Are Human Peripheral Nerves Sensitive To X-ray Imaging?

Diagnostic imaging techniques play an important role in assessing the exact location, cause, and extent of a nerve lesion, thus allowing clinicians to diagnose and manage more effectively a variety of pathological conditions, such as entrapment syndromes, traumatic injuries, and space-occupying lesions. Ultrasound and nuclear magnetic resonance imaging are becoming useful methods for this purpose, but they still lack spatial resolution. In this regard, recent phase contrast x-ray imaging experiments of peripheral nerve allowed the visualization of each nerve fiber surrounded by its myelin sheath as clearly as optical microscopy. In the present study, we attempted to produce high-resolution x-ray phase contrast images of a human sciatic nerve by using synchrotron radiation propagation-based imaging. The images showed high contrast and high spatial resolution, allowing clear identification of each fascicle structure and surrounding connective tissue. The outstanding result is the detection of such structures by phase contrast x-ray tomography of a thick human sciatic nerve section. This may further enable the identification of diverse pathological patterns, such as Wallerian degeneration, hypertrophic neuropathy, inflammatory infiltration, leprosy neuropathy and amyloid deposits. To the best of our knowledge, this is the first successful phase contrast x-ray imaging experiment of a human peripheral nerve sample. Our long-term goal is to develop peripheral nerve imaging methods that could supersede biopsy procedures.

Capsaicin-sensitive nerves and neurokinins modulate non-neuronal nNOS expression in lung

We investigated the effects of substance P (SP) and neurokinin A (NKA) infusion and acute stimulation of capsaicin-sensitive sensory nerves fibers (CAP) on lung recruitment of neuronal nitric oxide synthase (nNOS)-positive inflammatory and respiratory sepithelial (RE) cells in guinea-pigs. We evaluated if the effects of CAP stimulation were maintained until 14 days and had functional pulmonary repercussions. After 24 h of CAP and 30 min after SP and NKA infusions there was an increase in nNOS-positive eosinophils and mononuclear cells compared to controls (P < 0.05). SP group presented an increase in nNOS-positive RE (P < 0.05). After 14 days of CAP stimulation, there was a reduction in resistance (R-rs) and elastance (E-rs) of respiratory system in capsaicin pre-treated animals. We noticed a correlation between nNOS-positive eosinophils (R = -0.644, P < 0.05) and mononuclear cells (R = -0.88, P < 0.001) and R-rs. Concluding, CAP and neurokinins increase nNOS expression by inflammatory and RE cells. The increase in nNCS expression induced by low and high doses stimulation of CAP is longstanding and correlated to pulmonary mechanical repercussions. (c) 2007 Elsevier B.V. All rights reserved.

Effect of hyperbaric oxygen on the regeneration of experimental crush injuries of nerves

Hyperbaric oxygen has been successfully used on treatment of acute ischemic injuries involving soft tissues and chronic injuries. In nerve crush injuries, the mechanisms involved are very similar to those found in ischemic injuries. Consequently, it is logical to hypothesize that hyperbaric oxygen should improve nerve repair, which is a critical step on functional recovery. In the present study, we created standard nerve crush injuries on sciatic nerves of rats, which underwent treatment with hyperbaric oxygen. Results were assessed by functional evaluation using walking-track analysis. The functional recovery indexes observed did not differ from control group. We concluded that hyperbaric oxygen therapy, in the schedule used, had no influence on functional recovery after nerve crush injuries.

Endogenous opioid peptides in parasympathetic, sympathetic and sensory nerves in the guinea-pig heart.

Research has suggested that exogenous opioid substances can have direct effects on cardiac muscle or influence neurotransmitter release via presynaptic modulation of neuronal inputs to the heart. In the present study, multiple-labelling immunohistochemistry was employed to determine the distribution of endogenous opioid peptides within the guinea-pig heart. Approximately 40% of cardiac ganglion cells contained immunoreactivity for dynorphin A (1-8), dynorphin A (1-17) and dynorphin B whilst 20% displayed leu-enkephalin immunoreactivity. Different populations of opioid-containing ganglion cells were identified according to the co-existence of opioid immunoreactivity with immunoreactivity for somatostatin and neuropeptide Y. Immunoreactivity for prodynorphin-derived peptides was observed in many sympathetic axons in the heart and was also observed, though to a lesser extent, in sensory axons. Leu-enkephalin immunoreactivity was observed in occasional sympathetic and sensory axons. No immunoreactivity was observed for met-enkephalin-arg-gly-leu or for beta-endorphin. These results demonstrate that prodynorphin-derived peptides are present in parasympathetic, sympathetic and sensory nerves within the heart, but suggest that only the prodynorphin gene is expressed in guinea-pig cardiac nerves. This study has shown that endogenous opioid peptides are well placed to regulate cardiac function via both autonomic and sensory pathways.

Myotonic dystrophy transgenic mice exhibit pathologic abnormalities in diaphragm neuromuscular junctions and phrenic nerves

Myotonic dystrophy Type 1 (DM-1) is caused by abnormal expansion of a (CTG) repeat located in the DM protein kinase gene. Respiratory problems have long been recognized to be a major feature of this disorder. Because respiratory failure can be associated with dysfunction of phrenic nerves and diaphragm muscle, we examined the diaphragm and respiratory neural network in transgenic mice carrying the human genomic DM-1 region with expanded repeats of more than 300 CTG, a valid model of the human disease. Morphologic and morphometric analyses revealed distal denervation of diaphragm neuromuscular junctions in DM-1 transgenic mice indicated by a decrease in the size and shape complexity of end-plates and a reduction in the concentration of acetyl choline receptors on the postsynaptic membrane. More importantly, there was a significant reduction in numbers of unmyelinated, but not of myelinated, fibers in DM-1 phrenic nerves; no morphologic alternations of the nerves or loss of neuronal cells were detected in medullary respiratory centers or cervical phrenic motor neurons. Because neuromuscular junctions are involved in action potential transmission and the afferent phrenic unmyelinated fibers control the inspiratory activity, our results suggest that the respiratory impairment associated with DM-1 may be partially due to pathologic alterations in neuromuscular junctions and phrenic nerves.

Pathology of intracardiac nerves in experimental Chagas disease

Severe destruction of intrinsic cardiac nerves has been reported in experimental acute Chagas myocarditis, followed by extensive regeneration during the chronic phase of the infection. To further study this subject, the sympathetic and para-sympathetic intracardiac nerves of mice infected with a virulent Trypanosoma cruzi strain were analyzed, during acute and chronic infection, by means of histological, histochemical, morphometric and electron microscopic techniques. No evidences of destructive changes were apparent. Histochemical demonstration for acetylcholinesterase and catecholamines did not reveal differences in the amount and distribution of intracardiac nerves, in mice with acute and chronic Chagas myocarditis or in non-infected controls. Mild, probably reversible ultrastructural neural changes were occasionally present, especially during acute myocarditis. Intrinsic nerves appeared as the least involved cardiac structure during the course of experimental Chagas disease in mice.

New sonographic measures of peripheral nerves: a tool for the diagnosis of peripheral nerve involvement in leprosy

To evaluate ultrasonographic (US) cross-sectional areas (CSAs) of peripheral nerves, indexes of the differences between CSAs at the same point (∆CSAs) and between tunnel (T) and pre-tunnel (PT) ulnar CSAs (∆TPTs) in leprosy patients (LPs) and healthy volunteers (HVs). Seventy-seven LPs and 49 HVs underwent bilateral US at PT and T ulnar points, as well as along the median (M) and common fibular (CF) nerves, to calculate the CSAs, ∆CSAs and ∆TPTs. The CSA values in HVs were lower than those in LPs (p < 0.0001) at the PT (5.67/9.78 mm2) and T (6.50/10.94 mm2) points, as well as at the M (5.85/8.48 mm2) and CF (8.17/14.14 mm2) nerves. The optimum CSA- receiver operating characteristic (ROC) points and sensitivities/specificities were, respectively, 6.85 mm2 and 68-85% for the PT point, 7.35 mm2 and 71-78% for the T point, 6.75 mm2 and 62-75% for the M nerve and 9.55 mm2 and 81-72% for the CF nerve. The ∆CSAs of the LPs were greater than those of the HVs at the PT point (4.02/0.85; p = 0.007), T point (3.71/0.98; p = 0.0005) and CF nerve (2.93/1.14; p = 0.015), with no difference found for the M nerve (1.41/0.95; p = 0.17). The optimum ∆CSA-ROC points, sensitivities, specificities and p-values were, respectively, 1.35, 49%, 80% and 0.003 at the PT point, 1.55, 55-85% and 0.0006 at the T point, 0.70, 58-50% and 0.73 for the M nerve and 1.25, 54-67% and 0.022 for the CF nerve. The ∆TPT in the LPs was greater than that in the HVs (4.43/1.44; p <0.0001). The optimum ∆TPT-ROC point was 2.65 (90% sensitivity/41% specificity, p < 0.0001). The ROC analysis of CSAs showed the highest specificity and sensitivity at the PT point and CF nerve, respectively. The PT and T ∆CSAs had high specificities (> 80%) and ∆TPT had the highest specificity (> 90%). New sonographic peripheral nerve measurements (∆CSAs and ∆TPT) provide an important methodological improvement in the detection of leprosy neuropathy.

Critical role for the lactate transporter, monocarboxylate transporter 1 (mct1), in the regeneration of peripheral nerves

Axons, and particularly regenerating axons, have high metabolic needs in order to maintain critical functions such as axon transport and membrane depolarization. Though some of the required energy likely comes form extracellular glucose and ATP generated in the soma, we and others hypothesize that some of the energy may be supplied by lactate. Unlike glucose that requires glycolytic enzymes to produce pyruvate, lactate can be converted directly to pyruvate by lactate dehydrogenase and transported into mitochondria for oxidative metabolism. In order to be transported into or out of cells, lactate requires specific monocarboxylate transporters (MCTs), the most abundant of which is MCT1. If MCT1 and lactate are critical for nerve function and regeneration, we hypothesize that MCT1 heterozygote null mice, which appear phenotypically normal despite having approximately 40% MCT1 as compared to wildtype littermate mice, would have reduced capacity for repair following nerve injury. To investigate this, adult MCT1 heterozygote null mice or wild-type mice underwent unilateral sciatic nerve crush in the proximal thigh. We found that regeneration of the sciatic nerve, as measured by recovery of compound muscle action potentials (CMAP) in the lateral plantar muscles following proximal sciatic nerve stimulation, was delayed from a median of 21 days in wildtype mice to 38.5 days in MCT1 heterozygote mice. In fact, half of the MCT1 heterozygote null mice had no recovery of CMAP by the endpoint of the study at 42 days, while all of the wild-type mice had recovered. In addition, the maximal amplitude of CMAP recovery in MCT1 heterozygote mull mice was reduced from a mean of 3 mV to 0.5 mV. As would be expected, the denervated gastrocnemius muscle of MCT1 heterozygote null mice remained atrophic at 42 days compared to wild-type mice. Our experiments show that lactate supplied through MCT1 is necessary for nerve regeneration. Experiments are underway to determine whether loss of MCT1 prevents nerve regrowth directly due to reduced energy supply to axons or indirectly by dysfunctional Schwann cells normally dependent on lactate supply through MCT1.

Magnetization transfer-based 3D visualization of foot peripheral nerves.

PURPOSE: To investigate magnetization transfer (MT) effects as a new source of contrast for imaging and tracking of peripheral foot nerves. MATERIALS AND METHODS: Two sets of 3D spoiled gradient-echo images acquired with and without a saturation pulse were used to generate MT ratio (MTR) maps of 260 μm in-plane resolution for eight volunteers at 3T. Scan parameters were adjusted to minimize signal loss due to T2 dephasing, and a dedicated coil was used to improve the inherently low signal-to-noise ratio of small voxels. Resulting MTR values in foot nerves were compared with those in surrounding muscle tissue. RESULTS: Average MTR values for muscle (45.5 ± 1.4%) and nerve (21.4 ± 3.1%) were significantly different (P < 0.0001). In general, the difference in MTR values was sufficiently large to allow for intensity-based segmentation and tracking of foot nerves in individual subjects. This procedure was termed MT-based 3D visualization. CONCLUSION: The MTR serves as a new source of contrast for imaging of peripheral foot nerves and provides a means for high spatial resolution tracking of these structures. The proposed methodology is directly applicable on standard clinical MR scanners and could be applied to systemic pathologies, such as diabetes.

Beta-adrenoceptor stimulation increases neuropeptide Y release from sympathetic nerves in intact rats.

This study was undertaken to assess in conscious normotensive rats the effects of beta-adrenoceptor stimulation on plasma neuropeptide Y (NPY) levels. Wistar rats were subjected to adrenal demedullation on the right side and were either adrenalectomized or sham-operated on the left side. Eleven days later, the conscious rats were infused i.v. for 30 min with either isoproterenol (10 ng/min) or its vehicle. Plasma NPY levels were significantly lower (23.8 +/- 2.6 pM, means +/- S.E.M., n = 12, P < 0.01) in vehicle-treated medullectomized rats than in corresponding sham-operated controls (36.7 +/- 4.1 pM, n = 12). The medullectomized rats infused with isoproterenol showed plasma NPY levels (36.7 +/- 3.3 pM, n = 11) comparable to those of sham-operated rats having received the vehicle. These data therefore demonstrate that plasma NPY levels are lower in rats without adrenal medulla and that in these animals isoproterenol increases NPY release, most likely by activating pre-synaptic beta-adrenoceptors.

Deficiency in monocarboxylate transporter 1 (MCT1) in mice delays regeneration of peripheral nerves following sciatic nerve crush.

Peripheral nerve regeneration following injury occurs spontaneously, but many of the processes require metabolic energy. The mechanism of energy supply to axons has not previously been determined. In the central nervous system, monocarboxylate transporter 1 (MCT1), expressed in oligodendroglia, is critical for supplying lactate or other energy metabolites to axons. In the current study, MCT1 is shown to localize within the peripheral nervous system to perineurial cells, dorsal root ganglion neurons, and Schwann cells by MCT1 immunofluorescence in wild-type mice and tdTomato fluorescence in MCT1 BAC reporter mice. To investigate whether MCT1 is necessary for peripheral nerve regeneration, sciatic nerves of MCT1 heterozygous null mice are crushed and peripheral nerve regeneration was quantified electrophysiologically and anatomically. Compound muscle action potential (CMAP) recovery is delayed from a median of 21days in wild-type mice to greater than 38days in MCT1 heterozygote null mice. In fact, half of the MCT1 heterozygote null mice have no recovery of CMAP at 42days, while all of the wild-type mice recovered. In addition, muscle fibers remain 40% more atrophic and neuromuscular junctions 40% more denervated at 42days post-crush in the MCT1 heterozygote null mice than wild-type mice. The delay in nerve regeneration is not only in motor axons, as the number of regenerated axons in the sural sensory nerve of MCT1 heterozygote null mice at 4weeks and tibial mixed sensory and motor nerve at 3weeks is also significantly reduced compared to wild-type mice. This delay in regeneration may be partly due to failed Schwann cell function, as there is reduced early phagocytosis of myelin debris and remyelination of axon segments. These data for the first time demonstrate that MCT1 is critical for regeneration of both sensory and motor axons in mice following sciatic nerve crush.

Long-term in vivo regeneration of peripheral nerves through bioengineered nerve grafts.

Although autologous nerve graft is still the first choice strategy in nerve reconstruction, it has the severe disadvantage of the sacrifice of a functional nerve. Cell transplantation in a bioartificial conduit is an alternative strategy to improve nerve regeneration. Nerve fibrin conduits were seeded with various cell types: primary Schwann cells (SC), SC-like differentiated bone marrow-derived mesenchymal stem cells (dMSC), SC-like differentiated adipose-derived stem cells (dASC). Two further control groups were fibrin conduits without cells and autografts. Conduits were used to bridge a 1 cm rat sciatic nerve gap in a long term experiment (16 weeks). Functional and morphological properties of regenerated nerves were investigated. A reduction in muscle atrophy was observed in the autograft and in all cell-seeded groups, when compared with the empty fibrin conduits. SC showed significant improvement in axon myelination and average fiber diameter of the regenerated nerves. dASC were the most effective cell population in terms of improvement of axonal and fiber diameter, evoked potentials at the level of the gastrocnemius muscle and regeneration of motoneurons, similar to the autografts. Given these results and other advantages of adipose derived stem cells such as ease of harvest and relative abundance, dASC could be a clinically translatable route towards new methods to enhance peripheral nerve repair.

Stimulus-response curve of human motor nerves: multicenter assessment of various indexes.

The value of various indexes to characterize the stimulus-response curve of human motor nerves was assessed in 40 healthy subjects recruited from four European centers of investigation (Créteil, Lausanne, Liège, Marseille). Stimulus-response curves were established by stimulating the right median and ulnar motor nerves at the wrist, with stimulus durations of 0.05 and 0.5 ms. The following parameters were studied: the threshold intensity of stimulation to obtain 10% (I 10), 50% (I 50), and 90% (I 90) of the maximal compound muscle action potential, the ratios I 10/I 50, I 90/I 50, (I 90 - I 10)/I 10, (I 90-I 50)/I 50, and (I 50 - I 10)/I 10, and the slopes of the stimulus-response curves with or without normalization to I 50. For each parameter, within-center variability and reproducibility (in a test-retest study) were assessed and between-center comparisons were made. For most of the parameters, the results varied significantly within and between the centers. Within the centers, only the ratios I 10/I 50 and I 90/I 50 were found constant and reproducible. Between the centers, the absolute intensity thresholds (I 10, I 50, I 90) and the ratio I 90/I 50 did not show significant differences at stimulus duration of 0.5 ms, whatever the stimulated nerve. The reduced variability and good reproducibility of the ratios I 10/I 50 and I 90/I 50 open perspectives in neurophysiological practice for the use of these indexes of the stimulus-response curve, a rapid and noninvasive test.

Fast Blue and Diamidino Yellow as retrograde tracers in peripheral nerves: efficacy of combined nerve injection and capsule application to transected nerves in the adult rat

Capsule application of Diamidino Yellow (DY) to the cut end of the sciatic nerve immediately followed by capsule application of Fast Blue (FB) resulted in approximate to 95% double-labelled dorsal root ganglion neurones (DRGn) and motoneurones (Mn). Nerve injection of DY followed either immediately or 2 months later by capsule application of FB resulted in approximate to 90% double-labelled DRGn and Mn, indicating that DY and FB label similar populations of DRGn and Mn, and that insignificant DY fading occurred during this period. Inversing the order of application, however, i.e. nerve injection of FB followed immediately by capsule application of DY, resulted in double labelling in only approximate to 10% of the DRGn and Mn. These percentages increased to 70% of the DRGn and 60% of the Mn when the FB injection was followed 1 or 2 months after by the DY application, indicating that DY uptake is blocked by recent administration of FB. The results indicate that DY and FB might be useful for sequential labelling before and after nerve injury as a tool to investigate the accuracy of sensory and motor regeneration.