L4 Dorsal Root Ganglion Neurons Research Articles

Peripheral neural mechanism of visceral pain and acupoint sensitization in 2, 4, 6-trinitrobenzene sulfonic acid-induced colitis model mice.

To explore the neural mechanism of visceral pain and related somatic (acupoints) sensitization by using in vivo calcium imaging of dorsal root ganglia (DRG) neurons. Eight BALB/c mice were randomly divided into control and model groups, with 4 mice in each group. The colitis model was induced by colorectal perfusion of 2, 4, 6-trinitrobenzene sulfonic acid (TNBS) once daily for 7 days. Mice of the control group received colorectal perfusion of normal saline once daily for 7 days. The location and area of the somatic neurogenic inflammation (cutaneous exudation of Evans blue ［EB］) of the 2 groups of mice were observed after intravenous injection of EB. For pain behavioral tests, sixteen C57BL/6J mice were randomly divided into control and model groups, with 8 mice in each group, and a Von Frey filament was used to stimulate the referred somatic reactive regions in colitis mice, and the number of avoidance and paw withdraw reaction within 10 tests was recorded. For in vivo DRG calcium imaging tests, 24 Pirt-GCaMP6s transgenic mice were randomly and equally divided into control group and colitis model group. The responses of the neurons in L6 or L4 DRG to colorectal distension (CRD), lower back brushing, or mechanical stimulation at the hindpaw were observed using confocal fluorescence microscope. Compared with the control group, the area of EB exudation spot in the hindpaw and lower back regions was increased in the colitis model group (P<0.05), and the avoidance or paw withdraw numbers induced by Von Frey stimulation at the lower back and hindpaw were increased (P<0.01, P<0.05), indicating that colitis induced regional skin (acupoints) sensitization in the lower back and hindpaw regions. Compared with the control group, the percentage of L6 DRG neurons activated by 60 mm Hg CRD in the colitis model mice were apparently increased (P<0.01), the activated neurons mainly involved the medium-sized DRG neurons (P<0.01). In Pirt-GCaMP6s transgenic mice, following brushing the skin of the receptive field (lower back) of L6 DRG neurons, the fluorescence intensity of the brushing-activated DRG neurons and small, medium and large-sized neurons were significantly higher in the colitis model group than those in the control group (P<0.001, P<0.01, P<0.05). After brushing and clamping the skin of the right hindpaw (receptive field of L4 DRG neurons), the percentages of the activated L4 DRG neurons were obviously higher in the colitis model group than those in the control group (P<0.01, P<0.05), while there were no significant changes in the proportion of small, medium and large-sized neurons between the control and colitis model groups. Colitis may lead to body surface sensitization at the same and adjacent neuro-segments as well as to an increase of the number and activity of the responsive lumbar DRG neurons, among which the L6 DRG neurons at the same neuro-segment as the rectum colon showed an increase in the number of responders and intensity of calcium fluorescence signal while L4 DRG neurons at the level adjacent to the rectum colon showed an increase in the number of responders, suggesting that there may be different mechanisms of peripheral neural sensitization.

Peripheral Branch Injury Induces Oxytocin Receptor Expression at the Central Axon Terminals of Primary Sensory Neurons.

Considerable evidence suggests that oxytocin, as a regulatory nonapeptide, participates in modulatory mechanisms of nociception. Nonetheless, the role of this hypothalamic hormone and its receptor in the sensory pathway has yet to be fully explored. The present study performed immunohistochemistry, enzyme-linked immunosorbent assay, and RT-qPCR analysis to assess changes in the expression of the neuronal oxytocin receptor in female rats following tight ligation of the sciatic nerve after 1, 3, and 7 days of survival. Oxytocin receptor immunoreactivity was present in both dorsal root ganglia and lumbar spinal cord segments, but not accumulated at the site of the ligation of the peripheral nerve branch. We found a time-dependent change in the expression of oxytocin receptor mRNA in L5 dorsal root ganglion neurons, as well as an increase in the level of the receptor protein in the lumbar segment of the spinal cord. A peak in the expression was observed on day 3, which downturned slightly by day 7 after the nerve ligation. These results show that OTR expression is up-regulated in response to peripheral nerve lesions. We assume that the importance of OTR is to modify spinal presynaptic inputs of the sensory neurons upon injury-induced activation, thus to be targets of the descending oxytocinergic neurons from supraspinal levels. The findings of this study support the concept that oxytocin plays a role in somatosensory transmission.

NS5806 reduces carrageenan-evoked inflammation by suppressing extracellular signal-regulated kinase activation in primary sensory neurons and immune cells.

The compound NS5806 attenuates neuropathic pain via inhibiting extracellular signal-regulated kinase (ERK) activation in neuronal somata located at the dorsal root ganglion (DRG) and superficial spinal dorsal horn. NS5806 also reduces the expansion of DRG macrophages and spinal microglia several days after peripheral nerve injury, implying an anti-inflammatory effect. To test whether NS5806 inhibits inflammation, as a model we intraplantarly injected carrageenan into a hind paw of the rat. To examine whether NS5806 reduces carrageenan-evoked mechanical allodynia, thermal hyperalgesia, and edema, as well as ERK activation in the nerve fibres, mast cells, and macrophages in the hind paw skin, we used behavioural, immunohistochemical, and cytological methods. NS5806 did not impair motor function, affect basal nociception, or cause edema in naive rats. Six hours after carrageenan injection, mechanical allodynia, thermal hyperalgesia, and edema appeared in the rat's ipsilateral hind paw, and all were reduced by intraplantar co-injection of NS5806. NS5806 suppressed carrageenan-evoked ERK activation in the peripheral axons and somata of L4 DRG neurons, as well as mast cells and macrophages in the paw skin. NS5806 also reduced carrageenan-evoked mast cell degranulation and macrophage proliferation. NS5806 and the ERK pathway inhibitor PD98059 had a similar effect in inhibiting the proliferation of cultured RAW264.7 macrophages. Furthermore, all the in vivo anti-inflammatory effects of NS5806 were similar to those of PD98059. Acting like an ERK pathway inhibitor, NS5806 reduces inflammation-evoked mechanical allodynia, thermal hyperalgesia, and edema by suppressing ERK activation in primary sensory neurons, mast cells, and macrophages. Previous studies show that NS5806 only acts on neurons. This report unveils that NS5806 also acts on immune cells in the skin to exert its anti-inflammatory effects. Since NS5806 is lipid soluble for skin penetration, it suggests that NS5806 could also be developed into an anti-inflammatory drug for external use.

Aconitine – A promising candidate for treating cold and mechanical allodynia in cancer induced bone pain

Background and aimsPatients suffering from cancer induced bone pain (CIBP) have a poor quality of life that is exacerbated by the lack of effective therapeutic drugs. Monkshood is a flowering plant that has been used in traditional Chinese medicine where it has been used to relieve cold pain. Aconitine is the active component of monkshood, but the molecular mechanism for how this compound reduces pain is unclear. Methods and resultsIn this study, we employed molecular and behavioral experiments to explore the analgesic effect of aconitine. We observed aconitine alleviated cold hyperalgesia and AITC (allyl-isothiocyanate, TRPA1 agonist) induced pain. Interestingly, we found aconitine directly inhibits TRPA1 activity in calcium imaging studies. More importantly, we found aconitine alleviated cold and mechanical allodynia in CIBP mice. Both the activity and expression of TRPA1 in L4 and L5 DRG (Dorsal Root Ganglion) neurons were reduced with the treatment of aconitine in the CIBP model. Moreover, we observed aconiti radix (AR) and aconiti kusnezoffii radix (AKR), both components of monkshood that contain aconitine, alleviated cold hyperalgesia and AITC induced pain. Furthermore, both AR and AKR alleviated CIBP induced cold allodynia and mechanical allodynia. ConclusionsTaken together, aconitine alleviates both cold and mechanical allodynia in cancer induced bone pain via the regulation of TRPA1. This research on the analgesic effect of aconitine in cancer induced bone pain highlights a component of a traditional Chinese medicine may have clinical applications for pain.

Examination of the contribution of Nav1.7 to axonal propagation in nociceptors.

Nav1.7 is a promising drug target for the treatment of pain. However, there is a mismatch between the analgesia produced by Nav1.7 loss-of-function and the peripherally restricted Nav1.7 inhibitors, which may reflect a lack of understanding of the function of Nav1.7 in the transmission of nociceptive information. In the periphery, the role of Nav1.7 in transduction at nociceptive peripheral terminals has been comprehensively examined, but its role in axonal propagation in these neurons is less clearly defined. In this study, we examined the contribution of Nav1.7 to axonal propagation in nociceptors using sodium channel blockers in in vivo electrophysiological and calcium imaging recordings in mice. Using the sodium channel blocker tetrodotoxin (TTX) (1-10 µM) to inhibit Nav1.7 and other tetrodotoxin-sensitive sodium channels along the sciatic nerve, we first showed that around two-thirds of nociceptive L4 dorsal root ganglion neurons innervating the skin, but a lower proportion innervating the muscle (45%), are blocked by TTX. By contrast, nearly all large-sized cutaneous afferents (95%-100%) were blocked by axonal TTX. Many cutaneous nociceptors resistant to TTX were polymodal (57%) and capsaicin sensitive (57%). Next, we applied PF-05198007 (300 nM-1 µM) to the sciatic nerve between stimulating and recording sites to selectively block axonal Nav1.7 channels. One hundred to three hundred nanomolar PF-05198007 blocked propagation in 63% of C-fiber sensory neurons, whereas similar concentrations produced minimal block (5%) in rapidly conducting A-fiber neurons. We conclude that Nav1.7 is essential for axonal propagation in around two-thirds of nociceptive cutaneous C-fiber neurons and a lower proportion (≤45%) of nociceptive neurons innervating muscle.

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

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 Aδ-and Aβ-fiber nociceptors, and after-hyperpolarization duration in Aβ-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).

Calcium imaging in population of dorsal root ganglion neurons unravels novel mechanisms of visceral pain sensitization and referred somatic hypersensitivity.

Mechanisms of visceral pain sensitization and referred somatic hypersensitivity remain unclear. We conducted calcium imaging in Pirt-GCaMP6s mice to gauge responses of dorsal root ganglion (DRG) neurons to visceral and somatic stimulation in vivo. Intracolonic instillation of 2,4,6-trinitrobenzene sulfonic acid (TNBS) induced colonic inflammation and increased the percentage of L6 DRG neurons that responded to colorectal distension above that of controls at day 7. Colorectal distension did not activate L4 DRG neurons. TNBS-treated mice exhibited more Evans blue extravasation than did control mice and developed mechanical hypersensitivity in low-back skin and hind paws, which are innervated by L6 and L4 DRG neurons, respectively, suggesting that colonic inflammation induced mechanical hypersensitivity in both homosegmental and heterosegmental somatic regions. Importantly, the percentage of L4 DRG neurons activated by hind paw pinch and brush stimulation and calcium responses of L6 DRG neurons to low-back brush stimulation were higher at day 7 after TNBS than those in control mice. Visceral irritation from intracolonic capsaicin instillation also increased Evans blue extravasation in hind paws and low-back skin and acutely increased the percentage of L4 DRG neurons responding to hind paw pinch and the response of L6 DRG neurons to low-back brush stimulation. These findings suggest that TNBS-induced colitis and capsaicin-induced visceral irritation may sensitize L6 DRG neurons to colorectal and somatic inputs and also increase the excitability of L4 DRG neurons that do not receive colorectal inputs. These changes may represent a potential peripheral neuronal mechanism for visceral pain sensitization and referred somatic hypersensitivity.

Cav3.2 overexpression in L4 dorsal root ganglion neurons after L5 spinal nerve cutting involves Egr-1, USP5 and HMGB1 in rats: An emerging signaling pathway for neuropathic pain

Overexpression of Cav3.2 T-type Ca2+ channels in L4 dorsal root ganglion (DRG) participates in neuropathic pain after L5 spinal nerve cutting (L5SNC) in rats. The L5SNC-induced neuropathic pain also involves high mobility group box 1 (HMGB1), a damage-associated molecular pattern protein, and its target, the receptor for advanced glycation end-products (RAGE). We thus studied the molecular mechanisms for the L5SNC-induced Cav3.2 overexpression as well as neuropathic pain in rats by focusing on; 1) possible involvement of early growth response 1 (Egr-1), known to regulate transcriptional expression of Cav3.2, and ubiquitin-specific protease 5 (USP5) that protects Cav3.2 from proteasomal degradation, and 2) possible role of HMGB1/RAGE as an upstream signal. Protein levels of Cav3.2 as well as Egr-1 in L4 DRG significantly increased in the early (day 6) and persistent (day 14) phases of neuropathy after L5SNC, while USP5 protein in L4 DRG did not increase on day 6, but day 14. An anti-HMGB1-neutralizing antibody or a low molecular weight heparin, a RAGE antagonist, prevented the development of neuropathic pain and upregulation of Egr-1 and Cav3.2 in L4 DRG after L5SNC. L5SNC increased macrophages accumulating in the sciatic nerves, and the cytoplasm/nuclear ratio of immunoreactive HMGB1 in those macrophages. Our findings suggest that L5SNC-induced Cav3.2 overexpression in L4 DRG and neuropathic pain involves Egr-1 upregulation downstream of the macrophage-derived HMGB1/RAGE pathway, and that the delayed upregulation of USP5 might contribute to the persistent Cav3.2 overexpression and neuropathy.

Adjacent intact nociceptive neurons drive the acute outburst of pain following peripheral axotomy

Injury of peripheral nerves may quickly induce severe pain, but the mechanism remains obscure. We observed a rapid onset of spontaneous pain and evoked pain hypersensitivity after acute transection of the L5 spinal nerve (SNT) in awake rats. The outburst of pain was associated with a rapid development of spontaneous activities and hyperexcitability of nociceptive neurons in the adjacent uninjured L4 dorsal root ganglion (DRG), as revealed by both in vivo electrophysiological recording and high-throughput calcium imaging in vivo. Transection of the L4 dorsal root or intrathecal infusion of aminobutyrate aminotransferase inhibitor attenuated the spontaneous activity, suggesting that retrograde signals from the spinal cord may contribute to the sensitization of L4 DRG neurons after L5 SNT. Electrical stimulation of low-threshold afferents proximal to the axotomized L5 spinal nerve attenuated the spontaneous activities in L4 DRG and pain behavior. These findings suggest that peripheral axotomy may quickly induce hyperexcitability of uninjured nociceptors in the adjacent DRG that drives an outburst of pain.

Upregulation of interleukin-6 on Cav3.2 T-type calcium channels in dorsal root ganglion neurons contributes to neuropathic pain in rats with spinal nerve ligation

The T-type calcium channels Cav3.2, one of the low voltage-activated (LVA) calcium channels, have been found to play important roles in the neuronal excitability. Recently, we and others have demonstrated that accumulation of Cav3.2 channels in the dorsal root ganglion (DRG) neurons and sensory nerves contributes to neuropathic pain after peripheral nerve injury. In the present study, we aimed to further investigate the regulation of Cav3.2 channels by interleukin-6 (IL-6) in DRG neurons in neuropathic pain rats after spinal nerve ligation (SNL). The results showed that Cav3.2 channel protein expression in L5 DRG neurons was upregulated and blockade of this channel decreased the hyperexcitability of DRG neurons and mechanical allodynia in SNL neuropathic pain rats. Furthermore, inhibition of IL-6 trans-signaling reduced the upregulation of Cav3.2 T-type channel induced by FIL-6 (a fusion protein of IL-6 and sIL-6R) in primary cultured DRG neurons in vitro. In vivo, inhibition of IL-6 trans-signaling reversed the upregulation of Cav3.2, reduced the hyperexcitability of L5 DRG neurons and alleviated mechanical allodynia in SNL rats. Our results suggest that IL-6 upregulates Cav3.2 T-type channels expression and function through the IL-6/sIL-6R trans-signaling pathway in DRG neurons, thus contributes to the development of neuropathic pain in SNL rats.

Peripheral oxytocin restores light touch and nociceptor sensory afferents towards normal after nerve injury.

Oxytocin reduces primary sensory afferent excitability and produces analgesia in part through a peripheral mechanism, yet its actions on physiologically characterized, mechanically sensitive afferents in normal and neuropathic conditions are unknown. We recorded intracellularly from L4 dorsal root ganglion neurons characterized as low-threshold mechanoreceptors (LTMRs) or high-threshold mechanoreceptors (HTMRs) in female rats 1 week after L5 partial spinal nerve injury or sham control (n = 24 rats/group) before, during, and after ganglionic perfusion with oxytocin, 1 nM. Nerve injury desensitized and hyperpolarized LTMRs (membrane potential [Em] was -63 ± 1.8 mV in sham vs -76 ± 1.4 mV in nerve injury; P < 0.001), and sensitized HTMRs without affecting Em. In nerve-injured rats, oxytocin depolarized LTMRs towards normal (Em = -69 ± 1.9 mV) and, in 6 of 21 neurons, resulted in spontaneous action potentials. By contrast, oxytocin hyperpolarized HTMRs (Em = -68 ± 2.7 mV before vs -80 ± 3.2 mV during oxytocin exposure; P < 0.01). These effects were reversed after removal of oxytocin, and oxytocin had minimal effects in neurons from sham surgery animals. Sensory afferent neurons immunopositive for the vasopressin 1a receptor were larger (34 ± 6.3 μm, range 16-57 μm) than immunonegative neurons (26 ± 3.4 μm, range 15-43 μm; P < 0.005). These data replicate findings that neuropathic injury desensitizes LTMRs while sensitizing HTMRs and show rapid and divergent oxytocin effects on these afferent subtypes towards normal, potentially rebalancing input to the central nervous system. Vasopressin 1a receptors are present on medium to large diameter afferent neurons and could represent oxytocin's target.

Prostanoid EP4 Receptor-Mediated Augmentation of I h Currents in Aβ Dorsal Root Ganglion Neurons Underlies Neuropathic Pain.

An injury of the somatosensory system causes neuropathic pain, which is usually refractory to conventional analgesics, thus warranting the development of novel drugs against this kind of pain. The mechanism of neuropathic pain in rats that had undergone left L5 spinal nerve transection was analyzed. Ten days after surgery, these rats acquired neuropathic pain. The patch-clamp technique was used on the isolated bilateral L5 dorsal root ganglion neurons. The current-clamped neurons on the ipsilateral side exhibited significantly higher excitability than those on the contralateral side. However, only neurons with diameters of 40-50 μm on the ipsilateral side exhibited significantly larger voltage sags in response to hyperpolarizing current pulses than those on the contralateral side. Under the voltage clamp, only these neurons on the ipsilateral side showed a significantly larger density of an inward current at < -80 mV [hyperpolarization-activated nonselective cation (I h) current] with a rightward-shifted activation curve than that on the contralateral side. Ivabradine-an I h current inhibitor-inhibited I h currents in these neurons on both sides in a similar concentration-dependent manner, with an IC50 value of ∼3 μM. Moreover, the oral administration of ivabradine significantly alleviated the neuropathic pain on the ipsilateral side. An inhibitor of adenylyl cyclase or an antagonist of prostanoid EP4 receptors (CJ-023423) inhibited ipsilateral, but not contralateral I h, currents in these neurons. Furthermore, the intrathecal administration of CJ-023423 significantly attenuated neuropathic pain on the ipsilateral side. Thus, ivabradine and/or CJ-023423 may be a lead compound for the development of novel therapeutics against neuropathic pain.

Membrane potential oscillations are not essential for spontaneous firing generation in L4 Aβ-afferent neurons after L5 spinal nerve axotomy and are not mediated by HCN channels.

What is the central question of this study? Is spontaneous activity (SA) in L4 dorsal root ganglion (DRG) neurons induced by L5 spinal nerve axotomy associated with membrane potential oscillations in these neurons, and if so, are these membrane oscillations mediated by HCN channels? What is the main finding and its importance? Unlike injured L5 DRG neurons, which have been shown to be incapable of firing spontaneously without membrane potential oscillations, membrane potential oscillations are not essential for SA generation in conducting 'uninjured' L4 neurons, and they are not mediated by HCN channels. These findings suggest that the underlying cellular mechanisms of SA in injured and 'uninjured' DRG neurons induced by spinal nerve injury are distinct. The underlying cellular and molecular mechanisms of peripheral neuropathic pain are not fully understood. However, preclinical studies using animal models suggest that this debilitating condition is driven partly by aberrant spontaneous activity (SA) in injured and uninjured dorsal root ganglion (DRG) neurons, and that SA in injured DRG neurons is triggered by subthreshold membrane potential oscillations (SMPOs). Here, using in vivo intracellular recording from control L4-DRG neurons, and ipsilateral L4-DRG neurons in female Wistar rats that had previously undergone L5 spinal nerve axotomy (SNA), we examined whether conducting 'uninjured' L4-DRG neurons in SNA rats exhibit SMPOs, and if so, whether such SMPOs are associated with SA in those L4 neurons, and whether they are mediated by hyperpolarization-activated cyclic nucleotide gated (HCN) channels. We found that 7days after SNA: (a) none of the control A- or C-fibre DRG neurons showed SMPOs or SA, but 50%, 43% and 0% of spontaneously active cutaneous L4 Aβ-low threshold mechanoreceptors, Aβ-nociceptors and C-nociceptors exhibited SMPOs, respectively, in SNA rats with established neuropathic pain behaviors; (b) neither SMPOs nor SA in L4 Aβ-neurons was suppressed by blocking HCN channels with ZD7288 (10mgkg-1 , i.v.); and (c) there is a tendency for female rats to show greater pain hypersensitivity than male rats. These results suggest that SMPOs are linked to SA only in some of the conducting L4 Aβ-neurons, that such oscillations are not a prerequisite for SA generation in those L4 A- or C-fibre neurons, and that HCN channels are not involved in their electrogenesis.

Upregulation of N-type calcium channels in the soma of uninjured dorsal root ganglion neurons contributes to neuropathic pain by increasing neuronal excitability following peripheral nerve injury

N-type voltage-gated calcium (Cav2.2) channels are expressed in the central terminals of dorsal root ganglion (DRG) neurons, and are critical for neurotransmitter release. Cav2.2 channels are also expressed in the soma of DRG neurons, where their function remains largely unknown. Here, we showed that Cav2.2 was upregulated in the soma of uninjured L4 DRG neurons, but downregulated in those of injured L5 DRG neurons following L5 spinal nerve ligation (L5-SNL). Local application of specific Cav2.2 blockers (ω-conotoxin GVIA, 1–100 μM or ZC88, 10–1000 μM) onto L4 and 6 DRGs on the operated side, but not the contralateral side, dose-dependently reversed mechanical allodynia induced by L5-SNL. Patch clamp recordings revealed that both ω-conotoxin GVIA (1 μM) and ZC88 (10 μM) depressed hyperexcitability in L4 but not in L5 DRG neurons of L5-SNL rats. Consistent with this, knockdown of Cav2.2 in L4 DRG neurons with AAV-Cav2.2 shRNA substantially prevented L5-SNL-induced mechanical allodynia and hyperexcitability of L4 DRG neurons. Furthermore, in L5-SNL rats, interleukin-1 beta (IL-1β) and IL-10 were upregulated in L4 DRGs and L5 DRGs, respectively. Intrathecal injection of IL-1β induced mechanical allodynia and Cav2.2 upregulation in bilateral L4–6 DRGs of naïve rats, whereas injection of IL-10 substantially prevented mechanical allodynia and Cav2.2 upregulation in L4 DRGs in L5-SNL rats. Finally, in cultured DRG neurons, Cav2.2 was dose-dependently upregulated by IL-1β and downregulated by IL-10. These data indicate that the upregulation of Cav2.2 in uninjured DRG neurons via IL-1β over-production contributes to neuropathic pain by increasing neuronal excitability following peripheral nerve injury.

Upregulation of Cav3.2 T-type calcium channels in adjacent intact L4 dorsal root ganglion neurons in neuropathic pain rats with L5 spinal nerve ligation

Besides the injured peripheral dorsal root ganglion (DRG) neurons, the adjacent intact DRG neurons also have important roles in neuropathic pain. Ion channels including Cav3.2 T-type calcium channel in the DRG neurons are important in the development of neuropathic pain. In the present study, we aimed to examine the expression of Cav3.2 T-type calcium channels in the intact DRG neurons in neuropathic pain. A neuropathic pain model of rat with lumbar 5 (L5) spinal nerve ligation (SNL) was established, in which the L4 DRG was separated from the axotomized L5 DRG, and the molecular, morphological and electrophysiological changes of Cav3.2 T-type calcium channels in L4 DRG neurons were investigated. Western blotting showed that total and membrane protein levels of Cav3.2 in L4 DRG neurons increased, and voltage-dependent patch clamp recordings revealed an increased T-type current density with a curve shift to the left in steady-state activation in the acutely isolated L4 DRG neurons in neuropathic pain rats. Immunofluorescent staining further showed that the membrane expression of Cav3.2 increased in CGRP-, IB4-positive small neurons and NF200-positive large ones. In conclusion, the membrane expression and the function of Cav3.2 T-type calcium channels are increased in the intact L4 DRG neurons in neuropathic pain rats with peripheral nerve injury like SNL.

Plasticity of Select Primary Afferent Projections to the Dorsal Horn after a Lumbosacral Ventral Root Avulsion Injury and Root Replantation in Rats.

Injuries to the conus medullaris and cauda equina portions of the spinal cord result in neurological impairments, including paralysis, autonomic dysfunction, and pain. In experimental studies, earlier investigations have shown that a lumbosacral ventral root avulsion (VRA) injury results in allodynia, which may be ameliorated by surgical replantation of the avulsed ventral roots. Here, we investigated the long-term effects of an L6 + S1 VRA injury on the plasticity of three populations of afferent projections to the dorsal horn in rats. At 8 weeks after a unilateral L6 + S1 VRA injury, quantitative morphological studies of the adjacent L5 dorsal horn showed reduced immunoreactivity (IR) for the vesicular glutamate transporter, VGLUT1 and isolectin B4 (IB4) binding, whereas IR for calcitonin gene-related peptide (CGRP) was unchanged. The IR for VGLUT1 and CGRP as well as IB4 binding was at control levels in the L5 dorsal horn at 8 weeks following an acute surgical replantation of the avulsed L6 + S1 ventral roots. Quantitative morphological studies of the L5 dorsal root ganglia (DRGs) showed unchanged neuronal numbers for both the VRA and replanted series compared to shams. The portions of L5 DRG neurons expressing IR for VGLUT1 and CGRP, and IB4 binding were also the same between the VRA, replanted, and sham-operated groups. We conclude that the L5 dorsal horn shows selective plasticity for VGLUT1 and IB4 primary afferent projections after an L6 + S1 VRA injury and surgical repair.

Bulleyaconitine A preferably reduces tetrodotoxin-sensitive sodium current in uninjured dorsal root ganglion neurons of neuropathic rats probably via inhibition of protein kinase C.

Oral Bulleyaconitine A (BLA) is effective for treating neuropathic pain in human patients, but the underlying mechanism is poorly understood. Here, we tested whether BLA blocked voltage-gated sodium channels (VGSCs) in dorsal root ganglion (DRG) neurons. Compelling evidence shows that voltage-gated sodium channels are upregulated in uninjured DRG neurons but downregulated in injured ones following peripheral nerve injury. We found that BLA preferably inhibited Na currents in uninjured DRG neurons in neuropathic rats. Compared to sham rats, IC50 values for resting and inactivated Na currents were 113 and 74 times lower in injured and uninjured neurons of L4-6 DRGs in spared nerve injury (SNI) rats (4.55 and 0.56 nM) and were 688 and 518 times lower in the uninjured L4 and L6 DRG neurons of L5 spinal nerve ligation (L5-SNL) rats. The use-dependent blockage of BLA on Na currents was more potent in neuropathic rats compared to sham rats. Bulleyaconitine A facilitated the inactivation of Na channels in each group. IC50 values for resting and inactivated tetrodotoxin-sensitive (TTX-S) channels were 1855 and 1843 times lower than those for TTX-resistant channels in the uninjured neurons of L5 spinal nerve ligation rats. The upregulation of protein kinase C was associated with the preferable effect of BLA on TTX-S Na channels in the uninjured DRG neurons. Local application of BLA onto L4-6 DRGs at 0.1 to 10 nM dose-dependently alleviated the mechanical allodynia and thermal hyperalgesia in L5 spinal nerve ligation model. Thus, preferable blockage of TTX-S Na channels in uninjured DRG neurons may contribute to BLA's antineuropathic pain effect.

In vivo targeted gene delivery to peripheral neurons mediated by neurotropic poly(ethylene imine)-based nanoparticles.

A major challenge in neuronal gene therapy is to achieve safe, efficient, and minimally invasive transgene delivery to neurons. In this study, we report the use of a nonviral neurotropic poly(ethylene imine)-based nanoparticle that is capable of mediating neuron-specific transfection upon a subcutaneous injection. Nanoparticles were targeted to peripheral neurons by using the nontoxic carboxylic fragment of tetanus toxin (HC), which, besides being neurotropic, is capable of being retrogradely transported from neuron terminals to the cell bodies. Nontargeted particles and naked plasmid DNA were used as control. Five days after treatment by subcutaneous injection in the footpad of Wistar rats, it was observed that 56% and 64% of L4 and L5 dorsal root ganglia neurons, respectively, were expressing the reporter protein. The delivery mediated by HC-functionalized nanoparticles spatially limited the transgene expression, in comparison with the controls. Histological examination revealed no significant adverse effects in the use of the proposed delivery system. These findings demonstrate the feasibility and safety of the developed neurotropic nanoparticles for the minimally invasive delivery of genes to the peripheral nervous system, opening new avenues for the application of gene therapy strategies in the treatment of peripheral neuropathies.

Differing roles for parvalbumin neurons after nerve injury.

Neuropathic pain is abnormal, persistent pain that is caused by injury to the somatosensory system. It commonly involves damage to the peripheral nerves as a result of physical trauma, metabolic diseases such as diabetes, infections such as HIV or shingles, or toxicity induced neuropathies as a result of cancer treatments. Neuropathic pain is common, with epidemiological studies suggesting that around 18–30% of American, European and Australian adults suffer from chronic pain. This leads to significant disabilities and suffering. People with chronic pain complain of spontaneous pains that may be electric-shock like or continuous. Often coexisting with these are abnormal, evoked sensations such as tactile allodynia (pain in response to innocuous stimuli e.g., light brush or pressure) and cold allodynia (pain in response to innocuous cooling), or hyperalgesia (increase pain in response to an already noxious stimulus), as well as ataxia and proprioceptive deficits. The management of these conditions is often suboptimal for two main reasons: firstly, an incomplete knowledge of the mechanisms that are involved (and which of them are the most important). Secondly, patients often have compliance issues with pharmacological medications (Attal, 2012). Both peripheral and central neuronal mechanisms contribute to the pathophysiology and since the turn of the century particular emphasis has focussed on the role of glial cells in pain (West et al., 2015).

Increase of transcription factor EB (TFEB) and lysosomes in rat DRG neurons and their transportation to the central nerve terminal in dorsal horn after nerve injury

In the spinal dorsal horn (DH), nerve injury activates microglia and induces neuropathic pain. Several studies clarified an involvement of adenosine triphosphate (ATP) in the microglial activation. However, the origin of ATP together with the release mechanism is unclear. Recent in vitro study revealed that an ATP marker, quinacrine, in lysosomes was released from neurite terminal of dorsal root ganglion (DRG) neurons to extracellular space via lysosomal exocytosis. Here, we demonstrate a possibility that the lysosomal ingredient including ATP released from DRG neurons by lysosomal-exocytosis is an additional source of the glial activation in DH after nerve injury. After rat L5 spinal nerve ligation (SNL), mRNA for transcription factor EB (TFEB), a transcription factor controlling lysosomal activation and exocytosis, was induced in the DRG. Simultaneously both lysosomal protein, LAMP1- and vesicular nuclear transporter (VNUT)-positive vesicles were increased in L5 DRG neurons and ipsilateral DH. The quinacrine staining in DH was increased and co-localized with LAMP1 immunoreactivity after nerve injury. In DH, LAMP1-positive vesicles were also co-localized with a peripheral nerve marker, Isolectin B4 (IB4) lectin. Injection of the adenovirus encoding mCherry-LAMP1 into DRG showed that mCherry-positive lysosomes are transported to the central nerve terminal in DH. These findings suggest that activation of lysosome synthesis including ATP packaging in DRG, the central transportation of the lysosome, and subsequent its exocytosis from the central nerve terminal of DRG neurons in response to nerve injury could be a partial mechanism for activation of microglia in DH. This lysosome-mediated microglia activation mechanism may provide another clue to control nociception and pain.