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Summary Peripheral nociceptive stimuli from orofacial structures are largely transmitted by the trigeminal nerve. According to the peripheral noxious stimuli, neurons in the trigeminal ganglion (TG) produce neuropeptides such as substance P, and calcitonin-gene-related peptide, etc. Beside the production of neuropeptides, there exists unique non-synaptic interaction system between maxillary and mandibular neurons in the TG. Neurons in the TG are surrounded by satellite glial cells (SGCs), which initially receive the signal from TG neurons. These activated SGCs secrete a transmitter to activate adjacent SGCs or TG neurons, thereby amplifying the signal, for example, from mandibular neurons to maxillary neurons in the TG. Similar to the dorsal root ganglion, in the TG, microglia/macrophage-like cells (MLCs) are activated by uptake of a transmitter from TG neurons or SGCs. This communication between neurons, SGCs, and MLCs results in responses such as ectopic pain, hyperesthesia, or allodynia. The focus of this review is the cooperative interaction of the maxillary and mandibular nerves in the TG by neuropeptides, and adenosine 3′-phosphate (ATP) signaling from neurons to SGCs and MLCs. Stimulated neurons either secrete ATP by means of vesicular nucleotide transporters, or secrete neuropeptides from the neuronal cell body to mediate signal transmission.

Toll-like receptors (TLRs) orchestrate immune responses to a wide variety of danger- and pathogen-associated molecular patterns. Compared to the central nervous system (CNS), expression profile and function of TLRs in the human peripheral nervous system (PNS) are ill-defined. We analyzed TLR expression of satellite glial cells (SGCs) and microglia, glial cells predominantly involved in local immune responses in ganglia of the human PNS and normal-appearing white matter (NAWM) of the CNS, respectively. Ex vivo flow cytometry analysis of cell suspensions obtained from human cadaveric trigeminal ganglia (TG) and NAWM showed that both SGCs and microglia expressed TLR1-5, TLR7, and TLR9, although expression levels varied between these cell types. Immunohistochemistry confirmed expression of TLR1-TLR4 and TLR9 by SGCs in situ. Stimulation of TG- and NAWM-derived cell suspensions with ligands of TLR1-TLR6, but not TLR7 and TLR9, induced interleukin 6 (IL-6) secretion. We identified CD45LOW CD14POS SGCs and microglia, but not CD45HIGH leukocytes and CD45NEG cells as the main source of IL-6 and TNF-α upon stimulation with TLR3 and TLR5 ligands. In conclusion, human TG-resident SGCs express a broad panel of functional TLRs, suggesting their role in initiating and orchestrating inflammation to pathogens in human sensory ganglia.

Increasing evidence indicates that both microglia and satellite glial cell (SGC) activation play causal roles in neuropathic pain development after peripheral nerve injury; however, the activation mechanisms and their contribution to neuropathic pain remain elusive. To address this issue, we generated Ikkβ conditional knockout mice (Cnp-Cre/Ikkβ; cIkkβ) in which IKK/NF-κB-dependent proinflammatory SGC activation was abrogated. In these mice, nerve injury-induced spinal cord microglia activation and pain hypersensitivity were significantly attenuated compared to those in control mice. In addition, nerve injury-induced proinflammatory gene expression and macrophage infiltration into the dorsal root ganglion (DRG) were severely compromised. However, macrophages recruited into the DRG had minimal effects on spinal cord microglia activation, suggesting a causal effect for SGC activation on spinal cord microglia activation. In an effort to elucidate the molecular mechanisms, we measured Csf1 expression in the DRG, which is implicated in spinal cord microglia activation after nerve injury. In cIkkβ mice, nerve injury-induced Csf1 upregulation was ameliorated indicating that IKK/NF-κΒ-dependent SGC activation induced Csf1 expression in sensory neurons. Taken together, our data suggest that nerve injury-induced SGC activation triggers Csf1 induction in sensory neurons, spinal cord microglia activation, and subsequent central pain sensitization.

The P2X7 receptor mediates afferent nerve activation and is related to chronic neuropathic pain. Resveratrol (RES) has also been reported to exhibit anti-inflammatory effects. In this study, we investigated the neuroprotective effect of RES on the transmission of neuropathic pain mediated by the P2X7 receptor. The P2X7 mRNA and protein expression levels in L4-L5 dorsal root ganglia (DRG)s of the chronic constriction injury (CCI) group were significantly higher than those observed in the Ctrl + NS, Sham + RES and Sham groups. RES increased the threshold of thermal and mechanical hypersensitivity in rats with chronic neuropathic pain. The P2X7 mRNA and protein expression levels in the CCI + RES group were decreased compared with those in the CCI group. Our results showed that RES inhibited the upregulated co-expression of P2X7 and glial fibrillary acidic protein (GFAP, a marker of satellite glial cells) in satellite glial cells of DRG in the CCI group. The results demonstrated that the expression of GFAP was increased in the CCI group and that RES inhibited the upregulated expression of GFAP in the rats in the CCI group. In addition, the phosphorylation levels of p38 and extracellular regulated protein kinases (ERK)1/2 in the CCI group were markedly higher than those observed in the Ctrl + NS, Sham + RES and Sham groups, whereas the phosphorylation levels of p38 and ERK1/2 in CCI + RES group were markedly lower than those observed in the CCI group. RES inhibited BzATP-activated currents in DRG non-neurons in the CCI rats. Our data provide evidence that RES may suppress the transmission of neuropathic pain mediated by the P2X7 receptor in the satellite glial cells of dorsal root ganglia.

Background Pyridoxine (PDX; vitamin B6), is an essential vitamin. PDX deficiency induces various symptoms, and when PDX is misused it acts as a neurotoxicant, inducing severe sensory neuropathy. Results To assess the possibility of creating a reversible sensory neuropathy model using dogs, 150 mg/kg of PDX was injected subcutaneously into dogs for 7 days and body weight measurements, postural reaction assessments, and electrophysiological recordings were obtained. In addition, the morphology of dorsal root ganglia (DRG) and changes in glial fibrillary acidic protein (GFAP) immunoreactive satellite glial cells and ionized calcium-binding adapter molecule 1 (Iba-1) immunoreactive microglia/macrophages were assessed at 1 day, 1 week, and 4 weeks after the last PDX treatment. During the administration period, body weight and proprioceptive losses occurred. One day after the last PDX treatment, electrophysiological recordings showed the absence of the H-reflex in the treated dogs. These phenomena persisted over the four post-treatment weeks, with the exception of body weight which recovered to the pre-treatment level. Staining (CV and HE) results revealed significant losses of large-sized neurons in the DRG at 1 day and 1 week after PDX treatment cessation, but the losses were recovered at 4 weeks post-treatment. The Iba-1 and GFAP immunohistochemistry results showed pronounced increases in reactive microglia/macrophage and satellite glial cell at 1 day and 1 week, respectively, after the last PDX treatment, and thereafter, immunoreactivity decreased with increasing time after PDX treatment. Conclusions The results suggest that PDX-induced neuropathy is reversible in dogs; thus, dogs can be considered a good experimental model for research on neuropathy.

Some individuals recover from the pain of nerve trauma within 12 months or less whereas others experience life-long intractable pain. This transition between reversible pain and the establishment of chronic neuropathic pain is poorly understood. We examined the role of persistent inflammation in the dorsal root ganglia (DRG) in the long-term maintenance of mechanical allodynia; an index of neuropathic pain. Male Sprague-Dawley rats underwent chronic constriction injury (CCI), spared nerve injury (SNI) or sham surgery. Both CCI and SNI animals displayed robust mechanical allodynia in the ipsilateral paw at 7 d post-surgery; however, only SNI animals maintained mechanical allodynia at 42 d post-surgery. DRGs were extracted at 7 d or 42 d post-surgery to assess inflammation via rt-qPCR or immunohistochemistry to measure colony stimulating factor 1 (CSF1) expression, satellite glial cell (SGC) activation, presence of Iba1 positive macrophages and interleukin1 β (IL-1β) mRNA levels. Whereas DRGs from SNI animals continued to display inflammatory markers at 42 d, those from CCI animals did not. Moreover, the level of allodynia displayed by each individual animal correlated with the extent of DRG inflammation. These data support the hypothesis that the amount of CSF1 immunoreactivity and the persistence of inflammation in ipsilateral DRGs contribute to the difference between transient and persistent mechanical allodynia observed in the CCI and SNI models. We also suggest that feedback loops involving cytokines and neurotransmitters may contribute to increased DRG activity in chronic neuropathic pain. Consequently, targeting persistent CSF1 production and peripheral neuroinflammation may be an effective approach to the management of chronic neuropathic pain.

The cross talk between trigeminal ganglion (TG) neurons and satellite glial cells (SGCs) is crucial for the regulation of inflammatory orofacial pain. Substance P (SP) plays an important role by activating neurokinin (NK)-I receptors in this cross talk. The activation of extracellular signal-regulated kinase (ERK) 1/2, protein kinase A (PKA) and protein kinase C (PKC) in neurons and SGCs of peripheral ganglions by peripheral inflammation is associated with inflammatory hypersensitivity. This study tested the hypothesis that SP evoked SP-NK-I receptor positive feedback via the Renin-Angiotensin System/B-Protein Kinase A-Rapidly Accelerates Fibrosarcoma-MEK-Extracellular Signal-Regulated Kinase (RAS/PKA-RAF-MEK-ERK) pathway, which is involved in pain hypersensitivity. Inflammatory models were induced in vivo by injecting Complete Freund's adjuvant (CFA) into the whisker pad of rats. SP was administrated to SGCs in vitro for investigating, whether SP regulates the expression of NK-I receptor in the SGC nucleus. The effects of RAS-RAF-MEK, PKA and PKC pathways in this process were measured by co-incubating SGCs with respective Raf, PKA, PKC and MEK inhibitors in vitro and by pre-injecting these inhibitors into the TG in vivo. SP significantly upregulated NK-I receptor, p-ERK1/2, Ras, B-Raf, PKA and PKC in SGCs under inflammatory conditions. In addition, L703,606 (NK-I receptor antagonist), U0126 (MEK inhibitor), Sorafenib (Raf inhibitor) and H892HCL (PKA inhibitor) but not chelerythrine chloride (PKC inhibitor) significantly decreased NK-I mRNA and protein levels induced by SP. The allodynia-related behavior evoked by CFA was inhibited by pre-injection of L703,606, U0126, Sorafenib and H892HCL into the TG. Overall, SP upregulates NK-I receptor in TG SGCs via PKA/RAS-RAF-MEK-ERK pathway activation, contributing to a positive feedback of SP-NK-I receptor in inflammatory orofacial pain.

In the original article, there was a mistake in the legend for Figure 3 as published. The legend of Figure 3 (g-l) is incorrect. The correct legend appears below. FIGURE 3 | Photomicrographs of cryosections of canine cervical (C8) dorsal root ganglion showing cannabinoid receptor 2- (CB2), glial fibrillary acidic protein- (GFAP), and CD31-immunoreactivity. (a–c) Stars indicate NeuroTrace labeled (a) dorsal root ganglion sensory neurons which were CB2 receptor negative (b), as well as the satellite glial cells (white arrows). (d–f) Stars indicate sensory neurons encircled by satellite glial cells (white arrows) which were GFAP-immunoreactive (e) and CB2 receptor negative. CB2 receptor immunoreactivity was expressed by Schwann cells and neuronal nuclei (open arrow). (g–i) The empty arrow indicates one neuronal axon that bifurcates (T-junction) in its central and peripheral portions (large white arrows). The small arrows indicate the nuclei of Schwann cells. (j–l) Open arrows indicate smooth muscle cells (vessel on the left) and pericyte-like cells (elongated and thin blood vessel on the right) showing CB2 receptor immunoreactivity (j).White arrows indicate endothelial cells showing CD31 immunoreactivity (k). Bar: a–f, j–l = 50μm; g–i = 100 μm. The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.

Many trigeminal neuropathic pain patients suffer severe chronic pain. The neuropathic pain might be related with cross-excitation of the neighboring neurons and satellite glial cells (SGCs) in the sensory ganglia and increasing the pain signals from the peripheral tissue to the central nervous system. We induced trigeminal neuropathic pain by infraorbital nerve constriction injury (IONC) in Sprague-Dawley rats. We tested cytokine (CXCL2 and IL-10) levels in trigeminal ganglia (TGs) after trigeminal neuropathic pain induction, and the effect of direct injection of the anti-CXCL2 and recombinant IL-10 into TG. We found that IONC induced pain behavior. Additionally, IONC induced satellite glial cell activation in TG and cytokine levels of TGs were changed after IONC. CXCL2 levels increased on day 1 of neuropathic pain induction and decreased gradually, with IL-10 levels showing the opposite trend. Recombinant IL-10 or anti-CXCL2 injection into TG decreased pain behavior. Our results show that IL-10 or anti-CXCL2 are therapy options for neuropathic pain.

Peripheral tissue inflammation can alter the properties of somatic sensory pathways, causing behavioral hypersensitivity and resulting in increased responses to pain caused by noxious stimulation (hyperalgesia) and normally innocuous stimulation (allodynia). These hypersensitivities for nociception are caused by changes in the excitability of trigeminal ganglion (TG) neurons. These changes alter sensory information processing in the neurons in the medullary trigeminal nucleus of caudalis. Increasing information is becoming available regarding trigeminal neuron-neuron/neuron-satellite glial cells (SGCs) communication. The activation of intraganglionic communication plays an important role in the creation and maintenance of trigeminal pathological pain. Therefore, in this review, we focus on the recent findings for sensory functions and pharmacological modulation of TG neurons and SGCs under normal and pathological conditions, and we discuss potential therapeutic targets in glia-neuronal interactions for the prevention of trigeminal neuropathic and inflammatory pain.

cGMP-dependent kinase-I (cGKI) is known to regulate spinal pain processing. This enzyme consists of two isoforms (cGKIα and cGKIβ) that show distinct substrate specificity and tissue distribution. It has long been believed that the α isoform is exclusively expressed in the adult dorsal root ganglion. The aim of the present study was to reexamine the expression of cGKI isoforms in the adult mouse dorsal root ganglion using isoform-specific cGKI antibodies whose specificities had been validated in the previous studies. Immunoblot and immunohistochemical analyses revealed the presence of both isoforms in the dorsal root ganglion. Moreover, cGKIα was found to be mainly expressed within the cytoplasm of small- to medium-sized peptidergic and nonpeptidegic C-fibers, whereas cGKIβ was located within the nuclei of a wide range of dorsal root ganglion neurons. In addition, glutamine synthetase-positive satellite glial cells expressed both isoforms to varying degrees. Finally, using an experimental model for neuropathic pain produced by L5 spinal nerve transection, we found that cGKIα expression was downregulated in the injured, but not in the uninjured, dorsal root ganglion. In contrast, cGKIβ expression was upregulated in both the injured and uninjured dorsal root ganglions. Also, injury-induced cGKIβ upregulation was found to occur in small-to-medium-diameter dorsal root ganglion neurons. These data thus demonstrate the existence of two differently distributed cGKI isoforms in the dorsal root ganglion, and may provide insight into the cellular and molecular mechanisms of pain.

Sox2 is a transcriptional factor expressed in neural stem cells. It is known that Sox2 regulates cell differentiation, proliferation and survival of the neural stem cells. Our previous study showed that Sox2 is expressed in all satellite glial cells of the adult rat dorsal root ganglion. In this study, to examine the role of Sox2 in satellite glial cells, we establish a satellite glial cell-enriched culture system. Our culture method succeeded in harvesting satellite glial cells with the somata of neurons in the dorsal root ganglion. Using this culture system, Sox2 was downregulated by siRNA against Sox2. The knockdown of Sox2 downregulated ErbB2 and ErbB3 mRNA at 2 and 4 days after siRNA treatment. MAPK phosphorylation, downstream of ErbB, was also inhibited by Sox2 knockdown. Because ErbB2 and ErbB3 are receptors that support the survival of glial cells in the peripheral nervous system, apoptotic cells were also counted. TUNEL-positive cells increased at 5 days after siRNA treatment. These results suggest that Sox2 promotes satellite glial cell survival through the MAPK pathway via ErbB receptors.

Satellite glial cell (SGCs) in trigeminal and dorsal root ganglia are altered structurally and functionally under pathological conditions associated with chronic pain. These changes include reactive gliosis, augmented coupling by gap junctions, and increased responses to ATP via purinergic P2 receptors. Similar information for nodose ganglia (NG), which receive sensory inputs from internal organs via the vagus nerves, is missing. Here, we investigated changes in SGCs in mouse NG after the intraperitoneal administration of lipopolysaccharide (LPS), which induces systemic inflammation. Using calcium imaging we found that SGCs in intact, freshly isolated NG are sensitive to ATP, acting largely via purinergic P2 receptors (mixed P2X and P2Y), with threshold at 0.1 μM. A single systemic injection of LPS (2.5 mg/kg) induced a 6-fold increase in the responses to ATP, largely by augmenting the sensitivity of P2X receptors. Immunohistochemical analysis revealed that at 1-14 days post-LPS injection the expression of glial fibrillary acidic protein in SGCs was 2-3-fold greater than controls. The expression of pannexin 1 channels increased 2-fold at day 7 after LPS injection. Using intracellular labeling we examined dye coupling among SGCs around different neurons, and observed an over 2-fold higher incidence of dye coupling after the induction of inflammation. Incubating the ganglia with ATP increased dye coupling by acting on neuronal P2X receptors, suggesting a role for ATP in the LPS-induced changes. We conclude that inflammation induces prominent changes in SGCs of NG, which might have a role in vagal afferent functions, such as the inflammatory reflex. GLIA 2015;63:2121-2132.

Irritable bowel syndrome (IBS) and inflammatory bowel disease often display visceral hypersensitivity. Visceral nociceptors after inflammatory stimulation generate afferent nerve impulses through dorsal root ganglia (DRG) transmitting to the central nervous system. ATP and its activated-purinergic 2X7 (P2X7) receptor play an important role in the transmission of nociceptive signal. Purinergic signaling is involved in the sensory transmission of visceral pain. Moxibustion is a therapy applying ignited mugwort directly or indirectly at acupuncture points or other specific parts of the body to treat diseases. Heat-sensitive acupoints are the corresponding points extremely sensitive to moxa heat in disease conditions. In this study, we aimed to investigate the relationship between the analgesic effect of moxibustion on a heat-sensitive acupoint "Dachangshu" and the expression levels of P2X7 receptor in rat DRG after chronic inflammatory stimulation of colorectal distension. Heat-sensitive moxibustion at Dachangshu acupoint inhibited the nociceptive signal transmission by decreasing the upregulated expression levels of P2X7 mRNA and protein in DRG induced by visceral pain, and reversed the abnormal expression of glial fibrillary acidic protein (GFAP, a marker of satellite glial cells) in DRG. Consequently, abdominal withdrawal reflex (AWR) score in a visceral pain model was reduced, and the pain threshold was elevated. Therefore, heat-sensitive moxibustion at Dachangshu acupoint can produce a therapeutic effect on IBS via inhibiting the nociceptive transmission mediated by upregulated P2X7 receptor.

Aberrant sympathetic sprouting is seen in the uninjured trigeminal ganglia of transgenic mice that ectopically express nerve growth factor under the control of the glial fibrillary acidic protein promoter. These sympathetic axons form perineuronal plexuses around a subset of sensory somata in 2- to 3-month-old transgenic mice. Here, we show that aged transgenic mice (i.e., 11–14 and 16–18 months old) have dystrophic sympathetic plexuses (i.e., increased densities of swollen axons), and that satellite glial cells, specifically those in contact with dystrophic plexuses in the aged mice display strong immunostaining for tumor necrosis factor alpha. The colocalization of dystrophic plexuses and reactive satellite glial cells in the aged mice coincides with degenerative features in the enveloped sensory somata. Collectively, these novel results show that, with advancing age, sympathetic plexuses undergo dystrophic changes that heighten satellite glial cell reactivity and that together these cellular events coincide with neuronal degeneration.

Aims: To determine the involvement of tumor necrosis factor alpha (TNFα) signaling in the trigeminal ganglion (TG) in the mechanical hypersensitivity of the masseter muscle during temporomandibular joint (TMJ) inflammation. Methods: A total of 55 male Sprague-Dawley rats were used. Following injection of Complete Freund's Adjuvant into the TMJ, the mechanical sensitivities of the masseter muscle and the overlying facial skin were measured. Satellite glial cell (SGC) activation and TNFα expression in the TG were investigated immunohistochemically, and the effects of their inhibition on the mechanical hypersensitivity of the masseter muscle were also examined. Student t test or two-way repeated-measures analysis of variance followed by Bonferroni multiple comparisons test were used for statistical analyses. P < .05 was considered to reflect statistical significance. Results: Mechanical allodynia in the masseter muscle was induced without any inflammatory cell infiltration in the muscle after TMJ inflammation. SGC activation and an increased number of TNFα-immunoreactive cells were induced in the TG following TMJ inflammation. Intra-TG administration of an inhibitor of SGC activity or of TNFα- neutralizing antibody depressed both the increased number of TG cells encircled by activated SGCs and the mechanical hypersensitivity of the masseter following TMJ inflammation. Conclusion: These findings suggest that persistent masseter hypersensitivity associated with TMJ inflammation was mediated by SGC-TG neuron interactions via TNFα signaling in the TG.

Although many reports have demonstrated that ectopic pain develops in the orofacial region following tooth pulp inflammation, which often causes misdiagnosis and inappropriate treatment for patients with pulpitis, the precise mechanism remains unknown. In the present study, we hypothesized that the functional interaction between satellite glial cells and neurons mediated by interleukin 1β (IL-1β) in the trigeminal ganglion (TG) is involved in ectopic orofacial pain associated with tooth pulp inflammation. The digastric muscle electromyogram (D-EMG) activity elicited by capsaicin administration into the maxillary second molar tooth pulp was analyzed to evaluate the noxious reflex and was significantly increased in rats with inflammation of the maxillary first molar (M1) versus rats injected with saline. A significant increase in the expression of connexin43 (Cx43), a gap junction containing protein, was observed in activated satellite glial cells surrounding second molar-innervating neurons in the TG after M1 pulpitis. Daily administration of Gap26, a Cx43 mimetic peptide and inhibitor, in the TG significantly suppressed the enhancement of capsaicin-induced D-EMG activity and the percentage of Fluoro-Gold (FG)-labeled cells encircled by glial fibrillary acid protein-immunoreactive (IR) + Cx43-IR cells after M1 pulp inflammation ( P < 0.01). The percentage of FG-labeled cells encircled by glial fibrillary acid protein-IR + IL-1β-IR cells, IL-1 type I receptor-IR cells labeled with FG, and TRPV1-IR cells labeled with FG significantly increased after M1 pulp inflammation ( P < 0.01). Daily administration of IL-1ra, an IL-1 receptor antagonist, into the TG significantly reduced the enhancement of capsaicin-induced D-EMG activity and the percentage of TRPV1-IR neurons labeled with FG after M1 pulp inflammation ( P < 0.01). The present findings suggest that satellite glial cell is activated in the TG via activated gap junctions composed of Cx43 following tooth pulp inflammation, which leads to the hyperactivation of remote neurons via IL-1β mechanisms and results in ectopic tooth pulp pain in the adjacent tooth.

Recombinant adeno-associated viral (AAV)-mediated therapeutic gene transfer to dorsal root ganglia (DRG) is an effective and safe tool for treating chronic pain. However, AAV with various constitutively active promoters leads to transgene expression predominantly to neurons, while glial cells are refractory to AAV transduction in the peripheral nervous system. The present study evaluated whether in vivo satellite glial cell (SGC) transduction in the DRG can be enhanced by the SGC-specific GFAP promoter and by using shH10 and shH19, which are engineered capsid variants with Muller glia-prone transduction. Titer-matched AAV6 (as control), AAVshH10, and AAVshH19, all encoding the EGFP driven by the constitutively active CMV promoter, as well as AAV6-EGFP and AAVshH10-EGFP driven by a GFAP promoter (AAV6-GFAP-EGFP and AAVshH10-GFAP-EGFP), were injected into DRG of adult male rats. Neurotropism of gene expression was determined and compared by immunohistochemistry. Results showed that injection of AAV6- and AAVshH10-GFAP-EGFP induces robust EGFP expression selectively in SGCs, whereas injection of either AAVshH10-CMV-EGFP or AAVshH19-CMV-EGFP into DRG resulted in a similar in vivo transduction profile to AAV6-CMV-EGFP, all showing efficient transduction of sensory neurons without significant transduction of glial cell populations. Coinjection of AAV6-CMV-mCherry and AAV6-GFAP-EGFP induces transgene expression in neurons and SGCs separately. This report, together with our prior studies, demonstrates that the GFAP promoter rather than capsid tropism determines selective gene expression in SGCs following intraganglionic AAV delivery in adult rats. A dual AAV system, one with GFAP promoter and the other with CMV promoter, can efficiently express transgenes selectively in neurons versus SGCs.

BACKGROUND Neuronal remodeling in human heart disease is not well understood. METHODS Stellate ganglia from patients with cardiomyopathy (CMY) and refractory ventricular arrhythmias undergoing cardiac sympathetic denervation (n = 8), and from organ donors with normal hearts (n = 8) collected at the time of organ procurement were compared. Clinical data on all subjects were reviewed. Electron microscopy (EM), histologic, and immunohistochemical assessments of neurotransmitter profiles, glial activation and distribution, and lipofuscin deposition, a marker of oxidative stress, were quantified. RESULTS In CMY specimens, lipofuscin deposits were larger, and present in more neurons (26.3% ± 6.3% vs. 16.7% ± 7.6%, P < 0.043), than age-matched controls. EM analysis revealed extensive mitochondrial degeneration in CMY specimens. T cell (CD3+) infiltration was identified in 60% of the CMY samples, with one case having large inflammatory nodules, while none were identified in controls. Myeloperoxidase-immunoreactive neutrophils were also identified at parenchymal sites distinct from inflammatory foci in CMY ganglia, but not in controls. The adrenergic phenotype of pathologic samples revealed a decrease in tyrosine hydroxylase staining intensity compared with controls. Evaluation of cholinergic phenotype by staining for the vesicular acetylcholine transporter revealed a low but comparable number of cholinergic neurons in ganglia from both groups and demonstrated that preganglionic cholinergic innervation was maintained in CMY ganglia. S100 staining (a glial cell marker) demonstrated no differences in glial distribution and relationship to neurons; however, glial activation demonstrated by glial fibrillary acidic protein (GFAP) staining was substantially increased in pathologic specimens compared with controls. CONCLUSIONS Stellate ganglia from patients with CMY and arrhythmias demonstrate inflammation, neurochemical remodeling, oxidative stress, and satellite glial cell activation. These changes likely contribute to excessive and dysfunctional efferent sympathetic tone, and provide a rationale for sympathectomy as a treatment for arrhythmias in this population. FUNDING This work was made possible by support from NIH grants HL125730 to OAA, GM107949 to DBH, and HL084261 and OT2OD023848 to KS.

Sensitization and activation of the trigeminal ganglia have been implicated in the pathology of migraine. Satellite glial cells (SGCs), a specialized type of glial cells that ensheathe trigeminal neurons, may be critical for peripheral nociceptive sensitization. Tetrandrine (TET), an alkaloid extracted from a traditional Chinese herb, exerts an inhibitory effect on glial activation in vitro and has been used in various neurologic diseases. The current study investigated the effect of TET on nitroglycerin (NTG)-induced trigeminal sensitization and examined potential signaling pathways related to SGC activation in the model of migraine. We measured trigeminal nociceptive thresholds using electronic von Frey rigid tips before and after NTG injection in control rats and rats pretreated with TET, while expression and subcellular location of the inflammatory mediators S100B and activated phosphorylation extracellular signal-regulated kinase (p-ERK) were measured using real-time quantitative polymerase chain reaction, Western blotting, and double immunofluorescence staining. Pretreatment with TET caused a dose-dependent reversal of the trigeminal nociceptive hypersensitivity induced by NTG. In addition, TET pretreatment blocked the activation of S100B and p-ERK in trigeminal ganglion SGCs of NTG-treated rats. Reduced p-ERK activity can suppress the inflammation that leads to hyperexcitability of trigeminal ganglion neurons. Administration of TET may therefore be a safe and effective therapeutic treatment for the hyperalgesic symptoms of migraine.

Previous studies have shown that following peripheral nerve injury there was a downregulation of the gap junction protein connexin 36 (Cx36) in the spinal cord; however, it is not known whether Cx36 protein is expressed in the dorsal root ganglia (DRGs), nor if its levels are altered following peripheral nerve injuries. Here we address these aspects in the adult rat lumbar DRG. Cx36 mRNA was detected using qRT-PCR, and Cx36 protein was identified in DRG sections using immunohistochemistry (IHC) and immunofluorescence (IF). Double staining revealed that Cx36 co-localizes with both anti-β-III tubulin, a neuronal marker, and anti-glutamine synthetase, a satellite glial cell (SGC) marker. In neurons, Cx36 staining was mostly uniform in somata and fibers of all sizes and its intensity increased at the cell membranes. This labeling pattern was in contrast with Cx36 IF dots mainly found at junctional membranes in islet beta cells used as a control tissue. Co-staining with anti-Cx43 and anti-Cx36 showed that whereas mostly uniform staining of Cx36 was found throughout neurons and SGCs, Cx43 IF puncta were localized to SGCs. Cx36 mRNA was expressed in normal lumbar DRG, and it was significantly down-regulated in L4 DRG of rats that underwent sciatic nerve injury resulting in persistent hypersensitivity. Collectively, these findings demonstrated that neurons and SGCs express Cx36 protein in normal DRG, and suggested that perturbation of Cx36 levels may contribute to chronic neuropathic pain resulting from a peripheral nerve injury.

Excitability of neurons in the trigeminal ganglion (TG), trigeminal spinal subnucleus caudalis (Vc), and upper cervical spinal cord (C1-C2) is greatly enhanced after orofacial inflammation and trigeminal nerve injury, and TG, Vc, and C1-C2 neurons remain sensitized long after such episodes. Sensitized neurons generate various molecules, which are released from nociceptive neurons in these areas and are involved in modulating the excitability of TG, Vc, and C1-C2 nociceptive neurons. Hyperexcitable nociceptive neurons also activate satellite glial cells in the TG and microglial cells and astrocytes in the Vc and C1-C2. Glial cell activation spreads throughout the TG, Vc, and C1-C2 and triggers the release of various molecules involved in modulating nociceptive neurons in TG, Vc, and C1-C2 neurons. These findings suggest that functional interaction between neurons and glial cells is critical in persistent orofacial pain associated with orofacial inflammation and trigeminal nerve injury.

Iatrogenic trigeminal nerve injuries remain a common and complex clinical problem. Satellite glial cell (SGC) activation, associated phosphorylation of extracellular signal-regulated kinase (ERK), and neuropeptide expression in the trigeminal ganglion (TG) are known to be involved in trigeminal neuropathic pain related to trigeminal nerve injury. However, the involvement of these molecules in orofacial neuropathic pain mechanisms is still unknown. Phosphorylation of ERK1/2 in lingual nerve crush (LNC) rats was observed in SGCs. To evaluate the role of neuron-SGC interactions under neuropathic pain, calcitonin gene-related peptide (CGRP)-immunoreactive (IR), phosphorylated ERK1/2 (pERK1/2)-IR and glial fibrillary acidic protein (GFAP)-IR cells in the TG were studied in LNC rats. The number of CGRP-IR neurons and neurons encircled with pERK1/2-IR SGCs was significantly larger in LNC rats compared with sham rats. The percentage of large-sized CGRP-IR neurons was significantly higher in LNC rats. The number of CGRP-IR neurons, neurons encircled with pERK1/2-IR SGCs, and neurons encircled with GFAP-IR SGCs was decreased following CGRP receptor blocker CGRP8-37 or mitogen-activated protein kinase/ERK kinase 1 inhibitor PD98059 administration into the TG after LNC. Reduced thresholds to mechanical and heat stimulation to the tongue in LNC rats were also significantly recovered following CGRP8-37 or PD98059 administration. The present findings suggest that CGRP released from TG neurons activates SGCs through ERK1/2 phosphorylation and TG neuronal activity is enhanced, resulting in the tongue hypersensitivity associated with lingual nerve injury. The phenotypic switching of large myelinated TG neurons expressing CGRP may account for the pathogenesis of tongue neuropathic pain.

It has been proposed that glutamate serves as a mediator between neurons and satellite glial cells (SGCs) in sensory ganglia and that SGCs release glutamate. Using a novel method, we studied glutamate release from SGCs from murine trigeminal ganglia. Sensory neurons with adhering SGCs were enzymatically isolated from wild type and transgenic mice in which vesicular exocytosis was suppressed in glial cells. Extracellular glutamate was detected by microfluorimetry. After loading the cells with a photolabile Ca(2+) chelator, the intracellular Ca(2+) concentration was raised in SGCs by a UV pulse, which resulted in glutamate release. The amount of released glutamate was decreased in cells with suppressed exocytosis and after pharmacological block of hemichannels. The data demonstrate that SGCs of the trigeminal ganglion release glutamate in a Ca(2+)-dependent manner.

Pain is a serious and common problem with patients suffering from multiple sclerosis (MS). Very little has been done to investigate the peripheral mechanisms of pain in MS. Here we used a mouse model of experimental autoimmune encephalomyelitis (EAE) to investigate the possible contribution of satellite glial cells (SGCs) to pain in MS. EAE mice had reduced pain thresholds 10 days after disease induction. We examined dorsal root ganglia and found increased expression of glial fibrillary acidic protein in SGCs, a marker of SGC activation, and increased coupling among SGCs, a known component of activated SGCs. Activated SGCs have previously been shown to contribute to pain in other classical neuropathic pain models, suggesting that pain in multiple sclerosis has a peripheral component.

Neurons in sensory, sympathetic, and parasympathetic ganglia are surrounded by satellite glial cell (SGCs). There is little information on the effects of nerve damage on SGCs in autonomic ganglia. We studied the consequences of damage to sympathetic nerve terminals by 6-hydroxydopamine (6-OHDA) on SGCs in the mouse superior cervical ganglia (Sup-CG). Immunostaining revealed that at 1–30 d post-6-OHDA injection, SGCs in Sup-CG were activated, as assayed by upregulation of glial fibrillary acidic protein. Intracellular labeling showed that dye coupling between SGCs around different neurons increased 4-6-fold 1-14 d after 6-OHDA injection. Behavioral testing 1–7 d post-6-OHDA showed that withdrawal threshold to tactile stimulation of the hind paws was reduced by 65–85%, consistent with hypersensitivity. A single intraperitoneal injection of the gap junction blocker carbenoxolone restored normal tactile thresholds in 6-OHDA-treated mice, suggesting a contribution of SGC gap junctions to pain. Using calcium imaging we found that after 6-OHDA treatment responses of SGCs to ATP were increased by about 30% compared with controls, but responses to ACh were reduced by 48%. The same experiments for SGCs in trigeminal ganglia from 6-OHDA injected mice showed no difference from controls, confirming that 6-OHDA acted selectively on sympathetic nerves. However, systemic inflammation induced by lipopolysaccharide did not affect SGCs of Sup-CG, but did influence SGCs in trigeminal ganglia in the same manner as 6-OHDA did on SGCs in Sup-CG. We conclude that even though SGCs in sympathetic and sensory ganglia are morphologically similar, they are quite different functionally, particularly after damage.

Primary afferent nociceptors have been extensively characterized. However, like central neurons, they do not function in isolation. Within the dorsal root ganglia, satellite glial cells (SGCs) ensheath the cell bodies of primary afferents, allowing for bidirectional communication between neurons and SGCs. The mechanisms underlying this bidirectional communication and how SGCs can augment nociceptive signaling are not well understood. Ultimately, to understand the role of SGCs in nociceptive processing we have started developing a cellular profile of SGC-related markers during naive and inflamed states using flow cytometry (FC). To do this, female C57BL/6 mice received subcutaneous injections of Complete Freund's Adjuvant (CFA) or saline vehicle into the hairy skin of the right hind paw of. Behavioral sensitivity to mechanical stimulation was assessed using von Frey filaments at baseline, 2, 7, 14, 21, and 28 days following injection. We found that mice developed hypersensitivity at 7 days compared to vehicle controls. Next, we isolated L1-L3 DRGs at baseline, 2- and 7-days post-CFA for FC. We found that inflamed mice showed increased expression of glial fibrillary acidic protein (GFAP), and the calcium sensing protein, S100, 2 days following inflammation relative to naive controls. Seven days following inflammation we observed a decrease in GFAP, while S100 remained elevated. We also observed upregulation of glutamate converting enzyme, glutamine synthetase (GS), but not until 7 days following inflammation. These data suggest that peripheral inflammation may alter SGC-related markers in a temporally-specific manner that may contribute to hypersensitivity. Ongoing experiments are examining how inflammation impacts the expression of additional SGC-related proteins and genes, as well as the effect of anti-inflammatory administration on SGCs. We expect that our approach will provide an in-depth analysis of SGCs heterogeneity, and identify specific targets that are critical for driving SGC response to inflammation.

Sex-determining region Y (SRY)-box 2 (Sox2) is a member of the Sox family transcription factors. In the central nervous system, Sox2 is expressed in neural stem cells from neurogenic regions, and regulates stem cell proliferation and differentiation. In the peripheral nervous system, Sox2 is found only in the immature and dedifferentiated Schwann cells, and is involved in myelination inhibition or N-cadherin redistribution. In the present immunohistochemical study, we found that Sox2 is also expressed in other cells of the adult rat peripheral nervous system. Nuclear Sox2 was observed in all satellite glial cells, non-myelinating Schwann cells, and the majority of terminal Schwann cells that form lamellar corpuscles and longitudinal lanceolate endings. Sox2 was not found in myelinating Schwann cells and terminal Schwann cells of subepidermal free nerve endings. Satellite glial cells exhibit strong Sox2 immunoreactivity, whereas non-myelinating Schwann cells show weak immunoreactivity. RT-PCR confirmed the presence of Sox2 mRNA, indicating that the cells are likely Sox2 expressors. Our findings suggest that the role of Sox2 in the peripheral nervous system may be cell-type-dependent.

We have examined satellite glial cell (SGC) proliferation in trigeminal ganglia following chronic constriction injury of the infraorbital nerve. Using BrdU labeling combined with immunohistochemistry for SGC specific proteins we positively confirmed proliferating cells to be SGCs. Proliferation peaks at approximately 4 days after injury and dividing SGCs are preferentially located around neurons that are immunopositive for ATF-3, a marker of nerve injury. After nerve injury there is an increase GFAP expression in SGCs associated with both ATF-3 immunopositive and immunonegative neurons throughout the ganglia. SGCs also express the non-glial proteins, CD45 and CD163, which label resident macrophages and circulating leukocytes, respectively. In addition to SGCs, we found some Schwann cells, endothelial cells, resident macrophages, and circulating leukocytes were BrdU immunopositive.

Studies of the structural organization and functions of the cell body of a neuron (soma) and its surrounding satellite glial cells (SGCs) in sensory ganglia have led to the realization that SGCs actively participate in the information processing of sensory signals from afferent terminals to the spinal cord. SGCs use a variety ways to communicate with each other and with their enwrapped soma. Changes in this communication under injurious conditions often lead to abnormal pain conditions. "What are the mechanisms underlying the neuronal soma and SGC communication in sensory ganglia?" and "how do tissue or nerve injuries affect the communication?" are the main questions addressed in this review.

Most dorsal root ganglion neuronal somata (NS) are isolated from their neighbours by a satellite glial cell (SGC) sheath. However, some NS are associated in pairs, separated solely by the membrane septum of a common SGC to form a neuron-glial cell-neuron (NGlN) trimer. We reported that stimulation of one NS evokes a delayed, noisy and long-duration inward current in both itself and its passive partner that was blocked by suramin, a general purinergic antagonist. Here we test the hypothesis that NGlN transmission involves purinergic activation of the SGC. Stimulation of the NS triggered a sustained current noise in the SGC. Block of transmission through the NGlN by reactive blue 2 or thapsigargin, a Ca(2+) store-depletion agent, implicated a Ca(2+) store discharge-linked P2Y receptor. P2Y2 was identified by simulation of the NGlN-like transmission by puffing UTP onto the SGC and by immunocytochemical localization to the SGC membrane septum. Block of the UTP effect by BAPTA, an intracellular Ca(2+) scavenger, supported the involvement of SGC Ca(2+) stores in the signaling pathway. We infer that transmission through the NGlN trimer involves secretion of ATP from the NS and triggering of SGC Ca(2+) store discharge via P2Y2 receptors. Presumably, cytoplasmic Ca(2+) elevation leads to the release of an as-yet unidentified second transmitter from the glial cell to complete transmission. Thus, the two NS of the NGlN trimer communicate via a 'sandwich synapse' transglial pathway, a novel signaling mechanism that may contribute to information transfer in other regions of the nervous system.

The cytoarchitectonics of speech motor fields 44 and 45 of the brain were studied in five adult men and five women. Total frontal sections of thickness 20 μm were stained with cresyl violet for measurement of the thickness of the cortex and its layers, profile field areas of neurons in layers III and V, neuron density in layer III of field 45, satellite glial cell density, and the density of neurons surrounded by satellite glial cells. Both men and women showed a tendency to left-hemisphere dominance in the values of a number of cytoarchitectonic measures – the thickness of associative layer III, the profile field areas of neurons in this layer, and increases in the proportions of large and very large sizes in this layer. Interhemisphere differences in these par meters were more marked in men than women. A number of indictors of sexual dimorphism were identified in men and women. The most significant of these were increases in the densities of neurons, satellite glial cells, and neurons surrounded by satellite glial cells in women as compared with men.

Background Chemotherapy-induced peripheral neuropathy is a serious side effect in cancer treatment, a major manifestation being neuropathic pain that can be debilitating and can reduce the quality of life of the patient. Oxaliplatin and taxol are common anti-cancer drugs that induce neuropathic pain by an unknown mechanism. We tested the hypothesis that satellite glial cells in dorsal root ganglia (DRGs) are altered in chemotherapy-induced peripheral neuropathy models and contribute to neuropathic pain. Methods Mice were injected with either oxaliplatin or taxol and examined at 7–30 days. Glial fibrillary acidic protein (glial activation marker) expression was determined by immunohistochemistry. Satellite glial cells in isolated DRG were injected with the fluorescent dye Lucifer yellow and the incidence of dye coupling among these cells that surround different neurons was quantified. Results Taxol or oxaliplatin increased glial fibrillary acidic protein expression in satellite glial cells. Gap junction-mediated coupling between satellite glial cells was increased by up to fivefold after oxaliplatin and by up to twofold after taxol. This is consistent with work on other pain models showing that augmented satellite glial cell coupling contributes to chronic pain. Administration of the gap junction blocker carbenoxolone to chemotherapy-treated mice produced an analgesic-like effect. Conclusions We propose that increased coupling by gap junctions is part of satellite glial cell activation, and that augmented coupling contributes to the lowering of pain threshold in oxaliplatin- and taxol-treated mice. We further propose that gap junction blockers may have potential in treating chemotherapy-induced neuropathic pain.

Quantitative neurohistochemical parameters were used to study the comparative architectonics of two thalamic nuclei: the anterior dorsal and reticular. The cohort consisted of five cases from the collection of the Neurology Scientific Center, Russian Academy of Medical Sciences. Five women aged 19–33 years were studied; these were right-handed and had died suddenly without having suffered from neurological or mental disorders during life. The following parameters were measured: the densities of neurons, satellite glial cells, and neurons surrounded by satellite glial cells. Individual variability in neuron density was found to be greater in the reticular nucleus than the anterior dorsal nucleus. Variability in the density of satellite glial cells was greater in the anterior dorsal nucleus. Levels of variability in the density of neurons surrounded by satellite glial cells were similar in both nuclei. Interhemisphere asymmetry was demonstrated: neuron density and satellite glial cell density were greater in the anterior dorsal nuclei of the left hemisphere than that of the right hemisphere. The density of neurons surrounded by satellite glial cells in the anterior dorsal nucleus was greater in the left hemisphere, while that in the reticular nucleus was greater in the right hemisphere.

Somatic sensory neuron somata are located within the dorsal root ganglia (DRG) and are mostly ensheathed by individual satellite glial cell sheets. It has been noted, however, that a subpopulation of these DRG somata are intimately associated, separated only by a single thin satellite glial cell membrane septum. We set out to test whether such neuron-glial cell-neuron trimers (NGlNs) are also linked functionally. The presence of NGlNs in chick DRGs was confirmed by electron microscopy. Selective satellite glial cell immunostains were identified and were used to image the inter-neuron septa in DRG frozen sections. We used a gentle, dispase-based enzymatic method to isolate chick and rat NGlNs in vitro for double patch clamp recordings. In the majority of pairs tested, an action potential-like stimulus train delivered to one soma resulted in a delayed, noisy and long-duration response in its idle partner. The response to a second stimulus train given minutes later was markedly facilitated. Both bidirectional and unidirectional transmission was observed between the paired neurons. Transmission was chemical and block by the general purinergic blocker suramin implicated ATP as a neurotransmitter. We conclude that the two neuronal somata in the NGlN can communicate by chemical transmission, which may involve a transglial, bi-synaptic pathway. This novel soma-to-soma transmission reflects a novel form of processing that may play a role in sensory disorders in the DRG and interneuron communication in the central nervous system.

The effect of photodynamic therapy (PDT) on neurons is of critical importance when treating cancers within or adjacent to the nervous system. Neurons show reduced sensitivity to meta-tetrahydroxyphenyl chlorin (mTHPC) mediated PDT, so the aim of this study was to investigate whether neuron sparing is due to endogenous cellular antioxidant activity. Dorsal root ganglion (DRG) neurons and their associated satellite glia were subjected to mTHPC-PDT in a 3D co-culture system following incubation with antioxidant inhibitors: diethyl dithiocarbamate (DDC, SOD-1 inhibitor), 2-methoxyestradiol (2-MeOH(2), SOD-2 inhibitor) and L-buthionine sulfoximine (L-BSO, glutathione synthase inhibitor). Sensitivity of each cell type was assessed using a combination of live/dead staining and immunofluorescence. Pretreatment with DDC and with L-BSO significantly increased the sensitivity of neurons to mTHPC-PDT and also affected satellite glial cell viability, whereas 2-MeOE(2) caused only a small increase in neuron sensitivity (not significant). Pretreatment using a combination of DDC and L-BSO caused a near total loss of neuron and glial cell viability in treatment and control conditions. These findings suggest that the SOD-1 and glutathione pathways are likely to be involved in the neuronal sparing associated with mTHPC-PDT.

Satellite glial cells (SGCs) in sensory ganglia are altered structurally and biochemically as a result of nerve injury. Whereas there is ample evidence that P2 purinergic receptors in central glial cells are altered after injury, there is very little information on similar changes in SGCs, although it is well established that SGCs are endowed with P2 receptors. Using calcium imaging, we characterized changes in P2 receptors in SGCs from mouse trigeminal ganglia in short-term cultures. Seven days after the induction of submandibular inflammation with complete Freund's adjuvant, there was a marked increase in the sensitivity of SGCs to ATP, with the threshold of activation decreasing from 5 μM to 10 nM. A similar observation was made in the intact trigeminal ganglion after infra-orbital nerve axotomy. Using pharmacological tools, we investigated the receptor mechanisms underlying these changes in cultured SGCs. We found that in control tissues response to ATP was mediated by P2Y (metabotropic) receptors, whereas after inflammation the response was mediated predominantly by P2X (ionotropic) receptors. As the contribution of P2X1,3,6 receptors was excluded, and the sensitivity to a P2X7 agonist did not change after inflammation, it appears that after inflammation the responses to ATP are largely due to P2X2 and/or 5 receptors, with a possible contribution of P2X4 receptors. We conclude that inflammation induced a large increase in the sensitivity of SGCs to ATP, which involved a switch from P2Y to P2X receptors. We propose that the over 100-fold augmented sensitivity of SGCs to ATP after injury may contribute to chronic pain states.

In sensory ganglia each nerve cell body is usually enveloped by a satellite glial cell (SGC) sheath, sharply separated from sheaths encircling adjacent neurons by connective tissue. However, following axon injury SGCs may form bridges connecting previously separate perineuronal sheaths. Each sheath consists of one or several layers of cells that overlap in a more or less complex fashion; sometimes SGCs form a perineuronal myelin sheath. SGCs are flattened mononucleate cells containing the usual cell organelles. Several ion channels, receptors and adhesion molecules have been identified in these cells. SGCs of the same sheath are usually linked by adherent and gap junctions, and are functionally coupled. Following axon injury, both the number of gap junctions and the coupling of SGCs increase markedly. The apposed plasma membranes of adjacent cells are separated by 15–20 nm gaps, which form a potential pathway, usually long and tortuous, between connective tissue and neuronal surface. The boundary between neuron and SGC sheath is usually complicated, mainly by many projections arising from the neuron. The outer surface of the SGC sheath is covered by a basal lamina. The number of SGCs enveloping a nerve cell body is proportional to the cell body volume; the volume of the SGC sheath is proportional to the volume and surface area of the nerve cell body. In old animals, both the number of SGCs and the mean volume of the SGC sheaths are significantly lower than in young adults. Furthermore, extensive portions of the neuronal surface are not covered by SGCs, exposing neurons of aged animals to damage by harmful substances.

Background Intense nociceptive signaling arising from ongoing injury activates primary afferent nociceptive systems to generate peripheral sensitization. ERK1/2 phosphorylation in dorsal root ganglion can be used to visualize intracellular signal activity immediately after noxious stimulation. The aim of this study was to investigate spatiotemporal characteristics of ERK1/2 phosphorylation against tissue injury in the primary afferent neurons. Methods Plantar incisions were made in the hind paws of Sprague-Dawley rats (n =150). Levobupivacaine was injected into the plantar aspect of the paws and ankles, Mitogen-activated protein kinase kinase (MEK) inhibitor was injected into the paw, and carbenoxolone, dual inhibitor of the gap junction and pannexin channel, was intraperitoneally injected. Pain hypersensitivity was investigated by a behavioral study, while phosphorylated ERK1/2 was detected in dorsal root ganglion and hind paw using immunohistochemistry and Western blot. Results Phosphorylated ERK1/2 was induced in dorsal root ganglion (26.8 ± 2.9% at baseline, 65.6 ± 3.6% at 2 min, and 26.3 ± 3.4% at 2 h) after the incision. NF-200 positive A-fiber neurons and satellite glial cells were positive for phosphorylated ERK1/2. Injury-induced pain hypersensitivity was abolished by MEK inhibitor. Levobupivacaine treatment inhibited phosphorylated ERK1/2 induction, carbenoxolone treatment inhibited glial phosphorylated ERK1/2 at 2 min after the injury, and carbenoxolone inhibited pain hypersensitivity and neuronal phosphorylated ERK1/2 at 1 h after the injury. Conclusion ERK1/2 phosphorylation in A-fiber neurons and satellite glial cells immediately after injury contributes to the generation of pain hypersensitivity. Signal communication between neurons and satellite glial cells expands the duration of neuronal ERK1/2 phosphorylation and pain hypersensitivity at 1 h after tissue injury.

STUDY DESIGN Controlled, interventional, animal study. OBJECTIVE To observe the reaction of glial cells and endoneurial macrophages in the dorsal root ganglion (DRG) after application of nucleus pulposus (NP) and investigate whether activated DRG glial cells play a role in the pathogenesis of neuropathic pain. SUMMARY OF BACKGROUND DATA Peripheral nerve injury activated DRG and spinal cord glial cells and several cytokines and neurotrophins released from these activated glial cells might induce pain hypersensitivity. METHODS Adult male Sprague-Dawley rats were used. NP harvested from the tail was applied to the left L5 DRG. Behavioral testing was performed to investigate the mechanical withdrawal threshold. The numbers of activated satellite glial cells and endoneurial macrophages were counted, and the expressions of tumor necrosis factor-alpha (TNF-alpha) and glial cell-line derived neurotrophic factor (GDNF) were examined by double-labeled immunohistochemistry and immunoblotting. RESULTS The mechanical withdrawal threshold was significantly decreased for 28 days and then gradually recovered (P < 0.05). Long-term activation of endoneurial macrophages and satellite glial cells in the DRG was observed, and the reactions of these cells correlated well with pain-related behavior. TNF-alpha was expressed in both endoneurial macrophages and activated satellite glial cells, and TNF-alpha expression was significantly increased in the early stage (P < 0.05). Activated satellite glial cells also expressed GDNF, and its expression was significantly increased and persisted for 28 days (P < 0.05). CONCLUSION Activation of DRG glial cells and endoneurial macrophages plays an important role in the pathogenesis of the neuropathic pain state. TNF-alpha actively released from activated glial cells and endoneurial macrophages in the DRG might initiate and maintain the neuropathic pain together with TNF-alpha derived from the applied NP. In the recovery phase, persistent expression of GDNF from activated satellite glial cells might play an important role to restore the function of damaged neurons and recover from neuropathic pain.

During the development of peripheral ganglia, 50% of the neurons that are generated undergo apoptosis. How the massive numbers of corpses are removed is unknown. We found that satellite glial cell precursors are the primary phagocytic cells for apoptotic corpse removal in developing mouse dorsal root ganglia (DRG). Confocal and electron microscopic analysis revealed that glial precursors, rather than macrophages, were responsible for clearing most of the dead DRG neurons. Moreover, we identified Jedi-1, an engulfment receptor, and MEGF10, a purported engulfment receptor, as homologs of the invertebrate engulfment receptors Draper and CED-1 expressed in the glial precursor cells. Expression of Jedi-1 or MEGF10 in fibroblasts facilitated binding to dead neurons, and knocking down either protein in glial cells or overexpressing truncated forms lacking the intracellular domain inhibited engulfment of apoptotic neurons. Together, these results suggest a cellular and molecular mechanism by which neuronal corpses are culled during DRG development.

Background: The effect of photodynamic therapy (PDT) on neural cells is important when tumours are within or adjacent to the nervous system. The purpose of this study was to investigate PDT using the photosensitiser, meta-tetrahydroxyphenyl chlorin (mTHPC), on rat neurons and satellite glia, compared with human adenocarcinoma cells (MCF-7). Methods: Fluorescence microscopy confirmed that mTHPC was incorporated into all three cell types. Sensitivity of cells exposed to mTHPC-PDT (0–10 μg ml–1) was determined in a novel 3-dimensional collagen gel culture system. Cell death was quantified using propidium iodide and cell types were distinguished using immunocytochemistry. In some cases, neuron survival was confirmed by measuring subsequent neurite growth in monolayer culture. Results: MCF-7s and satellite glia were significantly more sensitive to PDT than neurons. Importantly, 4 μg ml–1 mTHPC-PDT caused no significant neuron death compared with untreated controls but was sufficient to elicit substantial cell death in the other cell types. Initially, treatment reduced neurite length; neurons then extended neurites equivalent to those of untreated controls. The protocol was validated using hypericin (0–3 μg ml–1), which caused neuron death equivalent to other cell types. Conclusion: Neurons in culture can survive mTHPC-PDT under conditions sufficient to kill tumour cells and other nervous system cells.

Satellite glial cells in the dorsal root ganglion (DRG), like the better-studied glia cells in the spinal cord, react to peripheral nerve injury or inflammation by activation, proliferation, and release of messengers that contribute importantly to pathological pain. It is not known how information about nerve injury or peripheral inflammation is conveyed to the satellite glial cells. Abnormal spontaneous activity of sensory neurons, observed in the very early phase of many pain models, is one plausible mechanism by which injured sensory neurons could activate neighboring satellite glial cells. We tested effects of locally inhibiting sensory neuron activity with sodium channel blockers on satellite glial cell activation in a rat spinal nerve ligation (SNL) model. SNL caused extensive satellite glial cell activation (as defined by glial fibrillary acidic protein [GFAP] immunoreactivity) which peaked on day 1 and was still observed on day 10. Perfusion of the axotomized DRG with the Na channel blocker tetrodotoxin (TTX) significantly reduced this activation at all time points. Similar findings were made with a more distal injury (spared nerve injury model), using a different sodium channel blocker (bupivacaine depot) at the injury site. Local DRG perfusion with TTX also reduced levels of nerve growth factor (NGF) in the SNL model on day 3 (when activated glia are an important source of NGF), without affecting the initial drop of NGF on day 1 (which has been attributed to loss of transport from target tissues). Local perfusion in the SNL model also significantly reduced microglia activation (OX-42 immunoreactivity) on day 3 and astrocyte activation (GFAP immunoreactivity) on day 10 in the corresponding dorsal spinal cord. The results indicate that early spontaneous activity in injured sensory neurons may play important roles in glia activation and pathological pain.

Migraine patients often report sinus pressure or pain and nasal congestion during severe migraine attacks and it has been suggested that sinus pathology can act as a trigger of migraine.1,2 Furthermore, patients suffering from acute allergic rhinitis experience headache and migraine at a much higher frequency than nonallergic subjects.3 While migraine and rhinosinusitis exhibit considerable comorbidity, the underlying cellular mechanisms are not well understood. However, it is well established that activation of trigeminal nerves and the peripheral and central release of neuropeptides are involved in mediating the inflammatory and nociceptive events characteristic of both migraine and rhinosinusitis.4–6 The trigeminal nerve consists of 3 major branches referred to as the ophthalmic (V1), maxillary (V2), and mandibular (V3) branches. Each branch provides somatosensory innervation of distinct regions of the head, face, nasal, and sinus cavities.7 Primary afferent neurons, whose cell bodies reside in the trigeminal ganglion, convey sensory information from peripheral tissues implicated in migraine and rhinosinusitis to the central nervous system (CNS). The pathophysiological events involved in migraine and rhinosinusitis involve both peripheral and central sensitization.4,8–10 Peripheral sensitization, which is the result of increased activity of trigeminal nociceptors, is thought to play a key role in the initiation of migraine and rhinosinusitis, while central sensitization, which involves enhanced excitability of second-order neurons, leads to pain.11 Peripheral sensitization is characterized by increased neuronal excitability and a lowering of the threshold for activation. In this context, activation is defined as causing changes in the cell that allow it to perform functions beyond those present in a basal state.12 It is now thought that glia cells that are closely associated with peripheral and central neurons can directly modulate the functional and excitability state of these neurons.12,13 Furthermore, neuronglia interactions are reported to be involved in all stages of inflammation and pain associated with several CNS diseases.14,15 Within the trigeminal ganglion, the cell bodies of neurons are completely surrounded by specialized glial cells known as satellite glia that together form distinct, functional units.13 Morphological studies have provided evidence that neurons and satellite glial cells extend processes that are thought to facilitate exchange of chemicals between neurons and glia.16,17 In addition, it was recently shown that trigeminal ganglion neurons and satellite glial cells can communicate directly via gap junctions.18 Gap junctions serve as intercellular conduits that allow for direct transfer of small molecular weight molecules, such as ions, that regulate cellular excitability, metabolic precursors, and second messengers.19,20 Gap junctions are found in most neurons and glial cells and function to facilitate neuron-neuron, glia-glia, and neuron-glia communication. Within the CNS, gap junctions are abundant and allow for extensive intercellular coupling between cells that form a communication network.19,21 Each cell contributes a hemichannel composed of 6 transmembrane proteins known as connexins. The connexin family includes more than 20 members.22 However, only 10 connexin proteins are known to be expressed by neuronal or glial cells.21 Connexins are dynamic membrane proteins that exhibit short half-lives.23 Changes in the expression of connexins and hence, communication through gap junctions, are associated with numerous CNS diseases including Alzheimer’s disease, as well as cortical spreading depression.19 Similarly, we have recently provided evidence of enhanced neuron to satellite glia communication occurring through gap junctions within trigeminal ganglion in response to inflammatory stimuli.18 The expression of connexin proteins involved in forming gap junctions between neuronal and satellite glial cells within the trigeminal ganglion under normal and disease states is not known. In addition, we have observed cross activation within the ganglion by which stimulation of neurons in one branch caused a rapid and sustained activation in the other branches, an example of intraganglionic communication.18 Based on our previous findings, we propose that neuronal-satellite glial cell signaling is involved in initiating and maintaining peripheral sensitization within the ganglion and, thus, contributes to the significant comorbidity reported for migraine, acute sinusitis, and allergic rhinitis. In this study, we used an in vivo animal model to test whether treatment of V2 neurons by tumor necrosis factor-alpha (TNF-α), a cytokine whose levels are elevated in nasal secretions during allergic rhinitis, can reduce the amount of stimulus required for cellular changes in neurons located in the V1 region, and thus act as a potential trigger. Increased neuron-satellite glia communication via gap junctions, as well as increased levels of connexin 26 and active p38, was observed in neurons and glia located in both V1 and V2 regions in response to cotreatment with TNF-α and capsaicin. Another significant finding from our study was that pretreatment with the anti-migraine drug tonabersat decreased gap junction communication and the level of connexin 26, and blocked p38 activation in both neurons and satellite glia.