Galanin diminishes cortical spreading depolarization across rodents - A candidate for treatment?

Galanin (Gal) is a neuropeptide with the potential to ameliorate cortical spreading depolarization (CSD), an electrophysiological phenomenon occurring after brain injury or in migraine aura. Gal is expressed in all cortical neurons both in rat and in mouse cortices. Here we investigated whether the effect of Gal on CSD previously described in the rat is conserved in the mouse cortex. In rats, the topical application of Gal to the cortex for 1 h did not induce any change in CSD amplitudes, propagation velocity, or threshold of elicitation. Rather, topical application of Gal for 3 h was necessary to obtain a significant decrease in these CSD parameters and to develop a remarkable increase in the KCl threshold to elicit a CSD in rat cortex. In contrast, the topical application of Gal on cortical surface for 1 h in mice was sufficient to significantly attenuate CSD amplitudes and increase threshold. A thinner cortex, a faster diffusion or different affinity/expression of receptors for Gal are possible reasons to explain this difference in the time course between rats and mice. Our data are relevant to postulate Gal as a potential target for inhibition of CSD under pathological situations such as stroke or ischemia. SIGNIFICANCE STATEMENT: The neuropeptide Galanin (Gal) is expressed in all neurons throughout the cerebral cortex, both in rats and mice, and is able to reduce or even inhibit Cortical Spreading Depolarization, thus, Gal has the potential to control neuronal excitability that may identify Gal as a target in drug development against CSD.

Neuropathological changes in the TASTPM mouse model of Alzheimer's disease and their relation to hyperexcitability and cortical spreading depolarization.

Although Alzheimer's disease (AD) is characterized by distinct pathological changes, their precise impact on cortical functions are not well understood. Here we used TASTPM mice as an AD model and asked whether the development of neurodegenerative changes has an impact on the extracellular space (ECS) and neuronal excitability, in particular cortical spreading depolarization (CSD) which requires intact neuron and glial functions. We studied wildtype (WT) and TASTPM mice (3, 6, and 12 months old). TASTPM mice showed progressive proliferation of neocortical Amyloid-beta (Aß) plaques between 3 and 12 months (more deposits in females than in males) and Aß accumulation in cortical vessels. As plaques proliferated, neuroinflammatory microglial reaction (CD68, CD39 and Galectin-3) and astrogliosis (GFAP) developed progressively. The cortical ECS volume shrank significantly to about half the size of the WT. CSD in both WT and TASTPM mice showed considerable heterogeneity but did not correlate with the histological changes. However, CSDs were easier to elicit in TASTPM than in WT mice at 3 months, and also compared to older TASTPM mice. Moreover, TASTPM mice showed more hyperexcitability manifested as clonic-tonic behavior after sodium thiopental anesthesia. Thus, AD pathology was associated with abnormal hyperexcitability but did not homogenously alter CSD susceptibility.

Importance of IL-6 trans-signaling and high autocrine IL-6 production in human osteoarthritic chondrocyte metabolism.

OBJECTIVE: Neutralization of Interleukin (IL)-6-signaling by antibodies is considered a promising tool for the treatment of osteoarthritis (OA). To gain further insight into this potential treatment, this study investigated the effects of IL-6-signaling and IL-6 neutralization on chondrocyte metabolism and the release of IL-6-signaling-related mediators by human chondrocytes. DESIGN: Chondrocytes were collected from 49 patients with advanced knee/hip OA or femoral neck fracture. Isolated chondrocytes were stimulated with different mediators to analyze the release of IL-6, soluble IL-6 receptor (sIL-6R) and soluble gp130 (sgp130). The effect of IL-6 and IL-6/sIL-6R complex as well as neutralization of IL-6-signaling on the metabolism was analyzed. RESULTS: OA chondrocytes showed high basal IL-6 production and release, which was strongly negatively correlated with the production of cartilage-matrix-proteins. Chondrocytes produced and released sIL-6R and sgp130. The IL-6/sIL-6R complex significantly increased nitric oxide, prostaglandin E2 and matrix metalloproteinase 1 production, decreased Pro-Collagen Type II and mitochondrial ATP production, and increased glycolysis in OA chondrocytes. Neutralization of IL-6-signaling by antibodies did not significantly affect the metabolism of OA chondrocytes, but blocking of glycoprotein 130 (gp130)-signaling by SC144 significantly reduced the basal IL-6 release. CONCLUSION: Although IL-6 trans-signaling induced by IL-6/sIL-6R complex negatively affects OA chondrocytes, antibodies against IL-6 or IL-6R did not affect chondrocyte metabolism. Since inhibition of gp130-signaling reduced the enhanced basal release of IL-6, interfering with gp130-signaling may ameliorate OA progression because high cellular release of IL-6 correlates with reduced production of cartilage-matrix-proteins.

Cortical spreading depolarization is a potential target for rat brain excitability modulation by Galanin.

The inhibitory neuropeptide Galanin (Gal) has been shown to mediate anticonvulsion and neuroprotection. Here we investigated whether Gal affects cortical spreading depolarization (CSD). CSD is considered the pathophysiological neuronal mechanism of migraine aura, and a neuronal mechanism aggravating brain damage upon afflictions of the brain. Immunohistochemistry localized Gal and the Gal receptors 1-3 (GalR1-3) in native rat cortex and evaluated microglial morphology after exposure to Gal. In anesthetized rats, Gal was applied alone and together with the GalR antagonists M40, M871, or SNAP 37889 locally to the exposed cortex. The spontaneous electrocorticogram and CSDs evoked by remote KCl pressure microinjection were measured. In rat cortex, Gal was present in all neurons of all cortical layers, but not in astrocytes, microglia and vessels. GalR2 and GalR3 were expressed throughout all neurons, whereas GalR1 was preponderantly located at neurons in layers IV and V, but only in about half of the neurons. In susceptible rats, topical application of Gal on cortex decreased CSD amplitude, slowed CSD propagation velocity, and increased the threshold for KCl to ignite CSD. In some rats, washout of previously applied Gal induced periods of epileptiform patterns in the electrocorticogram. Blockade of GalR2 by M871 robustly prevented all Gal effects on CSD, whereas blockade of GalR1 or GalR3 was less effective. Although microglia did not express GalRs, topical application of Gal changed microglial morphology indicating microglial activation. This effect of Gal on microglia was prevented by blocking neuronal GalR2. In conclusion, Gal has the potential to ameliorate CSD thus reducing pathophysiological neuronal events caused by or associated with CSD.

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.

Osteoarthritis-Induced Metabolic Alterations of Human Hip Chondrocytes.

Osteoarthritis (OA) alters chondrocyte metabolism and mitochondrial biology. We explored whether OA and non-OA chondrocytes show persistent differences in metabolism and mitochondrial function and different responsiveness to cytokines and cAMP modulators. Hip chondrocytes from patients with OA or femoral neck fracture (non-OA) were stimulated with IL-1ß, TNF, forskolin and opioid peptides. Mediators released from chondrocytes were measured, and mitochondrial functions and glycolysis were determined (Seahorse Analyzer). Unstimulated OA chondrocytes exhibited significantly higher release of IL-6, PGE2 and MMP1 and lower production of glycosaminoglycan than non-OA chondrocytes. Oxygen consumption rates (OCR) and mitochondrial ATP production were comparable in unstimulated non-OA and OA chondrocytes, although the non-mitochondrial OCR was higher in OA chondrocytes. Compared to OA chondrocytes, non-OA chondrocytes showed stronger responses to IL-1ß/TNF stimulation, consisting of a larger decrease in mitochondrial ATP production and larger increases in non-mitochondrial OCR and NO production. Enhancement of cAMP by forskolin prevented IL-1ß-induced mitochondrial dysfunction in OA chondrocytes but not in non-OA chondrocytes. Endogenous opioids, present in OA joints, influenced neither cytokine-induced mitochondrial dysfunction nor NO upregulation. Glycolysis was not different in non-OA and OA chondrocytes, independent of stimulation. OA induces persistent metabolic alterations, but the results suggest upregulation of cellular mechanisms protecting mitochondrial function in OA.

From spreading depolarization to epilepsy with neuroinflammation: The role of CGRP in cortex.

CGRP release plays a major role in migraine pain by activating the trigeminal pain pathways. Here we explored putative additional effects of CGRP on cortical circuits and investigated whether CGRP affects cortical excitability, cortical spreading depolarization (CSD), a phenomenon associated with migraine aura, blood-brain-barrier (BBB) and microglial morphology. We used immunohistochemistry to localize CGRP and the CGRP receptor (CGRP-R) in native cortex and evaluated morphology of microglia and integrity of the BBB after exposure to CGRP. In anesthetized rats we applied CGRP and the CGRP-R antagonist BIBN4096BS locally to the exposed cortex and monitored the spontaneous electrocorticogram and CSDs evoked by remote KCl pressure microinjection. In mouse brain slices CGRP effects on neuronal activity were explored by multielectrode array. CGRP immunoreactivity was detectable in intracortical vessels, and all cortical neurons showed CGRP-R immunoreactivity. In rat cortex in vivo, topical CGRP induced periods of epileptiform discharges, however, also dose-dependently reduced CSD amplitudes and propagation velocity. BIBN4096BS prevented these effects. CGRP evoked synchronized bursting activity in mouse cortical but not in cerebellar slices. Topical application of CGRP to rat cortex induced plasma extravasation and this was associated with reduced ramification of microglial cells. From these findings we conclude that CGRP induces a pathophysiological state in the cortex, consisting in neuronal hyperexcitability and neuroinflammation. Thus, CGRP may have a pronounced impact on brain functions during migraine episodes supporting the benefit of CGRP antagonists for clinical use. However, increased cortical CGRP may end the CSD-induced aura phase of migraine.

Substance P and Alpha-Calcitonin Gene-Related Peptide Differentially Affect Human Osteoarthritic and Healthy Chondrocytes.

Osteoarthritis (OA) is a degenerative joint disease that not only causes cartilage loss but also structural damage in all joint tissues. Joints are innervated by alpha-calcitonin gene-related peptide (αCGRP) and substance P (SP)-positive sensory nerve fibers. Alteration of sensory joint innervation could be partly responsible for degenerative changes in joints that contribute to the development of OA. Therefore, our aim was to analyze and compare the molecular effects of SP and αCGRP on the metabolism of articular chondrocytes from OA patients and non-OA cartilage donors. We treated the cells with SP or αCGRP and analysed the influence of these neuropeptides on chondrocyte metabolism and modulation of signaling pathways. In chondrocytes from healthy cartilage, SP had minimal effects compared with its effects on OA chondrocytes, where it induced inflammatory mediators, inhibited chondrogenic markers and promoted apoptosis and senescence. Treatment with αCGRP also increased apoptosis and senescence and reduced chondrogenic marker expression in OA chondrocytes, but stimulated an anabolic and protective response in healthy chondrocytes. The catabolic influence of SP and αCGRP might be due to activation of ERK signaling that could be counteracted by an increased cAMP response. We suggest that a switch between the G-subunits of the corresponding receptors after binding their ligands SP or αCGRP plays a central role in mediating the observed effects of sensory neuropeptides on chondrocytes.

Inhibition of Inducible Nitric Oxide Synthase Prevents IL-1ß-Induced Mitochondrial Dysfunction in Human Chondrocytes.

Interleukin (IL)-1ß is an important pro-inflammatory cytokine in the progression of osteoarthritis (OA), which impairs mitochondrial function and induces the production of nitric oxide (NO) in chondrocytes. The aim was to investigate if blockade of NO production prevents IL-1ß-induced mitochondrial dysfunction in chondrocytes and whether cAMP and AMP-activated protein kinase (AMPK) affects NO production and mitochondrial function. Isolated human OA chondrocytes were stimulated with IL-1ß in combination with/without forskolin, L-NIL, AMPK activator or inhibitor. The release of NO, IL-6, PGE2, MMP3, and the expression of iNOS were measured by ELISA or Western blot. Parameters of mitochondrial respiration were measured using a seahorse analyzer. IL-1ß significantly induced NO release and mitochondrial dysfunction. Inhibition of iNOS by L-NIL prevented IL-1ß-induced NO release and mitochondrial dysfunction but not IL-1ß-induced release of IL-6, PGE2, and MMP3. Enhancement of cAMP by forskolin reduced IL-1ß-induced NO release and prevented IL-1ß-induced mitochondrial impairment. Activation of AMPK increased IL-1ß-induced NO production and the negative impact of IL-1ß on mitochondrial respiration, whereas inhibition of AMPK had the opposite effects. NO is critically involved in the IL-1ß-induced impairment of mitochondrial respiration in human OA chondrocytes. Increased intracellular cAMP or inhibition of AMPK prevented both IL-1ß-induced NO release and mitochondrial dysfunction.

Impact of Diabetes Mellitus on Knee Osteoarthritis Pain and Physical and Mental Status: Data From the Osteoarthritis Initiative.

OBJECTIVE: Diabetes mellitus (DM) appears to increase osteoarthritic knee pain, which may be related to greater adiposity and more advanced disease status often observed in individuals with osteoarthritis (OA) and DM. We aimed to assess whether OA knee pain and health status are worse in individuals with OA and DM, independent of these potential confounders. METHODS: We included 202 OA participants with DM and 2,279 without DM from the Osteoarthritis Initiative. Knee pain was evaluated using the Knee Injury and Osteoarthritis Outcome Score (KOOS) and a numeric rating scale (NRS). Physical and mental status were assessed by the Medical Outcomes Study Short Form 12 (SF-12) questionnaire, physical component summary (PCS) score and mental component summary (MCS) score, and by the Center for Epidemiologic Studies Depression Scale (CES-D). Linear regression models assessed the influence of DM, adjusted for age, sex, body mass index (BMI), and radiographic severity. RESULTS: OA participants with DM reported worse knee pain and greater physical and mental issues compared with participants without DM. Individuals with DM had worse KOOS pain (ß = -4.72 [95% confidence interval (95% CI) -7.22, -2.23]) and worse NRS pain (ß = 0.42 [95% CI 0.04, 0.80]) independent of BMI, OA severity, age, and sex. The negative influence of DM was also apparent for SF-12 PCS (ß = -3.49 [95% CI -4.73, -2.25]), SF-12 MCS (ß = -1.42 [95% CI -2.57, -0.26]), and CES-D (ß = 1.08 [95% CI 0.08, 2.08]). CONCLUSION: Individuals with knee OA experience on average higher pain intensity and a worse physical and mental health status if they have DM. Linear regression models show that DM is a risk factor for higher pain, in addition to and independent of greater BMI and radiographic OA severity.

Pain-related behaviors associated with persistence of mechanical hyperalgesia after antigen-induced arthritis in rats.

Upon transient musculoskeletal diseases, some patients develop persistent pain while others recover from pain. Here, we studied whether such heterogeneity also occurs in rats after recovery from unilateral antigen-induced arthritis (AIA) in the knee joint, and which pain phenotype may predict the course of pain. Typically, inflammatory swelling lasts about 3 weeks. Pain-related behaviors were monitored for 84 days after AIA induction. Unbiased cluster analysis of intragroup differences at day 84 of AIA revealed that about one-third of the rats (cluster 1) showed persistent mechanical hyperalgesia at the injected knee joint, whereas the other rats (cluster 2) had recovered from pain. Retrograde analysis of pain-related behaviors revealed that cluster 1 rats exhibited more severe mechanical hyperalgesia at the injected knee from day 3 of AIA and mechanical hyperalgesia at the contralateral knee. Cluster 1 and 2 rats did not show different inflammatory swelling, secondary mechanical and thermal hyperalgesia at the ipsilateral hindpaw, guarding score, and asymmetry of weight bearing during AIA. Thus, in particular, early severe mechanical hyperalgesia in the inflamed joint and segmental contralateral mechanical hyperalgesia seem to be a risk factor for the development of persistent mechanical hyperalgesia pointing to the importance of spinal mechanisms. However, none of the rats showed an expression of ATF3 in dorsal root ganglion neurons, nor morphological spinal microglia activation thus not suggesting development of neuropathic pain. Both clusters showed a persistent upregulation of pCREB in dorsal root ganglion neurons, inversely correlated with mechanical hyperalgesia at the knee. The role of pCREB needs to be further explored.

Contribution of Inflammation and Bone Destruction to Pain in Arthritis: A Study in Murine Glucose-6-Phosphate Isomerase-Induced Arthritis.

OBJECTIVE: Arthritis is often characterized by inflammation and bone destruction. This study was undertaken to investigate the contribution of inflammation and bone destruction to pain. METHODS: Inflammation, bone resorption, pain-related behaviors, and molecular markers (activating transcription factor 3 [ATF-3], p-CREB, and transient receptor potential vanilloid channel 1) in sensory neurons were measured in murine glucose-6-phosphate isomerase (G6PI)-induced arthritis, a model of rheumatoid arthritis. Depletion of Treg cells before immunization changed self-limiting arthritis into nonremitting arthritis with pronounced bone destruction. Zoledronic acid (ZA) was administered to reduce bone resorption. RESULTS: Compared to nondepleted mice, Treg cell-depleted mice exhibited arthritis with more severe bone destruction and higher guarding scores (P < 0.05; n = 10 mice per group) as well as more persistent thermal hyperalgesia (P < 0.05), but displayed similar mechanical hyperalgesia at the hindpaws (n = 18-26 mice per group). These pain-related behaviors, as well as an up-regulation of the neuronal injury marker ATF-3 in sensory neurons (studied in 39 mice), appeared before the clinical score (inflammation) became positive and persisted in Treg cell-depleted and nondepleted mice. In the late stage of arthritis, Treg cell-depleted mice treated with ZA showed less bone resorption (<50%; P < 0.01) and less thermal hyperalgesia (P < 0.01) than Treg cell-depleted mice without ZA treatment (n = 15 mice per group), but ZA treatment did not reduce the clinical score and local mechanical hyperalgesia. CONCLUSION: Pain-related behaviors precede and outlast self-limiting arthritis. In nonremitting arthritis with enhanced bone destruction, mainly local thermal, but not local mechanical, hyperalgesia was aggravated. The up-regulation of ATF-3 indicates an early and persisting affection of sensory neurons by G6PI-induced arthritis.

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.

Mechanisms of Osteoarthritic Pain. Studies in Humans and Experimental Models.

Pain due to osteoarthritis (OA) is one of the most frequent causes of chronic pain. However, the mechanisms of OA pain are poorly understood. This review addresses the mechanisms which are thought to be involved in OA pain, derived from studies on pain mechanisms in humans and in experimental models of OA. Three areas will be considered, namely local processes in the joint associated with OA pain, neuronal mechanisms involved in OA pain, and general factors which influence OA pain. Except the cartilage all structures of the joints are innervated by nociceptors. Although the hallmark of OA is the degradation of the cartilage, OA joints show multiple structural alterations of cartilage, bone and synovial tissue. In particular synovitis and bone marrow lesions have been proposed to determine OA pain whereas the contribution of the other pathologies to pain generation has been studied less. Concerning the peripheral neuronal mechanisms of OA pain, peripheral nociceptive sensitization was shown, and neuropathic mechanisms may be involved at some stages. Structural changes of joint innervation such as local loss and/or sprouting of nerve fibers were shown. In addition, central sensitization, reduction of descending inhibition, descending excitation and cortical atrophies were observed in OA. The combination of different neuronal mechanisms may define the particular pain phenotype in an OA patient. Among mediators involved in OA pain, nerve growth factor (NGF) is in the focus because antibodies against NGF significantly reduce OA pain. Several studies show that neutralization of interleukin-1ß and TNF may reduce OA pain. Many patients with OA exhibit comorbidities such as obesity, low grade systemic inflammation and diabetes mellitus. These comorbidities can significantly influence the course of OA, and pain research just began to study the significance of such factors in pain generation. In addition, psychologic and socioeconomic factors may aggravate OA pain, and in some cases genetic factors influencing OA pain were found. Considering the local factors in the joint, the neuronal processes and the comorbidities, a better definition of OA pain phenotypes may become possible. Studies are under way in order to improve OA and OA pain monitoring.

Pain sensation in human osteoarthritic knee joints is strongly enhanced by diabetes mellitus.

The major burden of knee joint osteoarthritis (OA) is pain. Since in elder patients diabetes mellitus is an important comorbidity of OA, we explored whether the presence of diabetes mellitus has a significant influence on pain intensity at the end stage of knee OA, and we aimed to identify factors possibly related to changes of pain intensity in diabetic patients. In 23 diabetic and 47 nondiabetic patients with OA undergoing total knee arthroplasty, we assessed the pain intensity before the operation using the "Knee Injury and Osteoarthritis Outcome Score". Furthermore, synovial tissue, synovial fluid (SF), cartilage, and blood were obtained. We determined the synovitis score, the concentrations of prostaglandin E2 and interleukin-6 (IL-6) in the SF and serum, and of C-reactive protein and HbA1c and other metabolic parameters in the serum. We performed multivariate regression analyses to study the association of pain with several parameters. Diabetic patients had on average a higher Knee Injury and Osteoarthritis Outcome Score pain score than nondiabetic patients (P < 0.001). Knee joints from diabetic patients exhibited on average higher synovitis scores (P = 0.024) and higher concentrations of IL-6 in the SF (P = 0.003) than knee joints from nondiabetic patients. Multivariate regression analysis showed that patients with higher synovitis scores had more intense pain independent of all investigated confounders, and that the positive association between pain intensities and IL-6 levels was dependent on diabetes mellitus and/or synovitis. These data suggest that diabetes mellitus significantly increases pain intensity of knee OA, and that in diabetic patients higher pain intensities were determined by stronger synovitis.

Effects of interleukin-1ß on cortical spreading depolarization and cerebral vasculature.

During brain damage and ischemia, the cytokine interleukin-1ß is rapidly upregulated due to activation of inflammasomes. We studied whether interleukin-1ß influences cortical spreading depolarization, and whether lipopolysaccharide, often used for microglial stimulation, influences cortical spreading depolarizations. In anaesthetized rats, cortical spreading depolarizations were elicited by microinjection of KCl. Interleukin-1ß, the IL-1 receptor 1 antagonist, the GABAA receptor blocker bicuculline, and lipopolysaccharide were administered either alone or combined (interleukin-1ß + IL-1 receptor 1 antagonist; interleukin-1ß + bicuculline; lipopolysaccharide + IL-1 receptor 1 antagonist) into a local cortical treatment area. Using microelectrodes, cortical spreading depolarizations were recorded in a non-treatment and in the treatment area. Plasma extravasation in cortical grey matter was assessed with Evans blue. Local application of interleukin-1ß reduced cortical spreading depolarization amplitudes in the treatment area, but not at a high dose. This reduction was prevented by IL-1 receptor 1 antagonist and by bicuculline. However, interleukin-1ß induced pronounced plasma extravasation independently on cortical spreading depolarizations. Application of lipopolysaccharide reduced cortical spreading depolarization amplitudes but prolonged their duration; EEG activity was still present. These effects were also blocked by IL-1 receptor 1 antagonist. Interleukin-1ß evokes changes of neuronal activity and of vascular functions. Thus, although the reduction of cortical spreading depolarization amplitudes at lower doses of interleukin-1ß may reduce deleterious effects of cortical spreading depolarizations, the sum of interleukin-1ß effects on excitability and on the vasculature rather promote brain damaging mechanisms.

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.

Long-Lasting Activation of the Transcription Factor CREB in Sensory Neurons by Interleukin-1ß During Antigen-Induced Arthritis in Rats: A Mechanism of Persistent Arthritis Pain?

OBJECTIVE: In spite of successful treatment of immune-mediated arthritis, many patients still experience pain. We undertook this study to investigate whether antigen-induced arthritis (AIA) in rats triggers neuronal changes in sensory neurons that outlast the inflammatory process. METHODS: We induced unilateral AIA in the knee joint and assessed in sensory neurons the expression of CREB, a transcription factor that regulates genes involved in neuronal plasticity. We tested whether neutralization of the effects of tumor necrosis factor (TNF) by etanercept or infliximab or neutralization of the effects of interleukin-1ß (IL-1ß) by anakinra influences the up-regulation of phospho-CREB, and we studied the up-regulation of phospho-CREB by IL-1ß and TNF in cultured dorsal root ganglion (DRG) neurons. RESULTS: Unilateral AIA caused bilateral up-regulation of phospho-CREB in lumbar DRG neurons. While inflammation and pain subsided within 21 days, the up-regulation of phospho-CREB still persisted on day 42. At this time point mechanical hyperalgesia at the knee reappeared in the absence of swelling. TNF neutralization during AIA significantly reduced pain-related behavior but did not prevent phospho-CREB up-regulation. In contrast, anakinra, which only reduced thermal hyperalgesia, prevented phospho-CREB up-regulation, suggesting a role of IL-1ß in this process. In cultured DRG neurons the application of IL-1ß significantly enhanced phospho-CREB. CONCLUSION: Immune-mediated arthritis causes neuroplastic changes in sensory neurons that outlast the inflammatory phase. Such changes may facilitate the persistence or recurrence of pain after remission of arthritis. IL-1ß is an important trigger in this process, although its neutralization barely reduced mechanical hyperalgesia during inflammation.

Effects of differently activated rodent macrophages on sensory neurons: implications for arthritis pain.

OBJECTIVE: In arthritis, macrophages invade the affected joint. Experimental arthritis models have shown that macrophages also invade the dorsal root ganglia (DRGs) of the inflamed segments in which the perikarya of sensory neurons are located. It is unclear whether this macrophage invasion contributes to arthritis pain and/or furthers neuronal damage. The present study was undertaken to investigate how differently activated macrophages affect DRG neurons. METHODS: We determined the phenotype of macrophages in the DRGs of rats with antigen-induced arthritis (AIA). In a DRG neuron-macrophage coculture system, we investigated whether differently activated macrophages (stimulated with either lipopolysaccharide [LPS]/interferon-γ [IFNγ], tumor necrosis factor [TNF], or interleukin-4) damage DRG neurons and/or stimulate them to release the mediator calcitonin gene-related peptide (CGRP), which promotes pain and neurogenic inflammation. RESULTS: Macrophages in the DRGs of rats with AIA showed the phenotype of TNF-stimulated macrophages but did not express inducible nitric oxide synthase, which was found in cultured macrophages only after LPS/IFNγ activation. In neuron-macrophage cocultures, activation of macrophages stimulated DRG neurons to release CGRP within 1 hour, indicating neuronal activation by macrophages. Only 48-hour activation of macrophages with LPS/IFNγ increased the neuronal cell death rate in culture, provided that the macrophages were in direct contact with DRG neurons. This effect was dependent on nitric oxide. CONCLUSION: Macrophages have the potential to stimulate sensory neurons in the DRGs, and this may contribute to arthritis pain. If they are classically activated, such as after LPS/IFNγ stimulation, this may also further neuronal cell death. This is not the case in AIA but may occur in models involving damage of sensory neurons.