L5 Spinal Nerve Axotomy Induces Distinct Electrophysiological Changes in Axotomized L5- and Adjacent L4-Dorsal Root Ganglion Neurons in Rats In Vivo

Abstract

Peripheral neuropathic pain (PNP) is a major health problem for which effective drug treatment is lacking. Its underlying neuronal mechanisms are still illusive, but pre-clinical studies using animal models of PNP including the L5-spinal nerve axotomy (L5-SNA) model, suggest that it is partly caused by excitability changes in dorsal root ganglion (DRG) neurons. L5-SNA results in two DRG neuronal groups: (1) axotomized/damaged neurons in L5-plus some in L4-DRGs, and (2) ipsilateral L4-neurons with intact/uninjured fibers intermingling with degenerating L5-fibers. The axotomized neurons are deprived of peripherally derived trophic factors and degenerate causing neuroinflammation, whereas the uninjured L4-neuorns are subject to increased trophic factors and neuroinflammation associated with Wallerian degeneration of axotomized L5-nerve fibers. Whether these two groups of DRG neurons exhibit similar or distinct electrophysiological changes after L5-SNA remains unresolved. Conflicting evidence for this may result from some studies assuming that all L4-fibers are undamaged. Here, we recorded somatic action potentials (APs) intracellularly from C-and A-fiber L4/L5 DRG neurons in vivo, to examine our hypothesis that L5-SNA would induce distinct electrophysiological changes in the two populations of DRG neurons. Consistent with this hypothesis, we found (7 days post-SNA), in SNA rats with established pain hypersensitivity, slower AP kinetics in axotomized L5-neurons and faster AP kinetics in L4-nociceptive neurons including decreased rise time in Ad-and Ab-fiber nociceptors, and after-hyperpolarization duration in Ab-fiber nociceptors. We also found several changes in axotomized L5-neurons but not in L4-nociceptive neurons, and some changes in L4-nociceptive but not L5-neurons. The faster AP kinetics (decreased refractory period) in L4-nociceptive neurons that are consistent with their reported hyperexcitability may lead to repetitive firing and thus provide enhanced afferent input necessary for initiating and/or maintaining PNP development. The changes in axotomized L5-neurons may contribute to the central mechanisms of PNP via enhanced neurotransmitter release in the central nervous system (CNS).

Switching Rat Resident Macrophages from M1 to M2 Phenotype by Iba1 Silencing Has Analgesic Effects in SNL-Induced Neuropathic Pain

“…Five days after SNL, compared to sham conditions (n = 12), the recorded neurons showed a significantly reduced resting membrane potential (mV) (n = 13, p = 0.050), and action potentials (AP) characterized by: (1) reduced total amplitude (mV) (n = 13, p = 0.022); (2) slower kinetics revealed by prolonged duration at the base (ms) (n = 13, p < 0.001) and during falling phase (ms) (n = 13, p < 0.001) and increased area at the base (mV*ms) (n = 13, p < 0.001), due, in particular, to increased area and rate of the falling phase (n = 13, p < 0.001 for both); and (3) significantly faster after-hyperpolarization (n = 13, p = 0.034 at 80% recovery) (Figure 4E-K and Supplemental Table S4 for additional parameters of AP and complete comparisons between conditions). Our findings are consistent with those of previous studies showing that damaged L5 DRG neurons have broader somatic APs with slower kinetics after L5 SNL [43][44][45]. Intriguingly, the treatment with Iba1-siRNA did not reduce any of the parameters of AP (Figure 4E-K and Supplemental Table S4), suggesting that the analgesic effect of local L5 delivery of Iba1-siRNA might be initiated locally, but it acts remotely.…”

“…Contrary to our hypothesis that Iba1-siRNA will prevent clustering of Iba1 (+) macrophages around neurons, the macrophages continued to cluster around all DRG neurons, with a preference for large NF200 (+) neurons. Most likely, this response is the expression of an increased sensitivity to traumatic injury of large DRG neurons [43,45,67] that attracted more macrophages around them by a CCL2-or CSF1-dependent chemotaxis [15,68]. On the other hand, the reduction of CGRP (+) and IB4 (+) neurons could have also contributed (i.e., 50-75% for CGRP (+) neurons and up to 87% for IB4 (+) neurons in L5 DRG, two weeks after SNL) [69,70].…”

“…Iba1-silencing-induced M1 to M2 switch did not reduce the SNL-induced hyperexcitability of L5 DRG neurons, contradicting another part of our hypothesis. L5 DRG neurons after SNL, with their peripheral connections severed, are less likely to make a significant contribution to peripheral pain pathogenesis [45], while the neighboring intact L4 neurons become hyperexcitable and painful, possibly due to increased inflammatory mediators [6], to the availability/transport of NGF to their soma and lowered pH [45], or due to pro-nociceptive miRNAs shed by macrophages [8]. In our study, Iba1 silencing reduced the secretion of pro-inflammatory mediators and thus possibly contributed to the Iba1 silencing-induced analgesia by a reduced activation of intact L4 DRG neurons.…”

“…Intriguingly, the treatment with Iba1-siRNA did not reduce any of the parameters of AP (Figure 4E-K and Supplemental Table S4), suggesting that the analgesic effect of local L5 delivery of Iba1-siRNA might be initiated locally, but it acts remotely. Previously, Djouhri and collaborators have shown that in vivo, L5 DRG neurons after SNL, axotomized, disconnected from their peripheral targets and degenerating, are less likely to make a significant contribution to peripheral pain pathogenesis, while intact L4 DRG neurons, which become hyperexcitable after SNL, possibly due to an overabundance of pro-inflammatory mediators and NGF diffused locally, are more responsible for the enhanced afferent input necessary for initiating and/or maintaining traumatic neuropathic pain [45]. Starting from this observation and from the fact that macrophages have an intense pro-and anti-nociceptive dialogue with the neurons [8,9], we continued to investigate if macrophages were switched to an anti-inflammatory, pro-analgesic phenotype after Iba1 silencing.…”

Analgesia effect of lentivirus-siSCN9A infected neurons in vincristine induced neuropathic pain rats

“…The conduction and regulation of pain signals depend on the activity of ion channels on afferent fibers, in which voltage-gated channels and receptor-gated channels jointly regulate resting membrane potential and action potential [ 12 ]. It has been verified that some channel subtype genes may be used as potential drugs for the treatment of chronic pain in humans [ 13 ].…”