High-throughput segmentation of unmyelinated axons by deep learning.

Axonal characterizations of connectomes in healthy and disease phenotypes are surprisingly incomplete and biased because unmyelinated axons, the most prevalent type of fibers in the nervous system, have largely been ignored as their quantitative assessment quickly becomes unmanageable as the number of axons increases. Herein, we introduce the first prototype of a high-throughput processing pipeline for automated segmentation of unmyelinated fibers. Our team has used transmission electron microscopy images of vagus and pelvic nerves in rats. All unmyelinated axons in these images are individually annotated and used as labeled data to train and validate a deep instance segmentation network. We investigate the effect of different training strategies on the overall segmentation accuracy of the network. We extensively validate the segmentation algorithm as a stand-alone segmentation tool as well as in an expert-in-the-loop hybrid segmentation setting with preliminary, albeit remarkably encouraging results. Our algorithm achieves an instance-level [Formula: see text] score of between 0.7 and 0.9 on various test images in the stand-alone mode and reduces expert annotation labor by 80% in the hybrid setting. We hope that this new high-throughput segmentation pipeline will enable quick and accurate characterization of unmyelinated fibers at scale and become instrumental in significantly advancing our understanding of connectomes in both the peripheral and the central nervous systems.

Human organ donor-derived vagus nerve biopsies allow for well-preserved ultrastructure and high-resolution mapping of myelinated and unmyelinated fibers.

The vagus nerve provides motor, sensory, and autonomic innervation of multiple organs, and electrical vagus nerve stimulation (VNS) provides an adjunctive treatment option for e.g. medication-refractory epilepsy and treatment-resistant depression. The mechanisms of action for VNS are not known, and high-resolution anatomical mapping of the human vagus nerve is needed to better understand its functional organization. Electron microscopy (EM) is required for the detection of both myelinated and unmyelinated axons, but access to well-preserved human vagus nerves for ultrastructural studies is sparse. Intact human vagus nerve samples were procured intra-operatively from deceased organ donors, and tissues were immediately immersion fixed and processed for EM. Ultrastructural studies of cervical and sub-diaphragmatic vagus nerve segments showed excellent preservation of the lamellated wall of myelin sheaths, and the axolemma of myelinated and unmyelinated fibers were intact. Microtubules, neurofilaments, and mitochondria were readily identified in the axoplasm, and the ultrastructural integrity of Schwann cell nuclei, Remak bundles, and basal lamina was also well preserved. Digital segmentation of myelinated and unmyelinated axons allowed for determination of fiber size and myelination. We propose a novel source of human vagus nerve tissues for detailed ultrastructural studies and mapping to support efforts to refine neuromodulation strategies, including VNS.

Stain-Free Resolution of Unmyelinated Axons in Transgenic Mice Using Fluorescence Microscopy.

Though unmyelinated fibers predominate axon counts within peripheral nerves, they are frequently excluded in histomorphometric assessment as they cannot be readily resolved by light microscopy. Herein, we demonstrate stain-free resolution of unmyelinated axons in Sox10-Venus mice by widefield fluorescence imaging of sciatic nerve cryosections. Optional staining of cryosections using a rapid and nontoxic myelin-specific dye (FluoroMyelin Red) enables robust synchronous resolution of myelinated and unmyelinated fibers, comprising a high-throughput platform for neural histomorphometry.

Impaired mechanical, heat, and cold nociception in a murine model of genetic TACE/ADAM17 knockdown.

TNF-α-converting enzyme, a member of the ADAM (A disintegrin and metalloproteinase) protease family and also known as ADAM17, regulates inflammation and regeneration in health and disease. ADAM17 targets are involved in pain development and hypersensitivity in animal models of inflammatory and neuropathic pain. However, the role of ADAM17 in the pain pathway is largely unknown. Therefore, we used the hypomorphic ADAM17 (ADAM17ex/ex) mouse model to investigate the importance of ADAM17 in nociceptive behavior, morphology, and function of primary afferent nociceptors. ADAM17ex/ex mice were hyposensitive to noxious stimulation, showing elevated mechanical thresholds as well as impaired heat and cold sensitivity. Despite these differences, skin thickness and innervation were comparable to controls. Although dorsal root ganglia of ADAM17ex/ex mice exhibited normal morphology of peptidergic and nonpeptidergic neurons, a small but significant reduction in the number of isolectin ß-4-positive neurons was observed. Functional electrical properties of unmyelinated nociceptors showed differences in resting membrane potential, afterhyperpolarization, and firing patterns in specific subpopulations of sensory neurons in ADAM17ex/ex mice. However, spinal cord morphology and microglia activity in ADAM17ex/ex mice were not altered. Our data suggest that ADAM17 contributes to the processing of painful stimuli, with a complex mode of action orchestrating the function of neurons along the pain pathway.-Quarta, S., Mitric, M., Kalpachidou, T., Mair, N., Schiefermeier-Mach, N., Andratsch, M., Qi, Y., Langeslag, M., Malsch, P., Rose-John, S., Kress, M. Impaired mechanical, heat, and cold nociception in a murine model of genetic TACE/ADAM17 knockdown.

[Analysis of the quantitative differences in the thickness of the retinal nerve fibre layer between time-domain and spectral-domain optical coherence tomography in patients with relapsing-remitting multiple sclerosis]. / Analisis de las diferencias cuantitativas en el grosor de la capa de fibras nerviosas retiniana entre la tomografia de coherencia optica de dominio-tiempo y de dominio-espectral en pacientes con esclerosis multiple remitente recurrente.

INTRODUCTION: Optical coherence tomography (OCT) is a tool that is increasingly more commonly used in the study of neuro-degenerative diseases. AIMS: To analyse and correlate the thickness of the retinal nerve fibre layer (RNFL) by means of time-domain and spectral-domain OCT in patients with relapsing-remitting multiple sclerosis (MS), with and without a history of optic neuritis (ON). SUBJECTS AND METHODS: We conducted a cross-sectional study of the thickness (mean and by quadrants) of the RNFL of 15 disease-free subjects, 28 with MS with no prior history of ON and 18 with a history of ON. The full ophthalmologic examination included measurement of the RNFL by means of time-domain and spectral-domain tomography. RESULTS: Statistically significant differences are found between the two tomography scans on comparing the mean thickness of the RNFL of the control group (p = 0.000), the group with a history of ON (p = 0.000) and the group without ON (p = 0.000). We obtained a strong, statistically significant and directly proportional correlation between the mean thickness of the RNFL measured with the two types of tomography in the control group (rho = 0.842; p = 0.000), and the groups of eyes without ON (rho = 0.91; p = 0.000) and with ON (rho = 0.902; p = 0.000). CONCLUSIONS: There is a strong correlation between the two tomography scans in the measurement of the thickness of the RNFL in patients with MS, with and without a history of ON. Time-domain OCT quantifies greater thicknesses, and therefore both types of tomography have proven to be effective in the study of MS, although the results cannot be interchanged or extrapolated.

TITLE: Analisis de las diferencias cuantitativas en el grosor de la capa de fibras nerviosas retiniana entre la tomografia de coherencia optica de dominio-tiempo y de dominio-espectral en pacientes con esclerosis multiple remitente recurrente.Introduccion. La tomografia de coherencia optica (OCT) es una herramienta cada vez mas extendida en el estudio de las enfermedades neurodegenerativas. Objetivos. Analizar y correlacionar el grosor de la capa de fibras nerviosas retiniana (CFNR) mediante OCT de dominio-tiempo y dominio-espectral en pacientes con esclerosis multiple (EM) remitente recurrente, con y sin antecedente de neuritis optica (NO). Sujetos y metodos. Estudio transversal del grosor medio y por cuadrantes de la CFNR en 15 sujetos libres de enfermedad, 28 con EM sin historia previa de NO y 18 con antecedentes de NO. La exploracion oftalmologica completa incluia la medicion de la CFNR mediante tomografos de dominio-tiempo y dominio-espectral. Resultados. Existen diferencias estadisticamente significativas entre ambos tomografos al comparar el grosor medio de la CFNR en el grupo control (p = 0,000), el grupo con antecedentes de NO (p = 0,000) y el grupo sin NO (p = 0,000). Hemos obtenido una fuerte correlacion, estadisticamente significativa y directamente proporcional entre el grosor medio de la CFNR medido con ambos tomografos en el grupo control (rho = 0,842; p = 0,000), y los grupos de ojos sin NO (rho = 0,91; p = 0,000) y con NO (rho = 0,902; p = 0,000). Conclusiones. Existe una fuerte correlacion en la medicion del grosor de la CFNR entre ambos tomografos en pacientes con EM, con y sin antecedente de NO. La OCT de dominio-tiempo cuantifica grosores mayores, por lo que ambos tomografos se demuestran eficaces en el estudio de la EM, aunque los resultados no son intercambiables ni extrapolables.

A histomorphometric study of unmyelinated fibers of the fibular nerve in Wistar rats.

There are few histomorphometric studies on the unmyelinated fibers of the fibular nerve in rats, and the number of experimental studies using this nerve has been increasing in the last years. Sixty-two percent of the endoneurial area from 10 fibular nerves of adult Wistar rats was scanned by electron microscopy, and digitized. The total number of unmyelinated axons (1.882 ± 271) was significantly lesser, and their axon diameters (0.2 µm to 2.8 µm) significantly higher than that determined in previous studies. The histogram peaked at 1 µm. The differences could be due to the nerve sampled area, the number and the age of the animals evaluated, and the laboratory techniques used. This study brings new and referential data to be used in experimental investigations involving histomorphometric evaluation of the rat fibular nerve.

A histomorphometric study of unmyelinated fibers of the fibular nerve in Wistar rats / Histomorfometria das fibras amielínicas do nervo fibular em ratos Wistar

ABSTRACT There are few histomorphometric studies on the unmyelinated fibers of the fibular nerve in rats, and the number of experimental studies using this nerve has been increasing in the last years. Sixty-two percent of the endoneurial area from 10 fibular nerves of adult Wistar rats was scanned by electron microscopy, and digitized. The total number of unmyelinated axons (1.882 ± 271) was significantly lesser, and their axon diameters (0.2 µm to 2.8 µm) significantly higher than that determined in previous studies. The histogram peaked at 1 µm. The differences could be due to the nerve sampled area, the number and the age of the animals evaluated, and the laboratory techniques used. This study brings new and referential data to be used in experimental investigations involving histomorphometric evaluation of the rat fibular nerve.

RESUMO Embora o nervo fibular de ratos venha sendo incluído progressivamente em maior número de estudos experimentais nos últimos anos, há poucos estudos a respeito das suas fibras amielínicas. Os nervos fibulares de 10 ratos Wistar adultos foram avaliados através de microscopia óptica e eletrônica. Varredura sistemática através de microscopia eletrônica de transmissão das áreas fasciculares da porção distal no nervo foi realizada. Em média, 62% da área endoneural foi digitalizada. O número total de axônios amielínicos encontrados (1.882 ± 271) foi significativamente menor e as medidas dos diâmetros axonais (0,2 µm a 2,8 µm) maiores do que o determinado em estudos prévios. O pico do histograma foi constituído por fibras de 1µm. As diferenças podem ser devidas à amostragem de maior área endoneural, ao número e à idade dos animais avaliados, e as técnicas laboratoriais utilizadas. Os dados obtidos podem ser considerados referenciais para o nervo fibular de ratos Wistar adultos.

Electrophysiological evidence for the existence of a rare population of C-fiber low threshold mechanoreceptive (C-LTM) neurons in glabrous skin of the rat hindpaw.

The mammalian skin in innervated by distinct classes of low-threshold mechanoreceptive (LTM) primary afferent neurons that are classified as Aß-, Aδ- or C-LTMs according to their axonal conduction velocities (CVs). C-LTMs are thought to signal pleasant and erotic touch sensations in humans, and to exist only in the hairy skin of primates and other species. Using intracellular recordings from rat L4/L5 dorsal root ganglion (DRG) neurons that were classified in vivo as C-nociceptors or C-LTMs, according to their dorsal root CVs and their responses to mechanical and thermal stimuli, the present study provides the first electrophysiological evidence that C-LTMs exist in the glabrous skin of the rat's hindpaw. Indeed 6.4% (5/78) of the total sample of lumbar C-fiber DRG neurons with receptive fields in the glabrous skin of the rat hindpaw were C-LTMs. The electrophysiological properties of this rare subpopulation of C-fiber neurons (mean CV=0.48±0.06m/s) are distinct from those of C-fiber high threshold mechanoreceptors (HTMs). Indeed, their mean mechanical (1.7±1.1mN) and electrical (4.0±0.4V) thresholds was significantly different from that of C-HTMs. They also exhibited faster action potential and afterhyperpolarization kinetics than C-HTMs. The present study lends support to previous studies that have provided indirect evidence for the presence of C-LTMs in glabrous skin. If C-LTMs are present in human glabrous skin, they may, in this type of skin, represent a novel peripheral neuronal substrate for the pleasant/social touch sensation, and account for or contribute to touch hypersensitivity after injury.

Simultaneous Quantification of Unmyelinated Nerve Fibers in Sural Nerve and in Skin.

Peripheral polyneuropathies are common and their diagnosis may be challenging. We compared the results from sural-nerve and skin biopsies in 33 patients with a polyneuropathy and neuropathic pain examined in our hospital over a 6-year period. The biopsies were all from the same lower limb of each patient. Intraepidermal nerve fiber (IENF) densities in the skin were determined by fluorescence microscopy; unmyelinated fiber densities in sural-nerve biopsies (UFNB) were determined by electron microscopy. There was no correlation with age or gender in either biopsy type; there was a weak trend to correlation between UFNB density and IENF density, possibly because of the small sample size. The sensitivity of detection of quantitative abnormalities of unmyelinated fibers was better in the skin than in the nerves. Proximal and distal IENF densities were strongly correlated; and counts of UFNB were highly reproducible. Thus, quantification of unmyelinated fibers in sural-nerve and skin biopsies seem to be complementary. Sural-nerve biopsy may be required to confirm a specific diagnosis, to identify lesion mechanisms, and to devise therapeutic strategies, whereas skin biopsy seems to be more efficient in the follow-up of length-dependent polyneuropathies and in the diagnosis of neuropathic pain.

Perineural capsaicin induces the uptake and transganglionic transport of choleratoxin B subunit by nociceptive C-fiber primary afferent neurons.

The distribution of spinal primary afferent terminals labeled transganglionically with the choleratoxin B subunit (CTB) or its conjugates changes profoundly after perineural treatment with capsaicin. Injection of CTB conjugated with horseradish peroxidase (HRP) into an intact nerve labels somatotopically related areas in the ipsilateral dorsal horn with the exceptions of the marginal zone and the substantia gelatinosa, whereas injection of this tracer into a capsaicin-pretreated nerve also results in massive labeling of these most superficial layers of the dorsal horn. The present study was initiated to clarify the role of C-fiber primary afferent neurons in this phenomenon. In L5 dorsal root ganglia, analysis of the size frequency distribution of neurons labeled after injection of CTB-HRP into the ipsilateral sciatic nerve treated previously with capsaicin or resiniferatoxin revealed a significant increase in the proportion of small neurons. In the spinal dorsal horn, capsaicin or resiniferatoxin pretreatment resulted in intense CTB-HRP labeling of the marginal zone and the substantia gelatinosa. Electron microscopic histochemistry disclosed a dramatic, ∼10-fold increase in the proportion of CTB-HRP-labeled unmyelinated dorsal root axons following perineural capsaicin or resiniferatoxin. The present results indicate that CTB-HRP labeling of C-fiber dorsal root ganglion neurons and their central terminals after perineural treatment with vanilloid compounds may be explained by their phenotypic switch rather than a sprouting response of thick myelinated spinal afferents which, in an intact nerve, can be labeled selectively with CTB-HRP. The findings also suggest a role for GM1 ganglioside in the modulation of nociceptor function and pain.

Reduced dermal nerve fiber diameter in skin biopsies of patients with fibromyalgia.

Recent studies have provided evidence of pathology and functional abnormalities of small nerve fibers as a potential correlate of pain in the fibromyalgia syndrome. Here, we aimed to quantify dermal unmyelinated nerve fiber diameter at the electron microscopic level to find a potential morphological correlate of the functional disturbance. Thirty-two patients with fibromyalgia syndrome, 12 patients with small fiber neuropathy, and 24 healthy controls were prospectively recruited. Skin biopsies of the distal and proximal legs and index finger were taken and processed for immunofluorescence and for electron microscopy. We determined the diameter of small unmyelinated nerve fibers by measuring ten transversely cut axons of each biopsy. The mean axon diameter was reduced in patients with fibromyalgia syndrome compared with patients with small fiber neuropathy and controls (P < 0.05). Furthermore, we confirmed previous findings of disturbed small fiber function in quantitative sensory testing and of reduced intraepidermal nerve fiber density in patients with fibromyalgia. Our study provides further evidence of small fiber pathology in fibromyalgia syndrome and discloses differences compared with small fiber neuropathy, indicating that different pathomechanisms may lead to small fiber loss in the 2 disorders.

Novel finding of optic nerve central T2 hypointensity utilizing 3 Tesla MR imaging.

We sought to report a central T2 hypointensity within the optic nerve on 3 T MRI studies obtained as part of the NASA Flight Medicine Visual Impairment Intracranial Pressure Protocol that had not been described previously. Twenty-one astronauts, who had undergone MRI of both orbits with direct coronal T2 sequences between 2010 and 2012, were retrospectively included. Two of the astronauts did not have previous exposure to microgravity at the time of their scans. A central T2 hypointensity was observed in 100% of both right and left eyes. It was completely visualized throughout the nerve course in 15 right eyes (71.4%) and in 19 left eyes (90.5%).We describe a new finding seen in all study participants: a central T2 hypointensity in the epicenter of the optic nerve. We speculate that this T2 hypointensity may represent flow voids caused by the central retinal vessels.

Peri-implant bone innervation: histological findings in humans.

PURPOSE: The aim of the present study was to describe nerve fibres around osseointegrated implants in humans. MATERIALS AND METHODS: Twelve mechanically failed implants, retrieved from 10 patients were collected from three dental centres over a period of 5 years. After implant removal, decalcified semi-thin sections (0.5 µm) were stained with thionic methylene blue for light microscopic analysis. In addition, an ultrastructural analysis was performed on serial ultra-thin sections (0.06 µm) using transmission electron microscopy. RESULTS: Both myelinated and unmyelinated nerve fibres could be identified inside the Haversian canals of the osteonal bone near the implant threads. Myelinated fibres were also located at the woven bone around the implant. However, no differentiated nerve endings could be observed around the implants. CONCLUSIONS: This study shows the presence of nerve fibres in human peri-implant bone. Previous studies in animals showed that those fibres participate in the process of bone modelling and remodelling. Yet, the role of peri-implant bone innervation in the osseoperception phenomenon cannot be ruled out since the mechanism of mechanoreception in bone is not fully understood.

Selective impact of Tau loss on nociceptive primary afferents and pain sensation.

Tau protein hyperphosphorylation and consequent malfunction are hallmarks of Alzheimer's disease pathology; importantly, pain perception is diminished in these patients. In physiological conditions, Tau contributes to cytoskeletal dynamics and in this way, influences a number of cellular mechanisms including axonal trafficking, myelination and synaptic plasticity, processes that are also implicated in pain perception. However, there is no in vivo evidence clarifying the role of Tau in nociception. Thus, we tested Tau-null (Tau-/-) and Tau+/+ mice for acute thermal pain (Hargreaves' test), acute and tonic inflammatory pain (formalin test) and mechanical allodynia (Von Frey test). We report that Tau-/- animals presented a decreased response to acute noxious stimuli when compared to Tau+/+ while their pain-related behavior is augmented under tonic painful stimuli. This increased reactivity to tonic pain was accompanied by enhanced formalin-evoked c-fos staining of second order nociceptive neurons at Tau-null dorsal horn. In addition, we analyzed the primary afferents conveying nociceptive signals, estimating sciatic nerve fiber density, myelination and nerve conduction. Ultrastructural analysis revealed a decreased C-fiber density in the sciatic nerve of Tau-null mice and a hypomyelination of myelinated fibers (Aδ-fibers) - also confirmed by western blot analysis - followed by altered conduction properties of Tau-null sciatic nerves. To our knowledge, this is the first in vivo study that demonstrates that Tau depletion negatively affects the main systems conveying nociceptive information to the CNS, adding to our knowledge about Tau function(s) that might also be relevant for understanding peripheral neurological deficits in different Tauopathies.

Role of sodium channels in recovery of sciatic nerve-stretch injury in rats.

INTRODUCTION: To elucidate the mechanism of functional recovery after gradual nerve-stretch injury, we used rats in which the femur length was increased by 15 mm at 1.5 mm/day. METHODS: We performed electrophysiology, mRNA analysis of tetrodotoxin-resistant voltage-gated sodium channels (TTX-R VGSCs) in dorsal root ganglia, and histology of unmyelinated sciatic nerve fibers and examined pain thresholds at 1, 10, 20, and 30 days after cessation of lengthening. RESULTS: Electrophysiology revealed conduction block after cessation that recovered after 30 days. TTX-R VGSC levels decreased immediately after cessation but were restored after 10 (Nav1.9) or 20 (Nav1.8) days. Histology revealed that injured unmyelinated nerve fibers regenerate 30 days after cessation. Pain threshold decreased gradually during lengthening but had not recovered to the control group level after 30 days. CONCLUSIONS: Early restoration of TTX-R VGSC mRNA in dorsal root ganglia preceded functional recovery of stretched nerves before regeneration of injured unmyelinated nerve fibers.

D1-dopamine and α1-adrenergic receptors co-localize in dendrites of the rat prefrontal cortex.

Functional interactions between dopaminergic and noradrenergic systems occur in many brain areas, including the prefrontal cortex (PFC). Biochemical, electrophysiological and behavioral data indicate crosstalk between D1 dopamine receptor (D1R) and α1-adrenergic receptor (α1AR) signaling in the PFC. However, it is unknown whether these interactions occur within the same neurons, or between neurons expressing either receptor. In this study, we used electron microscopy immunocytochemistry to demonstrate that D1Rs and α1ARs co-localize in rat PFC neuronal elements, most prominently in dendrites (60-70%), but also significantly in axon terminals, unmyelinated axons and spines (∼20-30%). Our data also showed that the ratio of plasma membrane-bound to intracellular α1ARs is significantly reduced in D1R-expressing dendrites. Similar results were obtained using either a pan-α1AR or a selective α1bAR antibody to label noradrenergic receptors. Thus, these results demonstrate that D1Rs and α1ARs co-localize in PFC dendrites, thereby suggesting that the catecholaminergic effects on PFC function may be driven, at least in part, by cell-autonomous D1R-α1AR interactions.

Subtype identification in acutely dissociated rat nodose ganglion neurons based on morphologic parameters.

Nodose ganglia are composed of A-, Ah- and C-type neurons. Despite their important roles in regulating visceral afferent function, including cardiovascular, pulmonary, and gastrointestinal homeostasis, information about subtype-specific expression, molecular identity, and function of individual ion transporting proteins is scarce. Although experiments utilizing the sliced ganglion preparation have provided valuable insights into the electrophysiological properties of nodose ganglion neuron subtypes, detailed characterization of their electrical phenotypes will require measurements in isolated cells. One major unresolved problem, however, is the difficulty to unambiguously identify the subtype of isolated nodose ganglion neurons without current-clamp recording, because the magnitude of conduction velocity in the corresponding afferent fiber, a reliable marker to discriminate subtypes in situ, can no longer be determined. Here, we present data supporting the notion that application of an algorithm regarding to microscopic structural characteristics, such as neuron shape evaluated by the ratio between shortest and longest axis, neuron surface characteristics, like membrane roughness, and axon attachment, enables specific and sensitive subtype identification of acutely dissociated rat nodose ganglion neurons, by which the accuracy of identification is further validated by electrophysiological markers and overall positive predictive rates is 89.26% (90.04%, 76.47%, and 98.21% for A-, Ah, and C-type, respectively). This approach should aid in gaining insight into the molecular correlates underlying phenotypic heterogeneity of nodose ganglia. Additionally, several critical points that help for neuron identification and afferent conduction calibration are also discussed.

Ultrastructural and morphometric alterations in the aortic depressor nerve of rats due to long term experimental diabetes: effects of insulin treatment.

Most of the reports about an altered baroreflex attribute this condition to the diabetic efferent neuropathy of the aortic depressor nerve (ADN) (afferent arm of the baroreflex less explored). We evaluated the ADN ultrastructural alterations caused by long term experimental diabetes and the effects of insulin treatment. Wistar rats (N=14) received a single intravenous injection of streptozotocin (40 mg/kg) 12 weeks before the experiment. Control animals (N=9) received vehicle (citrate buffer). Insulin treated animals (N=8) received a single subcutaneous injection of insulin daily. Under pentobarbital anesthesia the ADNs were isolated and had their spontaneous activity recorded. Afterwards, proximal and distal segments of the nerves were prepared for transmission electron microscopy study. Morphometry of the unmyelinated fibers was carried out with the aid of computer software. ADN of the diabetic animals showed axonal atrophy for myelinated fibers, with more pronounced alterations of the myelin sheath, such as myelin infolding and out folding, presence of myelin balls and very thin myelin sheath in relation to the axonal size, particularly for the small myelinated fibers becoming evident. No differences were observed in myelinated fiber number and their density, as well as on the fascicular area. Unmyelinated fiber number was significantly larger in the diabetic group while fiber diameter was significantly smaller compared to control. This result suggests axonal atrophy or, if associated to the larger number of fibers present in this group, could indicate fiber sprouting. These alterations were more evident in the distal segments of the nerves and were moderated by insulin treatment.

Glutamate changes in healthy young adulthood.

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system and has been associated with several cognitive functions that are known to change with age. In rodents and humans age-related glutamate changes have been found in several brain areas. In this cross-sectional study the presence and extent of age-associated glutamate changes in the medial frontal cortex of healthy young adults were measured. Proton magnetic resonance spectroscopy ((1)H-MRS) and brain imaging were performed at 7 T in a 2 × 2 × 2 cm(3) voxel in 33 participants between 18 and 31 years old. Glutamate concentrations and grey and white matter volume could be successfully determined at an ultra-high magnetic field strength. Glutamate concentrations were lower in older individuals (0.33 mM/year). This decline is in line with grey matter thinning in the medial frontal cortex, but could not be explained by cortical thinning alone. Therefore, the decrease in glutamate in young adulthood may be due to physiological changes rather than anatomical changes.

Expression of transient receptor potential ankyrin 1 in human dental pulp.

INTRODUCTION: Transient receptor potential ankyrin 1 (TRPA1) is activated by noxious cold (<17°C) and contributes to cold and mechanical hypersensitivity after inflammation and nerve injury. METHODS: To investigate whether TRPA1 is involved in the mediation of nociception, including noxious cold and cold hypersensitivity in teeth, we examined the expression of TRPA1 and sodium channel Nav1.8 in human dental pulp using fluorescent and electron microscopic immunocytochemistry. RESULTS: TRPA1 was expressed in a large number of axons branching extensively in the peripheral pulp and in a few axons within the nerve bundles in the core of the coronal pulp and in the radicular pulp. Under electron microscopy, TRPA1 immunoreactivity was typically localized near the plasma membrane of unmyelinated axons in the peripheral pulp, suggesting that in these axons it may act as a functional receptor. The proportion of axons expressing TRPA1 in neurofilament 200-positive axons significantly increased in the painful pulp compared with the normal pulp. TRPA1 was also densely expressed in the processes and the cell body of odontoblasts. A large number of axons coexpressed TRPA1 and Nav1.8. CONCLUSIONS: These findings support the notion that TRPA1 is involved in the perception of noxious cold and cold hypersensitivity in human dental pulp and that TRPA1-mediated nociception is primarily mediated by axons and odontoblasts in the peripheral pulp.