Mutant RAS-driven Secretome Causes Skeletal Muscle Defects in Breast Cancer.

Cancer-induced skeletal muscle defects differ in severity between individuals with the same cancer type. Cancer subtype-specific genomic aberrations are suggested to mediate these differences, but experimental validation studies are very limited. We utilized three different breast cancer patient-derived xenograft (PDX) models to correlate cancer subtype with skeletal muscle defects. PDXs were derived from brain metastasis of triple-negative breast cancer (TNBC), estrogen receptor-positive/progesterone receptor-positive (ER+/PR+) primary breast cancer from a BRCA2-mutation carrier, and pleural effusion from an ER+/PR- breast cancer. While impaired skeletal muscle function as measured through rotarod performance and reduced levels of circulating and/or skeletal muscle miR-486 were common across all three PDXs, only TNBC-derived PDX activated phospho-p38 in skeletal muscle. To further extend these results, we generated transformed variants of human primary breast epithelial cells from healthy donors using HRASG12V or PIK3CAH1047R mutant oncogenes. Mutations in RAS oncogene or its modulators are found in approximately 37% of metastatic breast cancers, which is often associated with skeletal muscle defects. Although cells transformed with both oncogenes generated adenocarcinomas in NSG mice, only HRASG12V-derived tumors caused skeletal muscle defects affecting rotarod performance, skeletal muscle contraction force, and miR-486, Pax7, pAKT, and p53 levels in skeletal muscle. Circulating levels of the chemokine CXCL1 were elevated only in animals with tumors containing HRASG12V mutation. Because RAS pathway aberrations are found in 19% of cancers, evaluating skeletal muscle defects in the context of genomic aberrations in cancers, particularly RAS pathway mutations, may accelerate development of therapeutic modalities to overcome cancer-induced systemic effects. SIGNIFICANCE: Mutant RAS- and PIK3CA-driven breast cancers distinctly affect the function of skeletal muscle. Therefore, research and therapeutic targeting of cancer-induced systemic effects need to take aberrant cancer genome into consideration.

A human skeletal muscle stem/myotube model reveals multiple signaling targets of cancer secretome in skeletal muscle.

Skeletal muscle dysfunction or reprogramming due to the effects of the cancer secretome is observed in multiple malignancies. Although mouse models are routinely used to study skeletal muscle defects in cancer, because of species specificity of certain cytokines/chemokines in the secretome, a human model system is required. Here, we establish simplified multiple skeletal muscle stem cell lines (hMuSCs), which can be differentiated into myotubes. Using single nuclei ATAC-seq (snATAC-seq) and RNA-seq (snRNA-seq), we document chromatin accessibility and transcriptomic changes associated with the transition of hMuSCs to myotubes. Cancer secretome accelerated stem to myotube differentiation, altered the alternative splicing machinery and increased inflammatory, glucocorticoid receptor, and wound healing pathways in hMuSCs. Additionally, cancer secretome reduced metabolic and survival pathway associated miR-486, AKT, and p53 signaling in hMuSCs. hMuSCs underwent myotube differentiation when engrafted into NSG mice and thus providing a humanized in vivo skeletal muscle model system to study cancer cachexia.

Corrosion behavior of 316 stainless steel arc parts in liquid lead at 650 °C under high oxygen concentrations.

The corrosion of 316SS in contact with lead complicates the realization of high coolant temperature. To explore the corrosion behavior at high temperature, the corrosion test of 316SS was performed in liquid lead at a high temperature of 650 °C under Ar with oxygen levels of 10-2 wt% and 10-5 wt% by the static corrosion method. The mass changes after corrosion were determined; then, the corrosion depth and the oxide product formed were further characterized. A multi-oxide layer was formed on the 316SS alloy surface, and the thickness reached 17.5 µm over a period of 100 h at the oxygen level of ∼10-2 wt%. Fe oxide was the main product in the outer layer; the dense Fe-Cr oxide was formed in the inner layer and lead was isolated from the metal substrate. When the oxygen content was 10-5 wt%, corrosion by dissolution at a rather high rate was dominant, and the corrosion depth was as high as 50 µm for 100 h. It is speculated that the oxide layer is also formed at the initial stage but gets dissociated when there is no oxygen supply to sustain the oxide layer with prolonged exposure time. The oxygen content in the cover gas greatly influences the corrosion behavior of 316SS, thus directly affecting the application of 316SS immersed in liquid lead at high temperature.

Erratum: Skeletal muscle-specific overexpression of miR-486 limits mammary tumor-induced skeletal muscle functional limitations.

[This corrects the article DOI: 10.1016/j.omtn.2022.03.009.].

Skeletal muscle-specific overexpression of miR-486 limits mammary tumor-induced skeletal muscle functional limitations.

miR-486 is a myogenic microRNA, and its reduced skeletal muscle expression is observed in muscular dystrophy. Transgenic overexpression of miR-486 using muscle creatine kinase promoter (MCK-miR-486) partially rescues muscular dystrophy phenotype. We had previously demonstrated reduced circulating and skeletal muscle miR-486 levels with accompanying skeletal muscle defects in mammary tumor models. To determine whether skeletal muscle miR-486 is functionally similar in dystrophies and cancer, we performed functional limitations and biochemical studies of skeletal muscles of MMTV-Neu mice that mimic HER2+ breast cancer and MMTV-PyMT mice that mimic luminal subtype B breast cancer and these mice crossed to MCK-miR-486 mice. miR-486 significantly prevented tumor-induced reduction in muscle contraction force, grip strength, and rotarod performance in MMTV-Neu mice. In this model, miR-486 reversed cancer-induced skeletal muscle changes, including loss of p53, phospho-AKT, and phospho-laminin alpha 2 (LAMA2) and gain of hnRNPA0 and SRSF10 phosphorylation. LAMA2 is a part of the dystrophin-associated glycoprotein complex, and its loss of function causes congenital muscular dystrophy. Complementing these beneficial effects on muscle, miR-486 indirectly reduced tumor growth and improved survival, which is likely due to systemic effects of miR-486 on production of pro-inflammatory cytokines such as IL-6. Thus, similar to dystrophy, miR-486 has the potential to reverse skeletal muscle defects and cancer burden.

Tumor collection/processing under physioxia uncovers highly relevant signaling networks and drug sensitivity.

Preclinical studies of primary cancer cells are typically done after tumors are removed from patients or animals at ambient atmospheric oxygen (O2, ~21%). However, O2 concentrations in organs are in the ~3 to 10% range, with most tumors in a hypoxic or 1 to 2% O2 environment in vivo. Although effects of O2 tension on tumor cell characteristics in vitro have been studied, these studies are done only after tumors are first collected and processed in ambient air. Similarly, sensitivity of primary cancer cells to anticancer agents is routinely examined at ambient O2. Here, we demonstrate that tumors collected, processed, and propagated at physiologic O2 compared to ambient air display distinct differences in key signaling networks including LGR5/WNT, YAP, and NRF2/KEAP1, nuclear reactive oxygen species, alternative splicing, and sensitivity to targeted therapies. Therefore, evaluating cancer cells under physioxia could more closely recapitulate their physiopathologic status in the in vivo microenvironment.

Disorder of intestinal amino acid pathway in depression-like offspring rats induced by maternal separation.

To investigate the intestinal amino acids pathway in depression-like offspring rats induced by maternal separation. Sprague-Dawley (SD) female rats were randomly divided into a control group (=8) and a maternal separation group (=8). After normal delivery, the maternal rats were separated from offsprings for 14 consecutive days and 3 h per day in maternal separation group; while rats in the control group was received no interventions in postpartum. Depression-like behaviors of offspring rats were evaluated using the sucrose preference test, novelty suppressed feeding test, and forced swimming test. Amino acid analyzer was used to detect the changes of amino acid contents in the small intestine, and the expressions of alanine-serine-cysteine transporter 2 (ASCT2), solute carrier superfamily 6 member 19 (BAT1) and L-type amino acid transporter 1(LAT1) were detected by Western blot. The weight of the offspring rats in the maternal separation group was significantly lower than that of the control group at 21 and 28 d (=4.925 and 5.766, all <0.01). Compared with the control group, the percentage of sucrose preference of the offspring rats in the maternal separation group was significantly reduced (=2.709, <0.05), and the feeding latency was significantly prolonged (=-13.431, <0.01). The immobility time in FST of maternal separation group was significantly longer (=-3.616, <0.01).Increased concentration of aspartic acid (=-6.672, <0.01) and down-regulation of glutamine (=3.107, <0.01) and glycine (=9.781, <0.01) were observed in maternal separation group. Western blot analysis revealed that the protein expressions of ASCT2 (=6.734, <0.01) and BAT1 (=9.015, <0.01) in maternal separation group were reduced, while the expression of LAT1 was increased (=-8.942, <0.01). Maternal separation can induce the depression-like behavior in offspring rats; the amino acid contents and the amino acid transporter expression in the small intestine are reduced, which may be related to depression-like behavior induced by maternal separation.

Hormonally Regulated Myogenic miR-486 Influences Sex-specific Differences in Cancer-induced Skeletal Muscle Defects.

Cancer-induced skeletal muscle defects show sex-specific differences in severity with men performing poorly compared to women. Hormones and sex chromosomal differences are suggested to mediate these differences, but the functional skeletal muscle markers to document these differences are unknown. We show that the myogenic microRNA miR-486 is a marker of sex-specific differences in cancer-induced skeletal muscle defects. Cancer-induced loss of circulating miR-486 was more severe in men with bladder, lung, and pancreatic cancers compared to women with the same cancer types. In a syngeneic model of pancreatic cancer, circulating and skeletal muscle loss of miR-486 was more severe in male mice compared to female mice. Estradiol (E2) and the clinically used selective estrogen receptor modulator toremifene increased miR-486 in undifferentiated and differentiated myoblast cell line C2C12 and E2-inducible expression correlated with direct binding of estrogen receptor alpha (ERα) to the regulatory region of the miR-486 gene. E2 and toremifene reduced the actions of cytokines such as myostatin, transforming growth factor ß, and tumor necrosis factor α, which mediate cancer-induced skeletal muscle wasting. E2- and toremifene-treated C2C12 myoblast/myotube cells contained elevated levels of active protein kinase B (AKT) with a corresponding decrease in the levels of its negative regulator PTEN, which is a target of miR-486. We propose an ERα:E2-miR-486-AKT signaling axis, which reduces the deleterious effects of cancer-induced cytokines/chemokines on skeletal muscle mass and/or function.

Aging-associated skeletal muscle defects in HER2/Neu transgenic mammary tumor model.

BACKGROUND: Loss of skeletal muscle volume and resulting in functional limitations are poor prognostic markers in breast cancer patients. Several molecular defects in skeletal muscle including reduced MyoD levels and increased protein turn over due to enhanced proteosomal activity have been suggested as causes of skeletal muscle loss in cancer patients. However, it is unknown whether molecular defects in skeletal muscle are dependent on tumor etiology. METHODS: We characterized functional and molecular defects of skeletal muscle in MMTV-Neu (Neu+) mice (n= 6-12), an animal model that represents HER2+ human breast cancer, and compared the results with well-characterized luminal B breast cancer model MMTV-PyMT (PyMT+). Functional studies such as grip strength, rotarod performance, and ex vivo muscle contraction were performed to measure the effects of cancer on skeletal muscle. Expression of muscle-enriched genes and microRNAs as well as circulating cytokines/chemokines were measured. Since NF-κB pathway plays a significant role in skeletal muscle defects, the ability of NF-κB inhibitor dimethylaminoparthenolide (DMAPT) to reverse skeletal muscle defects was examined. RESULTS: Neu+ mice showed skeletal muscle defects similar to accelerated aging. Compared to age and sex-matched wild type mice, Neu+ tumor-bearing mice had lower grip strength (202±6.9 vs. 179±6.8 g grip force, p=0.0069) and impaired rotarod performance (108±12.1 vs. 30±3.9 seconds, P<0.0001), which was consistent with reduced muscle contractibility (p<0.0001). Skeletal muscle of Neu+ mice (n=6) contained lower levels of CD82+ (16.2±2.9 vs 9.0±1.6) and CD54+ (3.8±0.5 vs 2.4±0.4) muscle stem and progenitor cells (p<0.05), suggesting impaired capacity of muscle regeneration, which was accompanied by decreased MyoD, p53 and miR-486 expression in muscles (p<0.05). Unlike PyMT+ mice, which showed skeletal muscle mitochondrial defects including reduced mitochondria levels and Pgc1ß, Neu+ mice displayed accelerated aging-associated changes including muscle fiber shrinkage and increased extracellular matrix deposition. Circulating "aging factor" and cachexia and fibromyalgia-associated chemokine Ccl11 was elevated in Neu+ mice (1439.56±514 vs. 1950±345 pg/ml, p<0.05). Treatment of Neu+ mice with DMAPT significantly restored grip strength (205±6 g force), rotarod performance (74±8.5 seconds), reversed molecular alterations associated with skeletal muscle aging, reduced circulating Ccl11 (1083.26 ±478 pg/ml), and improved animal survival. CONCLUSIONS: These results suggest that breast cancer subtype has a specific impact on the type of molecular and structure changes in skeletal muscle, which needs to be taken into consideration while designing therapies to reduce breast cancer-induced skeletal muscle loss and functional limitations.

Systemic Actions of Breast Cancer Facilitate Functional Limitations.

Breast cancer is a disease of a specific organ, but its effects are felt throughout the body. The systemic effects of breast cancer can lead to functional limitations in patients who suffer from muscle weakness, fatigue, pain, fibromyalgia, or many other dysfunctions, which hasten cancer-associated death. Mechanistic studies have identified quite a few molecular defects in skeletal muscles that are associated with functional limitations in breast cancer. These include circulating cytokines such as TNF-α, IL-1, IL-6, and TGF-ß altering the levels or function of myogenic molecules including PAX7, MyoD, and microRNAs through transcriptional regulators such as NF-κB, STAT3, and SMADs. Molecular defects in breast cancer may also include reduced muscle mitochondrial content and increased extracellular matrix deposition leading to energy imbalance and skeletal muscle fibrosis. This review highlights recent evidence that breast cancer-associated molecular defects mechanistically contribute to functional limitations and further provides insights into therapeutic interventions in managing functional limitations, which in turn may help to improve quality of life in breast cancer patients.

[Changes of Wnt/ß-catenin signaling pathway in the hippocampus caused by prenatal stress induce depression- and anxiety-like behaviors in rats].

OBJECTIVE: To explore the pathogenic role of changes of Wnt/ß-catenin signaling pathway in the hippocampus in depression- and anxiety-like behaviors caused by prenatal stress (PS) in offspring rats. METHODS: Twelve female SpragueDawley rats weighing 240-260 g were randomly divided into control and restraint stress groups. The rats in the control group received no interventions, and those in restraint stress group were subjected to restraint stress (three times a day, 45 min each time) at the gestational age of 14-20 days. The 1-month-old offspring rats underwent open field test and forced swimming test to assess the anxiety- and depression-like behaviors, and the expressions of Wnt1, Gsk-3ß and ß-catenin in the hippocampus were detected using Western blotting. RESULTS: In open field test, the offspring rats with PS showed significantly decreased crossings of the center (P < 0.01) with reduced time spent in the center (P < 0.05) compared with control offspring rats. In forced swimming test, the offspring rats in PS group exhibited a significantly longer immobility time than in the control rats, and showed obvious depression- and anxiety-like behaviors. Compared with those in the control offspring rats, Gsk-3ß expression increased significantly while the expressions of ß-catenin and Wnt1 were significantly lowered in the hippocampus of the offspring rats in PS group (P < 0.01). CONCLUSIONS: PS causes changes in Wnt/ß-catenin signaling pathway in the hippocampus to contribute to the occurrence of depression-and anxiety-like behaviors in rats.

Screening of Chemical Dyes in Traditional Chinese Medicine by HPTLC-MS.

It has been uncovered that chemical dyes are illegally used in traditional Chinese medicines to brighten color and cover up inferiority, which threaten the safety of patients. In the present study, an HPTLC-MS method was developed for the effective screening of 11 chemical dyes (Sudan I, II, III, and IV; 808 Scarlet; Sudan Red 7B; malachite green; Basic Orange 2; auramine; Orange II; and erythrosine) in traditional Chinese medicine (TCM) raw materials and Chinese patent medicines. Firstly, unwashed HPTLC plates were chosen by comparing the background signals of the TLC plates used directly and prewashed with analytical grade and HPLC grade solvents. Twice developments were conducted to isolate chemical dyes of different polarity. Possible adulterants were preliminarily identified by comparing Rf values and in situ UV-Vis spectra with those of the references. Further confirmation was conducted by tandem MS analysis via an elution head-based TLC-MS interface. Sudan I and IV, 808 Scarlet, and Orange II were successfully detected in eight batches of TCM. The proposed method could be applied as a reliable technology for the screening of chemical dyes in TCM.

Inflammation-associated microRNA changes in circulating exosomes of heart failure patients.

OBJECTIVE: MiR-486 and miR-146a are cardiomyocyte-enriched microRNAs that control cell survival and self-regulation of inflammation. These microRNAs are released into circulation and are detected in plasma or in circulating exosomes. Little is known whether heart failure affects their release into circulation, which this study investigated. RESULTS: Total and exosome-specific microRNAs in plasma of 40 heart failure patients and 20 controls were prepared using the miRVana Kit. We measured exosomal and total plasma microRNAs separately because exosomes serve as cargos that transfer biological materials and alter signaling in distant organs, whereas microRNAs in plasma indicate the level of tissue damage and are mostly derived from dead cells. qRT-PCR was used to quantify miR-486, miR-146a, and miR-16. Heart failure did not significantly affect plasma miR-486/miR-16 and miR-146a/miR-16 ratio, although miR-146a/miR-16 showed a trend of elevated expression (2.3 ± 0.79, p = 0.27). By contrast, circulating exosomal miR-146a/miR-16 ratio was higher in heart failure patients (2.46 ± 0.51, p = 0.05). miR-146a is induced in response to inflammation as a part of inflammation attenuation circuitry. Indeed, Tnfα and Gm-csf increased miR-146a but not miR-486 in the cardiomyocyte cell line H9C2. These results, if confirmed in a larger study, may help to develop circulating exosomal miR-146a as a biomarker of heart failure.

Pharmacological Dual Inhibition of Tumor and Tumor-Induced Functional Limitations in a Transgenic Model of Breast Cancer.

Breast cancer progression is associated with systemic effects, including functional limitations and sarcopenia without the appearance of overt cachexia. Autocrine/paracrine actions of cytokines/chemokines produced by cancer cells mediate cancer progression and functional limitations. The cytokine-inducible transcription factor NF-κB could be central to this process, as it displays oncogenic functions and is integral to the Pax7:MyoD:Pgc-1ß:miR-486 myogenesis axis. We tested this possibility using the MMTV-PyMT transgenic mammary tumor model and the NF-κB inhibitor dimethylaminoparthenolide (DMAPT). We observed deteriorating physical and functional conditions in PyMT+ mice with disease progression. Compared with wild-type mice, tumor-bearing PyMT+ mice showed decreased fat mass, impaired rotarod performance, and reduced grip strength as well as increased extracellular matrix (ECM) deposition in muscle. Contrary to acute cachexia models described in the literature, mammary tumor progression was associated with reduction in skeletal muscle stem/satellite-specific transcription factor Pax7. Additionally, we observed tumor-induced reduction in Pgc-1ß in muscle, which controls mitochondrial biogenesis. DMAPT treatment starting at 6 to 8 weeks age prior to mammary tumor occurrence delayed mammary tumor onset and tumor growth rates without affecting metastasis. DMAPT overcame cancer-induced functional limitations and improved survival, which was accompanied with restoration of Pax7, Pgc-1ß, and mitochondria levels and reduced ECM levels in skeletal muscles. In addition, DMAPT restored circulating levels of 6 out of 13 cancer-associated cytokines/chemokines changes to levels seen in healthy animals. These results reveal a pharmacological approach for overcoming cancer-induced functional limitations, and the above-noted cancer/drug-induced changes in muscle gene expression could be utilized as biomarkers of functional limitations. Mol Cancer Ther; 16(12); 2747-58. ©2017 AACR.

Downregulation of FKBP14 by RNA interference inhibits the proliferation, adhesion and invasion of gastric cancer cells.

FK506 binding protein (FBBP) 14 belongs to the family of FKBPs. Altered expression of FKBPs are observed in several malignancies. The present study aimed to explore the expression and biological function of FKBP14 in gastric cancer. FKBP14 expression levels in 40 gastric cancer samples and matched control samples were evaluated using quantitative polymerase chain reaction. Cell proliferation was evaluated using Cell Counting kit-8 assay. A cell adhesion and a Transwell assay were performed to detect cell adhesion and invasion. Protein expression was determined using western blot analysis. It was found that FKBP14 expression in gastric cancer tissues was elevated compared with normal tissues. Silencing of FKBP14 expression in the gastric cancer MKN-45 and AGS cell lines, which have a higher expression level of FKBP14 compared with four other gastric cancer cell lines, significantly inhibited cellular proliferation, adhesion and invasion. In addition, the protein levels of proliferating cell nuclear antigen, matrix metalloproteinase 2 and the epithelial-mesenchymal-transition (EMT) markers ß-catenin, Snail1 and Twist were repressed in gastric cancer cells with FKBP14 silenced. In conclusion, FKBP14 may act as an oncogene by suppressing cellular proliferation, adhesion and invasion and EMT in gastric carcinogenesis. FKBP14 may be a diagnosis marker and potential therapeutic target in gastric cancer.

Aromatase inhibitors augment nociceptive behaviors in rats and enhance the excitability of sensory neurons.

Although aromatase inhibitors (AIs) are commonly used therapies for breast cancer, their use is limited because they produce arthralgia in a large number of patients. To determine whether AIs produce hypersensitivity in animal models of pain, we examined the effects of the AI, letrozole, on mechanical, thermal, and chemical sensitivity in rats. In ovariectomized (OVX) rats, administering a single dose of 1 or 5mg/kg letrozole significantly reduced mechanical paw withdrawal thresholds, without altering thermal sensitivity. Repeated injection of 5mg/kg letrozole in male rats produced mechanical, but not thermal, hypersensitivity that extinguished when drug dosing was stopped. A single dose of 5mg/kg letrozole or daily dosing of letrozole or exemestane in male rats also augmented flinching behavior induced by intraplantar injection of 1000nmol of adenosine 5'-triphosphate (ATP). To determine whether sensitization of sensory neurons contributed to AI-induced hypersensitivity, we evaluated the excitability of neurons isolated from dorsal root ganglia of male rats chronically treated with letrozole. Both small and medium-diameter sensory neurons isolated from letrozole-treated rats were more excitable, as reflected by increased action potential firing in response to a ramp of depolarizing current, a lower resting membrane potential, and a lower rheobase. However, systemic letrozole treatment did not augment the stimulus-evoked release of the neuropeptide calcitonin gene-related peptide (CGRP) from spinal cord slices, suggesting that the enhanced nociceptive responses were not secondary to an increase in peptide release from sensory endings in the spinal cord. These results provide the first evidence that AIs modulate the excitability of sensory neurons, which may be a primary mechanism for the effect of these drugs to augment pain behaviors in rats.

Navß4 regulates fast resurgent sodium currents and excitability in sensory neurons.

BACKGROUND: Increased electrical activity in peripheral sensory neurons including dorsal root ganglia (DRG) and trigeminal ganglia neurons is an important mechanism underlying pain. Voltage gated sodium channels (VGSC) contribute to the excitability of sensory neurons and are essential for the upstroke of action potentials. A unique type of VGSC current, resurgent current (INaR), generates an inward current at repolarizing voltages through an alternate mechanism of inactivation referred to as open-channel block. INaRs are proposed to enable high frequency firing and increased INaRs in sensory neurons are associated with pain pathologies. While Nav1.6 has been identified as the main carrier of fast INaR, our understanding of the mechanisms that contribute to INaR generation is limited. Specifically, the open-channel blocker in sensory neurons has not been identified. Previous studies suggest Navß4 subunit mediates INaR in central nervous system neurons. The goal of this study was to determine whether Navß4 regulates INaR in DRG sensory neurons. RESULTS: Our immunocytochemistry studies show that Navß4 expression is highly correlated with Nav1.6 expression predominantly in medium-large diameter rat DRG neurons. Navß4 knockdown decreased endogenous fast INaR in medium-large diameter neurons as measured with whole-cell voltage clamp. Using a reduced expression system in DRG neurons, we isolated recombinant human Nav1.6 sodium currents in rat DRG neurons and found that overexpression of Navß4 enhanced Nav1.6 INaR generation. By contrast neither overexpression of Navß2 nor overexpression of a Navß4-mutant, predicted to be an inactive form of Navß4, enhanced Nav1.6 INaR generation. DRG neurons transfected with wild-type Navß4 exhibited increased excitability with increases in both spontaneous activity and evoked activity. Thus, Navß4 overexpression enhanced INaR and excitability, whereas knockdown or expression of mutant Navß4 decreased INaR generation. CONCLUSION: INaRs are associated with inherited and acquired pain disorders. However, our ability to selectively target and study this current has been hindered due to limited understanding of how it is generated in sensory neurons. This study identified Navß4 as an important regulator of INaR and excitability in sensory neurons. As such, Navß4 is a potential target for the manipulation of pain sensations.

Artemin induced functional recovery and reinnervation after partial nerve injury.

Systemic artemin promotes regeneration of dorsal roots to the spinal cord after crush injury. However, it is unclear whether systemic artemin can also promote peripheral nerve regeneration, and functional recovery after partial lesions distal to the dorsal root ganglion (DRG) remains unknown. In the present investigation, male Sprague Dawley rats received axotomy, ligation, or crush of the L5 spinal nerve or sham surgery. Starting the day of injury, animals received intermittent subcutaneous artemin or vehicle across 2weeks. Sensory thresholds to tactile or thermal stimuli were monitored for 6weeks after injury. Immunohistochemical analyses of the DRG and nerve regeneration were performed at the 6-week time point. Artemin transiently reversed tactile and thermal hypersensitivity after axotomy, ligation, or crush injury. Thermal and tactile hypersensitivity reemerged within 1week of treatment termination. However, artemin-treated rats with nerve crush, but not axotomy or ligation, subsequently showed gradual return of sensory thresholds to preinjury baseline levels by 6weeks after injury. Artemin normalized labeling for NF200, IB4, and CGRP in nerve fibers distal to the crush injury, suggesting persistent normalization of nerve crush-induced neurochemical changes. Sciatic and intradermal administration of dextran or cholera toxin B distal to the crush injury site resulted in labeling of neuronal profiles in the L5 DRG, suggesting regeneration functional restoration of nonmyelinated and myelinated fibers across the injury site into cutaneous tissue. Artemin also diminished ATF3 and caspase 3 expression in the L5 DRG, suggesting persistent neuroprotective actions. A limited period of artemin treatment elicits disease modification by promoting sensory reinnervation of distal territories and restoring preinjury sensory thresholds.

Descending facilitation maintains long-term spontaneous neuropathic pain.

UNLABELLED: Neuropathic pain is frequently characterized by spontaneous pain (ie, pain at rest) and, in some cases, by cold- and touch-induced allodynia. Mechanisms underlying the chronicity of neuropathic pain are not well understood. Rats received spinal nerve ligation (SNL) and were monitored for tactile and thermal thresholds. While heat hypersensitivity returned to baseline levels within approximately 35 to 40 days, tactile hypersensitivity was still present at 580 days after SNL. Tactile hypersensitivity at post-SNL day 60 (D60) was reversed by microinjection of 1) lidocaine; 2) a cholecystokinin 2 receptor antagonist into the rostral ventromedial medulla; or 3) dorsolateral funiculus lesion. Rostral ventromedial medulla lidocaine at D60 or spinal ondansetron, a 5-hydroxytryptamine 3 antagonist, at post-SNL D42 produced conditioned place preference selectively in SNL-treated rats, suggesting long-lasting spontaneous pain. Touch-induced FOS was increased in the spinal dorsal horn of SNL rats at D60 and prevented by prior dorsolateral funiculus lesion, suggesting that long-lasting tactile hypersensitivity depends upon spinal sensitization, which is mediated in part by descending facilitation, in spite of resolution of heat hypersensitivity. PERSPECTIVE: These data suggest that spontaneous pain is present for an extended period of time and, consistent with likely actions of clinically effective drugs, is maintained by descending facilitation.

Alleviation of neuropathic pain hypersensitivity by inhibiting neuronal pentraxin 1 in the rostral ventromedial medulla.

Peripheral nerve injury causes spontaneous and long-lasting pain, hyperalgesia, and allodynia. Excitatory amino acid receptor-dependent increases in descending facilitatory drive from the brainstem rostral ventromedial medulla (RVM) contribute to injury-evoked hypersensitivity. Although increased excitability likely reflects changes in synaptic efficacy, the cellular mechanisms underlying injury-induced synaptic plasticity are poorly understood. Neuronal pentraxin 1 (NP1), a protein with exclusive CNS expression, is implicated in synaptogenesis and AMPA receptor recruitment to immature synapses. Its role in the adult brain and in descending pain facilitation is unknown. Here, we use the spared nerve injury (SNI) model in rodents to examine this issue. We show that SNI increases RVM NP1 expression and constitutive deletion or silencing NP1 in the RVM, before or after SNI, attenuates allodynia and hyperalgesia in rats. Selective rescue of RVM NP1 expression restores behavioral hypersensitivity of knock-out mice, demonstrating a key role of RVM NP1 in the pathogenesis of neuropathic pain.