Teased-fiber Techniques Research Articles

Peripheral nociceptive mechanisms in an experimental rat model of fibromyalgia induced by repeated cold stress

Fibromyalgia (FM) is a debilitating disease characterized by generalized and persistent musculoskeletal pain. Although central mechanisms are strongly implicated in the pathogenesis of FM, the involvement of peripheral mechanisms is poorly understood. To understand the peripheral nociceptive mechanisms, we examined muscular nociceptors in an FM model, which was made by exposing rats to repeated cold stress (RCS). A single muscle C-fiber nociceptors were identified through the teased fiber technique using ex vivo muscle-nerve preparations. Response properties of C-fibers to noxious stimuli were systematically analyzed. Messenger RNA expression of neurotrophic factors and inflammatory mediators were also studied in the muscle. In the RCS group, the mechanical response threshold of C-fibers, measured using a ramp mechanical stimulus, was significantly decreased, and the response magnitude was significantly increased in the RCS group when compared with the SHAM group, where the environmental temperature was not altered. The general characteristics of C-fibers and the responsiveness to noxious cold and heat stimuli were similar between the two groups. Messenger RNAs of neurotrophic factors and inflammatory mediators were not changed in the muscle during and after RCS. These results suggest that augmentation of the mechanical response of muscle C-fiber nociceptors contributes to hyperalgesia in the RCS model.

Nociception originating from the crural fascia in rats

Little is documented in the literature as to the function of muscle fascia in nociception and pain. The aim of this study was to examine the distribution of presumptive nociceptive nerve fibers, to characterize fascial thin-fiber sensory receptors, and to examine the spinal projection of nociceptive input from the rat crural fascia (CF). Nerve fibers labeled with specific antibodies to calcitonin gene-related peptide (CGRP) and peripherin were found to be densely distributed in the distal third of the CF. Thin-fiber receptors (Aδ- and C-fibers) responding to pinching stimuli to the CF with sharpened watchmaker’s forceps, identified in vivo with the teased fiber technique from the common peroneal nerve, exist in the CF. Forty-three percent of the mechano-responsive fascial C-fibers were polymodal receptors (nociceptors) responding to mechanical, chemical (bradykinin), and heat stimuli, whereas almost all Aδ-fibers were responsive only to mechanical stimuli. Repetitive pinching stimulus to the CF induced c-Fos protein expression in the middle to medial part of superficial layers ie, laminae I–II of the spinal dorsal horn at segments L2 to L4, peaking at L3. These results clearly demonstrate the following: 1) peptidergic and non-peptidergic axons of unmyelinated C-fibers with nerve terminals are distributed in the CF; 2) peripheral afferents responding to noxious stimuli exist in the fascia, and 3) nociceptive information from the CF is mainly processed in the spinal dorsal horn at the segments L2 to L4. These results together indicate that the “muscle fascia,” a tissue often overlooked in pain research, can be an important source of nociception under normal conditions.

Patterns of activity-dependent conduction velocity changes differentiate classes of unmyelinated mechano-insensitive afferents including cold nociceptors, in pig and in human

Activity-dependent slowing of conduction velocity (ADS) differs between classes of human nociceptors. These differences likely reflect particular expression and use-dependent slow inactivation of axonal ion channels and other mechanisms governing axonal excitability. In this study, we compared ADS of porcine and human cutaneous C-fibers. Extracellular recordings were performed from peripheral nerves, using teased fiber technique in pigs and microneurography in humans. We assessed electrically-induced conduction changes and responsiveness to natural stimuli. In both species, the group of mechano-insensitive C-fibers showed the largest conduction slowing (∼30%) upon electrical stimulation (2 Hz for 3 min). In addition, we found mechano-insensitive cold nociceptors in pig that slowed only minimally (<10% at 2 Hz), and a similar slowing pattern was found in some human C-fibers. Mechano-sensitive afferents showed an intermediate conduction slowing upon 2 Hz stimulation (pig: 14%, human 23%), whereas sympathetic efferent fibers in pig and human slowed only minimally (5% and 9%, respectively). In fiber classes with more pronounced slowing, conduction latencies recovered slower; i.e. mechano-insensitive afferents recovered the slowest, followed by mechano-sensitive afferents whereas cold nociceptors and sympathetic efferents recovered the fastest. We conclude that mechano-insensitive C-fiber nociceptors can be differentiated by their characteristic pattern of ADS which are alike in pig and human. Notably, cold nociceptors with a distinct ADS pattern were first detected in pig. Our results therefore suggest that the pig is a suitable model to study nociceptor class-specific changes of ADS.

Trpv1 mediates spontaneous firing and heat sensitization of cutaneous primary afferents after plantar incision

TrpV1, the receptor for capsaicin, contributes to nociception in animals but appears to be much more important for signaling increased behavioral sensitivity in the injured state. The current study examined the relationship between the marked reduction in heat hyperalgesia after incision in TrpV1 knockout (KO) mice and the activity of the nociceptors in these same mice. Also, the role of TrpV1 in spontaneous activity (SA) of afferents after incision was examined. Standard teased-fiber techniques were used to record from glabrous skin afferents from incised and control TrpV1 KO and C57Bl6 mice. The loss of TrpV1 had minimal effect on the responses of mechano-heat-sensitive C-fiber afferents in the normal and incised states. However, a different group of heat sensitive afferents, termed unclassified afferents, was sensitized to heat by incision and had markedly reduced sensitization in the TrpV1 KO mice. These unclassified afferents also developed SA after incision, and generally had a lower threshold temperature compared to unclassified afferents without SA. The rate of SA was inversely correlated to the threshold temperature for heat; afferents that exhibited a higher rate of SA had a lower heat threshold. The proportion of unclassified afferents with SA was also reduced in incised TrpV1 KO mice compared to incised C57Bl6 mice. We conclude that a distinct class of afferents outside the mechano-heat-sensitive afferent population likely contributes to heat hypersensitivity after plantar incision. KO of TrpV1 influences SA in these unclassified afferents in incised skin. SA in these afferents is perhaps a manifestation of heat sensitization.

Activity-dependent slowing of conduction velocity in uninjured L4 C fibers increases after an L5 spinal nerve injury in the rat

Growing evidence suggests that uninjured afferents may play an important role in neuropathic pain following nerve injury. The excitability of nociceptive neurons in the L4 spinal nerve appears to be enhanced following an injury to the adjacent L5 spinal nerve. In this study, we investigated whether the action-potential conduction properties of unlesioned, unmyelinated fibers are also altered. A teased-fiber technique was used to record from single C fibers from the L4 spinal nerve of the rat in vitro. Repeated electrical stimulation of the tibial nerve was used to investigate activity-dependent slowing of conduction velocity. Twin pulse stimulation at a 50 ms interpulse interval allowed investigation of supranormal conduction velocity. Blinded experiments were performed 8–10 days after sham surgery and after an L5 spinal nerve ligation (L5 SNL). Activity-dependent slowing revealed two populations of C fibers, a “nociceptor” population with a large degree of activity-dependent slowing and a “non-nociceptor” population with a smaller degree of activity-dependent slowing. Both populations showed enhanced activity-dependent slowing of conduction velocity and enhanced supranormal conduction velocities in lesioned animals compared to sham animals. Activity-dependent slowing was also enhanced after an L5 SNL in the mouse. These alterations in conduction velocity may reflect changes in expression of ion channels responsible for the membrane excitability. These data provide additional evidence that a nerve injury leads to persistent alterations in the properties of adjacent uninjured, unmyelinated fibers.

Fatigue and Paradoxical Enhancement of Heat Response in C-Fiber Nociceptors from Cross-Modal Excitation

Fatigue refers to the decrement of response seen with repeated stimulation and is a prominent attribute of nociceptors. Whether fatigue in nociceptors involves transduction, spike initiation, or conduction mechanisms is unknown. We investigated systematically how electrical, mechanical, and heat conditioning stimuli (eCS, mCS, hCS) affected the subsequent response to a test-heat stimulus applied 5 sec later to the receptive field of cutaneous nociceptors. Standard teased-fiber techniques were used to record from mechano-heat-sensitive C-fiber afferents in the anesthetized monkey. The eCS was applied to the nerve trunk, whereas the hCS and mCS were applied to the heat-test site. For the eCS, the number of pulses rather than frequency of stimulation determined the level of fatigue. Fatigue varied inversely with the time interval between the eCS and the test stimulus. For comparable responses from the CS, the magnitude of fatigue was less after the mCS than after the eCS. The mCS (but not the eCS) sometimes evoked a paradoxical increase in response to the test-heat stimulus. Recovery from fatigue was significantly faster after the eCS and mCS than the hCS. The paradoxical enhancement after the mCS probably results from temporal summation of generator potentials produced by mechanical and heat stimulation and suggests that the time constant of the generator potential is on the order of seconds. Concurrent enhancement-fatigue effects may also explain why fatigue was less after the mCS than the eCS. The dependency of recovery from fatigue on the modality of the CS suggests that fatigue results from transduction-spike initiation mechanisms.

Degeneration of Myelinated Efferent Fibers Induces Spontaneous Activity in Uninjured C-Fiber Afferents

We demonstrated recently that uninjured C-fiber nociceptors in the L4 spinal nerve develop spontaneous activity after transection of the L5 spinal nerve. We postulated that Wallerian degeneration leads to an alteration in the properties of the neighboring, uninjured afferents from adjacent spinal nerves. To explore the role of degeneration of myelinated versus unmyelinated fibers, we investigated the effects of an L5 ventral rhizotomy in rat. This lesion leads to degeneration predominantly in myelinated fibers. Mechanical paw-withdrawal thresholds were assessed with von Frey hairs, and teased-fiber techniques were used to record from single C-fiber afferents in the L4 spinal nerve. Behavioral and electrophysiological data were collected in a blinded manner. Seven days after surgery, a marked decrease in withdrawal thresholds was observed after the ventral rhizotomy but not after the sham operation. Single fiber recordings revealed low-frequency spontaneous activity in 25% of the C-fiber afferents 8-10 d after the lesion compared with only 11% after sham operation. Paw-withdrawal thresholds were inversely correlated with the incidence of spontaneous activity in high-threshold C-fiber afferents. In normal animals, low-frequency electrocutaneous stimulation at C-fiber, but not A-fiber, strength produced behavioral signs of secondary mechanical hyperalgesia on the paw. These results suggest that degeneration in myelinated efferent fibers is sufficient to induce spontaneous activity in C-fiber afferents and behavioral signs of mechanical hyperalgesia. Ectopic spontaneous activity from injured afferents was not required for the development of the neuropathic pain behavior. These results provide additional evidence for a role of Wallerian degeneration in neuropathic pain.

Mid-axonal tumor necrosis factor-alpha induces ectopic activity in a subset of slowly conducting cutaneous and deep afferent neurons

After injuries to the musculoskeletal system, peripheral nerve axons are exposed to numerous inflammatory mediators, including tumor necrosis factor-α (TNF). Exposure of sensory axons to TNF can cause behavioral hypersensitivity in the peripheral innervation territory of the affected axons. The hypothesis that TNF activates nociceptor axons was tested by using teased fiber techniques in the rat. Recordings were made of single nociceptors innervating both deep and cutaneous receptive fields supplied by the sciatic nerve. The axons proximal to the receptive field were exposed to ascending concentrations of TNF (0.01 to 1 ng/mL). In 21% of cutaneous and 9% of deep neurons, TNF rapidly evoked a transient response. There was no difference between deep and cutaneous nociceptors in the incidence of TNF responses. The majority of neurons responded to TNF injected into their receptive fields. Our data support that TNF can induce ectopic electrogenesis in a minority of nociceptor axons that innervate both deep and cutaneous tissues. This activity may correlate to the human perception of radiating pain that often accompanies neuritis. © 2002 by the American Pain Society

Responses of the Nociceptive Axons to the Inflammatory Mediators and Tumor Necrosis Factor-alpha

Background: Following musculoskeletal injuries, axons are exposed to tumor necrosis factor- (TNF-) and other inflammatory mediators. Exposure of axons to TNF- and complete Freund's adjuvant (CFA) can cause hyperalgesia or allodynia in the distribution of the affected axons. The hypothesis that TNF-, inflammatory mediators, and inflammation secondary to CFA activate nociceptor axons was tested using teased fiber techniques in the rat. Methods: Electrophysiologic recordings were made from single nociceptors innervating both deep and cutaneous receptive fields (RF) supplied by the sciatic nerve, The axons proximal to the of were exposed to either TNF-, a mixture of inflammatory mediators (histamine, serotonin, bradykinin, and prostaglandin), or CFA. Results: In a minority of nociceptors (15%), TNF- rapidly evoked a response that was dose-dependent and transient. There was no difference between deep and cutaneous nociceptors in the incidence of TNF- responses. The majority of neurons responded to TNF- injected into their RFs. No neurons responded to axonal application of either the mixed inflammatory mediators or CFA. Conclusions: Our data supports that TNF- can induce ectopic electrogenesis in nociceptor axons that innervate both deep and cutaneous tissues. This activity may correlate to the human perception of radicular pain that is often associated with neuritis.

Teased-fiber technique for peripheral myelinated nerves: methodology and interpretation.

Teased-fiber technique is the best approach for studying peripheral myelinated nerve fibers in their continuity. It enables the assessment of size of myelin segments formed by Schwann cells and characterization of pathologic changes affecting the internodia, the paranodal regions, and the invested axons. Fiber teasing is performed on prestained proximodistally oriented portions of peripheral nerves. Specimens about 10 mm long are stained for 24-48 hours in Sudan black and then transferred to glycerin, where, using a pair of fine forceps and a stereomicroscope, they are separated into smaller fiber bundles from which single fibers are isolated. The work is performed on a glass slide with an adhesive surface (albuminized or "superfrost"), on which the fibers are placed in strict proximodistal orientation. Following drying in an oven, the slides are mounted with glycerin-gelatine (same as used for frozen sections). The changes, when present, can usually be recognized during the preparation, but fibers are reexamined and changes confirmed in mounted slides. Photographic reconstruction of the fibers facilitates their assessment and enables the documentation of findings. The teased-fiber technique is auxiliary to histopathology, and to limit the workload and save costs, it can be performed on only a few specimens selected for better characterization of changes recognized or suspected in tissue sections. In particular, segmental demyelination and early stages of Wallerian or secondary axonal degeneration can be recognized in teased fibers. Segmental demyelination is characterized by loss of fully myelinated segments and their replacement by newly formed short and thin segments, remyelinating the preserved axon. The early stage of secondary axonal degeneration is recognized by formation of ovoidal fiber fragments in the midinternodal region.

Effects of oxygen radicals on nociceptive afferents in the rat skin in vitro

On the premises of clinical studies, a possible contribution of oxygen radicals has been considered to the development of inflammatory pain and hyperalgesia. In a rat skin-saphenous nerve preparation using standard teased-fiber techniques ( n = 57) hydrogen peroxide (1 mM, 10 mM and 50 mM) was applied in aqueous solution to cutaneous nerve endings of unmyelinated nociceptive afferents. Superoxide anion and hydroxyl radical were secondarily generated as reaction products from pyrogallol (1 and 10 mM) and from Fe-EDTA (1 mM) in hydrogen peroxide, respectively. None of these substances, except exceptionally, induced ongoing activity nor nociceptor sensitization to heat and mechanical stimuli. If occasionally there was a weak excitatory effect, the fibers were left with a profound desensitization to adequate stimulation. The addition of hydrogen peroxide did not enhance sustained responses to solutions of high proton concentration (pH 6.1). Responses to combined inflammatory mediators (bradykinin, serotonin, histamine and prostaglandin E 2, 10 μM) were increased, on average, when hydrogen peroxide was added but this effect did just not reach significance. These findings suggest that oxygen radicals do not play a major and specific role in nociceptor sensitization.

Chemosensitivity and sensitization of nociceptive afferents that innervate the hairy skin of monkey.

1. A large proportion of the cutaneous nociceptor population in monkey either does not respond to mechanical stimuli or has very high mechanical thresholds (> 6 bar). The goal of this study was to determine whether these mechanically insensitive nociceptive afferents (MIAs) differ from mechanically sensitive nociceptive afferents (MSAs) with regard to responses to chemical stimuli. 2. Teased-fiber techniques were used to record from 28 A delta-fiber (16 MIAs and 12 MSAs) and 23 C-fiber (10 MIAs and 13 MSAs) nociceptors in hairy skin of pentobarbital sodium-anesthetized monkeys. An electrocutaneous search technique was used to locate the putative receptive fields of the MIAs. The response to mechanical and heat stimuli was determined before and after intradermal injection of a standard mixture of algesic/inflammatory mediators (bradykinin, histamine, serotonin, and prostaglandin E1). 3. All 25 MSAs, but only 65% of the MIAs, responded to the chemical stimulus. The A delta-fibers, both MSAs and responsive MIAs, and the responsive C-fiber MIAs gave a robust discharge. In contrast, the C-fiber MSAs (the conventional polymodal C-fiber nociceptors) exhibited a significantly weaker response. Three MIAs responded only to the chemical mixture and not to mechanical or heat stimuli. 4. Before injection of the chemical mixture, a significantly smaller proportion of C-fiber MIAs (50%) than of C-fiber MSAs (92%) responded to heat stimuli, whereas a similar proportion (38%) of A delta-fiber MIAs and MSAs were heat sensitive. 5. Approximately one-half of the MIAs and MSAs were sensitized to mechanical stimuli after the chemical injection, as manifest by a decreased threshold and/or an enlarged receptive field. 6. The chemical injection sensitized 90% of A delta-fiber MSAs, but only 8% of A delta-fiber MIAs, to heat stimuli. In contrast, 38% of C-fibers were sensitized. 7. In 14 fibers, the chemical stimulus resulted in sensitization to mechanical stimuli without sensitization to heat stimuli, or vice versa. This dissociated sensitized state suggests that the molecular mechanisms of sensitization to heat and mechanical stimuli differ. 8. In conclusion, a large proportion of primate cutaneous nociceptors respond to intradermal injection of algesic/inflammatory mediators and may also become sensitized to mechanical and/or heat stimuli.

Comparison of heat and mechanical receptive fields of cutaneous C-fiber nociceptors in monkey

1. Receptive-field properties were investigated in cutaneous C-fiber nociceptive afferents (CMH) responsive to mechanical and heat stimuli. Teased-fiber techniques were used to record from 28 CMHs that innervated the hairy skin of upper or lower limb in anesthetized monkeys. 2. The response to mechanical stimuli was studied with the use of calibrated von Frey probes. The response to heat stimuli was studied with the use of a laser thermal stimulator that provided stepped increases in skin temperature with rise times to the desired temperature near 100 ms. The size of the receptive field (RF) for mechanical stimuli was determined by use of a suprathreshold stimulus that consisted of a 0.5-mm-diam probe that exerted a 200-mN force (10 bar). The size of the heat RF was determined by use of a 49 degrees C stimulus applied to a 7.5-mm-diam area for 1 s. 3. Heat thresholds were determined with an ascending series of stimulus intensities and were found to be stable over many hours: they ranged from 37 to 46 degrees C (mean, 41.1 degrees C). Mechanical thresholds ranged from 1.3 to 7.3 bar (mean, 3.3 bar). There was no correlation between mechanical and heat thresholds. Both thresholds extended well below the corresponding psychophysical pain thresholds in the literature. This suggests that spatial and/or temporal summation of C-fiber input are important for pain induced by either stimulus modality. 4. Mechanical RF diameters ranged from 3.3 to 9.6 mm (mean, 4.7 mm); heat RF diameters ranged from punctate (less than 1 mm) to 9.5 mm (mean, 4.3 mm). There was a significant linear correlation between mechanical and heat RF sizes with a slope of one. The distance between the center of the mechanical RF and the center of the heat RF along one axis ranged from 0 to 1.1 mm (mean, 0.4 mm). These data indicate that the heat RFs coincided with the mechanical RFs. 5. Within the mechanical RF determined with the suprathreshold stimuli, all CMHs had one or more punctate areas of maximal mechanical sensitivity where mechanical threshold was lowest. Heat excitability extended greater than 2 mm beyond these mechanically sensitive spots. Because lateral transmission of the heat stimulus is small, this indicates that heat transduction occurs outside the regions of maximal mechanical sensitivity. 6. Both the threshold to heat and the response magnitude at suprathreshold intensities depended on the percentage of the RF area overlapped by the heat stimulus. This indicates that multiple transducer sites probably contribute to the total evoked response.(ABSTRACT TRUNCATED AT 400 WORDS)

Responses to heat of C-fiber nociceptors in monkey are altered by injury in the receptive field but not by adjacent injury

We sought to determine the effects of a cut injury on the thermal responsiveness of C-fiber nociceptors sensitive to heat and mechanical stimuli (CMHs). Teased fiber techniques were used to record from single CMHs that innervated the hairy skin of the monkey arm. Responses to heat stimuli ranging from 41 to 49°C were compared before and after injury. In 11 CMHs, the injury was applied 4 mm peripheral to the edge of the receptive field. The response to the heat sequence was not significantly altered by this adjacent injury. In 16 CMHs, a cut was applied directly to the receptive field. This direct injury led to a significant increase in response to the sequence of heat stimuli (i.e., sensitization). It is concluded that spreading sensitization of C-fiber nociceptors to a cut injury does not occur in monkey.

Antidromic nerve stimulation in monkey does not sensitize unmyelinated nociceptors to heat

Spread of sensitization of nociceptors has been proposed as the mechanism for the secondary hyperalgesia in the region that surrounds a cutaneous injury. One possibility, tested in this study, is that antidromically propagated action potentials in nociceptors result in the release of substances at their cutaneous arborization which sensitize adjacent nociceptors. Using standard teased-fiber techniques, we recorded from single C-fiber nociceptive afferents innervating hairy skin in the monkey. Heat testing was performed before and after electrical stimulation of the parent nerve at strengths sufficient to activate C-fibers. The heat thresholds and total response to the heat sequences were not changed by the electrical stimulation. We conclude that antidromic stimulation of C-fibers in the monkey does not sensitize C-fiber nociceptors to heat stimuli.

Structure and function of urodele myelin lacking alpha-hydroxy fatty acid-containing galactosphingolipids: Slow nerve conduction and unusual myelin thickness

Myelin of several Caudata (Urodela) species appears to be unique in the fact that it lacks hydroxycerebrosides and hydroxysulfatides although it contains their non-hydroxy counterparts. Comparison of the nerve conduction velocities in the Urodeles Necturus (salamander) and Notophthalmus (newt) with that in a reptile, Anolis (chameleon) which contains hydroxycerebrosides and -sulfatides indicated that the values were significantly reduced in the urodeles. Furthermore, urodele myelin thickness remained uniformly the same regardless of the size of the nerve fiber. Despite these differences the myelins appeared structurally similar. Electron microscopic and X-ray diffraction studies did not disclose any structural difference between the two orders. A teased fiber technique established that the ratio of internodal distance and fiber diameter in urodele nerves was essentially similar to that in Anolis. These findings suggest that the absence of hydroxycerebroside and -sulfatide may be related to the reduction in nerve conduction velocity unusual myelin thickness in the urodele nervous system.