Cutaneous C-fibres in the rat and the rabbit - How efferent actions and axonal properties vary with functional class

The aim of this study was to investigate further some efferent actions and axonal properties of the unmyelinated fibres innervating rabbit and rat skin. This investigation can be separated into two parts. Firstly, single unit studies were carried out to determine which functional class(es) or sub-class(es) of C-fibre are responsible for antidromic vasodilatation in both rabbit and rat skin. The findings of these single unit studies were compared with the flare responses of the skin to noxious mechanical and heat stimuli. Secondly, activity-dependent slowing of conduction velocity and axonal spike shape were examined in identified cutaneous C-fibres in the rat in order to determine whether such axonal properties could be used to identify the different functional classes of C-fibre. For the antidromic vasodilatation study, fine filaments were dissected from the cut proximal end of the saphenous nerve in anaesthetized rabbits and rats. Individual C-fibres (conduction velocity <2m/s) were classified into functional groups according to their responses to mechanical and thermal stimulation. The threshold for electrical stimulation of individual C-fibres was determined using the collision technique. Filaments were antidromically electrically stimulated at intensities sufficient to excite the conducting C-fibres, and skin blood flow was monitored before, during and after filament stimulation using laser Doppler perfusion imaging or laser Doppler flowmetry. In both species, the only C-fibres capable of producing a detectable vasodilator response following antidromic stimulation were nociceptive in nature, and in all cases the area of vasodilatation coincided well with the afferent receptive field. However, not all nociceptors produced a detectable vasodilatation, and it seems that a sub-group of polymodal and heat nociceptors are responsible for the efferent action of antidromic vasodilatation in rabbit and rat skin. Flare responses in rabbit and rat skin were only detected following mechanical and heat stimuli within noxious ranges. The spread of the flare responses, together with the sizes of the afferent and efferent receptive fields of individual C-units, provide support for the axon reflex mechanism for the production of flare and antidromic vasodilatation. The percentage slowing of conduction velocity was calculated following a standard electrical tetanus in identified C-fibres dissected from the saphenous nerve in anaesthetized rats. Nociceptive C-fibres showed a greater slowing of conduction velocity than non-nociceptive fibres, and moreover, one could separate the two classes of non-nociceptive afferent C-fibres found in the rat saphenous nerve (the mechanoreceptors and cold thermoreceptors) on the basis of their conduction velocity slowing. In addition, activity-dependent slowing of conduction velocity could be used to differentiate between the afferent and non-afferent populations of inexcitable C-fibres. Spike shapes of functionally classified C-fibres were recorded extracellularly using standardized filter settings, and some variations in spike shape in relation to receptor type were found. Polymodal nociceptors displayed wider spikes than mechanoreceptors, and cold thermoreceptor units tended to have monophasic spikes. Also, the spontaneously active sympathetic efferent C-fibres tended to have spikes of relatively long duration. The use of axonal properties such as activity-dependent slowing of conduction velocity and spike shape to differentiate nociceptors from non-nociceptors has great potential in experiments where axons are isolated from their terminals

Perineural resiniferatoxin selectively inhibits inflammatory hyperalgesia

Resiniferatoxin (RTX) is an ultrapotent capsaicin analog that binds to the transient receptor potential channel, vanilloid subfamily member 1 (TRPV1). There is a large body of evidence supporting a role for TRPV1 in noxious-mediated and inflammatory hyperalgesic responses. In this study, we evaluated low, graded, doses of perineural RTX as a method for regional pain control. We hypothesized that this approach can provide long-term, but reversible, blockade of a portion of nociceptive afferent fibers within peripheral nerves when given at a site remote from the neuronal perikarya in the dorsal root ganglia. Following perineural RTX application to the sciatic nerve, we demonstrated a significant inhibition of inflammatory nociception that was dose- and time-dependent. At the same time, treated animals maintained normal proprioceptive sensations and motor control, and other nociceptive responses were largely unaffected. Using a range of mechanical and thermal algesic tests, we found that the most sensitive measure following perineural RTX administration was inhibition of inflammatory hyperalgesia. Recovery studies showed that physiologic sensory function could return as early as two weeks post-RTX treatment, however, immunohistochemical examination of the DRG revealed a partial, but significant reduction in the number of the TRPV1-positive neurons. We propose that this method could represent a beneficial treatment for a range of chronic pain problems, including neuropathic and inflammatory pain not responding to other therapies

Current status and future directions of botulinum neurotoxins for targeting pain processing.

Current evidence suggests that botulinum neurotoxins (BoNTs) A1 and B1, given locally into peripheral tissues such as skin, muscles, and joints, alter nociceptive processing otherwise initiated by inflammation or nerve injury in animal models and humans. Recent data indicate that such locally delivered BoNTs exert not only local action on sensory afferent terminals but undergo transport to central afferent cell bodies (dorsal root ganglia) and spinal dorsal horn terminals, where they cleave SNAREs and block transmitter release. Increasing evidence supports the possibility of a trans-synaptic movement to alter postsynaptic function in neuronal and possibly non-neuronal (glial) cells. The vast majority of these studies have been conducted on BoNT/A1 and BoNT/B1, the only two pharmaceutically developed variants. However, now over 40 different subtypes of botulinum neurotoxins (BoNTs) have been identified. By combining our existing and rapidly growing understanding of BoNT/A1 and /B1 in altering nociceptive processing with explorations of the specific characteristics of the various toxins from this family, we may be able to discover or design novel, effective, and long-lasting pain therapeutics. This review will focus on our current understanding of the molecular mechanisms whereby BoNTs alter pain processing, and future directions in the development of these agents as pain therapeutics

Impaired pain sensation in mice lacking prokineticin 2

Prokineticins (PKs), consisting of PK1 and PK2, are a pair of newly identified regulatory peptides. Two closely related G-protein coupled receptors, PKR1 and PKR2, mediate the signaling of PKs. PKs/PKRs participate in the regulation of diverse biological processes, ranging from development to adult physiology. A number of studies have indicated the involvement of PKs/PKRs in nociception. Here we show that PK2 is a sensitizer for nociception. Intraplantar injection of recombinant PK2 resulted in a strong and localized hyperalgesia with reduced thresholds to nociceptive stimuli. PK2 mobilizes calcium in dissociated dorsal root ganglion (DRG) neurons. Mice lacking the PK2 gene displayed strong reduction in nociception induced by thermal and chemical stimuli, including capsaicin. However, PK2 mutant mice showed no difference in inflammatory response to capsaicin. As the majority of PK2-responsive DRG neurons also expressed transient receptor potential vanilloid (TRPV1) and exhibited sensitivity to capsaicin, TRPV1 is likely a significant downstream molecule of PK2 signaling. Taken together, these results reveal that PK2 sensitize nociception without affecting inflammation

Sensitization of the Trigeminovascular Pathway: Perspective and Implications to Migraine Pathophysiology

Migraine headache is commonly associated with signs of exaggerated intracranial and extracranial mechanical sensitivities. Patients exhibiting signs of intracranial hypersensitivity testify that their headache throbs and that mundane physical activities that increase intracranial pressure (such as bending over or coughing) intensify the pain. Patients exhibiting signs of extracranial hypersensitivity testify that during migraine their facial skin hurts in response to otherwise innocuous activities such as combing, shaving, letting water run over their face in the shower, or wearing glasses or earrings (termed here cephalic cutaneous allodynia). Such patients often testify that during migraine their bodily skin is hypersensitive and that wearing tight cloth, bracelets, rings, necklaces and socks or using a heavy blanket can be uncomfortable and/or painful (termed her extracephalic cutaneous allodynia). This review summarizes the evidence that support the view that activation of the trigeminovascular pathway contribute to the headache phase of a migraine attack, that the development of throbbing in the initial phase of migraine is mediated by sensitization of peripheral trigeminovascular neurons that innervate the meninges, that the development of cephalic allodynia is propelled by sensitization of second-order trigeminovascular neurons in the spinal trigeminal nucleus which receive converging sensory input from the meninges as well as from the scalp and facial skin, and that the development of extracephalic allodynia is mediated by sensitization of third-order trigeminovascular neurons in the posterior thalamic nuclei which receive converging sensory input from the meninges, facial and body skin

Estrogen Signaling in Trigeminal Nociception

Migraine is much more common in women than in men, and painful episodes are linked to the hormonal fluctuations of the menstrual cycle. The MAP kinase extracellular-signal regulated kinase (ERK) is activated in experimental models of chronic pain, and is also activated by estrogen in sensory neurons. We used an established model of inflammatory trigeminal pain, injection of Complete Freund's adjuvant (CFA) into the masseter muscle, to determine whether ERK activation may play a role in hormone-related trigeminal pain disorders. We measured withdrawal responses to stimulation of the masseter (V3, primary allodynia) and whisker pad (V2, secondary allodynia) using graded monofilaments. Estrogen treatment in the presence of inflammation increased withdrawal response to stimulation of either masseter or whisker pad compared to inflammation alone, indicating an additive effect of inflammation and estrogen on both primary and secondary allodynia. We examined ERK activation in trigeminal ganglia from each treatment group using Western blot and immunohistochemistry. Both masseter inflammation and estrogen treatment increased ERK activation, and combined treatment had an additive effect. Both masseter inflammation and estrogen increased the percentage of pERK immunoreactive neurons in divisions 1 and 2 (V1/2), and combined treatment increased pERK immunoreactivity in V1/2 compared to inflammation alone. We stereotactically administered ERK antagonist U0126, or inactive control U0124, to the trigeminal ganglion of CFA+E2-treated rats. U0126 decreased withdrawal responses to mechanical stimulation of the whisker pad compared to U0124-treated rats. Because the secondary allodynia in V2 after inflammation in V3 was reduced by antagonizing ERK activation in the periphery, these data suggest a peripheral component to secondary trigeminal allodynia mediated through ERK activation. Cellular responses to estrogen can occur through both `genomic' and `nongenomic' pathways. The novel estrogen receptor GPR30 (G-protein coupled receptor 30) activates mediators of signal transduction, including ERK. A goal of this study was to determine which estrogen receptor is required for behavioral sensitization. In order to determine whether GPR30 is present in neurons of the trigeminal ganglion, we performed Western blots for GPR30 on trigeminal ganglion from ovariectomized female Sprague-Dawley rats. Our results show the presence of GPR30 protein in the trigeminal ganglion. Immunohistochemistry for GPR30 in trigeminal ganglion sections showed that GPR30 immunoreactivity was localized to neuronal cell bodies. To determine the subpopulation of neurons that express GPR30, we measured the diameters of GPR30 positive and negative neurons. Neurons expressing GPR30 were significantly smaller in diameter, suggesting that GPR30 is present in nociceptors. In order to investigate GPR30 expression within sub-populations, we co-localized GPR30 with peripherin and NFH using double label immunohistochemistry. GPR30 showed a high degree of overlap with peripherin and partial overlap with NFH, indicating that GPR30 is preferentially expressed in neurons with unmyelinated axons. ERK activation by estrogen in the trigeminal ganglion may occur through either the classical estrogen receptor ERα or through GPR30. In order to determine which estrogen receptor mediates ERK activation in trigeminal ganglion neurons, we examined ERK activation in primary cultures of trigeminal ganglion neurons treated with selective agonists for ERα (PPT), ERβ (DPN), and GPR30 (G-1). ERK was activated by selective agonists for ERα and GPR30, suggesting that activation occurs through either receptor. In order to determine which estrogen receptor mediates increased trigeminal sensitivity, we used a previously employed behavioral model of inflammatory trigeminal allodynia. Ovariectomized female Sprague-Dawley rats were injected in the masseter with CFA and subcutaneously administered G-1,PPT, or vehicle. Withdrawal response to stimulation of the whisker pad (V2) was measured using monofilaments. Either G-1 or PPT treatment increased secondary allodynia, indicating that both receptors function in trigeminal sensitization in vivo. Treatment with estrogen increased expression of ERα but not GPR30, while masseter inflammation increased GPR30 but not ERα. Differential modulation of these ERK-coupled receptors by estrogen and inflammation may play a role in increased trigeminal pain during periods of falling estrogen

Antidromic vasodilatation and the migraine mechanism

Despite the fact that an unprecedented series of new discoveries in neurochemistry, neuroimaging, genetics and clinical pharmacology accumulated over the last 20 years has significantly increased our current knowledge, the underlying mechanism of the migraine headache remains elusive. The present review article addresses, from early evidence that emerged at the end of the nineteenth century, the role of ‘antidromic vasodilatation’ as part of the more general phenomenon, currently defined as neurogenic inflammation, in the unique type of pain reported by patients suffering from migraine headaches. The present paper describes distinctive orthodromic and antidromic properties of a subset of somatosensory neurons, the vascular- and neurobiology of peptides contained in these neurons, and the clinical–pharmacological data obtained in recent investigations using provocation tests in experimental animals and human beings. Altogether, previous and recent data underscore that antidromic vasodilatation, originating from the activation of peptidergic somatosensory neurons, cannot yet be discarded as a major contributing mechanism of the throbbing head pain and hyperalgesia of migraine