Erythropoietin overrides the triggering effect of DNA platination products in a mouse model of cisplatin-induced neuropathy.

BACKGROUND: Cisplatin mediates its antineoplastic activity by formation of distinct DNA intrastrand cross links. The clinical efficacy and desirable dose escalations of cisplatin are restricted by the accumulation of DNA lesions in dorsal root ganglion (DRG) cells leading to sensory polyneuropathy (PNP). We investigated in a mouse model by which mechanism recombinant erythropoietin (rhEPO) protects the peripheral nervous system from structural and functional damage caused by cisplatin treatment with special emphasis on DNA damage burden. RESULTS: A cumulative dose of 16 mg cisplatin/kg resulted in clear electrophysiological signs of neuropathy, which were significantly attenuated by concomitant erythropoietin (cisplatin 32,48 m/s +/- 1,68 m/s; cisplatin + rhEPO 49,66 m/s +/- 1,26 m/s; control 55,01 m/s +/- 1,88 m/s; p < 0,001). The co-application of rhEPO, however, did not alter the level of unrepaired cisplatin-DNA lesions accumulating in DRG target cells. Micro-morphological analyses of the sciatic nerve from cisplatin-exposed mice showed damaged myelin sheaths and mitochondria. Co-administered rhEPO inhibited myelin sheaths from structural injuries and resulted in an increased number of intact mitochondria. CONCLUSION: The protective effect of recombinant erythropoietin is not mediated by reducing the burden of DNA platination in the target cells, but it is likely to be due to a higher resistance of the target cells to the adverse effect of DNA damage. The increased frequency of intact mitochondria might also contribute to this protective role.

Mode of action of cytokines on nociceptive neurons.

Cytokines are pluripotent soluble proteins secreted by immune and glial cells and are key elements in the induction and maintenance of pain. They are categorized as pro-inflammatory cytokines, which are mostly algesic, and anti-inflammatory cytokines, which have analgesic properties. Progress has been made in understanding the mechanisms underlying the action of cytokines in pain. To date, several direct and indirect pathways are known that link cytokines with nociception or hyperalgesia. Cytokines may act via specific cytokine receptors inducing downstream signal transduction cascades, which then modulate the function of other receptors like the ionotropic glutamate receptor, the transient vanilloid receptors, or sodium channels. This receptor activation, either through amplification of the inflammatory reaction, or through direct modulation of ion channel currents, then results in pain sensation. Following up on results from animal experiments, cytokine profiles have recently been investigated in human pain states. An imbalance of pro- and anti-inflammatory cytokine expression may be of importance for individual pain susceptibility. Individual cytokine profiles may be of diagnostic importance in chronic pain states, and, in the future, might guide the choice of treatment.

Effect of cytokines on neuronal excitability.

Numerous studies have shown that proinflammatory cytokines induce or facilitate pain and hyperalgesia in the presence of inflammation, injury to the nervous system or cancer. Besides acting as inflammatory mediators, increasing evidence indicates that cytokines may also specifically interact with receptor and ion channels regulating neuronal excitability, synaptic plasticity and injury under both physiological and pathological conditions. Here we summarize findings on two prototypical proinflammatory cytokines, tumor-necrosis factor-alpha and interleukin-1 beta, and their effects on neuronal excitability and ion channels with special regards to pain and hyperalgesia.

TNF-alpha differentially modulates ion channels of nociceptive neurons.

Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine involved in the development and maintenance of inflammatory and neuropathic pain conditions. The mechanisms by which TNF-alpha elicits pain behavior are still incompletely understood. Numerous studies suggest that TNF-alpha sensitizes primary afferent neurons. Most recently, it was shown that TNF-alpha induced an enhancement of TTX-R Na(+) current in dorsal root ganglion (DRG) cells. In the present study, we have tested the effect of acute application of TNF-alpha on voltage-gated potassium, calcium and sodium channel currents as well as its influence on membrane conductance in isolated rat DRG neurons. We report that voltage-gated potassium channel currents of nociceptive DRG neurons are not influenced by TNF-alpha (100 ng/ml), while voltage-gated calcium channel currents were decreased voltage-dependently by -7.73+/-6.01% (S.D.), and voltage-activated sodium channels currents were increased by +5.62+/-4.27%, by TNF-alpha. In addition, TNF-alpha induced a significant increase in IV ramps at a potential of +20 mV, which did not exist when the experiments were conducted in a potassium-free solution, indicating that this effect is mainly the result of a change in potassium conductance. These different actions of TNF-alpha might help to explain how it sensitizes primary afferent neurons after nerve injury and thus facilitates pain.

Diagnostic value of sural nerve biopsy in patients with suspected Borrelia neuropathy.

Peripheral neuropathy is a recognized but incompletely understood manifestation of borreliosis. As the pathology of this neuropathy has been described only in small case series, the value of nerve biopsy findings for the pathologic diagnosis of Borrelia-associated neuropathy is unclear. We collected and investigated 21 patients with peripheral neuropathy and with typical clinical and serologic signs of neuroborreliosis [Borrelia neuropathy (BN)]. Standard histology and immunohistochemistry were performed on sural nerve biopsies using antibodies to CD4, CD68 and membrane attack complex C5b-9, intercellular adhesion molecule (ICAM)-1, tumor necrosis factor (TNF)-alpha, interleukin (IL)-1, and IL-6. Nine patients with idiopathic vasculitic neuropathy (VN) and 14 with idiopathic axonal neuropathy (AN) served as disease controls. In BN, the characteristic histology was that of an AN with transmural or perivascular lymphocytic infiltration of nerve vessels. In BN, but less in VN and AN, perineurial thickening and neovascularization were observed. For BN but not for VN, this thickening correlated with increased perineurial immunoreactivity (IR) to TNF-alpha, C5b-9, and ICAM-1. In comparison to AN, both BN and VN displayed increased perineurial T-cell infiltration and human leukocyte antigen (HLA)-DR3-IR. In the endoneurium, cytokine (IL-1beta, IL-6, TNF-alpha), HLA-DR3, and ICAM-1 expression was more pronounced in VN but not in BN. The neuropathy in patients with neuroborreliosis resembles idiopathic VN but shows some distinctive features. None of the findings of this study are disease specific but as a pattern may help support the diagnosis of inflammatory neuropathy in patients with serological evidence for Borrelia infection.

Schwann cells promote post-traumatic nerve inflammation and neuropathic pain through MHC class II.

The activation of T helper cells requires antigens to be exposed on the surface of antigen presenting cells (APCs) via MHC class II (MHC-II) molecules. Expression of MHC-II is generally limited to professional APCs, but other cell types can express MHC-II under inflammatory conditions. However, the importance of these conditional APCs is unknown. We and others have previously shown that Schwann cells are potentially conditional APCs, but the functional relevance of MHC-II expression by Schwann cells has not been studied in vivo. Here, we conditionally deleted the MHC-II ß-chain from myelinating Schwann cells in mice and investigated how this influenced post-traumatic intraneural inflammation and neuropathic pain using the chronic constriction injury (CCI) model. We demonstrate that deletion of MHC-II in myelinating Schwann cells reduces thermal hyperalgesia and, to a lesser extent, also diminishes mechanical allodynia in CCI in female mice. This was accompanied by a reduction of intraneural CD4+ T cells and greater preservation of preferentially large-caliber axons. Activation of T helper cells by MHC-II on Schwann cells thus promotes post-traumatic axonal loss and neuropathic pain. Hence, we provide experimental evidence that Schwann cells gain antigen-presenting function in vivo and modulate local immune responses and diseases in the peripheral nerves.

Covariance among age, spinal p38 MAP kinase activation and allodynia.

UNLABELLED: This study examined effects of age (young rats, approximately 35 days, vs mature rats, approximately 75-110 days) on spinal nerve ligation (SNL)-induced tactile allodynia and phosphorylation of p38 (as measured by phospho-p38 MAP kinase [P-p38]) in dorsal root ganglia and spinal cord. Effects of SNL combined with spinal nerve transection also were assessed. Mature rats displayed milder SNL-induced allodynia than young rats. Addition of spinal nerve transection distal to the ligation in older animals resulted in an allodynia comparable to that seen in young animals. In DRG, both groups displayed early (5 h) and late (10 days) peaks in P-p38 following surgery as compared to naïve rats. Tight nerve ligation plus transection had no additional effect on P-p38 levels in DRG. In spinal cord, young rats had increased levels of P-p38 from 5 h to 3 days after SNL. Phosphorylated p38 levels then decreased, with a second peak at 10 days. In contrast, spinal cord from mature rats showed less early p38 phosphorylation, although they also displayed a late 10-day peak. Addition of a transection to the ligation produced restoration of the early peak along with intensification of allodynia. Alterations of spinal P-p38 at early time points thus seem to covary with intensity of tactile allodynia. PERSPECTIVE: Age and modifications to spinal nerve ligation, a common model of neuropathic pain, influence spinal p38 phosphorylation and allodynia. Early levels of spinal P-p38 seem to covary with allodynia intensity. This may mean that small variations of an injury could affect the therapeutic window of a p38 antagonist.

Anterograde transport of tumor necrosis factor-alpha in the intact and injured rat sciatic nerve.

Tumor necrosis factor-alpha (TNF) appears as a key player at both central and peripheral terminals in early degenerative pathology and pain behavior after peripheral nerve injury. Recent studies suggest that TNF may be axonally transported and thereby contribute to these central and peripheral actions. To characterize this transport, we used a double ligation (DL) procedure that distinguishes between anterograde and retrograde flow to visualize the axonal transport of endogenous TNF compared with the neurotrophin nerve growth factor (NGF) and to the neuropeptide calcitonin gene-related peptide (CGRP). In the intact nerve, TNF and CGRP immunoreactivity predominantly accumulated proximal to the DL (anterograde transport), whereas NGF displayed exclusive retrograde transport. At 20 hr after chronic constrictive injury (CCI), the anterograde transport of TNF and CGRP to the nerve injury site was dramatically increased. The results were corroborated by the analysis of axonal transport of exogenously applied 125I-TNF and 125I-NGF. After intraneural injection, 125I-TNF accumulated proximally to a DL, suggesting anterograde transport. In the unligated nerve, 125I-TNF was specifically transported anterogradely to the innervated muscle but not to skin. After CCI, 125I-TNF accumulated proximally to the peripheral nerve injury site, and endogenous TNF was exclusively increased in medium-sized and large dorsal root ganglion (DRG) neurons, suggesting that DRG neurons are a major contributing source of increased TNF traffic in the injured sciatic nerve. Our results suggest that anterograde transport of TNF plays a major role in the early neuronal response to peripheral nerve injury at sites distal to the cell body.

Tumor necrosis factor-alpha induces mechanical allodynia after spinal nerve ligation by activation of p38 MAPK in primary sensory neurons.

Tumor necrosis factor-alpha (TNF) is implicated in the initiation of neuropathic pain. In vitro, TNF activates p38 mitogen-activated kinase. Accordingly, we investigated whether TNF activates the p38 cascade in vivo to trigger pain behavior after spinal nerve ligation (SNL). Treatment starting 2 d before SNL with the TNF antagonist etanercept (1 mg, i.p., every third day) attenuated mechanical allodynia. Treatment starting 1 or 7 d after SNL was ineffective. Similarly, intrathecal infusion of a p38 inhibitor (SB203580, 4 mg/d) was effective only if it was started before but not 7 d after SNL. For both treatments, the cessation of therapy resulted in increased allodynia. In separate experiments using Western blots and immunohistochemistry, ipsilateral lumbar spinal cord and L5 and L6 DRG were analyzed for total and phosphorylated p38 after SNL alone or SNL combined with etanercept pretreatment. In DRG, activated p38 was transiently elevated 5 hr after SNL and returned to baseline by 1 d after SNL. Phosphorylated p38 was localized in small TNF-positive DRG neurons. In spinal cord, p38 was activated between 5 hr and 3 d after SNL and returned to baseline within 5 d. In DRG, but not spinal cord, etanercept pretreatment blocked p38 activation. These data indicate that after SNL treatment, phosphorylated p38 levels in spinal cord and DRG are transiently elevated. In DRG, p38 activation is blocked by systemic TNF inhibition. Parallel inhibition of p38 activation and allodynia may represent a clinically relevant therapeutic window. These data suggest a sequential role for TNF and p38 in the induction of neuropathic pain.

Increased sensitivity of injured and adjacent uninjured rat primary sensory neurons to exogenous tumor necrosis factor-alpha after spinal nerve ligation.

Tumor necrosis factor-alpha (TNF) is upregulated after nerve injury, causes pain on injection, and its blockade reduces pain behavior resulting from nerve injury; thus it is strongly implicated in neuropathic pain. We investigated responses of intact and nerve-injured dorsal root ganglia (DRG) neurons to locally applied TNF using parallel in vivo and in vitro paradigms. In vivo, TNF (0.1-10 pg/ml) or vehicle was injected into L5 DRG in naive rats and in rats that had received L5 and L6 spinal nerve ligation (SNL) immediately before injection. In naive rats, TNF, but not vehicle, elicited long-lasting allodynia. In SNL rats, subthreshold doses of TNF synergized with nerve injury to elicit faster onset of allodynia and spontaneous pain behavior. Tactile allodynia was present in both injured and adjacent uninjured (L4) dermatomes. Preemptive treatment with the TNF antagonist etanercept reduced SNL-induced allodynia by almost 50%. In vitro, the electrophysiological responses of naive, SNL-injured, or adjacent uninjured DRG to TNF (0.1-1000 pg/ml) were assessed by single-fiber recordings of teased dorsal root microfilaments. In vitro perfusion of TNF (100-1000 pg/ml) to naive DRG evoked short-lasting neuronal discharges. In injured DRG, TNF, at much lower concentrations, elicited earlier onset, markedly higher, and longer-lasting discharges. TNF concentrations that were subthreshold in naive DRG also elicited high-frequency discharges when applied to uninjured, adjacent DRG. We conclude that injured and adjacent uninjured DRG neurons are sensitized to TNF after SNL. Sensitization to endogenous TNF may be essential for the development and maintenance of neuropathic pain.

Intraneural injection of interleukin-1beta and tumor necrosis factor-alpha into rat sciatic nerve at physiological doses induces signs of neuropathic pain.

Proinflammatory cytokines are mediators of inflammatory and neuropathic pain. Here, we investigated pain-related behavior in rats after intraneural injection of different doses of rat recombinant interleukin-1beta (rrIL-1beta) and tumor necrosis factor-alpha (rrTNF) into the sciatic nerve. Doses ranged between 0.25 and 2500pg/ml for rrIL-1beta and 0.25-250pg/ml for rrTNF. Thermal hyperalgesia as measured according to the Hargreaves method was most prominent with 2.5pg/ml of rrIL-1beta or rrTNF. Mechanical allodynia as assessed using von Frey hairs was seen consistently with 2.5pg/ml of rrIL-1beta and 0.25-2.5pg/ml of rrTNF. Higher and lower doses had no significant effect on pain-related behavior. Morphometric analysis of semithin sections of the sciatic nerve 10 days after the injections revealed no significant fiber loss. The fiber size distribution was not significantly altered by any of the treatments. Particularly with injections of rrIL-1beta, an increase of epineurial macrophages was observed at all doses. The immunohistochemical expression of cellular markers of neuronal damage (activating transcription factor 3) or activation (phosphorylated p38 mitogen-activated kinase, NF-kappa B p65) in dorsal root ganglia (DRG) tended to increase with both cytokine injections. However, this did not reflect the extent of behavioral changes. In summary, we found a bell-shaped dose-response curve for the algesic effects of rrIL-1beta and rrTNF, peaking at doses equivalent to those of endogenous cytokines released locally after nerve injury. The absence of corresponding morphological changes in nerves supports the concept of a functional effect of the cytokines at these doses.

Spinal blockade of TNF blocks spinal nerve ligation-induced increases in spinal P-p38.

Spinal nerve ligation (SNL) results in a profound long lasting allodynia and increases in phosphorylated p38 in dorsal root ganglia (DRG) neurons and spinal cord microglia. We have previously shown that systemic etanercept, a tumor necrosis factor (TNF) antagonist, reduced allodynia by 42% and blocked SNL-induced increases in P-p38 levels in the L5 and L6 DRG, but not in the ipsilateral lumbar spinal cord. The present experiments demonstrated that intrathecal etanercept (100 microg) prevents SNL-induced increased levels of spinal P-p38. Pretreatment, but not posttreatment, with intrathecal etanercept (100 microg), given every third day, reduced mechanical allodynia by 50%. This therapeutic benefit was maintained for at least 7 days after cessation of treatment. Combined systemic and intrathecal administration of etanercept was no more effective than intrathecal treatment alone. These data imply that TNF provides the trigger for phosphorylation of p38 in both DRG neurons and spinal microglia.

EphA4 provides repulsive signals to developing cochlear ganglion neurites mediated through ephrin-B2 and -B3.

The ephrins and Eph receptors make up two large families of bi-directional signaling molecules that are known to play a role in the development of the nervous system. Recently, expression of EphA4 in the developing cochlea was shown, with strong expression in cells lining the osseous spiral lamina (OSL) through which afferent dendrites must pass to reach the organ of Corti (OC). It was also demonstrated that ephrin-B2 and -B3, both of which are known to interact with EphA4, are expressed by spiral ganglion (SG) neurons. To investigate the functional role of EphA4 in the development of inner ear neurons, neonatal rat SG explants were cultured for 72 hours on uniformly coated surfaces or near stripes of EphA4/IgG-Fc-chimera. Control explants were cultured on or near IgG-Fc and EphA1/IgG-Fc-chimera. To assess the roles of ephrin-B2 and -B3 in EphA4 signaling, SG explants were cultured with or without anti-ephrin-B2 and/or -B3 blocking antibodies. Growth patterns of SG neurites at the border of EphA4 receptor stripes showed repulsion, characterized by turning, stopping and/or reversal. In the case of IgG-Fc and EphA1, the neurites grew straight onto the stripes. Treatment with either anti-ephrin-B2 or -B3 blocking antibodies significantly reduced the repulsive effect of an EphA4 stripe. Moreover, when both antibodies were used together, neurites crossed onto EphA4 stripes with no evidence of repulsion. The results suggest that EphA4 provides repulsive signals to SG neurites in the developing cochlea, and that ephrin-B2 and -B3 together mediate this response.

Intramuscular injection of tumor necrosis factor-alpha induces muscle hyperalgesia in rats.

The role of proinflammatory cytokines in neuropathic and inflammatory pain is well established. Recent studies suggest that cytokines such as tumor necrosis factor-alpha (TNF) may also be involved in the development of muscle pain. To investigate the pathophysiology of intramuscular TNF, exogenous TNF (0.1-10 microg), formalin (9%) or vehicle was injected into the gastrocnemius or biceps brachii muscles of rats. To quantify muscle hyperalgesia, changes in forelimb grip force or withdrawal thresholds to increasing pressure applied to the gastrocnemius muscle were measured. TNF evoked a time- and dose-dependent muscle hyperalgesia within several hours after injection that was totally reversed by systemic treatment with the non-opioid analgesic metamizol. Paw withdrawal thresholds or latencies to mechanical and thermal stimuli, respectively, were unchanged after intramuscular injection of TNF or formalin. In contrast to formalin, which induced significant muscle tissue damage, macrophage infiltration, swelling and partial motor impairment demonstrated in rotarod tests, TNF induced neither histopathological tissue damage nor motor dysfunction. To investigate the effect of TNF and formalin on other potentially algesic mediators, muscles were analyzed for calcitonin-gene related peptide (CGRP), prostaglandin E2 (PGE2) and nerve growth factor (NGF) 1 day after injection. TNF and formalin evoked intramuscular upregulation of CGRP and NGF, whereas PGE2 was increased exclusively after TNF injection. These findings allow us to speculate that endogenous TNF may play a role in the development of muscle hyperalgesia. Targeting proinflammatory cytokines might be beneficial for the treatment of musculoskeletal pain syndromes.

Thalidomide treatment in chronic constrictive neuropathy decreases endoneurial tumor necrosis factor-alpha, increases interleukin-10 and has long-term effects on spinal cord dorsal horn met-enkephalin.

Thalidomide reduces thermal hyperalgesia and mechanical allodynia in chronic constrictive sciatic nerve injury (CCI). Since thalidomide mainly inhibits tumor necrosis factor alpha (TNF-alpha) synthesis with less well defined effects on other cytokines, we investigated the effect of the drug on the expression of the proinflammatory cytokines TNF-alpha, interleukin-1beta (IL-1beta) and interleukin 6 (IL-6), and of the anti-inflammatory cytokine interleukin-10 (IL-10) in the lesioned rat sciatic nerve. The increase of endoneurial TNF-alpha during the first week after CCI was reduced after thalidomide treatment, as shown with immunohistochemistry and enzyme-linked-immunosorbent assay. In contrast, endoneurial IL-1beta-immunoreactivity (IR) and IL-6-IR were not altered by thalidomide treatment, nor was macrophage influx. Recruitment of epineurial IL-10 immunoreactive macrophages as well as the recovery of injury-induced depletion of endoneurial IL-10-IR was enhanced by thalidomide treatment. To control for central plasticity as another factor for the effects of thalidomide, the spinal cord was analyzed for changes in neurotransmitters. The decrease in CGRP-IR and SP-IR in the dorsal horn of operated animals was not influenced by treatment. In contrast, the increase in met-enkephalin observed in the dorsal horn of operated animals was further enhanced in the thalidomide-treated animals. The study elucidates some of the complex alterations in CCI and its modulation by thalidomide, and provides further evidence for a possible therapeutic benefit of cytokine-modulating substances in the treatment of neuropathic pain.

Interaction of spiral ganglion neuron processes with alloplastic materials in vitro(1).

The cochlear implant (CI) involves the introduction of alloplastic materials into the cochlea. While current implants interact with cochlear neurons at a distance, direct interactions between spiral ganglion (SG) neurites and implants could be fostered by appropriate treatment with neurotrophic factors. The interactions of fibroblasts and osteoblasts with alloplastic materials have been well studied in vitro and in vivo. However, interactions of inner ear neurons with such alloplastic materials have yet to be described. To investigate survival and growth behavior of SG neurons on different materials, SG explants from post-natal day 5 rat SG were cultured for 72 h in the presence of neurotrophin-3 (10 ng/ml) on titanium, gold, stainless steel, platinum, silicone and plastic surfaces that had been coated with laminin and poly-L-lysine. Neurite outgrowth was investigated after immunohistological staining for neurofilament, by image analysis to determine neurite extension and directional changes. Neurite morphology and adhesion to the alloplastic material were also evaluated by scanning electron microscopy (SEM). On titanium, SG neurites reached the highest extent of outgrowth, with an average length of 662 microm and a mean of 31 neurites per explant, compared to 568 microm and 21 neurites on gold, 574 microm and 24 neurites on stainless steel, 509 microm and 16 neurites on platinum, 281 microm and 12 neurites on silicone and 483 microm and 31 neurites on plastic. SEM revealed details of adhesion of neurites and interaction with non-neuronal cells. The results of this study indicate that the growth of SG neurons in vitro is strongly influenced by alloplastic materials, with titanium exhibiting the highest degree of biocompatibility with respect to neurite extension. The knowledge of neurite interaction with different alloplastic materials is of clinical interest, as development in CI technology leads to closer contact of implanted electrodes with surviving inner ear neurons.

Postoperative magnetic resonance imaging findings after transtemporal and translabyrinthine vestibular schwannoma resection.

OBJECTIVES/HYPOTHESIS: Magnetic resonance imaging (MRI) has become the investigation of choice to follow up patients after vestibular schwannoma resection. STUDY DESIGN: Retrospective. METHODS: Postoperative MRI findings of 70 patients after vestibular schwannoma resection through a transtemporal (n = 48) and a translabyrinthine (n = 22) approach were reviewed. Time-dependent changes in intensity, size, and shape of enhancement in the internal auditory canal before and after contrast administration, postoperative temporal lobe gliosis, and changes of fat grafts were evaluated. RESULTS: After vestibular schwannoma resection, all patients showed signal enhancements in the internal auditory canal ranging from a faint to high signal intensity in the first postoperative MRI, 3 to 6 months after surgery. In the next MRI at 12 to 24 months after surgery, 30 patients (43%) showed a decreased signal, 35 patients (50%) a stable enhancement, and 5 patients (7%) an increased enhancement in the internal auditory canal depicted as an intense nodular or mass-like pattern. In patients with decreased or stable enhancement, a residual tumor could be excluded in the following MRI scans, whereas in all patients with increased enhancements after 12 to 24 months, signal enhancement further increased and residual tumors were detected. Different degrees of temporal lobe gliosis were found in 15 of 48 cases (31%) after transtemporal tumor removal. Enhancement of fat grafts used in 22 cases decreased to different degrees in 14 cases (64%). CONCLUSIONS: Differentiation of residual tumor from scar tissue in the internal auditory canal after vestibular schwannoma resection requires close, long-term follow-up. Nodular and progressive enhancements in the internal auditory canal indicate residual tumor. Linear enhancement in the internal auditory canal has been found to be a common finding after vestibular schwannoma resection not associated with residual tumor.

Single-fiber recording: in vivo and in vitro preparations.

This chapter focuses on in vivo and in vitro recording setups of extracellular single-unit recordings of peripheral sensory nerve or dorsal root fibers in rodents. Extracellular single-unit recording methods have been used to obtain a wealth of data about the properties of peripheral nervous system (PNS) and central nervous system (CNS) structures. The rationale for studying the activity of single-unit primary afferent fibers is predicated on the significance of relatively fine variations of fiber responsiveness to mechanical, chemical, and/or thermal stimuli. It involves microdissection of nerve fiber bundles until the electrical activity of a single fiber is isolated. Electrophysiological changes in thresholds and discharge rates of peripheral nociceptors to polymodal stimuli can provide neurophysiological correlation to behavioral hyperalgesia and allodynia as well as to cellular differences observable with immunohistochemistry. This chapter gives an overview about the necessary general and special equipment, details about the different setups and tissue preparations. Additionally, the chapter informs about the procedure of recording from single units, data acquisition and analysis including unit isolation criteria and techniques for spike discrimination techniques and fiber classification. It describes criteria for the classification of nociceptors and identification of cutaneous afferent units.

Myricetin reduces voltage activated potassium channel currents in DRG neurons by a p38 dependent mechanism.

Myricetin is a naturally occurring flavonoid known for its anti-neoplastic, anti-oxidant and anti-inflammatory effects. Currently, potential analgesic effects are proposed for several animal models of acute and chronic pain. Pilot studies show a flavonoid-induced modulation of intracellular mitogen-activated protein kinases (MAPK) as p38 and interactions with voltage activated potassium channel currents (I(K(V))). The aim of this study was to investigate the underlying modulation of I(K(V)) and the influence of MAPK phosphorylation in an in vitro cell model. Whole cell patch-clamp recordings of rat dorsal root ganglion neurons were performed and I(K(V)) isolated. I(K(V)) were concentration-dependently reduced by myricetin (1-75µM myricetin; reduction range 18-78%) with no voltage dependency (-80 to +60mV). The reduction of I(K(V)) was enhanced by blocking p38 with the p38 inhibitor SB203580 (40±20% without SB203580 vs. 62±5% with 5µM SB203580 or 83±7% with 10µM SB203580), but abolished by activation of p38 using anisomycin (40±20% without anisomycin vs. 0.73±17% with 5µM anisomycin). We conclude that myricetin reduces I(K(V)) by p38 dependent mechanisms in sensory neurons. Since a reduction of I(K(V)) rather increases neuronal excitability, it is unlikely that this effect of myricetin contributes to its analgesic effects.

Anti-allodynic effect of the flavonoid myricetin in a rat model of neuropathic pain: Involvement of p38 and protein kinase C mediated modulation of Ca²+ channels.

Flavonoids are increasingly ingested by the population as chemotherapeutic and anti-inflammatory agents. Myricetin is a naturally occurring flavonoid known for its anti-neoplastic and anti-inflammatory effects. Recently, behavioral studies indicate a potential analgesic effect in animal models of pain. Pilot studies suggest a flavonoid-induced modulation of intracellular protein kinases and interactions with voltage activated calcium channels. The aim of this study was to investigate the analgesic effect of myricetin in a neuropathic pain model (spinal nerve ligation, SNL) in rats. To identify potential mechanisms of action, in vitro whole cell patch-clamp recordings of isolated rat dorsal root ganglia (DRG) neurons were performed to analyze the modulation of voltage activated calcium channel currents (I(Ca(V))) and the influence of intracellular kinase phosphorylation such as p38 mitogen-activated protein kinase (p38) or protein kinase C (PKC). In vivo, a single injection of myricetin (0.1-10 mg/kg i.p.) reduced SNL-induced mechanical allodynia and thermal hyperalgesia lasting for several hours. In vitro, I(Ca(V)) (depolarization from -80 to 0 mV) were reduced (10-56%) by low (0.1-5 µM) concentrations of myricetin. This decrease was abolished by blockade of PKC (20 µM chelerythrine for 30 min), but not of p38 (10 µM SB203580 for 30 min). In contrast, higher (10-100 µM) concentrations of myricetin induced an increase of I(Ca(V)) (20-40%), which was blocked by inhibition of p38, but not of PKC. We conclude that myricetin transiently reduces established neuropathic pain behavior. This analgesic effect may be related to its PKC-induced decrease of I(Ca(V)) in DRG neurons.