Targeting Chondroitin Sulfate Reduces Invasiveness of Glioma Cells by Suppressing CD44 and Integrin ß1 Expression.

Chondroitin sulfate (CS) is a major component of the extracellular matrix found to be abnormally accumulated in several types of cancer tissues. Previous studies have indicated that CS synthases and modification enzymes are frequently elevated in human gliomas and are associated with poor prognosis. However, the underlying mechanisms of CS in cancer progression and approaches for interrupting its functions in cancer cells remain largely unexplored. Here, we have found that CS was significantly enriched surrounding the vasculature in a subset of glioma tissues, which was akin to the perivascular niche for cancer-initiating cells. Silencing or overexpression of the major CS synthase, chondroitin sulfate synthase 1 (CHSY1), significantly regulated the glioma cell invasive phenotypes and modulated integrin expression. Furthermore, we identified CD44 as a crucial chondroitin sulfate proteoglycan (CSPG) that was modified by CHSY1 on glioma cells, and the suppression of CS formation on CD44 by silencing the CHSY1-inhibited interaction between CD44 and integrin ß1 on the adhesion complex. Moreover, we tested the CS-specific binding peptide, resulting in the suppression of glioma cell mobility in a fashion similar to that observed upon the silencing of CHSY1. In addition, the peptide demonstrated significant affinity to CD44, promoted CD44 degradation, and suppressed integrin ß1 expression in glioma cells. Overall, this study proposes a potential regulatory loop between CS, CD44, and integrin ß1 in glioma cells, and highlights the importance of CS in CD44 stability. Furthermore, the targeting of CS by specific binding peptides has potential as a novel therapeutic strategy for glioma.

Melatonin Successfully Rescues the Hippocampal Molecular Machinery and Enhances Anti-oxidative Activity Following Early-Life Sleep Deprivation Injury.

Early-life sleep deprivation (ESD) is a serious condition with severe cognitive sequelae. Considering hippocampus plays an essential role in cognitive regulation, the present study aims to determine whether melatonin, a neuroendocrine beard with significant anti-oxidative activity, would greatly depress the hippocampal oxidative stress, improves the molecular machinery, and consequently exerts the neuro-protective effects following ESD. Male weanling Wistar rats (postnatal day 21) were subjected to ESD for three weeks. During this period, the animals were administered normal saline or melatonin (10 mg/kg) via intraperitoneal injection between 09:00 and 09:30 daily. After three cycles of ESD, the animals were kept under normal sleep/wake cycle until they reached adulthood and were sacrificed. The results indicated that ESD causes long-term effects, such as impairment of ionic distribution, interruption of the expressions of neurotransmitters and receptors, decreases in the levels of several antioxidant enzymes, and impairment of several signaling pathways, which contribute to neuronal death in hippocampal regions. Melatonin administration during ESD prevented these effects. Quantitative evaluation of cells also revealed a higher number of neurons in the melatonin-treated animals when compared with the saline-treated animals. As the hippocampus is critical to cognitive activity, preserving or even improving the hippocampal molecular machinery by melatonin during ESD not only helps us to better understand the underlying mechanisms of ESD-induced neuronal dysfunction, but also the therapeutic use of melatonin to counteract ESD-induced neuronal deficiency.

CHPF promotes malignancy of breast cancer cells by modifying syndecan-4 and the tumor microenvironment.

Breast cancer is the leading cause of cancer-related deaths in women worldwide. Several studies have indicated that abnormal chondroitin sulfate (CS) chains accumulate in breast cancer tissues; however, the functions and dysregulation of CS synthases are largely unknown. Here, we demonstrate that chondroitin polymerising factor (CHPF) is frequently upregulated in breast cancer tissues and that its high expression is positively associated with tumor metastasis, high stages, and short survival time. CHPF modulates CS formation in breast cancer cells. Additionally, we found that CHPF promotes tumor growth and metastasis accompanied by an increase in G-CSF levels and the number of myeloid-derived suppressor cells in tumor tissue. We revealed that tumor cell-derived G-CSF is co-localised with CS on the cell surface. Interestingly, our study is the first to identify that syndecan-4 (SDC4) is modified by CHPF and that it is involved in CHPF-mediated phenotypes. Moreover, breast cancer patients with high expression of both SDC4 and CHPF had worse overall survival compared to other subsets, which implied the synergistic effects of these two genes. In summary, our results indicated that the upregulation of CHPF in breast cancer contributes to the malignant behaviour of cancer cells, thereby providing novel insights on the significance of CHPF-modified SDC4 in breast cancer pathogenesis.

CHPF Regulates the Aggressive Phenotypes of Hepatocellular Carcinoma Cells via the Modulation of the Decorin and TGF-ß Pathways.

Aberrant composition of glycans in the tumor microenvironment (TME) and abnormal expression of extracellular matrix proteins are hallmarks of hepatocellular carcinoma (HCC); however, the mechanisms responsible for establishing the TME remain unclear. We demonstrate that the chondroitin polymerizing factor (CHPF), an enzyme that mediates the elongation of chondroitin sulfate (CS), is a critical elicitor of the malignant characteristics of HCC as it modifies the potent tumor suppressor, decorin (DCN). CHPF expression is frequently downregulated in HCC tumors, which is associated with the poor overall survival of HCC patients. We observed that restoring CHPF expression suppressed HCC cell growth, migration, and invasion in vitro and in vivo. Mechanistic investigations revealed that TGF-ß signaling is associated with CHPF-induced phenotype changes. We found that DCN, as a TGF-ß regulator, is modified by CHPF, and that it affects the distribution of DCN on the surface of HCC cells. Importantly, our results confirm that CHPF and DCN expression levels are positively correlated in primary HCC tissues. Taken together, our results suggest that CHPF dysregulation contributes to the malignancy of HCC cells, and our study provides novel insights into the significance of CS, which affects DCN expression in the TME.

Cross-Talk of Toll-Like Receptor 5 and Mu-Opioid Receptor Attenuates Chronic Constriction Injury-Induced Mechanical Hyperalgesia through a Protein Kinase C Alpha-Dependent Signaling.

Recently, Toll-like receptors (TLRs), a family of pattern recognition receptors, are reported as potential modulators for neuropathic pain; however, the desired mechanism is still unexplained. Here, we operated on the sciatic nerve to establish a pre-clinical rodent model of chronic constriction injury (CCI) in Sprague-Dawley rats, which were assigned into CCI and Decompression groups randomly. In Decompression group, the rats were performed with nerve decompression at post-operative week 4. Mechanical hyperalgesia and mechanical allodynia were obviously attenuated after a month. Toll-like receptor 5 (TLR5)-immunoreactive (ir) expression increased in dorsal horn, particularly in the inner part of lamina II. Additionally, substance P (SP) and isolectin B4 (IB4)-ir expressions, rather than calcitonin-gene-related peptide (CGRP)-ir expression, increased in their distinct laminae. Double immunofluorescence proved that increased TLR5-ir expression was co-expressed mainly with IB4-ir expression. Through an intrathecal administration with FLA-ST Ultrapure (a TLR5 agonist, purified flagellin from Salmonella Typhimurium, only the CCI-induced mechanical hyperalgesia was attenuated dose-dependently. Moreover, we confirmed that mu-opioid receptor (MOR) and phospho-protein kinase Cα (pPKCα)-ir expressions but not phospho-protein kinase A RII (pPKA RII)-ir expression, increased in lamina II, where they mostly co-expressed with IB4-ir expression. Go 6976, a potent protein kinase C inhibitor, effectively reversed the FLA-ST Ultrapure- or DAMGO-mediated attenuated trend towards mechanical hyperalgesia by an intrathecal administration in CCI rats. In summary, our current findings suggest that nerve decompression improves CCI-induced mechanical hyperalgesia that might be through the cross-talk of TLR5 and MOR in a PKCα-dependent manner, which opens a novel opportunity for the development of analgesic therapeutics in neuropathic pain.

Peripheral Neuropathic Pain: From Experimental Models to Potential Therapeutic Targets in Dorsal Root Ganglion Neurons.

Neuropathic pain exerts a global burden caused by the lesions in the somatosensory nerve system, including the central and peripheral nervous systems. The mechanisms of nerve injury-induced neuropathic pain involve multiple mechanisms, various signaling pathways, and molecules. Currently, poor efficacy is the major limitation of medications for treating neuropathic pain. Thus, understanding the detailed molecular mechanisms should shed light on the development of new therapeutic strategies for neuropathic pain. Several well-established in vivo pain models were used to investigate the detail mechanisms of peripheral neuropathic pain. Molecular mediators of pain are regulated differentially in various forms of neuropathic pain models; these regulators include purinergic receptors, transient receptor potential receptor channels, and voltage-gated sodium and calcium channels. Meanwhile, post-translational modification and transcriptional regulation are also altered in these pain models and have been reported to mediate several pain related molecules. In this review, we focus on molecular mechanisms and mediators of neuropathic pain with their corresponding transcriptional regulation and post-translational modification underlying peripheral sensitization in the dorsal root ganglia. Taken together, these molecular mediators and their modification and regulations provide excellent targets for neuropathic pain treatment.

Effects of Rutin on Wound Healing in Hyperglycemic Rats.

Long-term poor glycemic control negatively affects macrovascular and microvascular diseases, as well as wound restoration. Buckwheat is a good source of rutin (quercetin-3-O-rutoside) and has benefits in regulating blood sugar. This study was to evaluate the antioxidant and anti-inflammatory effects of rutin on wound healing in streptozotocin-induced hyperglycemic rats. Eighteen male Wistar rats were randomly divided into three groups: normal (NDM), hyperglycemic (DM), and hyperglycemic with rutin (DMR). After induction of hyperglycemia for 2 days, a 15 × 15 mm wound was induced on the back of each rat. Intraperitoneal injection of rutin significantly ameliorated diabetes-induced body weight loss and improved metabolic dysfunctions of hyperglycemic rats. Based on appearance and histopathological staining, rutin promotes wound healing and inhibits production of inflammatory cells. The immunoblotting data indicated that rutin promotes production of antioxidant enzymes induced by nuclear factor erythroid 2-related factor 2 (NRF2), inhibits the expression of matrix metalloproteinases (MMPs) regulated by NF-κB, and decreases the expression of vascular endothelial growth factor (VEGF). It also promotes the expression of neurogenic-related protein (UCH-L1). The aforementioned results indicated that rutin reduces oxidative stress and inflammatory response in hyperglycemic rats, promoting wound healing and subsequently reducing the risk of wound ulcers.

Chondroitin sulfate synthase 1 enhances proliferation of glioblastoma by modulating PDGFRA stability.

Chondroitin sulfate synthases, a family of enzyme involved in chondroitin sulfate (CS) polymerization, are dysregulated in various human malignancies, but their roles in glioma remain unclear. We performed database analysis and immunohistochemistry on human glioma tissue, to demonstrate that the expression of CHSY1 was frequently upregulated in glioma, and that it was associated with adverse clinicopathologic features, including high tumor grade and poor survival. Using a chondroitin sulfate-specific antibody, we showed that the expression of CHSY1 was significantly associated with CS formation in glioma tissue and cells. In addition, overexpression of CHSY1 in glioma cells enhanced cell viability and orthotopic tumor growth, whereas CHSY1 silencing suppressed malignant growth. Mechanistic investigations revealed that CHSY1 selectively regulates PDGFRA activation and PDGF-induced signaling in glioma cells by stabilizing PDGFRA protein levels. Inhibiting PDGFR activity with crenolanib decreased CHSY1-induced malignant characteristics of GL261 cells and prolonged survival in an orthotopic mouse model of glioma, which underlines the critical role of PDGFRA in mediating the effects of CHSY1. Taken together, these results provide information on CHSY1 expression and its role in glioma progression, and highlight novel insights into the significance of CHSY1 in PDGFRA signaling. Thus, our findings point to new molecular targets for glioma treatment.

Melatonin Promotes Nerve Regeneration Following End-to-Side Neurorrhaphy by Accelerating Cytoskeletal Remodeling via the Melatonin Receptor-dependent Pathway.

Acceleration of cytoskeletal remodeling in regenerated axons is crucial for a fully functional recovery following peripheral nerve injury (PNI). Melatonin plays important roles in cell differentiation and protection of cytoskeleton stability, thus, the present study aimed to investigate whether melatonin can enhance neurite outgrowth and promote cytoskeletal remodeling in a PNI animal model and in differentiated neurons. End-to-side neurorrhaphy (ESN) rat model was used for assessing cytoskeletal rearrangement in regenerated axon. Subject rats received 1 mg/kg/day melatonin injection for one month. The amplitude of compound muscle action potentials and the number of re-innervated motor end plates on target muscles were assessed to represent the functional recovery after ESN. Melatonin treatment enhanced functional recovery after ESN, compared to the saline treated group. Additionally, in spinal cord and peripheral nerve tissue, animals receiving melatonin displayed enhanced expression of GAP43 and ß3-tubulin one month after ESN, and an increased number of re-innervated motor end plates on their target muscle. In vitro analysis revealed that melatonin treatment significantly promoted neurite outgrowth, and increased expression of melatonin receptors as well as ß3-tubulin in mouse neuroblastoma Neuro-2a (N2a) cells. Treatment with a melatonin receptor antagonist, luzindole, significantly suppressed melatonin receptors and ß3-tubulin expression. Importantly, we found that melatonin treatment suppressed activation of calmodulin-dependent protein kinase II (CaMKII) in vitro and in vivo, suggesting that the ß3-tubulin remodeling may occur via CaMKII-mediated Ca2+ signaling. These results suggested that melatonin may promote functional recovery after PNI by accelerating cytoskeletal remodeling through the melatonin receptor-dependent pathway.

DSE regulates the malignant characters of hepatocellular carcinoma cells by modulating CCL5/CCR1 axis.

Abnormal expression of dermatan sulfate epimerase (DSE) has been found in many types of cancer, while its expression and biological functions in hepatocellular carcinoma (HCC) progression remains obscure. Here we report that DSE, the enzyme that catalyzes the conversion of chondroitin sulfate (CS) to dermatan sulfate (DS), is a critical mediator of malignant character in HCC, through regulation of CCL5 signaling. DSE mRNA and protein were downregulated frequently in HCC tumors, where these events were associated with advanced tumor stages, metastases, and poor survival. DSE-mediated tumor growth was evaluated in immune-deficient and immune-complement mice models. Restoring DSE expression in HCC cells suppressed tumor growth, as well as decreased IL-1ß and CCL5 levels in transplanted tumor tissue. Mechanistic investigations revealed that the expression of DSE altered CCL5 signaling and cell surface binding in HCC cells. Accordingly, DSE suppressed CCL5-induced cell growth, migration, and invasion, whereas silencing of DSE enhanced CCL5-triggered malignant phenotypes. Inhibiting CCR1 activity with BX471 decreased CCL5-induced malignant characters caused by siRNA-mediated knockdown of DSE in HCC cells, establishing the critical role of the CCL5/CCR1 axis in mediating the effects of DSE expression. Taken together, our results suggest that DSE dysregulation contributes to the malignant behavior of HCC cells. This provides novel insight into the significance of DSE in CCL5 signaling and HCC pathogenesis.

Phosphorylation of extracellular signal-regulated kinase 1/2 in subepidermal nerve fibers mediates hyperalgesia following diabetic peripheral neuropathy.

Peripheral neuropathy, a chronic complication of diabetes mellitus (DM), is often accompanied by the onset of severe pain symptoms that affect quality of life. However, the underlying mechanisms remain elusive. In the present study, we used Sprague-Dawley rats to establish a rodent model of the human type 1 DM by a single intraperitoneal (i.p.) injection with streptozotocin (STZ) (60 mg/kg). Hypersensitivity, including hyperalgesia and allodynia, developed in the STZ-induced diabetic rats. Cutaneous innervation exhibited STZ-induced reductions of protein gene product 9.5-, peripherin-, and neurofilament 200-immunoreactivity (IR) subepidermal nerve fibers (SENFs). Moreover, the decreases of substance P (SP)- and calcitonin gene-related peptide (CGRP)-IR SENFs were distinct gathered from the results of extracellular signal-regulated kinase 1 and 2 (ERK1/2)- and phosphorylated ERK1/2 (pERK1/2)-IR SENFs in STZ-induced diabetic rats. Double immunofluorescence studies demonstrated that STZ-induced pERK1/2-IR was largely increased in SENFs where only a small portion was colocalized with SP- or CGRP-IR. By an intraplantar (i. pl.) injection with a MEK inhibitor, U0126 (1,4-Diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene), hyperalgesia was attenuated in a dose-responsive manner. Botulinum toxin serotype A had dose-dependent analgesic effects on STZ-induced hyperalgesia and allodynia, which exhibited equivalent results as the efficacy of transient receptor potential vanilloid (TRPV) channel antagonists. Morphological evidence further confirmed that STZ-induced SP-, CGRP- and pERK1/2-IR were reduced in SENFs after pharmacological interventions. From the results obtained in this study, it is suggested that increases of pERK1/2 in SENFs may participate in the modulation of TRPV channel-mediated neurogenic inflammation that triggers hyperalgesia in STZ-induced diabetic rats. Therefore, ERK1/2 provides a potential therapeutic target and efficient pharmacological strategies to address hyperglycemia-induced neurotoxicity.

DSE promotes aggressive glioma cell phenotypes by enhancing HB-EGF/ErbB signaling.

Remodeling of the extracellular matrix (ECM) in the tumor microenvironment promotes glioma progression. Chondroitin sulfate (CS) proteoglycans appear in the ECM and on the cell surface, and can be catalyzed by dermatan sulfate epimerase to form chondroitin sulfate/dermatan sulfate (CS/DS) hybrid chains. Dermatan sulfate epimerase 1 (DSE) is overexpressed in many types of cancer, and CS/DS chains mediate several growth factor signals. However, the role of DSE in gliomas has never been explored. In the present study, we determined the expression of DSE in gliomas by consulting a public database and conducting immunohistochemistry on a tissue array. Our investigation revealed that DSE was upregulated in gliomas compared with normal brain tissue. Furthermore, high DSE expression was associated with advanced tumor grade and poor survival. We found high DSE expression in several glioblastoma cell lines, and DSE expression directly mediated DS chain formation in glioblastoma cells. Knockdown of DSE suppressed the proliferation, migration, and invasion of glioblastoma cells. In contrast, overexpression of DSE in GL261 cells enhanced these malignant phenotypes and in vivo tumor growth. Interestingly, we found that DSE selectively regulated heparin-binding EGF-like growth factor (HB-EGF)-induced signaling in glioblastoma cells. Inhibiting epidermal growth factor receptor (EGFR) and ErbB2 with afatinib suppressed DSE-enhanced malignant phenotypes, establishing the critical role of the ErbB pathway in regulating the effects of DSE expression. This evidence indicates that upregulation of DSE in gliomas contributes to malignant behavior in cancer cells. We provide novel insight into the significance of DS chains in ErbB signaling and glioma pathogenesis.

Nerve Decompression Improves Spinal Synaptic Plasticity of Opioid Receptors for Pain Relief.

Nerve decompression is an essential therapeutic strategy for pain relief clinically; however, its potential mechanism remains poorly understood. Opioid analgesics acting on opioid receptors (OR) within the various regions of the nervous system have been used widely for pain management. We therefore hypothesized that nerve decompression in a neuropathic pain model of chronic constriction injury (CCI) improves the synaptic OR plasticity in the dorsal horn, which is in response to alleviate pain hypersensitivity. After CCI, the Sprague-Dawley rats were assigned into Decompression group, in which the ligatures around the sciatic nerve were removed at post-operative week 4 (POW 4), and a CCI group, in which the ligatures remained. Pain hypersensitivity, including thermal hyperalgesia and mechanical allodynia, was entirely normalized in Decompression group within the following 4 weeks. Substantial reversal of mu- and delta-OR immunoreactive (IR) expressions in Decompression group was detected in primary afferent terminals in the dorsal horn. In Decompression group, mu-OR antagonist (CTOP, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 [Disulfide Bridge: 2-7]) and delta-OR antagonist (NTI, 17-(cyclopropylmethyl)-6,7-dehydro-4,5α-epoxy-3,14-dihydroxy-6,7-2',3'-indolomorphinan hydrochloride) re-induced pain hypersensitivity by intrathecal administration in a dose-responsive manner. Additionally, mu-OR agonist (DAMGO, [D-Ala2, NMe-Phe4, Gly-ol5]-enkephalin) and delta-OR agonist (SNC80, ((+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethyl-benzamide) were administrated intrathecally to attenuating CCI-induced chronic and acute pain hypersensitivity dose-dependently. Our current results strongly suggested that nerve decompression provides the opportunity for improving the synaptic OR plasticity in the dorsal horn and pharmacological blockade presents a novel insight into the therapeutic strategy for pain hypersensitivity.

CHSY1 promotes aggressive phenotypes of hepatocellular carcinoma cells via activation of the hedgehog signaling pathway.

Abnormal expression of chondroitin sulfate has been found in many types of cancer, while its biological functions in hepatocellular carcinoma (HCC) progression remain uninvestigated. Here, we report that chondroitin sulfate synthase 1 (CHSY1), the enzyme that mediates the polymerization step of chondroitin sulfate, is a critical mediator of malignant character in HCC that acts via modulating the activity of the hedgehog signaling. CHSY1 was up-regulated frequently in HCC where these events were associated with worse histologic grade and poor survival. Enforced expression of CHSY1 was sufficient to enhance cell growth, migration, invasion, and epithelial-mesenchymal transition, whereas silencing of CHSY1 suppressed these malignant phenotypes. Mechanistic investigations revealed that the increase of cell surface chondroitin sulfate by CHSY1 promoted sonic hedgehog binding and signaling. Inhibiting hedgehog pathway with vismodegib decreased CHSY1-induced migration, invasion, and lung metastasis of HCC cells, establishing the critical role of hedgehog signaling in mediating the effects of CHSY1 expression. Together, our results indicate that CHSY1 overexpression in HCC contributes to the malignant behaviors in cancer cells, we provide novel insights into the significance of chondroitin sulfate in hedgehog signaling and HCC pathogenesis.

Rearrangement of potassium ions and Kv1.1/Kv1.2 potassium channels in regenerating axons following end-to-end neurorrhaphy: ionic images from TOF-SIMS.

The voltage-gated potassium channels Kv1.1 and Kv1.2 that cluster at juxtaparanodal (JXP) regions are essential in the regulation of nerve excitability and play a critical role in axonal conduction. When demyelination occurs, Kv1.1/Kv1.2 activity increases, suppressing the membrane potential nearly to the equilibrium potential of K+, which results in an axonal conduction blockade. The recovery of K+-dependent communication signals and proper clustering of Kv1.1/Kv1.2 channels at JXP regions may directly reflect nerve regeneration following peripheral nerve injury. However, little is known about potassium channel expression and its relationship with the dynamic potassium ion distribution at the node of Ranvier during the regenerative process of peripheral nerve injury (PNI). In the present study, end-to-end neurorrhaphy (EEN) was performed using an in vivo model of PNI. The distribution of K+ at regenerating axons following EEN was detected by time-of-flight secondary-ion mass spectrometry. The specific localization and expression of Kv1.1/Kv1.2 channels were examined by confocal microscopy and western blotting. Our data showed that the re-establishment of K+ distribution and intensity was correlated with the functional recovery of compound muscle action potential morphology in EEN rats. Furthermore, the re-clustering of Kv1.1/1.2 channels 1 and 3 months after EEN at the nodal region of the regenerating nerve corresponded to changes in the K+ distribution. This study provided direct evidence of K+ distribution in regenerating axons for the first time. We proposed that the Kv1.1/Kv1.2 channels re-clustered at the JXP regions of regenerating axons are essential for modulating the proper patterns of K+ distribution in axons for maintaining membrane potential stability after EEN.

Redistribution of Cav2.1 channels and calcium ions in nerve terminals following end-to-side neurorrhaphy: ionic imaging analysis by TOF-SIMS.

The P/Q-type voltage-dependent calcium channel (Cav2.1) in the presynaptic membranes of motor nerve terminals plays an important role in regulating Ca2+ transport, resulting in transmitter release within the nervous system. The recovery of Ca2+-dependent signal transduction on motor end plates (MEPs) and innervated muscle may directly reflect nerve regeneration following peripheral nerve injury. Although the functional significance of calcium channels and the levels of Ca2+ signalling in nerve regeneration are well documented, little is known about calcium channel expression and its relation with the dynamic Ca2+ ion distribution at regenerating MEPs. In the present study, end-to-side neurorrhaphy (ESN) was performed as an in vivo model of peripheral nerve injury. The distribution of Ca2+ at regenerating MEPs following ESN was first detected by time-of-flight secondary ion mass spectrometry, and the specific localization and expression of Cav2.1 channels were examined by confocal microscopy and western blotting. Compared with other fundamental ions, such as Na+ and K+, dramatic changes in the Ca2+ distribution were detected along with the progression of MEP regeneration. The re-establishment of Ca2+ distribution and intensity were correlated with the functional recovery of muscle in ESN rats. Furthermore, the re-clustering of Cav2.1 channels after ESN at the nerve terminals corresponded with changes in the Ca2+ distribution. These results indicated that renewal of the Cav2.1 distribution within the presynaptic nerve terminals may be necessary for initiating a proper Ca2+ influx and shortening the latency of muscle contraction during nerve regeneration.

Intact subepidermal nerve fibers mediate mechanical hypersensitivity via the activation of protein kinase C gamma in spared nerve injury.

BACKGROUND: Spared nerve injury is an important neuropathic pain model for investigating the role of intact primary afferents in the skin on pain hypersensitivity. However, potential cellular mechanisms remain poorly understood. In phosphoinositide-3 kinase pathway, pyruvate dehydrogenase kinase 1 (PDK1) participates in the regulation of neuronal plasticity for central sensitization. The downstream cascades of PDK1 include: (1) protein kinase C gamma (PKCg) controls the trafficking and phosphorylation of ionotropic glutamate receptor; (2) protein kinase B (Akt)/the mammalian target of rapamycin (mTOR) signaling is responsible for local protein synthesis. Under these statements, we therefore hypothesized that an increase of PKCg activation and mTOR-dependent PKCg synthesis in intact primary afferents after SNI might contribute to pain hypersensitivity. RESULTS: The variants of spared nerve injury were performed in Sprague-Dawley rats by transecting any two of the three branches of the sciatic nerve, leaving only one branch intact. Following SNIt (spared tibial branch), mechanical hyperalgesia and mechanical allodynia, but not thermal hyperalgesia, were significantly induced. In the first footpad, normal epidermal innervations were verified by the protein gene product 9.5 (PGP9.5)- and growth-associated protein 43 (GAP43)-immunoreactive (IR) intraepidermal nerve fibers (IENFs) densities. Furthermore, the rapid increases of phospho-PKCg- and phosphomTOR-IR subepidermal nerve fibers (SENFs) areas were distinct gathered from the results of PGP9.5-, GAP43-, and neurofilament 200 (NF200)-IR SENFs areas. The efficacy of PKC inhibitor (GF 109203X) or mTOR complex 1 inhibitor (rapamycin) for attenuating mechanical hyperalgesia and mechanical allodynia by intraplantar injection was dose-dependent. CONCLUSIONS: From results obtained in this study, we strongly recommend that the intact SENFs persistently increase PKCg activation and mTOR-dependent PKCg synthesis participate in the initiation and maintenance of mechanical hypersensitivity in spared nerve injury, which represents as a novel insight into the therapeutic strategy of pain in the periphery.

Nerve demyelination increases metabotropic glutamate receptor subtype 5 expression in peripheral painful mononeuropathy.

Wallerian degeneration or nerve demyelination, arising from spinal nerve compression, is thought to bring on chronic neuropathic pain. The widely distributed metabotropic glutamate receptor subtype 5 (mGluR5) is involved in modulating nociceptive transmission. The purpose of this study was to investigate the potential effects of mGluR5 on peripheral hypersensitivities after chronic constriction injury (CCI). Sprague-Dawley rats were operated on with four loose ligatures around the sciatic nerve to induce thermal hyperalgesia and mechanical allodynia. Primary afferents in dermis after CCI exhibited progressive decreases, defined as partial cutaneous denervation; importantly, mGluR5 expressions in primary afferents were statistically increased. CCI-induced neuropathic pain behaviors through the intraplantar injections of 2-methyl-6-(phenylethynyl)-pyridine (MPEP), a selective mGluR5 antagonist, were dose-dependently attenuated. Furthermore, the most increased mGluR5 expressions in primary afferents surrounded by reactive Schwann cells were observed at the distal CCI stumps of sciatic nerves. In conclusion, these results suggest that nerve demyelination results in the increases of mGluR5 expression in injured primary afferents after CCI; and further suggest that mGluR5 represents a main therapeutic target in developing pharmacological strategies to prevent peripheral hypersensitivities.

Determinants of nerve conduction recovery after nerve injuries: Compression duration and nerve fiber types.

INTRODUCTION: The aims of this study were to determine the influences of: (1) timing of nerve decompression; and (2) nerve fiber types on the patterns of nerve conduction studies (NCS) after nerve injury. METHODS: Nerve conduction studies (NCS) were performed on 3 models of nerve injury: (1) crush injury due to transient nerve compression (crush group); (2) chronic constriction injury (CCI), or permanent compression (CCI group); and (3) CCI with removal of ligatures, or delayed nerve decompression (De-CCI group). RESULTS: There were distinct patterns of NCS recovery. The crush and De-CCI groups achieved similar motor nerve recovery, better than that of the CCI group. In contrast, recovery of sensory nerves was limited in the CCI and De-CCI groups and was lower than in the crush group. CONCLUSIONS: Immediate relief of compression resulted in the best recovery of motor and sensory nerve conduction. In contrast, delayed decompression restored only motor nerve conduction.

Redistribution of voltage-gated sodium channels after nerve decompression contributes to relieve neuropathic pain in chronic constriction injury.

Nerve decompression is an important therapeutic strategy to relieve neuropathic pain and promote the peripheral nerve regeneration. To address these issues, we investigated the effects of nerve decompression on relief of neuropathic pain behaviors, redistribution of voltage-gated sodium channels (VGSCs), and skin reinnervation with chronic constriction injury (CCI). At post-operative week (POW) 4, animals were divided into a decompression group, in which the ligatures were removed, and a CCI group, in which the ligatures remained. Thermal hyperalgesia and mechanical allodynia at POW 8 had distinct reductions in decompression group compared to CCI group. At that time in CCI group, morphological evidence of pan VGSCs (Pan Nav) and isoforms of VGSCs (Nav1.6, Nav1.9, except for Nav1.8) were shown the widely distribution along the injured sciatic nerve. All of the VGSCs in decompression group became clustering around the node of Ranvier, similar to the pattern of control sciatic nerve at POW 8. Skin reinnervation was demonstrated by epidermal nerve density (END) for protein gene product 9.5 (PGP 9.5)-immunoreactive (IR) nerve fibers and a significant difference between groups only at POW 24 (p=0.01). Growth-associated protein 43 (GAP-43) is participated in the nerve fiber growth and sprouting, a difference in END for GAP-43-IR nerve fibers at POW 24 between groups were also significant (p=0.02). These observations demonstrated that nerve decompression was accompanied with the disappearance of neuropathic pain behaviors after CCI. Morphological studies provided the evidence that redistribution of VGSCs along the injured sciatic nerve but still with an incomplete skin reinnervation. These significant findings demonstrated a role of VGSCs in the pathogenesis of neuropathic pain, and gave an approaching in pharmacological basis of therapeutics.