Combining micro-computed tomography with histology to analyze biomedical implants for peripheral nerve repair.

BACKGROUND: Biomedical implants used in tissue engineering repairs, such as scaffolds to repair peripheral nerves, can be too large to examine completely with histological analyses. Micro-computed tomography (micro-CT) with contrast agents allows ex vivo visualization of entire biomaterial implants and their interactions with tissues, but contrast agents can interfere with histological analyses of the tissues or cause shrinkage or loss of antigenicity. NEW METHOD: Soft tissue, ex vivo micro-CT imaging using Lugol's iodine was compatible with histology after using a rapid (48 h) method of removing iodine. RESULTS: Adult normal and repaired rat sciatic nerves were infiltrated ex vivo with iodine, imaged with micro-CT and then the iodine was removed by incubating tissues in sodium thiosulfate. Subsequent paraffin sections of normal nerve tissues showed no differences in staining with hematoxylin and eosin or immunostaining with multiple antibodies. Iodine treatment and removal did not alter axonal diameter, nuclear size or relative area covered by immunostained axons (p>0.05). Combining imaging modalities allowed comparisons of macroscopic and microscopic features of nerve tissues regenerating through simple nerve conduits or nerve conduits containing a titanium wire for guidance. COMPARISON WITH EXISTING METHODS: Quantification showed that treatment with iodine and sodium thiosulfate did not result in tissue shrinkage or loss of antigenicity. CONCLUSIONS: Because this combination of treatments is rapid and does not alter tissue morphology, this expands the ex vivo methods available to examine the success of biomaterial implants used for tissue engineering repairs.

An in vitro study of peptide-loaded alginate nanospheres for antagonizing the inhibitory effect of Nogo-A protein on axonal growth.

The adult mammalian central nervous system has limited ability to regenerate after injury. This is due, in part, to the presence of myelin-associated axon growth inhibitory proteins such as Nogo-A that bind and activate the Nogo receptor, leading to profound inhibition of actin-based motility within the growing axon tip. This paper presents an in vitro study of the use of a Nogo receptor-blocking peptide to antagonize the inhibitory effect of Nogo-A on axon growth. Alginate nanospheres were fabricated using an emulsion technique and loaded with Nogo receptor-blocking peptide, or with other model proteins. Protein release profiles were studied, and retention of the bioactivity of released proteins was verified. Primary dorsal root ganglion neurons were cultured and their ability to grow neurites was challenged with Nogo-A chimeric protein in the absence or presence of Nogo receptor antagonist peptide-loaded alginate nanospheres. Our results demonstrate that peptide released from alginate nanospheres could overcome the growth inhibitory effect of Nogo-A, suggesting that a similar peptide delivery strategy using alginate nanospheres might be used to improve axon regeneration within the injured central nervous system.

Experimental subarachnoid haemorrhage results in multifocal axonal injury.

The great majority of acute brain injury results from trauma or from disorders of the cerebrovasculature, i.e. ischaemic stroke or haemorrhage. These injuries are characterized by an initial insult that triggers a cascade of injurious cellular processes. The nature of these processes in spontaneous intracranial haemorrhage is poorly understood. Subarachnoid haemorrhage, a particularly deadly form of intracranial haemorrhage, shares key pathophysiological features with traumatic brain injury including exposure to a sudden pressure pulse. Here we provide evidence that axonal injury, a signature characteristic of traumatic brain injury, is also a prominent feature of experimental subarachnoid haemorrhage. Using histological markers of membrane disruption and cytoskeletal injury validated in analyses of traumatic brain injury, we show that axonal injury also occurs following subarachnoid haemorrhage in an animal model. Consistent with the higher prevalence of global as opposed to focal deficits after subarachnoid haemorrhage and traumatic brain injury in humans, axonal injury in this model is observed in a multifocal pattern not limited to the immediate vicinity of the ruptured artery. Ultrastructural analysis further reveals characteristic axonal membrane and cytoskeletal changes similar to those associated with traumatic axonal injury. Diffusion tensor imaging, a translational imaging technique previously validated in traumatic axonal injury, from these same specimens demonstrates decrements in anisotropy that correlate with histological axonal injury and functional outcomes. These radiological indicators identify a fibre orientation-dependent gradient of axonal injury consistent with a barotraumatic mechanism. Although traumatic and haemorrhagic acute brain injury are generally considered separately, these data suggest that a signature pathology of traumatic brain injury-axonal injury-is also a functionally significant feature of subarachnoid haemorrhage, raising the prospect of common diagnostic, prognostic, and therapeutic approaches to these conditions.

Peripheral Nerve Diffusion Tensor Imaging: Assessment of Axon and Myelin Sheath Integrity.

PURPOSE: To investigate the potential of diffusion tensor imaging (DTI) parameters as in-vivo biomarkers of axon and myelin sheath integrity of the median nerve in the carpal tunnel as validated by correlation with electrophysiology. METHODS: MRI examinations at 3T including DTI were conducted on wrists in 30 healthy subjects. After manual segmentation of the median nerve quantitative analysis of fractional anisotropy (FA) as well as axial, radial and mean diffusivity (AD, RD, and MD) was carried out. Pairwise Pearson correlations with electrophysiological parameters comprising sensory nerve action potential (SNAP) and compound muscle action potential (CMAP) as markers of axon integrity, and distal motor latency (dml) and sensory nerve conduction velocity (sNCV) as markers of myelin sheath integrity were computed. The significance criterion was set at P=0.05, Bonferroni corrected for multiple comparisons. RESULTS: DTI parameters showed a distinct proximal-to-distal profile with FA, MD, and RD extrema coinciding in the center of the carpal tunnel. AD correlated with CMAP (r=0.50, p=0.04, Bonf. corr.) but not with markers of myelin sheath integrity. RD correlated with sNCV (r=-0.53, p=0.02, Bonf. corr.) but not with markers of axon integrity. FA correlated with dml (r=-0.63, p=0.002, Bonf. corr.) and sNCV (r=0.68, p=0.001, Bonf. corr.) but not with markers of axon integrity. CONCLUSION: AD reflects axon integrity, while RD (and FA) reflect myelin sheath integrity as validated by correlation with electrophysiology. DTI parameters consistently indicate a slight decrease of structural integrity in the carpal tunnel as a physiological site of median nerve entrapment. DTI is particularly sensitive, since these findings are observed in healthy participants. Our results encourage future studies to evaluate the potential of DTI in differentiating axon from myelin sheath injury in patients with manifest peripheral neuropathies.

Ultrasound of the peripheral nerves in systemic vasculitic neuropathies.

INTRODUCTION: Ultrasound of the peripheral nerves (PNUS) can be used to visualize nerve pathologies in polyneuropathies (PNP). The aim of this study was to investigate, whether PNUS provides additional information in patients with proven systemic vasculitic neuropathies (VN). MATERIAL AND METHODS: Systematic ultrasound measurements of several peripheral nerves, the vagal nerve and the 6th cervical nerve root were performed in 14 patients and 22 healthy controls. Nerve conduction studies of the corresponding nerves were undertaken. Finally, the measured results were compared to a study population of demyelinating immune-mediated and axonal neuropathies. RESULTS: Patients with VN displayed significant smaller amplitudes of compound muscle action potentials (CMAP) (p<0.05) and sensory nerve action potentials (SNAP) compared to healthy controls, while conduction velocity did not differ between groups. The mean nerve cross-sectional areas (CSA) were increased in several peripheral nerves compared to the controls, most prominent in tibial and fibular nerve (p<0.01). PNUS revealed nerve enlargement in most of the clinically and electrophysiologically affected nerves (22 out of 31) in VN. Nerve enlargement was more often seen in vasculitic neuropathies than in other axonal neuropathies, but significantly rarer than in demyelinating neuropathies. CONCLUSION: Focal CSA enlargement in one or more nerves in electrophysiologically axonal neuropathies can be a hint for VN and thus facilitate diagnostic and therapeutic procedures.

Nonparametric pore size distribution using d-PFG: comparison to s-PFG and migration to MRI.

Here we present the successful translation of a pore size distribution (PSD) estimation method from NMR to MRI. This approach is validated using a well-characterized MRI phantom consisting of stacked glass capillary arrays (GCA) having different diameters. By employing a double pulsed-field gradient (d-PFG) MRI sequence, this method overcomes several important theoretical and experimental limitations of previous single-PFG (s-PFG) based MRI methods by allowing the relative diffusion gradients' direction to vary. This feature adds an essential second dimension in the parameters space, which can potentially improve the reliability and stability of the PSD estimation. To infer PSDs from the MRI data in each voxel an inverse linear problem is solved in conjunction with the multiple correlation function (MCF) framework, which can account for arbitrary experimental parameters (e.g., long diffusion pulses). This scheme makes no a priori assumptions about the functional form of the underlying PSD. Creative use of region of interest (ROI) analysis allows us to create different underlying PSDs using the same GCA MRI phantom. We show that an s-PFG experiment on the GCA phantom fails to accurately reconstruct the size distribution, thus demonstrating the superiority of the d-PFG experiment. In addition, signal simulations corrupted by different noise levels were used to generate continuous and complex PSDs, which were then successfully reconstructed. Finally, owing to the reduced q- or b- values required to measure microscopic PSDs via d-PFG MRI, this method will be better suited to biomedical and clinical applications, in which gradient strength of scanners is limited.

Giant scaffolding protein AHNAK1 interacts with ß-dystroglycan and controls motility and mechanical properties of Schwann cells.

The profound morphofunctional changes that Schwann cells (SCs) undergo during their migration and elongation on axons, as well as during axon sorting, ensheathment, and myelination, require their close interaction with the surrounding laminin-rich basal lamina. In contrast to myelinating central nervous system glia, SCs strongly and constitutively express the giant scaffolding protein AHNAK1, localized essentially underneath the outer, abaxonal plasma membrane. Using electron microscopy, we show here that in the sciatic nerve of ahnak1(-) (/) (-) mice the ultrastructure of myelinated, and unmyelinated (Remak) fibers is affected. The major SC laminin receptor ß-dystroglycan co-immunoprecipitates with AHNAK1 shows reduced expression in ahnak1(-) (/) (-) SCs, and is no longer detectable in Cajal bands on myelinated fibers in ahnak1(-) (/) (-) sciatic nerve. Reduced migration velocity in a scratch wound assay of purified ahnak1(-) (/) (-) primary SCs cultured on a laminin substrate indicated a function of AHNAK1 in SC motility. This was corroborated by atomic force microscopy measurements, which revealed a greater mechanical rigidity of shaft and leading tip of ahnak1(-) (/) (-) SC processes. Internodal lengths of large fibers are decreased in ahnak1(-) (/) (-) sciatic nerve, and longitudinal extension of myelin segments is even more strongly reduced after acute knockdown of AHNAK1 in SCs of developing sciatic nerve. Together, our results suggest that by interfering in the cross-talk between the transmembrane form of the laminin receptor dystroglycan and F-actin, AHNAK1 influences the cytoskeleton organization of SCs, and thus plays a role in the regulation of their morphology and motility and lastly, the myelination process.

A novel CaM kinase II pathway controls the location of neuropeptide release from Caenorhabditis elegans motor neurons.

Neurons release neuropeptides via the regulated exocytosis of dense core vesicles (DCVs) to evoke or modulate behaviors. We found that Caenorhabditis elegans motor neurons send most of their DCVs to axons, leaving very few in the cell somas. How neurons maintain this skewed distribution and the extent to which it can be altered to control DCV numbers in axons or to drive release from somas for different behavioral impacts is unknown. Using a forward genetic screen, we identified loss-of-function mutations in UNC-43 (CaM kinase II) that reduce axonal DCV levels by ∼90% and cell soma/dendrite DCV levels by ∼80%, leaving small synaptic vesicles largely unaffected. Blocking regulated secretion in unc-43 mutants restored near wild-type axonal levels of DCVs. Time-lapse video microscopy showed no role for CaM kinase II in the transport of DCVs from cell somas to axons. In vivo secretion assays revealed that much of the missing neuropeptide in unc-43 mutants is secreted via a regulated secretory pathway requiring UNC-31 (CAPS) and UNC-18 (nSec1). DCV cargo levels in unc-43 mutants are similarly low in cell somas and the axon initial segment, indicating that the secretion occurs prior to axonal transport. Genetic pathway analysis suggests that abnormal neuropeptide function contributes to the sluggish basal locomotion rate of unc-43 mutants. These results reveal a novel pathway controlling the location of DCV exocytosis and describe a major new function for CaM kinase II.

Relation between dopamine synthesis capacity and cell-level structure in human striatum: a multi-modal study with positron emission tomography and diffusion tensor imaging.

Positron emission tomography (PET) study has shown that dopamine synthesis capacity varied among healthy individuals. This interindividual difference might be due to a difference in the cell-level structure of presynaptic dopaminergic neurons, i.e., cellular density and/or number. In this study, the relations between the dopamine synthesis capacity measured by PET and the parameter estimates in diffusion tensor imaging (DTI) in striatal subregions were investigated in healthy human subjects. DTI and PET studies with carbon-11 labeled L-DOPA were performed in ten healthy subjects. Age-related changes in the above parameters were also considered. Fractional anisotropy showed a significant positive correlation with age in the posterior caudate. There was significant negative correlation between dopamine synthesis capacity and mean diffusivity in the posterior caudate and putamen. Assuming that mean diffusivity reflects the density of wide-spreading axonal terminals in the striatum, the result suggests that dopamine synthesis may be related to the density of dopaminergic neuronal fibers. It is evident that PET/DTI combined measurements can contribute to investigations of the pathophysiology of neuropsychiatric diseases involving malfunction of dopaminergic neurons.

Objective assessment of C-fiber function by electrically induced axon reflex flare in patients with axonal and demyelinating polyneuropathy.

INTRODUCTION: A simple test to evaluate the peripheral C-fiber function is the measurement of axon reflex flare area. In this study, we compared the flare area in healthy subjects and in two groups of patients with predominantly axonal or demyelinating polyneuropathy. MATERIALS AND METHODS: We examined 42 control subjects and 33 patients. The flare responses were elicited by the application of transcutaneous electrical stimulation and recorded by laser Doppler imaging. RESULTS: There was a significant reduction of electrically induced flare area in both groups of neuropathy patients (P < 0.001; analysis of covariance). Interestingly, patients with an axonal neuropathy had a significantly stronger reduction of flare size as compared to patients with demyelinating neuropathy (P = 0.03). CONCLUSIONS: The evaluation of the axon flare response in the arm can be used as a screening test of impaired C-fiber function in polyneuropathy patients with the advantages of simplicity of the procedure and time economy.

Assessing prenatal white matter connectivity in commissural agenesis.

Complete or partial agenesis of the corpus callosum are rather common developmental abnormalities, resulting in a wide spectrum of clinical neurodevelopmental deficits. Currently, a significant number of these cases are detected by prenatal sonography during second trimester screening examinations. However, major uncertainties about a detailed morphological diagnosis and the clinical significance do not allow accurate prenatal counselling. Here, we were able to demonstrate the 3D connectivity of aberrant commissural tracts in 16 cases with complete and four cases with partial callosal agenesis using the foetal magnetic resonance imaging techniques of diffusion tensor imaging and tractography in utero and in vivo between gestational weeks 20 and 37. The 'misguided' pre-myelinated callosal axons that represent the bundle of Probst were non-invasively visualized, and they showed a degree of structural integrity similar to that of the callosal pathways of age-matched foetuses without cerebral pathologies. In two foetuses, we were able to prove, by post-mortem histology, that diffusion tensor imaging allows the depiction of the bundle of Probst, even during early stages of pre-myelination at 20 and 22 gestational weeks. In cases with partial callosal agenesis, an aberrant sigmoid-shaped bundle was prenatally depicted, confirming the findings of heterotopic interhemispheric connectivity in adults with partial callosal agenesis. In addition to the corpus callosum, other white matter pathways were also involved, including somatosensory and motor pathways that showed significantly higher fractional anisotropy values in cases with callosal agenesis compared with control subjects. A detailed prenatal assessment of abnormal white matter connectivity in cases of midline anomalies will help to explain and understand the clinical heterogeneity in these cases, taking future foetal neurological counselling strategies to a new level.

Ultrasound as a useful tool in the diagnosis and management of traumatic nerve lesions.

OBJECTIVE: The possibility of depicting through ultrasound (US) the nerve and its surroundings should be very useful in traumatic nerve lesion (TNL) management. Our study aimed at evaluating the contribution of US as complementary tool in a neurophysiological laboratory for the diagnosis and management of TNL. METHODS: A total of 112 nerves from 98 consecutive patients with clinical suspicion of TNL were considered. Two independent and blinded clinicians, different from the examiners performing electrodiagnosis and US, classified clinical, neurophysiological and US findings and classified the contribution of US as follows: 'contributive' and 'non-contributive' if US confirmed the clinical and neurophysiological diagnosis or if US findings were unremarkable. RESULTS: US was 'contributive' (strongly modified the diagnostic and therapeutic path) in 58% of cases (n: 65) providing information on therapeutic approach (immediate or delayed surgery), diagnosis and follow-up. US specifically contributed to the (1) assessment of nerve continuity/discontinuity, hence neurotmesis/axonotmesis; (2) identification of aetiology; and (3) demonstration of multiple sites of damage. US was contributive mainly in cases with neurophysiological evidence of complete axonal damage. CONCLUSIONS: US should be used, when available, in all patients in whom TNL is suspected as it provides a more comprehensive diagnosis than neurophysiologic studies alone. Anatomical information is often crucial for choosing the most appropriate therapeutic strategies (and for surgical planning). SIGNIFICANCE: US can improve the outcome of TNL.

Transient cranial neuropathies as sequelae of Onyx embolization of arteriovenous shunt lesions near the skull base: possible axonotmetic traction injuries.

INTRODUCTION: Transarterial embolization with Onyx is a relatively safe and increasingly common method of treating cranial dural arteriovenous fistulas (DAVF) and arteriovenous malformations (AVM). Cranial neuropathy resulting from this procedure has been reported. CASE REPORTS: Three case histories are presented in two patients treated for a DAVF and an AVM near the skull base with heavily parasitized supply from external carotid artery branches. Transarterial embolization resulted in transient cranial neuropathies including two cases of lower facial nerve palsy and one case of trigeminal nerve mandibular segment (V3) neuralgia which resolved spontaneously over a few months. Treatment of the DAVF and AVM was otherwise successful. DISCUSSION: The most common cranial neuropathies following transarterial Onyx embolization procedures are facial nerve palsy and V3 neuralgia. The middle meningeal and internal maxillary arteries are common conduits used in dural-based and facial arteriovenous shunt lesions and are in proximity to the facial nerve and maxillary segment of the trigeminal nerve as they exit the skull base. Based on their relative frequency and anatomic proximity, it is surmised that these facial neuropathies are traction-type injuries related to microcatheter extraction from the Onyx cast. CONCLUSIONS: Cranial neuropathies, specifically facial nerve palsy and V3 neuralgia, following transarterial Onyx embolizations are probably axonotmetic traction injuries related to microcatheter extraction. These appear to be self-limiting and resolve over a few months.

Increased nerve vascularization detected by color Doppler sonography in patients with ulnar neuropathy at the elbow indicates axonal damage.

INTRODUCTION: The aim of this study was to establish the prevalence of increased intraneural vascularization detected by ultrasonography (IVUS) in patients with ulnar neuropathy at the elbow (UNE) and to determine its relationship to clinical, ultrasonographic, and electrodiagnostic findings. METHODS: High-resolution ultrasonography and color Doppler imaging were performed in 137 patients with confirmed UNE, 24 patient controls, and 70 healthy controls (HCs). RESULTS: IVUS was found in 21 (15%) of 137 patients with UNE, in 1 (4%) of 24 patient controls, and in 0 of 70 HCs (P = 0.001). Patients with IVUS were more likely to have severe weakness (P = 0.01), severe atrophy of ulnar-innervated muscles (P = 0.008), axonal damage (P = 0.001), and more pronounced nerve enlargement (P = 0.03) than those without IVUS. CONCLUSIONS: IVUS in the ulnar nerve can be detected in patients with UNE and is associated with nerve enlargement and clinical and electrodiagnostic severity. In addition, IVUS is associated with axonal damage.

Ultrasonography of ulnar neuropathy at the elbow: axonal involvement leads to greater nerve swelling than demyelinating nerve lesion.

OBJECTIVE: To evaluate nerve size parameters measured by ultrasound in patients with ulnar neuropathy at the elbow (UNE) and to correlate them with the type of nerve lesion. METHODS: The largest cross sectional area (CSA(max)) of the ulnar nerve around the elbow and the cubital-to-humeral nerve area ratio (CHR) were measured in 50 elbows with UNE and in 87 elbows of 50 healthy subjects. CSA(max) and CHR were compared between controls and patients with predominantly demyelinative and axonal nerve involvement. Subgroups of patients with pure sensory and mixed sensorimotor axonal lesion were also compared. RESULTS: In patients with axonal nerve involvement, a significantly larger CSA(max) and CHR were found when compared to those with predominantly demyelinating nerve lesion; both groups differed significantly from healthy controls. CSA(max) values in patients with sensorimotor axonal lesion were significantly higher than in those with pure sensory axonal involvement. CONCLUSION: CSA(max) and CHR highly correlate with the type of nerve pathology in UNE, with a significantly larger nerve swelling seen in axonal lesions, as compared to demyelinating lesions. SIGNIFICANCE: In addition to helping in the localization of nerve lesion, ultrasonography may also reflect the type and degree of nerve lesion as assessed by electrophysiological means.

Impact of DTI smoothing on the study of brain aging.

Diffusion tensor magnetic resonance imaging (DTI), a method for measuring the integrity of axon fiber tracts in the brain, plays an important role in clarifying brain changes that accompany aging and aging-associated neurodegenerative disease. While DTI smoothing methods theoretically have the potential to enhance such studies by reducing noise, it is unclear whether DTI smoothing has any practical impact on computed associations between fiber tract integrity and scientific variables of interest. Therefore we smoothed DTI images from 154 older adults using three kernel smoothing methods hypothesized to have differing strengths (the affine and log-Euclidean smoothers were hypothesized to enhance highly organized tracts better than the Euclidean smoother). Smoothing increased the strengths of expected associations between DTI and age, cognitive function, and the diagnosis of dementia. However, no particular smoothing method was uniformly superior in strengthening these associations. This data suggests that DTI smoothing enhances the sensitivity of studies of brain aging, but further research is needed to determine which smoothing technique is optimal.

Ultrasound imaging of regenerating rat sciatic nerves in situ.

Following clinical or veterinary peripheral nerve trauma, it is critical to localize the site of nerve injury, determine its type, whether a crush, maceration or transection, which will indicate whether and where surgical intervention is required, and subsequently to follow the process of axon regeneration. Typical surface ultrasound probes provide resolution of more than 1mm, sufficient detail for clinically relevant data from tissue in situ, such as heart valves, organs and fetal development. Higher resolution ultrasound nerve imaging yields data to the fascicular level and allows the following of the anatomical course of a nerve, but does not allow imaging of single axons or even groups of axons, which is required to study the process of axon regeneration, neurological recovery and other important clinical and basic science questions. More significant data could be acquired with even higher frequency, and therefore higher resolution, ultrasound imaging. The present study, using a rat sciatic nerve lesion model, was performed to determine whether a new ultrasound imaging device with 30 microm resolution would allow imaging of nerve anatomy and regenerating axons, and whether the data collected from a nerve in situ was the same as when the nerve was surgically exposed. Although the increased ultrasound resolution provided good anatomical detail on the location and type of nerve damage was nearly identical for nerves in situ and when exposed, the resolution was insufficient for imaging regenerating axons. Thus, an even higher resolution ultrasound device is required to allow non-invasive imaging of axons in situ.

Automatic identification and morphometry of optic nerve fibers in electron microscopy images.

The neuroanatomical morphology of the optic nerve is an important description for understanding different aspects like topological distribution of nerves. Manual identification and morphometry has been usually considered as tedious, time consuming, and susceptible to error. A method that automates the identification and analysis of axons from electron micrographic images is presented. First, using region growing approach binarizes the image by combining the feature information together with spatial information, and obtains a coarse classification between myelin and non-myelin pixels. Next, identifies the axon candidates by region labeling and remove false axons on the basis of the identification ruler. Then the connected myelin sheaths are separated from each other using the maximum gradient magnitude of the outer annulus. Finally, analyses the morphological data of fibers. The developed method has been tested on a number of optic nerve images and results were presented. Regional distributions of axon caliber were unimodal. The thickness of the myelin sheath was highly correlated with the fiber diameter; hence, myelin sheath width was also distributed in a unimodal manner.

Histological validation of ultrasound-guided neurography in early nerve regeneration.

Ultrasound-guided near-nerve neurography is a new tool that can be used to assess nerve regeneration before reinnervation occurs. In this study, ultrasound-guided near-nerve measurements were validated against axon diameter counts in rabbits during a 15-week regeneration period after a crush lesion of their peroneal nerve. The course of the nerve was determined ultrasonically, and the active near-nerve needle electrode was maneuvered just next to the nerve under ultrasound guidance. Measured action potentials were compared with axon diameter counts from histological sections of these same nerves. A moderate to good positive correlation was found, which reached a maximum of 0.7 at a cut-off of 3 microm, corresponding to the minimal size of the myelinated axons. Our results suggest that, following a similar validation study in humans, ultrasound-guided near-nerve neurography may be clinically useful when early evaluation of nerve activity is needed.