Antinociceptive effect of systemically administered dipyrone (metamizol), magnesium chloride or both in a murine model of cancer.

BACKGROUND: Opioid effectiveness to treat cancer pain is often compromised by the development of tolerance and the occurrence of undesirable side effects, particularly during long-term treatment. Hence, the search for more efficient analgesics remains a necessity. The main goal of this study was to relieve neuropathic symptoms associated with tumour growth by administering the non-opioid analgesic dipyrone (DIP) alone or in combination with magnesium chloride (MgCl2 ), an adjuvant that blocks the NMDA receptor channel. METHODS: Mice were inoculated with a melanoma cell line (B16-BL6) in the left thigh and two protocols were used to evaluate the effect of DIP (270 mg/kg), MgCl2 (200 mg/kg), or the combination DIP-MgCl2 . In the therapeutic protocol the drugs, alone or combined, were administered once tumour had promoted increased nociception. In the preventive protocol, drugs were administered prior to the appearance of the primary tumour. Tumour growth was assessed with a caliper and nociception was determined using behavioural tests. RESULTS: DIP promoted antinociception only at the beginning of both protocols due to the development of tolerance. The combination DIP-MgCl2 improved the antinociceptive effect, avoiding tolerance and reducing tumour growth in the preventive treatment, more efficiently than each compound alone. CONCLUSIONS: These results suggest that DIP-MgCl2 may represent a safe, affordable and accessible option to reduce tumour growth and to treat cancer pain avoiding the risk of tolerance, without the typical complications of opioids agents, particularly when long-term treatment is required. SIGNIFICANCE: This study shows a non-opioid analgesic combined with an adjuvant as a therapeutic option to treat cancer pain. The avoidance of antinociceptive tolerance when repeated administration is required, as well as tumor growth reduction, are additional advantages to be considered.

Systemic changes following carrageenan-induced paw inflammation in rats.

OBJECTIVE AND DESIGN: Carrageenan-induced paw edema has been described as a local and acute inflammatory process. In fact, little is known about the time course and systemic changes following a carrageenan injection. In this study, we examine the systemic changes that follow carrageenan injection in the paw. METHODS: Acute inflammation was produced by subplantar injection of carrageenan in a hind paw of Sprague-Dawley rats. Saline was used in control rats. Paw volume was measured with a plethysmometer. The hot plate latency test was used to quantify antinociception. C-reactive protein (CRP) levels were measured with a sandwich enzyme immunoassay. Fibrinogen concentration was measured using the gravimetric method. Lung morphometric analysis was performed using an image processing package. Lungs and paws were also examined for tissue factor (TF) and proinflammatory cytokines expression by immunohistochemistry. RESULTS: We found diverse systemic changes including increased levels of acute phase proteins, such as CRP and fibrinogen, and a lung inflammatory process characterized by lung edema, fibrin deposition, and leukocyte infiltration. An elevated expression of TF, IL-6, IL-1ß, and TNFα, was observed in paw and lung tissue sections by immunohistochemical methods. CONCLUSION: This study provides new evidence that a local carrageenan injection induces a systemic response.

Behavioral and electrophysiological evidence for tolerance to continuous morphine administration into the ventrolateral periaqueductal gray.

Repeated microinjections of morphine into the ventrolateral periaqueductal gray (vPAG) produce tolerance to the antinociceptive effect of morphine [Behav Neurosci 113 (1999) 833]. These results may be a direct effect of morphine on cells within the vPAG or be caused by cues linked to the microinjection procedure (i.e. associative tolerance). The objective of this paper was to determine whether continuous administration of morphine into the vPAG (i.e. no cues) would produce tolerance. Tolerance was assessed by measuring changes in behavior and changes in the activity of neurons in the rostral ventromedial medulla (RVM), the primary output target of the PAG. Rats were implanted with an osmotic minipump that released morphine (2.5 or 5 microg/h) or saline into the vPAG continuously. Continuous administration of morphine produced an increase in hotplate latency when measured 6 h after initiation of treatment. Tolerance to this antinociception was evident within 24 h. After 3 days, rats were anesthetized and the activity of RVM neurons was assessed. Although acute morphine administration into the RVM inhibits the activity of RVM on-cells and enhances the activity of off-cells, these neurons appeared normal following 3 days of continuous morphine administration. Systemic naloxone administration produced hyperalgesia that was associated with a marked increase in on-cell activity and a complete cessation of off-cell activity. The loss of morphine inhibition of nociception, measured behaviorally and electrophysiologically, demonstrates that tolerance is caused by a direct action of morphine on vPAG neurons.

Comparison of morphine and kainic acid microinjections into identical PAG sites on the activity of RVM neurons.

The rostral ventromedial medulla (RVM) modulates nociception through changes in the activity of two classes of neuron, ON- and OFF-cells. The activity of these neurons is regulated, in part, by input from the periaqueductal gray (PAG). The objective of this study was to determine whether PAG-mediated antinociception is associated with excitation of both ON- and OFF-cells in the RVM. Microinjection of morphine into the ventrolateral PAG produced antinociception at 50% of the injection sites. This antinociception was associated with continuous activation of RVM OFF-cells and inhibition of both the spontaneous and reflex-related activity of RVM ON-cells. Microinjection of kainic acid into the same injection sites produced antinociception 92% (37/40) of the time. Although kainic acid directly excites PAG output neurons, the changes in ON- and OFF-cell activity associated with microinjection of kainic acid into the ventrolateral PAG were the same as when morphine was injected. That is, ON-cells were inhibited and OFF-cells were activated. These data indicate that the excitatory connection between the PAG and RVM is directed at RVM OFF-cells specifically. In addition, these data suggest that direct activation of PAG output neurons, as occurs with kainic acid, is much more likely to produce antinociception than disinhibition of output neurons as occurs following morphine administration.

Tolerance to repeated microinjection of morphine into the periaqueductal gray is associated with changes in the behavior of off- and on-cells in the rostral ventromedial medulla of rats.

Although the administration of opioids is the most effective treatment for pain, their efficacy is limited by the development of tolerance. The midbrain periaqueductal gray matter (PAG) participates in opioid analgesia and tolerance. Microinjection of morphine into PAG produces antinociception, probably through neurons in the rostral ventromedial medulla (RVM), namely through the activation of off-cells, which inhibit nociception, and the inhibition of on-cells, which facilitate nociception. After its repeated microinjection into the PAG morphine loses effectiveness. The present study sought to determine whether tolerance to PAG morphine administration is associated with changes in the behavior of RVM neurons. Morphine (0.5 microg/0.4 microl) or saline (0.4 microl) was microinjected into the ventrolateral PAG twice daily. Initially morphine caused a latency increase in the hot plate test (antinociception) but this effect disappeared by day 3 (tolerance). On day 4, each rat was anesthetized with halothane and recordings were made from off- and on-cells in the RVM, i.e. from neurons that decrease or increase their firing, respectively, just before a heat-elicited tail flick. In contrast to saline-pretreated rats, PAG microinjection of morphine in tolerant animals did not change the baseline activity of off- or on-cells, did not prevent the off-cell pause or the on-cell activation upon tail heating, and did not lengthen the tail flick latency. However, microinjection of kainic acid into the PAG (1) caused off-cells to become continuously active and on-cells to become silent, and (2) prevented the tail flick, i.e. exactly what morphine did before tolerance developed. These results demonstrate a correspondence between neuronal and behavioral measures of tolerance to PAG opioid administration, and suggest that tolerance is mediated by a change in opioid-sensitive neurons within the PAG.

Circuitry underlying antiopioid actions of cholecystokinin within the rostral ventromedial medulla.

It is now well established that the analgesic actions of opioids can be modified by "anti-analgesic" or "antiopioid" peptides, among them cholecystokinin (CCK). Although the focus of much recent work concerned with CCK-opioid interactions has been at the level of the spinal cord, CCK also acts within the brain to modify opioid analgesia. The aim of the present study was to characterize the actions of CCK in a brain region in which the circuitry mediating the analgesic actions of opioids is relatively well understood, the rostral ventromedial medulla (RVM). Single-cell recording was combined with local infusion of CCK in the RVM and systemic administration of morphine in lightly anesthetized rats. The tail-flick reflex was used as a behavioral index of nociceptive responsiveness. Two classes of RVM neurons with distinct responses to opioids have been identified. OFF cells are activated, indirectly, by morphine and mu-opioid agonists, and there is strong evidence that this activation is crucial to opioid antinociception. ON cells, thought to facilitate nociception, are directly inhibited by opioids. Cells of a third class, NEUTRAL cells, do not respond to opioids, and whether they have any role in nociceptive modulation is unknown. CCK microinjected into the RVM by itself had no effect on tail flick latency or the firing of any cell class but significantly attenuated opioid activation of OFF cells and inhibition of the tail flick. Opioid suppression of ON-cell firing was not significantly altered by CCK. Thus CCK acting within the RVM attenuates the analgesic effect of systemically administered morphine by preventing activation of the putative pain inhibiting output neurons of the RVM, the OFF cells. CCK thus differs from another antiopioid peptide, orphanin FQ/nociceptin, which interferes with opioid analgesia by potently suppressing all OFF-cell firing.

Opioid tolerance induced by metamizol (dipyrone) microinjections into the periaqueductal grey of rats.

Non-opioid analgesics have been shown to elicit antinociception by an action upon central nervous system structures, in addition to their well known action upon peripheral tissues. Microinjection of metamizol (dipyrone), a widely used nonopioid analgesic, into the periaqueductal grey matter (PAG) of rats activates pain-modulating systems in the nucleus raphe magnus and inhibits spinal nociceptive neurons and the tail-flick reflex. Since these effects involve an activation of endogenous opioidergic systems, the possibility that metamizol induces opioid tolerance was investigated. Microinjection of metamizol into the ventrolateral PAG in awake rats induced antinociception, as demonstrated in the heat-elicited tail flick and hot plate tests. When microinjected into the ventrolateral PAG twice daily for 2 days, metamizol induced tolerance, i.e. a progressive loss of its antinociceptive effect. In contrast to rats repeatedly microinjected with saline, metamizol-tolerant rats were also tolerant to morphine microinjection into the same PAG site, and displayed signs of opioid withdrawal upon systemic administration of naloxone. These and other results suggest that metamizol activates endogenous opioid systems and that nonopioid analgesics may, by an action upon the central nervous system, lead to opioid tolerance and the risk of opioid withdrawal.

Tolerance to the antinociceptive effect of morphine microinjections into the ventral but not lateral-dorsal periaqueductal gray of the rat.

Tolerance to the antinociceptive effect of morphine is mediated at least in part by morphine's action within the periaqueductal gray (PAG). The objective of the present study was to determine whether both ventral and lateral-dorsal PAG regions contribute to the development of tolerance. It was found that the antinociceptive efficacy of microinjecting morphine (5 microg/0.4 microl) into the ventral but not the lateral-dorsal PAG diminished with successive injections. Control experiments indicated that this decrease was caused by tolerance to morphine and was not a result of cell death caused by repeated microinjections or habituation from repeated behavioral testing. The finding of greater susceptibility of the ventral compared with the lateral-dorsal PAG to the development of tolerance adds to a growing literature distinguishing antinociception from these two regions.

PAG-microinjected dipyrone (metamizol) inhibits responses of spinal dorsal horn neurons to natural noxious stimulation in rats.

In addition to their well-known peripheral and spinal effects, non-steroidal antiinflammatory drugs (NSAIDs) are believed to diminish nociceptive responses by acting supraspinally and activating descending modulatory systems. We have herein investigated whether this descending action involves a depression of spinal sensory neurons. In rats under barbiturate anesthesia, responses of lumbar wide-dynamic-range neurons to a noxious clamp in their receptive fields were depressed to 46% of baseline value by the microinjection of 100 microg dipyrone (metamizol) into the periaqueductal gray matter (PAG). These results show that PAG application of NSAIDs activates descending systems which depress the excitation of spinal sensory neurons by natural noxious stimuli.

Naloxone partial reversal of the antinociception produced by dipyrone microinjected into the periaqueductal gray of rats. Possible involvement of medullary off- and on-cells.

Medullary off- and on-cells have been proposed to inhibit and facilitate, respectively, nociceptive transmission. Upon heating the tail in lightly anesthetized rats, the tail flick (TF) reflex occurs only after off-cells decrease and on-cells increase their activity. Dipyrone (DIP) microinjection (100 micrograms/0.5 microliter) into the periaqueductal gray (PAG) caused retardation in the off-cell pause, on-cell burst and corresponding TF. This effect was partly reverted by naloxone given i.v. (l mg/kg) or microinjected into PAG (5 micrograms/0.5 microliter). These results suggest that endogenous opioids are partly responsible for the central antinociceptive action of DIP, and that such action involves medullary off- and on-cells.

Anti-nociception induced by systemic or PAG-microinjected lysine-acetylsalicylate in rats. Effects on tail-flick related activity of medullary off- and on-cells.

Previous experiments using metamizol have shown that this non-steroidal anti-inflammatory drug (NSAID) produces a central anti-nociceptive effect probably through neural substrates that also support the analgesic effects of opiates, such as the periaqueductal grey matter (PAG) and the off- and on-cells of the rostral ventromedial medulla (RVM). Off- and on-cells have been postulated to respectively inhibit and facilitate nociceptive transmission, since the heat-elicited tail flick reflex (TF) occurs only after off-cells have decreased (pause), and on-cells, have increased (burst) their activity. The aim of the present study was to examine whether the effect of metamizol upon TF and off- and on-cells responses could be generalized to other NSAIDs such as, in this case, lysine-acetylsalicylate (LASA). Fifty-nine off- and on-cells of the RVM were recorded in lightly anaesthetized rats. Systemic administration (200 and 300 mg/kg) or PAG microinjection (30, 50 and 100 micrograms) of LASA caused retardation of the heat-elicited off-cell pause, on-cell burst and the corresponding TF. Neuronal responses and TF retained their mutual time relationship but shifted simultaneously toward longer latencies. This anti-nociceptive effect of LASA was dose-dependent, present 5 min after administration and reached a maximum in 30 min for both administration methods. These data confirm that analgesics typically defined as peripherally-acting, such as metamizol and LASA in this study, may also have an anti-nociceptive effect by acting directly upon PAG, and suggest that this central effect involves the RVM off- and on-cells.

Interference with GABA transmission in the rostral ventromedial medulla: disinhibition of off-cells as a central mechanism in nociceptive modulation.

Blockade of GABA-mediated synaptic transmission in the rostral ventromedial medulla by local application of GABAA receptor antagonists produces antinociception, indicating that a GABA-mediated inhibition of some population of neurons in this region is normally required if nociceptive information is to be transmitted. The aim of the present study was to elucidate the medullary circuitry mediating this antinociception by recording the activity of putative nociceptive modulating neurons in the rostral ventromedial medulla before and after local infusion of the GABAA receptor antagonist bicuculline methiodide. It was thus possible to correlate changes in the activity of cells of different classes with the ability of the infusion to produce a behaviorally measurable antinociception. One class of medullary neurons, "off-cells," is identified by a pause in firing associated with the occurrence of nocifensor reflexes such as the tail flick evoked by noxious heat. These neurons are uniformly activated following systemic administration of morphine, and are thought to have a net inhibitory effect on nociception. Following local bicuculline administration, off-cells enter a prolonged period of continuous firing that is temporally linked with the period of tail flick inhibition. A second class of neurons, "on-cells," is identified by a burst of activity beginning just before the tail flick, and is directly inhibited by opioids. Unlike off-cells, cells of this class do not show a consistent change in activity associated with inhibition of the tail flick following bicuculline. These data indicate that alterations in the discharges of on-cells would not be able to explain the antinociceptive effect of bicuculline, and therefore point to disinhibition of off-cells as a sufficient basis for antinociception originating within the rostral ventromedial medulla.

Disinhibition of off-cells and antinociception produced by an opioid action within the rostral ventromedial medulla.

Activation of neurons in the rostral ventral medulla, by electrical stimulation or microinjection of glutamate, produces antinociception. Microinjection of opioid compounds in this region also has an antinociceptive effect, indicating that opioids activate a medullary output neuron that exerts a net inhibitory effect on nociception. When given systemically in doses sufficient to produce antinociception, morphine produces distinct, opposing responses in two physiologically identifiable classes of rostral medullary neurons. "Off-cells" are activated, and have been proposed to inhibit nociceptive transmission. "On-cells" are invariably depressed, and may have a pro-nociceptive role. Although on-cell firing is also depressed by iontophoretically applied morphine, off-cells do not respond to morphine applied in this manner. The present study used local infusion of the mu-selective opioid peptide Tyr-D-Ala-Gly-MePhe-Gly-ol-enkephalin (DAMGO) within the rostral medulla to determine whether off-cells are activated by an opioid action within this region that is sufficient to produce a behaviorally measurable antinociception. Activity of on- and off-cells was recorded before and after local infusion of DAMGO noxious heat-evoked tail flick reflex was inhibited in 17 of 28 cases. On-cell firing was profoundly depressed, and this occurred irrespective of the antinociceptive effectiveness of the injection. Off-cells were activated following DAMGO microinjections, but only in experiments in which the tail flick reflex was inhibited. Both reflex inhibition and neuronal effects were reversed following systemic administration of naloxone. These observations thus confirm the role of the on-cell as the focus of direct opioid action within the rostral medulla, and strongly support the proposal that disinhibition of off-cells is central to the antinociception actions of opioids within this region.

"Off" and "on" cells of the medulla oblongata as possible mediators of analgesia produced by mesencephalic and diencephalic stimulation in rats.

The present study shows the effects of electrical' stimulation of mesencephalic and diencephalic structures on the activity of two types of neuron from the rostral ventromedial medulla, which are related to the nocifensive reflex known as tail flick response (TF). One type of neuron, the off-cell, abruptly stops firing immediately before the tail is flicked, while the other type, the on-cell, increases firing just before the flick. When electrical stimulation was applied to mesencephalic and diencephalic structures the TF was inhibited and, simultaneously, both kinds of cells showed increments in their activities. On average, this increment was 81 +/- 22.09% for the off-cells, and 1563 +/- 257.66% for the on-cells. Quantitative analysis showed a directly proportional relationship between the activation of the firing rate of both kinds of cells and the intensity of the stimulation currents. Also, when the stimulation electrode was positioned more rostrally in the brain, greater currents were needed to reach the threshold of analgesia. The present work contributes to a large body of evidence indicating that off-cells, on-cells, or both, are involved in the complex mechanism of the control of nociceptive transmission and nocifensive reflexes.

"Off" and "On" cell of the medulla oblongata as possible mediators of analgesia produced by mesencephalic and diencephalic stimulation in rats / Las celulas \"OFF\" y \"ON\" del bulbo raquideo como posibles mediadoras de la analgesia producida por estimulacion mesencefalica y diencefalica en ratas

The present study shows the effects of electrical`stimulation of mesencephalic and diencephalic structures on the activity of two types of neuron from the rostral ventromedial medulla,which are related to the nocifensive reflex known as tail flick response (TF). One type of neuron, the off-cell abruptly stops firing immediately before the tail is flicked, while the other type, the on-cell, increases firing just before tyhe flick. When electrical stimulation was applied to mesencephalic and diencephalic structure the TF was inhibited and, simultancously, both kinds of cell showed increment in their activities. On average,this increment was 81ñ22.09%for the off-cell, and 1563ñ257.66%for the on cell. Quantitative analysis showed a directly proportional relationship between the activation of the firing rate of both kinds of cell and the intensy of the simulation currents. Also, when the simulation electrode was positioned more rostrally in the brain, greater currents were needed to reach the threshold of analgesia. The present work contributes to a large body of evidence indicating that off-cell, or both, are involved in the complex mechanism of the control of nociceptive tranmission and nicifensive reflexes