Myelinated Peripheral Nerve Fibers Research Articles

Myelin histology: a key tool in nervous system research.

The myelin sheath is a lipoprotein-rich, multilayered structure capable of increasing conduction velocity in central and peripheral myelinated nerve fibers. Due to the complex structure and composition of myelin, various histological techniques have been developed over the centuries to evaluate myelin under normal, pathological or experimental conditions. Today, methods to assess myelin integrity or content are key tools in both clinical diagnosis and neuroscience research. In this review, we provide an updated summary of the composition and structure of the myelin sheath and discuss some histological procedures, from tissue fixation and processing techniques to the most used and practical myelin histological staining methods. Considering the lipoprotein nature of myelin, the main features and technical details of the different available methods that can be used to evaluate the lipid or protein components of myelin are described, as well as the precise ultrastructural techniques.

Rapid, automated nerve histomorphometry through open-source artificial intelligence

We aimed to develop and validate a deep learning model for automated segmentation and histomorphometry of myelinated peripheral nerve fibers from light microscopic images. A convolutional neural network integrated in the AxonDeepSeg framework was trained for automated axon/myelin segmentation using a dataset of light-microscopic cross-sectional images of osmium tetroxide-stained rat nerves including various axonal regeneration stages. In a second dataset, accuracy of automated segmentation was determined against manual axon/myelin labels. Automated morphometry results, including axon diameter, myelin sheath thickness and g-ratio were compared against manual straight-line measurements and morphometrics extracted from manual labels with AxonDeepSeg as a reference standard. The neural network achieved high pixel-wise accuracy for nerve fiber segmentations with a mean (± standard deviation) ground truth overlap of 0.93 (± 0.03) for axons and 0.99 (± 0.01) for myelin sheaths, respectively. Nerve fibers were identified with a sensitivity of 0.99 and a precision of 0.97. For each nerve fiber, the myelin thickness, axon diameter, g-ratio, solidity, eccentricity, orientation, and individual x -and y-coordinates were determined automatically. Compared to manual morphometry, automated histomorphometry showed superior agreement with the reference standard while reducing the analysis time to below 2.5% of the time needed for manual morphometry. This open-source convolutional neural network provides rapid and accurate morphometry of entire peripheral nerve cross-sections. Given its easy applicability, it could contribute to significant time savings in biomedical research while extracting unprecedented amounts of objective morphologic information from large image datasets.

Optic, trigeminal, and facial neuropathy related to anti-neurofascin 155 antibody.

ObjectiveTo characterize the frequency and patterns of optic, trigeminal, and facial nerve involvement by neuroimaging and electrophysiology in IgG4 anti‐neurofascin 155 antibody‐positive (NF155+) chronic inflammatory demyelinating polyneuropathy (CIDP).MethodsThirteen IgG4 NF155+ CIDP patients with mean onset age of 34 years (11 men) were subjected to neurological examination, blink reflex, and visual‐evoked potential (VEP) testing, and axial and/or coronal T2‐weighted head magnetic resonance imaging (MRI).ResultsAmong 13 patients, facial sensory impairment, facial weakness, and apparent visual impairment were observed in three (23.1%), two (15.4%), and two (15.4%) patients, respectively. All 12 patients tested had blink reflex abnormalities: absent and/or delayed R1 in 11 (91.7%), and absent and/or delayed R2 in 10 (83.3%). R1 latencies had strong positive correlations with serum anti‐NF155 antibody levels (r = 0.9, P ≤ 0.0001 on both sides) and distal and F wave latencies of the median and ulnar nerves. Absent and/or prolonged VEPs were observed in 10/13 (76.9%) patients and 17/26 (65.4%) eyes. On MRI, hypertrophy, and high signal intensity of trigeminal nerves were detected in 9/13 (69.2%) and 10/13 (76.9%) patients, respectively, whereas optic nerves were normal in all patients. The intra‐orbital trigeminal nerve width on coronal sections showed a significant positive correlation with disease duration.InterpretationSubclinical demyelination frequently occurs in the optic, trigeminal, and facial nerves in IgG4 NF155+ CIDP, suggesting that both central and peripheral myelin structures of the cranial nerves are involved in this condition, whereas nerve hypertrophy only develops in myelinated peripheral nerve fibers.

Different patterns in age-related morphometric alteration of myelinated fibers and capillaries of the tibial nerve: a longitudinal study in normal rats.

Age-related regression of myelinated fibers in peripheral nerves of the lower limbs is strongly influenced by capillaries and results in balance dysfunction and falls. However, the temporal relationships between alteration patterns of myelinated fibers and capillaries have not yet been clarified. This study aimed to investigate age-related morphological and histological changes of both myelinated fibers and capillaries in peripheral nerves to clarify whether myelinated fibers or capillaries change earlier. Seven male Wistar rats each were randomly selected at 20weeks (young group), 70weeks (middle group), and 97weeks (old group) for histological evaluations. The left and right tibial nerves were removed morphologically and histologically to examine myelinated fibers and capillaries. Axon diameter and myelin thickness were almost unaltered in the middle group compared with the young group but were significantly reduced in the old group when compared with the other two groups. However, the capillary diameter and number of microvascular branch points were substantially reduced in the middle group. The current study demonstrates that myelinated fibers of peripheral nerves show signs of regression in elderly rats, whereas capillaries start to reduce in middle-aged animals. In normal aging of the tibial nerve, capillaries may regress before myelinated fibers.

Morphometric analysis of peripheral myelinated nerve fibers through deep learning.

Irrespective of initial causes of neurological diseases, these disorders usually exhibit two key pathological changes-axonal loss or demyelination or a mixture of the two. Therefore, vigorous quantification of myelin and axons is essential in studying these diseases. However, the process of quantification has been labor intensive and time-consuming because of the requisite manual segmentation of myelin and axons from microscopic nerve images. As a part of AI development, deep learning has been utilized to automate certain tasks, such as image analysis. This study describes the development of a convolutional neural network (CNN)-based approach to segment images of mouse nerve cross sections. We adapted the U-Net architecture and used manually-produced segmentation data accumulated over many years in our lab for training. These images ranged from normal nerves to those afflicted by severe myelin and axon pathologies; thus, maximizing the trained model's ability to recognize atypical myelin structures. Morphometric data produced by applying the trained model to additional images were then compared to manually obtained morphometrics. The former effectively shortened the time consumption in the morphometric analysis with excellent accuracy in axonal density and g-ratio. However, we were not able to completely eliminate manual refinement of the segmentation product. We also observed small variations in axon diameter and myelin thickness within 9.5%. Nevertheless, we learned alternative ways to improve accuracy through the study. Overall, greatly increased efficiency in the CNN-based approach out-weighs minor limitations that will be addressed in future studies, thus justifying our confidence in its prospects. Note: All the relevant code is freely available at https://neurology.med.wayne.edu/drli-datashairing.

Possible mechanism underlying analgesic effect of Tuina in rats may involve piezo mechanosensitive channels within dorsal root ganglia axon

OBJECTIVETo demonstrate the analgesic effect of Tuina mainly from mechanically sensitive ion channels in peripheral myelinated nerve fibers. METHODSA total of 40 healthy and pathogen-free adult male Sprague-Dawley rats were used in the study [weight: (220.0 ± 1.4) g, Shanghai Slac Laboratory Animal Co., Ltd., Shanghai, China; license No. Shanghai ICP 05033115]. The rats were housed in cages with free access to water and food in a temperature-controlled room [(22 ± 1) °C] and 12-h/12-h light-dark cycle. Thirty-two rats were randomly divided into five groups: naive, sham, chronic compression of dorsal root ganglion (CCD), Tuina (7 d) and Tuina (21 d). CCD rat model was established via unilateral DRG compression by “L” liked steel bar. Chinese Tuina treatment was accepted once per day. Behavior monitoring of paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were tested. The expression of Piezo1 and Piezo2 in myelinated nerve fiber were analyzed by immunohistochemistry and Western-blotting. RESULTSThere was a high expression of Piezo2 and a low expression of Piezo1 in the naive and CCD groups. In contrast, the expression of Piezo2 was down regulated and Piezo1 was increased after a period of Tuina. There was significant difference (P ≤ 0.05) between the groups. CONCLUSIONOur findings suggest that Tuina therapy can increase the expression of Piezo2 and decrease the expression of Piezo1 in the test rats. The different changes in the expressions of Piezo1 and Piezo2 may play an important role in alleviating CCD-induced allodynia and hyperalgesia.

Remodeling of myelinated fibers and internal capillaries in distal peripheral nerves following aerobic exercise in aged rats.

The aim of this study was to determine whether aerobic exercise (AE) in old age contributes to improving the morphologies of myelinated fibers (MFs) in peripheral nerves as well as capillaries. Furthermore, we investigated whether such processes are associated with complementary activity of brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) in the circulating blood and peripheral nerve tissue. Fourteen male Wistar rats (age: 95 wk) were randomly divided into moderate AE ( n = 8) and sedentary (SED; n = 6) groups. Rats in the AE group performed treadmill running for 1 h per day for 2 wk, following which the bilateral tibial nerves of the two groups were removed to examine MF and capillary structure. Levels of BDNF and VEGF in the serum and peripheral nerves were analyzed via enzyme-linked immunosorbent assay. Myelin thickness, axon diameter, and capillary luminal diameter were significantly larger in the AE group than in the SED group ( P < 0.0001). Levels of serum BDNF and VEGF were significantly lower and higher, respectively, in the AE group than in the SED group ( P < 0.001). Conversely, BDNF and VEGF levels in tibial nerve tissue were significantly higher, respectively, and lower in the AE group than in the SED group ( P < 0.001). In conclusion, our study indicates that regular AE induces enlargement of the capillaries and thickens the myelin in aged peripheral nerves, likely via a complementary process involving BDNF and VEGF. NEW & NOTEWORTHY Accumulating evidence indicates that age-related sarcopenia is accompanied by the degeneration of myelinated fibers (MFs) in peripheral nerves. Our study indicates that regular aerobic exercise contributes to increased thickness of the myelin surrounding MFs and enlargement of the capillaries, likely via a complementary process involving brain-derived neurotrophic factor and vascular endothelial growth factor. Our findings demonstrate that regular, moderate-intensity aerobic exercise may help to prevent and reverse peripheral nerve regression in older adults.

A GJA9 frameshift variant is associated with polyneuropathy in Leonberger dogs

BackgroundMany inherited polyneuropathies (PN) observed in dogs have clinical similarities to the genetically heterogeneous group of Charcot-Marie-Tooth (CMT) peripheral neuropathies in humans. The canine disorders collectively show a variable expression of progressive clinical signs and ages of onset, and different breed prevalences. Previously in the Leonberger breed, a variant highly associated with a juvenile-onset PN was identified in the canine orthologue of a CMT-associated gene. As this deletion in ARHGEF10 (termed LPN1) does not explain all cases, PN in this breed may encompass variants in several genes with similar clinical and histopathological features.ResultsA genome-wide comparison of 173 k SNP genotypes of 176 cases, excluding dogs homozygous for the ARHGEF10 variant, and 138 controls, was carried out to detect further PN-associated variants. A single suggestive significant association signal on CFA15 was found. The genome of a PN-affected Leonberger homozygous for the associated haplotype was sequenced and variants in the 7.7 Mb sized critical interval were identified. These variants were filtered against a database of variants observed in 202 genomes of various dog breeds and 3 wolves, and 6 private variants in protein-coding genes, all in complete linkage disequilibrium, plus 92 non-coding variants were revealed. Five of the coding variants were predicted to have low or moderate effect on the encoded protein, whereas a 2 bp deletion in GJA9 results in a frameshift of high impact. GJA9 encodes connexin 59, a connexin gap junction family protein, and belongs to a group of CMT-associated genes that have emerged as important components of peripheral myelinated nerve fibers. The association between the GJA9 variant and PN was confirmed in an independent cohort of 296 cases and 312 controls. Population studies showed a dominant mode of inheritance, an average age of onset of approximately 6 years, and incomplete penetrance.ConclusionsThis GJA9 variant represents a highly probable candidate variant for another form of PN in Leonberger dogs, which we have designated LPN2, and a new candidate gene for CMT disease. To date, approximately every third PN-diagnosed Leonberger dog can be explained by the ARHGEF10 or GJA9 variants, and we assume that additional genetic heterogeneity in this condition exists in the breed.

Reduced axonal diameter of peripheral nerve fibers in a mouse model of Rett syndrome

Rett syndrome (RTT) is a neurological disorder characterized by motor and cognitive impairment, autonomic dysfunction and a loss of purposeful hand skills. In the majority of cases, typical RTT is caused by de novo mutations in the X-linked gene, MECP2. Alterations in the structure and function of neurons within the central nervous system of RTT patients and Mecp2-null mouse models are well established. In contrast, few studies have investigated the effects of MeCP2-deficiency on peripheral nerves. In this study, we conducted detailed morphometric as well as functional analysis of the sciatic nerves of symptomatic adult female Mecp2+/− mice. We observed a significant reduction in the mean diameter of myelinated nerve fibers in Mecp2+/− mice. In myelinated fibers, mitochondrial densities per unit area of axoplasm were significantly altered in Mecp2+/− mice. However, conduction properties of the sciatic nerve of Mecp2 knockout mice were not different from control. These subtle changes in myelinated peripheral nerve fibers in heterozygous Mecp2 knockout mice could potentially explain some RTT phenotypes.

Age-related morphological regression of myelinated fibers and capillary architecture of distal peripheral nerves in rats.

BackgroundRegression of myelinated peripheral nerve fibers in the lower extremities contributes to sarcopenia and balance dysfunction in normal aging. This subclinical regression of myelinated fibers (MFs) is heavily influenced by alterations in microvasculature, though the mechanism underlying these age-related degenerative phenomena remains unclear. The aim of the present study was to examine age-related regressions in myelinated distal peripheral nerve fibers as well as capillary architecture in rats using both morphological and histochemical methods.ResultsMFs were categorized into tertiles of ‘large’, ‘medium’, and ‘small’ sizes based on the distribution of MF diameters. A two-way ANOVA was used to assess effects of fiber size (large/medium/small) and group (young/elderly) on myelin thickness, axon diameter, myelin perimeter, axon perimeter, and G-ratio (axon diameter/fiber diameter). Significant main effects were observed for both MF size and group with respect to all dimensions except for G-ratio. Values for fiber diameter (P < 0.01), myelin thickness (P < 0.01), axon diameter (P < 0.01), myelin perimeter (P < 0.01), and axon perimeter (P < 0.01) were significantly lower than those in the young group. Additionally, mean capillary diameter and number of microvascular branch points were significantly lower in the elderly group than in the young group.ConclusionsThe present study demonstrated that spontaneous age-related regression predominantly occurs for all fiber sizes in the distal peripheral nerves and the capillary architecture. The results of the present study further suggest that both the distal MFs and capillaries in the peripheral nerve may simultaneously regress with aging.

Unravelling crucial biomechanical resilience of myelinated peripheral nerve fibres provided by the Schwann cell basal lamina and PMP22.

There is an urgent need for the research of the close and enigmatic relationship between nerve biomechanics and the development of neuropathies. Here we present a research strategy based on the application atomic force and confocal microscopy for simultaneous nerve biomechanics and integrity investigations. Using wild-type and hereditary neuropathy mouse models, we reveal surprising mechanical protection of peripheral nerves. Myelinated peripheral wild-type fibres promptly and fully recover from acute enormous local mechanical compression while maintaining functional and structural integrity. The basal lamina which enwraps each myelinated fibre separately is identified as the major contributor to the striking fibre's resilience and integrity. In contrast, neuropathic fibres lacking the peripheral myelin protein 22 (PMP22), which is closely connected with several hereditary human neuropathies, fail to recover from light compression. Interestingly, the structural arrangement of the basal lamina of Pmp22−/− fibres is significantly altered compared to wild-type fibres. In conclusion, the basal lamina and PMP22 act in concert to contribute to a resilience and integrity of peripheral nerves at the single fibre level. Our findings and the presented technology set the stage for a comprehensive research of the links between nerve biomechanics and neuropathies.

The impact of internodal segmentation in biophysical nerve fiber models

Implementation of double cable models to simulate the behavior of myelinated peripheral nerve fibers requires defining a segmentation of the internode between successive nodes of Ranvier. The number of internodal segments is a model parameter that is not well agreed on, with values in the literature ranging from 1 to more than 500. Moreover, a lot of studies also lack a sensitivity study or a rationale behind the implementation used. In a model of a myelinated nerve fiber developed in our group, the segmentation scheme (i.e., the number of segments and their individual morphology) strongly influenced model outcomes such as action potential shape and velocity, stimulation threshold and absolute refractory period. In the present study these influences were investigated systematically in homogeneous neurons with different diameters. Uniformly segmented internodes were found to require several hundreds of segments (and associated computational power) to reach model outcomes differing by less than 1% from the asymptotic value. In fact, in the majority of segmentation schemes the main determinant is not the number of segments, but the length λ of the internodal segments directly adjacent to the nodes of Ranvier. If λ is larger than approximately 10 μm, model outcomes for the tested fibers are almost independent of the total number of segments. Furthermore, λ can be optimized to enable models using just three segments per internode, to reach physiologically relevant model outcomes with limited computational resources. However, to study anatomical or physiological details of the internode itself, an appropriately detailed segmentation scheme is crucial.

Segmentation of nerve fibers using multi-level gradient watershed and fuzzy systems

This paper presents an algorithm based on multi-level watershed segmentation combined with three fuzzy systems to segment a large number of myelinated nerve fibers in microscope images. The method can estimate various geometrical parameters of myelinated nerve fibers in peripheral nerves. It is expected to be a promising tool for the quantitative assessment of myelinated nerve fibers in related research. A novel multi-level watershed scheme iteratively detects pre-candidate nerve fibers. At each immersion level, watershed segmentation extracts the initial axon locations and obtains meaningful myelinated nerve fiber features. Thereafter, according to a priori characteristics of the myelinated nerve fibers, fuzzy rules reject unlikely pre-candidates and collect a set of candidates. Initial candidate boundaries are then refined by a fuzzy active contour model, which flexibly deforms contours according to the observed features of each nerve fiber. A final scan with a different set of fuzzy rules based on the a priori properties of the myelinated nerve fibers removes false detections. A particle swarm optimization method is employed to efficiently train the large number of parameters in the proposed fuzzy systems. The proposed method can automatically segment the transverse cross-sections of nerve fibers obtained from optical microscope images. Although the microscope image is usually noisy with weak or variable levels of contrast, the proposed system can handle images with a large number of myelinated nerve fibers and achieve a high fiber detection ratio. As compared to manual segmentation by experts, the proposed system achieved an average accuracy of 91% across different data sets. We developed an image segmentation system that automatically handles myelinated nerve fibers in microscope images. Experimental results showed the efficacy of this system and its superiority to other nerve fiber segmentation approaches. Moreover, the proposed method can be extended to other applications of automatic segmentation of microscopic images.

Sustained increase in the excitability of myelinated peripheral axons to depolarizing current is mediated by Nav1.6

Changes in the excitability of peripheral myelinated axons in response to long-lasting subthreshold depolarizing or hyperpolarizing currents (threshold electrotonus) are used as a complementary electrophysiological parameter in the study of peripheral nerve diseases in people. However, the contribution made by various axonal ion channels to specific components of threshold electrotonus remains incompletely understood. In this study, we have recorded threshold electrotonus responses from isolated nerve segments of sural nerve from control and Scn8amed mice, which lack functional Nav1.6 voltage-gated sodium channel. In med mice, the increase in axonal excitability produced by application of subthreshold depolarizing currents for 100–200ms was not sustained. In contrast, there was no difference in threshold electrotonus responses to subthreshold hyperpolarizing current application between Scn8amed and control mice. These data reveal the specific functional role of an identified subtype of voltage-gated sodium channel (Nav1.6) in mediating the depolarizing threshold electrotonus response of peripheral myelinated nerve fibers.

Sodium‐dependent vitamin C transporter 2 (SVCT2) is necessary for the uptake of L‐ascorbic acid into Schwann cells

Ascorbic acid has been shown to be an essential component for in vitro myelination and to improve the clinical and pathological phenotype of a mouse model of Charcot-Marie-tooth disease 1A. The mechanism of ascorbic acid uptake into peripheral nerves, however, has not been addressed so far. Hence, we studied the expression and activity of sodium-dependent vitamin C transporters 1 and 2 (SVCT1 and 2) in the peripheral nervous system. Using immunohistochemistry, immunoblotting, and reverse transcription PCR, we could show that SVCT1 and 2 were differentially expressed in myelinated peripheral nerve fibers and Schwann cell (SC) cultures. SVCT1 was expressed at very low levels confined to the axons, whereas SVCT2 was highly expressed both in the axons and in the SCs. SVCT2 was localized particularly in SC compartments of uncompacted myelin. Uptake assays using (14)C-labeled ascorbic acid showed transport of ascorbic acid into SC cultures. Ascorbic acid transport was dependent on the concentration of sodium, magnesium, and calcium in the extracellular medium. Treatment with the flavonoid phloretin, a known inhibitor of SVCT1 and 2, and specific RNA interference with SVCT2 caused significant reductions in ascorbic acid uptake into SCs. Phloretin-inhibited uptake of ascorbic acid was further shown in freshly dissected, cell-culture-naïve rat sciatic nerves. These results provide evidence for the first time that uptake of ascorbic acid in the peripheral nervous system is crucially dependent on the expression and activity of SVCT2.

Determining heat and mechanical pain threshold in inflamed skin of human subjects

In a previous article in the Journal of Visualized Experiments we have demonstrated skin microdialysis techniques for the collection of tissue-specific nociceptive and inflammatory biochemicals in humans. In this article we will show pain-testing paradigms that are often used in tandem with microdialysis procedures. Combining pain tests with microdialysis provides the critical link between behavioral and biochemical data that allows identifying key biochemicals responsible for generating and propagating pain. Two models of evoking pain in inflamed skin of human study participants are shown. The first model evokes pain with aid of heat stimuli. Heat evoked pain as described here is predominantly mediated by small, non-myelinated peripheral nociceptive nerve fibers (C-fibers). The second model evokes pain via punctuated pressure stimuli. Punctuated pressure evoked pain is predominantly mediated by small, myelinated peripheral nociceptive nerve fibers (A-delta fibers). The two models are mechanistically distinct and independently examine nociceptive processing by the two major peripheral nerve fiber populations involved in pain signaling. Heat pain is evoked with aid of the TSA II, a commercially available thermo-sensory analyzer (Medoc Advanced Medical Systems, Durham, NC). Stimulus configuration and delivery is handled with aid of specific software. Thermodes vary in size and shape but in principle consist of a metal plate that can be heated or cooled at various rates and for different periods of time. Algorithms assessing heat-evoked pain are manifold. In the experiments shown here, study participants are asked to indicate at what point they start experiencing pain while the thermode in contact with skin is heated at a predetermined rate starting at a temperature that does not evoke pain. The thermode temperature at which a subject starts experiencing pain constitutes the heat pain threshold. Mechanical pain is evoked with punctuated probes. Such probes are commercially available from several manufacturers (von Frey hairs). However, the accuracy of von Frey hairs has been criticized and many investigators use custom made punctuated pressure probes. In the experiments shown here eight custom-made punctuated probes of different weights are applied in consecutive order, a procedure called up-down algorithm, to identify perceptional deflection points, i.e., a change from feeling no pain to feeling pain or vice versa. The average weight causing a perceptional deflection constitutes the mechanical pain threshold.

Determining heat and mechanical pain threshold in inflamed skin of human subjects

In a previous article in the Journal of Visualized Experiments we have demonstrated skin microdialysis techniques for the collection of tissue-specific nociceptive and inflammatory biochemicals in humans. In this article we will show pain-testing paradigms that are often used in tandem with microdialysis procedures. Combining pain tests with microdialysis provides the critical link between behavioral and biochemical data that allows identifying key biochemicals responsible for generating and propagating pain. Two models of evoking pain in inflamed skin of human study participants are shown. The first model evokes pain with aid of heat stimuli. Heat evoked pain as described here is predominantly mediated by small, non-myelinated peripheral nociceptive nerve fibers (C-fibers). The second model evokes pain via punctuated pressure stimuli. Punctuated pressure evoked pain is predominantly mediated by small, myelinated peripheral nociceptive nerve fibers (A-delta fibers). The two models are mechanistically distinct and independently examine nociceptive processing by the two major peripheral nerve fiber populations involved in pain signaling. Heat pain is evoked with aid of the TSA II, a commercially available thermo-sensory analyzer (Medoc Advanced Medical Systems, Durham, NC). Stimulus configuration and delivery is handled with aid of specific software. Thermodes vary in size and shape but in principle consist of a metal plate that can be heated or cooled at various rates and for different periods of time. Algorithms assessing heat-evoked pain are manifold. In the experiments shown here, study participants are asked to indicate at what point they start experiencing pain while the thermode in contact with skin is heated at a predetermined rate starting at a temperature that does not evoke pain. The thermode temperature at which a subject starts experiencing pain constitutes the heat pain threshold. Mechanical pain is evoked with punctuated probes. Such probes are commercially available from several manufacturers (von Frey hairs). However, the accuracy of von Frey hairs has been criticized and many investigators use custom made punctuated pressure probes. In the experiments shown here eight custom-made punctuated probes of different weights are applied in consecutive order, a procedure called up-down algorithm, to identify perceptional deflection points, i.e., a change from feeling no pain to feeling pain or vice versa. The average weight causing a perceptional deflection constitutes the mechanical pain threshold.

The effects of oxaliplatin, an anticancer drug, on potassium channels of the peripheral myelinated nerve fibres of the adult rat

Oxaliplatin is a novel chemotherapeutic agent which is effective against advanced colorectal cancer, but at the same time causes severe neuropathy in the peripheral nerve fibres, affecting mainly the voltage-gated sodium (Na +) channels (VGNaCs), according to literature. In this study the effects of oxaliplatin on the peripheral myelinated nerve fibres (PMNFs) were investigated in vitro using the isolated sciatic nerve of the adult rat. The advantage of this nerve-preparation was that stable in amplitude evoked compound action potentials (CAP) were recorded for over 1000 min. Incubation of the sciatic nerve fibres in 25, 100 and 500 μM oxaliplatin, for 300–700 min caused dramatic distortion of the waveform of the CAP, namely broadening the repolarization phase, repetitive firing and afterhyperpolarization (AHP), related to the malfunction of voltage-gated potassium (K +) channels (VGKCs). At a concentration of 5 μM, oxaliplatin caused broadening of the repolarization phase of the CAP only, while the no observed effect concentration was estimated to be 1 μM. These findings are indicative of severe effects of oxaliplatin on the VGKCs. In contrast, the amplitude and the rise-time of the depolarization of the CAP did not change significantly, a clear indication that the VGNaCs of the particular nerve preparation were not affected by oxaliplatin. The effects of oxaliplatin on the PMNFs were similar to those of 4-aminopyridine (4-AP), a classical antagonist of VGKCs. These similarities in the pattern of action between oxaliplatin and 4-AP combined with the fact that the effects of oxaliplatin were more pronounced and developed at lower concentrations suggest that oxaliplatin acts as a potent VGKCs antagonist.

Binding of ɛ-toxin from Clostridium perfringens in the nervous system

Epsilon-toxin (ɛ-toxin), produced by Clostridium perfringens type D, is the main agent responsible for enterotoxaemia in livestock. Neurological disorders are a characteristic of the onset of toxin poisoning. ɛ-Toxin accumulates specifically in the central nervous system, where it produces a glutamatergic-mediated excitotoxic effect. However, no detailed study of putative binding structures in the nervous tissue has been carried out to date. Here we attempt to identify specific acceptor moieties and cell targets for ɛ-toxin, not only in the mouse nervous system but also in the brains of sheep and cattle. An ɛ-toxin-GFP fusion protein was produced and used to incubate brain sections, which were then analyzed by confocal microscopy. The results clearly show specific binding of ɛ-toxin to myelin structures. ɛ-Prototoxin-GFP and ɛ-toxin-GFP, the inactive and active forms of the toxin, respectively, showed identical results. By means of pronase E treatment, we found that the binding was mainly associated to a protein component of the myelin. Myelinated peripheral nerve fibres were also stained by ɛ-toxin. Moreover, the binding to myelin was not only restricted to rodents, but was also found in humans, sheep and cattle. Curiously, in the brains of both sheep and cattle, the toxin strongly stained the vascular endothelium, a result that may explain the differences in potency and effect between species. Although the binding of ɛ-toxin to myelin does not directly explain its neurotoxic effect, this feature opens up a new line of enquiry into its mechanism of toxicity and establishes the usefulness of this toxin for the study of the mammalian nervous system.

Discriminative touch and emotional touch.

Somatic sensation comprises four main modalities, each relaying tactile, thermal, painful, or pruritic (itch) information to the central nervous system. These input channels can be further classified as subserving a sensory function of spatial and temporal localization, discrimination, and provision of essential information for controlling and guiding exploratory tactile behaviours, and an affective function that is widely recognized as providing the afferent neural input driving the subjective experience of pain, but not so widely recognized as also providing the subjective experience of affiliative or emotional somatic pleasure of touch. The discriminative properties of tactile sensation are mediated by a class of fast-conducting myelinated peripheral nerve fibres--A-beta fibres--whereas the rewarding, emotional properties of touch are hypothesized to be mediated by a class of unmyelinated peripheral nerve fibres--CT afferents (C tactile)--that have biophysical, electrophysiological, neurobiological, and anatomical properties that drive the temporally delayed emotional somatic system. CT afferents have not been found in the glabrous skin of the hand in spite of numerous electrophysiological explorations of this area. Hence, it seems reasonable to conclude that they are lacking in the glabrous skin. A full understanding of the behavioural and affective consequences of the differential innervation of CT afferents awaits a fuller understanding of their function.