Axonal accumulation of hyperpolarization-activated cyclic nucleotide-gated cation channels contributes to mechanical allodynia after peripheral nerve injury in rat

Peripheral nerve injury causes neuropathic pain including mechanical allodynia and thermal hyperalgesia due to central and peripheral sensitization. Spontaneous ectopic discharges derived from dorsal root ganglion (DRG) neurons and from the sites of injury are a key factor in the initiation of this sensitization. Numerous studies have focused primarily on DRG neurons; however, the injured axons themselves likely play an equally important role. Previous studies of neuropathic pain rats with spinal nerve ligation (SNL) showed that the hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channel in DRG neuronal bodies is important for the development of neuropathic pain. Here, we investigate the role of the axonal HCN channel in neuropathic pain rats. Using the chronic constriction injury (CCI) model, we found abundant axonal accumulation of HCN channel protein at the injured sites accompanied by a slight decrease in DRG neuronal bodies. The function of these accumulated channels was verified by local application of ZD7288, a specific HCN blocker, which significantly suppressed the ectopic discharges from injured nerve fibers with no effect on impulse conduction. Moreover, mechanical allodynia, but not thermal hyperalgesia, was relieved significantly by ZD7288. These results suggest that axonal HCN channel accumulation plays an important role in ectopic discharges from injured spinal nerves and contributes to the development of mechanical allodynia in neuropathic pain rats.     

The effect of conduction block on the spinal release of immunoreactive-neuropeptide Y (ir-NPY) in the neuropathic rat

Peripheral nerve injury may result in significant changes in neuropeptide production and the development of neuropathic pain behaviour. Rats with a chronic constriction injury of one sciatic nerve were used to study the spinal release of immunoreactive neuropeptide Y (ir-NPY), using the antibody-coated microprobe technique. Previous work has shown an increase in NPY synthesis by large to medium-sized primary afferent neurones, as well as a new area of ir-NPY release in the deep dorsal horn on the side of nerve injury, when compared to uninjured rats. The stimulus for spontaneous ir-NPY release was unclear, but may have been due to ectopic neuronal discharges developing after nerve injury. This study used local anaesthetic to block all electrical input from the injured nerve. No change was found in the new zone of spontaneous release of ir-NPY in the deep dorsal horn ipsilateral to nerve injury. It appears therefore, that ir-NPY is released from the central termination of primary afferent neurones, without regulation from neuronal activity in the primary afferent neurones themselves.     

Effect of 2-(phosphono-methyl)-pentanedioic acid on allodynia and afferent ectopic discharges in a rat model of neuropathic pain.

Increased glutamate availability in the spinal cord and primary afferent nerves plays an important role in acute and chronic pain. Afferent ectopic discharges from the site of nerve injury constitute a source of abnormal sensory input to the spinal dorsal horn. The ectopic afferent activity is largely responsible for the development of hypersensitivity of dorsal horn neurons and neuropathic pain. Inhibition of glutamate carboxypeptidase II (GCP II) reduces glutamate release generated from N-acetyl-aspartyl-glutamate in nerve tissues and may have an analgesic effect on neuropathic pain. In the present study, we determined the effect of a GCP II inhibitor, 2-(phosphono-methyl)-pentanedioic acid (2-PMPA), on allodynia and ectopic afferent discharges in an animal model of neuropathic pain. Neuropathic pain was induced by partial ligation of the left sciatic nerve in rats. Tactile allodynia was assessed using von Frey filaments applied to the plantar surface of the injured hindpaw. Single-unit activity of ectopic discharges was recorded from the sciatic nerve proximal to the site of ligation. Intravenous injection of 50 to 100 mg/kg 2-PMPA significantly reduced allodynia in a dose-dependent manner. Furthermore, 2-PMPA dose-dependently attenuated the ectopic discharge activity of injured sciatic afferent nerves. At a dose of 100 mg/kg, 2-PMPA significantly inhibited the ectopic activity from 14.7 +/- 2.1 to 4.4 +/- 0.5 impulses/s without altering the conduction velocity of afferent nerves. Therefore, these data suggest that the antiallodynic effect of 2-PMPA may be mediated, at least in part, by inhibition of ectopic afferent discharges at the site of nerve injury.     

The role of injury discharge in the induction of neuropathic pain behavior in rats.

When sensory fibers are damaged, a discharge of impulses is emitted, which can last up to a few minutes. In the present study, we examined whether this injury discharge plays a role in triggering 'autotomy'--a behavior involving self-injury in animals that is induced by total denervation of a hind paw. Sensory input from the sciatic and saphenous neuroma is thought to elicit chronic pain sensations which cause the rat to injure the hind paw. In the present experiments: (1) injury discharge was prevented by using a local anesthetic block and (2) injury discharge was artificially prolonged by delivering 150 electrical pulses to the nerve just prior to transection, at a strength sufficient to drive A- and C-fibers. In one group of animals, the nerve was stimulated at 0.5 Hz at which frequency a synchronous, repetitive activity in C-fibers augments the response of some nociceptive dorsal horn neurons by temporal summation ('wind-up'). In 2 other groups, the sciatic nerve was stimulated at 0.1 Hz and 10 Hz. The results show that blocking injury discharge significantly delayed the average time of onset of autotomy and suppressed it in magnitude compared to control rats. In contrast, electrical stimulation, especially at the 'wind-up' frequency, significantly shortened the onset of autotomy and enhanced its severity. Thus, in spite of its short duration, injury discharge affects the subsequent development of neuropathic pain related behavior.     

Primary afferent input critical for maintaining spontaneous pain in peripheral neuropathy

Central sensitization after peripheral nerve injury may result in ectopic neuronal activity in the spinal cord dorsal horn, implying a potential autonomous pain-generating mechanism. This study used peripheral nerve blockade and systemic lidocaine administration, with detailed somatosensory assessment, to determine the contribution of primary afferent input in maintaining peripheral neuropathic pain. Fourteen patients with neuropathic pain (7 with unilateral foot pain due to peripheral nerve injury and 7 with bilateral pain in the feet due to distal polyneuropathy) underwent comprehensive characterization of somatosensory function by quantitative sensory testing. Patients were then administered an ultrasound-guided peripheral nerve block with lidocaine and intravenous lidocaine infusion in randomized order. The effect of these interventions on spontaneous pain intensity and on evoked cold, warm, pinprick, and brush responses was assessed at each session. All patients had sensory disturbances at baseline. The peripheral nerve block resulted in a complete abolition of ipsilateral pain within 10 min (median) in all patients, with lidocaine plasma concentrations being too low to account for a systemic effect of the drug. Intravenous lidocaine infusion reduced the spontaneous pain by 45.5% (±31.7%), and it reduced mechanical and thermal hypersensitivity in most patients who displayed such signs. However, the improvement in evoked hypersensitivity was not related to the effect of the drug on spontaneous pain intensity. This study demonstrated that regardless of the individual somatosensory phenotype and signs of central sensitization, primary afferent input is critical for maintaining neuropathic pain in peripheral nerve injury and distal polyneuropathy.     

Dorsal root section elicits signs of neuropathic pain rather than reversing them in rats with L5 spinal nerve injury

Mechanical allodynia- and hyperalgesia-like behavior which develops in rats after L5 spinal nerve lesion has been suggested to be due to ectopic activity in the lesioned afferent neurons originating at the lesion site and/or in the dorsal root ganglion because it is eliminated by section of the dorsal root. Here we reevaluated the effect of a dorsal rhizotomy in rats after L5 spinal nerve lesion. Using calibrated von Frey hairs, paw withdrawal threshold to single stimuli and paw withdrawal incidence to repetitive stimulation were tested before and after nerve section. Neuropathic pain behavior of similar time course and magnitude also developed after cutting the L5 dorsal root, and L5 spinal nerve lesion-induced abnormal behavior could not be reversed by dorsal rhizotomy. The neuropathic pain behavior elicited by dorsal root section also developed when impulse conduction in the dorsal root axons was blocked during rhizotomy by a local anesthetic, i.e. when the immediate injury discharge was prevented from reaching the spinal cord. These results challenge the widely accepted idea that neuropathic pain behavior developing after spinal nerve lesion is dependent on ectopic activity in the lesioned afferent neurons. However, the present results do not rule out the possibility that after the two nerve lesions the mechanisms generating neuropathic pain behavior are different. After dorsal rhizotomy neuropathic pain behavior may be related to deafferentation whereas after spinal nerve lesion it may be caused by ectopic activity.     

Contrasting thermal sensitivity of spontaneously active A- and C-fibers in experimental nerve-end neuromas

Injured afferent A- and C-fibers ending in experimental neuromas in the rat sciatic nerve generate a substantial spontaneous discharge. We show that for individual axons the rate and percent incidence of spontaneous discharge are sensitive to neuroma temperature. Within the range of 14–43°C, firing rate of all of the myelinated fibers examined increased as temperature rose, and decreased as temperature fell. For fibers with a tonic rhythmic discharge pattern, Q₁₀ averaged 1.64 at 34–42°C. Some fibers that were initially silent began to fire as the neuroma was warmed, and some fibers active at baseline temperature fell silent when the neuroma was cooled. Unmyelinated fibers behaved quite differently, showing either no response to temperature changes (44% of fibers sampled), or an increase in discharge rate upon cooling (56%). These effects are probably not secondary to vascular changes, but rather reflect thermal sensitivity of the ectopic neuroma impulse generator sites. This thermal sensitivity may account for the aggravation of phantom limb pain and other neuralgias during cold weather (i.e., post-traumatic cold intolerance).     

Parasystole and its variants.

Various mechanisms that cause deviations from the classical manifestations of a parasystolic rhythm are reviewed and illustrated by selected clinical electrocardiograms. They consist of: (1) Transient or continued fixed coupling of the ectopic beats, due to (a) synchronization of basic and parasystolic rhythms; (b) reversed coupling of the basic to the ectopic rhythm (unidirectional protection); (c) the operation of supernormal phase of excitability; and (d) intermittent parasystole, due to gap in the protection of the parasystolic center. (2) Irregularities in response to a regular parasystolic discharge may be caused by a second degree exit block, usually of Mobitz type II, rarely of type I. An electrophysiologic basis for the emergence and maintenance of parasystolic rhythms appears to be abnormal states of spontaneous diastolic (phase 4) depolarization in otherwise latent subsidiary cardiac pacemakers.     

Characteristics of ectopic discharges in a rat neuropathic pain model

Injured afferent neurons produce spontaneous activity that is generated away from the normal impulse generation site. Since this activity, referred to as ectopic discharges, may play a significant role in neuropathic pain, it is important to systematically analyze the activity in various pain states. The present study used the segmental spinal nerve injury model of neuropathic pain to quantify the ectopic discharges from injured afferents in the neuropathic rat under various conditions. All aspects of measured ectopic discharges declined as postoperative time lengthened. Neuropathic pain behaviors declined in a similar fashion over the same time period. Surgical sympathectomy on neuropathic animals lowered the level of ectopic discharges along with neuropathic pain behaviors. The data indicate that the level of ectopic discharges is well correlated with that of pain behaviors in a rat neuropathic pain model, and this reinforces the supposition that ectopic discharges are important to the maintenance of neuropathic pain behaviors. The data suggest that there are two components of ectopic discharge generator mechanisms: sympathetically dependent and sympathetically independent components.     

Pathophysiologic mechanisms of neuropathic pain

New animal models of peripheral nerve injury have facilitated our understanding of neuropathic pain mechanisms. Nerve injury increases expression and redistribution of newly discovered sodium channels from sensory neuron somata to the injury site; accumulation at both loci contributes to spontaneous ectopic discharge. Large myelinated neurons begin to express nociceptive substances, and their central terminals sprout into nociceptive regions of the dorsal horn. Descending facilitation from the brain stem to the dorsal horn also increases in the setting of nerve injury. These and other mechanisms drive various pathologic states of central sensitization associated with distinct clinical symptoms, such as touch-evoked pain.     

Cutaneous pain threshold changes after sympathetic block in reflex dystrophies.

In a group of 30 subjects suffering from sympathetic reflex dystrophies of the limbs, the sympathetic ganglia of the affected side were blocked with a local anesthetic. Using an original method, we measured the cutaneous pain threshold before the block and at prefixed intervals after the block during a period of 2 days. In all subjects the cutaneous pain threshold showed damped oscillations both in the limb ipsilateral to the block and in the contralateral one. The analysis of these oscillations showed: (a) that the sympathetic control of the cutaneous pain threshold may be exerted through a negative feedback loop (skin-afferent input-CNS-sympathetic output-skin); (b) that the afferent discharge of a limb controls the contralateral sympathetic output through central mechanisms.     

Genetic impairment of interleukin-1 signaling attenuates neuropathic pain, autotomy, and spontaneous ectopic neuronal activity, following nerve injury in mice

Peripheral nerve injury may lead to neuropathic pain, which is often associated with mechanical and thermal allodynia, ectopic discharge of from injured nerves and from the dorsal root ganglion neurons, and elevated levels of proinflammatory cytokines, particularly interleukin-1 (IL-1). In the present study, we tested the role of IL-1 in neuropathic pain models using two mouse strains impaired in IL-1 signaling: Deletion of the IL-1 receptor type I (IL-1rKO) and transgenic over-expression of the IL-1 receptor antagonist (IL-1raTG). Neuropathy was induced by cutting the L5 spinal nerve on one side, following which mechanical and thermal pain sensitivity was measured. Wild-type (WT) mice and the parent strains developed significant allodynia and hyperalgesia in the hind-paw ipsilateral to the injury compared with the contralateral hind-paw. The mutant strains, however, did not display decreased pain threshold in either hind-paw. Pain behavior was also assessed by cutting the sciatic and saphenous nerves and measuring autotomy scores. WT mice developed progressive autotomy, beginning at 7 days post-injury, whereas the mutant strains displayed delayed onset of autotomy and markedly reduced severity of the autotomy score. Electrophysiological assessment revealed that in WT mice a significant proportion of the dorsal root axons exhibited spontaneous ectopic activity at 1, 3, and 7 days following spinal nerve injury, whereas in IL-1rKO and IL-1raTG mice only a minimal number of axons exhibited such activity. Taken together, these results suggest that IL-1 signaling plays an important role in neuropathic pain and in the altered neuronal activity that underlies its development.     

Ectopic mechanosensitivity in injured sensory axons arises from the site of spontaneous electrogenesis

Injured sensory axons trapped in a neuroma or freely regenerating in the distal nerve stump, frequently display ectopic mechanosensitivity, spontaneous impulse discharge or both. This abnormal neural activity is thought to contribute to spontaneous and movement-evoked neuropathic paraesthesias, dysaesthesias and pain, as well as to allodynia and hyperalgesia. The present paper examines the relationship between mechanosensitivity and spontaneous discharge in three distinct sciatic nerve injury models in the rat: nerve transection (neuroma), nerve crush and chronic nerve constriction injury (CCI). Impulse pattern analysis was used to determine that the sites of mechanosensitivity and of spontaneous electrogenesis are either identical or very close to one another. This suggests that mechanosensitivity and spontaneous firing are aspects of a single underlying pathophysiological process.     

Key role of the dorsal root ganglion in neuropathic tactile hypersensibility.

Cutting spinal nerves just distal to the dorsal root ganglion (DRG) triggers, with rapid onset, massive spontaneous ectopic discharge in axotomized afferent A-neurons, and at the same time induces tactile allodynia in the partially denervated hindlimb. We show that secondary transection of the dorsal root (rhizotomy) of the axotomized DRG, or suppression of the ectopia with topically applied local anesthetics, eliminates or attenuates the allodynia. Dorsal rhizotomy alone does not trigger allodynia. These observations support the hypothesis that ectopic firing in DRG A-neurons induces central sensitization which leads to tactile allodynia. The question of how activity in afferent A-neurons, which are not normally nociceptive, might induce allodynia is discussed in light of the current literature.     

Gabapentin suppresses ectopic nerve discharges and reverses allodynia in neuropathic rats

Repetitive ectopic discharges from injured afferent nerves play an important role in initiation and maintenance of neuropathic pain. Gabapentin is effective for treatment of neuropathic pain but the sites and mechanisms of its antinociceptive actions remain uncertain. In the present study, we tested a hypothesis that therapeutic doses of gabapentin suppress ectopic afferent discharge activity generated from injured peripheral nerves. Mechanical allodynia, induced by partial ligation of the sciatic nerve in rats, was determined by application of von Frey filaments to the hindpaw. Single-unit afferent nerve activity was recorded proximal to the ligated sciatic nerve site. Intravenous gabapentin, in a range of 30 to 90 mg/kg, significantly attenuated allodynia in nerve-injured rats. Furthermore, gabapentin, in the same therapeutic dose range, dose-dependently inhibited the ectopic discharge activity of 15 injured sciatic afferent nerve fibers through an action on impulse generation. However, the conduction velocity and responses of 12 normal afferent fibers to mechanical stimulation were not affected by gabapentin. Therefore, this study provides electrophysiological evidence that gabapentin is capable of suppressing the ectopic discharge activity from injured peripheral nerves. This action may contribute, at least in part, to the antiallodynic effect of gabapentin on neuropathic pain.     

Projected pain from noxious heat stimulation of an exposed peripheral nerve – A case report

Distinct sensory properties of unmyelinated axons in the isolated rat sciatic nerve have previously been revealed by measuring stimulated neuropeptide (CGRP) release in response to noxious stimuli. Axonal sensitization to heat by inflammatory mediators has been demonstrated and shown to depend on the heat‐ and proton‐activated ion channel TRPV1. Recently, we have demonstrated in vitro that heat stimulation of nociceptive axons generates ectopic action potential discharge which resembles the heat response of the corresponding cutaneous nerve endings. It remained however, to be established whether adequate axonal stimulation could also generate projected sensations in a conscious human subject. In a singular human trial, the superficial radial nerve (SR) was exposed and stimulated mechanically as well as with noxious cold (3°C). These stimuli were unable to induce any conscious local or projected sensations. However, controlled radiant heat applied to the nerve resulted in intense slowly adapting burning pain sensations projected into the center of the SR innervation area. No local sensation was reported. Thus, presumably activated nervi nevorum in the sheath of a healthy nerve do not cause conscious sensations, while axons of passage in mid‐nerve exhibit a sensory transduction capacity for noxious heat though not for mechanical and cold stimulation. Axonal heat transduction may therefore become a source of ectopic discharge and neuropathic pain when heat threshold drops to body temperature as is the case with peripheral nerve endings in inflamed skin.     

Gunshot versus nongunshot spinal cord injury: acute care and rehabilitation outcomes

OBJECTIVE: To examine the impact of gunshot-caused spinal cord injury on acute and rehabilitative care outcome using a case control design. DESIGN: Two groups (i.e., gunshot- vs. nongunshot-caused spinal cord injury) of 212 individuals were matched case-for-case on age (i.e., within 10 yr), education, gender, race, marital status, primary occupation, impairment level, and Model System region. Outcome measures included length of hospital stay, functional status (FIM), treatment charges, and home discharge rates. RESULTS: The two groups did not differ in the length of stay during acute and rehabilitative care, charges during rehabilitative care, or postrehabilitation discharge placement. Several significant between-group differences in treatment procedures were noted (e.g., prevalence of spinal surgery), which may, in part, account for the higher acute-care charges among those persons with nongunshot-caused spinal cord injury. CONCLUSION: Once an individual is stabilized and admitted for rehabilitative care, gunshot etiology of spinal cord injury seems largely unrelated to the initial rehabilitation outcome.     

Hyperpolarization-activated, cation-nonselective, cyclic nucleotide-modulated channel blockade alleviates mechanical allodynia and suppresses ectopic discharge in spinal nerve ligated rats.

Abnormal spontaneous firing is well described in axotomized sensory neurons and likely contributes to nerve injury-induced pain. The hyperpolarization-activated current I(h) initiates spontaneous, rhythmic depolarization in the sinoatrial node and central neurons. This study was undertaken to investigate the possible contribution of I(h) to primary afferent ectopic discharge and pain behavior in nerve-injured rats. Nerve injury was produced by tight ligation of lumbar spinal nerves (L5/6). Two weeks later, rats showed marked mechanical allodynia. Withdrawal thresholds were measured before and after administration of saline or the specific I(h) antagonist ZD7288 (1, 3, or 10 mg/kg, intraperitoneally). ZD7288 dose-dependently reversed mechanical allodynia. In a second experiment, we performed both in vivo and in vitro extracellular single unit recordings from teased dorsal root fascicles. Intravenous infusion (2.5 or 5 mg/kg) of ZD7288 during a period of 10 minutes significantly blocked ectopic discharges in vivo. Perfusion (25 to 100 mumol/L) of ZD7288 for 5 minutes in vitro almost completely blocked ectopic discharges from large myelinated fibers (Abeta) while partially suppressing ectopic discharge from thinly myelinated fibers (Adelta). We conclude from these data that in axotomized sensory neurons, a ZD7288-sensitive current contributes to spontaneous discharges in myelinated fibers. Thus, I(h) might substantially contribute to the pathophysiology of nerve injury-related neuropathic pain. PERSPECTIVE: The current study investigated the mechanism of abnormal spontaneous discharges (ectopic discharges) from axotomized sensory afferents. Ectopic discharges are a main driving source of nerve injury-induced neuropathic pain. Understanding the mechanism of ectopic discharges and identifying how to control them will be useful toward developing new therapies.     

Corticosteroids suppress ectopic neural discharge originating in experimental neuromas

Some injured sensory fibers ending in an experimental neuroma in the rat sciatic nerve discharge spontaneously. Furthermore, many become sensitive to a range of physical and chemical stimuli. The resulting afferent barrage is thought to contribute to paresthesias and pain associated with peripheral nerve injury. We report that the development of such ectopic neuroma discharge is largely prevented when the freshly cut nerve end is treated with any of 3 commercially available corticosteroid preparations including two in depot form, triamcinolone hexacetonide (Lederspan) and triamcinolone diacetate (Ledercort), and one in soluble form, dexamethasone (Dexacort). These corticosteroids also produce a rapid and prolonged suppression of ongoing discharge in chronic neuromas that have already become active. The kinetics of corticosteroid suppression of neuroma discharge suggest a direct membrane action rather than an anti-inflammatory action.     

Effect of local and intravenous lidocaine on ongoing activity in injured afferent nerve fibers

Lidocaine applied systemically or locally attenuates neuropathic pain in patients. Here we tested the hypothesis that ectopic activity in injured afferent A- or C-fibers is suppressed by lidocaine. In rats the sural nerve (skin nerve) or lateral gastrocnemius-soleus nerve (muscle nerve) was crushed. Four to 11 days after crush lesion afferent fibers were isolated from the lesioned nerves in bundles rostral to the injury site. Ongoing ectopic activity was recorded from 75 A-fibers (muscle N = 43, skin N = 32) and 69 C-fibers (muscle N = 30, skin N = 39). Most afferent fibers were functionally characterized by their responses to mechanical and thermal (mostly heat) stimuli applied at or distal to the nerve injury site. Low-threshold cold-sensitive cutaneous C-fibers were excluded from the analysis [34] and [35]. Lidocaine was either applied to the nerve at or distal to the injury site in concentrations of 1 to 1000 μg/mL or injected i.v. in doses of 0.09 to 9 mg/kg (skin) or 0.047 to 4.7 mg/kg (muscle). Local application of lidocaine depressed ectopic activity in A- and C-fibers dose-dependently. Depression was weaker in C- than in A-fibers. Intravenous application of lidocaine depressed ongoing ectopic activity in A- and C-fibers dose-dependently. Responses to heat or mechanical stimulation of the injured nerve were not suppressed at the highest concentrations of lidocaine. The results support the hypothesis that decrease of neuropathic pain following local or systemic application of a local anesthetic is related to decrease of ectopic ongoing activity in injured afferent nerve fibers.