Long-term tactile hypersensitivity after nerve crush injury in mice is characterized by the persistence of intact sensory axons.

ABSTRACT: Traumatic peripheral nerve injuries are at high risk of neuropathic pain for which novel effective therapies are urgently needed. Preclinical models of neuropathic pain typically involve irreversible ligation and/or nerve transection (neurotmesis). However, translation of findings to the clinic has so far been unsuccessful, raising questions on injury model validity and clinically relevance. Traumatic nerve injuries seen in the clinic commonly result in axonotmesis (ie, crush), yet the neuropathic phenotype of "painful" nerve crush injuries remains poorly understood. We report the neuropathology and sensory symptoms of a focal nerve crush injury using custom-modified hemostats resulting in either complete ("full") or incomplete ("partial") axonotmesis in adult mice. Assays of thermal and mechanically evoked pain-like behavior were paralleled by transmission electron microscopy, immunohistochemistry, and anatomical tracing of the peripheral nerve. In both crush models, motor function was equally affected early after injury; by contrast, partial crush of the nerve resulted in the early return of pinprick sensitivity, followed by a transient thermal and chronic tactile hypersensitivity of the affected hind paw, which was not observed after a full crush injury. The partially crushed nerve was characterized by the sparing of small-diameter myelinated axons and intraepidermal nerve fibers, fewer dorsal root ganglia expressing the injury marker activating transcription factor 3, and lower serum levels of neurofilament light chain. By day 30, axons showed signs of reduced myelin thickness. In summary, the escape of small-diameter axons from Wallerian degeneration is likely a determinant of chronic pain pathophysiology distinct from the general response to complete nerve injury.

Expression of Piezo2 in the Dental Pulp, Sensory Root, and Trigeminal Ganglion and Its Coexpression with Vesicular Glutamate Transporters.

INTRODUCTION: Information on the type of vesicular glutamate transporter (VGLUT) that is expressed in the Piezo2-positive (Piezo2+) neurons in the trigeminal ganglion (TG) and on the type of Piezo2+ axons and their distribution in the dental pulp is important for understanding dental pain elicited by mechanical stimuli and developing new therapeutic strategies. METHODS: We examined the expression of Piezo2 and its coexpression with VGLUT1 and VGLUT2 in rat TG, the sensory root, and human dental pulp using light and electron microscopic immunohistochemistry and quantitative analysis. RESULTS: VGLUT1 and VGLUT2 were expressed in the TG neurons. Piezo2 was expressed in axons of all types but primarily in small myelinated (Aδ) axons in the sensory root. In the dental pulp, Piezo2 was expressed densely in the numerous axons that form a plexus in the peripheral pulp. Piezo2+ axons in the peripheral pulp were mostly unmyelinated, and Piezo2 immunoreactivity was often concentrated near the axolemma, suggesting that it may represent functional receptors. CONCLUSIONS: These findings suggest that VGLUT1 and VGLUT2 are involved in the glutamate signaling in Piezo2+ neurons, Piezo2 may be primarily activated by noxious mechanical stimuli, and Piezo2-mediated dental mechanotransduction may be primarily elicited in the peripheral pulp.

Expression of Piezo1 in the Trigeminal Neurons and in the Axons That Innervate the Dental Pulp.

Information on the neurons and axons that express the mechanosensitive channel Piezo1 and its expression in axons innervating the dental pulp may help understand the nature of the Piezo1-mediated mechanosensation and the underlying mechanism of dentin sensitivity elicited by mechanical stimuli. For this, we here investigated the neurochemical properties of the neurons in the rat trigeminal ganglion (TG) and their axons in its sensory root that express Piezo1 and the expression of Piezo1 in the rat and human dental pulp by light and electron microscopic immunohistochemistry and quantitative analysis. Piezo1 was expressed mainly in medium-sized and large TG neurons. Piezo1-immunopositive (+) neurons frequently coexpressed the marker for neurons with myelinated axons, NF200, but rarely the markers for neurons with unmyelinated axons, CGRP or IB4. In the sensory root of TG, Piezo1 was expressed primarily in small myelinated axons (Aδ, 60.2%) but also in large myelinated (Aß, 24.3%) and unmyelinated (C, 15.5%) axons. In the human dental pulp, Piezo1 was expressed in numerous NF200+ axons, which formed a network in the peripheral pulp and often "ascended" toward the dentin. Most Piezo1+ myelinated axons in the radicular pulp became unmyelinated in the peripheral pulp, where Piezo1 immunoreaction product was associated with the axonal plasma membrane, suggesting a functional role of Piezo1 in the peripheral pulp. These findings suggest that Piezo1 is involved primarily in mediating the acute pain elicited by high-threshold mechanical stimuli, and that the Piezo1-mediated dental mechanotransduction occurs primarily in the axons in the peripheral pulp.

Exploratory fall risk and preventive intervention in acute vestibular neuritis.

OBJECTIVE: To analyze recovery from dizziness in patients with acute vestibular neuritis (AVN) after applying the "Dizziness & Fall Risk Assessment and Intervention (DFRAI)". METHODS: This prospective study involved patients with AVN who underwent a survey of dizziness and fall risk. The patients received medical treatment and customized vestibular rehabilitation, and vestibular function was evaluated at the initial attack and 3 months later. RESULTS: Forty-one patients underwent subjective questionnaire assessments, which showed significant improvement in visual analog scale-dizziness handicap inventory-fear of falling (VAS-DHI-FOF) results from the initial vertigo attack to 3 months later. In the sensory organization test (SOT), the initial composite score was 63 ± 13.1, which improved to 77.5 ± 4.9 3 months later. In caloric testing, the canal paresis (CP) score was 42.9 ± 35.2, which improved to 29.9 ± 23.5 3 months later. CONCLUSIONS: Subjective improvement in dizziness and objective recovery of vestibular function were confirmed. DFRAI is a comprehensive solution for dizziness, and appropriate application of the DFRAI is expected to have a positive effect on recovery from dizziness and fall prevention in patients with AVN.

Ablation of dynamin-related protein 1 promotes diabetes-induced synaptic injury in the hippocampus.

Dynamin-related protein 1 (Drp1)-mediated mitochondrial dysfunction is associated with synaptic injury in the diabetic brain. However, the dysfunctional mitochondria by Drp1 deletion in the diabetic brain are poorly understood. Here, we investigated the effects of neuron-specific Drp1 deletion on synaptic damage and mitophagy in the hippocampus of a high-fat diet (HFD)/streptozotocin (STZ)-induced diabetic mice. HFD/STZ-induced diabetic mice exhibited metabolic disturbances and synaptic damages. Floxed Drp1 mice were crossed with Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα)-Cre mice, to generate neuron-specific Drp1 knockout (Drp1cKO) mice, which showed marked mitochondrial swelling and dendritic spine loss in hippocampal neurons. In particular, diabetic Drp1cKO mice exhibited an increase in dendritic spine loss and higher levels of oxidative stress and neuroinflammation compared with diabetic wild-type (WT) mice. Diabetic WT mice generally displayed increased Drp1-induced small mitochondrial morphology in hippocampal neurons, but large mitochondria were prominently observed in diabetic Drp1cKO mice. The levels of microtubule-associated protein 1 light-chain 3 and lysosomal-associated membrane protein 1 proteins were significantly increased in the hippocampus of diabetic Drp1cKO mice compared with diabetic WT mice. The inhibition of Drp1 adversely promotes synaptic injury and neurodegeneration in the diabetic brain. The findings suggest that the exploratory mechanisms behind Drp1-mediated mitochondrial dysfunction could provide a possible therapeutic target for diabetic brain complications.

SLC6A20 transporter: a novel regulator of brain glycine homeostasis and NMDAR function.

Glycine transporters (GlyT1 and GlyT2) that regulate levels of brain glycine, an inhibitory neurotransmitter with co-agonist activity for NMDA receptors (NMDARs), have been considered to be important targets for the treatment of brain disorders with suppressed NMDAR function such as schizophrenia. However, it remains unclear whether other amino acid transporters expressed in the brain can also regulate brain glycine levels and NMDAR function. Here, we report that SLC6A20A, an amino acid transporter known to transport proline based on in vitro data but is understudied in the brain, regulates proline and glycine levels and NMDAR function in the mouse brain. SLC6A20A transcript and protein levels were abnormally increased in mice carrying a mutant PTEN protein lacking the C terminus through enhanced ß-catenin binding to the Slc6a20a gene. These mice displayed reduced extracellular levels of brain proline and glycine and decreased NMDAR currents. Elevating glycine levels back to normal ranges by antisense oligonucleotide-induced SLC6A20 knockdown, or the competitive GlyT1 antagonist sarcosine, normalized NMDAR currents and repetitive climbing behavior observed in these mice. Conversely, mice lacking SLC6A20A displayed increased extracellular glycine levels and NMDAR currents. Lastly, both mouse and human SLC6A20 proteins mediated proline and glycine transports, and SLC6A20 proteins could be detected in human neurons. These results suggest that SLC6A20 regulates proline and glycine homeostasis in the brain and that SLC6A20 inhibition has therapeutic potential for brain disorders involving NMDAR hypofunction.

Nationwide Analysis of Mortality Rates and Related Surgical Procedures in Hearing Disability Patients in South Korea.

BACKGROUND AND OBJECTIVES: Hearing loss (HL) and its repercussions are major problems in today's society. There are limited data on the relationship between degree of HL and otologic disorders. The aim of this study is to estimate mortality rates, rates of sudden idiopathic HL and related otologic surgical procedures in hearing disability patients in South Korea. SUBJECTS AND METHODS: Retrospective medical data for 160,205 patients with hearing disability was extracted. Mortality rates, rates of sudden idiopathic HL and related otologic surgical procedures were compared with a normal control group consisting of 865,475 people; approximately 5 times the number of hearing disability patients. RESULTS: According to the Korean National Disability Registry (NDR), 0.458% of the population in South Korea suffered from hearing disability in 2015. Higher rates of mortality and sudden idiopathic HL were reported in hearing disability patients, increasing up to a maximum of 1.594 times and 1,039.695 times, respectively, compared to the normal control group. Mastoidectomy surgery was 2.5 times more frequently performed and pressure equalizing (PE) tube insertion was about 15 times more frequently performed in hearing disability patients. CONCLUSIONS: Hearing disability is related to higher risks of mortality, sudden idiopathic HL and otologic surgical procedures, including mastoidectomy and PE tubing.

Psychiatric Distress as a Common Risk Factor for Tinnitus and Joint Pain: A National Population-Based Survey.

OBJECTIVES: This study aimed to investigate the relationship between tinnitus and joint pain from representative samples of Koreans. METHODS: The demographics and the responses to a questionnaire about tinnitus and joint pain severity and mental health status of adults aged ≥50 years in the 2010-2012 Korean National Health and Nutrition Examination Survey were analyzed. RESULTS: Among 9,032 individuals, 26.7% reported experiencing tinnitus within the past year. Participants with tinnitus were more frequently older, hearing loss, and had lower education levels, income, and body weight. Participants with regular exercise and sleep had a lower tinnitus prevalence. The incidences of stress, depressed mood, and suicidal ideation were significantly higher in the tinnitus group and participants with joint pain. The rates of participants with tinnitus according to the number of joint pain sites (zero, one, two, and three) was 22.1%, 31.4%, 33.3%, and 44.2%, and those of participants with severely annoying tinnitus according to the number of joint pain sites (zero, one, two, and three) were 3.3%, 6.8%, 7.9%, and 10.7%, respectively. CONCLUSION: Tinnitus prevalence and severity were significantly related to joint pain, and both conditions were related to psychiatric distress. Thus, the authors suggest that psychiatric distress as a common risk factor for tinnitus and joint pain should be considered when deciding treatment strategies and in guiding public health policy.

The optimal and safe intensity for facial nerve stimulation during intraoperative neuromonitoring in middle ear surgery.

OBJECTIVE: This study aimed to investigate the optimal and safe intensity for facial nerve stimulation during middle ear surgery. METHODS: Thirty-seven patients who had their facial nerve exposed prior to surgery were prospectively enrolled in this study, and electromyography (EMG) recordings were obtained from the orbicularis oculi and orbicularis oris muscles. Four pigs were also enrolled in an animal study, and continuous stimulation was performed on the facial nerves of the pigs for 10 minutes. The EMG responses were measured and the pathologic outcomes of the facial nerve after stimulation were determined. RESULTS: In the human study, the mean intensity of the minimal electrical stimulation threshold was 0.21 mA (range: 0.1-0.3 mA). A linear correlation was observed between stimulus intensity and response amplitude for intensities below 0.4 mA. Response amplitudes reached a plateau between 0.4 mA and 1.0 mA. The minimal stimulus intensity that could generate a maximal response was 0.4 mA in the orbicularis oculi (244 µV) and orbicularis oris (545 µV). In the animal study, there were no observed changes in EMG or nerve damage incidence after the continuous stimulation of 3.0 mA. CONCLUSIONS: 0.4 mA is considered to be the optimal intensity of facial nerve stimulation during middle ear surgery, and it was estimated through the animal study that a stimulation of 3.0 mA is safe from facial nerve damage.

Decreased expression of type I (IFN-ß) and type III (IFN-λ) interferons and interferon-stimulated genes in patients with chronic rhinosinusitis with and without nasal polyps.

BACKGROUND: Little is known about antiviral responses in the sinonasal mucosal tissue of patients with chronic rhinosinusitis (CRS). OBJECTIVE: we investigated the presence of virus and the expression of Toll-like receptor (TLR) 3, TLR7, and interferon and interferon-stimulated genes (ISGs) in healthy mucosal tissue of control subjects and the inflammatory sinus mucosal tissue of CRS patients, and evaluated whether levels of interferons and ISGs might be affected by CRS-related cytokines and by treatment with macrolides, dexamethasone, or TLR3 and TLR7 agonists. METHODS: The presence of virus in the sinonasal mucosa was evaluated with real-time PCR. The expression of interferons and ISGs in the sinonasal mucosa and in cultured epithelial cells treated with TH1 and TH2 cytokines, macrolides, dexamethasone, or TLR3 and TLR7 agonists were evaluated with real-time PCR and Western blotting. The expression of TLR3 and TLR7 in the sinonasal mucosa were evaluated with immunohistochemistry. RESULTS: Respiratory viruses were detected in 15% of samples. Interferons and ISGs are expressed in normal mucosa, but their levels were decreased in patients with CRS. Interferon and ISG levels were upregulated in cells treated with macrolides, dexamethasone, or TLR3 agonist, but some were decreased in cytokine-treated cells. TLR3 and TLR7 levels showed no significant difference between normal and inflammatory sinus mucosal tissue. CONCLUSION: These results suggest that decreased levels of interferons and ISGs in patients with CRS might contribute to impairment of the antiviral innate response in inflammatory sinonasal epithelial cells. Macrolides and glucocorticoids might provide positive effects on the treatment of CRS by upregulating interferon and ISG expression.

Shank3 Mice Carrying the Human Q321R Mutation Display Enhanced Self-Grooming, Abnormal Electroencephalogram Patterns, and Suppressed Neuronal Excitability and Seizure Susceptibility.

Shank3, a postsynaptic scaffolding protein involved in regulating excitatory synapse assembly and function, has been implicated in several brain disorders, including autism spectrum disorders (ASD), Phelan-McDermid syndrome, schizophrenia, intellectual disability, and mania. Here we generated and characterized a Shank3 knock-in mouse line carrying the Q321R mutation (Shank3 Q321R mice) identified in a human individual with ASD that affects the ankyrin repeat region (ARR) domain of the Shank3 protein. Homozygous Shank3 Q321R/Q321R mice show a selective decrease in the level of Shank3a, an ARR-containing protein variant, but not other variants. CA1 pyramidal neurons in the Shank3 Q321R/Q321R hippocampus show decreased neuronal excitability but normal excitatory and inhibitory synaptic transmission. Behaviorally, Shank3 Q321R/Q321R mice show moderately enhanced self-grooming and anxiolytic-like behavior, but normal locomotion, social interaction, and object recognition and contextual fear memory. In addition, these mice show abnormal electroencephalogram (EEG) patterns and decreased susceptibility to induced seizures. These results indicate that the Q321R mutation alters Shank3 protein stability, neuronal excitability, repetitive and anxiety-like behavior, EEG patterns, and seizure susceptibility in mice.

Neutrophil extracellular traps in nasal secretions of patients with stable and exacerbated chronic rhinosinusitis and their contribution to induce chemokine secretion and strengthen the epithelial barrier.

BACKGROUND: Neutrophil extracellular traps (NETs) participate in innate immunity by trapping microorganisms. Their pathophysiological implications have not been defined in chronic rhinosinusitis (CRS). OBJECTIVE: We investigated the presence of NETs in nasal secretion of patients with stable or exacerbated CRS and evaluated whether NETs participate in the secretion of chemokines in sinonasal epithelial cells, the epithelial permeability, and transendothelial leucocyte migration, and elucidate whether NETs are released by macrolides and dexamethasone. METHODS: The presence of NETs in nasal secretion and the release of NETs from neutrophils stimulated with macrolides or dexamethasone were evaluated by dsDNA Assay kit and fluorescence microscope. The chemokine secretion, epithelial permeability, and transendothelial leucocyte migration were measured in cultured cells incubated with NETs, the supernatant of unstimulated neutrophils (unstim), NETs inhibitor (DPI), or H3Cit, where the expression of junctional complex proteins and ICAM-1 was evaluated by real-time PCR, Western blots, and confocal microscope. RESULTS: The amount of NETs and NETs-forming neutrophils in nasal secretion increased in exacerbated CRS. Epithelial cells treated with NETs or H3Cit secreted chemokines and showed decreased permeability associated with up-regulated junctional complex proteins. Increased transendothelial leucocyte migration associated with up-regulated ICAM-1 was noted in endothelial cells treated with NETs or H3Cit. These findings were not found in cells treated with unstim, or DPI. NETs were released by macrolides, but not by dexamethasone. CONCLUSIONS AND CLINICAL RELEVANCE: NETs formation increased in exacerbated CRS, inducing chemokine secretion, strengthening the epithelial barrier, and promoting the neutrophils infiltration. Therefore, the release of NETs in CRS might be beneficial or detrimental to CRS patients.

Primary sensory neurons expressing tropomyosin receptor kinase A in the rat trigeminal ganglion.

Tropomyosin receptor kinase A (trkA), a high affinity receptor for nerve growth factor (NGF), has been implicated in neuronal survival, neurite outgrowth and inflammatory pain. So far, the characterization of the primary sensory neurons that express trkA, and are thus potentially affected by NGF, has remained incomplete. The goal of this study was to investigate the trkA-expressing neurons and fibers in the rat trigeminal ganglion and its sensory root using light- and electron-microscopic immunohistochemistry and quantitative analysis. TrkA-immunopositive (+) trigeminal neurons varied from small to large. Double immunofluorescent staining showed that about 28%, 33% and 3% of the trkA(+) neurons coexpressed SP, CGRP and IB4, respectively. About 11% of the trkA(+) neurons also coexpressed parvalbumin. Electron microscopy revealed that trkA was expressed in all types of fibers: While the large majority of the trkA(+) fibers were unmyelinated (35.3%) and small myelinated (<20 µm2 in cross-sectional area; 45.5%), a still considerable fraction (19.2%) was large myelinated. These findings indicate that all types of trigeminal neurons (ones with unmyelinated, small myelinated or large myelinated fibers) may be regulated by NGF/trkA signaling.

Impact of germination on the structures and in vitro digestibility of starch from waxy brown rice.

The in vitro digestibility as well as the molecular and crystalline structures of waxy rice starches isolated from brown rice, germinated brown rice (GBR), ultrasonicated GBR, and heat-moisture treated GBR were investigated. The germinated brown rice starch (GBRS) had a lower average molecular weight and a higher proportion of DP 6-12 in amylopectin than brown rice starch (BRS). The relative crystallinity, intensity ratio of the band at 1,047 cm(-1) and 1,022 cm(-1), gelatinization temperature and pasting temperature of waxy rice starch were reduced by germination. However, the ultrasonication and heat-moisture treatment of GBRS increased the relative crystallinity and gelatinization temperature. The digestibility of starch from brown waxy rice was increased by germination. The rapidly digestible starch (RDS), slowly digestible starch (SDS) and resistant starch (RS) contents were 50.5%, 42.4%, and 7.1% in BRS, and 69.0%, 27.9% and 3.1% in GBRS, respectively. The ultrasonication and heat-moisture treatment of GBRS reduced RDS content and increased RS content in raw and gelatinized starches. The decrease in starch digestibility of cooked GBR was more pronounced after heat-moisture treatment than after ultrasonication.

Rice varieties in relation to rice bread quality.

BACKGROUND: It is difficult to predict rice bread quality only from the amylose content (AC) or dough characteristics of new lines produced by rice breeding programmes. This study investigated the AC relative to bread baking quality of rice varieties developed in Korea, and identified specific characteristics that contribute to rice bread quality. RESULTS: Manmibyeo, Jinsumi, Seolgaeng and Hanareumbyeo were classified as low AC, YR24088 Acp9, Suweon517, Chenmaai and Goamibyeo as intermediate AC and Milyang261 as high AC. Suweon517, Milyang261 and Manmibyeo had a high water absorption index (WAI), while Goamibyeo, YR24088 Acp9, Jinsumi, Seolgaeng, Hanareumbyeo and Chenmaai had a low WAI. The gelatinisation enthalpy of flour varied from 9.2 J g(-1) in Milyang261 to 14.8 J g(-1) in YR24088 Acp9. After 7 days of storage the rate of flour retrogradation and crumb firmness were weakly correlated, with the exception of Jinsumi. Bread volumes of Jinsumi, Chenmaai, YR24088 Acp9 and Goamibyeo were comparable to that of wheat flour, but the rest were unsuited to bread making because of their low volume and hard crumb texture. CONCLUSION: Based on volume, texture and crumb firmness ratio, Chenmaai and Goamibyeo were the most appropriate varieties for making bread. An intermediate AC and low WAI were the primary indicators of rice bread flour quality.

Effect of phenolic acids on the rheological properties and proteins of hard wheat flour dough and bread.

BACKGROUND: The effects of different phenolic acids on the rheological properties and gluten proteins of hard wheat flour dough and bread were investigated. Caffeic, ferulic, syringic and gallic acids were each blended with hard wheat flour at a concentration of 4.44 µmol L(-1) g(-1) flour. RESULTS: Mixing time and tolerance were reduced with the addition of phenolic acids. The phenolic acids reduced the maximum resistance to extension (R(max)) and increased the extensibility of dough, with effects in the following order: gallic < syringic < ferulic < caffeic acid. The effect on R(max) was more pronounced in overmixed dough. Loaf volume was most significantly decreased with the addition of caffeic acid. Extraction of sodium dodecyl sulfate-soluble high-molecular-weight proteins was increased in both mixed and fermented doughs by the addition of ferulic and caffeic acids. The order of influence of the phenolic acids on the rheological properties and protein structure of dough and bread was consistent with that of their antioxidant activity. CONCLUSION: The addition of caffeic and ferulic acids reduced R(max) and increased the extensibility of hard wheat flour dough by modifying the high-molecular-weight gluten, which resulted in decreased bread volume.

Antioxidant activity of hard wheat flour, dough and bread prepared using various processes with the addition of different phenolic acids.

BACKGROUND: The purpose of this study was to evaluate the effect of baking process on the antioxidant activity of different phenolic acids. Antioxidant potential was determined using the ß-carotene-bleaching activity assay, and free phenolic acid levels were determined by high-performance liquid chromatography. Four phenolic acids, caffeic acid, ferulic acid, syringic acid and gallic acid, were mixed with wheat flour at a concentration of 4.44 µmol/g of flour. RESULTS: Type of phenolic acid and processing affected antioxidant activity. Of the phenolic acids, caffeic acid had the most pronounced antioxidant effect. The ranking of phenolic acids in terms of their antioxidant activity in fermented dough and bread was similar to that before processing, i.e. syringic acid < gallic acid < ferulic acid < caffeic acid. The content of ferulic acid was greater than that of the other phenolic acids after baking. Antioxidant activity and free phenolic acid content were reduced by mixing but recovered after fermentation and baking. Phenolic acid recovery after baking was 74-80%. CONCLUSION: Phenolic acids retain their antioxidant activity after the baking process, which has potential health benefits for consumers. Elucidation of interactions between the baking process and phenolic acids is important for the development of functional foods.