Proportions of four distinct classes of sensory neurons are retained even when axon regeneration is enhanced following peripheral nerve injury.

Introduction: Recovery from peripheral nerve injuries is poor because axon regeneration is slow and inefficient. Experimental therapies that increase signaling of neuronal brain-derived neurotrophic factor (BDNF) through its TrkB receptor or through its downstream effectors enhance axon regeneration, increasing the number of motor and sensory neurons whose axons successfully regenerate and reinnervate muscle targets. The goal of this study was to compare the proportions of four different classes of sensory (dorsal root ganglion, DRG) neurons that successfully reinnervate two different muscle targets in control mice and mice treated pharmacologically to enhance axon regeneration. Methods: Following sciatic nerve transection and repair, C57BL/6 J mice were treated for 2 weeks, either with R13, a prodrug that releases the small molecule TrkB ligand, 7,8-dihydroxyflavone, with compound 11 (CP11), an inhibitor of asparaginyl endopeptidase (δ-secretase), or with a control vehicle. Four weeks after injury, different fluorescent retrograde tracers were injected into the gastrocnemius and tibialis anterior muscles to mark DRG neurons that had successfully reinnervated these muscles. Using immunofluorescence, retrogradely labeled DRG neurons also expressing markers of four different sensory neuronal classes were counted. Results and discussion: Treatments with R13 or CP11 resulted in muscle reinnervation by many more DRG neurons than vehicletreated controls, but neurons expressing proteins associated with the different classes of DRG neurons studied were largely in the same proportions found in intact mice.

Cardio-omentopexy requires a cardioprotective innate immune response to promote myocardial angiogenesis in mice.

Objective: The pedicled greater omentum, when applied onto stressed hearts using omentopexy, has been shown to be protective in humans and animals. The mechanisms underlying cardioprotection using omentopexy remain elusive. This study examined whether macrophage-mediated angiogenesis accounts for the cardioprotective effect of omentopexy in mice. Methods: C57BL/6 mice were subjected to minimally invasive transverse aortic constriction for 6 weeks and subsequent cardio-omentopexy for 8 weeks. Control mice underwent the same surgical procedures without aortic constriction or cardio-omentopexy. Results: Transverse aortic constriction led to left ventricular concentric hypertrophy, reduced mitral E/A ratio, increased cardiomyocyte size, and myocardial fibrosis in the mice that underwent sham cardio-omentopexy surgery. The negative effects of transverse aortic constriction were prevented by cardio-omentopexy. Myocardial microvessel density was elevated in the mice that underwent aortic constriction and sham cardio-omentopexy surgery, and cardio-omentopexy further enhanced angiogenesis. Nanostring gene array analysis uncovered the activation of angiogenesis gene networks by cardio-omentopexy. Flow cytometric analysis revealed that cardio-omentopexy triggered the accumulation of cardiac MHCIIloLyve1+TimD4+ (Major histocompatibility complex class IIlow lymphatic vessel endothelial hyaluronan receptor 1+ T cell immunoglobulin and mucin domain conataining 4+) resident macrophages at the omental-cardiac interface. Intriguingly, the depletion of macrophages with clodronate-liposome resulted in the failure of cardio-omentopexy to protect the heart and promote angiogenesis. Conclusions: Cardio-omentopexy protects the heart from pressure overload-elicited left ventricular hypertrophy and dysfunction by promoting myocardial angiogenesis. Cardiac MHCIIloLyve1+TimD4+ resident macrophages play a critical role in the cardioprotective effect and angiogenesis of cardio-omentopexy.

Chronic Pain Causes Peripheral and Central Responses in MIA-Induced TMJOA Rats.

Chronic pain is the predominant symptom that drives temporomandibular joint osteoarthritis (TMJOA) patients to seek medical care; however, currently used treatment modalities remain less effective. This study aimed to investigate chronic pain and the peripheral and central responses in monoiodoacetate (MIA)-induced TMJOA rats. First, the appropriate dose of MIA was determined based on pain behavior assessment in rats. Alterations of the condylar structure in TMJOA rats were evaluated by histological staining and micro-computed tomography (micro-CT). Second, the period of TMJOA chronic pain was further explored by assessing the numbers of glial fibrillary acidic protein (GFAP)-positive astrocytes and ionized calcium-binding adaptor molecule 1 (IBA-1)-positive microglia in the trigeminal spinal nucleus (TSN) and performing nonsteroidal anti-inflammatory drug (NSAID) efficacy experiments. Finally, the expression of neurofilament 200 (NF200), calcitonin gene-related peptide (CGRP), and isolectin B4 (IB4) in the trigeminal ganglion (TG) and TSN was assessed by immunofluorescence. MIA at 4 mg/kg was considered an appropriate dose. Gradual MIA-induced alterations of the condylar structure were correlated with temporomandibular joint (TMJ) pain. The numbers of GFAP- and IBA-1-positive cells were increased at 2, 3, and 4 weeks after MIA injection. NSAIDs failed to alleviate pain behavior 10 days after MIA injection. CGRP and IB4 levels in the TG and TSN were upregulated at 2 and 4 weeks. These results suggest that TMJOA-related chronic pain emerged 2 weeks after MIA injection. CGRP- and IB4-positive afferents in both the peripheral and central nervous systems may be involved in MIA-induced TMJOA-related chronic pain in rats.

Silencing the enhancer of zeste homologue 2, Ezh2, represses axon regeneration of dorsal root ganglion neurons.

Recovery from injury to the peripheral nervous system is different from that of the central nervous system in that it can lead to gene reprogramming that can induce the expression of a series of regeneration-associated genes. This eventually leads to axonal regeneration of injured neurons. Although some regeneration-related genes have been identified, the regulatory network underlying axon regeneration remains largely unknown. To explore the regulator of axon regeneration, we performed RNA sequencing of lumbar L4 and L5 dorsal root ganglion (DRG) neurons at different time points (0, 3, 6, 12 hours, 1, 3 and 7 days) after rat sciatic nerve crush. The isolation of neurons was carried out by laser capture microscopy combined with NeuN immunofluorescence staining. We found 1228 differentially expressed genes in the injured sciatic nerve tissue. The hub genes within these differentially expressed genes include Atf3, Jun, Myc, Ngf, Fgf2, Ezh2, Gfap and Il6. We verified that the expression of the enhancer of zeste homologue 2 gene (Ezh2) was up-regulated in DRG neurons after injury, and this up-regulation differed between large- and small-sized dorsal root ganglion neurons. To investigate whether the up-regulation of Ezh2 impacts axonal regeneration, we silenced Ezh2 with siRNA in cultured DRG neurons and found that the growth of the newborn axons was repressed. In our investigation into the regulatory network of Ezh2 by interpretive phenomenal analysis, we found some regulators of Ezh2 (including Erk, Il6 and Hif1a) and targets (including Atf3, Cdkn1a and Smad1). Our findings suggest that Ezh2, as a nerve regeneration-related gene, participates in the repair of the injured DRG neurons, and knocking down the Ezh2 in vitro inhibits the axonal growth of DRG neurons. All the experimental procedures approved by the Administration Committee of Experimental Animals of Jiangsu Province of China (approval No. S20191201-201) on March 21, 2019.

Sensory-specific peripheral nerve pathology in a rat model of Fabry disease.

Fabry disease (FD) causes life-long pain, the mechanisms of which are unclear. Patients with FD have chronic pain that mirrors symptoms of other painful peripheral neuropathies. However, it is unclear what underlying damage occurs in FD peripheral nerves that may contribute to chronic pain. Here, we characterized myelinated and unmyelinated fiber pathology in peripheral nerves of a rat model of FD. Decreased nerve fiber density and increased nerve fiber pathology were noted in unmyelinated and myelinated fibers from FD rats; both observations were dependent on sampled nerve fiber modality and anatomical location. FD myelinated axons exhibited lipid accumulations that were determined to be the FD-associated lipid globotriaosylceramide (Gb3), and to a lesser extent lysosomes. These findings suggest that axonal Gb3 accumulation may drive peripheral neuron dysfunction and subsequent pain in FD.

Co-localization of nociceptive markers in the lumbar dorsal root ganglion and spinal cord of dromedary camel.

Nociceptive markers in mice have been identified in two distinct peptidergic and nonpeptidergic neurons in the dorsal root ganglion (DRG) and distributed in different laminae of the dorsal horn of the spinal cord. Recently, however, a study in humans showed a significant overlapping in these two populations. In this study, we investigated the distribution of various nociceptive markers in the lumbar DRG and spinal cord of the dromedary camel. Immunohistochemical data showed a remarkable percentage of total neurons in the DRG expressed IB4 binding (54.5%), calcitonin gene-related peptide (CGRP; 49.5%), transient receptor potential vanilloid 1 (TRPV1; 48.2%), and nitric oxide synthase (NOS; 30.6%). The co-localization data showed that 89.6% and 74.0% of CGRP- and TRPV1-labeled neurons, respectively, were IB4 positive. In addition, 61.6% and 84.2% of TRPV1- and NOS-immunoreactive neurons, respectively, were also co-localized with CGRP. The distribution of IB4, CGRP, TRPV1, substance P, and NOS immunoreactivities in the spinal cord were observed in lamina I and outer lamina II (IIo). Quantitative data showed that 82.4% of IB4-positive nerve terminals in laminae I and IIo were co-localized with CGRP, and 86.0% of CGRP-labeled terminals were co-localized with IB4. Similarly, 85.1% of NOS-labeled nerve terminals were co-localized with CGRP. No neuropeptide Y (NPY) or cholecystokinin (CCK) immunoreactivities were detected in the DRG, and no co-localization between IB4, NPY, and CCK were observed in the spinal cord. Our results demonstrate marked convergence of nociceptive markers in the primary afferent neurons in camels, which is similar to humans rather than the mouse. The data also emphasizes the importance of interspecies differences when selecting ideal animal models for studying nociception and treating chronic pain.

Oxaliplatin Depolarizes the IB4- Dorsal Root Ganglion Neurons to Drive the Development of Neuropathic Pain Through TRPM8 in Mice.

Use of chemotherapy drug oxaliplatin is associated with painful peripheral neuropathy that is exacerbated by cold. Remodeling of ion channels including TRP channels in dorsal root ganglion (DRG) neurons contribute to the sensory hypersensitivity following oxaliplatin treatment in animal models. However, it has not been studied if TRP channels and membrane depolarization of DRG neurons serve as the initial ionic/membrane drives (such as within an hour) that contribute to the development of oxaliplatin-induced neuropathic pain. In the current study, we studied in mice (1) in vitro acute effects of oxaliplatin on the membrane excitability of IB4+ and IB4- subpopulations of DRG neurons using a perforated patch clamping, (2) the preventative effects of a membrane-hyperpolarizing drug retigabine on oxaliplatin-induced sensory hypersensitivity, and (3) the preventative effects of TRP channel antagonists on the oxaliplatin-induced membrane hyperexcitability and sensory hypersensitivity. We found (1) IB4+ and IB4- subpopulations of small DRG neurons displayed previously undiscovered, substantially different membrane excitability, (2) oxaliplatin selectively depolarized IB4- DRG neurons, (3) pretreatment of retigabine largely prevented oxaliplatin-induced sensory hypersensitivity, (4) antagonists of TRPA1 and TRPM8 channels prevented oxaliplatin-induced membrane depolarization, and (5) the antagonist of TRPM8 largely prevented oxaliplatin-induced sensory hypersensitivity. These results suggest that oxaliplatin depolarizes IB4- neurons through TRPM8 channels to drive the development of neuropathic pain and targeting the initial drives of TRPM8 and/or membrane depolarization may prevent oxaliplatin-induce neuropathic pain.

Contribution of TRESK two-pore domain potassium channel to bone cancer-induced spontaneous pain and evoked cutaneous pain in rats.

Cancer-associated pain is debilitating. However, the mechanism underlying cancer-induced spontaneous pain and evoked pain remains unclear. Here, using behavioral tests with immunofluorescent staining, overexpression, and knockdown of TRESK methods, we found an extensive distribution of TRESK potassium channel on both CGRP+ and IB4+ nerve fibers in the hindpaw skin, on CGRP+ nerve fibers in the tibial periosteum which lacks IB4+ fibers innervation, and on CGRP+ and IB4+ dorsal root ganglion (DRG) neurons in rats. Moreover, we found a decreased expression of TRESK in the corresponding nerve fibers within the hindpaw skin, the tibial periosteum and the DRG neurons in bone cancer rats. Overexpression of TRESK in DRG neurons attenuated both cancer-induced spontaneous pain (partly reflect skeletal pain) and evoked pain (reflect cutaneous pain) in tumor-bearing rats, in which the relief of evoked pain is time delayed than spontaneous pain. In contrast, knockdown of TRESK in DRG neurons produced both spontaneous pain and evoked pain in naïve rats. These results suggested that the differential distribution and decreased expression of TRESK in the periosteum and skin, which is attributed to the lack of IB4+ fibers innervation within the periosteum of the tibia, probably contribute to the behavioral divergence of cancer-induced spontaneous pain and evoked pain in bone cancer rats. Thus, the assessment of spontaneous pain and evoked pain should be accomplished simultaneously when evaluating the effect of some novel analgesics in animal models. Also, this study provides solid evidence for the role of peripheral TRESK in both cancer-induced spontaneous pain and evoked cutaneous pain.

Neurochemical and Ultrastructural Characterization of Unmyelinated Non-peptidergic C-Nociceptors and C-Low Threshold Mechanoreceptors Projecting to Lamina II of the Mouse Spinal Cord.

C-nociceptors (C-Ncs) and non-nociceptive C-low threshold mechanoreceptors (C-LTMRs) are two subpopulations of small unmyelinated non-peptidergic C-type neurons of the dorsal root ganglia (DRGs) with central projections displaying a specific pattern of termination in the spinal cord dorsal horn. Although these two subpopulations exist in several animals, remarkable neurochemical differences occur between mammals, particularly rat/humans from one side and mouse from the other. Mouse is widely investigated by transcriptomics. Therefore, we here studied the immunocytochemistry of murine C-type DRG neurons and their central terminals in spinal lamina II at light and electron microscopic levels. We used a panel of markers for peptidergic (CGRP), non-peptidergic (IB4), nociceptive (TRPV1), non-nociceptive (VGLUT3) C-type neurons and two strains of transgenic mice: the TAFA4Venus knock-in mouse to localize the TAFA4+ C-LTMRs, and a genetically engineered ginip mouse that allows an inducible and tissue-specific ablation of the DRG neurons expressing GINIP, a key modulator of GABABR-mediated analgesia. We confirmed that IB4 and TAFA4 did not coexist in small non-peptidergic C-type DRG neurons and separately tagged the C-Ncs and the C-LTMRs. We then showed that TRPV1 was expressed in only about 7% of the IB4+ non-peptidergic C-Ncs and their type Ia glomerular terminals within lamina II. Notably, the selective ablation of GINIP did not affect these neurons, whereas it reduced IB4 labeling in the medial part of lamina II and the density of C-LTMRs glomerular terminals to about one half throughout the entire lamina. We discuss the significance of these findings for interspecies differences and functional relevance.

Inhibition of PTEN activity promotes IB4-positive sensory neuronal axon growth.

Traumatic nerve injuries have become a common clinical problem, and axon regeneration is a critical process in the successful functional recovery of the injured nervous system. In this study, we found that peripheral axotomy reduces PTEN expression in adult sensory neurons; however, it did not alter the expression level of PTEN in IB4-positive sensory neurons. Additionally, our results indicate that the artificial inhibition of PTEN markedly promotes adult sensory axon regeneration, including IB4-positive neuronal axon growth. Thus, our results provide strong evidence that PTEN is a prominent repressor of adult sensory axon regeneration, especially in IB4-positive neurons.

CDDO-Me Inhibits Microglial Activation and Monocyte Infiltration by Abrogating NFκB- and p38 MAPK-Mediated Signaling Pathways Following Status Epilepticus.

Following status epilepticus (SE, a prolonged seizure activity), microglial activation, and monocyte infiltration result in the inflammatory responses in the brain that is involved in the epileptogenesis. Therefore, the regulation of microglia/monocyte-mediated neuroinflammation is one of the therapeutic strategies for avoidance of secondary brain injury induced by SE. 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid methyl ester (CDDO-Me; RTA 402) is an activator of nuclear factor-erythroid 2-related factor 2 (Nrf2), which regulates intracellular redox homeostasis. In addition, CDDO-Me has anti-inflammatory properties that suppress microglial proliferation and its activation, although the underlying mechanisms have not been clarified. In the present study, CDDO-Me ameliorated monocyte infiltration without vasogenic edema formation in the frontoparietal cortex (FPC) following SE, accompanied by abrogating monocyte chemotactic protein-1 (MCP-1)/tumor necrosis factor-α (TNF-α) expressions and p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation. Furthermore, CDDO-Me inhibited nuclear factor-κB (NFκB)-S276 phosphorylation and microglial transformation, independent of Nrf2 expression. Similar to CDDO-Me, SN50 (an NFκB inhibitor) mitigated monocyte infiltration by reducing MCP-1 and p38 MAPK phosphorylation in the FPC following SE. Therefore, these findings suggest, for the first time, that CDDO-Me may attenuate microglia/monocyte-mediated neuroinflammation via modulating NFκB- and p38 MAPK-MCP-1 signaling pathways following SE.

A simplified aortic ring assay: A useful ex vivo method to assess biochemical and functional parameters of angiogenesis.

We present a simplified method for conducting aortic ring assays which yields robust sprouting and high reproducibility targeted towards matrix biologists studying angiogenesis and extracellular matrix signaling. Main adjustments from previously established protocols include embedding aortic rings between two layers of 3D type I collagen matrix and supplementing with vascular endothelial media. We also introduce a concise and effective staining protocol for obtaining high-resolution images of intracellular and extracellular matrix proteins along with a more accurate protocol to quantify angiogenesis. Importantly, we present a novel method to perform biochemical analyses of vessel sprouting without contamination from the aortic ring itself. Overall, our refined method enables detection of low abundance and phosphorylated proteins and provides a straightforward ex vivo angiogenic assay that can be easily reproduced by those in the matrix biology field.

Roscovitine Attenuates Microglia Activation and Monocyte Infiltration via p38 MAPK Inhibition in the Rat Frontoparietal Cortex Following Status Epilepticus.

Under physiological conditions, microglia are unique immune cells resident in the brain that is isolated from the systemic immune system by brain-blood barrier. Following status epilepticus (SE, a prolonged seizure activity), microglia are rapidly activated and blood-derived monocytes that infiltrate the brain; therefore, the regulations of microglia activation and monocyte infiltration are one of the primary therapeutic strategies for inhibition of undesirable consequences from SE. Roscovitine, a potent (but not selective) cyclin-dependent kinase 5 (CDK5) inhibitor, has been found to exert anti-inflammatory and microglia-inhibiting actions in several in vivo models, although the underlying mechanisms have not been clarified. In the present study, roscovitine attenuated SE-induces monocyte infiltration without vasogenic edema formation in the frontoparietal cortex (FPC), accompanied by reducing expressions of monocyte chemotactic protein-1 (MCP-1) and lysosome-associated membrane protein 1 (LAMP1) in resident microglia, while it did not affect microglia transformation to amoeboid form. Furthermore, roscovitine ameliorated the up-regulation of p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation, but not nuclear factor-κB-S276 phosphorylation. Similar to roscovitine, SB202190, a p38 MAPK inhibitor, mitigated monocyte infiltration and microglial expressions of MCP-1 and LAMP1 in the FPC following SE. Therefore, these findings suggest for the first time that roscovitine may inhibit SE-induced neuroinflammation via regulating p38 MAPK-mediated microglial responses.

High-Content Imaging of Immunofluorescently Labeled TRPV1-Positive Sensory Neurons.

Studying TRP channel expressing nociceptors requires the identification of the respective subpopulations as well as the quantification of dynamic cellular events. However, the heterogeneity of sensory neurons and associated nonneuronal cells demands the analysis of large numbers of cells to reflect the distribution of entire populations. Here we report a detailed workflow how to apply high-content screening (HCS) microscopy to signaling events in TRPV1-positive neurons as well as an approach to use the selective elimination of TRPV1 positive cells from dissociated rat sensory ganglia as base for transcriptomic analysis of TRPV1-positive cells and/or as control for TRPV1 antibody specificity.

Non-Peptidergic Nociceptive Neurons Are Essential for Mechanical Inflammatory Hypersensitivity in Mice.

Small nerve fibers that bind the isolectin B4 (IB4+ C-fibers) are a subpopulation of primary afferent neurons that are involved in nociceptive sensory transduction and do not express the neuropeptides substance P and calcitonin-gene related peptide (CGRP). Several studies have attempted to elucidate the functional role of IB4+-nociceptors in different models of pain. However, a functional characterization of the non-peptidergic nociceptors in mediating mechanical inflammatory hypersensitivity in mice is still lacking. To this end, in the present study, the neurotoxin IB4-Saporin (IB4-Sap) was employed to ablate non-peptidergic C-fibers. Firstly, we showed that intrathecal (i.t.) administration of IB4-Sap in mice depleted non-peptidergic C-fibers, since it decreased the expression of purinoceptor 3 (P2X3) and transient receptor potential cation channel subfamily V member 1 (TRPV1) in the dorsal root ganglia (DRGs) as well as IB4 labelling in the spinal cord. Non-peptidergic C-fibers depletion did not alter the mechanical nociceptive threshold, but it inhibited the mechanical inflammatory hypersensitivity induced by glial cell-derived neurotrophic factor (GDNF), but not nerve growth factor (NGF). Depletion of non-peptidergic C-fibers abrogated mechanical inflammatory hypersensitivity induced by carrageenan. Finally, it was found that the inflammatory mediators PGE2 and epinephrine produced a mechanical inflammatory hypersensitivity that was also blocked by depletion of non-peptidergic C-fibers. These data suggest that IB4-positive nociceptive nerve fibers are not involved in normal mechanical nociception but are sensitised by inflammatory stimuli and play a crucial role in mediating mechanical inflammatory hypersensitivity.

Non-peptidergic nociceptive neurons are essential for mechanical inflammatory hypersensitivity in mice

Small nerve fibers that bind the isolectin B4 (IB4+ C-fibers) are a subpopulation of primary afferent neurons that are involved in nociceptive sensory transduction and do not express the neuropeptides substance P and calcitonin-gene related peptide (CGRP). Several studies have attempted to elucidate the functional role of IB4+-nociceptors in different models of pain. However, a functional characterization of the non-peptidergic nociceptors in mediating mechanical inflammatory hypersensitivity in mice is still lacking. To this end, in the present study, the neurotoxin IB4-Saporin (IB4-Sap) was employed to ablate non-peptidergic C-fibers. Firstly, we showed that intrathecal (i.t.) administration of IB4-Sap in mice depleted non-peptidergic C-fibers, since it decreased the expression of purinoceptor 3 (P2X3) and transient receptor potential cation channel subfamily V member 1 (TRPV1) in the dorsal root ganglia (DRGs) as well as IB4 labelling in the spinal cord. Non-peptidergic C-fibers depletion did not alter the mechanical nociceptive threshold, but it inhibited the mechanical inflammatory hypersensitivity induced by glial cell-derived neurotrophic factor (GDNF), but not nerve growth factor (NGF). Depletion of non-peptidergic C-fibers abrogated mechanical inflammatory hypersensitivity induced by carrageenan. Finally, it was found that the inflammatory mediators PGE2 and epinephrine produced a mechanical inflammatory hypersensitivity that was also blocked by depletion of non-peptidergic C-fibers. These data suggest that IB4-positive nociceptive nerve fibers are not involved in normal mechanical nociception but are sensitised by inflammatory stimuli and play a crucial role in mediating mechanical inflammatory hypersensitivity.

Medullary neural circuit regeneration after trigeminal nerve transection.

The inferior alveolar nerve (IAN) comprises several types of sensory fibers. To clarify whether each type of primary afferent is regenerated comparably after injury, we developed a model of complete IAN transection (IANX) in mice. A retrograde tracer, fluoro-gold, injected into the mental skin was transferred to the cell bodies of a subset of isolectin B4 (IB4)-binding (non-peptidergic C) or CGRP-positive (peptidergic C) neurons at 2 weeks post-axotomy, indicating that the injured C afferents had regenerated anatomically. IANX led to a decrease of IB4-binding and CGRP immunoreactivity (IR) in the trigeminal ganglion (TG) and within the trigeminal spinal subnucleus caudalis (Vc) (i.e. terminals of the central branch of TG neurons). Two weeks after IANX, the reduction in IB4-binding activity and CGRP expression in the TG recovered to the control level; however, IB4-binding within the Vc did not, suggesting that central branch non-peptidergic neurons remained impaired. Two weeks after IANX, pinching or heat stimulus-induced extracellular signal-regulated kinase phosphorylation (pERK) was restored to the control level, but in the case of pinch stimulation the distribution pattern of pERK-IR cells was altered in the Vc. Taken together, our results support the possibility that peptidergic neurons regenerate more efficiently than non-peptidergic neurons after trigeminal nerve injury.

Involvement of advillin in somatosensory neuron subtype-specific axon regeneration and neuropathic pain.

Advillin is a sensory neuron-specific actin-binding protein expressed at high levels in all types of somatosensory neurons in early development. However, the precise role of advillin in adulthood is largely unknown. Here we reveal advillin expression restricted to isolectin B4-positive (IB4+) neurons in the adult dorsal root ganglia (DRG). Advillin knockout (KO) specifically impaired axonal regeneration in adult IB4+ DRG neurons. During axon regeneration, advillin was expressed at the very tips of filopodia and modulated growth cone formation by interacting with and regulating focal-adhesion-related proteins. The advillin-containing focal-adhesion protein complex was shed from neurite tips during neurite retraction and was detectable in cerebrospinal fluid in experimental autoimmune encephalomyelitis, oxaliplatin-induced peripheral neuropathy, and chronic constriction injury of the sciatic nerve. In addition, advillin KO disturbed experimental autoimmune encephalomyelitis-induced neural plasticity in the spinal-cord dorsal horn and aggravated neuropathic pain. Our study highlights a role for advillin in growth cone formation, axon regeneration, and neuropathic pain associated with IB4+ DRG neurons in adulthood.

Organization of alpha-transducin immunoreactive system in the brain and retina of larval and young adult Sea Lamprey (Petromyzon marinus), and their relationship with other neural systems.

We employed an anti-transducin antibody (Gαt-S), in combination with other markers, to characterize the Gαt-S-immunoreactive (ir) system in the CNS of the sea lamprey, Petromyzon marinus. Gαt-S immunoreactivity was observed in some neuronal populations and numerous fibers distributed throughout the brain. Double Gαt-S- and opsin-ir neurons (putative photoreceptors) are distributed in the hypothalamus (postoptic commissure nucleus, dorsal and ventral hypothalamus) and caudal diencephalon, confirming results of García-Fernández et al. (Cell and Tissue Research, 288, 267-278, 1997). Singly Gαt-S-ir cells were observed in the midbrain and hindbrain, increasing the known populations. Our results reveal for the first time in vertebrates the extensive innervation of many brain regions and the spinal cord by Gαt-S-ir fibers. The Gαt-S innervation of the habenula is very selective, fibers densely innervating the lamprey homologue of the mammalian medial nucleus (Stephenson-Jones et al., Proceedings of the National Academy of Sciences of the United States of America, 109, E164-E173, 2012), but not the lateral nucleus homologue. The lamprey neurohypophysis was not innervated by Gαt-S-ir fibers. We also analyzed by double immunofluorescence the relation of this system with other systems. A dopaminergic marker (TH), serotonin (5-HT) or GABA do not co-localize with Gαt-S-ir neurons although codistribution of fibers was observed. Codistribution of Gαt-S-ir fibers and isolectin-labeled extrabulbar primary olfactory fibers was observed in the striatum and hypothalamus. Neurobiotin retrograde transport from the spinal cord combined with immunofluorescence revealed spinal-projecting Gαt-S-ir reticular neurons in the caudal hindbrain. Present results in an ancient vertebrate reveal for the first time a collection of brain targets of Gαt-S-ir neurons, suggesting they might mediate non-visual modulation by light in many systems.

Mediation of Movement-Induced Breakthrough Cancer Pain by IB4-Binding Nociceptors in Rats.

Cancer-induced bone pain is characterized by moderate to severe ongoing pain that commonly requires the use of opiates. Even when ongoing pain is well controlled, patients can suffer breakthrough pain (BTP), episodic severe pain that "breaks through" the medication. We developed a novel model of cancer-induced BTP using female rats with mammary adenocarcinoma cells sealed within the tibia. We demonstrated previously that rats with bone cancer learn to prefer a context paired with saphenous nerve block to elicit pain relief (i.e., conditioned place preference, CPP), revealing the presence of ongoing pain. Treatment with systemic morphine abolished CPP to saphenous nerve block, demonstrating control of ongoing pain. Here, we show that pairing BTP induced by experimenter-induced movement of the tumor-bearing hindlimb with a context produces conditioned place avoidance (CPA) in rats treated with morphine to control ongoing pain, consistent with clinical observation of BTP. Preventing movement-induced afferent input by saphenous nerve block before, but not after, hindlimb movement blocked movement-induced BTP. Ablation of isolectin B4 (IB4)-binding, but not TRPV1+, sensory afferents eliminated movement-induced BTP, suggesting that input from IB4-binding fibers mediates BTP. Identification of potential molecular targets specific to this population of fibers may allow for the development of peripherally restricted analgesics that control BTP and improve quality of life in patients with skeletal metastases.SIGNIFICANCE STATEMENT We present a novel preclinical measure of movement-induced breakthrough pain (BTP) that is observed in the presence of morphine controlling ongoing pain. Blockade of sensory input before movement prevented BTP, whereas nerve block after movement failed to reverse BTP. These observations indicate that blocking peripheral sensory input may prevent BTP and targeting central sites may be required for pain relief once BTP has been initiated. Preventing sensory input from TRPV1-expressing fibers failed to alter movement-induced BTP. In contrast, preventing sensory input from isolectin B4 (IB4)-binding fibers blocked movement-induced BTP. Therefore, examining molecular targets on this population of nociceptive fibers may prove useful for developing an improved strategy for preventing BTP in cancer patients with skeletal metastases.