrathecal (ITH) injection of CaCl2. While ITH CaCl2 was devoid of any effect on the analgesia induced by ITH morphine, it did antagonize the analgesic effect produced by ITH injection of dynorphin A or (D-Pen2,D-Pen5)-enkephalin (DPDPE). In accordance with this, the uptake of 45Ca by synaptosomes prepared from the dorsal column of rat spinal cord was suppressed by dynorphin A or DPDPE, but not by morphine. The results imply that different kinds of opioid ligands might have different mechanisms of action at discrete areas of the CNS. Blockade of neuronal Ca2+ uptake may serve as an important mechanism for morphine analgesia in the brain as well as DPDPE and dynorphin A analgesia in the spinal cord.     

Decreased neuropeptide content in the spinal cord of aged rats: the effect of GM1 ganglioside

This study investigated the status of substance P (SP), methionine-enkephalin (Met-Enk) and dynorphin A(1-13) (Dyn A) in the spinal cord of aged Sprague-Dawley rats and the effect of GM1 ganglioside on these neuropeptides. SP and Met-Enk, but not Dyn A, were decreased in both dorsal and ventral horns of the aged spinal cord. Treatment with GM1 ganglioside (30 mg/kg i.p., daily for 30 days) restored, in part, the neuropeptide deficits in the ventral horns, but not in the dorsal horns. This information might be important for understanding the sensory and motor deficits associated with ageing, and how the spinal cord neuropeptides might be amplified in the aged spinal cord.     

Dynorphin increases extracellular levels of excitatory amino acids in the brain through a non-opioid mechanism.

Administration of dynorphin A-(1-17) (Dyn 1-17), through a microdialysis probe stereotaxically placed into rat hippocampus, caused marked increases in the extracellular levels of glutamate and aspartate. The degree and duration of elevation of these excitatory amino acids (EAA) induced by Dyn 1-17 were dose dependent but were not modified by the centrally active opioid receptor antagonist nalmefene. At comparable doses, Dyn 2-17, which is inactive at the opioid receptor, produced similar alterations in EAA as Dyn 1-17, whereas Dyn 1-8 caused significantly smaller changes of glutamate. Dynorphin and EAAs have each been implicated as pathophysiological factors in brain or spinal cord injuries, with dynorphin's actions shown to involve both opioid and non-opioid components. The present observations indicate a direct potential linkage between dynorphin and excitotoxin mechanisms of CNS injury and provide further support for the concept that dynorphin's pathophysiologic effects may include non-opioid actions of this peptide.     

兔脊髓损伤后脊髓运动诱发电位D波与后肢运动功能的关系

目的 探讨兔脊髓损伤模型损伤后双后肢肌力和脊髓运动诱发电位D波之间的关系.方法 45只兔按随机数字表法分为打击组(显露硬脊膜并打击,并按打击能量大小分为50gcf组、75 gcf组、100gcf组、125 gcf组、150gcf组、175 gcf组、200 gcf组、250 gcf组)和对照组(显露硬脊膜但不打击).打击后和第4周末记录兔双后肢肌力、运动诱发电位D波的潜伏期和波幅,并在第4周末取家兔脊髓行神经微丝(NF)免疫组化染色及病理形态观察,测量NF吸光度值.结果 脊髓损伤当时和第4周末脊髓硬膜外运动诱发电位D波波幅下降的幅度与兔后肢运动功能呈线性相关关系(R2=0.98,P＜0.001,回归曲线Y=0.33+4.82X;R2=0.99,P＜0.001,回归曲线Y=0.04+0.51X),而D波潜伏期的变化则与术后肌力的变化无相关关系(R2=-0097,P=0.573;R2=-0.165,P=0.337);NF吸光度值随着D波波幅的下降而下降,两者呈线性相关关系(R2=0.87,P＜0.001,回归曲线Y=0.12+0.58X).结论 在脊髓损伤中可以通过脊髓硬膜外运动诱发电位D波波幅的下降程度作为判断脊髓损伤运动功能障碍程度的指标.

Abstract：

Objective To study the relationship between D-wave of thc spinal motor evoked potential and hindlimb motor function status (myodynamia) of rabbits with experimental spinal cord injury. Methods Forty-five rabbits were randomly divided into a control group and 8 injured groups (receiving Allen's injury of50, 75, 100, 125, 150, 175, 200 and 250 gcf, respectively). The myodynamia of hindlimbs and the latency and amplitude of D-wave of the spinal motor evoked potential were recorded right after the injury and in the 4th week of injury, respectively. Simultaneously in the 4th week of injury,the rabbits' spinal sections were stained with neurofilament (NF) immunohistochemistry and their pathological morphologies were observed; and optical densities of NF were measured. Statistic analysis of correlation between myodynamia of hindlimbs and latency and amplitude of D-wave was performed.Results A statistic linear correlation was found between amplitudes declination of D-wave of the spinal motor evoked potential and modified Tarlov's scores right after the injury and in the 4th week of injury (R2=0.98, P＜0.00, regression curve: Y=0.33+4.82X; R=0.99, P＜0.001, regression curve: Y=0.04+0.5 1X),but no statistical correlation was noted between myodynamia and the latency of D-wave of the spinal motor evoked potential at the same time periods(R2=-0.097, P=0.573; R2=-0.165, P=0.337). And the optical density of NF decreased following the amplitude declination of D-wave of the spinal motor evoked potential with linear correlation (R2=0.87, P＜0.001, regression curve: Y= 0.12+0.58X).Conclusion Amplitude declination of D-wave of the spinal motor evoked potential could be used to evaluate the severity of dyskinesia in the injured spinal cord.     

Differential effects of N-peptidyl-O-acyl hydroxylamines on dynorphin-induced antinociception in the mouse capsaicin test

In the capsaicin test, intrathecal (i.t.) dynorphins are antinociceptive. Cysteine protease inhibitors such as p-hydroxymercuribenzoate (PHMB) given i.t. augment and prolong their activity. The effect of two novel cysteine protease inhibitors, N-peptidyl-O-acyl hydroxylamines, on the antinociception induced by i.t. administered dynorphin A or dynorphin B has been investigated. When administered i.t. 5 min before the injection of capsaicin (800 ng) into the plantar surface of the hindpaw, dynorphin A (62.5-1000 pmol) or dynorphin B (0.5-4 nmol) produced a dose-dependent and significant antinociceptive effect. The effect of dynorphin A (1 nmol) and dynorphin B (4 nmol) disappeared completely within 180 and 60 min, respectively. PHMB (2 nmol) and Boc-Tyr-Gly-NHO-Bz (BYG-Bz) (2 nmol) co-administered with dynorphin A or dynorphin B significantly prolonged antinociception induced by both. On the other hand, Z-Phe-Phe-NHO-Bz (ZFF-Bz) (1 and 2 nmol) only prolonged antinociception induced by dynorphin A. The results suggest that Z-Phe-Phe-NHO-Bz is an inhibitor of cysteine proteases preferring cleavage of dynorphin A, with less specificity towards dynorphin B in the mouse spinal cord.     

Differential effects of 5-HT1 and 5-HT2 receptor agonists on hindlimb movements in paraplegic mice

The effects induced by serotonergic (5-HT) agonists of the 5-HT1 and 5-HT2 subclasses were examined on hindlimb movement generation in adult mice completely spinal cord transected at the low thoracic level. One week postspinalization, intraperitoneal injection (0.5-10 mg/kg) of meta-chlorophenylpiperazine (m-CPP; 5-HT(2B/2C) agonist) or trifluoromethylpiperazine (TFMPP; 5-HT(1B) agonist) failed to induce locomotor-like movements. However, dose-dependent nonlocomotor movements were induced in air-stepping condition or on a motor-driven treadmill. In contrast, hindlimb locomotor-like movements were found after the injection of quipazine (5-HT(2A/2C) agonist; 1-2 mg/kg). Combined with L-DOPA (50 mg/kg, i.p.), low doses of quipazine but not of m-CPP and TFMPP produced locomotor-like and nonlocomotor movements in air-stepping condition or on the treadmill. Subsequent administration of m-CPP or TFMPP significantly reduced and often completely abolished the hindlimb movements induced by quipazine and L-DOPA. Altogether, these results demonstrate that 5-HT(2A/2C) receptor agonists promote locomotion while 5-HT(1B) and 5-HT(2B/2C) receptor agonists interfere with locomotor genesis in the hindlimbs of complete paraplegic mice. These results suggest that only subsets of spinal 5-HT receptors are specific to locomotor rhythmogenesis and should be activated to successfully induce stepping movements after spinal cord injury.     

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. The lower levels of dynorphin (1–13) in spinal cord may be responsible for the potentiation of morphine effects by κ-opiate agonist in morphine tolerant dependent rodents.     

Dynorphin A (1-13) neurotoxicity in vitro: opioid and non-opioid mechanisms in mouse spinal cord neurons

Dynorphin A is an endogenous opioid peptide that preferentially activates kappa-opioid receptors and is antinociceptive at physiological concentrations. Levels of dynorphin A and a major metabolite, dynorphin A (1-13), increase significantly following spinal cord trauma and reportedly contribute to neurodegeneration associated with secondary injury. Interestingly, both kappa-opioid and N-methyl-D-aspartate (NMDA) receptor antagonists can modulate dynorphin toxicity, suggesting that dynorphin is acting (directly or indirectly) through kappa-opioid and/or NMDA receptor types. Despite these findings, few studies have systematically explored dynorphin toxicity at the cellular level in defined populations of neurons coexpressing kappa-opioid and NMDA receptors. To address this question, we isolated populations of neurons enriched in both kappa-opioid and NMDA receptors from embryonic mouse spinal cord and examined the effects of dynorphin A (1-13) on intracellular calcium concentration ([Ca2+]i) and neuronal survival in vitro. Time-lapse photography was used to repeatedly follow the same neurons before and during experimental treatments. At micromolar concentrations, dynorphin A (1-13) elevated [Ca2+]i and caused a significant loss of neurons. The excitotoxic effects were prevented by MK-801 (Dizocilpine) (10 microM), 2-amino-5-phosphopentanoic acid (100 microM), or 7-chlorokynurenic acid (100 microM)--suggesting that dynorphin A (1-13) was acting (directly or indirectly) through NMDA receptors. In contrast, cotreatment with (-)-naloxone (3 microM), or the more selective kappa-opioid receptor antagonist nor-binaltorphimine (3 microM), exacerbated dynorphin A (1-13)-induced neuronal loss; however, cell losses were not enhanced by the inactive stereoisomer (+)-naloxone (3 microM). Neuronal losses were not seen with exposure to the opioid antagonists alone (10 microM). Thus, opioid receptor blockade significantly increased toxicity, but only in the presence of excitotoxic levels of dynorphin. This provided indirect evidence that dynorphin also stimulates kappa-opioid receptors and suggests that kappa receptor activation may be moderately neuroprotective in the presence of an excitotoxic insult. Our findings suggest that dynorphin A (1-13) can have paradoxical effects on neuronal viability through both opioid and non-opioid (glutamatergic) receptor-mediated actions. Therefore, dynorphin A potentially modulates secondary neurodegeneration in the spinal cord through complex interactions involving multiple receptors and signaling pathways.     

Dynorphin A: in vivo release in the spinal cord of the cat

The antibody microprobe technique was used to study the release of immunoreactive dynorphin A within the lower lumbar spinal cord of anaesthetised cats. A basal release was observed in the dorsal horn, centered in the region of lamina I, but was abolished by spinal cord transection at the thoracolumbar junction. Release of dynorphin A in the lamina I region was evoked by high-frequency electrical stimulation of unmyelinated primary afferent fibres, whereas stimulation of myelinated (including A delta) afferents was ineffective. Evidence was also obtained for release in laminae V-VI and at the spinal cord surface. These results suggest that in the lumbar spinal cord of the cat, dynorphin A is released in the superficial dorsal horn by impulses in descending pathways and in somatic unmyelinated primary afferent fibres.     

A strychnine-sensitive site is involved in dynorphin-induced paralysis and loss of the tail-flick reflex

Dynorphin A(1-13) administered intrathecally to rats induces a reversible hindlimb paralysis and permanent loss of the tail-flick reflex in a dose-dependent and all-or-none manner. The loss of the tail-flick reflex has been determined to result from neurotoxicity linked to the N-methyl-D-aspartate (NMDA) receptor. Recently, it has been reported that NMDA antagonists attenuate irreversible paralysis induced by dynorphin A(1-17) and dynorphin A(2-17). In the present studies, we examined whether repeated injections of dynorphin A(1-13) acetate salt could change the characteristics of the reversible paralysis. Injections repeated every 48 h resulted in hindlimb paralysis upon each injection which was not different in terms of magnitude or duration (P greater than 0.60). Injections repeated at 2 h intervals resulted in desensitization of the paralytic effects (P less than 0.05). We also examined if strychnine sulfate, a glycine antagonist would alter the paralytic response to dynorphin. Strychnine protected rats from paralysis (P less than 0.01) and loss of the tail-flick reflex with an ED50 of 7 nmol. We conclude that the reversible paralysis induced by dynorphin A(1-13) is repeatable which suggests that the paralysis results from nontoxic or subtoxic actions of dynorphin. Desensitization to the paralytic effects occurs with closely spaced injections by some unknown mechanism. In addition, we conclude that the spinal glycinergic inhibitory system may participate in the induction of the paralysis because strychnine antagonizes dynorphin-induced paralysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization and localization of immunoreactive dynorphin, alpha-neo-endorphin, Met-enkephalin and substance P in human spinal cord

By use of specific antisera, the distributions of immunoreactive dynorphin (ir-DYN), alpha-neo-endorphin (ir-alpha-NEO), Met-enkephalin (ir-MET) and substance P (ir-SP) were evaluated in discrete regions of human spinal cord and spinal ganglia. The relative concentrations of immunoreactive peptides in particular regions were as follows: sacral greater than lumbar greater than cervical greater than thoracic. Concentrations of ir-DYN, ir-alpha-NEO and ir-SP were 2-10-fold, but of ir-MET 1-2-fold, higher in the dorsal as compared to the ventral parts of cervical, lumbar and sacral cord. The concentrations of all peptides (when examined in discrete areas of thoracic cord) were found to be highest in the substantia gelatinosa. All peptides were present in the gray matter but only ir-MET was found in white matter. Gel-permeation chromatography of dorsal sacral spinal cord extracts revealed two major ir-DYN peaks. The smaller molecular weight peak, eluted at the position of synthetic dynorphin1-17. ir-alpha-NEO and ir-SP comigrated exactly with their respective synthetic marker peptides. Substantial amounts of ir-SP and also, as confirmed by high pressure liquid chromatography, ir-MET, were found in the dorsal and ventral roots and spinal ganglia, and very low concentrations of ir-DYN or ir-alpha-NEO were also detected in these tissue. These results suggest that dynorphin and alpha-neo-endorphin, in addition to enkephalins, may be involved in transmission of somatosensory information in the human spinal cord.     

Loss of antiallodynic and antinociceptive spinal/supraspinal morphine synergy in nerve-injured rats: restoration by MK-801 or dynorphin antiserum

The co-administration of morphine at spinal (i.th.) and supraspinal (i.c.v.) sites to the same rat produces antinociceptive synergy, a phenomenon which may underlie the clinical analgesic utility of this drug. In animals with peripheral nerve injury, however, the antinociceptive potency and efficacy of i.th. morphine is significantly decreased. Here, the possible loss of spinal/supraspinal morphine antinociceptive synergy and relationship to elevation of spinal dynorphin content was studied. Ligation of lumbar spinal nerves resulted in elevated dynorphin in the ipsilateral lumbar and sacral spinal cord. In sham-operated rats supraspinal/spinal co-administration of morphine produced synergistic antinociception which was unaffected by i.th. MK-801 or dynorphin A((1-17)) antiserum. In nerve-injured rats, i.th. morphine was inactive against tactile allodynia and showed diminished in potency against acute nociception without supraspinal/spinal antinociceptive synergy. Antiserum to dynorphin A((1-17)) or the non-competitive NMDA antagonist MK-801 increased the antinociceptive potency of i.th. morphine, restored supraspinal/spinal morphine antinociceptive synergy and elicited a dose-related i.th. morphine antiallodynic action. These agents did not demonstrate antinociceptive or antiallodynic activity alone and did not alter morphine actions in sham-operated animals. The loss of spinal/supraspinal antinociceptive synergy and lack of antiallodynic activity of spinal morphine appear to be due to the elevation across multiple spinal segments of dynorphin following nerve injury. Pathological actions of elevated dynorphin may directly or indirectly modulate the NMDA receptor, result in a loss of supraspinal/spinal morphine synergy and may thus account for the decreased clinical analgesic efficacy of morphine in peripheral neuropathies.     

Kappa opioid receptor antagonist and N-methyl-D- aspartate receptor antagonist affect dynorphin- induced spinal cord electrophysiologic impairment

The latencies of motor-and somatosensory-evoked potentials were prolonged to different degrees, and wave amplitude was obviously decreased, after injection of dynorphin into the rat subarachnoid cavity. The wave amplitude and latencies of motor-and somatosensory-evoked potentials were significantly recovered at 7 and 14 days after combined injection of dynorphin and either the kappa opioid receptor antagonist nor-binaltorphimine or the N-methyl-D-aspartate receptor antagonist MK-801. The wave amplitude and latency were similar in rats after combined injection of dynorphin and nor-binaltorphimine or MK-801. These results suggest that intrathecal injection of dynorphin causes damage to spinal cord function. Prevention of N-methyl-D-aspartate receptor or kappa receptor activation lessened the injury to spinal cord function induced by dynorphin.     

Lasting reduction of severe spasticity after ending chronic treatment with intrathecal baclofen.

OBJECTIVE--To investigate whether the dose of intrathecal baclofen necessary for a sufficient reduction of muscle tone and spasms changes during treatment of severe spasticity. METHODS--A group of 27 patients received intrathecal baclofen for 61 (SD 18) months. RESULTS--Spasticity remained absent or strongly reduced after stopping the intrathecal baclofen infusion in seven patients. The dose of baclofen could be reduced to 40% of that dose which was originally necessary in 10 patients. The dose remained the same or increased slightly in 10 patients. Possible reasons for the continuing reduction of spasticity after terminating long term intrathecal baclofen infusion in some patients could be: lasting morphological changes in spinal cord neurons by second messenger controlled modulation of gene expression, a toxic effect of baclofen on spinal neurons, muscular atrophy, inflammation due to the catheter, or progression of multiple sclerosis. CONCLUSIONS--A higher initial daily dose of intrathecal baclofen might lead to a faster, lasting suppression of spasticity and the development of spastic symptoms might even be prevented by pre-emptive treatment with baclofen in patients with newly acquired lesions of the spinal cord.     

Dynorphin B and spinal analgesia: induction of antinociception by the cannabinoids CP55,940, Delta(9)-THC and anandamide

The endogenous opioid dynorphin B was evaluated for its role in cannabinoid-induced antinociception. Previous work in our laboratory has shown that the synthetic, bicyclic cannabinoid, CP55,940, induces the release of dynorphin B whilst the naturally occurring cannabinoid, Delta(9)-tetrahydrocannabinol (Delta(9)-THC), releases dynorphin A. The dynorphins contribute in part to the antinociceptive effects of both cannabinoids at the level of the spinal cord. The present study compares dynorphin B released from perfused rat spinal cord in response to acute administration of anandamide (AEA), Delta(9)-THC and CP55,940 at two time points, 10 min and 30 min post administration, and attempts to correlate such release with antinociceptive effects of the drugs. Dynorphin B was collected from spinal perfusates of rats pretreated with Delta(9)-THC, CP55,940 or AEA. The supernatant was lyophilized and the concentrations of dynorphin B were measured via radioimmunoassay. At a peak time of antinociception (10 min), CP55,940 and Delta(9)-THC induced significant two-fold increases in the release of dynorphin B. AEA did not significantly release dynorphin B. Upon a 30-min pretreatment with the drugs, no significant dynorphin B release was observed, although antinociceptive effects persisted for CP55,940 and Delta(9)-THC. Previous work indicates that Delta(9)-THC releases dynorphin A while AEA releases no dynorphin A. This study confirms that although all three test drugs produced significant antinociception at 10 min, the endocannabinoid, AEA, does not induce antinociception via dynorphin release. Thus, our data indicate a distinct mechanism which underlies AEA-induced antinociception.