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BACKGROUND:Monoclonal antibodies (mAbs) towards CGRP or the CGRP receptor show good prophylactic antimigraine efficacy. However, their site of action is still elusive. Due to lack of passage of mAbs across the blood-brain barrier the trigeminal system has been suggested a possible site of action because it lacks blood-brain barrier and hence is available to circulating molecules. The trigeminal ganglion (TG) harbors two types of neurons; half of which store CGRP and the rest that express CGRP receptor elements (CLR/RAMP1). METHODS:With specific immunohistochemistry methods, we demonstrated the localization of CGRP, CLR, RAMP1, and their locations related to expression of the paranodal marker contactin-associated protein 1 (CASPR). Furthermore, we studied functional CGRP release separately from the neuron soma and the part with only nerve fibers of the trigeminal ganglion, using an enzyme-linked immunosorbent assay. RESULTS:Antibodies towards CGRP and CLR/RAMP1 bind to two different populations of neurons in the TG and are found in the C- and the myelinated Aδ-fibers, respectively, within the dura mater and in trigeminal ganglion (TG). CASPR staining revealed paranodal areas of the different myelinated fibers inhabiting the TG and dura mater. Double immunostaining with CASPR and RAMP1 or the functional CGRP receptor antibody (AA58) revealed co-localization of the two peptides in the paranodal region which suggests the presence of the CGRP-receptor. Double immunostaining with CGRP and CASPR revealed that thin C-fibers have CGRP-positive boutons which often localize in close proximity to the nodal areas of the CGRP-receptor positive Aδ-fibers. These boutons are pearl-like synaptic structures, and we show CGRP release from fibers dissociated from their neuronal bodies. In addition, we found that adjacent to the CGRP receptor localization in the node of Ranvier there was PKA immunoreactivity (kinase stimulated by cAMP), providing structural possibility to modify conduction activity within the Aδ-fibers. CONCLUSION:We observed a close relationship between the CGRP containing C-fibers and the Aδ-fibers containing the CGRP-receptor elements, suggesting a point of axon-axon interaction for the released CGRP and a site of action for gepants and the novel mAbs to alleviate migraine.

Existing methods identify only ≈10 Aδ-fibers in human sensory nerves per recording. This study examines methods to increase the detection of Aδ-fibers. Two to 20 averages of 500 replicate responses to epidermal nerve stimulation are obtained. Pairs of different averages are constructed. Each pair is analyzed with algorithms applied to amplitude and frequency to detect replication of responses to stimulation as "simultaneous similarities in two averages" (SS2AVs) at ≥99.5th percentile of control. In a pair of averages the latencies of amplitude and frequency SS2AVs for the same response to stimulation may differ by ≤0.25 ms. Therefore, Aδ-fibers are identified by the 0.25 ms moving sum of SS2AV latencies of the pairs of averages. Increasing averages increases pairs of different averages and detection of Aδ-fibers: from 2 to 10 Aδ-fibers with two averages (one pair) to >50 Aδ-fibers with 12–20 averages (66–190 pairs). Existing methods identify ≤10 Aδ-fibers in 10 averages/45 pairs with the medians of amplitude and frequency algorithms applied to all 45 pairs. This study identifies Aδ-fibers (i) by applying these algorithms at the 99.5th percentile of control, (ii) to each pair of averages and (iii) by the 0.25 ms sum of algorithm identified events (SS2AVs) in all pairs. These three changes significantly increase the detection of Aδ-fibers, e.g., in 10 averages/45pairs from 10 to 45. Three modifications of existing methods can increase the detection of Aδ-fibers to an amount suitable (>50 with ≥12 averages) for statistical comparison of different nerves. • Aδ-fibers are identified in near nerve recordings from sensory nerves. • Multiple records increase pairings of records and detection of Aδ-fibers. • 50 or more Aδ-fibers may be identified with 12 or more records. [ABSTRACT FROM AUTHOR]

Abstract Background Monoclonal antibodies (mAbs) towards CGRP or the CGRP receptor show good prophylactic antimigraine efficacy. However, their site of action is still elusive. Due to lack of passage of mAbs across the blood-brain barrier the trigeminal system has been suggested a possible site of action because it lacks blood-brain barrier and hence is available to circulating molecules. The trigeminal ganglion (TG) harbors two types of neurons; half of which store CGRP and the rest that express CGRP receptor elements (CLR/RAMP1). Methods With specific immunohistochemistry methods, we demonstrated the localization of CGRP, CLR, RAMP1, and their locations related to expression of the paranodal marker contactin-associated protein 1 (CASPR). Furthermore, we studied functional CGRP release separately from the neuron soma and the part with only nerve fibers of the trigeminal ganglion, using an enzyme-linked immunosorbent assay. Results Antibodies towards CGRP and CLR/RAMP1 bind to two different populations of neurons in the TG and are found in the C- and the myelinated Aδ-fibers, respectively, within the dura mater and in trigeminal ganglion (TG). CASPR staining revealed paranodal areas of the different myelinated fibers inhabiting the TG and dura mater. Double immunostaining with CASPR and RAMP1 or the functional CGRP receptor antibody (AA58) revealed co-localization of the two peptides in the paranodal region which suggests the presence of the CGRP-receptor. Double immunostaining with CGRP and CASPR revealed that thin C-fibers have CGRP-positive boutons which often localize in close proximity to the nodal areas of the CGRP-receptor positive Aδ-fibers. These boutons are pearl-like synaptic structures, and we show CGRP release from fibers dissociated from their neuronal bodies. In addition, we found that adjacent to the CGRP receptor localization in the node of Ranvier there was PKA immunoreactivity (kinase stimulated by cAMP), providing structural possibility to modify conduction activity within the Aδ-fibers. Conclusion We observed a close relationship between the CGRP containing C-fibers and the Aδ-fibers containing the CGRP-receptor elements, suggesting a point of axon-axon interaction for the released CGRP and a site of action for gepants and the novel mAbs to alleviate migraine.

Introduction: Cold-induced sweating syndrome type 1 (CISS1), is a rare, severe, autosomal recessive disease. It is characterized by morphological alterations and profuse sweating when ambient temperature is <22 °C. Although some individuals with CISS1 have decreased pain perception, no study has been conducted to evaluate thermal and pain sensations in these patients. The aim of this study was to assess the function of the nociceptive Aδ-fibers and warmth C-fibers by using CO2 laser-evoked potentials (LEPs) in patients affected by CISS1.Methods: Four patients were studied. Laser pulses were applied to the skin of the right hand and the perioral region at painful intensity to record Aδ-LEPs, and at non-painful intensity to obtain C-LEPs. Fifteen healthy subjects were studied for control purposes.Results: No significant difference in latencies or amplitudes of either Aδ- or C-LEPs was found between the 2 groups.Conclusion: Cutaneous nociceptive and warmth pathway functions are normal in CISS1. Muscle Nerve 54: 100-103, 2016. [ABSTRACT FROM AUTHOR]

Abstract: Brief noxious heat stimuli activate Aδ- and C-fibers and allow contact heat-evoked potentials (CHEPs) to be recorded from the scalp. Under normal conditions, only late responses related to Aδ-fibers can be recorded. This study aimed to demonstrate C-fiber responses to contact heat stimuli. A preferential A-fiber compression blockade of the superficial radial nerve was applied in 22 healthy subjects. Quality and intensity of heat-evoked pain and CHEPs were examined at baseline, during nerve compression, and during nerve compression with simultaneous application of topical capsaicin (5%). During the A-fiber block, three subjects had CHEPs with latencies below 400ms, eight subjects within 400–800ms and six subjects (29%) later than 800ms. Pain intensity to contact heat stimuli after compression was reduced and fewer subjects reported the heat stimuli as stinging. Following nerve compression and capsaicin application, ultralate CHEPs with latencies >800ms could be recorded in 13 subjects (62%), pain intensity to the contact heat stimuli was increased and the warm/hot-burning pain quality became more intense. The main results of our study are the demonstration of ultralate C-fiber-related CHEPs following A-fiber blockade in 29% of healthy subjects increasing to 62% when the blockade was combined with capsaicin. After blockade of Aδ-fibers we recorded responses with latencies in the range between the latencies of Aδ- and C-fibers suggesting release of Aδ-fibers with slower conduction velocity than normally recorded with CHEPs. [Copyright &y& Elsevier]

In clinical practice, small myelinated sensory fibers conveying pain and other sensations, Aδ-fibers, cannot be examined with available nerve conduction study techniques. Equipment available in clinical neurophysiology laboratories is used to record from human sensory nerves multiple averaged responses to non-painful stimulation of intraepidermal nerves. Ten averaged responses are analyzed in all possible pair combinations with an algorithm applied to a 0.45 ms period of amplitude and frequency (power spectrum). The median of the algorithms is compared to control data to identify potentials generated as response to intraepidermal stimulation. Median analysis of the algorithm applied to amplitude and frequency of multiple record pairs identifies potentials with conduction velocities of Aδ-fibers. The analysis of frequency (power spectrum) adds data to the analysis of amplitude. Median analysis of multiple record pairs yields more data than analysis of one pair of alternate averages with the same algorithms. At present, analysis of one pair of alternate average records with an algorithm is the only method to identify Aδ-fiber generated potentials. Median analysis of the same algorithm applied to the amplitude of multiple record pairs increases the number of Aδ-fiber generated potentials identified. Neither median analysis of amplitude nor frequency of multiple records pairs has ever been used for conduction studies of nerve fibers, including Aδ-fibers. Stimulation, recording and data analysis methods used in this study can be applied in the clinical EMG laboratory to identify the conduction velocities of Aδ-fibers in human sensory nerves. • Aδ-fiber recordings are obtained with non-painful stimulation. • Amplitude analysis of record pairs identifies Aδ-fiber generated potentials. • Frequency analysis of record pairs adds data to amplitude analysis. • Aδ-fiber conduction velocities provide a histogram of mainly nociceptive Aδ-fibers. [ABSTRACT FROM AUTHOR]

Abstract: Physiological studies suggest visceral spinal afferents are generally small diameter, unmyelinated C-fibers or myelinated Aδ-fibers, but little is known about the size and chemical phenotypes of visceral sensory neurons supplying the small intestine. This study examines the size and expression patterns of transient receptor potential vanilloid 1 (TRPV1), calcitonin gene-related peptide (CGRP), substance P (SP), neuronal nitric oxide synthase (NOS) and isolectin B4-binding (IB4) in dorsal root ganglion (DRG) neurons projecting to the gastrointestinal tract. The spinal afferent innervation of mouse jejunum and distal colon was investigated with retrograde neuronal tracing and multi-label immunohistochemistry. Expression of histochemical markers and soma sizes of retrogradely labeled DRG profiles were determined with confocal microscopy. Most (>75%) jejunal and colonic afferent neurons were medium- and large-sized cells. The majority (82%) of jejunal afferents expressed TRPV1, but few bound IB4. All retrogradely labeled jejunal afferents expressing NOS-immunoreactivity (64%) also expressed TRPV1 and CGRP and most expressed SP. Most labeled colonic afferents expressed TRPV1 (62%) and half expressed NOS. Taken together these data demonstrate that the spinal afferent supply of the jejunum and colon is largely from medium and large sensory neurons, suggesting most intestinal afferent axons are A fibers. The various chemically-defined subpopulations of afferents may play multiple roles in sensory innervation of the jejunum apart from nociceptive transduction. Additionally, we have identified a unique chemical code, TRPV1/NOS/CGRP/SP, that distinguishes many spinal afferent terminals from those of enteric neurons. [Copyright &y& Elsevier]

Abstract: Objective: Laser-evoked potentials have been shown to be clinically useful for the electrophysiological assessment of nociceptive pathways. Contact heat evoked potentials (CHEP) are less established but might be advantageous for clinical purposes. This study aimed at determining the conduction velocity (CV) of central pain (spinothalamic tract, STT) pathways using contact heat stimulation in order to replicate previous findings using laser stimulation. Methods: Contact heat stimulation 3°C higher than the pain threshold was applied at different body locations in 20 subjects. Results: The CHEP latencies correlated significantly with the respective pain thresholds. Without normalization for this effect no significant linear regression between distance to the brain and the latencies was found. Conversely, if thresholds were considered, the regression was significant and the CV of the STT (ranging between 11.2 and 13.4m/s) was comparable to CVs estimated after laser stimulation. Conclusions: Pain thresholds seem crucial in interpreting CHEP latencies. It is suggested that the rather low heating rate is responsible for the dependence of latencies on the pain thresholds. Significance: This study shows the importance of pain thresholds and their control to attain valid CV of the STT after contact heat stimulation in healthy subjects. [Copyright &y& Elsevier]

Abstract: By co-activating Aδ- and C-nociceptors, a brief and intense infrared laser stimulus may produce a double sensation referred to as ‘first’ and ‘second’ pain, evoke late laser-evoked brain potentials (LEPs) related to the processing of Aδ-fiber input, but fails to evoke any consistent activity related to C-fibers. Yet, ultra-late LEPs may be recorded if C-nociceptors are activated in isolation. Bromm and Treede (1985) reported that if a subject selectively focused his attention towards second pain, co-activation of Aδ- and C-nociceptors could elicit both late and ultra-late LEPs (Bromm B, Treede RD. Electroencephalogr Clin Neurophysiol 1985;61). They hypothesized that for C-fiber input to elicit consistent ultra-late LEPs, attention must be specifically focused towards that sensory channel. However, the significance of this study was limited by the fact it relied on results of a single subject and used latency-correction algorithms. In an attempt to replicate these findings, this study examined LEPs recorded while trained subjects focused their attention either towards first pain or towards second pain. Whether or not subjects attended second pain, laser stimuli co-activating Aδ- and C-nociceptors failed to elicit ultra-late LEPs, indicating that focusing attention towards C-fiber sensory input is not a sufficient condition for that input to evoke LEPs. However, selectively attending to first or second pain significantly modulated Aδ-fiber related late LEPs. When subjects attended first pain, a late parietal P3 component was recorded, possibly related to task closure. When subjects attended second pain, a prefrontal positive enhancement of the P2 component was observed. Whether it could reflect brain processes related to response-inhibition is discussed. [Copyright &y& Elsevier]

Little is known about the sensory characteristics and underlying mechanisms behind secondary hyperalgesia (HA) (2° HA). The aim of the present study was to investigate the relationships between two different noxious stimuli, mechanical and cold on capsaicin-induced 2° HA. Fourteen healthy volunteers were exposed to three different cold stimuli (20, 10, 0°C) 30 s each, on both forearms. The cold stimuli were applied before (baseline) and 8 min after intradermal administration of 50-μg capsaicin to the forearm, distally to the injection site in the inspected area of 2° HA. Pain intensities were assessed immediately after each cold stimulus by means of a visual analogue scale (cold-VAS). Additionally, areas of mechanical HA (cm2) were assessed distally and proximally to the injection site at three different time points: 5, 8 (right after the second series of cold stimuli), and 30 min after the injection. No significant differences in cold-VAS were found between pre- and post-capsaicin injection at the tested forearm ( P= 0.334), whereas significant reduction from pre- to post-injection was found in cold-VAS in the control forearm ( P= 0.024). Further, 8 min after the injection, the cold stimulation led to an expansion of 2° HA area (from 5.1±1.38 to 11.4±1.72 cm2) to punctuate stimuli distally but not proximally to the injection site ( P<0.05). It is concluded that there is no HA to cold stimuli within the area of mechanical 2° HA. However, cooling acts as a conditioning stimulus and expands the area of capsaicin-induced punctuate HA. [ABSTRACT FROM AUTHOR]

Abstract: The present study examined the influence of innocuous skin cooling on the perception and neurophysiological correlates of brief noxious CO2 laser stimuli. In nine normal subjects, brief CO2 laser pulses of four different intensities (duration 50 ms; diameter 5 mm; intensity range 5.8–10.6 mJ/mm2) were delivered at random every 5–10 s on the dorsum of the hand. Innocuous skin cooling was performed by a thermode (20 °C; 3×3 cm) with a central hole for the laser test stimuli. Quality and intensity (VAS) of perceptions, reaction times and laser evoked potentials (LEPs) were examined. Signal detection theory analysis was performed to evaluate discrimination performance and decision criterion. During innocuous skin cooling, detection threshold increased from 4.8±1.81 to 8.2±1.05 mJ/mm2 and pain threshold from 8.7±1.53 to 13.5±1.57 mJ/mm2. proportion of detected stimuli decreased from 87% to 48% and pain reports from 42% to 10%. The well localized ‘pricking’ sensation mediated by Aδ-nociceptors almost vanished. The intensity of sensations (VAS scores) was considerably reduced. Sensory discriminative performance was significantly depressed but decision criterion remained unchanged. Reaction times were delayed. The late-LEPs, correlates of Aδ-nociceptor activations, were also significantly depressed while the ultra-late LEPs, correlates of C-nociceptors, were not affected. Taken together, these results strongly suggest that innocuous skin cooling interfered with the sensory processing of laser heat stimuli and more prominently with those related to Aδ-nociceptive input. [Copyright &y& Elsevier]

Co-activation of Aδ- and C-fiber nociceptors by brief cutaneous laser heat stimuli may induce a dual sensation composed of first and second pain but evokes only a single, Aδ-fiber related, late laser-evoked potential (LEP). Yet, when concomitant activation of Aδ-nociceptors is avoided, C-nociceptor activation evokes an ultra-late LEP. As cumulating evidence indicates that late and ultra-late LEPs may share common generators, investigators have hypothesized that when Aδ-fibers trigger a late LEP, the later arriving C-fiber afferent volley cannot trigger an ultra-late LEP because underlying generators are in a ‘refractory state’. Better understanding of these interactions could have important consequences regarding the functional significance of LEPs. Therefore, this hypothesis was tested by applying two consecutive laser stimuli to the hand dorsum such as to produce a second Aδ-nociceptor afferent volley arriving at generators during their expected ‘refractory period’. Results showed that late LEPs evoked by the second stimulus were not altered and consequently that this hypothesis does not hold. In addition, when stimuli ended the sensory detection task, an ample P600 component was recorded. Studies have shown that this component is probably related to the P3b component described in other sensory modalities. This result provides support to the ‘context closure’ model hypothesizing that this component reflects the closure of information processing occurring when expectations are terminated. Altogether, these results suggest that late and ultra-late LEPs reflect very general processes, which are mainly related to detection and orientation and constitute only a fraction of the central processing of both nociceptive inputs. [Copyright &y& Elsevier]

The expression of capsaicin (VR1) receptor by A-fiber primary afferent neurons has been investigated by double immunohistochemical staining with VR1 and 200 kD neurofilament (NF200; an A-fiber marker) antibodies, and by VR1 immunohistochemical staining in combination with cholera toxin B subunit (CTB; also an A-fiber marker) retrograde tracing. Approximately 30% of the VR1-positive dorsal root ganglion (DRG) neurons were NF200-positive. Intra-sciatic nerve injection of CTB labeled over 30% of the VR1-positive neurons in the L5 DRG. Size frequency distribution analysis revealed that these VR1 and NF200, or VR1 and CTB, double-labeled neurons were predominantly small and medium sized. These results suggest that capsaicin receptors are likely to be expressed by Aδ-fiber neurons as well as C-fiber neurons. [Copyright &y& Elsevier]

Antitumor agents, as taxanes and platinum compounds, induce peripheral neuropathies which can hamper their use for cancer treatment. The study of chemotherapy-induced neuropathies in humans is difficult because of ethical reasons, differences among administration protocols and intrinsic characteristics of patients. The aim of the present study is to compare the neuropathic signs induced by individual or combined administration of paclitaxel and oxaliplatin. Oxaliplatin and paclitaxel were administered individually and combined to induce peripheral neuropathy in rats, sensory neuropathic signs were assessed in the hind limbs and orofacial area. The in vitro skin-saphenous nerve preparation was used to record the axonal activity of Aδ sensory neurons. Animals treated with the combination developed mechanical allodynia in the paws and muscular hyperalgesia in the orofacial area, which was similar to that in animals treated with monotherapy, the latter also developed cold allodynia in the paws. Aδ-fibers of the rats treated with the combination were hyperexcited and presented hypersensitivity to pressure stimulation of the innervated skin, also similar to that recorded in the fibers of the animals treated with monotherapy. Our work objectively demonstrates that the combination of a platinum compound with a taxane does not worsen the development of sensorial neuropathies in rats, which is an interesting data to take into account when the combination of antitumor drugs is necessary. Co-administration of antitumor drugs is more effective in cancer treatment without increasing the risk of the disabling neuropathic side effects. • Oxaliplatin or paclitaxel induce a chronic painful peripheral neuropathy in the hind limbs and orofacial region in rats. • When both antitumorals are co-administered, the peripheral neuropathy is not worse than when individually administered. • In the paws, impaired mechanical perception correlates with a hyperexcited state of the Aδ-fibers of the saphenous nerve. • The risk of inducing a disabling neuropathy does not increase when the combination is needed to improve the effectiveness. [ABSTRACT FROM AUTHOR]

Intense radiant heat pulses concomitantly activate Aδ- and C-fiber skin nociceptors, and elicit a typical double sensation: an initial Aδ-related pricking pain is followed by a C-related prolonged burning sensation. It has been repeatedly reported that C-fiber laser-evoked potentials (C-LEPs) become detectable only when the concomitant activation of Aδ-fibers is avoided or reduced. Given that the saliency of the eliciting stimulus is a major determinant of LEPs, one explanation for these observations is that the saliency of the C-input is smaller than that of the preceding Aδ-input. However, even if the saliency of the C-input is reduced because of the preceding Aδ-input, a C-LEP should still be visible even when preceded by an Aδ-LEP response. Here we tested this hypothesis by applying advanced signal processing techniques (peak alignment and time-frequency decomposition) to electroencephalographic data collected in two experiments conducted in 34 and 96 healthy participants. We show that, when using optimal stimulus parameters (delivering >80 stimuli within a small skin territory), C-LEPs can be reliably detected in most participants. Importantly, C-LEPs are observed even when preceded by Aδ-LEPs, both in average waveforms and single trials. By providing quantitative information about several response properties of C-LEPs (latency jitter, stimulus-response and perception-response functions, dependency on stimulus repetitions and stimulated area), these results define optimal parameters to record C-LEPs simply and reliably. These findings have important clinical implications for assessing small-fiber function in neuropathies and neuropathic pain., (Copyright © 2014 the authors 0270-6474/14/3411439-13$15.00/0.)