Ectopic discharges from injured nerve fibers are highly correlated with tactile allodynia only in early, but not late, stage in rats with spinal nerve ligation

It is widely accepted that ectopic discharges originated from injured sites and dorsal root ganglion (DRG) neurons after peripheral nerve injury contribute to neuropathic pain. However, it has been recently shown that ectopic discharges were not always necessary for neuropathic pain. In the present study, we aim to further examine the role of ectopic discharges in neuropathic pain in a spinal nerve ligation (SNL) model. With teased fiber recordings in vivo, the characteristics of ectopic discharges were observed over 14 days after SNL, and the correlation between ectopic discharges and tactile allodynia was analyzed. It was observed that ectopic discharges have three firing patterns (tonic, bursting, and irregular) after SNL, and proportions of these three patterns changed dynamically over time. The tonic and bursting types were dominant in the first 24 h following SNL, while the irregular type became the only pattern in the late stage (day 14). The average frequencies of ectopic discharges and the percentage of active filaments also changed over time, reaching the peak 24 h after SNL and then declined gradually. Ectopic discharges were highly correlated with tactile allodynia in the first 24 h following SNL, but surprisingly, not in the late stage of days 1 to 14. These findings suggest that ectopic discharges may be crucial in the triggering of neuropathic pain in the early stage, but their importance become more limited over time.     

Three types of sodium channels in adult rat dorsal root ganglion neurons

Several types of Na+ currents have previously been demonstrated in dorsal root ganglion (DRG) neurons isolated from neonatal rats, but their expression in adult neurons has not been studied. Na+ current properties in adult dorsal root ganglion (DRG) neurons of defined size class were investigated in isolated neurons maintained in primary culture using a combination of microelectrode current clamp, patch voltage clamp and immunocytochemical techniques. Intracellular current clamp recordings identified differing relative contributions of TTX-sensitive and -resistant inward currents to action potential waveforms in DRG neuronal populations of defined size. Patch voltage clamp recordings identified three distinct kinetic types of Na+ current differentially distributed among these size classes of DRG neurons. 'Small' DRG neurons co-express two types of Na+ current: (i) a rapidly-inactivating, TTX-sensitive 'fast' current and (ii) a slowly-activating and -inactivating, TTX-resistant 'slow' current. The TTX-sensitive Na+ current in these cells was almost completely inactivated at typical resting potentials. 'Large' cells expressed a single TTX-sensitive Na+ current identified as 'intermediate' by its inactivation rate constants. 'Medium'-sized neurons either co-expressed 'fast' and 'slow' current or expressed only 'intermediate' current. Na+ channel expression in these size classes was also measured by immunocytochemical techniques. An antibody against brain-type Na+ channels (Ab7493)10 labeled small and large neurons with similar intensity. These results demonstrate that three types of Na+ currents can be detected which correlate with electrogenic properties of physiologically and anatomically distinct populations of adult rat DRG neurons.     

Single-channel analysis of tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels in rat dorsal root ganglion neurons

Tetrodotoxin-sensitive and tetrodotoxin-resistant single sodium channel currents were recorded from rat dorsal root ganglion neurons. The two types of sodium channel currents could be distinguished by the effects of predepolarization, 10 nM tetrodotoxin, and the inactivation during depolarization. Single-channel conductances were calculated to be 6.3 and 3.4 pS in the tetrodotoxin-sensitive and tetrodotoxin-resistant channels, respectively.     

Differential sensitivity of tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels to the insecticide allethrin in rat dorsal root ganglion neurons

The pyrethroid insecticides are known to modify neuronal sodium channels to cause a prolongation of whole cell current. The sodium channels expressed in the dorsal root ganglion neurons of the rat are of two types, one highly sensitive to tetrodotoxin and the other highly resistant to tetrodotoxin. The pyrethroid allethrin exerted profound effects on tetrodotoxin-resistant sodium channels while causing minimal effects on tetrodotoxin-sensitive sodium channels. Currents derived from tetrodotoxin-resistant sodium channels were greatly prolonged during a step depolarization; the tail currents upon repolarization were also augmented and prolonged. In the tetrodotoxin-sensitive sodium channel currents, these changes caused by allethrin were much smaller or negligible. The activation and inactivation voltages of tetrodotoxin-resistant peak sodium currents were not significantly altered by allethrin. The differential action of allethrin on the two types of sodium channels would be important not only in identifying the target molecular structure but also in interpreting the symptoms of poisoning in mammals.     

Development of purinergic sensitivity in sensory neurons after peripheral nerve injury in the rat

Purinoceptors are present in the cell bodies as well as in both peripheral and central terminals of many sensory neurons, where they may play a role in sensory transmission, including pain. After peripheral nerve injury at the spinal nerve level, some axotomized afferent neurons develop ongoing discharges (ectopic discharges) that originate in the dorsal root ganglion (DRG). In the present study, we attempted to determine whether or not purinergic sensitivity develops in injured sensory neurons which display ectopic discharges, as well as in silent units. The L(4) and L(5) spinal nerves were ligated in Sprague-Dawley rats. Four to 21 days after the surgery, the DRGs with attached dorsal roots and spinal nerves were removed and ectopic discharges were recorded from teased dorsal root fascicles using an in vitro recording set-up. The results showed that 75.6 and 65.1% of the chronically axotomized DRG neurons displaying ectopic discharges enhanced their activity after application of adenosine 5'-triphosphate (ATP, 1 mM) or alpha,beta-methylene ATP (mATP, 100 microM), respectively. In addition, application of these purinoceptor agonists evoked activity in 7 of 28 axotomized DRG neurons, which did not show ongoing discharges. In contrast, only 1 of 34 DRG neurons acutely isolated from normal rats (no previous spinal nerve ligation) responded to either mATP or ATP. In most of the tested units, mATP-induced enhancement of ectopic discharges was blocked by non-specific P2X receptor antagonists, PPADS or suramin. The data from the present study suggest that purinergic sensitivity develops in DRG neurons after chronic axotomy and that this purinergic sensitivity is likely to be mediated by P2X purinoceptors. This acquired purinergic sensitivity may play an important functional role in the enhancement of ectopic discharges and exacerbation of pain upon sympathetic activation in the neuropathic pain state.     

p38 activation in uninjured primary afferent neurons and in spinal microglia contributes to the development of neuropathic pain induced by selective motor fiber injury

Compelling evidence shows that the adjacent uninjured primary afferents play an important role in the development of neuropathic pain after nerve injury. The underlying mechanisms, however, are largely unknown. In the present study, the selective motor fiber injury was performed by L5 ventral root transection (L5 VRT), and p38 activation in dorsal root ganglia (DRG) and L5 spinal dorsal horn was examined. The results showed that phospho-p38 immunoreactivity (p-p38-IR) was increased in both L4 and L5 DRGs, starting on day 1 and persisting for nearly 3 weeks (P<0.05) following L5 VRT and that the activated p38 was confined in neurons, especially in IB4 positive C-type neurons. L5 VRT also induced p38 activation in L5 spinal dorsal horn, occurred at the first day after the lesion and lasted for 2 weeks (P<0.05). The activated p38 is restricted entirely in spinal microglia. In contrast, selective injury of sensory neurons by L5 dorsal root transection (L5 DRT) failed to induce behavioral signs of neuropathic pain and activated p38 only in L5 DRG but not in L4 DRG and L5 spinal dorsal horn. Intraperitoneal injection of thalidomide, an inhibitor of TNF-alpha synthesis, prevented p38 activation in DRG and spinal cord. Intrathecal injection of p38 inhibitor SB203580, starting before L5 VRT, inhibited the abnormal pain behaviors. Post-treatment with SB203580 performed at the 1st day or at the 8th day after surgery also reduced established neuropathic pain. These data suggest that p38 activation in uninjured DRGs neurons and in spinal microglia is necessary for the initiation and maintenance of neuropathic pain induced by L5 VRT.     

Antisense knock down of TRPA1, but not TRPM8, alleviates cold hyperalgesia after spinal nerve ligation in rats

Patients with neuropathic pain frequently experience hypersensitivity to cold stimulation. However, the underlying mechanisms of this enhanced sensitivity to cold are not well understood. After partial nerve injury, the transient receptor potential ion channel TRPV1 increases in the intact small dorsal root ganglion (DRG) neurons in several neuropathic pain models. In the present study, we precisely examined the incidence of cold hyperalgesia and the changes of TRPA1 and TRPM8 expression in the L4 and L5 DRG following L5 spinal nerve ligation (SNL), because it is likely that the activation of two distinct populations of TRPA1- and TRPM8-expressing small neurons underlie the sensation of cold. We first confirmed that L5 SNL rats developed cold hyperalgesia for more than 14 days after surgery. In the nearby uninjured L4 DRG, TRPA1 mRNA expression increased in trkA-expressing small-to-medium diameter neurons from the 1st to 14th day after the L5 SNL. This upregulation corresponded well with the development and maintenance of nerve injury-induced cold hyperalgesia of the hind paw. In contrast, there was no change in the expression of the TRPM8 mRNA/protein in the L4 DRG throughout the 2-week time course of the experiment. In the injured L5 DRG, on the other hand, both TRPA1 and TRPM8 expression decreased over 2 weeks after ligation. Furthermore, intrathecal administration of TRPA1, but not TRPM8, antisense oligodeoxynucleotide suppressed the L5 SNL-induced cold hyperalgesia. Our data suggest that increased TRPA1 in uninjured primary afferent neurons may contribute to the exaggerated response to cold observed in the neuropathic pain model.     

Changes in sodium channel expression following trigeminal nerve injury

We have investigated the expression of TTX-sensitive (TTXs) and TTX-resistant (TTXr) sodium channel subtypes following injury to the inferior alveolar nerve (IAN), in order to determine their potential role in the development of trigeminal neuropathic pain. In seven anaesthetised ferrets, fluorogold (2%) was injected into the left IAN to identify cell bodies with axons in this nerve. In four animals, the nerve was sectioned distal to the injection site and the remaining three served as controls. After 3 days, the animals were perfused with 4% paraformaldehyde. The left and right IANs and trigeminal ganglia were processed using indirect immunofluorescence with specific primary antibodies to TTXs subtypes Na(v)1.3 and Na(v)1.7 and TTXr subtypes Na(v)1.8 and Na(v)1.9. Image analysis was used to quantify the percentage area of staining (PAS) in the nerves. In the ganglia, counts were made of positively labelled cells in the fluorogold population. PAS for Na(v)1.8 and Na(v)1.9 was significantly greater in injured nerves than in either contralateral or control nerves. After injury, significantly fewer cells in the ganglia expressed Na(v)1.3 (controls 36.9%; injured 13.1%), Na(v)1.7 (controls 17.0%; injured 8.1%) and Na(v)1.9 (controls 60.3%; injured 29.0%) (p<0.05, unpaired t test). These changes are different from those previously reported in the dorsal root ganglion following damage to peripheral nerves of spinal origin. As they occur at a time of known high abnormal neural discharge, it seems likely that changes in sodium channel expression may play a role in nerve injury-induced trigeminal pain.     

Effects of peripheral nerve injuries and tissue inflammation on the levels of neuropeptide Y-like immunoreactivity in rat primary afferent neurons

Changes in neuropeptide Y-like immunoreactivity (NPYir) in the rat L₄ and L₅ spinal cord and dorsal root ganglia (DRG) were examined after different sciatic nerve injuries (transection, loose ligation, and crush) and a localized, painful inflammation of the hind paw. Inflammation had no effect on NPYir. All the nerve injuries produced comparable increases in NPYir in ipsilateral laminae III–V axons and varicosities, and induction of NPYir in many DRG cells. Most NPYir DRG cells were medium to large (mean diameters: 40–45 μm); less than 2% of the cells had diameters of 25 μm or less. We conclude that the nerve injury-evoked increase in NPYir occurs mostly in the somata and intraspinal arbors of low-threshold mechanoreceptors; very few, if any, C-fiber afferents are involved. Nerve injury, rather than a painful condition, appears to be the stimulus for the induction of NPYir synthesis.