Carolina Roza

The Tetrodotoxin‐Resistant Na+ Channel Nav1.8 is Essential for the Expression of Spontaneous Activity in Damaged Sensory Axons of Mice

The tetrodotoxin‐resistant sodium channel α subunit, Nav1.8, is exclusively expressed in primary sensory neurons and is suggested to play a role in the generation of ectopic action potentials after axonal injury and thereby contribute to neuropathic pain. Here we investigated the involvement of Nav1.8 in ectopic impulse generation in damaged axons by examining spontaneous activity and mechanosensitivity in neuromas formed by section of the saphenous nerve in Nav1.8 null mice and in their wild‐type littermates. We recorded 522 identified units from 24 neuromas in vitro at two time points, 8–11 days (median 10 days) and 19–29 days (median 22 days) post‐operatively. At ≈10 days, neither genotype showed spontaneous activity, but a significantly higher proportion of fibres were mechanosensitive in wild‐type (54 %) compared to Nav1.8 null neuromas (18 %). At ≈22 days, 19 % of fibres recorded in wild‐type neuromas showed spontaneous activity, whereas only one fibre of the 238 (0.4 %) recorded in neuromas taken from null mice showed ongoing activity. In recordings at ≈22 days, a similar proportion of fibres were mechanosensitive in wild‐type and Nav1.8 null neuromas (51 and 46 %, respectively). We conclude that Nav1.8 is essential for the expression of spontaneous activity in damaged sensory axons, and may also contribute to the development of ectopic mechanosensitivity.

Role of central and peripheral tachykinin NK1 receptors in capsaicin-induced pain and hyperalgesia in mice.

&NA; Substance P and its receptor (NK1) are thought to play an important role in pain and hyperalgesia. Here we have further examined this role by comparing the behavioural responses to intradermal capsaicin of mutant mice with a disruption of the NK1 receptor (NK1 KO) and wild‐type (WT) mice. We have also evaluated the contribution of peripheral NK1 receptors to capsaicin‐evoked behaviour by selective blockade of peripheral NK1 receptors in WT mice using a non‐brain penetrant NK1 receptor antagonist. Injection of 6 &mgr;g capsaicin into the heel evoked paw licking with the same latency in WT and KO mice, but a significantly longer duration in WT mice. A higher dose (30 &mgr;g) evoked a similar duration of licking in both groups. There were no differences in mechanical sensitivity tested with von Frey hairs between WT and KO mice before capsaicin. Both capsaicin doses resulted in pronounced increases in responses to von Frey hairs (hyperalgesia) and novel responses to cotton wisps (allodynia) applied to the digits of the injected paw in WT mice, but no significant changes from baseline in KO mice. Selective blockade of peripheral NK1 receptors in WT mice resulted in a complete inhibition of capsaicin‐evoked plasma extravasation, but the mechanical hyperalgesia induced by 30 &mgr;g capsaicin intraplantar was still significantly greater than that seen in KO mice. We conclude that the response to intradermal capsaicin is still present but abbreviated in mice lacking NK1 receptors, such that secondary hyperalgesia is not observed even after a high dose. Further, the lack of secondary hyperalgesia in NK1 KO mice is largely due to the loss of central rather than peripheral NK1 receptors. The phenotype of the NK1 KO mice is consistent with a loss of function of mechanically‐insensitive nociceptors, and thus we propose that substance P may be expressed by this group of primary sensory neurones and required for their function.

Characterization of mechanical withdrawal responses and effects of μ-, δ- and κ-opioid agonists in normal and μ-opioid receptor knockout mice

Abstract Clinical and experimental observations suggest that opiates can exert different influences on the perception of stimuli from distinct sensory modalities. Thermally-induced nociception is classically responsive to opiate agonists. μ-Opioid receptor-deficient transgenic mice are more sensitive to thermal nociceptive stimuli and morphine fails to attenuate the nociceptive responses to thermal stimuli in these animals. To enhance our understanding of opiate influences on mechanical sensitivity, we have examined withdrawal responses to a sequence of ascending forces of mechanical stimuli in mice with normal (wild type), half-normal (heterozygous) and absent (homozygous) μ-opioid receptor levels. We report data from mice examined without drug pretreatment or following pretreatment with morphine, the selective κ-opioid agonist, U50488H, and the selective δ-opioid agonist, DPDPE. Saline-pretreated mice of each genotype displayed similar, monotonically increasing frequency of withdrawal responses to the graded stimuli. Subcutaneously administered morphine produced a dose-dependent reduction in withdrawal responses in wild type and heterozygous mice, but had no significant effect in homozygous mice. Intraventricular administration of DPDPE also reduced the frequency of paw withdrawal (FPW) in wild type mice, but not in homozygous mice. In contrast, systemic U50488H produced a dose-dependent attenuation of paw withdrawal in both wild type and homozygous mice. These findings suggest that (1) interactions of endogenous peptides with μ-opioid receptors may not play a significant role in the response to mechanical stimuli in drug-free animals, and (2) deficiency of μ-opioid receptors has no functional consequence on the response to the prototypical κ-opioid receptor agonist, but decreases responses to the prototypical μ- and δ-opioid receptor agonists.

Retigabine, the specific KCNQ channel opener, blocks ectopic discharges in axotomized sensory fibres.

&NA; The M‐current has been proposed as a potential target for analgesia under neuropathic pain conditions. M‐currents and/or their molecular correlates, KCNQ proteins, have been demonstrated in key elements of the nociceptive system including spinal and dorsal root ganglion neurons. Here we demonstrate that retigabine, a selective KCNQ channel opener, applied at neuromatose endings modulates the excitability of axotomized fibres inhibiting ectopic discharges. Responses to mechanical and chemical stimulation were obtained from intact and previously axotomized Aδ‐ and C‐fibres using in vitro preparations and extracellular electrophysiological recording techniques. Application of retigabine (10 μM) produced an estimated ∼80% reduction in the number of discharges produced by mechanical and chemical stimulation of most axotomized fibres tested (24/27). The electrical threshold of stimuli applied to the neuroma was found to increase in the presence of retigabine (+17.5 ± 2.3%) and to decrease in the presence of a high potassium medium (−16.5 ± 3.7%). This indicates that retigabine produces a hyperpolarization and a subsequent reduction of the excitability in aberrant sensory endings. Application of XE‐991 (10 μM), a KCNQ channel blocker, had no effect on responses to stimulation of the neuroma but blocked the effects of retigabine indicating a specific involvement of KCNQ channels. In contrast to the strong effects on ectopic discharges, retigabine did not change responses to stimulation recorded from intact receptors. Results indicate that KCNQ channel opening at axotomized endings may constitute a novel and selective mechanism for modulation of some neuropathic pain symptoms.

Proteomic profiling of neuromas reveals alterations in protein composition and local protein synthesis in hyper-excitable nerves

Neuropathic pain may arise following peripheral nerve injury though the molecular mechanisms associated with this are unclear. We used proteomic profiling to examine changes in protein expression associated with the formation of hyper-excitable neuromas derived from rodent saphenous nerves. A two-dimensional difference gel electrophoresis (2D-DIGE) profiling strategy was employed to examine protein expression changes between developing neuromas and normal nerves in whole tissue lysates. We found around 200 proteins which displayed a >1.75-fold change in expression between neuroma and normal nerve and identified 55 of these proteins using mass spectrometry. We also used immunoblotting to examine the expression of low-abundance ion channels Nav1.3, Nav1.8 and calcium channel α2δ-1 subunit in this model, since they have previously been implicated in neuronal hyperexcitability associated with neuropathic pain. Finally, S35methionine in vitro labelling of neuroma and control samples was used to demonstrate local protein synthesis of neuron-specific genes. A number of cytoskeletal proteins, enzymes and proteins associated with oxidative stress were up-regulated in neuromas, whilst overall levels of voltage-gated ion channel proteins were unaffected. We conclude that altered mRNA levels reported in the somata of damaged DRG neurons do not necessarily reflect levels of altered proteins in hyper-excitable damaged nerve endings. An altered repertoire of protein expression, local protein synthesis and topological re-arrangements of ion channels may all play important roles in neuroma hyper-excitability.

The NSAID dexketoprofen trometamol is as potent as μ-opioids in the depression of wind-up and spinal cord nociceptive reflexes in normal rats

The aim of this study was to examine the potency of the antinociceptive effects of the non-steroidal antiinflammatory drug (NSAID), Dexketoprofen Trometamol (the active enantiomer of ketoprofen) on spinal cord nociceptive reflexes. These effects were compared with those of the mu-opioid receptor agonist fentanyl in normal animals. The experiments were performed in male Wistar rats anaesthetised with alpha-chloralose. The nociceptive reflexes were recorded as single motor units in peripheral muscles, activated by mechanical and electrical stimulation. Both dexketoprofen and fentanyl inhibited responses evoked by mechanical and electrical stimulation with doses in the same nanomolar range (dexketoprofen ID50s: 100 and 762 nmol kg-1 and fentanyl: 40 and 51 nmol kg-1, respectively). Dexketoprofen and fentanyl also significantly inhibited wind-up. Since fentanyl has been shown to be some 1000 times more potent than morphine in this type of experiments, we conclude that dexketoprofen has central analgesic actions in normal animals and depresses nociceptive responses with a potency similar to that of mu-opioid agonists.

Pressor responses to distension of the ureter in anaesthetised rats: characterisation of a model of acute visceral pain.

We have characterised pressor responses to stimulation of the ureter in anaesthetised rats (n = 20) as a model of acute visceral pain. The left ureter was cannulated close to the bladder and graded stimuli applied (5-90 mmHg, 30 s). The threshold was approximately 25 mmHg. Suprathreshold pressures evoked responses proportional to the stimulus intensity, which were little altered when stimulation of the kidney was prevented by ligation of the ureteric-pelvic junction. The stimulus response curve was dose-dependently attenuated by morphine (1-3 mg kg-1 i.v.), in a naloxone reversible manner. The characteristics of the responses observed correlate well with pain sensation in man and with the properties of ureteric primary afferent neurones in animals.

Accumulation of Kv7.2 channels in putative ectopic transduction zones of mice nerve-end neuromas

BackgroundModulation of M-type currents has been proposed as a new strategy for the treatment of neuropathic pain due to their role in regulating neuronal excitability. Using electrophysiological techniques we showed previously that the opening of Kv7 channels with retigabine, blocked ectopic discharges from axotomized fibers but did not alter transduction at intact skin afferents. We hypothesized that after nerve damage, accumulation of Kv7 channels in afferent fibers may increase M-type currents which then acquired a more important role at regulating fiber excitability.FindingsIn this study, we used an immunohistochemical approach to examine patterns of expression of Kv7.2 channels in afferent fibers after axotomy and compared them to patterns of expression of voltage gated Na+ channels (Nav) which are key electrogenic elements in peripheral axons known to accumulate in experimental and human neuromas.Axotomy induced an enlargement and narrowing of the nodes of Ranvier at the proximal end of the neuroma together with a dramatic demyelination and loss of structure at its distal end in which naked accumulations of Nav were present. In addition, axotomy also induced accumulations of Kv7.2 that co-localized with those of Nav channels.ConclusionsWhilst Nav channels are mandatory for initiation of action potentials, (i.e. responsible for the generation/propagation of ectopic discharges) an increased accumulation of Kv7.2 channels after axotomy may represent a homeostatic compensation to over excitability in axotomized fibers, opening a window for a peripheral action of M-current modulators under conditions of neuropathy.

Effects of centrally acting analgesics on spinal segmental reflexes and wind‐up

The spinal cord is a prime site of action for analgesia. Here we characterize the effects of established analgesics on segmental spinal reflexes. The aim of the study was to look for the pattern of action or signature of analgesic effects on these reflexes.

DREAM regulates BDNF-dependent spinal sensitization

BackgroundThe transcriptional repressor DREAM (downstream regulatory element antagonist modulator) controls the expression of prodynorphin and has been involved in the modulation of endogenous responses to pain. To investigate the role of DREAM in central mechanisms of pain sensitization, we used a line of transgenic mice (L1) overexpressing a Ca2+- and cAMP-insensitive DREAM mutant in spinal cord and dorsal root ganglia.ResultsL1 DREAM transgenic mice showed reduced expression in the spinal cord of several genes related to pain, including prodynorphin and BDNF (brain-derived neurotrophic factor) and a state of basal hyperalgesia without change in A-type currents. Peripheral inflammation produced enhancement of spinal reflexes and increased expression of BDNF in wild type but not in DREAM transgenic mice. The enhancement of the spinal reflexes was reproduced in vitro by persistent electrical stimulation of C-fibers in wild type but not in transgenic mice. Exposure to exogenous BDNF produced a long-term enhancement of dorsal root-ventral root responses in transgenic mice.ConclusionsOur results indicate that endogenous BDNF is involved in spinal sensitization following inflammation and that blockade of BDNF induction in DREAM transgenic mice underlies the failure to develop spinal sensitization.