Impact of Interleukin-6 Activation and Arthritis on Epidermal Growth Factor Receptor (EGFR) Activation in Sensory Neurons and the Spinal Cord.

In tumor cells, interleukin-6 (IL-6) signaling can lead to activation of the epidermal growth factor receptor (EGFR), which prolongs Stat3 activation. In the present experiments, we tested the hypothesis that IL-6 signaling activates EGFR signaling in peripheral and spinal nociception and examined whether EGFR localization and activation coincide with pain-related behaviors in arthritis. In vivo in anesthetized rats, spinal application of the EGFR receptor blocker gefitinib reduced the responses of spinal cord neurons to noxious joint stimulation, but only after spinal pretreatment with IL-6 and soluble IL-6 receptor. Using Western blots, we found that IL-6-induced Stat3 activation was reduced by gefitinib in microglial cells of the BV2 cell line, but not in cultured DRG neurons. Immunohistochemistry showed EGFR localization in most DRG neurons from normal rats, but significant downregulation in the acute and most painful arthritis phase. In the spinal cord of mice, EGFR was highly activated mainly in the chronic phase of inflammation, with localization in neurons. These data suggest that spinal IL-6 signaling may activate spinal EGFR signaling. Downregulation of EGFR in DRG neurons in acute arthritis may limit nociception, but pronounced delayed activation of EGFR in the spinal cord may be involved in chronic inflammatory pain.

Spinal pain processing in arthritis: Neuron and glia (inter)actions.

Diseases of joints are among the most frequent causes of chronic pain. In the course of joint diseases, the peripheral and the central nociceptive system develop persistent hyperexcitability (peripheral and central sensitization). This review addresses the mechanisms of spinal sensitization evoked by arthritis. Electrophysiological recordings in anesthetized rats from spinal cord neurons with knee input in a model of acute arthritis showed that acute spinal sensitization is dependent on spinal glutamate receptors (AMPA, NMDA, and metabotropic glutamate receptors) and supported by spinal actions of neuropeptides such as neurokinins and CGRP, by prostaglandins, and by proinflammatory cytokines. In several chronic arthritis models (including immune-mediated arthritis and osteoarthritis) spinal glia activation was observed to be coincident with behavioral mechanical hyperalgesia which was attenuated or prevented by intrathecal application of minocycline, fluorocitrate, and pentoxyfylline. Some studies identified specific pathways of micro- and astroglia activation such as the purinoceptor- (P2 X7 -) cathepsin S/CX3 CR1 pathway, the mobility group box-1 protein (HMGB1), and toll-like receptor 4 (TLR4) activation, spinal NFκB/p65 activation and others. The spinal cytokines TNF, interleukin-6, interleukin-1ß, and others form a functional spinal network characterized by an interaction between neurons and glia cells which is required for spinal sensitization. Neutralization of spinal cytokines by intrathecal interventions attenuates mechanical hyperalgesia. This effect may in part result from local suppression of spinal sensitization and in part from efferent effects which attenuate the inflammatory process in the joint. In summary, arthritis evokes significant spinal hyperexcitability which is likely to contribute to the phenotype of arthritis pain in patients.

Spinal interleukin-1ß induces mechanical spinal hyperexcitability in rats: Interactions and redundancies with TNF and IL-6.

Both spinal tumor necrosis factor (TNF) and interleukin-6 (IL-6) contribute to the development of "mechanical" spinal hyperexcitability in inflammatory pain states. Recently, we found that spinal sensitization by TNF was significantly reduced by blockade of spinal IL-6 signaling suggesting that IL-6 signaling is involved in spinal TNF effects. Here, we explored whether spinal interleukin-1ß (IL-1ß), also implicated in inflammatory pain, induces "mechanical" spinal hyperexcitability, and whether spinal IL-1ß effects are related to TNF and IL-6 effects. We recorded the responses of spinal cord neurons to mechanical stimulation of the knee joint in vivo and used cellular approaches on microglial and astroglial cell lines to identify interactions of IL-1ß, TNF, and IL-6. Spinal application of IL-1ß in anesthetized rats modestly enhanced responses of spinal cord neurons to innocuous and noxious mechanical joint stimulation. This effect was blocked by minocycline indicating microglia involvement, and significantly attenuated by interfering with IL-6 signaling. In the BV2 microglial cell line, IL-1ß, like TNF, enhanced the release of soluble IL-6 receptor, necessary for spinal IL-6 actions. Different to TNF, IL-1ß caused SNB-19 astrocytes to release interleukin-11. The generation of "mechanical" spinal hyperexcitability by IL-1ß was more pronounced upon spinal TNF neutralization with etanercept, suggesting that concomitant TNF limits IL-1ß effects. In BV2 cells, TNF stimulated the release of IL-1Ra, an endogenous IL-1ß antagonist. Thus, spinal IL-1ß has the potential to induce spinal hyperexcitability sharing with TNF dependency on IL-6 signaling, but TNF also limited IL-1ß effects explaining the modest effect of IL-1ß.

Oncostatin M induces hyperalgesic priming and amplifies signaling of cAMP to ERK by RapGEF2 and PKA.

Hyperalgesic priming is characterized by enhanced nociceptor sensitization by pronociceptive mediators, prototypically PGE2 . Priming has gained interest as a mechanism underlying the transition to chronic pain. Which stimuli induce priming and what cellular mechanisms are employed remains incompletely understood. In adult male rats, we present the cytokine Oncostatin M (OSM), a member of the IL-6 family, as an inducer of priming by a novel mechanism. We used a high content microscopy based approach to quantify the activation of endogenous PKA-II and ERK of thousands sensory neurons in culture. Incubation with OSM increased and prolonged ERK activation by agents that increase cAMP production such as PGE2 , forskolin, and cAMP analogs. These changes were specific to IB4/CaMKIIα positive neurons, required protein translation, and increased cAMP-to-ERK signaling. In both, control and OSM-treated neurons, cAMP/ERK signaling involved RapGEF2 and PKA but not Epac. Similar enhancement of cAMP-to-ERK signaling could be induced by GDNF, which acts mostly on IB4/CaMKIIα-positive neurons, but not by NGF, which acts mostly on IB4/CaMKIIα-negative neurons. In vitro, OSM pretreatment rendered baseline TTX-R currents ERK-dependent and switched forskolin-increased currents from partial to full ERK-dependence in small/medium sized neurons. In summary, priming induced by OSM uses a novel mechanism to enhance and prolong coupling of cAMP/PKA to ERK1/2 signaling without changing the overall pathway structure.

Involvement of Spinal IL-6 Trans-Signaling in the Induction of Hyperexcitability of Deep Dorsal Horn Neurons by Spinal Tumor Necrosis Factor-Alpha.

UNLABELLED: During peripheral inflammation, both spinal TNF-α and IL-6 are released within the spinal cord and support the generation of inflammation-evoked spinal hyperexcitability. However, whether spinal TNF-α and IL-6 act independently in parallel or in a functionally dependent manner has not been investigated. In extracellular recordings from mechanonociceptive deep dorsal horn neurons of normal rats in vivo, we found that spinal application of TNF-α increased spinal neuronal responses to mechanical stimulation of knee and ankle joints. This effect was significantly attenuated by either sgp130, which blocks IL-6 trans-signaling mediated by IL-6 and its soluble receptor IL-6R (sIL-6R); by an antibody to the IL-6 receptor; or by minocycline, which inhibits the microglia. IL-6 was localized in neurons of the spinal cord and, upon peripheral noxious stimulation in the presence of spinal TNF-α, IL-6 was released spinally. Furthermore, TNF-α recruited microglial cells to provide sIL-6R, which can form complexes with IL-6. Spinal application of IL-6 plus sIL-6R, but not of IL-6 alone, enhanced spinal hyperexcitability similar to TNF-α and the inhibition of TNF-α-induced hyperexcitability by minocycline was overcome by coadministration of sIL-6R, showing that sIL-6R is required. Neither minocycline nor the TNF-α-neutralizing compound etanercept inhibited the induction of hyperexcitability by IL-6 plus sIL-6R. Together, these data show that the induction of hyperexcitability of nociceptive deep dorsal horn neurons by TNF-α largely depends on the formation of IL-6/sIL-6R complexes that are downstream of TNF-α and requires the interactions of neurons and microglia orchestrated by TNF-α. SIGNIFICANCE STATEMENT: Both spinal TNF-α and IL-6 induce a state of spinal hyperexcitability. We present the novel finding that the full effect of TNF-α on the development of spinal hyperexcitability depends on IL-6 trans-signaling acting downstream of TNF-α. IL-6 trans-signaling requires the formation of complexes of IL-6 and soluble IL-6 receptor. Spinal TNF-α furthers the release of IL-6 from neurons in the spinal cord during peripheral noxious stimulation and recruits microglial cells to provide soluble IL-6 receptor, which can form complexes with IL-6. Therefore, a specific interaction between neurons and microglia is required for the full development of TNF-α-induced hyperexcitability of nociceptive deep horsal horn neurons.

Neuronal prostaglandin E2 receptor subtype EP3 mediates antinociception during inflammation.

The pain mediator prostaglandin E2 (PGE2) sensitizes nociceptive pathways through EP2 and EP4 receptors, which are coupled to Gs proteins and increase cAMP. However, PGE2 also activates EP3 receptors, and the major signaling pathway of the EP3 receptor splice variants uses inhibition of cAMP synthesis via Gi proteins. This opposite effect raises the intriguing question of whether the Gi-protein-coupled EP3 receptor may counteract the EP2 and EP4 receptor-mediated pronociceptive effects of PGE2. We found extensive localization of the EP3 receptor in primary sensory neurons and the spinal cord. The selective activation of the EP3 receptor at these sites did not sensitize nociceptive neurons in healthy animals. In contrast, it produced profound analgesia and reduced responses of peripheral and spinal nociceptive neurons to noxious stimuli but only when the joint was inflamed. In isolated dorsal root ganglion neurons, EP3 receptor activation counteracted the sensitizing effect of PGE2, and stimulation of excitatory EP receptors promoted the expression of membrane-associated inhibitory EP3 receptor. We propose, therefore, that the EP3 receptor provides endogenous pain control and that selective activation of EP3 receptors may be a unique approach to reverse inflammatory pain. Importantly, we identified the EP3 receptor in the joint nerves of patients with painful osteoarthritis.

Spinal interleukin-6 is an amplifier of arthritic pain in the rat.

OBJECTIVE: Significant joint pain is usually widespread beyond the affected joint, which results from the sensitization of nociceptive neurons in the central nervous system (central sensitization). This study was undertaken to explore whether the proinflammatory cytokine interleukin-6 (IL-6) in the joint induces central sensitization, whether joint inflammation causes the release of IL-6 from the spinal cord, and whether spinal IL-6 contributes to central sensitization. METHODS: In anesthetized rats, electrophysiologic recordings of spinal cord neurons with sensory input from the knee joint were made. Neuronal responses to mechanical stimulation of the rat knee and leg were monitored. IL-6 and soluble IL-6 receptor (sIL-6R) were applied to the knee joint or the spinal cord. Spinal release of IL-6 was measured by enzyme-linked immunosorbent assay. Soluble gp130, which neutralizes IL-6/sIL-6R, was spinally applied during the development of joint inflammation or during established inflammation. RESULTS: A single injection of IL-6/sIL-6R into the rat knee joint as well as application of IL-6/sIL-6R to the rat spinal cord significantly increased the responses of spinal neurons to mechanical stimulation of the knee and ankle joint, i.e., induced central sensitization. Application of soluble gp130 to the rat spinal cord attenuated this effect of IL-6. The development of knee inflammation in the rat caused spinal release of IL-6. Spinal application of soluble gp130 attenuated the development of inflammation-evoked central sensitization but did not reverse it. CONCLUSION: Our findings indicate that the generation of joint pain in the rat involves not only IL-6 in the joint but also IL-6 released from the spinal cord. Spinal IL-6 contributes to central sensitization and thus promotes the widespread hyperalgesia observed in the course of joint disease.

Prostaglandin EP3 receptor activation is antinociceptive in sensory neurons via PI3Kγ, AMPK and GRK2.

BACKGROUND AND PURPOSE: Prostaglandin E2 is considered a major mediator of inflammatory pain, by acting on neuronal Gs protein-coupled EP2 and EP4 receptors. However, the neuronal EP3 receptor, colocalized with EP2 and EP4 receptor, is Gi protein-coupled and antagonizes the pronociceptive prostaglandin E2 effect. Here, we investigated the cellular signalling mechanisms by which the EP3 receptor reduces EP2 and EP4 receptor-evoked pronociceptive effects in sensory neurons. EXPERIMENTAL APPROACH: Experiments were performed on isolated and cultured dorsal root ganglion (DRG) neurons from wild type, phosphoinositide 3-kinase γ (PI3Kγ)-/- , and PI3Kγkinase dead (KD)/KD mice. For subtype-specific stimulations, we used specific EP2, EP3, and EP4 receptor agonists from ONO Pharmaceuticals. As a functional readout, we recorded TTX-resistant sodium currents in patch-clamp experiments. Western blots were used to investigate the activation of intracellular signalling pathways. EP4 receptor internalization was measured using immunocytochemistry. KEY RESULTS: Different pathways mediate the inhibition of EP2 and EP4 receptor-dependent pronociceptive effects by EP3 receptor stimulation. Inhibition of EP2 receptor-evoked pronociceptive effect critically depends on the kinase-independent function of the signalling protein PI3Kγ, and adenosine monophosphate activated protein kinase (AMPK) is involved. By contrast, inhibition of EP4 receptor-evoked pronociceptive effect is independent on PI3Kγ and mediated through activation of G protein-coupled receptor kinase 2 (GRK2), which enhances the internalization of the EP4 receptor after ligand binding. CONCLUSION AND IMPLICATIONS: Activation of neuronal PI3Kγ, AMPK, and GRK2 by EP3 receptor activation limits cAMP-dependent pain generation by prostaglandin E2 . These new insights hold the potential for a novel approach in pain therapy.

Joint pain.

Both inflammatory and degenerative diseases of joints are major causes of chronic pain. This overview addresses the clinical problem of joint pain, the nociceptive system of the joint, the mechanisms of peripheral and central sensitization during joint inflammation and long term changes during chronic joint inflammation. While the nature of inflammatory pain is obvious the nature and site of origin of osteoarthritic pain is less clear. However, in both pathological conditions mechanical hyperalgesia is the major pain problem, and indeed, both joint nociceptors and spinal nociceptive neurons with joint input show pronounced sensitization for mechanical stimulation. Molecular mechanisms of mechanical sensitization of joint nociceptors are addressed with an emphasis on cytokines, and molecular mechanisms of central sensitization include data on the role of excitatory amino acids, neuropeptides and spinal prostaglandins. The overview will also address long-term changes of pain-related behavior, response properties of neurons and receptor expression in chronic animal models of arthritis.

The analgesic potential of cytokine neutralization with biologicals.

Many acute and chronic inflammatory diseases, cancer and neuropathic disorders are accompanied by severe pain states reducing drastically the life quality of the patients. Biologicals which preferentially target cytokines often reduce the disease processes by influencing immune cells, tissue healing, inflammatory aspects and other typical processes of the diseases. Remarkably the effect of biologicals in pain and nociception is often neglected or insufficiently explored. However, because of the dense interaction of the nociceptive system with the surrounding peripheral or central tissue, targeting cytokines has the potential to treat pain at the same time as the other symptoms of the disease. The following review shows mainly results from animal experiments (with some parallels to human studies). It depicts where and how cytokines are involved in nociceptive processing and pain and also indicates possible target strategies. It concentrates on the excitatory cytokines IL-6, TNF-α, IL-1ß, IFN-γ and IL-17.

The potential of substance P to initiate and perpetuate cortical spreading depression (CSD) in rat in vivo.

The tachykinin substance P (SP) increases neuronal excitability, participates in homeostatic control, but induces brain oedema after stroke or trauma. We asked whether SP is able to induce cortical spreading depression (CSD) which often aggravates stroke-induced pathology. In anesthetized rats we applied SP (10-5, 10-6, 10-7, or 10-8 mol/L) to a restricted cortical area and recorded CSDs there and in remote non-treated areas using microelectrodes. SP was either applied in artificial cerebrospinal fluid (ACSF), or in aqua to perform a preconditioning. Plasma extravasation in cortical grey matter was assessed with Evans Blue. Only SP dissolved in aqua induced self-regenerating CSDs. SP dissolved in ACSF did not ignite CSDs even when excitability was increased by acetate-preconditioning. Aqua alone elicited as few CSDs as the lowest concentration of SP. Local pretreatment with 250 nmol/L of a neurokinin 1 receptor antagonist prevented the SP-induced plasma extravasation, the initiation of CSDs by 10-5 mol/L SP diluted in aqua, and the initiation of CSDs by aqua alone, but did not suppress KCl-induced CSD. Thus neurokinin 1 receptor antagonists may be used to explore the involvement of SP in CSDs in clinical studies.

The role of spinal nuclear factor-kappa B in spinal hyperexcitability.

In behavioral experiments, inhibition of nuclear factor-kappaB activation by systemic administration of the IkappaB kinase inhibitor S1627 has been shown to attenuate inflammatory and neuropathic pain. Here, we specifically investigated with electrophysiological recordings in anesthetized rats whether spinal application of S1627 influences hyperexcitability of dorsal horn neurons during an acute knee joint inflammation. Spinal application of S1627 before and early during development of inflammation totally prevented spinal hyperexcitability suggesting an important role of spinal nuclear factor-kappaB in this process. During established inflammation, however, S1627 did not reduce the responses of neurons to mechanical stimulation of the inflamed knee within 2.5 h after spinal administration, thus suggesting that spinal hyperexcitability is not maintained by continuous nuclear factor-kappaB activation.

Changes in the effect of spinal prostaglandin E2 during inflammation: prostaglandin E (EP1-EP4) receptors in spinal nociceptive processing of input from the normal or inflamed knee joint.

Inflammatory pain is caused by sensitization of peripheral and central nociceptive neurons. Prostaglandins substantially contribute to neuronal sensitization at both sites. Prostaglandin E2 (PGE2) applied to the spinal cord causes neuronal hyperexcitability similar to peripheral inflammation. Because PGE2 can act through EP1-EP4 receptors, we addressed the role of these receptors in the spinal cord on the development of spinal hyperexcitability. Recordings were made from nociceptive dorsal horn neurons with main input from the knee joint, and responses of the neurons to noxious and innocuous stimulation of the knee, ankle, and paw were studied after spinal application of recently developed specific EP1-EP4 receptor agonists. Under normal conditions, spinal application of agonists at EP1, EP2, and EP4 receptors induced spinal hyperexcitability similar to PGE2. Interestingly, the effect of spinal EP receptor activation changed during joint inflammation. When the knee joint had been inflamed 7-11 hr before the recordings, only activation of the EP1 receptor caused additional facilitation, whereas spinal application of EP2 and EP4 receptor agonists had no effect. Additionally, an EP3alpha receptor agonist reduced responses to mechanical stimulation. The latter also attenuated spinal hyperexcitability induced by spinal PGE2. In isolated DRG neurons, the EP3alpha agonist reduced the facilitatory effect of PGE2 on TTX-resistant sodium currents. Thus pronociceptive effects of spinal PGE2 can be limited, particularly under inflammatory conditions, through activation of an inhibitory splice variant of the EP3 receptor. The latter might be an interesting target for controlling spinal hyperexcitability in inflammatory pain states.

Noradrenergic agonists and antagonists influence migration of cortical spreading depression in rat-a possible mechanism of migraine prophylaxis and prevention of postischemic neuronal damage.

Cortical spreading depression (CSD) is thought to be a neuronal mechanism that expands the penumbra zone after focal brain ischemia and that causes migraine aura. Both adrenergic agonists and antagonists significantly influence the size of the penumbra zone and decline the frequency of migraine. To study whether these compounds act by influencing CSD, we applied different drugs topically to an area of the exposed cortex of anesthetized adult rats and observed the migration of CSD-related DC potential deflections across the treated area. The adrenergic agonist norepinephrine (1 mmol/L) and the alpha(2)-agonist clonidine (0.56 mmol/L) blocked reversibly the migration of CSD. The beta-blocker propranolol (250 micromol/L to 1 mmol/L) dose-dependently diminished migration velocity or even blocked migration of CSD. The CSD blockade by the alpha(2)-antagonist yohimbine (1.75 mmol/L) was because of its action on inhibitory 5-HT(1A) receptors. None of the substances in the concentrations used had influence on regional cerebral blood flow or on systemic arterial blood pressure. The data suggest that the interference of these compounds with CSD may contribute to their beneficial therapeutic effect. The effect of beta-receptor antagonists in human migraine needs further exploration, since these drugs also work in migraine without aura.

Nociceptive neurons in the rat caudal trigeminal nucleus respond to blood plasma perfusion of the subarachnoid space: the involvement of complement.

The meninges of the brain are innervated by afferent nerve fibres containing SP and CGRP, two typical peptides found in sensory neurons. These fibres project to the trigeminal nuclear complex and the cervical dorsal horn. Discharge of the afferents may provide a physiological basis for some types of headaches. Considerable speculation surrounds the possible causes of meningeal afferent activation. Blood-borne substances released during subarachnoid haemorrhage are one possibility and there is a possibility that these also play a role in migraine. In the case of migraine, blood components, e.g. from platelets, cannot be excluded. To investigate the possible effects of platelets and plasma factors, the subarachnoid space of the rat was continuously perfused with artificial cerebrospinal fluid during extracellular recordings from single units of the caudal trigeminal nucleus. Washed and concentrated suspensions of adenosindiphosphate (ADP)--activated platelets and plasma, from which platelets had been removed--were introduced as a bolus into the continuous flow. Neurons in the caudal nucleus of the trigeminal complex receiving input from the meninges were stimulated. They did not respond to the activated platelet suspensions but showed intense responses to plasma. Plasma completely lost its ability to excite trigeminal neurons after heat inactivation (30 min, 56 degrees C). It is concluded that the complement system may be involved in the excitatory nociceptive effect of platelet-poor plasma.

Mechanisms of pain in arthritis.

Inflammation in the joint causes peripheral sensitization (increase of sensitivity of nociceptive primary afferent neurons) and central sensitization (hyperexcitability of nociceptive neurons in the central nervous system). The processes of sensitization are thought to be the basis of arthritic pain that appears as spontaneous pain (joints at rest) and hyperalgesia (augmented pain response on noxious stimulation and pain on normally nonpainful stimulation). Sensitization also facilitates efferent neuronal processes through which the nervous system influences the inflammatory process. Peripheral sensitization is produced by the action of inflammatory mediators such as bradykinin, prostaglandins, neuropeptides, and cytokines which activate corresponding receptors in proportions of nerve fibers. In addition, the expression of receptors, for example, bradykinin and neurokinin 1 receptors, is upregulated during inflammation. The development of hyperexcitability of spinal cord neurons is produced by various transmitter/receptor systems that constitute and modulate synaptic activation of the neurons. The key transmitter is glutamate that activates N-methyl-d-aspartate (NMDA) and non-NMDA receptors on spinal cord neurons. Blockade of these receptors prevents and reduces central sensitization. Excitatory neuropeptides (substance P and calcitonin gene-related peptide) further central sensitization. Central sensitization also is facilitated by mediators that have complex actions (e.g., prostaglandin E(2)). Spinal PGE(2) binds to receptors at presynaptic endings of primary afferent neurons (thus influencing synaptic release) and to receptors on postsynaptic spinal cord neurons. The administration of PGE(2) to the spinal cord surface produces changes of responsiveness of spinal neurons similar to peripheral inflammation, and spinal indomethacin to the spinal cord attenuates development of hyperexcitability significantly.

Blockade of voltage-gated calcium channels in rat inhibits repetitive cortical spreading depression.

Blockers of L-, N-, and P/Q-type voltage-gated calcium channels (VGCCs) were topically applied to the cortical surface of anaesthetized adult rats to study their role in cortical spreading depression (CSD), a correlate of the migraine aura. By pricking the brain, single CSD could still be elicited after blockade of the three different types of VGCCs as in the untreated brain. Topical KCl application to the untreated cortex resulted in repetitive CSD. However, after application of blockers at either L-, or N-, or P/Q-type VGCCs to the cortical surface, application of KCl elicited only one or very few CSD, and their repetition rate was dramatically reduced. The results suggest that cortical excitability resulting in repetitive CSD is markedly influenced by N- and P/Q-type VGCCs and less by L-type VGCCs.

Involvement of peripheral and spinal tumor necrosis factor α in spinal cord hyperexcitability during knee joint inflammation in rats.

OBJECTIVE: Tumor necrosis factor α (TNFα) is produced not only in peripheral tissues, but also in the spinal cord. The purpose of this study was to address the potential of peripheral and spinal TNFα to induce and maintain spinal hyperexcitability, which is a hallmark of pain states in the joints during rheumatoid arthritis and osteoarthritis. METHODS: In vivo recordings of the responses of spinal cord neurons to nociceptive knee input under normal conditions and in the presence of experimental knee joint inflammation were obtained in anesthetized rats. TNFα, etanercept, or antibodies to TNF receptors were applied to either the knee joint or the spinal cord surface. RESULTS: Injection of TNFα into the knee joint cavity increased the responses of spinal cord neurons to mechanical joint stimulation, and injection of etanercept into the knee joint reduced the inflammation-evoked spinal activity. These spinal effects closely mirrored the induction and reduction of peripheral sensitization. Responses to joint stimulation were also enhanced by spinal application of TNFα, and spinal application of either etanercept or anti-TNF receptor type I significantly attenuated the generation of inflammation-evoked spinal hyperexcitability, which is characterized by widespread pain sensitization beyond the inflamed joint. Spinally applied etanercept did not reduce established hyperexcitability in the acute kaolin/carrageenan model. In antigen-induced arthritis, etanercept decreased spinal responses on day 1, but not on day 3. CONCLUSION: While peripheral TNFα increases spinal responses to joint stimulation, spinal TNFα supports the generation of the full pattern of spinal hyperexcitability. However, established spinal hyperexcitability may be maintained by downstream mechanisms that are independent of spinal TNFα.

Enhanced neuronal excitability in adult rat brainstem causes widespread repetitive brainstem depolarizations with cardiovascular consequences.

The brainstem of the adult rat is relatively resistant to spreading depolarization (SD) but after enhancement of excitability SD can be evoked by local application of KCl. In the present experiments, we observed that the enhanced excitability even triggers prolonged periods of repetitive depolarizations (RDs), which elicit significant cardiovascular changes. In contrast to KCl-evoked SDs with amplitudes of ∼24 mV and spreading velocity of 4 mm/min, spontaneous RDs had amplitudes of 7 to 12 mV, propagated up to 30 times faster than KCl-evoked SDs, and depolarized larger brainstem areas including the contralateral side. Similarly as SD, RDs depended on glutamatergic neurotransmission and were blocked by MK-801 or by the calcium channel blocker agatoxin. They depended on sodium channels and were blocked by tetrodotoxin. Functionally, the invasion of RDs into the spinal trigeminal and other nuclei evoked bursts of action potentials, indicating that specific neuronal systems are affected. In fact, during episodes of RDs the blood pressure and the local blood flow at the surface of the brainstem and the cortex increased substantially. Brainstem RDs did not propagate into the cerebral cortex. We propose to consider brainstem RPs as a pathophysiological mechanism whose significance for brainstem disease states should be further explored.