Human Nerve Fibers Research Articles

Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers.

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to isolate nerve-specific mitochondria and evaluate disease-induced alterations of mitochondria in the distal tip of sensory nerves. The protocol combines fluorescence immunohistochemistry, confocal microscopy and 3D image analysis techniques to visualize and quantify nerve-specific mitochondria. Detailed parameters are defined throughout the procedures in order to provide a concrete example of how to use these techniques to isolate nerve-specific mitochondria. Antibodies were used to label nerve and mitochondrial signals within tissue sections of skin punch biopsies, which was followed by indirect immunofluorescence to visualize nerves and mitochondria with a green and red fluorescent signal respectively. Z-series images were acquired with confocal microscopy and 3D analysis software was used to process and analyze the signals. It is not necessary to follow the exact parameters described within, but it is important to be consistent with the ones chosen throughout the staining, acquisition and analysis steps. The strength of this protocol is that it is applicable to a wide variety of circumstances where one fluorescent signal is used to isolate other signals that would otherwise be impossible to study alone.

Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to isolate nerve-specific mitochondria and evaluate disease-induced alterations of mitochondria in the distal tip of sensory nerves. The protocol combines fluorescence immunohistochemistry, confocal microscopy and 3D image analysis techniques to visualize and quantify nerve-specific mitochondria. Detailed parameters are defined throughout the procedures in order to provide a concrete example of how to use these techniques to isolate nerve-specific mitochondria. Antibodies were used to label nerve and mitochondrial signals within tissue sections of skin punch biopsies, which was followed by indirect immunofluorescence to visualize nerves and mitochondria with a green and red fluorescent signal respectively. Z-series images were acquired with confocal microscopy and 3D analysis software was used to process and analyze the signals. It is not necessary to follow the exact parameters described within, but it is important to be consistent with the ones chosen throughout the staining, acquisition and analysis steps. The strength of this protocol is that it is applicable to a wide variety of circumstances where one fluorescent signal is used to isolate other signals that would otherwise be impossible to study alone.

Sensitivity to ischaemia of single sympathetic nerve fibres innervating the dorsum of the human foot

Changes in nerve conduction velocity following an impulse (i.e. velocity recovery cycles) reflect after-potentials, and can provide an indication of altered nerve membrane properties. This study used microneurography to assess the effects of ischaemia on single human sympathetic fibres innervating the dorsum of the foot. It was found that velocity recovery cycles can distinguish whether a sympathetic nerve fibre is depolarized or not. The method may be used to detect membrane depolarization of sympathetic nerve fibres in human patients when autonomic neuropathy is suspected. The aim of this study was to determine whether velocity recovery cycles (VRCs) could detect the effects of ischaemia on sympathetic nerve fibres. VRCs of human sympathetic nerve fibres of the superficial peroneal nerve innervating the dorsum of the foot were recorded by microneurography in seven healthy volunteers. Sympathetic nerve fibres were identified by studying their response to manoeuvres increasing sympathetic outflow and by measuring activity-dependent slowing at 2Hz stimulation. VRCs were assessed at rest, during 30min of induced limb ischaemia and during 20min of recovery after ischaemia. From each VRC was measured the relative refractory period (RRP), the supernormality and the time to peak supernormality (SN@). During ischaemia, RRP increased from the baseline value of 37.4±8.7ms (mean±SEM) to 67.1±12.1ms (P<0.01) and SN@ increased from 68.6±9.8ms to 133.8±11.0ms (P<0.005). The difference between SN@ and RRP separated ischaemic from non-ischaemic sympathetic nerve fibres. It is concluded that these sympathetic nerve fibres are sensitive to ischaemia, and that VRCs provide a method to study changes of axonal membrane potential of human sympathetic nerve fibres invivo.

Corneal Nerve Fiber Structure, Its Role in Corneal Function, and Its Changes in Corneal Diseases.

Recently, in vivo confocal microscopy is used to examine the human corneal nerve fibers morphology. Corneal nerve fiber architecture and its role are studied in healthy and pathological conditions. Corneal nerves of rats were studied by nonspecific acetylcholinesterase (NsAchE) staining. NsAchE-positive subepithelial (stromal) nerve fiber has been found to be insensitive to capsaicin. Besides, NsAchE-negative but capsaicin-sensitive subbasal nerve (leash) fibers formed thick mesh-like structure showing close interconnections and exhibit both isolectin B4- and transient receptor potential vanilloid channel 1- (TRPV1-) positive. TRPV1, TRPV3, TRPA (ankyrin) 1, and TRPM (melastatin) 8 are expressed in corneal nerve fibers. Besides the corneal nerve fibers, the expressions of TRPV (1, 3, and 4), TRPC (canonical) 4, and TRPM8 are demonstrated in the corneal epithelial cell membrane. The realization of the importance of TRP channels acting as polymodal sensors of environmental stresses has identified potential drug targets for corneal disease. The pathophysiological conditions of corneal diseases are associated with disruption of normal tissue innervation, especially capsaicin-sensitive small sensory nerve fibers. The relationships between subbasal corneal nerve fiber morphology and neurotrophic keratopathy in corneal diseases are well studied. The recommended treatment for neurotrophic keratopathy is administration of preservative free eye drops.

Membrane properties in small cutaneous nerve fibers in humans

Assessment of membrane properties is important for understanding the mechanisms of painful peripheral neuropathy, developing new diagnostic techniques, and screening/profiling of analgesics that target ion channels. Small cutaneous nerves were activated electrically by small diameter (0.2 mm) cathodes, and large nerves were activated by ordinary patch electrodes. This new perception threshold tracking method combines perception threshold assessment and stimulation paradigms from conventional threshold tracking. The strength-duration time-constant of large fibers (580 µs ± 160 µs) was lower than the time constant of small fibers (1060 µs ± 690 µs; P < 0.01, paired t-test). Threshold electrotonus showed similar threshold reductions to sub-threshold prepulses, except for 80 ms hyperpolarizing prepulses, to which small fibers showed less threshold reduction than large fibers (repeated-measures analysis of variance, Bonferroni, P = 0.006). This is a reliable method to investigate the membrane properties of small cutaneous nerve fibers in humans and may be used in clinical settings as a diagnostic or profiling tool. Muscle Nerve 55: 195-201, 2017.

Can cross-talk occur in human myelinated nerve fibers?

The possibility that impulse cross-talk can occur between myelinated human nerve fibers was explored. Instances of impulse conduction without decrement were found, and published recordings of compound action potentials of functionally homogeneous fibers were scrutinized. Both analytical approaches yielded results consistent with cross-talk occurring in some nerves after electrical stimulation. The possible ionic current paths in and out of neighboring fibers, which could be responsible for the phenomenon, have been considered in the light of seminal work on unmyelinated single axons. Muscle Nerve 54: 361-365, 2016.

Theoretical predication of temperature effect on conducting processes in simulated amyotrophic lateral sclerosis at 20-40[Formula: see text]C.

The present study investigates action potential abnormalities in previously simulated cases of amyotrophic lateral sclerosis, termed as ALS1, ALS2 and ALS3, respectively, when the temperature is changed from 20[Formula: see text]C to 42[Formula: see text]C.These ALS cases are modeled as three progressively severe axonal abnormalities. The effects of temperature on the kinetics of currents, defining action potentials in the normal and abnormal cases, are also given and discussed. These computations use our temperature-dependent multi-layered model of human motor nerve fibers. The results show that the classical "transient" sodium current ([Formula: see text]) contributes mainly to the nodal action potential generation in the normal and abnormal cases for the temperature range of 20-39[Formula: see text]C, as the contribution of fast and slow potassium currents ([Formula: see text] and [Formula: see text]) to the total ionic current ([Formula: see text]) is negligible. However, the contribution of [Formula: see text] and [Formula: see text] to the membrane repolarization is enhanced at temperatures higher than 39[Formula: see text]C, especially at 42[Formula: see text]C, and the after-potentials are hyperpolarized in the normal and ALS1 cases, while, they are re-depolarized in the ALS2 and ALS3 cases. The ionic channels beneath the myelin sheath are insensitive to the short-lasting current stimuli and do not contribute to the internodal action potential generation for the normal and abnormal cases in the whole investigated temperature range. Nevertheless that the uniform axonal dysfunction progressively increases in the nodal and internodal segments of each next simulated ALS case, the action potentials cannot be regarded as definitive indicators for the progressive degrees of this disease, when the temperature is changed from 20[Formula: see text]C to 42[Formula: see text]C. However, the results are essential for the interpretation of mechanisms of action potential measurements in ALS patients with symptoms of cooling, warming and fever, which can result from alteration in body temperature. Our results also suggest that the conducting processes in patients with ALS are in higher risk during hyperthermia ([Formula: see text]C) than hypothermia ([Formula: see text]C).

Hyperglycemia- and neuropathy-induced changes in mitochondria within sensory nerves.

ObjectiveThis study focused on altered mitochondrial dynamics as a potential mechanism for diabetic peripheral neuropathy (DPN). We employed both an in vitro sensory neuron model and an in situ analysis of human intraepidermal nerve fibers (IENFs) from cutaneous biopsies to measure alterations in the size distribution of mitochondria as a result of hyperglycemia and diabetes, respectively.MethodsNeurite- and nerve-specific mitochondrial signals within cultured rodent sensory neurons and human IENFs were measured by employing a three-dimensional visualization and quantification technique. Skin biopsies from distal thigh (DT) and distal leg (DL) were analyzed from three groups of patients; patients with diabetes and no DPN, patients with diabetes and confirmed DPN, and healthy controls.ResultsThis analysis demonstrated an increase in mitochondria distributed within the neurites of cultured sensory neurons exposed to hyperglycemic conditions. Similar changes were observed within IENFs of the DT in DPN patients compared to controls. This change was represented by a significant shift in the size frequency distribution of mitochondria toward larger mitochondria volumes within DT nerves of DPN patients. There was a length-dependent difference in mitochondria within IENFs. Distal leg IENFs from control patients had a significant shift toward larger volumes of mitochondrial signal compared to DT IENFs.InterpretationThe results of this study support the hypothesis that altered mitochondrial dynamics may contribute to DPN pathogenesis. Future studies will examine the potential mechanisms that are responsible for mitochondrial changes within IENFs and its effect on DPN pathogenesis.

Expression of 5-HT3 receptors and TTX resistant sodium channels (Na(V)1.8) on muscle nerve fibers in pain-free humans and patients with chronic myofascial temporomandibular disorders.

BackgroundPrevious studies have shown that 5-HT3-antagonists reduce muscle pain, but there are no studies that have investigated the expression of 5-HT3-receptors in human muscles. Also, tetrodotoxin resistant voltage gated sodium-channels (NaV) are involved in peripheral sensitization and found in trigeminal ganglion neurons innervating the rat masseter muscle. This study aimed to investigate the frequency of nerve fibers that express 5-HT3A-receptors alone and in combination with NaV1.8 sodium-channels in human muscles and to compare it between healthy pain-free men and women, the pain-free masseter and tibialis anterior muscles, and patients with myofascial temporomandibular disorders (TMD) and pain-free controls.MethodsThree microbiopsies were obtained from the most bulky part of the tibialis and masseter muscles of seven and six healthy men and seven and six age-matched healthy women, respectively, while traditional open biopsies were obtained from the most painful spot of the masseter of five female patients and from a similar region of the masseter muscle of five healthy, age-matched women. The biopsies were processed by routine immunohistochemical methods. The biopsy sections were incubated with monoclonal antibodies against the specific axonal marker PGP 9.5, and polyclonal antibodies against the 5-HT3A-receptors and NaV1.8 sodium-channels.ResultsA similar percentage of nerve fibers in the healthy masseter (85.2%) and tibialis (88.7%) muscles expressed 5-HT3A-receptors. The expression of NaV1.8 by 5-HT3A positive nerve fibers associated with connective tissue was significantly higher than nerve fibers associated with myocytes (P < .001). In the patients, significantly more fibers per section were found with an average of 3.8 ± 3 fibers per section in the masseter muscle compared to 2.7 ± 0.2 in the healthy controls (P = .024). Further, the frequency of nerve fibers that co-expressed NaV1.8 and 5-HT3A receptors was significantly higher in patients (42.6%) compared to healthy controls (12.0%) (P < .001).ConclusionsThis study showed that the 5-HT3A-receptor is highly expressed in human masseter and tibialis muscles and that there are more nerve fibers that express 5-HT3A-receptors in the masseter of women with myofascial TMD compared to healthy women. These findings indicate that 5-HT3-receptors might be up-regulated in myofascial TMD and could serve as potential biomarkers of chronic muscle pain.

Reduced expression of Munc13-1 in human and porcine diabetic peripheral nerve

Peripheral neuropathy (PN) involves widespread peripheral nerve disorders affecting a large human population worldwide. In Europe and the United States, the first single most prominent cause of peripheral neuropathy is diabetes, affecting 60–70% patients with long-term diabetes followed by idiopathic neuropathy, peripheral nerve damage of unknown etiology, diagnosed in 10–40% of all patients admitted to hospitals with symptoms of peripheral nerve damage. The molecular mechanisms underlying the pathogenesis of this disorder are not yet fully understood, however a few potential molecular contributors, such as Munc13-1, have been recently suggested. Munc13-1 is a diacylglycerol (DAG) receptor and a multifunction active zone protein essential for synaptic vesicle priming and crucial for insulin release from pancreatic beta cells. Here, for the first time, we focused on the comparative expression of Munc13-1 in human and porcine peripheral nerves. Our results revealed significantly reduced number of Munc13-1 in human (64.26%±6.68%) and porcine (84.09%±2.21%) diabetic nerve fibers and lower number of the double stained, neuronal marker, Neurofilament (NF) and Munc13-1 positive, human (56.83%±3.77%) and porcine (65.87%±4.86%) nerve fibers. Optical density quantification of Western blots showed similar results. Our study indicates that Munc13-1, on account of its role in both insulin and neurotransmitter exocytosis and through its binding properties, may be an important factor contributing to the development or progression of diabetic neuropathy.

Mean retinal ganglion cell axon diameter varies with location in the human retina

We examined retinal ganglion cell (RGC) axon diameters within the human retinal nerve fibre layer in order to provide information about the possible effects of axonal stimulation when a retinal prosthesis includes the nonfoveal regions. Five pairs of eyes were obtained from donors aged from 48 to 84 years. Fixation delay ranged from 5 to 22 h. Tissue was processed for electron microscopy. Inferior and/or nasal RGC axons were on average larger than superior and/or temporal axons. The inferior retina contained some very large axons. Foveal axons were on average smaller than nonfoveal axons and had a size distribution that suggested different size groupings not seen in other samples. Peripheral versus central axons within the superior and inferior nasal retinal samples were compared; peripheral axons were significantly larger, unlike the inferior temporal samples or the samples nasal to the optic disc. Stimulus-current thresholds will change as a retinal prosthesis increases in size and encroaches on the nonfoveal axons. These changes can be anticipated based on mean axon diameter. Knowing in advance the possible outcome of electrical stimulation of the axonal population may help refine prosthetic procedures and patient training.

The relationship between Bell’s palsy and morphometric aspects of the facial nerve

Epidemiological data of Bell's palsy (BP) have been reported. For example, the annual incidence of BP is 15-30 per 100,000 persons, with equal numbers of men and women affected, and there is no predilection for either side of the face. However, details of the relationship between BP and morphometric aspects of the facial nerve have not been available in textbooks. We performed a morphometric analysis of human facial nerve fibers and estimated the total number of myelinated axons (TN) and average transverse area of myelinated axons (ATA). The facial nerve showed a significant decrease of TN with increasing age (r = -0.77; p < 0.01), but showed no significant changes of ATA with age (r = -0.01; p = 0.96). We supposed that the TN decrease with age was a factor in the delayed recovery from BP seen in the elderly. Moreover, the TN and ATA showed no significant differences between female and male specimens (p < 0.05), or between the right and left side specimens (p < 0.05). Our present results seem to explain the absence of significant sex and affected side differences in BP.

Threshold predictions of different pulse shapes using a human auditory nerve fibre model containing persistent sodium and slow potassium currents

The ability of a human auditory nerve fibre computational model to predict threshold differences for biphasic, pseudomonophasic and alternating monophasic waveforms was investigated. The effect of increasing the interphase gap, interpulse interval and pulse rate on thresholds was also simulated. Simulations were performed for both anodic-first and cathodic-first stimuli. Results indicated that the model correctly predicted threshold reductions for pseudomonophasic compared to biphasic waveforms, although reduction for alternating monophasic waveforms was underestimated. Threshold reductions were more pronounced for cathodic-first stimuli compared to anodic-first stimuli. Reversal of the phases in pseudomonophasic stimuli suggested a threshold reduction for anodic-first stimuli, but a threshold increase in cathodic-first stimuli. Inclusion of the persistent sodium and slow potassium currents in the model resulted in a reasonably accurate prediction of the non-monotonic threshold behaviour for pulse rates higher than 1000 pps. However, the model did not correctly predict the threshold changes observed for low pulse rate biphasic and alternating monophasic waveforms. It was suggested that these results could in part be explained by the difference in the refractory periods between real and simulated auditory nerve fibres, but also by the lack of representation of stochasticity observed in real auditory nerve fibres in our auditory nerve model.

Reproducibility and influence of test modality order on thermal perception and thermal pain thresholds in quantitative sensory testing

Objective To evaluate the reproducibility of quantitative sensory testing (QST), performed with the method-of-limits (MLI) at different test intervals, by assessing the inter- and intra-individual variation of thermal cold (CT) and warm (WT) perception thresholds, and of thermal cold- (CPT) and heat pain (HPT) thresholds. Methods QST with the MLI was performed in 38 healthy subjects in three repeated and pseudo-randomized test sessions, done at three occasions (days 1, 2 and 7). Results At repeated testing, none of the thermal threshold estimates showed systematic significant differences, neither between days nor between sessions within the same day, when determined as first tests (FT), and for CT and WT also after thermal pain assessment (aTPA). However, when determined directly aTPA, both CT and WT were noted significantly higher. Also the coefficients of variation and repeatability showed increased values aTPA. Conclusions The high reproducibility show that the MLI is a feasible method for thermal QST, with reproducible results both at shorter and longer test intervals, on condition that temperature thresholds are determined before any painful thermal stimuli are given, as the latter influence both CT and WT assessments. Significance The findings show that QST with the MLI is a reliable tool for indirect evaluation of human small nerve fiber function.

Effect of external tissue resistivity on threshold level of myelinated nerve fiber

AbstractThe rise of the threshold of electric current stimulation to generate compound action potential of nerve conduction study is considered to have a relationship to malfunctions of the nerve. The effect of the decrease of the resistivity of the external tissue and the thickness of myelin sheaths was investigated by computer simulation. A myelinated human nerve fiber dipped in the homogeneous conductor was stimulated with a monopolar cathode located outside the axon. The rise of the threshold by demyelination was found to be comparable to the effect of the decrease of the resistivity of the external tissue by a few Ωm when the external resistivity is about 10 Ωm. Actually the reduction of the thickness of the myelin sheaths also reduces the resistivity of the external tissue. Therefore, the contribution of both effects in the case of demyelination was estimated. As a result, the contributions of each effect were antagonized. As one of the causes of the rise of the threshold of nerve activation, the decrease of the resistivity of the external tissue is considerable. © 2010 Wiley Periodicals, Inc. Electron Comm Jpn, 93(2): 50–56, 2010; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/ecj.10065

Effects of ultraviolet radiation on human cutaneous nerve fibres

Due to an increasing number of skin diseases as a result of exposure to ultraviolet (UV) radiation, it is necessary to evaluate the effectiveness of new skin care formulations with broad-spectrum sunscreens. This study aims to assess the status of nerve fibres in healthy human skin, to quantify effects of UV radiation on nerve endings, and to evaluate neuroprotective effects of new skin care formulations against UV exposure damage. Samples were obtained from 34 female patients enrolled for plastic surgery and were immediately treated (10 min) with three emulsions: Cream 1, Cream 2 (placebo) and a sunscreen with sun protection factor 15 (SPF15). Control samples and those treated with the cream emulsions were exposed to UVA and UVB for 60 min. Nerve fibres were identified by immunofluorescence using a monoclonal antibody (anti-human CD56/NCAM). Cell damage was assessed by image analysis. Several cellular nervous structures were identified in the skin samples, including free nerve endings. UVA and UVB significantly decreased (40-60%) density of nerve endings in the control samples and those treated with placebo (Cream 2) or SPF15 (all P < 0.001). Cream 1 completely blocked effects of UV radiation on nerve endings (P > 0.05 vs. control). Quantification of cell damage induced by UV radiation provides useful information for identification of new skin care compounds with neuroprotective properties.

Higher sensitivity of human auditory nerve fibers to positive electrical currents

Most contemporary cochlear implants (CIs) stimulate the auditory nerve with trains of amplitude‐modulated, symmetric biphasic pulses. Although both polarities of a pulse can depolarize the nerve fibers and generate action potentials, it remains unknown which of the two (positive or negative) phases has the stronger effect. Animal experiments have shown that cathodic (negative) current flows are more effective than anodic (positive) ones in eliciting neural responses, and this finding has motivated the development of novel speech‐processing algorithms. Here we show electrophysiologically and psychophysically that the human auditory system exhibits the opposite pattern, being more sensitive to anodic stimulation. We measured electrically evoked compound action potentials in CI listeners for phase‐separated pulses, allowing us to tease out the responses to each of the two opposite‐polarity phases. At an equal stimulus level, the anodic phase yielded the larger response. This finding was corroborated by a measure of psychophysical masking patterns and may relate to a particular orientation of the nerve fibers relative to the electrode or to a substantial degeneration/demyelination of the peripheral processes. Potential applications to improve CI speech‐processing strategies are discussed.

Retigabine reduces the excitability of unmyelinated peripheral human axons

Enhancement of membrane K + conductance may reduce the abnormal excitability of primary afferent nociceptive neurons in neuropathic pain. It has been shown that retigabine, a novel anticonvulsant, activates Kv7 (KCNQ/M) channels in the axonal/nodal membrane of peripheral myelinated axons. In this study, we have tested the effects of retigabine on excitability parameters of C-type nerve fibers in isolated fascicles of human sural nerve. Application of retigabine (3–10 μM) produced an increase in membrane threshold. This effect was pronounced in depolarized axons and small in hyperpolarized axons. This finding indicates that retigabine produces a membrane hyperpolarization which is limited by the K + equilibrium potential. The retigabine-induced reduction in excitability was accompanied by modifications of the post-spike recovery cycle. Most notable is the development of a late subexcitability at 250–400 ms following a short burst of action potentials. All effects of retigabine were blocked in the presence of XE991 (10 μM). The data show that Kv7 channels are present on axons of unmyelinated, including nociceptive, peripheral human nerve fibers. It is likely that activation of these channels by retigabine may reduce the ectopic generation of action potentials in neuropathic pain.

Higher Sensitivity of Human Auditory Nerve Fibers to Positive Electrical Currents

Most contemporary cochlear implants (CIs) stimulate the auditory nerve with trains of amplitude-modulated, symmetric biphasic pulses. Although both polarities of a pulse can depolarize the nerve fibers and generate action potentials, it remains unknown which of the two (positive or negative) phases has the stronger effect. Understanding the effects of pulse polarity will help to optimize the stimulation protocols and to deliver the most relevant information to the implant listeners. Animal experiments have shown that cathodic (negative) current flows are more effective than anodic (positive) ones in eliciting neural responses, and this finding has motivated the development of novel speech-processing algorithms. In this study, we show electrophysiologically and psychophysically that the human auditory system exhibits the opposite pattern, being more sensitive to anodic stimulation. We measured electrically evoked compound action potentials in CI listeners for phase-separated pulses, allowing us to tease out the responses to each of the two opposite-polarity phases. At an equal stimulus level, the anodic phase yielded the larger response. Furthermore, a measure of psychophysical masking patterns revealed that this polarity difference was still present at higher levels of the auditory system and was therefore not solely due to antidromic propagation of the neural response. This finding may relate to a particular orientation of the nerve fibers relative to the electrode or to a substantial degeneration and demyelination of the peripheral processes. Potential applications to improve CI speech-processing strategies are discussed.

Morphometric evaluation of the aging process in various human nerve fibers

We carried out a morphometric comparison of tissue sections from the human spinal cord, medulla oblongata, cranial nerves, autonomic nerves and spinal nerves with the help of a very accurate method that includes two-step fixation, nitrocellulose embedding and discriminative staining. We conducted morphometric evaluations to compare various axonal areas between different individuals using a combination of an image analyzer and a high power microscope. Our study showed a negative correlation between age and axonal area in all nerves except the greater splanchnic nerve. We believe that such data would not have been discovered without the use of our sophisticated electronic equipment together with the special preparation method we employed. Recourse to such methodology will allow more precise study of neurohistology and lead to better understanding of the aging process in human beings.