The role of neuroinflammation in the transition of acute to chronic pain and the opioid-induced hyperalgesia and tolerance.

Current evidence suggests that activation of glial and immune cells leads to increased production of proinflammatory mediators, creating a neuroinflammatory state. Neuroinflammation has been proven to be a fundamental mechanism in the genesis of acute pain and its transition to neuropathic and chronic pain. A noxious event that stimulates peripheral afferent nerve fibers may also activate pronociceptive receptors situated at the dorsal root ganglion and dorsal horn of the spinal cord, as well as peripheral glial cells, setting off the so-called peripheral sensitization and spreading neuroinflammation to the brain. Once activated, microglia produce cytokines, chemokines, and neuropeptides that can increase the sensitivity and firing properties of second-order neurons, upregulating the signaling of nociceptive information to the cerebral cortex. This process, known as central sensitization, is crucial for chronification of acute pain. Immune-neuronal interactions are also implicated in the lesser-known complex regulatory relationship between pain and opioids. Current evidence suggests that activated immune and glial cells can alter neuronal function, induce, and maintain pathological pain, and disrupt the analgesic effects of opioid drugs by contributing to the development of tolerance and dependence, even causing paradoxical hyperalgesia. Such alterations may occur when the neuronal environment is impacted by trauma, inflammation, and immune-derived molecules, or when opioids induce proinflammatory glial activation. Hence, understanding these intricate interactions may help in managing pain signaling and opioid efficacy beyond the classical pharmacological approach.

Concomitant Antihyperalgesic and Antitumor Effects of Gabapentin in a Murine Cancer Pain Model.

Cancer pain may be the consequence of physical nerve compression by a growing tumor. We employed a murine model to study whether gabapentin was able to regulate tumor growth, in addition to controlling hyperalgesic symptoms. A fluorescent melanoma cell line (B16-BL6/Zs green) was inoculated into the proximity of the sciatic nerve in male C57BL/6 mice. The tumor gradually compressed the nerve, causing hypersensitivity. Tumor growth was characterized via in vivo imaging techniques. Every other day, gabapentin (100 mg/Kg) or saline was IP administered to each animal. In the therapeutic protocol, gabapentin was administered once the tumor had induced increased nociception. In the preventive protocol, gabapentin was administered before the appearance of the positive signs. Additionally, in vitro experiments were performed to determine gabapentin's effects on cell-line proliferation, the secretion of the chemokine CCL2, and calcium influx. In the therapeutically treated animals, baseline responses to noxious stimuli were recovered, and tumors were significantly reduced. Similarly, gabapentin reduced tumor growth during the preventive treatment, but a relapse was noticed when the administration stopped. Gabapentin also inhibited cell proliferation, the secretion of CCL2, and calcium influx. These results suggest that gabapentin might represent a multivalent strategy to control cancer-associated events in painful tumors.

Functional relationship between brainstem putative pain-facilitating neurons and spinal nociceptfive neurons during development of inflammation in rats.

The so-called on- and off-cells of the rostral ventromedial medulla (RVM) send their axons to the spinal dorsal horn. Activation of on-cells precedes and coincides with a facilitation, and activation of off-cells coincides with an inhibition, of withdrawal reflexes elicited by noxious agents. Considerable evidence supports the notion that on- and off-cells modulate nocifensive reflexes during opioid and non-opioid action and also during normal circumstances and during peripheral neuropathy and inflammation. Yet it is unclear whether on- and off-cells act upon sensory spinal circuits that might lead to ascending projections and the experience of pain. Here, in deeply anesthetized rats we recorded single unit discharges from pairs of one on-like or off-like cell in RVM and a nociceptive neuron in the spinal dorsal horn with input from a hind paw. Both ongoing activity and responses to a calibrated noxious stimulus applied to the paw were documented during basal conditions and during development of paw inflammation. Probably due to the strong barbiturate anesthesia, off-like cells were depressed and did not yield interpretable results. However, we showed for the first time that during the increase in neuronal activity that results from paw inflammation the activity of spinal nociceptive neurons reflects the activity of their partner on-like cells in a highly correlated manner. This implies a tight relationship between spinal sensory and RVM modulatory functions that may underlie inflammation-induced hyperreflexia and clinically relevant hyperalgesia.

Role of RVM neurons in capsaicin-evoked visceral nociception and referred hyperalgesia.

Most forms of visceral pain generate intense referred hyperalgesia but the mechanisms of this enhanced visceral hypersensitivity are not known. The on-cells of the rostral ventromedial medulla (RVM) play an important role in descending nociceptive facilitation and can be sensitized to somatic mechanical stimulation following peripheral nerve injury or hindpaw inflammation. Here we have tested the hypothesis that visceral noxious stimulation sensitizes RVM ON-like cells, thus promoting an enhanced descending facilitation that can lead to referred visceral hyperalgesia. Intracolonic capsaicin instillation (ICI) was applied to rats in order to create a hyperalgesic state dependent on noxious visceral stimulation. This instillation produced acute pain-related behaviors and prolonged referred hyperalgesia that were prevented by the RVM microinjection of AP5, an NMDA selective antagonist. In electrophysiological experiments, ON-like RVM neurons showed ongoing spontaneous activity following ICI that lasted for approximately 20 min and an enhanced responsiveness to von Frey and heat stimulation of the hindpaw and to colorectal distention (CRD) that lasted for at least 50 min post capsaicin administration. Moreover, ON-like cells acquired a novel response to CRD and responded to heat stimulation in the innocuous range. OFF-like neurons responded to capsaicin administration with a brief (<5 min) inhibition of activity followed by an enhanced inhibition to noxious stimulation and a novel inhibition to innocuous stimulation (CRD and heat) at early time points (10 min post capsaicin). These results support the hypothesis that noxious visceral stimulation may cause referred hypersensitivity by promoting long-lasting sensitization of RVM ON-like cells.

NSAIDs, Opioids, Cannabinoids and the Control of Pain by the Central Nervous System.

Nonsteroidal anti-inflammatory drugs (NSAIDs) act upon peripheral tissues and upon the central nervous system to produce analgesia. A major central target of NSAIDs is the descending pain control system. The rostral structures of the descending pain control system send impulses towards the spinal cord and regulate the transmission of pain messages. Key structures of the descending pain control system are the periaqueductal gray matter (PAG) and the rostral ventromedial region of the medulla (RVM), both of which are critical targets for endogenous opioids and opiate pharmaceuticals. NSAIDs also act upon PAG and RVM to produce analgesia and, if repeatedly administered, induce tolerance to themselves and cross-tolerance to opioids. Experimental evidence shows that this is due to an interaction of NSAIDs with endogenous opioids along the descending pain control system. Analgesia by NSAIDs along the descending pain control system also requires an activation of the CB1 endocannabinoid receptor. Several experimental approaches suggest that opioids, NSAIDs and cannabinoids in PAG and RVM cooperate to decrease GABAergic inhibition and thus enhance the descending flow of impulses that inhibit pain.

Tolerance to non-opioid analgesics in PAG involves unresponsiveness of medullary pain-modulating neurons in male rats.

Opiate analgesia can be hampered by a reduction in pharmacological effectiveness (tolerance), and this crucially depends on the periaqueductal gray matter (PAG). Non-opioids like metamizol (dipyrone) or aspirin also induce PAG-dependent analgesia and tolerance, but the neuronal bases of this tolerance are unknown. Metamizol is a pyrazolon derivative and cyclooxygenase inhibitor with widespread use as an analgesic in Europe and Latin America. Metamizol was microinjected into the PAG of awake male rats, and antinociception was assessed by the tail flick (TF) and hot plate (HP) tests. Microinjection twice daily for 2.5 days caused tolerance to metamizol. The rats were then anesthetized and recordings from pain-facilitating on-cells and pain-inhibiting off-cells of the rostral ventromedial medulla (RVM) were performed. PAG microinjection of morphine or metamizol depresses on-cells, activates off-cells and thus inhibits nociception, including TF and HP. In metamizol-tolerant rats, however, PAG microinjection of metamizol failed to affect on- or off-cells, and this is interpreted as the reason for tolerance. In metamizol-tolerant rats morphine microinjection into PAG also failed to affect RVM neurons or nociception (cross-tolerance). In naïve, non-tolerant rats the antinociceptive effect of PAG-microinjected metamizol or morphine was blocked when CTOP, a mu-opioid antagonist, was previously microinjected into the same PAG site. These results emphasize a close relationship between opioid and non-opioid analgesic mechanisms in the PAG and show that, like morphine, tolerance to metamizol involves a failure of on- and off-cells to, respectively, disfacilitate and inhibit nociception. Cross-tolerance between non-opioid and opioid analgesics should be important in the clinical setting.

Mecanismos del dolor neuropático: Del laboratorio a la clínica / Mechanisms of neuropathic pain: from the laboratory to the clinic

El dolor neuropático es un síndrome de muy difícil manejo en la práctica clínica. Suele originarse a consecuencia de lesiones o enfermedades que afectan al sistema somatosensorial. Lesiones de nervios periféricos, la diabetes, el virus herpes zoster, entre otras, son ejemplos de entidades clínicas que causan dolor neuropático. Este tipo de dolor supone modificaciones de la fisiología normal de las neuronas que integran la vía de transmisión nociceptiva y que operan tanto a nivel periférico, como central. Entre estos mecanismos se incluye la generación de descargas ectópicas, cambios en el genoma de las neuronas involucradas, alteración de canales iónicos, pérdida de las actividades inhibitorias endógenas, activación anormal del sistema inmunitario y sensibilización tanto periférica, como central. Este artículo resume los mecanismos moleculares y neuroplásticos que ocurren en situaciones de dolor neuropático y revisa diferentes modelos experimentales para su estudio. El desarrollo de estos modelos ha permitido dilucidar los mecanismos subyacentes de esta patología, el diseño de nuevas estrategias terapéuticas y el diagnóstico acertado de la enfermedad

Neuropathic pain is a very complex clinical syndrome that results as a consequence of lesions or diseases affecting the somatosensory system. Lesion of peripheral nerves, diabetes, herpes zoster virus, among others, are clinical entities that may cause neuropathic pain. Neuropathic pain also reflects changes of the normal physiology of the nociceptive transmission neurons that may happen at peripheral and central levels. These mechanisms include the generation of ectopic discharges, genomic changes of the neurons involved, alterations of ionic channels and of the endogenous inhibition of pain, abnormal activation of the immune system as well as peripheral and central sensitization. This article review the molecular and neuroplastic mechanisms associated to neuropathic pain and discuss different experimental models for its study. The development of these models has been helpful to understand the subjacent mechanisms of this pathology, and has contributed to design new therapeutical approaches and the correct diagnose of the disease

Critical role of the rostral ventromedial medulla in early spinal events leading to chronic constriction injury neuropathy in rats.

UNLABELLED: Neuropathic pain is a major clinical problem, and several animal models have been developed to investigate its mechanisms and its treatment. In this report, the role of the rostral ventromedial medulla (RVM) in the early events of the chronic constriction injury (CCI) model was investigated in behavioral and electrophysiological experiments. Placing the 4 CCI ligatures around the sciatic nerve induced large discharges and residual ongoing activity in spinal nociceptive neurons. Two weeks after CCI ligation, the rats showed behavioral hyperalgesia and allodynia as well as increased ongoing activity and responsiveness of spinal nociceptive neurons to innocuous and noxious stimuli. Blockade of excitatory synapses in the RVM by a kynurenate microinjection (2 nmol in 0.5 muL) 5 minutes before placement of the sciatic ligatures had no immediate effect on spinal neuronal activity but largely prevented the activation of spinal neurons. In kynurenate microinjected rats, behavioral hyperalgesia and allodynia developed slowly and incompletely, which corresponded with an incompletely developed hyperexcitability of spinal neurons. To the best of our knowledge, these results show for the first time that the initial response to nerve damage requires facilitation from the RVM. PERSPECTIVE: The present and previous findings indicate that descending facilitation from brainstem nuclei critically contributes to the spinal hyperexcitability that underlies neuropathic pain. The present results indicate that this contribution begins at the very moment the nerve is damaged and should be prevented and treated accordingly.

Dolor y malestar: un ejemplo experimental de interacción neuro-inmune / Pain and sickness: an experimental example of neuro-immune interaction

Bajo la definición de malestar general se agrupan una constelación de signos y síntomas tales como fiebre, disminución de la actividad, pérdida del apetito, disminución de la libido, trastornos del sueño y modificaciones en el umbral del dolor, entre otros. Estas manifestaciones son necesarias para hacer frente a procesos infecciosos y son iniciadas por el sistema inmunitario y controladas por el sistema nervioso, por lo que representan un ejemplo ideal de interacción entre ambos componentes. En el presente estudio se analizaron los cambios conductuales inducidos en el umbral del dolor luego de la administración i.p. de lipopolisacárido (LPS), una endotoxina que forma parte de la pared de bacterias gram negativas. Adicionalmente, se determinaron los cambios que ocurren en los patrones de descarga de dos poblaciones de neuronas de la formación reticular bulbar, las células “off” y “on”, luego de la administración de la endotoxina. Estas células aparentemente constituyen los brazos ejecutores del sistema de modulación endógena del dolor. Los resultados obtenidos indican que dependiendo de la dosis de LPS empleada (100 o 200 μg/kg) se pueden activar diferentes mecanismos de modulación, que a su vez parecen depender de las dos poblaciones de neuronas bulbares. La dosis de 100 μg/kg de LPS produjo antinocicepción, la cual estuvo asociada a incrementos en la actividad de las células “off” y a la disminución de la actividad de las “on”. La dosis de 200 μg/kg de LPS produjo hiperalgesia, la cual se acompañó de una disminución de la actividad de las celulas off”y un aumento en la actividad de las células on.

The definition of sickness/illness behavior involves a constellation of signs and symptoms such as fever, decrease of physical activity, anorexia, decrease of libido, insomnia and changes of the pain threshold, among others. These manifestations are necessary to face infectious processes and are apparently initiated and controlled by the immune and the nervous system, so that they represent an ideal example of interaction between both components. In the present study the behavioral changes in the pain threshold induced by the i.p. administration of lipopolysaccharide, an endotoxin derived from gram-negative bacterial cell walls, were analyzed. Additionally, changes in the discharge pattern of two neuronal populations of the medullary reticular formation, the off- and on-cells, which apparently act as the executory arm of the endogenous pain modulatory system were determined after endotoxin administration. The results indicate that depending on the LPS dose (100 or 200 μg/kg) different modulatory mechanisms might be activated, which in turn seem to be the consequence of the activation of the two different classes of medullary neurons. Antinociception was produced after administration of 100 μg/kg of LPS. That was associated to an increase of the off-cell firing and a reduction of the on-cell activity. Hyperalgesia was produced after administration of 200 μg/kg of LPS. That effect was accompanied by an inhibition of the off-cell activity and an increase of the on-cell firing. These findings suggest that changes of the pain threshold that occur during sickness/illness involve the differential participation of the two control options of the endogenous pain modulatory system and of specialized mechanisms of communication between the immune and the nervous system.

Involvement of cholecystokinin in the opioid tolerance induced by dipyrone (metamizol) microinjections into the periaqueductal gray matter of rats.

The analgesic effect of non-steroidal anti-inflammatory drugs (NSAIDs) is partly due to an action upon the periaqueductal gray matter (PAG), which triggers the descending pain control system and thus inhibits nociceptive transmission. This action of NSAIDs engages endogenous opioids at the PAG, the nucleus raphe magnus and the spinal cord. Repeated administration of NSAIDs such as dipyrone (metamizol) and acetylsalicylate thus induces tolerance to these compounds and cross-tolerance to morphine. Since cholecystokinin plays a key role in opioid tolerance, the present study in rats investigated whether PAG cholecystokinin is also responsible for tolerance to PAG-microinjected dipyrone. Microinjection of cholecystokinin (1 ng/0.5 microl) into PAG blocked the antinociceptive effect of a subsequent microinjection of dipyrone (150 microg/0.5 microl) into the same site, as evaluated by the tail flick and hot plate tests. Microinjection of proglumide (0.4 microg/0.5 microl), a non-selective cholecystokinin antagonist, into PAG prevented the development of tolerance to subsequent microinjections of dipyrone, as well as cross-tolerance to microinjection of morphine (5 microg/0.5 microl) into the same site. In rats tolerant to PAG dipyrone, a PAG microinjection of proglumide restored the antinociceptive effect of a subsequent microinjection of dipyrone or morphine. These results suggest that PAG-microinjected dipyrone triggers and/or potentiates local opioidergic circuits leading to descending inhibition of nociception, on the one hand, and to a local antiopioid action by cholecystokinin, on the other. Reiteration of these events would then result in an enhancement of cholecystokinin's antiopioid action and thus tolerance to opioids and dipyrone in the PAG.

Induction of opioid tolerance by lysine-acetylsalicylate in rats.

The analgesic effect of non-steroidal antiinflammatory drugs (NSAIDs) is due to their action upon the peripheral damaged tissues, the spinal cord, and brain stem structures of the 'descending pain-control system' such as the periaqueductal gray matter (PAG) and the nucleus raphe magnus (NRM). The NSAID dipyrone (metamizol) has been shown to engage opioidergic circuits at the PAG, the NRM and the spinal cord, but it is unknown whether this can be generalized to typical NSAIDs and to systemic administration. In the present study lysine-acetylsalicylate (LASA), an injectable form of the prototypical NSAID aspirin, was microinjected into the PAG (100 microg/0.5 microl) in freely moving rats to induce inhibition of tail flick and hot plate responses. This antinociception was reverted by naloxone (1 mg/kg i.p.). PAG microinjection of LASA twice daily for three days induced tolerance to LASA (i.e. a progressive loss of effectiveness) and cross-tolerance to PAG-microinjected morphine (5 microg/0.5 microl). The antinociceptive effect of systemically administered LASA (300 mg/kg i.p., equivalent to the 1000 mg analgesic dose for humans) was also abolished by naloxone. Intraperitoneal injection of LASA twice daily induced tolerance to LASA and cross-tolerance to i.p. morphine (1 or 5 mg/kg). LASA-tolerant rats showed opioid withdrawal signs when injected with naloxone. These findings support the notion that the contribution of the PAG and downstream pain-control structures to the analgesic effect of NSAIDs involves opioidergic mechanisms, and suggest that repeated therapeutic administration of NSAIDs may induce tolerance, cross-tolerance to opiates, and susceptibility to a withdrawal syndrome.

Involvement of local cholecystokinin in the tolerance induced by morphine microinjections into the periaqueductal gray of rats.

The ventrolateral periaqueductal gray (PAG) is a key structure for the development of opioid tolerance. An increased activity of 'anti-opioids' like cholecystokinin (CCK) has been proposed as a possible mechanism for opioid tolerance. The present study evaluates the role of PAG-located CCK in the opioid tolerance induced by repeated microinjections of morphine (MOR) into PAG. Male rats were implanted with chronic guide cannulae aimed at the PAG. Microinjection of MOR (0.5 microg in 0.5 microl) into PAG caused antinociception as quantified with the tail flick and the hot plate tests. When MOR microinjection was repeated twice daily, the antinociceptive effect disappeared within 2 days (tolerance). However, if each MOR microinjection was preceded (within 15 min) by a microinjection of the non-selective CCK receptor antagonist proglumide (PRO), (0.4 microg in 0.5 microl) into the same PAG site, the microinjections of MOR always produced antinociception and did not induce tolerance. If PRO microinjections were suspended, subsequent MOR microinjections induced tolerance. In MOR-tolerant rats, a single PRO microinjection into the same PAG site was enough to restore the antinociceptive effect of MOR. On the other hand, if CCK (1 ng in 0.5 microl) was microinjected into PAG, then MOR microinjection administered 15 min later into the same PAG site did not elicit antinociception. These results show that CCK has anti-opioid activity in PAG and that tolerance to MOR in PAG can be prevented or reversed if CCK receptors are blocked with PRO. Finally, opioid tolerance induced by repeated systemic MOR injections (5mg/kg intraperitoneal ) was reversed by a single microinjection of PRO into PAG. This emphasizes the central importance of PAG in the MOR/CCK interactions that lead to opioid tolerance.

Opioidergic effects of nonopioid analgesics on the central nervous system.

1. The analgesic effect of nonsteroidal anti-inflammatory drugs (NSAIDs) is partly due to the fact that they act upon the periaqueductal gray matter (PAG) and the rostral ventromedial medulla of the brain stem and thus activate the descending pain-control system, which inhibits nociceptive transmission at the spinal dorsal horn. 2. The analgesic action of dipyrone (metamizol) and of lysine-acetylsalicylate (LASA), two well-known NSAIDs. whether microinjected into the PAG or given systemically, can be reverted by naloxone. Repeated administration of dipyrone or LASA induces tolerance to their antinociceptive effect, with cross-tolerance to morphine, and a withdrawal syndrome upon naloxone administration. Dipyrone tolerance can be reverted by proglumide, a cholecystokinin antagonist. 3. These findings reveal a close association between the central action of NSAIDs and endogenous opioids.

Putative role of medullary off- and on-cells in the antinociception produced by dipyrone (metamizol) administered systemically or microinjected into PAG.

Recent investigations have shown that non-steroidal antiinflammatory drugs (NSAIDs) may exert an antinociceptive effect when administered at or within the central nervous system (CNS). This might be due to the engagement of CNS substrates that support the analgesic effects of opiates, including the periaqueductal gray matter (PAG) and the rostral ventromedial medulla (RVM). The off- and on-cells of the RVM have been proposed to inhibit and facilitate, respectively, nociceptive transmission. Accordingly, upon heating of a rat's tail the tail-flick (TF) reflex occurs only after off-cells have decreased, and on-cells have increased, their activity. In the present study, i.v. administration (200 and 400 mg/kg) or PAG microinjection (25, 50, 100 and 250 micrograms) of dipyrone (metamizol) to lightly anesthetized rats caused a dose-related retardation of the heat-elicited off-cell pause, on-cell discharge and corresponding TF. Neuronal response and TF retained their mutual time relationship but shifted pari passu toward longer latencies. This antinociception was apparent already 5 min post-injection and reached a maximum in 50-60 min for i.v. administration and 30-35 min for PAG microinjection. These results confirm other authors' findings of the direct antinociceptive action of NSAIDs upon PAG, and provide the first evidence for a plausible involvement of RVM off- and on-cells in such antinociceptive effect.